Annelida, Clitellata) Using Scanning Electron Microscopy

C. CARAMELO, E. MARTÍNEZ-ANSEMIL

Turk J Zool 2012; 36(1): 1-14 © TÜBİTAK Research Article doi:10.3906/zoo-1002-25

Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy

Carlos CARAMELO, Enrique MARTÍNEZ-ANSEMIL* Departamento de Bioloxía Animal, Bioloxía Vexetal e Ecoloxía, Universidade da Coruña, 15071 A Coruña - SPAIN

Received: 09.02.2010

Abstract: Over the past several years, the authors of this work have investigated external structures of microdrile oligochaetes using scanning electron microscopy (SEM). Both published and still-unpublished data have revealed new structures of interest, especially those associated with mating, ciliate sense receptors, chaetae, and dorsal pores. Mating systems can be classifi ed in 3 categories: grasping, coupling, and embracing. Ciliate sense receptors can be classifi ed as poorly defi ned (unelevated), sensory buds, or papillae, and are provided with blunt cilia and/or sharp cilia. Th ey are present along the whole body (including clitellum, budding, and regeneration zones), scattered on the prostomium, peristomium, and pygidium, arranged in transversal rows and scattered in chaetal segments. Somatic chaetae show great variability when observed by SEM. Hair chaetae are at least potentially provided with denticulations. Dorsal pores were observed in aquatic microdriles. SEM proves to be an invaluable tool to discover important structures associated with functional anatomy in microdriles.

Key words: Functional anatomy, microdrile oligochaetes, morphology, SEM, ciliate sense receptors, mating structures, somatic chaetae, dorsal pores

Introduction A descriptive study dealing with the external Before the year 2000, the observation by scanning structures involved in attachment and sperm electron microscopy (SEM) of external structures transfer in some freshwater microdriles using SEM in microdriles was uncommon. Only a few studies was published by Cuadrado and Martínez-Ansemil have been published, most of them referring to (2001). More recently, Yáñez et al. (2006) and observations on ciliate sense receptors (Chapman, Caramelo and Martínez-Ansemil (2010) contributed 1979; Farnesi et al., 1982a, 1982b; Smith, 1983; to the knowledge of ciliate sense receptors in Römbke and Schmidt, 1999) and somatic chaetae microdriles, providing a signifi cant amount of new (Harman and McMahan, 1975; Milbrink, 1983; data as well as analyses of the typology and patterns Smith, 1985; Chapman and Brinkhurst, 1986; Grimm, of distribution in the main taxonomic groups. 1986, 1987, 1988; Finogenova and Poddubnaja, 1990; Th e aim of the present work was to provide an Römbke and Schmidt, 1990; Ohtaka, 1995, Bouché overview of the functional anatomy of microdrile et al., 1999). oligochaetes as revealed by SEM. New data are

* E-mail: [email protected]

1 Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy

provided mainly on systems related with mating, falciformis, 2 specimens: Porto do Cabo, 02/04/83. somatic chaetae, and dorsal pores. Tubifi cinae: Krenedrilus realis, 1 specimen: Arbón reservoir, Navia river, Asturias, Spain, leg. M. Real, 07/08/88. Limnodrilus udekemianus, 2 specimens: Materials and methods Porto do Cabo, (1 specimen), 07/02/07; Sar, (1 Th e biological material used in the present study specimen), 06/09/07. Potamothrix bavaricus, 2 was examined previously in the studies by Cuadrado specimens: Ebrón stream, Valencia, Spain, leg. S. and Martínez-Ansemil (2001), Yáñez et al. (2006), Pérez, 12/05/96. Potamothrix heuscheri, 1 specimen: and Caramelo and Martínez-Ansemil (2010). Most Ebrón stream, Valencia, Spain, leg. S. Pérez, of the samples were collected from habitats in Galicia 08/02/96. Psammoryctides barbatus, 2 specimens: (NW Iberian Peninsula); detailed information about Ebrón stream, Teruel, Spain, leg. S. Pérez, 16/11/95. each species discussed in this paper is distributed Spirosperma velutinus, 3 specimens: Porto do among the aforementioned articles and the current Cabo, 07/02/07. Phreodrilidae: Insulodrilus sp., one (i.e. when dealing with individuals for which 3 specimens: Knockmoyle, Ireland, leg. Wisdom, new data are provided). All the fi gures in this paper 11/08. Parvidrilidae: Parvidrilus spelaeus, 1 are original. specimen: Pajsarjeva cave, Vhrnika, Slovenia, leg. B. Material studied Sambugar and F. Gasparo, 26/05/97. Lumbriculidae: Th e material studied comprises a total of 36 Lumbriculus variegatus, 5 specimens: Sar, 06/09/07. species belonging to the microdrile families Naididae Stylodrilus heringianus, 4 specimens: Sar, 17/06/08. (9 Naidinae, 2 Pristininae, 5 Rhyacodrilinae, 1 Enchytraeidae: Enchytraeus albidus, 2 specimens: Phallodrilinae, and 9 Tubifi cinae), Phreodrilidae culture original from Bull Island, Dublin, Ireland, (1), Parvidrilidae (1), Lumbriculidae (4), and leg. R. Schmelz and R. Collado, 06/95. Fridericia Enchytraeidae (4), as well as Eiseniella tetraedra monochaeta, 2 specimens: A Zapateira, A Coruña, (Savigny, 1826)—a lumbricid very common in Spain, leg. R. Schmelz, 27/01/09. Lumbricillus freshwaters. sp., 2 specimens: culture original from littoral of Material providing new data North Sea, Tjärnö, Sweden, leg. R. Schmelz and R. Collado, 09/94. Lumbricidae: Eiseniella tetraedra, 2 Most specimens were collected in the 2 following specimens: Porto do Cabo, (1 specimen), 07/02/07; streams of A Coruña (Galicia): Porto do Cabo stream Sar, (1 specimen), 17/07/08. at Moeche and Sar stream at Codesido (Rois). In the list below we will refer to them, respectively, as Porto Methods do Cabo and Sar. Specimens collected in the fi eld were brought Naidinae: Nais alpina, 2 specimens: Porto do alive to the laboratory and identifi ed using a light Cabo, 07/02/07. Nais elinguis, 3 specimens: Sar, microscope equipped with diff erential interference 10/09/03. Ophidonais serpentina, 3 specimens: contrast (mounted in a drop of water). Individuals Sar, 15/05/08. Stylaria lacustris, 3 specimens: Sar, were anesthetized with 7.5% magnesium chloride 02/03/02. Vejdovskyella comata, 3 specimens: added progressively to water or with ethanol and fi xed Sar, 02/03/02. Pristininae: Pristina aequiseta, 6 with 2% glutaraldehyde and 1% paraformaldehyde specimens: Porto do Cabo, 07/02/07. Pristina in 0.1M cacodylate buff er for 2 h. Aft er fi xation, longiseta, 6 specimens: Sar, 12/11/08. Rhyacodrilinae: specimens were washed 3 times in 0.1M cacodylate Bothrioneurum vejdovskyanum, 2 specimens: buff er for 10 min, postfi xed with 1% osmium Sar, 06/09/07. Branchyura sowerbyi, 3 specimens: tetroxide for 2 h in the same buff er, and dehydrated Cazalegas reservoir, Alberche river, Spain, leg. N. through increasing acetone series. All specimens Prat. Peristodrilus montanus, 3 specimens: Porto were mounted on stubs, and sputter-coated with do Cabo, 07/02/07. Protuberodrilus tourenqui, 1 gold. Observations were carried out using a Jeol JSM- specimen: Porto do Cabo, 07/02/07. Rhyacodrilus 6400 SEM at the SAI (Universidade da Coruña).

2 C. CARAMELO, E. MARTÍNEZ-ANSEMIL

Results and discussion naidids have similar penial chaetae that—based on Systems related with mating (Figures 1 and 2) form, location and orientation—might also have an anchorage function. Although they could anchor In observations conducted using SEM, Cuadrado directly on the body wall, new anchorage structures and Martínez-Ansemil (2001) described some can be expected in these species when more extensive structures and mechanisms used by naidids for observations through SEM are performed. attachment and sperm transfer. Some of these systems appear to be similar to those already known Coupling. Many microdriles are provided with in earthworms (see Jamieson, 2006) whereas others complementary mating structures that fi t each represent anatomical novelties that could be found other and are used both for attachment and sperm exclusively in microdriles. All mating systems revealed transfer. Th is seems to be the case for Rhyacodrilus by Cuadrado and Martínez-Ansemil (2001) imply falciformis, whose falciform penial chaetae fi t into the apposition of male and spermathecal pores. Aft er the lateral oblong spermathecal pores. Similarly, observing by SEM several other species belonging in Protuberodrilus tourenqui, the male porophore to the families Enchytraeidae, Lumbriculidae, brings the 2 male pores (close to the tip) to fi t with and Phreodrilidae, and aft er comparing these the 2 spermathecal pores of the partner, which are observations with previous results, we now propose placed near the median ventral line of the body in that the way to achieve the apposition of genital pores a depression fl anked by prominent chaetophores and to maintain the attachment of partners during (Cuadrado and Martínez-Ansemil, 2001). Th e the sperm transfer in microdriles can be included in prominent pendant penes of Stylodrilus heringianus, 1 of the following 3 categories: oriented towards the rear part of the worm, could Grasping. Th e most elaborate system described also help in holding partners when they penetrate by Cuadrado and Martínez-Ansemil (2001) belongs deeply into the large spermathecal pores (Figure 1E). to this category. It is the system utilized by the Erséus (1979) and Erséus and Baker (1982) rhyacodriline Peristodrilus montanus (Figure 1A- interpreted the giant penial chaetae of Adelodrilus C), in which the combined action of a complex Cook and Inanidrilus Erséus as structures involved system of muscles allow the penial chaetae (placed in sperm transfer. Cuadrado and Martínez-Ansemil between male pores) to enter the lateral sides of a singular ‘anchorage bridge’ located between the (2001) consider their probable participation also in spermathecal pores. Th e apposition of the male and the holding of the partners. When protracting penes the spermathecal pores, and the fi rm holding of the 2 are long and surrounded by cuticular sheaths (e.g., partners, would easily allow sperm transfer to occur. Limnodrilus Claparède, Aktedrilus Knöllner), their A dense ciliated area on the prominent outer part of intrusion far into the spermathecal ducts might the tegument surrounding the penial chaetal bundles help, not only in the sperm transfer, but also in the may facilitate the fi nding of the correct anchorage attachment of the mates (Cuadrado and Martínez- place. Ansemil, 2001). Whether or not attachment is the primary function, anatomically all these structures According to Cuadrado and Martínez-Ansemil (2001), similar systems could be present in at least of the male genitalia somehow seem to play a role in 4 of the 5 species of the tubifi cine genus Krenedrilus the holding of the partners, especially when they are Dumnicka, and in the phallodriline Bathydrilus strongly curved and the spermathecal pores occupy a rohdei (Jamieson, 1977). In Krenedrilus the bundles lateral or a dorsal position (e.g., Aktedrilus). However, of penial chaetae are arranged with the tips close besides their eventual participation in sperm transfer, together and directed towards each other, and a small another possible role could be associated with sperm epidermal papilla is present in the mid-ventral line competition (i.e. the emptying of full spermathecae of the spermathecal segment. B. rohdei is provided from a previous mating—prior to sperm transfer with an ‘X-shaped mid-ventral slit’ that, according by the new mate), a phenomenon already known in to Erséus (1981), corresponds to the location of the other taxonomical groups (e.g., Cordero et al., 2004; tips of the penial chaetae of the mate. Many aquatic Velando et al., 2008).

3 Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy

A 10 m m ab 100 sp

sp sp

p 100 m

D pc pc C

100 m 20 m E

Figure 1. Mating structures. (A-C) Peristodrilus montanus: A, anchorage bridge; B, male pores and penial chaetae; C, detail of the penial chaetae and the ciliature. (D) Protuberodrilus tourenqui: genital chaeta. (E) Stylodrilus heringianus: general view of the genital region. ab, anchorage bridge; mp, male pore; p, penis; pc, penial chaetae; sp, spermathecal pore.

Some of the coupling systems also seem to be Th e second mechanism, described by Schmelz (2003) aided by gland secretions, such as in P. tourenqui (see for enchytraeids in the genus Fridericia Michaelsen, Cuadrado and Martínez-Ansemil, 2001). consists of the eversion of the bursae, embracing Embracing. Th e embrace seems to be a common the partner as 2 narrow claspers, thus allowing the procedure to achieve and maintain the apposition contact of male and lateral spermathecal pores during of male and spermathecal pores during the sperm mating. According to Schmelz (2003), most of the transfer in many of the microdriles, especially glandular secretions of the male copulatory organs those that are devoid of grasping penial chaetae or are probably adhesive slimes. Figure 2E-G illustrates elaborated coupling structures. Embrace allows the this embrace system. alignment of male and spermathecal pores even According to Cuadrado and Martínez-Ansemil if the latter have a lateral position and it can be (2001), the typical gutter-shaped spermathecal performed by at least 2 diff erent mechanisms. Th e chaetae (Figure 1D), present in many aquatic fi rst mechanism involves a great fl exibility of the body microdriles, would not participate directly in the wall and a complex muscular system at the genital attachment and sperm transfer, but would act as region, that permit the protrusion and retraction of piercing chaetae involved in some mechanical or diff erent areas to ensure the apposition of the pores chemical stimulation during sperm transfer. If during sperm transfer, as has been described by some mechanical stimulation occurs, the secretions Cuadrado and Martínez-Ansemil (2001) for some of the large glands associated with these genital tubifi cines. Similarly, it has now been clearly shown chaetae would serve to fi rmly attach the partners for the enchytraeid Enchytraeus albidus (Figure 2A, while they protract and retract. Until biochemical B) and presumed for Insulodrilus sp. (Figure 2C, D). and physiological studies are performed, the exact

4 C. CARAMELO, E. MARTÍNEZ-ANSEMIL

A C

fp mp

100 m 20 m B F D

10 m E 2 m 10 m co vd

gb gb

bs ag mp 100 m G mb

Figure 2. Mating structures. (A, B) Enchytraeus albidus: A, general view of the genital region; B, detail of a male pore. (C, D) Insulodrilus sp.: C, general view of the genital region; D, detail of a male pore. (E, F) Fridericia monochaeta: E, general view of genital region; F, spermathecal pore and intersegmental furrow. (G) Fridericia: schematic drawing of the male copulatory organs (modifi ed from Schmelz, 2003). ag, area glareosa; bs, bursal slit; co, copulatory organs; fp, female pore; gb; glandular body; mb, medial side of bursa; mp, male pore; vd, vas deferens. role of these structures remains unknown. Th us, Ciliate sense receptors (Figure 3) Koene et al. (2005) proposed that the chaetal glands A summary of the typology and distribution in earthworms may produce an allohormone that of the external ciliated sense receptors according manipulates the reproductive physiology of the to Yáñez et al. (2006) and Caramelo and Martínez- mating partner. Ansemil (2010) is provided below. Th e only novelties

5 Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy

are some micrographs helping in the interpretation a transversal chaetal row and scattered or organized of the diff erent categories and patterns of distribution in other transversal rows in each chaetal segment proposed by the aforementioned authors. (Figure 3G-I). Ciliate sense receptors persist in the Typology clitellar region at maturity and are present in budding and regeneration zones (Figure 3J-M). Th e density of Th e observations by SEM allow identifi cation of ciliate sense receptors is greater at the anterior region diff erent types of receptors according to the shape of (especially on the prostomium and peristomium), the cilia and to the shape of the receptors. less in the subsequent chaetal segments, and increases Types according to the shape of the cilia again in the posterior segments. Receptors of blunt cilia, generally multi-ciliated Except in the case of the composed receptors, the and shorter than 6 μm long (Figure 3A-C); aquatic families and subfamilies of microdriles show Receptors of sharp cilia, with 1-5 cilia more than 6 the same types of ciliated sense receptors and with a μm long (mostly 8-14 μm long) (Figure 3E); similar distribution along the body. However, a longer size in blunt and sharp cilia and a smaller number Composed receptors, with a variable number of of cilia in the multi-ciliated receptors of blunt cilia blunt and sharp cilia together (Figure 3D, F). could be related to a more epibenthic way of life (see Both blunt and sharp cilia are present in all Caramelo and Martínez-Ansemil, 2010, Table 1). Th e microdriles studied, with the only exception in the number of cilia per receptor, at least in the anterior amphibiotic family Enchytraeidae. Out of the 8 body region, is generally greater in the amphibiotic species studied in this family (Römbke and Schmidt, family Enchytraeidae and especially in the lumbricid 1999; Caramelo and Martínez-Ansemil, 2010), only E. tetraedra. Higher numbers have been observed in Cognettia sphagnetorum seems to have sharp cilia in terrestrial megadriles (see Aros et al., 1978; Moment addition to the characteristic receptors of very short and Johnson, 1979). blunt cilia of this family. Although diff erent kinds of cilia and ciliate sense Types according to the shape of the receptor receptors are presumed to play diff erent functions, additional ultrastructural and physiological data are Poorly defi ned (unelevated) (Figure 3A, E, F, I, J) needed to accurately assign a role to a particular kind Sensory buds (epithelial bumps) (Figure 3B, C, G, of sense receptor. Th e limited information available H) Papillae (Figure 3D) to date generally suggests that the penetrative Th e most common type of sense receptor in uniciliate sensory cells act as mechanoreceptors microdriles is the poorly defi ned one. Sensory buds and the multiciliate sensory cells with emergent are common among the Enchytraeidae. Ciliate cilia act as chemoreceptors (see Welsch et al., 1984). papillae appear to be restricted to the chaetal segments Nonetheless, penetrative multiciliate sense receptors of a few species of Naididae. Th e characteristic multi- can be produced by uniciliated and/or multiciliated ciliated receptors of blunt cilia of the enchytraeids sensory cells. According to Yáñez et al. (2006), the deserve special mention (Figure 3C). Papillae are distribution in rows of most of the multiciliated always composed receptors, with the sharp cilia receptors of blunt cilia at the chaetal segments could be occupying the inner part. Many sensory buds of related to a tactile function in relation to locomotion, Eiseniella tetraedra, the most common lumbricid whereas the presence of receptors with long cilia found in freshwaters, are also composed receptors, in freshwater families of microdriles together with but here the sharp cilia are located at the periphery of their absence in earthworms could indicate that the organ (Figure 3F). they act as rheoreceptors. Th e recent observation of long sharp cilia in aquatic or semiaquatic worms Patterns of distribution of terrestrial origin (E. tetraedra and probably also External ciliate sense receptors are scattered on C. sphagnetorum) is interpreted by Caramelo and the prostomium (including proboscis when present), Martínez-Ansemil (2010) as the expression of an on the peristomium and pygidium, and arranged in ancient character.

6 C. CARAMELO, E. MARTÍNEZ-ANSEMIL

2 m 2 m 2 µm

B A 2 m C 2 m E F

20 m

D 2 m 20 m H 20 m 2 m dp

G J I

20 m 50 m 5 m

ec K L M

Figure 3. Ciliate sense receptors. (A) Scarcely defi ned sense receptor of blunt cilia: Lumbriculus variegatus; (B, C) Sensory buds: B, Protuberodrilus tourenqui; C, Lumbricillus sp.; (D) Papilla: Ophidonais serpentina; (E) Sense receptor of sharp cilia: Insulodrilus sp.; (F) Composed receptor: Eiseniella tetraedra; (G). Sense receptors on prostomium and peristomium: Pristina longiseta; (H) Sense receptors on pygidium: Enchytraeus albidus; (I) Transversal chaetal row: Peristodrilus montanus; (J) Cilia at the clitellum: Stylodrilus heringianus; (K) Sense receptors at a budding zone: P. l ong i s eta ; (L, M) Sense receptors on a regeneration zone: Lumbriculus variegatus, L, general view; M, detail of sense receptors. dp, dorsal pore; ec, emerging chaeta.

7 Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy

Somatic chaetae (Figure 4) defi ned pattern (Figure 4A-H). Th us, the view of the Somatic chaetae are generally used to identify hair chaetae as generally smooth structures must microdriles at the family level, and sometimes also at be challenged by that of hair chaetae as structures the subfamily, generic, or specifi c levels. Th us, the type at least potentially provided with some kind of of the chaetae, their particular shape, and number denticulations. in dorsal and ventral bundles are always included Crotchets (Figure 4K-O) in the description of oligochaete taxa. Most of the A number of more recent references have reported information about the somatic chaetae in microdriles the presence of intermediate teeth in chaetae that has been provided by optical microscopy. Our historically were considered bifi d (mostly ventral observations by SEM, combined with the available crotchets) (e.g., Brinkhurst, 1963; Brinkhurst and literature, reveal particular traits that we briefl y Jamieson 1971; Smith, 1985; Martínez-Ansemil summarize and discuss in the following 3 sections and Giani, 1986; Chapman and Brinkhurst, devoted to hair chaetae, crotchets, and needles. Th e 1987; Grimm, 1987). Similarly, our observations presence or absence of denticulations in hair chaetae (Figure 4K-O) confi rm that pectination is more and the presence or absence of intermediate teeth widespread than had been previously thought. in those chaetae currently considered to be bifi d Currently, pectination in bifi d chaetae has already crotchets are the most relevant questions. been documented in at least one species in each of Hair chaetae (Figure 4A-H) the following genera: Nais, Dero Oken, Branchiura Hair chaetae are present in many aquatic Beddard, Peristodrilus, Rhyacodrilus Bretscher, oligochaetes, commonly in dorsal bundles Potamothrix, Psammoryctides, Tubifex Lamarck, and and, rarely, in ventral ones (Capilloventridae, Varichaetadrilus Brinkhurst and Kathman. Parvidrilidae). Traditionally, the hair chaetae of In most cases, the presence of intermediate teeth microdriles were reported to be smooth; thus, with in typical bifi d chaetae is not characteristic of a only a few exceptions, the mentions of denticulations particular species but rather appears to occur under in hair chaetae were limited to the Naidinae (Ripistes certain environmental conditions (see Chapman and Dujardin, Stylaria Lamarck, and Vejdovskyella Brinkhurst, 1987). In our opinion, the development Michaelsen), and Pristininae (Pristina Ehrenberg), of intermediate teeth in typical bifi d crotchets reveals but even in these groups (the formerly family a certain degree of plasticity in the phenotypic Naididae) smooth hair chaetae were considered the expression of the chaetoblasts, particularly in the general rule (Sperber, 1948). transition between typical bifi d crotchets and typical A limited number of studies have dealt with the pectinate chaetae when following the evolutionary observation of hair chaetae by means of SEM, but, models shown in Brinkhurst (1984). interestingly, they generally report the observation Needles (Figure 4H, I) of denticulations in hair chaetae of taxa that were formerly considered devoid (e.g., Smith, 1985; Needles are acicular dorsal chaetae whose bifi d Chapman and Brinkhurst, 1986; Grimm, 1987; or simple-pointed condition is sometimes diffi cult Ohtaka, 1995). We have also found some kind to ascertain. Yet, when observed using SEM, minute of denticulations (Figure 4A-H) in at least some teeth can show remarkable diff erences (Figure 4H, I). chaetae in most of the genera studied (Nais Müller, Some variability in the shape of the chaetae Stylaria, Vejdovskyella, Pristina, Peristodrilus Baker was generally accepted in the literature, but aft er and Brinkhurst, Krenedrilus, Potamothrix Vejdovský the insightful study by Loden and Harman (1980) and Mrázek, Psammoryctides Hrabĕ, Spirosperma “Ecophenotypic variation in setae of Naididae Randolph, and Parvidrilus Erséus). Although the (Oligochaeta)”, the shape of the chaetae as a valid treatment of the samples might sometimes infl uence criterion to distinguish species from one another was the appearance of denticulations, it seems now that seriously questioned. Furthermore, the environment probably all hair chaetae have at least the potential infl uences not only the shape but also the presence to develop denticulations according to a more or less and number of a particular kind of chaetae as has

8 C. CARAMELO, E. MARTÍNEZ-ANSEMIL

1 m 1 m 5 m 2 m 1 m 5 m

A B C D E F

2 m 5 m 2 m 2 m 2 m

G H I J K 2 m 1 m N O 2 m

L M 1 m

Figure 4. Somatic chaetae. (A-G) Denticulations on hair chaetae: A-B, Pristina aequiseta; C-D, Pristina longiseta; E, Nais elinguis; F, Vejdovskyella comata; G, Peristodrilus montanus. (H, I). Bifi d needle chaetae: H, V. comata; I, P. l ong i s eta . (J) Bifi d hair chaeta: Stylaria lacustris. (K-O) Pectination on crotchets: K, P.montanus, ventral crotchet; L, Branchiura sowerbyi, dorsal crotchet; M-N, Rhyacodrilus falciformis ventral crotchets; O, Nais alpina ventral crotchets. been demonstrated by Chapman and Brinkhurst some morphological diff erences between taxa, as (1987) in their work eloquently entitled “Hair could be the case of diff erent types of denticulations today, gone tomorrow: induced chaetal variation in in hair chaetae. However, due to the high plasticity tubifi cid oligochaetes”. Th e SEM could help to reveal and homoplasy concerning the expression and the

9 Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy

evolution of the chaetae among microdriles, SEM Peristodrilus montanus. (Figure 3I, Figure 5J, K). would probably be a more useful tool to detect Dorsal pores observed only at 3/4 and 4/5. Diameter, abnormalities produced by environmental stresses as 13-20 μm. shown by Milbrink (1983) than to reveal phylogenetic Protuberodrilus tourenqui. (Figure 5E, F). Dorsal relationships. pores present from 5/6 to at least 7/8. Diameter, 8-12 Dorsal pores (Figures 3I and 5) μm. In many oligochaetes, the coelomic cavity SEM proves to be a useful tool for the observation communicates with the exterior through small pores of dorsal pores in microdriles. Interestingly, all 4 located in the mid-dorsal line of the body. Th ese aquatic species of microdriles showing dorsal pores unpaired structures are named dorsal or coelomic belong to the Rhyacodrilinae, an ancient subfamily pores. Until now, dorsal pores were generally of naidids. Historically, it has been accepted that the functions of dorsal pores were connected with their considered present only in earthworms (megadriles) presence in species from terrestrial habitats and their and the enchytraeids of the genus Fridericia absence in aquatic species (i.e. protection against (Stephenson, 1930; Brinkhurst and Jamieson, 1971; desiccation, keeping the surface wall moist for Bouché, 1972; Schmelz, 2003). Nevertheless, the respiratory exchanges, protection against bacteria and presence of dorsal pores had already been reported parasites) (see Stephenson, 1930). Before introducing in 2 aquatic species of microdriles: the rhyacodrilines new biological hypotheses related to the presence Monopylephorus auklandicus (Benham, 1909) and of dorsal pores in aquatic microdriles, we think it Bothrioneurum vejdovskyanum (see Chekanovskaya, appropriate to investigate their presence in other 1962; Timm, 1979). microdriles, especially other rhyacodriline genera Th e observation of species belonging to the and their closest relatives. It is worth highlighting families Enchytraeidae, Naididae, Lumbriculidae, the diff erent number and distribution of dorsal pores Lumbricidae, Phreodrilidae, and Parvidrilidae in the 4 species of rhyacodrilines, and that the fi rst revealed the presence of dorsal pores—not only in dorsal pore is located in a more anterior segment taxa where they should be expected (the lumbricid than had been reported previously in megadriles and Fridericia. Eiseniella tetraedra and the enchytraeid Fridericia monochaeta) (Figure 5A-D) but also in some Other structures (Figure 6) species of typical freshwater microdriles. Although In addition to the structures described above, not all the material was observed in dorsal view, we have occasionally observed some structures that it must be pointed out that the only freshwater could also provide additional information from a microdriles where we have found dorsal pores are taxonomical or a biological perspective if they were the rhyacodrilines B. vejdovskyanum, Peristodrilus systematically observed. Th us, it is noteworthy that montanus, and Protuberodrilus tourenqui. Th e study the accurate position and diameter of the nephridial of a poorly preserved specimen of Parvidrilus spelaeus pores is easily observed by SEM and could be (Parvidrilidae) also revealed the presence of pores in informative (Figures 6A, B). Furthermore, it is the mid-dorsal line of the body, but these are probably interesting to note the porosity observed at the body not truly dorsal (coelomic) ones but the pores of wall (e.g., Lumbriculus variegatus: Figures 6C, D). the mid-dorsal glandular pouches characteristic of Finally, we note the observation of female marks the family (see Martínez-Ansemil et al., 2002). Th e (depressions in the body wall) with diff erent shapes in mature (well developed male pores and clitellum) distribution and diameter of the dorsal pores that enchytraeids (Fridericia monochaeta) (Figures 6E, F) we observed in the 3 species of rhyacodrilines are as and naidids (Limnodrilus udekemianus) (Figure 6G); follows: these depressions correspond with the position of Bothrioneurum vejdovskyanum. (Figure 5G-I). the female pores. Perhaps the shape of female marks Dorsal pores present from 3/4 to the posterior end. is highly variable and consequently not very useful Diameter, 8-13 μm. as a taxonomical character (e.g., comparison of the

10 C. CARAMELO, E. MARTÍNEZ-ANSEMIL

B E 6/7

20 m

A 500 m D C 7/8

50 m

5 m F

30 m 6/7 G

5 m 10 m 100 m J I K

10 m 10 m 200 m

Figure 5. Dorsal pores. (A, B) Eiseniella tetraedra: A, dorsal pores at anterior body region; B, detail of a pore. (C, D) Fridericia monochaeta: C, pore at 6/7; D, detail. (E, F) Protuberodrilus tourenqui: E, pores at 6/7 and 7/8; F, detail. (G-I) Bothrioneurum vejdovskyanum: G, dorsal pores at anterior body region; H, detail of the pore 37/38; I, coelomocytes fl owing through a pore. (J, K) Peristodrilus montanus: J, pores at 3/4 and 4/5; K, detail of the pore 4/5.

diff erent shape of the 2 marks of the same individual pores and clitellum are well developed—provide us in L. udekemianus: Figure 6H, I). Regardless, these with insight into how protandric worms likely avoid female marks—devoid of female pores when male self-fertilization.

11 Morphological investigations of microdrile oligochaetes (Annelida, Clitellata) using scanning electron microscopy

50 m B 5 m 50 m

2 m

A D C

50 m F 5 m

fm E H 10 m 50 m I 10 m

fm mp G

Figure 6. Nephridial pores. (A, B) Lumbricillus sp.: A, nephridial pores at anterior body region; B, detail of the nephridial pore of VII. Epidermal pores. (C, D) Lumbriculus variegatus in regeneration: C, general view; D, detail. Female marks. (E, F) Fridericia monochaeta: E, general view of the genital region; F, detail of a female mark. (G-I) Limnodrilus udekemianus: G, general view of the genital region; H-I, detail of the 2 female marks from the same individual. co, copulatory organ; fm, female mark; mp, male pore.

Conclusion and has resulted in the broader understanding and SEM is an invaluable tool that has increased the discovery of several important structures. However, knowledge of the functional anatomy on microdriles, additional studies and observations using SEM are still

12 C. CARAMELO, E. MARTÍNEZ-ANSEMIL

needed, especially with regard to mating structures. Acknowledgements Furthermore, the results obtained during this study Th is work was supported by the Spanish Ministry encourage us to pursue new investigations using other of Education and Science and FEDER (Project No. techniques (e.g., CLSM, TEM, microscopic sections) CGL.2006-13417). Th e authors wish to thank R. in order to broaden our knowledge on the muscular Schmelz for providing enchytraeids and phreodrilids, complex and glands involved in the diff erent mating S. Cuadrado for his scientifi c collaboration, M. systems, the relationships between the ciliate sense Quintela for proofreading, and Mark J. Wetzel for receptors and the nervous system, and the muscular valuable scientifi c comments and revising the text. and nervous systems that control the opening of the Two anonymous reviewers improved the quality of dorsal pores. the paper.

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