PhD thesis

Ecophysiology of marine invertebrate planktonic larvae: species and community level approach

Eco
siologia de larves planctòniques d’invertebrats marins: aproximació a nivell d’espècies i comunitat

Rodrigo Almeda García

Barcelona - January 2011

Institut de Ciències del Mar (CSIC) Universitat de Barcelona

Ecophysiology of marine invertebrate planktonic larvae: species and community level approach

Ecofisiología de larvas planctónicas de invertebrados marinos: aproximación a nivel de especies y comunidad

Ecofisiologia de larves planctòniques d'invertebrats marins: aproximació a nivell d'espècies i comunitat

Rodrigo Almeda García

Memòria presentada per Rodrigo Almeda García per optar al grau de Doctor per la Universitat de Barcelona. Tesi realitzada al Institut de Ciències del Mar e inscrita al Departament d
Ecologia, Facultat de Biologia, Universitat de Barcelona. Programa de doctorat de Ciències del Mar (Bienni 2003 ‐2005).

Vist i plau Vist i plau Vist i plau del codirector del codirector de la tutora

Rodrigo Almeda Dr. Miquel Alcaraz Dr. Albert Calbet Dra. Montserrat Vidal Doctorando Professor d
investigació Investigador científic Professora titular ICM ‐CSIC ICM ‐CSIC ICM ‐CSIC UB

Barcelona  Gener 2011

A mi abuela Paula ...

A la meva àvia Paula ...

To my grandmother Paula ... Cover : Copepod larva (“nauplius”). Digital image of Wim van Egmond. Contents

Contents 1 General introduction & thesis outline 5 Hypotheses & objectives 33 Results Chapter 1 Seasonal abundance and vertical distribution of zooplankton in NW Mediterranean 39 coastal waters: importance of small planktonic metazoans ( article I ) Chapter 2 Trophic role and carbon budget of metazoan microplankton in northwest Mediterranean 75 coastal waters ( article II ) Chapter 3 Feeding rates and abundance of marine invertebrate planktonic larvae under harmful algal 95 bloom conditions o Vancouver Island ( article III ) Chapter 4 Ecophysiology of early developmental stages of the cyclopoid copepod Oithona davisae 4.1. Eects of temperature and food concentration on survival, development and growth rates of naupliar stages of Oithona davisae (Copepoda, Cyclopoida) ( article IV ) 115 4.2. Feeding rates and gross growth eciencies of larval developmental stages of Oithona 133 davisae (Copepoda, Cyclopoida) ( article V ) 4.3. Metabolic rates and carbon budget of early developmental stages of the marine cyclopoid copepod Oithona davisae ( article VI ) 149 Chapter 5 Ecophysiology of planktonic larvae of the spionid polychaete Polydora ciliata 5.1. Swimming behavior and prey retention of the polychaete larvae Polydora ciliata 167 (Johnston) ( article VII ) 5.2. Feeding and growth kinetics of the planktotrophic larvae of the spionid 181 polychaete Polydora ciliata (Johnston) ( article VIII ) 5.3. Larval growth in the dominant polychaete Polydora ciliata is food-limited in a 193 eutrophic Danish estuary (Iseord) ( article IX )

General discussion 209 Main conclusions 221 Resum/ Resumen (Catalan & Spanish summaries) 225 Reference list 281 Report of the directors 297 Acknowledgements/ Agraïments/ Agradecimientos 305 Annex- Image gallery 309

1

General introduction & thesis outline

“I am quite tired having worked all day at the produce of my net. e number of animals that the net collects is very great & fully explains the manner so many animals of a large size live so far from land. Many of these creatures so low in the scale of nature are most exquisite in their forms & rich in colours. It creates a feeling of wonder that so much beauty should be apparently created for such little purpose.” Johannes Müller’s trip diary, 11 th January 1845

Introduction & thesis outline

General introduction

Most marine invertebrates have a complex life history involving stages of planktonic larval development between the egg and the adult form (Thorson 1950; Strathmann 1987, 1993). These larvae may differ from adults in size, form, habitat, mode of nutrition, and/or ability to disperse (Barnes et al. 1988; Young 2002). The environmental conditions experienced during larval development can have profound effects on the subsequent performance of individuals and cohorts (Pechenik et al. 1998, 2002). Survival and growth of marine invertebrate larval stages can influence species recruitment success and population connectivity, distribution and abundance (Roughgarden et al. 1988; Eckert 2003). However, in spite of the obvious importance of larvae in the life cycles of most marine animals, the ecophysiology of larvae of many ecologically relevant invertebrates remains poorly understood in comparison to our knowledge of the adult phases. Additionally, as important components of zooplankton communities, the trophic role of planktonic larvae in marine food webs should not be neglected.

Brief introduction to the history of invertebrate larval biology

The first crustacean larvae were probably observed by Van Leeuwenhoek in 1699, who appreciated the important morphological diffe rences between the newly hatched forms and the adult stages of the copepod Cyclops (Gurney 1942). However, these larval forms were not initially recognized as such and were classified as the crustacean genus Nauplius by O.F. Müller (1776). Martinus Slabber (1778) was likely the first to correctly identify and illustrate several larvae of benthic invertebrates, but his observations were largely ignored (Fig.1). John Vaughan Thompson (1828, 1830) discovered that small planktonic organisms could be collected in fine mesh nets and he presented the first evidence of metamorphosis in larvae of crabs and barnacles from laboratory observations. After the discoveries of Thompson, the descriptions of larval anatomy and metamorphosis increased in the following decades (e.g. Milne-Edwards 1834-1840; 1842; Sars 1844; Nordman 1846). Many organisms that were previously described as genera (e.g. Nauplius -Müller 1776; Zoea-Bosc 1802; Megalopa -Leach 1815) or minuscule adults (e.g. veliger larvae) were then recognized as marine invertebrate larval stages (MacDonald 1858 ). Johannes Müller (1846, 1850), apparently unaware of Thompson’s findings, improved and

5 Introduction & thesis outline developed the use of net-tows for plankton sampling and discovered and illustrated a wide number of planktonic larval forms from the North Sea. In the following decades, compound microscopes and marine laboratory facilities improved rapidly, which facilitated the study of larval development of representatives of most marine phyla (e.g. Kowalevsky 1867, 1883; Agassiz 1877; Prouho 1892). The discovery of larval forms played a pivotal role in the studies of general evolution and phylogeny. For example, Darwin (1851) employed evidence from larval forms to fully accept that barnacles were crustaceans, and Ernst Haeckel’s biogenetic law (Haeckel 1866) was based largely on larval anatomy. In the early years of the 20 th century, notable contributions included detailed studies of larval morphogenesis and metamorphosis of a large number of species from temperate habitats (e.g. Grandori 1912; Mortensen 1921; Gurney 1930) and, descriptions of larval behavior (e.g. habitat selection by meroplanktonic larvae, Mortensen 1921; Wilson 1932,1948). The Danish marine biologist, Gunnar Thorson (1946, 1950, 1964) provided a detailed description of larval forms and distributions and a comprehensive categorization of larval developmental patterns. Thorson was the first to compile the previous studies on larval biology and put them into an environmental context; he is considered the pioneer of current discipline of larval ecology (Young 2002).

Figure 1. Illustrationsll off planktonic l k llarvae bby Martinus Slabber (from Slabber 1778).

Research on larval ecology, biochemistry and physiology has expanded rapidly in recent decades ( see reviews in McEdward 1995; Anger 2001; Hadfield & Paul 2001). Despite increased contributions to the knowledge of larval biology many areas in this field remain poorly understood.

6 Introduction & thesis outline

The definition of 'larva'. Classification and diversity of marine invertebrate larvae

Animal development that includes larval stages is termed ‘indirect development’; this is in contrast to ‘direct development’ where the embryo develops directly into a juvenile, which is typically a miniature, sexually immature version of the adult. Biologists have used the term 'larva' in a number of different ways and there is no generally accepted definition at this time (Strathmann 1987; McEdward & Jaines 1993; Young 2002). The definition that is used depends on whether the research focus is on morphology, evolutionary history, developmental progression, or ecological aspects (McEdward & Jaines 1993; Young et al. 2002). In this thesis, we have used the concept of larva described by Hickman (1999): 'the larva is a structural state or series of states that occurs between the onset of the divergent morphogenesis following embryonic development and the onset of metamorphosis to the adult body plan'.

Marine invertebrate’s larvae show an impressive diversity of body forms (Fig. 2) and many larval body plans have been given specific names (Table 1). The external morphology and developmental sequences of larvae are not only relevant for the description of the ontogeny of individual species, but may reflect also evolutionary relationships between taxa (Haeckell 1866; Jägersten 1972; Bininda-Emonds et al. 2002).

Marine larvae have been classified by site of development, nutritional mode, dispersal potential, and morphogenesis (Thorson 1950; Mieikovsky 1971; Levin & Bridges 1995). From an ecological perspective and for the purposes of this thesis, we have focused on the following categories:

- Planktonic larvae, whose development occur in the water column, vs. benthonic larvae, whose development takes place on or within the seafloor. - Holoplanktonic larvae, which are larvae of organisms that spend their entire life in the plankton, vs. meroplanktonic larvae, which are planktonic larval stages of benthic marine invertebrates. - Planktotrophic larvae, which feed on plankton, vs. lecithotrophic larvae, which derive nutrition exclusively from yolk provisioned from the egg.

This thesis is focused mainly on planktotrophic larvae. In the next sections we provide a brief description of the holoplaktonic larval stages of copepods and the meroplanktonic larval stages of representative coastal marine benthic invertebrates.

7 Introduction & thesis outline

Table 1 . Types of marine invertebrate larvae (from Young 2002).

Taxon Larval forms amphiblastula, parenchymula, petrosiid larva, cinctoblastula, hoplitomella, clavablastula, Phylum Porifera dispherula, trichimella, calciblastula Phylum Cnidaria Hydrozoa planula, actinula Anthozoa planula, Edwardsian larva, halcampoides larva, Semper's larva (zoanthina, zoamhella) Phylum Ctenophora cydippid, planuloid larva Phylum Platyhelminthes Turbellaria Müller's larva, Gotte's larva, Luther's larva Monogenea onchomiricidia Trematoda miricidia, redia, cercaria Cesroda Coracidium Phylum Dicyemida infusoriform larva, vermiform larva Phylum Orthonectida orthonectid larva Phylum Nemertea pilidium larva, planuliform larva, cephalothricid larva, Desor's larva Phylum Loricifera Higgin's larva Phylum Priapulida priapulid larva Phylum Acanrhocephala acanthor, acanthella Phylum Cycliophora pandora larva, chordoid larva, male larva Phylum Annelida trochophore, nectochaeta, endolarva, exolarva, mitraria, aulophora Phylum Mollusca Polyplacophora trochophore Aplacophora trochophore, pericalymma Bivalvia trochophore, bivalved veliger, pediveliger, pericalymma, , Gastropoda trochophore, veliger, echinospira, polyrrochous larva Scaphopoda trochophore, veliger, stenocalymma Cephalopoda Paralarva Phylum Arthropoda Merosromata trilobite larva Pycnogonida protonymphon nauplius, zoea, megalopa, phyllosoma, puerulus, cyprid, gaucothoe, protozoea, mysis, Crustacea elaphocaris, acanthosoma, camptopsis, furcilia, amphion, cryropia, mastigopus, eryoneicus, amizoea, pseudozoea, ketrogon, trichogon Phylum Echiura trochophore Phylum Sipuncula trochophore, pelagosphera Phylum Bryowa coronate larva, cyphonautes Phylum Enroprocta trochophore Phylum Phoronida actinotroch, demersallecithotrophic larva Phylum Brachiopod tripartite (articulate) larva, glottidia (inarriculate) larva Phylum Echinodermata Crinoidea doliolaria Ophiuroidea vitellaria, ophioplureus Asteroidea bipinnaria, brachiolaria, barrel-shaped larva Echinoidea echinopluteus, vitellaria Holothuroidea vitellaria, doliolaria, auricularia, pemactula Müller stage tornaria, Heider stage tornaria, Metschnikoff stage tornaria, Phylum Hemichordata Krohn stage tornaria, Aqassiz stage tornaria planktosphera, pterobranch larva Phylum Chordata Urochordata tadpole Cephalochordata amphioxides

8 Introduction & thesis outline

Figure 2. Examples of planktonic larvae of marine invertebrates (from Levin & Bridges 1995). (a) Sponge amphiblastula; (b) cnidarian planula; (c) ctenophore cydippid; (d) turbellarian Muller's larva; (e) rhynchoel (nemertean) pilidium; (f) loriciferan Higgins larva; (g) priapulan larva; (h) spionid polychaete larva; (i) polychaete trochophore; (j) polyplacophoran trochophore; (k) gastropod veliger; (I) bivalve veliger; (m) crustacean (barnacle) nauplius; (n) crustacean (brachyuran decapod) zoea; (0) crustacean (decapod) megalopa;

9 Introduction & thesis outline

Figure 2 (cont.). Examples of larvae of marine benthic invertebrate planktonic larvae (from Levin & Bridges 1995). (p) pycnogonid protonymphon; (q) sipunculan pelagosphera; (r) echiuran trochophore; (s) bryozoan coronate; (t) bryozoan cyphonautes; (u) entoproct trochophore; (v) phoronid actinotroch; (w) articulate brachiopod larva; (x) asteroid bipinnaria; (y) ophiuroid ophiopluteus; (z) echinoid echinopluteus; (aa) holothuroid doliolaria; (bb) enteropneust tornaria; (cc) ascidian tadpole. Scale bar units are in µm.

10 Introduction & thesis outline

Life cycle of planktonic copepods and description of their larval stages

The copepods are a very diverse group of crustaceans with over 11500 known species, most of which are marine (Humes 1994, Fig.3). Among marine invertebrates, they are the largest, most dominant and diverse group of metazooplankton (i.e. multicellular zooplankton) in marine environments (Longhurst 1985; Verity & Smetacek 1996) (Fig.3). They contribute a substantial fraction of the secondary production in most marine planktonic communities and are considered the key link between primary producers and higher trophic levels in pelagic food webs (Cushing 1989; Williams et al. 1994).

Figure 3. Copepods drawn by Ernst Haeckel in 1904 ( from Kunstformen der Natur , Art Forms in Nature, plate 56)

11 Introduction & thesis outline

Copepod taxonomy is based mainly on external morphology (Fig. 4). Among the different orders of copepods, the most common in marine environments are Calanoida, Cyclopoida, Poecilostomatoida, and Harpacticoida (Fig. 4). Harpactacoids are primarily benthic whereas calanoids and cyclopoids are mainly planktonic. Poecilostomatoida include parasitic copepods and free-living planktonic copepods (Boxshall & Halsey 2004).

Figure 4 . Schematic representations of the external morphology of the main groups of marine copepods (drawings from Rose 1933 )

Most planktonic copepods are dioecious and have sexual reproduction (Gilbert & Williamson 1983). Copepods are sexually dimorphic; adult males of many copepod species have geniculate antennae and are typically smaller than females (Gilbert & Williamson 1983; Mauchline 1998; Ohtsuka & Huys 2001). Copepod mating behavior frequently includes encounter, pursuit, capture and copulation (Buskey 1988, Titelman et al. 2007). Females release chemicals (i.e pheromones) or use hydrodynamic signals to attract the males (Bagøien & Kiørboe 2005a, 2005b). Copepods do not have a special copulatory organ for internal fertilization; the term copulation refers to the attachment of a spermatophore (i.e, a container filled with sperm and various secretions) produced internally by the male onto or close to the female's genital opening. The sperm is released from the spermatophore and passed into the seminal receptacle (spermatheca) where the eggs are fertilized (Ohtsuka & Huys 2001). Most copepod species produce subitaneous eggs, i.e. embryos that usually develop within hours or a few days; some calanoid species also produce resting eggs, i.e. eggs that contain diapausing embryos that remain dormant for long periods of time under adverse environmental conditions (Zillioux & Gonzalez 1972; Mauchline 1998). Cyclopoids bear two sacks of eggs attached to their genital segment. Some marine calanoid copepods release their eggs into the water column whereas in other calanoid species females carry a single clutch of eggs (Gilbert & Williamson 1983).

12 Introduction & thesis outline

After a certain period of embryonic development, the first free-swimming larvae, the nauplius, hatches. The postembryonic development of planktonic copepods is generally characterized by 11 larval developmental stages, six naupliar stages (designated as nauplius I to nauplius VI, NI to NVI) and five copepodid stages (namely copepodid I to copepodid V, CI to CV) (Fig. 4).

Figure 4. Generalized holoplanktonic life cycle schematic of free-living planktonic copepods. Planktonic copepods generally have complex life cycles characterized by 13 life stages, including the egg, six naupliar stages (NI to NVI), five copepodid stages (CI to CV) and an adult stage. Lower-cases indicate life cycle processes; upper-cases identify the major life cycle stages; bold upper-cases indicate classification categories of marine organisms according to their habitat and mobility (‘life mode’).

The nauplius is the most ancestral type of crustacean larva and has been used as key characteristic that unites the entire subphylum Crustacean (Cisne 1982; Dahms 2000; Harvey et al. 2002). In terms of numbers, the nauplius has been called ‘the most abundant type of multicellular animal on earth’ (Fryer 1987). The basic body plan of nauplius larvae is highly conservative across crustaceans (Dahms 2000). The newly hatched nauplius has an unsegmented body and only three pairs of cephalic appendages: antennules (or first antennae), antennae (or second antennae), and mandibles (Fig. 5). The antennules are uniramous whereas

13 Introduction & thesis outline the antennae and mandibles are typically biramous (Harvey et al. 2002). Nauplii have usually a characteristic single median frontal eye (“naupliar eye”) that persists throughout development (Dahms 2000).

Figure 5. Generalized nauplius, ventral view (Redrawn from Sverdrup et al. 1942)

Nauplii belonging to the 4 major planktonic copepod orders can be distinguished by their morphology and anatomy (Fig. 6). In copepods, as in other crustaceans, ontogenetic processes are linked to molting events that take place between stages (Williamson 1982). The nauplii of most free-swimming copepods typically molt five times (from stages NI to NVI). Trunk segments and additional appendages are added to the larval body through the subsequent molts between stages NII-NVI (Ogilvie 1953; Koga 1984). Each of the naupliar stages can be recognized by changes in the number of setae on the antennules and by the number of spines on the posterior end of the body (caudal armature) (Klein Breteler 1982; Koga 1984). When the naupliar VI stage molts into the copepodid I stage, important morphological changes occur. The copepodite I have prosome and urosome differentiated and begins to resemble the adult stage. Through the subsequent molts (CII-CV), the number of body segments and functional appendages is increased. Finally, after the fifth molt, adulthood is reached.

14 Introduction & thesis outline

Figure 6 : Examples of copepod naupliar stages of cyclopoida ( Oithona davisae ), calanoida ( Acartia clausi ), poecilostomatoida ( Oncaea venusta ) and harpacticoida ( Longipedia brevispinosa ). Scale =0.1 mm (From Koga 1984)

Benthic marine invertebrate life cycles and their meroplanktonic larvae

Approximately 70-80% of benthic marine invertebrates have planktonic larval stages that spend varying amounts of time, from minutes to months, in the water column (Thorson 1946, 1950; Strathmann 1993; Young 2002). For many benthic invertebrate species, especially sedentary species, significant dispersal is achieved only by free-swimming larvae (Strathmann 1985, 1990; Levin & Bridges 1995). Life cycles that include separate planktonic larval phases followed by benthic juveniles and adult phases are commonly referred to as biphasic life cycles. A simplified biphasic life cycle is illustrated in Figure 7. Reproductively mature, benthic adults release gametes into the water column where fertilization and larval development occur. The length of the dispersal period in the water column depends on the species. Lecithotrophic larvae have a short pelagic larval duration and do not disperse long distances; whereas planktotrophic

15 Introduction & thesis outline larvae generally have a rather long planktonic lifespan (Thorson 1950; Jagersten 1972). Dispersal is accompanied by growth and development of one or more larval stages. Planktotrophic larvae traits comprise mainly morphological but also behavioral features related to feeding and locomotion, representing adaptations to a planktonic lifestyle and permitting the exploitation of planktonic food (Strathmann 1993; Peterson et al. 1997). The end of the larval period approaches when the organism is physiologically competent to settle and take on a benthic existence. Methamorphosis to the benthic form occurs once the larvae accept a settlement location. The lifecycle is completed as these marine invertebrates pass through benthic juvenile stages to reproductively ma ture adult stages.

Figure 7. Schematic idealized life-history cycle of benthic invertebrates with meroplanktonic larvae. Lower-cases indicate life cycle processes; upper-cases identify the major life cycle stages; bold upper- cases indicate classification categories of marine organisms according to their habitat and mobility (‘life mode’).

Although larvae of crustaceans and chordates are unciliated and move by muscular locomotion, the majority of larval forms are ciliated (Young 2002). Among ciliated larvae, two archetypical larval body plans are distinguished: the trochophora and the dipleurula larvae (Fig.

16 Introduction & thesis outline

8), which have features that are hypothetically ancentral for the larval forms of protostomes (e.g. annelids, mollusks) and deuteromestems (e.g. echinoderms, hemichordates), respectively (Hatschek 1878; Nielsen 1985, 1995, 1998). Trochophore and dipleurula larvae are mainly defined by the arrangement and function of bands of cilia; the typical trochophora is characterized by the presence of two opposed bands of cilia, the prototroch (pre-oral ciliary band) and the metatroch (post-oral ciliary band), which are used as a downstream collection feeding system (Strathmann et al 1972, Nielsen 1995, Rouse 1999) (Fig. 8). In many species, the trochophora larva has also a posterior pre-anal band of cilia, the teletroch (Fig. 8). The ancestral dipleurula larva typically has a single continuous circum-oral/peri-oral band of cilia, the neotroch, which is used as an upstream collection feeding system (Strathmann et al 1972; Nielsen 1994) (Fig. 8). Not all marine larvae can be forced in the trochophore-dipleurula dichotomy because, as mentioned before, marine invertebrate larvae are very diverse, reflecting the specific adaptations required for planktonic feeding and locomotion (Strathmann 1978; Rouse 1999; Pernet et al. 2002).

Figure 8 . Larval type and ciliary band of protostome (trochophora larva with downstream-collecting band) and deuterostome (dipleurula larva with upstream-collecting band). Drawing by Claus Nielsen (science.jrank.org/pages/48723/protostome–deuterostome-origins.html).

17 Introduction & thesis outline

Among the diverse number of benthic marine invertebrates with meroplanktonic larvae, we briefly describe the development and larval types of each of the groups which we focus on in this thesis: spionid and serpulid polychaetes, bivalves, gastropods, cirripedes, and echinoids.

Adult spionids (Polychaeta, Spionidae), commonly known as “palp worms”, are suspension and deposit feeding polychaetes characterized by the presence of two tentacular palps used for gathering and collecting food particles. Generally all species live in mucus-lined burrows or tubes (Ruppert & Barnes 1994). Spionids often dominate benthic communities in coastal and shelf habitats, where some of these species may occur at very high densities (Anger et al. 1986). Various developmental types are present among the different spionid species, including broadcast spawning followed by planktotrophic and/or lecitototrophic larvae, brooding in capsules and cocoons, and direct development (Giese & Pearse 1974). In polychaetes with indirect development, two larval stages are easily distinguished: unsegmented trochophores and segmented larvae (segmented larvae with only a few segments are generally known as metatrochophores) (Pernet et al. 2002). Segments that bear chaetae are called chaetigers and larval chaetiger number is often used to indicate developmental stage (Pernet et al. 2002). Embryogenesis and early larval development often take place in egg capsules; larvae are then released from the egg capsules into the plankton at the three-chaetiger stage (Daro & Polk 1973; Blake & Arnofsky 1999). In species with planktotrophic larvae, the number of chaetigers increases during planktonic development until reaching aprox. 15 chaetigers at which point metamorphosis usually occurs. Larvae of spionids are generally the most common developmental stages of polychaetes in the plankton. During the reproductive season they may become the dominant members of coastal metazooplankton (Thorson 1950; Daro & Polk, 1973; Anger et al. 1986).

Serpulids (Serpulidae, Polychaeta), commonly named “plume worms” are suspension feeding polychaetes that inhabit calcareous tubes (Ruppert & Barnes 1994). These polychaetes are characterized by the presence of an anterior branchial crown that is used for respiration and filter-feeding and an operculum that is used to block the tube entrance for protection and water retention (Ruppert & Barnes 1994). Serpulids show a high variety of developmental modes, including species with planktotrophic or lecitotrophic larvae, and direct development (Geese & Pearse 1974; Wilson 1991; Kupriyanova et al. 2001). Serpulids are commonly broadcast spawners, which release gametes into the water column, with embryonic and larval development that takes place in the plankton (Pernet et al. 2002). The first swimming larval

18 Introduction & thesis outline

stage is a small trochophore. Subsequent segmented larvae have a relatively long planktonic period before metamorphosis and settlement (Pernet et al. 2002).

Gastropods (Mollusca, Gastropods), comprise among other groups, the well-known “snails” (prosobranch) and “slugs” (opisthobranchs) (Strathmann 1987, Ruppert & Barnes 1994, Goddard 2001). Most marine gastropod species are benthic, exhibit a wide range of feeding modes and may be found at a wide variety of depths, from the neritic to the abyssal zone (Goddard 2001, Buckland-Nicks et al. 2002). Marine gastropods produce two main types of larvae, trochophores and veligers (Barnes 1982; Strathmann 1987). Trochophores can occur as a free-swimming stage or, more commonly, they remain within egg capsules while developing into veliger larvae that then hatch into the pelagic stage (Goddard 2001; Buckland-Nicks et al. 2002). Veligers are unique to gastropods and marine bivalves and have a wide variety of forms (Buckland-Nicks et al. 2002). Characteristically, gastropod veligers posses a shell (protoconch) and a bilobed velum: a ciliated organ used for feeding, locomotion and respiration (Fretter 1967; Goddard 2001; Buckland-Nicks et al. 2002). The free-swimming veliger may exist from a few days to over a year, as is seen in some caeonogastropods (Scheltema 1966; Goddard 2001; Buckland-Nicks et al. 2002). Extensive ornamentation and increased size of the protoconch reflect a long pelagic life (Buckland-Nicks et al. 2002). Competent veligers become negatively phototatic and settle to the benthos where they search out a suitable substrate for juvenile life (Barnes 1982; Goddard 2001; Buckland-Nicks et al. 2002).

The bivalves or lamellibranchs (Mollusca, bivalvia or Lamellibranchia) include the clams, mussels, scallops, and oysters (Ruppert & Barnes 1994). Most bivalves have a biphasic life cycle and reproduce by broadcast spawning (Stratmann 1987). However, in several species the fertilization occurs in the mantle cavity where eggs are brooded until they become veligers (Brink 2001; Zardus & Martel 2002). Generally, a free swimming trochophore is succeeded by a free-swimming and planktotrophic veliger larva (Barnes 1982; Brink 2001; Zardus & Martel 2002). Bivalve veligers are distinguished by a hinged (bipartite) shell (prodissoconch) and a round or oval disk-shaped velum (Waller 1981; Brink 2001; Zardus & Martel 2002). Bivalve veligers resemble tiny clams, especially when the velum is retracted. The first shell forms within 1-3 days of life and has a “D” shape (D-stage veliger, Zardus & Martel 2002). Similar to gastropod veligers, bivalve veligers may remain in the plankton from days to months (Brink 2001; Zardus & Martel 2002). Prior to metamorphosis, many species enter a pediveliger stage

19 Introduction & thesis outline

(Carriker 1956), characterized by a well developed foot which is used to explore and taste the substratum (Bayne 1965; Zardus & Martel 2002).

Cirripeds (Crustacean, Cirripedia) include sessile filter feeders such as barnacles (Thecostraca) and crustacean parasites (Rhizocephala) (Ruppert & Barnes 1994). The adult barnacle body is enclosed within a bivalved carapace that is covered and protected by calcareous plates. They have six feathery and very long pairs of thoracic limbs, referred to as "cirri", which are used to filter food (Strathmann 1987; Rupert & Barnes 1994). Most adult barnacles are hermaphroditic and commonly reproduce by cross-fert ilization (Arnsberg 2001 ). Embryonic development takes place in sacs inside the carapace (Strathmann 1987). The pelagic phase of the barnacle life cycle includes 2 different types of planktonic larvae: the nauplius and the cyprid (Strathmann 1987; Arnsberg 2001; Harvey et al. 2002). Cirripede naupliar stages are recognizable by two characteristic front-lateral horns and a single caudal spine (Strathmann 1987; Arnsberg 2001; Harvey et al. 2002). There are six naupliar stages. Nauplius I is released from the adult carapace into the plankton. Feeding begins at stage II and continues through stage VI. During this time the nauplii accumulate large quantities of lipid droplets. The naupliar phase concludes with a metamorphic molt from NVI to a cypris larva (Arnsberg 2001; Harvey et al. 2002). The cypris larva possesses six pairs of thoracic appendages for swimming and a well- developed pair of antennules, which are used to walk in the substrate in search of a suitable attachment site (Arnsberg 2001; Harvey et al. 2002). The cypris larvae do not feed, but live off of the energy reserves stored during the naupliar phases. Several hours after attachment the cyprids molt into the first juvenile instar (Arnsberg 2001; Harvey et al. 2002).

Echinoids (Echinodermata, Echinoidea) which comprise the well-know sea urchins, most of which are dioecious broadcast spawners (Strathmann 1987; Miller 2001; Emlet et al. 2002). Approximately 70% of sea urchin species have planktotrophic larvae (Emlet et al. 2002). After fertilization, embryogenesis proceeds rapidly and the gastrula transforms into a prism stage (Miller 2001; Emlet et al. 2002). This stage develops into the typical feeding larva of echinoids, the echinopluteus (Miller 2001). This larvae is characterized by a bilaterally symmetrical arrangement of ciliated arms (epidermal extensions supported by calcareous spicules), which are used for feeding and swimming (Miller 2001). Larval development involves the increase in size and in the number of arms (Miller 2001; Emlet et al. 2002). Echinoplutei may live in the plankton from weeks to months before undergoing metamorphosis (Miller 2001; Emlet et al. 2002).

20 Introduction & thesis outline

Planktonic larvae are particularly vulnerable stages in the life cycles of marine invertebrates, since larval mortality often exceeds 90% (Rumrill 1990). However, a dispersing larval stage is advantageous for animals that are sessile as adults. Planktonic larvae provide a means to colonize new suitable habitats either in local or geographically distant areas, hence enlarging biogeographical distributions. Enhanced dispersal can also facilitate the establishment of new colonization sites following disturbances, reducing the risk of localized extinctions (Hansen et al. 2002; Petersen et al. 2002; Eckert 2003). Larval dispersion may also favor connectivity and increased gene flow between populations (Roughgarden et al. 1998). Therefore, knowledge of larval ecology is an essential part of understanding the population and community dynamics of benthic marine invertebrates.

Introduction to the ecophysiology of marine invertebrate planktonic larvae

Ecophysiology or environmental physiology is the biological discipline that studies the influence of environmental factors on processes or functions in an organism or in any of its parts. Physiology and bioenergetics are closely allied to species fitness and overall success. Given the crucial role of larval recruitment for the stability of populations and communities, understanding larval physiology is essential for comprehension of the biological dynamics of marine invertebrate species. Moreover, in the present context of rapid anthropogenic-induced environmental change (Hoegh-Guldberg & Bruno 2010), information about the effects of environmental factors (temperature, food availability) on larval physiology is of particular relevance to comprehend the impac t of global-ocean change on marine invertebrate species.

Many field and laboratory studies have demonstrated that water temperature and food concentration are the most important environmental factors that influence survival development, growth, feeding and metabolism in copepods (Huntley & Boyd 1984; Huntley & Lopez 1992; Hirst & Lampitt 1998; Hirst & Kiørboe 2002). However, most studies have focused on adults or late copepodites of calanoids (Ikeda et al. 2001; Hirst & Bunker 2003), while naupliar life stages have received less attention, particularly those belo nging to small copepods such as those belonging to the cyclopoid genus Oithona. During last decade, Oithona has received special attention because of its high abundance and ubiquity; it is likely the most important copepod in all oceans (Galliene & Robins 2001). Despite the importance of Oithona , little is

21 Introduction & thesis outline known about the physiology of its early developmental stages, which may be crucial to the explanation of their evolutionary success.

One of the goals of this thesis was to assess the influence of intrinsic (stage, body weight) and environmental (temperature, fo od) factors on different physiological processes (growth, development, feeding, respiration, excretion) of Oithona davisae early developmental stages ( articles IV, V, and VI ).

Larval physiology has been extensively studied in some benthic invertebrate species, particularly those of interest to aquaculture (e.g. Mytilus edulis -Widdows 1991; Sprung 1984a, 1984b, 1984c). However, little is known about the ecophysiology of some ecologically important invertebrates, for example, many species of polychaetes. Laboratory studies and field observations have demonstrated that reproductive season and geographic range often coincide with the embryonic and larval tolerance of a species to the environmental factors such as temperature (Orton 1920; Thorson 1950; Kinne 1970). Temperature change may have important consequences on larval survival and dispersion through retardation or acceleration of rates of development and growth (Thorson 1950; Costlow et al. 1966, Hoegh-Guldberg & Pearse 1995). Food quality and quantity have been cited as major factors in determining survival, growth and development of planktotrophic larvae (Paulay et al. 1985; Pechenik 1987; Rumrill 1990; Basch 1996). Anthropogenic climate change has triggered a global reduction in the production of ocean phytoplankton (Behrenfeld et al. 2006) and changes in the structure and the phenologies of planktonic communities (Edwards & Richardson 2004). For instance, recent warming trends have led to earlier spawning events, but not early spring phytoplankton blooms, resulting in a temporal mismatch between larval production and food supply (Edwards & Richardson 2004 Hoegh-Guldberg & Bruno, 2010). Low food availability during larval development can lead directly to larval mortality by starvation or indirectly by causing decreased growth rates, prolonging the planktonic period of the larvae and further exposing larvae to additional sources of mortality, such as predation (Thorson 1950). Many meroplanktonic larvae have been shown to be food limited during their development, i.e. natural food concentrations are below those required to support maximum rates of growth and development (Olson & Olson 1989; Yu 2009), which emphasizes the relevance of understanding the physiological basis and ecological significance of food limited growth on marine benthic invertebrate larvae.

22 Introduction & thesis outline

The influence of food on the swimming behavior and physiology of meroplanktonic larvae of the polychaete Polydora ciliata was studied ( articles VII and VIII ). Food limitation in larval growth was examined in a typical Danish eutrophic estuary, Isefjord ( article IX )

The estimation of ingestion, growth, respiration and egestion rates allows us to determine the energy or carbon budgets and energy transformation efficiencies of animals. The basic energy budget model may be shown as: I = G + M+ E where I, G ,M and E are the rates of ingestion, growth, metabolism (respiration), and egestion, respectively. These rates are frequently expressed in energy or carbon units.

From an economic perspective, the population dynamics of larvae affects recruitment of species that are exploitable resources of great importance, or are part of the fouling organisms. The abundance of copepod larvae (nauplii) contributes decisively to the recruitment of fish stocks of commercially important species (Castonguay et al. 2008). In addition, copepod nauplii are preferred and nutritious food sources for many farm-raised marine fish and shrimp larvae (Hernández-Molejón & Álvarez-Lajonchère 2003). Therefore, Information about the physiology and carbon budget of larval stages under different environmental conditions is relevant not only in basic scientific disciplines such as larval ecology, but also in applied investigations in the context of aquaculture and fisheries.

Carbon budget and net growth efficiencies and of Oithona davisae nauplii were estimated ( article VI ). We also determined the optimal temperature range and food concentration for the production of O. davisae nauplii in the laboratory (articles IV and V).

Marine planktonic food webs and the role of invertebrate larvae

By definition, “plankton” comprises all organisms suspended in the water column, whose mobility cannot counteract the marine hydrodynamics (Hensen 1887). These organisms are extremely diverse in terms of taxonomy, size and trophism (Sieburth et al. 1978, Fig.9).

23 Introduction & thesis outline

Femto− Pico− Nano− Micro− Meso− Macro− Mega− PLANKTON 0.02−02 0.2−2 2−20 20−200 0.2−20 2−20 20−200 µm µm µm µm mm cm cm Virio− plankton

Bacterio− plankton

Myco− plankton

Phyto− plankton

Protozoo− plankton

Metazoo− plankton

Figure 9. Distribution of different taxonomic-trophic components of the plankton according to their size (redrawn from Sierburth et al. 1978)

All planktonic components are linked to one another through predator-prey interactions influenced by ecological preferences, and top-down or bottom-up effects (Zöllner et al. 2009). The view of marine planktonic food webs has changed considerably within the last four decades with the recognition of microbial processes in the functioning of planktonic systems (Pomeroy 1974; Azam et al. 1983; Platt et al. 1983). Initially the planktonic food web was described as a linear relationship between the large phytoplankton (e.g. diatoms and dinoflagellates) and mesozooplankton (e.g. copepods) that transfer photosynthetic carbon to higher trophic levels (“classical food chain”, Fig. 10). The microbial loop theory (Azam 1983) showed that the dissolved organic matter from all organisms is recovered into the food web by the circuit of heterotrophic bacteria to heterotrophic flagellates to microzooplankton (Fig. 10). Contemporary studies highlighted the importance of the small phytoplankton fractions (picoplankton) in marine food webs (Pomeroy 1974; Platt et al. 1983; Chisholm et al. 1988). Later the role of marine viruses as significant agents in the control of bacteria and phytoplankton was recognized (Proctor & Furhman 1990; Bratbak et al. 1990). At this time, it is generally accepted that the trophic structure of marine pelagic ecosystems involves complex and highly interactive webs, where the microbial food web plays an important role in the carbon flow and nutrient recycling (Karl 1999; Sherr & Sherr 1994, 2000; Calbet & Landry 2004) (Fig. 10).

24 Introduction & thesis outline

Figure 10. Simplified diagram of a marine planktonic food web showing the components of the classical food web (in blue) and the microbial loop (in red). Solid lines represent the flux of particulate matter and the dashed lines represent the fluxes of dissolved organic matter and inorganic nutrients. DOM: dissolved organic matter. All organisms, not just bacteria, can be infected by viruses. Draws by R. Almeda and from Tracey Saxby, IAN Image Library (ian.umces.edu/imagelibrary/)

Zooplankton includes protozoans (protozooplankton, heterotrophic single-celled eukaryotes) and metazoans (metazooplankton, multicellular heterotrophs). Metazooplankton occupy a key position in marine pelagic food webs because their role in the transfer matter and energy to higher trophic levels (Fig. 10). In addition, metazooplankton directly influences microbial food webs by predating upon the protists which fall into their prey size spectrum such as heterotrophic nanoflagellates, dinoflagellates and ciliates (Stoecker & Capuzzo 1990; Gifford

25 Introduction & thesis outline

& Dagg 1991), and indirectly via trophic cascade effects and nutrient regeneration (Calbet & Landry 1999). Among the different size categories of metazooplankton (Fig. 9), metazoan microplankton have traditionally been undersampled due to the use of plankton nets with mesh size ≥ 200 µm (Calbet et al. 2001; Galliene & Robins 2001; Turner 2004).

The composition, abundance and biomass of metazoan microplankton were estimated in different marine systems, NW Mediterranean co astal waters along a seasonal cycle ( article II ) and in west coast of Vancouver Island (Canadian Pacific coast) during the summer ( article III ).

The metazoan microplankton is mainly composed by planktonic larvae such as copepod nauplii, copepodites and meroplanktonic larvae. The lack of knowledge regarding the metazoan microplankton community contrasts with their importance in terms of abundance, biomass and productivity in marine environments (Hopcroft et al. 2001; Turner 2004; Zervoudaki et al. 2007). In addition, copepod nauplii are the main prey of many fish larvae (Last 1980; Conway et al. 1991, 1998) and as previously mentioned, their abundance may determine the recruitment of commercially important fish species (Castonguay et al. 2008). Therefore, the bias on the quantification and function of small metazoans obviously causes important underestimations in the production of metazooplankton, their trophic impact on primary producers and zooplankton mediated fluxes and, in general, of their importance in marine planktonic food webs (Turner 2004).

The trophic role, growth rates, respiration rates and growth efficiencies of the metazoan microplankton community was determined in NW Mediterranean coastal waters along an annual cycle ( article II ).

Besides their small size, the trophic role of meroplanktonic larvae in marine food webs has been largely ignored because of their temporality in the plankton. However, most meroplanktonic larvae are planktotrophic and their nourishment depends upon the existing plankton community. In fact, the release of larvae is often coupled to phytoplankton blooms to maximize the exposure of larvae to an abundant food supply (Thorson 1946, 1950; Starr et al. 1990, 1991, 1994). This synchronicity often leads to meroplanktonic larvae becoming the dominant members of the coastal zooplankton community during the reproductive season of benthic invertebrates (Thorson 1946; Williams & Collins 1986; Andreu & Duarte 1996); however,

26 Introduction & thesis outline

the possible trophic function of planktonic larvae in the marine carbon flow also remains largely unknown.

We determined the abundance, feeding rates, prey selection and trophic impact of different meroplanktonic larvae under algal blo om conditions on the west coast of Vancouver Island ( article III )

Therefore, information concerning abundance, biomass and the trophic role of planktonic larvae is crucial for our increased understanding of food webs and biogenic fluxes in pelagic marine environments.

Thesis outline

The thesis has been structured following the normative for a PhD thesis as a compendium of publications (Comissió de Doctorat, Facultat de Biologia, Universitat de Barcelona, 2010). The general aim of the present thesis was to gain new knowledge on the ecology and physiology of marine invertebrate planktonic larvae and their trophic role in pelagic food webs.

In this general introduction we provided a general review of marine invertebrate larval biology and the importance of these larval stages in marine life cycles and species success. We also briefly introduced the main topics of the thesis i.e. larval ecophysiology and the trophic role of planktonic larvae in marine food webs.

The results of this thesis are presented as nine scientific articles (referred by roman numerals, see below list of publications) organized in five central chapters , as follow:

In Chapter 1 , “Seasonal abundance and vertical distribution of zooplankton in NW Mediterranean coastal waters: importance of small planktonic metazoans” (article I ), we examined the abundance, composition and vertical distribution of the zooplankton community in a coastal area of the northwestern Mediterranean throughout an annual cycle, with special emphasis on the small planktonic metazoans, including planktonic larvae. We also evaluated the influence of environmental conditions on the seasonal and vertical distribution of

27 Introduction & thesis outline

zooplankton. The efficiency of different zooplankton sampling methods (Van Dorn bottles and microplankton net) and the contribut ion of metazoan microplankton and planktonic larvae to total zooplankton biomass were also assessed.

In Chapter 2 , “Trophic role and carbon budget of metazoan microplankton in northwest Mediterranean coastal waters” ( article II ), we ascertained the physiology and trophic role of microplanktonic larvae from a community approach. We determined the feeding rates and trophic impact of the entire natural community of metazoan microplankton (<200 µm) from a coastal site in the northwestern Mediterranean along a seasonal cycle. This metazooplankton size fraction is mainly composed of planktonic larvae (nauplii, copepodites and meroplanktonic larvae). Additionally we determined the growth, respiration and growth efficiencies of the metazoan microplankton community in different seasons.

In Chapter 3 , “Feeding rates and abundance of marine invertebrate planktonic larvae under harmful algal bloom conditions off Vancouver Island” (article III ), we determined the feeding rates, prey selection and trophic impact of different meroplanktonic larvae on the natural bloom of Heterosigma akashiwo and Prorocentrum triestinum which occurred on the west coast of Vancouver Island in July of 2006. We also estimated the abundance, biomass and composition of the zooplankton community during this harmful algal bloom.

In Chapter 4 , “Ecophysiology of early developmental stages of the copepod Oithona davisae”, we conducted a complete laboratory study on the ecophysiology of early developmental stages of the copepod Oithona davisae . We studied different physiological processes in relation to intrinsic and environmental factors.

This chapter includes 3 scientific publications, as follow:

4.1. “Effects of temperature and food concentration on survival, development and growth rates of naupliar stages of Oithona davisae (Copepoda, Cyclopoida)” ( article IV ) where the survival, development, and growth of the naupliar stages of O. davisae under different temperature regimes and food concentrations were determined.

4.2. “Feeding rates and gross growth efficiencies of larval developmental stages of Oithona davisae (Copepoda, Cyclopoida)” ( article V ). In this article we estimated the feeding (clearance and ingestion rates) and gross growth efficiencies of early developmental stages of O. davisae in relation to stage, body weight, temperature, and food concentration.

28 Introduction & thesis outline

4.3. ” Metabolic rates and carbon budget of the early developmental stages of the marine cyclopoid copepod Oithona davisae” ( article VI ). In this publication we reported the respiration and excretion rates, as related to stage, body weight, temperature and food availability, and the C:N:P metabolic ratios, net growth efficiencies, and carbon budget of O. davisae nauplii .

In Chapter 5 , “Ecophysiology of planktonic larvae of the spionid polychaete Polydora ciliate”. We conducted field and laboratory studies to determine the influence food on swimming behavior and physiology of Polydora ciliata larvae. The field studies were conducted in the eutrophic estuary Isefjord (Denmark), where this species is one the dominant benthic invertebrates

This chapter involved 3 publications:

5.1 . “Swimming behavior and prey retention of the polychaete larvae Polydora ciliata (Johnston)” ( article VII ), where the swimming behavior of P. ciliata larvae under different prey regimens was described and a simple swimming model was developed. Ontogenic changes in food size spectra and grazing rates in natural food assemblages were also determined.

5.2. “Feeding and growth kinetics of the planktotrophic larvae of the spionid polychaete Polydora ciliata (Johnston)” ( article VIII ). In this paper we determined the effect of food concentration on the feeding rates, growth rates and gross growth efficiencies of different developmental stages of P. ciliata.

5.3. “Larval growth in the dominant polychaete, Polydora ciliata , is food-limited in a eutrophic Danish estuary (Isefjord)” ( article IX ). In this last publication, we examined the food limitation in larval growth of P. ciliata . In the field we determined the larval abundance and the food availability over different periods. In the laboratory, we compared the specific growth of larvae reared on natural food suspensions with those of larvae reared on phytoplankton- enriched food suspensions.

In the general discussion , we relate the results obtained from the preceding chapters, evaluate the initial hypothesis of the thesis, and discuss the meaning of these findings in the context of marine invertebrate larval ecology and the role of larvae in marine planktonic food webs. To conclude, we highlight the main conclusions of the thesis.

29 Introduction & thesis outline

List of scientific articles/publications

I. Almeda R. , Alcaraz M., Calbet A., Saiz E., Trepat I. Seasonal abundance and vertical distribution of zooplankton in NW Mediterranean coas tal waters: importance of small planktonic metazoans. In preparation to be submitted to Progress in Oceanography .

II. Almeda R. , Calbet A., Alcaraz M., Saiz E., Trepat I., Arin L., Movilla J.I., Saló V. (2011). Trophic role and carbon budget of metazoan microplankton in northwest Mediterranean coastal waters. Limnology and Oceanography 56(1): 415-430.

III. Almeda R., Messmer A., Sampedro N., Gosselin L.A. (2011). Feeding rates and abundance of marine invertebrate planktonic larvae under harmful algal bloom conditions off Vancouver Island. Harmful Algae 10: 194-206.

IV. Almeda R. , Alcaraz M., Calbet A., Yebra L., Saiz E. (2010). Effects of temperature and food concentration on survival, development and growth rates of naupliar stages of Oithona davisae (Copepoda, Cyclopoida). Marine Ecology Progress Series 410 : 97-109.

V. Almeda R. , Augustin C.B., Alcaraz M., Calbet A., Saiz E. (2010). Feeding rates and gross growth efficiencies of larval developmental stages of Oithona davisae (Copepoda, Cyclopoida). Journal of Experimental Marine Biology and Ecology 387: 24-35.

VI. Almeda R. , Alcaraz M., Calbet A., Saiz E. (2011). Metabolic rates and energy budget of the early developmental stages of the marine cyclopoid copepod Oithona davisae . Limnology and Oceanography 56(1): 403-414.

VII. Hansen B.W., Jakobsen H.H., Andersen A., Almeda R ., Pedersen T.M., Christensen A.M., Nilsson B. (2010). Swimming behavior and prey retention of the polychaete larvae Polydora ciliata (Johnston). Journal of Experimental Biology 213: 3237-3246.

VIII. Almeda R ., Pedersen T.M., Jakobsen H.H., Alcaraz M., Calbet A., Hansen B.W. (2009). Feeding and growth kinetics of the planktotrophic larvae of the spionid polychaete Polydora ciliata (Johnston). Journal of Experimental Marine Biology and Ecology 382: 61-68.

IX. Pedersen T.M., Almeda R ., Fotel F.L., Jakobsen H.H., Mariani P., Hansen B.W. (2010). Larval growth of the dominant polychaete, Polydora ciliata, is indeed food limited in a eutrophic Danish estuary, Isefjord. Marine Ecology Progress Series 407: 99-110 .

30 Hypotheses & objectives

Hypotheses & objectives

Hypotheses & objectives

The main objective of the present thesis was to improve our knowledge on the ecology and physiology of marine invertebrate planktonic larvae including their trophic role in pelagic food webs. For that purpose, we conducted field and laboratory studies with holo ‐ and meroplanktonic larvae of ecologically important marine invertebrates, using both a species ‐ based and community ‐level approach.

The general hypothesis of this thesis was that invertebrate planktonic larvae play a key role in the functioning of marine ecosystems . In particular, the following specific hypotheses were formulated:

1. Planktonic invertebrate larvae (holo ‐ and meroplanktonic larvae), including those belonging to the small size fractions (<200 µm, metazoan microplancton), exert an important trophic impact on marine planktonic food webs, which may noticeably influence phytoplankton and protozoan dynamics in coastal waters.

2. The ecological success of many species of marine invertebrates is in part explained by the physiological characteristics of their planktonic larval stages.

The hypothesis 1 was tested through addressing the following objectives :

1. Determination of the seasonal abundance and vertical distribution of a whole zooplankton community in a coastal area of the northwestern Mediterranean throughout an annual cycle, with special emphasis on metazoan microplankton and planktonic larvae (article I).

2. Estimation of feeding rates, trophic impact and growth efficiencies of the metazoan microplankton community along a seasonal cycle in NW Mediterranean coastal waters (article II ).

3. Assessment of feeding rates, prey selection and trophic impact of different meroplanktonic larvae on the natural harmful bloom of Heterosigma akashiwo and Prorocentrum triestinum, which occurred on the west coast of Vancouver Island in July of 2006 (article III ).

33 Hypotheses & objectives

The hypothesis 2 was tested through addressing the following objectives :

4. Determination of different physiological processes of Oithona davisae early larvae (nauplii and early copepodites) in relation to intrinsic (stage, body weight) and environmental (temperature, food) factors, as follow: survival, development and growth rates (article IV ), feeding rates (article V), and respiration and excretion rates (article VI ).

5. Evaluation of the influence of food on swimming behavior (article VII ), feeding (articles VII and VIII ) and growth rates and gross growth efficiencies (articles VIII and IX ) of planktonic larval stages of the polychaete Polydora ciliate.

34 Results

“Knowledge advances by steps and not by leaps ” Lord Macaulay (1800-1859)

Seasonal abundance and vertical distribution of zooplankton in NW Mediterranean coastal waters: importance of small planktonic metazoans

Chapter 1

To be submitted to Progress in Oceanography

Seasonal abundance and vertical distribution of zooplankton in NW Mediterranean coastal waters: importance of small planktonic metazoans

Rodrigo Almeda*, Miquel Alcaraz, Albert Calbet, Enric Saiz, Isabel Trepat

Institut de Ciències del Mar (CSIC), P. Marítim de la Barceloneta 37–49, 08003 Barcelona, Spain

*[email protected]

Abstract Our understanding of zooplankton function in marine ecosystems is limited due to the scarce information regarding the small planktonic metazoans. We examined the seasonal succession and vertical distribution patterns of the whole metazooplankton community in a coastal area of the northwestern Mediterranean throughout an annual cycle, with special em phasis on the sm all size fractions.
e study included the use of tw o sam pling m ethods for m etazooplankton (m icroplankton net and Van Dorn bottles) to adequately cover the m ain range of m etazooplankton size-fractions, from 50-200 µm (m e t a z o a n m i c r o p l a n k t o n ) t o 200-2000 µm (m e s o z o o p l a n k t o n ). W e a l s o e v a l u a t e t h e i n u e n c e o f t h e m a i n abiotic and biological factors on the seasonally and vertical structure of m etazooplankton com m unity. Copepod nauplii and copepodites w ere num erically dom inant during m ost of the studied period, w ith densities that ranged from ~ 620 to 23900 in d3 . am n d f r o m ~ 265 t o3 , 10000 respectively. i n d . m In general, the patterns of vertical distribution of m esozooplankton and m etazoan m icroplankton w ere quite sim ilar.
e vertical gradients in abundance and biom ass of m etazooplankton w ere m ore im portant in sum m er and autum n and tended to follow those of their potential m icroplankton prey (ciliates, dinoagellates, diatom s). M etazoan m icroplankton and m esozooplankton abundance appeared to be directly related to tem perature. M inim um values in the abundance of copepods and their larvae (nauplii and copepodites) w ere observed during the spring phytoplankton bloom . Larval developm ental stages (including holo-and m eroplanktonic larvae) as e s t i m a t e d f r o m p l a n k t o n n e t a n d Va n Do r n b o t t l e s c o m p r i s e d , r e s p e c t i v e l y , t h e 45% (r a n g e : 24%-75%) a n d 55% (r a n g e : 26%-69%) o f t h e t o t a l m e t a z o o p l a n k t o n c a r b o n .
e m e t a z o a n m i c r o p l a n k t o n s i z e f r a c t i o n r e p r e s e n t e d t h e 34% (r a n g e : 15%-54%) of the total m etazooplankton carbon w hen Van Dorn bottles are used for sam pling.
erefore, although the m esozooplankton dom inates in term s of biom ass, the m etazoan m icroplankton represents a considerable fraction of the total m etazooplankton biom ass. M oreover, as physiological rates of sm all organism s are generally higher than those of larger organism s, the exclusion of m etazoan m icroplankton m ay lead to im portant underestim ations of zooplankton s e conda ry p roduction in m a rine s y s te m s .

1. Introduction a l . 2001; Ga l l ie nne a nd Rob ins 2001; T urne r 2004; Ricca rdi 2010).
e com m on us e of re l a tive l y l a rge M e ta zoop l a nkton occup y a ke y p os itionm in e s the h-s ize ne ts (≥ 200 μm ) in zoop l a nkton s a m p l ing m a rine p e l a gic food we b s b e ca us e thel ir e arol ds e to in s the ignica nt e s tim a tion e rrors of zoop l a nkton b ioge oche m ica l cycl e s a nd in the tra ns fea r bof unda m a nce tte ra nd com p os ition (Ca l b e t e t a l . 2001; T urne r a nd e ne rgy to highe r trop hic l e ve l s . Howe ve2004) r, our b e ca us e m os t cop e p od de ve l op m e nta l s ta ge s unde rs ta nding of the rol e of zoop l a nkton in m(na a up rine l ii a nd cop e p odite s ), m e rop l a nktonic l a rva e , e cos ys te m s is l im ite d b y the s ca rcity of a inform nd e ve a n tion s om e s m a l l a dul t cop e p ods (i.e ., Oithonids , on s m a l l zoop l a nkte rs s uch a s cop e p od deOnca ve l e op ida m e ) e a nta re l < 200 µm . M e ta zoa n m icrop l a nkton s ta ge s (na up l ii a nd cop e p odite s ) a nd mm e rop a y l b a nktonice im p orta nt in te rm s of a b unda nce , b iom a s s a nd l a rva e . In the ge ne ra l l y a cce p te d p l a nkton-sp roduction ize cl a in s s m e s a rine e nvironm e nts (Hop cro e t a l . a s p rop os e d b y S ie b urth e t a l . (1978 ), m e2001; ta zoop T urne l a nktonr 2004). Cop e p od na up l ii a re the m a in p re y wa s cons ide re d to e xte nd m a inl y from m e sof op m l a a nkton ny s up h l a rva e (La s t 198 0; Conwa y e t a l . 1991, 1998 ) (> 200 µm ), s o tha t the com m unity occup yinga s nd m the a l ir l ea r b unda nce m a y de te rm ine the re cruitm e nt of s ize cl a s s (m icrop l a nkton) wa s s up p os ecom d to m incl e ude rcia l l y im p orta nt s h s p e cie s (Ca s tongua y e t a l . a l m os t e xcl us ive l y unice l l ul a r e uka ryote2008 s (p ). In rim a ddition, a ry the dyna m ics of p l a nktonic l a rva e , p roduce rs a nd he te rotrop hic p rotozoa ns ). Ne veb rthe oth hol l e o- s sa , nd m e rop l a nktonic, a re crucia l to de te rm ine the re l e va nce of m e ta zoa n m icrop l a nktonthe ha s ucces b s e s e n of the corre s p onding a dul t p op ul a tions . incre a s ingl y re cognize d in the l a s t de ca de
e through re fore , inform a tion on this im p orta nt, b ut p oorl y the us e of a p p rop ria te s a m p l ing m e thodss tudie (Ca d, com l b e tp e one t nt of m e ta zoop l a nkton is crucia l for

39 Chapter 1

our understanding of dynamics of marine ecosystems. su rf ac e u sing a mic roplank ton net ( 5 0 µm mesh, 36 c m In a similar context, the structure of zooplankton diameter) . A er plank ton net w as w ashed, the c ontent communities presents a high spatial and temporal of the c od end w as pou red into a 5 0 0 mL plastic b ottle, variability in coastal systems. Zooplankton communities maintanined in a c ool b ox ( ab ou t 4 ºC) and x ed in may respond to environmental variability through b orax - b u ered f ormalin ( 4 % nal c onc entration) at the changes in abundance, biomass and composition. retu rn to the lab oratory . Understanding the seasonal abundance and spatial e stu dy of the v ertic al distrib u tion of z ooplank ton heterogeneity (patchiness) of zooplankton in relation w as made on disc rete z ooplank ton samples tak en at 1, to environmental conditions (physico-chemical and 5 , 10 , 20 and 30 m depth w ith a transparent dou b le biological factors) is essential for a better compression Van Dorn b ottle as desc rib ed in A lc araz ( 19 7 7 , 19 8 3) . of the dyna m ics m a rine e cos ys te m s .
eSampling a b unda w as c nce ondu c ted approx imately at 12:0 0 pm. 30 s ucce s s ion a nd ve rtica l s tructure of zoopL l a w nkton ater samples f rom eac h sampling depth w ere ltered com m unitie s in p a tche s or l a ye rs ha s b eb y e n grav com ity m throu onl y gh 5 0 µm- mesh large su rf ac e Nitex ® s tudie d for the l a rge s ize fra ction (m alters. cro-a nd e lters w ith the retained organisms w ere m e s ozoop l a nkton, e .g. Longhurs t 1976; Al ca ramaintained z 1985; in a c ool b ox as in the c ase of net samples Ande rs on e t a l . 2004). Howe ve r, the p a tte rns ofu s ntil e a proc s ona essed l in the lab oratory . is c onsisted on a a b unda nce a nd ve rtica l s tructure of the mc aref e ta u zoa lly nw ashing of the lters and the c ollec tion of the m icrop l a nkton com m unity a re p oorl y known.organism in 25 0 ml plastic b ottles. e samples w ere In the p re s e nt s tudy, we e xa m ine d the preserv s ucce ed s sin ion b orax - b u ered f ormaldehy de at 4 % nal a nd ve rtica l dis trib ution of the whol e zoop lc a onc nkton entration. Similarly , w ater samples f or the analy sis com m unity in a coa s ta l a re a of the northweof phy s toplank te rn ton ( c hlorophy ll) b iomass and mic rob ial Me dite rra ne a n a l ong a n a nnua l cycl e , withc ommu s p e nitycia l w ere c ollec ted w ith the Van Dorn b ottle e m p ha s is on m e ta zoa n m icrop l a nkton aand nd gently p l a transf nktonic erred to 2.3 L acid w ashed b ottles. larvae. e specic objectives of the study w ere: 1) to assess the seasonal changes in 2.2.abundance Sample analysis andof calculationsholo- and m eroplanktonic larvae; 2) to determ ine the vertical We estimate the concentration of chlorophyll ( total structure of the m etazooplankton com m unity in relation and > 10 µm) in the w ater, and the ab undance ( cells to dierent physical, chem ical and biological variables; L-1 ) of ciliates, diatoms, and dinoagellates f rom w ater 3) to evaluate the capture eciency of large volum e Van samples of all depths. For the sample corresponding to Dorn bottles as com pared to m icroplankton nets for 5 m depth w e conducted a more detailed analysis of the sam pling dierent size fractions of m etazooplankton; 4) microb ial community, including b iomass estimations of to estim ate the contribution of m etazoan m icroplankton picoplank ton ( heterotrophic b acteria, Prochlorococcus , and the w hole planktonic larvae com m unity to total Synechococcus , picoeuk aryotes) , heterotrophic metaz oop lank ton b iomass. nanoagellates ( H NF) , phototrophic nanoagellates ( PNF) , dinoagellates, diatoms, and ciliates. 2. Material and methods Total and > 10 μm Chl a w ere analysed in 7 5 to 5 0 0 mL w ater samples ltered through GF/F Whatman 2.1. Field sampling ( total) and 10 μm ( > 10 µm) pore-siz e polycarb onate We c ondu c ted a monthly sampling in a NWNuclepore lters, respectiv ely. e lters w ere f roz en at Mediterranean c oastal site ( 1.5 Km o Barc elona, 4 0–8 0 °C and later analyz ed uorimetrically aer acetone m depth) f rom Septemb er 20 0 5 to Septemb er 20 0ex 6 traction b ef ore and aer acidication according to b etw een 9 :0 0 and 12:0 0 pm. H ow ev er, the samplingParsons of et al. ( 19 8 4 ) . Dec emb er 20 0 5 and Feb ru ary 20 0 6 had to b e c anc elledSamples ( 2 ml) f or heterotrophic b acteria, du e to tec hnic al prob lems. A ltogether, the annu al stuProchlorococcus dy , Synechococcus, and picoeuk aryotes w as c onstru c ted on the b asis of 12 monthly samplings.w ere preserv ed in paraf ormaldehyde + glutaraldehyde Vertic al proles of temperatu re, salinity and( 1 + 0 .0 5 % nal concentrations, respectiv ely) and stored u oresc enc e w ere ob tained u sing a Sea Bird 25 CTDat -8 0 °C until analysis. Flow Cytometry analyses w ere ( Seab ird Elec tronic s I nc , Bellev u e, WA ) , equ ippedconducted w ith a FA CScalib ur ow cytometer f ollow ing w ith a Sea- Tec h u orometer ( Sea- Tec h I nc , Corv allis,the procedures of Gasol and del Giorgio ( 20 0 0 ) . OR) . I n tw o oc c asions ( samplings c orresponding to theH eterotrophic b acteria b iomass w as estimated f rom months of A pril and Ju ly ) the CTD w as no av ailab le,v olume determinations ( V, µm 3) using the relationship so temperatu re and salinity w ere measu red only f at g C = 120 V 0.72 ( Norland 19 9 3) . Prochlorococcus 5 m depth w ith an YSI - 30 portab le temperatu re andand Synechococcus b iomass w ere determined aer salinity meter. We u sed tw o methods f or the estimation assuming a carb on content of 0.123 pg C μm –3 and of z ooplank ton ab u ndanc e: plank ton nets ( integrated equiv alent spherical diameters ( ESD) of , respectiv ely, w ater c olu mn) and Van Dorn b ottle ( depth- disc rete0.6 0 and 1.0 μm ( Waterb ury et al. 19 8 6 ) . Picoeuk aryots samples) . Depth- integrated net samples w ere ob tained b iomass w as estimated using a nominal siz e ( ESD) of 1.5 b y v ertic al tow s f rom near the b ottom ( ~38 m) to the µm and a conv ersion f actor of 0.22 pg C μm –3 ( Børsheim

40 Chapter 1

and Bratbak 1987). patterns of vertical distribution in all the months. While For n a n o
a gella t es, 40 t o 1 00 mL sa mpleschlorophyll wer ewas homogeneously distributed in the water pr eser ved in glut a r a ld ehyd e (1 % n a l concolumn cen in t rwinter a t ion (November ) , 2005 - January 2006, Fig. lt er ed on t o 2 μm por e-size b la ck polyca1) , during r b on most a t eof year we observed signicant vertical memb r a n e lt er s a n d st a in ed wit h–1 DAPI n a (5 l gradientsμg mL (Fig. 1) . con cen t r a t ion ) for 5 min (Por t er a n d Feig 19e 8 0) vertical . At lea distribution st of protistan microplankton 200 cells wer e coun t ed b y epi
uor escen ce micr oscopyis shown is Fig. 2. Diatoms, ciliates, and dino
agellates a t a ma gn ica t ion of ×1000 a n d cla ssied werea s a present ut o- or throughout the year. In most months we het er ot r ophic a ccor d in g t o t heir
uor escenobserved ce for important vertical gradients (Fig. 2) . e chlor ophyll un d er b lue light . Fiy cells wer e sizedconcentration a n d of ciliates ranged from less than 1 cell con ver t ed in t o ca r b on usin g a con ver sionmL fa-1 ct to or 10 ofcells 0. 22mL -1 (Fig. 2) . e maximum abundance pgC μm –3 (B ør sheim a n d B r a t b a k 19 8 7 ) . values were usually found with the rst 10 m depth in the To d et er min e t he con cen t r a t ion of d inmoths o
a gella of water t es, stratication, except in July1 and August cilia t es a n d d ia t oms, 250 mL sa mples wer e xed2006 wit (Fig. h 2) . e abundance of dino
agellates varied 1% a cid ic Lugol’s solut ion , a n d a llowed t o set tfrom le for 1 48 cell hmL -1 to ca. 25 cells mL -1 (Fig. 2) , the vertical in 100 mL Ut er möhl cha mb er s. e whole cha mbdistribution er for pattern showing strong gradients. During cilia t es a n d d in o
a gella t es, a n d a t lea st early40 micr summer oscopic the maximum values were observed at eld s (or 200 cells) for d ia t oms, wer e coun t ed usinsurface, g a whereas n from September 2005 to May 2006 the in ver t ed micr oscope (Nik on DIAPHOT 200) a thighest 200x concentrations corresponded to sub-surface ma gn ica t ion . Fiy r a n d omly-chosen cells depths for ea (Fig. ch 2) . e abundance of diatoms was also gr oup wer e sized a n d con ver t ed in t o ca rhighly b on variable, usin g ranging from less than 5 cell mL -1 to t he con ver sion fa ct or s of 0. 19 a n d 0. 053–3 pg1500 C cells μm mL -1 , with maxima located at 10-30 m depth for oligot r ich cilia t es (Put t a n d St oeck er 19(Fig. 8 92) .) In a spring n d (March and April 2006) there was a t in t in n id s (Ver it y a n d La n gd on 19 8 4) bloomr espect of ively,the colonial a n d autotrophic
agellate Phaeocystis t he equa t ion s of pg C Din-1 o = cell 0.7 60 × volume 0.819 for sp. During the bloom, the highest concentrations of d in o
a gella t es a n d pg C-1 Dia = 0.288 t cell x volume 0.811 Phaeocystis occurred at surface in March and near the for d ia t oms (Men d en -Deuer a n d Lessabottom r d 2000) in April . (Fig.2) . B eca use micr ozoopla n k t on sa mples wer e pr eser ved wit h a cid ic Lugol’s solut ion , n o d ist in ct ion b3.2. et Seasonalween dynamics, composition and biomass st r ict het er ot r ophs a n d a ut o- /mixot r ophs waof s mathe dmicrobial e for community cilia t es a n d d in o
a gella t es. For t he est ima t ion of zoopla n k t on a b un de a n average-integrated ce a n d values of chlorophyll a and composit ion , b ot h t ype of sa mples (fr om n etmicroplankton s a n d are shown in Figure 3. e highest Va n Dor n b ot t les) wer e d ivid ed in t o t woconcentrations n omin a of l chlorophyll a were observed in early size fr a ct ion s b y lt r a t ion t hr ough a 200 spring µm sieve. (March 2006 and April 2006) and the minimum e t wo fr a ct ion s ob t a in ed in clud ed values r espect in ively summer and January 2006 (Fig. 3A) . e or ga n isms fr om 50 t o 200 µm, (her ea er cacontribution lled of the phytoplankton >10 µm to the total micr opla n k t on ) a n d or ga n isms > 200 µmphytoplankton (her ea er biomass (chlorophyll a) was 40% in ca lled mesozoopla n k t on ) . Two a liquot s per average, fr a ct ion ranging from 10% in September 2006 to 65% in wer e coun t ed un d er a st er eomicr oscope. ForMarch b ioma 2006 ss (Fig. 3A) . e maximum chlorophyll values d et er min a t ion , t he b od y len gt h of a t lea st corresponded60 or ga n isms to the occurrence of the Phaeocystis sp chosen a t r a n d om wa s mea sur ed on d igitbloom a l pict and ur to es the increase in diatoms concentration ma d e un d er a un d er a micr oscope (X 100) usin(Fig. g t he3 A, fr B ee ) . e peak in diatom concentration (more -1 ima ge a n a lysis sowa r e Ima geJ®. e in d ivid uathan l ca 1000 r b cells on mL , Fig. 3B ) was due to a bloom of the weight wa s ca lcula t ed b y a pplyin g b od y size-casmall r diatoms b on Pseudo-nitzschia and Leptocilindrus. con ten t r ela tion ships fr om the liter a tur e (Ta b leDino
agellates 1) . peaked in June-July 2006, with maximum values of 15 cells mL -1 (Fig. 3C) . e highest concentrations of ciliates were observed in spring and 3. Results summer (Fig. 3C) . 3.1. Vertical proles of abiotic factors, chlorophyll e composition and biomass of the entire microbial and protistan microplankton community at 5 m depth is shown in Figure 4. e highest values of picoplankton carbon corresponded to We found a homogenous water column in terms spring and summer (Fig. 4A) . Heterotrophic bacteria of temperature and salinity in winter and early spring dominated this size fraction during all the studied (Fig. 1) . During the rest of the year, we observed the period (Fig. 4A) , whereas picouekaryots, although establishment of vertical temperature and salinity occurred also the year round, had a minor contribution gradients, in occasions with thermoclines and to picoplankton carbon (Fig. 4A) . Synechococcus haloclines located at dierent depths (Fig. 1) . In general, spp. ware abundant throughout the warmer months, the chlorophyll >10 µm and <10 µm showed similar resulting an important contributor to autotrophs carbon

41 Chapter 1

in midsummer (Fig. 4A), while Prochlorococcus spp. abundance w as generally low ( Table 2 ) . Meroplanktonic only occurred in September through January, though larv ae w ere a common component of th is zooplankton in l ow b iom a s s (F ig. 4A).
e p icop l a nktonsize fra fraction ction th rough out th e year. e most abundant re p re s e nte d in a ve ra ge a b out 47% of tota lgroups ca rb w on ere of polych aete, biv alv e and gastropod larv ae the m icrob ia l com m unity (ra nge : 29%-70%),( Table with 2 ) . eir density w as generally low as compared m inim um a nd m a xim um va l ue s in wintetor copepoda nd s um dev m elopmental e r, stages, alth ough in A pril re s p e ctive l y (F ig. 4). 2 0 0 6 polych aete larv ae w ere dominant ( Table 2 ) . Na noa ge l l a te s occurre d throughout a l l the yeRegarding a r, a nd mesozooplankton estimated from th e the ir contrib ution to the m icrob ia l ca rb on ra ngenet d samples,from copepodite and adult copepods w ere 15% to 47% (F ig. 4B), a nd dinoa ge l l a te s re pth re e s most e nte abundant d groups, w ith densities ranging from 1% to 11% of m icrob ia l C, with m a xim umfrom va ~5 0 l 0 ue to s 660 0 and from ~ 60 0 to 65 0 0 ind m 3, in June w hen unidentied sm allrespectiv dinoagellates ely ( Table 3) . A lth ough peakedcopepod nauplii w ere (Fig. 4B). e contribution of diatomalso present, s th to eir abundance total wcarbon as about one order of w as m ore im portant during springmagnitude lowand er th anautum in th e fraction n (16%< 2 0 0 µm ( Table -31% ) w ith m axim um values reached2 and 3) .in Oth erMay, h oloplanktonic w hen components of th e th e c h ain-f o r m ing diatoP s eu m do s -nitzs cs ph ia . andmesozooplankton community w ere appendicularians, Dac tyl io s o l en fr agil isb s l imu o o smed ( F ig. 4 B ) . cladocerans, e pteropods, ch aetognath s and doliolids c o ntr ib u tio n o f c il iates to mic r o b ial C( Table c o mmu3) . nity e abundance w as of th ese groups w as generally v er y v ar iab l e ( 1 % -2 3% ) w ith max imu m v al u eslow in as A p compared r il , to th ose of copepod and copepodites c o inc iding w ith a bl o o m o f th e l ar ge mix o trex o cept p h ic in csome il iate summer month s, w h en cladocerans Labo ea s p . ( F ig. 4 B ) . To tal mic r o bial bio masreach s ed rconcentrations anged of ~5 7 0 0 ind. m 3 ( Table 3) . e fr o m 30 to c a. 12 4-1 , µg w ith C L max imu m v al u esdensities in of polych aete, biv alv e and gastropod larv ae spring and summer ( F ig. 4 C ) . in th e > 2 0 0 µm-size category w ere low er th an in th e < 2 0 0 µm fraction. C ontrarily, th e concentrations of 3.3. Vertical distribution of zooplankton ech inoderms, cirriped and decapod larv ae w ere one order of magnitude h igh er th an in th e < 2 0 0 µm fraction Dierences in th e v ertical distribution of metazoan ( Table 3) . microplankton abundance and biomass w ere more C opepods and th eir dev elopmental stages w ere important during summer ( Table 4 and F ig. 5 ) . In late present th rough out th e year, w ith max imum and spring and summer th e micrometazoans occurred minimum concentrations in summer and early spring mainly in surface w aters ( 1-10 m) w ith some ex ceptions respectiv ely ( F ig. 7 ) . C opepod community w as mainly as in J uly 2 0 0 6, w h en an important peak in biomass and composed of calanoid copepods in w inter and spring, abundance of biv alv e larv ae occurred at 30 m ( Table and of cyclopoid ( Oith ona ) and poecilostomatoid 4 and F ig. 5 ) . In w inter, w h en th e w ater column w as ( Oncaea ) copepods in summer. C ladocerans, pteropods mix ed, th e v ertical gradients in abundance and biomass and rotifers sh ow ed a marked seasonality ( F ig. 7 ) . w ere low er ( Table 4 and F ig. 5 ) . Mesozooplankton depth During most of th e year cladocerans ( mainly Penilia distribution ( Table 5 and F ig. 6) seemed to follow th at of av irostris) w ere absent in th e w ater column. In early metazoan microzooplankton, ex cept during spring and summer ( J ully 1-2 0 0 6) started to appear and reach ed w inter ( Table 4 and F ig. 5 ) . peak v alues at th e end of month , persisting in th e In general, th e dierent groups of metazoan plankton until S eptember ( F ig.7 ) . Pteropods reach ed microplankton and mesozooplankton follow ed similar th eir max imum concentration during summer w h ile patterns of v ertical distribution ( F ig. 5 and 6) . e th eir presence diminish ed to almost th eir complete max imum gradient in copepods and copepodites absence in w inter and spring ( F ig.7 ) . Rotifers w ere biomass ( mesozooplankton, F ig. 6) w as observ ed in absent most of year and ex h ibited an abrupt peak in A pril 2 0 0 6. C lodocerans sh ow ed strong gradients of J une ( F ig.7 ) . A ppendicularians w ere present along abundance and biomass during summer ( J ul2 -0 6) w ith all th e studied period but w e did not observ ed a clear max imal abundance and biomass at surface w aters seasonal pattern in abundance. C h aetognath s, alth ough ( Table 5 and F ig. 6) . w ere more abundance in summer and autumn neith er sh ow ed a clear seasonal pattern ( F ig.7 ) . 3.4. Metazooplankton seasonal distribution patterns e total biomass of copepod nauplii w ith in th e e densities of th e dierent groups of metazoan study period w as h igh ly v ariable, ranging from less th an microplankton ( 5 0 -2 0 0 µm) as estimated from integrated 0 .1 µg C L -1 in A pril 2 0 0 6 to more th an 0 .8 µg C L -1 in plankton net h auls are sh ow n in Table 2 . C opepod nauplii May 2 0 0 6. C opepods and copepodites w ere th e most and copepodites w ere numerically dominant during important groups in term of biomass th rough out th e most of th e studied period, w ith densities ranging from year ex cept in J uly 1-2 0 0 6 w h en cladocerans dominated ~ 62 0 to 2 39 0 0 ind. m 3 and from ~ 2 65 to 10 0 0 0 ind. m 3, ( F ig. 7 ) . e minimum biomass v alues of copepod respectiv ely ( Table 2 ) . Oth er h oloplankton components nauplii and copepodites w ere observ ed in early spring. w ere pteropods, appendicularians, and rotifers but th eir e max imum v alues of pteropods and cladocerans

42 Chapter 1

biomass were observed during summer and early to follow , w ith som e tim e lag, that of protozoan autumn. Appendicularians and rotifers biomass peaked m icroplankton, particularly dinoagellates (Fig 3 and in Ma r 2006 a nd Jun 2006 re s p e ctive l y (Fig.7).
eFig. s ize11). e m inim um abundance of copepods and ca te gory < 200 µm (m e ta zoa n m icrop l a nkton)their conta developm ine d ental stages (nauplii and copepodites) a n im p orta nt fra ction of the b iom a s s of cop eoccurred p od na during up l ii the spring phytoplankton peak (M arch (a ve ra ge : 72%, ra nge : 36%-93%), cop e p odite sand (a veA pril ra ge2006) w hen a Phaeocystis bloom occurred 38%, ra nge : 18%-53%), rotife rs (99 %, ra nge 91%-100%)(Fig. 3 an d Fig. 7). C ontrarily, polychaete an d decapod a nd p te rop ods (a ve ra ge : 14%, ra nge 0%-100%),larvae whe re peaked a s during the spring phytoplankton bloom for othe r hol op l a nktonic group s wa s ve ry l ow(Fig. (<5%) 3 and 8). e abundance m axim um of rotifers and (Fig.7). d i n o a g e l l a t e s c o i n c i d e d ( J u n e 20 0 6 , F i g . 3 a n d F i g . 7 ) . Biva l ve , p ol ycha e te a nd ga s trop od l a rva e we ere vertical the distribution of m etazooplankton w as in m os t a b unda nt group s of m e rop l a nkton part during determ the in ed by the hydrograph ic prole of the w ater s tudie d p e riod, with a cl e a r s ucce s s ion in the acolum b unda n, nce w ith m axim um gradients of m etazooplankton p e a ks for the m a in group s (Fig. 8). Echinode rmabundance l a rva eduring m onths of m axim um gradients p e a ke d in a b unda nce in winte r (ca . 3003), ind.of tem m perature and salinity. e patterns of vertical p ol yche ta a nd de ca p oda l a rva e in e a rl y sdistribution p ring (~1420 of m etazoan m icroplankton (Fig. 5) and a nd 70 ind. m3, re s p e ctive l y), b iva l ve a nd cirripm e d esozooplanktonl a rva e (Fig. 6) seem ed to be unrelated to in e a rl y s um m e r (1780 a nd 5303 re ind. s p m e ctive lchlorophyll y) concentration (Fig. 1). H ow ever, during a nd ga s trop od l a rva e in l a te s um m e r/esum a rl y m a utum er and nautum n (from Septem ber and October (810 ind. m 3).
e < 200 µm s ize ca te gory conta ine2005, d aand n from June to Septem ber 2006) the vertical im p orta nt fra ction of the b iom a s s of b iva l vedistribution , ga s trop of od m etazoan m icroplankton (Fig. 5) tends a nd p ol ycha e ts (a ve ra ge : 56%, 37%, 24%, re s pto efollow ctive l y)those of their potential m icroplankton prey whe re a s the contrib ution of e chinode rm s , cirrip(ciliates, e d a din nd oagellates, diatom s), w ith som e exception s decapod larvae was considerably lower (<5%) (Fig. 8). (Fig. 2). M esozooplankton biom ass depth distribution (Fig. 6) w as inversely related to the Phaeocystis spp 3.5. Relations between zooplankton and abiotic vertical distribution (M arch and A pril 2006, Fig. and biological factors 2). Sim ilar to m etazoan m icroplankton, the vertical distribution of m esozooplankton biom ass (Fig. 6)
e tem poral variability on the biom ass of the seem s to b e related th at of ciliates an d d in oagellates at m ain com ponents of the planktonic com m unity and least during som e m onths (Septem ber 2005, July 2-2006 the ratio heterotrophs/ autotrophs biom ass (H/A) are a n d Se p t e mb e r 20 0 6 ( F i g . 2) . shown in Table 6.
e ratio was generally > 1, except in March (Table 6).
ere were not clear relationships 3.6. Comparison of plan kton n et vs. bottles for between tem perature and the biom ass of the m icrobial com ponents (Table 6), or that of m etazoans and eitherquantitative zooplan kton samplin g. bacteria or protozoan biom ass (Table 6). However, e seasonal pattern of metazooplankton abundance phytoplankton was directly related to bacteria and an d biomass as estimated w ith b oth samp lin g meth o d s protozoan biom ass (Fig. 9A, B).
e biom ass of thew as quite similar (F ig. 11). H ow ever, th e abun d an ce an d entire m etazooplankton com m unity was negativelybiomass of micrometazooan plan kton as estimated by correlated to total phytoplankton biom ass (Fig. 9C). th e in tegration of zooplan kton samples from Van D orn
e abundance and biom ass of m etazoanbottles w ere higher than those obtained from plan kton m icroplankton increased exponentially with the n ets samp les b y a facto r o f 2 ( ran ge: 1.2-4.8) an d 2.3 rise in tem perature (Fig. 10A, B). In the case of (ran g e: 1 -5 .5 ) resp ectively, (F ig . 1 1 A , C). Co n trarily, Van m esozooplankton, we found the sam e exponentialD ohrn bottles samples underestimated the abundance relationship with tem perature in term s of abundanceand biomass of mesozooplankton as compared to but not in biom ass (Fig. 10C, D). Regarding the plan kton n et samples except in M arch an d A pril 2006 (3 dierent zooplankton groups, the abundance anof d all2 times h igh er, resp ectively, F ig.11 B , D ). E xclud in g holoplanktonics groups follow ed an exponentialthe samplin gs on M arch an d A pril 2006, the abun dan ce relationship w ith tem perature2 = 0.44-0.71) (r exceptand biomass estimations of mesozooplankton from for apendicularians, rotifers and chaetognats .n e et samples w ere 1.7 times h igh er (ran ge 1-3.5) th an abundance of bivalve and gastropod larvae wth ere ose from Van D oh rn bottles (Fig. B , D ). e slop es positively correlated to tem perature2 = 0 .(r 3 2 a n d 0 . 5 1 , corresponding to the linear regressions betw een the resp ectively). number of organism obtained w ith plankton net and e total zooplankton abundance and biombottles ass w ere > 1 for metazoan microplan kton , w hile for did not show a seasonal pattern follow ing theme s o z o o p l a n k t o n t he s l o p e w a s < 1 ( F i g . 1 2) . phytoplankton bloom s (chlorophyll). C onversely, m axim um abundance of m etazooplankon occurred aer the dinoagellate abundance peak, and the 3.7. Con tribution of the metazo an microp lan kton seasonal abundance of total m etazooplankton tendsa n d p l a n k t o n i c l a r va e t o t o t a l z o o p l a n k t o n b i o ma s s

43 Chapter 1

Metazoan microplankton represented the 18% of plank ton up to mesoz ooplank ton ( Gasol et al. 19 9 7) . the total metazooplankton carbon when considering One of the main sources of dissolv ed organic carb on net samples (range: 3%-37%, Fig. 13A), while in bottle f or b acteria is the phytoplank ton exudations ( A z am et samples micrometazoans constituted the 34% (range: al. 19 8 3; Furhman 19 9 2) , w hich explains the ob serv ed 15%-54%) (Fig. 13B). correlation b etw een b acteria and autotrophs b iomass Larval developmental stages (including holo-and in accordance w ith other eld ob serv ations ( Cole et al. meroplanktonic larvae) as estimated from plankton 19 8 8 ; White et al. 19 9 1; Moran et al. 2002) net and bottle comprised, respectively, the 45% Similar to other Mediterranean areas ( Satta et (range: 24%-75%) and 55% (range: 26%-69%) of the al. 19 9 6 ) , the study site w as a heterotrophic system total metazooplankton carbon (Fig. 13 C, D). In both during most of the year ( H:A > 1) , primary producers plankton net and bottles samples, larvae constituted the b iomass exceeding or equalling heterotroph b iomass dominant component of the micrometazoan fraction; in only during phytoplank ton b looms. e predominance average 93% (range: 55%-100%) and 95% (range 77%- of heterotrophy suggests z ooplank ton community 100%) for plankton net and bottles samples, respectively depends on sources other than primary producers to (Fig. 13 C, D).
e contribution of larval stages to the sustain the organic carb on demands and a major role total m esozooplankton carbon was quite sim ilar inof m icrobial carbon uxes (m icrobial loop, A zam 1983) plankton and bottles sam ples, in average ~34% (13%- in the coastal N W M editerranean Sea (G asol et al. 61%) (Fig.13 C, D). 1997). Meroplanktonic larvae estim ated from plankton net and bottles represented, respectively, the 20% 4.2. Com position, seasonal abundance and vertical (range: 5%-60%) and 25% (range: 8%-54%) of distribution of the m etazooplan kton com m un ity in the total m etazooplankton biom ass (Fig.13 E, F).relation to abiotic an d biological factors
e contribution of m eroplankton to the total m esozooplankton carbon was quite sim ilar in plankton Copepod nauplii w ere num erically the dom inant and bottles sam ples, in average ~21% (3-53%) (Fig.13 com ponents of the m etazooplankon com m unity as E,F ). Meroplankton represented the 26% and 32% of d escribed in previous stud ies in coastal areas (Calbet et the m etazoan m icroplankton biom ass from planktonal. 2001; L opez et al. 2008). e n auplii densities w ere net and bottles sam ples, respectively (Fig. 13 E, F ). in the sam e range than those reported for oligo- and m esotroph ic w aters (Ro et al. 1995; Calbet et al. 2001; Que v e d o a n d A n a d Û n 20 0 2; P e d e r s e n e t a l . 20 0 5 , L o pe z 4. Discussion et al. 2008), but low er than in m ore productive areas (Lucic et al. 2003; Uye et al. 1996). Regarding biom ass, 4. 1. Physical-chemical and biological variables as observed in previous studies adult copepods and e seasonal v ariab ility and v ertical proles of copepodites w ere the dom inant com ponents in biom ass temperature and salinity w ere the expected ones f or NW o f m e ta zo o pl a n kto n c o m m un i ty , e x c e pt i n s um m e r Mediterranean coastal w aters ( Masó and Duarte 19 8 9 ; w hen clodocerans dom inated (A lcaraz 1970; A tienza Salat 19 9 6 ) . Primary producers ( chlorophyll) f ollow ed 20 0 7 ) . the typical mid-latitude annual cycle, w ith a relativ e W e are aw are that a m onthly sam pling during maximum in autumn and a more intense b looming in a single year does not faithfully describe a seasonal late w inter-spring ( Mura et al. 19 9 6 ; Duarte et al. 19 9 6 ) . cycle. H ow ever, som e m ain features can be identied, Besides others f actors ( e.g. irradiance, temperature) , w hich in light of previous studies, m ay help to better phytoplank ton b looms are promoted b y the increase ch aracterize th e season ality in th e plan kton of th e area. in nutrient supply w hen v ertical stab ility of w ater In this regard, w e have to consider generalizations are column decreases ( Morel and A ndre 19 9 1; Estrada et risky because zooplankton seasonal abundance m ay al. 19 8 5 ; Duarte et al. 19 9 6 ) . eref ore, the periods of dier am ongst M editerranean coastal areas and be w ater column mixing in autumn-w inter, and thermal variable am ongst annual cycles (Kim or and Berdugo stratication in summer, explained in part the ev olution 1967; Regn er 1985; Christou 1998, Calbet et al. 2001). of the depth-integrated and v ertical distrib ution pattern N evertheless, w e found seasonal patterns for som e of phytoplank ton b iomass. A lthough the scarce depth hol ozoopl ankton g r oups ( Cl ad oc e r ans , pte r opod s , of the study area prev ented the f ormation of the typical Oithona , d ol iol id s ) q uite s im il ar as d e s c r ib e d in summer deep phytoplank ton maximum at the b ase of pr e v ious s tud ie s in M e d ite r r ane an c oas tal ar e as ( Cal b e t the thermocline, usually b etw een 4 0 and 100 m ( Estrada e t al . 20 0 1 ; Kate c hakis e t al . 20 0 2; A tie nza e t al . 20 0 7 ) et al. 19 8 5 ) , the sub -surf ace relativ e maxima ob serv ed Te m pe r atur e s e e m s to inue nc e d e c is iv e l y on the in May and June w ere clearly related to temperature s e as onal ab und anc e patte r n of hol ozoopl ankton ( b oth ( density) gradients. m e tazoan m ic r opl ankton and m e s ozoopl ankton) in A s expected, heterotrophic b acteria constituted an M e d ite r r ane an c oas tal ar e as in ag r e e m e nt w ith pr e v ious important f raction of the plank ton b iomass, not only s tud ie s ( Cal b e t e t al . 20 0 1 ) . E g g pr od uc tion, and regarding the microb ial components, b ut f or the w hole c ons e q ue ntl y the ab und anc e of naupl ii, is c ontr ol l e d b y

44 Chapter 1

water temperature in many copepods species (Mauchline Uchim a 1 9 8 8 ; B r og l io et al . 2 0 0 4; A tien z a et al . 2 0 0 6; 1998). Field observations support the positive relation Hen r ik sen et al . 2 0 0 7 ) an d con sequ en tl y d iatom s ar e between nauplii abundance and temperature (Lopez et n ot an ap p r op r iate f ood f or this ab u n d an t sp ecies. al. 2008). Besides copepods, the control de populations I n con tr ast to total hol op l an k ton , m er op l an k ton ic of some holoplanktonic organisms such as clodocerans l ar v ae show ed their m axim al ab u n d an ce d u r in g the is directly correlate with temperature (Onbe and Ikeda p hytop l an k ton b l oom , as ob ser v ed in other stu d ies in 1995, Calbet et al 2001, Atienza et al. 2007).
is link the M ed iter r an ean an d A tl an tic coastal w ater s ( A n d r eu between temperature and the cladocerans population an d Du ar te 1 9 9 6; C al b et et al . 2 0 0 1 ; Hig hel d et al . dynamics may be related to the inuence of warm 2010). temperature on benthic resting eggs, which may induce M icr op l an k ton p r otoz oan ar e com m on l y p ar t of resting eggs hatching (Eglo et al. 1997) m etaz oop l an k ton d iet ( Stock er an d C ap u z z o 1 9 9 0 ; It is com m only stated that, in tem perateC al b et an zones, d Saiz 2 0total 0 5 , A l m ed a et al . 2 0 1 1 ) . I n f act, the zooplankton abundance is directly dependentv er tical d istr on ib uthe tion of m etaz oan s ob ser v ed d u r in g the phytoplankton biom ass, thus zooplanktonstu d y tenabundance d s to f ol l ow that of m icr op r otoz oan s, an d the peaks follow the phytoplankton bloom sseason w ith al absom u n d an ece tim of total e m etaz oop l an k ton seem s to lag (zooplankton production is slow er)b e(e.g. cor el atedDauby w ith the season al ab u n d an ce of p r otoz oan 1980; Krause and ram s 1983). How ever, asm observed icr op l an k ton , p ar ticu l ar l y d in o ag el l ates. e in other studies in the M editerraneanm w etaz aters oop l an k(Regner ton m axim al ab u n d an ce p eak w as m ain l y 1985; C hristou 1998; C albet et al. 2001), thecon seasonal stitu ted b y sm al l cop ep od s of the g en u s O ithon a. A s pattern of total zooplankton abundancem en during tion ed p rour ev iou sl y, O ithon a f ed on l y on m otil e p r ey, study w as not directly related to thean dphytoplankton m ain l y on a siz e r an g e of n an o- an d m icr op l an k ton ; biom ass (chlorophyll). Several studieshen ce have d in o found ag el l ates an d cil iates w ou l d ap p r op r iate the m inim al m etazooplankton concentrationsf ood f or theses under cop ep od s ( Uchim a an d Hir an o 1 9 8 6; phytoplankton bloom conditions in agreemB r og l io et ent al . w 2 ith 0 0 4; Hen r ik sen et al . 2 0 0 7 ) . ese our ndings (Harvey et al. 1935; C albet etob al. ser v2001). ation s con r m the im p or tan ce of p l an k ton ic Negative correlation betw een nauplii abundancep r otoz oan an d mand etaz oan tr op hic in ter action s in m ar in e chlorophyll concentration has alsop l an kbeen ton ic com observed m u n ities ( San d er s an d Wick m an 1 9 9 3) . (Lopez et al. 2008). During our study, spring bloomM ar in e - l if e has tr ad ition al l y b een d iv id ed in to form ing phytoplankton groups were diatomp l an k ton s an d and b en thos, w ithou t con sid er in g that m ost of Phaecystis sp. colonies. A lthough somthese e or gzooplankters an ism s ar e n ot tr u l y in d ep en d en t ( Gian g r an d e can fed eciently on Phaecystis, m anyet al . studies 1 9 9 4; B oer ohave et al . 1 9 9 6) . I n f act, the p l an k ton show n that Phaeocystis bloom s producean d b ennegative thos com m eects u n ities ar e r ecip r ocal l y con n ected (e.g. reduced feeding and abundance) on zooplanktonb y b oth f u n ction al ( en er g y u xes) an d str u ctu r al (review ed by Nejstgaard et al. 2007), including( l if e cycl poor es d yn am ics) r el ation ship s, p ar ticu l ar l y in fecundity in som e copepod species coastal(Turner ar eas et ( B oeral. o et al . 1 9 9 6) . O u r n d in g s con r m 2002), w hich m ay explain the low m etazooplanktonthe tim in g b etw een p hytop l an k ton b l oom s an d the ab u n d an ce d u r in g the Phaecystis bloom. ap e p ear an ce of som e m er op l an k ton ic l ar v ae, w hich v er tical d istr ib u tion p atter n s of Phaeocystis spp an d m axim iz e the exp osu r e of l ar v ae to an ab u n d an t f ood cop ep od s w er e in v er sel y r el ated , w hich su p p or t this su p p l y ( or son 1 9 46, 1 9 5 0 ) . F or som e b en thic sp ecies, p hytop l an k ton sp ecies m ay hav e p oten tial n eg ativ e p hytop l an k ton b l oom s act as d ir ect in d u ctor s of the eects on the cop ep od ab u n d an ce. I n g en er al , in NW r el ease of g am etes or l ar v ae b y either d ir ect con tact M ed iter r an ean coastal w ater s, the m ain com p on en t of ( settl em en t of p hytop l an k ton p ar ticl es) or chem ical the sp r in g p hytop l an k ton b l oom s ar e d iatom s. Diatom s tr ig g er in g ( Star r et al . 1 9 9 0 , 1 9 9 1 , 1 9 9 4) . Hen ce, ar e n u tr ition al l y in su cien t f or the r ep r od u ction of b esid es other f actor s ( e. g . , tem p er atu r e, p r ed ation ) , som e cop ep od sp ecies ( Stattr u p an d Jen sen 1 9 9 0 ; this b en thic-p el ag ic cou p l in g can d r iv e the p atter n s Kl ep p el 1 9 9 3) an d sev er al au thor s hav e ar g u ed that they of som e m er op l an k ton ic l ar v ae in coastal w ater s. O n m ay p r od u ce toxic or d el eter iou s eects in the cop ep od the sam e han d , the ob ser v ed r el ation ship s b etw een the em b r yon ic an d n au p l iar d ev el op m en t ( B an et al . 1 9 9 7 ; com p osition an d ab u n d an ce of m icr ob ial p l an k ton ic I an or a et al . 2 0 0 4) . How ev er , el d an d l ab or ator y stu d ies com m u n ity an d som e “hol op l an k ton ic esp ecies” hav e d em on str ated that d iatom s hav e n ot n eg ativ e eects ( top l an k ton b l oom -cal an oid s; d in o ag el l ates-r otif er s) on cal an oid cop ep od eg g hatchin g su ccess ( Jon asd ottir su g g est that p l an k ton com m u n ity m ay act as tr ig g er an d Kior b oe 1 9 9 6; I r ig oien et al . 2 0 0 2 ) . L ik el y, the sig n al f or hatchin g of r estin g eg g s of som e z oop l an k ton sp r in g p hytop l an k ton m ay b e n u tr ition al l y p oor or g r ou p s. er ef or e, ou r el d ob ser v ation s em p hasis in ap p r op r iate f or the r ep r od u ction an d d ev el op m en t the r el ev an ce of u n d er statin g b en to-p el ag ic cou p l in g of som e z oop l an k ter s ( e. g . cal n oid s) b u t n ot f or other p r ocesses to com p r ehen d the p op u l ation d yn am ics of g r ou p s. is is the case of O ithon a sp ecies ( in cl u d in g b oth p l an k ton ic an d b en thon ic m ar in e system s. n au p l ii an d cop ep od ites) that f ed on l y on m otil e p r ey ( Uchim a an d Hir an o 1 9 8 6; Tsu d a an d Nem oto 1 9 8 8 ;

45 Chapter 1

4.3. Bottle versus net samples: comparison of the organisms are generally higher than those of larger c atc hing e
c ienc y organisms, the exclusion of metazoan microplankton may lead to important subestimations of zooplankton In most plankton studies there are severe secondary production, with consequent eects on restrictions imposed by the lack of universal sampling marine biogeochemical models. gears able to provide reliable samples for the whole plankton components. is has occasionally resulted in neglecting plankton groups that are placed, by their 5. References size or special characteristics, in no man’s land between Alcaraz, M. 1970. Ciclo anual de los cladóceros en aguas more evident plankton components. Plankton nets, de Castellón (Mediterráneo occidental). Invest. Pesq. 34, 281- even those able to take multiple depth samples, have 290. an integrative character and certain unpredictability Alcaraz, M. 1977. Muestreo cuantitativo de zooplancton: (Winsor and Clarke 1940; Hansen and Andersen 1962; Análisis comparativo de la ecacia de mangas y botellas en un Grandperrin and Michel 1969). Other sampling gears sistema estuárico. Invest. Pesq. 41, 285-294. Alcaraz, M. 1982. Zooplankton biomass and its relation like pumps, large volume bottles, etc., that can provide to total particulate carbon and nitrogen in Northwest Africa. more discrete samples have at turn specic problems Rapp. P.-V. Rtun. Cons. Inl Explor. Mer. 180, 270-273. (Sameoto et al. 2000). Alcaraz, M. 1983. Coexistence and segregation of e sampling methodology used in this study congeneric pelagic copepods: Spatial distribution of the (simultaneous use of microplankton nets and large Acartia complex in the ria of Vigo (NW of Spain). J. Plankton volume water samples at discrete depths) had been Res. 5, 891-900. already demonstrated to be adequate in coastal areas Alcaraz, M. 1985. Vertical distribution of zooplankton to quantitatively sample the minor components of biomass during summer stratication in the western plankton. e coe
cient of variation between repeated Mediterranean. In: P.E. Gibbs (ed.) Proceedings of the19th EMBS, Plymouth. Cambridge University Press. bottle samples (18 %, Alcaraz 1982), better than that Alcaraz, M., Domínguez, M. 1985. Larvas de moluscos obtained in net hauls (Winsor and Clarke 1940), and lamelibranquios en la ría de Pontevedra (NO de España): ciclo the possibility for unequivocally relate the organisms anual. Invest. Pesq. 49, 165-173. sampled to the physical, chemical and biological Almeda, R., Calbet, A., Alcaraz, M., Saiz, E., Trepat, characteristics of the water collected by the bottle I., Arin, L., Movilla, J., Saló, V. 2011. Trophic role and are major advantages (Alcaraz 1985; Alcaraz and carbon budget of metazoan microplankton in northwest Dominguez 1985). e higher abundance and biomass Mediterranean coastal waters. Limnol. Oceanogr. 56(1), 415- of metazoan microplankton as obtained by integrating 430. bottle samples as compared to net hauls coincides with Almeda, R., Messmer, A., Sampedro, N., Gosselin, L.A. 2011. Feeding rates and abundance of marine invertebrate previous studies (Alcaraz 1977). eir lower e
ciency planktonic larvae under harmful algal bloom conditions o of plankton bottles to collect larger zooplankton (> 200 Vancouver Island. Harmful Algae 10, 194-206. µm) could be due to the higher swimming capacity Andersen, V., Devey, C., Gubanova, A., Picheral, M., of mesozooplankton organisms, and to their patchy Melnikov ,V., Tsarin, S., Prieur, L. 2004. Vertical distribution distribution; the exceptions observed in March and of zooplankton across the Almery-Oran frontal zone April 2006 are clearly due to the clogging of the net (Mediterranean Sea). J. Plankton Res. 26, 275-293. due to the Phaeocystis bloom that reduced the ltering Andreu, P., Duarte, C.M. 1996. Zooplankton seasonality e
ciency of the net. Our results reect the importance of in Blanes Bay (northwest Mediterranean). Publ. Esp. Inst. Esp. appropriate sampling methods for accurate estimations Oceanogr. 22, 47-54. Atienza, D., Calbet, A., Alcaraz, M., Saiz, E., Trepat, I. of the zooplankton community structure. 2006. Trophic impact, metabolism, and biogeochemical role of the marine cladoceran Penilia avirostris and the codominant 4.4. Relative importance of metazoan microplankton in copepod Oithona nana in NW Mediterranean coastal waters. term of biomass Mar. Biol. 150, 221.235. Atienza, D., Calbet, A., Saiz, E., Lopes, R. 2007. Ecological Most copepod nauplii and meroplanktonic larvae success of the cladoceran Penilia avirostris in the marine were < 200 µm, which emphasize the importance of using environment: feeding performance, gross growth e
ciencies ne mesh plankton nets for adequate metazooplankton and life history. Mar. Biol. 151, 1385-1396. sampling (Calbet et al. 2001; Galliene and Robins 2001; Azam, F., Fenchel, T., Field, J.G., Gray, J.S., Meyerreil, Turner 2004; Lopez et al. 2008). Our results conrm L.A., ingstad, F. 1983. e ecological role of water-column that the exclusion of metazoan microplankton suppose microbes in the sea. Mar. Ecol. Prog. Ser. 10, 257-26. the loss of ~ one-third of the metazooplankton biomass Ban, S., Burns, C., Castel, J., Chaudron ,Y., Christou ,E., et (Galliene and Robins 2001; Riccardi 2010; Almeda et al. 1997. e paradox of diatom-copepod interactions. Mar. al. 2011). erefore, although the mesozooplankton Ecol. Prog. Ser. 157, 287-293. Berggreen, U., Hansen, B., Kiorboe, T. 1988. Food size generally dominates in terms of biomass, the metazoan spectra, ingestion and growth of the copepod Acartia tonsa microplankton represents a considerable fraction of the during development: implications for determination of total metazooplankton carbon in most marine pelagic copepod production. Mar. Biol. 99, 341-352. systems. Moreover, as physiological rates of small Boero, E., Belmonte, G., Fanelli, G., Piraino. S., Rubino, F.

46 Chapter 1

1996.
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A ldeh yde suppression of copepod recruitment in e Mediterranean climate as a template for Mediterranean blooms of a ubiq uitous planktonic diatom. N ature 42 9 , 40 3- marine ecosystems, th e ex ample of th e N E S panish littoral. 40 7 . Prog. Oceanogr. 44, 2 45 -2 7 0 . J onasdottir, S .H., K iorboe, T. 19 9 6. C opepod recruitment E glo, D.A ., F ofono, P.W ., Onbé , T. 19 9 7 . Reproductiv e and food composition: do diatoms aect h atch ing success? biology of marine cladocerans. A dv . Mar. B iol. 31, 7 9 -168. Mar. B iol. 12 5 , 7 43-7 5 E strada, M. 19 85 . Deep ph ytoplankton and ch oloryph yll K rause, M., rams, J . 19 83. Zooplankton dynamics max ima in th e w estern during F LE X ’7 6. In: S ündermann, J . and Lenz, W . ( eds) , Mediterranean. In: M. Moraitou-A postolopoulou and V. N orth S ea Dynamics. S pringer Verlag, B erlin, pp. 632 -661. K iortsis ( eds.) : Lopez, E ., Viesca, L., A nadón, R. 2 0 0 7 . S easonal v ariation Mediterranean Marine E cosystems, pp. 2 47 -2 7 7 . Plenum in abundance and feeding rates of th e rst stages of copepods Press, N ew Y ork. in a temperate sea. Mar. E col. Prog. S er. 35 2 , 161-17 5 F otel, F .L., J ensen N .J ., W ittrup, L., Hansen B .W . 19 9 9 . López, E ., A nadón, R. 2 0 0 8. C opepod communities along In situ and laboratory grow th by a population of blue mussel an A tlantic Meridional Transect: A bundance, size structure, larv ae ( Mytilus edulis L.) from a Danish embayment, K nebel and grazing rates. Deep-S ea Res. I, 5 5 ( 10 ) 137 5 -139 1. Vig. J . E x p. Mar. B io. E col. 2 33, 2 13-2 30 . Lucic, D., N jire, J ., Morov ic M., et al. 2 0 0 3. F uh rman, J . 19 9 2 . B acterioplankton roles in cycling of Microzooplankton in th e open w aters of th e north ern A driatic organic matter: th e microbial food w eb. In: P.G . F alkow ski S ea from 19 9 0 to 19 9 3: e importance of copepod nauplii and A .D. 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47 Chapter 1

Katechakis, A., Stibor, H., Sommer, U., Hansen, T. 2002. Riccardi, N . 2010. Selectivity of plankton nets over Changes in the phytoplankton community and in the microbial m esozooplankton taxa: im plications for abundance, biom ass food web of Blanes Bay (Catalan Sea, NW Mediterranean) and diversity estim ation J. Lim nol. 69 (2), 287-296. under prolonged grazing pressure by doliolids (Tunicata), R o et al., 1995 J.C . R o, J.T. Turner, M .K . W ebber and cladocerans or copepods (Crustacea). Mar. Ecol. Progr. Ser., R .R . H opcro, B acterivory by tropical copepod nauplii: extent 234, 55-69. and possible signicance, A quatic M icrobial Ecology 9 (1995), Kimor, B., Berdugo, V. 1967. Cruise to the Eastern pp. 165-175 Mediterranean, Cyprus 03 Plankton reports. Sea Fish. Res. St. Quevedo, M ., A nadón, R . 2000. Spring m icrozooplankton Halif. Bull. 45, 6-12. com position, biom ass and potential grazing in the C entral King, K.R. 1980.
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Publ. Esp. Inst. Esp. Stoecker, D .K ., C apuzzo, J. 1990. Predation on protozoa: O ceanogr. 22, 23-29. Its im portance to zooplankton. J. Plankton R es. 12, 891-908, O nbé , T., Ikeda, T.1995. M arine cladocerans in Toyam a orson, G . 1946. Reproduction and larval developm ent Bay, southern Japan Sea: seasonal occurrence and day-night of D anish m arine bottom invertebrates. M eddr. Kom m n. vertical distributions. J. Plankton R es. 17, 595-609. D anm . Fisk.-og H avunders. Ser. Plankton. 4(1), 1-523. Parsons, T.R ., M aita, Y., Lalli C .M . 1984. A m anual of orson, G . 1950. Reproductive and larval ecology of chem ical and biological m ethods for sea w ater analysis. m arine bottom invertebrates. B iol. R ev. C am b. Philos. Soc. 25, Pergam on, O xford. 1-45. Pedersen, S.A . R ibergaard, M .H ., Sim onsen, C .S. 2005. Tsuda,A ., N em oto, T. 1988. Feeding of copepods on M icro- and m esozooplankton in Southw est G reenland w aters natural suspended particles in Tokyo B ay, Japan. J. O ceanogr. in relation to environm ental factors. J. M ar. Syst. 56, 85-112. Soc. Jpn. 44: 217-227. Porter, K .G ., Feig, Y.S. 1980. e use of D A P I for identifying Turner, J.T., Ianora, A ., Esposito, F., C arotenuto, Y., and counting aquatic m icroora. Lim nol. O ceanogr. 25, 943- M iralto, A . 2002. Zooplankton feeding ecology: does a diet 948. of Phaeocystis support good copepod grazing, survival, egg Putt, M ., Stoecker, D . K . 1989. A n experim entally production and egg hatching success? J. Plankton R es. 24, determ ined carbon: volum e ratio for m arine ‘oligotrichous’ 1185-1195. ciliates from estuarine and coastal w aters. Lim nol. O ceanogr. Turner, J.T. 2004. e im portance of sm all planktonic 34, 1097-1103. copepods and their roles in pelagic m arine food w ebs. Zool. Regner, D .1985. Seasonal and m ultiannual dynam ics of Stud. 43, 255-266. copepods in the m iddle A driatic. A cta A driat. 26, 11-99.

48 Chapter 1

Verity, P.G., Langdon, C. 1984. Relationships between White, P.A., Kal, J., Rasussen, J.B., Gasol, J.M. 1991. lorica volume, carbon, nitrogen, and ATP content of tintinnids e eect of tem perature and algal biom ass on bacterial in Narragansett Bay. J. Plankton Res. 6, 859-868. production and specic grow th rate in freshw ater and m arine High
eld, J.M., Eloire, D., Conway D.V.P., et al. 2010. habitats. M icrob. E col. 21, 99-118. Seasonal dynamics of meroplankton assemblages at Station W insor, C . P., C larke, G .L. 1940. A statistical study of L4. J. Plankton Res. 32, 681-691 v a r i a ti on i n th e ca tch of p la nkton nets. J . M a r i ne R es. 3 , 1 - 3 4 . Uye, S.I., Nagano, N., Tamaki, H. 1996. Geographical and seasonal variations in abundance, biomass and estimated Acknowledgments production rates of microzooplankton in the Inland Sea of Japan. J. Oceanog. 52, 689-703. i s wor k wa s f u nded b y th e S p a ni sh M i ni str y of S ci ence Waterbury, J.B., Watson, S.W., Valois, F.W., Franks, D.G. a nd I nnov a ti on (M I C I N N ) th r ou gh a P h . D . f ellowsh i p (B E S - 1986. Biological and ecological characterization of the marine 2 0 0 5 - 7 4 9 1 )to R . A. a nd th e r esea r ch p r ojects C T M 2 0 0 4 - unicellular cyanobacterium Synechococcus. Can. Bull. Fish. 0 2 7 7 5 to A. C . , C T M 2 0 0 6 - 1 2 3 4 4 - C 0 2 - 0 1 /M AR to M . A. , a nd Aquat. Sci. 214: 71-120. C TM 2007-60052 to E.S.

49 Chapter 1

TABLE 1

Table 1. Length vs. carbon content regressions used to provide biomass estimates for the different groups of zooplankton. Individual biomass (µg C) = a × L (µm) b.

Organism Measurement a b Reference

Copepods Length of prosome 4.27×10 -9 3.07 Uye (1982) Copepodites Length of prosome 1.11×10 -8 2.92 Berggreen et al. (1988) Copepod nauplii Body length (without setae) 3.18×10 -9 3.31 Berggreen et al. (1988) Appendicularia Trunk length (without tail) 7.33×10 -8 2.63 King (1980) Cladoceran Total length 1.82×10 -13 4.51 Uye (1982) as Penilia Rotifers Body length 1.06×10 -7 2.74 Hansen et al. (1997) Chaetognata Total length (mm) 5.1×10 -2 3.16 Uye (1982) Tunicata Total length (mm) 1.62 1.93 Heron et al. (1988) as Thalia Pteropoda Total length 3.06×10 -8 2.88 Fotel et al (1999) as bivalve Polychaeta larvae Maximal length 1.58×10 -4 1.38 Hansen (1999) as Polydora Bivalvia larvae Maximal length 3.06×10 -8 2.88 Fotel et al (1999) Gastropoda larvae Maximal length 2.31×10 -5 2.05 Hansen and Ockelmann (1991) Echinoderma larvae Total length 3.06×10 -8 2.88 Fotel et al (1999) as bivalve Cirripede nauplii Body length (without spine) 2.20×10 -10 3.72 Turner et al. (2001) Decapoda larvae Carapace length 4.01×10 -12 4.43 Hirota and Fukuda (1985)* Other larvae Total length 3.06×10 -8 2.88 Fotel et al (1999) as bivalve

* Calculated using length and carbon content data from table 1 (r 2 = 0.91)

50 Chapter 1 ) -3 TABLE 2 TABLE Holoplankton Meroplanktonic larvae Nauplii Nauplii Copepodit. Append. Rotifers Pterop. Others Polych. Bivalve Gastrop. Echin. Cirrip. Others Date Metazoan microplankton (Ind m Metazoan microplankton SEP-05 SEP-05 05 14 Sep OCT-05 05 17 Oct 10042 NOV-05 05 29 Nov 8495 JAN-06 2790 1436 06 18 Jan MAR-06 1985 06 15 Mar 12090 APR-06 333 66 2759 06 Apr 4 MAY-06 1573 06 16 May 67 JUN-06 11469 619 811 0 67 06 14 Jun JUL1-06 85 06 6 Jul 1420 0 5254 JUL2-06 265 155 96 06 31 Jul 25 AUG-06 19121 0 3659 06 29 Aug 23897 40 78 0 SEP-06 118 10031 14594 52 8 06 28 Sep 305 7563 459 135 0 0 12570 6782 20 89 0 0 628 2174 71 315 3515 0 0 36 34 0 36 246 0 0 393 309 107 36 0 36 155 65 0 36 0 0 95 359 838 71 0 302 907 20 249 12 0 212 888 17 39 40 107 361 0 89 22 1255 23 32 11 71 18 0 0 1420 36 44 106 22 0 11 25 14 1101 142 36 426 0 0 0 0 178 64 0 249 0 533 106 0 0 0 0 320 36 0 0 0 36 18 0 0 0 0 0 36 Table 2.Table towsmicroplankton from 200 net µm) (< microplankton metazoan of composition and Abundance

51 Chapter 1 ) -3 TABLE 3TABLE Mesozooplankton (Ind m Holoplankton Meroplanktonic larvae Copep. Copepd. Naup. Append. Clado. Quet. Pterop. Others Polych. Bival. Gastr. Echin. Cirrip. Decap. Date SEP-05 SEP-05 05 14 Sep OCT-05 2885 05 17 Oct NOV-05 3278 05 29 Nov 2834 JAN-06 2014 1093 3739 06 18 Jan MAR-06 1558 3465 256 06 15 Mar 3995 APR-06 1093 1081 376 2834 2851 06 Apr 4 MAY-06 170 154 819 06 16 May 1502 34 603 JUN-06 2014 444 478 137 0 06 17 14 Jun JUL1-06 1980 489 85 2595 285 06 6 Jul 17 85 JUL2-06 973 68 114 3175 171 06 31 Jul 3584 11 AUG-06 34 0 68 34 205 6555 06 29 Aug 563 239 6293 SEP-06 11 3926 17 5019 17 188 290 119 825 20 06 28 Sep 0 6567 717 0 5582 341 341 0 17 300 1753 70 0 0 3687 205 85 290 455 119 0 163 51 0 922 0 5668 17 0 150 0 1058 17 273 34 0 307 256 0 0 0 0 17 34 0 148 102 17 990 296 90 205 57 171 615 17 6 512 0 0 307 205 0 46 102 34 0 151 85 182 137 34 273 0 17 17 361 102 119 34 205 34 41 0 10 285 0 185 34 23 35 46 17 324 34 68 489 68 23 51 17 34 34 0 17 0 17 0 Table 3. Abundance and composition of mesozooplankton (> 200 µm) from microplankton net tows. net microplankton from µm) 200 (> mesozooplankton of composition and Abundance 3. Table

52 Chapter 1 ) -3 TABLE 4TABLE Metazoan microplankton (Ind m Holoplankton Meroplanktonic larvae Nauplii Nauplii Copit. Append. Rotifers Pterop. Others Polychaeta Bivalve Gastropod Echino. Cirriped Others 1 52832 5704 744 0 248 0 496 0 992 0 0 0 1 3382 2255 113 0 113 0 338 225 0 451 0 0 1 2367 225 0 0 0 0 113 113 0 0 0 0 1 9018 1240 113 0 1353 0 113 338 113 0 0 0 5 27055 3382 5 113 41442 3589 0 5 653 1465 3034 1319 0 5 0 0 6764 0 338 2677 5 0 0 141 225 7891 0 1240 653 0 789 5 113 0 979 3617 0 0 0 775 1187 1305 0 0 0 0 132 0 564 0 0 0 0 0 141 113 0 132 0 0 451 0 0 0 338 1938 0 1421 0 0 775 0 0 0 0 0 0 1 ------1 3112 692 0 0 0 0 115 0 0 0 0 0 10 20 30 28295 14316 3269 12513 1578 10 2255 20 113 30 113 25792 113 23560 7192 5208 0 4464 10 0 20 248 2976 0 30 248 2029 4284 10709 225 0 1691 0 0.2 3044 0 10 - 0 0.1 20 0 30 9300 0 789 0 0 0 16486 - 0 789 19868 3664 4368 10 0 7891 789 0 0 0 20 1691 141 282 - 0 676 30 9920 141 8793 451 0 1916 113 0 2367 496 11498 0 0 1240 1015 338 10 - 3044 0 113 20 0 225 0 1488 0 30 3480 0 0 0 496 113 0 0 2643 - 0 1031 0 1916 225 225 225 1034 0 0 0 0 0 0 0 496 129 225 0 - 0 225 345 0 113 113 338 0 564 0.1 0 0 0 564 248 0 113 0 0.2 282 0 113 - 0 0.1 0 141 0 0 0 282 0 0.1 248 282 338 0 0 - 0 0 0 0 338 248 338 0 0 0 441 676 2191 0 0 113 0 564 919 338 - 0 282 141 113 773 0 0 113 113 2068 113 0 0 0 - 0 387 1465 230 0 0 0 0 0 - 0 0 113 0 0 0 129 - 0 0 0 0 0 0 0 0 0 (m) (m) Depth 05 05 06 05 06 06 SEP SEP JAN APR OCT Date NOV MAR Table 4. Composition and vertical distribution of metazoan microplankton bottles. Dorn Van with (<200 µm) from samples obtained

53 Chapter 1 ) -3 Metazoan microplankton (Ind m Holoplankton Meroplanktonic larvae (cont.) . Composition and vertical distribution of metazoan microplancton. * Cladoceran Cladoceran * microplancton. metazoan of distribution vertical and Composition . (cont.) Tabla 4 Tabla Nauplii Nauplii Copepodites Append. Rotifers Pterop. Others Polychaeta Bivalve Gastropod Echinoderm Cirriped Others 1 31141 1973 705 141 0 0 564 0 1 0 29236 4145 0 109 0 0 0 436 0 436 545 1200 0 0 0 1 53585 22551 1 127811 558 10756 112 558 0 223 422 1476 0 0 2344 1116 0 6116 0 844 0 0 0 0 0 5 17332 2677 5 7215 141 5 4171 141 49025 1578 0 15120 5 6200 0 65673 802 0 141 41675 344 5 0 1091 44640 115 0 1353 115 218 10371 5 1127 0 1091 229 28789 0 4851 789 3436 4851 0 0 0 1375 0 105 451 1091 0 0 0 113 0 1527 0 0 211 0 0 0 0 0 451 633 0 902 738 0 0 2425 0 0 338 0 105 1 49036 8455 0 0 451 0 0 338 902 0 113 338 1 14316 8116 2367 3607 0 0 902 1240 0 113 113 0 10 20 30 10286 9441 15077 2536 10 20 2677 3382 30 4617 141 5189 7200 282 705 2111 10 0 3607 20 2945 51251 30 0 141 2638 19636 0 13437 564 12865 10 0 1583 109 0 8836 20 2255 5527 0 30 1898 3709 0 873 0 15491 0 436 0 30109 211 433 2059 141 10 12873 0 0 564 24109 282 20 0 0 0 17473 30 0 845 109 0 12738 132 0 845 218 423 10258 0 7440 1127 0 10 0 4847 0 0 1187 436 20 4171 0 0 0 676 0 0 18455 30 211 0 0 436 19727 1964 0 0 15331 0 3182 0 0 0 0 2742 0 0 2142 0 0 0 0 0 1916 109 0 0 1898 0 0 0 338 225 655 0 0 0 1200 141 867 338 0 0 0 0 0 0 0 0 0 396 34364 0 0 0 327 113 0 0 225 0 2400 0 218 113 0 545 0 0 109 338 0 0 0 109 327 108 338 0 113 0 0 0 0 0 0 0 0 564 422 0 0 564 0 225 451 0 0 0 535 113 0 338 0 451 0 0 113 745 0 564 113 113 0 0 0 0 0 0 0 0 0 0 0 0 113 105 0 0 (m) (m) Depth 06 06 06 06 06 JUN Date AUG JUL1 JUL2 MAY SEPT 06

54 Chapter 1 ) -3 TABLE 5 TABLE Mesozooplankton (Ind Mesozooplankton m Holoplankton Meroplanktonic larvae ------0 ------0 - 655 545 164 764 0 0 0 0 164 0 0 55 0 0 0 873 655 218 545 0 0 0 0 55 0 55 2291 0 55 0 764 655 764 818 709 545 0 273 164 273 164 0 0 945 0 425 273 0 0 318 0 850 55 0 0 982 212 0 0 0 0 0 0 0 273 2230 429 0 212 0 107 0 0 267 0 197 85 113 0 0 0 0 0 0 0 0 0 0 0 0 Cop. Copepodi. Nauplii Append. Clado. Quet. Pterop. Others Polych. Bival. Gastrop. Echinod. Cirrip. Deca. Others 5673 2182 3927 1636 1418 3120 218 2280 764 3480 2040 218 2640 8291 3000 840 436 1309 2160 1091 109 120 327 600 927 720 218 545 120 120 0 382 120 0 1527 327 0 360 273 873 1364 0 0 0 240 0 927 0 0 109 0 218 1691 0 2400 109 164 0 676 109 0 3491 218 327 1309 360 0 818 2727 0 564 0 0 0 0 0 873 1031 0 0 545 240 902 2727 480 0 644 0 1636 0 0 1200 273 0 0 1182 0 0 0 0 129 0 0 0 0 0 0 273 0 1418 0 0 109 0 0 0 109 0 636 120 0 0 0 0 273 0 0 55 0 258 55 0 0 0 0 0 120 0 0 0 0 0 0 109 0 0 764 0 0 0 0 327 0 113 0 3480 55 109 0 0 113 0 327 0 0 109 0 1727 113 0 727 0 0 0 327 0 0 273 55 55 109 55 0 0 91 55 0 0 0 0 0 800 182 133 91 3709 2727 1418 3158 327 2211 109 1737 545 947 109 158 109 1036 0 218 600 0 0 109 1091 436 0 273 818 158 0 0 327 0 1091 0 0 218 800 158 0 0 0 133 0 0 0 1600 0 0 0 0 55 0 133 0 0 0 0 164 0 0 0 109 1600 55 267 0 133 164 0 113 0 0 133 55 133 55 133 1364 868 744 620 124 248 0 0 124 0 0 0 0 0 0 1127 1864 789 113 0 225 225 0 113 0 0 0 0 0 0 1964 1333 133 533 0 0 0 0 0 0 0 0 133 133 0 5 5 5 5 5 5 1 1 1 1 1 1 10 20 30 2400 10 1309 20 30 109 436 10 20 30 0 218 10 20 327 30 0 10 327 20 30 0 10 436 20 30 0 0 0 0 (m) Depth 05 05 06 05 06 06 SEP SEP JAN Date APR OCT NOV MAR Table 5. Composition and vertical distribution of mesozooplankton from samples obtained with Van Dorn bottles. bottles. Dorn Van with obtained samples from distribution of mesozooplankton vertical and Composition 5. Table

55 Chapter 1 Others Deca. ) -3 Mesozooplankton (Ind m (Ind Mesozooplankton Holoplankton Meroplanktonic larvae Composition and vertical distribution of mesozooplankton from samples obtained with Van Dorn bottles. Dorn Van with obtained samples from mesozooplankton of distribution vertical and Composition 436 382 382 0 0 0 0 0 0 0 0 0 0 0 0 982 1255 327 545 273 0 0 55 0 0 0 0 55 0 0 818 709 164 0 0 0 0 0 55 0 0 0 0 0 0 764 927 99 545 55 0 982 0 1200 0 0 0 109 0 545 55 109 218 0 0 436 0 0 0 0 218 109 0 0 0 Cop. Copepodi. Nauplii Append. Clado. Quet. Pterop. Others Polych. Bival. Gastrop. Echinod. Cirrip. 5673 3055 873 764 2618 0 218 0 0 0 109 0 109 0 0 1091 1855 545 109 218 109 436 0 0 0 0 0 0 0 0 3213 1636 2649 1255 620 1121 1091 1253 1145 0 327 982 66 0 0 4364 3927 982 6873 791 2400 0 0 4255 2291 264 55 218 0 0 1309 327 0 4145 982 0 0 764 6764 1855 0 0 3273 0 218 5236 2073 1527 0 0 0 0 218 0 0 109 1418 0 2400 0 0 0 0 0 0 0 3164 0 0 0 0 2509 0 764 0 436 6655 0 0 0 655 0 109 0 3164 0 436 0 0 109 0 0 0 0 66 109 655 1200 0 0 0 0 0 0 0 0 0 0 764 0 0 109 0 0 218 109 2509 0 0 0 264 0 218 0 109 0 109 0 0 0 0 0 0 109 655 545 0 0 1304 0 109 0 0 1091 0 0 218 0 0 655 0 218 0 0 0 109 0 0 0 0 0 0 0 0 0 109 0 0 0 0 109 109 0 109 0 0 0 0 0 0 2291 3273 327 2509 982 1964 1309 218 0 436 109 2400 0 0 109 109 327 0 436 0 0 327 0 436 0 0 0 0 0 0 1364 1473 164 4145 1418 6000 109 109 8727 0 1964 836 0 2073 218 218 55 3164 0 1091 4691 55 25091 0 109 218 0 8182 0 327 0 5236 0 655 764 109 1527 0 0 0 436 0 327 2727 273 0 109 0 0 55 0 0 327 55 0 109 109 0 0 218 0 0 0 0 0 0 218 0 0 0 109 0 109 0 0 1 1 1 1 1 1 5 5 5 5 5 5 10 20 30 955 10 727 20 30 455 10 818 20 30 0 10 0 20 30 0 10 0 20 30 182 3073 0 10 3927 20 30 982 3164 0 3273 1964 973 1855 0 545 109 327 0 0 436 873 2182 218 0 1418 109 109 0 0 327 0 109 0 0 0 218 109 109 0 0 0 0 109 218 0 0 0 0 0 0 0 0 0 0 (m) Depth Tabla 5 (cont.) 5 Tabla 06 06 06 06 06 06 JUN Date AUG JUL1 JUL2 MAY SEPT

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Table 6

Table 6. Contribution of autotrophs, bacteria, protozoans and metazoans to the plankton carbon along the studied annual cycle (µg C L -1 ). T: temperature. Phyto.: phytoplankon (Prochlorococcus + Synechococcus + picoeukaryotes + PNF + diatoms + dinoflagellates). H:A is the ratio of total heterotrophic (bacteria + protozoans + metazoans) biomass to total autotrophic (phytoplankton) biomass. Microbial community biomass was estimated at 5 m depth ( see Methods). Because no distinction between strict heterotrophs and auto- /mixotrophs was made for picoeukaryotes, dinoflagellates, ciliates, we considered as heterotrophic the 50% of biomass of dinoflagellates and picoukatyotes, and the 100% of the biomass of ciliates except in April 2006, when the mixotrophic cilate Laboea dominated and 50% of the biomass of ciliates was considered as autotrophic.

Date T Phyto. Bacteria Protozoans Metazoans H:A SEP-05 23.5 11.1 10.7 7.9 10.8 2.7 OCT-05 21.3 19.6 12.3 18.1 8.0 2.0 NOV-05 16.1 13.2 15.5 3.4 4.2 1.8 JAN-06 12.6 11.9 9.7 12.7 6.2 2.4 MAR-06 12.3 48.7 33.4 8.9 3.9 0.9 APR-06 14.0 46.6 46.6 25.6 1.8 1.6 MAY-06 17.8 61.0 43.8 14.8 4.2 1.0 JUN-06 20.8 34.7 48.5 21.1 4.7 2.1 JUL-06 24.5 36.5 33.4 10.0 4.0 1.3 JUL2-06 24.1 33.8 51.1 18.3 12.1 2.4 AUG-06 24.1 19.3 31.8 8.7 7.5 2.5 SEP-06 21.9 30.1 22.2 23.3 7.4 1.8

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Figure 1

Figure 1. Vertical profiles of temperature, salinity, fluorescence, and chlorophyll (>10 µm and <10 µm) along the annual cycle.

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Figure 2

Figure 2. Vertical distribution of ciliates, dinoflagellates, diatoms and Phaeocystis along the annual cycle.

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Figure 3

Figure 3. Integrated concentration of chlorophyll (A), diatoms and Phaeocystis (B), and ciliates and dinoflagellates (C) throughout the studied period.

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Figure 4

Figure 4. Biomass composition of picoplankton (A), nanoplankton and microplankton unicellular (B), and total microbial biomass and temperature (C) at 5 m depth along the seasonal cycle.

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Figure 5

Figure 5 . Vertical distribution and composition of metazoan microplankton biomass

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Figure 6

Figure 6. Vertical distribution and composition of mesozooplankton biomass

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Figure 7

Figure 7. Annual pattern of abundance (Ind m 3) and biomass (µg C L -1 ) of holozooplankton from net tows. Dots and lines represent the total abundance, left axis) and bars the contribution of the < 200µm (metazoan microplankton) and >200 µm (mesozooplankon) fractions to the total biomass (right axis).

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Figure 8

Figure 8. Annual pattern of abundance (Ind m 3) and biomass (µg C L -1 ) of meroplankton from net tows. Dots and lines represent the total abundance, left axis) and bars the contribution of the < 200µm (metazoan microplankton) and >200 µm (mesozooplankon) fractions to the total biomass (right axis).

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Figure 9

Figure 9. Relationships between bacterial biomass (A), protozooplankton (B) and

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Figure 10

Figure 10. Relationships between micrometazoan abundance (A), micrometazoan biomass (B), mesozooplankton abundance (C) and mesozooplankton biomass (D) with temperature. ns: no significant.

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Figure 11

Figure 11. Comparison of plankton net vs. bottles for quantitative zooplankton sampling.

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Figure 12

Figure 12. Relationships between the numbers of organisms obtained with plankton net and bottles for metazoan microplankton and mesozooplankton.

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Figure 13

Figure 13. Contribution of the metazoan microplankton (A, B), planktonic larvae (C, D) and meroplankton (D, F) to the total zooplankton biomass along a seasonal cycle. Left panel correspond with samples obtained with plankton net tows and right panels with integrated values from Van Dorn bottles samples.

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Resum de l’article I – Article I summary– Catalan version

Abundància estacional i distribució vertical del zooplàncton en aigües costaneres del nord-oest Mediterrani: importància dels m etazous planctònics de petita grandària

Rodrigo Almeda, Miquel Alcaraz, Albert Calbet, Enric Saiz, Isabel Trepat Article en preparació per ser enviat a Progress in Oceanography

La nostra comprensió sobre la funció del zooplàncton en els ecosistemes marins és limitat a causa de l'escassa informació sobre els metazous planctònics de petita grandària. Al llarg d'un cicle anual, es va examinar la successió estacional i la distribució vertical de tota la comunitat de metazooplàncton d'una zona costanera del nord-oest del Mediterrani, amb especial interès en les fraccions de grandària petita. L'estudi va incloure l'ús de dos mètodes de mostreig per zooplàncton, xarxa de microplàncton i ampolles Van Dorn, per cobrir adequadament les principals fraccions de grandària del metazooplàncton, de 50 a 200 µm (microplancton metazou) i de 200 a 2000 µm (mesozooplàncton). També es va avaluar la influència dels principals factors abiòtics i biològics sobre l'estructura estacional i vertical de la comunitat de metazooplàncton. Els nauplis de copèpodes i copepodits van ser numèricament dominants durant la major part del període d'estudi, amb densitats que van oscil·lar aproximadament entre 620 a 23900 ind. m -3 i de 265 a 10.000 ind. m -3 , respectivament. En general, els patrons de distribució vertical del microplàncton metazou i del mesozooplàncton van ser similars. Els gradients verticals d'abundància i biomassa de metazooplàncton van ser més importants a l'estiu i la tardor, i van tendir a seguir els de les seves preses microplanctónicas potencials (ciliats, dinoflagel·lats, diatomees). L'abundància del metazou microplanctònic i del mesozooplàncton sembla estar relacionada directament amb la temperatura. Els valors mínims d'abundància de copèpodes i les seves larves (nauplis i copepodits) es van observar durant la proliferació primaveral de fitoplàncton. Segons les estimacions a partir de les mostres de la xarxa de microplancton i les ampolles Van Dorn, les fases larvàries planctòniques (incloent larves holo- i meroplanctòniques), van representar respectivament, el 45% (rang 24%-75%) i 55% (rang 26%-69%) del carboni total del metazooplàncton. La fracció de grandària corresponent al metazou microplanctònic va representar, en mitjana, el 34% (rang 15%-54%) del carboni total del metazooplàncton quan es va mostrejar amb ampolles Van Dorn. Per tant, encara que el mesozooplàncton domina en termes de biomassa, el microplancton metazou representa una fracció considerable de la biomassa total del metazooplàncton.

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Resumen del artículo I – Article I summary– Spanish version

Abundancia estacional y distribución vertical del zooplancton en aguas costeras del noroeste del Mediterráneo: importancia de los metazoos planctónicos de pequeño tamaño

Rodrigo Almeda, Miquel Alcaraz, Albert Calbet, Enric Saiz, Isabel Trepat Artículo en preparación para ser enviado a Progress in Oceanography

Nuestra comprensión sobre la función del zooplancton en los ecosistemas marinos es limitada debido a la escasa información acerca de los metazoos planctónicos pequeño tamaño. A lo largo de un ciclo anual, se examinó la sucesión estacional y la distribución vertical de toda la comunidad de metazooplancton de una zona costera del noroeste del Mediterráneo, con especial interés en las fracciones de tamaño pequeño. El estudio incluyó el uso de dos métodos de muestreo (red de microplacton y botellas Van Dorn) para cubrir adecuadamente las principales fracciones de tamaño del metazooplancton, de 50 a 200 µm (microplancton metazoo) y de 200 a 2000 µm (mesozooplancton). También evaluamos la influencia de los principales factores abióticos y biológicos sobre la estacionalidad y la distribución vertical de la comunidad metazooplancton. Los nauplios de copépodos y copepoditos fueron numéricamente dominantes durante la mayor parte del período de estudio, con densidades que oscilaron aproximadamente entre 620 a 23900 ind. m 3 y de 265 a 10.000 ind. m 3, respectivamente. Los gradientes verticales de abundancia y biomasa de metazooplancton fueron más importantes en verano y otoño, y tendían a seguir los de sus presas microplanctónicas potenciales (ciliados, dinoflagelados, diatomeas). La abundancia del metazoo microplanctónico y del mesozooplancton parece estar relacionada directamente con la temperatura. Según las estimaciones a partir de las muestras de la red de microplancton y las botellas Van Dorn, las fases larvarias planctónicas (incluyendo larvas holo- y meroplanctónicas), representaron respectivamente, el 45% (rango 24%-75%) y 55% (rango 26%-69%) del carbono total del metazooplancton. La fracción de tamaño correspondiente al metazoo microplanctónico representó, en promedio, el 34% (rango 15%-54%) del carbono total del metazooplancton cuando se muestreó con botellas Van Dorn. Por lo tanto, aunque el mesozooplancton domina en términos de biomasa, el microplancton metazoo representa una fracción considerable de la biomasa total del metazooplancton. Además, como las tasas fisiológicas de los organismos pequeños son generalmente más altas que los de los organismos más grandes, la exclusión de metazoos microplanctónicos puede dar lugar a importantes subestimaciones de la producción secundaria del zooplancton en los ecosistemas marinos.

72 Trophic role and carbon budget of metazoan microplankton in northwest Mediterranean coastal waters

Chapter 2

Limnol. Oceanogr., 56(1), 2011, 415–430 E 2011, by the American Society of Limnology and Oceanography, Inc. doi:10.4319/lo.2011.56.1.0415

Trophic role and carbon budget of metazoan microplankton in northwest Mediterranean coastal waters

Rodrigo Almeda, * Albert Calbet, Miquel Alcaraz, Enric Saiz, Isabel Trepat, Laura Arin, Juancho Movilla, and Violeta Salo´

Institut de Cie`ncies del Mar (CSIC), Barcelona, Spain

Abstract We determined the feeding rates, trophic effect, and growth efficiencies of natural assemblages of metazoan microplankton from a coastal site in the northwest (NW) Mediterranean over a seasonal cycle in laboratory incubations. Micrometazoans, i.e., multicellular heterotrophic plankters between 20 and 200 mm, were mainly constituted by invertebrate larval stages. Copepod nauplii and copepodites dominated the community, except in April, when polychaete larvae dominated. We analyzed the grazing pressure of micrometazoans on chlorophyll a (Chl a; total and . 10 mm), nanoflagellates, phototrophic nanoflagellates, dinoflagellates, diatoms, and ciliates. Micrometazoans grazed on all the prey groups, with carbon-specific ingestion rates ranging from 0.31 to 1.24 d 21. The gross growth efficiencies for the entire metazoan microplankton community, calculated as the slope of the linear regression relating specific growth rates vs. specific ingestion rates, varied between 0.27 and 0.39. The respiratory carbon losses of micrometazoans depended on temperature and ranged from 0.16 to 0.36 d 21, with a Q10 5 2. The average net growth efficiency, 0.41, was independent of temperature and food availability. Overall micrometazoans have higher specific growth rates than, but similar food conversion efficiencies to, mesozooplankton. The grazing effect on the standing stock of the different prey was , 1% d21 for Chl a (total and . 10 mm) and , 2.5 % d21 for the other studied prey, which seems insufficient to exert relevant control on phytoplankton and protozoan dynamics. The inclusion of micrometazoans did not change appreciably our current view of the role of metazooplankton in marine trophic webs of NW Mediterranean coastal waters.

Historically, research on zooplankton has focused on the underrepresented in oceanographic studies because of the study of the large size fractions ( . 200 mm, mesozoo- common use of . 200- mm mesh nets for metazooplankton plankton; Sieburth et al. 1978), whereas the smaller sampling (Calbet et al. 2001; Gallienne and Robins 2001). zooplankters ( , 200 mm) have received less attention However, recent investigations have documented the (Paffenho¨fer 1998; Gallienne and Robins 2001; Turner importance of micrometazoans in terms of abundance, 2004). It was not until the establishment of the dilution biomass, and production in marine environments (Hop- technique in 1982 (Landry and Hassett 1982) that the role croft et al. 2001; Turner 2004; Zervoudaki et al. 2007). of microzooplankton sensu lato was acknowledged, and Metazoan microplankton may feed efficiently on a wide nowadays, there is little doubt microzooplankton occupy a range of prey sizes, from nano- to microzooplankton (Uye key position in marine food webs as major consumers of and Kasahara 1983) and their specific ingestion rates can primary production (Calbet and Landry 2004), and as an be three to four times higher than those of mesozooplank- important link between low and high trophic levels of the ton organisms (White and Roman 1992; Saiz and Calbet food web (Calbet and Saiz 2005). 2007). Therefore, these small metazoans may be important The grazing activity of microzooplankton on primary intermediaries between the classical and microbial food producers as assessed by the dilution technique (Landry webs because of their small size, high abundance, and and Hassett 1982) provides an estimation of the trophic ability to feed on small particles (Berggreen 1988; Turner effect of the entire microzooplankton community (both and Roff 1993). Data on metazoan microplankton feeding metazoans and protozoans), but cannot assess the relative on natural communities are scarce (White and Roman contribution of its different components. Although proto- 1992; Merrell and Stoecker 1998; Calbet et al. 2009) and zoans dominate the microzooplankton, small metazoans, most of the experimental studies of micrometazoan feeding represented mainly by copepod nauplii, copepodites, (both in oligotrophic and in more productive waters) rotifers, and meroplanktonic larvae, may become impor- seldom address the trophic effect of the whole metazoan tant contributors to the microzooplankton (Beers and microplankton community. The majority of studies include Stewart 1970; Paffenho¨fer 1998). The metazoan micro- single predator species feeding on laboratory-cultured plankton are composed mostly of invertebrate develop- phytoplankton (Berggreen et al. 1988; Almeda et al. 2009, mental stages whose dynamics are crucial to determine the 2010 b), thus precluding a rigorous extrapolation of the data success of adult populations, and constitute a fundamental obtained to natural conditions and the correct evaluation trophic link for the recruitment of commercially important of the importance of metazoans in marine food webs. fish species (Castonguay et al. 2008). Despite their In the present study we examined the trophic role of relevance, these small planktonic metazoans have been metazoan microplankton in meso-oligotrophic coastal waters along an annual cycle. The specific objectives of * Corresponding author: [email protected] the study were (1) to quantify the metazoan microplankton 415

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416 Almeda et al.

abundance and composition in northwest (NW) Mediter- ranean coastal waters along an annual cycle, (2) to determine the feeding rates and trophic effect of the metazoan microplankton community under different tro- phic conditions, and (3) to assess the carbon budget of the metazoan microplankton community in different seasons.

Methods

Field sampling— We conducted monthly experiments throughout an annual cycle, from September 2005 to September 2006, the samples being collected at a coastal station in the NW Mediterranean (1.5 km off Barcelona, 40- m water-column depth). The water for the experiments was collected at 5-m depth with 15-liter transparent hydrograph- ic bottles, gently transferred to 40-liter carboys, and transported to the laboratory within 2 h of collection. We measured the light intensity at the depth of water collection with a LI-COR radiation sensor (LI-193SA spherical quantum sensor), and the salinity and the temperature with an YSI 30 portable salinity and temperature meter. The samples for metazooplankton abundance and composition assessment were obtained by vertical tows from near the bottom ( , 38 m) to the surface using a Fig. 1. Schematic representation of the used methodology to microplankton net (50- mm mesh, 36-cm diameter). After obtain the metazoan microzooplankton concentrate. (A) The the plankton net was washed, the contents of the cod end fraction , 200 mm was obtained by reverse filtration screening sample were entirely poured into a 500-mL plastic bottle through a 200- mm mesh sieve. (B) The fraction , 50 mm was and preserved in borax-buffered formaldehyde at 4 % final removed by reverse flow screening through a 50- mm mesh sieve. concentration. (C) The result was a concentrate of plankton of size ranging from The zooplankton for feeding experiments were collected 50 to 200 mm that was composed mainly of micrometazoans. by slow-speed vertical tows using the same plankton net as for the abundance quantification, but using a 10-liter four extra bottles without predators (two initial and two plastic bag as non-filtering cod end in order to minimize the control). To avoid nutrient enrichment effects due to capture stress and to avoid physical damage of the zooplankton excretion in the grazing bottles, the experi- organisms. Once onboard, the plastic bags containing the mental water was amended with a nutrient mixture 21 21 samples were tied without trapping air to avoid organisms (15 mmol L NH 4Cl and 1 mmol L Na 2HPO 4). From sticking to the air–water interface, and kept in an each initial bottle we took subsamples to determine the Chl isothermal container previously filled with water at in situ a concentration (total Chl a and Chl a . 10 mm) and the temperature until our return to the laboratory. abundance of the different components of the microbial community ( see Sample processing and calculations). We Experimental design— We analyzed the grazing pressure should note here that each experimental concentration had of micrometazoans on phytoplankton (Chl a) and on the its own initials to account for the remains of phytoplankton main components of the microbial planktonic community. The experiments consisted of 24-h incubations of natural Table 1. Increasing concentrations of metazoan micro- 21 seawater with added concentrated metazoan microplank- plankton (ind. L ) used in the experimental bottles. For each concentration there were two replicates. Notice that only the ton (50–200 mm) as grazers. Once in the laboratory, natural lowest density providing significant grazing was used for water was siphoned into a 50-liter bucket, mixed carefully, calculations ( see text and Table 3). Expts, experiments. and transferred sequentially using silicon tubing into acid- washed 2.3-liter polycarbonate bottles. Zooplankton sam- Expts Concentration (ind. L 21) ples collected for experiments were carefully poured into a Sep 05 40 81 162 container. The concentrate of plankton of size ranging Oct 05 515 1030 2061 from 50 to 200 mm, composed mainly of micrometazoans, Nov 05 107 213 426 was obtained by first reverse-filtration screening though a Jan 06 227 454 908 200- mm-mesh sieve, and then removing the , 50- mm Mar 06 — — 4750 fraction (Fig. 1). Aliquots of this concentrate were then Apr 06 511 1022 2043 added to produce a concentration gradient in 2.3-liter May 06 267 534 1068 bottles of the whole (unfiltered) seawater (Table 1). We Jun 06 417 835 1670 added three different concentrations of the metazoan Jul 06 941 1883 3765 microzooplankton concentrate to sets of four bottles (two Aug 06 534 1068 2136 Sep 06 197 395 790 initial and two experimental), and we also prepared a set of

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Trophic role of metazoan microplankton 417

Table 2. Equation coefficients used to calculate the carbon content for the different groups of micrometazoans as a function of individual length (L). Biomass ( mg C) 5 a 3 L ( mm) b.

Organism Measurement a b Reference Copepod nauplii Body length (without setae) 3.18 310 29 3.31 Berggreen et al. (1988) Copepodites Length of prosome 1.11 310 28 2.92 Berggreen et al. (1988) Polychaeta larvae Maximal length 1.58 310 24 1.38 Hansen (1999) as Polydora Bivalvia larvae Maximal length 3.06 310 28 2.88 Fotel et al. (1999) Gastropoda larvae Maximal length 2.31 310 25 2.05 Hansen and Ockelmann (1991) Echinoderma larvae Maximal length 3.06 310 28 2.88 Fotel et al. (1999) as bivalve Cirripedia nauplii Body length (without spine) 2.20 310 210 3.72 Turner et al. (2001) Appendicularia Trunk length (without tail) 7.33 310 28 2.63 King (1980) Rotifers Body length 1.06 310 27 2.74 Hansen et al. (1997) Pteropoda Total length 3.06 310 28 2.88 Fotel et al. (1999) as bivalve and protozoans within the 50–200 mm size fractions. The was calculated by applying body size–carbon content prey concentrations in the experimental bottles were in the relationships from the literature (Table 2). same order of magnitude than in situ conditions, except in The concentrations of total and . 10- mm Chl a were March 2006 when it was one order of magnitude higher. determined by filtering from 75 to 300 mL and from 100 to All bottles (except initials) were incubated at in situ 500 mL of water through GF/F Whatman and 10- mm pore temperature in a large (600-liter) outdoor incubator with size polycarbonate Nuclepore filters, respectively. The open-circuit water running from the coastal seawater intake filters were frozen at 280 uC and later analyzed fluorime- at the Institut de Cie`ncies del Mar, Barcelona, Spain. trically after acetone extraction before and after acidifica- Natural sunlight was dimmed with appropriate neutral tion according to Parsons et al. (1984). plastic mesh to mimic the light intensity at 5-m depth, The microbial components studied in the feeding experi- usually between 33 % and 50 % of the irradiance at the ments included heterotrophic nanoflagellates (HNF), pho- surface. To minimize the settling of algae, and to ensure the totrophic nanoflagellates (PNF), dinoflagellates, diatoms, homogeneity of the light conditions, we gently mixed the and ciliates. For nanoflagellates, 40–100-mL samples were bottles by repeatedly turning them upside down and preserved in glutaraldehyde (1 % final concentration), moving them around the incubator ca. three times per filtered onto 2- mm pore size black polycarbonate mem- day. After the incubation time (24 h), we took subsamples brane filters, and stained with 4 9,6-diamidino-2-phenylin- to determine changes in prey abundance, and for the dole (5 mg mL 21 final concentration) for 5 min (Porter and experimental bottles we took an additional 1-liter sample to Feig 1980). At least 200 cells were counted by epifluores- determine the abundance, biomass, and growth rates of cence microscopy and classified as auto- or heterotrophic micrometazoans during the incubation. according to their fluorescence for chlorophyll. Fifty cells In some of the samplings respiration rates of micro- were sized and converted into carbon using a conversion metazoans were measured in parallel following the classical factor of 0.22 pg C mm23 (Børsheim and Bratbak 1987). incubation method (Omori and Ikeda 1984). Each exper- To determine the concentration of dinoflagellates, iment consisted of three to six initial and final bottles with ciliates, and diatoms, 250-mL samples were fixed with 1 % the metazoan microplankton community concentrate acidic Lugol’s solution, and allowed to settle for 48 h in (experimental bottles) and three to six initial and control 100 mL Utermo¨hl chambers. The whole chamber for bottles only with the fraction , 50 mm (Fig. 1). The ciliates and dinoflagellates, and at least 40 microscopic incubations were conducted in 65-mL Winkler bottles for fields (or 200 cells) for diatoms, were counted using an , 20 h. The biomass of micrometazoans in the experimen- inverted microscope (Nikon Diaphot 200) at 200 3 tal bottles ranged from 47 to 174 mg C bottle 21 depending magnification. Fifty randomly chosen cells for each group on the experiment. The bottles were covered with were sized and converted into carbon using the conversion aluminum foil (dark conditions) and placed in the same factors of 0.19 and 0.053 pg C mm23 for oligotrich ciliates incubator as the feeding experiment bottles. (Putt and Stoecker 1989) and tintinnids (Verity and Langdon 1984), respectively, and the equations of Men- Sample processing and calculations— For the estimation den-Deuer and Lessard (2000) for dinoflagellates (pg C Dino 21 0.819 21 of metazoan microplankton abundance and composition, cell 5 0.760 3 volume ) and diatoms (pg C Diat cell net samples were divided into two nominal size fractions 5 0.288 3 volume 0.811 ). Because microplankton samples (50–200 mm, . 200 mm) by filtering through a 200- mm sieve. were preserved with acidic Lugol’s solution, no distinction Two aliquots per fraction (at least 350 organisms per between strict heterotrophs and auto- or mixotrophs was aliquot) were counted under a stereomicroscope. For made for ciliates and dinoflagellates. biomass determination, the body len gth of at least 50 The 1-liter subsamples for the estimation of microme- organisms randomly chosen was measured on digital tazoan abundance, biomass, and growth rates during the pictures made under a microscope ( 3100) using image experiments were concentrated onto a 37- mm sieve and analysis software (ImageJ H). The individual carbon weight preserved in borax-buffered formaldehyde at 4 % (final

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concentration). Abundance, biomass, and composition of was made for microprotozoans, for the application of experimental metazoan microplankton were estimated as Nejstgaard et al. (2001) equations we assumed that 50 % of described above. Carbon-specific growth rates (G, d 21) of the dinoflagellate biomass and 100 % of the ciliate biomass micrometazoans were determined for each experimental was heterotrophic (i.e., grazers). bottle according to the equation We examined the relationships between ingestion rates and food concentration (functional responses) for each G~Ln(W =W )=t 1 2 1 ð Þ prey type and for total prey. To obtain the functional responses, regression equations were fitted to the data where t is the duration of incubation (days) and W 1 and W 2 are the initial and final carbon content of the microme- following standard least squares procedures (Sigma plot tazoans, respectively. software 9.0). The trophic effect by the metazoan micro- Clearance and ingestion rates on each size fraction of zooplankton community for each prey type was calculated as the percentage of biomass of the standing stock grazed Chl a and prey type were calculated using Frost’s (1972) daily assuming a homogenous prey and predator distribu- equations. We used the average biomass of predators tion in the water column. We calculated the potential during the incubation to calculated specific feeding rates trophic effect using the expected ingestion rates at the in (except for the September 2005 experiment, where we used situ prey abundance according to the functional responses the initial biomass values because metazoan final samples obtained in this study. were not collected). From the three different metazoan microplankton concentrations used in each feeding exper- Dissolved oxygen concentration was analyzed by Wink- iment (Table 1), we took into account for further calcula- ler titration method using a Mettler Toledo DL50 Graphix tions only those that accomplished the following criteria: Titrator to determine the titration endpoint. Oxygen the grazing was significant, i.e., the prey net growth rates in consumption by micrometazoans was determined as the grazing bottles were significantly lower than in the controls difference between initial and final oxygen concentration (ANOVA, p , 0.05); and the reduction of food during the after the subtraction of the oxygen consumed in the control bottles. Respiration rates were converted to carbon incubation was the lowest, always , 40 %. The larval demands using a respiration coefficient (RQ) of 0.97 densities and experimental conditions of each experiment (Omori and Ikeda 1984). are shown in Table 1. The gross growth efficiency (GGE, i.e., the efficiency by The underestimation of micrometazoan grazing rates on which ingested feed is converted into body weight) was phytoplankton (Chl a) due to trophic interactions was calculated for each experiment as corrected according to Nejstgaard et al. (2001). Dilution experiments (Landry and Hassett 1982) were conducted GGE ~G=I 6 simultaneously with the micrometazoan feeding experi- ð Þ ments and under similar incubation conditions. The the assimilation efficiency (AE, i.e., the percentage of experimental details and results are reported in Calbet et ingested food that is digested) was estimated as al. (2008). The correction factor (K p) for the effects of AE ~(G zR) =I 7 micrometazoan predation on phytoplankton (total Chl a ð Þ and Chl a . 10 mm) was derived according to the equations and the net growth efficiency (NGE; i.e., the percentage of (Nejstgaard et al., 2001) assimilated food converted into growth) was calculated as

gcorr,p ~gmetazoan,p zKp 2 NGE ~G=(G zR) 8 ð Þ ð Þ K ~g (C {C =C) 3 where G, R, and I are the daily carbon-specific growth, p protozoan,p à ð Þ respiration, and ingestion rates, respectively. In addition, GGE was also calculated as the slope of the {1 C~(C t{C0)Ln(C t=C0) 4 linear regression relating specific growth rates (d 21) vs. ð Þ specific ingestion rates (d 21). {1 C ~(C t {C0)Ln(C t =C0) 5 à à à ð Þ Results where g corr,p is the corrected micrometazoan grazing coefficient on the prey p; g metazoan,p is the uncorrected Planktonic community abundance and composition— Mi- micrometazoan grazing coefficient on the prey p calculated crometazoan abundance ranged from 2 to 33 individuals 21 21 according to Frost (1972); g protozoan ,p is the microzooplank- (ind.) L (equivalent to , 0.13–2.02 mg C L ), with ton grazing coefficient on the prey p determined from maximum values in summer (Table 3). During the studied simultaneous dilution experiments (Calbet et al. 2008); C period, copepod nauplii and copepodites were the domi- and C* are the average microzooplankton biomass ( mg C nant components of metazoan microplankton, except for 21 L ) in the control and grazing bottles, respectively; C 0 is the April, when polychaete larvae were the most abundant initial microzooplankton biomass; and C t and C t* are the group (Fig. 2). In general, calanoid copepod nauplii and final concentrations of microzooplankton in the control and copepodites dominated in abundance during spring and in the grazing bottles, respectively. Because no distinction winter, whereas oithonid nauplii and copepodites were between strict heterotrophs and auto- or mixotrophs dominant in summer and quite abundant in autumn.

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Table 3. Experimental conditions and micrometazoans densities in nature (in situ) and in the feeding experiments (expts). T, temperature; S, salinity; Biom., biomass; Abund., abundance. The experimental densities of micrometazoans correspond to those used for the calculation of feeding rates according to the criteria described in the Methods section.

Abund. in situ Biom. in situ Abund. expts Biom. expts Expts Date T ( uC) S (ind. L 21) (mg C L 21) (ind. L 21) (mg C L 21) Sep 05 14 Sep 05 23.5 38.0 14.5 1.15 162 11.6 Oct 05 17 Oct 05 21.5 37.4 11.4 0.76 2061 119.2 Nov 05 29 Nov 05 16.1 38.2 2.0 0.13 426 31.6 Jan 06 18 Jan 06 13.0 38.3 14.5 1.19 227 17.2 Mar 06 15 Mar 06 12.5 38.2 4.2 0.32 4750 398.1 Apr 06 04 Apr 06 14.2 37.2 2.3 0.39 511 84.4 May 06 16 May 06 18.1 37.9 15.0 1.25 267 23.0 Jun 06 14 Jun 06 21.1 38.0 13.2 1.41 417 64.8 Jul 06 31 Jul 06 24.4 37.9 33.0 1.65 941 63.3 Aug 06 29 Aug 06 24.4 38.0 22.7 2.02 534 48.8 Sep 06 28 Sep 06 22.2 37.9 16.9 1.19 197 12.5

We found highly contrasted situations during the study followed by a slow decline over the subsequent samplings in terms of plankton biomass and composition. The annual (Table 4). During spring and autumn, . 10- mm phyto- cycle was characterized by the development of two plankton contributed to more than 40 % of total phyto- phytoplankton blooms in October 2005 and March 2006, plankton biomass (chlorophyll), whereas the rest of the

Fig. 2. In situ composition in (A) abundance and (B) carbon biomass of the metazoan microzooplankton community along an annual cycle in the coastal NW Mediterranean. Composition in (C) abundance and (D) carbon biomass of metazoan microzooplankton in the different feeding experiments.

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Table 4. Abundance (Abun., cells mL 21) and biomass (Biom., mg C L 21) of the different components of the natural microbial community (nano- and microplankton) at 5-m depth. Chl a, total Chl a in mg L 21; Chl a .10, Chl a .10 mm in mg L 21; Dinoflag., dinoflagellates; TOT., total edible prey biomass in mg C L 21.

HNF PNF Ciliates Dinoflag. Diatoms TOT. Expts Chl a Chl a.10 Abun. Biom. Abun. Biom. Abun. Biom. Abun. Biom. Abun. Biom. Biom. Sep 05 0.18 0.03 738 5.6 1238 6.44 1.6 2.78 2.4 2.00 6.3 0.77 17.59 Oct 05 1.54 0.69 714 3.5 2749 10.97 3.4 4.8 3.3 2.55 145.9 15.36 37.18 Nov 05 0.97 0.38 324 1.4 1143 4.11 2.5 1.09 1.2 1.18 38.0 7.38 15.16 Jan 06 0.47 0.09 914 10.9 1924 8.51 2.5 3.51 1.7 1.42 24.9 4.69 29.03 Mar 06 1.66 0.92 937 5.8 3460 27.31 1.5 1.17 3.0 3.59 154.2 19.16 57.03 Apr 06 1.13 0.59 612 5.4 1605 13.20 4.9 28.8 5.1 8.32 132.1 20.10 75.82 May 06 0.95 0.38 450 2.6 3497 15.05 2.4 3.35 3.1 0.98 1180.5 33.16 55.14 Jun 06 0.49 0.06 635 6.7 3657 21.01 3.8 5.26 21.2 11.07 24.0 2.22 46.26 Jul 06 0.39 0.08 1267 10.3 2857 9.80 3.1 2.14 3.3 2.04 131.0 12.52 36.8 Aug 06 0.31 0.03 648 2.8 2610 8.35 4.6 3.75 3.9 1.82 16.8 0.86 17.58 Sep 06 0.73 0.05 730 2.8 6728 12.36 8.2 10.65 5.1 5.01 31.4 1.43 32.25

year, but mainly in summer, , 10- mm phytoplankton significantly from those of Chl a . 10 mm (ANOVA, p . dominated the community (Table 4). 0.05), with the exception of some summer experiments Nanoflagellates, mainly composed of flagellates between (Fig. 3C). Carbon-specific ingestion rates on chlorophyll 2 and 5 mm in size, were the dominant component of the were highly variable, ranging from 0 to close to 60 ng Chl microbial community during most of the year ( . 50 % of mg C 21d21. The maximum ingestion rate was found in total carbon; Table 4). The contribution of diatoms and March 2006, coinciding with the highest Chl a concentra- dinoflagellates to the total biomass was more important tion. The contribution of Chl a . 10 mm to the ingestion during spring and autumn. Ciliate biomass was very was very variable, and ranged from 0 % to 66 % (mean 5 variable and ranged from 2 % to 38 % of the total microbial 37 %). biomass depending on the year period. Remarkable The initial total biomass and the relative contributions of features of plankton species composition were the blooms the different prey in the feeding experiments are shown in of the colonial Phaeocystis sp. in March–April, the ciliate Fig. 4A. Metazoan microplankton fed on all studied prey Laboea sp. in April, the chain-forming diatoms Pseudo- (Fig. 4B). The specific clearance rates ranged from less than nitzschia sp. and Dactyliosolen fragilissimus in May, and 1 to more than 80 mL mg C 21 d21 depending on the feeding unidentified small dinoflagellates in June. experiment and prey type (Fig. 5). We found significant differences ( p , 0.05; ANOVA, Tukey test) for some prey Feeding rates— The concentration and biomass of types within each experiment. Although in some cases more metazoan microplankton used in the feeding experiments than one prey was cleared at maximal rates, the highest were generally one order of magnitude higher than the one clearance rates were observed mainly on ciliates and HNF in situ, except for the months with high Chl a concentration (Fig. 5). Specific ingestion rates varied from , 0.3 to more (October and November 2005 and March and April 2006), than 1.2 d 21 (Fig. 4B). Although during summer nano- when they were two to three orders of magnitude higher flagellates were an important part of the diet, diatoms and (Table 1). The composition of the metazoan microzoo- dinoflagellates were the main component during the rest of plankton community used in the experiments was quite the experiments. Ciliates contributed to a small fraction of similar to that of the in situ conditions in spite of the the diet ( , 10 %), except in the April 2006 experiment, screening process (Fig. 3). when they contributed more than 30 % to the diet (Fig. 4B). The initial Chl a concentration in the experiments and In most of the experiments we observed a positive the clearance and ingestion rates of Chl a (total and . correlation between prey abundance and their relative 10 mm), with and without applying Nejstgaard equations contribution to the micrometazoan diet ( r2 5 0.51–0.99, correction, are shown in Fig. 3. Clearance rates were very depending on the experiments). variable and, in general, negatively related to Chl a concentration. Negative values of uncorrected clearance Functional responses— The relationship between inges- and ingestion rates (represented as 0) were observed in tion rates and food concentration for total Chl a, Chl a . January and May 2006 (Fig. 3B,D). The corrected feeding 10 mm, diatoms, dinoflagellates, and total prey was well rates were from 1.01 to 6 times higher than the uncorrected described by the type II functional response model (Fig. 6). ones (mean 5 2.7) for total Chl a, and between 1 and 2.3 On the contrary, the pattern of ingestion rates of HNF and times higher (mean 5 1.2) for Chl a . 10 mm (Fig. 3). PHF seemed to be unrelated to food concentration Exceptionally high clearance rates were observed in (Fig. 6C,D). The relationships between ingestion rates of September 2005 ( , 40 and 80 mL mg C 21 d21 for Chl a ciliates and their concentration appeared to be adequately total and Chl a . 10 mm, respectively), the rates being described by linear regression, without any apparent generally higher during the summer months. In most of the saturation threshold within the concentrations of the study experiments, the clearance rates of total Chl a did not differ (Fig. 6E). According to the obtained functional responses,

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Fig. 3. Feeding rates of metazoan microzooplankton on phytoplankton (Chl a total and . 10 mm). (A) Initial chlorophyll concentration in the feeding experiments, (B) clearance rates without applying Nejstgaard equations correction, (C) uncorrected ingestion rates without applying Nejstgaard equations correction, (D) corrected clearance rates applying Nejstgaard equations correction, and (E) corrected ingestion rates applying Nejstgaard equations correction. Bars represent SE. the maximum carbon-specific ingestion rates of chlorophyll 0.1 % to 0.9 % for Chl a . 10 mm. The trophic effect on the were 81 and 74 ng Chl mg C 21 d21 for total Chl a and Chl a standing stocks of nanoflagellates, ciliates, dinoflagellates, . 10, respectively (Fig. 6A,B). Considering total prey, the and diatoms varied from 0 % to 6.1 % and was generally , maximum specific ingestion rate was 1.09 d 21 and feeding 4% (Table 5). saturation was reached at , 280 mg C L 21 (Fig. 6H). Carbon budget: growth, respiration, and growth efficien- Trophic effect— The potential grazing pressure of meta- cies— Specific growth rates of the main components of the zoan microplankton for each prey type, as percentage of metazoan microplankton community are shown in Table 6. the standing stock removed daily, is shown in Table 5. The Specific growth rates of copepod nauplii and copepodites mean grazing effect of metazoan microplankton was , 1% ranged from 0.01 to 0.49 d 21 and from 0.03 to 0.38 d 21, for Chl a (total and . 10 mm) of standing stocks, with respectively (Table 6). Meroplanktonic larvae showed values ranging from 0.1 % to 1.7 % for total Chl a and from specific growth rates significantly lower than nauplii and

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Fig. 4. (A) Initial contribution in biomass of potential prey (left axis) and total biomass of potential prey (right axis) in the feeding experiments. (B) Contribution of the different prey to the carbon-specific ingestion rates. copepodites (two-way ANOVA, Tukey test, p , 0.05; specific respiration rates exceeded ingestion rates and Table 6). Specific growth rates of total metazoan micro- efficiencies were not calculated (Table 7). Assimilation zooplankton community ranged from 0.02 to 0.40 d 21 efficiencies varied from 0.39 to 0.87, with the higher values depending on the experiment (Table 7). Growth rates corresponding to higher temperatures (May and September followed a saturation curve model as a function of prey 2006). NGE was quite similar for all experiments, with a concentration (Fig. 7), although the model is mostly driven mean value of 0.41 (Table 7). by the very high values. The GGEs, calculated specifically for each experiment, were very variable and ranged from Discussion 0.06 to 0.38 (Table 7). GGE calculated as the slope of the linear regression relating carbon-specific growth rates with Metazoan microplankton abundance and community carbon-specific ingestion rates were 0.27 when all growth composition— Copepod developmental stages were the data were considered (Fig. 8A) and 0.39 when only growth dominant group of micrometazoans in agreement with rates of experiments dominated by copepod larvae were previous studies in most marine environments (Hopcroft et included (Fig. 8B). Respiration rates ranged from 0.25 to al. 2001). The densities for the whole micrometazoan 21 21 0.72 mL O 2 mg C d and increased exponentially with community was in the same range reported for oligo- and temperature (Fig. 9). These respiration rates were equiva- mesotrophic waters (Calbet et al. 2001), but lower than in lent to respiratory carbon losses of 0.16 and 0.36 d 21, more productive areas (Lucˇic´ et al. 2003). During our respectively (Table 7). The Q 10 value for the experimental study, the abundance of copepod nauplii and copepodites temperature range (13–24.4 uC) was 2.0. In August 2006, was not related to the phytoplankton blooms as observed

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Fig. 5. Weight-specific clearances of metazoan microzooplankton on the studied prey in the different experiments. in previous studies for mesozooplankton (Calbet et al. because commonly smaller organisms exhibit higher 1996). Conversely, polychaete larvae peaked during the specific feeding rates than larger ones (Saiz and Calbet spring phytoplankton bloom, confirming the link between 2007). However, in field conditions, previous studies report phytoplankton blooms and the appearance of some both higher (White and Roman 1992; Roman and Gauzens meroplanktonic larvae (Starr et al. 1990). For some benthic 1997) and similar or lower (Lo´pez et al. 2007; Calbet et al. species, phytoplankton blooms act as direct inductors of 2009) specific ingestion rates of metazoan microzooplank- the release of gametes or larvae by either direct contact ton as compared to mesozooplankton at similar tempera- (settlement of phytoplankton particles) or chemical trig- ture. Aside from body size and temperature, this variability gering (Starr et al. 1990). Hence, besides other factors (e.g., may be caused by the influence of other factors on the temperature, predation), this benthic–pelagic coupling can ingestion rates of metazoans, such as taxonomic composi- drive the patterns of some meroplanktonic larvae in coastal tion (Henriksen et al. 2007; Almeda et al. 2010 b) and food waters. availability (Saiz and Calbet 2007, 2010).

Feeding rates— Clearance and ingestion rates of zoo- Diet composition— Metazooplankton, including plank- plankton depend on many factors, such as body size, food tonic developmental stages, feed as omnivores, ingesting a concentration, prey size, food quality, and temperature variety of autotrophic, heterotrophic, and detrital food (Berggreen et al. 1988; Almeda et al. 2009, 2010 b). Carbon- sources (Kleppel et al. 1988; Turner 2004). As occurs for specific clearance and ingestion rates observed in this study mesozooplankton (Broglio et al. 2004), metazoan micro- were in the range reported in the literature for metazoan plankton exhibited a diverse diet composition and were microplankton at similar food concentrations and temper- able to switch between a preferably herbivorous diet to an atures (Uitto 1996; Roman and Gauzens 1997; Calbet et al. omnivorous one in response to the seasonal variation of the 2009) and for copepod nauplii in laboratory studies (Berg- available food items. The ability to use different food green et al. 1988; Henriksen et al. 2007; Almeda et al. 2010 b). sources (opportunistic feeders) may enhance the probabil- It is important to note that feeding rates could be affected by ity of obtaining a nutritionally complete ration in variable, several potential sources of bias. For instance, bottle effects dilute food environments (Kleppel 1993), thus increasing and crowding (Peters and Downing 1984) and the lack of the probability of the success of the population. It is turbulence during the incubations may result in lower important to note that other food resources not considered feeding rates than under natural conditions (Saiz et al. in this study (e.g., picoplankton, detritus) may also contri- 2003). Nevertheless, predator biomass used in the feeding bute to the diet of metazoan microzooplankton. However, experiments was in the range commonly used in incubation although some copepod nauplii and meroplanktonic larvae experiments with larger zooplankton (Nejstgaard et al. 2001; have been reported to feed upon bacteria (Turner and Broglio et al. 2004) Another source of error could be Tester 1992), other studies demonstrated that free bacte- inclusion of all nauplii stages as predators for the calculation rioplankton is too small to be efficiently ingested by most of feeding rates, because some calanoid nauplii start feeding copepod developmental stages (Uye and Kasahara 1983; at naupliar stage II or III (Landry 1975). Berggreen et al. 1988). The specific ingestion rates of metazoan microplankton The composition of the diet may depend on the taxo- are expected to be higher than those of mesozooplankton nomic composition of the micrometazoan community. As

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Fig. 6. Relationships between ingestion rates and the concentration of the different prey: (A) Total Chl a; (B) Chl a . 10 mm; (C) HNF; (D) PNF; (E) ciliates; (F) dinoflagellates; (G) diatoms; (H) total prey. The lines correspond to the function fitted to the data. r2 5 coefficient of determination. ns 5 not significant.

example, some ambush feeders, such as Oithona species, 1982), copepod nauplii and meroplanktonic larvae usually including their naupliar stages, fed only on motile prey, not only exhibit different food size spectra than mesozoo- whereas many calanoid copepod species are suspension plankton (Turner and Roff 1993) but also have a different feeders and can feed on a larger variety of prey (Henriksen diet and feeding selectivity (Poulet 1977). The dietary et al. 2007). Although for some copepod nauplii their differences between the different copepod developmental feeding niche coincides with that of adult stages (Conover stages would represent an advantage when food is scarce

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Table 5. Metazoan microplankton trophic effect (estimated as percentage of standing stock consumed daily) upon the different prey along the seasonal cycle. Chl a, Total Chl a; Chl a .10, Chl a .10 mm; Dinoflag., dinoflagellates.

Expts Chl a Chl a.10 HNF PNF Ciliates Dinoflag. Diatoms Sep 05 1.0 0.5 2.1 2.9 2.3 2.7 2.1 Oct 05 0.6 0.3 1.9 1.2 1.5 1.7 1.0 Nov 05 0.1 0.1 0.7 0.5 0.3 0.3 0.2 Jan 06 1.0 0.6 1.5 2.3 2.4 2.9 2.0 Mar 06 0.2 0.1 0.6 0.2 0.6 0.7 0.4 Apr 06 0.3 0.2 0.7 0.5 0.8 0.6 0.5 May 06 1.0 0.6 4.0 1.4 2.5 3.1 1.2 Jun 06 1.2 0.7 2.4 1.2 2.8 2.0 2.5 Jul 06 1.4 0.8 2.2 2.8 3.3 3.8 2.3 Aug 06 1.7 0.9 6.1 4.0 4.0 4.7 3.7 Sep 06 0.9 0.6 3.6 1.6 2.4 2.3 2.2

Table 6. Carbon-specific growth rates (G, d 21 6 SE) of the main groups of metazoan microplankton in the feeding experiments. Average carbon content (W, ng C ind 21) during the incubation (stated as geometric mean between the initial and final carbon content) is also provided.

Copepod nauplii Copepodites Meroplankton Expts G (d 21) W (ng C ind. 21) G (d 21) W (ng C ind. 21) G (d 21) W (ng C ind. 21) Oct 05 0.32 60.02 48.18 0.19 60.07 129.17 0.03 60.03 230.05 Nov 05 0.17 60.07 53.51 0.18 60.04 152.24 0.15 60.03 92.14 Jan 06 0.08 60.04 66.45 0.13 60.05 156.42 0.11 60.01 176.12 Mar 06 0.49 60.06 55.68 0.38 60.07 176.59 0.26 60.06 199.14 Apr 06 0.38 60.09 49.07 0.29 60.14 254.80 0.15 60.03 220.37 May 06 0.30 60.05 66.07 0.22 60.15 186.87 0.03 60.02 198.70 Jun 06 0.18 60.07 74.36 0.16 60.09 187.59 0.07 60.05 163.92 Jul 06 0.04 60.03 36.01 0.13 60.07 145.22 0.05 60.03 127.19 Aug 06 0.01 60.09 38.26 0.03 60.06 189.74 20.08 60.10 127.97 Sep 06 0.29 60.05 45.16 0.21 60.06 165.96 0.14 60.03 124.09 because they reduce intraspecific competition. According to through their subsequent ingestion by animals at higher the obtained data, metazoan microplankton exhibit high trophic levels (Hopcroft et al. 2001). clearance rates on small nanoplankton ( , 2–5 mm), a size Our results showed that ciliates and HNF are frequent fraction in which most mesozooplankton feed inefficiently components in the micrometazoan diet, corroborating the (Berggreen et al. 1988), with some exceptions like tunicates relevance of the heterotrophic link between the microbial and cladocerans (Atienza et al. 2006 a). By feeding on food webs and the classical food chain (Calbet and Saiz particles smaller than those edible by mesozooplankton, 2005; Saiz and Calbet in press). Protozoans are considered micrometazoans drive microbial food web energy, usually more nutritious and richer in nitrogen-containing com- unavailable to larger metazoans, into the classical food pounds than diatoms, and also they are a source of web, either by natural growth into larger stages and/or essential lipids, not always available in phytoplankton, for

Table 7. Carbon-specific growth rates (G), carbon-specific ingestion rates (I), carbon-specific respiration rates (R), GGE, AE, and NGE of metazoan microzooplankton in the different experiments along a seasonal cycle. Expts, experiments.

Expts G (d 21) 6SE I (d 21) 6SE R (d 21) 6SE GGE 6SE AE 6SE NGE 6SE Sep 05 — 0.87 60.02 — — — — Oct 05 0.25 60.02 0.66 60.01 — 0.38 60.01 — — Nov 05 0.13 60.04 0.64 60.03 0.21 60.02 0.21 60.05 0.54 60.02 0.39 60.02 Jan 06 0.12 60.04 0.71 60.06 0.16 60.02 0.16 60.04 0.39 60.01 0.42 60.01 Mar 06 0.40 60.05 1.23 60.04 — 0.32 60.03 — — Apr 06 0.21 60.01 1.24 60.09 — 0.17 60.02 — — May 06 0.20 60.06 0.57 60.05 0.30 60.02 0.34 60.08 0.87 60.01 0.40 60.01 Jun 06 0.13 60.05 0.64 60.08 — 0.19 60.06 — — Jul 06 0.09 60.01 0.39 60.01 — 0.23 60.01 — — Aug 06 0.02 60.01 0.31 60.01 0.36 60.01 0.06 60.05 * * Sep 06 0.24 60.04 0.81 60.12 0.33 60.01 0.30 60.01 0.70 60.01 0.42 60.01 * Specific respiration rates exceed ingestion and efficiencies cannot be calculated.

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al. 2010 b). These observations suggest that micrometazoans are frequently food limited in nature, but the degree of food limitation depends on taxonomic composition.

Growth, respiration, and carbon budget— Specific growth rates of metazoan microplankton found in this study were in the range reported for copepod developmental stages and meroplanktonic larvae under different food concen- tration and temperature regimes (Almeda et al. 2009, 2010 a,b). Similar to the ingestion rates, the in situ food concentrations during our study (Table 4) would be insufficient to support the maximum growth rates observed in the incubation experiments (Fig. 7), suggesting food- limited growth of micrometazoans in nature. Specific growth rates of metazoan microplankton were higher than those commonly reported for mesozooplankton (copepods) by the egg production method in Mediterranean waters (Saiz et al. 2007). Therefore, ignoring micrometazoan growth may result in an underestimation of secondary production particularly in oligo-mesotrophic systems. Carbon-specific respiration rates (d 21) were similar to those observed for copepod nauplii in the laboratory (Ko¨ster et al. 2008; Almeda et al. in press). As expected, temperature positively affected respiration rates and the observed Q 10 value was similar to that reported for calanoid copepods (1.8–2.1; review in Ikeda et al. 2001). The carbon-specific ingestion rates were enough to Fig. 7. Effect of food concentration on specific growth rates compensate for the carbon respiratory losses of micro- of metazoan microzooplankton. The line corresponds to the metazoans during all experiments, except in August 2006 function fitted to the data. r2 5 coefficient of determination. (Table 7). The GGE of metazoan microplankton changed according to food availability, temperature, and metazoan metazoans (Stoecker and Capuzzo 1990; but see Broglio et composition, in agreement with previous reports of al. 2004). Hence, although the biomass contribution of laboratory experiments with copepod nauplii and mero- protozoans to the diet may be very variable (Saiz and planktonic larvae (Almeda et al. 2009, 2010 b). Neverthe- Calbet in press), protozoans may be qualitatively important less, the general GGE of metazoan microplankton, as in the metazoan microzooplankton diet because they are estimated by the slope of the regression equation relating able to fill the nutritional deficit created by feeding solely specific ingestion rates vs. specific growth rates, was quite on phytoplankton (Kleppel 1993). similar to the values reported for copepods and mesozoo- plankton in general (0.35; Mullin and Brooks 1970) but Functional responses— The functional responses observed slightly higher than observed for Oithona (0.21; Almeda et in this study should be considered cautiously because of the al. 2010 b) and the median values reported by Straile (1997) scarcity of data at high food concentrations. The saturating for copepods (0.22). Hence, although the GGE of food concentration (food concentration required for metazooplankton may range between 0.20 and 0.40 achieving the maximal specific ingestion rate), when total depending on the species and stage composition, our prey concentration is considered, was quite similar to that results support than an average value of 0.30 may be observed for copepod nauplii in temperate coastal waters assumed for detailing the carbon flow through metazoo- when feeding on phytoplankton ( , 240 mg C L 21; Lo´pez et plankton in marine planktonic food webs. al. 2007) and for Oithona davisae nauplii and copepodites The AE of metazooplankton commonly ranges from feeding on optimal prey (200–320 mg C L 21; Almeda et al. 10 % to close to 100 %, depending on the species, 2010 b). However, the saturating food concentrations development stage, food quality, and food concentration reported for calanoid developmental stages in laboratory (Conover 1978). The AEs found in our study were in the experiments are usually higher than those observed here ( $ range expected for metazooplankton feeding in an omniv- 500 mg C L 21; Berggreen et al. 1988). The in situ food orous diet (Conover 1966). Previous studies had reported concentrations during our study period (Table 3) would be that NGEs of zooplankton increase with increasing insufficient to support the maximum ingestion rates of temperature (Ikeda et al. 2001) and food concentration metazoan microplankton found in the feeding experiments (Vidal 1980). However, we found an almost constant NGE (Fig. 6H). The maximum specific ingestion rates observed kept despite the differences in temperature and food in this study were lower than those commonly found for availability. A possible explanation would be a similar calanoid nauplii in the laboratory but close to those dependence on temperature and food availability of the reported for Oithona spp. (Saiz and Calbet 2007; Almeda et different physiological processes, resulting in a similar

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x x

Fig. 8. Carbon-specific growth rates vs. carbon-specific ingestion rates of metazoan microzooplankton. (A) Linear regression including all growth data. (B) Linear regression including only growth rates of experiments dominated by copepod larvae (excluded data are represented as empty circles). allocation of assimilated materials. Similar results have Potential trophic effect of metazoan microplankton on been found in laboratory studies with O. davisae nauplii food web structure— The degree to which zooplankton (Almeda et al. in press). The NGE found in this study were predation may regulate population of primary producers is in the range of the common values reported for adult still a subject of debate. Some studies had reported that copepods (0.29–0.61, Conover 1978; 0.21–0.54, Ikeda et al. phytoplankton can be controlled by the metazooplankton 2001), indicating that metazoan microplankton exhibit grazing (Roman and Gauzens 1997), whereas others similar food conversion efficiency as compared to meso- indicate metazoans exert little grazing pressu re on phyto- zooplankton (copepods) and an equivalent carbon transfer plankton ( , 5%; Broglio et al. 2004; Atienza et al. 2006 b). efficiency to higher trophic levels. However, most of these studies excluded the micrometazo- ans and consequently underestimated the trophic effect by metazooplankton. The scarce studies including microme- tazoans show a wide range of grazing effects, from low (0.3–10 %; Roman and Gauzens 1997; Calbet et al. 2009) to high values (e.g., 23–54 %, White and Roman 1992; Lonsdale et al. 1996). In our study, the inclusion of metazoan microplankton in the metazooplankton grazing doubled the trophic effect considering only the mesozoo- plankton community according to previous studies in the same area (Broglio et al. 2004). However, even including the micrometazoans, the grazing pressure by metazoo- plankton was low in terms of reduction of standing stock of phytoplankton ( , 5%). On the other hand, the trophic effect by the entire microzooplankton community ranged from 9 % to 97 % for total Chl a and from 0 % to 130 % for Chl a . 10 mm according to the dilution experiments conducted simultaneously with the present study (Calbet et al. 2008; Fig. 10). Therefore, the contribution of micro- metazoans to the total grazing on phytoplankton is quite low, and very likely protists must be the main organisms responsible for the predatory pressure on primary produc- ers in Mediterranean coastal waters, at least during some seasons (Fig. 10). Some studies suggest that the effects of micrometazoans grazing on phytoplankton may be medi- Fig. 9. Effect of temperature on respiration rates of metazo- ated mostly by their control of microzooplankton popula- an microzooplankton. The line corresponds to the function fitted tions (Fessenden and Cowles 1994). However, our results to the data. r2 5 coefficient of determination. indicate that their grazing pressure on protozoans appeared

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phytoplankton or protozoan standing stock in NW Mediterranean coastal waters.

Acknowledgments We thank Dolors Vaque´, Nagore Sampedro, Albert Ren˜e´, and Hassina Illoul for their help in the plankton identification. This work was funded by the Spanish Ministry of Science and Inno- vation (MICINN) through a Ph.D. fellowship (BES-2005-7491) to R.A. and the research projects CTM2004-02775 to A.C., CTM2006-12344-C02-01/MAR and CTM2009-05476-E/MAR to M.A., and CTM2007-60052 to E.S.

References

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POULET , S. A. 1977. Grazing of marine copepod development TURNER , J. T. 2004. The importance of small planktonic copepods stages on naturally occurring particles. J. Fish Res. Board and their roles in pelagic marine food webs. Zool. Stud. 43: Can. 34: 2381–2387. 255–266. PUTT , M., AND D. K. S TOECKER . 1989. An experimentally ———, H. L EVINSEN , T. G. N IELSEN , AND B. W. H ANSEN . 2001. determined carbon : volume ratio for marine ‘‘oligotrichous’’ Zooplankton feeding ecology: Grazing on phytoplankton and ciliates from estuarine and coastal waters. Limnol. Oceanogr. predation on protozoans by copepod and barnacle nauplii in 34: 1097–1103, doi: 10.4319/lo.1989.34.6.1097 Disko Bay, West Greenland. Mar. Ecol. Prog. Ser. 221: ROMAN , M. R., AND A. L. G AUZENS . 1997. Copepod grazing in the 209–219, doi: 10.3354/meps221209 equatorial Pacific. Limnol. Oceanogr. 42: 623–634, ———, AND J. C. R OFF . 1993. Trophic levels and trophospecies in doi: 10.4319/lo.1997.42.4.0623 marine plankton: Lessons from the microbial food web. Mar. SAIZ , E., AND A. C ALBET . 2007. Scaling of feeding in marine cala- Microb. Food Webs 7: 225–248. noid copepods. Limnol. Oceanogr. 52: 668–675, doi: 10.4319/ ———, AND P. A. T ESTER . 1992. Zooplankton feeding ecology: lo.2007.52.2.0668 Bacterivory by metazoan microzooplankton. J. Exp. Biol. ———, AND ———. In press. Copepod feeding in the ocean: 160: 149–167, doi: 10.1016/0022-0981(92)90235-3 Scaling patterns, composition of their diet and the bias of UITTO , A. 1996. Summertime herbivory of coastal mesozooplank- estimates due to microzooplankton grazing during incuba- ton and metazoan microplankton in the northern Baltic. Mar. tions. Hydrobiologia, doi: 10.1007/s10750-010-0421-6 Ecol. Prog. Ser. 132: 47–56, doi: 10.3354/meps132047 YE AND ASAHARA ———, ———, D. A TIENZA , AND M. A LCARAZ . 2007. Feeding and U , S. I., S. K . 1983. Grazing of various production of zooplankton in the Catalan Sea, northwestern developmental stages of Pseudodiaptomus marinus (Copepo- Mediterranean. Prog. Oceanogr. 74: 313–328, doi: 10.1016/ da: Calanoida) on naturally occurring particles. Bull. Plank- j.pocean.2007.04.004 ton Soc. Jpn. 30: 147–158. VERITY , P. G., AND C. L ANGDON . 1984. Relationships between ———, ———, AND E. B ROGLIO . 2003. Effects of small-scale turbulence on copepods: The case of Oithona davisae . Limnol. lorica volume, carbon, nitrogen, and ATP content of Oceanogr. 48: 1304–1311, doi: 10.4319/lo.2003.48.3.1304 tintinnids in Narragansett Bay. J. Plankton Res. 6: 859–868, doi: 10.1093/plankt/6.5.859 SIEBURTH , J. M., V. S METACEK , AND J. L ENZ . 1978. Pelagic VIDAL , J. 1980. Physioecology of zooplankton. IV. Effects of ecosystem structure: Heterotrophic compartments of the phytoplankton concentration, temperature, and body size on plankton and their relationships to plankton size fractions. the net production efficiency of Calanus pacificus . Mar. Biol. Limnol. Oceanogr. 23: 1256–1263, doi: 10.4319/lo.1978. 56: 203–211, doi: 10.1007/BF00645344 23.6.1256 WHITE , J. R., AND M. R. R OMAN . 1992. Seasonal study of grazing STARR , M., J. H. H IMMELMAN , AND J. C. T HERRIAULT . 1990. Direct by metazoan zooplankton in the mesohaline Chesapeake Bay. coupling of marine invertebrate spawning with phytoplankton Mar. Ecol. Prog. Ser. 86: 251–261, doi: 10.3354/meps086251 blooms. Science 247: 1071–1074, doi: 10.1126/science. ZERVOUDAKI , S., AND oTHERS . 2007. The importance of small-sized 247.4946.1071 copepods in a frontal area of the Aegean Sea. J. Plankton Res. STOECKER , D. K., AND J. C APUZZO . 1990. Predation on protozoa: 29: 317–338, doi: 10.1093/plankt/fbm018 Its importance to zooplankton. J. Plankton Res. 12: 891–908, doi: 10.1093/plankt/12.5.891 STRAILE , D. 1997. Gross growth efficiencies of protozoan and Associate editor: Thomas Kiørboe metazoan zooplankton and their dependence on food concen- tration, predator–prey weight ratio, and taxonomic group. Received: 31 July 2010 Limnol. Oceanogr. 42: 1375–1385, doi: 10.4319/lo.1997.42.6. Accepted: 25 October 2010 1375 Amended: 22 November 2010

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Resum de l’article II – Article II summary – Catalan version

Funció tròfica i balanç de carboni dels metazous microplanctònics en aigües costaneres del nord-oest de la Mediterrània .

Rodrigo Almeda, Albert Calbet, Miquel Alcaraz, Enric Saiz, Isabel Trepat, Laura Arin, Juancho Movilla, and Violeta Saló. Article publicat a Limnology and Oceanography (2011, 56(1):415-430).

Durant un cicle estacional es van determinar, mitjançant incubacions de laboratori, les taxes d'alimentació, l'impacte tròfic i les eficiències de creixement dels assemblatges naturals de metazous microplanctònics procedents d'un àrea costanera en el nord-oest del Mediterrani. Els micrometazous, és a dir els organismes pluricel·lulars planctònics heterotròfics entre 20 i 200 µm, van estar constituïts principalment per fases larvàries d'invertebrats. Els nauplis de copèpodes i copepodits van dominar la comunitat, excepte a l'abril quan les larves de poliquets van ser el grup més abundant. Es va analitzar la pressió per depredació dels micrometazous sobre clorofil·la a (Chl a, total i > 10 µm), nanoflagel.lats heterotròfics, nanoflagel.lats fototròfics, dinoflagel.lats, diatomees i ciliats. Els micrometazous van depredar tots els grups estudiats, amb taxes d'ingestió específiques en carboni que van variar entre 0.31 i 1.24 d -1 . Les eficiències brutes de creixement per al conjunt de la comunitat de metazous microplanctònics, calculades com el pendent de la regressió lineal que relaciona les taxes específiques de creixement versus les taxes especifiques d'ingestió, va variar entre 0.27 i 0.39. Les perdudes respiratòries en carboni dels micrometazous van dependre de

-1 la temperatura i van variar entre 0.16 i 0.36 d , amb un Q 10 = 2. L'eficiència mitjana de creixement net, 0.41, va ser independent de la temperatura i la disponibilitat d'aliment. Els micrometazous en conjunt tenen taxes específiques de creixement més altes que les del mesozooplàncton, però eficiències de conversió de l'aliment similars. L'efecte per predació en el “stock” existent de les diferents preses va ser < 1% d -1 per Chl a (total i > 10 µm) i < 2.5% d -1 per a les altres preses estudiades, el que sembla insuficient per exercir un control rellevant sobre la dinàmica del fitoplàncton i els protozous. La inclusió dels micrometazous no canvia apreciablement la visió actual sobre el paper del metazooplàncton a les xarxes tròfiques marines en aigües costaneres del nord- oest Mediterrani.

91 Chapter 2

Resumen del artí culo II – Article II summary– Spanish version

Función trófica y balance de carbono del metazoo microplanctónico en aguas costeras del noroeste del Mediterráneo.

Rodrigo Almeda, Albert Calbet, Miquel Alcaraz, Enric Saiz, Isabel Trepat, Laura Arin, Juancho Movilla, and Violeta Saló. Articulo publicado en Limnology and Oceanography (2011, 56(1):415-430).

Durante un ciclo estacional se determinaron, mediante incubaciones de laboratorio, las tasas de alimentación, el impacto trófico y las eficiencias de crecimiento de los ensamblajes naturales de metazoos microplanctónicos procedentes de un área costera del noroeste del Mediterráneo. Los micrometazoos, es decir los organismos pluricelulares planctónicos heterotróficos entre 20 y 200 µm, estuvieron constituidos principalmente por fases larvarias de invertebrados. Los nauplios de copépodos y copepoditos dominaron la comunidad, excepto en abril cuando las larvas de poliquetos fueron el grupo más abundante. Se analizó la presión por depredación de los micrometazoos sobre clorofila a (Chl a, total y > 10 µm), nanoflagelados heterotróficos, nanoflagelados fototróficos, dinoflagelados, diatomeas y ciliados. Los micrometazoos predaron sobre todos los grupos estudiados, con tasas de ingestión específicas en carbono que variaron entre 0.31 y 1.24 d -1 . Las eficiencias brutas de crecimiento para el conjunto de la comunidad de metazoos microplanctónicos, calculadas como la pendiente de la regresión lineal que relaciona las tasas específicas de crecimiento versus las tasas especificas de ingestión, varió entre 0.27 y 0.39. Las pérdidas respiratorias en carbono de los micrometazoos dependieron de la temperatura y variaron entre 0.16

-1 y 0.36 d , con un Q 10 = 2. La eficiencia de crecimiento neto promediada, 0.41, fue independiente de la temperatura y la disponibilidad de alimento. Los micrometazoos en conjunto tienen tasas específicas de crecimiento más altas que las del mesozooplancton, pero eficiencias de conversión del alimento similares. El efecto por predación en el “stock” existente de las diferentes presas fue < 1% d -1 para Chl a (total y > 10 µm) y < 2.5% d -1 para las otras presas estudiadas, lo que parece insuficiente para ejercer un control relevante sobre la dinámica del fitoplancton y los protozoos. La inclusión de los micrometazoos no cambia apreciablemente la visión actual sobre el papel de metazooplanct on en las redes tróficas marinas en aguas costeras del noroeste Mediterráneo.

92 Feeding rates and abundance of marine invertebrate planktonic larvae under harmful algal bloom conditions off Vancouver Island

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Harmful Algae 10 (2011) 194–206

Contents lists available at ScienceDirect

Harmful Algae

journal homepage: www.elsevier.com/locate/hal

Feeding rates and abundance of marine invertebrate planktonic larvae under harmful algal bloom conditions off Vancouver Island

Rodrigo Almeda a,*, Amber M. Messmer b,c, Nagore Sampedro a, Louis A. Gosselin b,c a Institut de Cie `ncies del Mar (CSIC), Barcelona, Catalonia, Spain b Thompson Rivers University, Kamloops, British Columbia, Canada c University of Victoria, Victoria, British Columbia, Canada

ARTICLE INFO ABSTRACT

Article history: The interactions between toxic phytoplankton and their potential grazers are poorly understood aspects Received 17 August 2010 of the ecology of harmful algal blooms. In this study, we determined the feeding rates, prey selection and Received in revised form 30 September 2010 trophic impact of different marine invertebrate planktonic larvae on the natural bloom of Heterosigma Accepted 30 September 2010 akashiwo and Prorocentrum triestinum which occurred on the west coast of Vancouver Island in July of 2006. Additionally, we estimated the abundance, biomass and composition of zooplankton before and Keywords: during the harmful algal bloom. Feeding Feeding experiments were performed with polychaete ( Serpula columbiana ), echinoderm ( Stronglyo- Food selection centratus purpuratus ) and cirripede ( Balanus crenatus ) larvae obtained from laboratory cultures, and Harmful algae bloom Heterosigma akashiwo bivalve and gastropod larvae collected from the study site by plankton tows. All larvae fed on H. akashiwo Meroplanktonic larvae whereas only cirripede nauplii and echinoderm larvae fed on P. triestinum . H. akashiwo was the main Prorocentrum triestinum component of all larval diets ( >64%). We observed a positive relationship between prey availability in Trophic impact the food assemblages and their contribution to all larval diets. The potential trophic impact of meroplanktonic larvae on bloom forming phytoplankton species was low ( <1.5%). The ingestion of bloom forming phytoplankton did not appear to have any adverse effects on the studied grazers after 48 h of incubation. In contrast, field abundance of planktonic larvae and other zooplankton continuously decreased throughout the progression of the bloom, with losses approaching 75% in comparison to their pre-bloom abundance. The presence of H. akashiwo negatively affected the abundance of meroplanktonic larvae, despite efficient grazing of these larvae. Therefore, grazing pressure was reduced which likely contributed to the growth and persistence of the bloom. The reduction in meroplanktonic larvae and other zooplankton abundance associated with the H. akashiwo bloom may have potential impacts on benthic recruitment and energy transfers to higher trophic levels in marine food webs. ß 2010 Elsevier B.V. All rights reserved.

1. Introduction 1986; Nagasaki et al., 1994; Mayali and Azam, 2004 ). Among these interactions, the grazing pressure of zooplankton on bloom- Harmful algal blooms (HABs, commonly called ‘red tides’) are a forming phytoplankton species remains poorly understood. common phenomenon in coastal waters and their occurrence has Studies of zooplankton grazing on natural food assemblages become more frequent worldwide over recent decades ( Anderson, during HABs are scarce ( Turner and Anderson, 1983; Turner and 1989; Smayda, 1990; Hallegraeff, 1993 ). The formation and Tester, 1997; Calbet et al., 2003; Turner and Borkman, 2005 ) and persistence of HABs is partly controlled by several physical– most of our knowledge on the interaction between HAB and chemical factors (often referred to as ‘‘bottom-up’’ controls) such zooplankton is provided from laboratory studies using unnaturally as column stratification, nutrient concentration, temperature and high concentrations of unialgal diets that are difficult to salinity ( Anderson and Lindquist, 1985; Connell and Jacobs, 1999 ). extrapolate to natural conditions ( Turner et al., 1998 ). Some In addition, biological factors including viral or bacterial infection, harmful bloom-forming phytoplankton exhibit chemical charac- parasitism and zooplankton grazing (‘‘top-down’’ controls) may teristics that make them less palatable to zooplankton ( Huntley et contribute to the decline of some HABs ( Watras et al., 1985; Uye, al., 1986; Teegarden, 1999 ) or have an adverse effect on zooplankton abundance and survival ( Turner et al., 1998; Turner, 2006 ). Reduced grazing pressure by zooplankton could contribute * Corresponding author. to the formation and persistence of some harmful phytoplankton E-mail address: [email protected] (R. Almeda). blooms ( Smayda and Villareal, 1989; Gilbert, 1990 ). Therefore,

1568-9883/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi: 10.1016/j.hal.2010.09.007

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information on the effects of HABs on grazer populations and of different meroplanktonic larvae during the HAB; and (3) to zooplankton–toxic phytoplankton trophic interactions (e.g. graz- evaluate the potential trophic impact (top-down control) of ing rates, feeding selection patterns, trophic impact) are required meroplanktonic larvae on the HAB. for a better understanding of the ecology and dynamics of harmful phytoplankton blooms. 2. Materials and methods Most available information on the interactions of zooplankton grazers with harmful phytoplankton blooms has focused on 2.1. Bloom event and identification of bloom-forming phytoplankton copepods and recently on microprotozoans ( Turner and Tester, species 1997; Turner, 2006 ). Little is known regarding the interactions between marine invertebrate planktonic larvae (meroplankton) Sampling was conducted at the mouth of Bamfield Inlet (N 48 and harmful bloom-forming phytoplankton. It is important to 50.343 0 W 123 08.152 0), in Barkley Sound, Vancouver Island, study these interactions because meroplanktonic larvae may Canada during a coastal phytoplankton bloom in July 2006 ( Fig. 1 ). frequently be the dominant component of the coastal metazoo- Chlorophyll concentration was used as an indicator of phytoplank- plankton during the reproductive season ( Blanner, 1982; Anger ton biomass during the phytoplankton bloom. Chlorophyll and et al., 1986; Andreu and Duarte, 1996 ) and are the key factor that water temperature data were extracted from SeaWifs satellite determines the success of adult populations of benthic inverte- images. Seawater samples were collected with a Niskin bottle at brates ( Thorson, 1946, 1950 ). For example, phytoplankton blooms 5 m depth from the sampling site to identify the dominant bloom- act to directly induce the release of gametes or larvae in some forming phytoplankton species. Seawater subsamples (200 ml) benthic invertebrate species either through direct contact (phyto- were fixed with Lugol’s solution (1%) for light field microscopy and plankton particle settlement) or chemical triggering ( Starr et al., with glutaraldehyde (1%) for epifluorescence microscopy (see 1990, 1991, 1994 ). This results in a high abundance of mer- procedure details below). Cells were examined at 200 with a  oplanktonic larvae during phytoplankton blooms ( Thorson, 1946; Nikon Diaphot TMD inverted microscope and at 1000 magnifi-  Olson, 1987; Andreu and Duarte, 1996 ). Low abundance of cation with an Olympus BX61 epifluorescence microscope under copepods and a high abundance of meroplanktonic larvae blue and UV wavelength excitation. Aliquots from Lugol’s samples (polychaete larvae) have been observed during blooms of some (20 ml) were filtered using a vacuum pump onto polycarbonate toxic phytopla nkton species (e.g. Alexandrium spp.) ( Turner and Nuclepore filters (0.8 mm pore size and 25 mm diameter) for Anderson, 1983; Watras et al., 1985 ). Therefore, the associated Scanning Electron Microscopy (SEM). The filters were air dried and feeding interactions and the potential capability of marine stored under partial vacuum in hermetically closed boxes until invertebrate planktonic larvae to control harmful phytoplankton preparation for the SEM. A part of the filter was placed on a SEM blooms should be not neglected. stub and coated with a film (of about 150 A ˚ ) of gold–palladium to In this study, we examine the trophic interactions between avoid electric charges; the sputter coater used was a Polaron SC- representative marine invertebrate planktonic larvae and natural 500. The examination and microphotographs of the specimens food assemblages during a phytoplankton bloom that occurred on were made with a Hitachi S-3500N scanning electron microscope the west coast of Vancouver Island in July 2006. The specific operating at 5 kV. The identification of bloom-forming phyto- objectives of this study were: (1) to determine the abundance, plankton species was based on morphology and ultrastructure biomass and composition of zooplankton before and during the (Hara et al., 1985; Hara and Chihara, 1987; Dodge, 1982; Steidinger HAB; (2) to estimate the feeding rates and food selection behavior and Tangen, 1996 ).

Fig. 1. (A) Satellite image of the west coast of Vancouver Island during the harmful phytoplankton bloom in July of 2006. Note that besides the Heterosigma akashiwo / Prorocentrum triestinum bloom (red color), we can also observe some remains of a coccolithophorid bloom (green color) which previously occurred in the same area. (B) Sampling s tation in the Bamfield inlet ( $). Map courtesy of Canadian Hydrographic Services. Ottawa, Canada. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

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Table 1 Length vs. carbon content regressions used to provide biomass estimates for the different groups of zooplankton. Individual biomass ( mg C) = a L (mm) b. Â Organism Measurement a b Reference

9 Copepods Length of prosome 4.27 10 À 3.07 Uye (1982)  8 Copepodites Length of prosome 1.11 10 À 2.92 Berggreen et al. (1988)  9 Copepod nauplii Body length (without setae) 3.18 10 À 3.31 Berggreen et al. (1988)  8 Appendicularia Trunk length (without tail) 7.33 10 À 2.63 King (1980)  10 Cladocera Total length 4.57 10 À 3.46 Uye (1982)  7 Rotifers Body length 1.06 10 À 2.74 Hansen et al. (1997a)  4 Polychaeta larvae Maximal length 1.58 10 À 1.38 Hansen (1999) as Polydora sp  8 Bivalvia larvae Maximal length 3.06 10 À 2.88 Fotel et al. (1999)  5 Gastropoda larvae Maximal length 2.31 10 À 2.05 Hansen and Ockelmann (1991)  8 Echinoderma larvae Total length 3.06 10 À 2.88 Fotel et al. (1999) as bivalve  10 Cirripedia nauplii Body length (without spine) 2.20 10 À 3.72 Turner et al. (2001)  12 * Decapoda larvae Carapace length 4.01 10 À 4.43 Hirota and Fukuda (1985)  8 Other larvae Total length 3.06 10 À 2.88 Fotel et al. (1999) as bivalve  * Calculated using length and carbon content data from Table 1 (r2 = 0.91).

2.2. Estimation of abundance, biomass and composition of suspension though a 25 mm screen and the eggs were rinsed in a zooplankton during the HAB bath of 0.2 mm autoclaved filtered seawater (FSW). The eggs were then placed in a beaker with FSW for 24 h to allow the embryos to The abundance, biomass and composition of zooplankton in the develop to the trochophore stage. S. columbiana larvae were study site (Bamfield Inlet) were determined periodically through- provided with the flagellate Isochrysis galbana as a food source for out July 2006. Zooplan kton samples were obtained by duplicate 4 d until the beginning of the experiments. vertical tows from 30 m to the surface using a microplankton net Adult B. crenatus were exposed to bright light after 6 h of  (64 mm mesh, 30 cm diameter). During sample collection, the emersion resulting in the release of nauplius I larvae. The barnacle plankton net was rinsed and the entire cod end sample was poured larvae were transferred to a beaker of autoclaved 0.2 mm FSW for into a 500 ml plastic bottle and preserved in borax-buffered 24 h before the beginning of the experiment, during which time formaldehyde at 4% final concentration. To estimate the abun- they molted to the nauplius II stage. No food was provided during dance and composition of metazooplankton, these samples were this period because these larvae develop through the nauplius I divided into two nominal size fractions (64–200 mm, >200 mm) by stage without feeding ( Strathmann, 1987 ). filtering through a 200 mm sieve. Two aliquots per fraction (at least To induce spawning in S. purpuratus , 0.5 M KCL solution was 350 organisms per aliquot) were counted under a stereomicro- injected into the intracoelomic cavity in 3–6 adults of each sex scope. Body length measurements were made for >60 randomly (Strathmann, 1987 ). Sperm were collected with a glass pipette and selected individuals from each zooplankton size fraction to kept at 4 8C until use and the oocytes were collected and determine biomass. Measurements were taken from digital images transferred to autoclaved 0.2 mm FSW. An aliquot of sperm was from a stereomicroscope using image analysis (ImageJ software). added to the oocytes for 5 min. The remaining sperm were Each individual’s carbon weight was calculated by applying body removed by filtering the suspension though a 25 mm screen and size–carbon content relationships from the literature ( Table 1 ). then rinsing the eggs in a bath of 0.2 mm filtered seawater (FSW). The eggs were then placed in a beaker with 0.2 mm FSW and 2.3. Sources of larvae for meroplankton maintained for 76 h to allow development to the early pluteus feeding experiment stage (four-armed stage).

We obtained larvae of Serpula columbiana (Polychaeta), 2.4. Design of the feeding experiment Stronglyocentrotus purpuratus (Echinoderma), and Balanus crenatus (Cirripedia) through laboratory cultures. Mature individuals of The feeding experiment consisted of bottle incubations of the each species were collected from different locations in Trevor naturally blooming microplanktonic community with and Channel; S. columbiana specimens were obtained from the without the addition of meroplanktonic larvae as grazers. intertidal zone of Dixon Island, S. purpuratus from the subtidal Details of the larvae and the larval densities used in the feeding zone in Scott’s Bay, and B. crenatus from the subtidal zone in experiments are described in Table 2 . At the start of the Bamfield Inlet. experiment, subsamples of larvae of each group were fixed with Bivalve and gastropod larvae were collected 24 h before the start formaldehyde at 4% final concentration for later biomass of the experiment from Trevor Channel using a 64 mm-mesh size determinations. Approximately 40 photographs of each group plankton net. Bivalve larvae of similar size (mainly Mytilus spp.) of larvae were taken using an inverted microscope (100 ), from  were carefully pipetted from each plankton sample, repeatedly which body lengths ( L, mm) were measured by image analysis rinsed by transferring them through a series of Petri dishes filled (Image J software). The average individual carbon weight was with 0.2 mm filtered seawater (FSW), and maintained in autoclaved calculated by applying the body size–carbon content relation- 0.2 mm FSW at 16 8C until commencement of the experiments. A ships given in Table 1 . similar procedure was used for the collection of gastropod larvae. Seawater and plankton for the feeding experiment were Some types (morphology) and sizes of gastropod veligers (mainly collected at 5 m depth with a Niskin bottle. The seawater samples nudibranchia) were used in the feeding experiment. were gently transferred to carboys and transported to the To obtain S. columbiana larvae, 5 adult worms of each sex were laboratory within 1 h of collection. Seawater was screened through induced to spawn by breaking their tubes, starting at the posterior a 25 mm mesh by reverse filtration to exclude metazoan end, until the soft body of the polychaete could be touched. This consumers. The filtered seawater was carefully siphoned into caused the animal to contract and releases its gametes. An aliquot 700 ml acid-washed glass bottles in a three-step filling procedure of sperm suspension was mixed with oocytes for 5 min for to ensure homogeneity between replicates. The experiment fertilization. The remaining sperm were removed by filtering the comprised three types of treatments: (1) initial plankton conditions

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Table 2 Characteristics of the marine invertebrate larvae used in the feeding experiment at commencement of incubation. Each larval type was used separately.

1 1 Taxon Larval stage Species Length SE ( mm) Biomass SE ( mg C Ind À ) Density (Ind l À ) Æ Æ Polychaeta Trochophore Serpula columbiana 149.2 2.1 0.16 0.01 50 Æ Æ Echinoidea Four-armed echinopluteus Strongylocentrotus purpuratus 261.9 2.2 0.28 0.01 30 Æ Æ Cirripedia Nauplii II Balanus crenatus 287.8 6.1 0.33 0.02 30 Æ Æ Bivalvia Late veliger Mytilus spp. 298.5 11.2 0.45 0.05 29 Æ Æ Gastropoda Late veliger Unidentified 362.1 18.9 4.26 0.42 7 Æ Æ

(3 replicate bottles of the above 25 mm filtered seawater, sampled rates in the grazing bottles were different and lower than in the immediately at the start of the experiment); (2) control (3 replicate controls (one-way ANOVA test, p < 0.05). bottles of 25 mm filtered seawater, sampled after 48 h); and (3) Selective feeding by metazoan microzooplankton was evaluat- experimental (25 mm filtered seawater, with added larvae, sampled ed using the electivity index ( E*) of Vanderploeg and Scavia (1979) .

after 48 h, 3 replicate bottles for each larval type). The experimental Electivity index of the i food type ( Ei) was calculated as treatments therefore involved a total of 15 bottles (5 larval types 3 Â replicate bottles per larval type). Wi 1=n The bottles were placed in situ in mesh bags suspended at a 5 m Ei À ð Þ ¼ Wi 1=n depth in Bamfield Inlet off of the Bamfield Marine Science Center þ ð Þ dock. The incubations were carried out for 48 h and the bottles with n as the total number of prey types in a given experiment, and

were gently mixed by hand every 2–3 h. The water temperature the coefficient Wi defined by during incubation was recorded every 24 h. Water subsamples were taken from the bottles at the beginning and at the end of the Fi experiments to estimate the initial and final abundance and Wi ¼ Fi biomass of planktonic organisms. P where Fi is the clearance rate of the i food type, and SFi is the sum 2.5. Sample processing and calculations of clearance rates on all food types. The electivity index ( E) ranges between 1 and +1, where 0 signifies no electivity (no selective À Grazing was measured by quantitative microscopic analysis of grazing), negative values correspond to avoidance and positive the microbial community abundance in initial, control and values represent selection. The use of this index has been experimental treatments. The microbial components studied in especially recommended in cases where different food types the feeding experiments included the bloom-forming phytoplank- are not equally abundant ( Lechowicz, 1982 ). The selectivity ton species, heterotrophic nanoflagellates (HNF), phototrophic coefficients were computed within each experiment based on nanoflagellates (PNF), dinoflagellates, diatoms and ciliates. average clearance values (between replicate bottles) for each For nanoflagellates, 50 ml samples were preserved in glutaral- considered prey type. dehyde (1% final concentration). Duplicate 20 ml subsamples were The trophic impact of meroplanktonic larvae on each prey type gravity-filtered at low pressure onto 0.8 mm pore-size black was calculated as the % of biomass of the standing stock grazed 1 polycarbonate membrane filters and stained with DAPI (5 mg ml À daily assuming a homogenous prey and predator distribution in final concentration) for 5 min ( Porter and Feig, 1980 ). Filters were the water column. We calculated the potential impact from in situ 1 mounted on glass slides and stored at 20 8C until analysis. At least larvae biomass and their specific ingestion rates (d À ) obtained À 600 cells were counted by epifluorescence microscopy at a from the feeding experiments. magnification of 1000 and classified as auto- or heterotrophic  according their fluorescence for chlorophyll under blue light. Fifty 3. Results cells were sized and converted into carbon using a conversion 3 factor of 0.22 pgC mmÀ for HNF ( Børsheim and Bratbak, 1987 ) and 3.1. Bloom event and bloom-forming phytoplankton species 1 0.991 the equation pgC cell À = 0.109 volume for PNF ( Montagnes  et al., 1994 ). The bloom event that ocurred on the west coast of Vancouver To determine the concentration of dinoflagellates, ciliates and Island in July 2006 ( Fig. 1 ) was composed of the raphydophicean diatoms, 200 ml samples were fixed with 1% acidic Lugol’s solution Heterosigma akashiwo (Hada) Hada ex Y. Hara & Chihara (= and allowed to settle for 48 h in 50 ml Utermo¨hl chambers. An Heterosigma carterae , previously referred to as Olisthodiscus luteus ) inverted microscope (Nikon DIAPHOT 200) at 200 magnification and the dinoflagellate Prorocentrum triestinum Schiller ( Fig. 2 ). The  was used to count all ciliates and dinoflagellates in each chamber cell density of H. akashiwo and P. triestinum reached values higher 6 4 1 and diatoms were counted in at least 50 microscopic fields (or 200 than 10 and 10 cells l À , respectively ( Table 3 ). During the cells). Sixty randomly chosen cells for each group were sized and phytoplankotn bloom, water temperature increased from 13 to  converted into carbon using the conversion factors of 0.19 and 17 8C and chlorophyll (Chl) concentration increased from 2 to 3 1  0.053 pgC mmÀ for oligotrich ciliates ( Putt and Stoecker, 1989 ) 6 mg Chl l À (Fig. 3 A and B). and tintinnids ( Verity and Langdon, 1984 ), respectively, and the 1 0.819 equations of pgC Dino cell À = 0.760 volume for dinoflagel- 3.2. Abundance, biomass and composition of zooplankton during the 1  0.811 lates and pgC cell À = 0.288 volume for diatoms ( Men- HAB Diat  den-Deuer and Lessard, 2000 ). Because microzooplankton were preserved with acidic Lugol’s solution, no distinction between Zooplankton abundance decreased progressively from 40 to 1  strict heterotrophs and auto-/mixotrophs was made for ciliates 10 ind l À (Fig. 3 C) as the bloom developed. The zooplankton  and dinoflagellates. biomass was dominated by the mesozooplankton fraction ( Fig. 4 A). Clearance and ingestion rates were calculated for each prey Meroplanktonic larvae represented 22–37% of the total metazoo- type according to Frost (1972) after verification that prey growth plankton biomass ( Fig. 4 B). Adult calanoid copepods and copepod

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Fig. 2. Electronic and fluorescence microscope images used for the identification of the bloom-forming phytoplankton species. (A) and (B) Heterosigma akashiwo ; (C) and (D) Prorocentrum triestinum . F: flagella; N: nucleus; Cp: chloroplasts; P: pores. larvae (nauplii and copepodites) were the most important groups 3.4. Feeding rates, feeding selectivity and trophic impact in terms of carbon biomass ( Fig. 4 C). Polychaete, gastropod and bivalve larvae were the major contributors of biomass to the There were no apparent adverse effects of H. akashiwo on larvae meroplankton community ( Fig. 4 D). All zooplankton groups/ after the 48 h incubation period, grazer survival being 100%. fractions decreased in abundance and biomass with the develop- Clearance and ingestion rates varied significantly (ANOVA, ment of the HAB ( Fig. 4 ), although this decrease was more drastic in p < 0.05) depending on the larvae and the prey type ( Tables 4 1 1 some groups (e.g. rotifers, appendicularia) than others ( Fig. 4 ). and 5 ). Clearance rates ranged from 0.34 ml larvae À dÀ for early 1 1 polychaete larvae feeding on HNF to 6.36 ml larvae À dÀ for late 3.3. Microbial community abundance and compostion in the feeding gastropod larvae feeding on ciliates ( Table 4 ). All studied larvae fed experiment on H. akashiwo but only B. crenatus nauplii and S. purpuratus echinopluteus grazed on P. triestinum (Tables 4 and 5 ). We did not The abundance, size and biomass of the different microbial detect significant feeding on pico-flagellates ( Tables 4 and 5 ). The groups (excluding bacterioplankton) at the beginning of the carbon-specific ingestion rate of B. crenatus nauplii was signifi- feeding experiment (July 16, 2006), are shown in Table 3 . The cantly (ANOVA, p < 0.01) higher than the ingestion rates observed initial microbial community was dominated, in terms of biomass, in other larvae ( Fig. 5 ). by H. akashiwo and P. triestinum , which comprised 74% of total Heterosigma akashiwo was the main component of the diet  carbon biomass ( Table 3 ). Other important contributors to the (>64%) of all studied larvae ( Fig. 5 ). Prorocentrum triestinum microbial community were pico- and nanoflagellates ( 20%). In represented 25 and 20% of the diet of B. crenatus nauplii and   contrast, ciliates (represented by non-loricate ciliates such as S. purpuratus echinopluteus, respectively. The biomass contri- Strombidium spp., Mesodinium spp.), dinoflagellates (mostly bution of ciliates, dinoflagellates and diatoms to the larval diet Heterocapsa spp., Scripsiella spp. Protoperidinium bipes ) and was low ( <8%), whereas HNF and PNF represented 20–25% of  diatoms (mainly Leptocylindrus spp. and) represented a small the diet of bivalve, gastropod and polychaete larvae ( Fig. 5 ). We percentage of total microbial biomass ( <6%, Table 3 ). observed a positive relationship between prey availability in

Table 3 Initial abundance, size, and biomass of the different studied potential prey from the microbial community. HNF: heterotrophic nanoflagellates; PNF: phototrophic nanoflagellates except Heterosigma akashiwo ; Dinoflag.: dinoflagellates except Prorocentrum triestinum ; M: major axis, m: minor axis. The volume of the organism was assumed to be equivalent to that of the corresponding ellipsoid except for HNF, PNF and picoflagellates which were considered spherical [ M = m = equivalent spherical diameter, ESD ( mm)]. A nominal size of 1.5 mm was assumed for picoflagellates ( <2 mm).

1 3 1 1 Prey type Abundance SE (cells ml À ) M SE ( mm) m SE ( mm) Biovolume SE ( mm cell À ) Biomass SE ( mg C l À ) Æ Æ Æ Æ Æ Picoflagellates 9297 489 1.5 1.5 1.77 3.6 0.2 Æ Æ HNF 3579 155 2.4 0.1 2.4 0.1 7.3 0.01 5.7 0.2 Æ Æ Æ Æ Æ PNF 6965 539 4.0 0.3 4.0 0.3 33.5 0.01 24.6 1.9 Æ Æ Æ Æ Æ Diatoms 102 7 31.2 2.1 2.5 0.1 107 16 1.6 0.2 Æ Æ Æ Æ Æ H. akashiwo 1027 48 14.2 0.3 9.3 0.2 661 33 98.5 4.6 Æ Æ Æ Æ Æ Dinoflag. 23 2 15.8 0.5 11.4 0.4 1159 99 5.6 0.5 Æ Æ Æ Æ Æ Ciliates 5.2 0.2 19.2 1.2 14.6 0.9 2318 393 2.7 0.1 Æ Æ Æ Æ Æ P. triestinum 64 5 36.4 0.4 10.7 0.3 2323 132 26.9 2.1 Æ Æ Æ Æ Æ

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on dinoflagellates and ciliates. Gastropod and bivalve veligers exhibited positive selection for nanoflagellates, ciliates and dinoflagellates except for P. triestinum . The trophic impact (% of biomass of the standing stock grazed daily) of each larval type was very low ( <1%) for all prey types (Table 6 ). The cumulative potential trophic impact on H. akashiwo and P. triestinum by all studied larvae was 1.29% and 0.99% per day, respectively.

4. Discussion

4.1. Bloom event and bloom-forming phytoplankton species

Heterosigma akashiwo is a noxious flagellate that frequently causes heavy and extensive blooms in temperate coastal waters in Pacific and Atlantic Oceans ( Hara and Chihara, 1987 ). Blooms of H. akashiwo are frequent in early summer on the west coast of Vancouver Island where they can become extensive ( Taylor and Haigh, 1993 ). Consistent with previous field observations ( Taylor and Haigh, 1993 ), the increased water temperature observed during this study period seemed to be a crucial factor in bloom formation since the germination of H. akashiwo cysts is known to be successful above 15 8C ( Imai and Itakura, 1999 ). The extent and intensity of a bloom of this species depends on several factors, including water stratification, salinity and nutrient concentration (Honjo, 1990, 1993; Taylor and Haigh, 1993 ). Taylor and Haigh (1993) indicate that strong HABs occur after early spring diatom blooms that cause surface water to become nitrate depleted. A bloom of coccolithophorids did occur in Trevor Channel ( Fig. 1 ) previous to our study; this may have aided in the removal of nitrate, providing optimal conditions for the intense bloom of H. akashiwo observed in July 2006. Prorocentrum triestinum is a cosmopolitan bloom-forming dinoflagellate considered as poten- tially harmful ( Anderson, 1995 ).

4.2. Effects of harmful algal bloom on the plankton community

Heterosigma akashiwo is known mainly for its lethal effects on wild and cultivated fish and shellfish ( Black et al., 1991; Honjo, 1993; Smayda, 1998 ), resulting in large economic losses for aquaculture industries and further depletion of endangered wild species ( Horner et al., 1991 ). In fact, fish kills (farmed salmon) due to Heterosigma akashiwo were documented in close areas (North Puget Sound, BC) during the studied bloom ( Rensel, 2007 ). However, information concerning the effects of naturally occurring H. akashiwo blooms on zooplankton communities is scarce. Our field results show that the abundance of meroplanktonic larvae and other zooplankton are negatively affected by the H. akashiwo bloom; this may impact benthic recruitment and energy transfers to higher trophic levels in marine food webs. Previous fields studies that were focused on microzooplankton have also documented negative effects of H. akashiwo blooms on copepod nauplii (Kamiyama, 1995 ) and tintinnids ( Verity and Stoecker, 1982; Kamiyama, 1995; Kamiyama et al., 2000 ), which is consistent with our findings. The effects of H. akashiwo on zooplankton are expected to be site-specific since toxicity level of this species Fig. 3. Temporal progression of temperature (A), chlorophyll a concentration (B) depends on strain and environmental conditions ( Ono et al., 2000; and zooplankton abundance (C) under the harmful phytoplankton bloom Haque and Onoue, 2002 ). conditions during the study period. Heterosigma akashiwo produces an allelopathic substance, identified as a polysaccharide-protein complex ( Yamasaki et al., the food assemblages and their contribution to larval diets 2009 ) that may cause dramatic changes in the abundance and (Fig. 6 ). composition of the phytoplankton community ( Pratt, 1966; Honjo Selection indexes ( E*) varied depending on the larvae and prey et al., 1978; Yamasaki et al., 2009 ). The allelopathic substance type ( Fig. 7 ). All larvae showed positive selection of H. akashiwo produced by H. akashiwo can inhibit diatom growth (e.g. (Fig. 7 ). Polychaete larvae preferentially fed on nanoflagellates Skeletonema costatum ) and stimulate or enhance dinoflagellate whereas echinoderm larvae and cirripede nauplii preferentially fed growth, for example, Prorocentrum spp. ( Pratt, 1966; Honjo et al.,

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Fig. 4. Zooplankton biomass and composition under the harmful phytoplankton conditions during the study period. Error bars represent the standard error.

1978; Yamasaki et al., 2009 ). The phytoplankton composition omorii do not exhibit apparent adverse effects from ingesting H. observed during the studied bloom is in agreement with the akashiwo (Uye and Takamatsu, 1990 ). Egg hatching success in expected composition due to these allelopathic effects. some invertebrate species is negatively related to the H. akashiwo Laboratory studies have reported that the effects of Heterosigma cell concentration and the embryos and early larvae of some akashiwo on zooplankton may vary depending on species, invertebrates seem to be more susceptible to H. akashiwo than later developmental stage, cell concentration and exposure time. H. stages ( Wang et al., 2006 ). The negative effects of H. akashiwo on akashiwo has adverse effects on the survival, feeding, growth and/ zooplankton are frequently time-dependent ( Wang et al., 2006; Yu or reproduction of some species of copepods ( Tomas and Deason, et al., 2010 ). Little ( <10%) to no lethal effects of H. akashiwo on 1981; Yu et al., 2010 ), rotifers ( Xie et al., 2008 ) and on early stages invertebrate larvae have been observed after incubation 48 h  of gastropod larvae ( Wang et al., 2006 ). However, other (Botes et al., 2003; Wang et al., 2006 ). In contrast, mortality zooplankters such as the copepods Calanus pacificus and Acartia increased significantly ( >70%) after 96 h exposure of gastropod

Table 4 1 1 Clearance rates (ml larvae À dÀ ) standard error (SE) of the studied meroplanktonic larvae on the different potential prey. HNF: heterotrophic nanoflagellates; PNF: phototrophic Æ nanoflagellates except Heterosigma akashiwo ; Dinoflag.: dinoflagellates except Prorocentrum triestinum. 0 indicates no significant ingestion (ANOVA, p > 0.05) and (*) that the concentration in the experimental bottles was significantly higher than in the control bottles (ANOVA, p < 0.05).

Prey Polychaeta larvae Echinoderm larvae Cirripede larvae Bivalve larvae Gastropod larvae

Picoflagellates * * * ** HNF 0.336 0.030 0 * 1.859 0.315 4.077 1.394 Æ Æ Æ PNF 0.534 0. 142 0 0.974 0.131 1.898 0.187 4.442 0.570 Æ Æ Æ Æ Diatoms 0 1.292 0.154 * 0 * Æ H. akashiwo 0.475 0.045 1.135 0.214 3.556 0.351 1.615 0.227 5.775 0.831 Æ Æ Æ Æ Æ Dinoflag. 0 2.158 0.453 3.935 0.422 1.689 0.323 5.636 1.164 Æ Æ Æ Æ Ciliates 0 2.579 0.415 4.471 0.499 1.571 0.397 6.305 0.462 Æ Æ Æ Æ P. triestinum 0 1.224 0.230 5.123 0.501 0 0 Æ Æ

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Table 5 1 1 Ingestion rates (cells larvae À dÀ ) standard error (SE) by the studied meroplanktonic larvae on the different potential prey. HNF: heterotrophic nanoflagellates; PNF: Æ phototrophic nanoflagellates except Heterosigma akashiwo ; Dinoflag.: dinoflagellates except Prorocentrum triestinum. 0 indicates no significant ingestion (one way-ANOVA, p > 0.05) and (*) that the concentration in the experimental bottles was higher than in the control bottles (one way-ANOVA, p < 0.05).

Prey Polychaeta larvae Echinoderm larvae Cirripede larvae Bivalve larvae Gastropod larvae

Picoflagellates * * * ** HNF 1549 135 0 * 8044 1247 18580 7169 Æ Æ Æ PNF 5832 1509 0 10647 2543 19829 1775 48393 5949 Æ Æ Æ Æ Diatoms 0 195 22 * 0 * Æ H. akashiwo 698 64 1635 290 4571 375 2275 294 8250 609 Æ Æ Æ Æ Æ Dinoflag. 0 52 10 88 8 42 5 142 34 Æ Æ Æ Æ Ciliates 0 9.4 1.5 15.2 1.4 5.9 1.4 24.0 1.7 Æ Æ Æ Æ P. triestinum 0 122 21 428 28 0 0 Æ Æ

larvae ( Wang et al., 2006 ). This is consistent with our feeding the metabolic activity of mammalian cells and induce apoptotic experiment results, in which no meroplankton mortality was cell death by inhibiting the plasma membrane Ca 2+ -ATPase observed after 48 h of exposure. The larvae produced in our transporter ( Twiner et al., 2004, 2005 ). As previously mentioned, laboratory cultures had never been exposed to any toxic H. akashiwo produces polysaccharide-protein complexes (APPCs), phytoplankton and the larvae collected in plankton nets (bivalve analogous to a glycocalyx, which has allelopathic effects on the and gastropod larvae) were washed and maintained in filtered phytoplankton community ( Yamasaki et al. (2009) . The inhibitory seawater for 24 h before the experiment. Zooplankton that ingest effect of APPCs on the growth of diatoms has been attributed to harmful phytoplankton species may become inhibited only after APPCs that bind to the cell surfaces of target species. Similarly, several days of continuous ingestion ( da Costa and Ferna ´ndez, several studies have demonstrated that these APPCs on cell 2002; Guisande et al., 2002 ); therefore, long-term incubation membranes ( Honjo, 1993; Oda et al., 1998 ) cause adherence of H. experiments are required to more effectively evaluate the harmful akashiwo cells to the zooplankton body, resulting in strongly effects of H. akashiwo on zooplankton. inhibited swimming ability and consequently decreased food The mechanisms of Heterosigma akashiwo toxicity remain ingestion, development, reproduction and survival ( Yan et al., controversial and unresolved ( Twiner et al., 2001; Rensel and 2003; Wang et al., 2006; Xie et al., 2008; Yu et al., 2010 ). The Whyte, 2003 ). The toxic mechanisms of H. akashiwo include the attachment of H. akashiwo cells to zooplankton might promote or discharge of mucus or other lectin-like polysaccharide substances activate the release of endogenous toxic substances from the algae. (Nakamura et al., 1998; Oda et al., 1998; Smayda, 1998 ), the Additional field and laboratory studies are required to better production of brevetoxin-like neurotoxins ( Khan et al., 1997; understand the effects and mechanisms of H. akashiwo toxicity on Haque and Onoue, 2002 ) and the production of reactive oxygen zooplankton. species ( Honjo, 1994 ). Recently, it has been reported that H. Although there are no reports on the toxicity of Prorocentrum akashiwo produces extracellular organic compounds that can alter triestinum , this species is considered as potentially harmful (Anderson, 1995 ). This species is not noxious for some copepods (da Costa et al., 2005 ) but negatively affects the survival of bivalve larvae after 4 days of incubation ( Lee, 2003 ). More research is needed to assess the mechanism by which P. triestinum is harmful to some marine invertebrate larvae.

4.3. Feeding rates, food selection and diet compostion

Clearance and ingestion rates of zooplankton vary widely depending on many factors including body size, predator density, prey size, food concentration, food quality and temperature ( Frost, 1972; Hansen, 1991a; Hansen et al., 1997; Almeda et al., 2009, 2010 ). The clearance rates measured in this study are in agreement with the range of values reported in the literature for this type of meroplanktonic larvae feeding on natural food assemblages (Rassoulzadegan and Fenaux, 1979; Baldwin and Newell, 1991; Turner et al., 2001; Vargas et al., 2006 ) and in the upper limit or higher than those commonly reported from laboratory studies (Strathmann, 1971; Jeong et al., 2004; Almeda et al., 2009 ). The high C-specific ingestion rates of cirripede nauplii as compared to other meroplanktonic larvae are not only related to body size, since larvae of similar size differed significantly (ANOVA, p < 0.01) in specific ingestion rates. These differences in ingestion rates may reflect important differences in feeding behavior and physiology between crustacean and ciliated meroplanktonic larvae as supported by previous studies ( Hansen et al., 1997; Vargas et al., 2006 )

1 Fig. 5. Carbon-specific ingestion rates (d À , left axis) and diet composition (% Information regarding zooplankton feeding rates on P. triesti- biomass, right axis) of the studied larvae feeding on natural food assemblages under num is limited, although this species is considered as a non-toxic the harmful phytoplankton bloom conditions. food suitable for some zooplankton ( da Costa et al., 2005 ). The

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Fig. 6. Relationships between ingestion rates of each prey type and biomass concentration.

interactions between zooplankton grazers and H. akashiwo seem to However, in agreement with our results for meroplanktonic larvae, be species-specific. Some species of copepods (e.g. Acartia H. akashiwo is accepted as food by some species of copepods (e.g. hudsonica , A. tonsa —Tomas and Deason, 1981 ), rotifers (e.g. Acartia omorii —Uye and Takamatsu, 1990 ; Schmackeria inopinus — Synchaeta cecilia —Egloff, 1986 ) and tintinnids ( Favella sp.— Verity Yu et al., 2010 ), and rotifers (e.g. Brachionus plicatilis —Xie et al., and Stoecker, 1982 ) show no or little feeding on H. akashiwo . 2008 ). Although in some cases feeding on H. akashiwo results in

Table 6 Potential trophic impact (% biomass of the standing stock grazed daily) by the studied larvae upon the different prey during the harmful phytoplankton bloom. The last column indicates the total trophic impact by the studied larvae as a whole. HNF: heterotrophic nanoflagellates; PNF: phototrophic nanoflagellates except Heterosigma akashiwo ; Dinoflag.: dinoflagellates except Prorocentrum triestinum.

Prey Polychaeta larvae Echinoderm larvae Cirripede larvae Bivalve larvae Gastropod larvae All larvae

HNF 0.18 0.00 0.00 0.18 0.10 0.46 PNF 0.34 0.00 0.20 0.23 0.14 0.91 Diatoms 0.00 0.12 0.00 0.00 0.00 0.12 H. akashiwo 0.28 0.10 0.58 0.18 0.16 1.29 Dinoflag. 0.00 0.14 0.49 0.15 0.12 0.90 Ciliates 0.00 0.11 0.38 0.09 0.09 0.67 P. triestinum 0.00 0.12 0.87 0.00 0.00 0.99

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Fig. 7. Electivity index ( E*) of the studied meroplanktonic larvae feeding on natural food assemblages under harmful phytoplankton bloom conditions.

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3 low ingestion efficiencies ( Xie et al., 2008; Yu et al., 2010 ), the 50 mg C m À (Campbell et al., 2005 )]. Since bloom-forming specific ingestion rates observed in this study were in the range of phytoplankton species were accepted as food by the meroplankton those commonly found for meroplankton feeding on non-toxic larvae in the present study, the small trophic impact may mainly prey ( White and Roman, 1992; Hansen et al., 1997 ). be due to low grazer abundance. In conclusion, meroplanktonic Prey selectivity by zooplankton may be the result of differential larvae seem to have a minimal effect on H. akashiwo /P. triestinum vulnerability of prey species (passive selection, e.g. size-based blooms on the west coast of Vancouver Island. selection) or of choice exercised by the predator in accepting or rejecting a prey type (active selection). In contrast to other Acknowledgments zooplankton species that actively reject H. akashiwo as food (Taniguchi and Takeda, 1988; Uye and Takamatsu, 1990 ), the This work is based on research performed at the Bamfield larvae examined in this study selectively ingested this species. Marine Science Center (Vancouver Island). We thank Lluisa Cross, However, this selection may due to the high abundance of this prey Marta Estrada, and Santiago Fraga for their help in the identifica- (passive selection) instead of an active selection. Passive selection tion of the bloom forming-phytoplankton species. Thanks to Irene is frequently a size-based selection that could be explained partly Forn and Jose ´ Manuel Fortun˜o for their help in obtaining the by the morphology of the predator’s filtering structures ( Boyd, fluorescence and electronic microscope images, respectively. We 1976 ). The size of H. akashiwo is within or at the upper limit would also like to thank Arancha Lana for her help in the obtaining (depending on larvae type) of the spectrum of particle sizes of data from satellite images and the Ocean Biology Processing commonly ingested by meroplanktonic larvae ( Rassoulzadegan Group (Code 614.2) at the GSFC, Greenbelt, MD 20771, for the and Fenaux, 1979; Hansen, 1991a; Raby et al., 1997; Jeong et al., production and distribution of the ocean color data. A Ph.D. 2004; Vargas et al., 2006; Hansen et al., 2010 ). However, P. fellowship to RA (BES-2005-7491) from the Spanish Ministry of triestinum seems to be too large to be captured by veliger and Science and Innovation (MICINN) and a TRU CUEF award to AMM trochophore larvae, whereas the available diatom species may be funded this work. The research was also supported by MICROROL too thin to be efficiently captured by cirripede nauplii and too long (CTM2004-02775) research project from MICINN to RA and an to be ingested by veliger and trochophore larvae. In agreement NSERC Discovery grant to LAG.[SS] with other studies for some zooplankton ( Parsons et al., 1967; Raby et al., 1997; Turner and Borkman, 2005 ), when prey were of the References appropriate size and were not actively avoided, the diet composi- tion was concentration dependent. Biochemical composition (food Almeda, R., Pedersen, T.M., Jakobsen, H.H., Alcaraz, M., Calbet, A., Hansen, B.W., quality) plays an important role in active food selection by 2009. Feeding and growth kinetics of the planktotrophic larvae of the spionid polychaete Polydora ciliata (Johnston). J. Exp. Mar. Biol. Ecol. 382, 61–68. zooplankton ( Gallagher, 1988; Paffenho¨fer and Lewis, 1990 ). Some Almeda, R., Augustin, C.B., Alcaraz, M., Calbet, A., Saiz, E., 2010. Feeding rates and authors have proposed that the ability to reject H. akashiwo by gross growth efficiencies of larval developmental stages of Oithona davisae some zooplankters is chemically mediated ( Uye and Takamatsu, (Copepoda, Cyclopoida). J. Exp. Mar. Biol. Ecol. 387, 24–35. Anderson, D.M., 1989. Toxic algal blooms: a global perspective. In: Okaichi, T., 1990 ). However, these mechanisms and the intra- and extracellu- Anderson, D.M., Nemoto, T. (Eds.), Red Tides: Biology, Environmental Science lar compounds have not been fully elucidated (see below). and Toxicology. Elsevier, New York, pp. 11–16. Picoplankton concentrations significantly increased in all our Anderson, D.M., Lindquist, N.L., 1985. Time-course measurements of phosphorus depletion and cyst formation in the dinoflagellate Gonyaulax tamarensis Lebour. experimental treatments, suggesting a positive effect of these J. Exp. Mar. Biol. Ecol. 86, 1–13. larvae on picoplankton growth. The mechanism responsible for Anderson, D.M., 1995. The Ecology and Oceanography of Harmful Algal Blooms. A this effect is not clear. However, the larvae used in this study may National Research Agenda. Woods Hole Oceanographic Institution, Woods Hole, MA. have removed other potential grazers of picoplankton (e.g. ciliates, Andreu, P., Duarte, C.M., 1996. Zooplankton seasonality in Blanes Bay (northwest dinoflagellates, HNF— Hansen, 1991b; Sherr and Sherr, 2002 ), Mediterranean). In: Duarte, C.M. (Ed.), Seasonality in Blanes Bay—A Paradigm of resulting in relaxed protozoan grazing pressure on these small the NW Medlterranean Littoral. Publ. Espec. lnst. Esp. Oceanogr. 22. pp. 47–54. prey, in turn, overshadowing the effects of meroplanktonic larval Anger, K., Anger, V., Hagmeier, E., 1986. Laboratory studies on larval growth of Polydora ligni , Polydora ciliata , and Pygospio elegans (Polychaeta, Spionidae). grazing. Although some of the meroplanktonic larvae that we have Helgol. 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Taxes d'alimentació i abundància de larves planctòniques d'invertebrats marins sota condicions de proliferació d'algues nocives enfront de l’Illa de Vancouver.

Rodrigo Almeda, Amber Messmer , Nagore Sampedro, Louis A. Gosselin. Article publicat a Harmful algae (2011, 10: 194-206)

Les interaccions entre el fitoplàncton tòxic i els seus predadors potencials són aspectes poc entesos de l'ecologia de les proliferacions d'algues nocives. En aquest estudi, es van determinar les taxes d'alimentació, la selecció de preses i l'impacte tròfic de diferents larves planctòniques d'invertebrats marins sobre la proliferació natural d’ Heterosigma akashiwo i Prorocentrum triestinum que va tenir lloc en la costa oest de la illa de Vancouver al juliol del 2006. A més, es van estimar l'abundància, la biomassa i la composició del zooplàncton abans i durant la proliferació d'algues nocives. Els experiments d'alimentació es van realitzar amb larves de poliquet ( Serpula columbiana ), d'equinoderm ( Stronglyocentratus purpuratus ) i de cirrípede ( Balanus crenatus ) obtingudes mitjançant cultius de laboratori, i amb larves de bivalves i gasteròpodes recol·lectades a la zona d'estudi mitjançant arrossegaments amb xarxes de plàncton. Mentre que totes les larves es van alimentar amb H. akashiwo , només els nauplis de cirrípede i larves d'equinoderm es van alimentar amb P. triestinum . H. akashiwo va ser el component principal en la dieta de totes les larves (> 64%). Es va observar una relació positiva entre la disponibilitat de la presa en els assemblatges d'aliment i la seva contribució a la dieta de totes les larves. L'impacte tròfic potencial de les larves meroplanctòniques sobre les espècies fitoplanctòniques responsables de la proliferació va ser baix (<1.5%). Després de 48 hores d'incubació, la ingestió de les espècies de fitoplàncton que van formar la proliferació no va tenir aparentment cap efecte advers sobre els predadors estudiats. Per contra, l'abundància de larves planctòniques i un altre zooplàncton en el camp va disminuir de manera contínua a llarg de la progressió de la proliferació, amb pèrdues de fins a un 75% en comparació a la seva abundància abans de la proliferació. La presència d' H. akashiwo va afectar negativament a l'abundància de larves meroplanctòniques, malgrat l'eficient predació d'aquestes larves. Per tant, la pressió per depredació va ser reduïda, la qual cosa probablement va contribuir al creixement i la persistència de la proliferació. La reducció de l'abundància de larves meroplanctòniques i un altre zooplàncton associada amb la proliferació d' H. akashiwo podria tenir un impacte potencial sobre el reclutament bentònic i les transferències d'energia a nivells tròfics superiors a les xarxes tròfiques marines.

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Resumen del artículo III – Article III summary– Spanish version

Tasas de alimentación y abundancia de larvas planctónicas de invertebrados marinos bajo condiciones de proliferación de algas nocivas frente a la Isla de Vancouver

Rodrigo Almeda, Amber Messmer , Nagore Sampedro, Louis A. Gosselin. Artículo publicado en Harmful algae (2011, 10: 194-206)

Las interacciones entre el fitoplancton tóxico y sus predadores potenciales son aspectos poco entendidos de la ecología de las proliferaciones de algas nocivas. En este estudio, se determinó las tasas de alimentación, la selección de presas y el impacto trófico de diferentes larvas planctónicas de invertebrados marinos sobre la proliferación natural de Heterosigma akashiwo y Prorocentrum triestinum que tuvo lugar en la costa oeste de la Isla de Vancouver en julio del 2006. Además, se estimó la abundancia, la biomasa y la composición del zooplancton antes y durante la proliferación de algas nocivas. Los experimentos de alimentación se realizaron con larvas de poliqueto ( Serpula columbiana ), de equinodermo ( Stronglyocentratus purpuratus ) y de cirripedo ( Balanus crenatus ) obtenidas mediante cultivos de laboratorio, y con larvas de bivalvos y gasterópodos colectadas en la zona de estudio mediante arrastres con redes de plancton. Mientras que todas las larvas se alimentaron de H. akashiwo, sólo los nauplios de cirrípedo y larvas de equinodermos se alimentaron de P. triestinum. H. akashiwo fue el componente principal en la dieta de todas las larvas (> 64%). Se observó una relación positiva entre la disponibilidad de la presa en los ensamblajes de alimento y su contribución a la dieta de todas las larvas. El impacto trófico potencial de las larvas meroplanctónicas sobre las especies fitoplanctónicas responsables de la proliferación fue bajo (<1.5%). Después de 48 horas de incubación, la ingestión de las especies de fitoplancton que formaron la proliferación no tuvo aparentemente ningún efecto adverso sobre los predadores estudiados. Por el contrario, la abundancia de larvas planctónicas y otro zooplancton en el campo disminuyeron de manera continua a largo de la progresión de la proliferación, con pérdidas de hasta un 75% en comparación con su abundancia antes de la floración. La presencia de H. akashiwo afectó negativamente a la abundancia de larvas meroplanctónicas, a pesar de la eficiente predación de estas larvas. Por lo tanto, la presión por depredación se redujo, lo que probablemente contribuyó al crecimiento y la persistencia de la proliferación fitoplanctónica. La reducción en la abundancia de larvas meroplanctónicas y otro zooplancton en relación con la proliferación de H. akashiwo podría tener un impacto potencial sobre el reclutamiento bentónico y las transferencias de energía a niveles tróficos superiores en las redes tróficas marinas.

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Ecophysiology of early developmental stages of the copepod Oithona davisae

Effects of temperature and food concentration on survival, development and growth rates of naupliar stages of Oithona davisae (Copepoda, Cyclopoida)

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Vol. 410: 97–109, 2010 MARINE ECOLOGY PROGRESS SERIES Published July 14 doi: 10.3354/meps08625 Mar Ecol Prog Ser

Effects of temperature and food concentration on the survival, development and growth rates of naupliar stages of Oithona davisae (Copepoda, Cyclopoida)

Rodrigo Almeda*, Albert Calbet, Miquel Alcaraz, Lidia Yebra, Enric Saiz

Institut de Ciències del Mar (CSIC), P. Marítim de la Barceloneta 37–49, 08003 Barcelona, Spain

ABSTRACT: Oithona spp. are probably the most abundant and ubiquitous copepods in the world’s oceans. However, knowledge of their development and growth rates is scarce compared to that of calanoid copepods. In the present laboratory study, we determined the survival, development and growth rates of the naupliar stages of Oithona davisae under different temperature regimes and food concentrations. Naupliar survival was reduced to approximately 60% at the lowest food concentra- tion tested (11 µg C l –1 after 7 d at 20°C). The development of O. davisae nauplii was equipropor- tional, but not isochronal. Food concentrations required for maximum development and growth rates –1 were 56 and 87 µg C l , respectively. The Q10 values for development and growth depended on the temperature range. O. davisae nauplii showed similar developmental times, but lower growth rates and food requirements than values reported in the literature for calanoid copepods. We suggest that these differences may help to explain the ubiquity of Oithona spp. in oceanic environments.

KEY WORDS: Nauplii · Oithona davisae · Growth · Development · Survival · Food · Temperature

Resale or republication not permitted without written consent of the publisher

INTRODUCTION laboratory studies have shown that water tem- perature and food concentration are the most impor- Copepod nauplii are under-studied components of tant environmental factors influencing development, plankton communities, even though they are the most growth and survival of copepods (see reviews by abundant metazoans on the planet (Björnberg 1986, Huntley & Boyd 1984, Huntley & Lopez 1992, Hirst & Fryer 1986) and the main prey for many fish larvae of Lampitt 1998, Hirst & Kiørboe 2002). However, most commercially important species (Last 1980). The lack studies have focused on adults or late copepodites of information about naupliar ecology is partially be- (Hart 1990, Hirst & Bunker 2003), while naupliar life cause they have been under-sampled by conventional stages have received less attention. Therefore, infor- methods, such as plankton nets of 200 µm mesh (Alca- mation about copepod naupliar ecophysiology is cru- raz 1977, Calbet et al. 2001, Gallienne & Robins 2001). cial in order to gain a better understanding of the role With the use of appropriately sized plankton nets, nau- of zooplankton in marine biogenic fluxes. From an plii have frequently been found to outnumber late economic perspective, copepod nauplii are preferred copepodites and adults by several orders of magnitude and nutritious food sources for many farm-raised (Calbet et al. 2001, Turner 2004). They can also some- marine fish and shrimp larvae (Hernández-Molejón & times represent a comparable or higher fraction of Álvarez-Lajonchère 2003 and references therein). copepod biomass than older life stages (Castellani et Therefore, knowledge about the food requirements al. 2007). and experimental conditions for optimal growth and Knowledge of copepod life history, such as larval production of nauplii in the laboratory may be useful survival, development and growth rates, under differ- for aquaculture operations. ent environmental conditions is essential to compre- Among marine planktonic copepods, the genus hending their population dynamics. Many field and Oithona (Cyclopoida) is probably the most abundant

*Email: [email protected] © Inter-Research 2010 · www.int-res.com

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and ubiquitous copepod in the world’s oceans (Galli- ml –1 , equivalent to 11, 44, 88,132 and 265 µg C l –1 , re- enne & Robins 2001). Nevertheless, knowledge about spectively). The O. marina volume was converted to C the biology and ecology of these small copepods is lim- content using a conversion factor of 0.123 pg C µm –3 ited compared to the vast body of research devoted to provided by Pelegri et al. (1999). The O. marina cul- larger calanoid copepods (Paffenhöfer 1993, Tu rner tures used in our experiments were not fed the day 2004). Information is available on Oithona spp. devel- before use, to ensure that the dinoflagellate depleted opment and fecundity (Sabatini & Kiørboe 1994, Uye & all the Rhodomonas salina and that only O. marina was Sano 1995, Peterson 2001, Castellani et al. 2005a). offered as prey. The absence of R. salina in stock bot- However, studies are scarce on growth rates of Oithona tles was verified with a Coulter Multisizer particle spp. nauplii as a function of environmental variables, counter. Nauplii were incubated for 7 d at 20 ± 1°C in a such as food concentration and temperature (Sabatini & temperature-controlled room under a 12 h light:12 h Kiørboe 1994, Hopcroft & Roff 1998). To partially fill this dark cycle. Time was measured relative to the starting knowledge gap, we have examined the effects of tem- time of incubations of egg-bearing females. perature and food concentration on survival, develop- Food concentrations were checked daily using a ment and growth rates of Oithona davisae nauplii in the Coulter Multisizer III particle counter. Variations in laboratory. This species inhabits eutrophic embay- food concentrations between daily adjustments were ments and may occasionally dominate the copepod frequently <20% and never exceeded 40% of the cor- community (Uye & Sano 1995). It is indigenous to west- responding food level. Each experimental container ern Pacific, coastal areas, but is also an invasive species was sampled every 24 h by filtering an aliquot of water along the United States west coast (Ferrari & Orsi 1984), through a 32 µm sieve. This isolated at least 200 indi- southern Chilean coast (Hirakawa 1988) and the Span- viduals for subsequent counting, measuring and stag- ish Mediterranean (Saiz et al. 2003). ing. Naupliar samples were preserved with Lugol’s solution. The original water volume in the experimen- tal containers was kept constant by daily adjustments MATERIALS AND METHODS with either fresh Oxyrrhis marina from stock cultures or alternatively by adding filtered (0.2 µm) seawater. Experimental organisms. Oithona davisae speci- A second experiment was conducted to examine mens came from a continuous culture maintained in the influence of temperature on survival, development our laboratory since October 2000 (Saiz et al. 2003). and growth of Oithona davisae naupliar stages (i.e. Specifically, specimens were grown in 20 l methacryl- temperature-effects experiment). Five temperatures ate tanks at 20 ± 1°C in a room maintained at constant were tested (12, 16, 20, 24 and 28°C) under satiating temperature and under a 12 h light:12 h dark cycle. food concentrations (>2000 cells ml –1 ). Approximately Copepod cultures were fed ad libitum a suspension of 8000 nauplii from each temperature tested were the heterotrophic dinoflagellate Oxyrrhis marina placed in 2 l Pyrex glass bottles. These individuals (equivalent spherical diameter [ESD] = 15 µm). O. were fed Oxyrrhis marina for 7 d under a 12 h light: marina were fed the cryptophyte Rhodomonas salina 12 h dark cycle. Temperatures were maintained at (ESD = 8 µm). Prey sizes were measured by a Coulter ±0.2°C of the desired temperature using water-baths Multisizer III particle counter (Beckman Coulter). controlled by thermoregulators. Daily food adjust- To obtain cohorts of nauplii, we removed adults ments were made to maintain food satiation during (including egg-bearing females) from the stock culture the experiment. All other procedures were the same with a 132 µm sieve and placed them in a new tank as those described for the food-effects experiment. where they were fed Oxyrrhis marina ad libitum (i.e. Body length–weight relationships. To estimate >3000 cells ml –1 , equivalent to >660 µg C l –1 ). Adults weight-specific growth rates (see below), we deter- were removed with a 100 µm sieve after 20 h, and the mined the body length–weight relationships of Oitho- hatched nauplii were transferred to a new tank. To iso- na davisae nauplii and copepodites. Samples from late the dislodged egg sacs, we allowed them to settle naupliar cohorts were taken over the course of their for 2 h before siphoning the nauplii into new tanks. development. These samples were concentrated using Experimental design and general procedures. We 37 and 60 µm sieves for nauplii and copepodites, re- examined the survival, development and growth of spectively, and rinsed thoroughly in autoclaved and fil- Oithona davisae naupliar life stages as they relate to tered (0.2 µm) seawater. Three aliquots containing food concentrations (i.e. food-effects experiment). A 1000 individuals each were filtered onto pre-inciner- cohort of nauplii was split into aliquots of about 4000 ated (450°C, 6 h) glass-fibre filters (GF/A grade), after nauplii each that were placed into 10 l methacrylate which the filters were dried (60°C, 24 h) and stored in tanks containing Oxyrrhis marina suspensions at dif- a vacuum desiccator (with silica gel) for further analy- ferent concentrations (50, 200, 400, 600 and 1200 cells sis of organic C and N content. Three additional ali-

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quots of ~100 individuals per replicate) were fixed in a to moult to the following stage (Landry 1983). The 4% borax-buffered formaldehyde solution for the MDT for a given developmental stage was estimated counting of larvae and length determinations. The C by fitting a sigmoidal Hill function to the cumulative and N content of the larvae were determined by a proportion of that stage over time: Perkin-Elmer 2400 CHN Elemental Analyzer. Total lar- y = a tb/(MDT b + tb) (2) va e per aliquot were counted with an inverted micro- × scope (40 × magnification). Body sizes ( L, µm) of O. where y is the cumulative percentage of each stage, t is davisae larvae (copepodites: prosome length; nauplii: the time (d) since females were separated, a is the total length) were measured by capturing digital pic- highest y-axis value (i.e. 100% when the entire cohort tures of at least 50 individuals under a microscope is in early developmental stages), MDT is the t value

(100 × magnification) and using ImageJ software for that produces 50% of the y-axis value and b is the quantitative analysis. Relationships between body shape coefficient. weight (measured as C and N mass) and body size for Stage-specific development rates ( D, d –1 ) were calcu- both nauplii and copepodites were calculated by lated as the inverse of the MDT. The stage duration was regression analysis. estimated as the difference in MDT of 2 consecutive Estimates of survival, development and growth developmental stages. The equiproportional rule of rates. Larval survival ( S) in each incubation container copepods (i.e. the duration of a given life-history stage was estimated daily by subsampling as described occupies a constant proportion of the total or egg de- above and applying the following calculation: velopmental time regardless of temperature; Corkett 1984) was tested considering the proportion of time that S (%) = [( N V /V + N )/ N ] 100 (1) x x × tank aliqout R i × each stage occupied with respect to the total naupliar where Nx is the current number of live larvae esti- development time (time between egg hatching and mated from the aliquot at Day x, NR is the number of moult into the first copepodite stage [CI]). larvae removed on previous days, N i is the number of For specific growth rate determinations, the body initial larvae and Vtank and Valiqout are the tank and lengths of 50 random individuals from each cohort aliquot volume, respectively. were measured daily, and their C weights were calcu- Samples were counted using an inverted microscope lated by applying body size to C content relationships. at 40 × magnification, and the developmental stages of Additionally, we measured 30 individuals per treat- at least 40 individuals per incubation container were ment at each developmental stage (from Nauplus determined with microscopy at 100 × magnification ac- Stage I to VI, NI to NVI) to estimate the effects of tem- cording to Uchima (1979). Stage-specific median de- perature and food concentration on naupliar body velopment time (MDT) and stage duration (SD) were length. In all cases, body length was determined using calculated from observed changes in stage frequency ImageJ software, as mentioned previously. Weight- over time (see Table 1 for terminology and definitions). specific growth rates ( G, d –1 ) were calculated as the The stage-specific MDT was calculated as the time slope of regression lines relating the natural logarithm required for 50% of the individual organisms in culture of C biomass to incubation time.

Table 1. Terminology and units

Symbol Term Unit Explanation

MDT Median developmental time d Time at which 50% of the larvae in the culture has moulted to the following stage SD Stage duration d Difference in MDT of 2 consecutives stages D Developmental rates d –1 Inverse of MDT –1 Dmax Maximum development rate d From Ivlev’s equation W Weight ng L Length µm G Specific growth rates d –1 –1 Gmax Maximum specific growth rates d From Ivlev’s equation C Food concentration µg C l –1 –1 CD Satiating food concentration for development µg C l Food concentration required to achieve 95% of Dmax –1 CG Satiating food concentration for growth µg C l Food concentration required to achieve 95% of Gmax

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Temperature coefficient ( Q10 ) values for the temper- Survival ature-effects experiment were calculated as: Naupliar survival was high (>91%) at all food levels, Q = ( M /M )10/( T2 – T 1) (3) 10 2 1 except at the lowest food concentration (11 µg l –1 ),

where M2 and M1 are the rates of the studied process at where survivorship declined to 58% by the end of the temperatures T2 and T1 (°C), respectively. incubation (Fig. 2A). Regarding the temperature- All statistical analyses and regressions were con- effects experiment, naupliar survival was also high (85 ducted with SPSS 17.0 software. All curves were fitted to 93%) at all temperatures and decreased only by standard least-squares procedures using Sigma plot slightly with rising temperature (Fig. 2B). In both ex- 9.0 software. periments, differences between treatments started to be evident after the fifth incubation day (Fig. 2). We observed a slight increase in mortality in treatments RESULTS where nauplii underwent metamorphosis to the CI developmental stage (Fig. 2). Elemental composition (C, N) of Oithona davisae nauplii and copepodites Development rates The relationships between body weight ( W, organic C and N) and body length ( L, µm) of nauplii and cope- The cumulative abundance percentage of each nau- podites are shown in Fig. 1A, B. The fitted equations pliar stage over time at the different temperatures and were: food conditions is presented in Fig. 3. In all cases, these 2.14 2 data fitted the Hill functions well (p < 0.05). W (ng C) = 0.0021 × L , r = 0.96 for nauplii (4) Effects of food concentration. The cumulative per- W (ng N) = 7.47 10 –6 L2.92 , r 2 = 0.98 for nauplii (5) × × centage fits among the low-food treatments covered a W (ng C) = 0.0318 L1.61 , r 2 = 0.94 for copepodites (6) × wider range than the higher food concentrations (Fig. 1.58 2 W (ng N) = 0.0091 × L , r = 0.99 for copepodites (7) 3, right panels) because of the slower development at these food levels. Cohorts reached CI within the 7 d The C:N ratio decreased during larval development of incubation time at all food concentrations except the Oithona davisae (Fig. 1C). Nauplii showed gradual lowest. MDT for each naupliar stage decreased with decreases from 8.4 (NI to NII) to 5.4 (NV to NVI) in increasing food concentrations, leading to a decrease their C:N ratios with increasing age/size. In contrast, in the developmental stage duration (Table 2). Nau- C:N ratios in copepodites varied only slightly between pliar development was not isochronal, because the developmental stages, with an average value of 4.12. duration of the late naupliar stage, particularly NVI, The relationship between the C:N ratio and body was longer than other developmental stages. Naupliar length ( L, µm) of nauplii O. davisae is described by the development was nearly equiproportional at high food equation (Fig. 1C): –1 –1 levels ( ≥ 88 µg C l ). Development rates ( D, d ) related –0.74 2 –1 C:N = 239 × (1 + L) , r = 0.96 (8) to food concentration ( C, µg C l ) were fitted to the

Body length

Fig. 1. Oithona davisae . Relationships between body length (total length for nauplii and prosome for copepodites) and (A) body car- bon weight, (B) nitrogen weight and (C) the ratio of carbon to nitrogen (C:N) of the developmental stages. Error bars represent ±SE

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where a and α are constants (Corkett et al. 1986). The fitted Belehrádek’s func- tion for each naupliar stage and the applied temperature range are shown in Table 5. The equation parameter a increased gradually with the age of developmental stages (Table 5). Across

all temperatures tested, Q10 values for development ranged from approxi- mately 2.4 for NII and NIII stages (12 to 28°C) to 1.6 for CI stages (20 to 28°C). At temperatures <20°C, no individuals reached the CI developmental stage.

Within stages, Q10 values were temper- Fig. 2. Oithona davisae . Survival rates of early developmental stages reared for 7 d ature dependent; for example, the NII at (A) 5 food concentrations and (B) 5 temperatures. +: cohort was dominated by produced Q10 values of 4.41 at from 12 Naupliar Stage VI (NVI) and Copopodite Stage I (CI) (NVI–CI metamorphosis) to 20°C, but 1.19 at from 20 to 28°C. Using a common range of temperature

Table 2. Oithona davisae . Effect of food concentration on the median develop- (20 to 28°C) for all stages, Q10 values ment time (MDT, d) and stage duration (SD, d) of early larval stages at 20°C. NII increased from 1.2 for Stages NII and to NVI: Naupliar Stages II to VI; CI: Copepodite Stage I NIII to 1.6 for Stages NIV to CI.

Food concentration (µg C l –1 ) 11 44 88 132 265 MDT SD MDT SD MDT SD MDT SD MDT SD Body length and growth rates

NII 2.54 1.22 1.14 0.98 1.16 0.94 1.16 0.80 1.17 0.84 Effects of food concentration. Nau- NIII 3.76 1.54 2.12 0.81 2.09 0.88 1.96 0.97 2.01 0.89 pliar body length decreased at the low- NIV 5.30 2.93 0.94 2.97 0.79 2.94 0.75 2.90 0.85 NV 3.87 1.33 3.76 1.12 3.68 1.08 3.75 0.98 est food concentration (ANOVA, p < NVI 5.20 1.83 4.89 1.72 4.76 1.62 4.74 1.56 0.01; Tukey’s test, p < 0.01), with CI 7.03 6.60 6.39 6.30 reductions ranging from 3% in Stage NII to 15% in Stage NV (Fig. 5A). Growth rates were exponential and Ivlev’s equation (Fig. 4A): consistent across food levels, except at the lowest food

(– C ) concentration (Fig. 6A; ANCOVA, F = 107.34, p < D = D (1 – e β ) (9) max 0.01). Naupliar specific growth rates ( G, d –1 ) relative –1 –1 where Dmax is the maximum development rate (d ) and to food concentrations ( C, µg C l ) followed a satura-

β is a constant (rate at which development approaches tion curve (Ivlev’s equation) expressed by the function the maximum development rate). The parameters for the (Fig. 7A): fitted Ivlev’s function across different stages are pre- sented in Table 3. Food concentrations required to G = 0.296(1 – e (–0.034 C ) ), r 2 = 0.97 (11) achieve 95% of the maximum development rate ( CD, sa- tiating food concentration) ranged from 40 to 56 µg C l –1 , where 0.296 is the maximum growth rate (d –1 ) and depending on the naupliar stage (Table 3). 0.034 is a constant that indicates the rate at which Effects of temperature. Temperature had a clear growth approaches the maximum rate. The satiating effect on development times; MDT and all stage dura- food concentration ( CG) at which maximum naupliar tions decreased with rising temperatures (Fig. 3, growth became limited was 87 µg C l –1 . Table 4). Cohorts only reached CI within the 7 d in- Effects of temperature. Temperature had a signifi- cubation time at high temperatures (20 to 28°C). Simi- cant effect and was inversely correlated to body length lar to the food concentration experiments, naupliar (Fig. 5B; ANOVA, p < 0.05). In early stages (NII to development of Oithona davisae was equiproportional, NIV), body size differences were only significant but not isochronal. The functional relationships between the lowest and highest temperatures. For between temperature ( T, °C) and MDT (d –1 ) were well example, the body length for NIII individuals ranged described (p < 0.05) by Belehrádek’s function (Fig. 4B): from 114 µm at 12°C to 107 µm at 28°C (Tukey’s test, p < 0.05), with differences becoming more evident at –2.05 MDT = a(T – α) (10) later stages (NV to NVI) (Fig. 5B).

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Fig. 3. Oithona davisae . Cumulative abundance percentages of all animals that have not yet passed a given stage, plotted against time. Left panels: for food concentration treatments; right panels: for temperature treatments. Successive stages from Naupliar Stage I (NI) to Copepodite Stage I (CI) are indicated by different symbols. Hill functions (Eq. 2) were fitted to the data, including all 100 and 0% values, which for clarity are not shown in the graphs except one, immediately before and after the occurrence of a stage, respectively. Stages older than CI are not shown

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Table 3. Oithona davisae . Parameters of the Ivlev’s equation (Eq. 9) used to describe the relationship between food con- –1 –1 centration (µg C l ) and developmental rates ( Dmax and β, d ) –1 for early developmental stages. SD (µg C l ): the satiating food concentration for maximum development; r 2: coefficient of determination; SE: standard error. NII to NVI: Naupliar Stages II to VI; CI: Copepodite Stage I

2 Dmax (±SE) β (±SE) r SD

NII 0.87 (±0.02) 0.061 (±0.008) 0.96 49 NIII 0.49 (±0.05) 0.070 (±0.005) 0.98 43 NIV 0.34 (±0.04) 0.073 (±0.005) 0.99 41 NV 0.27 (±0.02) 0.074 (±0.008) 0.80 40 NVI 0.21 (±0.02) 0.056 (±0.004) 0.93 53 CI 0.16 (±0.02) 0.053 (±0.005) 0.88 56

DISCUSSION

Elemental composition (C, N)

For ecophysiology studies, such as ours and espe- cially for those examining growth, it is important to express data in biomass-specific units to facilitate com- parison across studies. It is also crucial to have con- current, accurate and reliable estimates of biomass, or at least robust body length–weight relationships, since using literature relationships may produce severe bias. Intra-specific differences, origin and condition of or- ganisms, previous food intake and the method of sam- ple preservation can result in different body length– weight relationships, especially when only longitudinal measurements are considered. For instance, the rela- Fig. 4. Oithona davisae . (A) Relationships between food con- tionships we measured between body length and C centration and development rates for early larval stages. The weight for Oithona davisae copepodites differed from fitted curves are described by Ivlev’s functions. (B) Relation- ships between temperature and median development time for observations of the same species by Uye & Sano (1998). early larval stages under satiating food conditions. The fitted The C:N ratios observed in Oithona davisae nauplii curves are described by Belehrádek functions are consistent with those ratios commonly reported for

Growth rates were exponential and Table 4. Oithona davisae . Effect of temperature on median development time dependent on temperature (Fig. 6A; (MDT, days) and stage duration (SD, d) of early larval stages under satiating ANCOVA, F = 13.81, p < 0.01). Relation- food conditions. NII to NVI: Naupliar Stages II to VI; CI: Copepodite Stage I ships between naupliar specific growth rates ( G, d –1 ) and temperatures ( T, °C) Temperature (°C) were established by the model (Fig. 7B): 12 16 20 24 28 MDT SD MDT SD MDT SD MDT SD MDT SD 2 log G = (0.053 × T) – 1.721, r = 0.87 (12) NII 4.12 2.91 1.90 1.64 1.26 0.70 1.18 0.59 1.09 0.51 Q values for growth declined with ris- 10 NIII 7.03 3.54 1.23 1.96 0.91 1.77 0.70 1.60 0.47 ing temperatures (e.g. at 12 to 24°C NIV 4.77 1.16 2.87 0.73 2.47 0.73 2.07 0.47 Q = 4.8; at 16 to 24°C Q = 2.8; at 20 to 10 10 NV 5.93 3.60 1.10 3.20 0.76 2.54 0.71 28°C Q10 = 1.6). The Q10 value was 3.6 NVI 4.69 1.50 3.96 1.11 3.25 0.96 across the range of temperatures tested CI 6.19 5.07 4.20 in the experiments (12 to 28°C).

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Table 5. Oithona davisae . Parameters of the Belehrádek function (Eq. 10) used to describe the relationship between median development time (MDT, d) and temperature ( T, °C) for early development stages. r 2: coefficient of determina- tion; SE: standard error; NII to NVI: Naupliar Stages II to VI; CI: Copepodite Stage I

2 a (±SE) α (±SE) r Temp. range (°C)

NII 650 (±231) 0.04 (±2.30) 0.94 12–28 NIII 1114 (±230) 0.8 (±1.4) 0.97 12–28 NIV 2423 (±893) –5.2 (±4.2) 0.94 16–28 NV 3181 (±1156) –5.8 (±4.3) 0.95 16–28 NVI 9721 (±1446) –21.4 (±3.2) 0.99 20–28 CI 11049 (±226) –18.6 (±0.4) 0.99 20–28

marine copepods (~3 to 14; Omori 1969, Postel et al. 2000). Since C:N ratios are related to lipid and protein ratios, the decrease in the C:N ratio observed with O. davisae development may be indicative of the gradual decrease in yolk lipids as development proceeds.

Survival

Mortality in early developmental stages strongly affects population dynamics among marine copepods (Peterson & Kimmerer 1994, Plourde et al. 2009). Stage- specific mortality depends on both intrinsic and exter- nal factors. Intrinsic factors that may affect early stage survival of copepods include shifts from yolk-related, Fig. 6. Oithona davisae . Time course of carbon biomass of early endogenous foods to exogenous food sources. This food larval stages in relation to (A) food concentration (µg C l –1 ) and shift may explain the high, early mortality observed (B) temperature. Error bars represent ±SE

Fig. 5. Oithona davisae. (A) Effect of food concentration on length of naupliar stages at 20°C. (B) Effect of temperature on length of naupliar stages under satiating food conditions

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Fig. 7. Oithona davisae . (A) Specific growth rates (±SE) of naupliar stages as a function of food concentration (µg C l –1 ). Fitted curves correspond to Ivlev’s equation (see ‘Results’). (B) Specific growth rates (±SE) of naupliar stages as a function of tempera- 2 ture. Fitted curves correspond to exponential models. The equations are: log G = (0.053 × T) – 1.721, r = 0.99, for temperatures 2 between 20 and 28°C (continuous line) and log G = (0.053 × T) – 1.721, r = 0.87 (dashed line), for the total range of temperature (12 to 20°C), where G is the specific growth rate (d –1 ), T is the temperature (°C) and r 2 is the correlation coefficient among some species (Trujillo-Ortiz & Arroyo-Ortega nauplii due to its morphology (i.e. optimal size; Saiz et 1991). However, we did not observe this type of early al. unpubl. data), biochemical composition (essential mortality in Oithona davisae , which is consistent with lipids; Klein Breteler et al. 1999), motility (Uchima & observations that Oithona spp. nauplii start to feed after Hirano 1986) and digestibility (without a theca). Previ- hatching (Uchima & Hirano 1986). Another intrinsic ously reported incidences of 100% mortality of O. da- factor affecting early mortality relates to the large visae nauplii at high food concentrations (Uchima & morphological and physiological changes associated Hirano 1986) are in complete disagreement with our re- with copepod development, particularly with the meta- sults. The high mortality observed by Uchima & Hirano morphosis from nauplii to copepodite (Epp & Lewis (1986) was probably a consequence of the poor nutri- 1980, Ferrari & Dahms 2007). This could be the cause of tive quality of the prey used in their experiments ( Du- increased mortality observed in our experiments during naliella spp.; Klein Breteler et al. 1999, Dahl et al. 2009). O. davisae transitions from NVI to CI, as occurs in other We did not observe any temperature-related effects, copepods (Lonsdale 1981). except for faster development at higher temperatures. Regarding external factors, in addition to predation This observation suggests an accelerated transition from and disease (virus, bacteria, parasites, etc.), copepod NVI to CI stages, along with its associated mortality. mortality is mainly affected by starvation and temper- ature. The following conditions can influence these factors: famine duration, lipid reserves, food quality Development and growth rates and developmental stage (Paffenhöfer 1971, Calbet & Alcaraz 1997, Ismar et al. 2008). Food concentrations Post-embryonic development in copepods can follow that induce relevant mortality in Oithona davisae nau- different patterns. Isochronal development (i.e. con- plii (~11 µg C l –1 ) were lower than those commonly re- stant stage duration across the entire lifespan) has ported for calanoid nauplii studied in laboratory set- been frequently reported in calanoid copepods (Miller tings (~36 to 100 µg C l –1 ; Paffenhöfer 1971, Berggreen et al. 1977, Klein Breteler et al. 1982) and in the cyclo- et al. 1988, Cook et al. 2007, Ismar et al. 2008). More- poid Oithona similis (Sabatini & Kiørboe 1994). How- over, mortality of O. davisae nauplii was only signifi- ever, isochronal development was not observed in O. cant after 5 d, suggesting that this species can with- davisae nauplii. We noted 2 differences in the duration stand relatively long periods of poor food conditions. of naupliar stages relative to other copepods. First, in As previously stated, food quality may influence nau- most calanoid copepods, pre-feeding stages are rela- pliar mortality during development (Paffenhöfer 1971). tively short, but first-feeding stages (NII or NIII) are The prey used in the present study ( Oxyrrhis marina) longer (Landry 1975, 1983). In contrast, early stages are considered a high-quality food for Oithona davisae (NII) and late stages (NIV and NV) of O. davisae nau-

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plii were of similar duration, which is probably be- higher (0.20 d –1 at 15°C; Sabatini & Kiørboe 1994). The cause feeding starts just after hatching. Second, O. smaller size of Oithona spp. would lead one to deduce davisae undergo long NVI stages, unlike other cope- higher weight-specific metabolic rates (Lynch 1977, pods in which NVI passes rapidly (Uchima 1979, Peter- Ikeda et al. 2001). However, the growth rates of Oitho- son 2001). Our results support the rule of equipropor- na spp. nauplii appear to be lower than those fre- tional development (Corkett 1984), as opposed to the quently reported for calanoids (0.27 to 0.50 d –1 ; Kiør- isochronal rule, which seems to be a pattern generally boe & Sabatini 1995, Calbet & Alcaraz 1997, Leandro found in copepod development (Miller et al. 1977, Hart et al. 2006) and those predicted from global models 1990, Peterson 2001). (Huntley & Lopez 1992, Hirst & Lampitt 1998, Hirst & As typically occurs in copepods (McLaren 1978), the Bunker 2003).

development time of Oithona davisae nauplii de- The influence of temperature ( Q10 ) on growth rates creased with rising temperatures. The naupliar devel- was similar to the pattern mentioned above for nau- opment time estimated by Uye & Sano (1998) based on pliar development (NI to CI). Consistent with the Ar-

theoretical assumptions (equiproportional rule) were rhenius equation, the Q10 is temperature dependent 2 d longer than those stemming from our experimental and decreases with rising temperatures. Therefore, the

results. At similar temperatures and under satiating typical application of uniform values of Q10 calculated food conditions, O. davisae development time (NI to across different temperature ranges can result in errors CI) is comparable to that of other marine cyclopoid and in determining temperature effects on physiological calanoid copepods, even at larger sizes (Hart 1990, rates. It is surprising that the relationship of growth Paffenhöfer 1993, Sabatini & Kiørboe 1994, Peterson rates to temperature did not follow the exponential 2001). This is consistent with previous studies (Hart model (i.e. Van’t Hoff-Arrhenius law). As Oithona da- 1990, Sabatini & Kiørboe 1994, Peterson 2001), which visae is a thermophilic species experiencing its highest concluded that copepod development times in the population densities during warm seasons (Uye & Sano presence of unlimited food resources were indepen- 1995, Nakane et al. 2008), we think that the lower tem- dent of adult body size. Therefore, despite the differ- peratures used in our experiments (12 and 16°C) might ences in size between cyclopoid and calanoid cope- be sub-optimal, resulting in deviation from the expo- pods, generation times and the potential for nential model. The exponential model seems to fit best recruitment might be similar (Peterson 2001). How- when high experimental temperatures are considered ever, O. davisae development levels off at food con- (Fig. 7). centrations (~50 µg C l –1 at 20°C) lower than those commonly reported for coastal calanoid nauplii (>200 µg C l –1 at 15°C; Klein Breteler et al. 1982). Ecological implications While further confirmation is needed, this observation suggests a competitive advantage of O. davisae over Laboratory experiments under controlled conditions other coastal copepods under low food conditions. are a fundamental tool for understanding the effects Conversely, O. davisae showed higher satiating food of environmental variables on the distribution and concentrations for growth than for development, which activity of marine zooplankton. Nonetheless, caution is was consistent with findings for other copepod species required when extrapolating laboratory results to (Ban 1994, Campbell et al. 2001). Hence, nauplii could the field (e.g. bottle effects, crowding and lack of maintain maximum developmental rates at food con- turbulence). centrations that were limiting for somatic growth (i.e. In addition to factors such as predation and salinity, development may be less sensitive than growth to food development and growth rates of wild copepods can concentrations). This suggests that development and, be affected by both temperature and food availability. consequently, recruitment may be prioritised over Development and growth of marine copepods are fre- somatic growth for some copepod species. quently food-limited in natural settings (Hirst & Bun- It is difficult to compare our estimated naupliar ker 2003, Saiz & Calbet 2007). The degree of food lim- growth rates with those from the literature, because itation can be highly variable among groups/species available data are scarce and mostly examine cala- and across life-history stages (Hirst & Bunker 2003, noids (Calbet & Alcaraz 1997, Campbell et al. 2001, Finlay & Roff 2006). Food concentrations producing Lee et al. 2003, Leandro et al. 2006). In the case of satiation in Oithona davisae nauplii were lower than in oithonids, our understanding is that naupliar growth calanoid nauplii studied in laboratories (Klein Breteler rates under saturating food conditions have only been et al. 1982, Berggreen et al. 1988, Calbet & Alcaraz reported for Oithona similis examined under labora- 1997, Leandro et al. 2006). Therefore, we expect that tory conditions (Sabatini & Kiørboe 1994). These re- O. davisae would achieve maximum growth and de- sults were similar to our observations, albeit slightly velopment rates in nature with less food than calanoid

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nauplii. The satiating food concentration for the growth vironments, such as oceans, with substantial predation and development of O. davisae nauplii is relatively low (Verity & Smetacek 1996), reduced size and low motil- given the high food concentrations (~2 to 50 µg chloro- ity may offer a competitive advantage that reduces phyll a l–1 ) of eutrophic coastal habitats (Uye & Sano predation risk. In summary, our study suggests that 1995, Gifford et al. 2007, Nakane et al. 2008). This food Oithona spp. in marine ecosystems may have an concentration is equivalent to 94 to 1410 µg C l –1 , using advantage over other copepods because of differences an average conversion factor of 47 (Riemann et al. in food requirements that maximise survival and rates 1989). In addition to food quantity, food quality (e.g. of development and growth. size, palatability, motility, nutritional value) should be considered when expressing food limitation in nature. Acknowledgements. We thank P. Jimenez, E. Velasco and O. davisae nauplii and adults only feed on relatively J.Movilla for their help in maintaining the algae and copepod large and motile prey (Uchima & Hirano 1986, Tsuda & cultures. This work was funded by a PhD fellowship to R.A. Nemoto 1988, Uchima 1988, Broglio et al. 2004, (BES-2005-7491) from the Spanish Ministry of Education and Science and supported by MICROROL (CTM2004-02775), Atienza et al. 2006, Henriksen et al. 2007), which sub- OITHONA (CTM2007-60052), Intramural (200630I226) and stantially reduces the amount of food available for PERFIL (CTM2006-12344) research projects from the same ingestion. During warm seasons, the biomass of poten- ministry. tial prey in some eutrophic waters inhabited by O. davisae is frequently close to their critical food concen- LITERATURE CITED tration for development (~50 µg C l –1 ; Gifford et al. 2007 and >500 cells ml –1 ; Nakane et al. 2008). In addi- Alcaraz M (1977) Muestreo cuantitativo de zooplankton: tion, field naupliar growth rates reported for other análisis comparativo de la eficacia de mangas y botellas en un sistema estuárico. Investig Pesq 41:285–294 Oithona species (Hopcroft & Roff 1998) are similar to ➤ Atienza D, Calbet A, Saiz E, Alcaraz M, Trepat I (2006) those reported for O. davisae nauplii under satiating Trophic impact, metabolism, and biogeochemical role of food conditions and at similar temperatures (0.49 d –1 at the marine cladoceran Penilia avirostris and the co-domi- 28°C). The low satiating threshold exhibited by nant copepod Oithona nana in NW Mediterranean coastal waters. Mar Biol 150:221–235 Oithona spp. nauplii would provide a competitive ➤ Ban S (1994) Effect of temperature and food concentration on advantage over other copepods in these environments post embryonic development, egg production and adult and under oligotrophic conditions. body size of calanoid copepod Eurytemora affinis . J Plank- Many hypotheses have been offered as to why oitho- ton Res 16:721–735 nids are so abundant and ubiquitous relative to calano- ➤ Berggreen U, Hansen B, Kiørboe T (1988) Food size spectra, ingestion and growth of the copepod Acartia tonsa during ids (Lampitt 1978, Lampitt & Gamble 1982, Paffenhöfer development: implications for determination of copepod 1993, Castellani et al. 2005b). Copepod life strategies production. Mar Biol 99:341–352 are adapted to food fluctuations. For example, when Björnberg TKS (1986) The rejected nauplius: a commentary. primary production declines, many calanoid species Syllogeus 58:232–236 ➤ Broglio E, Saiz E, Calbet A, Trepat I, Alcaraz M (2004) Trophic produce resting eggs that accumulate in sediments impact and prey selection by crustacean zooplankton on and hatch prior to the onset of the spring bloom. Oitho- the microbial communities of an oligotrophic coastal area nids do not produce resting eggs and must adapt to tol- (NW Mediterranean Sea). Aquat Microb Ecol 35:65–78 erate periods of low food availability and to maintain ➤ Calbet A, Alcaraz M (1997) Growth and survival rates of early their populations throughout the year. developmental stages of Acartia grani (Copepoda: Cala- noida) in relation to food concentration and fluctuations in The success of oithonids over calanoids has been at- food supply. Mar Ecol Prog Ser 147:181–186 tributed to their wide array of prey preferences (Lam- ➤ Calbet A, Landry M, Scheinberg R (2000) Copepod grazing in pitt 1978, Uchima 1988, González & Smetacek 1994, a subtropical bay: species-specific responses to a midsum- Calbet et al. 2000) and low metabolic rates (Lampitt & mer increase in nanoplankton standing stock. Mar Ecol Prog Ser 193:75–84 Gamble 1982, Paffenhöfer 1993, Castellani et al. 2005). ➤ Calbet A, Garrido S, Saiz E, Alcaraz A, Duarte CM (2001) Differences in the motion/feeding behaviour may con- Annual zooplankton succession in coastal NW Mediter- tribute to differences in metabolic requirements be- ranean waters: the importance of the smaller size frac- tween calanoids and oithonids. Our study shows that tions. J Plankton Res 23:319–331 development rates of Oithona spp. nauplii are similar ➤ Campbell RG, Wagner MM, Teegarden GJ, Boudreau CA, Durbin EG (2001) Growth and development rates of the to those of calanoid nauplii, but growth rates of Oitho- copepod Calanus finmarchicus reared in the laboratory. na spp. nauplii are lower than those of calanoid nau- Mar Ecol Prog Ser 221:161–183 plii. In addition, naupliar development of Oithona spp. ➤ Castellani C, Irigoien X, Harris RP, Lampitt RS (2005a) Feed- saturates at lower concentrations. Thus, Oithona spp. ing and egg production of Oithona similis in the North Atlantic. Mar Ecol Prog Ser 288:173–182 development is completed sooner and results in ➤ Castellani C, Robinson C, Smith T, Lampitt RS (2005b) Tem- smaller sized individuals than that of calanoid cope- perature affects respiration rate of Oithona similis . Mar pods inhabiting oligotrophic regions. Moreover, in en- Ecol Prog Ser 285:129–135

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Mar Biol 3:4–10 lence on copepods: the case of Oithona davisae . Limnol ➤ Paffenhöfer GA (1971) Grazing and ingestion of nauplii, cope- Oceanogr 48:1304–1311 podids and adults of the marine planktonic copepod Cala- Trujillo-Ortiz A, Arroyo-Ortega JE (1991) Analysis of mortal- nus helgolandicus . Mar Biol 11:286–298 ity and expectation of life of Acartia californiensis Trinast ➤ Paffenhöfer GA (1993) On the ecology of marine cyclopoid (Calanoid: Copepod) under laboratory conditions. Cienc copepods (Crustacea, Copepoda). J Plankton Res 15:37–55 Mar 17:11–18 ➤ Pelegri S, Dolan J, Rassoulzadegan F (1999) Use of high tem- ➤ Tsuda A, Nemoto T (1988) Feeding of copepods on natural perature catalytic oxidation (HTCO) to measure carbon suspended particles in Tokyo Bay, Japan. J Oceanogr Soc content of microorganisms. Aquat Microb Ecol 16:273–280 Jpn 44:217–227 ➤ Peterson WT (2001) Patterns in stage duration and develop- Turner JT (2004) The importance of small planktonic cope- ment among marine and freshwater calanoid and cyclopoid pods and their roles in pelagic marine food webs. Zool copepods: a review of rules, physiological constraints, and Stud 43:255–266 evolutionary significance. Hydrobiologia 453/454:91–105 Uchima M (1979) Morphological observation of developmen- Peterson WT, Kimmerer WJ (1994) Processes controlling tal stages in Oithona brevicornis (Copepoda, Cyclopoida). recruitment of the marine calanoid copepod Temora longi- Bull Plankton Soc Japan 26:59–76 cornis in Long Island Sound: egg production, egg mortality, ➤ Uchima M (1988) Gut content analysis of neritic copepods and cohort survival rates. Limnol Oceanogr 39:1594–1605 Acartia omorii and Oithona davisae by a new method. Mar Plourde S, Maps F, Joly P (2009) Mortality and survival in Ecol Prog Ser 48:93–97 early stages control recruitment in Calanus finmarchicus . Uchima M, Hirano R (1986) Food of Oithona davisae (Cope- J Plankton Res 31:371–388 poda: Cyclopoida) and the effect of food concentration at Postel L, Fock H, Hagen W (2000) Biomass and abundance. first feeding on the larval growth. Bull Plankton Soc Japan In: Harris RP et al. (eds) ICES zooplankton methodology 33:21–28 manual. Academic Press, San Diego, p 83–192 ➤ Uye S, Sano K (1995) Seasonal reproductive biology of the ➤ Riemann B, Simonsen P, Stensgaard L (1989) The carbon and small cyclopoid copepod Oithona davisae in a temperate chlorophyll content of phytoplankton from various nutri- eutrophic inlet. Mar Ecol Prog Ser 118:121–128 ent regimes. J Plankton Res 11:1037–1045 ➤ Uye S, Sano K (1998) Seasonal variations in biomass, growth ➤ Sabatini M, Kiørboe T (1994) Egg production, growth and rate and production rate of the small cyclopoid copepod development of the cyclopoid copepod Oithona similis . Oithona davisae in a temperate eutrophic inlet. Mar Ecol J Plankton Res 16:1329–1351 Prog Ser 163:37–44 Saiz E, Calbet A (2007) Scaling of feeding in marine calanoid ➤ Verity PG, Smetacek V (1996) Organism life cycles, preda- copepods. Limnol Oceanogr 52:668–675 tion, and the structure of marine pelagic ecosystems. Mar Saiz E, Calbet A, Broglio E (2003) Effects of small-scale turbu- Ecol Prog Ser 130:277–293

Editorial responsibility: Hans Heinrich Janssen, Submitted: October 28, 2009; Accepted: April 15, 2010 Oldendorf/Luhe, Germany Proofs received from author(s): June 22, 2010

Er ratum

• On page 105, caption to Fig. 7, one of the equations for the fitted curves was incorrect. The equation for the continuous

line in (B) should have been: log G = (0.021 × T) – 0.926. Also, the total range of temperature was given incorrectly. The complete figure with its corrected caption is reproduced here.

Fig. 7. Oithona davisae . (A) Specific growth rates (±SE) of naupliar stages as a function of food concentration (µg C l –1 ). Fitted curves correspond to Ivlev’s equation (see ‘Results’). (B) Specific growth rates (±SE) of naupliar stages as a function of temp era- 2 ture. Fitted curves correspond to exponential models. The equations are: log G = (0.021 × T) – 0.926, r = 0.99, for temperatures 2 between 20 and 28°C (continuous line) and log G = (0.053 × T) – 1.721, r = 0.87 (dashed line), for the total range of temperature (12 to 28°C), where G is the specific growth rate (d –1 ), T is the temperature (°C) and r 2 is the correlation coefficient

127 Chapter 4

Resum de l’article IV – Article IV summary– Catalan version

Efectes de la temperatura i la concentració d'aliment en la supervivència, desenvolupament i creixement de les fases naupliares de Oithona davisae (Copepoda, Cyclopoida)

Rodrigo Almeda, Miquel Alcaraz, Albert Calbet, Lidia Yebra, and Enric Saiz. Article publicat en Marine Ecology Progress Series (2010, 410:97-109)

Els copèpodes del gènere Oithona són probablement els més abundants i ubics dels oceans. No obstant això, el coneixement sobre les seves taxes de desenvolupament i creixement és molt escàs comparat amb el de copèpodes calanoides. En aquest estudi de laboratori, es van determinar la supervivència i les taxes de desenvolupament i creixement de les fases naupliares d’ Oithona davisae sotmeses a diferents règims de temperatura i concentracions d'aliment. La supervivència dels nauplis es va reduir aproximadament un 60% a la concentració d'aliment més baixa provada (11 µg C L -1 , després de 7 dies i a 20 ºC). El desenvolupament dels nauplis d’ O.davisae va ser equiproporcional però no isocronal. Les concentracions d'aliment requerides per obtenir taxes màximes de desenvolupament i creixement van ser 56 and 87 µg C L -1 , respectivament. Els valors de

la Q 10 per a desenvolupament i creixement van dependre del rang de temperatura. Els nauplis d' O.davisae van mostrar temps de desenvolupament similars, però taxes de creixement i requeriments d'aliment més baixos que els valors trobats per nauplis de calanoides a la literatura. Suggerim que aquestes diferències contribuirien a explicar la ubiqüitat d’ Oithona spp . en els ambients oceànics.

128 Chapter 4

Resumen del artículo IV – Article IV summary– Spanish version

Efectos de la temperatura y la concentración de alimento en la supervivencia, el desarrollo y el crecimiento de las fases naupli ares de Oithona davisae (Copepoda, Cyclopoida)

Rodrigo Almeda, Miquel Alcaraz, Albert Calbet, Lidia Yebra, and Enric Saiz. Artículo publicado en Marine Ecology Progress Series (2010, 410:97-109)

Los copépodos del género Oithona son probablemente los más abundantes y ubicuos de los océanos. Sin embargo, el conocimiento sobre sus tasas de desarrollo y crecimiento es muy escaso comparado con el de copépodos calanoides. En este estudio de laboratorio, determinamos la supervivencia y las tasas de desarrollo y crecimiento de las fases naupliares de Oithona davisae sometidas a distintos regímenes de temperatura y concentraciones de alimento. La supervivencia de los nauplios se redujo aproximadamente un 60% en la concentración de alimento más baja probada (11 µg C L -1 , después de 7 días y a 20 ºC). El desarrollo de los nauplios de O. davisae fue equiproporcional pero no isocronal. Las concentraciones de alimento requeridas para obtener tasas máximas de desarrollo y crecimiento fueron 56 and 87 µg C L -1 , respectivamente. Los valores de la

Q10 para desarrollo y crecimiento dependieron del rango de temperatura. Los nauplios de O. davisae mostraron tiempos de desarrollo similares, pero tasas de crecimiento y requerimientos de alimento más bajos que los valores encontrados para nauplios de calanoides en la literatura. Sugerimos que estas diferencias contribuirían a explicar la ubiquidad de Oithona spp. en los ambientes oceánicos.

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Feeding rates and gross growth efficiencies of larval developmental stages of Oithona davisae (Copepoda, Cyclopoida)

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Feeding rates and gross growth ef ficiencies of larval developmental stages of Oithona davisae (Copepoda, Cyclopoida)

Rodrigo Almeda a,⁎, Christina B. Augustin b, Miquel Alcaraz a, Albert Calbet a, Enric Saiz a a Institut de Ciències del Mar (ICM, CSIC), P. Marítim de la Barceloneta 37-49, 08003, Barcelona, Spain b Leibnitz Institute for Baltic Sea Research Warnemünde, Seestrasse 15, 18119 Rostock, Germany a r t i c l e i n f o a b s t r a c t

Article history: Among marine planktonic copepods, the genus Oithona is probably the most abundant and ubiquitous Received 7 December 2009 copepod in the world's oceans. However, knowledge about the ecophysiology of Oithonids is very scarce Received in revised form 1 March 2010 compared to calanoid copepods, particularly for their larval stages. We determined feeding rates and gross Accepted 1 March 2010 growth ef ficiencies of different developmental stages of the cyclopoid copepod Oithona davisae as related to food concentration, body weight and temperature in the laboratory. Keywords: The feeding rates of nauplii and copepodites of Oithona davisae in relation to food concentration followed a Copepodite type III functional response, with feeding threshold concentrations ranging from 50 to 75 g C L −1, Feeding ∼ μ Gross growth ef ficiency depending on the developmental stage. All feeding parameters varied according to body weight/age. The −1 Growth food concentration required to achieve the maximum ingestion rates increased from 200 μg C L in early Nauplii nauplii to 320 μg C L −1 in copepodites. Speci fic ingestion rates (d − 1) increased with increasing temperature,

Oithona davisae with a Q10 =2.45. Growth rates were negatively related to larval size and positively related to food concentration and temperature. Gross growth ef ficiency ranged from 0.16 to 0.60 depending on the developmental stage, food availability and temperature. Oithona davisae developmental stages exhibited much lower maximum speci fic ingestion rates than calanoid nauplii but exhibited quite similar gross growth ef ficiencies. This indicates that Oithona nauplii should display lower metabolic losses and consequently lower food requirements than calanoid nauplii. Together with other factors, this feeding/energetic strategy may contribute to the success of the Oithona species in marine ecosystems of contrasting trophic characteristics. © 2010 Elsevier B.V. All rights reserved.

1. Introduction frequently outnumbered late copepodites and adults by several orders of magnitude ( Turner, 1982, 1994, 2004; Hansen et al., 1999; Calbet Copepods are usually the major component of marine metazoo- et al., 2001 ) and sometimes, they represent a higher fraction of plankton ( Longhurst, 1985; Verity and Smetacek, 1996 ) and the key biomass than late stages ( Castellani et al., 2007 ). Moreover, nauplii can link from primary producers to fish production in pelagic food webs ingest small nanoplankton ( Uye and Kasahara, 1983; Berggreen et al., (Cushing, 1989 ). In recent decades, feeding rates of marine copepods 1988 ) and their speci fic ingestion rates can be three to four times have been extensively studied both in the laboratory and in the field higher than those of adults ( Paffenhöfer, 1971; White and Roman, (reviewed by Saiz and Calbet, 2007 ). However, most of the research 1992; Lonsdale et al., 1996 ). Thus, copepod nauplii may be important has been focused on copepodites and adult stages, and copepod nauplii intermediaries between the classical and microbial food webs because have received comparatively little attention. This lack of information of their small size, high abundance and ability to feed on small particles contrasts with the fact that copepod nauplii are the most abundant (Turner and Roff, 1993 ). forms of metazoans on the planet ( Björnberg, 1986; Fryer, 1986 ) and In addition to other factors (e.g., food quality), copepod feeding the main prey of most fish larvae ( Last, 1980; Conway et al., 1991, rates are mainly affected by body size, temperature and food 1998 ). Copepod nauplii have been historically undersampled by concentration ( Marshall and Orr, 1955; Conover, 1956; Mullin, conventional methods such as 200- μm mesh plankton nets ( Alcaraz, 1963; Ikeda, 1977; Frost, 1972 ). The effects of these factors on feeding 1977; Calbet et al., 2001; Gallienne and Robins, 2001 ). When parameters (e.g., maximum rates of ingestion and clearance and appropriate fine meshes of plankton nets have been employed, nauplii saturating food concentrations) are basic inputs for ecological models. Previous studies have reported that copepod ingestion rates are positively related to food concentration and body size ( Paffenhöfer, ⁎ Corresponding author. Postal address: P. Marítim de la Barceloneta 37-49, 08003. Barcelona, Spain. Tel.: +34 93 2309500; fax: +34 93 2309555. 1971; Frost, 1972; Peters and Downing, 1984 ). Although temperature E-mail address: [email protected] (R. Almeda). is known to accelerate the feeding rates of several copepod species

0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi: 10.1016/j.jembe.2010.03.002

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(Conover, 1956; Anraku, 1964 ), there is a disagreement about the To obtain cohorts of nauplii (NI), we removed adults (including actual effect of temperature in the field (reviewed in Saiz and Calbet, egg-bearing females) from the stock culture with a 132- μm mesh size 2007 ). Copepod feeding behaviour differs between life stages of the sieve and placed them in a new tank, where they were fed ad libitum same species ( Fernández, 1979 ) and similarly, the effect of environ- Oxyrrhis marina (N3000 cells mL − 1, equivalent to N660 μg C L − 1). mental factors would be different depending on the developmental After 20 h, adults were removed with a 100- μm mesh, leaving the stage. Besides feeding rates, the gross growth ef ficiency (GGE) is hatched nauplii in a new tank. In order to remove the dislodged sacs of another important measurement for detailing the flow of material eggs, we allowed them to settle for 2 h before siphoning the nauplii to from prey to predator. For the application of ecological models, GGE of a new tank. zooplankton is commonly assumed to be constant (median ∼30%) for all types of zooplankton and during all the cycle life (reviewed by 2.2. Functional feeding responses Straile, 1997; Buitenhuis et al., 2006 ). However, GGE may vary signi ficantly depending on the developmental stage and food We determined the grazing response to food concentration for concentration ( Jones et al., 2002; Rey-Rassat et al., 2002 ). Conse- different developmental stages of Oithona davisae in five feeding quently, general patterns of copepod feeding and growth ef ficiencies experiments ( Table 1 ). For each experiment, a cohort of newly based on late stages should not be extrapolated to naupliar ones. hatched nauplii were kept at saturating food conditions ( N3000 Therefore, knowledge about feeding rates and growth ef ficiency of cells mL − 1, Oxyrrhis marina ) during the time required to reach the copepod nauplii is crucial for a better understanding of the role of desired developmental stage ( Table 1 ). Identi fication of developmen- copepods in planktonic food webs. tal stages (at least 40 individuals) was conducted with an inverted Among marine copepods, cyclopoids of the genus Oithona are microscope (×100) based on morphological observation according to considered the most abundant and ubiquitous copepods in the Uchima (1979) . world's oceans ( Gallienne and Robins, 2001 ). However, knowledge The feeding rates were obtained by incubating larvae in triplicate about the feeding and ecology of these small copepods is very scarce bottles with the food suspension (experimental bottles) and measur- compared to the vast number of experimental studies devoted to ing the change in prey concentration relative to that in the food calanoid copepods ( Paffenhöfer, 1993; Turner, 2004; Saiz and Calbet, suspension without larvae (triplicate control bottles) after the 2007 ). We found some information in the literature about the feeding incubation time (approximately 24 h). The food concentrations used rates of adult stages of Oithona in the laboratory ( Lampitt, 1978; for the experiments covered a range of approximately 100 to 1600 Lampitt and Gamble, 1982; Saiz et al., 2003 ) and in the field cells mL − 1, equivalent to 22 and 352 μg C L − 1, respectively. Food (Nakamura and Turner, 1997; Lonsdale et al., 2000; Gifford et al., concentration and cell prey size were veri fied by checking with a 2007; Castellani et al., 2005 ). However, there are few references on Coulter Multisizer particle counter. Oxyrrhis marina volume was feeding rates of Oithona nauplii ( Henriksen et al., 2007 ). To our converted to carbon content using the conversion factor of 0.123 pg knowledge, there are no previous studies on the effects of temper- C μm− 3 (Pelegri et al., 1999 ). The culture of O. marina was not fed ature and body size on feeding rates and growth energetics of Oithona Rhodomonas salina the day before the experiments began in order to nauplii. ensure that the dino flagellate depleted all the R. salina and that only O. The present study attempts to provide basic information con- marina was offered as prey. The absence of R. salina in stock bottles cerning the feeding rates and growth ef ficiencies of the early was veri fied by checking with a Coulter Multisizer particle counter. developmental stages of cyclopoid copepods belonging to the genus Before preparing the food suspension, the stock culture of O. marina Oithona . The model used was Oithona davisae , a dominant copepod in was filtered through a 10- μm mesh to remove cell aggregations and some eutrophic embayments ( Uye and Sano, 1995 ). This species was other particles. Food suspensions were prepared in 1-L jars by typically distributed in the west Paci fic coastal waters, but nowadays successive dilution of stock cultures with 0.2 μm filtered seawater it can also be found in coastal waters of the US west coast, southern (0.2 μm-FSW). Food suspensions were amended with a nutrient

Chile and the Spanish Mediterranean as an invasive species; Oithona mixture (15 μM NH 4Cl and 1 μM Na 2HPO 4) to compensate for nutrient davisae is considered an example of species colonisation by transoce- enrichment due to larval excretion. Incubations were conducted in anic ships through ballast water ( Razouls et al., 2005 –2009 ). The 72 mL transparent plastic cell culture bottles. For each food level, speci fic objectives of this study were the following: (1) to determine triplicate initial, experimental and control bottles were filled with the the effect of food concentration on feeding rates for different larval prey suspension in a three-step filling procedure to ensure homoge- stages, (2) to estimate the effects of body size and temperature on neity between replicates. feeding parameters, and (3) to assess the effects of temperature, body Prior to the feeding experiments ( ∼ 30 min), each larval cohort size/stage and food availability on GGE. was concentrated with 37- μm mesh (60- μm mesh for copepodites) and washed several times with 0.2 μm-FSW in order to remove the food particles. Larvae in the concentrate were counted per triplicate 2. Material and methods by microscopy, and aliquots were added to experimental bottles to obtain the desired larval density. Equivalent volumes of 0.2 μm-FSW 2.1. Experimental organisms

Oithona davisae specimens came from a continuous culture kept in Table 1 Characteristics of the developmental stages of Oithona davisae used in the feeding and our institute since October 2000. The culture was created from growth experiments. Parameters correspond to the conditions at the beginning of the zooplankton samples collected from the harbour of Barcelona, Spain experiments. The last column indicates the larval density ranges used in the (Saiz et al., 2003 ). Oithona davisae was grown in 20-L methacrylate experimental bottles for the functional responses. tanks, at 20±1 °C in a constant temperature room under a 12 h Dominant larval Cohort age Length±SE Weight±SE Larval density light:12 h dark cycle. Copepod cultures were routinely fed ad libitum a stages (hours) (μm) a (ng C ind − 1) (ind ml − 1) suspension of the heterotrophic dino flagellate Oxyrrhis marina NI –NII 40 89.4±0.7 31.6±0.8 1 –6 (equivalent spherical diameter, ESD=16 μm). In turn, O. marina NI –NII 48 92.3±1.1 33.9±0.8 1 –8 was fed the cryptophyte Rhodomonas salina (ESD =8 μm) grown in NII –NIII 72 101.1±0.6 41.1±0.7 1 –4 f/2 medium ( Guillard, 1975 ). Prey sizes were measured by a Coulter NV –NVI 109 147.0±2.3 92.5±2.9 1 –4 Multisizer III particle counter (Beckman Coulter, USA). O. marina was C2 –C3 188 237.7±3.3 213.4±4.7 0.5 –3 also used as prey in all feeding experiments. a Total body length for nauplii and prosome length for copepodites.

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were added to the control and initial bottles. Larval densities ranged where M2 and M1 are the rates of the studied process at temperatures − 1 from 0.5 to 8 larvae mL depending on larval stage and food T2 and T1 (in °C), respectively. concentration ( Table 1 ). The bottles were sealed with plastic foil to prevent air-bubble formation, mounted on a plankton wheel 2.4. Growth rates and gross growth ef ficiency (0.2 rpm) and incubated at 20±1 °C with a 12 h light:12 h dark cycle for about 24 h. We estimated the gross growth ef ficiency (GGE; the ef ficiency by Initial and final food concentrations were determined by triplicate which ingested food is converted into body mass) as related to food counting using a Coulter Multisizer particle counter. Clearance and concentration, body size/stage and temperature. For each develop- ingestion rates were calculated for each food concentration according mental stage, we calculated the GGE under the lowest and the highest to Frost (1972) after veri fication that prey growth rates in grazing food concentrations used in the functional feeding responses. bottles were signi ficantly different and lower than in the controls Moreover, the effect of temperature in GGE was estimated for nauplii (t-test p b0.05). Carbon-speci fic ingestion rates and clearance rates II –III under satiating food concentration. were calculated by dividing either ingestion rates (in μg C Larva −1 d−1) From each experimental bottle (three and four replicates in the or clearance rates (in mL larva −1 d−1) by the initial larval biomass in μg. case of the functional responses and ‘temperature effect experiment ’, The carbon weight ( W) of nauplii and copepodites was calculated by respectively), 25 individuals per replicate (total=75 –100 individuals applying length-carbon content relationships ( Almeda et al., 2009, per treatment) were photographed with a camera attached to an submitted for publication ): inverted microscope (×100). Length measurements and carbon content estimations were conducted as described above. 1 2:14 2 Daily speci fic growth rate ( G, d − ) was calculated as W ng C = 0 :0021* L r = 0 :96 for nauplii 1 ð Þ ð Þ G = ln W = W = t 4 ð ð 2 1ÞÞ ð Þ W ng C = 0 :0318* L1:61 r2 = 0 :94 for copepodites 2 ð Þ ð Þ where t is the duration of incubation (days) and W1 and W2 are the initial and final carbon contents of the larvae, respectively. where L (µm) is the total body length for nauplii and the prosome Carbon-speci fic ingestion rates under the lowest and highest food length for copepodites. levels were calculated by dividing ingestion rates (in μg C Larva −1 d−1) Digital pictures of nauplii were taken with a camera attached to an by the average larval biomass (in μg C) during the incubation. inverted microscope (×100 or 200). The initial length of larvae for The gross growth ef ficiency (GGE) was calculated as each functional response was measured from digital pictures of at least 50 individuals (initial nauplii) through image analysis by ImageJ s GGE = G = I 5 software. ð Þ All statistical analyses and regressions were conducted with SPSS s where G and I are the daily carbon-speci fic growth and the carbon- 17.0 software. All curves were fitted by standard least-squares speci fic ingestion rates, respectively. Additionally, we estimated a procedures using Sigma plot software 9.0. general GGE for Oithona davisae larvae as the slope of the linear regression relating speci fic growth rates (d −1) versus speci fic ingestion 2.3. Effect of temperature on feeding rates rates (d −1).

We examined the in fluence of temperature on feeding rates of 3. Results Oithona davisae nauplii (NII –NIII) under satiating food concentration ‘ ’ ( temperature effect experiment ). The four following temperatures 3.1. Clearance and ingestion rates were tested: 16, 20, 24 and 28 °C. Nauplii were preacclimated for approximately 3 h to the experimental temperatures. A cohort of NIII Ingestion rates increased asymptotically with increasing food was concentrated and split into four aliquots (one per temperature), concentrations in all feeding experiments ( Fig. 1 ). When the range of placed in 125 mL Pyrex glass bottles and kept at satiating food food concentrations used in the experiment included food concentra- − 1 concentration ( N3000 cells mL , O. marina , Almeda et al., 2009, 1 1 tions b200 cells mL − (b60 μg C L − ), clearance rates peaked at submitted for publication ). moderately low food concentrations and declined at lower and higher Food suspensions were prepared from stock cultures at 3000 concentrations ( Fig. 2 A, D, E). Unfortunately, in experiments in which − 1 cells mL . Triplicate initial and four replicates of control and 1 the tested food concentrations were not below 200 cells mL − , no experimental bottles were filled with the prey suspension in a decline of clearance rates at the lower food concentrations was three-step filling procedure to ensure homogenous concentration and observed ( Fig. 2 B, C). For consistency, we fitted all data to a type III acclimated for approximately 2 h at each temperature. (sigmoid) functional response model using the following functions: After preconditioning, nauplii at each temperature were concen- trated and washed with FSW at the experimental temperature. The 2 2 2 I = I * C = C + K 6 nauplii in each concentrate were counted per triplicate by microscopy max ð mÞ ð Þ and aliquots were added to experimental bottles to obtain the desired 2 2 − 1 F = I * C = C + K ; so F = I = 2K 7 larval density (from 2.6 to 5.4 nauplii mL , depending on the max ð t Þ max max t ð Þ temperature conditions). Volumes of 0.2 μm-FSW equivalent to those − 1 − 1 added with the nauplii were added to control and initial bottles. where I is the ingestion rate (cells ind d ), F is the clearance rate − 1 − 1 − 1 Bottles were incubated at the different temperatures (±0.1 °C) in (mL ind d ), C is the concentration of prey (cells mL ), Imax is − 1 − 1 water baths controlled by thermoregulators with a 12 h light:12 h the maximum ingestion rate (cells ind d ), Km is the half- dark cycle for approximately 24 h. Feeding rates were calculated as saturation food concentration and Kt is the food concentration at described previously for the functional feeding responses. which maximum clearance rate ( Fmax ) is reached. This functional response model fit well to the data in all studied developmental stages The effect of temperature on feeding rate was estimated by Q10 b approximation: (p 0.01). The feeding parameters obtained for the different larval stages from the fitted functions are shown in Tables 2 and 3 for ingestion and 10 = T2−T1 Q = M =M 3 fi − 1 10 ð 2 1Þ ð Þ clearances rates, respectively. Maximum speci c ingestion rates (d )

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Fig. 1. Relationship between food concentration ( Oxyrrhis marina , cells mL − 1) and ingestion rates (cells ind − 1 d− 1) for different developmental stages of Oithona davisae : NI –NII (A), NI –NII (B), NII –NII (C), NV –NVI (D), and CII –CIII (E). Each point is the mean value of three replicates and the error bars represent the standard error (SE). The continuous lines correspond to the model fitted to the data.

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Fig. 2. Relationship between food concentration ( Oxyrrhis marina , cells mL − 1) and clearance rates (mL ind − 1 d− 1) for different developmental stages of Oithona davisae: NI –NII (A), NI –NII (B), NII –NII (C), NV –NVI (D), and CII –CIII (E). Each point is the mean value of three replicates and the error bars represent the standard error (SE). The continuous lines correspond to the model fitted to the data.

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Table 2 Parameters of the type III functional response model used to describe the relationships between ingestion rates and food concentration (Eq. (6)) for different developmental stages of s Oithona davisae . Imax : maximum ingestion rates, I max : maximum speci fic ingestion rates, Km: half-saturation food concentration, Ks: saturating food concentration, SE: standard error, r2: correlation coef ficient.

s 2 Larval Imax ±SE Imax ±SE I max ±SE Km ±SE Ks ±SE r Stages (cells ind − 1 d− 1) (ng C ind − 1 d− 1) (d − 1) (cells mL − 1) (cells mL − 1)

NI –NII 121±4 33.7±1.2 1.05±0.04 160±14 699±60 0.97 NI –NII 141±3 39.2±0.8 1.14±0.02 180±10 785±44 0.98 NII –NIII 153±5 42.6±1.3 1.04±0.03 161±16 701±70 0.94 NV –NVI 282±18 78.7±5.0 0.85±0.05 246±12 1071 ±51 0.99 C2 –C3 541±23 150.9±6.3 0.71±0.03 259±21 1128 ±91 0.97

decreased through larval development from ∼1.10 in early nauplii growth to body size and temperature are given in Tables 4 and 5 , 1 (NI –NII) to ∼0.71 in copepodites ( Table 2 ). On the other hand, the respectively. Low food levels ( ∼30 –40 μg C L − ) reduced speci fic saturating food concentration ( Ks) increased with development stage growth rates in all larval stages ( ∼2 times lower for early nauplii and 1 1 from ∼700 cells mL − (∼200 µg C L − ) in early nauplii to ∼1130 ∼3 times lower for late nauplii and copepodites; Table 6 ). 1 1 cells mL − (∼320 µg C L − ) in copepodites. Maximum clearance rates Gross growth ef ficiency varied depending on larval stage, 1 1 increased during development from ∼0.4 mL ind − d− for early temperature and food concentration ( Table 6 ). GGEs were signi fi- 1 1 nauplii to ∼1.10 mL ind − d− for copepodites. However, speci fic cantly lower at 16 °C than at higher temperatures (ANOVA, Tukey test, clearance rates of early nauplii doubled those of late stages ( Table 3 ). pb0.01). However, between 20 and 28 °C, no signi ficant differences

Maximum clearance rates ( Fmax ) occurred below the food saturation were observed (ANOVA, Tukey test N0.05) and GGE had a mean value conditions, indicating that larvae began to decrease their clearance of 0.22 ( Table 6 ). Under food satiating conditions, the GGEs ranged rates while still under limiting food conditions ( Table 3 ). At saturating between ∼0.33 for NI –NII nauplii and ∼0.22 for later stages ( Table 6 ). food conditions, clearance rates were approximately 2.5-fold lower For a particular larval stage, GGEs were lower at low than at high food than the maximum values. concentrations. The GGE under low food levels varied between 0.24 All feeding parameters varied according to body weight as shown and 0.60. The effect of food concentration on GGE was particularly 1 in Fig. 3 . Clearance and ingestion rates in relation to body weight fit evident at food concentrations of ∼30 –40 μg C L − , almost doubling well to power functions ( Fig. 3 A–C), and the logarithmic form of the the GGE observed under high food levels ( Table 6 ). The general GGE equations are given in Table 4 . However, other feeding parameters obtained as the slope of the linear regression relating carbon-speci fic fi fi such as Km, Kt and Kc as related to body weight were t to saturation growth rates with carbon-speci c ingestion rates was ∼0.21 ( Fig. 6 ). curves ( Fig. 3 D–F, see equations in Table 4 ). These last feeding parameters exhibited a maximum value when body weight was 4. Discussion 1 ∼100 μg C ind − , and further increase in body weight did not support higher values. The maximum values for Km, Kt and Kc were 259, 250, 4.1. Clearance and ingestions rates and 1130 cells mL − 1 (72, 75, and 328 μg C L − 1), respectively (Table 4 ). The feeding response of copepods in relation to food concentration Feeding rates of Oithona davisae nauplii (NII –NIII) increased with has been commonly described by one of three models: rectilinear, increasing temperature under food saturating conditions ( Fig. 4 ). curvilinear or sigmoidal (types I, II and III, respectively, Holling, 1959 ). 1 1 Maximum ingestion rates increased from 75 to 275 cells ind − d− at The three models give essentially the same information about the 1 16 °C and 28 °C, respectively. Similarly, speci fic ingestion rates (d − ) range at which ingestion is limited, the maximum ingestion rates and increased from 0.51 to 1.49 at 16 and 28 °C, respectively ( Q10 =2.45). the sensitivity of the ingestion to changes in food concentration. The Clearance rates under saturation conditions ( Fs) increased from 0.03 main differences among the models occur at low food concentrations −1 − 1 to 0.11 mL ind d at 16 º C and 28 °C, respectively ( Q10 =2.95). (Frost, 1975; Båmstedt et al., 2000 ). In type I, clearances rates are Feeding rates in relation to temperature were fitted to logarithmic constant at food concentrations below saturating concentrations. The functions ( Fig. 4 ) and the obtained equations are given in Table 5 . type III functional response differs from type II in the presence of a ‘feeding threshold ’, i.e., a prey concentration below which the copepod 3.2. Growth rates and gross growth ef ficiency stops feeding ( Wlodarczyk, 1988 ) or reduces its clearance rates (Kiørboe et al., 1985 ). Type III clearance curves have been observed in Under food saturating conditions, speci fic growth rates decreased some calanoid copepods ( Kiørboe et al., 1985; Paffenhöfer and Stearns, with increasing body weight/stage and increased with increasing 1988 ; Durbin and Durbin, 1992 ). The presence of lower feeding temperature ( Fig. 5 ). Speci fic growth rates ranged from 0.33 d − 1 in thresholds can be interpreted as an adaptation to conserve energy at early nauplii to 0.12 d − 1 in copepodites ( Fig. 5 ). In nauplii (NII –NIII), low food concentrations because the energetic cost of collecting food speci fic growth rates (d − 1) increased from 0.08 at 16 °C to 0.34 at at very low concentrations would not be compensated by the energetic

28 °C with a Q10 =3.34 ( Fig. 5 ). Fitted equations relating speci fic gain ( Price and Paffenhöfer, 1985 ). Feeding thresholds vary depending

Table 3 Parameters of the type III functional response model used to describe the relationships between clearances rates and food concentration (Eq. (7)) for different developmental stages s of Oithona davisae . Imax : maximum ingestion rates, Fmax : maximum clearance rates, Fmax : maximum speci fic clearance rates, Kt: feeding threshold food concentrations, Fs: clearance rates at satiating food concentrations, SE: standard error, r2: correlation coef ficient.

s 2 Larval Imax ±SE Kt ±SE Fmax ±SE Fmax ±SE Fs ±SE r Stages (cells ind − 1 d− 1) (cells mL − 1) (ml ind − 1 d− 1) (ml μg C d − 1) (ml ind − 1d− 1)

NI –NII 118±6 153±8 0.38±0.02 12.2±0.6 0.16±0.01 0.97 NI –NII 140±3 179±7 0.39±0.01 11.5±0.3 0.17±0.01 0.99 NII –NIII 157±8 172±17 0.46±0.02 11.1±0.6 0.21±0.01 0.98 NV –NVI 296±19 260±16 0.57±0.04 6.2±0.4 0.26±0.02 0.93 C2 –C3 517±25 235±12 1.11±0.05 5.2±0.3 0.44±0.02 0.92

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Fig. 3. Oithona davisae larvae. Relationships between body weight ( W, ng C ind − 1) and the feeding parameters calculated from the functional feeding responses: maximum ingestion s rates, Imax (A), maximum clearance rates, Fmax (B), maximum speci fic ingestion rates, I max (C), feeding threshold food concentrations, Kt (D), half-saturation food concentration, Km

(E) and satiating food concentration i.e. food concentration that correspond to 95% of the maximum ingestion rate, Ks (F). Error bars represent the standard error (SE). The continuous lines correspond to the model fitted to the data (see Table 4 ).

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Table 4 Table 5 Regression equations relating feeding rates and feeding parameters to body weight Regression equations relating feeding and growth rates to temperature ( T a, °C) for − 1 (ng C ind ) for the developmental stages of Oithona davisae at 20 °C. Imax : maximum Oithona davisae nauplii (NII –NIII) under food satiating conditions. Imax : maximum s S ingestion rates, Fmax : maximum clearance rates, I max : maximum speci fic ingestion rates, ingestion rates, Fs: clearance rates at satiating food concentrations, I max : maximum

G: speci fic growth rates at satiating food concentrations, Km: the half-saturation food speci fic ingestion rates, G: speci fic growth rates at satiating food concentrations, 2 concentration; Ks: saturating food concentration; Kt: feeding threshold concentration, r : correlation coef ficient. r2: correlation coef ficient. Parameter Equation r2 2 Parameter Equation r − 1 − 1 Imax (cells ind d ) Imax =−900.5+353*ln( T) 0.99 − 1 − 1 S − 1 S Imax (cells ind d ) Log Imax =0.948+0.766*(Log W) 0.99 Imax (d ) Imax =−4.256+1.733 *ln( T) 0.98 S − 1 S − 1 − 1 Imax (d ) Log Imax =0.393 –0.234*(Log W) 0.96 Fs (ml ind d ) Fs =−0.372+0.145*ln( T) 0.99 − 1 − 1 − 1 Fmax (ml ind d ) Log Fmax =−1.223+0.530*(Log W) 0.96 G (d ) G= −1.186+0.459*ln( T) 0.99 − 1 G (d ) Log Gmax =0.1934 –0.486*(Log W) 0.87 (0.029 W) Km Km =259*(1 −e ) 0.92 (0.034 W) Kt Kt =246*(1 −e ) 0.85 (0.029 W) The absence of a feeding threshold in some of our functional responses Ks Kt =1128*(1 −e ) 0.92 (Fig. 2 B, C) can be explained because the nauplii were not exposed to low enough food concentrations. Therefore, we determined that the on copepod species ( Wlodarczyk et al., 1992 ) but are also in fluenced appropriate model to describe the functional responses of Oithona by other variables such as the developmental stage and the size and davisae larval stages was type III (sigmoidal). quality of the prey ( Paffenhöfer, 1970; Frost, 1975 ; Fernández, 1979 ; According to our results, early developmental stages of copepods Schnack, 1983 ). In the case of Oithona davisae , feeding thresholds were (NI –NIII) have a lower feeding threshold than late stages (CII –CIII), as observed in the functional responses under food concentrations lower previously noted by Frost (1975) for nauplii and adults of Calanus 1 1 than 200 cells mL − (∼50 μg C L − ) for early nauplii and approxi- paci ficus . Feeding thresholds decrease with increasing prey size ( Frost, 1 1 mately 260 cells mL − (∼75 μg C L − ) for late stages and copepodites. 1975 ) and generally are higher for natural particle spectra than for

s Fig. 4. Oithona davisae nauplii (NII –NIII). Relationships between temperature ( T, °C) and maximum ingestion rates, Imax (A), maximum speci fic ingestion rates, I max (B) and clearance

rates at satiating food conditions, Fs (D). Error bars represent the standard error (SE). The continuous lines correspond to the model fitted to the data (see Table 5 ).

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Fig. 6. Carbon-speci fic growth rates ( G, d − 1) vs. carbon-speci fic ingestion rates ( I, d − 1) of Oithona davisae developmental stages. Fitted equation: G = 0.208 * I + 0.036, r2 =0.71.

cultured prey, perhaps because natural food assemblages contain higher amounts of non-suitable preys in terms of quality, size or palatability ( Parsons et al., 1967; McAllister, 1970 ). Oithona davisae showed higher feeding threshold concentrations compared with other copepod nauplii and copepodites (6 –47 μg C L −1 for C. paci ficus 1 (NIII –NV) ( Frost, 1975; Fernández, 1979 ) and ∼4.7 μg C L − for Acartia clausii (CI –CV) ( Takahashi and Tiselius, 2005 )) and some copepod adult stages ( b31 μg C L − 1 for Acartia hudsonica (Durbin and Durbin, 1992; Wlodarczyk et al., 1992 ) and 5 μg C L − 1 for Centropages hamatus (Saage et al., 2009 )). The high feeding threshold for Oithona davisae indicates that this species is adapted to inhabit environments with high food concentrations (inshore waters, bays and estuaries), which is in agreement with field observations ( Uye and Sano, 1995, 1998 ). Following this reasoning, it would be expected that offshore and oceanic Oithona species show feeding thresholds (and/or maximum clearance rates) at much lower food concentrations than Oithona davisae as noted previously in some calanoid copepods fi − 1 Fig. 5. Oithona davisae larvae. (A): Relationship between speci c growth rates ( G, d ) (Paffenhöfer and Stearns, 1988 ). Hence, the feeding behaviour in and body weight ( W, ng C ind − 1) for different larval stages incubated at 20 °C and under satiating food concentrations. (B): Relationship between speci fic growth rates response to food concentration helps to explain the copepod dis- (G, d − 1) of nauplii NII –NIII and temperature under satiating food concentrations. tribution in near to offshore environments. It is interesting to point

Table 6 Growth energetics of Oithona davisae in relation to larval stage, temperature and food availability. W: initial body weight, T: temperature, L: low food level, H: high food level, C: food concentration, Is: speci fic ingestion rate, G: speci fic growth rates, GGE: gross growth ef ficiency, SE: standard error.

Stage W T Food level C I±SE G±SE GGE±SE (ng C ind − 1) (°C) (μg C L − 1) (d − 1) (d − 1)

NI –II 31.6 20.5 L 30 0.27±0.03 0.16±0.01 0.60±0.08 H 441 0.94±0.02 0.33±0.01 0.34±0.02 NI –II 33.9 20.5 L 65 0.54±0.07 0.20±0.02 0.39±0.06 H 270 0.95±0.04 0.33±0.02 0.33±0.02 NII –III 39.8 16.0 H 749 0.51±0.04 0.08±0.01 0.16±0.02 39.9 20.5 H 728 1.01±0.03 0.21±0.01 0.21±0.01 41.1 20.5 L 68 0.57±0.06 0.13±0.01 0.24±0.02 H 280 1.13±0.01 0.23±0.01 0.20±0.01 41.4 24.0 H 717 1.26±0.02 0.27±0.01 0.22±0.01 41.6 28.1 H 657 1.49±0.06 0.34±0.01 0.23±0.02 NV –VI 92.5 20.5 L 37 0.15±0.03 0.05±0.01 0.36±0.07 H 291 0.75±0.01 0.17±0.01 0.23±0.01 CII –CIII 213.4 20.5 L 34 0.14±0.02 0.04±0.01 0.33±0.07 H 263 0.66±0.01 0.12±0.01 0.19±0.01

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out that observed feeding thresholds, at which clearance rates are and body weight for calanoid copepods ( Hansen et al., 1997; Saiz and maximal, correspond to the food concentration values required Calbet, 2007 ) largely overestimate the ingestion rates when applied to for maximum developmental rates of Oithona davisae nauplii Oithonids. 1 (∼50 μg C L − ; Almeda et al., submitted for publication ). It is well known that speci fic ingestion rates generally decrease In contrast to most calanoid copepods, Oithonids are ambush with increasing development stage/body size ( Paffenhöfer, 1971, feeders that rely on detecting prey by hydromechanical signals 1976; Lonsdale et al., 1996; Rey et al., 2001 ). As expected, nauplii of O. (Hwang and Turner, 1995; Kiørboe and Visser, 1999; Svensen and davisae showed higher speci fic ingestion rates (d − 1) than adult stages Kiørboe, 2000 ). The ef ficiency of prey detection may determine the of the same species ( Saiz et al., 2003 ) and Oithona nana adults when copepod species distribution in low or high food environments and feeding on a protozoan diet ( Lampitt and Gamble, 1982 ). A general their feeding thresholds. For Oithonids, because an increase in leaping model describing the effect of body weight on speci fic ingestion rates frequency would not be energy-ef ficient, a higher sensitivity of the (d − 1) that includes data of larvae and adult stages of O. davisae is mechanoreceptor sensors (a highly ef ficient sensory system) would shown in Fig. 7 . The maximum speci fic ingestion rates of Oithona be the strategy to increase encountering food particles ( Paffenhöfer, davisae under satiating food conditions seemed to conform to the 1998 ). There are clear differences in morphological structures general trend of three-quarters scaling to body mass ( −0.25 if we associated with mechanoreception between Oithona species, e.g., consider weight-speci fic rates, Peters 1983 ; Hansen et al., 1997 ). nauplii of Oithona plumifera , a typical oceanic species, have more and Regarding temperature effects, it is expected that there is an longer setae than Oithona davisae nauplii. The differences in the exponential increase in feeding rates with increasing temperature relative length of antennules with respect to the cephalothorax and within the thermal tolerance range of a species (Arrhenius rate law; the number and length of setae are particularly evident between Carlotti et al., 2000 ). At the experimental temperatures we used, copepodites and adult stages of both species. In fact, Oithona species Oithona davisae nauplii feeding rates did not fit well to exponential that only live in inshore waters, such as Oithona davisae , O. nana and functions and the data were fitted to predictive models ( Table 5 ). O. aruensis , exhibit shorter antennules (with respect to cephalothorax Oithona davisae is considered to be a thermophilic species because its length) than species that can also live in offshore waters, such as maximum population density in natural systems occurs during the O. similis , O. atlantica and O. plumifera . Therefore, although compar- warm seasons ( TN20 °C; Uye and Sano, 1995; Nakane et al., 2008 ). ative studies about the sensorial system of Oithona are required, we Therefore, the lowest temperature used in our experiments (16 °C) suggest that Oithona davisae has a sensory system less ef ficient than was probably a suboptimal temperature (or near to lower limits) for offshore Oithona species. This may explain why Oithona davisae is this species, which produced a deviation from the expected restricted to inshore environments with high food concentrations. exponential model relating feeding rates and temperature. −1 Maximum speci fic ingestion rates (d ) observed here were similar The food satiating concentration ( Ks, food concentration required to those reported by Henriksen et al. (2007) for early nauplii of O. davisae for achieving the maximal speci fic ingestion rate) estimated in this feeding on Heterocapsa sp. However, the maximum clearance rates study was quite similar to that observed by Henriksen et al. (2007) for observed in our study were almost 40% higher than those reported by nauplii (NIII) of the same species at a similar experimental Henriksen et al. (2007) . This could be due to the differences in prey size temperature (20 °C). The satiating food concentrations for O. davisae between both studies. The prey used in this study, Oxyrrhis marina larvae are lower than those reported for calanoid larval stages (125 – (16 μm, ESD), is within optimal prey size for early nauplii of Oithona 1000 μg C L − 1 for Calanus paci ficus (NII –NVI) at 15 °C ( Fernández, davisae (Saiz et al., unpublished), and lower clearance rates are expected 1979 ); 500 μg C L − 1 for Acartia tonsa (NII –CV) at 17 °C ( Berggreen for smaller prey such as Heterocapsa (12.8 μm, ESD). Speci fic ingestion et al., 1988 ); 600 and 800 μg C L − 1 for Calanus typicus and rates (d −1) of O. davisae nauplii were lower by a factor of 3 than calanoid C. helgolandicus (NIV –NV), respectively, at 15 °C ( López et al., nauplii with similar body weight and at the same temperature ( Acartia 2007 )). Even some Oithona adult stages require lower food concen- grani , Henriksen et al., 2007 ) and unexpectedly, even lower than those trations for maximum speci fic ingestion than some calanoid nauplii −1 reported for large calanoid nauplii ( Saiz and Calbet, 2007 cf. table A 1.3, (Oithona davisae , Ks ∼120 μg C L at 21 °C ( Saiz et al., 2003 ) and − 1 temperature correction using Q10 =2.95). This suggests that, aside from Oithona nana , Ks b100 μg C L at 10 °C ( Lampitt and Gamble, 1982 )). body size, other factors should be considered to explain the differences in Therefore, although more data are required, we suggest that the speci fic ingestion rates between cyclopoids and calanoids. In fact, ingestion rates of calanoid larvae in natural systems may be more available models describing the relationships between feeding rates limited by food than those of Oithona nauplii. According to our results, late larval stages (CII –CIII) required

higher food concentrations for achieving maximum feeding rates ( Ks) than early nauplii (NI –NIII). Hence, it is expected that adult stages

would have higher a KS than nauplii. However, satiating food concentrations reported for adult stages of Oithona davisae by Saiz − 1 et al. (2003) were lower ( Ks ∼120 μg C L ) than those observed here for nauplii. Nevertheless, our results suggest that late stages of a given copepod species are more likely to be food-limited in nature than their younger stages, which is in agreement with results of field studies with Oithona spp. ( Hopcroft and Roff, 1998a,b; Hopcroft et al., 1998 ).

4.2. Growth rates and gross growth ef ficiency

As with many metazooplankters, speci fic growth rates of the different developmental stages were negatively related to size ( Sprung, 1984; Paffenhöfer, 1976 ) and positively related to food concentration and temperature ( Vidal, 1980; Huntley and Boyd, 1984; Huntley and Fig. 7. Oithona davisae . Relationship between body weight ( W) and maximum speci fic Lopez, 1992 ). As explained previously for ingestion rates, the lowest ingestion rates ( Is) including data from previous studies. All values standardized to 20 °C temperature used in our experiments was probably a suboptimal s 2 using a Q10 =2.45. Fitted equation (log form): log I =−0.265*(log W)+0.454, r =0.97. temperature (or near to lower limits) for this species, which produced a

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34 R. Almeda et al. / Journal of Experimental Marine Biology and Ecology 387 (2010) 24 –35 deviation from the expected exponential model relating speci fic growth food becomes limiting for calanoid copepods ( Paffenhöfer, 1993; rates and temperature. Speci fic growth rates of O. davisae nauplii were Castellani et al., 2005 ). Therefore, we suggest that, in addition to other slightly lower than those reported for Oithona similis (0.20 d −1 at 15 °C factors, the feeding/energetic strategy of Oithona species contributes to (Sabatini and Kiørboe, 1994 )) and much lower than those commonly their success in marine ecosystems of contrasting trophic characteristics. reported for calanoids (0.27 d −1–0.50 d −1 (Kiørboe and Sabatini, 1995; Calbet and Alcaraz, 1997; Leandro et al., 2006 )). Acknowledgements The gross growth ef ficiency (GGE) of Oithona davisae larvae showed a wide range of values (from 0.16 to 0.60) depending on We thank P. Jiménez and E. Velasco for their help in maintaining the developmental stage, food availability and temperature. GGE of algae and copepods cultures. This work was funded by a PhD fellowship Oithona davisae changed during larval development in agreement to RA (BES-2005-7491) from the Spanish Ministry of Education and with patterns observed for other copepods ( Petipa, 1967; Paffenhöfer, Science and supported by MICROROL (CTM2004-02775) and OITHONA 1976 ). However, there was not a clear pattern in the relationship (CTM2007-60052) research projects from the same ministry and a between GGE of O. davisae and larval age/body weight. The high GGE contract to C.B Augustin by EUR-OCEANS Network of Excellence for in nauplii NI –NII compared to the other larval stages may be a Ocean Ecosystems Analysis. [SS] consequence of using the yolk reserved for growing. The decrease in GGE with increasing food concentration observed References in O. davisae larvae has also been reported for other copepods (Conover, 1964; Mullin and Brooks, 1970; Paffenhöfer, 1976; Heinle et Alcaraz, M., 1977. Muestreo cuantitativo de zooplankton: análisis comparativo de la al., 1977 ) and some meroplanktonic larvae ( Thompson and Bayne, eficacia de mangas y botellas en un sistema estuárico. Invest. Pesq. 41, 285 –294. Almeda, R., Pedersen, T., Jakobsen, H.H., Alcaraz, M., Calbet, A., Hansen, B.W., 2009. 1974; Almeda et al., 2009 ). GGE can be expressed as the product of net Feeding and growth kinetics of the planktotrophic larvae of the spionid polychaete growth ef ficiency and assimilation ef ficiency. In many metazooplank- Polydora ciliata (Johnston). J. Exp. Mar. Biol. Ecol. 382, 61 –68. ters, the assimilation ef ficiency declines with increasing food Almeda, R., Calbet, A., Alcaraz, M., Yebra L., Saiz, E. Effect of temperature and food concentration on survival, development and growth rates of naupliar stages of Oithona concentrations ( Straile, 1997 , and references therein). An increase davisae (Copepoda, Cyclopoida). Mar. Ecol. Prog. Ser. (submitted for publication). in the amount of food ingested under higher food concentrations Anraku, M., 1964. In fluence of the Cape Cod Canal on the hydrography and on the usually decreases gut passage time, resulting in a decline in copepods in Buzzards Bay and Cape Cod Bay, Massachusetts. II. Respiration and assimilation ef ficiency ( Dagg and Walser, 1986; Butler and Dam, feeding. Limnol. Oceanogr. 9, 195 –206. Båmstedt, U., Gifford, D.J., Irigoien, X., Atkinson, A., Roman, M.R., 2000. Feeding. In: 1994; Sandier and Van Den Bosch, 1994 ). Another possible explana- Harris, R.P., Wiebe, P., Lenz, J., Skjoldal, H.R., Huntley, M. (Eds.), ICES Zooplankton tion for the observed decrease in GGE may be due to the increase in Methodology Manual. London Academic Press, pp. 297 –400. the accumulation rates of carbon reserves under increasing food Berggreen, U., Hansen, B., Kiørboe, T., 1988. Food size spectra, ingestion and growth of the copepod Acartia tonsa during development: implications for determination of levels. This would reduce the proportion of the ingested food used for copepod production. Mar. Biol. 99, 341 –352. growing in length and consequently, the GGE. Björnberg, T.K.S., 1986. The rejected nauplius: a commentary. Syllogeus 58, 232 –236. fl The effect of temperature on GGE of zooplankton is unclear. Buitenhuis, E., Quere, C.L., Aumont, Q., Beaugrand, G., et al., 2006. Biogeochemical uxes through mesozooplankton. Global Biogeochem. Cycles 20, GB2003. doi:10.1029/ Organisms are generally adapted to ambient temperatures and no 2005GB002511 . strong dependency of GGE on temperature is to be expected in situ Butler, M., Dam, H.G., 1994. Production rates and characteristics of fecal pellets of the (Straile, 1997 ). 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Effects of small-scale turbulence on copepods: the Jones, R.H., Flynn, K.J., Anderson, T.R., 2002. Effect of food quality on carbon and case of Oithona davisae . Limnol. Oceanogr. 48, 1304 –1311. nitrogen growth ef ficiency in the copepod Acartia tonsa . Mar. Ecol. Prog. Ser. 235, Sandier, B., Van Den Bosch, E., 1994. Herbivorous nutrition of Cyclops vicinus : the effect 147 –156. of a pure algal diet on feeding, development, reproduction and life cycle. J. Plankton Kiørboe, T., Sabatini, M., 1995. Scaling of fecundity, growth and development in marine Res. 16, 171 –195. planktonic copepods. Mar. Ecol. Prog. Ser. 120, 285 –298. Schnack, S.B., 1983. On the feeding of copepods on Thalassiosira partheneia from the Kiørboe, T., Visser, A.W., 1999. Predator and prey perception in copepods due to Northwest Africa upwelling area. Mar. Ecol. Prog. Ser. 11, 49 –54. hydromechanical signals. Mar. Ecol. Prog. Ser. 179, 81 –95. Sprung, M., 1984. 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Deep-Sea Res. 32 (12A), 1535 –1570. 261 –274. Lonsdale, D.J., Cosper, E.M., Doall, M., 1996. Effects of zooplankton grazing on Turner, J.T., 1994. Planktonic copepods of Boston harbor, Massachusetts Bay and Cape phytoplankton size-structure and biomass in the lower Hudson river estuary. Cod Bay, 1992. Hydrobiologia 292 (293), 405 –413. Estuaries 19, 874 –889. Turner, J.T., 2004. The importance of small planktonic copepods and their roles in Lonsdale, D.J., Caron, D.A., Dennet, M.R., Schaffner, R., 2000. Predation by Oithona spp. pelagic marine food webs. Zool. Stud. 43, 255 –266. on protozooplankton in the Ross Sea, Antarctica. Deep-Sea Res. II 47, 3273 –3328. Turner, J.T., Roff, J.C., 1993. Trophic levels and trophospecies in marine plankton: López, E., Anadón, R., Harris, R.P., 2007. Functional responses of copepod nauplii using a lessons from the microbial food web. Mar. Microb. Food Webs 7, 225 –248. high ef ficiency gut fluorescence technique. Mar. Biol. 150, 1047 –1048. Uchima, M., 1979. Morphological observation of developmental stages in Oithona Marshall, S.M., Orr, A.P., 1955. The Biology of a Marine Copepod, Calanus finmarchicus brevicornis (Copepoda, Cyclopoida). Bull. Plankton Soc. Jap. 26, 59 –76. (Gunnerus). Oliver and Boyd, Edinburgh, p. 180. Uchima, M., Hirano, R., 1988. Swimming behavior of the marine copepod Oithona McAllister, C.D., 1970. Zooplankton rations, phytoplankton mortality and the davisae : internal control and search for environment. Mar. Biol. 99, 47 –56. estimation of marine production. In: Steele, J.H. (Ed.), Marine Food Chains. Oliver Uye, S., Kasahara, S., 1983. Grazing of various developmental stages of Pseudodiaptomus and Boyd, Edinburgh, pp. 419 –457. marinus (Copepoda: Calanoida) on naturally occurring particle. Bull. Plankton Soc. Mullin, M.M., 1963. Some factors affecting the feeding of marine copepods of the genus Jap. 30, 147 –158. Calanus . Limnol. Oceanogr. 8, 239 –250. Uye, S., Sano, K., 1995. Seasonal reproductive biology of the small cyclopoid copepod Mullin, M.M., Brooks, E.R., 1970. The effect of concentration of food on body weight, Oithona davisae in a temperate eutrophic inlet. Mar. Ecol. Prog. Ser. 118, 121 –128. cumulative ingestion, and rate of growth of the marine copepod Calanus Uye, S., Sano, K., 1998. Seasonal variations in biomass, growth rate and production rate helgolandicus . Limnol. Oceanogr. 15, 748 –755. of the small cyclopoid copepod Oithona davisae in a temperate eutrophic inlet. Mar. Nakamura, Y., Turner, J.T., 1997. Predation and respiration by the small cyclopoid Ecol. Prog. Ser. 163, 37 –44. copepod Oithona similis : how important is feeding on ciliates and heterotrophic Verity, P.G., Smetacek, V., 1996. Organism life cycles, predation, and the structure of flagellates? J. Plankton Res. 19, 1275 –1288. marine pelagic ecosystems. Mar. Ecol. Prog. Ser. 130, 277 –293. Nakane, T., Nakata, K., Bouman, H., Platt, T., 2008. Environmental control of short-term Vidal, J., 1980. Physioecology of zooplankton I. Effects of phytoplankton concentration, variation in the plankton community of inner Tokyo Bay, Japan. Estuar. Coast. Shelf. temperature, and body size on the growth rate of Calanus paci ficus and Sci. 78, 796 –810. Pseudocalanus sp. Mar. Biol. 56, 111 –134. Paffenhöfer, G.A., 1970. Cultivation of Calanus helgolandicus under controlled conditions. White, R., Roman, M.R., 1992. Seasonal study of grazing by metazoan zooplankton in the Helgoländer Meeresunters. 20, 346 –359. mesohaline Chesapeake Bay. Mar. Ecol. Prog. Ser. 86, 251 –261. Paffenhöfer, G.A., 1971. Grazing and ingestion of nauplii, copepodids and adults of the Wlodarczyk, E., 1988. Diel feeding, threshold feeding, and gut evacuation Irate in the marine planktonic copepod Calanus helgolandicus . Mar. Biol. 11, 286 –298. marine copepod Acartia hudsonica from Narragansett Bay, Rhode Island. M.S. thesis, Paffenhöfer, G.A., 1976. Feeding, growth, and food conversion of the marine planktonic Univ. Rhode Island. copepod Calanus helgolandicus . Limnol. Oceanogr. 21, 39 –50. Wlodarczyk, E., Durbin, A.G., Durbin, E.G., 1992. Effect of temperature on lower feeding Paffenhöfer, G.A., 1993. On the ecology of marine cyclopoid copepods (Crustacea, thresholds, gut evacuation rate, and diel feeding behavior in the copepod Acartia Copepoda). J. Plankton Res. 15, 37 –55. hudsonica . Mar. Ecol. Prog. Ser. 85, 93 –106.

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Resum de l’article V– Article V summary– Catalan version

Taxes d'alimentació i eficiències brutes de creixement de les fases de desenvolupament larvari de Oithona davisae (Copepoda, Cyclopoida)

Rodrigo Almeda, Christina B. Augustin, Miquel Alcaraz, Albert Calbet, Enric Saiz Article publicat a Journal of Experimental Marine Biology and Ecolog y (2010, 387: 24-35)

Entre els copèpodes planctònics marins, els de el gènere Oithona són probablement els més abundants i ubics dels oceans del planeta. No obstant això, el coneixement sobre la ecofisiologia de Oithona és molt escàs comparat amb el de copèpodes calanoides, particularment durant el seu desenvolupament larvari. Mitjançant incubacions de laboratori, es van determinar les taxes d'alimentació i les eficiències brutes de creixement de diferents fases de desenvolupament del copèpode ciclopoide Oithona davisae en relació a la concentració d'aliment, el pes corporal i la temperatura. Les taxes d'alimentació de nauplis i copepodits d’Oithona davisae en relació a la concentració aliment van seguir una resposta funcional tipus III, amb concentracions llindars d'aliment entre 50 i 75 µg C L -1 , depenent de la fase de desenvolupament. Tots els paràmetres d’alimentació varien segons el pes corporal/edat. La concentració d'aliment necessària per aconseguir les taxes d'ingestió màximes va variar des de 200 µg C L -1 en nauplis primerencs a 320 µg C L -1 en copepodits. Les taxes específiques d'ingestió (d -1 ) es van incrementar en augmentar la temperatura, amb un Q 10 = 2.45. Les taxes de creixement es van relacionar negativament amb la grandària de les larves i positivament amb la concentració d'aliment i la temperatura. L'eficiència bruta de creixement va variar entre 0.16 i 0.60 en funció de l'etapa de desenvolupament, la disponibilitat d'aliment i la temperatura. Les fases de desenvolupament d’ Oithona davisae van exhibir taxes especifiques d'ingestió molt més baixes que les de nauplis de calanoides però eficiències brutes de creixement similars. Això indica que els nauplis d’ Oithona haurien de tenir pèrdues metabòliques més baixes i, per tant, requeriments d'aliment més baixos que els de nauplis de calanoides. Juntament amb altres factors, aquesta estratègia alimentària/ energètica pot contribuir a l'èxit de l'espècies d’ Oithona en ecosistemes marins de diferents característiques tròfiques.

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Resumen del artículo V – Article V summary– Spanish version

Tasas de alimentación y eficiencias brutas de crecimiento de las fases de desarrollo larvario de Oithona davisae (Copepoda, Cyclopoida)

Rodrigo Almeda, Christina B. Augustin, Miquel Alcaraz, Albert Calbet, Enric Saiz Artículo publicado a Journal of Experimental Marine Biology and Ecolog y (2010, 387: 24-35)

Entre los copépodos planctónicos marinos, los del género Oithona son probablemente los más abundantes y ubicuos de los océanos del planeta. Sin embargo, el conocimiento sobre la ecofisiología de Oithona es muy escaso comparado con el de copépodos calanoides, particularmente durante su desarrollo larvario. Mediante incubaciones de laboratorio, se determinaron las tasas de alimentación y las eficiencias brutas de crecimiento de diferentes fases de desarrollo del copépodo ciclopoide Oithona davisae en relación a la concentración de alimento, el peso corporal y la temperatura. Las tasas de alimentación de nauplios y copepoditos de Oithona davisae en relación a la concentración alimento siguieron una respuesta funcional tipo III, con concentraciones umbrales de alimento entre 50 y 75 µg C L -1 , dependiendo de la fase de desarrollo. Todos los parámetros de alimentación varían según el peso corporal/edad. La concentración de alimento necesaria para alcanzar las tasas de ingestión máximas varió desde 200 µg C L -1 en nauplios tempranos a 320 µg C L -1 en copepoditos. Las tasas específicas de ingestión (d -1 ) se

incrementaron al aumentar la temperatura, con un Q 10 = 2.45. Las tasas de crecimiento se relacionaron negativamente con el tamaño de las larvas y positivamente con la concentración de alimento y la temperatura. La eficiencia bruta de crecimiento varió entre 0.16 y 0.60 en función de la etapa de desarrollo, la disponibilidad de alimento y la temperatura. Las fases de desarrollo de Oithona davisae exhibieron tasas especificas de ingestión mucho más bajas que las de nauplios de calanoides pero eficiencias brutas de crecimiento similares. Esto indica que los nauplios de Oithona deberían tener pérdidas metabólicas más bajas y, por consiguiente, requerimientos de alimento más bajos que los de nauplios de calanoides. Junto con otros factores, esta estrategia alimentaria/ energética puede contribuir al éxito de la especies de Oithona en ecosistemas marinos de diferentes características tróficas.

146 Metabolic rates and energy budget of the early developmental stages of the marine cyclopoid copepod Oithona davisae

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Limnol. Oceanogr., 56(1), 2011, 403–414 E 2011, by the American Society of Limnology and Oceanography, Inc. doi:10.4319/lo.2011.56.1.0403

Metabolic rates and carbon budget of early developmental stages of the marine cyclopoid copepod Oithona davisae

Rodrigo Almeda, * Miquel Alcaraz, Albert Calbet, and Enric Saiz

Institut de Cie`ncies del Mar (CSIC), Barcelona, Spain

Abstract The genus Oithona has been considered the most abundant and ubiquitous copepod in the world’s oceans. However, despite its importance, the metabolism of its developmental stages (nauplii and copepodites), crucial to explain their evolutionary success, is almost unknown. We determined respiration rates, ammonium and phosphate excretion rates, and the net growth efficiencies of early developmental stages of Oithona davisae as related to stage, body weight, temperature, and food availability. Respiration and excretion rates increased with increasing body weight and were positively related to temperature and food. Specific respiration rates of nauplii and copepodites varied from 0.11 to 0.55 d 21 depending on stage, body weight, temperature, and food availability. Metabolic C : N ratios were higher than 14, indicating lipid-oriented metabolism. Assimilation efficiencies and net growth efficiencies ranged from 65 % to 86 % and from 23 % to 32 %, respectively, depending on body weight, stage, and temperature. Assimilation efficiencies and net growth efficiencies estimated using the respiration rates of nauplii with food were 1.7 times higher and 0.6 times lower, respectively, than those calculated using respiration rates of nauplii without food. Therefore, the use of respiration rates measured in filtered seawater led to substantial bias on the estimations of zooplankton carbon budget. O. davisae developmental stages exhibited similar assimilation and growth efficiencies but lower carbon-specific respiratory losses than calanoid copepods. Hence, the low metabolic costs of Oithona compared with calanoids may be one reason for their success in marine ecosystems.

Among the factors that regulate the success of copepod naupliar stages that allows the corroboration of this populations in the oceans, energetic balance is of prime hypothesis. Given the importance of the larval stages for importance. The assessment of metabolic budgets may the success of population recruitment, understanding the reveal important differences in the cost of maintenance and variability and the control exerted by the environmental in the efficiency of food utilization by different organisms (temperature, food availability, etc.) and inherent (age, size, that help to understand their evolutionary success. For this etc.) factors on their metabolic activity is of major reason, respiration and excretion rates of copepods have importance to comprehend the capacity of Oithona to been extensively investigated during the last century (for exploit diverse marine ecosystems. review, Marshall 1973; Ikeda 1985; Ikeda et al. 2001). The present study will provide basic information However, most of the available information stems from concerning the metabolic rates (respiration and excretion studies devoted to late copepod stages while copepod rates) of the naupliar and early copepodite stages of the nauplii have traditionally been ignored. Copepod nauplii genus Oithona. We used as a model the species Oithona are the most abundant forms of metazoans on the planet davisae that commonly inhabits productive embayments, (Fryer 1986) and the main prey of most fish larvae (Last where it can be the most abundant copepod (Uye and Sano 1980); therefore, their production contributes significantly 1995). The specific objectives of this study were to to the recruitment of commercially important fish species determine the respiration and ammonium and phosphate (Castonguay et al. 2008). Given that copepod nauplii may excretion rates of O. davisae early developmental stages in stand for an important fraction of the biomass of relation to body weight, temperature, and food availability, metazooplankton in spite of their small size (Calbet et al. and to estimate the assimilation and net growth efficiencies 2001) and that the specific metabolic rates are inversely deduced by combining the present respiration rates and the related to body mass (Ikeda 1985; Ikeda et al. 2001), the ingestion and growth rates reported from this and previous potential importance of nauplii in the energy flow of studies (Almeda et al. 2010 b). marine food webs must be stressed. Among copepods, the genus Oithona has been consid- Methods ered the most abundant and ubiquitous in the world’s oceans (Gallienne and Robins 2001). The success of Experimental organisms— The specimens of O. davisae oithonids over calanoids in some marine systems has been were obtained from a laboratory culture maintained in the attributed, among other reasons, to their comparatively Institut de Cie`ncies del Mar (CSIC, Barcelona, Spain) since low respiration rates (Lampitt and Gamble 1982; Paffen- October 2000. Oithona davisae was grown in 20-liter ho¨fer 1993; Castellani et al. 2005). However, there is no Plexiglas tanks, at 20 6 1uC in a constant-temperature information regarding the respiration rates of the Oithona room and under a 12 : 12 light : dark cycle. Copepod cultures were routinely fed a suspension of the heterotro- * Corresponding author: [email protected] phic dinoflagellate Oxyrrhis marina (equivalent spherical 403

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404 Almeda et al.

Table 1. Cohort composition (dominant stages) at the beginning of the experiments. Size (total body length for nauplii and prosome length for copepodites) and estimated body weight (W) of the Oithona davisae developmental stages used in the study are also provided. Carbon content was calculated from size using the equation reported in Almeda et al. (2010 a).

Cohort age Developmental Length 6 SE W 6 SE (h) stage (mm) (ng C ind. 21) 20 NI 84.4 60.6 28.3 60.4 48 NI–NII 93.4 60.8 34.7 60.7 72 NII–NIII 102.2 61.1 42.9 61.0 76 NII–NIII 107.3 61.2 46.9 61.1 83 NIII–NIV 114.5 61.2 54.8 61.2 96 NIV–NV 127.2 61.0 68.4 61.2 180 CII–CIII 210.9 61.5 176.0 62.1

oxygen concentration measurements was 1 min. Ammonia (NH 4-N) and phosphate (PO 4-P) concentrations were determined by the reactions of Berthelot and molybdate, respectively (Hansen and Koroleff 1999), using a Double Beam spectrophotometer (VarianCary H). The chemical Fig. 1. Schematic representation of incubation chambers used for the estimation of metabolic rates. analyses were made immediately after taking samples. Body weight effect on respiration and excretion rates— diameter [ESD] 5 16 mm). In turn, Oxyrrhis marina were Several cohorts of newly hatched nauplii were kept at fed the cryptophyte Rhodomonas salina (ESD 5 8 mm) saturating food conditions ( . 3000 cells mL 21, Ox. marina ) grown in f/2 medium. Prey sizes were measured using a during the time required to reach the desired developmental Coulter Multisizer III particle counter (Beckman Coulter). stage (Table 1). Developmental stages (at least 30 individuals To obtain cohorts of nauplii, adults (including egg-bearing [ind.]) were identified under an inverted microscope ( 3100) females) from the stock culture were removed with a 132- according to Uchima (1979). Prior to the experiments ( , mm-mesh-size sieve and placed in a new tank, where they 30 min), the individuals from each cohort were concentrated were fed ad libitum with Ox. marina (. 3000 cells mL 21, using a 37- mm mesh for nauplii and a 60- mm mesh for equivalent to . 660 mg C L 21). After 20 h, adults were copepodites, and thoroughly rinsed with autoclaved 0.2- mm- separated from the hatched nauplii with a 100- mm mesh. In filtered seawater (0.2- mm-FSW) to remove food and other order to remove the dislodged sacs of eggs, we allowed particles. We counted the organisms in each cohort by them to settle to the bottom of the tank, and subsequently triplicate under the microscope, and aliquots were added to the upper water containing only the nauplii were siphoned the experimental bottles (three replicates) to obtain the out to a new tank. desired density of experimental organisms (from 10 to 60 ind. mL 21, depending on the developmental stage). The bottles General experimental procedures— We conducted three were incubated in darkness at 20 uC ( 6 0.1 uC) in temperature- series of experiments in order to determine the influence of controlled water baths. Three additional bottles with only body weight, temperature, and food availability on the 0.2- mm-FSW were set as controls. The temperature of the metabolic rates of O. davisae early developmental stages. In water baths was continuously monitored during the incuba- all cases, respiration and excretion rates were estimated by the tion by an external sensor (ENDECO H). classical incubation method in closed chambers. These At the end of the incubation, three replicate water chambers consisted of Pyrex bottles (130-mL total volume) samples for ammonium and phosphate analysis were taken capped by silicon stoppers pierced by the oxygen probes and from each experimental and control bottle by siphoning by a syringe needle to compensate for pressure changes due to with a silicone tube with the submerged tip fitted with a 37- small temperature oscillations (Fig. 1). mm-mesh gauze to avoid losing experimental organisms. The respiration rates of copepod nauplii were measured The remaining water was sieved through a 37- mm mesh and using optical oxygen sensors (‘‘oxygen optodes,’’ Oxygen the experimental organisms concentrated and fixed with Dipping Probe DP-PSt3, Presens H), which consist of a fiber Lugol’s solution. Subsamples of nauplii were counted optic cable with a distal tip coated with a luminophore. The under an inverted microscope ( 340) to estimate their light emitted from the source (blue light-emitting diode) is concentration during the incubation, and 50 individuals transmitted through the fiber optic cable to the lumino- were measured by image analysis in order to estimate their phore. The intensity, lifetime, and modulation of phase carbon contents using the equation provided by Almeda et angle of the resulting fluorescence signal depend on the al. (2010 a). The mortality of nauplii or copepodites at the oxygen concentration (Holst et al. 1997). The frequency of end of the incubation experiments was always negligible.

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Metabolism of Oithona davisae nauplii 405

Temperature effect on respiration and excretion rates— ed as described above, with the exception of the incubation Oxygen consumption and ammonia and phosphate time that was shorter (16 h). excretion rates of O. davisae nauplii (NIII–NIV) were The specific ingestion and growth rates of fed nauplii simultaneously determined at four different temperatures were simultaneously determined as described below. (16 uC, 20 uC, 24 uC, 28 uC) in 0.2- mm-FSW. The tempera- tures used in this experiment are into the temperature Calculation of weight-specific respiration and excretion range experienced by this species in nature (8.9–28.2 uC, rates— The oxygen consumption rates were calculated as Uye and Sano 1995). A cohort of nauplii (NIII–NIV), the slopes of the linear regression equations relating obtained as described above, was divided into aliquots and incubation time and dissolved oxygen concentration for conditioned for , 2 h at each temperature under food each experimental condition. When the decrease in oxygen satiating conditions ( Ox. marina , . 3000 cells mL 21). concentration was not linear along the incubation After acclimation, nauplii were concentrated again using a (Fig. 2A,B), respiration rates were calculated using the 37- mm-mesh sieve rinsed in 0.2- mm-FSW. The experimen- linear decrease in oxygen during the first hours of tal concentration of nauplii ranged from 90 to 140 ind. incubation before the change of the slope. mL 21 and their length averaged 114.5 6 1.2 mm SE Respiration rates of starving nauplii (incubated without (54.8 ng C ind. 21). Control and experimental bottles were food) and Ox. marina bottles were determined after incubated in water baths at the corresponding tempera- subtracting the oxygen consumption rate in the control tures. The water-bath temperatures during the incubations bottles with only filtered seawater. The respiration rates of were continuously monitored by an external sensor feeding nauplii were calculated after taking into account also (ENDECO H). Water samples for ammonia and phosphate the oxygen consumption in the incubation bottles due to Ox. analysis and nauplii were collected at the end of the marina . In order to do that, the initial and final concentration incubation and processed as described above. The effect of of Ox. marina (cells mL 21) in the bottles with only Ox. temperature on respiration and excretion rates was marina and the bottles with the feeding nauplii (i.e., with Ox. estimated by the Q 10 value: marina ) were measured with a Coulter Multisizer, and the respective average concentrations during the incubation were 10 = T2 {T1 Q10 ~ M2=M1 ð Þ 1 determined according to Frost’s (1972) equations. Concen- ð Þ ð Þ trations were converted into carbon units from cell volume where M 2 and M 1 are the rates of the studied process at according to Pelegri et al. (1999). Carbon-specific respiration temperatures T2 and T1 (in uC), respectively. rates of Ox. marina were calculated in the Ox. marina bottles (without nauplii), and then were used to subtract the oxygen Food availability effects on respiration and excretion consumption by Ox. marina in the feeding nauplii bottles rates— The experimental setup consisted of measuring the using the average concentration of Ox. marina in them. The metabolic rates of (1) O. davisae nauplii (NII–NIII) with respiration rate of the feeding nauplii was estimated from the food ( Ox. marina ), (2) nauplii without food (in 0.2- mm- remaining oxygen consumption rate divided by the number FSW), (3) Ox. marina alone, and (4) control bottles with of nauplii incubated. only 0.2- mm-FSW. There were three to four replicates per Individual excretion rates were calculated as the difference treatment. The density of nauplii was , 15 ind. mL 21 on in NH 4-N and PO 4-P concentrations between experimental average. We used an Ox. marina concentration of 11,000 and control bottles at the end of the incubation divided by the cells mL 21 (2.75 mg C mL 21) to maintain saturating number of nauplii. In the experiments with feeding nauplii, conditions during the incubation. Nauplii were concentrat- excretion rates were corrected for the presence of Ox. marina ed, counted, and added to the experimental bottles as in the incubations similarly as described above for respiration described before. All these incubations were conducted at rates. constant temperature (20 uC). Prior to preparing the food Per capita respiration and excretion rates were converted suspensions for the experiment, the stock culture of Ox. into carbon-specific rates using the carbon content of marina was filtered through a 10- mm mesh to remove cell Oithona nauplii as estimated described above. Oxygen aggregation and other particles. The culture of Ox. marina consumption rates were transformed into carbon losses using was not fed the day before the experiments began in order a respiratory quotient of 0.97 (Omori and Ikeda 1984). The to ensure that all Rhodomonas salina were depleted by Ox. C : N (respired C : excreted N), N : P (excreted N : excreted P), marina . The absence of R. salina in stock bottles was and C : P (respired C : excreted P) metabolic ratios (by atoms) verified by checking with a Coulter Multisizer particle were calculated for each experiment in order to determine the counter. In order to reduce the number of bacteria in the stoichiometric composition of metabolic products as well as stock cultures, Ox. marina was concentrated by exposure to the type of catabolism (Omori and Ikeda 1984). low temperatures (4 uC), so that the dinoflagellates settled In the food effect experiment, the clearance and by reducing their swimming activity. After that, the ingestion rates were estimated according to Frost (1972) supernatant was carefully siphoned out and the dinoflagel- and the specific ingestion rates were calculated using the late resuspended again in filtered seawater; this washing average biomass of nauplii during the incubation. Specific protocol was repeated two times. Prior to the experiments, growth rates (G, mg C mg C 21 d21) were calculated as Ox. marina was acclimated to 20 uC and their swimming activity checked under an inverted microscope ( 3200). Incubation conditions and other procedures were conduct- G~ ln W =W =t 2 ½ ð 2 1ފ ðÞ

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where t is the duration of incubation (d), and W 1 and W 2 are the initial and final carbon content of the nauplii, respectively. Carbon content of nauplii was estimated as described above.

Metabolic balance: assimilation and net growth efficien- cies— The carbon budget corresponding to the larval stages of Oithona davisae was calculated by combining the metabolic rates and the feeding and growth rates deter- mined in the present study with the growth and ingestion rates under saturating food conditions estimated according to Almeda et al. (2010 b). The assimilation efficiency (AE), i.e., the percentage of ingested food that is digested, can be expressed as

AE ~ GzRÃ I|100 3 C ð Þ where G is the carbon-specificÀ growth Á rates ( mg C mg C 21 d21), 21 21 RC* is the carbon-specific respiration rates ( mg C mg C d ), and I is the carbon-specific ingestion rates ( mg C mg C 21 d21) under food saturation conditions. The net growth efficiency (NGE), i.e., the percentage of assimilated food converted into growth, was calculated as

NGE ~G GzRÃ |100 4 C ð Þ When carbon-specific respirationÀ Á rates were measured in filtered seawater (R C), the expected carbon-specific respi- ration rates under food saturation conditions (R C*) were estimated using the correction factor obtained in the food effect experiment ( see below).

Results

Effects of body weight and stage on respiration and excretion rates— Respiration rates increased potentially with 21 21 increasing body weight from , 0.015 mL O 2 ind. d in 21 21 early nauplii to , 0.07 mL O 2 ind. d in copepodites (Fig. 3A). Under similar conditions (at 20 uC and without food), weight-specific respiration rates declined with increas- ing body weight and stage from , 0.29 to 0.18 d 21 (Table 2). Ammonium and phosphate excretion rates (nmol ind. 21 d21) were potentially related to body weight (Fig. 3B,C). In contrast to weight-specific respiration rates, weight-specific excretion rates did not follow a clear pattern in relation to body weight or stage (Table 2). Under similar conditions (at 20 uC, without food, and incubation time . 20 h), the C : N and C : P metabolic ratios tended to decrease with increasing body weight and stage; in contrast, N : P ratios were positively related to body weight and stage, increasing from 9 to 21 (Table 2).

Effect of temperature on respiration and excretion rates— Fig. 2. Some illustrative examples of the temporal evolution Respiration rates of nauplii NIII–NV (54.8 ng C ind. 21) of oxygen concentration in the experimental chambers under followed an exponential increase as a function of temper- different experimental conditions. Control chambers are bottles ature (Fig. 4A), except for the experiments at 16 uC, which with only 0.2- mm-FSW, and experimental chambers correspond to rendered lower values ( see Discussion). Weight-specific (A) nauplii incubated in filtered seawater at 20 uC, (B) nauplii respiration rates increased from 0.10 d 21 at 16 uC to incubated in filtered seawater at 28 uC, and (C) nauplii incubated 21 0.35 d at 28 uC (Table 2). The calculated Q 10 values for with food ( Oxyrrhis marina ) at 20 uC. respiration decreased as increasing temperature (Q 10 [16– 19.3 uC] 5 6.5; Q 10 [19.3–24 uC] 5 2.0; Q 10 [24–28 uC] 5 1.7)

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and rendered a value of 2.64 considering the total range of temperature (16–28 uC). Both ammonium and phosphate excretion rates were fitted to an exponential model in the temperature range of 19.3 uC to 28 uC (Fig. 4B,C); as for the respiration rates, excretion at 16 uC, particularly for phosphate, was much lower than expected and excluded from the fitting. Weight-specific ammonium and phosphate excretion rates increased from 0.0011 to 0.0094 mmol NH 4- 21 21 N mmol C d and from 0.00010 to 0.00038 mmol PO 4-P 21 21 mmol C d at, respectively, 16 uC and 28 uC. The Q 10 for ammonium excretion decreased with increasing temperature (Q 10 [16–19.3 uC] 5 70.8; Q 10 [19.3–24 uC] 5 3.6; Q 10 [24– 28 uC] 5 1.7). The Q 10 for phosphate excretion were close to 1 at high temperatures (Q 10 [16–19.3 uC] 5 34.8; Q 10 [19.3– 24 uC] 5 1.1; Q 10 [24–28 uC] 5 1.2). The C : N metabolic ratio was significantly higher at 16 uC (ANOVA, Tukey test, F3,8 5 27.9, p , 0.01), whereas no significant differences were found among metabolic C : N ratios at 19.3 uC, 24.5 uC, and 28 uC. The N : P metabolic ratio increased with increasing temper- ature and C : P metabolic ratio did not follow any clear pattern with temperature (Table 2).

Effect of food availability on respiration and excretion rates— The average specific respiration rates of Ox. marina at 20 uC was 0.27 d 21, whereas specific ammonium and phosphate excretion rates of Ox. marina were 2.37 and 0.69 nmol mg C 21 d21, respectively. These values were used as described in the Methods section to correct the values obtained in incubations with feeding copepods. Feeding activity significantly increased the respiration rates of O. davisae nauplii by a factor of 2.3 in comparison with those estimated without food (ANOVA, F1,6 5 11.5, p , 0.05, Fig. 5A; Table 2). Similarly, the ammonium and phosphate excretion rates increased by a factor of 1.4 and 11.0, respectively (ANOVA, F1,6 5 19.5, p , 0.01 and F1,6 5 75.0, p , 0.01, respectively; Fig. 5B,C; Table 2). The C : N metabolic ratio for feeding nauplii was significantly higher than for those without food (ANOVA, F1,6 5 6.6, p , 0.05; Table 2); on the contrary, N : P and C : P metabolic ratios were significantly lower for fed than for unfed animals (ANOVA, F1,6 5 621.0, p , 0.01 and F1,6 5 163.8, p , 0.01, respectively; Table 2). Starvation effects were evident in experimental treat- ments without food because the decrease in oxygen concentration was not constant along the incubation time and depended on temperature (Fig. 2A,B). On the con- trary, the rate of oxygen consumption corresponding to nauplii feeding on Ox. marina was nearly constant (Fig. 2C). Excretion rates of unfed nauplii were negatively related to incubation time (Table 2). Ammonium and phosphate excretion rates at 20 uC decreased from 17 to 21 21 , 3 nmol NH 4-N mmol C Zoo d , and from , 0.86 21 21 Fig. 3. Relationships between carbon body weight and (A) to 0.41 nmol PO 4-P mmol C Zoo d , respectively, with respiration rate, (B) ammonium excretion rate, and (C) phosphate increasing incubation time from 16 to 25 h (Table 2). excretion rate for Oithona davisae larval developmental stages incubated in filtered seawater at 20 uC. Filled circles correspond to naupliar stages and open circles to copepodites. The continuous lines correspond to the allometric functions fitted to the data. W is r 21 21 21 21 the body weight (ng C ind. ), R is the respiration rate ( mL O 2 ind. d ), E P is the phosphate excretion rate (nmol PO 4-P ind. 21 21 21 2 ind. d ), E N is the ammonium excretion rate (nmol NH 4-N d ), and r is the coefficient of determination.

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The ingestion and clearance rates (avg. 6 SE) of nauplii NII–NIII (46.9 ng C ind. 21) were 243 6 10 cells ind. 21 d21 21 21 SE) and 0.023 6 0.001 mL ind. d , respectively. Specific C : P 6 21 21 ( ingestion and growth rates (d ) were 1.09 6 0.05 d and

), phosphate 21

1 0.22 6 0.03 d , respectively (Table 3). 2 d 1 . W, carbon body

2 Metabolic balance: assimilation and net growth efficien- SE)

Zoo cies— For the calculation of AE and NGE, carbon-specific N : P 6 ( respiration rates under food saturation conditions (R C*, mg C mg C 21 d21) were estimated as follows: mol C m

-N RÃ ~RC|2:3 5

Oithona davisae C 4

SE) ð Þ Methods, Food effect experiment). 6 C : N ( where R C is the carbon-specific respiration rates measured see with filtered seawater ( mg C mg C 21 d21 ) and 2.3 is the mol NH

m correction factor obtained in the food effect experiment. ,

N AE ranged from 65 % to 86 % and NGE varied from 23 % to

as food ( 32 % depending on body weight, stage, and temperature

SE) (Table 3). The AE was higher in early nauplii (NI–NIII) 6 ( than in later stages (NIV–CIII), and the NGE decreased P with increasing body size and stage (Table 3). AE tended to increase with increasing temperature (Table 3). With the exception of the value at 16 uC, the NGE kept nearly Oxyrrhis marina constant as related to temperature (Table 3). Figure 6 shows the carbon budget of nauplii in the food effect experiment where ingestion, growth, and respiration rates

SE) E were measured simultaneously under food saturation ), ammonia excretion rates (E 1 6 ( 2 conditions. According to this carbon budget, the carbon d N

1 losses corresponding to egestion and sloping feeding would 2 represent , 15 ng C d 21 (, 28 % of ingested C, Fig. 6). Zoo Discussion mol C m Effect of body weight and stage— Metabolic rates seem to SE) E

6 follow general scaling laws (Peters 1983), and examples for ( mol C m C allometric scaling in biological systems can be found from ,

C the cell level to the ecosystem level (West and Brown 2005). The question about the exact value of the power exponent was reopened in recent years and nowadays metabolic rates in many ectotherms are widely accepted to follow a three- quarters power law to body mass (power exponent 5 0.75,

C); t, incubation time (h); SE, standard error. All metabolic rates are from incubations conducted with filtered seawater, Peters 1983; West and Brown 2005). Similar relationships ), and metabolic quotients (C : N, N : P, C : P, by atoms) of different larval stages of u 1

2 between metabolic rates and body weight have been also

d found in copepods (Ikeda et al. 2001). However, these 1

2 relationships commonly exclude naupliar developmental

Zoo stages (Ikeda 1985; Ikeda et al. 2001). In the case of Oithona davisae , respiration rates of their developmental

mol C stages conform to the general three-quarters power law m scaling to body mass (Fig. 3A, power exponent 5 0.77). -P 4 Surprisingly, excretion rates as a function of body weight in our experiments showed a power exponent $ 1, probably due to the very low rates recorded for the smallest nauplii. mol PO m 28.334.742.968.4 NI NI–NII NII–NIII NIV–NV 20.0 20.0 20.0 20.0 20.3 23.3 25.3 21.4 0.258(0.008) 0.265(0.009) 0.288(0.021) 0.222(0.008) 0.00498(0.00018) 0.00352(0.00045) 0.01226(0.00094) — 0.00032(0.00002) 0.00041(0.00005) 0.00070(0.00009) 53(3) 84(9) 19(1) 16(1) 18(1) 9(2) — 832(55) 328(39) 718(86) — — — 54.854.854.8 NIII–NIV54.8 NIII–NIV NIII–NIV 16.046.9 NIII–NIV 19.3 24.5 20.2 28.0 NII–NIII 20.0 19.8 20.0 0.109(0.011) 19.6We 0.201(0.007) 0.290(0.011) cannot 16.2 0.00110(0.00013) 0.349(0.013) 0.00467(0.00014) *0.553(0.090) 0.00775(0.00044) discard 0.00010(0.00002) 0.00945(0.00036) 0.00033(0.00002) *0.02418(0.00160) 0.00035(0.00002) 101(11) 0.00038(0.00002) possible *0.00955(0.00100) 43(2) 38(4) 11(1) 37(3) 23(3) artifacts 14(0.3) 22(0.4) 1108(176) 25(0.3) 604(22) 3(0.3)to 845(74) 938(77) explain 62(16) such low ); T, temperature of incubation ( , 176.0 CII–CIII 20.0 20.1 0.185(0.008) 0.01176(0.00084) 0.00057(0.00005) 17(2) 21(0.5) 320(41) 1 P

2 values, for instance a strong reduction of excretion rates after 24 h of starvation. Average values of weight-specific respiration rates (R The metabolic quotients depend on the metabolic substrate of the animal (proteins, carbohydrates, or lipids; reviewed in Omori and Ikeda 1984). According to Ikeda Table 2. effect effect (1974), an O : N metabolic ratio of 24 (C : N 5 12) indicates Experiment W Stage T t R with the exception of those values indicated with an asterisk that were carried out with a suspension of weight (ng C ind. Body weight excretion rates (E Temperature Food effect 46.9that NII–NIIIprotein 20.0 16.2 and lipid 0.240(0.013) are 0.01685(0.00045) metabolized 0.00086(0.00004) in 14(0.5) equal 20(1) quantities 278(6) at

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the same time, whereas an O : N metabolic ratio , 24 (C : N , 12) indicates protein-oriented metabolism, and a ratio . 24 (C : N . 12) indicates lipid-oriented metabolism (Omori and Ikeda 1984). The C : N metabolic ratios of O. davisae developmental stages were higher than 12 (range 14–101, Table 2), hence indicating a lipid-based metabo- lism. The decrease in C : N metabolic ratios during the development of O. davisae may be a consequence of the differences in biochemical composition between stages. Early copepod nauplii usually have significant amounts of lipid reserves from yolk. Almeda et al. (2010 a) provided evidence that C : N composition ratio of O. davisae declined along their development, probably because early nauplii exhibit higher lipid reserves (remains of yolk) than later stages. This C : N composition pattern is partly reflected by the decrease in C : N metabolic ratios along the development (Table 2, body size effect experiment). The low N : P ratios are also characteristic of a lipid-based metabolism (Ikeda 1977; Omori and Ikeda 1984). However, differences on incubation time and starvation effects between experiments could mask some of these effects, as indicated by the relatively low C : N metabolic ratios obtained in short incubations ( , 16 h, Table 2). The effects of body size on the AE and NGE of zooplankton are unclear in the literature and different patterns have been reported depending on the species (Conover 1966, 1978) and developmental stage (Vidal 1980). Ontogenetic differences in AE and NGE of copepods may reflect differences in size-specific rates of anabolic and catabolic processes (Vidal 1980). At a given temperature (20 uC) and similar conditions (body weight effect, Table 3), the NGE of O. davisae larvae appeared inversely related to body weight following the trend observed in other copepods (Vidal 1980) and confirming the global pattern of NGE of marine copepods reported by Ikeda et al. (2001).

Effect of temperature on respiration and excretion rates— For poikilotherms like copepods one would expect metabolic rates to be higher at increasing temperature because of the dependence of biochemical kinetics on temperature (Arrhenius law). In our experiments respira- tion rates of O. davisae nauplii followed that law, in agreement with previous observations for calanoids (Ikeda et al. 2001) and adult stages of other oithonids (Castellani et al. 2005). This contrasts with Hiromi et al. (1988), who found no effect of temperature on respira- tion activity of adult stages of O. davisae over a wide temperature range (5–30 uC). Nevertheless, some copepods, mainly estuarine species, have been reported to exhibit the ability of dampening changes in their metabolic rates (homeostasis) in spite of temperature changes (low Q 10 ) as an adaptation to rapid temperature fluctuations (Gaudy et al. 2000). Fig. 4. Relationships between temperature and (A) respira- tion rates, (B) ammonium excretion rate, and (C) phosphate excretion rate for Oithona davisae nauplii (NIII–NIV) incubated r 21 21 in filtered seawater. The continuous lines correspond to the fitted d ), E N is the ammonium excretion rate (nmol NH 4-N ind. 21 21 21 exponential functions for temperatures between 19.3 uC and 28 uC. d ), E P is the phosphate excretion rate (nmol PO 4-P ind. d ), 21 2 T is the temperature ( uC), R is the respiration rate ( mL O 2 ind. and r is coefficient of correlation.

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We have noticed, however, that in our experiments, the lowest temperature (16 uC) rendered very low metabolic rates, likely reflecting the thermophilic character of this species (Uye and Sano 1995). O. davisae is a perennial species but its population density varies remarkably with season. This species is very scarce during winter and spring (when temperatures are , 20 uC), whereas it is very abundant during the warm seasons, with temperatures between 20 uC and 28 uC. Hence, 16 uC might be considered as suboptimal temperature for this species. With the exception of our lowest experimental temperature, the Q10 values obtained for respiration and ammonium excretion ( , 2.5) are within the range reported for other copepods (Ikeda et al. 2001; Castellani et al. 2005). Phosphate excretion rates showed a less clear pattern. Because the main component of yolk-sack lipovitellin of crustacean is phosphatidylcholine (Lee et al. 2006), the exhaustion of this reserve under starvation conditions and at higher temperatures may result in low phosphate excretion rates. The effect of temperature on the metabolic ratios of O. davisae also reflected effects of starvation. The C : N metabolic ratios decreased and the N : P increased as a function of temperature, indicating a protein-oriented metabolism at higher temperature, the result of a higher degree of starvation after the exhaustion of lipid reserves (Mayzaud and Conover 1988). The effects of temperature on assimilation efficiencies of zooplankton are controversial in the literature, with reports of both positive and negative as well as the lack of effects (Conover 1966; Chervin 1978). According to the global pattern of NGE of epipelagic marine copepods reported by Ikeda et al. (2001), the NGE is expected to decrease with increasing temperature. However, for a given species, the effects of temperature on NGE may differ depending on the developmental stage (Vidal 1980) and the range of thermal tolerance of species (Iguchi and Ikeda 2005). For O. davisae nauplii, with the exception of the value at 16 uC, the NGE kept nearly constant as related to temperature. According to this, the allocation of assimilated materials of O. davisae nauplii did not vary with a change in temperature, reflecting similar temperature dependence between physio- logical processes. It is supported by previous results showing that ingestion and growth rates (Almeda et al. 2010 b) have quite similar Q 10 values than respiration rates at similar temperature range.

Effect of food availability— Body size and temperature are considered to be the main factors influencing zoo- plankton metabolic rates. In their review, Ikeda et al. (2001) concluded that body weight and temperature explained 93–96 % of the variance in respiration rates and , 84 % and 87 % of the ammonium and phosphate excretion rates. However, it is important to note that most of the previous measurements of metabolic rates of Fig. 5. Effect of food concentration on (A) respiration rates, (B) ammonium excretion rates, and (C) phosphate excretion rates r 21 21 of Oithona davisae nauplii (NII–NIII) incubated at 20 uC. R is the excretion rate (nmol PO 4-P ind. d ). The columns represent 21 21 respiration rate ( mL O 2 ind. d ), E N is the ammonium the mean value of four replicates and the error bars the 21 21 excretion rate (nmol NH 4-N ind. d ), and E P is the phosphate corresponding standard error (SE).

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Table 3. Assimilation efficiencies (AE, %) and net growth efficiencies (NGE, %) of Oithona davisae developmental stages. W, carbon body weight; T, temperature ( uC); I, carbon-specific ingestion rates ( mg C mg C 21 d21); G, carbon-specific growth rates ( mg C mg C 21 d21); 21 21 RC, specific respiration rates with filtered seawater ( mg C mg C d ); R C*, specific respiration rates under food satiating concentrations (mg C mg C 21 d21). I and G were calculated from Almeda et al. (2010 b) except those from the ‘‘Food effect experiment’’ that were estimated from this study. Note that AE and NGE calculations were based on R C* ( see Methods).

Experiment W Stage T I G R C RC* AE NGE Body weight effect 28.3 NI 20.0 1.13 0.31 0.29 0.67 86 32 34.7 NI–NII 20.0 1.08 0.28 0.26 0.60 81 32 42.9 NII–NIII 20.0 1.03 0.25 0.27 0.62 85 29 68.4 NIV–NV 20.0 0.92 0.20 0.22 0.51 77 28 176.0 CII–CIII 20.0 0.74 0.13 0.19 0.44 77 23 Temperature effect 54.8 NIII–NIV 16.0 0.53 0.09 0.11 0.25 65 26 54.8 NIII–NIV 19.3 0.87 0.19 0.20 0.46 75 29 54.8 NIII–NIV 24.5 1.29 0.29 0.29 0.67 74 30 54.8 NIII–NIV 28.0 1.36 0.35 0.35 0.81 85 30 Food effect 46.9 NII–NIII 20.0 1.09 0.23 0.24 0.55 72 29

copepods were obtained in the absence of food. Food food may result in an increase of energetic expenditure availability appears to be the main factor driving copepod (Paffenho¨fer 1993, 2006). The respiration rates under feeding rates in the field (Saiz and Calbet 2007) and, starved conditions have been considered somewhere consequently, it must influence their metabolism. An between basal and routine metabolism (Prosser 1973; Ikeda increase in metabolic rates in fed animals has been et al. 2001), whereas the respiration rates in the presence of previously reported for calanoid copepods (Kiørboe et al. food may be closer to active metabolism. The use of filtered 1985). In the case of oithonids, respiration rates of fed adult seawater simplifies the experiments from a technical stages of O. davisae were between 1.4 and 2.8 times higher viewpoint, but it may result in an important underestima- than for starved animals (Nakata and Nakane 1987; tion of copepod metabolic rates when extrapolated to field Hiromi 1994) in clear agreement with our results for conditions. Moreover, not only the presence–absence of food naupliar stages. The observed increase of respiration rates is relevant for the correct estimation of the respiration rates in association with feeding activity is commonly referred to of copepods. The quantity and quality of food are also as ‘‘specific dynamic action’’ (SDA) and it is attributed to important factors (Conover 1966; Ikeda 1977) and, therefore, the cost of biosynthesis of new tissue from ingested food it should be considered for a better understanding of copepod (Kiørboe et al. 1985). However, besides SDA, the increase metabolism in nature. Similarly to respiration rates, zoo- on swimming and feeding activity under the presence of plankton excretion rates are positively affected by the presence of food (Takahashi and Ikeda 1975; Ikeda 1977). In O. davisae , the increase in ammonium excretion of fed nauplii was lower than previous reports for other zooplank- ton (factors of , 2 to 6, Takahashi and Ikeda 1975). However, the increase on phosphate excretion of fed nauplii was particularly high and it may be an artifact produced by the loss and fragmentation of food during the ingestion (sloppy feeding, Saba et al. 2009). Laboratory experiments under controlled conditions are a fundamental tool for understanding the effects of environmental variables on activity of marine zooplankton. Nonetheless, caution is required when extrapolating laboratory results to the field (e.g., crowding effects, lack of turbulence, etc.). Long incubation time in experiments with filtered seawater may result in starvation effects that translate into a progressive decrease in the metabolic rates of copepods (Mayzaud 1973, 1976; Ikeda 1977). In the case of O. davisae nauplii, starvation effects were evident in all experiments with filtered seawater since the decrease in oxygen consumption was nonlinear during the incubation Fig. 6. Carbon budget of Oithona davisae nauplii (NII–NIII) (Fig. 5), in agreement with previous reports for other at 20 uC and under saturating food conditions ( Oxyrrhis marina ). Egestion was calculated as the difference between ingested and zooplankton (Mayzaud 1976; Kiørboe et al. 1985). The assimilated carbon biomass. Carbon budget was calculated using observed changes in respiration rates were temperature the average carbon biomass of nauplii during the incubation dependent and may be related to changes in metabolic (50.7 ng C ind. 21). substrates under starving conditions (Conover 1978).

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Although some authors reported higher metabolic rates at factors, differences in thermal tolerance, prey preferences, the beginning of incubations as likely artifacts due to and sensory systems efficiency may explain the differences handling stress, our data show that oxygen consumption of in distribution and abundance among Oithona species. fed nauplii was linear along the incubation. The respiration However, it has been proposed that the common feature rates of marine invertebrates are also affected by the that explains the success of oithonids in marine environ- concentration of dissolved oxygen in the water (Prosser ments is their low metabolic requirements compared with 1973). For some copepods, a rapid decrease in the calanoids (Lampitt and Gamble 1982; Paffenho¨fer 1993; respiration rates is observed when the O 2 concentration Castellani et al. 2005). Although it is difficult to compare decreases to below a critical point (Marshall 1973). In the respiration rates between naupliar stages of oithonids and case of fed O. davisae nauplii, we observed a linear decrease calanoids due to the scarcity of data (Klekowski et al. 1977; in oxygen concentration until 60 % (Fig. 2C), indicating Ko¨ster et al. 2008), they appear to be significantly lower. that the changes of unfed nauplii respiration rates above When respiration rates (R) of nauplii of the calanoid this concentration are related to starvation and not to the copepod Eucalanus pileatus (R 5 0.297 d 21, body weight decrease in oxygen concentration. The possibility of [W] 5 2 mg C ind. 21, Ko¨ster et al. 2008), using the same detecting changes in the rate of oxygen consumption methodology and under fed conditions, are corrected for 0.75 during the incubation, either as a consequence of starvation size (R/W ) and temperature (Q 10 5 2.5), specific or by a limitation in the substrate (oxygen concentration), respiration rates of O. davisae happen to be . 5 times is a clear advantage of continuous measurement systems (as lower than those for E. pileatus nauplii. In addition, optodes) as compared to the classical Winkler bottle respiration rates of O. davisae nauplii (at 20 uC) were on method. average , 2.2 times lower than the values predicted by the Excretion rates of zooplankton decrease along the equation provided by Ikeda et al. (2001) for determining incubation time under starvation conditions (Mayzaud respiration rates of calanoid copepods (adults and cope- 1973), in agreement with our observations for O. davisae podites) as a function of body weight and temperature. nauplii. As mentioned above, excretion rates under This is in accordance with the fact that O. davisae nauplii, starvation will depend on the biochemical composition of as well as adult stages of other Oithona spp., have much the organism, those with high lipid contents being less lower ingestion and growth rates than similarly size susceptible to starvation in short incubation time (May- copepods (Lampitt 1978; Paffenho¨fer 1993; Saiz and Calbet zaud 1976). We were not able to correct the starvation 2007). These differences between the metabolic rates of effects on excretion rates due to the lack of continuous Oithona and calanoid copepods are not accompanied by monitoring of ammonium and phosphate concentrations differences in the AE and NGE. The majority of AE values that could allow establishing the relationships between reported here for O. davisae developmental stages fall excretion rates and incubation time. within the range of common values observed in calanoid The increase in C : N metabolic rates observed for fed O. copepods feeding on heterotrophic prey (between 70 % and davisae nauplii indicates a slightly higher lipid catabolism, 90 %, Conover 1978). Similarly, NGE of O. davisae larvae likely due to food used in our experiments ( Ox. marina ) that were within the range of the common values reported for has a high content and high capacity of producing essential adult stages of calanoid copepods (21–54 %, Ikeda et al. lipids that are efficiently supplied to copepods by trophic 2001). Therefore, O. davisae developmental stages exhibit transfer (Veloza et al. 2006). However, the low N : P and C : P quite similar food conversion efficiency compared to adult ratio when food was available may be the result of the calanoid copepod and copepodites. overestimation of phosphate excretion rates mentioned above. A likely explanation about the reasons of the low The AE and NGEs of zooplankton vary depending on metabolic requirements of Oithona as compared to food concentration and food quality (Paffenho¨fer and calanoids could be the differences in swimming and feeding Ko¨ster 2005). The use of respiration rates in filtered behavior (Paffenho¨fer 1993). In contrast to most calanoids, seawater for calculating metabolic balance may result in oithonids (nauplii and adults) move with occasional leaps important bias on the estimation of AE and NGE. For O. and rely on detecting prey by hydromechanical signals davisae developmental stages, the AE and NGE calculated (ambush feeders, Paffenho¨fer 1993; Svensen and Kiørboe using respiration rates in filtered seawater would range 2000; Paffenho¨fer and Mazzocchi 2002). As an example, in from 38 % to 53 % and from 41 % to 52 %, respectively. the absence of prey, Oithona davisae nauplii spent 98 % of Therefore, AE and NGE estimated using the respiration their time sinking, unmotile, and only under the presence of rates of nauplii with food were 1.7 times higher and 0.6 motile prey increase the jumping frequency (Henriksen et times lower, respectively, than those calculated using al. 2007). This feeding and swimming strategy of Oithona is respiration rates of nauplii without food. hypothesized to be more energetic–efficient than that of most calanoids (Paffenho¨fer 1993). Metabolic requirements and NGEs of O. davisae early In summary, two important conclusions can be high- developmental stages compared with other copepods— lighted from this work. First, as a general methodological Oithona is able to grow well both in low and high food aspect in zooplankton physiology, the importance of concentration environments (Calbet and Agustı´ 1999), measuring respiration rates with food. Respiration rates whereas calanoid broadcasters are frequently dominant without food result in a significant underestimation of only in high productive areas or seasonally during the copepod metabolic rates and, in addition, the use of phytoplankton blooms (Peterson 1998). Besides other respiration rates measured in filtered seawater led to

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Metabolism of Oithona davisae nauplii 413 important errors in the estimations of assimilation and GALLIENNE , C. P., AND D. B. R OBINS . 2001. Is Oithona the most net growth efficiencies. Therefore, measurements of zoo- important copepod in the world’s oceans? J. Plankton Res. 23: plankton respiration rates with food are required for a 1421–1432, doi: 10.1093/plankt/23.12.1421 correct evaluation of the importance of metazoan zoo- GAUDY , R., G. C ERVETTO , AND M. P AGANO . 2000. Comparison of plankton in marine carbon fluxes. Second, particularly for the metabolism of Acartia clause and A. tonsa : Influence of temperature and salinity. J. Exp. Mar. Biol. Ecol. 247: 51–63, the studied copepod genus, our study shows that the AEs doi: 10.1016/S0022-0981(00)00139-8 and growth efficiencies of Oithona nauplii are similar to HANSEN , H. P., AND F. K OROLEFF . 1999. Determination of nutrients, those exhibited by similarly sized calanoids, whereas their p. 159–228. In K. Grasshoff, K. Kremling, and M. Ehrhardt specific respiratory losses are comparatively lower; our [eds.], Methods of seawater analysis. Wiley-VCH. results, therefore, support the hypothesis that the wide HENRIKSEN , C. I., E. S AIZ , A. C ALBET , AND B. W. H ANSEN . 2007. distribution and high degree of ecological success of Feeding activity and swimming patterns of Acartia grani and Oithona in marine ecosystems is in part explained by their Oithona davisae nauplii in the presence of motile and non- low metabolic losses. motile prey. Mar. Ecol. Prog. Ser. 331: 119–129, doi: 10.3354/ meps331119 HIROMI , J. 1994. Further studies on respiration of the small Acknowledgments planktonic copepod Oithona davisae with special reference to We thank E. Velasco and P. Jime´nez for their help in maintaining the effect of feeding. Bull. Coll. Agric. Vet. Med. Nihon Univ. the algae and copepod cultures. This work was funded by the 51: 149–153. Spanish Ministry of Science and Innovation (MICINN) through a ———, T. N AGATA , AND S. K ADOTA . 1988. Respiration of the Ph.D. fellowship to R.A. (BES-2005-7491) and the research projects small planktonic copepod Oithona davisae at different CTM2004-02775 and Intramural-200630I226 to A.C., CTM2007- temperatures. Bull. Plankton Soc. Jpn. 35: 143–148. 60052 to E.S., and CTM2006-12344 to M.A. HOLST , G., R. N. G LUD , M. K U¨ HL , AND I. K LIMANT . 1997. A microoptode array for fine-scale measurement of oxygen References distribution. Sens. Actuators B Chem. 38–39: 122–129, doi: 10.1016/S0925-4005(97)80181-5 ALMEDA , R., M. A LCARAZ , A. C ALBET , L. Y EBRA , AND E. S AIZ . IGUCHI , N., AND T. I KEDA . 2005. Effects of temperature on 2010 a. Effect of temperature and food concentration on metabolism, growth and growth efficiency of Thysanoessa survival, development and growth rates of naupliar stages of longipes (Crustacea:Euphausiacea) in the Japan Sea. J. Oithona davisae (Copepoda, Cyclopoida). Mar. Ecol. Prog. Plankton Res. 27: 1–10, doi: 10.1093/plankt/fbh146 Ser. 410: 97–109, doi: 10.3354/meps08625 IKEDA , T. 1974. Nutritional ecology of marine zooplankton. Mem. ———, C. B. A UGUSTIN , M. A LCARAZ , A. C ALBET , AND E. S AIZ . Fac. Fish. Hokkaido Univ. 22: 1–97. 2010 b. Feeding rates and gross growth efficiencies of larval ———. 1977. The effect of laboratory conditions on the developmental stages of Oithona davisae (Copepoda, Cyclo- extrapolation of experimental measurements to the ecology poida). J. Exp. Mar. Biol. Ecol. 387: 24–35, doi: 10.1016/ of marine zooplankton. IV. Changes in respiration and j.jembe.2010.03.002 excretion rates of boreal zooplankton species maintained CALBET , A., AND S. A GUSTI´. 1999. Latitudinal changes of copepod under fed and starved conditions. Mar. Biol. 41: 241–252, egg production rates in Atlantic waters: Temperature and doi: 10.1007/BF00394910 food availability as the main driving factors. Mar. Ecol. Prog. ———. 1985. Metabolic rates of epipelagic marine zooplankton Ser. 181: 155–162, doi: 10.3354/meps181155 as a function of body mass and temperature. Mar. Biol. 85: ———, S. G ARRIDO , E. S AIZ , A. A LCARAZ , AND C. M. D UARTE . 1–11, doi: 10.1007/BF00396409 2001. Annual zooplankton succession in coastal NW Medi- ———, Y. K ANNO , K. O ZAKI , AND A. S HINADA . 2001. Metabolic terranean waters: The importance of the smaller size fractions. rates of epipelagic marine copepods as a function of body J. Plankton Res. 23: 319–331, doi: 10.1093/plankt/23.3.319 mass and temperature. Mar. Biol. 139: 587–596. CASTELLANI , C., C. R OBINSON , T. S MITH , AND R. S. L AMPITT . 2005. KIØRBOE , T., F. M OHLENBERG , AND K. H AMBURGER . 1985. Temperature affects respiration rate of Oithona similis . Mar. Bioenergetics of the planktonic copepod Acartia tonsa : Ecol. Prog. Ser. 285: 129–135, doi: 10.3354/meps285129 Relation between feeding, egg production and respiration, CASTONGUAY , M., S. P LOURDE , D. R OBERT , J. A. R UNGE , AND L. and composition of specific dynamic action. Mar. Ecol. Prog. FORTIER . 2008. Copepod production drives recruitment in a Ser. 26: 85–97, doi: 10.3354/meps026085 marine fish. Can. J. Fish. Aquat. Sci. 65: 1528–1531, doi: 10.1139/ KLEKOWSKI , R. Z., I. V. K UKINA , AND N. I. T UMANSEVA . 1977. F08-126 Respiration in the microzooplankton of the equatorial CHERVIN , M. B. 1978. Assimilation of particulate organic carbon upwellings in the eastern Pacific Ocean. Pol. Arch. Hydrobiol. by estuarine and coastal copepods. Mar. Biol. 49: 265–275, (suppl.) 24: 467–489. doi: 10.1007/BF00391139 KO¨ STER , M., C. K RAUSE , AND G. A. P AFFENHO ¨ FER . 2008. Time- CONOVER , R. J. 1966. Assimilation of organic matter by series measurements of oxygen consumption of copepod zooplankton. Limnol. Oceanogr. 11: 338–345, doi: 10.4319/ nauplii. Mar. Ecol. Prog. Ser. 353: 157–164, doi: 10.3354/ lo.1966.11.3.0338 meps07185 ———. 1978. Transformation of organic matter, p. 221–500. In LAMPITT , R. S. 1978. Carnivorous feeding by a small marine O. Kinne [ed.], Marine ecology, v. 4. Wiley and Sons. copepod. Limnol. Oceanogr. 23: 1228–1231, doi: 10.4319/ FROST , B. W. 1972. Effects of size and concentration of food lo.1978.23.6.1228 particles on the feeding behaviour of the marine planktonic ———, AND J. C. G AMBLE . 1982. Diet and respiration of the small copepod Calanus pacificus . Limnol. Oceanogr. 17: 805–815, planktonic marine copepod Oithona nana . Mar. Biol. 66: doi: 10.4319/lo.1972.17.6.0805 185–190, doi: 10.1007/BF00397192 FRYER , G. 1986. Structure, function and behaviour, and the LAST , J. M. 1980. The food of twenty species of fish larvae in the elucidation of evolution in copepods and other crustaceans. west-central North Sea. Fish. Res. Tech. Rep. 60. MAFF Syllogeus 58: 150–157. Directorate of Fisheries Research, Lowestoft.

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LEE , R. F., W. H AGEN , AND G. K ATTNER . 2006. Lipid storage in SABA , G. K., D. K. S TEINBERG , AND D. A. B RONK . 2009. Effects of marine zooplankton. Mar. Ecol. Prog. Ser. 307: 273–306, diet on release of dissolved organic and inorganic nutrients by doi: 10.3354/meps307273 the copepod Acartia tonsa . Mar. Ecol. Prog. Ser. 386: MARSHALL , S. M. 1973. Respiration and feeding in copepods. Adv. 147–161, doi: 10.3354/meps08070 Mar. Biol. 11: 57–120, doi: 10.1016/S0065-2881(08)60268-0 SAIZ , E., AND A. C ALBET . 2007. Scaling of feeding in marine MAYZAUD , P. 1973. Respiration and nitrogen excretion of zoo- calanoid copepods. Limnol. Oceanogr. 52: 668–675, plankton. II. Studies of the metabolic characteristics of starved doi: 10.4319/lo.2007.52.2.0668 animals. Mar. Biol. 21: 19–28, doi: 10.1007/BF00351188 SVENSEN , C., AND T. K IØRBOE . 2000. Remote prey detection in ———. 1976. Respiration and nitrogen excretion of zooplankton: Oithona similis : Hydromechanical versus chemical cues. J. IV. The influence of starvation on the metabolism and the Plankton Res. 22: 1155–1166, doi: 10.1093/plankt/22.6.1155 biochemical composition of some species. Mar. Biol. 37: TAKAHASHI , M., AND T. I KEDA . 1975. Excretion of ammonia and 47–58, doi: 10.1007/BF00386778 inorganic phosphorus by Euphausia pacifica and Metridia ———, AND R. J. C ONOVER . 1988. O : N ratio as a tool to describe paeifiea at different concentrations of phytoplankton. J. Fish. zooplankton metabolism. Mar. Ecol. Prog. Ser. 45: 289–302, Res. Board Can. 32: 2189–2195. doi: 10.3354/meps045289 UCHIMA , M. 1979. Morphological observation of developmental NAKATA , K., AND T. N AKANE . 1987. Respiration of plankton in the stages in Oithona brevicornis (Copepoda, Cyclopoida). Bull. Mikawa Bay. Pollut. Contr. 22: 281–294. Plankton Soc. Jpn. 26: 59–76. OMORI , M., AND T. I KEDA . 1984. Methods in marine zooplankton UYE , S., AND K. S ANO . 1995. Seasonal reproductive biology of the ecology. John Wiley & Sons. small cyclopoid copepod Oithona davisae in a temperate PAFFENHO ¨ FER , G. A. 1993. On the ecology of marine cyclopoid eutrophic inlet. Mar. Ecol. Prog. Ser. 118: 121–128, copepods (Crustacea, Copepoda). J. Plankton Res. 15: 37–55, doi: 10.3354/meps118121 doi: 10.1093/plankt/15.1.37 VELOZA , A. J., F. L. E. C HU , AND K. W. T ANG . 2006. Trophic ———. 2006. Oxygen consumption in relation to motion in modification of essential fatty acids by heterotrophic marine planktonic copepods. Mar. Ecol. Prog. Ser. 317: 187–192, doi: 10.3354/meps317187 protists and its effects on the fatty acid composition of the copepod Acartia tonsa. Mar. Biol. 148: 779–788, ———, AND M. K O¨ STER . 2005. Digestion of diatoms by planktonic copepods and doliolids. Mar. Ecol. Prog. Ser. doi: 10.1007/s00227-005-0123-1 297: 303–310, doi: 10.3354/meps297303 VIDAL , J. 1980. Physioecology of zooplankton. IV. Effects of phytoplankton concentration, temperature, and body size on ———, AND M. G. M AZZOCCHI . 2002. On some aspects of the behaviour of Oithona plumifera (Copepoda: Cyclopoida). J. the net production efficiency of Calanus pacificus . Mar. Biol. Plankton Res. 24: 129–135, doi: 10.1093/plankt/24.2.129 56: 203–211, doi: 10.1007/BF00645344 PELEGRI , S., J. D OLAN , AND F. R ASSOULZADEGAN . 1999. Use of high WEST , G. B., AND J. H. B ROWN . 2005. The origin of allometric temperature catalytic oxidation (HTCO) to measure carbon scaling laws in biology from genomes to ecosystems: Towards content of microorganisms. Aquat. Microb. Ecol. 16: a quantitative unifying theory of biological structure and 273–280, doi: 10.3354/ame016273 organization. J. Exp. Biol. 208: 1575–1592, doi: 10.1242/ PETERS , R. H. 1983. Ecological implications of body size. jeb.01589 Cambridge Univ. Press. PETERSON , W. 1998. Life cycle strategies of copepods in coastal upwelling zones. J. Mar. Syst. 15: 313–326, doi: 10.1016/ Associate editor: Mikhail V. Zubkov S0924-7963(97)00082-1 PROSSER , C. L. 1973. Oxygen: Respiration and metabolism, p. 165– Received: 01 June 2010 211. In C. L. Prosser [ed.], Comparative animal physiology. Accepted: 19 October 2010 W. B. Saunders. Amended: 08 November 2010

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Resum de l’article VI– Article VI summary– Catalan version

Taxes metabòliques i balanç energètic de les fases primerenques de desenvolupament del copèpode ciclopoide marí Oithona davisae

Rodrigo Almeda, Miquel Alcaraz, Albert Calbet, Enric Saiz Article publicat a Limnology and Oceanography (2011, 56(1): 403-414)

El gènere de copèpodes Oithon a ha estat considerat el més abundant i ubic dels oceans del planeta. No obstant això, malgrat la seva importància, el metabolisme de les seves fases de desenvolupament (nauplis i copepodits), crucial per explicar el seu èxit evolutiu, és gairebé desconegut. Es van determinar les taxes de respiració, les taxes d'excreció d'amoni i fosfat, i les eficiències de creixement net de les fases primerenques de desenvolupament d’ Oithona davisae en funció de la fase de desenvolupament, el pes corporal, la temperatura i la disponibilitat d'aliment. La respiració i les taxes d'excreció es van incrementar amb l'augment de pes corporal i es van relacionar positivament amb la temperatura i l'aliment. Les taxes específiques de respiració de nauplis i copepodits van variar entre 0.11 i 0.55 d -1 en funció de la fase de desenvolupament, el pes corporal, la temperatura i la disponibilitat d'aliment. Els ràtios de C: N metabòlics van ser majors de 14, la qual cosa indica un metabolisme basat en lípids. Les eficiències d'assimilació i les eficiències netes de creixement van variar entre 65% i 86% i entre 23% i 32%, respectivament, depenent del pes corporal, la fase de desenvolupament i la temperatura. Les eficiències d'assimilació i les eficiències netes de creixement calculades usant les taxes de respiració de nauplis amb aliment van ser 1.7 vegades més altes i 0.6 més baixes, respectivament, que les estimades usant les taxes respiratòries de nauplis sense aliment. Per tant, l'ús de taxes respiratòries mesurades en aigua de mar filtrada condueix a errors substancials en les estimacions del balanç del carboni del zooplàncton. Les fases de desenvolupament d' O. davisae presenten eficiències d'assimilació i creixement similars a les copèpodes calanoides, però pèrdues respiratòries específiques en carboni més baixes. Per tant, els baixos costos metabòlics d’ Oithona en comparació dels de calanoides pot ser una de la raons del seu èxit en els ecosistemes marins.

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Resumen del artículo VI – Article VI summary– Spanish version

Tasas metabólicas y balance energético de las fases tempranas de desarrollo del copépodo ciclopoide marino Oithona davisae

Rodrigo Almeda, Miquel Alcaraz, Albert Calbet, Enric Saiz Article publicat a Limnology and Oceanography (2011, 56(1): 403-414)

El género de copépodos Oithona ha sido considerado el más abundante y ubicuo de los océanos del planeta. Sin embargo, a pesar de su importancia, el metabolismo de sus fases de desarrollo (nauplios y copepoditos), crucial para explicar su éxito evolutivo, es casi desconocido. Se determinaron las tasas de respiración, las tasas de excreción de amonio y fosfato, y las eficiencias de crecimiento neto de las fases tempranas de desarrollo de Oithona davisae en función de la fase de desarrollo, el peso corporal, la temperatura y la disponibilidad de alimento. La respiración y las tasas de excreción se incrementaron con el aumento de peso corporal y se relacionaron positivamente con la temperatura y el alimento. Las tasas específicas de respiración de nauplios y copepoditos variaron entre 0.11 y 0.55 d -1 en función de la fase de desarrollo, el peso corporal, la temperatura y la disponibilidad de alimento. Los ratios de C: N metabólicos fueron mayores de 14, lo que indica un metabolismo basado en lípidos. Las eficiencias de asimilación y las eficiencias netas de crecimiento variaron entre 65% y 86% y entre 23% y 32%, respectivamente, dependiendo del peso corporal, la fase de desarrollo y la temperatura. Las eficiencias de asimilación y las eficiencias netas de crecimiento calculadas usando las tasas de respiración de nauplios con alimento fueron 1.7 veces más altas y 0.6 más bajas, respectivamente, que las estimadas usando las tasas respiratorias de nauplios sin alimento. Por consiguiente, el uso de tasas respiratorias medidas en agua de mar filtrada conduce a errores sustanciales en las estimaciones del balance del carbono del zooplancton. Las fases de desarrollo de O. davisae presentan eficiencias de asimilación y crecimiento similares a las copépodos calanoides, pero pérdidas respiratorias específicas en carbono más bajas. Por lo tanto, los bajos costes metabólicos de Oithona en comparación con los de calanoides puede ser una de la razones de su éxito en los ecosistemas marinos.

162 Ecophysiology of planktonic larvae of the spionid poly chaete Poly dora ciliata

Swimming behavior and prey retention of the poly chaete larvae Poly dora ciliata (Johnston)

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The Journal of Experimental Biology 213, 3237-3246 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jeb.038810

Swimming behavior and prey retention of the polychaete larvae Polydora ciliata (Johnston)

B. W. Hansen 1, H. H. Jakobsen 2, *, A. Andersen 3, R. Almeda 4, T. M. Pedersen 1, A. M. Christensen 1 and B. Nilsson 1 1Roskilde University, Department of Environmental, Social and Spatial Change, P O Box 260, DK-4000 Roskilde, Denmark, 2National Institute of Aquatic Resources, Charlottenlund Slot, Jægersborg Allé 1, DK-2920 Charlottenlund, Denmark, 3Department of Physics and Center for Fluid Dynamics, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark and 4Institut de Ciències del Mar, CSIC P. Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain *Author for correspondence at present address: National Environmental Research Institute, Aarhus University, Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmark ([email protected])

SUMMARY The behavior of the ubiquitous estuarine planktotrophic spionid polychaete larvae Polydora ciliata was studied. We describe ontogenetic changes in morphology, swimming speed and feeding rates and have developed a simple swimming model using low Reynolds number hydrodynamics. In the model we assumed that the ciliary swimming apparatus is primarily composed of the prototroch and secondarily by the telotroch. The model predicted swimming speeds and feeding rates that corresponded well with the measured speeds and rates. Applying empirical data to the model, we were able to explain the profound decrease in specific feeding rates and the observed increase in the difference between upward and downward swimming speeds with larval size. We estimated a critical larval length above which the buoyancy-corrected weight of the larva exceeds the propulsion force generated by the ciliary swimming apparatus and thus forces the larva to the bottom. This modeled critical larval length corresponded to approximately 1 mm, at which, according to the literature, competence for metamorphosis and no more length increase is observed. These findings may have general implications for all planktivorous polychaete larvae that feed without trailing threads. We observed bell shaped particle retention spectra with a minimum prey size of approximately 4 m equivalent spherical diameter, and we found that an ontogenetic increase in maximum prey size add to a reduction in intra-specific food competition in the various larval stages. In a grazing experiment using natural seawater, ciliates were cleared approximately 50% more efficiently than similar sized dinoflagellates. The prey sizes retainable for P. ciliata larvae covers the microplankton fraction and includes non-motile as well as motile prey items, which is why the larvae are trophically positioned among the copepods and dinoflagellates. Not only do larval morphology and behavior govern larval feeding, prey behavior also influences the feeding efficiency of Polydora ciliata . Key words: Polydora ciliata , low Reynolds number hydrodynamics, swimming behavior, prey selection.

INTRODUCTION three-quarters of the head circumference, and secondarily by a band Spionid polychaetes are some of the most common benthic of cilia around the terminal segment (the telotroch). Many spionids invertebrates of neritic environments. Hence, during certain periods, lack feeding cilia (metatroch) and food groove, and must therefore thei r planktotrophic larvae are very abundant in coastal waters. They capture particles using alternative mechanisms (Pernet and reach concentrations >1000 l
1 and are a major component of the McArthur, 2006). This is most likely the case for P. ciliata larvae. zooplankton community (Anger et al., 1986; Zajac, 1991; Bochert Data on particle size retention in polychaete larvae are scarce. and Brick, 1996; Pedersen et al., 2008). Spionids are one of the most For example, the upper limit for ingestible particle size increased common and widespread polychaete families in the Oslofjord, with larval size for Streblospio benedicti (Pernet and McArthur, Norway (Schram, 1968) and in the Iseford complex, Denmark 2006) and also for a gastropod veliger. For the gastropod veliger it (Rasmussen, 1973) from which the present larval material was was simply explained by changes in morphological dimensions of collected. Among spionids, Polydora ciliata is one of the dominant the feeding apparatus (velum structures) (Hansen, 1991). The species, with a wide geographic distribution in Northwest Europe, linkage between particle retention mechanism and the morphology and it is also found in Australian and Japanese waters as an introduced of the ciliary feeding apparatus are not reported for Polydora larvae. species (NIMPIS, 2002). P. ciliata larvae may be the single most In the spionid Marenzelleria cf . viridis phytoplankton <20  m were dominant polychaete larvae in neritic waters and reach concentrations the principal dietary component and a maximum prey diameter of >500 l
1 and their potential trophic role cannot be neglected (Daro 80  m is described for larvae with 6
10 segments (setigers) and Polk, 1973, Pedersen et al., 2008; Pedersen et al., 2010). (Burckhart et al., 1997). Polydora has been reported to have been Fig. 1 shows P. ciliata in the early, intermediate, and late larval reared on microalgae (Anger et al., 1986), and Daro and Polk (Daro stage. Polydora spp. larval morphology is described in detail by and Polk, 1973) reported small phytoplankters and even invertebrate Wilson (Wilson, 1928), Hannertz (Hannertz, 1956), Blake (Blake, larvae as prey. No comprehensive uptake efficiencies of a coherent 1969) and Plate and Husemann (Plate and Husemann, 1994). Based broad regime of prey sizes are reported for any spionid larvae. on the detailed sketches by Wilson (Wilson, 1928), we presume Here we address prey size selectivity and the swimming patterns that the ciliary swimming apparatus is primarily composed of a band of Polydora larvae following offerings of different sized prey either of closely spaced cilia (the prototroch), surrounding approximately as single species or as mixtures of in situ prey. To facilitate the

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Fig. 1. Differential interference contrast (DIC) microscope images of early, intermediate and late Polydora ciliata larvae. The images are composed of multiple layers from different focal planes sliced through the larvae and software- merged into the final images. The larvae were fixated with a few drops of saturated MgCl 2. Apart from some body contraction, the fixation did not seem to damage the larvae.

interpretation of the observed development in ontogenetic swimming Swimming behavior behaviors we developed a simple swimming model based on low Swimming of P. ciliata larvae ( N58) of various lengths was studied Reynolds number hydrodynamics. Using the modeling approach we in a 15 ϫ15 ϫ15 cm 3 acrylic aquarium at a temperature of 20°C and further interpreted our observations of the maximum clearance rate a salinity of 15 PSU. The setup allowed us to visualize swimming and its dependence on larval size. Thus our aim was to explore the trajectories spatially and estimate true three-dimensional swimming interaction between functional morphology and behavioral velocities and behaviors. Briefly, a mirror was mounted transversely components in spionid larval suspension feeding. inside the aquarium allowing direct observations of larvae and their mirrored images simultaneously. Illumination was provided by an MATERIALS AND METHODS infrared light-emitting diode placed perpendicular to the camera. Larval collection The diode was positioned in the focal point of a Fresnel lens Live zooplankton was collected from surface water (0
2.5 m) with (diameter 16 cm), thus creating parallel (collimated) light beams in a 50  m WP-2 net equipped with a non-filtering cod-end by the experimental aquarium and in turn mirrored into a monochrome horizontal trawling at slow speed. The sampling station analogue CCD video camera (Minitron MTV-1802CD, Minitron Søminestationen (55° 44 ЈN, 11°48 ЈE) is situated in the Isefjord, Enterprise Company, Taipei, Taiwan) fitted with a 105 mm lens. Denmark, and it is shallow with muddy bottom sediments and Size calibration was done by filming a ruler and converting pixels eutrophic waters. Once the net was retrieved, zooplankton was gently to millimeters. Video observations were recorded on a VHS tape transferred to an isothermic container, diluted with surface water, recorder. After recording the video sequences of interest were and transported to the laboratory within 2 h of collection. In the digitized using a Pinnacle movie box TM (Pinnacle Systems, laboratory, P. ciliata larvae were concentrated using a cold fiber Mountain View, CA, USA) into QuickTime TM . Movies and motion optic light source, sorted with a pipette under the dissecting tracks were analyzed using a motion analysis software package microscope and placed in a beaker with 0.2  m filtered seawater (LabTrack, Bioras.dk, Nivå, Denmark). After the motion tracks were (FSW) from the sampling area. In order to reduce contamination digitized, three-dimensional tracks were reconstructed by merging by other planktonic organisms, the larvae were washed repeatedly each motion track with the mirror track manually. Larval swimming by transferring them through a series of Petri dishes with 0.2  m was studied in three prey regimes: filtered seawater, filtered seawater FSW until ambient phytoplankton were absent. with Rhodomonas salina added as small prey, and filtered seawater

Table 1. Polydora ciliata larval swimming speed in three different prey regimes

–1 –1 –1 Prey species (size, ESD) Vdown (mm s ± s.d.) N V up (mm s ± s.d.) N V (mm s ± s.d.) N Filtered seawater 1.03±0.39 5 0.43±0.11 4 0.66±0.25 2 Rhodomonas salina (9 m) 0.82±0.46 16 0.35±0.22 8 0.45±0.10 9 Akashiwo sanguinea (53 m) 1.02±0.43 15 0.46±0.19 12 0.62±0.19 8 ESD, equivalent spherical diameter.

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Table 2. Characteristics of the Polydora ciliata larval stages used in the feeding experiments Larval stage Length ( m) Number of setigers Biomass ( g C)** Prototroch cilia length ( m) Telotroch cilia length ( m) Early 220±18 3.5±0.8 0.270 26.8±1 25±8 Intermediate 561±27 8.9±1.7 0.982 44.5±8 32.3±8.8 Late* 919±16 13.3±1.9 1.941 48.1±3 32.3±11 *With palps. **Larval carbon biomass was calculated based on length (Hansen, 1999). Values are means ± s.d. with Akashiwo sanguinea added as large prey (Table 1). Length and of air bubbles. Bottles were incubated for 24 h on a slowly rotating diameter of live animals ( N57) were estimated from size-calibrated plankton wheel (0.5 r.p.m.) in a temperature-controlled room at 16°C video frames in the software package ImageJ (http://rsbweb.nih.gov/ and constant dim light (<5  mol photons m
2 s
1 ). ij/index.html). Cell concentrations in grazing and control trials were determined by a CytoBuoy scanning flow cytometer (CSFC; CytoBuoy b.v., Cilia measurements Woerden, The Netherlands). The CSFC is designed to process live A sub-sample of ( N25) live larvae of a wide range of development phytoplankton particles larger than 1  m. In this study we counted sizes was sedated with a few drops of MgCl 2 in a small 4 mm deep around 0.5 ml samples in triplicate. The prey cell in the experimental micro-well and lengths of the prototroch, the telotroch and the larval trials was counted by leaving the CSFC sample suction tube directly body were measured. Larvae were separated in three groups into the incubation bottles. During counting, the trial bottles were according to size (Table 2). The MgCl 2 was applied until the larvae carefully stirred with a mechanical stirrer set at the lowest r.p.m. ceased moving. Apart from inducing body shrinkage, MgCl 2 seemed Prior to starting a series of grazing trials, we compared the counting not to damage the larvae, as they recovered after a short period of performance of CSFC to the electronic particle counter and found time and resumed swimming. The larvae were photographed under no significant deviation between the instruments (Almeda et al., a microscope at 100 ϫ magnification and the images were 2009). Clearance and ingestion rates were calculated for each food subsequently analyzed using the software package ImageJ. concentration according to Frost (Frost, 1972) after the verification that prey growth rates in grazing bottles were significantly lower Maximum clearance and ingestion rates than in the prey growth controls (one-way ANOVA, P<0.05) and The functional response for three different development stages of P. the maximum rates derived from the functional response equations. ciliata larvae fed the diatom Thalassiosira weissflogii was determined. The full data-sets are presented elsewhere (Almeda et al., 2009). Particle size selection Larvae were separated into three groups according to size (Table 2). We conducted experiments to examine larval feeding efficiency as Subsamples of larvae from each size group ( N25
30) were fixed in a function of particle size for early and late larval stages. The two 5% acid lugol solution, and the average lengths and numbers of stages were separated from collected larvae according to their size. setigers were determined using an inverted microscope. Initial grazing Larvae were offered potential prey of different size (ESD) ranging trials with aged tank seawater turned out unsuccessfully and instead from 4 to 53  m (Table 3). Otherwise the experimental procedure 0.2  m filtered seawater from the sampling area was used. was as described above. The experiments were performed with one Thalassiosira weissflogii were provided from a stock culture growing phytoplankton species per experimental treatment and with an equal in B1 medium (Hansen, 1989). Stock phytoplankton concentrations initial phytoplankton biomass (0.2  gCml
1 and 1  gCml
1 for early and the size (equivalent spherical diameter; ESD) of Thalassiosira and late larval stages, respectively). The prey retention efficiencies weissflogii were determined by a Multiziser III Coulter Counter electronic particle counter (Beckman Coulter, Miami, FL, USA). The
1 cell carbon quota used was 131 pg Ccell (Dutz et al., 2008). Each 1.20 0.06 grazing trial was executed in triplicate grazing bottles with parallel triplicate phytoplankton growth controls. Average concentrations of 1.00 0.05 larvae ranged from 2.6 to 1.0 larva ml
1 for early and late larval stage, ) 0.04 3 respectively. The phytoplankton suspensions were amended with a 0. 80 nutrient mixture to compensate for nutrient enrichment due to larvae excretion. The incubations were carried out in 70 ml blue cap glass 0.0 3 0.60 bottles mounted with plastic film before closing to prevent inclusions 0.02 0.40 Table 3. Phytoplankton species used in the prey size selection 0.01 Body vol experiment and their dimensions

Prototroch 0.20 0 Phytoplankton species ESD ( m) Cell volume ( m3) u Isochrysis galbana 4 34 0 me (mm Rhodomonas salina 9 357 0.2 0.4 0.6 0. 8 1.0 1.2 ba Thalassiosira weissflogii 13 1124 Body length (mm) nd length (mm) Heterocapsa triquetra 19 3591 Akashiwo sanguinea 53 79,730 Fig. 2. Morphometric relationships of Polydora ciliata larvae. Larval circumference (prototroch cilia band length) is plotted versus larval body Particle sizes were estimated as outlined in Materials and methods. length (white circles; left axis), and larval body volume is plotted versus ESD, equivalent spherical diameter. larval body length (black circles; right axis).

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Fig. 3. Four examples of reconstructed three- dimensional swimming paths of Polydora ciliata larvae showing the typical helical structure of some 0 0 paths and the large overall path variation. –0.2 –0.4 0.2 –0.6

–0. 8

m m m 0.4 –1.0 m –1.2 1. 8 –1.4 2. 3 1.6 0.6 2.2 –1.6 1.4 2.1 –1. 8 1.2 2.0 1.0 m 0. 8 1. 8 1.6 0. 8 m 1.9 m 1.4 3.1 m 1.2 0.6 3.0 2.9 1. 8 mm 1.0 2. 8 2.7 1.7 mm 2.6

0 0.2

0.5 0.4

m m 0.6

1.0

m m 0. 8 1.5 2.0 2.4 2.0 2. 3 2.0 1.0 1.9 2.2 2.0 2.1 1.9 1.9 m 2.5 2.4 m 1. 8 1. 8 m 2.4 2.0 m 1. 81.7 2. 3 1.7 1.6 1. 8 2. 3 2. 3 1.9 mm 1.6 mm 2.2

are given as the clearance rates on the different sized prey, according RESULTS to Frost (Frost, 1972). Morphometrics of live larvae The body length ( l) of the larvae that we used for motion analysis Grazing on natural prey ranged from 0.3 mm to 1.0 mm (Fig. 2). The diameter of the larval head The experiments consisted of incubations of natural microplanktonic and hence the circumference of the prototroch cilia band increased as communities with the addition of P. ciliata larvae for the the larval body length increased. The number of setigers increased experimental treatments. Natural seawater (NSW) for grazing from typically three to 13 during growth (Fig. 1 and Table 2). The incubations was collected at 1 m depth with a Niskin bottle and diameter of the larvae ( d) ranged from 0.16 mm to 0.26 mm and l/d screened through a 200  m mesh by reverse filtration in order to increased by a factor of two during growth from l/d1.9 on average exclude mesozooplankton. The water was amended with a nutrient for the early larval stage to l/d3.8 on average for the late larval stage mixture to compensate for nutrient enrichment due to larval and hence the larvae did not grow isometrically (Fig. 2). The prototroch excretion. Twelve 70 ml blue cap bottles were filled with NSW. cilia were of comparable length in the intermediate and late larval Then 75 late-stage larvae approximately 900  m long, were added stages and in these stages the cilia were significantly longer than the to six of the NSW-containing bottles. Two bottles with larvae and cilia in the early stage (one-way ANOVA, P<0.001; Table 2). The two without larvae were fixed with 1% acidic Lugols solution to telotroch cilia, however, seemed not to increase in length significantly determine the initial microplankton abundance. The remaining eight during larval ontogeny and remained statistically invariable during bottles consisting of four grazing bottles and four control bottles the larval development (one-way ANOVA, P0.356). were incubated for 24 h on a plankton wheel (0.5 r.p.m.) in a temperature-controlled room at 16°C and constant dim light. Swimming patterns and speeds Ciliate, dinoflagellate and diatom abundance was estimated by The larvae were swimming in helical paths and showed large settling 10 ml aliquots in Utermöhl chambers for 24 h in two individual variations (Fig. 3). A typical swimming path is a sequence replicates per treatment and counted under an inverted microscope. of downwards, upwards, and horizontal trajectories. We tested Digital images of each cell type were taken and sized with the swimming speed of the general swimming groups, i.e. horizontal, open source software package ImageJ. Cell volumes were downwards and upwards swimming against prey and filtered converted into carbon content using a factor of 0.19 pg C m
3 (Putt seawater and found no significant differences in the swimming speed and Stoecker, 1989) whereas the carbon of dinoflagellates and when different prey was offered (one-way ANOVA, P>0.05; diatoms were estimated accordingly to the recommendations of Table 1). Furthermore we did not see any differences in swimming Menden-Deuer and Lessard (Menden-Deuer and Lessard, 2000). pattern and the data sets were pooled into one data set consisting Grazing was calculated if prey types in grazing trails were of swimming paths divided into three groups based on the overall significantly different from controls (one-way ANOVA, P<0.05). swimming direction: horizontal, downwards and upwards Clearance and ingestion rates were calculated for each prey type swimming. The average of all our measured horizontal swimming according to Frost (Frost, 1972). speeds ( V) is 0.50±0.21 mm s
1 and we observed that the average

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2.5 lengthwise with constant speed at low Reynolds number is [see Happel and Brenner (Happel and Brenner, 1983), pp. 154-156]: π η ) 2.0 = 8 c V –1 D , (3) τ 2 + −1τ − τ ( 0 1)coth 0 0 where c is a geometrical parameter that is defined in the following 1.5 way: c = a2 − b2 , (4) 1.0 and where t0 is another geometrical parameter with the definition:

S 0.5 wimming t0  a/c . (5) Eqn 3 is exact and valid for all values of a/b, i.e. it is valid for both 0

s prolate spheroids with low a/b and slender prolate spheroids with

peed (mm 0.2 0.4 0.6 0. 8 1.0 1.2 high a/b. To explore the drag model for a/b in the range between Body length (mm) 2 and 4, which is relevant for P. ciliata larvae, we would like to have a more simple analytical expression to work with than the exact Fig. 4. Swimmings speeds of Polydora ciliata versus larval body length. Eqn 3. The often-used slender spheroid approximation [see Happel Measured swimming speeds: downward speed V (white circles); down and Brenner (Happel and Brenner, 1983), p. 156; and Berg (Berg, upward speed Vup (black circles); horizontal speed V (grey diamonds). The solid line indicates the theoretical model (Eqn 10) of the horizontal 1993), pp. 55-58]: swimming speed. 4 π η a V D = , (6) ln(2 a / b) − 1 / 2 horizontal swimming speed decreases slightly with increasing is not applicable in the present study, since it overestimates the drag body length (Fig. 4). The larvae swam downwards significantly considerably in the a/b range from 2 to 4 and is only a valid
1 faster than they swam upwards: Vdown 0.93±0.40 mm s versus approximation of Eqn 3 when a/b is larger than 10. Instead we
1 Vup 0.42±0.19 mm s (t-test, t5.6, d.f. 58, P<0.001) and the consider the following approximation [see White (White, 2006), difference between the average upward and downward swimming pp. 171-172]: speed increased with increasing body length. In the following we 6 π (4 + a / b) η b V D = . (7) show that our observations on the swimming behavior can be 5 explained using a simple swimming model based on low Reynolds number hydrodynamics and taking the effect of gravity on the Eqn 7 gives a good approximation with less than 1% relative error slightly negatively buoyant larvae into account. in the a/b ratio range from 2 to 4 in comparison with the exact drag expression (Eqn 3), and since approximation (Eqn 7) is easy Swimming model to work with and analyze we shall use it in the following. Inserting In our hydrodynamic model we assumed that each individual larva Eqn 7 into Eqn 2 and rearranging, we see that the drag coefficient swims lengthwise with constant velocity due to the propulsion force becomes: generated by the ciliary swimming apparatus, and that the π + = 6 (4 a / b) propulsion force is balanced by the drag on the larval body. We CD , (8) will not attempt to describe the helical swimming paths of the 5 a / b larvae, but only the observed average swimming speed. For which decreases from CD(3/5) 6 p with a/b2 (early larval stage) simplicity we model the cigar shaped body of the larva as a prolate to CD(2/5) 6 p with a/b4 (late larval stage) . We note for spheroid with polar radius abody length/2 and equatorial radius comparison the well-known result that CD6p for a sphere. bbody diameter/2. The propulsion force F and the drag force D must have the same From the measured body lengths ( l) and horizontal swimming magnitude if the larva swims with constant velocity. We presume speeds ( V) we can estimate the Reynolds number of the flow past that the ciliary swimming apparatus consists primarily of the the swimming larvae. We define the Reynolds number as: prototroch and secondarily of the telotroch cilia band. We do not have detailed observations of the metachronal wave motion in the ρl V Re = , (1) cilia bands, and we therefore make some simplifying assumptions η about the propulsion force. In the following we assume that F is where r is the density of the water and  is the dynamic viscosity. proportional to the length of the cilia bands as suggested by Hansen For the larvae that swim horizontally we find that the Reynolds (Hansen, 1991) and we model the strength of the total cilia force number ranges from Re 0.12 to Re 0.46. Since the typical Reynolds as: number is fairly low, it is a reasonable approximation to model the F 2 C b U , (9) flow using Stokes equation. The drag D on the larva is therefore  p F  proportional to both the larval size and the swimming speed: where CF is a dimensionless propulsion force coefficient that depends on geometry and distribution of the cilia and U is an D C aV , (2)  D  effective time-averaged speed of the cilia motion. The propulsion where CD is a dimensionless drag coefficient that only depends on force per unit cilia band length most likely increases as the larva the geometrical shape of the larva. The drag D on a prolate spheroid grows and each cilium gets longer, but for simplicity we assume with polar radius a and equatorial radius b, which is moving that CFU is constant during the larval development. By balancing

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3.0 6 ) –1

) 1. 8 10 –1 1.6 2.5 d –1 a 1.4 y 2.0 1.2 10 5 1.0 1.5 0. 8 Force (nN) 1.0 0.6 10 4 0.4 0.5 0.2

0 M 0 10 3 S 0.2 0.4 0.6 0. 8 1.0 1.2 a x. cle 0 0.2 0.4 0.6 0. 8 1.0 1.2 pecific cle Body length (mm) Body length (mm) a r a Fig. 5. Theoretical curves for propulsion force F (solid line) and buoyancy- nce r Fig. 6. Maximum clearance rate of Polydora ciliata larvae plotted versusa r corrected weight mЈg (dashed line) versus larval body length of Polydora a a larval body length. Black circles (left axis) show the maximum clearancence r ciliata. Note that the critical larval body length, Imax , corresponds to the rate (ml larva te (ml l –1 day –1 ); white circles (right axis) show the body volume

point where the curves for F and mЈg intersect. specific clearance rate (body volume h–1 ). Error bars indicate ±s.d. Opena te (

triangles showa the model of the maximum clearance rate (Eqn 14). rv b ody vol a u

the propulsion force F and the drag force D we obtain the swimming band CFU and decreases as one over the square root of theme h excess speed: density rs
r. To compare the model with observations we plotted 5 CF U F and mЈg versus larval body length ( l) using the excess density V = . (10)
3
2 + a b rs
r5kg m , g9.8 ms , l/d from our fit of the morphometrics 3 (4 / )
6
1 of live larvae (Fig. 2), and CFU2.0 ϫ10 Nm from our fit of The simple model (Eqn 10) predicts a slow decrease of the the horizontal swimming speeds (Fig. 4). The propulsion force is swimming speed in agreement with the observed horizontal large compared with the buoyancy-corrected weight at small body swimming speeds (Fig. 4). Our fit of Eqn 10 to the measured length, whereas the reverse is true at large body length (Fig. 5). Using horizontal swimming speeds determines the propulsion force per the above parameters we find the maximum larval body length
6
1 unit cilia band length to CFU2.0 ϫ10 N m , where we have lmax 0.95 mm in good agreement with the observed body lengths used  1.00 ϫ10
3 Pa s and a/b from our fit to the morphometrics for the largest free swimming larvae reported in nature. We return of live larvae (Fig. 2). to the value of the excess density in the Discussion.

Influence of gravity on swimming behavior Maximum clearance and ingestion rates The larvae are slightly negatively buoyant, and the upward The maximum clearance rate Qmax increased approximately swimming speed Vup is therefore smaller than the downward proportionally to the larval body length, and the body volume swimming speed Vdown (Table 1 and Fig. 4). In addition to the specific clearance rate therefore showed a dramatic 10-fold decrease propulsion force F and the drag force D, the larvae are influenced from 10 5 body volume h
1 in the early larval stage to by gravity, i.e. the buoyancy-corrected weight mЈg, where mЈ is the

buoyancy-corrected mass of the larva and g denotes the acceleration )

4 –1 due to gravity. If we again model the larva body as a prolate spheroid ) 12,000 10 –1 we have:

d 10,000 –1 4 a

′ = π ρ − ρ 2 y 3 m ( s ) a b , (11) 10 3 8000

where rs is the average density of the larva. The larva will evidently sink to the bottom if mЈg is larger than F. The buoyancy-corrected 6000 10 2 weight grows cubically with size, and if the propulsion force grows 4000 linearly with size, there will be a critical maximum body length lmax for which F and mЈg have the same magnitude. When the larva 10 1 exceeds the critical body length, gravity will ultimately force the 2000 animal towards the sea floor. Using Eqns 9 and 11 and the force M 0 10 0 balance FmЈg we obtain the following expression: a S x. inge 0 0.2 0.4 0.6 0. 8 1.0 1.2 pecific inge η Body length (mm) 6 ( lmax / dmax ) CF U l = . (12) s max ρ − ρ tion r ( s ) g Fig. 7. Maximum ingestion rate of Polydora ciliata larvae plotted versus s larval bodya length. Black circles (left axis) show the maximum ingestion tion r Eqn 12 shows that the maximum larval body length lmax increases rate (cell te (cell llarva –1 day –1 ); white circles (right axis) show the body volume as the square root of the propulsion force per unit length of the cilia specific ingestion rate (body volume day –1 ). Error bars indicate ±s.d. a te ( a rv b

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0. 30 2.5 Table 4. Clearance and ingestion rates obtained in Polydora ciliata larval incubation experiments in natural seawater (Isefjorden) ) ) –1 –1 2.0 Property Dinoflagellates Ciliates d d Initial concentration (cells ml –1 ) 411±45 4.04±0.74 –1 –1 a a y 0.20 y Initial biomasscell volume ( g C (µmml –13) 4745±132 29693±65 1.5 Avg. Biomass (ng C ml –1 ) 0.77±0.02 10.09±0.03 Clearance rates (ml larva –1 day –1 ) 1.06±0.13 2.65±0.268 Ingestion rates (cells larva –1 day –1 ) 277±19 3.5±0.1 1.0 –1 –1 0.10 Clearance rates (ml larva day )* 1.17±0.12 3.08±0.37 Ingestion rates ( g C larva –1 day –1 ) 0.224±0.012 0.037±0.001 –1 0.5 Carbon specific ingestion (% day ) 13.0±0.7 2.15±0.05 Volume specific ingestion (% day –1 ) 5.4±0.3 0.045±0.004

Cle Cle Values are based on the most abundant prey groups found. Note that 0 0 a a clearance values marked with an asterisk are based on the per capita r r

a 5 10 15 20 25 30 35 40 45 50 55 60 a

nce r nce r change in cell numbers whereas unmarked clearance rates are calculated Prey E SD ( µm) from changes in prey biomass.

a a Values are means±s.d. te (ml l te (ml l Fig. 8. Food size spectra for Polydora ciliata larvae. Clearance rate of early larvae (black circles; left axis) and late larvae (white circles; right axis) a a plotted versusrv the equivalent spherical diameter (ESD; m) of the prey.rv a a Error bars indicate ±s.d. retention increased with particle size until a maximum somewhere between 19  m and 53  m was reached.

10 4 body volume h
1 found in the late larval stage (Fig. 6). The same Grazing on natural prey characteristics are exhibited by the maximum ingestion rate Imax The protist plankton community was initially dominated by the that increased from approximately 2 ϫ10 3 Thalassiosira weissflogii dinoflagellate Heterocapsa triquetra with more than cells per larva per day to approximately 10 4 T. weissflogii cells per 400 cells ml
1 , and its abundance exceeded the abundance of larva per day. A dramatic decrease in the body volume specific ciliates of approximately 4 cells ml
1 with a factor of 100. The ingestion rate, similar to the decrease in the body volume specific ciliate community consisted mainly of Strombidium spp. Diatoms clearance rate was likewise observed (Fig. 7). were scarce with only 0.1 cells ml
1 and they consisted mainly of To better understand the observed trends of the maximum Ditylum cf. brightwellii and Coscinodiscus spp. Feeding rates clearance rate Qmax we modeled it as the product of the clearance on dinoflagellates and ciliates are shown in Table 4. The area and the flow speed relative to the larva averaged over the dominance of H. triquetra made dinoflagellates the most clearance area. To estimate Qmax we assume that the maximum important source of food for P. ciliata in this situation. However, clearance rate is equal to the product of the area of the prototroch ciliates that made a much less abundant food source were in fact cilia band and the swimming speed of the larva: cleared at a rate that was 2.5-fold higher than the clearance rate on H. triquetra . Qmax  p  (2 b + lc) lc V , (13) where we have used lc to denote the prototroch cilia length and DISCUSSION where the dimensionless number  is used to take into account that Swimming patterns and speeds the prototroch does not encircle the larva completely. Using the No systematic differences in the patterns of the swimming paths model expression for the swimming speed in Eqn 10 we obtain the related to larval size or food particle regime were observed, and we expression: found similar swimming paths even when the larvae were exposed to 0.2  m filtered seawater. Earlier observations, which were . 5 π α C (2 b + l ) l U Q = F c c . (14) interpreted as raptorial feeding behavior, are most likely reactions max + 3 (4 a / b) to physical obstruction by a large particle as shown for gastropod Our observations of the prototroch cilia length, lc, for the various veligers and calanoid copepodites (Hansen et al., 1991). Moreover, larval stages gave lc27  m for the early stage, lc45  m for the at any given larval development stage, the larval swimming paths intermediate stage, and lc48  m for the late stage (Table 2). With of individuals were very variable (Fig. 3). This is also described by these values for lc in the three larval stages and 0.75 as suggested Wilson (Wilson, 1928) where the larvae frequently swims slowly from sketches by Wilson (Wilson, 1928), we find that the model rotating on their longitudinal axes, sometimes in circles about one of Qmax using Eqn 14 overestimates the measured maximum place, and sometimes they curl up and erect their body spines when clearance rate for the early stage, agrees well with the measured disturbed. We have speculated that the adaptive significance of an maximum clearance rate for the intermediate stage, and apparently non-predictable swimming path besides the spine erection underestimates the measured maximum clearance rate for the late could be a means of predator avoidance, but this remains to be stage (Fig. 6). verified. The horizontal swimming speed decreases slowly with body Particle size selection length, which indicates that the propulsion force due to the ciliary The larvae of P. ciliata are able to feed on prey ranging in size from swimming apparatus increases slower with body length than the 4 m to 53  m (Fig. 8). Clearance rates varied with prey size and drag on the body. Wilson (Wilson, 1928) suggested that body spines larval length. The early larval stage displayed a maximum retention contribute negligibly to the drag, and that the notochord moderately (optimal prey size) for particles of 12.9  m and showed a reduced or not at all contributes to the swimming force. Hence, we modeled retention for smaller and larger particles. The late larval stage the propulsion force as generated by the prototroch and the telotroch

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solely. The decrease of the measured average horizontal speed is to measure the excess density, since the larvae contract upon fixation, well described by the simple swimming model (Eqn 10 and Fig. 4) and we have not been able to find values for the excess density of which takes into account that the larvae become relatively more polychaete larvae in the literature. slender (Figs 1 and 2) during their ontogenetic development.
6
1 However, we speculate that our fit CFU2.0 ϫ10 Nm may be Maximum clearance rates an underestimate of the cilia force per unit length of the cilia band, The propulsion force generated by the cilia apparatus of the larva and also that the cilia force per unit length is not constant but balances the drag on the body of the larva when the larva is increases slowly throughout larval development as each cilium gets swimming freely with constant velocity. The net force exerted on longer. There are two main reasons for the possible underestimate the water by the larva is therefore zero and the flow relative to the of the propulsion force. First, in the simple model we balance the larva is well approximated by a velocity field with parallel propulsion force due to the cilia swimming apparatus by the drag streamlines and constant flow speed equal to the swimming speed on a passively sinking prolate spheroid. In general the flow around (Visser, 2001). The flow generated by a freely swimming larva a freely swimming larva is different from the flow around a passively differs from the flow generated by a larva that is tethered either by sinking larva, and we note that our modeling approach does not gravity as discussed by Strickler (Strickler, 1982) and Emlet and take this into account. Langlois et al. (Langlois et al., 2009) Strathman (Emlet and Strathman, 1985) or by mucous threads as considered a simple model of a self-propelled flagellate consisting explored by Fenchel and Ockelmann (Fenchel and Ockelmann, of a spherical body with a cylindrical flagellum, and showed that 2002). However, in general, the effect of tethering on the maximum the drag on the body is larger for a self-propelled flagellate relative clearance rate depends on the prey capture mechanism and the to the drag on a passively sinking sphere. We presume that our interaction between prey and cilia as discussed by Emlet (Emlet, estimate of the propulsion force for the swimming larvae similarly 1990). We have not found evidence that P. ciliata larvae have underestimates the true propulsion force, since we do not take into mucous threads or that gravity, despite the observed effect on the account the increase of the flow speed near the larval body due to upward and downward swimming speeds of P. ciliata larvae, should the beating cilia. Second, the drag on the larval body may be larger have a qualitative influence on the flow structure and result in an than the prediction obtained from the prolate spheroid expression increase of the maximum clearance rate in the late larval stage. in Eqn 3, since this expression is derived for an object with a smooth The maximum specific clearance and ingestion rates should be surface and ignores the rough surface and setae structures on the measured using prey with size as close as possible to the optimal larval body. This could lead to an underestimate of the propulsion prey size, especially when comparing different developmental force. Presumably this effect is more pronounced in the late larval stages of given larvae. Here it was recorded on Thallassiosira stage, since the larvae at this stage begin to develop large palps weisflogii , which were close to the optimal prey size for both early (Fig. 1). Both a detailed morphological description of the cilia band and late larvae (Fig. 8). It was recorded to be higher than reported structures and close observations of the metachronal wave of the for Mediomastus fragile and Marenzelleria cf. viridis (Hansen, 1993; beating cilia in swimming larvae in combination with direct Burckardt et al., 1997), but similar to other ciliated larvae (Jespersen numerical simulations of the flow past the larva would be needed and Olsen, 1982; Hansen and Ockelmann, 1991). We assume that to fully address these issues. the particle capture is by the prototroch and maybe more ciliary Planktotrophic larvae display actively phototactic behavior after structures in combination as described by Strathmann (Strathmann, hatching and this is also described for P. ciliata (Wilson, 1928). 1971) and Hart (Hart, 1996). The measured maximum clearance This is an adaptation to swim towards the photic zone of the water rates are described reasonably well by Eqn 14, using 27  m as the column where phytoplankton availability is highest. Later when prototroch cilia length ( lc) for the early larval stage, 45  m for the approaching competence for metamorphosis they become negatively intermediate larval stage, and 48  m for the late larval stage phototactic, which is interpreted as an adaptation to seeking (Table 2; Fig. 6). The maximum clearance rate Qmax increased settlement on the sea floor (Thorson 1950; Thorson, 1964). The approximately proportionally to the larval body length, and the body relationship between propulsion force and gravity suggests that the volume specific clearance rate therefore showed a dramatic 10-fold hydrodynamic constraints could play a direct role in the settlement decrease. Increasing clearance and ingestion rates as well as process (Fig. 5). The buoyancy-corrected weight of the slightly increasing optimal prey size has been demonstrated for crustaceans, negatively buoyant larva becomes more and more important in e.g. the shrimp Sergestes similis and the copepod Acartia tonsa comparison with the propulsion force as the larval size increases, (Omori, 1979; Berggreen et al., 1988). However, specific food and gravity therefore puts a final constraint on how large the larva uptake rates declined with body weight (Omori, 1979) and optimal can be without sinking. Eqn 12 suggests that the maximum body prey size remained constant for a ciliated gastropod veliger (Hansen length lmax increases with increasing propulsion force per unit length and Ockelmann, 1991). Moreover, a thorough compilation of 13 of the cilia band CFU and decreases with increasing density echinoderm larval species reveals a substantial variation in feeding difference rs
r. In our estimate we used the value capabilities among larvae of similar size and shows that some are
6
1 CFU2.0 ϫ10 Nm obtained from our fit of the swimming model better suspension feeders than the others. Hence, no clear pattern prediction to the measured horizontal swimming speed. We assumed in size-related clearance rate was detected (Hart, 1996). that the larvae on average are 0.5% denser than water and found good agreement with the observation that the largest free swimming Particle size selection larvae have a body length of approximately 1.0 mm, which is The ontogenetic retention spectrum exhibits a constant sized supported by the findings by Hansen (Hansen, 1999). The larvae minimum prey, an increased optimal prey size and also an increased in the present study had up to 13 setigers where they at maximum maximum prey size with larval body size. This is most probably can obtain 18 setigers and still be pelagic (Thorson, 1946). However, related to the morphological constraints and the ontogenetic changes the largest possible value of the average larval density would be in the ciliary apparatus (Hansen, 1991; Riisgaard et al., 2000). larger if the actual value of the cilia force per unit length is larger However, in contrast to the opposed band veliger the maximum than our present estimate as discussed above. It is very challenging prey size for late P. ciliata larvae seems to be larger, which is

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Swimming behavior of P. ciliata 3245 supported by late stage larvae when capturing motile oligotrich ACKNOWLEDGEMENTS ciliates in the natural prey regime. This could be due to the increasing We would like to thank an anonymous reviewer and Dr R. R. Strathmann for constructive criticism and suggestions for changes of an earlier version of this length of the prototroch cilia, dimensions of the mouth structure, manuscript. This work is based on research performed at Roskilde University’s or a contribution from the tentacles. Oral diameter is, for instance, field station “Søminestationen”. The project was supported by the Danish National described as being the upper limit for the size of prey for single Science Research Council grant (no. 272-07-0485) to B.W.H., an instrument grant to DTU AQUA (H.H.J.) from the VELUX Foundation, and grant CTM2004- band echinoplutei larvae (e.g. Rassoulzadegan et al., 1984). Since 02575/MAR from the Spanish Ministry of Education and Science to R.A. J. R. the principle behind food particle capture is not thoroughly Andersen gave valuable Photoshop assistance that improve the appearance of understood we suggest that there should be further investigation of Fig. 1. the morphology and functional capability of P. ciliata larvae. REFERENCES However, no matter how prey capture takes place the resulting Almeda, R., Pedersen, M. T., Jakobsen, H. H. and Hansen, B. W. (2009). Feeding principal ontogenetic changes in particle retention add to a reduction and growth kinetics by the planktotrophic larvae of the spionid polychaete Polydora in intraspecific food competition among the various larval stages ciliata (Johnston). J. Exp. Mar. Biol. Ecol . 382 , 61-68. Anger, K., Anger, V. and Hagmeir, E. (1986). Laboratory studies of larval growth of as suggested for a calanoid copepod (Berggreen et al., 1988). Polydora ligni , Polydora ciliata , and Pygospio elegans (Polychaeta, Spionidae). Helgoländer Meeresunters. 40 , 377-395. Berg, H. C. (1993). Random Walks in Biology Expanded Edition. Princeton, New Grazing on natural prey Jersey, USA: Princeton University Press. When larvae were exposed to a natural prey field, ciliates were Berggreen, U., Hansen, B. and Kiørboe, T. (1988). Food size spectra, ingestion and growth of the copepod Acartia tonsa during development: implications for cleared significantly more than dinoflagellates, even though the determination of copepod production. Mar. Biol. 99 , 341-352. dinoflagellate concentration was two orders of magnitude higher Blake, J. A. (1969). Reproduction and larval development of Polydora from northern New England (Polychaeta: Spionidae). Ophelia 7, 1-63. than the ciliate concentration (Table 4). Since dinoflagellates swim Bochert, R. and Bick, A. (1996). Reproduction and larval development of slower than ciliates (Hansen et al., 1997) and diatoms are non-motile Marenzelleria viridis (Verril, 1973) (Polychaeta: Spionidae). Mar. Biol. 123 , 763-773. Burckhardt, R., Schumann, R. and Bochert, R. (1997). Feeding biology of the in the water column, the higher clearance rates observed on ciliates, pelagic larvae of Marenzelleria cf. viridis (Polychaeta: Spionidae) from the Baltic may be the results of a swimming-velocity-driven increased Sea. Aquat. Ecol. 31 , 149-162. Daro, H. M. and Polk, P. (1973). The autecology of Polydora ciliata along the Belgian encounter probability of ciliates over the alternative prey particles coast. Neth. J. Sea. Res. 6, 130-140. (Jakobsen, 2005). Dutz, J., Koski, M. and Jonasdottir, S. H. (2008). Copepod reproduction is unaffected by diatom aldehydes or lipid composition. Limnol. Oceanogr. 53 , 225- The larva did not display any specialized behavior as a response 235. to the ambient prey regime. Not only do larval morphology and Emlet, R. B. (1990). Flow fields around ciliated larvae: effects of natural and artificial tethers. Mar. Ecol. Prog. Ser. 63 , 211-225. behavior govern larval feeding, prey behavior such as swimming Emlet, R. B. and Strathman, R. R. (1985). Gravity, drag, and feeding currents of speed also influence the feeding efficiency of P. ciliata . The larvae small zooplankton, Science 228 , 1016-1017. Fenchel, T. (1988). Marine plankton food chains. Ann. Rev. Ecol. Syst. 19 , 19-38. ontogeny seems to favor high prey uptake rates in the early stage Fenchel, T. and Ockelmann, K. W. (2002). Larva on a string, Ophelia 56 , 171-178. with a remarkable 10-fold decrease of the specific clearance rate Frost, B. W. (1972). Effects of size and concentration of food particles on the feeding 5 4
1 behaviour of the marine planktonic copepod Calanus pacificus . Limnol. Oceanogr. from 10 to 10 body volumes h during ontogeny (Fig. 6). The same 17 , 805-815. phenomenon is reported for larvae of Philine aperta with a factor Hannertz, L. (1956). Larval development of the polychaete families Spionidae Sars, Disomidae Mesnil, and Poicilochaetidae n. fam. In the Gullmar Fjord (Sweden). Zool. of five decrease in maximum clearance during ontogeny (Hansen Bidrag. Uppsala 31 , 1-204. and Ockelmann, 1991). Additionally, a decrease in maximum Hansen, B. (1991). Feeding behaviour in larvae of the opisthobranch Philine aperta . II. Food size spectra and particle selectivity in relation to larval behaviour and specific clearance rate with size of zooplankton within the morphology of the velar structures. Mar. Biol . 111 , 263-270. 2
2000 µm body size range reveals a common scaling factor of
0.23 Hansen, B. (1993). Aspects of feeding, growth and stage development by trochophora larvae of the boreal polychaete Mediomastus fragile (Rasmussen) (Capitellidae). J. (Hansen et al., 1997). Exp. Mar. Biol. Ecol. 166 , 273-288. Hansen, B. W. (1999). Cohort growth of planktotrophic polychate larvae - are they food limited? Mar. Ecol. Prog. Ser. 178 , 109-119. LIST OF SYMBOLS AND ABBREVIATIONS Hansen, B. and Ockelmann, K. W. (1991). Feeding behaviour in larvae of the a larva model, polar radius (mm) opisthobranch Philine aperta (L.) I. Growth and functional response at different b larva model, equatorial radius (mm) developmental stages. Mar. Biol . 111 , 255-261. c larva model, geometric parameter (mm) Hansen, B. W., Hansen, P. J. and Nielsen, T. G. (1991). Effects of large nongrazable particles on clearance and swimming behaviour of zooplankton. J. Exp. Mar. Biol. CD drag coefficient (dimensionless) Ecol. 152 , 257-269. CF propulsion force coefficient (dimensionless) Hansen, P. J. (1989). The red tide dinoflagellate Alexandrium tamarense : Effects d larva diameter (mm) behaviour and growth of a tintinnid ciliate. Mar. Ecol. Prog. Ser. 53 , 105-116. Hansen, P. J., Bjørnsen, P. K. and Hansen, B. W. (1997). Zooplankton grazing and dmax maximum larva diameter (mm) growth: scaling within the 2-2000 m body range. Limnol. Oceanogr. 42 , 687-704. D drag (nN) Happel, J. and Brenner, H. (1983). Low Reynolds Number Hydrodynamics , 2nd F propulsion force (nN) Edition. The Hague, The Netherlands: Martinus Nijhoff Publishers. g acceleration due to gravity (m s
2 ) Hart, M. W. (1996). Variation in suspension feeding rates among larvae of some temperate, eastern Pacific echinoderms. Invertebrate Biol. 115 , 30-45.
1
1 Imax maximum ingestion rate (cell larva day ) Jakobsen, H. H., Halvorsen, E., Hansen, B. W. and Visser, A. W. (2005). Effects of l larva length (mm) prey motility and concentration on feeding in Acartia tonsa and Temora longicornis : l prototrochal cilia length (mm) the importance of feeding modes. J. Plankton Res. 27 , 775-785. c Jespersen, H. and Olsen, K. (1982). Bioenergetics in veliger larvae of Mytilus edulis lmax maximum larva length (mm) L. Ophelia 21 , 101-113. mЈ buoyancy-corrected mass of larva (kg) Langlois, V. J., Andersen, A., Bohr, T., Visser, A. W. and Kiørboe, T. (2009).
1
1 Significance of swimming and feeding currents for nutrient uptake in osmotrophic Qmax maximum clearance rate (ml larva day )
1 and interception feeding flagellates. Aquatic Microbial Ecol. 54 , 35-44. U effective speed of the beating cilia (m s ) Menden-Deuer, S. and Lessard, E. J. (2000). Carbon to volume relationships for
1 V swimming speed, horizontal (mm s ) dinoflagellates, diatoms, and other protist plankton. Limnol. Oceanogr. 45 , 569-579.
1 Vdown swimming speed, downward (mm s ) NIMPIS (2002). Polydora ciliata species summary. National Introduced Marine Pest V swimming speed, upward (mm s
1 ) Information System (ed. Hewitt, C. L., Martin, R. B., Sliwa, C., McEnnulty, F. R., up Murphy, N. E., Jones, T. and Cooper, S.). Web publication:  prototroch, geometric parameter (dimensionless) http://crimp.marine.csiro.au/nimpis.  dynamic viscosity (Pa s) Omori, M. (1979). Growth, feeding, and mortality of larval and early postlarval stages r density of water (kg m
3 ) of the oceanic shrimp Sergestes similis Hansen. Limnol. Oceanogr . 24 , 273-288.
3 Pedersen, T. M., Josefson, A., Hansen, B. W. and Hansen, J. S. (2008). Mortality rs average density of larva (kg m ) through ontogeny of soft bottom marine invertebrates with planktonic larva. J. Mar. t0 larva model, geometric parameter (dimensionless) Syst. 73 , 185-207.

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Pedersen, T. M., Almeda, R., Fotel, F. L., Jakobsen, H. H., Mariani, P. and Strathmann, R. R. (1971). The feeding behavior of planktotrophic echinoderm larvae: Hansen, B. W. (2010). Larval growth in the dominant ploycheate Polydora ciliata is mechanisms, regulation, and rates of suspension feeding. J. Exp. Mar. Biol. Ecol. 6, food-limited in an eutrophic Danish estuary (Isefjord). Mar. Ecol. Prog. Ser. 407 , 99- 109-160. 110. Strickler, J. R. (1982). Calanoid copepods, feeding currents, and the role of gravity, Pernet, B. and McArthur, L. (2006). Feeding by larvae of two different developmental Science 218 , 158-160. modes in Streblospio benedicti (Polychaeta: Spionidae). Mar. Biol. 149 , 803-811. Thorson, G. (1946). Reproduction and larval development of Danish marine bottom Plate, S. and Husemann, E. (1994). Identification guide to the planktonic polychaete invertebrates, with special reference to the planktonic larvae in the sound (Øresund). larvae around the island Helgoland (German Bight). Helgoländer Meeresunters. 48 , Medd. Kom. Dan. Fisk. Hav. 4, 1-523. 1-58. Thorson, G. (1950). Reproductive and larval ecology of marine bottom invertebrates. Putt, M. and Stoecker, D. (1989). An experimentally determined carbon volume ratio Biol. Rev. Camb. Philos. Soc. 25 , 1-45. for marine “oligotrichous” ciliates from eustraine and coastal waters. Limnol. Thorson, G. (1964). Light as an ecological factor in the dispersal and settlement of Oceanogr. 34 , 1097-1103. larvae of marine bottom invertebrates. Ophelia 1, 167-208. Rasmussen, E. (1973). Systematics and ecology of the Isefjord fauna. Ophelia 11 , 1- Visser, A. W. (2001). Hydromechanical signals in the plankton. Mar. Ecol. Prog. Ser. 495. 222 , 1-24. Rassoulzadegan, F., Fenaux, L. and Strathmann, R. R. (1984). Effect of flavor and White, F. M. (2006). Viscous Fluid Flow, 3rd Edition. New York City, USA: McGraw- size on selection of food by suspension feeding plutei. Limnol. Oceanogr . 29 , 357- Hill. 361. Wilson, D. P. (1928). The larvae of Polydora ciliata Johnston and Polydora hoplura Riisgaard, H. U., Nielsen, C. and Larsen, P. S. (2000). Downstream collecting in Claparède. J. Mar. Biol. Ass. UK 15 , 567-603. ciliary suspension feeders: the catch-up principle. Mar. Ecol. Prog. Ser. 207 , 33-51. Zajac, R. N. (1991). Population ecology of Polydora ligni (Polychaeta: Spionidae). Schram, T. A. (1968). Studies on the meroplankton in the inner Oslofjord I. Composition Seasonal variation in population characteristics and reproductive activity. Mar. Ecol. of the plankton at Nakkeholmen during a whole year. Ophelia 5, 221-243. Prog. Ser . 77 , 197-206.

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Resum de l’article VII– Article VII summary– Catalan versión

Comportament natatori i retenció de preses de les larves del poliquet Polydora ciliata (Johnston)

Benni W. Hansen, Hans H. Jakobsen., Anders Andersen, Rodrigo Almeda, Troels M. Pedersen, Anette M. Christensen, Birgitte Nielsso n. Article publicat a Journal of Experimental Biology (2010, 213: 3237-3246)

Es va estudiar el comportament de les larves estuàriques planctotrófiques del poliquet espiònid Polydora ciliata . Descrivim els canvis ontogènics en la morfologia, la velocitat de natació i les taxes d'alimentació i desenvolupem un senzill model usant la hidrodinàmica de nombre de Reynolds petit. En el model es va assumir que l'aparell ciliar de natació està compost fonamentalment per la prototroca i de manera secundària per la teletroca. El model va predir velocitats de natació i taxes d'alimentació concordes amb les velocitats i les taxes mesurades experimentalment. Amb l'aplicació de dades empíriques al model, vam ser capaços d'explicar la important disminució en les taxes específiques d'alimentació i l'augment en la diferència entre les velocitats de natació cap amunt i cap avall a causa de l'increment de grandària de les larves. Es va estimar una longitud larvària crítica a partir de la qual el pes amb correcció de la flotabilitat de la larva excedeix la força de propulsió generada per l'aparell ciliar de natació i per tant força a la larva cap al fons. D'acord amb el model, aquesta longitud larvària crítica correspon a aproximadament a 1 mm, longitud a la qual, segons la literatura, les larves són competents per a la metamorfosi i no incrementen més la seva longitud. Aquestes troballes podrien tenir implicacions generals per a totes les larves de poliquets planctotròfiques que s'alimenten sense guies de ruta. Es va observar un espectre de grandària de partícules amb forma de campana, una grandària mínima de presa d’aproximadament 4 µm de diàmetre esfèric equivalent, i un augment en la grandària òptim de presa amb l'ontogènia que podria reduir la competència intraespecífica per l'aliment entre fases larvàries. En els experiments d'alimentació amb aigua de mar natural, els ciliats van ser filtrats un 50% més eficientment que els dinoflagel·lats de grandària similar. Les grandàries de presa que poden ser filtrades per la larves de P. ciliata cobreixen la fracció microplanctónica i inclou preses amb i sense motilitat, per la qual cosa les larves estarien posicionades tròficament entre els copèpodes i dinoflagel·lats. No només la morfologia de les larves i el comportament determinen l'alimentació larvària, sinó que el comportament de la preses podria també influir en l'eficiència d'alimentació de les larves de Polydora ciliata.

177 Chapter 5

Resumen del artículo VII – Article VII summary– Spanish versión

Comportamiento natatorio y retención de presas de las larvas del poliqueto Polydora ciliata (Johnston)

Hansen B.W., Jakobsen H.H., Andersen A., Almeda R., Pedersen T.M., Christensen A.M., Nilsson B. Artículo publicado en Journal of Experimental Biology (2010, 213: 3237-3246)

Se estudió el comportamiento de las larvas estuáricas planctotróficas del poliqueto espiónido Polydora ciliata . Describimos los cambios ontogénicos en la morfología, la velocidad de natación y las tasas de alimentación y desarrollamos un sencillo modelo usando la hidrodinámica de número de Reynolds pequeño. En el modelo se asumió que el aparato ciliar de natación está compuesto fundamentalmente por la prototroca y de manera secundaria por la teletroca. El modelo predijo velocidades de natación y tasas de alimentación acordes con las velocidades y las tasas medidas experimentalmente. Con la aplicación de datos empíricos al modelo, fuimos capaces de explicar la importante disminución en las tasas específicas de alimentación y el aumento en la diferencia entre las velocidades de natación hacia arriba y hacia abajo debido al incremento de tamaño de las larvas. Se estimó una longitud larvaria crítica a partir del cual el peso con corrección de la flotabilidad de la larva excede la fuerza de propulsión generada por el aparato ciliar de natación y por lo tanto fuerza a la larva hacia el fondo. De acuerdo con el modelo, esta longitud larvaria crítica corresponde a aproximadamente a 1 mm, longitud a la cual, según la literatura, las larvas son competentes para la metamorfosis y no incrementan más su longitud. Estos hallazgos podrían tener implicaciones generales para todas las larvas de poliquetos planctívoras que se alimentan sin guías de ruta. Se observó un espectro de tamaño de partículas con forma de campana, un tamaño mínimo de presa de aproximadamente 4 µm de diámetro esférico equivalente, y un aumento en el tamaño optimo de presa con la ontogenia que podría reducir la competencia intraespecífica por el alimento entre fases larvarias. En los experimentos de alimentación con agua de mar natural, los ciliados fueron filtrados un 50% más eficientemente que los dinoflagelados de tamaño similar. Los tamaños de presa que pueden ser filtradas por la larvas de P. ciliata cubren la fracción microplanctónica e incluye presas con y sin motilidad, por lo que las larvas estarían posicionadas tróficamente entre los copépodos y dinoflagelados. No sólo la morfología de las larvas y el comportamiento determinan la alimentación larvaria, sino que el comportamiento de la presas puede también influir en la eficiencia de alimentación de las larvas de Polydora ciliata.

178 Feeding and growth kinetics of the planktotrophic larvae of the spionid polychaete Polydora ciliata (Johnston)

Chapter 5

Journal of Experimental Marine Biology and Ecology 382 (2009) 61 –68

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Journal of Experimental Marine Biology and Ecology

journal homepage: www.elsevier.com/locate/jembe

Feeding and growth kinetics of the planktotrophic larvae of the spionid polychaete Polydora ciliata (Johnston)

Rodrigo Almeda a,⁎, Troels Møller Pedersen b, Hans Henrik Jakobsen c, Miquel Alcaraz a, Albert Calbet a, Benni Winding Hansen b a Institut de Ciències del Mar (ICM, CSIC). P. Marítim de la Barceloneta 37-49, 08003 Barcelona, Spain b Roskilde University, Department of Environmental, Social and Spatial Change, PO Box 260, DK-4000, Roskilde, Denmark c National Institute of Aquatic Resources, Technical University of Denmark, Kavalergården 6, DK-2920, Charlottenlund, Denmark a r t i c l e i n f o a b s t r a c t

Article history: We studied the effect of food concentration on the feeding and growth rates of different larval developmental Received 23 August 2009 stages of the spionid polychaete Polydora ciliata. We estimated larval feeding rates as a function of food Received in revised form 27 September 2009 abundance by incubation experiments with two different preys, presented separately, the cryptophyte Rhodo- Accepted 28 September 2009 monas salina (ESD=9.7 µm) and the diatom T. weiss flogii (ESD=12.9 µm). Additionally, we determined larval growth rates and gross growth ef ficiencies (GGE) as a function of R. salina concentration. Keywords: P. ciliata Feeding larvae exhibited a type II functional response. Clearance rates decreased continuously with increasing Functional response food concentration, and ingestion rates increased up to a food saturation concentration above which ingestion Gross growth ef ficiency remained fairly constant. The food concentration at which feeding became saturated varied depending on the 1 1 Growth food type, from ca. 2 µg C mL − when feeding on T. weiss flogii to ca. 5 µg C mL − when feeding on R. salina. The Polychaete larvae maximum carbon speci fic ingestion rates were very similar for both prey types and decreased with increasing Polydora ciliata larval size/age, from 0.67 d −1 for early larvae to 0.45 d −1 for late stage larvae. Growth rates as a function of food concentration ( R. salina ) followed a saturation curve; the maximum speci fic growth rate decreased slightly during larval development from 0.22 to 0.17 d −1. Maximum growth rates were reached at food concentrations ranging from 2.5 to 1.4 µg C mL −1 depending on larval size. The GGE, estimated as the slope of the regression equations relating speci fic growth rates versus speci fic ingestion rates, were 0.29 and 0.16 for early and intermediate larvae, respectively. The GGE, calculated speci fically for each food level, decreased as the food concentration increased, from 0.53 to 0.33 for early larvae and from 0.27 to 0.20 for intermediate larval stages. From an ecological perspective, we suggest that there is a trade-off between larval feeding/growth kinetics and larval dispersal. Natural selection may favor that some meroplanktonic larvae, such as P. ciliata , present low filtration ef ficiency and low growth rates despite inhabiting environments with high food availability. This larval performance allows a planktonic development suf ficiently long to ensure ef ficient larval dispersion. © 2009 Elsevier B.V. All rights reserved.

1. Introduction larval ecophysiology and biology generally are less well understood in comparison to the adult phases of the life cycles. Regarding their trophic Approximately 80% of marine benthic invertebrates (about 90,000 role, benthic invertebrate larvae participate both as grazers (plankto- species) have a biphasic life cycle and produce a planktonic larvae stage trophic larvae) and as prey in marine food webs. In coastal areas, (meroplankton) that spend a variable period of time, from minutes to planktotrophic invertebrate larvae are frequently the dominant compo- months, in the water column ( Thorson, 1950, 1964 ). For many benthic nent of the metazooplankton community during the reproductive invertebrate species, signi ficant dispersal is achieved only during this season ( Thorson, 1950; Blanner, 1982; Pedersen et al., in revision ). free-swimming larval stage ( Strathmann, 1985, 1990; Levin and Bridges, Notwithstanding, the possible trophic function of planktonic larvae in 1995; Pechenik, 1999 ), and planktonic larval population dynamics (e.g. the marine carbon flow also remains largely unknown. mortality, dispersion, development time) may determine the success of Spionid polychaetes are amongst the most common invertebrates the adult population by controlling recruitment. Despite the obvious in neritic benthic environments. Their planktotrophic larvae are importance of larval fitness for a given population success, planktonic periodically very abundant in coastal waters and may be the major component of zooplankton ( Anger et al., 1986 and references therein; Zajac, 1991; Pedersen et al., 2008 ). Polydora ciliata is one of the most ⁎ Corresponding author. Tel.: +34 93 2309500; fax: +34 93 2309555. abundant spionid polychaete species in northern European coasts and E-mail address: [email protected] (R. Almeda). it has also been found in coastal waters of Australia and Japan as an

0022-0981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi: 10.1016/j.jembe.2009.09.017

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introduced species ( NIMPIS, 2002; Ruellet 2004 ). P. ciliata is a serious Table 1 pest of important commercial bivalve species, as it burrows into Classi fication of Polydora ciliata larval stages according to their age/size. shells, weakening them and making them more prone to predation Larval stage Length range, μm Avg. initial length, μm Setigers (Kent, 1979, 1981 ; Boscolo and Giovanardi, 2002 ). Moreover, this Early 180 –400 232 3 –6 species may become a potential competitor for food resources of the Intermediate 400 –700 550 7 –10 suspension feeders where they settle on ( Ropert and Goulletquer, ⁎Late 700 –1200 902 11 –15

1999 ). P. ciliata populations are able to alter habitats by increasing the ⁎Larva with palps. amount of mud in an area (due to burrowing activities). These layers of mud can change the conditions locally thus outcompeting, or virtually eliminating the co-occurring fauna and flora. Hence, dense Initially, subsamples of larvae from each size/age group were fixed P. ciliata may be considered as a potential threat to the native fauna with 5% acid Lugol's solution and their average length and number of and ecological balance in some biotopes ( Daro and Polk, 1973 ). setigers were determined using an inverted microscope (100× Planktotrophic larvae depend on plankton as food source. As magni fication). The phytoplankton species used as prey was the plankton abundance and composition are temporally and spatially cryptophyte Rhodomonas salina (equivalent spherical diameter, variable, larval development may be limited by the food availability and ESD=9.7 µm) and the diatom T. weiss flogii (ESD=12.9 µm). quality during their pelagic life stage. Insuf ficient or inappropriate food Feeding rates were obtained by incubating larvae in triplicate bottles supply during larval development can lead to larval mortality by with the food suspension (experimental bottles) and measuring the starvation as proposed by Petersen et al. (2002) , and indirectly by change in prey concentration relative to that in the food suspension causing slow growth rates, prolonging the planktonic period of the without larvae (triplicate control bottles) after a certain period of larvae, and thus exposing larvae further to other sources of mortality incubation time (approximately 24 h for T. weiss flogii and 48 h for such as predation ( Thorson, 1950 ). Therefore, knowledge of grazing and R. salina ). growth response of marine invertebrate larvae against food availability is The food concentrations used for the functional response experi- of great importance in larval ecology. To our knowledge, there are only ments with T. weiss flogii ranged from 300 to 70,000 cells mL − 1 some partial studies on feeding and growth of polychaete larvae ( Blake (approx. from 0.03 to 6.0 µg C mL − 1) and in the case of R. salina from and Kudenov, 1981; Anger et al., 1986; Qian and Chia, 1991,1993 ) and 5000 to 100,000 cells mL −1 (approx. from 0.3 to 7.2 µg C mL − 1). complete functional feeding responses (grazing response to different These food ranges cover the food levels observed in the study area food concentrations) have been only reported for the trochophora larva (Pedersen et al., in revision ). Food suspensions were prepared in 1 L of the boreal capitellid Mediomastus fragile (Hansen, 1993 ) and for larvae jars using 0.2 µm FSW from the sampling area and phytoplankton of the spionid polychaete Marenzelleria cf. viridis (Burckhardt et al., from stock cultures as described in Hansen (1989) . Before preparing 1997 ). the food suspension each phytoplankton stock culture was filtered Therefore, the objectives of this study were: through a 20 µm mesh to remove potential cell aggregates. The phytoplankton suspensions were amended with a nutrient mixture 1) To determine the functional feeding responses of different P. ciliata (final concentration: 15 µM NH 4Cl and 1 µM Na 2HPO 4) to compensate larval stages feeding on two different preys separately (a cryptophyte the controls for nutrient enrichment due to larval excretion in the and a diatom). grazing trials. For each food level, triplicate initial, experimental and fi 2) To estimate the growth rates and gross growth ef ciencies of control bottles were filled by sequentially adding small amounts of P. ciliata larvae as a function of food concentration. the food suspension to each bottle. 3) To provide essential physiological background to understand distri- In the experimental bottles, larval densities ranged from 1 to 3 bution and life history traits of the species. larvae mL −1 depending on larval stage and food concentration. The incubations were carried out in acid washed 50 mL Pyrex glass bottles. 2. Material and methods The bottles were filled to the top ( final vol. 70 mL) and mounted with a plastic film to prevent any inclusion of air bubbles. Bottles were incubated 2.1. Larval collection on a rotating plankton wheel (0.5 rpm) in a temperature-controlled room at 16 °C at constant dim light (<5 µmol photons s −1 m−2). Live zooplankton samples were collected from surface waters (0 – Algae cell size (ESD, µm) was measured with an electronic particle 2.5 m) using a 50 µm-mesh WP-2 net, equipped with a non- filtering counter (Multisizer Coulter Counter, Beckman Coulter, Miami, FL, USA). cod-end, trawled horizontally at slow speed. The sampling station The cell volume was calculated and converted into carbon content using ‘Søminestationen ’ (55° 44 ′ N, 11° 48 ′ E) is situated in the Isefjord the appropriate equations described in Menden-Deuer and Lessard (Denmark), characterized by low depth, muddy bottom sediments and (2000) . The cell concentrations were determined by a Cytobuoy® eutrophic waters ( Rasmussen 1973; Novana 2004 ). Once the net was scanning flow cytometer (CSFC) ( http://www.cytobuoy.com/ ). The retrieved, the content of the cod-end was gently transferred to an CSFC is particularly designed to process live phytoplankton particles in isothermal container, diluted with surface water, and transported to the the size range of 1 µm and larger including diatom chains and particle laboratory within 2 h of collection. colonies. In this study we counted around 0.5 mL of sample in triplicate. In the laboratory, P. ciliata larvae were concentrated from the The prey cells in the experiment trials were counted by inserting the container by light attraction using a cold fiber optic light source, CSFC sample suction tube directly into the incubation bottles. During sorted with a pipette under the dissecting microscope and placed in a counting the trial bottles were carefully stirred with a mechanical stirrer beaker with 0.2 µm filtered seawater (FSW) from the sampling area. set at the lowest RPM. Prior to starting the series of grazing trials, we To reduce the presence of other planktonic organisms in the samples, compared the counting performance of CSFC to a Beckmann Coulter the larvae were repeatedly washed by transferring them through a Multi seizer III electronic particle counter, and found a linear counting series of Petri dishes filled with 0.2 µm FSW. relationship between the two instruments ( Fig. 1 ). Clearance and ingestion rates were calculated for each food 2.2. Functional feeding responses concentration according to Frost (1972) after the veri fication that prey concentrations in grazing bottles always were signi ficantly lower We determined the grazing response to different food concentra- than that in the controls (ANOVA test, P<0.05, software: SPSS 15.0). tions of different larval development stages of P. ciliata . We sorted The reduction in algal concentration in experimental bottles never and classi fied larvae in three groups according to their size ( Table 1 ). exceeded 40%.

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biomass during the incubation period. Additionally, we obtained the GGE for a particular larval stage as the slope of the linear regression of speci fic growth rates versus speci fic ingestion rates. To obtain functional relationships between physiological para- meters and food concentration, a set of regression equations was fitted to experimental data, following standard least squares proce- dures (Sigma plot software 9.0). The best fitting models are presented (highest correlation coef ficient, the lowest residual sum of squares).

3. Results

3.1. Functional feeding responses

Clearance rates ( F, mL larva − 1 d−1) of P. ciliata larvae on both species of phytoplankton followed an exponential decay function (curvilinear model, Figs. 2 and 3 ):

Fig. 1. Calibration of the Cytobuoy Scanning Flow Cytometer (CSFC) used in the feeding −αC F = F eð Þ 4 experiments. Live R. salina cells were counted in the CSFC and in the Multisizer III max ð Þ Coulter Counter (MIII). The slope of 1.02, P<0.0001 indicates that the two instruments count identically.

Carbon speci fic ingestion rates and clearance rates were calculated by dividing either ingestion rates (in µg C larva −1 d−1) or clearance rates (in mL larva −1 d−1) by the average larval biomass in µg C (see below).

2.3. Growth rates and ef ficiency of growth

Early larvae (3 –4 setigers) were collected as described above and incubated in 70 ml acid washed glass bottles (2 –3 larvae mL − 1) and fed during 8 days with five different food concentrations of R. salina. The food concentrations ranged from ca. 5000 to 100,000 cells ml − 1 (from 0.3 to 7.2 µg C mL − 1). Additional experiments were conducted incubating larvae for 48 h in 0.2 µm FSW to assess residual larval growth in absence of food. The incubation conditions were otherwise identical to those described in the feeding experiments section above. We took subsamples of larvae from each experimental bottle after 2, 6 and 8 days to estimate the growth rates during the incubation. The contents of the experimental bottles were carefully sieved through a 37 µm mesh and 25 –30 larvae were randomly collected and fixed immediately with 5% acid Lugol's solution. The remaining larvae were gently returned to the bottles with renewed phytoplank- ton suspensions. To measure the larval body length, we placed the preserved larvae under a dissection microscope (40× magni fication) and took images with a digital camera. The images were analyzed using the ImageJ software ( Abramoff et al., 2004 ). Larval body length (L, µm) was converted to carbon biomass ( W, µg) according to the equation ( Hansen, 1999 ):

W = 1 :5810 −4 × L1:38 1 ð Þ Larval speci fic somatic growth ( G, d − 1) was calculated as

G = 24 = T × Ln W = W 2 ð Þ ð 2 1Þ ð Þ where T is the duration of incubation (hours) and W1 and W2 are the initial and final carbon biomass of the larvae, respectively. The gross growth ef ficiency (GGE; ef ficiency by which ingested feed is converted into body weight) for each food concentration was calculated as

GGE = G = Is 3 ð Þ where G and Is are the daily carbon-speci fic growth and the carbon- Fig. 2. Relationship between food concentration ( T. weiss flogii , µg C mL − 1) and speci fic ingestion, respectively. We estimated the carbon-speci fic clearance rates ( ●; left axis) and ingestion rates ( ○; right axis) of three Polydora ciliata larval developmental stages: early (A), intermediate (B) and late (C). Each point is the ingestion rates for each food concentration at the beginning (0 –2 days) mean value of three replicates and the error bars represent the standard error (SE). The fi and at the end (6 –8 days) of the experiment. Carbon-speci c ingestion continuous lines correspond to the model fitted to the data (see equations in the text). rates were calculated taking into account the average larval carbon Notice, the different scale on Y-axis.

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were higher when feeding on T. weiss flogii than when feeding on R. salina . Obviously, this was also evident in the maximum speci fic − 1 − 1 clearance rates (mL µg C larva d ), which were, in average, 0.89±0.07 (SE) for T. weiss flogii and 0.49±0.01 (SE) for R. salina. The ingestion rates increased with food concentration up to a saturation food level of above which ingestion remained fairly constant ( Figs. 2 and 3 ), following a functional response type II .The best fit was for the Ivlev (1961) equation:

−βC I = Imax 1−eð Þ 5 ð Þ ð Þ

− 1 − 1 where I is the ingestion rate (cells larva d ), Imax is the asymptotic maximum ingestion (cells larva − 1 d− 1), β is a constant (rate at which ingestion approaches the maximum ingestion rate) and C is the phytoplankton concentration (µg C mL − 1). The parameters of the Ivlev functions for the different larval stages

tested are shown in Table 3 . The Imax increased with larval size; Imax on T. weiss flogii ranged from ca 2000 cells d − 1 for early larvae to − 1 10000 cells d for late larvae. Imax with R. salina as prey increased from 4000 to 10000 cells larva − 1 d− 1 for early and intermediate larval stages, respectively ( Table 3 ). Maximum speci fic ingestion rates s − 1 (I max , d ) were quite similar for both prey types and decreased with increasing larval size from 0.67 to 0.45 d − 1. The coef ficient β (rate at which ingestion approaches the maximum ingestion) was quite similar for early and intermediate larval stages. For T. weiss flogii saturation was reached at lower concentrations for late Fig. 3. Relationship between food concentration ( R. salina , µg C mL − 1) and clearance than for intermediate and early larval stages. rates ( ●; left axis) and ingestion rates ( ○; right axis) of two Polydora ciliata larval Saturating food concentrations ( SI, here de fined as the food developmental stages: early (A), intermediate (B). Each point is the mean value of three replicates and the error bars represent the standard error (SE). The continuous lines concentration corresponding to an ingestion equivalent to 95% of correspond to the model fitted to the data (see equations in the text). Notice, the Imax ) did not vary much for the different larval stages, but they were different scale on Y-axis. quite different for both preys, being always higher for R. salina both in cells and in carbon units ( Table 3 ).

3.2. Growth rates and ef ficiency of growth Table 2 Maximum clearance rates of different larval stages of Polydora ciliata . Larval growth rates responded to food concentration following a s 2 Larval stage Food type Fmax (±SE) F max (±SE) α (±SE) r curvilinear model ( Fig. 4 ). We used Ivlev models to describe the Early T. weiss flogii 0.215 (±0.016) 0.867(±0.064) 0.518 (±0.174) 0.89 relationship between speci fic growth rates ( G, d −1) and food concentra- Early R. salina 0.171 (±0.009) 0.476(±0.033) 0.235 (±0.033) 0.97 tion ( C, µgC mL −1): Intermediate T. weiss flogii 1.011 (±0.103) 1.029 (±0.105) 1.011 (±0.337) 0.82 Intermediate R. salina 0.402 (±0.251) 0.512 (±0.046) 0.173 (±0.028) 0.95 Late T. weiss flogii 1.490 (±0.087) 0.788 (±0.047) 0.566 (±0.105) 0.95 −γC G = Gmax 1−eð Þ 6 ð Þ ð Þ Fmax and α are the parameters of the exponential decay functions (Eq. (4) ) used to 1 describe the relationships between food concentration ( μg C mL − ) and clearance rates − 1 where Gmax is the maximum carbon-speci fic growth (d ) and γ is a (mL larva − 1d− 1). s 1 1 constant (rate at which growth approaches the maximum growth F (ml µg C − d− ) = maximum carbon speci fic clearance rates, SE = standard max larva fi error. rate). The parameters of the tted Ivlev function for three periods during the incubation are presented in Table 4 . The maximum speci fic − 1 growth rates ( Gmax , d ) decreased during the development, from − 1 − 1 − 1 where Fmax (mL larva d ) is the maximum clearance rate, C 0.22 to 0.17 d . The coef ficient γ increased during the larval (µg C mL − 1) is the food concentration and α is a stage and prey speci fic development ( Table 4 ). Early larvae were able to grow slightly constant. (0.025±0.015 d − 1) under starving conditions. The parameters of the fitted functions for the different larval stages The saturating food concentration required to obtain a maximum

investigated are shown in Table 2 . Fmax increased with increasing growth rate ( SG, here de fined as the food concentration required to get larval size. For the same larval stage, the maximum clearance rates 95% of maximal growth rate (G max )) was higher in early than in late

Table 3 Maximum ingestion rates for different larval stages of Polydora ciliata.

s 2 Larval stage Food type Imax (± SE ) I max (± SE ) β (± SE ) r SI Early T. weiss flogii 1866 (±29) 0.645 (±0.010) 1.242 (±0.066) 0.99 2.41 Early R. salina 3953 (±130) 0.675 (±0.017) 0.627 (±0.056) 0.99 4.77 Intermediate T. weiss flogii 6624 (±306) 0.579 (±0.010) 1.275 (±0.149) 0.98 2.35 Intermediate R. salina 9576 (±693) 0.583 (±0.027) 0.624 (±0.127) 0.95 4.80 Late T. weiss flogii 9889 (±264) 0.448 (±0.010) 2.063 (±0.193) 0.99 1.45

− 1 − 1 − 1 Imax (cells larva d ) and β are the parameters of the Ivlev equation (Eq. (5) ) used to describe the relationships between food concentration (µg C mL ) and ingestion rates (cells larva − 1d− 1). s − 1 − 1 I max = maximum carbon speci fic ingestion rates, d ; SI = saturating food concentration in µg C mL ; SE = standard error.

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Table 5 In fluence of food concentration (µgC mL − 1) on gross growth ef ficiency (GGE= G/Is) of two larval developmental stages of Polydora ciliata .

Larval stage Food conc. G I s GGE ⁎GGE

Early 0.00 0.025 0.000 0.34 0.083 0.159 0.53 0.74 0.126 0.268 0.47 0.29 1.68 0.180 0.455 0.40 3.30 0.220 0.630 0.35 7.29 0.219 0.662 0.33 Intermediate 0.46 0.052 0.192 0.27 1.13 0.075 0.303 0.25 2.03 0.100 0.507 0.20 0.16 4.06 0.116 0.556 0.21 8.10 0.115 0.575 0.20

G = carbon speci fic growth rates (d − 1), Is = carbon speci fic ingestion rates (d − 1). ⁎GGE calculated as the slope of the linear regression relating G to Is for each particular larval stage (see Fig. 5 ). Fig. 4. Carbon-speci fic growth rates of Polydora ciliata larvae (mean value±SE) at different food concentration ( R. salina ) for three periods during the incubation: after 2 days (0 –2 d), after 6 days (0 –6 d) and over the entire incubation time (0 –8 d). 4. Discussion

Table 4 4.1. Functional feeding responses Parameters of the equations (Eq. (6) ) used to describe the relationships between food concentration (µg C mL − 1) and carbon speci fic growth rates (d − 1) of Polydora ciliata P. ciliata larvae did not present a decline on clearance rates at low larvae in three incubation periods (days). food concentrations, in contrast to other meroplanktonic larvae ( Sprung, 2 Incubation period Gmax (±SE) γ (±SE) r SG 1984b; Burckhardt et al., 1997 ) and some copepods ( Frost, 1975; 0–2 0.218 (±0.010) 1.190 (±0.066) 0.97 2.51 Kiørboe et al., 1985 ). The absence of a feeding threshold may either be a 0–6 0.187 (±0.003) 1.930 (±0.140) 0.96 1.55 result of a de ficient experimental set up (the food concentrations used 0–8 0.167 (±0.004) 2.120 (±0.193) 0.93 1.41 not being low enough) or due to physiological (behavioral) reasons. SE = standard error. Since the lowest food concentrations used in the functional response SG = food concentration required to get 95% of maximal growth rate ( Gmax ), γ is a experiment were in the range of the lowest food concentrations found in constant (rate at which growth approaches the maximum growth rate). natural larval habitat ( Pedersen et al., in revision ) we suggest that the second hypothesis has stronger arguments. In most polychaete larvae including P. ciliata , the ciliary apparatus is used for both feeding and larval stages, ranging from 2.51 to 1.41 µg C mL − 1 (from ~35000 to propulsion ( Hansen et al., submitted for publication ). This may result in a 20000 cells mL − 1). constant feeding even at low food concentrations. Gross growth ef ficiencies (GGE) calculated as the slope of the linear Another important observation was that ingestion rates did not regression relating carbon-speci fic growth rates with carbon-speci fic decrease at very high food concentrations, as opposed to other ciliated ingestion rates, were 0.29 for early larvae and 0.16 for intermediate meroplanktonic larvae such as bivalves ( Riisgard et al., 1980; Riisgard, larval stages ( Fig. 5 ). GGE, calculated speci fically for each food level, 1988; Beiras and Pérez Camacho, 1994 ). A decrease in ingestion rates decreased as the food concentration increased, from 0.53 to 0.33 for at very high particle concentrations has been proposed to be a result fi early stages and from 0.27 to 0.20 for intermediate stages ( Table 5 ). from interference and clogging of the larval ltration apparatus (Gallager, 1988 ). This suggests that P. ciliata larvae are fit to heavily eutrophic environments with high particle concentrations by being able to clear their ciliary bands ef ficiently. The functional response model of P. ciliata larvae was similar to those described for other meroplanktonic larvae ( Jespersen and Olsen, 1982; Crisp et al., 1985; Hansen and Ockelmann, 1991 ) and for holoplanktonic zooplankters ( Mullin, 1963; Frost, 1972; Gaudy, 1974 ). Maximum speci fic clearance and ingestion rates of P. ciliata larvae were higher than those reported for other polychaete larvae (Mediomastus fragile — Hansen, 1993 ; Marenzelleria cf. viridis — Burckhardt et al., 1997 ), but similar to those observed in other meroplanktonic larvae with similar body size (bivalve — Jespersen and Olsen, 1982 ; gastropod — Hansen and Ockelmann, 1991 ). It is interesting to note that the maximum speci fic feeding rates of polychaete larvae, and other meroplanktonic larvae, are commonly lower than for holoplanktonic larvae of similar size and at same temperature, such as copepods nauplii and copepodites ( Paffenhöfer 1971 ; Berggreen et al., 1998; Hansen et al., 1997 ). This indicates a less ef ficient feeding mechanism in these ciliary feeders. Fig. 5. Carbon-speci fic growth rates ( G, d −1) versus carbon-speci fic ingestion rates ( Is, d −1) Maximum speci fic clearance rates of P. ciliata larvae feeding on of Polydora ciliata larvae for two experimental periods. Error bars represent the standard T. weiss flogii almost doubled those for R. salina . These results corroborate errors. Gross growth ef ficiency (GGE) for a particular larval stage was calculated as the slope the data of Hansen et al. (submitted for publication) , that showed that of the linear regression relating speci fic growth rates to speci fic ingestion rates. Regressions are: G=0.035+0.293 I s, r2=0.98 for 0 –2 days and G=0.024+0.161 I s, r2=0.98 for 6 – the optimal prey size for early larval stages (230 µm long ±30, SD) of 8 days. P. ciliata was ca. 13 µm (corresponding to the T. weiss flogii ESD), lower

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prey sizes being retained at reduced ef ficiencies and maximal prey size 4.3. Is P. ciliata larvae growth limited by food in nature? increasing during the larval development. Similarly, the saturating food concentration was approximately 2 times higher when feeding on Larvae are food limited when natural food concentrations are below R. salina than when feeding on T. weiss flogii . In the case of larvae of those required to support maximal rates of growth and development polychaete Marenzelleria cf. viridis , the low clearance rates reported (Olson and Olson, 1989; Fenaux et al., 1994 ). In the current study site, could be due to the use of a very small prey, Chlorella vulgaris (usually 2 – the available food concentrations ( Pedersen et al., in revision ) would be 6 µm ESD). This small prey may not be ef ficiently retained by a relatively insuf ficient to support the maximum growth rates of P. ciliata larvae. In large larva (760 µm of length). As a result, feeding saturation was general, the food concentrations typically observed in coastal eutrophic reached at very high food concentrations at 118 µg C m l−1 (Burckhardt waters ( Huntley and Boyd, 1984 ) are much lower than the saturating et al., 1997 ). food concentrations required for both maximal ingestion and growth A decrease of maximum speci fic ingestion rates with increasing rates of P. ciliata larvae. This suggests that growth of P. ciliata larva is body size has also been reported for most marine zooplankters frequently limited by food in nature. (Hansen et al., 1997 ). It is expected that small larvae have higher The food concentration required for maximal ingestion rates of ingestion demands per unit biomass than larger ones since speci fic P. ciliata larvae was within the range generally reported for mero- metabolic requirements decrease with increasing body size ( Ikeda et planktonic ciliated larvae ( Pechenik and Fischer, 1979; Sprung 1984a; al., 2001 ), Crisp et al., 1985; Riisgard, 1988; Hansen and Ockelmann, 1991; Pérez- Camacho et al., 1994 ). Maximal speci fic growth rates were achieved at 4.2. Growth rates and ef ficiency of growth food concentrations similar to those reported for other ciliated meroplanktonic larva and ranged from 1 to 3 µgC mL −1 (Bayne, 1965; Maximum speci fic growth rates observed in this study were quite Wilson, 1979; Crisp et al., 1985; Beiras and Pérez Camacho, 1994; Pérez- similar to those reported for P. ciliata and other polychaete larvae Camacho et al., 1994; Lee, 2002 ), with some exceptions ( Sprung, 1984a; (Hansen, 1999 ). In general, P. ciliata maximal speci fic growth rates MacDonald, 1988; Hansen, 1993 ). With respect to holoplanktonic were comparable to other meroplanktonic larvae and holoplanktonic larvae, such as copepod nauplii and copepodites, maximum growth 1 metazoans ( Hansen et al., 1997 and references therein). However, the rates are commonly reached at lower food levels, ≤0.5 µg C mL − carbon speci fic growth of P. ciliata larvae was decreasing with (Vidal, 1980 a; Berggreen et al., 1998; Calbet and Alcaraz, 1997; Finlay increasing body size. This trend was also noted for the same species and Roff, 2006 ), than meroplanktonic ciliated larvae. Therefore, by Hansen (1999) and has been commonly found among metazoo- although food limited growth seems to be a general rule for marine plankters ( Sprung, 1984b; Paffenhöfer, 1976 ). This could due to the zooplankton ( Lampert 1985; Hirst and Bunker, 2003 ), most meroplak- change in the ratio between energy used for maintenance and growth, tonic larvae are likely to be limited by food to a higher degree than which increases with body size, resulting in a decrease in growth holoplanktonic larvae (e.g. copepod nauplii). ef ficiency ( Fenchel, 1974 ). Early larvae (3 –4 setigers) displayed residual growth under 4.4. Ecological signi ficance starvation conditions, for at least 2 days. This indicating that larvae carbon reserves can sustain the metabolic requirement and even add Traditionally food limitation, temperature and predation have been to larval length growth but most likely not in terms of carbon growth. considered the main factors controlling the survival of meroplanktonic Thus, newly hatched larvae are able to tolerate temporary starvation larvae, which are the key for understanding variations in recruitment of periods by using their carbon storages and, probably, exhibiting low benthic marine communities ( Thorson, 1946, 1964, 1966 ). metabolic carbon losses (low energy maintenance requirements) as Besides larval survival, the reproductive strategy can contribute to suggested by Hansen (1993) for Mediomastus fragile polychaete the variations in the recruitment of marine benthic invertebrates. The larvae. This starvation tolerance increases the probability of survival reproductive pattern of P. ciliata may facilitate their success in in a highly fluctuating food environment where a risk for a mis-match recruitment since they start to reproduce just one month after between food availability and larval requirements are common. settlement and spawn several times (4 –5) per year. Some polychaetes Gross growth ef ficiencies (GGE) of P. ciliata larvae were similar to species produce larvae with different nutrition modes (lecitotrophic, those reported for other meroplanktonic larvae ( Sprung, 1984c; Hansen, adelphophagy, planktotrophy) in one single spawning ( Levin and 1993 ) but were in the lower end of that reported for holoplanktonic Huggett, 1990 ). This could be facilitated the adaptation to differences metazoans ( Kiørboe et al., 1985; Hansen et al., 1997 ). The GGE was higher trophic levels and constrains. As far we know, this strategy has not been in earlier larvae than in intermediate and late larval stages, a pattern observed in of P. ciliata since their larvae are only planktotrophic. exhibited by other meroplankton such as bivalve larvae ( Bayne et al., P. larvae exhibit relatively low clearance rates which probably limit 1976 ). Nevertheless, we could face an artifact due to the carryover of yolk its dispersion in oligotrophic waters. In fact, the species only presents reserves by early larval stages that ultimately provides a surplus for very dense population in highly productive coastal waters and estuaries. growth. The planktonic period for P. ciliata larvae has been estimated varying The decrease in gross growth ef ficiency (GGE) with increasing food between 2 to 6 weeks ( Wilson, 1928; Anger et al., 1986 ). Food limitation concentration observed in P. ciliata larvae has been also reported for other is considered a disadvantage for larval survival, because it extends the zooplankters ( Mullin and Brooks, 1970; Thompson and Bayne, 1974; development time and consequently increases the probability of Paf fenhöfer, 1976 ). GGE can be expressed as the product of net growth predation mortality ( Rumrill, 1990 ). However, as mentioned before, ef ficiency and assimilation ef ficiency. In many metazooplankters the most of meroplaktonic larvae display a high degree of food limitation and assimilation ef ficiency declines with increasing food concentrations thus slow growth rates in nature. Slow larval growth rates lead to a (Straile, 1997 , and references therein). An increase in the amount of prolongation of the planktonic period, which enables larval dispersion food ingested under higher food concentrations usually decreases the and hence the spreading of the species. A higher dispersion of the turnover time of food in the gut, resulting in a decline in assimilation organism may apparently be of more selective relevance for the overall ef ficiency ( Dagg and Walser, 1986; Butler and Dam, 1994; Sandier and survival of the species than a potential increase of larval mortality due to Van Den Bosch, 1994 ). Another possible explanation for the observed longer planktonic period. This hypothesis is supported by two arguments: decrease in GGE may be due to the increase in the accumulation rates of carbon reserves under increasing food levels. This would reduce the 1) Despite the suggestions that pelagic predation could be an important proportion of the ingested food used for growing and consequently the source of mortality (Thorson, 1946), recent experimental studies using GGE. natural densities of meroplanktonic larvae as prey have shown

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surprisingly low predation rates ( Johnson and Shanks, 2003; Metaxas Dagg, M.J., Walser Jr., W.E., 1986. The effect of food concentration on fecal pellet size of marine copepods. Limnol. Oceanogr. 31, 1066 –1071. and Burdett-Coutts, 2006 ). Physical, chemical and behavioral larval Daro, M.H., Polk, P., 1973. The autocology of Polydora ciliata along the Belgian coast. defenses to deter planktonic predators may lead to these relatively low Neth. J. Sea Res. 6, 130 –140. rates (e.g. sabellariid polychaetes larvae, Wilson, 1929; Pennington Eckert, G.L., 2003. Effects of the planktonic period on marine population fluctuations. Ecology 84, 372 –383. and Chia, 1984 ). Fenaux, L., Strathmann, M.F., Strathmann, R.R., 1994. Five tests of food-limited growth 2) Larval dispersion is crucial for the survival of marine benthic of larvae in coastal waters by comparisons of rates of development and form of invertebrate species because it promotes the connectivity between echinoplutei. Limnol. Oceanogr. 39 (1), 84 –98. populations, which is important for the persistence of the popula- Fenchel, T., 1974. Intrinsic rate of natural increase: the relationship with body size. Oecologia 14, 317 –326. tions ( Roughgarden et al., 1988 ), facilitates the establishment of sites Finlay, K., Roff, J.C., 2006. Ontogenetic growth rate responses of temperate marine following disturbances ( Hansen et al., 2002; Petersen et al., 2002 ), copepods to chlorophyll concentration and light. Mar. Ecol. Prog. Ser. 313, and favors the flow of genetic information ( Palumbi, 2003; 145 –156. Frost, B.W., 1972. Effects of size and concentration of food particles on the feeding Trakhtenbrot et al., 2005 ). Marine populations with longer plank- behaviour of the marine planktonic copepod Calanus paci ficus . Limnol. Oceanogr. tonic period may decrease the adult population fluctuations, 17, 805 –815. reducing local extinction risks ( Eckert, 2003 ). The analyses of fossil Frost, B.W., 1975. A threshold feeding behaviour in Calanus paci ficus . Limnol. Oceanogr. 20, 263 –266. shells showed that some marine species with planktotrophic larval Gallager, S.M., 1988. Visual observations of particle manipulation during feeding in (longer planktonic period) stages had lower probability of extinction larvae of a bivalve mollusc. Bull. Mar. Sci. 43, 344 –365. than species with non-feeding larval stages due to lower dispersion Gaudy, R., 1974. Feeding four species of pelagic copepods under experimental conditions. Mar. Biol. 25, 125 –141. capabilities ( Jablonski and Lutz, 1983 ). Hansen, P.J., 1989. The red tide dino flagellate Alexandrium tamarense : effects behaviour and growth of a tintinnid ciliate. Mar. Ecol. Prog. Ser. 53, 105 –116. Therefore, we suggest that there is a trade-off between larval Hansen, B., 1993. Aspects of feeding, growth and stage development by trochophora feeding/growth kinetics and dispersal. Natural selection seems to favor larvae of the boreal polychaete Mediomastus fragile (Rasmussen) (Capitellidae). fi J. Exp. Mar. Biol. Ecol. 166, 273 –288. that some meroplanktonic larvae, such as P. ciliata , present low ltration Hansen, B.W., 1999. Cohort growth of planktotrophic polychaete larvae — are they food and growth rates despite inhabiting environments with extremely high limited? Mar. Ecol. Prog. Ser. 178, 109 –119. food. This larval performance allows a planktonic development Hansen, B., Ockelmann, K.W., 1991. Feeding behaviour in larvae of the opistobranch fi fi Philine aperta I.Growth and functional response at different developmental stages. suf ciently long to ensure ef cient larval dispersion. Mar. Biol. 111, 255 –261. Hansen, P.J., Bjørnsen, P.K., Hansen, B., 1997. Zooplankton grazing and growth: scaling Acknowledgements within the 2 –2000 µm body size range. Limnol. Oceanogr. 42 (4), 687 –704. Hansen, B.W., Stenalt, E., Petersen, J.K., Ellegaard, C., 2002. Invertebrate re-colonisation in Mariager Fjord (Denmark) after severe hypoxia. I. Zooplankton and settlement. This work is based on a research performed at Roskilde University Ophelia 56 (3), 197 –213. field station Søminestationen. We thank T.F. Sørensen and G. Drillet for Hansen, B. W., Jakobsen, H. H., Andersen, A., Almeda R., Pedersen, T. M., Christensen, A. M., their assistance in the field work. This work was supported by a PhD Nilsson, B., submitted for publication. Swimming behavior and prey retention of the polychaete larvae Polydora ciliata (Johnston). J. Exp. Biol. fellowship to R.A. (BES-2005-7491) from the Spanish Ministry of Hirst, A.G., Bunker, A.J., 2003. Growth of marine planktonic copepods: global rates and Education and Science, grant no.272-07-0485 to B.W.H. from the Danish patterns in relation to chlorophyll a, temperature, and body weight. Limnol. Oceanogr. National Science Research Council, and grant CTM2004-02575/MAR 48, 1988 –2010. Huntley, M., Boyd, C., 1984. 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Resum de l’article V III– Article VIII summary– Catalan versión

Alimentació i cinètica del creixement de larves planctotròfiques del poliquet espiònid Polydora ciliata (Johnston)

Rodrigo Almeda, Troels M. Pedersen, Hans H. Jakobsen, Miquel Alcaraz, Albert Calbet, Benni W. Hansen. Article publicat a Journal of Experimental Marine Biology and Ecology (2009, 382: 61-68) .

Es va estudiar l'efecte de la concentració d'aliment en les taxes d'alimentació i creixement de diferents fases de desenvolupament larvari del poliquet espiònid Polydora ciliata . Es va estimar les taxes d'alimentació de les larves en funció de la concentració d'aliment mitjançant experiments d'incubació amb dues preses diferents ofertes per separat, el criptòfit Rhodomonas salina (ESD = 9.7 µm) i la diatomea Thalassiosira weissflogii (ESD = 12.9 µm). A més, es van determinar les taxes de creixement larvari i l'eficiència de creixement brut (GGE) en funció de la concentració de R. salina . Les larves de P. ciliata van mostrar una resposta funcional de tipus II. Les taxes d’aclariment van disminuir contínuament amb l'augment de concentració d'aliment, i les taxes d'ingestió van augmentar fins a una concentració de saturació per sobre de la cual la ingestió es va mantenir relativament constant. La concentració d'aliment a la qual la ingestió es saturava va variar depenent del tipus d'aliment, des d'aprox. 2 µg C mL -1 quan s'alimentavan de T. weissflogii a 5 µg C mL -1 quan s'alimentavan de R. salina . Les taxes d'ingestió específiques màximes van ser molt similars pels dos tipus de preses i van disminuir amb l'augment de grandària i l'edat de les larves, de 0.67 -1 en larves primerenques a 0.45 d -1 en larves de fase tardana. Les taxes màximes de creixement específic van disminuir lleugerament durant el desenvolupament larvari de 0.22 a 0.17 d -1 . Les taxes màximes de creixement es van aconseguir a concentracions d'aliment que van anar des de 2.5 fins a 1.4 µg C mL 1, depenent de la grandària de les larves. La GGE, estimada com la pendent de les equacions de regressió que relacionen les taxes de creixement amb les taxes d'ingestió específica, va anar de 0.29 i 0.16 per a fases larvàries primerenques i intermèdies, respectivament. La GGE, calculada específicament per a cada nivell d'aliment, va disminuir conforme augmentava la concentració d'aliment, de 0.53 a 0.33 per a larves primerenques i de 0.27 a 0.20 per a fases larvàries intermèdies. Des d'una perspectiva ecològica, suggerim que existeix una solució de compromís entre l'alimentació/cinètica del creixement larvari i la dispersió de les larves. La selecció natural pot afavorir que algunes larves meroplanctòniques, tals com P. ciliata , presentin una baixa eficiència de filtració i baixes taxes de creixement malgrat habitar en sistemes amb alta disponibilitat d'aliment. Aquest funcionament de les larves permet un desenvolupament planctònic prou llarg per assegurar una dispersió larvària eficient.

189 Chapter 5

Resumen del artículo VIII – Article VIII summary– Spanish versión

Alimentación y cinética del crecimiento de larvas planctotróficas del poliqueto espiónido Polydora ciliata (Johnston).

Rodrigo Almeda, Troels M. Pedersen, Hans H. Jakobsen, Miquel Alcaraz, Albert Calbet, Benni W. Hansen. Artículo publicado en Journal of Experimental Marine Biology and Ecology (2009, 382: 61-68) .

Se estudió el efecto de la concentración de alimento en las tasas de alimentación y crecimiento de diferentes fases de desarrollo larvario del poliqueto espiónido Polydora ciliata . Estimamos las tasas de alimentación de larvas en función de la concentración de alimento mediante experimentos de incubación con dos presas diferentes ofrecidas por separado, la criptofita Rhodomonas salina (ESD = 9.7 µm) y la diatomea Thalassiosira weissflogii (ESD = 12.9 µm). Además, se determinaron las tasas de crecimiento larvario y la eficiencia de crecimiento bruto (GGE) en función de la concentración de R. salina. Las larvas de P. ciliata mostraron una respuesta funcional de tipo II. Las tasas de aclaramiento disminuyeron continuamente con el aumento de concentración de alimento, y las tasas de ingestión aumentaron hasta una concentración de saturación por encima del cual la ingestión se mantuvo relativamente constante. La concentración de alimento a la cual la ingestión se saturaba varió dependiendo del tipo de alimento, desde aprox. 2 µg C mL -1 cuando se alimentaban de T. weissflogii a 5 µg C mL -1 cuando se alimentaban de R. salina . Las tasas de ingestión específicas máximas fueron muy similares para ambos tipos de presas y disminuyeron con el aumento de tamaño y la edad de las larvas, de 0.67 -1 en larvas tempranas a 0.45 d -1 en larvas de fase tardía. Las tasas máximas de crecimiento específico disminuyeron ligeramente durante el desarrollo larvario de 0.22 -1 a 0.17 d -1 . Las tasas máximas de crecimiento se alcanzaron a concentraciones de alimento que fueron desde 2.5 hasta 1.4 µg C mL -1 , dependiendo del tamaño de las larvas. La GGE, estimada como la pendiente de las ecuaciones de regresión que relacionan las tasas de crecimiento con las tasas de ingestión específica, fue de 0.29 y 0.16 para fases larvarias tempranas e intermedias, respectivamente. La GGE, calculada específicamente para cada nivel de alimento, disminuyó conforme aumentó la concentración de alimento, de 0.53 a 0.33 para larvas tempranas y de 0.27 a 0.20 para fases larvarias intermedias. Desde una perspectiva ecológica, sugerimos que existe una solución de compromiso entre la alimentación/cinética del crecimiento larvario y la dispersión de las larvas. La selección natural puede favorecer que algunas larvas meroplanctónicas, tal como P. ciliata , presenten una baja eficiencia de filtración y bajas tasas de crecimiento a pesar de habitar sistemas con alta disponibilidad de alimento. Este funcionamiento de las larvas permite un desarrollo planctónico lo suficientemente largo para asegurar una dispersión larvaria eficiente.

190 Larval growth of the dominant polychaete, Polydora ciliat a, is indeed food limited in a eutrophic Danish estuary, Isefjord.

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Vol. 407: 99–110, 2010 MARINE ECOLOGY PROGRESS SERIES Published May 20 doi: 10.3354/meps08563 Mar Ecol Prog Ser

Larval growth in the dominant polychaete Polydora ciliata is food-limited in a eutrophic Danish estuary (Isefjord)

Troels Møller Pedersen 1, Rodrigo Almeda 2, Frank Lech Fotel 1, 4 , Hans Henrik Jakobsen 3, Patrizio Mariani 3, Benni Winding Hansen 1, *

1Roskilde University, Department of Environmental, Social and Spatial Change, PO Box 260, 4000, Roskilde, Denmark 2Institut de Ciènces del Mar, CSIC, P. Marítim de la Barceloneta 37-49, 08003 Barcelona, Catalunya, Spain 3National Institute of Aquatic Resources, Technical University of Denmark, Kavalergården 6, 2920 Charlottenlund, Denmark 4Present address: DHI Water & Environment, Agern Allé 5, 2970 Hørsholm, Denmark

ABSTRACT: Food limitation in larval growth of the spionid polychaete Polydora ciliata was examined in a typical eutrophic estuary, Isefjord, in Denmark. In the field, food availability and the energetic requirements of the P. ciliata larval population were measured during 2 different periods in 2004 and 2007 that together cover the productive part of the year for plankton. In the laboratory, specific growth rates (µ) of larvae reared on natural food suspensions (~0.10 d –1 ) were always lower than those of larvae reared on phytoplankton-enriched food suspensions (100% retention efficiency for Rhodomonas salina ; ~0.21 d –1 ). Total meroplankton biomass (average: 3.72 µg C l –1 , range: 0.11 to 26.05 µg C l –1 ) was frequently similar to or exceeded that of holoplankton (average: 5.70 µg C l –1 , range: 0.08 to 29.89 µg C l –1 ), suggesting a trophic significance of meroplankton in the estuary. P. cil- iata was commonly the dominant meroplanktonic larvae (average: 0.5 µg C l –1 , range: 0.03 to 2.51 µg C l –1 ). The available food in the optimal prey size fraction (2004, average: <20 µm; range: 99 to 274 µg C l –1 ; 2007, average: 7 to 18 µm; estimated carbon demand: 119 µg C l –1 ; range: 19 to 474 µg C l –1 ) seemed to be sufficient to cover the energetic carbon requirements of the population through- out the study (0.09 to 3.15 µg C l –1 d–1 ), but insufficient to attain the maximum specific growth rates reported in previous laboratory experiments. This suggests that P. ciliata larvae probably exhibit a low feeding efficiency and their maximum specific growth rates are consequently attained at food concentrations even higher than those found in this eutrophic environment.

KEY WORDS: Food limitation · Larval growth · Polydora ciliata · Larvae

Resale or republication not permitted without written consent of the publisher

INTRODUCTION tion prolongs the planktonic period and increases the exposure of larvae to other sources of mortality such as Spawning of benthic invertebrates, including Poly- predation (Thorson 1950). Consequently, food limita- dora ciliata , results in the release of a large number of tion during the planktonic larval stages is suggested to planktonic larvae (meroplankton) which spend a vari- decrease recruitment into benthic populations (Thor- able period of time, from minutes to months, in the son 1950, Vance 1973, Olson & Olson 1989). water column (Thorson 1950, 1964). Since planktonic Beside food quantity, food quality should be taken into food resources are highly patchy, it is likely that larvae account as well when evaluating potential food-limited are often food limited in natural environments, i.e. nat- growth in nature. Plankton cells may be too large or ural food concentrations are below those required to small for larvae to catch and ingest and can vary consid- support maximum rates of growth and development erably in nutritional quality (Pechenik 1987). Bivalve, (Olson & Olson 1989, Fenaux et al. 1994). Food limita- gastropod and many capitellid polychaete larvae retain

*Corresponding author. Email: [email protected] © Inter-Research 2010 · www.int-res.com

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food particles in the nano-fraction (2 to 20 µm) from a few zooplankton (200 to 2000 µm) (Schram 1968, Anger et to 15 µm equivalent spherical diameter (ESD) (Riisgård al. 1986 and references therein, Zajac 1991, Pedersen et et al. 1980, Hansen 1993, Raby et al. 1997). In contrast, al. 2008), suggesting an important role as grazers on small spionid polychaete larvae (5 to 10 setigers) are able primary producers. However, the possible trophic func- to ingest large centric diatoms, e.g. Coscinodiscus spp. tion in the marine carbon flow of polychaete larvae, as (Anger et al. 1986). Recent experiments indicate that the well as other meroplanktonic larvae, remains largely particle regime which Polydora ciliata can exploit in- unknown. In order to fully understand the function of creases during the larval ontogeny from 4 to 50 µm ESD, planktotrophic larvae in the ecosystem, their temporal and the optimal food size for small larvae (230 ± 30 µm in and spatial abundance as well as their physiological length) is around 12 µm (Hansen et al. unpubl.). A simi- requirements must be assessed. lar ontogenetic development of the retention spectra has The objectives of the present study were to first test previously been reported in gastropod veliger larvae if larvae of the spionid polychaete Polydora ciliata (Hansen 1991). Thus, food limitation is not solely due to exhibited food-limited growth in situ throughout the a general low food concentration; i.e. a match between entire productive part of year in plankton. To address available food size and the physiological capabilities of this topic we carried out 2 experimental approaches: the larvae is also crucial for optimal feeding and growth. (1) we determined the food availability (in terms of Food limitation is thought to be more important for quantity and quality [size]) for P. ciliata larvae in its crustacean larvae than for the ciliated larvae of bivalves habitat and evaluated if it was enough to support the and echinoderms (Olson & Olson 1989). Furthermore, maximum growth rates in situ as observed in previous food limitation is expected to be less important in laboratory experiments (Almeda et al. 2009); (2) we coastal waters than in oceanic waters (Huntley & Boyd assessed the degree of food limitation by comparing 1984). However, food limitation in coastal waters has growth rates of larvae reared on natural food suspen- been reported for bivalve (Fotel et al. 1999) and echino- sions with those reared on natural food suspensions derm larvae (Fenaux et al. 1994, Eckert 1995). In case of enriched with cultivated phytoplankton ( Rhodomonas polychaete larvae, studies are very scarce but the exist- salina) in excess in the laboratory. The second objec- ing research suggests food-limited growth during sum- tive of the present study was to estimate the energy mer (Paulay et al. 1985, Hansen 1999). requirements of P. ciliata larval populations and their Spionid polychaetes, and very often Polydora spp., trophic significance. are among the most common invertebrates in neritic benthic environments. During certain periods, their planktotrophic larvae MATERIALS AND METHODS can be the major component of meso- Field site and sampling. The present study was conducted during 2 periods in 2004 (April to September) and 2007 (September to November), covering the entire productive part of the year, at the field station Søminestationen (Fig. 1) situated in the Isefjord, Denmark. The field site has a depth of 4 m and is characterized by muddy bottom sedi- ments and eutrophic waters (Rasmus- sen 1973), being a relevant example of a boreal shallow water estuary (Conley et al. 2000). Quantitative zooplankton samples were collected every 3 to 4 wk by dupli- cate hauls in 2004 and more frequently (every second day to second week) by single hauls in 2007. The hauls were conducted from bottom to surface with a 45 µm mesh size WP-2 net equipped with a closed cod-end and a digital flow Fig. 1. Søminestationen sample station ( Q) in the Danish estuarine system, Isefjord meter (Hydro Bios, model 438 110). (55° 42’ 44.45” N, 11° 47’ 45.48” E) Samples were gently concentrated on a

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45 µm screen and stored in containers with 80 to 96% tered the metric dimensions of the particles and ethanol in final concentration. assigned a unique identifier to each which was used Live zooplankton for growth experiments were gen- later for image retrieval and sample analysis (Sieracki tly collected by trawling the WP-2 net equipped with a et al. 1998). We used the autoimage mode data acqui- 1 l closed cod-end at slow speed through surface water sition in the software package VisualSpreadsheet (0 to 2.5 m). Immediately after the net was retrieved, (VISP, v 1.5.16, www.fluidimaging.com) with a mini- zooplankton were diluted by surface water and mal particle size of 5 µm. The procedure allowed us to brought to the laboratory in a 50 l thermo box. derive individual particle width ( W) and length ( L) , Seawater samples for food availability studies and while particle volume ( V) was calculated assuming growth experiments were collected every second day prolate spheroid particle shapes: during growth incubation experiments. The discrete W2 L V π × × (1) samples were taken at 0, 1, 2, 3 and 4 m depth by a 3 l = 6 heart-valve water bottle sampler pooled in a 30 l The aspect ratio ( W/L) was used to differentiate bucket; these were considered integrated water col- between chain-formed and spherical particles. The umn samples. Temperature was also measured every relevant aspect ratio was decided for each sample second day during growth incubation experiments by run, but it was typically around 0.4. The individual a thermometer attached to the sampling bottle, and particle volumes were converted to cell carbon using salinity was determined by a hand-held refractometer the carbon:volume relationship given by Montagnes (ATAGO) with a resolution of 0.5 salinity units. et al. (1994) for autotrophic flagellates (spherical par- Food availability. According to an ongoing study ticles). The chain-formed particles presented a differ- (Hansen et al. unpubl.), the particle regime Polydora ent challenge. Because chains are formed by smaller ciliata can exploit increases during the larval develop- cells with similar carbon content, we applied a con- ment from 4 to 50 µm ESD, and the optimal food size stant carbon:volume relationship. This fixed value for small larvae (230 ± 30 µm in length) is around was based on inspections of the images generated by 12 µm (range: 7 to 18 µm). Therefore, large phyto- the FlowCAM which revealed dominance of diatoms plankton chains or colonies can most likely not be cap- of the species Skeletonema cf. costatum during peri- tured by P. ciliata larvae. We used 2 experimental ap- ods when chain-formed particles were present. Typi- proaches to determine the food availability for P. ciliata cally, these cells had a size of 20 µm which corre- larvae: (1) fractioned chlorophyll a (chl a) measure- sponds to a carbon:volume ratio of 0.06 using the ments and (2) plankton community analysis. appropriate isometric carbon:volume relationship for Fractioned chl a measurements: We fractionated diatoms (Montagnes et al. 1994). collected seawater through Nitex screens and into dif- Total phytoplankton biomass (µg C l –1 ) was regres- ferent size fractions (<20, 20–50, 50–200 and >200 µm) sed against the measured chl a (µg chl a l–1 ) to obtain a in order to assess the available phytoplankton biomass conversion factor. This conversion factor was applied for Polydora ciliata larvae. Water samples (50 ml) of to calculate the available phytoplankton biomass in each fraction were filtered in triplicate through GF/F term of carbon from the chl a measurements. filters by use of syringes and filter capsules. The filters Growth experiments. We carried out 9 growth ex- with retained particles were extracted overnight in periments during the 2 study periods (Table 1). Poly- 96% ethanol as the extraction solvent (according to dora ciliata larvae were concentrated from the con- Jespersen & Christoffersen 1987). If necessary, the ex- tainer by light attraction using a cold fiber optic light tract was filtered through 0.2 µm filter capsules or cen- source. Early larvae (<300 µm in length) were sorted trifuged to get rid of particles. Chl a and phaeopig- with a pipette under a dissecting microscope and incu- ments in the extractions were measured in quartz glass bated within a few hours from collection. Larvae were cuvettes on a Turner Designs 700 fluorometer. incubated for 5 d in 2 types of food treatments (3 to 5 Plankton community analysis: We analysed the size replicates per treatment except in September 2004, see structure and morphological composition of the plank- ‘Results’): (1) natural seawater from the sampling site ton community in order to assess the available food for (screened through 200 µm), i.e. natural food suspen- Polydora ciliata . Samples of natural seawater were sions; and (2) natural seawater (screened through fixed in acid Lugol’s to a final concentration of 5%, 200 µm) with added culture phytoplankton ( Rhodo- stored in brown glass bottles and analysed in an auto- monas salina , ~9 to 10 µm ESD) in excess, i.e. enriched mated microscope (FlowCAM ®, www.fluidimaging. food suspensions. com) within 48 h after fixation. Each sample was Excess of food (~40 000 cells ml –1 ) was obtained from analysed in triplicate for 20 min. Images of plankton published saturation levels of functional food re- particles were continuously acquired by a video cam- sponses according to Almeda et al. (2009). Incubations era. The automated microscope counted and regis- were conducted in 70 ml acid-washed glass bottles

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Table 1. Polydora ciliata. Larval concentration and initial and final larval length in the growth experiments (*converted from number of setigers according to Hansen 1999). Percent cleared in the bottles per day is calculated from the clearance rate versus body length relationship presented in Hansen et al. (unpubl.). The minimum and maximum clearance is based on the initial and final body length, respectively, and the intermediate is an average of the 2, each in natural food suspension. Estimates of the pelagic life span are based upon the growth rates in natural and enriched food suspension. In these estimates the initial length was set to 2.5 setigers and the length at metamorphosis was set to 17 setigers (Anger et al. 1986)

Year Experiment Larval Initial larval Final larval length Bottle volume cleared Pelagic life span concentration length (µm) (% d –1 ) (d) (larvae ml –1 ) (µm) Natural Enriched Min. Intermediate Max. Natural Enriched

2004 May 0.5 388.8 ± 18.5* 645.8 ± 17.2* 801.8 ± 50.7* 37.5 48.8 60.0 42.8 27.4 Jun 0.5 245.9 ± 16.4* 421.7 ± 33.4* 678.5 ± 42* 21.5 31.0 40.5 47.9 19.7 Aug 0.5 127.3 ± 0* 342.2 ± 95.9* 663.1 ± 50* 11.5 21.0 30.5 23.9 12.3 Sep 0.5 241.3 ± 23* 462.1 ± 58.9* 488.1 ± 25.8* 21.0 32.0 43.0 46.4 38.9

2007 Sep (initial) 1.43 229.4 ± 14.5 283.6± 17.2 524.2± 12.1 57.1 64.3 71.4 57.8 15.0 Sep (terminal 1) 1.43 217.7 ± 25.3 313.4 ± 12.7 435.7 ± 17.6 54.3 68.6 82.9 32.8 17.2 Sep (terminal 2) 0.86 212.9 ± 60.6 283.7 ± 8.5 350.5 ± 7.8 31.7 37.3 42.9 29.8 17.1 Oct 0.71 295.2 ± 52.5 452.4 ± 5.6 612.5 ± 33.1 38.6 49.6 60.7 27.0 16.0 Nov 0.57 244.1 ± 1.8 318 ± 14.6 490.4 ± 7.8 24.0 28.9 33.7 49.5 17.0

placed on a plankton wheel at 0.5 rpm in dim light at average larval length ( L, µm) to carbon biomass ( W, 16°C. Larval densities varied depending on the exper- µg) according to the equation (Hansen 1999): iment (Table 1). In the September terminal 1 experi- W = (1.58 10 –4 )L1.38 , R 2 = 0.996 (4) ment, we conducted an additional growth experiment × (September terminal 2) with a different larval density Setiger counting was carried out under an inverted

in order to assess possible crowding effects. An initial microscope (100 ×). To measure body length, preserved subsample of larvae from each experiment was taken larvae were placed under a dissecting microscope and fixed for length and setiger number determination. equipped with a digital camera and at least 40 images

Every second day, 80% of the food suspension was of random chosen larvae were taken per bottle (40 × or

removed by inverse filtration through 45 µm mesh and inverted microscope [100 ×], depending on the experi- the suspension renewed from pre-screened integrated ment). These images were used with ImageJ software in situ seawater collected the same day from the sam- to measure length. pling area (for natural food suspensions) or by Rhodo- Zooplankton abundance and energy requirements. monas salina in excess (enriched food suspensions). Quantitative zooplankton samples were split by a Fol- At the end of the incubation, larvae from each experi- som plankton splitter and a minimum of 300 individu- mental bottle were concentrated and fixed with acid als were counted and identified. Carbon biomass of the Lugol’s solution for later size measurements. individual zooplankton taxa, including Polydora ciliata Larval specific growth rates were estimated from the larvae, was estimated using the formulas reported by increases in soma tic biomass according to the expres- Nielsen et al. (2007, their Table 5.1). sion for logarithmic growth: Energy requirements of the population of Polydora ciliata larvae were determined in terms of estimated = (24/ T) ln( W /W ) (2) µ × f i carbon demand (ECD) according to equation: where T is the duration of incubation (h) and W and W i f ECD = ( P )/GGE (5) are the initial and final carbon biomasses of the larvae µ (µg), respectively. where P is the population biomass of P. ciliata larvae In 2004, larval biomasses were estimated by count- (µg C l –1 ), µ is the specific growth rate (d –1 ) and GGE ing the number of setigers (~30 larvae replicate –1 ). (= specific carbon growth/specific carbon ingestion) is Average number of setigers ( S) was converted to car- the gross growth efficiency assumed equal to 0.29, R 2 = bon biomass ( W, µg) according to the equation 0.98 (Almeda et al. 2009). The population biomass of (Hansen 1999): P. ciliata larvae was calculated as an average for each relevant month and the specific growth rates deter- W = (6.81 10 –3 )S2.03 , R 2 = 0.990 (3) × mined in our growth experiments were used. In 2007, larval biomasses were estimated by measur- Growth data were not normally distributed because ing body length (~40 larvae replicate –1 ). We converted of 2 outliers. Levine’s test indicated that variances

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were homogeneous, so further analysis was conducted RESULTS on untransformed data. A 2-way ANOVA (Systat v.11) was conducted to determine the effects of treatment, Abiotic factors and food availability time of growth experiment and their interaction on the specific growth rates. Multiple comparisons of signifi- Temperature showed a typical seasonal pattern with cant effects were conducted with Bonferroni-corrected spring temperatures of ~10°C, increasing in summer to probabilities for planned comparisons, in particular around 20°C, then declining throughout fall to ~5°C comparing treatment for each experiment and sea- (Fig. 2A). The salinity differed by ~5 units between sonal differences within each treatment. September 2004 and September 2007 (Fig. 2A). Total

25 25 A 2004 2007

20 20

15 15

Temp Salinity Salinity Temperature (°C) Temperature 10 10

5 5 5-Jul 7-Jul 9-Jul 1-Oct 8-Oct 2-Aug 4-Aug 6-Aug 1-Sep 3-Sep 6-Sep 8-Sep 8-Nov 20-Apr 23-Oct 26-Oct 29-Oct 31-Oct 14-Jun 16-Jun 18-Jun 30-Aug 13-Sep 23-Sep 26-Sep 12-Nov 15-May 19-May

16 B 14 2004 2007

12 Size (µm) >200 200 10 50 )

–1 20 8 (µg l a

Chl 6

4

2

0 5-Jul 7-Jul 9-Jul 1-Oct 8-Oct 1-Sep 3-Sep 4-Sep 6-Sep 8-Sep 2-Aug 4-Aug 6-Aug 8-Nov 20-Apr 23-Oct 26-Oct 29-Oct 31-Oct 14-Jun 16-Jun 18-Jun 30-Aug 13-Sep 23-Sep 26-Sep 12-Nov 27-Nov 17-May 19-May 21-May

Fig. 2. (A) Integrated temperature and salinity and (B) size composition of the in situ phytoplankton community in Isefjord during the experimental periods in 2004 and 2007

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chl a (Fig. 2B) reflects the bell-shaped pattern of the organisms were higher in May to August 2004 than in water temperature generated by the global radiation, September to November 2007. High densities of with spring values of 2 to 3 µg chl a l–1 ; values Rotifera were observed in October 2007. The holo- increased during summer, reaching ~10 µg chl a l–1 , plankton was dominated, both in density and biomass, and then declined with some fluctuations through the by Calanoida (Copepoda) throughout the sampling fall. The <20 µm phytoplankton size fraction was dom- periods, with the exception of 30 August 2004 and inant most of the time from April to September 2004 October 2007, when Rotifera showed high biomass and (Fig. 2B) except 21 May, when the >200 µm size frac- densities. The meroplankton was dominated both in tion was prevalent. In September 2007, phytoplankton density and biomass by Polychaeta in May 2004. From were more or less equally distributed among 3 size June to August 2004, biomass was mostly dominated classes (<20 µm, 20–50 µm, 50–200 µm) except on 23 by Gastropoda, whereas density was dominated by and 26 September, when the 50–200 µm size fraction Bivalvia. Throughout the rest of the sampling period dominated. During October and November 2007, the (September to November 2007), Polychaeta dominated 20–50 µm phytoplankton size fraction became more the meroplankton. dominant until late November, when the phytoplank- ton present was either <20 or 50–200 µm. Phytoplank- ton >200 µm were mostly found in August and the A beginning of September 2004. 600 Plankton biomass dynamics in 2007 estimated by the FlowCAM are shown in Fig. 3. The study site was )

highly dynamic in terms of plankton abundance and –1 composition (Fig. 3A). Biomass fluctuations showed an 400 initial dominance in chain-formed phytoplankton fol- lowed by 2 smaller biomass peaks of chain-formed par- ticles, most likely Skeletonema cf. costatum , separated 200 by ~20 to 30 d. The phytoplankton biomass outside the S. cf. costatum peaks was characterized by smaller Biomass (µg C l ellipsoid particles. In September, most of the non- chained particles were dinoflagellates such as Hetero- 0 capsa triquetra , unidentified spheroid dinoflagellates 1000 of unknown trophy, cell doublets of S. cf. costatum and B various unidentified flagellates. After the last pulse of ) –1

S. cf. costatum , most of the particles were H. triquetra l

C

and small unidentified algae, while S. cf. costatum was 100 g µ

present below the detection level. Since small larvae (

s

have much narrower prey size spectra than larger lar- s vae (Hansen et al. unpubl.), we isolated the biomass a m o fraction available for newly hatched larvae in the i 10 B range of 7 to 18 µm (Fig. 3B). Chl a was plotted against carbon, and a linear re- gression (±SE) rendered a carbon:chl a conversion 2 factor of 47 (C = 47(±11)chl a – 81(±78); R = 0.52, 1 p = 0.0054). 26-Sep 23-Sep 13-Sep 12-Nov 27-Nov 29-Oct 26-Oct 6-Sep 8-Sep 8-Nov Available carbon biomass for the prey size ranges 4-Sep 1-Oct that can be captured by Polydora ciliata larvae in the study area, corresponding to the date of the growth Fig. 3. Food availability in Isefjord during fall 2007 estimated experiment, is shown in Table 2. by the FlowCAM. (A) Structural diversity of the standing plankton biomass. Closed circles are the total plankton bio- mass and the black bars are biomass of round particles (parti- cles with aspect ratio >0.4). The difference between total car- Zooplankton community and biomass of Polydora bon and round particles is mostly due to the biomass of the ciliata larvae chain-formed diatom Skeletonema cf. costatum (B) Biomass of plankton particles in the 7–18 µm size range representing the optimal prey size spectrum of Polydora ciliata small larvae, The density and biomass of both holo- and mero- according to Hansen et al. (unpubl.). Black bars are round planktonic organisms are depicted in Fig. 4. Generally, particles (flagellates) and gray bars are elongated particles both density and biomass of holo- and meroplanktonic (chain-formed particles)

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Table 2. Polydora ciliata . Available carbon biomass for the prey size ranges that Polydora ciliata was the most domi- can be captured by P. ciliata larvae in the study area corresponding to date of nant polychaete larvae; it constituted 20 the growth experiments. Phytoplankton biomass was calculated by fractioned to 100% (average: 71%; Fig. 6) of the chlorophyll a measurements and plankton community by FlowCam analysis. na: not available total polychaete larval population and was found in densities from 0.03 larvae l–1 in late October (31 Oct) to 8.05 larvae Year Experiment Phytoplankton biomass Plankton community –1 (µg C l –1 ) (µg C l –1 , 7–18 µm) l in May (17 May). On average, P. cili- 0–50 µm <20 µm ata represented 38.5% of the total meroplanktonic biomass. 2004 May 117 99 na 2004 Jun 153 149 na 2004 Aug 290 274 na 2004 Sep 276 249 na Growth experiments 2007 Sep (initial) 238 113 89 2007 Sep (terminal 1) 169 93 262 The 2-way ANOVA showed that 2007 Oct 256 119 95 there was interaction between time of 2007 Nov 152 73 31 the growth experiments and treatment (F = 12.69, p << 0.005). This indicates The relative proportion of holo- and meroplankton that the effect of added food on larval growth differed biomass is shown in Fig. 5. The meroplankton repre- during the course of the year. Specific growth rates of sented more than 50% of the planktonic biomass on 7 Polydora ciliata larvae were higher when offered out of 18 sampling days, more than 80% on 3 sampling enriched food suspensions (Fig. 7). Based on multiple days and more than 90% of the total biomass on 26 comparisons, larval growth was significantly different September 2007. Hence, meroplankton indeed episod- between treatments (p < 0.05) in all experiments

ically dominated the zooplankton community. except in May and September 2004 (p ≤ 0.14 and p ≤ 1,

Holoplankton Meroplankton 60 60 2004 2007 2004 2007 50 50 ) –1 40 40

30 30

20 20 Abundance (ind. l 10 10

0 0 35 35 Harpacticoida Echinodermata 30 Cyclopoida Cirripedia 30 Calanoida Polychaeta )

–1 25 Ostracoda Bivalvia 25 Cladocera Gastropoda 20 Rotifera Bryozoa 20

15 15

10 10 Biomass (µg C l 5 5

0 0 26-Sep 26-Sep 30-Aug 30-Aug 23-Sep 23-Sep 17-May 17-May 12-Nov 12-Nov 29-Oct 29-Oct 26-Oct 26-Oct 23-Oct 23-Oct 14-Jun 14-Jun 31-Oct 31-Oct 6-Sep 6-Sep 8-Sep 8-Sep 8-Nov 8-Nov 4-Sep 4-Sep 2-Aug 2-Aug 8-Oct 8-Oct 1-Oct 1-Oct 5-Jul 5-Jul Fig. 4. Composition of the holoplankton and meroplankton in Isefjord, based on both abundance and biomass, for the 2 sampling periods in 2004 and 2007

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2004 2007 showed that there was no consistent 100 effect of enrichment. For example, in 2007, there were no significant differ- ences between the specific growth 80 rates from the enriched experiments, while there were significant differences between many of the specific growth 60 rates in natural food suspensions. Furthermore, there was no significant difference between the specific growth 40 rate in natural food suspension in May 2004 and November 2007, but there % of total biomass was a significant effect of treatment in 20 November 2007 in contrast to May 2004. The overall conclusion is that en- richment stimulated the specific growth 0 rate of P. ciliata larvae. Estimates of Polydora ciliata clear- 5-Jul 1-Oct 8-Oct 2-Aug 4-Sep 8-Nov 8-Sep 6-Sep ance in the experimental containers 31-Oct 14-Jun 23-Oct 26-Oct 29-Oct 12-Nov 17-May 23-Sep 30-Aug 26-Sep under natural food conditions were Fig. 5. Fraction of holoplankton (gray bars) and meroplankton (black bars) of the total zoo planktonic biomass in Isefjord for the 2 sampling periods in based upon the relationship between 2004 and 2007 body length and maximum clearance rate from Hansen et al. (unpubl.). Max- 2004 2007 100 imum clearance ranged between 30.5 and 82.9% of bottle volume per day (Table 1). Using initial larvae length, 80 the minimum clearance ranged be- tween 11.5 and 57.1% of bottle volume per day (Table 1). The intermediate clearance was an average of the mini- 60 mum and the maximum volume cleared and ranged from 21 to 68.6% (Table 1). The number of setigers of newly 40 hatched larvae (2 to 3 setigers) and the number of setigers of larvae ready for metamorphosis (17 setigers, according 20 to Anger et al. 1986) were used to esti- mate the pelagic life span (Table 1) under natural and enriched food condi- 0 tions. An average reduction of pelagic % lifespan under optimal food conditions P. ciliata (i.e. enriched) as compared to natural 26-Sep 23-Sep 30-Aug 17-May 12-Nov 29-Oct 26-Oct 23-Oct 14-Jun 31-Oct 6-Sep 4-Sep 8-Nov 8-Sep 2-Aug 8-Oct 1-Oct

5-Jul food conditions was estimated to be as Fig. 6. Polydora ciliata . Percentage P. ciliata (black bars) of the total polychaete much as 19.6 d.

larval biomass larval polychaete of total biomass (grey bars) in Isefjord for the 2 sampling periods in 2004 and 2007

respectively). The September 2004 growth experiment Estimated carbon demand was only based on 2 replicates for each treatment, which put some constrains on the interpretation of the The ECD of Polydora ciliata larval population for the statistical output. Average specific growth rates on different growth experiments is shown in Table 3, and natural food suspensions were ~0.10 d –1 , 2-fold lower ranged from 0.07 to 1.23 µg C l –1 . During the entire than that in enriched suspensions (average: ~0.21 d –1 ). study period, the available carbon biomass (in relevant Larval survival was visually checked at the end of the size fractions, Table 2) exceeded by several orders of incubation and we did not observe evident mortality magnitude the food requirements to support the maxi- in any treatment. The multiple comparisons also mum growth of the population (Table 3).

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0.35 not grow at their maximum rates at the 2004 * 2007 Isefjord study site, which is a typical 0.30 example of a Danish estuary. Consider- ) –1 Natural * ing the typical food concentration of Enriched * 0.25 * * * Danish estuaries around the year (Con- * ley et al. 2000), one can extrapolate 0.20 these findings to other Danish coastal environments and to the remaining part 0.15 of the year. Since newly hatched larvae only ingest a rather narrow prey size 0.10 between 7 and 18 µm ESD (Hansen et

Specific growth rate (d rate growth Specific al. unpubl.), recruitment of larger indi- 0.05 viduals is limited to the availability of particles within this size range. That is, 0.00 at periods such as 26 September 2007, May Jun Aug Sep Sep Sep Sep Oct Nov (initial) (terminal 1) (terminal 2) when we recorded the highest phyto- plankton biomass, the particle window Fig. 7. Polydora ciliata . Specific growth rate (d –1 , ±SD) of P. ciliata larvae in natural between 7 and 18 µm ESD only reached and enriched food suspensions in Isefjord for the 2 sampling periods in 2004 and 480 µg C l –1 , which is close to one-fifth 2007. N = 35 to 100 larvae per incubation bottle. *Significant (p < 0.05) difference of the required biomass for maximum between treatments growth by the early larval stage.

Table 3. Polydora ciliata . Estimated carbon demand (ECD) of P. ciliata based upon biomass of larval population and the specific growth rates in natural and Growth with natural vs. enriched food enriched food suspensions (see Eq. 5). Trophic impact equals ECD (natural) as suspensions % of the plankton community biomass in the size fraction that can be grazed by larvae (0–50 µm) The larval specific growth patterns between the 2 years were obviously dif- Year Experiment ECD (µg C l –1 ) Trophic impact (%) Natural Enriched Natural ferent (Fig. 7). In 2004, specific growth in natural food suspensions fluctuated 2004 May 0.81 1.23 0.69 more than it did in 2007, and growth in 2004 Jun 0.33 0.79 0.21 the enriched food suspensions steadily 2004 Aug 0.04 0.07 0.01 increased during 2004, whereas it was 2004 Sep 0.03 0.04 0.01 2007 Sep (initial) 0.02 0.07 0.01 more similar and high throughout 2007. 2007 Sep (terminal 1) 0.21 0.39 0.12 Environmental characteristics varied 2007 Sep (terminal 2) 0.23 0.40 0.14 between the 2 years: temperature de- 2007 Oct 0.30 0.50 0.13 velopment was very different, salinity 2007 Nov 0.03 0.08 0.02 was much lower in 2007 and available food steadily increased during 2004 and was more constant during 2007. Since DISCUSSION growth incubations were performed at constant tem- perature in the laboratory both years, temperature Food availability and growth rate most likely cannot explain the observed larval growth differences. Differences in salinity were presumably According to recent laboratory studies (Almeda et al. not the cause either, since Polydora ciliata is a well- 2009), maximum growth rates of Polydora ciliata larvae adapted euryhaline species. The effect of food avail- are reached, with optimal food size, at food concentra- ability, however, may be reflected in the basic physio- tions ranging from 1410 to 2510 µg C l –1 depending on logical condition of the larval populations. A gradual larval size. During the present study periods, the change in food concentration was reflected in a steady phytoplankton concentrations were always lower than growth increase during 2004 for both natural and these critical food levels. Moreover, if we consider the enriched food suspensions (except for the September size fraction of plankton that can be effectively cap- experiment). It is more difficult, based on food avail- tured by larvae, the available carbon biomass is one ability, to explain the growth pattern observed in 2007. order of magnitude lower than the critical food levels Therefore, the different growth patterns in both 2004 observed in the laboratory. Therefore, P. ciliata may and 2007 could be due to a combination of the effects

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of these environmental variables, or another factor reach metamorphosis was greatly reduced when ex- altogether. cess food was offered (Table 1). Enriching the avail- Specific growth rates of larvae reared on natural and able food resulted in an average reduction of the pela- enriched food suspensions indicated that larvae were gic life span of 19.6 d, corresponding to a 47.6% not growing at their maximum rates in nature. Maxi- reduction on average. On the one hand, this reduces mum growth rates observed in the enrichment treat- the dispersal ratio of the propagules; on the other ments were similar to maximum growth rates of larvae hand, it potentially reduces larval mortality and observed under food satiating conditions in the labora- thereby increases benthic recruitment of Polydora tory (Almeda et al. 2009). However, artifacts from lab- ciliata larvae. oratory bottle incubation conditions are possible, e.g. the decline in food concentration during the 2 d be- tween the water changes. The estimated clearances Energetic carbon demand and trophic significance shown in Table 1 indicate that the larvae in 2 of the growth experiments (September initial and terminal 1 The available carbon was enough to support the food 2007) could have experienced starvation through the demand considering the food requirements (ECD) of whole experiment since the percent bottle volume Polydora ciliata . However, these larvae were not able cleared based upon the initial length was above 50% to grow at maximum growth rates at in situ food con- (57.1 and 54.3%, respectively). Furthermore, some lar- centrations. This suggests a low feeding efficiency vae in 4 of the 9 growth experiments (May 2004 and and, hence, that the food limitation of larvae is func- September initial, September terminal 1 and October tional. The food requirements of Polydora ciliata larvae 2007) may have experienced starvation at the end of constituted only negligible fractions of the standing the experiment. But if one compares September termi- stock of primary producers; therefore, the P. ciliata nal 1 and 2 (Fig. 7), there was only a slight difference population had a very slight effect on the plankton in growth rates in natural food suspension. Moreover, community (low trophic impact, <1%, on total primary growth rates in natural food suspensions in experi- producers in energetic requirements, see Table 3). The ments with high larval densities (e.g. September initial daily food requirements (ECD) of the Polydora ciliata and terminal 1 2007) were not different to those in population corresponds well with the finding of Han- experiments with low larval density (e.g. November sen et al. (1999) in a Greenlandic study, where mero- 2007). Therefore, an eventual crowding effect cannot plankton (mainly bivalves and gastropods) daily explain the differences in growth between treatments, ingested just 0.12 µg C l –1 , equivalent to 0.32% of the and the depletion of food in the experimental contain- average phytoplankton biomass. ers was expected to be minimally important. Hence we The trophic impacts as well as the bottom-up control conclude that the observed differences in growth be- exerted by microzooplankton are more important fac- tween treatments are not due to the depletion of food tors controlling phytoplankton communities. However, between the water changes. larval grazing pressure on protozoans (Hansen et al. In addition to food quantity, food nutritional quality unpubl.) and associated cascading trophic effects may influence larval growth rates of Polydora ciliata . could be a key factor structuring the planktonic micro- The copepod Temora longicornis fed on Skeletonema bial assemblage in coastal areas during periods with cf. costatum at rates comparable to feeding rates on high larval abundances. This trophic interaction re- Rhodomonas salina in the laboratory. However, the mains to be studied quantitatively. copepod egg production and somatic growth was sig- Meroplankton, including polychaete larvae, is an nificantly reduced compared to controls fed with important fraction of mesozooplankton, even periodi- R. salina (Dutz et al. 2008). There is growing evidence cally exceeding that of holoplankton in boreal estuar- that diatoms including S. cf. costatum contain biomole- ies (Blanner 1982, Hansen et al. 2002, present study). cules aimed at defending the algae against predation The trophic impact of other meroplanktonic larvae has (Pohnert et al. 2002, Pohnert 2005) and it is very likely been reported in Isefjord previously by Jørgensen that the increased growth in the R. salina treatments (1981), where, based on ECD, a cohort of bivalve lar- by P. ciliata larvae is a result of improved food quality. vae ( Mytilus edulis) daily cleared 40 to 50% of the sur- But to answer such question we need a thorough rounding water mass for small particles (probably fla- analysis of phytoplankton growth and nutritional sta- gellates). Although more information about the trophic tus as well as other factors influencing the functional impact of meroplankton on microbial assemblages is food limitation and potential active food selection of required, meroplanktonic larvae potentially play an meroplanktonic organisms. important trophic role in terms of both their direct con- Comparing the specific growth rates in both natural trol of the microbial community and as a source of cas- and enriched food suspensions, the time required to cading effects in the microbial plankton food web

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(Martin et al. 1996). Hence we propose that mero- Dutz J, Koski M, Jonasdottir SH (2008) Copepod reproduction planktonic larvae need more attention in terms of is unaffected by diatom aldehydes or lipid composition. future research and as key components structuring Limnol Oceanogr 53:225–235 ➤ Eckert GL (1995) A novel larval feeding strategy of the tropical the planktonic microbial assemblage in boreal coastal sand dollar, Encope michelili (Agassiz): adaption to food waters. limitation and an evolutionary link between planktotrophy and lecithotrophy. J Exp Mar Biol Ecol 187:103–128 Fenaux L, Strathmann MF, Strathmann RR (1994) Five tests of food-limited growth of larvae in coastal waters by compar- CONCLUSIONS isons of rates of development and form of echinoplutei. Limnol Oceanogr 39:84–98 The planktotrophic larvae of Polydora ciliata are ➤ Fotel FL, Jensen NJ, Wittrup L, Hansen BW (1999) In situ and functionally food limited regardless of the time of year laboratory growth by a population of blue mussel larvae (i.e. even during the productive part of the year), even (Mytilus edulis L.) from a Danish embayment, Knebel Vig. J Exp Mar Biol Ecol 233:213–230 though they inhabit a heavily eutrophic estuary. Food- ➤ Gosselin LA, Qian PY (1997) Juvenile mortality in benthic limited growth appears to be a general premise for marine invertebrates. Mar Ecol Prog Ser 146:265–282 boreal planktotrophic meroplankton (Burckhardt et al. ➤ Hansen B (1991) Feeding behaviour in larvae of the opistho- 1997, Hansen 1999, Hansen et al. 2002, Petersen et al. branch Philine aperta . II. Food size spectra and particle selectivity in relation to larval behaviour and morphology 2002). The ecological consequences of growth-limited of the velar structures. Mar Biol 111:263–270 larvae could lead to a less fit juvenile population and ➤ Hansen B (1993) Aspects of feeding, growth and stage devel- poor post-metamorphic performance (Pechenik et al. opment by trochophora larvae of the boreal polychaete 1996, McEdwards & Qian 2001). The juvenile stage for Mediomastus fragile (Rasmussen) (Capitellidae). J Exp benthic marine invertebrates is a vulnerable stage in Mar Biol Ecol 166:273–288 ➤ Hansen BW (1999) Cohort growth of planktotrophic poly- the life cycle, and high mortality rates are often chaete larvae — are they food limited? Mar Ecol Prog Ser observed (Gosselin & Qian 1997, Pedersen et al. 2008). 178:109–119 This could be due to carry-over effects from the nutri- ➤ Hansen BW, Nielsen TG, Levinsen H (1999) Plankton commu- tional condition of the recruits, the larvae. nity structure and carbon cycling on the western coast of Greenland during the stratified summer situation. III. Mesozooplankton. Aquat Microb Ecol 16:233–249 Hansen BW, Stenalt E, Petersen JK, Ellegaard C (2002) Inver- Acknowledgements. This work is based on research per- tebrate re-colonisation in Mariager Fjord (Denmark) after formed at Roskilde University’s field station Søminestationen. a severe hypoxia. I. Zooplankton and settlement. Ophelia We thank T. F. Sørensen and G. Drillet for assistance in the 56:197–213 field, A. B. Faarborg and B. Søborg for chlorophyll measure- ➤ Huntley M, Boyd C (1984) Food-limited growth of marine zoo- ments, E. M. Pedersen, K. T. Jensen and A. Winding for com- plankton. Am Nat 124:455–478 ments on earlier versions of the manuscript, and A. M. Palm- Jespersen AM, Christoffersen K (1987) Measurements of kvist and G. Banta for statistical assistance. This project was chlorophyll a from phytoplankton using ethanol as extrac- supported by the Danish National Science Research Council tion solvent. Arch Hydrobiol 109:445–454 (grant no. 272-07-0485) to B.W.H., an instrument grant to Jørgensen CB (1981) Mortality, growth and grazing impact of DTU AQUA supported by the VELUX foundation to H.H.J. a cohort of bivalve larvae, Mytilus edulis L. Ophelia 20: and by a mobility grant to R.A. from the Spanish Ministry of 185–192 Education and Science. ➤ Martin D, Pinedo S, Sardá R (1996) Grazing by meroplank- tonic polychaete larvae may help to control nanoplankton in the NW Mediterranean littoral: in situ experimental evi- LITERATURE CITED dence. Mar Ecol Prog Ser 143:239–246 McEdward LR, Qian PY (2001) Effects of the duration and ➤ Almeda R, Pedersen TM, Jakobsen HH, Alcaraz A, Calbet A, timing of starvation during larval life on the metamorpho- Hansen BW (2009) Feeding and growth kinetics of the sis and initial juvenile size of the polychaete Hydroides planktotrophic larvae of the spionid polychaete Polydora elegans (Haswell). J Exp Mar Biol Ecol 261:185–197 ciliata (Johnston). J Exp Mar Biol Ecol 382:61–68 Montagnes DJS, Berges JA, Harrison PJ, Taylor FJR (1994) ➤ Anger K, Anger V, Hagmeier E (1986) Laboratory studies on Estimating carbon, nitrogen, protein, and chlorophyll a larval growth of Polydora ligni , Polydora ciliata , and Pygo- from volume in marine phytoplankton. Limnol Oceanogr spio elegans (Polychaeta, Spionidae). Helgol Meeres- 39:1044–1060 unters 40:377–395 Nielsen TG, Ottosen LDM, Hansen BW (2007) Structure and Blanner P (1982) Composition and seasonal variation of the function of the pelagic ecosystem in Young Sound, NE zooplankton in the Limfjord (Denmark) during 1973–1974. Greenland. In: Rysgaard S, Glud RN (eds) Carbon cycling Ophelia 21:1–40 in Arctic marine ecosystems: case study Young Sound. ➤ Burckhardt R, Schumann R, Bochert R (1997) Feeding biology BioScience 58:87–207 of the pelagic larvae of Marenzelleria cf. viridis (Poly- Olson RR, Olson MH (1989) Food limitation of planktotrophic chaeta: Spionidae) from the Baltic Sea. Aquat Ecol 31: marine invertebrate larvae: Does it control recruitment 149–162 success? Annu Rev Ecol Syst 20:225–247 ➤ Conley DJ, Kaas H, Møhlenberg F, Rasmussen B, Windolf J ➤ Paulay G, Boring L, Strathmann RR (1985) Food limited (2000) Characteristics of Danish estuaries. Estuaries 23: growth and development of larvae: experiments with nat- 820–837 ural sea water. J Exp Mar Biol Ecol 93:1–10

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Pechenik JA (1987) Environmental influences on larval sur- bivalve larvae in a temperate embayment. Mar Biol 127: vival and development. In: Giese AC, Pearse JS, Pearse 665–672 VB (eds) Reproduction of marine invertebrates, Vol. IX. Rasmussen E (1973) Systematics and ecology of the Isefjord Boxwood Press, Pacific Grove, CA, p 551–608 marine fauna (Denmark). Ophelia 11:1–495 ➤ Pechenik JA, Hammer K, Weise C (1996) The effect of star- Riisgård HU, Randløv A, Kristensen PS (1980) Rates of water vation on acquisition of competence and post-metamor- processing, oxygen consumption and efficiency of particle phic performance in the marine prosobranch gastropod retention in veligers and young post-metamorphic Mytilus Crepidula fornicate (L.). J Exp Mar Biol Ecol 199: edulis . Ophelia 19:37–47 137–152 Schram TA (1968) Studies on meroplankton in the inner ➤ Pedersen TM, Hansen JLS, Josefson AB, Hansen BW (2008) Oslofjord I. Composition of the plankton at Nakkeholmen Mortality through ontogeny of soft-bottom marine inverte- during a whole year. Ophelia 5:221–243 brates with planktonic larvae. J Mar Syst 73:185–207 ➤ Sieracki CK, Sieracki ME, Yentsch CM (1998) An imaging- Petersen JK, Stenalt E, Hansen BW (2002) Invertebrate re- in-flow system for automated analysis of marine micro- colonisation in Mariager Fjord (Denmark) after a severe plankton. Mar Ecol Prog Ser 168:285–296 hypoxia. II. Blue mussels ( Mytilus edulis L.). Ophelia 56: ➤ Thorson G (1950) Reproductive and larval ecology of marine 215–226 bottom invertebrates. Biol Rev Camb Philos Soc 25:1–45 ➤ Pohnert G (2005) Diatom/copepod interactions in plankton: Thorson G (1964) Light as an ecological factor in the dispersal the indirect chemical defense of unicellular algae. Chem- and settlement of larvae of marine bottom invertebrates. BioChem 6:946–959 Ophelia 1:167–208 ➤ Pohnert G, Lumnieau O, Cueff A, Adolph S, Cordevant C, ➤ Vance RR (1973) On the reproductive strategies in marine Lange M, Poulet SA (2002) Are volatile unsaturated alde- benthic invertebrates. Am Nat 107:339–352 hydes from diatoms the main line of chemical defence ➤ Zajac RN (1991) Population ecology of Polydora ligni (Poly- against copepods? Mar Ecol Prog Ser 245:33–45 chaeta: Spionidae). I. Seasonal variation in population ➤ Raby D, Mingelbier M, Dodson JJ, Klein BW, Lacadeuc Y, characteristics and reproductive activity. Mar Ecol Prog Legendre L (1997) Food-particle size and selection by Ser 77:197–206

Editorial responsibility: Steven Morgan, Submitted: February 24, 2009; Accepted: February 28, 2010 Bodega Bay, California, USA Proofs received from author(s): May 3, 2010

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Resum de l’article IX– Article IX summary– Catalan versión

El creixement larvari del poliquet dominant, Polydora ciliata , està limitat per aliment en un estuari danès eutròfic, Isefjord.

Troels M. Pedersen, Rodrigo Almeda, , Frank L. Fotel, Hans H. Jakobsen, Patrizio Mariani, Benni W. Hansen Article acceptat a Marine Ecology Progress Series (2010, 407: 99-110).

La limitació per aliment en el creixement larvari del poliquet espiònid Polydora ciliata va ser examinada en un estuari eutròfic típic, Isefjord, a Dinamarca. En el camp, es van mesurar la disponibilitat d'aliment i els requeriments energètics de la població de larves de P. ciliata durant dos períodes diferents en 2004 i 2007 que, en conjunt, cobreixen la part productiva de l'any per al plàncton. En el labo ratori, les taxes de creixement específic ( μ) de les larves criades en suspensions d'aliment natural (0.10 d -1 ) van ser sempre inferiors a les de les larves criades en suspensions d'aliment natural enriquides amb fitoplàncton (eficiència de retenció del 100% per Rhodomonas salina ; 0.21 d -1 ). La biomassa total de meroplàncton (mitjana: 3.72 µg C L -1 , rang: de 0.11 a 26.05 µg C L -1 ) va ser amb freqüència similar o superior a la del holoplàncton (mitjana: 5.70 µg C L -1 , rang de 0.08 a 29.89 µg C L -1 ), la qual cosa suggereix que el meroplàncton pot tenir una importància tròfica en l'estuari. Les larves de P. ciliada van ser generalment les larves meroplanctòniques dominants (mitjana: 0.5 µg C L -1 , rang: de 0.03 a 2.51 µg C L -1 ). L'aliment disponible en la fracció de la grandària de presa òptima (2004, mitjana: <20 µm; rang: 99 a 274 µg C L -1 ; 2007, mitjana: 7 a 18 µm; la demanda de carboni estimada: 119 µg C L -1 , rang : 19 a 474 µg C L -1 ) sembla ser suficient per cobrir les necessitats energètiques de carboni de la població al llarg del període d'estudi (de 0.09 a 3.15 µg C L -1 d -1 ), però insuficient per aconseguir les taxes de creixement específic obtingudes en experiments de laboratori previs . Això suggereix que les larves de P. ciliata probablement presenten una eficiència de l'alimentació baixa i que, per tant les seves taxes màximes de creixement específic són aconseguides a concentracions d'aliment fins i tot superiors a les quals es troben en aquest ambient eutròfic.

205 Chapter 5

Resumen del artículo IX – Article IX summary– Spanish versión

El crecimiento larvario del poliqueto dominante, Polydora ciliata , está limitado por alimento en un estuario danés eutrófico, Isefjord.

Troels M. Pedersen, Rodrigo Almeda, , Frank L. Fotel, Hans H. Jakobsen, Patrizio Mariani, Benni W. Hansen Artículo aceptado en Marine Ecology Progress Series (2010, 407: 99-110).

La limitación por alimento en el crecimiento larvario del poliqueto espiónido Polydora ciliata fue examinada un típico estuario eutrófico, Isefjord, en Dinamarca. En el campo, se midieron la disponibilidad de alimento y los requerimientos energéticos de la población de larvas de P. ciliata durante dos períodos diferentes en 2004 y 2007 que, en conjunto, cubren la parte productiva del año para el plancton. En el lab oratorio, las tasas de crecimiento específico ( μ) de las larvas criadas en suspensiones de alimento natural (~ 0.10 d -1 ) fueron siempre inferiores a los de las larvas criadas en suspensiones de alimento natural enriquecidas con fitoplancton (eficiencia de retención del 100% para Rhodomonas salina ; ~ 0.21 d -1 ). La biomasa total de meroplancton (promedio: 3.72 µg C L -1 , rango: de 0.11 a 26.05 µg C L -1 ) fue con frecuencia similar o superior a la del holoplankton (promedio: 5.70 µg C L -1 , rango de 0.08 a 29.89 µg C L -1 ), lo que sugiere que el meroplancton puede tener una importancia trófica en el estuario. Las larvas de P. ciliada fueron las generalmente las larvas meroplanctónicas dominantes (promedio: 0.5 µg C L -1 , rango: de 0.03 a 2.51 µg C L -1 ). El alimento disponible en la fracción del tamaño de presa óptimo (2004, promedio: <20 µm; rango: 99 a 274 µg C L-1 ; 2007, promedio: 7 a 18 µm; la demanda de carbono estimada: 119 µg C L -1 , rango : 19 a 474 µg C L-1 ) parece ser suficiente para cubrir las necesidades energéticas de carbono de la población a lo largo del periodo de estudio (de 0.09 a 3.15 µg C L -1 d -1 ), pero insuficiente para alcanzar las tasas de crecimiento específico obtenidas en experimentos de laboratorio previos . Esto sugiere que las larvas de P. ciliata probablemente presentan una eficiencia de la alimentación baja y que, por consiguiente sus tasas máximas de crecimiento específico son alcanzadas a concentraciones de alimento incluso superiores a las que se encuentran en este ambiente eutrófico.

206 General discussion

“Nunca se alcanza la verdad total, ni nunca se está totalmente alejado de ella” Aristóteles (384 AC - 322 AC)

General discussion

General discussion

The results included in this thesis have been discussed in detail in their respective articles. The aim of this general discussion is to relate/associate the main results obtained from each study and evaluate the initial hypothesis.

Importance of invertebrate planktonic larvae in terms of abundance and biomass

Planktonic larvae (including holo ‐ and meroplanktonic larvae) were numerically the most important components of the metazooplankton communities in both the oligo ‐mesotrophic coastal waters of the NW Mediterranean throughout the year, and the eutrophic waters off Vancouver Island during the summer (articles I and III ). Copepod nauplii and copepodites were frequently the most abundant metazoans, although meroplanktonic larvae occasionally dominated the metazooplankon community (articles I, II and IX ). The contribution of meroplanktonic larvae to the total zooplankton biomass was often more important in the eutrophic boreal estuary, Isefjord, than in the coastal systems that we studied (articles I, III and IX ). Planktonic larvae (including holo ‐ and meroplanktonic larvae) represented a considerable fraction of total metazooplankton carbon biomass (in average ~50%, articles I, III and IX ) and may episodically become the dominant component of metazooplankton communities (articles I and IX ). Specific growth rates of planktonic larvae (e.g. copepod nauplii) were higher than those commonly reported for mesozooplankton (adult copepods) by the egg production method (Kiørboe & Johansen 1986; Poulet et al. 1995) (articulo II ). Therefore, ignoring larval growth may result in significant underestimations of secondary production in marine planktonic systems (Calbet et al. 2000; Rey ‐Rassat et al. 2004).

The abundance and biomass of small planktonic metazoans has been traditionally underestimated due to the use of coarse plankton nets (>200 µm) (Banse 1962; Turner 1994). However, the importance of small planktonic metazoans (<200 µm, metazoan microplankton) has been increasingly recognized in the last decade through the use of appropriate sampling methods (Calbet et al. 2001; Gallienne & Robins 2001; Turner 2004; Riccardi 2010). We found that, in coastal waters, the small size fraction was mainly composed of planktonic larvae,

209 General discussion

including copepod nauplii, copepodites and meroplanktonic larvae (articles I and III ). In NW Mediterranean coastal waters, the metazoan microplankton represented, on average over the year, between 18% and 34% of the total metazoan biomass, depending on sampling method (plankton net or Van Dorn bottles) (article I). The abundance of small metazoans was higher when Van Dorn bottles were used for sampling; this reflects the importance of appropriate sampling methods for accurate estimations of zooplankton community structure (article I). The contribution of metazoan microplankton to the total metazooplankotn biomass was ca. 20 ‐25% at the Vancouver Island studied site (article III ). Therefore, although the mesozooplankton dominates in terms of biomass, the metazoan microplankton represents a considerable fraction of the total metazooplankton biomass. Moreover, as physiological rates of small organisms are generally higher than those of larger organisms, the exclusion of metazoan microplankton may lead to important underestimations of zooplankton secondary production (Gallienne & Robins 2001; Turner 2004).

Trophic role of invertebrate planktonic larvae in pelagic food webs

Although the degree to which metazooplankton predation may regulate primary producers is still a subject of debate, there is increasing evidence that metazoans exert little pressure on phytoplankton and protistan zooplankton (e.g. Broglio et al. 2004; Atienza et al. 2006). However, most metazooplankton grazing studies have excluded micro ‐metazoans and consequently underestimated the trophic effect of planktonic metazoans on primary producers. Additionally, most feeding experiments have been focused on holoplanktonic organisms (mainly adult copepods) and the trophic role of meroplanktonic organisms has been largely ignored. In the present thesis, the trophic role of larvae on planktonic food webs was assessed using both species and community level approaches. In the oligo ‐mesotrophic NW Mediterranean coastal waters, we conducted grazing experiments with the metazoan microplankton community (microplanktonic larvae) that fed on natural food assemblages throughout an annual cycle (article I). In the eutrophic waters of the West coast of Vancouver Island (article III ) and in the heavily eutrophic boreal estuary, Isefjord, in Denmark, (article VII) grazing experiments were conducted using individual species of meroplanktonic larvae. As a whole, this work provides us with a knowledge base on which to evaluate the trophic role of larvae on plankton food webs under contrasting environmental (trophic) conditions.

210 General discussion

In NW Mediterranean coastal waters, the trophic impact (estimated as the % of standing stock grazed per day) exerted by the microplanktonic larval community (metazoan microplankton) on the microbial community was very low, < 5% (article II) . According to the experiments for the estimation of total microzooplankton grazing (including protozoans and metazoans) by dilution technique (Landry & Hassett 1982), which were conducted simultaneously with the metazoan microplankton feeding experiments, the contribution of metazoans to the total microzooplankton grazing on phytoplankton was quite low (Calbet et al. 2008; article II ). Hence, protists are likely the main source of predation pressure on phytoplankton in Mediterranean coastal waters, at least during certain times of the year (Calbet et al. 2008; article II ). The small effect of micrometazoans in comparison to protozoans may be due to both to their lower biomass in the field (one or two orders of magnitude lower in NW Mediterranean coastal waters, article II ) and their lower specific ingestion rates (Jacobson & Anderson 1993)

With focus on meroplankonic larvae, populations of Polydora ciliata have a very slight trophic effect (< 1%) on primary producers in highly eutrophic waters (article IX ). Similarly, when considering the carbon specific ingestion rates of Polydora ciliata larvae on natural food suspensions (article VII ) and their carbon biomass in nature (article IX ), their potential trophic impact on dinoflagellates and ciliates was very low (<1%). The grazing pressure of meroplanktonic larvae on the microbial community did not influence harmful phytoplankton blooms of Heterosigma akashiwo and Prorocentrum triestinum in the coastal waters Vancouver Island (trophic impact < 2%) (article III ). The negative effect of Heterosigma akashiwo on the abundance of meroplanktonic larvae (as well as other zooplankton) may act to reduce the grazing pressure on the harmful phytoplankton bloom (article III ).

Our results suggests that, even including the metazoan microplankton community and meroplanktonic larvae, the trophic role of metazooplankton in planktonic food webs seems to be generally insufficient to control phytoplankton and protozoan population dynamics.

Hypothesis 1: NOT VALID

Planktonic larvae (holo ‐ and meroplanktonic larvae), including those belonging to the small size fractions (<200 µm, metazoan microplankton), exert an important trophic impact on marine planktonic food webs, which may noticeably influence phytoplankton and protozoan dynamics in coastal waters.

211 General discussion

Ecophysiology of larval developmental stages of the copepod Oithona davisae

Small planktonic copepods of the genus Oithona are among the most ubiquitous and abundant metazoans in the world oceans, from polar to tropical latitudes (Gallienne & Robins 2001). The success of a given species is determined by their recruitment success; consequently, understanding the physiology of larval developmental stages is essential for comprehension of species fitness.

Physiological processes in larvae are regulated by endogenous/intrinsic and environmental factors. Similar to other zooplankton (Vidal 1980, Vidal & Whitledge 1982; Ikeda 1985; Hansen et al. 1997; Gillooly 2000; Ikeda et al. 2001), we found that body size (or body weight) and larval stage were important intrinsic factors that influenced the ingestion, growth and respiration rates of Oithona davisae nauplii (articles V and VI ). Under similar conditions of temperature and food, physiological rates of Oithona davisae nauplii followed allometric scaling laws (articles V and VI ) and, in most cases, confirmed the three ‐quarters power law to body mass (power exponent ~ 0.75; Kleiber 1932; Peters 1983; Hansen et al. 1997) (articles V and VI ). A general model describing the relationship between respiration rate and body size which includes data from this thesis (article VI ) and from previous studies is shown in Figure 1. In this case, the obtained power exponent (0.98) deviated from the three ‐quarters power law. This deviation may be due to differences in sensitivity between species or stages to starvation effects or to methodological variability. Regardless, the resulting regression model indicates that body mass accounts for 97 % of the variance in respiration rates of Oithona at a given temperature under filtered seawater conditions (Fig. 1).

Temperature and food availability are two of the main environmental factors that influence the distribution and physiology of zooplankton (Huntley & Boyd 1984, Huntley & Lopez 1992, Gillooly 2000; Ikeda et al. 2001). The different physiological rates (growth, ingestion, respiration, excretion) studied in Oithona davisae larval stages following exponential models in relation to temperature between 20 ‐28 ºC (articles IV, V and VI ). In contrast, all physiological rates were very low at temperatures ≤ 16 ºC, which resulted in deviation from the exponential model (articles IV, V and VI ). This likely reflects the thermophilic nature of this species (Uye & Sano 1995); O. davisae is very scarce during the winter and spring seasons (when temperatures are < 20 ºC), whereas it is very abundant during the warmer seasons when

212 General discussion

temperatures are commonly between 20 and 28 ºC. Therefore, we might conclude that this warmer temperature range (20 ‐28 ºC) is likely the optimum for Oithona davisae .

The Q10 values obtained for growth (article IV) ingestion (article V), and respiration rates

(article VI ) were all quite similar between 16 ‐28 ºC (~Q 10 = 2.4), which suggests that all of these physiological processes has a similar dependence on water temperature. The Q10 values obtained from our study were within the accepted range reported for other copepods (Ikeda et al. 2001; Castellani et al. 2005)

Figure 1. Relationship between body weight (W, ng C ind ‐1) and respiration rate (R, µl ind ‐1 d‐1) for

Oithona . All values standardized to 20 ºC using a Q10 = 3.1 for O.similis (Castellani et al. 2005) and a Q10 = 2.64 (from this study, article VI) for the other Oithona species. Data from other studies are indicated with numbers:1) Marshall & Orr (1966), 2) Klekowski et al. (1977), 3) Lampitt & Gamble (1982), 4) Nakata & Nakane (1987), 5) Hiromi et al. (1988) 6) Nakamura & Turner (1997), 7) Castellani et al. 2005; 8) Atienza et al. 2006. Fitted equation (log form ): Log R= 0.978 × logW ‐ 3.288 r2 = 0.97.

213 General discussion

Oithona is able to grow well both in oligotrophic oceanic environments and in coastal eutrophic waters under conditions where food has become a limited resource for calanoid copepods (Calbet & Agustí 1999). Copepod life strategies are adapted to fluctuations in food availability, for example, many calanoid species produce resting eggs that accumulate in sediments and hatch prior to the onset of the spring bloom (Marcus 1996; Peterson 1998). In contrast, Oithonids do not produce resting eggs and must adapt to tolerate periods of low food availability and to maintain their populations throughout the year. We have identified several physiological features of O. davisae developmental stages that suggest competitive advantages over calanoid copepods when and where food is scarce, as follows:

- Food concentrations that induce relevant mortality in O. davisae nauplii were lower than those concentrations commonly reported for calanoid nauplii (article IV ). O. davisae nauplii can also withstand relatively long periods (5 days) of poor food conditions without significant mortality (article IV )

- Oithona davisae nauplii showed similar developmental rates but required less food than what has been reported for calanoid nauplii (article IV ).

- Naupliar development is less sensitive than growth to food concentration, suggesting that development and consequently recruitment may be prioritized over somatic growth in O. davisae (article IV ).

- Oithona davisae nauplii and copepodites showed a type III functional feeding response. (article VI ). This type of functional response, which has also been observed in some calanoid copepods is characterized by the presence of a lower feeding threshold i.e a prey concentration below which the copepod stops feeding or reduces its clearance rates (article VI ). The presence of lower feeding thresholds can be interpreted as an adaptation to conserve energy at low food concentrations because the energetic cost of collecting food at very low concentrations would not be compensated by the energetic gain (article VI ).

- The satiating food concentrations of O. davisae larvae were lower than those commonly reported for calanoid larval stages (article VI ). Some adult Oithona stages require lower food concentrations for maximum specific ingestion than some calanoid nauplii (article VI ). This suggests that the ingestion rates of calanoid larvae in natural systems may be more limited by food than the ingestion rates of Oithona (article VI ).

214 General discussion

- Oithona davisae developmental stages exhibited similar gross growth efficiencies (GGE), assimilation efficiencies (AE) and net growth efficiencies (NGE), but lower specific ingestion rates and specific carbon respiratory losses than calanoid copepods (articles V and VI ).

These results confirm that food requirements and respiratory losses of Oithona nauplii are lower than those of calanoid copepods (Lampitt & Gamble 1982, Paffenhöfer 1993); these physiological differences may partially explain the success of Oithona in marine environments. The differences in food requirements and metabolic carbon needs between calanoids and oithonids are likely related to the differences in swimming and feeding behavior between these groups of copepods. In contrast to most calanoids (Titelman & Kiorboe 2003; Henriksen et al. 2007), oithonids (including nauplii, copepodites and adults) move with occasional leaps and rely on detecting prey by hydromechanical signals (ambush ‐feeders, Paffenhöfer 1993; Svensen & Kiørboe 2000; Paffenhofer & Mazzocchi 2002). As an example, in the absence of prey, Oithona davisae nauplii spent 98% of their time sinking, unmotile, and only in the presence of motile prey increase their jumping frequency (Henriksen et al. 2007). The feeding and swimming strategy of Oithona is hypothesized to be more energyefficient than that of most calanoids (Paffenhöfer 1993), which may help to explain the wide distribution and high degree of ecological success of Oithona in marine ecosystems. In addition, a low mobility may contribute to low mortality in the ocean since encounter rates with predators are expected to be lower in unmoving
oithonids when compared with suspension feeding copepods that move actively for feeding (Eiane & Ohman 2004).

Physiological basis and ecological significance of food limited growth on meroplanktonic larvae: the case of Polydora ciliata

The marine environment is nutritionally dilute and highly heterogeneous in the distribution of planktonic food resources. Food limited growth seems to be a general rule for marine zooplankton (Huntley & Boyd 1984; Lampert 1985; Hirst & Bunker 2003). However, we suggest that most meroplaktonic larvae are likely to be limited by food to a higher degree than holoplanktonic larvae (e.g. copepod nauplii). It has been shown that many species of meroplanktonic larvae are food limited during their development (Olson & Olson 1989; Yu 2009). However, physiological basis and ecological significance of food limitation on meroplanktonic larval development still remain understudied and poorly understood.

215 General discussion

Polydora ciliata is one of the most abundant spionid polychaete species on the northern European coast; their planktotrophic larvae are common to coastal waters and may frequently become the major components of metazooplankton communities (Anger et al. 1986; Pedersen et al. 2008) (article IX ). The growth rate of P. ciliata larvae is limited by food despite this species inhabiting heavily eutrophic waters (Isefjord, Denmark). This conclusion is supported by 2 experimental approaches:

- Laboratory studies showed that maximum growth rates, with an optimal food size, were reached at concentrations ranging from 1410 to 2510 µg C L‐1 depending on larval size (article VIII ). In the field, the available food in the optimal prey size fraction was insufficient to attain the maximum specific growth rates observed in the laboratory (articles VII and IX ).

- Specific growth rates of larvae reared on natural food suspensions were lower than the growth rates of larvae fed on enriched food suspensions; this indicates that larvae in the natural environment may not grow at their maximum potential (article IX ). Maximum growth rates observed in the enriched diet treatments were similar to the maximum growth rates observed under food satiating conditions in the laboratory (articles VII and IX ).

Maximal growth rates were comparable between similarly sized Polydora ciliata and Oithona davisae larvae under satiating food conditions (articles V and VIII ). However, the food requirements for maximal growth were much higher for P. ciliata larvae than for Oithona davisae developmental stages. Similar results have been reported for other larvae meroplanktonic and copepod larvae (see article VIII discussion section). Along an annual cycle in NW Mediterranean coastal waters, the meroplanktonic larval community showed specific growth rates that were significantly lower than nauplii and copepodites living under similar food availability conditions (article II ). Therefore, growth rates of meroplanktonic larvae in nature are expected to be lower than growth rates of copepod nauplii as is suggested from our findings (articles II and VIII ). The differences in the degree of food limited growth between mero ‐ and holoplanktonic larvae may be due to physiological differences between these groups of planktonic organisms. This is supported by our results, as follows:

- Maximum specific clearance rates in Polydora ciliata larvae (article VIII ), as well as in other meroplanktonic larvae (e.g. Jaspersen & Olsen 1982; Hansen & Ockelmann 1991), were lower than the maximum specific clearance rates in holoplanktonic larvae, such as copepod larvae (Paffenhöfer 1971; Berggreen et al. 1998; Hansen et al. 1997). For example, maximum specific clearance rates of O. davisae nauplii were one order of magnitude higher than those

216 General discussion

of P. ciliata larvae (articles V and VIII ). This difference suggests that P. ciliata larvae may employ a less efficient feeding mechanism than O. davisae nauplii; a trend that may help to explain why the maximum specific growth rates of P. ciliata larvae are attained at food concentrations even higher than those found in heavily eutrophic environments (articles VIII and IX ).

- Polydora ciliata larvae show similar swimming behavior (swimming speed and paths) in the presence or absence of food (article VII ). In contrast, as previously mentioned, the swimming and feeding activity of O. davisae nauplii increases only in the presence of prey (Henriksen et al. 2007). Similarly, some calanoid nauplii increase the percentage of time spent swimming as food concentration increases (van Duren & Videler 1995). Continuous swimming is usually associated with high energetic costs and consequently with high food requirements; hence the non ‐specialized behavior of P. ciliata larvae in response to prey regime may be less energetically efficiency than the swimming and feeding behavior of copepod nauplii.

From an ecological perspective, food limitation causes decreased growth rates, which prolongs the larval life period and thus further exposes larvae to additional sources of mortality such as predation (Thorson 1950); however, food limitation increases pelagic life period and consequently, the duration of larval dispersion. Low satiating food concentration requirements favor rapid larval development and consequently adult recruitment is expected to be a competitive advantage for holoplanktonic organisms such as Oithona davisae (article IV ). For planktonic copepods, in which all life stages disperse in the water column, a longer larval life period should not provide any selective advantages. However, for many benthic invertebrates, particularly those that are sessile during the adult phase, larvae act as dispersal mechanisms (Leving & Bridges 1995). Larval dispersal is crucial for species survival and persistence because it promotes connectivity among populations (Roughgarden et al. 1988), favors the flow of genetic information (Palumbi 2003; Trakhtenbrot et al. 2005), facilitates the recolonization of sites following disturbances (Hansen et al. 2002; Petersen et al. 2002). Marine invertebrate species with longer planktonic periods may counteract fluctuations in adult population density, which reduces the risk of localized extinctions (Eckert 2003). We suggest that, for many benthic invertebrates there is a trade ‐off between larval feeding/growth kinetics and dispersal. Natural selection seems to favor that some meroplanktonic larvae, such as P. ciliata , possess low filtration and growth rates despite inhabiting environments with extremely high food availability (article IX ). In this case, what may be initially perceived as suboptimal larval

217 General discussion

performance, may actually function to allow planktonic development long enough to ensure efficient larval dispersion.

Hypothesis 2: VALID

The ecological success of many species of marine invertebrates is in part explained by the physiological characteristics of their planktonic larval stages

218 Main conclusions

Main conclusions

Main conclusions

I. Planktonic invertebrate larvae, including both holo ‐ and meroplanktonic larvae, represent a considerable fraction of the total metazooplankton biomass in coastal waters.

II. Copepod nauplii and copepodites are numerically the dominant components of the metazooplankton community in NW Mediterranean coastal waters.

III. The total abundance of metazoan microplankton and mesozooplankton in NW Mediterranean coastal waters is positively correlated with water temperature along a seasonal cycle.

IV. The seasonal abundance pattern and vertical distribution of total metazoan microplankton and mesozooplankton is not directly related to phytoplankton biomass (chlorophyll) in NW Mediterranean coastal waters.

V. The occurrence of some meroplanktonic larvae (polychaete larvae) in coastal waters is linked to the spring phytoplankton blooms.

VI. Metazoan microplankton fed efficiently on nanoplankton and microplankton prey (nanoflagellates, diatoms, dinoflagellates and ciliates).

VII. The trophic impact of microplanktonic larvae (<200 µm, metazoan microplankton) is insufficient to control phytoplankton and protozoan dynamics in NW Mediterranean coastal waters

VIII. The grazing pressure of meroplankotnic larvae has a minimal effect on Heterosigma akashiwo /Prorocentrum triestinum harmful blooms.

221 Main conclusions

IX. Heterosigma akashiwo /Prorocentrum triestinum harmful blooms negatively affect the field abundance of planktonic larvae and other zooplankton.

X. Overall, metazoan microplankton have higher specific growth rates, but similar food conversion efficiencies, than mesozooplankton

XI. Feeding activity significantly increases the respiration rates of copepod nauplii, hence the assessment of respiration rates without food results in a significant underestimation of their metabolic rates.

XII. The food requirements and C respiratory losses of Oithona nauplii are lower than those calanoid copepods; these physiological differences may partially explain the success of Oithona in marine environments.

XIII. Despite inhabiting eutrophic waters, the larval growth of Polydora ciliata shows a high degree of food limitation compared with holoplanktonic organisms.

XIV. The special characteristics of the ecophysiology of some benthic marine invertebrate larvae, such as P. ciliata , allow for extended planktonic development, which ensure efficient larval dispersion.

222 Resum/Resumen Catalan & Spanish summaries

Resum de la tesi

Resum de la tesi Thesis summary ‐ Catalan version

La gran majoria dels invertebrats marins tenen un cicle de vida complex que inclou fases larvàries planctòniques entre la fase embrionària i la fase adulta (Thorson 1950; Strathmann 1987, 1993). Aquestes larves poden diferenciar ‐se dels adults en grandària, forma, hàbitat, forma de nutrició, i/o la capacitat de dispersió (Barnes et al. 1988; Young 2002). La supervivència i el creixement de les fases larvàries poden influir en l'èxit del reclutament de les espècies, i en la connectivitat, distribució i abundància de les poblacions d'invertebrats marins (Roughgarden et al. 1988; Eckert 2003). No obstant això, malgrat l'òbvia importància de les larves en els cicles de vida de la majoria dels animals marins, el nostre coneixement sobre la ecofisiologia larvària segueix sent escàs en comparació amb el de les fases adultes. A més, com components importants de les comunitats de zooplàncton, el paper tròfic de les larves planctòniques a les xarxes alimentàries marines no hauria de ser ignorat. Aquesta tesi doctoral té com a objectiu principal contribuir al coneixement de l'ecologia i la fisiologia de larves planctòniques d'invertebrats marins, incloent el seu paper a les xarxes tròfiques planctòniques.

INTRODUCCIÓ

La definició de "larva". Classificació de les larves d'invertebrats marins

El desenvolupament animal que inclou fases larvàries es denomina "desenvolupament indirecte". Per contra, en el "desenvolupament directe" l'embrió es desenvolupa directament en un juvenil, el qual sol ser una versió en miniatura però sexualment immadura de l'adult. El terme "larva" s'ha utilitzat de moltes maneres diferents depenent de l'enfocament i la disciplina científica i, en aquest moment, no existeix una definició acceptada de manera general (Strathmann 1987; McEdward & Jaines 1993; Young 2002). En aquesta tesi, s'ha utilitzat el concepte de larva descrit per Hickman (1999): "la larva és un estat estructural o una sèrie d'estats que es produeix entre l
inici de la morfogènesis divergent després del desenvolupament embrionari i l
inici de la metamorfosi que comporta al pla corporal adult.

225 Resum de la tesi

Les larves d'invertebrats marins mostren una impressionant diversitat de formes corporals, moltes de les quals han rebut noms específics. Les larves han estat classificades pel lloc de desenvolupament, la manera de nutrició, el potencial de dispersió, i la morfogènesis (Thorson 1950; Mieikovsky 1971; Levin & Bridges 1995). Des d'una perspectiva ecològica i per a les finalitats d'aquesta tesi, destaquem les següents categories: - Larves planctòniques, el desenvolupament de les quals es produeix en la columna d'aigua, enfront a larves bentòniques, el desenvolupament de les quals té lloc sobre o en el fons marí. - Larves holoplanctòniques, que són les larves d'organismes que passen tot el seu cicle vida en el plàncton, enfront a larves meroplanctòniques, que són les fases larvàries planctòniques d'invertebrats marins bentònics. - Larves planctotròfiques, que s'alimenten de plàncton, enfront a larves lecitotròfiques, la nutrició de les quals es deriva exclusivament de reserves vitel ∙lines procedents de l'ou.

Cicle de vida dels copèpodes planctònics i descripció de les seves fases larvàries

Els copèpodes són un grup molt divers de crustacis, amb més d'11.500 espècies conegudes, la majoria de les quals són marines (Humes 1994). Entre els invertebrats marins, els copèpodes són el grup dominant i més divers del metazooplàncton (és a dir, zooplàncton multicel ∙lular) en ambients marins (Longhurst 1985; Verity & Smetacek 1996). Aporten una fracció considerable de la producció secundària en la majoria de les comunitats planctòniques marines i es consideren la peça clau entre els productors primaris i els nivells tròfics superiors a les xarxes tròfiques pelàgiques (Cushing 1989).

La majoria dels copèpodes planctònics són dioics i presenten reproducció sexual (Gilbert & Williamson 1983). Els cicles de vida dels copèpodes planctònics són complexos i, en general, es caracteritzen per 13 fases de vida, incloent l'ou, sis fases larvàries denominades nauplis (NI ‐ NVI), cinc fases larvàries denominades copepodits (CI ‐CV) i la fase adulta (Fig. 1). La larva naupli és el tipus de larva més ancestral en crustacis i s'ha utilitzat com a principal característica que uneix a tot el subfilo Crustacis (Cigne 1982; Dahms 2000; Harvey et al. 2002). En termes d'abundància numèrica, els nauplis s'han considerat "la forma més abundant d'animal multicel ∙lular de la terra" (Fryer 1987). En copèpodes, com en altres crustacis, els processos ontogenètics estan vinculats a mudes que tenen lloc entre les diferents etapes vitals (Williamson 1982). Els nauplis de la majoria de copèpodes de vida lliure muden cinc vegades (de la fase NI a NVI). Quan els nauplis VI muden a copepodits I es produeixen importants canvis morfològics. En

226 Resum de la tesi

el copepodit I es diferencien el prosoma i l
urosoma i la seva morfologia comença a assemblar ‐ se a la de la fase adulta. A través de les següents mudes (CII ‐CV), el nombre de segments corporals i apèndixs funcionals s'incrementa. Finalment, després de la cinquena muda, s'aconsegueix l'edat adulta.

Figura 1. Esquema del cicle de vida típic en copèpodes holoplanctonics de vida lliure. Les lletres minúscules indiquen processos del cicle de vida; les lletres majúscules identifiquen les principals fases del cicle de vida; les lletres majúscules en negreta indiquen categories de classificació dels organismes marins d'acord amb el seu hàbitat i la mobilitat ("manera de vida").

Cicle de vida d'invertebrats marins bentònics: les larves meroplanctòniques

Aproximadament el 70 ‐80% dels invertebrats marins bentònics tenen larves planctòniques que passen un cert temps, de minuts a mesos, en la columna d'aigua (Thorson 1946, 1950; Strathmann 1993; Young 2002). Per a moltes espècies d'invertebrats bentònics, especialment en espècies sedentàries, una dispersió significativa solament té lloc durant la fase larvària de natació lliure (Strathmann 1985, 1990; Levin & Bridges 1995). Els cicles de vida que

227 Resum de la tesi

inclouen larves planctòniques seguit de juvenils i adults bentònics es denomina comunament cicle de vida bifàsic (Fig. 2).

Figura 2. Esquema idealitzat del cicle de vida d'invertebrats bentònics amb larves meroplanctòniques. Les lletres minúscules indiquen processos en el cicle de vida, les lletres majúscules identifiquen les principals fases del cicle de vida, les lletres majúscules en negreta indiquen categories de classificació dels organismes marins d'acord amb el seu hàbitat i la mobilitat ("manera de vida").

Els adults bentònics, madurs sexualment, alliberessin els gàmetes en la columna d'aigua on té lloc la fertilització i el desenvolupament embrionari i larvari. La durada del període de dispersió en la columna d'aigua depèn de l
espècie. Les larves lecitotròfiques tenen una vida pelàgica curta i no es dispersen a llargues distàncies, mentre que les larves planctotròfiques generalment tenen una vida planctònica relativament llarga (Thorson 1950; Jagersten 1972). La dispersió és acompanyada pel creixement i el desenvolupament d'una o més fases larvàries. Les propietats de les larves planctotròfiques inclouen tant característiques morfològiques com de comportament en relació a l'alimentació i la locomoció, que representen adaptacions a un estil de vida planctònic i que permeten l'explotació de les fonts d'aliment planctòniques (Strathmann 1993; Peterson et al. 1997). El final del període larvari ocorre quan l'organisme està fisiològicament competent per assentar ‐se i portar una existència bentònica. La metamorfosi a

228 Resum de la tesi

la forma bentònica es produeix una vegada que les larves han acceptat el lloc d'assentament. El cicle de vida es completa quan els invertebrats marins bentònics passar a través de les etapes juvenils i arriben a la maduresa sexual en la fase adulta.

A continuació es descriuen breument els principals tipus de larves meroplanctòniques dels grups d'invertebrats bentònics en els quals s'ha centrat aquesta tesi: poliquets (spiònids i serpúlids), bivalves, gasteròpodes, cirrípedes i equinoïdeus. En poliquets amb desenvolupament indirecte, es distingeixen principalment dues fases larvàries: trocòfores i larves segmentades (les larves amb pocs segments es coneixen generalment com metatrocòfores) (Pernet et al. 2002). Els segments que porten quetes (setes) es diuen setígers i freqüentment el nombre setígers en la larva s'utilitza per indicar la fase de desenvolupament (Pernet et al. 2002). En spiònids, la embriogènesis i el desenvolupament primerenc de les larves solen tenir lloc en càpsules d'ous, i la primera fase de desenvolupament que s'allibera al plàncton és la larva amb 3 setígers (Daro & Polk 1973; Blake & Arnofsky 1999). Per contra, els poliquets serpúlids solen alliberar els gàmetes en la columna d'aigua i tot el desenvolupament embrionari i larvari ocorre en el plàncton (Pernet et al. 2002). Els gasteròpodes marins tenen dos tipus principals de larves: trocòfores i velígeres (Barnes 1982; Strathmann 1987). Les trocòfores poden ser de natació lliure o, comunament, mantenir ‐se dins de càpsules d'ous mentre es desenvolupen a larves velígeres, les quals són alliberades al plàncton (Goddard 2001; Buckland ‐Nicks et al. 2002). Les larves velígeres són exclusives de gasteròpodes i bivalves marins i presenten una gran diversitat morfològica (Buckland ‐Nicks et al. 2002). Les velígeres de gasteròpodes es caracteritzen per posseir una petxina (protoconcha) i un vel bilobulat: un òrgan ciliat que és utilitzat per a l'alimentació, la locomoció i la respiració (Fretter 1967; Goddard de 2001, Buckland ‐Nicks et al. 2002). En bivalves, encara que en algunes espècies les trocòfores poden ser incubades en la cavitat del mantell, és freqüent que tant les trocòfores com les vèligeres siguin de natació lliure (Barnes 1982; Brink 2001; Zardus i Martel 2002). Les velígeres de bivalves es caracteritzen per una petxina amb xarnera (prodisoconcha) i un vel ovalat o arrodonit (Waller 1981; Brink 2001).

Les larves planctòniques són etapes del cicle de vida dels invertebrats marins particularment vulnerables, ja que la mortalitat larvària sovint supera el 90% (Rumrill 1990). No obstant això, una fase de dispersió larvària és avantatjosa per a animals que són sèssils com a adults. Les larves planctòniques proporcionen un medi de colonització de nous hàbitats, promouen el flux d'informació genètica entre poblacions, i permeten l'establiment de nous llocs de colonització després d'alteracions ambientals, la qual cosa redueix el risc d'extincions locals

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(Roughgarden et al. 1998; Hansen et al. 2002; Petersen et al. 2002; Eckert 2003). Per tant, el coneixement de l'ecologia larvària és essencial per entendre la dinàmica de les poblacions i comunitats dels invertebrats marins bentònics.

Introducció a l’ecofisiologia d'invertebrats marins larves planctòniques

L
ecofisiologia o fisiologia ambiental és la disciplina de la biologia que estudia la influència de factors ambientals en els processos o funcions d'un organisme o de qualsevol de les seves parts. La fisiologia i la bioenergètica estan estretament relacionades amb l'eficiència biològica de les espècies i el seu èxit evolutiu. Donat el paper crucial del reclutament larvari en l'estabilitat de les poblacions i comunitats, la comprensió de l
ecofisiologia larvària és essencial per entendre la dinàmica biològica dels invertebrats marins. A més, en el context actual de ràpids canvis ambientals d'origen antropogènic (Hoegh ‐Guldberg i Bruno 2010), l'estudi dels efectes dels factors ambientals (temperatura, disponibilitat d'aliments) sobre la fisiologia de la larves és d'especial rellevància per predir l'impacte del canvi climàtic sobre els invertebrats marins.

Nombrosos estudis de camp i de laboratori han demostrat que la temperatura de l'aigua i concentració d'aliment són els factors ambientals que més afecten al desenvolupament, supervivència, creixement, alimentació i metabolisme en copèpodes (Huntley & Boyd, 1984; Huntley & López 1992; Hirst & Lampitt 1998; Hirst & Kiørboe 2002). No obstant això, la majoria d
aquests estudis s'han centrat en adults o fases tardanes de copepodits de calanoides (Ikeda et al. 2001;. Hirst & Bunker 2003), mentre que els nauplis han rebut menys atenció, particularment els de copèpodes petits, com els pertanyents al gènere de Oithona (Cyclopoida). Durant l'última dècada, Oithona ha rebut una atenció especial a causa de la seva gran abundància i ubiqüitat, i probablement es tracti del gènere de copèpodes més important de tots els oceans (Galliene & Robins 2001). Malgrat la importància d
Oithona, se sap molt poc sobre la fisiologia de les seves fases larvàries, la qual cosa pot ser crucial per entendre el seu èxit evolutiu.

Un dels objectius d'aquesta tesi va ser avaluar la influència de factors intrínsecs (fase de desenvolupament, pes corporal) i ambientals (temperatura, aliment), en diferents processos fisiològics (creixement, desenvolupament, alimentació, respiració i excreció) en nauplis d’Oithona davisae (articles IV , V i VI )

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La fisiologia de les larves ha estat àmpliament estudiada en algunes espècies d'invertebrats bentònics, especialment els de interès en aqüicultura (per exemple, en Mytilus edulis ‐Widdows 1991; Sprung 1984a, 1984b, 1984c). No obstant això, poc se sap sobre la ecofisiologia d'alguns invertebrats d'importància ecològica, per exemple, moltes espècies de poliquets. Els estudis de laboratori i observacions de camp han demostrat que tant l'època de reproducció i com la distribució geogràfica coincideixen sovint amb la tolerància dels embrions i larves d'una espècie als factors ambientals tals com la temperatura (Orton 1920; Thorson 1950; Kinne 1970). Els canvis de temperatura poden tenir conseqüències importants per a la supervivència i dispersió de les larves mitjançant el retard o l'increment de les taxes de desenvolupament i creixement (Thorson 1950; Costlow et al. 1966, Hoegh ‐Guldberg & Pearse, 1995). La quantitat i qualitat d'aliment són factors clau en la supervivència, desenvolupament, i creixement de larves planctotròfiques (Paulay et al. 1985; Pechenik 1987; Rumrill 1990; Basch 1996). El canvi climàtic ha provocat una reducció global en la producció de fitoplàncton marí (Behrenfeld et al. 2006) així com canvis en l'estructura i la fenologia de les comunitats planctòniques (Edwards & Richardson 2004). Per exemple, la tendència actual d'increment de temperatura està produint l'avançament dels esdeveniments de fresi de certes espècies, però no així la proliferació de fitoplàncton primaveral, donant lloc a un desajustament temporal entre la producció de larves i la disponibilitat d'aliment (Edwards & Richardson 2004; Hoegh ‐Guldberg & Bruno 2010). La limitació d'aliment durant el desenvolupament larvari pot comportar de forma directa a la mortalitat de les larves per inanició o, indirectament, produir una reducció en les taxes de creixement, perllongant el període planctònic i augmentant l'exposició a fonts addicionals de mortalitat, tals com la depredació (Thorson 1950). Són moltes les espècies de larves planctotròfiques en les quals s'ha demostrat un alt grau la limitació per aliment durant el desenvolupament (Olson & Olson 1989; Yu 2009), pel que resulta d'especial interès d'entendre las bases fisiològiques i el significat ecològic de la limitació per aliment durant el desenvolupament larvari de invertebrats bentònics.

Es va estudiar la influència d'aliment en el comportament natatori i la fisiologia de les larves meroplanctòniques del poliquet Polydora ciliata (articles VII i VIII ). Es va avaluar el grau de limitació per aliment en el creixement larvari de P. ciliata en un estuari eutròfic danès, Isefjord (article IX )

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L'estimació de les taxes d'ingestió, creixement, respiració i egestió ens permet determinar els balanços d'energia o carboni i l'eficiència en la transformació d'energia dels animals. El model bàsic de balanç d'energia pot expressar ‐se com: I = G + M + I on I, G, M i I són les taxes d'ingestió, creixement, metabolisme (respiració), i egestió, respectivament, expressades en unitats d'energia o carboni.

Des d'una perspectiva econòmica, la dinàmica de les poblacions de larves afecta al reclutament d'espècies que són recursos explotables de gran importància, o bé formen part dels organismes incrustants (fouling). L'abundància de larves de copèpodes (nauplis) contribueix de manera decisiva al reclutament de les poblacions d'espècies de peixos de gran interès comercial (Castonguay et al. 2008). A més, els nauplis de copèpodes són una font d'aliment preferent i altament nutritiva per a moltes larves de peixos marins i gambetes criades en explotacions aqüícoles (Hernández ‐Molejón & Álvarez ‐Lajonchere 2003). Per tant, l'estudi de la fisiologia i el balanç de carboni de les fases larvàries sota diferents condicions ambientals no només és rellevant en disciplines científiques bàsiques, tals com l'ecologia, sinó que també és important en investigacions aplicades com en el camp de l'aqüicultura i les pesqueries.

Es van estimar les eficiències de creixement net i el balanç de carboni en nauplis de Oithona davisae (article VI ), així com el rang òptim de temperatura i de concentració d'aliment per a la producció de nauplis d' O. davisae en laboratori (articles IV i V)

Les xarxes tròfiques planctòniques marines i el paper de les larves

Per definició, el "plàncton" comprèn a tots els organismes suspesos en la columna d'aigua, la mobilitat del quals no pot contrarestar la hidrodinàmica marina (Hensen 1887). Aquests organismes són molt diversos en termes de la taxonomia, grandària i tipus de alimentació (Sieburth et al. 1978, Fig. 3). Tots els components planctònics estan relacionats entre si a través d'interaccions predador ‐presa influenciades per preferències ecològiques i efectes top ‐down i bottom ‐up (Zöllner et al. 2009).

El zooplàncton inclou tant protozous (protozooplàncton, eucariotes unicel ∙lulars heterotròfics) com metazous (metazooplàncton, organismes multicel ∙lulars heteròtrofs) (Fig.3). El metazooplàncton ocupa una posició clau a les xarxes tròfiques pelàgiques a causa de la seva

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funció en la transferència de matèria i energia a nivells tròfics superiors. A més, el metazooplàncton influeix directament a les xarxes tròfiques microbianes depredant sobre els protozous que es troben dins del seu espectre de grandària de presa, per exemple nanoflagel ∙lats heterotròfics, dinoflagel ∙lats i ciliats (Stoecker & Capuzzo 1990; Gifford & Dagg 1991), i indirectament a través d'efectes de cascada tròfica i la regeneració de nutrients (Calbet & Landry 1999).

Femto − Pico − Nano − Micro − Meso − Macro − Mega − PLÀNCTON 0.02 −02 0.2 −2 2−20 20 −200 0.2 −20 2−20 20 −200 µm µm µm µm mm cm cm Virio − plàncton

Bacterio − plàncton

Mico − plàncton

Fito − plàncton

Protozoo − plàncton

Metazoo − plàncton

Figura 3. Distribució dels diferents components del plàncton en funció de la seva grandària (from Sierburth et al. 1978)

Tradicionalment, entre les diferents categories de grandària del metazooplàncton (Fig. 3), el microplàncton metazou ha estat mostrejat incorrectament a causa de l'ús de xarxes de plàncton amb llum de malla ≥ 200 µm (Calbet et al. 2001, Galliene & Robins 2001; Turner 2004).

La composició, abundància i biomassa del microplancton metazou van ser estimades en diferents sistemes marins, en aigües costaneres del nord ‐oest del Mediterrani al llarg d'un cicle anual (article II ) i en la costa oest de la illa de Vancouver (costa del Pacífic canadenc) durant l'estiu (article III ).

El microplancton metazou es compon principalment de larves planctòniques tals com nauplis de copèpodes, copepodits i larves meroplanctòniques. La falta de coneixement dels metazous microplanctònics contrasta amb la seva importància en termes d'abundància, biomassa i la productivitat en ambients marins (Hopcroft et al. 2001; Turner 2004; Zervoudaki et

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al. 2007). Les larves de copèpodes són numèricament el component majoritari del metazooplàncton. A més, els nauplis de copèpodes són la principal presa d'un gran nombre de larves de peixos (Last 1980; Conway et al. 1991, 1998) i, com es va esmentar anteriorment, la seva abundància pot determinar el reclutament de moltes espècies de peixos de gran valor comercial (Castonguay et al. 2008). Per tant, la inconsistència en la quantificació i la funció dels metazous planctònics de petita grandària suposa un important biaix en les estimacions d'abundància, biomassa i producció del zooplàncton, de l'impacte sobre els productors primaris, fluxos de matèria i energia intervinguts pel zooplàncton i, en general de la seva importància a les xarxes tròfiques planctòniques (Turner 2004).

El paper tròfic, les taxes de creixement, les taxes de respiració i les eficiències de creixement de la comunitat de microplancton metazou es va determinar en aigües costaneres del nord ‐oest del Mediterrani al llarg d'un cicle anual (article II )

A més de la seva petita grandària, el paper de les larves meroplanctòniques a les xarxes tròfiques marines ha estat ignorat en gran mesura a causa de la seva temporalitat en el plàncton. No obstant això, les majoria de les larves meroplanctòniques són planctotròfiques i la seva alimentació depèn de la comunitat de plàncton existent. De fet, l'alliberament de les larves coincideix sovint amb les proliferacions de fitoplàncton per així maximitzar l'exposició de les larves a una disponibilitat alta d'aliment (Thorson 1946, 1950; Starr et al. 1990, 1991, 1994). Aquesta sincronia sovint comporta al fet que certes larves meroplanctòniques siguin els membres dominants de la comunitat del zooplàncton costaner durant la temporada reproductiva d'invertebrats bentònics (Thorson 1946; Williams & Collins 1986; Andreu & Duarte 1996). No obstant això, la possible funció tròfica de les larves planctòniques en els fluxos de carboni marins segueix sent en gran part desconeguda.

Es va determinar l'abundància, les taxes d'alimentació, la selecció de preses i l'impacte tròfic de diferents larves meroplanctòniques sota condicions de proliferació de fitoplàncton esdevingudes en la costa oest de la illa de Vancouver (article III )

Per tant, informació relativa a la abundància, biomassa i el paper tròfic de les larves planctòniques és crucial per a una major comprensió les xarxes tròfiques i els fluxos biogènics en els ecosistemes pelàgics marins.

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HIPÒTESIS I OBJECTIUS

El principal objectiu d'aquesta tesi va ser millorar el nostre coneixement sobre l'ecologia i la fisiologia de larves planctòniques d'invertebrats marins, incloent el seu paper a les xarxes tròfiques planctòniques . Amb aquesta finalitat, es van realitzar experiments de camp i de laboratori amb larves holo ‐ i meroplanctòniques amb una aproximació experimental a nivell d'espècie i comunitat.

La hipòtesi general de la tesi va ser que les larves planctòniques d'invertebrats juguen un paper clau en el funcionament dels ecosistemes marins . Concretament, es van formular les següents hipòtesis específiques:

1. Les larves planctòniques (larves holo ‐ i meroplanctòniques), incloent les que pertanyen a fraccions de petita grandària (< 200 µm, microplancton metazou), exerceixen un impacte tròfic important sobre les xarxes tròfiques planctòniques marines, que pot influir notablement en la dinàmica del fitoplàncton i dels protozous en aigües costaneres.

2. L'èxit ecològic de moltes espècies d'invertebrats marins es deu en part a les característiques fisiològiques de les fases larvàries planctòniques.

Per comprovar la hipòtesi 1 es van marcar els següents objectius :

1. Determinar de l'abundància estacional i la distribució vertical de tota la comunitat de zooplàncton en un àrea costanera del nord ‐oest mediterrani al llarg d'un cicle anual, amb especial interès en les larves planctòniques incloses les pertanyents al microplancton metazou (articulo I)

2. Estimar les taxes d'alimentació, l'impacte tròfic i les eficiències de creixement de la comunitat de microplancton metazou al llarg d'un cicle estacional en aigües del nord ‐oest mediterrani (articulo II )

3. Determinar l'abundància, les taxes d'ingestió, la selecció de preses i l'impacte tròfic de diferents larves meroplanctòniques sobre la proliferació natural nociva de Heterosigma akashiwo i Prorocentrum triestinum, que va tenir lloc en la costa oest de la Illa de Vancouver al juliol del 2006 (article III ).

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Per contrastar la hipòtesi 2 es van marcar els següents objectius :

4. Determinació de diferents processos fisiològics en larves primerenques de Oithona davisae (nauplis i copepodits primerencs) en funció de factors intrínsecs (fase de desenvolupament, pes corporal) i ambientals (temperatura, aliment) tals com: la supervivència, les taxes de desenvolupament i creixement (article IV ), les taxes d'alimentació (article V) i les taxes de respiració i excreció (article VI )

5. Avaluació de l'efecte de l'aliment sobre el comportament natatori (article VII ), l'alimentació (articles VII i VIII ) i les taxes de creixement (articles VIII i IX ) de les fases larvàries planctòniques del poliquet espiónid Polydora ciliata

RESULTATS

Els resultats d'aquesta tesi estan presentat com 9 publicacions/articles científics (indicats amb nombres romans) que s'han organitzat en 5 capítols principals . A continuació s'exposa una breu síntesi dels diferents capítols.

CAPITUL 1: Abundància estacional i distribució vertical del zooplàncton en aigües costaneres del nord ‐oest Mediterrani: importància dels metazous planctònics de petita grandària (article I)

Nostra comprensió sobre la funció del zooplàncton en els ecosistemes marins és limitat a causa de l'escassa informació sobre els metazous planctònics petita grandària. Al llarg d'un cicle anual, es va examinar la successió estacional i la distribució vertical de tota la comunitat de metazooplàncton d'una zona costanera del nord ‐oest del Mediterrani, amb especial interès en les fraccions de grandària petita. L'estudi va incloure l'ús de dos mètodes de mostreig per zooplàncton, xarxa de microplàncton i ampolles Van Dorn, per cobrir adequadament les principals fraccions de grandària del metazooplàncton, de 50 a 200 µm (microplancton metazou) i de 200 a 2000 µm (mesozooplàncton). També es va avaluar la influència dels principals factors abiòtics i biològics sobre l'estructura estacional i vertical de la comunitat de metazooplàncton. Els nauplis de copèpodes i copepodits van ser numèricament dominants durant la major part del període d'estudi, amb densitats que van oscil ∙lar aproximadament entre 620 a 23900 ind. m‐3 i de 265 a 10.000 ind. m‐3, respectivament. En general, els patrons de distribució vertical del microplàncton metazou i del mesozooplàncton van ser similars. Els

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gradients verticals d'abundància i biomassa de metazooplàncton van ser més importants a l'estiu i la tardor, i van tendir a seguir els de les seves preses microplanctónicas potencials (ciliats, dinoflagel ∙lats, diatomees). L'abundància del metazou microplanctònic i del mesozooplàncton sembla estar relacionada directament amb la temperatura. Els valors mínims d'abundància de copèpodes i les seves larves (nauplis i copepodits) es van observar durant la proliferació primaveral de fitoplàncton. Segons les estimacions a partir de les mostres de la xarxa de microplancton i les ampolles Van Dorn, les fases larvàries planctòniques (incloent larves holo ‐ i meroplanctòniques), van representar respectivament, el 45% (rang 24% ‐75%) i 55% (rang 26% ‐69%) del carboni total del metazooplàncton. La fracció de grandària corresponent al metazou microplanctònic va representar, en mitjana, el 34% (rang 15% ‐54%) del carboni total del metazooplàncton quan es va mostrejar amb ampolles Van Dorn. Per tant, encara que el mesozooplàncton domina en termes de biomassa, el microplancton metazou representa una fracció considerable de la biomassa total del metazooplàncton.

CAPITUL 2: Funció tròfica i balanç de carboni del metazou microplanctònic en aigües costaneres del nord ‐oest del Mediterrani (article II)

Durant un cicle estacional es van determinar, mitjançant incubacions de laboratori, les taxes d'alimentació, l'impacte tròfic i les eficiències de creixement dels assemblatges naturals de metazous microplanctònics procedents d'un àrea costanera del nord ‐oest del Mediterrani. Els micrometazous, és a dir els organismes pluricel ∙lulars planctònics heterotròfics entre 20 i 200 µm, van estar constituïts principalment per fases larvàries d'invertebrats. Els nauplis de copèpodes i copepodits van dominar la comunitat, excepte a l'abril quan les larves de poliquets van ser el grup més abundant. Es va analitzar la pressió per depredació dels micrometazous sobre clorofil ∙la a (Chl a, total i > 10 µm), nanoflagel ∙lats heterotròfics, nanoflagel ∙lats fototròfics, dinoflagel ∙lats, diatomees i ciliats. Els micrometazous van depredar sobre tots els grups estudiats, amb taxes d'ingestió específiques en carboni que van variar entre 0.31 i 1.24 d‐1. Les eficiències brutes de creixement per al conjunt de la comunitat de metazous microplanctònics, calculades com el pendent de la regressió lineal que relaciona les taxes específiques de creixement respecte a les taxes especifiques d'ingestió, va variar entre 0.27 i 0.39. Les pèrdues respiratòries en carboni dels micrometazous van dependre de la temperatura i van variar entre 0.16 i 0.36 d‐1, amb un Q10 = 2. L'eficiència mitjana de creixement net , 0.41, va ser independent de la temperatura i la disponibilitat d'aliment. Els micrometazous en conjunt tenen taxes específiques de creixement més altes que les del mesozooplàncton, però eficiències

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de conversió de l'aliment similars. L'efecte per predació en el estoc existent de les diferents preses va ser < 1% d‐1 per Chl a (total i > 10 µm) i < 2.5% d‐1 per a les altres preses estudiades, la qual cosa sembla insuficient per exercir un control rellevant sobre la dinàmica del fitoplàncton i els protozous. La inclusió dels micrometazous no canvia apreciablement la visió actual sobre el paper de metazooplàncton a les xarxes tròfiques marines en aigües costaneres del nord ‐oest Mediterrani.

CAPÍTOL 3: Abundància i taxes d'alimentació de larves planctòniques d'invertebrats marins sota condicions de floració d'algues nocives enfront de la Illa de Vancouver (article III)

Les interaccions entre el fitoplàncton tòxic i els seus predadors potencials són aspectes poc entesos de l'ecologia de les proliferacions d'algues nocives. En aquest estudi, es va determinar les taxes d'alimentació, la selecció de preses i l'impacte tròfic de diferents larves planctòniques d'invertebrats marins sobre la proliferació natural d
Heterosigma akashiwo i Prorocentrum triestinum que va tenir lloc en la costa oest de la illa de Vancouver al juliol del 2006. A més, es va estimar l'abundància, la biomassa i la composició del zooplàncton abans i durant la proliferació d'algues nocives. Els experiments d'alimentació es van realitzar amb larves de poliquet (Serpula columbiana ), d'equinoderm (Stronglyocentratus purpuratus ) i de cirríped (Balanus crenatus ) obtingudes mitjançant cultius de laboratori, i amb larves de bivalves i gasteròpodes recol ∙lectades a la zona d'estudi mitjançant arrossegaments amb xarxes de plàncton. Mentre que totes les larves es van alimentar amb H. akashiwo , només els nauplis de cirríped i larves d'equinoderm es van alimentar amb P. triestinum . H. akashiwo va ser el component principal en la dieta de totes les larves (> 64%). Es va observar una relació positiva entre la disponibilitat de la presa en els assemblatges d'aliment i la seva contribució a les dietes de totes les larves. L'impacte tròfic potencial de les larves meroplanctòniques sobre les espècies fitoplanctòniques responsables de la proliferació va ser baix (<1,5%). La ingestió de les espècies de fitoplàncton que van formar la proliferació no va tenir aparentment cap efecte advers sobre els predadors estudiats després de 48 hores d'incubació. Per contra, l'abundància de larves planctòniques i un altre zooplàncton en el camp van disminuir de manera contínua a llarg de la progressió de la proliferació, amb pèrdues de fins a un 75% en comparació de la seva abundància abans de la proliferació. La presència d' H. akashiwo va afectar negativament a l'abundància de larves meroplanctòniques, malgrat l'eficient predació d'aquestes larves. Per tant, la pressió per depredació va ser reduïda, la qual cosa probablement va contribuir al creixement i la persistència de la proliferació. La reducció de l'abundància de larves

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meroplanctòniques i un altre zooplàncton associada amb la proliferació d' H. akashiwo podria tenir un impacte potencial sobre el reclutament bentònic i les transferències d'energia a nivells tròfics superiors a les xarxes tròfiques marines.

CAPÍTOL 4: Ecofisiología de les fases de desenvolupament primerenques del copèpode Oithona davisae (articles IV, V, VI)

Entre els copèpodes planctònics marins, els de el gènere Oithona són probablement els més abundants i ubics dels oceans del món. No obstant això, el nostre coneixement sobre l
ecofisiologia d
Oithona és molt escàs en comparació del de copèpodes calanoides, en particular per a les seves fases larvàries (nauplis i copepodits). Determinem les taxes d'alimentació, creixement, respiració i excreció de les larves de Oithona davisae en relació la temperatura, i la concentració d'aliment. A més, estimem els coeficients metabòlics C:N:P, l'eficiència de creixement, i el balanç de carboni en nauplis d' O. davisae . Les taxes d'ingestió, el creixement i la respiració dels nauplis augmenta amb l'augment de temperatura, amb un Q10 d
aproximament 2.5 en un rang de temperatura de 16 ‐28 ºC (articles IV, V, VI). La supervivència dels nauplis es va reduir aproximadament un 60% a la concentració d'aliment més baixa provada en els experiments (11 µg C L‐1, després de 7 dies i a 20 ºC) (article IV). El desenvolupament dels nauplis d' O. davisae va ser equiproporcional però no isocronal (Article IV). Les concentracions d'aliment requerides per obtenir taxes màximes de desenvolupament i creixement a 20 º C van ser 56 and 87 µg C L‐1, respectivament (article IV). Les fases larvàries d' O. davisae van mostrar una resposta funcional alimentària de tipus III, amb concentracions llindars d'aliment entre 50 i 75 µg C L‐1, depenent de la fase de desenvolupament (article V). Tots els paràmetres d'alimentació varien segons el pes corporal/edat. La concentració d'aliment necessària per aconseguir les taxes d'ingestió màximes va variar des de 200 µg C L‐1 en nauplis primerencs a 320 µg C L‐1 en copepodits. (article V). L'eficiència bruta de creixement va variar entre 0.16 i 0.60 en funció de l'etapa de desenvolupament, la disponibilitat d'aliment i la temperatura (article V). Les taxes específiques de respiració de nauplis i copepodits van variar entre 0.11 i 0.55 d‐1 en funció de la fase de desenvolupament, el pes corporal, la temperatura i la disponibilitat d'aliment (article VI). Els ràtios de C:N metabòlics van ser majors de 14, la qual cosa indica un metabolisme basat en lípids (article VI). Les eficiències d'assimilació i les eficiències netes de creixement van variar entre 65% i 86% i entre 23% i 32%, respectivament, depenent del pes corporal, la fase de desenvolupament i la temperatura (article VI). Els nauplis O. davisae presenten taxes de desenvolupament i eficiències de creixement similars a les

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copèpodes calanoides, però perdudes respiratòries especifiques i requeriments d'aliment més baixos. Per tant, els baixos costos metabòlics de Oithona comparats amb calanoides poden ser una de la raons el seu èxit evolutiu en els ecosistemes marins.

CAPÍTOL 5: Ecofisiologia de les larves planctòniques del poliquet espiònid Polydora ciliata (articles VI, VII and VIII)

Polydora ciliata és una de les espècies de poliquets espiònids mes comuns i abundants en ecosistemes litorals bentònics del nord d'Europa. Les seves larves planctòniques són molt freqüents en aigües costaneres, i en ocasions poden arribar a ser el component dominant del metazooplàncton. Es va estudiar la influència de l'aliment en el comportament natatori, les taxes d'alimentació i creixement i l'eficiència de creixement de les larves de P. ciliata . A més, es va avaluar el grau de limitació per aliment de les poblacions larvàries de P. ciliata en aigües eutròfiques d'un estuari danès, Isefjord. Els patrons natatoris no van canviar en relació a la grandària de les larves ni amb la disponibilitat o el tipus d'aliment (article VI). La grandària òptima de presa es va incrementar durant el desenvolupament larvari de 13 µm a 20 ‐50 µm (article VI). En experiments d'alimentació amb suspensions de preses naturals, els ciliats van ser filtrats més eficientment que els dinoflagel ∙lats (article VI). Les larves de P. ciliata van mostrar una resposta funcional tipus II amb taxes màximes d'ingestió de 0.40 d‐1 i 0.46 d‐1 alimentant ‐se de Thalassiosira weissflogii i Rhodomonas salina , respectivament (article VII). Les taxes màximes de creixement van ser aconseguides a concentracions d'aliment que van variar entre 1.4 ‐2.5 µg C ml ‐1 depenent de la grandària de les larves (article VII). L'eficiència bruta de creixement vari de 0.47 en larves primerenques a 0.28 en fases larvàries intermèdies i tardanes (article VII). Les larves de P. ciliata van ser el component principal del meroplàncton a l'àrea d'estudi durant el període de mostreig (article IX). Les taxes de creixement van ser significativament mes altes en larves incubades amb aigua de mar natural enriquida amb fitoplàncton de cultiu (R.salina ) que en larves incubades en aigua de mar natural, en mitjana 0.215 d‐1 i 0.107 d‐1, respectivament (article IX). En condicions naturals, durant el període d'estudi, no es van trobar concentracions d'aliment prou altes com per permetre taxes màximes de creixement (article IX). En conjunt, aquests estudis mostren que el creixement larvari de P. ciliata està limitat per l'aliment malgrat habitar en aigües altament eutròfiques. Des d'una perspectiva ecològica, suggerim que existeix una solució de compromís (compensació) entre l'alimentació/cinètica del creixement i la dispersió larvària . La selecció natural pot afavorir que algunes larves meroplanctòniques, tal com P. ciliata , presenten baixa eficiència de filtració i baixes taxes de creixement malgrat

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habitar en sistemes amb alta disponibilitat d'aliment. Aquestes característiques fisiològiques de les larves permeten un desenvolupament planctònic prou llarg com per assegurar una dispersió larvària eficient.

DISCUSSIÓ GENERAL

Els resultats inclosos en aquesta tesis han estat discutits detalladament en els seus respectius articles. El propòsit d'aquesta discussió general és relacionar els principals resultats obtinguts en els diferents estudis i avaluar les hipòtesis inicials.

Importància de les larves planctòniques en termes d'abundància i biomassa

Les larves planctòniques (incloent larves holo ‐ i meroplanctòniques) van ser numèricament els components més importants de les comunitats de metazooplàncton tant en aigües costaneres oligo ‐mesotrófiques del Mediterrani nord ‐occidental al llarg de tot l'any com en aigües eutròfiques de la illa de Vancouver durant l'estiu (articles I i III). Els nauplis de copèpodes i copepodits van ser freqüentment els metazous més abundants, encara que ocasionalment les larves meroplanctòniques van dominar la comunitat de metazooplàncton (articles I, II i IX). La contribució de les larves meroplanctòniques a la biomassa total de zooplàncton va ser sovint més important en l'estuari eutròfic boreal, Isefjord, que en els sistemes costaners estudiats (articles I, III i IX). Les larves planctòniques van representar una fracció considerable de la biomassa total de carboni de metazooplàncton (en mitjana de 50%, Articles I, III i IX) i episòdicament pot constituir el component dominant de les comunitats de metazooplàncton (articles I i IX). Les taxes específiques de creixement de les larves planctòniques (nauplios de copèpodes) van ser superiors a les observades comunament pel mesozooplàncton (copèpodes adults) mitjançant el mètode de producció d'ous (Kiørboe & Johansen 1986; Poulet et al. 1995) (article II). Per tant, ignorar el creixement larvari pot resultar en important subestimacions de la producció secundària en els sistemes marins planctònics (Calbet et al. 2000; Rei ‐Rassat et al. 2004)

Tradicionalment, l'abundància i biomassa de petits metazous planctònics s'ha subestimat a causa de la utilització de xarxes de plàncton amb llum de malla gruixuda (>200 µm ) (Banse 1962; Turner 1994). No obstant això, la importància dels metazous planctònics petits (<200 µm, microplàncton metazou) ha estat reconeguda cada vegada més en l'última dècada gràcies a l'ús

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de mètodes apropiats de mostreig (Calbet et al. 2001; Gallienne & Robins 2001, Turner 2004, Riccardi 2010). En els nostres estudis trobem que, en aigües costaneres, la fracció de grandària petita es compon principalment de larves planctòniques, incloent nauplis de copèpodes, copepodits i larves meroplanctòniques (articles I i III). En aigües costaneres del nord ‐oest Mediterrani, i depenent del mètode de mostreig (xarxa de plàncton o ampolles Van Dorn), el microplàncton metazou va representar, en mitjana durant tot l'any, entre el 18% i el 34% de la biomassa total de metazous (article I). L'abundància de metazous petits va ser major quan van ser utilitzades ampolles Van Dorn per al mostreig, la qual cosa reflecteix la importància de l'ús de mètodes apropiats de mostreig per a una estimació precisa de l'estructura comunitat zooplanctònica (article I). A l'àrea d'estudi a la Illa de Vancouver, la contribució del microplàncton metazou a la biomassa total de metazooplàncton va ser aproximadament 20 ‐ 25% (article III). Per tant, encara que el mesozooplàncton domina en termes de biomassa, el microplàncton metazoo representa una fracció considerable de la biomassa total del metazooplàncton. A més, com les taxes fisiològiques dels organismes petits són generalment més altes que els de els organismes més grans, l'exclusió de metazous microplanctònics pot donar lloc a importants subestimacions de la producció de secundària del zooplàncton (Gallienne & Robins 2001, Turner 2004).

.

Paper de les larves planctòniques d'invertebrats a les xarxes tròfiques pelàgiques

Encara que el grau en què la depredació per metazooplàncton pot regular als productors primaris segueix sent un tema de debat, cada vegada hi ha més evidències que els metazous exerceixen poca pressió sobre les dinàmiques del fitoplàncton i el zooplàncton unicel ∙lular (per exemple, Broglio et al. 2004; Atienza et al. 2006). No obstant això, en la majoria dels estudis sobre depredació del metazooplàncton han estat exclosos els metazous microplanctònics, i per tant, es subestima l'impacte tròfic dels metazous sobre els productors primaris. A més, la majoria d'experiments de depredació s'han centrat en els organismes holoplanctònics (principalment copèpodes adults) i el paper tròfic del meroplàncton ha estat ignorat en gran mesura. En aquesta tesi, la funció de les larves a les xarxes tròfiques planctòniques es va avaluar mitjançant aproximacions a nivell d'espècies i de comunitat. En aigües costaneres oligo ‐ mesotròfiques del nord ‐oest del Mediterrani i al llarg d'un cicle anual, duem a terme experiments de depredació amb la comunitat de metazous microplanctònics (constituïda majoritàriament per larves microplanctòniques) alimentant ‐se d'assemblatges naturals

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d'aliment (article II). En aigües eutròfiques de la costa oest de la illa de Vancouver (article III) i en l'estuari boreal altament eutròfic, Isefjord, a Dinamarca, (article VII), els experiments de depredació es van realitzar amb determinades espècies de larves meroplanctòniques. En conjunt, aquests treballs ens ofereixen una base de coneixements solida que ens permet avaluar el paper de les larves a les xarxes tròfiques planctòniques en condicions ambientals (tròfiques) contrastades.

En aigües costaneres del nord ‐oest del Mediterrani, l'impacte tròfic (mesurat com el percentatge de la biomassa estoc consumida per dia) exercit per la comunitat de larves microplanctòniques sobre la comunitat microbiana va ser molt baix, menor de 5% (article II). D'acord amb els experiments de dilució que es van dur a terme simultàniament amb els experiments de depredació del metazou microplanctònic, la contribució dels metazous a la depredació total del microzooplàncton sobre el fitoplàncton va ser molt baixa (Calbet et al. 2008; Article II). Per tant, els protists són probablement la principal font de pressió de depredació sobre el fitoplàncton en aigües costaneres del Mediterrani, almenys durant certes èpoques de l'any (Calbet et al. 2008; article II). El baix impacte tròfic dels metazous microplanctònics comparat amb el dels protozous pot ser hagut de tant a la seva menor biomassa en el camp (un o dos ordres de magnitud menor en el nord ‐oest de les aigües costaneres del Mediterrani, article II) com a les seves taxes d'ingestió específiques més baixes (Jacobson & Anderson 1993).

Respecte a les larves meroplanctòniques, les poblacions de Polydora ciliata van tenir un efecte tròfic molt lleu (<1%) sobre els productors primaris en aigües altament eutròfiques (article IX). De la mateixa manera, si considerem les taxes d'ingestió especifiques de les larves Polydora ciliata sobre suspensions d'aliment natural (article VII) i la seva biomassa en carboni en la naturalesa (article IX), el seu impacte tròfic potencial sobre dinoflagel ∙lats i ciliats és molt baix (<1%). La pressió per depredació de les larves meroplanctòniques sobre la comunitat microbiana no va influir en la proliferació algal d
Heterosigma akashiwo i Prorocentrum triestinum en aigües costaneres de la Illa de Vancouver (impacte tròfic <2%) (article III). L'efecte negatiu d
Heterosigma akashiwo sobre l'abundància de larves meroplanctòniques (així com sobre un altre zooplàncton) contribuiria a reduir la pressió tròfica sobre la proliferació de fitoplàncton nociu (article III).

Els nostres resultats suggereixen que, fins i tot incloent la comunitat de metazous microplanctònics i les larves meroplanctòniques, la funció tròfica del metazooplàncton a les

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xarxes tròfiques planctòniques sembla ser, en general, insuficient per controlar la dinàmica de les poblacions del fitoplàncton i els protozous.

Hipòtesi 1: NO ÉS VALIDA

Les larves planctòniques (larves holo ‐ i meroplanctòniques), incloent les que pertanyen a fraccions de petita grandària (< 200 µm, microplancton metazou), exerceixen un impacte tròfic important sobre les xarxes tròfiques planctòniques marines, que pot influir notablement en la dinàmica del fitoplàncton i dels protozous en aigües costaneres

Ecofisiología de larves d'estadis de desenvolupament del copèpode Oithona davisae

Els petits copèpodes planctònics del gènere Oithona es troben entre els metazous més ubics i abundants dels oceans del món, des de latituds polars a tropicals (Gallienne & Robins 2001). L'èxit d'una espècie donada està determinat pel seu èxit en el reclutament i, per tant, el coneixement de la fisiologia de les fases larvàries és essencial per entendre l'eficàcia biològica d'una espècie.

Els processos fisiològics en les larves estan regulats per factors ambientals i endògens/intrínsecs. Igual que en un altre zooplàncton (Vidal 1980; Vidal & Whitledge 1982; Ikeda 1985; Hansen et al. 1997; Gillooly 2000; Ikeda et al. 2001), en el nostre estudi trobem que la grandària corporal (o pes corporal) i la fase de desenvolupament són factors intrínsecs que influeixen de manera important en les taxes d'ingestió, creixement i respiració en les larves d
Oithona davisae (articles V i VI). Sota condicions similars de temperatura i aliment, les taxes fisiològiques de les larves d
Oithona davisae van mostrar relacions al ∙lomètriques respecte al pes corporal (articles V i VI). La temperatura i la disponibilitat d'aliment són dos dels principals factors ambientals que afecten a la distribució i la fisiologia del zooplàncton (Huntley & Boyd 1984, Huntley & López 1992, Gillooly 2000, Ikeda et al. 2001). Les diferents taxes fisiològiques estudiades en les larves d'O. davisae (creixement, ingestió, respiració, excreció) van seguir models exponencials en relació a la temperatura a temperatures entre 20 i 28 ºC (articles IV, V i VI). Per contra, totes les taxes fisiològiques van ser molt baixes a temperatures ≤ 16 ºC, la qual cosa donava lloc a desviacions del model exponencial (articles IV, V i VI). Aquests resultats probablement reflecteixen el caràcter termòfil d'aquesta espècie (Uye & Sayo 1995); O. davisae

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és molt escassa durant l'hivern i la primavera (quan les temperatures són <20 º C), mentre que és molt abundant durant les estacions més càlides, quan la temperatura de l'aigua es troben entre 20 i 28 ºC. Per tant, podem concloure que 20 ‐28 ºC és probablement el rang de temperatura òptim para O. davisae . Entre 16 ‐28 ºC, els valors de Q10 per a les taxes de creixement (article IV) ingestió (article V) i respiració (article VI) van ser totes molt similars (≈Q10 = 2.4), la qual cosa suggereix que tots aquests processos fisiològics mostren una dependència similar a la temperatura (articles IV, V i VI). Els valors de la Q10 obtinguts en el nostre estudi (articles IV, V i VI) es troben dins del rang de valors que s'observen normalment en altres copèpodes (Ikeda et al. 2001; Castellani et al. 2005). Oithona és capaç de créixer tant en ambients oligotròfics oceànics com en aigües costaneres eutròfiques quan l'aliment arriba a ser un recurs limitat per als copèpodes calanoides (Calbet & Agustí 1999). Les estratègies de vida dels copèpodes estan adaptades a les fluctuacions en la disponibilitat d'aliment, per exemple, moltes espècies de calanoides produeixen ous de resistència que s'acumulen en els sediments que eclosionen just abans de la proliferació fitoplanctònica primaveral (Marcus 1996; Peterson 1998). Per contra, Oithona no produeixen ous de resistència i ha d'adaptar ‐se a tolerar períodes de baixa disponibilitat d'aliment i mantenir les seves poblacions durant tot l'any. En els nostres estudis, hem identificat diverses característiques fisiològiques en les larves d' O. davisae que suggereixen avantatges competitius sobre els copèpodes calanoides quan i on el menjar és escàs, tal com: - Les concentracions d'aliment que indueixen una mortalitat rellevant en nauplis d' O. davisae van ser inferiors a les concentracions comunament observades en nauplis calanoides (article IV). A més, els nauplis d' O. davisae poden suportar períodes relativament llargs (5 dies) en condicions de molt poc aliment sense una mortalitat significativa (article IV) - Els nauplis d' O. davisae van mostrar taxes de desenvolupament similars a les observades per nauplis de calanoides , però requeriments d'aliment més baixos (article IV). - El desenvolupament dels nauplis en O. davisae és menys sensible a concentració d'aliment que el creixement, la qual cosa suggereix que el desenvolupament i per tant el reclutament pot ser prioritari enfront del creixement somàtic (article IV). - Els nauplis i copepodits d
O. davisae van mostrar una resposta funcional alimentària de tipus III (article VI). Aquest tipus de resposta funcional, que també s'ha observat en alguns copèpodes calanoides, es caracteritza per la presència d'un "llindar de baixa alimentació ", és a dir una concentració d'aliment per sota de la qual el copèpode deixa d'alimentar ‐se o

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redueix les seves taxes d
aclariment (article VI). La presència d'aquests llindars pot ser interpretat com una adaptació per conservar energia en concentracions d'aliments baixes a causa que el cost energètic de l'adquisició d'aliments no seria compensat pel guany energètic (article VI). - Les concentracions d'aliment saturants per a larves d' O. davisae són inferiors al fet que s'han descrit per a fases lavaries de calanoides (article VI). Això suggereix que en els sistemes naturals les taxes d'ingestió de les larves calanoides poden estar limitades per aliment en un major grau que les de les larves d
Oithona (article VI). - Les larves d' O. davisae van mostrar eficiències de creixement brut, eficiències d'assimilació i eficiències de creixement net similars a les de copèpodes calanoides però taxes específiques d'ingestió i pèrdues respiratòries especifiques més baixes (articles V i VI).

En conjunt els nostres resultats confirmen que els requeriments d'aliment i les pèrdues respiratòries especifiques en nauplis d' O. davisae són inferiors a les de copèpodes calanoides (Lampitt & Gamble 1982; Paffenhöfer 1993); aquestes diferències fisiològiques poden explicar en part l'èxit ecològic d
Oithona en els ambients marins. Les diferències en els requeriments d'aliment i les perdudes metabòliques de carboni entre calanoides i Oithona probablement està relacionat amb les diferències en la natació i la conducta alimentària entre aquests dos grups de copèpodes. En contrast amb la majoria calanoides (Titelman & Kiorboe 2003; Henriksen et al. 2007), Oithona spp (incloent nauplis, copepodits i adults) es mouen amb salts ocasionals i usen senyals hidromecàniques per a la detecció de preses (Paffenhöfer 1993; Svensen & Kiørboe 2000; Paffenhofer & Mazzocchi 2002). A manera d'exemple, en absència de preses, els nauplis d
Oithona davisae passen el 98% del seu temps immòbils i només davant la presència de preses mòbils augmenten la seva freqüència de salts (Henriksen et al. 2007). L'estratègia d'alimentació i la natació d
Oithona pot considerar ‐se més eficient energèticament que la de la majoria de calanoides (Paffenhöfer 1993), el que contribueix a explicar l'àmplia distribució i èxit ecològic d
Oithona en els ecosistemes marins.

Bases fisiològiques i significat ecològic de la limitació per aliment del creixement larvari en larves meroplanctòniques: el cas de Polydora ciliata

La limitació per aliment del creixement, és a dir, que les concentracions d'aliment en la naturalesa estan per sota de les necessàries per proporcionar taxes màximes de creixement,

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sembla ser una regla general pel zooplàncton marí (Huntley & Boyd, 1984; Lampert 1985; Hirst & Búnquer 2003). No obstant això els nostres resultats suggereixen que el creixement de les larves meroplanctòniques podria trobar ‐se limitat per l'aliment en un major grau que el de les larves holoplanctòniques (per exemple, els nauplis de copèpodes). Les taxes de creixement de les larves de Polidora ciliata van estar limitades per aliment malgrat aquesta espècie habita en aigües altament eutròfiques. Aquesta conclusió es recolza en dues aproximacions experimentals: - Els estudis de laboratori van mostrar que les taxes màximes de creixement, amb una grandària òptima dels aliment, es van aconseguir a concentracions que van variar entre 1410 i 2510 µg C L‐1 depenent de la grandària de les larves (article VIII). A l'àrea d'estudi, la disponibilitat d'aliment va ser insuficient per proporcionar les taxes màximes de creixement larvari observades en el laboratori (articles VII i IX). - Les taxes específiques de creixement de les larves criades en suspensions d'aliment naturals van ser inferiors a les taxes de creixement de larves alimentades amb suspensions d'aliment natural enriquides, la qual cosa indica que les larves en el mitjà natural no pot créixer al seu màxim potencial (article IX).

Les taxes màximes de creixement de les larves de Polydora ciliata i Oithona davisae d'una grandària semblant van ser bastant similars sota condicions saturants d'aliment (article V i VIII). No obstant això, els requeriments d'aliment per aconseguir taxes de creixement màximes van ser molt més alts per a les larves de P. ciliata que per les d' O. davisae . Resultats similars han estat observats en altres larves meroplanctòniques i en larves de copèpodes (vegeu la discussió de l'article VIII). Al llarg d'un cicle anual aigües costaneres del nord ‐oest del Mediterrani, taxes de creixement de la comunitat de larves meroplanctòniques van ser significativament més baixes que els de nauplis i copepodits en condicions similars de disponibilitat d'aliment (article II). Per tant, els nostres resultats suggereixen que en la naturalesa taxes de creixement de les larves meroplanctòniques podrien ser inferiors a les taxes de creixement de les larves holoplanctòniques (nauplis de copèpodes) (articles II i VIII). Les diferències en el grau de limitació per aliment del creixement entre larves mero ‐ i holoplanctòniques pot deure's a diferències fisiològiques entre aquests grups d'organismes planctònics, tal com: - Les taxes especifiques màximes de filtració (aclariment) de les larves de Polydora ciliata (article VIII), així com les d'altres larves meroplanctòniques (Jaspersen & Olsen 1982; Hansen & Ockelmann 1991), van ser inferiors a les de larves holoplanctòniques tals com nauplis de copèpodes (Paffenhöfer 1971; Berggreen et al. 1998;. Hansen et al. 1997). Per

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exemple, les taxes màximes especifiques de filtració dels nauplis d' O. davisae van ser un ordre de magnitud superior al de les larves de P. ciliata (articles V i VIII). Aquesta diferència suggereix que les larves de P. ciliata presenten un mecanisme d'alimentació menys eficient que el de nauplis d' O. davisae , la qual cosa explica perquè són necessàries concentracions d'aliment tan altes (fins i tot superiors a les quals es troba en ambients altament eutròfics) per permetre que les larves de P. ciliata puguin aconseguir les seves taxes màximes de creixement (articles VIII i IX). - Les larves de P. ciliata mostren un comportament natatori similar davant la presència o absència d'aliment (article VII). Per contra, com es va esmentar anteriorment, l'activitat natatòria i d'alimentació dels nauplis d' O. davisae augmenta només davant la presència de preses (Henriksen et al. 2007). De la mateixa manera, alguns nauplis de calanoides augmenten el temps de natació a mesura que augmenta la concentració d'aliment (van Durin & Videler 1995). Una natació contínua s'associa generalment a costos d'energètics alts i, en conseqüència, a necessitats d'aliment altes. Això suggereix que el comportament poc especialitzat de les larves de P. ciliata en relació al règim alimentari poden ser energèticament menys eficient que el de nauplis de copèpodes.

Des d'una perspectiva ecològica, la limitació per aliment causa una disminució de les taxes de creixement i per tant perllonga el període de vida planctònic, amb el que s'exposa a les larves a fonts addicionals de mortalitat addicionals com la depredació (Thorson 1950); d'altra banda, l'augment en el període de vida planctònic facilita la dispersió larvària. Quan es requereixen baixes concentracions d'aliment per a unes taxes de creixement màxim, s'afavoreix un desenvolupament larvari ràpid i en conseqüència el reclutament a la fase adult; això és un avantatge adaptatiu per a organismes holoplanctònics com Oithona davisae (article IV). Per a copèpodes planctònics, en els quals totes les fases del cicle vital es dispersen en la columna d'aigua, un període de vida larvari més llarg no suposa aparentment cap avantatge selectiu. No obstant això, per a molts invertebrats bentònics, especialment els que són sèssils durant la fase adulta, les larves actuen com el seu únic mecanisme de dispersió (Levin & Bridges 1995). La dispersió larvària és crucial per a la supervivència de l'espècie i la seva persistència, ja que promou la connectivitat entre poblacions afavorint el flux genètic (Roughgarden et al. 1988., Palumbi 2003; Trakhtenbrot et al. 2005), facilita la recolonització dels llocs després d'alteracions ambientals (Hansen et al. 2002;. Petersen et al 2002.), i contraresta les fluctuacions de densitat en poblacions adultes, la qual cosa redueix el risc d'extincions locals (Eckert 2003). Suggerim que, per a molts invertebrats bentònic hi ha un solució de compromís entre el creixement i la

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dispersió. La selecció natural podria afavorir que algunes larves meroplanctòniques, tal com a P. ciliata , posseeixin taxes de creixement baixes tot hi que habiten en ambients amb molt alta disponibilitat d'aliment (article IX). En aquest cas, el que inicialment pot ser percebut com un rendiment subòptim de les larves, en realitat pot funcionar per permetre un desenvolupament planctònic prou llarg com per assegurar una dispersió larvària eficient.

Hipòtesi 2: SI ÉS VALIDA

L'èxit ecològic de moltes espècies d'invertebrats marins es deu en part a les característiques fisiològiques de les seves larves planctòniques

CONCLUSIONS PRINCIPALS

I. Les larves planctòniques d'invertebrats, incloent tant larves holoplanctòniques com meroplanctòniques, representen una fracció considerable de la biomassa total de metazooplàncton en aigües costaneres.

II. Els nauplis de copèpodes i els copepodits són numèricament els components dominants de la comunitat de metazooplàncton en aigües costaneres del nord ‐oest del Mediterrani.

III. L'abundància total del microplancton metazou i del mesozooplàncton en aigües costaneres del nord ‐oest del Mediterrani es correlaciona positivament amb la temperatura de l'aigua al llarg d'un cicle estacional.

IV. El patró d'abundància estacional i la distribució vertical del microplancton metazou i del mesozooplàncton no estan directament relacionats amb la biomassa de fitoplàncton (clorofil ∙la) en aigües costaneres del nord ‐oest del Mediterrani.

V. L'aparició d'algunes larves meroplanctòniques (poliquets) en aigües costaneres està vinculada a les proliferacions fitoplanctòniques primaverals

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VI. El microplancton metazou depreda de manera eficient sobre preses nano ‐ i microplanctòniques (nanoflagel ∙lats, diatomees, dinoflagel ∙lats i ciliats).

VII. L'impacte tròfic de les larves microplanctòniques (<200 µm, microplancton metazou) és insuficient per controlar la dinàmica del fitoplàncton i protozous en aigües costaneres del nord ‐oest del Mediterrani.

VIII. La pressió depredadora exercida per les larves meroplanctòniques té un efecte mínim sobre la proliferacions nocives de Heterosigma akashiwo / Prorocentrum triestinum .

IX. Les proliferacions nocives de Heterosigma akashiwo / Prorocentrum triestinum afecten negativament a l'abundància de les larves planctòniques i la resta del zooplàncton

X. En el seu conjunt, el microplancton metazou té taxes de creixement específic més altes que el mesozooplàncton, però eficiències de conversió d'aliment similars.

XI. L'activitat alimentària augmenta significativament les taxes de respiració dels nauplis de copèpodes, per tant l'estimació de taxes de respiració sense aliment comporta a importants subestimes de les taxes metabòliques de nauplis de copèpodes.

XII. Les necessitats d'aliment i les pèrdues respiratòries de carboni dels nauplis de Oithona són més baixos que els de copèpodes calanoides; aquestes diferències fisiològiques poden explicar, en part, l'èxit ecològic de Oithona en ambients marins.

XIII. Malgrat habitar en aigües eutròfiques, el desenvolupament larvari de Polydora ciliata mostra un alt grau de limitació per l'aliment en comparació amb els organismes holoplanctònics.

XIV. Les característiques especials de la ecofisiologia d'algunes larves d'invertebrats marins bentònics, tals com P. ciliata , permeten un desenvolupament planctònic perllongat, que garanteix una dispersió larvària eficient.

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Resumen de la tesis Thesis summary ‐ Spanish version

La gran mayoría de los invertebrados marinos tienen un ciclo de vida complejo que incluye fases larvarias planctónicas entre la fase embrionaria y la fase adulta (Thorson 1950; Strathmann 1987, 1993). Estas larvas pueden diferenciarse de los adultos en tamaño, forma, hábitat, forma de nutrición, y/o la capacidad de dispersión (Barnes et al. 1988; Young 2002). La supervivencia y el crecimiento de las fases larvarias pueden influir en el éxito del reclutamiento de las especies, y en la conectividad, distribución y abundancia de las poblaciones de invertebrados marinos (Roughgarden et al. 1988; Eckert 2003). Sin embargo, a pesar de la obvia importancia de las larvas en los ciclos de vida de la mayoría de los animales marinos, nuestro conocimiento sobre la ecofisiología larvaria sigue siendo escaso en comparación con el de las fases adultas. Además, como importantes componentes de las comunidades de zooplancton, el papel trófico de las larvas planctónicas en las redes alimentarias marinas no debería ser ignorado. Esta tesis doctoral tiene como objetivo principal contribuir al conocimiento de la ecología y la fisiología de larvas planctónicas de invertebrados marinos, incluyendo su papel en las redes tróficas planctónicas.

INTRODUCCIÓN

La definición de "larva". Clasificación de las larvas de invertebrados marinos

El desarrollo animal que incluye fases larvarias se denomina "desarrollo indirecto". Por el contrario, en el "desarrollo directo" el embrión se desarrolla directamente en un juvenil, el cual suele ser versión en miniatura, pero sexualmente inmadura, del adulto. El término "larva" se ha utilizado de muchas maneras diferentes dependiendo del enfoque y la disciplina científica y, en este momento, no existe una definición aceptada de manera general (Strathmann 1987; McEdward & Jaines 1993; Young 2002). En esta tesis, se ha utilizado el concepto de larva descrito por Hickman (1999): "la larva es un estado estructural o una serie de estados que se produce entre el inicio de la morfogénesis divergente después del desarrollo embrionario y el inicio de la metamorfosis que conlleva al plan corporal adulto.

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Las larvas de invertebrados marinos muestran una impresionante diversidad de formas corporales, muchas de las cuales han recibido nombres específicos. Las larvas marinas han sido clasificadas por el lugar de desarrollo, el modo de nutrición, el potencial de dispersión, y la morfogénesis (Thorson 1950; Mieikovsky 1971; Levin & Bridges 1995). Desde una perspectiva ecológica y para los propósitos de esta tesis, destacamos las siguientes categorías: - Larvas planctónicas, cuyo desarrollo se produce en la columna de agua, frente a larvas bentónicas, cuyo desarrollo tiene lugar sobre o en el fondo marino. - Larvas holoplanctónicas, que son las larvas de organismos que pasan toda su ciclo vida en el plancton, frete a larvas meroplanctónicas, que son las fases larvarias planctónicas de invertebrados marinos bentónicos. - Larvas planctotróficas, que se alimentan de plancton, frente a larvas lecitotróficas, cuya nutrición se derivan exclusivamente de reservas vitelinas procedentes del huevo.

Ciclo de vida de los copépodos planctónicos y descripción de sus fases larvarias

Los copépodos son un grupo muy diverso de crustáceos, con más de 11.500 especies conocidas, la mayoría de las cuales son marinas (Humes 1994). Entre los invertebrados marinos, los copépodos son el grupo dominante y más diverso del metazooplancton (zooplancton multicelular) en ambientes marinos (Longhurst 1985; Verity & Smetacek 1996). Los copépodos aportan una fracción considerable de la producción secundaria en la mayoría de las comunidades planctónicas marinas (Cushing 1989) y, además, constituye un eslabón clave entre los productores primarios y los niveles tróficos superiores en las redes tróficas pelágicas.

La mayoría de los copépodos planctónicos son dioicos y presentan reproducción sexual (Gilbert & Williamson 1983). Los ciclos de vida de los copépodos planctónicos son complejos y, en general, se caracterizan por 13 fases de vida, incluyendo el huevo, seis fases larvarias denominadas nauplios (NI ‐NVI), cinco fases larvarias denominadas copepoditos (CI ‐CV) y la fase adulta (Fig.1). La larva nauplio es el tipo de larva más ancestral en crustáceos y se ha utilizado como principal característica que une a todo el subfilo Crustáceos (Cisne 1982; Dahms 2000; Harvey et al. 2002). En términos de abundancia numérica, los nauplios se han considerado "la forma más abundante de animal multicelular de la tierra" (Fryer 1987). En copépodos, como en otros crustáceos, los procesos ontogenéticos están vinculados a mudas que tienen lugar entre las distintas etapas vitales (Williamson 1982). Los nauplios de la mayoría de copépodos de vida libre mudan cinco veces (de la fase NI a NVI). En la muda de nauplio VI a copépodito I se

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producen importantes cambios morfológicos. En el copepodito I se diferencian el prosoma y el urosoma y su morfología comienza a asemejarse a la de la fase adulta. A través de las siguientes mudas (CII ‐CV), se incrementa el número de segmentos corporales y apéndices funcionales. Finalmente, después de la quinta muda, se alcanza la fase adulta.

Figura 1. Esquema del ciclo de vida holoplanktónico típico en copépodos de vida libre. Las letras minúsculas indican procesos del ciclo de vida; las letras mayúsculas identifican las principales fases del ciclo de vida; las letras mayúsculas en negrita indican categorías de clasificación de los organismos marinos de acuerdo con su hábitat y la movilidad ("modo de vida").

Ciclo de vida de invertebrados marinos bentónicos: la larva meroplanctónica.

Aproximadamente el 70 ‐80% de los invertebrados marinos bentónicos tienen larvas planctónicas que pasan un cierto tiempo, de minutos a meses, en la columna de agua (Thorson 1946, 1950; Strathmann 1993; Young 2002). Para muchas especies de invertebrados bentónicos, especialmente en especies sedentarias, la dispersión solo tiene lugar durante la fase larvaria de natación libre (Strathmann 1985, 1990; Levin & Bridges 1995). Comúnmente, los ciclos de vida

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que incluyen larvas planctónicas seguido de juveniles y adultos bentónicos se denominan ciclos de vida bifásicos (Figura 2).

Figura 2. Esquema idealizado del ciclo de vida de invertebrados bentónicos con larvas meroplanctónicas. Las letras minúsculas indican procesos en el ciclo de vida, las letras mayúsculas identifican las principales fases del ciclo de vida, las letras mayúsculas en negrita indican categorías de clasificación de los organismos marinos de acuerdo con su hábitat y la movilidad ("modo de vida").

Los adultos bentónicos sexualmente maduros liberaran los gametos en la columna de agua donde tiene lugar la fertilización y el desarrollo embrionario y larvario. La duración del período de dispersión en la columna de agua depende de la especie. Las larvas lecitotróficas tienen una vida pelágica corta y no se dispersan a largas distancias, mientras que las larvas planctotróficas generalmente tienen una vida planctónica relativamente larga (Thorson 1950; Jagersten 1972). La dispersión larvaria viene acompaña del crecimiento y el desarrollo de una o más fases larvarias. Las larvas planctotróficas presenta características morfológicas y de comportamiento que representan adaptaciones a un estilo de vida planctónico y les permiten la explotación de las fuentes de alimento planctónicas (Strathmann 1993; Peterson et al. 1997). El final del periodo larvario ocurre cuando el organismo se encuentra fisiológicamente competente

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para asentarse y llevar una existencia bentónica. La metamorfosis a la forma bentónica se produce una vez que las larvas han aceptado el lugar de asentamiento. El ciclo de vida se completa cuando los invertebrados marinos bentónicos pasar a través de las etapas juveniles y alcazan la madurez sexual en la fase adulta.

A continuación se describen brevemente los tipos de larvas meroplanctónicas de los grupos de invertebrados bentónicos con los que se ha trabajado en esta tesis: poliquetos (spiónidos y serpúlidos), bivalvos, gasterópodos, cirrípedos y equinoideos. En poliquetos con desarrollo indirecto, se distinguen principalmente dos fases larvarias: trocóforas y larvas segmentadas (las larvas con pocos segmentos se conocen generalmente como metatrocóforas) (Pernet et al. 2002). Los segmentos que portan quetas (setas) se llaman setígeros y frecuentemente el número setígeros se utiliza para indicar la fase de desarrollo larvario (Pernet et al. 2002). En espiónidos, la embriogénesis y el desarrollo temprano de las larvas suelen tener lugar en cápsulas de huevos, y la primera fase de desarrollo que se libera al plancton es una larva con 3 setígeros (Daro & Polk 1973; Blake & Arnofsky 1999). Por el contrario, los poliquetos serpúlidos suelen liberar los gametos en la columna de agua y todo el desarrollo embrionario y larval tiene lugar en el plancton (Pernet et al. 2002). Los gasterópodos marinos tiene dos tipos principales de larvas: trocóforas y velígeras (Barnes 1982; Strathmann 1987). Las trocóforas puede ser de natación libre o, comúnmente, mantenerse dentro de cápsulas de huevos mientras se desarrollan a larvas velígeras, las cuales son liberadas al plancton (Goddard 2001; Buckland ‐ Nicks et al. 2002). Las larvas velígeras son exclusivas de gasterópodos y bivalvos marinos y presentan una gran diversidad morfológica (Buckland ‐Nicks et al. 2002). Las velígeras de gasterópodos se caracterizan por poseer una concha (protoconcha) y un velo bilobulado: un órgano ciliado que es utilizado para la alimentación, la locomoción y la respiración (Fretter 1967; Goddard 2001, Buckland ‐Nicks et al. 2002). En bivalvos, aunque en algunas especies las trocóforas pueden ser incubadas en la cavidad del manto, es frecuente que tanto las trocóforas como las veligeras sean de natación libre (Barnes 1982; Brink 2001; Zardus & Martel 2002). Las velígeras de bivalvos se caracterizan por una concha con charnela (prodisoconcha) y un velo ovalado o redondeado (Waller 1981; Brink 2001).

Las larvas planctónicas son etapas del ciclo de vida de los invertebrados marinos particularmente vulnerables, ya que la mortalidad larvaria a menudo supera el 90% (Rumrill 1990). Sin embargo, una fase de dispersión larvaria es ventajosa para animales que son sésiles como adultos. Las larvas planctónicas proporcionan un medio colonización de nuevos hábitats,

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promueven el flujo de información genética entre poblaciones, y permiten el establecimiento en nuevos sitios de colonización tras alteraciones ambientales, lo que reduce el riesgo de extinciones locales (Roughgarden et al. 1998; Hansen et al. 2002; Petersen et al. 2002; Eckert 2003). Por lo tanto, el conocimiento de la ecología larvaria es esencial para entender la dinámica de las poblaciones y comunidades de los invertebrados marinos bentónicos.

Introducción a la ecofisiología de invertebrados marinos larvas planctónicas

La ecofisiología o fisiología ambiental es la disciplina de la biología que estudia la influencia de factores ambientales en los procesos o funciones de un organismo o de cualquiera de sus partes. La fisiología y la bioenergética están estrechamente relacionadas con la eficiencia biológica de las especies y su éxito evolutivo. Dado el papel crucial del reclutamiento larvario en la estabilidad de las poblaciones y comunidades, la comprensión de la ecofisiología larvaria es esencial para entender la dinámica biológica de los invertebrados marinos. Además, en el contexto actual de rápidos cambios ambientales inducidos antropogénicamente (Hoegh ‐ Guldberg & Bruno 2010), el estudio de los efectos de los factores ambientales (temperatura, disponibilidad de alimentos) sobre la fisiología de la larvas es de especial relevancia para predecir el impacto del cambio climático sobre los invertebrados marinos.

Numerosos estudios de campo y de laboratorio han demostrado que la temperatura del agua y la concentración de alimento son factores ambientales decisivos en la supervivencia, el desarrollo, el crecimiento, la alimentación y el metabolismo de copépodos (Huntley & Boyd, 1984; Huntley & López 1992; Hirst & Lampitt 1998; Hirst & Kiørboe 2002). Sin embargo, la mayoría estos estudios se han centrado en adultos o fases tardías de copepoditos de calanoides (Ikeda et al. 2001; Hirst & Bunker 2003), mientras que las fases naupliares han recibido menos atención, particularmente los nauplios de los copépodos pequeños, como los pertenecientes al género de ciclopoides Oithona . Durante la última década, Oithona ha recibido una atención especial debido a su gran abundancia y ubicuidad, y probablemente se trate del género de copépodos más abundante del mundo (Galliene & Robins 2001). Sin embrago, a pesar de la posible importancia ecológica de Oithona , se sabe muy poco acerca de la fisiología de sus fases larvarias, lo cual puede ser crucial para entender su éxito evolutivo.

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Uno de los objetivos de esta tesis fue evaluar la influencia de factores intrínsecos (fase de desarrollo, peso corporal) y ambientales (temperatura, alimento), en diferentes procesos fisiológicos (crecimiento, desarrollo, alimentación, respiración y excreción) en fases larvarias de Oithona davisae (artículos IV, V y VI ).

La fisiología de las larvas ha sido ampliamente estudiado en algunas especies de invertebrados bentónicos, especialmente los de interés en acuicultura (por ejemplo, Mytilus edulis ‐Widdows 1991; Sprung 1984a, 1984b, 1984c). Sin embargo, poco se sabe sobre la ecofisiología de algunos invertebrados de importancia ecológica, por ejemplo, muchas especies de poliquetos. Los estudios de laboratorio y de campo han demostrado que tanto la época de reproducción y como la distribución geográfica coinciden a menudo con la tolerancia de las embriones y larvas de una especie a los factores ambientales tales como la temperatura (Orton 1920; Thorson 1950; Kinne 1970). Los cambios de temperatura pueden tener consecuencias importantes para la supervivencia y dispersión de las larvas a través del retraso o el incremento de las tasas de desarrollo y crecimiento (Thorson 1950; Costlow et al. 1966, Hoegh ‐Guldberg & Pearse 1995). La cantidad y calidad de alimento son factores clave en la supervivencia, desarrollo, y crecimiento de las larvas planctotróficas (Paulay et al. 1985; Pechenik 1987; Rumrill 1990; Basch 1996). El cambio climático ha provocado una reducción global en la producción de fitoplancton marino (Behrenfeld et al. 2006) así como cambios en la estructura y la fenología de las comunidades planctónicas (Edwards & Richardson 2004). Por ejemplo, la actual tendencia de aumento de temperatura está produciendo el adelanto de los eventos de desove de ciertas especies, pero no así la proliferación de fitoplancton primaveral, dando lugar a un desajuste temporal entre la producción de larvas y la disponibilidad de alimento (Edwards & Richardson 2004; Hoegh ‐Guldberg & Bruno 2010). La limitación de alimento durante el desarrollo larvario puede conllevar de forma directa a la mortalidad por inanición o, indirectamente, producir una reducción en las tasas de crecimiento, prolongando el período planctónico y aumentando la exposición de las larvas a fuentes adicionales de mortalidad, tales como la depredación (Thorson 1950). Son muchas las especies de larvas meroplanctónicas en las que se ha demostrado un alto grado de limitación por alimento durante el desarrollo (Olson & Olson 1989; Yu, 2009), por lo que resulta de especial interés entender las bases fisiológicas y el significado ecológico de la limitación por alimento en el desarrollo larvario de las invertebrados bentónicos.

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Estudiamos la influencia de alimento en el comportamiento natatorio y la fisiología de las larvas meroplanctónicas del poliqueto Polydora ciliata (artículos VII y VIII ). Se evaluó el grado de limitación por alimento en el crecimiento larvario de P. ciliata en un estuario eutrófico danés, Isefjord (artículo IX )

La estimación de las tasas de ingestión, crecimiento, respiración y egestión nos permite determinar los balances de energía o carbono y la eficiencia en la transformación de energía de los animales. El modelo básico de balance de energía puede expresarse como: I = G + M + E donde I, G, M y E son las tasas de ingestión, crecimiento, metabolismo (respiración), y egestión, respectivamente, expresadas en unidades de energía o carbono.

Desde una perspectiva económica, la dinámica de las poblaciones larvarias afecta al reclutamiento de especies que son recursos explotables de gran importancia, o bien forman parte de organismos incrustantes (fouling). La abundancia de las larvas de copépodos contribuye de manera decisiva al reclutamiento de las poblaciones de especies de peces de gran interés comercial (Castonguay et al. 2008). Además, los nauplios de copépodos son una fuente de alimento preferente y altamente nutritiva para el criado de larvas de peces y camarones en explotaciones acuícolas (Hernández ‐Molejón & Álvarez ‐Lajonchere 2003). Por lo tanto, el estudio de la fisiología y el balance de carbono de las fases larvarias bajo diferentes condiciones ambientales no sólo es relevante en disciplinas científicas básicas, tales como la ecología, sino que también es importante en investigaciones aplicadas como por ejemplo en el campo de la acuicultura y las pesquerías.

Se estimaron las eficiencias de crecimiento neto y el balance de carbono en nauplios de Oithona davisae (artículo VI ), así como el rango óptimo de temperatura y de concentración de alimento para la producción de nauplios de O. davisae en laboratorio (artículos IV y V)

Las redes tróficas planctónicas marinas y el papel de las larvas

Por definición, el "plancton" comprende a todos los organismos suspendidos en la columna de agua, cuya movilidad no pueden contrarrestar la hidrodinámica marina (Hensen 1887). Estos organismos son muy diversos en términos de la taxonomía, tamaño y tipo de alimentación (Sieburth et al. 1978, Fig. 3) Todos los componentes planctónicos están

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relacionados entre sí a través de interacciones predador ‐presa influenciadas por preferencias ecológicas y efectos top ‐down y bottom ‐up (Zöllner et al. 2009). El zooplancton incluye tanto protozoos (protozooplancton, eucariotas unicelulares heterotróficos) como metazoos (metazooplancton, organismos multicelulares heterótrofos) (Fig.3). El metazooplancton ocupa una posición clave en las redes tróficas pelágicas debido a su función en la transferencia de materia y energía a niveles tróficos superiores. Además, el metazooplancton influye directamente en las redes tróficas microbianas depredando sobre los protistas que se encuentran dentro de su espectro de tamaño de presa, por ejemplo nanoflagelados heterotróficos, dinoflagelados y ciliados (Stoecker & Capuzzo 1990; Gifford & Dagg 1991), e indirectamente a través de efectos de cascada trófica y la regeneración de nutrientes (Calbet & Landry 1999).

Femto − Pico − Nano − Micro − Meso − Macro − Mega − PLANCTON 0.02 −02 0.2 −2 2−20 20 −200 0.2 −20 2−20 20 −200 µm µm µm µm mm cm cm Virio − plancton

Bacterio − plancton

Mico − plancton

Fito − plancton

Protozoo − plancton

Metazoo − plancton

Figure 3. Distribución de los diferentes componentes del plancton en función de su tamaño (Sierburth et al. 1978)

Tradicionalmente, entre las diferentes categorías de tamaño del metazooplancton (Fig. 3), el metazoo microplanctónico (20 ‐200 µm) ha sido muestreado incorrectamente debido al uso de redes de plancton con luz de malla ≥ 200 µm (Calbet et al. 2001, Galliene & Robins 2001; Turner 2004).

La composición, abundancia y biomasa del microplancton metazoo fueron estimadas en distintos sistemas marinos, en aguas costeras del NW Mediterráneo a lo largo de un ciclo anual (artículo II ) y en la costa oeste de la isla de Vancouver (costa del Pacifico canadiense) durante el verano (artículo III ).

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El microplancton metazoo se compone principalmente de larvas planctónicas tales como nauplios de copépodos, copepoditos y larvas meroplanctónicas. La falta de conocimiento sobre microplancton metazoo contrasta con su importancia en términos de abundancia, biomasa y la productividad en ambientes marinos (Hopcroft et al. 2001; Turner 2004; Zervoudaki et al. 2007). Las larvas de copépodos son numéricamente el componente mayoritario del metazooplancton. Además, los nauplios de copépodos son la principal presa de un gran número de larvas de peces (Last 1980; Conway et al. 1991, 1998) y, como se mencionó anteriormente, su abundancia puede determinar el reclutamiento de muchas especies de peces de gran valor comercial (Castonguay et al. 2008). Por lo tanto, la inconsistencia en la cuantificación y la función de los metazoos microplanctónicos supone un importante sesgo en las estimaciones de abundancia, biomasa y producción del zooplancton, del impacto sobre los productores primarios, flujos de materia y energía mediados por el zooplancton y, en general de su importancia en las redes tróficas planctónicas marinas (Turner 2004).

El papel trófico, las tasas de crecimiento y de respiración, y las eficiencias de crecimiento de la comunidad de microplancton metazoo se determinaron en aguas costeras del noroeste del Mediterráneo a lo largo de un ciclo anual (artículo II ).

Además de por su pequeño tamaño, el papel de las larvas meroplanctónicas en las redes tróficas marinas ha sido ignorado en gran medida a causa de su temporalidad en el plancton. Sin embargo, la mayoría de las larvas meroplanctónicas son planctotróficas y su alimentación depende de la comunidad de plancton existente. De hecho, la liberación de larvas coincide a menudo con las proliferaciones de fitoplancton con la finalidad de maximizar la exposición de las larvas a una disponibilidad alta de alimento (Thorson 1946, 1950; Starr et al. 1990, 1991, 1994). A menudo, esta sincronía conlleva a que ciertas larvas meroplanctónicas lleguen a ser los miembros dominantes del zooplancton costero durante la época reproductiva de invertebrados bentónicos (Thorson 1946; Williams & Collins 1986; Andreu & Duarte 1996). Sin embargo, la posible función trófica de las larvas planctónicas en los flujos de carbono marinos sigue siendo en gran parte desconocida.

Se determinó la abundancia, las tasas de alimentación, la selección de presas y el impacto trófico de diferentes larvas meroplanctónicas bajo condiciones de proliferación de fitoplancton acontecidas en la costa oeste de la isla de Vancouver (artículo III )

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Por lo tanto, información relativa a la biomasa y el papel trófico de las larvas planctónicas es crucial para una mayor comprensión las redes tróficas y los flujos biogénicos en los ecosistemas pelágicos marinos.

HIPÓTESIS Y OBJETIVOS

El principal objetivo de esta tesis fue mejorar nuestro conocimiento sobre la ecología y la fisiología de larvas planctónicas de invertebrados marinos, incluyendo su papel en las redes tróficas planctónicas. Con esta finalidad, se realizaron experimentos de campo y de laboratorio con larvas holo ‐ y meroplanctónicas de invertebrados marinos de alto valor ecológico, con una aproximación experimental a nivel de especie y comunidad.

La hipótesis general de la tesis fue que las larvas planctónicas de invertebrados juegan un papel clave en el funcionamiento de los ecosistemas marinos . Concretamente, se formularon las siguientes hipótesis específicas:

1. Las larvas planctónicas (larvas holo ‐ y meroplanctónicas), incluyendo las que pertenecen a fracciones de pequeño tamaño (< 200 µm, microplancton metazoo), ejercen un importante impacto trófico sobre las redes tróficas planctónicas marinas, que puede influir notablemente en la dinámica del fitoplancton y de los protozoos en aguas costeras.

2. El éxito ecológico de muchas especies de invertebrados marinos se debe en parte a las características fisiológicas de las fases larvarias planctónicas.

Para comprobar la hipótesis 1 se marcaron los siguientes objetivos:

1. Determinar la abundancia estacional y la distribución vertical de toda la comunidad de zooplancton en un área costera del noroeste mediterráneo a lo largo de un ciclo anual, con especial interés en larvas planctónicas incluidas las pertenecientes al microplancton metazoo (articulo I)

2. Estimar las tasas de alimentación, el impacto trófico y las eficiencias de crecimiento de la comunidad de microplancton metazoo a lo largo de un ciclo estacional en aguas del noroeste mediterráneo (articulo II )

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3. Determinar la abundancia, las tasas de ingestión, la selección de presas y el impacto trófico de diferentes larvas meroplanctónicas sobre la proliferación fitoplanctónica nociva de Heterosigma akashiwo y Prorocentrum triestinum, que tuvo lugar en la costa oeste de la Isla de Vancouver en Julio del 2006 (artículo III ).

Para contrastar la hipótesis 2 se marcaron los siguientes objetivos:

4. Determinación de diferentes procesos fisiológicos en larvas tempranas de Oithona davisae (nauplii y copepoditos tempranos) en función de factores intrínsecos (fase de desarrollo, peso corporal) y ambientales (temperatura, alimento) tales como: la supervivencia, las tasas de desarrollo y crecimiento (artículo IV ), las tasas de alimentación (artículo V) y las tasas de respiración y excreción (artículo VI)

5. Evaluación del efecto del alimento sobre el comportamiento natatorio (artículo VII) , la alimentación (artículos VII y VIII) y las tasas de crecimiento (artículos VIII y IX) de las fases larvarias planctónicas del poliqueto espiónido Polydora ciliata

RESULTADOS

Los resultados de esta tesis están presentados en 9 artículos científicos/publicaciones (indicados con números romanos) organizados en 5 capítulos principales . A continuación se expone una breve síntesis de los distintos capítulos.

CAPITULO 1: Abundancia estacional y distribución vertical del zooplancton en aguas costeras del noroeste Mediterráneo: importancia de los metazoos planctónicos de pequeño tamaño (artículo I)

Nuestra comprensión sobre la función del zooplancton en los ecosistemas marinos es limitada debido a la escasa información acerca de los metazoos planctónicos pequeño tamaño. A lo largo de un ciclo anual, examinamos la sucesión estacional y la distribución vertical de toda la comunidad de metazooplancton de una zona costera del noroeste del Mediterráneo, con especial interés en las fracciones de tamaño pequeño. El estudio incluyó el uso de dos métodos de muestreo para zooplancton, red de microplacton y botellas Van Dorn) para cubrir adecuadamente las principales fracciones de tamaño del metazooplancton, de 50 a 200 µm

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(microplancton metazoo) y de 200 a 2000 µm (mesozooplancton). También evaluamos la influencia de los principales factores abióticos y biológicos sobre la estructura estacional y vertical de la comunidad de metazooplancton. Los nauplios de copépodos y copepoditos fueron numéricamente dominantes durante la mayor parte del período de estudio, con densidades que oscilaron aproximadamente entre 620 a 23900 ind. m3 y de 265 a 10.000 ind. m3, respectivamente. En general, los patrones de distribución vertical del microplancton metazoo y del mesozooplancton fueron similares. Los gradientes verticales de abundancia y biomasa de metazooplancton fueron más importantes en verano y otoño, y tendieron a seguir los de sus presas microplanctónicas potenciales (ciliados, dinoflagelados, diatomeas). La abundancia del metazoo microplanctónico y del mesozooplancton parece estar relacionada directamente con la temperatura. Los valores mínimos de abundancia de copépodos y sus larvas (nauplios y copepoditos) se observaron durante la proliferación fitoplanctónica primaveral. Según las estimaciones a partir de las muestras de la red de microplancton y las botellas Van Dorn, las fases larvarias planctónicas (incluyendo larvas holo ‐ y meroplanctónicas), representaron respectivamente, el 45% (rango 24% ‐75%) y 55% (rango 26% ‐69%) del carbono total del metazooplancton. La fracción de tamaño correspondiente al metazoo microplanctónico representó, en promedio, el 34% (rango 15% ‐54%) del carbono total del metazooplancton cuando se muestreó con botellas Van Dorn. Por lo tanto, aunque el mesozooplancton domina en términos de biomasa, el microplancton metazoo representa una fracción considerable de la biomasa total del metazooplancton. Además, como las tasas fisiológicas de los organismos pequeños son generalmente más altas que los de los organismos más grandes, la exclusión de los metazoos microplanctónicos puede dar lugar a importantes subestimaciones de la producción de secundaria del zooplancton en los ecosistemas marinos.

CAPITULO 2: Función trófica y balance de carbono del metazoo microplanctónico en aguas costeras del noroeste del Mediterráneo (artículo II)

Durante un ciclo estacional se determinaron, mediante incubaciones de laboratorio, las tasas de alimentación, el impacto trófico y las eficiencias de crecimiento de los ensamblajes naturales de metazoos microplanctónicos procedentes de un área costera del noroeste del Mediterráneo. Los micrometazoos, es decir los organismos pluricelulares planctónicos heterotróficos entre 20 y 200 µm, estuvieron constituidos principalmente por fases larvarias de invertebrados. Los nauplios de copépodos y copepoditos dominaron la comunidad, excepto en

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abril cuando las larvas de poliquetos fueron el grupo más abundante. Se analizó la presión por depredación de los micrometazoos sobre clorofila a (Chl a, total y > 10 µm), nanoflagelados heterotróficos, nanoflagelados fototróficos, dinoflagelados, diatomeas y ciliados. Los micrometazoos predaron sobre todos los grupos estudiados, con tasas de ingestión específicas en carbono que variaron entre 0.31 y 1.24 d‐1. Las eficiencias brutas de crecimiento para el conjunto de la comunidad de metazoos microplanctónicos, calculadas como la pendiente de la regresión lineal que relaciona las tasas específicas de crecimiento versus las tasas especificas de ingestión, varió entre 0.27 y 0.39. Las pérdidas respiratorias en carbono de los micrometazoos ‐1 dependieron de la temperatura y variaron entre 0.16 y 0.36 d , con un Q10 = 2. La eficiencia de crecimiento neto promedio, 0.41, fue independiente de la temperatura y la disponibilidad de alimento. Los micrometazoos en conjunto tienen tasas específicas de crecimiento más altas que las del mesozooplancton, pero eficiencias de conversión del alimento similares. El efecto por predación en el stock existente de las diferentes presas fue < 1% d‐1 para Chl a (total y > 10 µm) y < 2.5% d‐1 para las otras presas estudiadas, lo que parece insuficiente para ejercer un control relevante sobre la dinámica del fitoplancton y los protozoos. La inclusión de los micrometazoos no cambia apreciablemente la visión actual sobre el papel de metazooplancton en las redes tróficas marinas en aguas costeras del noroeste Mediterráneo.

CAPÍTULO 3: Tasas de alimentación y abundancia de larvas planctónicas de invertebrados marinos bajo condiciones de proliferación de algas nocivas frente a la Isla de Vancouver (artículo III)

Las interacciones entre el fitoplancton tóxico y sus predadores potenciales son aspectos poco entendidos de la ecología de las proliferaciones de algas nocivas. En este estudio, se determinó las tasas de alimentación, la selección de presas y el impacto trófico de diferentes larvas planctónicas de invertebrados marinos sobre la proliferación natural de Heterosigma akashiwo y Prorocentrum triestinum que tuvo lugar en la costa oeste de la Isla de Vancouver en julio del 2006. Además, se estimó la abundancia, la biomasa y la composición del zooplancton antes y durante la proliferación de algas nocivas. Los experimentos de alimentación se realizaron con larvas de poliqueto (Serpula columbiana ), de equinodermo (Stronglyocentratus purpuratus ) y de cirrípedo (Balanus crenatus ) obtenidas mediante cultivos de laboratorio, y con larvas de bivalvos y gasterópodos colectadas en la zona de estudio mediante arrastres con redes de plancton. Mientras que todas las larvas se alimentaron de H. akashiwo, sólo los nauplios de

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cirrípedo y larvas de equinodermos se alimentaron de P. triestinum. H. akashiwo fue el componente principal en la dieta de todas las larvas (> 64%). Se observó una relación positiva entre la disponibilidad de la presa en los ensamblajes de alimento y su contribución a las dietas de todas las larvas. El impacto trófico potencial de las larvas meroplanctónicas sobre las especies fitoplanctónicas responsables de la proliferación fue bajo (<1,5%). La ingestión de las especies de fitoplancton que formaron la proliferación no tuvo aparentemente ningún efecto adverso sobre los predadores estudiados después de 48 horas de incubación. Por el contrario, la abundancia de larvas planctónicas y otro zooplancton en el campo disminuyeron de manera continua a largo de la progresión de la proliferación, con pérdidas de hasta un 75% en comparación con su abundancia antes de la floración. La presencia de H. akashiwo afectó negativamente a la abundancia de larvas meroplanctónicas, a pesar de la eficiente predación de estas larvas. Por lo tanto, la presión por depredación fue reducida, lo que probablemente contribuyó al crecimiento y la persistencia de la proliferación fitiplanctónica . La reducción en la abundancia de larvas meroplanctónicas y otro zooplancton en relación con la proliferación de H. akashiwo podría tener un impacto potencial sobre el reclutamiento bentónico y las transferencias de energía a niveles tróficos superiores en las redes tróficas marinas .

CAPÍTULO 4: Ecofisiología de las fases de desarrollo larvarias del copépodo Oithona davisae (artículos IV, V, VI).

Entre los copépodos planctónicos marinos, los del género Oithona son probablemente los más abundantes y ubicuos de los océanos del mundo. Sin embargo, nuestro conocimiento acerca de la ecofisiología de Oithona es muy escaso en comparación con el de copépodos calanoideos, en particular para sus fases larvarias (nauplios y copepoditos). Determinamos las tasas de alimentación, crecimiento, respiración y excreción de las larvas de Oithona davisae en relación la temperatura, y la concentración de alimento. Además, se estimaron los coeficientes metabólicos C: N: P, la eficiencia de crecimiento, y el balance de carbono de nauplios de O. davisae. Las tasas de ingestión, el crecimiento y la respiración de los nauplios aumentaron con el incremento de temperatura, con un Q10 de aprox. 2.5 para un rango de temperatura de 16 ‐28 ºC (artículos IV, V, VI ). La supervivencia de los nauplios se redujo aproximadamente un 60% a la concentración de alimento más baja probada en los experimentos (11 µg C L‐1, después de 7 días y a 20 ºC) (artículo IV ). El desarrollo de los nauplios de O. davisae fue equiproporcional pero no isocronal (artículo IV ). Las concentraciones de alimento requeridas para obtener tasas máximas

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de desarrollo y crecimiento a 20 ºC fueron 56 and 87 µg C L‐1, respectivamente (artículo IV ). Las fases larvarias de O. davisae mostraron una respuesta funcional alimentaria de tipo III , con concentraciones umbrales de alimento entre 50 y 75 µg C L‐1, dependiendo de la fase de desarrollo (artículo V). Todos los parámetros de alimentación varían según el peso corporal/edad. La concentración de alimento necesaria para alcanzar las tasas de ingestión máximas varió desde 200 µg C L‐1 en nauplios tempranos a 320 µg C L‐1 en copepoditos (artículo V). La eficiencia bruta de crecimiento varió entre 0.16 y 0.60 en función de la etapa de desarrollo, la disponibilidad de alimento y la temperatura (artículo V) . Las tasas específicas de respiración de nauplios y copepoditos variaron entre 0.11 y 0.55 d‐1 en función de la fase de desarrollo, el peso corporal, la temperatura y la disponibilidad de alimento (artículo VI) . Los ratios de C: N metabólicos fueron mayores de 14, lo que indica un metabolismo basado en lípidos (artículo VI) . Las eficiencias de asimilación y las eficiencias netas de crecimiento variaron entre 65% y 86% y entre 23% y 32%, respectivamente, dependiendo del peso corporal, la fase de desarrollo y la temperatura (artículo

VI) . Los nauplios O. davisae presentan tasas de desarrollo y eficiencias de crecimiento similares a las copépodos calanoides, pero perdidas respiratorias especificas y requerimientos de alimento más bajos. Por lo tanto, los bajos costes metabólicos de Oithona comparados con los de calanoides pueden ser una de la razones su éxito evolutivo en los ecosistemas marinos.

CAPÍTULO 5: Ecofisiología de las larvas planctónicas del poliqueto espiónido Polydora ciliata (artículos VI, VII and VIII)

Polydora ciliata es una de las especies de poliquetos espiónidos más comunes y abundantes en ecosistemas litorales bentónicos del norte de Europa. Sus larvas planctónicas son muy frecuentes en aguas costeras, y en ocasiones puede convertirse en el componente mayoritario del metazooplancton. Estudiamos la influencia del alimento en el comportamiento natatorio, las tasas de alimentación y crecimiento y la eficiencia de crecimiento de larvas de P. ciliata . Además, se evaluó el grado de limitación por alimento de las poblaciones larvarias de P. ciliata en aguas eutróficas de un estuario danés, Isefjord. Los patrones natatorios no cambiaron en relación al tamaño de las larvas ni con la disponibilidad o el tipo de alimento (artículo VI ). El tamaño optimo de presa se incremento durante el desarrollo larvario de 13 µm a 20 ‐50 µm (artículo VI). Los ciliados fueron filtrados más eficientemente que los dinoflagelados en experimentos de alimentación con suspensiones de presas naturales (artículo VI ). Las larvas de P. ciliata mostraron una respuesta funcional tipo II con tasas máximas de ingestión de 0.40 d‐1 y

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0.46 d‐1 alimentándose de Thalassiosira weissflogii y Rhodomonas salina , respectivamente (artículo VII ). Las tasas máximas de crecimiento fueron alcanzadas a concentraciones de alimento que variaron entre 1.4 y 2.5 µg C mL ‐1 dependiendo del tamaño de las larvas (artículo VII ). La eficiencia bruta de crecimiento varió de 0.47 en larvas tempranas a 0.28 en fases larvarias intermedias y tardías (artículo VII ). Las larvas de P. ciliata fueron el componente principal del meroplancton en el área de muestro durante el periodo de estudio (artículo IX ). Las tasas de crecimiento fueron significativamente mayores en larvas incubadas con agua de mar natural enriquecida con fitoplancton de cultivo (R. salina ) que en larvas incubadas en agua de mar natural, en promedio 0.215 d‐1 y 0.107 d‐1, respectivamente (artículo IX ). En condiciones naturales, durante el período de estudio, no se encontraron concentraciones de alimento saturantes (artículo IX ). En conjunto, nuestros resultados muestran que el crecimiento larvario de P. ciliata está limitado por el alimento a pesar de habitar en aguas altamente eutróficas. Desde una perspectiva ecológica, sugerimos que existe una solución de compromiso (compensación) entre la alimentación/cinética del crecimiento y la dispersión de las larvas mero planctónicas. La bajas tasas de crecimiento larvario de P. ciliata , a pesar de habitar en sistemas con alta disponibilidad de alimento, permiten un desarrollo planctónico lo suficientemente largo como para asegurar una dispersión larvaria eficiente.

DISCUSIÓN GENERAL

Los resultados incluidos en estas tesis han sido discutidos en detalle en sus respectivos artículos. El propósito de esta discusión general es relacionar los principales resultados obtenidos en los diferentes estudios y evaluar las hipótesis iniciales.

Importancia de las larvas planctónicas en términos de abundancia y biomasa

Las larvas planctónicas (incluyendo larvas holo ‐ y meroplanctónicas) fueron numéricamente los componentes más importantes de las comunidades de metazooplancton tanto en aguas costeras oligo ‐mesotróficas del Mediterráneo noroccidental a lo largo de todo el año como en aguas eutróficas de la isla de Vancouver durante el verano (artículos I y III ). Los nauplios de copépodos y copepoditos fueron frecuentemente los metazoos más abundantes, aunque ocasionalmente las larvas meroplanctónicas dominaron la comunidad de metazooplancton (artículos I, II y IX ). La contribución de las larvas meroplanctónicas a la

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biomasa total de zooplancton fue a menudo más importante en el estuario eutrófico boreal, Isefjord, que en los sistemas costeros estudiados (artículos I, III y IX ). Las larvas planctónicas representaron una fracción considerable de la biomasa total de carbono de metazooplancton (en promedio apox. 50%, artículos I, III y IX ) y episódicamente puede constituir el componente dominante de las comunidades de metazooplancton (artículos I y IX ). Las tasas específicas de crecimiento de las larvas planctónicas (nauplios de copépodos) fueron superiores a las estimadas comúnmente para el mesozooplancton (copépodos adultos) mediante el método de producción de huevos (Kiørboe y Johansen 1986; Poulet et al 1995) (artículo II ). Por lo tanto, ignorar el crecimiento larvario puede resultar en importante subestimaciones de la producción secundaria en los sistemas marinos planctónicos (Calbet et al. 2000; Rey ‐Rassat et al. 2004)

Tradicionalmente, la abundancia y biomasa de pequeños metazoos planctónicos se ha subestimado debido a la utilización de redes de plancton con luz de malla gruesa (>200 µm) (Banse 1962, Turner 1994). Sin embargo, la importancia de los pequeños metazoos planctónicos (microplancton metazoo) ha sido reconocida cada vez más en la última década gracias al uso de métodos apropiados de muestreo (Calbet et al. 2001; Gallienne & Robins 2001, Turner 2004, Riccardi 2010). En nuestros estudios encontramos que, en aguas costeras, la fracción de pequeño tamaño se compone principalmente de larvas planctónicas, incluyendo nauplios de copépodos, copepoditos y larvas meroplanctónicas (artículos I y III ). En aguas costeras del noroeste Mediterráneo, y dependiendo del método de muestreo (red de plancton o botellas Van Dorn), el microplancton metazoo representó, en promedio durante todo el año, entre el 18% y el 34% de la biomasa total de metazoos (artículo I). La abundancia de pequeños metazoos fue mayor cuando fueron utilizadas botellas Van Dorn, lo que refleja la importancia del uso de métodos apropiados de muestreo para una estimación precisa de la estructura comunidad zooplanctónica (artículo I). En el área de estudio en la Isla de Vancouver, la contribución del microplancton metazoo a la biomasa total de metazooplankton fue aproximadamente 20 ‐25% (artículo III ). Por lo tanto, aunque el mesozooplancton domina en términos de biomasa, el microplancton metazoo representa una fracción considerable de la biomasa total del metazooplancton. Además, como las tasas fisiológicas de los organismos pequeños son generalmente más altas que los de los organismos más grandes, la exclusión de metazoos microplanctónicos puede dar lugar a importantes subestimaciones de la producción de secundaria del zooplancton en los ecosistemas marinos (Gallienne & Robins 2001; Turner 2004).

268 Resumen de la tesis

Papel de las larvas planctónicas de invertebrados en las redes tróficas pelágicas

Aunque el grado en que la depredación por metazooplancton puede regular a los productores primarios sigue siendo un tema de debate, cada vez hay más evidencias de que los metazoos ejercer poca presión sobre las dinámicas del fitoplancton y el zooplancton unicelular (por ejemplo, Broglio et al. 2004; Atienza et al. 2006). Sin embargo, en la mayoría de los estudios sobre depredación del metazooplancton han sido excluidos los metazoos microplanctónicos, y por lo tanto, se subestiman el impacto trófico de los metazoos planctónicos sobre los productores primarios. Además, la mayoría de experimentos de depredación se han centrado en los organismos holoplanctónicos (principalmente copépodos adultos) y el papel trófico del meroplancton ha sido ignorado en gran medida. En esta tesis, la función de las larvas en las redes tróficas planctónicas se evaluó mediante aproximaciones a nivel de especie y de comunidad. En aguas costeras oligo ‐mesotróficas del noroeste del Mediterráneo y a lo largo de un ciclo anual, llevamos a cabo experimentos de depredación con la comunidad de metazoos microplanctónicos (constituida mayoritariamente por larvas microplanctónicas) alimentándose de ensamblajes naturales de alimento (artículo I). En aguas eutróficas de la costa oeste de la isla de Vancouver (artículo III ) y en el estuario boreal altamente eutrófico, Isefjord, en Dinamarca, (artículo VII ), los experimentos de depredación se realizaron con especies determinadas de larvas meroplanctónicas. En conjunto, estos trabajos nos ofrecen una base de conocimiento solida que nos permite evaluar el papel de las larvas en las redes tróficas planctónicas en condiciones ambientales (tróficas) contrastadas. En aguas costeras del noroeste del Mediterráneo, el impacto trófico (medido como el porcentaje de la biomasa stock consumida por día) ejercido por la comunidad de larvas microplanctónicas sobre la comunidad microbiana fue muy bajo, menor de 5% (artículo II ). De acuerdo con los experimentos de estima de la depredación del microzooplancton total (incluidos protozoos y metazoo) mediante la técnica de las diluciones (Landry & Hassett 1982), los cuales se llevaron a cabo simultáneamente a los experimentos de depredación del metazoo microplanctónico, la contribución de los metazoos a la depredación total del microzooplancton sobre el fitoplancton fue muy baja (Calbet et al. 2008; artículo II ). Por lo tanto, los protistas son probablemente la principal fuente de presión de depredación sobre el fitoplancton en aguas costeras del Mediterráneo, al menos durante ciertas épocas del año (Calbet et al. 2008; artículo II ). El bajo impacto trófico de los metazoos microplanctónicos comparado con el de los protozoos puede ser debido tanto a su menor biomasa en el campo (uno o dos órdenes de

269 Resumen de la tesis

magnitud menor en el noroeste de las aguas costeras del Mediterráneo, Artículo II ) como a sus tasas de ingestión específica más bajas (Jacobson & Anderson 1993). Respecto a las larvas meroplanctónicas, las poblaciones de Polydora ciliata tuvieron un efecto trófico muy leve (<1%) sobre los productores primarios en aguas altamente eutróficas (artículo IX ). Del mismo modo, si consideramos las tasas de ingestión especificas de las larvas Polydora ciliata sobre suspensiones de alimento natural (artículo VII ) y su biomasa en carbono en la naturaleza (artículo IX ), su impacto trófico potencial sobre dinoflagelados y ciliados es muy bajo (<1%).La presión por depredación de las larvas meroplanctónicas sobre la comunidad microbiana no influyó en la proliferacion fitoplanctónica de Heterosigma akashiwo y Prorocentrum triestinum en aguas costeras de la Isla de Vancouver (impacto trófico <2%) (artículo III ). El efecto negativo de Heterosigma akashiwo sobre la abundancia de larvas meroplanctónicas (así como sobre otro zooplancton) contribuiría a reducir la presión trófica sobre la proliferación de fitoplancton nocivo (artículo III ). Nuestros resultados sugieren que, incluso incluyendo la comunidad de metazoos microplanctónicas y las larvas meroplanctónicas, la función trófica de metazooplancton en las redes tróficas planctónicas parece ser, en general, insuficiente para controlar la dinámica de las poblaciones del fitoplancton y los protozoos.

Hipótesis 1: NO ES VALIDA

Las larvas planctónicas (larvas holo ‐ y meroplanctónicas), incluyendo las que pertenecen a fracciones de pequeño tamaño (< 200 µm, microplancton metazoo), ejercen un importante impacto trófico sobre las redes tróficas planctónicas marinas, que puede influir notablemente en la dinámica del fitoplancton y de los protozoos en aguas costeras.

Ecofisiología de las fases larvarias de desarrollo del copépodo Oithona davisae Los pequeños copépodos planctónicos del género Oithona se encuentra entre los metazoos más ubicuos y abundantes de los océanos del mundo, desde latitudes polares a tropicales (Gallienne & Robins 2001). El éxito de una especie dada está determinado por su éxito en el reclutamiento y, por lo tanto, el conocimiento de la fisiología de las fases larvarias es esencial para entender la eficacia biológica de una especie.

270 Resumen de la tesis

Los procesos fisiológicos en las larvas están regulados por factores ambientales y endógenos/intrínsecos. Al igual que en otro zooplancton (Vidal 1980; Vidal & Whitledge 1982; Ikeda 1985; Hansen et al. 1997; Gillooly 2000; Ikeda et al. 2001), en nuestro estudios encontramos que el tamaño corporal (o peso corporal) y la fase de desarrollo son factores intrínsecos que influyen de manera importante en las tasas de ingestión, crecimiento y respiración en las larvas de Oithona davisae (artículos V y VI ). Bajo condiciones similares de temperatura y alimento, las tasas fisiológicas de las larvas de O. davisae mostraron relaciones alométricas respecto al peso corporal (artículos V y VI ). La temperatura y la disponibilidad de alimento son dos de los principales factores ambientales que afectan a la distribución y la fisiología del zooplancton (Huntley & Boyd 1984, Huntley & López 1992, Gillooly 2000, Ikeda et al. 2001). Los diferentes tasas fisiológicas estudiados en las larvas de O. davisae (crecimiento, ingestión, respiración, excreción) siguieron modelos exponenciales en relación a la temperatura entre 20 y 28 ºC (artículos IV, V y VI ). Por el contrario, todas las tasas fisiológicas fueron muy bajas a temperaturas ≤ 16 ºC, lo que da lugar a desviaciones del modelo exponencial (artículos IV, V y VI ). Estos resultados probablemente reflejan el carácter termófilo de esta especie (Uye & Sano 1995); O. davisae es muy escasa durante el invierno y la primavera (cuando las temperaturas son <20 ºC), mientras que es muy abundante durante las estaciones más cálidas, cuando la temperatura del agua se encuentran entre 20 y 28 ºC. Por lo tanto, podemos concluir que 20 ‐28 ºC es probable el rango de temperatura óptimo para O. davisae . Entre 16 ‐28 ºC, los valores de Q10 para las tasas de crecimiento (artículo IV ) ingestión (artículo V) y respiración

(artículo VI ) fueron todas muy similares (~Q 10 = 2.4), lo que sugiere que todos estos procesos fisiológicos muestras una dependencia similar respecto a la temperatura. Los valores de la Q10 obtenidos en nuestro estudio (artículos IV , V y VI ) se encuentran dentro del rango de valores que se observa normalmente en copépodos (Ikeda et al. 2001; Castellani et al. 2005).

Oithona es capaz de crecer bien tanto en ambientes oligotróficos oceánicos como en aguas costeras eutróficos cuando el alimento llega a ser recurso limitado para los copépodos calanoideos (Calbet & Agustí 1999). Las estrategias vida de copépodos están adaptadas a las fluctuaciones en la disponibilidad de alimento, por ejemplo, muchas especies de calanoideos producen huevos de resistencia que se acumulan en los sedimentos y eclosiona justo antes de la proliferación fitoplanctónica primaveral (Marcus 1996; Peterson 1998). Por el contrario, Oithona no producen huevos de resistencia y debe adaptarse a tolerar períodos de baja disponibilidad de alimento y mantener sus poblaciones durante todo el año. En nuestros estudios, hemos identificado varias características fisiológicas en las larvas de O. davisae que

271 Resumen de la tesis

sugieren ventajas competitivas sobre los copépodos calanoideos cuando y donde la comida es escasa, tales como: ‐ Las concentraciones de alimento que inducen una mortalidad relevante en nauplios de O. davisae fueron inferiores a las concentraciones comúnmente observadas en nauplios calanoideos (artículo IV ). Además, los nauplios de O. davisae puede soportar períodos relativamente largos (5 días) en condiciones alimento pobres sin una mortalidad significativa (artículo IV ) ‐ Los nauplios de O. davisae mostraron tasas de desarrollo similares a las observadas para nauplios de calanoideos, pero requerimientos de alimento más bajos (artículo IV ). ‐ El desarrollo naupliar en O. davisae es menos sensible a concentración de alimento que el crecimiento, lo que sugiere que el desarrollo y por consiguiente el reclutamiento puede estar priorizado frente al crecimiento somático (artículo IV ). ‐ Los nauplios y copepoditos de Oithona davisae mostraron una respuesta funcional alimentaria de tipo III (artículo VI ). Este tipo de respuesta funcional, que también se ha observado en algunos copépodos calanoideos, se caracteriza por la presencia de un "umbral de baja alimentación ", es decir una concentración de alimento por debajo de la cual el copépodo deja de alimentarse o reduce sus tasas de aclaramiento (artículo VI ). La presencia de estos umbrales puede ser interpretado como una adaptación para conservar energía en concentraciones de alimentos bajas debido a que el coste energético de la adquisición de alimentos no sería compensado por la ganancia energética (artículo VI ). ‐ Las concentraciones de alimento saturantes para larvas de O. davisae son inferiores a que se han descrito para fases larvarias de calanoideos estadios (artículo VI ). Esto sugiere que en los sistemas naturales las tasas de ingestión de las larvas calanoideos pueden estar limitadas por alimento en un mayor grado que las de las larvas de Oithona (artículo VI ). ‐ Las larvas O. davisae mostraron eficiencias de crecimiento bruto, eficiencias de asimilación y las eficiencias de crecimiento neto similares a las de copépodos calanoideos pero tasas específicas de ingestión y pérdidas respiratorias especificas más bajas (artículos V y VI ).

En conjunto nuestros resultados confirman que los requerimientos de alimento y las pérdidas respiratorias especificas en nauplios de O. davisae son inferiores a las de copépodos calanoideos (Lampitt & Gamble 1982, Paffenhöfer 1993); estas diferencias fisiológicas puede explicar en parte el éxito ecológico de Oithona en los ambientes marinos. Las diferencias en los requerimientos de alimento y las pérdidas metabólicas de carbono entre calanoides y oitónidos probablemente están relacionadas con las diferencias en la natación y la conducta alimentaria

272 Resumen de la tesis

entre estos grupos de copépodos. En contraste con la mayoría calanoides (Titelman & Kiorboe 2003; Henriksen et al. 2007), los oitonidos (incluyendo nauplios, copepoditos y adultos) se mueven con saltos ocasionales y usan señales hidromecánicas para la detección de presas (Paffenhöfer 1993; Svensen & Kiørboe 2000; Paffenhofer & Mazzocchi 2002). A modo de ejemplo, en ausencia de presas, los nauplios de Oithona davisae pasan el 98% de su tiempo inmóviles y sólo ante la presencia de presas móviles aumentan su frecuencia de saltos (Henriksen et al. 2007). La estrategia de alimentación y la natación de Oithona puede considerarse energéticamente más eficiente que la de la mayoría calanoides (Paffenhöfer 1993), lo q contribuye a explicar la amplia distribución y éxito ecológico de Oithona en los ecosistemas marinos.

Bases fisiológicas y significado ecológico de la limitación por alimento del crecimiento larvario en larvas meroplanctónicas: el caso de Polydora ciliata

La limitación por alimento del crecimiento, es decir, que las concentraciones de alimento en la naturaleza están por debajo de las necesarias para proporcionar tasas máximas de crecimiento, parece ser una regla general para el zooplancton marino (Huntley & Boyd, 1984; Lampert 1985; Hirst & Bunker 2003). Sin embrago, nuestro resultados sugieren que el crecimiento de las larvas meroplanctonicas podría encontrarse limitado por el alimento en un mayor grado que el de las larvas holoplanctónicas (por ejemplo, los nauplios de copépodos).

Las tasas de crecimiento larvario de Polidora ciliata estuvieron limitadas por alimento a pesar de esta especie habita en aguas altamente eutróficas. Esta conclusión se apoya en dos aproximaciones experimentales: - Los estudios de laboratorio mostraron que las tasas de crecimiento máximo, con un tamaño óptimo de los alimento, se alcanzaron a concentraciones que variaron entre 1410 y 2510 µg C L‐1 dependiendo del tamaño de las larvas (artículo VIII ). En el área de estudio, la disponibilidad de alimento fue insuficiente para proporcionar las tasas máximas de crecimiento larvario observadas en el laboratorio (artículos VII y IX ). - Las tasas específicas de crecimiento de las larvas criadas en suspensiones de alimento naturales fueron inferiores a las tasas de crecimiento de larvas alimentadas con suspensiones de alimento natural enriquecidas, lo que indica que las larvas en el medio natural no puede crecer a su máximo potencial (artículo IX ).

273 Resumen de la tesis

Las tasas máximas de crecimiento de las larvas de Polydora ciliata y Oithona davisae de un tamaño parecido fueron bastante similares bajo condiciones saturantes de alimento (artículo V y VIII ). Sin embargo, los requerimientos de alimento para alcanzar tasas de crecimiento máximas fueron mucho más altos para las larvas de P. ciliata que para las de O. davisae . Resultados similares han sido observados en otras larvas meroplanctónicas y en larvas de copépodos (véase la discusión del artículo VIII ). A lo largo de un ciclo anual aguas costeras del noroeste del Mediterráneo, tasas de crecimiento de la comunidad de larvas meroplanctónicas fueron significativamente más bajas que los de nauplios y copepoditos en condiciones similares de disponibilidad de alimento (artículo II ). Por lo tanto, nuestros resultados sugieren que en la naturaleza tasas de crecimiento de las larvas meroplanctónicas podrían ser inferiores a las tasas de crecimiento de las larvas holoplanctónicas (nauplios de copépodos) (artículos II y VIII ). Las diferencias en el grado de limitación por alimento del crecimiento entre larvas mero ‐ y holoplanctónicos puede deberse a diferencias fisiológicas entre estos grupos de organismos planctónicos, como sugieren nuestros resultados: - Las tasas máximas especificas de filtración (aclaramiento) de las larvas de Polydora ciliata (artículo VIII ), así como las de otras larvas meroplanctónicas (Jaspersen & Olsen 1982; Hansen & Ockelmann 1991), fueron inferiores a las de larvas holoplanctonicas tales como nauplios de copépodos (Paffenhöfer 1971; Berggreen et al. 1998; Hansen et al. 1997). Por ejemplo, las tasas máximas especificas de filtración de los nauplios de O. davisae fueron un orden de magnitud superior al de las larvas de P. ciliata (artículos V y VIII ). Esta diferencia sugiere que las larvas de P. ciliata presentan un mecanismo de alimentación menos eficiente que el de nauplios de O. davisae , lo que explica porque son necesarias concentraciones de alimento tan altas (incluso superiores a las que se encuentra en ambientes altamente eutróficos) para permitir que las larvas de P. ciliata puedan alcanzar sus tasas máximas de crecimiento (artículos VIII y IX ). - Las larvas de P. ciliata muestran un comportamiento natatorio similar ante la presencia o ausencia de alimento (artículo VII ). Por el contrario, como se mencionó anteriormente, la actividad natatoria y de alimentación de los nauplios de O. davisae aumenta sólo ante la presencia de presas (Henriksen et al. 2007). Del mismo modo, algunos nauplios de calanoideos aumentar el tiempo de natación a medida que aumenta la concentración de alimento (Van Duren & Videler 1995). Una natación continua se asocia generalmente a costes de energéticos altos y, en consecuencia, a necesidades de alimento altas. Esto sugiere

274 Resumen de la tesis

que el comportamiento poco especializado de las larvas de P. ciliata en relación al régimen alimentario pueden ser energéticamente menos eficiente que el de nauplios de copépodos.

Desde una perspectiva ecológica, la limitación por alimento causa una disminución de las tasas de crecimiento y por lo tanto prolonga el periodo de vida planctónico, con lo que se exponen a la larvas a fuentes adicionales de mortalidad como la predación (Thorson 1950), por otro lado, el aumento en el periodo de vida planctónico facilita la dispersión larvaria. Cuando se requieren bajas concentraciones de alimento para unas tasas de crecimiento máximo, se favorece un desarrollo larvario rápido y en consecuencia el reclutamiento a la fase adulto; esto supone es una ventaja adaptativa para organismos holoplanctónicos como Oithona davisae (artículo IV ). Para copépodos planctónicos, en los que todas las fases del ciclo vital se dispersan en la columna de agua, un periodo de vida larvario más largo no supone, aparentemente, ninguna ventaja selectiva. Sin embargo, para muchos invertebrados bentónicos, especialmente los que son sésiles durante la fase adulta, las larvas actúan como su único mecanismo de dispersión (Levin & Bridges 1995). La dispersión larvaria es crucial para la supervivencia de la especie y su persistencia, ya que promueve la conectividad entre poblaciones favoreciendo el flujo genético (Roughgarden et al. 1988., Palumbi 2003; Trakhtenbrot et al. 2005), facilita la recolonización de los lugares después de alteraciones ambientales (Hansen et al. 2002; Petersen et al. 2002), y contrarresta las fluctuaciones de densidad en poblaciones adultas, lo que reduce el riesgo de extinciones locales (Eckert 2003). Sugerimos que, para muchos invertebrados bentónicos hay un solución de compromiso entre las alimentación/ crecimiento y la dispersión. La selección natural podría favorecer de que algunas larvas meroplanctónicas, tales como P. ciliata , posean tasas de y crecimiento a pesar de que habitan en ambientes con muy alta disponibilidad de alimento (artículo IX ). En este caso, lo que inicialmente puede ser percibido como un rendimiento larvario subóptimo, en realidad puede funcionar para permitir un desarrollo planctónico lo suficientemente prolongado como para asegurar una dispersión larvaria eficiente.

Hipótesis 2: SI ES VALIDA

El éxito ecológico de muchas especies de invertebrados marinos se debe en parte a las características fisiológicas de las fases larvarias planctónicas

275 Resumen de la tesis

CONCLUSIONES PRINCIPALES

I. Las larvas planctónicas de invertebrados, incluyendo tanto larvas holoplanctónicas como meroplanctónicas, representan una fracción considerable de la biomasa total de metazooplancton en las aguas costeras.

II. Los nauplios de copépodos y los copepoditos son numéricamente los componentes dominantes de la comunidad de metazooplancton en aguas costeras del noroeste del Mediterráneo.

III. La abundancia total del microplancton metazoo y del mesozooplancton en aguas costeras del noroeste del Mediterráneo se correlaciona positivamente con la temperatura del agua a lo largo de un ciclo estacional.

IV. El patrón de abundancia estacional y la distribución vertical del microplancton metazoo y del mesozooplancton no están directamente relacionados con la biomasa de fitoplancton (clorofila) en aguas costeras del noroeste del Mediterráneo.

V. La aparición de algunas larvas meroplanctónicas (poliquetos) en aguas costeras está vinculada a las proliferaciones fitoplanctónicas primaverales.

VI. El microplancton metazoo depreda de manera eficiente sobre presas nano ‐ y microplanctónicas (nanoflagelados, diatomeas, dinoflagelados y ciliados).

VII. El impacto trófico de las larvas microplanctónicas (<200 µm, microplancton metazoo) es insuficiente para controlar la dinámica del fitoplancton y protozoos en aguas costeras del noroeste del Mediterráneo.

VIII. La presión depredadora ejercida por las larvas meroplanctónicas tiene un efecto mínimo sobre la proliferaciones nocivas de Heterosigma akashiwo / Prorocentrum triestinum.

IX. Las proliferaciones nocivas de Heterosigma akashiwo / Prorocentrum triestinum afectan negativamente a la abundancia de las larvas planctónicas y el resto de zooplancton.

X. En su conjunto, el microplancton metazoo tiene tasas de crecimiento específico más altas que el mesozooplancton, pero eficiencias de conversión de alimento similares.

276 Resumen de la tesis

XI. La actividad alimentaria aumenta significativamente las tasas de respiración de los nauplios de copépodos, por lo tanto la estimación de tasas de respiración sin alimento conlleva a importantes subestimas de las tasas metabólicas de nauplios de copépodos

XII. Las necesidades de alimentos y las pérdidas respiratorias de carbono de los nauplios de Oithona son más bajos que los de copépodos calanoides; estas diferencias fisiológicas pueden explicar en parte el éxito ecológico de Oithona en ambientes marinos.

XIII. A pesar de habitar en aguas eutróficas, el desarrollo larvario de Polydora ciliata muestra un alto grado de limitación por el alimento en comparación con organismos holoplanctónicos.

XIV. Las características especiales de la ecofisiología de algunas larvas de invertebrados marinos bentónicos, tales como P. ciliata , permiten un desarrollo planctónico prolongado, que garantiza una dispersión larvaria eficiente.

277

Reference list

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294 Report of the thesis directors

Report of the thesis directo rs

Report of the thesis directors

Dr. Miquel Alcaraz and Dr. Albert Calbet , as the co ‐directors of the PhD thesis entitled Ecophysiology of marine invertebrate planktonic larvae: species and community level approach developed by the PhD candidate Rodrigo Almeda, inform of the impact factor of the journals and the implication of the PhD candidate in each scientific article included in thesis.

Article I: Seasonal abundance and vertical distribution of marine zooplankton in NW Mediterranean coastal waters: importance of small planktonic metazoans  by R. Almeda , M. Alcaraz, A. Calbet, E. Saiz, I. Trepat, in preparation to be submitted for publication to Progress in oceanography , with an impact factor of 3.582 (5 years: 4.215 ), and located in the first quartile (rank 3 out of 56 journals) in the category Oceanography. In this article, we examined the succession and vertical distribution of the whole zooplankton community in relation to environmental factors in a coastal area of the northwestern Mediterranean throughout an annual cycle, with special emphasis on metazoan microplankton and planktonic larvae. The sampling program and the variables needed were discussed together with the PhD candidate, the directors and E. Saiz. Rodrigo Almeda carried out the field sampling, preparation, and sample analysis in the laboratory. I. Trepat provided chlorophyll analysis. The PhD student has performed the data analysis and elaborated the MS under the comments and supervision of the co ‐directors and E. Saiz, and the consensus and agreement of the other co ‐authors. The PhD is the first author of this article, and the data included have not been used for the elaboration of any other doctoral thesis.

Article II:
Trophic role and carbon budget of metazoan microplankton in northwest Mediterranean coastal waters by R. Almeda , A. Calbet, M. Alcaraz, E. Saiz, I. Trepat, L. Arín, J. Movilla, and V. Saló, published in Limnology and Oceanography (2011, 56 (1): 415 ‐430), with an impact factor of 3.545 (5 years: 4.099 ), and located in the first quartile (rank 4 out of 56 journals) in the category Oceanography. This article is a successful attempt to fill the gap in the knowledge of the ecological role of metazoan microplankton in NW Mediterranean coastal marine systems. The abundance, biomass, feeding rates, trophic impact and growth efficiencies of the metazoan microplancton

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community were determined along a year cycle. The study demonstrates that micro ‐metazoans graze on all the prey groups and that the grazing impact on the standing stock of the different preys is insufficient to control both phytoplankton and protozoan dynamics in NW Mediterranean coastal waters. The sampling program, the variables needed and the laboratory setup to perform the study were discussed together with the PhD student, the directors and E. Saiz. R. Almeda carried out the sampling, incubation experiments, sample analysis and the estimates of feeding, growth and respirations of metazoan microplancton. I. Trepat and L. Arín provided respectively chlorophyll analysis and taxonomic help for phytoplankton identification, and J Movilla and V. Saló contributed to taking samples and analysis and acquisition of environmental variables. The PhD student has performed the data analysis and elaborated the MS under the comments and supervision of the co ‐directors and E. Saiz, and the consensus and agreement of the other co ‐authors. The PhD is the first author of this article, and the data included have not been used for the elaboration of any other doctoral thesis.

Article III: Abundance and feeding rates of marine invertebrate planktonic larvae under harmful algal bloom conditions off Vancouver Island by R. Almeda, A. Messmer A, N. Sampedro, L. A. Gosselin, published in Harmful Algae (2011, 10: 194 ‐206), with an impact of 2.50 (5 years: 2.572 ), and located in the first quartile (rank 12 of 88) in the category Marine and Freshwater Biology. This study addresses the interaction between toxic phytoplankton and their potential grazers, in this case an almost complete representation of the most important meroplanktonic larvae, including polychaetes, echinoderms, lamellibranch molluscs, gastropods and cirripede nauplii. The study area was a site in the west coast of Vancouver Island (Canada). The presence of toxic phytoplankton species negatively affected the abundance of meroplanktonic larvae, despite their efficient grazing. The reduction in micro ‐ meroplanktonic larvae and other zooplankton abundance associated with the noxious algal blooms may have potential impacts on benthic recruitment and energy transfer to higher trophic levels in marine food webs. R. Almeda discussed the objectives, performed the feeding experiments and data analysis, and elaborated the MS with the comments and agreement of the other co ‐authors (A. Messmer and L.A. Gosselin). A. Sampedro contributed to taxonomic identification and discussion of the results obtained. R. Almeda is the first author, and the data included have not been used for the elaboration of any other doctoral thesis.

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Article IV:
Effect of temperature and food concentration on survival, development and growth rates of naupliar stages of Oithona davisae (Copepoda, Cyclopoida) by Almeda R., Alcaraz M., Calbet A., Yebra L., Saiz E., published in Marine Ecology Progress Series (2010, 410: 97109), with an impact of 2.51 (5 years 3.023 ), and located in the first quartile (rank 8 of 56) in the category Oceanography. The study is a valuable contribution to explain the role played by temperature and food concentration variability on the success of the most abundant and ubiquitous, albeit hardly known, copepod genus Oithona . Almost nothing is known regarding the ecological characteristics of its naupliar stages, as they are systematically lost using traditional sampling methods for zooplankton. In the present laboratory study the survival, development and growth rates of the naupliar stages of Oithona davisae under different temperature regimes and food concentrations are discussed. The conclusion is that their success in the exploitation of almost all marine planktonic environments is partly due to their lower food requirements as compared to calanoid copepods. These differences may help to partially explain the ubiquity of Oithona spp. in the world ocean. The experimental setup to perform the study was discussed together with R. Almeda, the co ‐directors of the thesis and E. Saiz. The PhD student also performed the corresponding incubation experiments, analysed the samples and made the data treatment and elaborated the MS under the comments, discussion and supervision of the co ‐directors and E. Saiz and L. Yebra R. Almeda is the first author, and the data included have not been used for the elaboration of any other doctoral thesis.

Article V: Feeding rates and gross growth efficiencies of larval developmental stages of Oithona davisae (Copepoda, Cyclopoida) by Almeda R., Augustin C.B., Alcaraz M., Calbet A., Saiz E., published in Journal of Experimental Marine Biology and Ecolog y (2010, 387 (1 ‐2): 24 ‐35 ) with an impact of 2.116 (5 years 2.433 ), and located in the first quartile (rank 17 of 88) in the category Marine and Freshwater Biology. Again in this paper the study is focused on the early developmental stages of the marine planktonic copepod Oithona . The idea was to ascertain the reasons for their success in all the marine environments, and therefore to study the feeding rates and gross growth efficiencies for different developmental stages as related to food concentration, body weight and temperature in the laboratory. The success of Oithona nauplii would depend of their lower metabolic losses and consequently lower food requirements as compared to calanoid nauplii. Together with other factors, this feeding/energetic strategy will contribute to the success of

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the Oithona species in marine ecosystems of contrasting trophic characteristics. The experimental setup to perform the study was discussed together with R. Almeda, the co ‐ directors of the Thesis and E. Saiz. The PhD candidate also performed the corresponding incubation experiments, analysed the samples and made the data treatment and elaborated the MS under the comments, discussion and supervision of the co ‐directors and E. Saiz, while C.B Augustin collaborated in the ingestion experiments. R. Almeda is the first author, and the data included have not been used for the elaboration of any other doctoral thesis.

Article VI:
Metabolic rates and energy budget of the early developmental stages of the marine cyclopoid copepod Oithona davisae  by Almeda R., Alcaraz M., Calbet A., Saiz E., published in Limnology and oceanography (2011, 56 (1): 403 ‐414), with an impact factor of 3.545 (5 years: 4.099 ), and located in the first quartile (rank 4 out of 56 journals) in the category Oceanography. This paper closes the study of the ecological role of copepod nauplii and early copepodite stages, the main components of metazoan microplankton, frequently forgotten in zooplankton studies. We determined respiration rates, ammonium and phosphate excretion rates and the net growth efficiencies of early developmental stages of Oithona davisae as related to stage, body weight, temperature and food availability. O. davisae developmental stages exhibited similar assimilation efficiency and growth efficiencies, but lower specific carbon respiratory losses than calanoid copepods. The low metabolic costs of Oithona compared with calanoids may be one reason for their success in marine ecosystems. The experimental approach was discussed together with R. Almeda, the thesis co ‐directors. M. Alcaraz supervised the respiration and excretion measurements. The Ph D student also performed the corresponding incubation experiments, analysed the samples and made the data treatment, conclusions drawn and elaborated the MS under the comments the co ‐directors of the thesis and E. Saiz. R. Almeda is the first author, and the data included have not been used for the elaboration of any other doctoral thesis.

Article VII:
Swimming behavior and prey retention of the polychaete larvae Polydora ciliata (Johnston) by Hansen B. W., Jakobsen H. H., Andersen A., Almeda R., Pedersen T. M., Christensen, A. M., Nilsson B, published in the Journal of Experimental Biology (2010, 213: 3237 ‐3246), with an impact factor of 2.722 (5 years 3.254 ), and located in the first quartile (rank 18 out of 73 journals) in the category Biology.

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This article focuses in the relationships between swimming characteristics of meroplanktonic larvae (the ubiquitous, estuarine planktotrophic spionid polychaete larvae Polydora ciliata ) in relation to its feeding characteristics. The study of the ontogenetic changes in morphology, swimming speed and feeding rates have allowed to develop a simple swimming model using low Reynolds number hydrodynamics. The model predicted swimming speeds and feeding rates that corresponded well with the measured speeds and rates. There is a critical larval length, above which the buoyancy ‐corrected weight of the larva exceeds the propulsion force generated by the ciliary swimming apparatus and thus forces the larva to the bottom. These findings may have general implications for all planktivorous polychaete larvae that feed without trailing threads. Not only do larval morphology and behavior govern larval feeding, but prey behavior also influences the feeding efficiency of Polydora ciliata. The experimental approach was discussed together with R. Almeda, B Hansen and the other co ‐authors. R. Almeda participated in the larval collection and video ‐recording of swimming tracks of Polydora larvae, and conducted the feeding experiments (particle size selection, ontogenic changes in feeding rates, grazing on natural prey) with the collaboration of T.M Peterson. The other co ‐ authors analysed the swimming tracks of larvae, and A. Andersen and HH Jakobsen concentrated in the model. R. Almeda also contributed to data analysis and manuscript elaboration. R. Almeda is the fourth co ‐author, and the data included have not been used for the elaboration of any other doctoral thesis.

Article VIII:
Feeding and growth kinetics of the planktotrophic larvae of the spionid polychaete Polydora ciliata (Johnston) by Almeda R., Pedersen T.M., Jakobsen H.H., Alcaraz M., Calbet A., Hansen B.W., published in the Journal of Experimental Marine Biology and Ecolog y, (2009, 382: 61 ‐68), with an impact factor of 2.166 (5 years 2.433 ), and located in the first quartile (rank 17 out of 88 journals) in the category Marine and Freshwater Biology. In this study the growth rates and gross ‐growth efficiency (GGE) of planktonic larval stages of P. ciliata has been studied in relation to the food abundance and type of prey (the cryptophyte Rhodomonas salina (ESD = 9.7 µm) and the diatom T. weissflogii (ESD = 12.9 µm). The GGE, calculated specifically for each food level, decreased as the food concentration increased. There is a trade ‐off between larval feeding/growth kinetics and larval dispersal. Natural selection may favor that some meroplanktonic larvae exhibit low filtration efficiency and low growth rates despite inhabiting environments with high food availability. This larval performance allows a planktonic development long enough to ensure efficient larval dispersion.

301 Report of the thesis directors

The experimental approach was discussed together with R. Almeda, B Hansen, Jakobsen H.H and the thesis co ‐directors. R. Almeda conducted the field sampling, feeding experiments and estimates of growth rates of Polydora larvae with the collaboration of T.M Peterson. The other co ‐authors contributed to the sampling, maintenance of the experimental organisms and sample analysis. R. Almeda elaborated the MS with the comments and agreement of the other co ‐authors. R. Almeda is the first author, and the data included have not been used for the elaboration of any other doctoral thesis.

Article IX :
Larval growth of the dominant polychaete, Polydora ciliata, is indeed food limited in a eutrophic Danish estuary, Isefjord by Pedersen T.M., Almeda, R., Fotel F.L., Jakobsen H.H., Mariani P., Hansen B.W., published in Marine Ecology Progress Series (2010, 407:99 ‐110), with an impact factor of 2.519 (5 years 3.023 ), and located in the first quartile (rank 8 out of 56 journal) in the category Oceanography. In this article, the food limitation in larval growth of P. ciliata in a eutrophic estuary, Isefjord, Denmark was studied. In the field we determined the larval abundance and the food availability during different periods. In the laboratory, we compared the specific growth rates of larvae reared in natural food suspensions with those of larvae reared on phytoplankton ‐ enriched food suspensions. Specific growth rates of larvae reared on natural food suspensions were always lower than those of larvae reared on phytoplankton ‐enriched food suspensions. For P. ciliata, commonly the dominant meroplanktonic larva, the available in situ food seemed to be sufficient to cover the energetic carbon requirements of the population, but insufficient to attain the maximum specific growth rates reported in previous laboratory experiments. This suggests that P. ciliata larvae probably exhibit a low feeding efficiency and their maximum specific growth rates are consequently attained at food concentrations even higher than those found in this eutrophic environment. The experimental approach was discussed together with R. Almeda, B Hansen and the other co ‐authors. R. Almeda conducted the field sampling, sample analysis, growth experiments and results elaboration together with the other coauthors. R. Almeda also contributed to data conclusion drawn and manuscript writing and revision. R. Almeda is the second co ‐author, and the data included have not been used for the elaboration of any other doctoral thesis.

302 Acknowledgements Agraïments/Agradecimientos

Acknowledgements

Acknowledgements Agraïments/Agradecimientos

Para empezar quiero agradecer a mis directores de tesis, Miquel y Albert, el haberme dado la oportunidad de realizar esta tesis doctoral, poner a mi disposición todas las infraestructuras y el material necesarios para poder llevarla cabo con éxito, y además, permitirme participar en campañas oceanográficas que han sido un sueño hecho realidad. Quiero dar las gracias a Enric, que ha sido casi como un tercer director de tesis, por valorar mi trabajo, por su ayuda y sus ánimos durante estos últimos años. Agradezco especialmente a todos los que han formado parte del grupo del zooplancton y con los que he tenido la oportunidad de trabajar más estrechamente; gracias a Tina, por haber sido tan buena compañera de trabajo, por cuidarme, y por aquellos inolvidables pasteles, a Patri (La Jimenez), por haber traído tanta alegría al grupo y por tantas veces que me has hecho reír, a Lidia por tu compañía en congresos, en el laboratorio, etc, a Dacha, por tu apoyo y consejos, a Sara, Isabel, Juancho, Matina, Meri, Romi, Eva, Águeda, Rodrigo, muchas gracias a todos por haberme hecho el trabajo más fácil y por vuestro compañerismo.

També vull agrair a totes les persones de l'Institut de les Ciències del Mar la seva ajuda durant aquest temps, especialment als companys del departament de biologia marina i oceanografia. Moltes gràcies a Cesc, Anna Sabata, Estela amb els quals vaig donar els meus primers passos en el camp de la microbiologia marina durant el DEA, i que em van ensenyar que aquell munt de punts que es veien en la mostra de dapi eren bacteris i flagel ∙lats, a Jordi Felipe per la seva ajuda amb el citómetro, a Dolors Vaqué, sempre disposada a ajudar tothom, que em va ensenyar a identificar protozous i que em va explicar que allò que semblava un sol era un ciliat Strobilidium , a tot el grup de fitoplàncton, especialment a Laura, Nagore, Albert Reñe, Hassina, amb els quals he compartit molta hores de microscopi durant els primers anys de la tesi i em van ajudar sempre que he necessitat, a Roser Ventosa, per la teva ajuda amb l'anàlisi de nutrients, a Lluïsa, Jose Manuel, Irene, Ted, Marta, Celia, Rafel, Batis, Evarist, Elisa, etc, gràcies a tots de debò, sense dubta he tingut els millors mestres que podria tenir.

305 Acknowledgements

Quiero agradecer especialmente a Julia, Clara, Arancha, Andrés, Bego, Pati, Itziar, Meli, Ana Mari, con los que he pasado muchísimas horas compartiendo despacho y siempre me han escuchado y apoyadocreo que he sido afortunado por tener a esta buena gente a mi lado.Gracias a mis compañeros del ICM, Ero, Silvia, Estela, Imelda, Gemma, Martí, Gisela, Cristina, Juancho, Raquel, Montse, Elisabet, Eva, Clara, Vane, Thomas, Bea, Albert, Ida, Uxue, Andrea, Irene, Claudio, Massimo, Montse, etc a los doctorandos, gracias por compartir esta aventura (peligrosa) que es hacer una tesis doctoral mucho ánimo a todos los que estáis a punto de acabar

Dzi ękuj ę bardzo Julia! desde que llegué a Barcelona y coincidimos en ese tren, nuestros caminos se han cruzado y siempre me has demostrado tu amistad y cariño, muchas gracias, de verdad.

Very special thanks to the colleagues that I meet during my research stays in foreign countries, Benni, Troels, Amber, Louis, Hans, Jenny, etc for their work and motivation and making of these stays an extraordinary experience.

A mis amigos que siempre me han mostrado su apoyo, y con los que he compartido las alegrías y las penas y que a pesar de la distancia siempre me han demostrado su amistad incondicional, especialmente a C. Fabio (el bicho), Fran, Javi, Darío y Virginia. Gràcies Oriol per ser un dels meus millors amics a Barcelona i per tants moments bons. Muchas gracias porque sin vosotros no hubiera llegado hasta aquí!

Por último, quiero dar las gracias a mi familia, especialmente a mi madre, mi abuela, mi hermana y mi sobrino, por todo su cariño y apoyo

Gràcies a tots ¡ Gracias a todos! Thanks to all!

306 Annex Image gallery

Acknowledgements

A B C

D E F

I G H

L JK L M Plate 1 Marine copepods A-F : adult stages; J-K : copepodites A: calanoid copepod ( Acartia ), B: cyclopoid copepod ( Oithona ), C: siphonostomatoid copepod (Caligus ), D: ex-poecilostomatoid (= cyclopoid) copepod ( Oncaea ), E: ex-poecilostomatoid (=cyclopoid) copepod ( Farranula ), F: harpacticoid copepod ( Microsetella ), G: harpacticoid copepod (Euterpina ), H: calanoid copepodite ( Acartia ), I: calanoid copepodite ( Temora ), J: cyclopoid copepodite (Oithona ), K: ex-poecilostomatoid (= cyclopoid) copepodite ( Oncaea ).

309 Acknowledgements

A B C

D E F

G

H I J N

L K L M Plate 2 Marine copepod nauplii A-D : calanoid nauplii (undetermined species), E-F : calanoid nauplii ( Acartia ), G-H : cyclopoid nauplii ( Oithona ), I-J : ex-poecilostomatoid (=cyclopoid) nauplii ( Oncaea ), K-L : harpacticoid nauplii (Microsetella ), M-N : harpacticoid nauplii ( Euterpina ).

310 Acknowledgements

A B

C D

E F G

Plate 3 Oithona davisae (Copepoda, cyclopoida). A-D : adult females, E: egg sac, F-G : adult males

311 Acknowledgements

A B C D

C E F G H

J

I K

Plate 4 Oithona davisae developmetal stages A-H: nauplii; I-K: copepodites . A: newly hatched nauplius. B: naupliar stages ; C: Nauplius I , D: nauplius II, E: nauplius III, F: nauplius IV, G: nauplis V, H: nauplius VI, I: copepodite II, J: copepodite III, K: copepodite V moulting.

312 Acknowledgements

A B C

E F

D G

J

H I

N K

L M O P Plate 5 A-B : Appendicularia , A: Oikopleura , B: Fritillaria. C-F: Rotifera . C: Synchaeta, D: Trichocerca , E: cf. bdelloida, F: Synchaeta. G-K: Cladocera. G: Podon , H-I: Penilia , J-K: Evadne. L: Chaetognatha ( Sagitta ) M: Tunicata (Doliolum ). N-P: Pteropoda ( Clio )

313 Acknowledgements

A B C

D

F G

E

F

I H

K

J L M

Plate 6 Polychaete larvae A-E. larvae of Spionidae, A-C : Polydora ciliata, D: undetermined species; E: Polydora sp . F-J: larvae of Sabellaridae. K: larva of Phyllodocidae; L: larva of Glyceridae, M: larva of Nephtyidae (Images D and E courtesy of L.A. Gosselin)

314 Acknowledgements

Sperm AA B C

Fertilization envelope

Eggs D E F

H I G

J K L Plate 7 Serpula columbiana (Polychaeta, Serpulidae) A-B : adult stages, C: male spawing, D: female spawning, E: fertilized eggs (zygote), F: early embryos, G-H : trochophores, I-J : early segmeted larvae (metatrochopores), K-L : early juveniles. (Images B, G, K and L courtesy of L.A. Gosselin)

315 Acknowledgements

A B

C D

E F G

Plate 8 Echinoderm developmental stages A: larva of Ophiuroidea (Ophiopluteus), B: larva of Echinoidea (late echinopluteus); C: larvae of Echinoidea (early echinopluteus); D: skeleton of Ophiuroidea larva. E-F : early juveniles

316 Acknowledgements

A B C

D E F

G H I

J K L

Plate 9 Strongylocentratus purpuratus (Echinodermata, Echinoidea) A: adult male spawing, B: female spawning, C: eggs, D: early embryos , E: 4-cell embryo: F: blastula, G-H : early echinopluteus, K-L : late echinopluteus (Images K and L courtesy of L.A. Gosselin)

317 Acknowledgements

A B C D

E F G

H I

Plate 10 Crustacean larvae A-E : nauplii; F-G : zoea stages. A-B : decapod nauplii, C: euphausid nauplius, D-E : cirrepede nauplii, F: zoea, G: decapod zoea ( Carcinus ), H: decapod zoea ( Pagurus bernhardus, “hermit crab”), I: decapod zoea.

318 Acknowledgements

A B

C D E

FG H

I J

Plate 11 Balanus creantus (Crustacea, Cirripedia ) A: adult stages attached to the mussel shells, B: nauplius I, C : nauplius II, D: nauplius III, E: nauplius V, F: nauplius VI, G-H : cyprid larvae, I-J : early juveniles

319 Acknowledgements

A B C D

E F G H

I J K

L

M N O P

Q R S

Plate 12 Molluscan larvae A-K : gastropod veliger larvae, I-S : bivalve veliger larvae

320 Acknowledgements

A

B

C

F D E

G H

K

I J L M

Plate 13 A: larva of Phoronida (“actinotrocha larva”), B-D : larvae of platyhelminthes (Müller’s larva), E: polychaete larva; F-H : anthozoan larvae (“actinula”), I: terebellidae polychaete larva (Lanice conchilega ), J: bryozoan larva (“cyphonautes”), K: nemertean larva (“pilidium”), L: enteropneusta larva (“tornaria”), M: Ascidian larva (“tadpole”).

321 Acknowledgements

Back cover: tunicata larva, digital image of Wim van Egmond Courtesy of Michael W. Davidson www.microscopy.fsu.edu

322