Advances in MARINE BIOLOGY

VOLUME 46 ThisPageIntentionallyLeftBlank Advances in MARINE BIOLOGY

Edited by A. J. SOUTHWARD

Marine Biological Association, The Laboratory, Citadel Hill, Plymouth, PL1 2PB, UK

P. A. TYLER

School of Ocean and Earth Science, University of Southampton, Southampton Oceanography Centre, European Way, Southampton, SO14 3ZH, UK

C. M. YOUNG

Oregon Institute of Marine Biology, University of Oregon P.O. Box 5389, Charleston, Oregon 97420, USA and L. A. FUIMAN

Marine Science Institute, University of Texas at Austin, 750 Channel View Drive, Port Aransas, Texas 78373, USA

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030405060708MP987654321 LIST OF CONTRIBUTORS

BARBARA E. BROWN Department of Marine Sciences and Coastal Management, University of Newcastle on Tyne, Newcastle on Tyne NE1 7RU, UK; Present address: Ling Cottage, Mickleton, Barnard Castle, Co. Durham DL12 OLL, UK S. L. COLES, Department of Natural Sciences, Bishop Museum, 1525 Bernice St., Honolulu, HI 96734, USA ANNE-JOHANNE TANG DALSGAARD, University of Copenhagen, c/o Danish Institute for Fisheries Research, Charlottenlund Castle, DK-2920 Charlottenlund, Denmark. ANDREW J. GOODAY, Southampton Oceanography Centre, European Way, Southampton SO14 3ZH, UK V. GUNAMALAI, Unit of Invertebrate Reproduction and Aquaculture, Department of Zoology, University of Madras, Guindy Campus, Chennai – 600 025, India. WILHELM HAGEN, Universita¨t Bremen (NW2A), Postfach 330440, D-28334 Bremen, Germany GERHARD KATTNER, Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany. DO¨ RTHE MU¨ LLER-NAVARRA, University of Hamburg, Center for Marine and Climate Research, Institute for Hydrobiology and Fisheries Research, Olbersweg 24, D-22767 Hamburg, Germany. MICHAEL ST.JOHN, University of Hamburg, Center for Marine and Climate Research, Institute for Hydrobiology and Fisheries Research, Olbersweg 24, D-22767 Hamburg, Germany. T. SUBRAMONIAM, Unit of Invertebrate Reproduction and Aquaculture, Department of Zoology, University of Madras, Guindy Campus, Chennai – 600 025, India

v ThisPageIntentionallyLeftBlank CONTENTS

CONTRIBUTORS TO VOLUME 46...... v SERIES CONTENTS FOR LAST TEN YEARS ...... ix

Benthic Foraminifera (Protista) as Tools in Deep-water Palaeoceanography: Environmental Influences on Faunal Characteristics

Andrew J. Gooday

1. Introduction ...... 3 2. Deep-sea Environments ...... 5 3. Methodology: Sieve Sizes, Sampling Devices and Replication ...... 6 4. Aspects of Deep-sea Foraminiferal Ecology ...... 7 5. Faunal Approaches to Reconstructing Palaeoceanography ...... 15 6. Organic Matter Fluxes ...... 18 7. Oxygen Concentrations ...... 33 8. Bottom-water Hydrography ...... 39 9. Water Depth ...... 43 10. Species Diversity Parameters as Tools in Palaeoceanography ...... 45 11. Summary of Environmental Influences on Live Assemblages ...... 54 12. Relationship of Modern and Fossil Assemblages ...... 56 13. Problems and Future Directions ...... 62 Acknowledgements ...... 69 References ...... 70

Breeding Biology of the Intertidal Sand Crab, Emerita (Decapoda: Anomura)

T. Subramoniam and V. Gunamalai

1. Introduction ...... 92 2. Distribution and Natural History ...... 93 3. Sex Ratio and Size at Sexual Maturity ...... 95 4. Neoteny ...... 96 5. Protandric Hermaphroditism in E. asiatica ...... 99 6. Mating Habits ...... 104 7. Spermatophores and Sperm Transfer ...... 106 8. Moulting Pattern of E. asiatica—A Case Study ...... 112 9. Reproductive Cycle ...... 122

vii viii Contents

10. Interrelationship Between Moulting and Reproduction ...... 135 11. Biochemistry of Eggs ...... 139 12. Yolk Utilisation ...... 146 13. Larval Development ...... 161 14. Emerita as Indicator Species ...... 168 15. Conclusions ...... 170 Acknowledgements ...... 171 References ...... 172

Coral Bleaching – Capacity for Acclimatization and Adaptation

S. L. Coles and Barbara E. Brown

1. Introduction ...... 184 2. Coral Upper Temperature Tolerance Thresholds ...... 186 3. The Coral Bleaching Process ...... 188 4. Coral Bleaching Protective Mechanisms ...... 190 5. Coral and Zooxanthellae Thermal Acclimation, Acclimatization, and Adaptation: Empirical Observations ...... 195 6. Coral Bleaching Recovery ...... 201 7. Bleaching and Coral Disease, Reproduction, and Recruitment ...... 204 8. Long-Term Ecological Implications of Coral Bleaching ...... 207 9. Conclusions ...... 209 Acknowledgements ...... 211 References ...... 212

Fatty Acid Trophic Markers in the Pelagic Marine Environment

Johanne Dalsgaard, Michael St. John, Gerhard Kattner, Do¨ rthe Mu¨ ller-Navarra and Wilhelm Hagen

1. Introduction ...... 227 2. Fatty Acid Dynamics in Marine Primary Producers ...... 238 3. Fatty Acid Dynamics in Crustaceous Zooplankton ...... 255 4. Fatty Acid Dynamics in Fish ...... 269 5. Applications of Fatty Acid Trophic Markers in Major Food Webs ...... 278 6. Summary and Conclusions ...... 313 Acknowledgements ...... 318 References ...... 318

Taxonomic Index ...... 341 Subject Index ...... 347 Series Contents for Last Ten Years*

VOLUME 30, 1994. Vincx, M., Bett, B. J., Dinet, A., Ferrero, T., Gooday, A. J., Lambshead, P. J. D., Pfannku¨ che, O., Soltweddel, T. and Vanreusel, A. Meiobenthos of the deep Northeast Atlantic. pp. 1–88. Brown, A. C. and Odendaal, F. J. The biology of oniscid Isopoda of the genus Tylos. pp. 89–153. Ritz, D. A. Social aggregation in pelagic invertebrates. pp. 155–216. Ferron, A. and Legget, W. C. An appraisal of condition measures for marine fish larvae. pp. 217–303. Rogers, A. D. The biology of seamounts. pp. 305–350. VOLUME 31, 1997. Gardner, J. P. A. Hybridization in the sea. pp. 1–78. Egloff, D. A., Fofonoff, P. W. and Onbe´ , T. Reproductive behaviour of marine cladocerans. pp. 79–167. Dower, J. F., Miller, T. J. and Leggett, W. C. The role of microscale turbulence in the feeding ecology of larval fish. pp. 169–220. Brown, B. E. Adaptations of reef corals to physical environmental stress. pp. 221–299. Richardson, K. Harmful or exceptional phytoplankton blooms in the marine ecosystem. pp. 301–385. VOLUME 32, 1997, Vinogradov, M. E. Some problems of vertical distribution of meso- and macroplankton in the ocean. pp. 1–92. Gebruk, A. K., Galkin, S. V., Vereshchaka, A. J., Moskalev, L. I. and Southward, A. J. Ecology and biogeography of the hydrothermal vent fauna of the Mid-Atlantic Ridge. pp. 93–144. Parin, N. V., Mironov, A. N. and Nesis, K. N. Biology of the Nazca and Sala y Gomez submarine ridges, an outpost of the Indo-West Pacific fauna in the eastern Pacific Ocean: composition and distribution of the fauna, its communities and history. pp. 145–242. Nesis, K. N. Goniatid squids in the subarctic North Pacific: ecology, biogeography, niche diversity and role in the ecosystem. pp. 243–324. Vinogradova, N. G. Zoogeography of the abyssal and hadal zones. pp. 325–387. Zezina, O. N. Biogeography of the bathyal zone. pp. 389–426. Sokolova, M. N. Trophic structure of abyssal macrobenthos. pp. 427–525.

ix x Series Contents for Last Ten Years

Semina, H. J. An outline of the geographical distribution of oceanic phytoplankton. pp. 527–563.

VOLUME 33, 1998. Mauchline, J. The biology of calanoid copepods. pp. 1–660.

VOLUME 34, 1998. Davies, M. S. and Hawkins, S. J. Mucus from marine molluscs. pp. 1–71. Joyeux, J. C. and Ward, A. B. Constraints on coastal lagoon fisheries. pp. 73–199. Jennings, S. and Kaiser, M. J. The effects of fishing on marine ecosystems. pp. 201–352. Tunnicliffe, V., McArthur, A. G. and McHugh, D. A biogeographical perspective of the deep-sea hydrothermal vent fauna. pp. 353–442.

VOLUME 35, 1999. Creasey, S. S. and Rogers, A. D. Population genetics of bathyal and abyssal organisms. pp. 1–151. Brey, T. Growth performance and mortality in aquatic macrobenthic invertebrates. pp. 153–223. VOLUME 36, 1999. Shulman, G. E. and Love, R. M. The biochemical ecology of marine fishes. pp. 1–325. VOLUME 37, 1999. His, E., Beiras, R. and Seaman, M. N. L. The assessment of marine pollution – bioassays with bivalve embryos and larvae. pp. 1–178. Bailey, K. M., Quinn, T. J., Bentzen, P. and Grant, W. S. Population structure and dynamics of walleye pollock, Theragra chalcogramma. pp. 179–255. VOLUME 38, 2000. Blaxter, J. H. S. The enhancement of marine fish stocks. pp. 1–54. Bergstro¨ m, B. I. The biology of Pandalus. pp. 55–245. VOLUME 39, 2001. Peterson, C. H. The ‘‘Exxon Valdez’’ oil spill in Alaska: acute indirect and chronic effects on the ecosystem. pp. 1–103. Johnson, W. S., Stevens, M. and Watling, L. Reproduction and develop- ment of marine peracaridans. pp. 105–260. Rodhouse, P. G., Elvidge, C. D. and Trathan, P. N. Remote sensing of the global light-fishing fleet: an analysis of interactions with oceanography, other fisheries and predators. pp. 261–303. Series Contents for Last Ten Years xi

VOLUME 40, 2001. Hemmingsen, W. and MacKenzie, K. The parasite fauna of the Atlantic cod, Gadus morhua L. pp. 1–80. Kathiresan, K. and Bingham, B. L. Biology of mangroves and mangrove ecosystems. pp. 81–251. Zaccone, G., Kapoor, B. G., Fasulo, S. and Ainis, L. Structural, histo- chemical and functional aspects of the epidermis of fishes. pp. 253–348.

VOLUME 41, 2001. Whitfield, M. Interactions between phytoplankton and trace metals in the ocean. pp. 1–128. Hamel, J.-F., Conand, C., Pawson, D. L. and Mercier, A. The sea cucumber Holothuria scabra (Holothuroidea: Echinodermata): its biology and exploitation as beche-de-Mer. pp. 129–223.

VOLUME 42, 2002. Zardus, J. D. Protobranch bivalves. pp. 1–65. Mikkelsen, P. M. Shelled opisthobranchs. pp. 67–136. Reynolds, P. D. The scaphopoda, pp. 137–236. Harasewych, M. G. Pleurotomarioidean gastropods. pp. 237–294.

VOLUME 43, 2002. Rohde, K. Ecology and biogeography of marine parasites. pp. 1–86. Ramirez Llodra, E. Fecundity and life-history strategies in marine invertebrates. pp. 87–170. Brierley, A. S. and Thomas, D. N. Ecology of southern ocean pack ice. pp. 171–276. Hedley, J. D. and Mumby, P. J. Biological and remote sensing perspectives of pigmentation in coral reef organisms. pp. 277–317.

VOLUME 44, 2003. Hirst, A. G., Roff, J. C. and Lampitt, R. S. A Synthesis of growth rates in epipelagic invertebrate zooplankton. pp. 3–142. Boletzky, S. von. Biology of early life stages in cephalopod molluscs. pp. 143–203. Pittman, S. J. and McAlpine, C. A. Movements of marine fish and decapod crustaceans: process, theory and application. pp. 205–294. Cutts, C. J. Culture of harpacticoid copepods: potential as live feed for rearing marine fish. pp. 295–315.

VOLUME 45, 2003. Cumulative and Subject Index. ThisPageIntentionallyLeftBlank Benthic Foraminifera (Protista) as Tools in Deep-water Palaeoceanography: Environmental Influences on Faunal Characteristics

Andrew J. Gooday

Southampton Oceanography Centre, European Way, Southampton SO14 3ZH, UK E-mail: [email protected]

1. Introduction ...... 3 2. Deep-sea Environments ...... 5 3. Methodology: Sieve Sizes, Sampling Devices and Replication ...... 6 4. Aspects of Deep-sea Foraminiferal Ecology ...... 7 4.1. Introduction ...... 7 4.2. Small-scale patterns ...... 8 4.3. Regional patterns ...... 14 5. Faunal Approaches to Reconstructing Palaeoceanography ...... 15 6. Organic Matter Fluxes ...... 18 6.1. General considerations ...... 18 6.2. Reconstructing annual flux rates ...... 19 6.3. Responses to seasonally varying fluxes ...... 29 6.4. Are calcareous species more responsive than other foraminifera? . . . . . 31 7. Oxygen Concentrations ...... 33 7.1. General considerations ...... 33 7.2. Qualitative approaches ...... 35 7.3. Quantitative approaches ...... 37 8. Bottom-water Hydrography ...... 39 8.1. General considerations ...... 39 8.2. Carbonate undersaturation ...... 40 8.3. Current flow ...... 41 9. Water Depth ...... 43

ADVANCES IN MARINE BIOLOGY VOL 46 Copyright ß 2003 Academic Press 0-12-026146-4 All rights of reproduction in any form reserved 2 ANDREW J. GOODAY

10. Species Diversity Parameters as Tools in Palaeoceanography ...... 45 11. Summary of Environmental Influences on Live Assemblages ...... 54 12. Relationship of Modern and Fossil Assemblages ...... 56 13. Problems and Future Directions ...... 62 13.1. Relationship between environmental factors and spatial scales ...... 62 13.2. Calibration of proxies ...... 64 13.3. Microhabitat studies ...... 66 13.4. Problems in taxonomy ...... 67 13.5. Biological–geological synergy in foraminiferal research? ...... 68 Acknowledgements ...... 69 References ...... 70

Foraminiferal research lies at the border between geology and biology. Benthic foraminifera are a major component of marine communities, highly sensitive to environmental influences, and the most abundant benthic organisms preserved in the deep-sea fossil record. These characteristics make them important tools for reconstructing ancient oceans. Much of the recent work concerns the search for palaeoceanographic proxies, particularly for the key parameters of surface primary productivity and bottom-water oxygenation. At small spatial scales, organic flux and pore-water oxygen profiles are believed to control the depths at which species live within the sediment (their ‘microhabitats’). Epifaunal/shallow infaunal species require oxygen and labile food and prefer relatively oligotrophic settings. Some deep infaunal species can tolerate anoxia and are closely linked to redox fronts within the sediment; they consume more refractory organic matter, and flourish in relatively eutrophic environments. Food and oxygen availability are also key factors at large (i.e. regional) spatial scales. Organic flux to the sea floor, and its seasonality, strongly influences faunal densities, species compositions and diversity parameters. Species tend to be associated with higher or lower flux rates and the annual À2 flux range of 2–3 g Corg m appears to mark an important faunal boundary. The oxygen requirements of benthic foraminifera are not well understood. It has been proposed that species distributions reflect oxygen concentrations up to fairly high values (3 ml lÀ1 or more). Other evidence suggests that oxygen only begins to affect community parameters at concentrations <0.5 ml l À1. Different species clearly have different thresholds, however, creating species successions along oxygen gradients. Other factors such as sediment type, hydrostatic pressure and attributes of bottom-water masses (particularly carbonate undersaturation and current flow) influence foraminiferal distribu- tions, particularly on continental margins where strong seafloor environmental gradients exist. Epifaunal species living on elevated substrata are directly exposed to bottom-water masses and flourish where suspended food particles are advected by strong currents. Biological interactions, e.g. predation and competition, must also play a role, although this is poorly understood and difficult to quantify. Despite often clear qualitative links between BENTHIC FORAMINIFERA 3 environmental and faunal parameters, the development of quantitative foraminiferal proxies remains problematic. Many of these difficulties arise because species can tolerate a wide range of non-optimal conditions and do not exhibit simple relationships with particular parameters. Some progress has been made, however, in formulating proxies for organic fluxes and bottom- water oxygenation. Flux proxies are based on the Benthic Foraminiferal Accumulation Rate and multivariate analyses of species data. Oxygen proxies utilise the relative proportions of epifaunal (oxyphilic) and deep infaunal (low-oxygen tolerant) species. Yet many problems remain, particularly those concerning the calibration of proxies, the closely interwoven effects of oxygen and food availability, and the relationship between living assemblages and those preserved in the permanent sediment record.

1. INTRODUCTION

The oceans are of fundamental importance to the functioning of the planet and its ecosystems. The global climate is closely coupled with the thermohaline circulation and surface productivity of the oceans. This climate/ocean system has fluctuated radically in the geological past, most recently during the last 2.6 million years (my), the period of the late Pliocene and Quaternary ice ages. Recently, it has become apparent that major changes in the earth’s climate have occurred over time scales as short as decades or even years (Alley et al., 1993; Committee on Abrupt Climate Change, 2002) and that such changes can rapidly impact the ocean floor environment through deep-water production (Dokken and Jansen, 1999; Scho¨ nfeld et al., in press). Present concerns about global warming have heightened awareness of these rapid climatic oscillations and the need to understand them. This, in turn, has promoted attempts to decipher the history of the oceans, as revealed by records preserved in the deep-sea sediments (Clark et al., 1999; Wefer et al., 1999; Scha¨ fer et al., 2001). Benthic foraminifera convey a substantial amount of information about conditions on the ocean floor and have played an important part in efforts to understand these conditions. Indeed, much of the recent research by geologists on modern deep-sea faunas has been driven by a desire to develop reliable tools for use in palaeoceanography. Many earlier approaches were qualitative or semi-quantitative and aimed at obtaining a general under- standing of past environmental conditions. They were often based on ‘‘total’’ (live plus dead) assemblages that may not have represented the living fauna accurately (Douglas and Woodruff, 1981). In more recent years, there has been a spate of publications describing ‘‘live’’ (rose Bengal stained) deep-sea faunas. These have been more quantitative in nature and focussed 4 ANDREW J. GOODAY on the development of proxies (Loubere, 1994; Mackensen et al., 1995; Murray, 2001), particularly for primary productivity and organic matter fluxes to the sea floor. Planktonic foraminiferal assemblages have long been used to estimate sea-surface temperatures over the last few hundred thousand years (Imbrie and Kipp, 1971; Hale and Pflaumann, 1999). Despite their more complex ecology, benthic foraminifera also have the potential to be good proxies. They are widely distributed, highly sensitive to environmental conditions, and are by far the most abundant benthic organisms preserved in the Cenozoic and Cretaceous deep-sea sediments. There are two contrasting types of proxy based on benthic foraminifera. The first utilises faunal characteristics such as species and species assemblages, diversity parameters and test morphotypes; the second depends on the elemental and isotopic chemistry of calcareous tests. Only the former is considered here. This review developed from an investigation, conducted under the U.K. Natural Environment Research Council’s BENBO (BENthic BOundary layer study) programme, of foraminifera and their test geochemistry at three oceanographically dissimilar sites situated at water depths of 1100 m, 1950 m, and 3600 m on the UK continental margin. Results from the BENBO study are used to illustrate some of the points discussed below. The discussion builds on recent accounts of foraminiferal ecology by Bernhard and Sen Gupta (1999), Jorissen (1999), van der Zwaan et al. (1999) and Loubere and Fariduddin (1999b), Murray’s (2001) critical examination of some of the basic concepts underlying the use of foraminifera in palaeoceanographic reconstructions, and Mackensen’s (1997) review of the application of benthic foraminiferal proxies in high latitude palaeoceanography. Some of the ideas developed in this paper were prompted by Levin et al.’s (2001) synthesis of regional diversity patterns in the deep sea. The overall goal is to present an overview of faunal approaches based on benthic foraminifera and to discuss factors that generate and modify the proxy signal. Unlike previous reviews, I have attempted, where possible, to integrate observations made at large and small spatial scales and utilise the insights of benthic ecologists into the responses of organisms to environmental gradients. Foraminifera are sarcodine protists characterised by a network of pseudopodia that contain numerous granules (termed granuloreticulate pseudopodia) and by complex life cycles that often involve sexual and asexual generations (Goldstein, 1999). Although naked taxa exist (Pawlowski et al., 2002), the cell body is usually enclosed within a single- chambered (monothalamous) or multi-chambered (polythalamous) test (‘shell’) composed of agglutinated particles collected from the surrounding environment or of organic material or calcium carbonate (usually calcite) secreted by the organism. The main subdivisions of the foraminifera are based almost entirely on test characteristics, particularly the composition BENTHIC FORAMINIFERA 5 and structure of the test wall (Loeblich and Tappan, 1987, 1989; Sen Gupta, 1999). In modern oceans, the most important orders are the following: Allogromiida: organic wall, usually monothalamous Astrorhizida: agglutinated wall, organic cement, monothalamous Textulariida: agglutinated wall, calcitic cement, polythalamous Lituolida: agglutinated wall, organic or calcitic cement, polythalamous Trochamminida: agglutinated wall, organic cement, trochospiral arrangement of chambers Miliolida: wall often with a white, ‘‘porcellaneous’’ appearance in reflected light, composed of high-Mg calcite, imperforate, usually polythalamous Lagenida: wall glassy (‘hyaline’) when fresh, composed of low-Mg calcite, monolamellar, perforate, monothalamous or polythalamous Robertinida: wall glassy when fresh, composed of aragonite, perforate, multilocular Buliminida: wall glassy when fresh, composed of low-Mg calcite, bilamellar, perforate, multilocular; chamber arrangement high trochospiral, triserial, biserial or uniserial; aperture often with toothplate Rotaliida: wall glassy when fresh, composed of low-Mg calcite, bilamellar, perforate, multilocular; chamber arrangement low trochospiral, planispiral, or irregular. It is important to note that, according to recent molecular studies, there is no phylogenetic distinction between the organic-walled and agglutinated monothalamous taxa, traditionally referred to the orders Allogromiida and Astrorhiziida respectively (Pawlowski et al., 2001; papers in Cedhagen et al., 2002). These foraminifera are represented by a series of evolutionary lineages, many of which include both wall types.

2. DEEP-SEA ENVIRONMENTS

The deep sea lies beyond the shelf break (usually located at around 200 m water depth) and, in a general sense, is a more uniform environment than the continental shelf. It is characterised by a lack of light, high pressures, generally low temperatures and constant salinities (Tyler, 1995). Primary production is confined to chemosynthetic communities located around vents and cold seeps. The vast majority of organisms are sustained by organic matter derived from phytoplankton primary production settling through the water column or by laterally advected material. Although sometimes considered as a single habitat, substantial environmental differences exist within the deep sea, particularly between continental margins and abyssal plains (Berger and Wefer, 1992; Gooday and Rathburn, 1999; 6 ANDREW J. GOODAY

Etter and Mullineaux, 2000). Bathyal continental slopes and rises are physically much more heterogeneous than abyssal plains. They are often topographically complex (Mellor and Paull, 1994) and subject to vigorous current activity and catastrophic mass movements (Masson et al., 1996). Compared with abyssal plains, sedimentation rates are usually higher on the continental slope and the sediments are more heterogeneous, less well oxidised and richer in animal life (Etter and Grassle, 1992; Bett, 2001). Continental slopes have experienced dramatic oceanographic changes linked to global climatic fluctuations in the geologic past, particularly during the Pliocene and Pleistocene glacial cycles. By comparison, the abyssal sea floor is relatively uniform and quiescent with gently undulating topography. Because the amount of organic matter reaching the ocean floor decreases with increasing depth, abyssal environments are typically more food limited than continental margins that also receive laterally advected organic matter from the continental shelf. High productivity areas associated with upwelling or major river discharges are more common on continental margins (Diaz and Rosenburg, 1995; Rogers, 2000). These environmental contrasts have ecological consequences for benthic communities. In a general sense, one would expect sediment types, near-bottom currents and oxygen depletion coupled with organic enrichment to exert a greater influence on continental margins (e.g. Schaff et al., 1992; Schmiedl et al., 1997; Levin et al., 2000), and patterns of food inputs derived from surface production to be more important on abyssal plains (e.g. Loubere, 1991; Smith et al., 1997).

3. METHODOLOGY: SIEVE SIZES, SAMPLING DEVICES AND REPLICATION

Most analyses of living foraminiferal faunas are based on sieved residues stained with rose Bengal which colours the protoplasm red. Wet sorting (i.e. in a dish of water) makes it easier to see stained protoplasm and therefore yields more accurate results than dry sorting. Sieve sizes strongly influence the abundance of individual species and hence assemblage composition and diversity. Many studies are based on sediment fractions >125, >150 or even >250 mm, which can be analysed relatively quickly. However, some dominant species are small and therefore concentrated in the finer (63–125 or 63–150 mm) residues (Schro¨ der et al., 1987; Sen Gupta et al., 1987; Rathburn and Corliss, 1994; Kurbjeweit et al., 2000). In the ice-covered central Arctic, the average size of foraminiferal tests is 70 mm and many of the important species pass through a 125 mm mesh (Wollenburg and Mackensen, 1998). Small epifaunal species may be very abundant and important for detecting responses to freshly deposited, labile organic matter BENTHIC FORAMINIFERA 7

(e.g. Gooday, 1988, 1996; Gooday and Lambshead, 1989; Mackensen et al., 2000; Rathburn et al., 2001; Gooday and Hughes, 2002) (see Figure 7 on page 57). To ensure maximum comparability, studies ideally should be based on several different size fractions (>150, 125–150, 63–125 mm). Because fine fractions are very time consuming to analyse, it may be necessary to split samples. Wet samples can be split using the Asko¨ splitter of Elmgren (1973). The more elaborate device designed by Jensen (1982) is also very effective. Many of the earlier ecological studies on deep-sea benthic foraminifera were based on box core or even Van Veen grab samples. More recently, the use of hydraulically dampened multiple corers (‘multicorers’) of different design (e.g. Barnett et al., 1984) has become widespread. This is an important technical advance since multicorers retain light, flocculent surface material such as phytodetritus that is rarely present in box cores (Thiel et al., 1989). Bett et al. (1994) showed that multicorers sample metazoan meiofauna much more efficiently than box corers. Recent work on the UK continental margin suggests that box corers even underestimate macrofaunal densities by a factor of >2 compared with multicorers 10 cm in diameter (Bett, in press). These differences presumably arise because lighter-bodied, surface-dwelling organisms are blown away by the bow wave generated by the box corer. Further faunal losses from box corers may occur as the overlying water is drained on deck. Nevertheless, box corers retain sandy sediments more reliably than multicorers and their greater surface area permits the recognition of sedimentary features, biogenic and other habitat structures that may be important for interpreting foraminiferal assemblages (Scho¨ nfeld, 2002a, 2000c). Because populations often exhibit considerable small-scale patchiness (e.g. Gooday and Lambshead, 1989), samples for living foraminifera should ideally be replicated, for example, by taking one multicore from each of several deployments. One solution to the additional sorting load imposed by replication is to take several subcores from a standard multicore using a cut-off syringe. A 20 ml syringe has a cross-sectional area of 3.45 cm2 compared to 25.5 cm2 in the case of a 57 mm internal diameter multicorer tube.

4. ASPECTS OF DEEP-SEA FORAMINIFERAL ECOLOGY

4.1. Introduction

Foraminifera are one of the principle eukaryotic life forms in the deep sea and often constitute a substantial proportion of benthic biomass (Snider 8 ANDREW J. GOODAY et al., 1984; Altenbach and Sarnthein, 1989; Gooday et al., 1992; Kro¨ ncke et al., 2000). Where bottom waters are well oxygenated, live assemblages are highly diverse, often with well over 100 morphospecies occurring in relatively small volumes of surface sediment (Gooday et al., 1998). These assemblages include taxa with organic, agglutinated and calcareous test walls. The proportion of calcareous foraminifera tends to decline with increasing water depth (Douglas, 1981; Jorissen et al., 1998; Hughes et al., 2000), probably reflecting a decrease in the organic carbon flux to the sea floor. At great depths, carbonate dissolution becomes important (Berger, 1979) and below the Carbonate Compensation Depth (CCD: generally >4000–5500 m, but considerably shallower in some areas around Antarctica), faunas consist almost entirely of taxa with agglutinated or organic tests (Saidova, 1967). Many of them are undescribed soft-walled forms belonging to groups such as the Komokiacea (Tendal and Hessler, 1977; Schro¨ der et al., 1989; Gooday, 1990) which disintegrate rapidly after death. Foraminifera play an important role in deep-sea ecology, for example, by processing of fresh organic material deposited on the sea floor (Moodley et al., 2002), as prey for other organisms (Gooday et al., 1992), and by providing habitat structure (Levin, 1991). The use of benthic foraminifera in palaeoceanography is based on ecological observations made at spatial scales ranging from centimetres (e.g. sediment microhabitats) to 100–1000 km2 (regional distributions). One overriding factor, the organic matter flux to the sea floor, pervades much of the recent literature on deep-sea foraminiferal ecology (Jorissen, 1999). The organic flux delivers food to the benthos. It is also inversely related to bottom-water oxygenation and controls oxygen profiles and other geo- chemical gradients within the sediment. These, in turn, influence foramini- fera and other sediment-dwelling organisms. In some areas, regional faunal patterns also clearly reflect other factors, notably the imprint of bottom- water hydrography.

4.2. Small-scale patterns

During the 1980s, it was recognised that species tend to occupy distinct horizontal levels within the sediment profile rather than being confined to the surface layer (Basov and Khusid, 1983; Corliss, 1985; Gooday, 1986). Various terms have been used to categorise these microhabitats; for example, epifaunal (0–1 cm), shallow (0–2 cm), intermediate infaunal (1–4 cm), transitional (0–4 cm), deep infaunal (>4 cm) (Corliss, 1991; Rathburn and Corliss, 1994; Rathburn et al., 1996; Mackensen, 1997). Jorissen (1999) considers these schemes too rigid and recognises instead four basic patterns: (1) type A – population maximum near sediment surface, (2) BENTHIC FORAMINIFERA 9 type B – fairly stable populations in the upper part (several centimetres) of the sediment column followed by a fairly sharp decline in deeper layers, (3) type C – one or more subsurface maxima, (4) type D – an irregular pattern with a surface maximum and one or more subsurface maxima. Comparisons between these faunal patterns and geochemical profiles suggest that they reflect differential species responses to geochemical gradients (e.g. pore-water oxygen, H2S) within the sediment and therefore, ultimately, the flux of organic matter to the sea floor. Other factors that may be involved in controlling foraminiferal microhabitats, but for which there is little direct evidence, include the intensity of competitive interactions, the redistributing effects of bioturbation, the creation of microhabitats by burrowing macro- and mega-fauna, and possibly sequences of different bacterial food types related to redox boundaries (Moodley et al., 1998b; Jorissen 1999; Scho¨ nfeld, 2001; Fontanier et al., 2002). Foraminiferal microhabitats are not necessarily static (Linke and Lutze, 1993). Direct observations of specimens in aquaria (e.g. Gross, 2000), and analyses of carbon isotopes in carbonate shells (Mackensen et al., 2000), indicate that some deep-sea species move within the sediments. Species that are deeply infaunal in well-oxygenated settings occur close to the sediment surface in eutrophic, oxygen-depleted environments (Mackensen and Douglas, 1989; Kitazato, 1994; Rathburn and Corliss, 1994). Infaunal species also move up and down in the sediment in response to seasonal fluctuations in the food supply and corresponding changes in the depth of the oxygenated layer (Barmawidjaja et al., 1992; Kitazato and Ohga, 1995; Ohga and Kitazato, 1997). These field observations are supported by laboratory studies such as those of Nomaki (2002) and Nomaki, pers. comm. who demonstrated that infaunal species from Sagami Bay, Japan (1426 m water depth) migrate vertically within the sediment profile following a food pulse. These movements may be responses either to the availability of food at the sediment surface, or to changes in oxygen concentrations within sediment pore-waters. The experiments of Heinz et al. (2002), using sediment from 919 m water depth in the Mediterranean Sea, suggest that oxygen availability is the main factor. They found that, when pore-water oxygen levels remained constant, foraminiferal distributions did not change following a food pulse. Earlier experiments based on samples from coastal waters also suggested that shallow-water, infaunal species respond to changing oxygen gradients (Alve and Bernhard, 1995; Moodley et al., 1998b). These kinds of observations, and the earlier studies of Shirayama et al. (1984), Corliss and Emerson (1990) and Loubere et al. (1993), were conceptualised in the TROX model of Jorissen et al. (1995) which relates microhabitat occupancy to a balance between the relative availability of food and oxygen (Figure 1). According to this model, oligotrophic systems are food limited and species are concentrated near the surface where most of 10 ANDREW J. GOODAY

Figure 1 TROX model of Jorissen (1999; based on Jorissen et al., 1995), combined with parabolic curve depicting changes in local species diversity with increasing productivity (Levin et al., 2001, Figure 10A therein). Diversity is depressed in highly oligotrophic areas, such as the ice-covered central Arctic Ocean (Wollenburg and Mackensen, 1998) and the modern eastern Mediterranean Sea (Schmiedl et al., 1998), where the food supply is too low to sustain many species. Diversity is highest in well-oxygenated bathyal and abyssal settings, for example Porcupine Seabight and Porcupine Abyssal Plain (Gooday et al., 1998). Diversity is again depressed in highly eutrophic areas such as the Arabian Sea OMZ (Oman and Pakistan margins) and Santa Barbara Basin (Gooday et al., 2000) where stress caused by oxygen depletion eliminates many species. Local species diversity will also be influenced by other factors, such as disturbance of the sediment surface by current flow and the size of the regional species pool. The diagram also shows approximate levels of foraminiferal standing crops (straight diagonal line) in these different settings. High densities in eutrophic regions are believed to reflect an abundance of food combined with reduced macro- and mega-faunal predation. When oxygen depletion becomes very severe, densities fall again to low values (not shown). This BENTHIC FORAMINIFERA 11 the food is located. Eutrophic systems are oxygen limited and species are concentrated near the surface into order to avoid anoxic conditions deeper in the sediment profile. Maximum penetration is found in intermediate (‘mesotrophic’) settings where both food and oxygen are available well below the sediment/water interface. This basic scheme has been refined by Jorissen et al. (1998), Jorissen (1999), van der Zwaan et al. (1999) and Fontanier et al. (2002) who make the following suggestions: (1) the organic flux is the pre-eminent parameter controlling foraminiferal microhabitats; (2) Oxygen is not a limiting factor for deep infaunal (Type C) species that occur below the subsurface oxic/anoxic interface. These species may be more closely linked to subsurface accumulations of organic matter (Rathburn and Corliss, 1994) or to populations of anaerobic bacteria associated with redox boundaries (Jorissen et al., 1998; Fontanier et al., 2002), (3) Biological interactions, particularly competition for labile food material, play a role in determining where foraminifera live within the sediment profile. The TROX model and its successors provide a useful framework for understanding how various factors may interact to control foraminiferal microhabitats, although they are qualitative and cannot be used to reconstruct values for parameters such as organic fluxes directly. Corliss and colleagues (Corliss, 1985, 1991; Corliss and Chen, 1988; Roscoff and Corliss, 1991; Rathburn and Corliss, 1994) related microhabitat preferences to calcareous test morphotypes. (1) ‘‘Epifaunal’’ species (those living in the top 1 cm of sediment, i.e. shallow infaunal of some authors) tend to have either milioline coiling, trochospiral tests with rounded, plano- convex or biconvex shapes and pores either absent or confined to one side of the test (Figures 2A–F, 3H–I). (2) Infaunal species (those living at >1 cm depth) tend to have tests that are rounded and planispiral or flattened ovoid, flattened tapered, tapered and cylindrical or spherical in shape with pores present all over the test (Figure 3A–G). There are many exceptions to these generalisations, and microhabitats cannot always be predicted from morphotypes (Jorissen 1999), but assignments seem to be accurate in most (75%) cases (Buzas et al., 1993). Thus the linkage between test morphotypes and microhabitats, although imperfect, provides a basis for analysing relationships between foraminiferal faunas, depth in the sediment, and hence food and oxygen availability. version of TROX model reproduced from ‘‘Modern Foraminifera’’ (editor B.K. Sen Gupta), 1999, p. 175, Benthic foraminiferal microhabitats below the sediment–water interface, F. Jorissen, Figure 10.9, with kind permission of Kluwer Academic Publishers. The original version of the TROX model was published in Marine Micropaleontology Vol. 26, F.J. Jorissen, H.C. de Stigter, J.G. Widmark, A conceptual model explaining benthic foraminiferal microhabitats, pp. 3–15, 1995, with permission from Elsevier Science. 12 ANDREW J. GOODAY

Figure 2 Light photographs taken using the PalaeoVision system. A, B. Cibicidoides wuellerstorfi (Schwager). C, D. Hoeglundina elegans (d’Orbigny), from BENBO Site A, 52 54.10N, 16 55.30W, 3576 m depth. E, F. Epistominella exigua (Brady), from Madeira Abyssal Plain, 31 5.500 N, 21 10.00W, 4940 m depth. BENTHIC FORAMINIFERA 13

Figure 3 Light photographs taken using the PalaeoVision system. A. Globobulimina auriculata (Bailey), from Oman margin, 19 18.70N, 58 15.50E, 662 m water depth. B, C. Melonis barleeanum (Williamson), BENBO Site B, 57 25.60N, 15 41.00W, 1100 m depth. D. Chilostomella oolina Schwager, from Oman margin, 19 14.10N, 58 31.30E, 1254 m depth. E. Trifarina angulosa (Williamson), from BENBO Site C, 57 06.00N, 12 30.80W, 1926 m depth. F. Rectuvigerina cylindrica (d’Orbigny), Oman margin, 19 22.180N, 58 11.440E, 95 m depth. G. Bulimina aculeata d’Orbigny, from Antarctic Peninsula shelf, 65 100S, 64 460W, 560 m water depth. H.I. Nuttallides umbonifer (Cushman), from Madeira Abyssal Plain, 31 5.500N, 21 10.00W, 4940 m depth. 14 ANDREW J. GOODAY

4.3. Regional patterns

At regional scales, foraminiferal species distributions are influenced by a variety of environmental factors, including temperature, salinity, food and oxygen availability, sediment type, current and wave action (Murray, 1991). These often vary spatially and temporally, particularly in complex, energetic, continental shelf and coastal settings, making it difficult to identify straightforward, predictable relationship between species and single parameters. In the deep sea, where the physico-chemical environment is generally more uniform, it is often easier to recognise the influence of a few variable parameters on foraminiferal distributions (Murray, 2001). During the last two decades, the view has become popular that the organic matter flux to the ocean floor is a crucial parameter in this food-limited environment (e.g. Grassle and Morse-Porteous, 1987; Nees and Struck, 1999; Loubere and Fariduddin, 1999b; van der Zwaan et al., 1999; Wollenburg and Kuhnt, 2000; Morigi et al., 2001). Both the intensity of the flux and its seasonal variations appear to be important (Loubere and Fariduddin, 1999a). Work conducted in the 1970s and 1980s off the NW African margin by G.F. Lutze and colleagues at Kiel University (Germany) generated a vast body of faunal data and played a major part in the development of this paradigm (Lutze, 1980; Lutze and Coulbourne, 1984; Lutze et al., 1986; Altenbach, 1988; Altenbach and Sarnthein 1989; Altenbach et al., 1999). Earlier researchers also made contributions but based on much smaller databases (e.g. Osterman and Kellogg, 1979; Sen Gupta et al., 1981; Miller and Lohmann, 1982). Where organic fluxes are high, or circulation restricted, oxygen depletion in the bottom water and sediment pore water becomes a significant ecological factor. Foraminifera are more tolerant of oxygen depletion than most metazoan taxa (Josefson and Widbom, 1988; Moodley et al., 1997), but the degree of tolerance varies substantially between species. Tolerant species usually have ‘‘infaunal’’ morphologies and occur in deeper, oxygen- depleted or anoxic sediment layers. In dysoxic, organically enriched settings, epifaunal/shallow infaunal species disappear and deep infaunal species take advantage of the enhanced food supply and reduced macrofaunal predation to develop dense, low-diversity populations close to the sediment–water interface. The current emphasis on food and oxygen availability should not obscure the impact of other factors on foraminiferal species distributions, particu- larly on continental slopes. Mackensen et al. (1993), Mackensen (1997) and Schnitker (1994) focus on the influence of hydrography and suggest that epifaunal species assemblages reflect the characteristics of bottom-water masses. This is particularly true of foraminifera living on ‘elevated epifaunal’ microhabitats above the sediment surface. In the deep sea, substrates include BENTHIC FORAMINIFERA 15 stones, manganese nodules (Mullineaux, 1987; Scho¨ nfeld, 2002a), sponges, hydroids, corals and other sessile animals (Lutze and Thiel, 1989; Rogers, 1999; Beaulieu, 2001), and even mobile animals such as pycnogonids and isopods (Linke and Lutze, 1993; Svavarsson and O´ lafsdo´ ttir, 2000). These species are in direct contact with the bottom water and clearly respond to hydrographic factors, particularly current flow and the associated flux of food particles. Other parameters that may help to explain deep-sea species distributions include sediment characteristics (e.g. grain size and porosity), temperature, water depth (i.e. hydrostatic pressure) (Hermelin and Shimmield, 1990; Kurbjeweit et al., 2000; Hayward et al., 2002) and the disturbance of sediment communities by ‘‘benthic storms’’, turbidity currents and volcanic ash falls (Kaminski, 1985; Hess and Kuhnt, 1996; Hess et al., 2001). Biotic factors are also likely to play a role. Predation, for example, may limit foraminiferal standing stocks in areas where deposit feeders are abundant (Douglas, 1981; Buzas et al., 1989).

5. FAUNAL APPROACHES TO RECONSTRUCTING PALAEOCEANOGRAPHY

Observations made at these different spatial scales contribute to the use of foraminifera in palaeoceanographic reconstructions. Faunas are usually analysed at the species level and abundance patterns attributed to the influence of one or more environmental factors. This approach is easily applicable to Quaternary sediments where extant species are common. Analyses of test morphotypes and diversity parameters can also yield information about palaeoenvironments and are particularly useful in older deposits where most species are extinct. In addition to these qualitative approaches, a considerable effort has been devoted to developing foraminiferal proxies for key environmental factors, particularly organic carbon fluxes to the sea floor (Mackensen and Bickert, 1999; Wefer et al., 1999; Weinelt et al., 2001). In the following sections, I review some of the environmental attributes that are believed to control the abundance, composition and diversity of foraminiferal assemblages. Faunal indicators that have proved useful for reconstructing these parameters are summarised in Table 1. Some are related to bottom-water hydrography, others either directly or indirectly to the organic flux to the sea floor. In all cases, a central problem concerns the development of reliable, quantitative relationships (transfer functions) between environmental parameters and faunal attri- butes. The review focusses on parameters that are used widely in palaeoceanographic studies and is not intended to be comprehensive. 16

Table 1 Characteristics of benthic foraminiferal faunas that have been used in palaeoceanographic reconstructions.

Environmental parameter/ Faunal indicator Remarks References property

Surface primary productivity/ Abundance of foraminiferal Transfer function links Herguera and Berger (1992) organic matter flux to tests >150 mm ‘‘benthic foraminiferal sea floor accumulation rate’’ (BFAR) to productivity Organic matter flux to sea floor Assemblages of ‘‘high produc- Assemblages indicate high Sarnthein and tivity’’ taxa (e.g. organic matter flux to Altenbach (1995); Globobulimina, Melonis) sea floor, with or without Altenbach et al. (1999) corresponding decrease in oxygen concentrations; high percentages of some species characteristic of particular flux ranges Organic matter flux to sea floor Ratio between infaunal and Infaunal morphotypes tend to Corliss and Chen (1988) epifaunal morphotypes dominate in high produ- ctivity areas Surface ocean productivity and Principle components analysis Requires large dataset for Loubere (1991, 1994, 1996); organic carbon flux to of species abundance data calibration Loubere and Fariduddin sea floor (1999a) Seasonality in organic matter Relative abundance of ‘‘phyto- Reflects seasonally pulsed Thomas et al. (1995) flux detritus species’’ inputs of labile organic GOODAY J. ANDREW matter to sea floor Seasonality in surface ocean Discriminant function Loubere (1998); Loubere and productivity and organic analysis of assemblage data Fariduddin (1999a) carbon flux to sea floor from E. Pacific Ocean (low seasonality) and Indian Ocean (highly seasonal) Oxygen-deficient bottom- and (i) Characteristic species asso- (i) Species not consistently (i) Sen Gupta and Machain- FORAMINIFERA BENTHIC pore-water ciations associated with particular Castillo (1993), Bernhard range of oxygen concen- et al. (1997) trations and also found in high productivity areas (ii) Transfer function based on (ii) Proportion of different (ii) Kaiho (1991, 1994, 1999); relative frequency of infau- morphotypes also related Van der Zwaan et al. nal and epifaunal morpho- to organic flux (in Kouwenhoven, 2000) types (iii) Patterns of species diver- (iii) Oxygen-deficient environ- (iii) Den Dulk et al. (1998); sity and dominance ments characterised by Gooday et al. (2000) low diversity/high domi- nance assemblages CaCO3 corrosive bottom Abundance of Nuttallides Distribution of N. umbonifer (i) Mackensen et al. (1995) water/oligotrophic umbonifer linked to (i) corrosive bot- (ii) Loubere (1991) conditions tom water (broadly corre- sponds to Antarctic Bottom Water); (ii) highly oligo- trophic conditions. Current flow Characteristic associations of Species are suspension feeders Mackensen et al. (1995); sessile epifaunal species that capture food particles Scho¨ nfeld (1997, 2002a,c) living on raised substrates advected by currents Water depth (i) Bathymetric ranges of abun- (i) Ranges depend on organic (i) Phleger (1960); Phlumm and dant species in modern matter fluxes to sea floor Frerichs (1976); Culver oceans and therefore largely local, (1988) although broad distinction between shelf, slope and abyssal depth zones is pos- sible. (ii) Ratio between planktonic (ii) Ratio is independent of flux (ii) Van der Zwaan et al. (1990, and benthic tests intensity; estimates become 1999) less accurate with increasing 17 water depth 18 ANDREW J. GOODAY

In particular, sediment characteristics, temperature, substrate disturbance, and biotic interactions are not treated in detail.

6. ORGANIC MATTER FLUXES

6.1. General considerations

The search for proxies of particulate organic matter (POM) fluxes to the sea floor is a major goal in palaeoceanography. Much of the recent geologically orientated research on deep-sea benthic foraminifera has addressed this issue (e.g. Jorissen et al., 1998; papers in Jorissen and Rohling, 2000; Morigi et al., 2001). On continental margins, refractory organic material is transported down the continental slope by various mechanisms, including nepheloid layers, turbidity currents and down- canyon currents. A large proportion of the labile POM arriving at the ocean floor, however, originates from phytoplankton primary production in the overlying water column. This is particularly true in central oceanic areas where the POM flux largely reflects the intensity of surface primary production and lateral advection from slope and shelf areas is not a significant factor. The material that settles out below the zone of winter mixing constitutes the long-term export production to the ocean interior (Berger and Wefer, 1990). In open-ocean settings, only a small fraction (0.01–1.0%) of this exported material reaches the bottom and this fraction decreases with increasing water depth (Suess, 1980; Berger et al., 1988, 1989; Berger and Wefer, 1992). The flux at 2000 m shows a linear relation with levels of primary production below production levels of 200 g CmÀ2 yÀ1, but at higher levels the flux remains constant, for reasons that are not well understood (Lampitt and Antia, 1997). Although the complex processes by which organic matter derived from surface production is delivered to the ocean floor (‘bentho-pelagic coupling’) are understood in general terms, actual rates of supply are more difficult to determine accurately (Berger and Wefer, 1992; Murray, 2001). Estimates are often derived from empirical equations that incorporate primary produc- tion, export production, and flux rate data obtained from sediment traps (Suess, 1980; Pace et al., 1987; Berger et al., 1988, 1989; Berger and Wefer, 1990, 1992). These parameters are not necessarily well constrained. In particular, primary productivity estimates may vary by a factor of 2–3 and exhibit considerable variability, both spatially and temporally (Berger et al., 1988; Herguera, 2000). Oxygen fluxes across the sediment–water interface, obtained by measuring either sediment pore water oxygen profiles or sediment community oxygen consumption (SCOC), provide a more direct BENTHIC FORAMINIFERA 19 and time-averaged measure of POM fluxes (e.g. Loubere et al., 1993; Graf et al., 1995; Jahnke, 1996, 2002; Rowe et al., 1997; Sauter et al., 2001). Even this approach is not without problems since oxygen fluxes reflect inputs of refractory carbon (e.g. redeposited material) of limited nutritional value to foraminifera, as well as labile material. These data are still relatively scarce, although they can be extrapolated using other measures as proxies (Jahnke, 1996). Thus, despite considerable improvements in our knowledge of ocean- wide and global patterns of POC fluxes, values at particular localities will often be subject to substantial uncertainties, a fact that complicates the task of calibrating flux proxies (Berger et al., 1994). Another complicating factor is that primary production and export flux usually have a more or less distinct seasonal component (Berger and Wefer, 1990; Lampitt and Antia, 1997) that is transmitted down through the oceanic water column (Asper et al., 1992; Turley et al., 1995), leading to the seasonally pulsed deposition of phytodetritus on the sea floor (Billett et al., 1983). In the temperate abyssal NE Atlantic Ocean, these deposits deliver an estimated 2–4% of spring-bloom production to the benthos (Turley et al., 1995). The strength of seasonality in the vertical flux is related to the nature of the pelagic ecosystem (Lampitt and Antia, 1997), i.e. the ‘‘plankton climate’’ provinces of Longhurst (1996, 1998). It is most intense at high latitudes and least intense in tropical regions (Fischer et al., 1988; Berger and Wefer, 1990; Wefer and Fischer, 1991; Ramseier et al., 1999). Berger and Wefer (1990) suggest that export production is higher in strongly seasonal systems compared with more constant ones, although this is not confirmed by sediment trap data (Lampitt and Antia, 1996).

6.2. Reconstructing annual flux rates

6.2.1. Species abundances

Total foraminiferal standing stocks reflect food availability (Phleger, 1964, 1976; Douglas, 1981) while particular species tend to be associated with either higher or lower levels of organic flux (e.g. Lutze, 1980; Rathburn and Corliss, 1994; Mackensen, 1997; Altenbach et al., 1999; Fontanier et al., 2002). So-called ‘‘high productivity assemblages’’ have received particular attention (Table 2). They occur in areas that receive a strong and relatively continuous input of organic matter, usually derived from intense primary production associated with upwelling, hydrographic fronts, or major rivers discharges (although material from the latter source is usually dominated by refractory material of limited food value). Characteristic taxa include Bulimina spp., Bolivina spp., Cassidulina spp., Chilostomella oolina Schwager 1878, Globobulimina spp., Melonis barleeanum (Williamson), M. zaandami 20

Table 2 Some examples of modern foraminiferal species and assemblages associated with high productivity areas. Ammobaculites agglutinans and Hormosina dentaliniformis are agglutinated, all other species are calcareous.

Area (water depth) Size fraction Oxygen (ml lÀ1) Characteristic species Reference

NW African margin off Cap >250 mm >1.0 Bulimina marginata, Lutze (1980), Barbas & Cap Blanc Chilostomella oolina, Lutze and Coulbourne Globobulimina spp., (1984) Uvigerina peregrina NW Africa off Cap Blanc >150 mm 4.5 Globobulimina pyrula, Melonis Jorissen et al. (1998) barleeanum, Uvigerina peregrina Tropical Atlantic >63 mm 5.0 Alabaminella weddellensis Fariduddin and Loubere (1997) Eastern South Atlantic: >125 mm 2.7–5.1 Bulimina spp., Uvigerina Schmiedl and lower slope off Cunene auberiana, Fursenkoina Mackensen (1997) River (800–2000 m) mexicana, Valvulineria laevigata GOODAY J. ANDREW Lower slope off Cunene River >125 mm 5.2 Melonis spp., U. peregrina, Schmiedl and (3000–4000 m) Globobulimina turgida, Mackensen (1997) Chilostomella oolina, Nonionella opima, Cassidulina reniforme ETI FORAMINIFERA BENTHIC East Pacific Rise >63 mm 3.3–3.7 Hispid Uvigerina; Melonis Loubere (1991) barleeanum Eastern equatorial and North >63 mm 1.8–3.5 A. weddellensis, Bulimina alazi- Loubere (1996) Pacific Ocean nensis, Chilostomella oolina, Globobulimina sp., Sphaeroidina bulloides, Stainforthia sp. NE US slope (350–500 m) >250 mm 3.0 Globobulimina spp., Bulimina Miller and Lohmann (1982) aculeata North Carolina slope off Cape >300 mm 4.0 Globobulimina auriculata Gooday et al. (2001) Hatterras (850 m) dominant; Ammobaculites agglutinans, Hormosina dentaliniformis also important 21 22 ANDREW J. GOODAY

(Van Voorthuyen), Nonionella stella Cushman & Moyer, Sphaeroidina bulloides Deshayes, and Uvigerina spp. (usually U. peregrina Cushman) (Figure 3A–G). High productivity taxa are infaunal and tolerate varying degree of oxygen depletion. Some (e.g. Globobulimina spp., Bolivina spp., Brizalina spp.) withstand dysoxic or anoxic conditions better than others (e.g. Uvigerina spp.) (Miller and Lohmann, 1982; Sen Gupta et al., 1981; Corliss et al., 1986; Rathburn and Corliss, 1994; Bernhard et al., 1997; Schmiedl et al., 1997). Species of Melonis apparently prefer more degraded food material than Bulimina exilis Brady (Caralp, 1989). Evidence from strongly dysoxic or anoxic settings, and from environments where a strong organic flux is combined with well-oxygenated bottom water, suggests that Chilostomella oolina and Nonion scaphum (Fitchel & Moll) are associated with labile organic carbon inputs, Globobulimina affinis (d’Orbigny) and Melonis barleeanum with more refractory material (Fontanier et al., 2002). Laboratory experiments in which algae were added to sediments recovered from the centre of Sagami Bay, Japan, tend to contradict these field observations (Nomaki, 2002; Nomaki, pers. comm.). Another Chilostomella species, C. ovoidea Reuss, did not respond at all whereas G. affinis migrated upwards in the sediment following the addition of food, and ingested fresh algae. In situ feeding experiments at the same locality using 13C labelled algae support these results (Nomaki, 2002; Kitazato et al., in press) and suggest that C. ovoidea and G. affinis may have different diets in Sagami Bay. Other foraminifera, many of them epifaunal or shallow infaunal, are associated with lower flux rates. Such species include Cibicidoides wuellerstorfi (Schwager), Hoeglundina elegans (d’Orbigny), Oridorsalis umbonatus (Reuss), Nuttallides umbonifer (Cushman), Globocassidulina subglobosa (Brady) (Figures 2A–F, 3H–I) (Altenbach, 1988; Sarnthein and Altenbach, 1995; Altenbach et al., 1999; Loubere and Fariddudin, 1999b; Morigi et al., 2001). As discussed below, these species are believed to feed largely on fresh POM and are relatively intolerant of dysoxic conditions. Can species abundances be used as indicators of absolute flux rates? Altenbach et al. (1999) addressed this question by analysing the relationship between flux to the sea floor and percentage species abundances in 382 samples from the equatorial eastern Atlantic to the Arctic. Species occurred over a range of annual flux values spanning between 1 and 3 orders of magnitude, and only 4–64% of total abundance was explained by flux rates. When only high percentage occurrences were considered, however, the range was much smaller. Thus, the abundant occurrence of particular species (presumably reflecting their optimum habitat) may be typical of particular flux regimes (Table 3), although mere occurrences, or even moderate abundances, are of little significance. The percentage abundance of a few species (e.g. Cibicidoides wuellerstorfi in the >250 mm fraction) can be used to BENTHIC FORAMINIFERA 23

Table 3 Relationship between dominant foraminiferal species, organic flux to the seafloor and surface primary production; based on data from NE Atlantic Ocean in Altenbach et al. (1999) supplemented by data from Sarnthein and Altenbach (1995) and Wollenburg and Kuhnt (2000) (Arctic Ocean). Note that all the species included in this table occur in smaller numbers over much wider flux ranges than shown in this table. Cribrostomoides subglobosum and Adercotryma glomeratum are agglutinated, all other species are calcareous.

Organic Primary Typical flux to productivity bathymetric seafloor (g mÀ2yrÀ1) setting (g mÀ2yrÀ1)

Higher-flux species Trifarina fornasinii 10–30 100–300 Inner and outer shelf Uvigerina mediterranea 2–9 150–250 Slope (200–1000 m) Uvigerina peregrina 2–20 100–300 Lower slope (700–2000 m) Hoeglundina elegans 2.5–15 200–280 Lower slope (400–2000 m) Sphaeroidina bulloides 3–12 90–300 Slope (700–1000 m) Bolivina albatrossi 5–15 100–300 Slope (300–1000 m) Cibicidoides 2.5–20 90–300 Slope (250–1500 m) pseudoungerianus Globobulimina spp. >3 Slope Intermediate-flux species Cibicidoides kullenbergi 1–4 80–250 Lower slope/rise (2000–4000 m) Lower-flux species Cibicidoides wuellerstorfi 0.2–3.0 15–100 Lower slope – abyssal 1Pyrgo rotalaria 0.2–2.5 15–200 Lower slope – abyssal Eponides tumidulus <0.4 <10–25 Abyssal 2Epistominella arctica 0.03–2.0 Upper slope, Arctic Ocean 2Stetsonia hovarthi <0.4 <10–25 Abyssal Oridorsalis umbonatus <1.5 Abyssal Nuttallides umbonifera <1.5 Abyssal Species spanning wide flux range Epistominella exigua 0.9–100 Slope-abyssal Melonis zaandami 2–7 Slope-abyssal Cribrostomoides 0.4–10 Slope-abyssal subglobosum Adercotryma glomeratum 0.03–12 Slope-abyssal, Arctic Ocean

1Often reported as Pyrgo murrhina or Pyrgo murrhyna; Pyrgo rotalaria is the senior synonym (Thies, 1991). 2Epistominella arctica and Stetsonia hovarthi are considered synonyms by Scott and Vilks (1991). 24 ANDREW J. GOODAY

À2 estimate flux rates <2 g Corg m with reasonable confidence. In addition, some infaunal species (e.g. Uvigerina mediterranea Hofker 1932) tend to be associated with a specific range of flux values irrespective of their abundance. Sarnthein and Altenbach (1995) and Altenbach et al. (1999) suggest that the À2 annual flux range of 2–3 g Corg m marks an upper threshold of dominance for many species characteristic of oligotrophic abyssal settings and a lower threshold of dominance for species adapted to more eutrophic shelf and bathyal environments. Other authors have also reported evidence for an ecological boundary around the same flux levels (De Rijk et al., 2000; Jian et al., 1999; Morigi et al., 2001; Weinelt et al., 2001).

6.2.2. Morphotype approaches

These approaches rely on the relationship between organic fluxes and the relative abundance of infaunal and epifaunal morphotypes. The use of morphotypes as flux indicators is complicated by the fact that they are also related to oxygen availability, at least at low oxygen concentrations. This is discussed in Section 7. Corliss and Chen (1988) reanalysed the data of Mackensen et al. (1985) from the Norwegian margin (dead assemblage, 0–1 cm layer, >125 mm fraction), assigning the species to either infaunal or epifaunal morphotypes. Both categories occurred between 200 m and 500 m water depth, infaunal morphotypes predominated between 500 m and 1500 m, epifaunal morpho- types were most abundant below this depth (Corliss and Chen, 1988). A similar pattern was observed by Roscoff and Corliss (1991) in the Greenland-Norwegian Sea. Below 800 m depth, these patterns correlated well with the organic carbon content of surface sediments; high organic carbon values were associated with dominance by infaunal morphotypes, low carbon values with epifaunal morphotypes. Corliss and Chen (1988) suggest that the switch from infaunal to epifaunal dominance occurs within À2 the yearly organic carbon flux range 3–6 g Corg m . Despite the imperfect relationship between microhabitats and morphotypes referred to above, the Corliss and Chen (1988) approach can provide a general indication of organic fluxes levels. The quality of the available food is also important. Deep infaunal species living below the level at which oxygen disappears from the sediment pore water apparently consume more degraded organic matter than epifaunal and shallow-infaunal species (Goldstein and Corliss, 1994; Fontanier et al., 2002). The abundance of the former and scarcity of the latter at a site off Cap Blanc (NW African margin) overlain by well- oxygenated bottom water (4.5 ml/l) was attributed by Jorissen et al. (1998) to the lack of freshly deposited labile detritus compared to the relatively large amounts of more degraded material available deeper in the sediment. BENTHIC FORAMINIFERA 25

Thus, as van der Zwaan et al. (1999) conclude, infaunal morphotypes reflect the abundance of organic matter stored within the sediment, rather than the flux of fresh material.

6.2.3. Benthic Foraminiferal Accumulation Rate (BFAR)

The population density and biomass of different components of the deep-sea benthic fauna, from bacteria to megafauna, are related to food availability (Rowe, 1983; Lampitt et al., 1986; Altenbach, 1988; Lochte, 1992; Tietjen, 1992; van der Zwaan et al., 1999; Wollenburg and Kuhnt, 2000; Fontanier et al., 2002). This relationship provides the basis for an equation, proposed by Herguera and Berger (1991), linking the abundance, or more accurately the accumulation rate, of benthic foraminifera (BFAR) to the total organic matter flux reaching the sea floor (Figure 4) (see also Berger and Herguera, 1992; Herguera, 1992). BFAR is the number of foraminiferal tests >150 mm that accumulate per cm2 per 103 years [¼ (no. benthic foraminifera g of dry

Figure 4 Relationship between the benthic foraminiferal (BF) accumulation rate (BFAR) and the organic matter flux to the sea floor (Jsf), based on data from the Ontong Java Plateau. Filled circles are core-top (i.e. modern) samples; diamonds are from sediment deposited during the last glacial maximum; squares are from sediments deposited during the transition from glacial to interglacial conditions. Small white symbols inset into larger black symbols show water depths; the two inset open circles (above the line) indicate depths from 4000–4500 m, the inset open triangles indicate depths >4500 m. Reproduced from Geology Vol. 19, J.C. Herguera and W.H. Berger, Paleoproductivity from benthic foraminifera abun- dance: glacial to post-glacial change in west-equatorial Pacific, p. 1175, Figure 2, with thanks to the Geological Society of America. 26 ANDREW J. GOODAY sedimentÀ1) x (sed. rate in cm kyrÀ1) x (dry bulk density in g cmÀ3)]. The calculation depends on the sedimentation rate being constant during the time interval examined. Struck (1992) used the term INDAR (Individual Accumulation Rate) in much the same sense as BFAR. By applying equations that describe the loss or decomposition of organic particles during their passage through the water column (Herguera and Berger, 1991), BFAR can be used to estimate surface primary productivity, although such estimates involve important assumptions and considerable errors (Herguera, 2000). The BFAR approach appears to work adequately in well-oxygenated sediments (Herguera and Berger, 1991; Nees et al., 1997; Schmiedl and Mackensen, 1997; Nees and Struck, 1999; Herguera, 2000) but fails to yield realistic palaeoproductivity estimates where oxygen depletion is a limiting factor (Naidu and Malmgren, 1995). It can also be compromised by post- mortem taphonomic processes such as the dissolution of calcareous tests (Loubere and Fariduddin, 1999b). For example, Wollenburg and Kuhnt (2000) report that highest BFAR values in the Arctic Ocean were derived from areas under permanent ice where the Corg flux was lowest whereas seasonally ice-free areas subject to carbonate dissolution yielded dispro- portionately low values. Severe dissolution substantially limits the use of BFAR to reconstruct Quaternary and Holocene paleoproductivity in this region (Wollenburg et al., 2001). An additional problem is that BFAR assumes a steady rain of sinking material (Murray, 2001). In fact, a high proportion of the flux may be delivered episodically in the form of phytodetritus aggregates (reviewed by Beaulieu, 2002), larger phytoplankton mats (Kemp et al., 2000), large faecal pellets (Pfannkuche and Lochte, 1993) and animal carcasses (e.g. Christiansen and Boetius, 2000). Because they sink rapidly, these particles have a greater food value and therefore support a higher benthic biomass than more refractory, slowly descending particles. Small (<150 mm), opportunistic foraminifera are often abundant in areas where the organic flux is strongly pulsed. Much of the foraminiferal production supported by this labile organic material will therefore pass through a 150 mm sieve. If finer fractions (<150 mm) are incorporated into BFAR calculations, the organic flux values will be overestimated since small foraminifera have a much smaller biomass than larger ones (Ohkushi et al., 2000). Nevertheless, there is evidence that BFAR values are sensitive to differences in the quality of deposited organic matter. Guichard et al. (1997) found a generally good correlation between BFAR (>150 mm fraction) and flux rates for organic carbon (gC cmÀ2 10À3 years) in a core from the NW African upwelling area. Deviations observed in certain horizons were interpreted as reflecting fluctuations in the quality of organic material reaching the sea floor. BFAR values that were disproportionately high in relation to the Corg flux values were probably due to strong BENTHIC FORAMINIFERA 27 phytodetrital inputs derived from intense spring blooms while dispropor- tionately low BFAR values probably reflected periods when the flux was of poor food quality due to enhanced lateral advection. Schmiedl and Mackensen (1997) also reported deviations from a linear relation between BFAR (>125 mm fraction) and palaeo-export in the eastern South Atlantic. BFAR values during glacial stage 12 were more than ten times higher than interglacial values and translated into unrealistically high palaeoproductiv- ity estimates. These results suggest that opportunistic epifaunal species that respond to phytodetrital pulses add dead shells to the sediments at a higher rate than deeper infaunal species (de Stigter et al., 1999). Refinements of the BFAR method will need to take account of different rates of production (Scho¨ nfeld, 2002b).

6.2.4. Multivariate analysis of assemblage data

Multivariate statistical techniques such as Principal Components Analysis and Factor Analysis have been widely used to extract environmental signals from large fauna datasets (Loubere and Qian, 1997). Loubere and colleagues used multivariate analyses of relative species abundances in surficial sediments to directly estimate ocean surface productivity (reviewed by Loubere and Fariduddin, 1999b). In order to minimise the effects of other environmental parameters, samples were selected from a relatively narrow range of water depths. Regression of modern species abundances against estimated average annual surface productivity, based on synthetic maps, satellite measurements of surface ocean pigment concentrations, and sediment trap data, yielded transfer functions with r2 values of 0.97 (eastern Pacific) and 0.89 (World Ocean). This approach was first developed on a transect along the East Pacific Rise where surface productivity is the only significant variable (Loubere, 1991). It was later applied in the eastern equatorial Pacific (Loubere, 1994), the Atlantic Ocean (Fariduddin and Loubere, 1997), the Indian Ocean (Loubere, 1998) and the World Ocean (Loubere and Fariduddin, 1999a). The functions obtained yielded estimates of surface productivity that could be tested by comparing them with observed values (Loubere, 1994; Loubere and Fariduddin, 1999a). Analyses were based on >63 mm fractions that included small opportunists, making it possible also to differentiate (using Discriminant Function Analysis) between assemblages associated with high and low degrees of seasonality (Loubere, 1998; Loubere and Fariduddin, 1999b). Recently, Loubere (2000) has applied this method to cores in the eastern Equatorial Pacific to infer fluctuations in palaeoproductivity over the last 130,000 years. A similar approach was used by Kuhnt et al. (1999) to investigate organic carbon flux rates in the South China Sea. In this case, 28 ANDREW J. GOODAY

Correspondence Factor Analysis (AFC) of selected species (those with a potential fossil record) from the dead assemblages (>150 mm fraction of surficial box-core sediments) was used to derive a number of factors. Factor 1 depended mainly on the relative abundance of calcareous species and was correlated with water depth and organic carbon flux. Negative values were due to the abundance of high productivity taxa (Uvigerina spp.) while positive values were associated with oligotrophic taxa. Kuhnt et al. (1999) used factor 1 values derived from two gravity cores as a proxy for carbon fluxes in the South China Sea during the Holocene. In a later paper, Jian et al. (2001) reconstructed Late Quaternary changes in monsoon-driven upwelling intensity in the same area based on BF flux values [estimated carbon flux values in g mÀ2 calculated from AFC factor 1 using regression in Kuhnt et al. (1999, Figure 4A)]. Wollenburg and Kuhnt (2000) used AFC to examine the relation between benthic foraminiferal assemblages and organic carbon flux, based on a large faunal dataset from the Arctic Ocean. Again, there was a close correlation between factor 1 and the organic flux, suggesting that this relationship may prove to be a reliable transfer function for palaeoproductivity. Wollenburg et al. (2001) applied it to two sediment cores collected to the north of Svalbard (81–82N) and reconstructed a convincing palaeopro- ductivity record for the last 145 kyr. The interpretation of ancient assemblages using the multivariate approaches requires very large datasets based on modern faunas from which to derive the multiple regression equations. These methods work fairly well if the fossil assemblages have counterparts, or their close equivalents, in the modern calibration dataset (Loubere and Qian, 1997; Wollenburg et al., 2001). However, where there is no counterpart (‘no-analogue’ conditions), substantial errors may occur in the estimation of ancient environmental parameters such as palaeoproductivity. These situations are not easy to recognise and present methods for extrapolating from the calibration dataset to no-analogue conditions are less than satisfactory (Mekik and Loubere, 1999). Also, because the transfer function must be calibrated using modern surface ocean productivity values that are difficult to measure accurately, the multivariate methods are better suited to reconstructing relative changes in palaeoproductivity rather than absolute values (Loubere, 2000). An additional problem is that productivity is estimated over short time scales (a few years at most) whereas dead foraminiferal assemblages in surface sediments represent hundreds or thousands of years of accumulation. There is no certainty that modern productivity values, even if measured accurately, are representative of the entire period over which the dead assemblage has accumulated. BENTHIC FORAMINIFERA 29

6.3. Responses to seasonally varying fluxes

As well as the overall scale of the flux (i.e. the annual flux rate), the degree of seasonality in its delivery to the sea floor is an important parameter (Gooday, 2002; Hayward et al., 2002; Loubere and Fariddudin, 1999b). Seasonal delivery of food leads to temporal fluctuation in the population densities of some benthic foraminiferal species that are not reflected in dead assemblages (Douglas et al., 1980). Indeed, seasonality generally is a difficult parameter to detect in the palaeoceanographic record (Smart et al., 1994; Thomas and Gooday, 1996). Pulsed fluxes of phytodetritus, usually reflecting seasonal surface production, occur in the temperate North Atlantic Ocean (Hecker, 1990b; Rice et al., 1994), the Greenland-Norwegian Sea (Graf et al., 1995), the Southern Ocean (Mackensen et al., 1993), the monsoon-influenced Arabian Sea (Pfannkuche et al., 2000) and the NE Pacific Ocean (Smith and Druffel, 1998). Beaulieu (2002) provides a detailed review of the occurrence, composition and origin of phytodetritus, its geochemical significance and fate on the seafloor. A limited number of small foraminiferal species are physically associated with phytodetrital deposits. In open-ocean areas, where the overall organic matter flux is not too high, the best-known are Epistominella exigua (Brady) (Figure 2E–F) and Alabaminella weddellensis (Earland), both species with cosmopolitan distributions at abyssal depths. In the temperate NE Atlantic Ocean, individuals are commonly found living within phytodetrital aggregates (Gooday, 1988, 1993, 1996). Epistominella exigua is also associated with strong seasonality in primary production in the Indian Ocean (Loubere, 1998). These ‘‘phytodetritus species’’ are probably enrichment opportunists that undergo rapid population increase when presented with a good food supply (Gooday and Rathburn, 1999). Experiments suggest that shallow-water foraminiferal species grow con- tinuously and rapidly when adequate food is available but slowly when food is scarce (Bradshaw, 1961). Deep-water species also undergo rapid population growth when presented with a pulse of algal food under experimental conditions (Heinz et al., 2001, 2002). On bathyal continental margins, where conditions are more eutrophic, other small benthic species respond to phytodetritus. In the NE Atlantic Ocean, the two most common are Nonionella iridea Heron-Allen & Earland and Eponides pusillus Parr (¼ Eilohedra nipponica (Kuwano) of Wollenburg and Mackensen, 1998), both of which are abundant at BENBO Site C (1900 m water depth) and at a 1340-m site in the Porcupine Seabight (Gooday and Lambshead, 1989; Gooday and Hughes, 2002). In the high Arctic, Epistominella arctica Green 1960 is an opportunist that reproduces rapidly during short-pulsed, local phytoplankton blooms (Wollenburg and Kuhnt, 2000). It apparently prefers more oligotrophic conditions than either 30 ANDREW J. GOODAY

E. exigua or Eponides pusillus. Mackensen et al. (1993, 1995) described a Northern High Productivity fauna dominated by Bulimina aculeata d’Orbigny (Figure 3G) from parts of the Southern Ocean characterised by highly seasonal production leading to a large pulse of phytodetritus deposited within a very short period of time. This assemblage occurs at depths <2600 m between the Polar Front and southern boundary of Antarctic Circumpolar Current, an area with fine-grained sediments and low current velocities. In Sagami Bay, Japan (1430 m water depth), calcareous (Bolivina pacifica Cushman & McCulloch, Stainforthia apertura (Uchio)) and agglutinated (Textularia kattegatensis Ho¨ glund) species colonise the phytodetrital layer (Ohga and Kitazato, 1997; Kitazato et al., 2000). Tiny juvenile specimens (2 chambers) were common in the phytodetritus during May but rare at other times of the year, suggesting a reproductive response (Kitazato et al., 2000). Rathburn et al. (2001) reported dramatic increases in the abundance of Nonionella fragilis, corresponding to periods of enhanced surface production, at a 900-m site in the Southern Californian Bight. Other examples were reviewed by Gooday and Rathburn (1999). The recognition of ‘phytodetritus species’ was based largely on detailed studies of fine sieve fractions (>63 or >32 mm) conducted at single sites (Gooday and Rathburn, 1999; Kitazato et al., 2000). Regional studies suggest that Epistominella exigua is adapted to a greater range of productivity values than many deep-sea taxa (Altenbach et al., 1999), possibly reflecting its opportunistic life history. Because of its small size (mean test diameter 120–130 mm), the apparent distribution of this species is strongly influenced by the sieve fraction analysed (Kurbjeweit et al., 2000). Generally, it avoids areas of high productivity. In the South Atlantic, an E. exigua assemblage is associated with the core of highly saline North Atlantic Deep Water (NADW) above the lysocline. It typically replaces high productivity species in areas where the organic flux is diminished, and is replaced in turn by the Nuttallides umbonifer assemblages in highly oligotrophic regions between the lysocline and the CCD (Mackensen et al., 1993, 1995). Off SW Africa, the E. exigua assemblage coincides with areas of low and seasonally fluctuating organic matter fluxes on the flanks of Walvis Ridge and lower part of the continental slope (Schmiedl et al., 1997). This species appears to prefer lower carbon flux levels in the SW Pacific Ocean east of New Zealand (Hayward et al., 2002). Kurbjeweit et al. (2000) recognised an E. exigua assemblage in the western, northern and central Arabian Sea. Population densities fluctuated seasonally and were positively correlated with the organic carbon flux. Epistominella exigua is abundant in the eastern equatorial Pacific Ocean (Loubere, 1994, 1996) where phytodetritus deposition is spread over a longer time period and is not strongly seasonal (Smith, 1994; Smith et al., 1996). Thus E. exigua appears to flourish in areas where a good supply of fresh phytodetritus, seasonally pulsed or otherwise, is combined with a BENTHIC FORAMINIFERA 31 modest annual flux intensity. Other ‘‘phytodetritus species’’ may have rather different ecological requirements. Statistical analyses of samples from the Atlantic (Fariduddin and Loubere, 1997), Pacific (Loubere, 1996; Hayward et al., 2002), and Indian (Loubere, 1998) Oceans tend to group Alabaminella weddellensis with high productivity species, rather than with E. exigua. Fossil occurrences support the view that A. weddellensis and E. exigua have different ecological characteristics (Nees and Struck, 1999; Okhushi et al., 2000). Species do not always exhibit the same response to pulsed food inputs across their entire range. Epistominella arctica is an opportunist in the High Arctic (Wollenburg and Kuhnt, 2000) but at the temperate BENBO Site C it shows only a modest numerical increase in post-bloom (July 1998) samples with phytodetritus compared to pre-bloom (May 1998) samples devoid of phytodetritus, and is never associated directly with these deposits (Gooday and Hughes, 2002). If this tiny species is adapted to very low productivity combined with extremely short pulses of phytodetritus, as suggested by Thomas et al. (1995), then conditions at Site C are probably not ideal for it. Similarly, Stainforthia fusiformis (Williamson) occurs in relatively low number in ‘‘live’’ assemblages at this seasonal bathyal site but is reported to be highly opportunistic in shallower, continental shelf settings (Alve, 1994). These observations suggest that foraminiferal species may exhibit different life-history characteristics in different areas. Where conditions are optimal, they may react opportunistically to a fluctuating food supply. Near the edges of their range, however, factors close to the tolerance limit exert strong controls that dampen opportunistic responses.

6.4. Are calcareous species more responsive than other foraminifera?

In general terms, hyaline calcareous foraminifera (orders Rotaliida and Buliminida) appear to be more closely linked to organic matter fluxes than agglutinated and allogromiid taxa. This probably explains the generally observed decrease in abundance of calcareous taxa with increasing water depth beyond the shelf break (Jorissen et al., 1998; Hughes et al., 2000; Kurbjeweit et al., 2000). Many phytodetrital species are rotaliids (Gooday, 1988; Gooday and Lambshead, 1989; Gooday and Hughes, 2002). At two bathyal NE Atlantic sites, the Porcupine Seabight and BENBO Site C (1345 m and 1960 m water depth respectively), hyaline species increased in absolute and relative abundance in samples collected in the summer (i.e. after the spring bloom) compared with samples collected in the spring (before the bloom) (Figure 5). There was no increase, however, in the case of agglutinated and allogromiid foraminifera; indeed, some groups (saccamminids/psammosphaerids, hormosinaceans, Lagenammina spp.) 32 ANDREW J. GOODAY

Figure 5 Percentage abundance of stained benthic foraminiferal taxa in multi- corer samples (0–1 cm layer >63 mm fraction) collected during spring (light ornamentation) and summer (dark ornamentation). Upper panel: Porcupine Seabight (51 360N13000W; 1345 m water depth); spring samples from April 1983, summer samples from July 1983. In each case, values are means of 7 samples; asterisks indicate significant differences ( p<0.05) between live and dead abun- dances. Lower panel: BENBO Site C (57 07.500N1230.300W; 1950 m water depth); Single samples from May 1998 and July 1998. Note that in both cases the rotaliids are the only group to show a substantial increase in abundance after the spring bloom. were more abundant in the spring than in the summer. In a study of foraminifera colonising artificial substrates on Cross Seamount (water depth 800–2000 m) in the Central Pacific, Bertram and Cowen (1999) reported that agglutinated taxa settled on the plates at a uniform rate whereas the settlement rates of other foraminifera (predominately calcareous) varied over time. Higher rates corresponded to periods of enhanced particle flux. Since taphonomic processes usually (although not always) lead to the destruction of many agglutinated foraminifera, these observation imply that the ‘‘productivity signal’’ conveyed by calcareous taxa will be enhanced in the palaeoceanographic record (Gooday and Hughes, 2002). In order to utilise the food available in organically enriched areas, calcareous foraminifera are often exposed to oxygen-depleted bottom water or sediment pore water. As a group, they tolerate these conditions better than most soft-shelled and other non-calcareous foraminifera (Moodley BENTHIC FORAMINIFERA 33 et al., 1997; Gooday et al., 2000). However, calcareous foraminifera living in low-oxygen environments must also withstand the acidic conditions often associated with organic enrichment. In contrast, metazoans with calcareous hard parts (e.g. molluscs and echinoderms) are often rare in dysoxic environments, presumably because they find it difficult to prevent the dissolution of their shells and skeletons (Rhoades and Morse, 1971; Thompson et al., 1985).

7. OXYGEN CONCENTRATIONS

7.1. General considerations

Modern oceans are generally well oxygenated and persistent large-scale oxygen depletion is confined to coastal, continental shelf and slope settings. These extreme conditions are usually associated with high productivity or with weak bottom-water circulation; for example, off large rivers, Oxygen Minimum Zones (OMZs), silled basins and fjords (Diaz and Rosenberg, 1995). Bottom-water oxygen concentrations are tightly coupled with organic matter fluxes, making it difficult to separate the effects of these two variables on benthic communities (Levin and Gage, 1998). Many of the ideas about how oxygen affects faunal parameters have been developed by macrofaunal ecologists (e.g. Sanders, 1969; Pearson and Rosenberg, 1978; Levin and Gage, 1998; Levin et al., 2000, 2001, 2002). Macro- and mega-faunal animals usually live on the sediment surface, or can extend body parts above the sediment–water interface, and are directly affected by oxygen concentrations in the bottom water. Foraminifera attached to elevated substrata are also in direct contact with bottom water (Lutze and Thiel, 1989; Scho¨ nfeld, 2002a, 2002c). However, in the case of foraminifera and other meiofauna living within the sediments, it is the concentration of oxygen in the sediment pore water that matters (Gooday et al., 2000; Pike et al., 2001; Scho¨ nfeld, 2001). Because of oxygen consumption within the sediment, deeper infaunal species will typically encounter dysoxic or anoxic conditions, even when the bottom water is well oxygenated (Murray, 2001; Fontanier et al., 2002). Unlike organic matter fluxes, it is relatively easy to measure sediment pore-water oxygen profiles directly and accurately, although processes such as bioturbation, and the possibility that foraminifera extend their pseudopodia into overlying, more strongly oxygenated sediment layers (Bernhard and Sen Gupta, 1999), make it difficult to determine the amount of oxygen actually available to individual specimens. 34 ANDREW J. GOODAY

There is considerable debate about the importance of oxygen as a limiting factor for foraminifera. Early distributional studies indicated that changes in the abundance of certain species was linked to bottom-water oxygen values within the range 4–6 ml lÀ1 (e.g. Lohmann, 1978; Streeter and Shackelton, 1979). Hayward et al. (2002) consider that the dissolved oxygen content of bottom waters was an important influence on benthic foraminiferal distributions at sites in the SW Pacific where values ranged from 3.7 to 6.2 ml lÀ1. The large-scale studies of Kaiho (1994), and small- scale studies of foraminiferal occurrences in relation to sediment pore-water oxygen profiles (Scho¨ nfeld, 2001), suggested that some oxyphilic species have lower oxygen tolerance limits in the region of 1.5 and 3.0 ml l À1.On the California Borderland, four Bolivina species exhibited distinct patterns of distribution in relation to a bottom-water oxygen gradient ranging from <1 ml lÀ1 to 5–6 ml lÀ1 (Douglas, 1979, 1981). Those associated with lower oxygen concentrations had thinner walls, less ornamentation, and more compressed cross-sectional outlines than those from better oxygenated sites. In contrast, the level at which oxygen concentrations begin to affect the community structure of foraminifera and other benthic organisms is <1 ml lÀ1 and perhaps considerably less than this value (Jorissen et al., 1995; Bernhard et al., 1997; Levin et al., 2000, 2001). Below this threshold, increasing physiological stress associated with progressively lower oxygen concentrations (Moodley et al., 1997), sometimes combined with the toxic effects of sulphides (Moodley et al., 1998a; Bernhard, 1993), acts as a barrier to many species (van der Zwaan et al., 1999). Although foraminifera disappear completely when bottom-water anoxia is permanent or persists for very long periods (Alve, 1990; Bernhard and Riemers, 1991; Moodley et al., 1998b), there is increasing evidence that some species can reside in anoxic sediment layers (Loubere et al., 1993; Rathburn and Corliss, 1994; Jannink et al., 1998; Jorissen et al., 1998; Jannink, 2001; Fontanier et al., 2002). Experiments have demonstrated tolerance of anoxia for considerable periods of time, tolerance to sulphidic conditions for periods of weeks (Moodley and Hess, 1992; Bernhard, 1993; Moodley et al., 1998a; Bernhard and Sen Gupta, 1999; van der Zwaan et al., 1999), and subsurface movement through anoxic sediments (Moodley et al., 1998b). A possible mechanism for tolerating sulphides is the development of endosymbiotic relationships with sulphide-oxidising bacteria (Bernhard and Sen Gupta, 1999; Bernhard, 2002). Deep-infaunal species living in anoxic layers often develop large populations. For these species, food rather than oxygen availability seems to be the main agent controlling abundance (Fontanier et al., 2002). Different tolerances to oxygen depletion create successions of forami- niferal species along gradients of oxygen concentrations (Bernhard et al., 1997). There are corresponding changes in community parameters. BENTHIC FORAMINIFERA 35

As oxygen concentrations decrease, abundance and dominance increase, species richness decreases, the proportion of hyaline calcareous taxa increases and the proportion of agglutinated and allogromiid taxa decreases (Phleger and Soutar, 1973; Phleger, 1976; Douglas, 1981; Mullins et al., 1985; Alve, 1990; Perez-Cruz and Machain-Castillo, 1990; Sen Gupta and Machain-Castillo, 1993; Gooday et al., 2000; Levin et al., 2002). Many of the calcareous species have small, thin-walled tests. Similar community trends are reported for macrofaunal metazoans and seem to reflect the distinct but interwoven influences of food and oxygen availability (Levin and Gage, 1998; van der Zwaan et al., 1999; Levin et al., 2000, 2002). Species richness is probably related to oxygen concentrations; i.e. dysoxia eliminates the more oxyphilic species (Figure 6). The sharp reduction in species numbers observed in low-oxygen settings probably also reflects the toxic effects of hydrogen sulphide where this is present (Moodley et al., 1998a). Dominance is influenced mainly by food availability; i.e. a few dysoxia-tolerant species flourish in response to an abundant food supply. The absence of macro- and mega-faunal animals in severely dysoxic regions may also facilitate the development of large foraminiferal populations (Phleger and Soutar, 1973; Douglas, 1981). Reduced predation and competition pressure, combined with an enhanced food supply, has been proposed as an explanation for the high abundance of nematodes (meiofaunal metazoans) where oxygen concentrations fall below 0.2 ml lÀ1 on the Peru Margin (Neira et al., 2001).

7.2. Qualitative approaches

Many of the foraminifera found in oxygen-depleted, organically enriched settings belong to the calcareous orders Rotaliida (particularly the families Chilostomellidae and Nonionidae) and Buliminida (reviewed by Sen Gupta and Machain-Castillo, 1993; Kaiho, 1994; Bernhard, 1996; Bernhard and Sen Gupta, 1999; Holbourn et al., 2001a). In modern environments, they often have small, thin-walled tests characterised by a variety of infaunal morphologies; these include flattened, elongate biserial/triserial (e.g. Bolivina, Bulimina, Fursenkoina, Stainforthia, Uvigerina), planispiral/lenti- cular (e.g. Cassidulina, Chilostomella, Epistominella, Lenticulina, Nonion, Nonionella), or globular (e.g., Globobulimina) (Figure 3A–G). Buliminid and bolivinid morphotypes occur in organically enriched sediments as far back as the mid- and Late Cretaceous (Holbourn et al., 1999, 2001a,b). Miliolids are rarely reported but certain agglutinated taxa (e.g. Bathysiphon spp., Reophax spp., Trochammina spp.) may be fairly common in modern OMZ settings, provided dysoxia is not too intense (i.e. <0.1 ml lÀ1). These statements are generalisations and it is important to emphasise that a broad 36 ANDREW J. GOODAY

Figure 6 Schematic representation of the effect of an oxygen gradient on foraminiferal species richness. Each horizontal line represents the distribution of a single hypothetical species in relation to oxygen concentrations; the filled circle represents its lower oxygen tolerance limit. Most of the hypothetical species are found À1 in oxic environments, but one or two are absent where O2 values exceed 0.5–1.0 ml l . Note that there is currently no evidence that obligate dysaerobic species actually exist, although low-oxygen tolerant species may be eliminated by competition with oxyphilic species at higher oxygen concentrations. There is good evidence, however, that species are progressively eliminated when oxygen concentrations fall below a certain threshold (1.0–0.5 ml lÀ1). Foraminifera disappear entirely when regional anoxia is persistent (Alve, 1990). The presence of sulphides may be another factor eliminating species at low oxygen concentrations. The oxygen gradient is unspecified in this diagram; it may be on small spatial scale (i.e. across-sediment pore water profiles) or on regional scales (e.g. across an oxygen minimum zone). Oxygen gradients may be associated with different food types; for example, well-oxygenated, sediment surface microhabitats are characterised by labile material, deep-infaunal microhabitats by more refractory material. The availability of food will influence the abundance of particular species. The quality of the food (labile near the sediment surface, more refractory in deeper layers) may influence rates of reproduction and test production (Ohga and Kitazato, 1997; de Stigter et al., 1999; Jorissen and Wittling, 1999). Note that oxygen itself does not influence abundance, except by providing an increased living space for oxyphilic species (Jannink, 2000). BENTHIC FORAMINIFERA 37 range of test morphotypes occurs in dysoxic environments (Holbourn et al., 2001a). Our present understanding suggests that no species or test morphotype is confined to sediments overlain by oxygen-depleted bottom water (Sen Gupta and Machain-Castillo, 1993; Bernhard and Sen Gupta, 1999; van der Zwaan et al., 1999). The taxa mentioned above also occur in organically enriched settings where the bottom water is relatively well oxygenated. They are probably related to food availability and sediment geochemical profiles rather than to oxygen concentrations in the overlying water (Rathburn and Corliss, 1994; Rathburn and Miao, 1995; Rathburn et al., 1996; Fontanier et al., 2002). This is an important consideration when interpreting the palaeoenvironmental signal conveyed by fossil assemblages. Despite these caveats, there are good examples from the palaeoceano- graphic record of ‘‘dysoxic’’ foraminiferal assemblages that seem to reflect fluctuations in bottom-water oxygenation. In late Quaternary Santa Barbara Basin sediments, Cannariato et al. (1999) detected rapid faunal shifts back and forth between assemblages that were inferred to reflect oxic and dysoxic conditions. They attributed these changes to major climatic oscillations that altered thermohaline circulation and ventilation. Scho¨ nfeld et al. (in press) report a sharp increase in the abundance of Globobulimina affinis (a low- oxygen tolerant species) off the Iberian Peninsula during Heinrich Event H1 at the onset of the last deglaciation (around 17,000 years ago) and the earlier Heinrich Events H4 (around 40,000 years ago). During these periods of rapid climatic change, massive injections of meltwater associated with iceberg surges are believed to have led to the supression of deep-water production and the development of dysoxic bottom water. Qualitative interpretations of bottom-water oxygenation are enhanced by a multiproxy approach, i.e. the use of foraminiferal evidence in conjunction with other palaeoenvironmental indicators, particularly those based on trace fossils (Baas et al., 1998) and redox-sensitive elements (e.g. von Rad et al., 1999).

7.3. Quantitative approaches

Efforts to develop quantitative proxies for bottom-water oxygen concentra- tions have focussed on the proportion of infaunal morphotypes. Kaiho (1991, 1994) recognised oxic, suboxic and dysoxic indicator species and defined a dissolved oxygen index (BFOI: the number of ‘‘oxic’’ specimens as a proportion of the ‘‘oxic’’ þ ‘‘dysoxic’’ total, i.e. excluding the ‘‘suboxic’’ category). For the range 0–1.5 ml lÀ1, he proposed a similar index based on the proportion of dysoxic indicators. Species were placed in these categories on the basis of test characteristics that were inferred to reflect preferences for different oxygen levels and microhabitats. Oxic species include large, thick-walled, epifaunal morphotypes; dysoxic species include thin-walled, 38 ANDREW J. GOODAY elongate, flattened, infaunal morphotypes. Kaiho (1994) found a good correlation between BFOI values derived from modern samples and corresponding dissolved bottom-water oxygen levels. He suggested that the indexes discriminate among five ranges of dissolved oxygen values, from anoxic (0–0.1 ml lÀ1) to high oxic (3–6 ml lÀ1). In a later paper, Kaiho (1999) explored correlations between BFOI values and two indicators of food inputs, primary production and organic flux to the sea floor. Correlations with productivity and fluxes (r ¼ 0.74 and 0.71, respectively) were much lower than with bottom-water dissolved oxygen (r ¼ 0.90). Baas et al. (1998) applied a slightly modified version of the BFOI index to cores obtained off the Portuguese margin in order to reconstruct bottom-water oxygen concen- trations during Late Glacial Heinrich events. BFOI minima, corresponding to high percentages of Globobulimina affinis (and occasionally Chilostomella ovoidea), were inferred to reflect dysoxic bottom water. They were closely associated with Heinrich events, minima in benthic foraminiferal 13C, and abundance maxima of trace fossils believed to represent dysoxic conditions. The BFOI approach is rather problematic for several reasons. First, the evidence that benthic foraminifera are sensitive to changes in bottom-water oxygenation above 1.0 ml l À1 is not strong. Second, the pore-water oxygen concentrations experienced by infaunal foraminifera may be entirely unrelated to bottom-water oxygen values, which can be influenced by current activity and other factors (Jorissen, 1999). Third, recent evidence suggests that the proportion of deep infaunal foraminifera depends largely on food supply rather than oxygen availability (Jorissen et al., 1998; Morigi et al., 2001). As a result, deep infaunal taxa are sometimes abundant in sediments overlain by well-oxygenated bottom water (Gooday et al., 2001; Fontanier et al., 2002). In contrast to Kaiho’s (1999) results, Morigi et al. (2001) found a stronger correlation between BFOI and organic flux (r ¼ 0.82) than between BFOI and bottom-water oxygenation (r ¼ 0.64) in samples from off NW Africa (19–27N; 506–3314 m water depth). However, the correlation between % deep infaunal species and bottom-water oxygen concentrations was better than the correlation with the organic flux, at least up to values of 3mll À1. In an earlier paper, Kaiho (1994) suggested that the significant correlation between BFOI and bottom-water oxygenation broke down above values of 3.2 ml lÀ1. Further evidence that BFOI may reflect oxygen values up to 3.0 ml lÀ1 comes from Scho¨ nfeld’s (2001) study of foraminiferal distributions in relation to sediment oxygen profiles. He reports peaks around 1.5 and 3.0 ml lÀ1 in the frequency distribution of the lower oxygen range limits of species. These values correspond to the high oxic/low oxic and low oxic/suboxic boundaries, respectively, of Kaiho (1994). Van der Zwaan et al. (in Kouwenhoven, 2000) have recently developed a transfer function for bottom-water oxygen concentrations based on the percentage abundance of calcareous epifaunal (oxyphilic) species: [Oxygen BENTHIC FORAMINIFERA 39 content mmol lÀ1] ¼ 7.9602 þ 5.95 [% oxyphilic species]. This proxy, which was calibrated using modern foraminiferal data from the Mediterranean Sea and Atlantic and Indian Oceans, depends on the fact that oxyphilic species require free oxygen and therefore are confined to fully oxic microenviron- ments close to the sediment surface. Kouwenhoven (2000) used this method to provide a generally convincing reconstruction of oxygenation history of the late Miocene (6.3–8.1 million years ago) Mediterranean (Monte del Casino section in Northern Italy). However, in places, it yielded oxygen estimates that were either unrealistically low or peaked to unrealistically high values, depending on whether or not particular species were included in the calculations. Jannink et al. (in Jannink, 2001) proposed a very similar transfer function [Oxygen content mmol lÀ1] ¼ 7.23 þ 5.62 [% oxyphilic species] with an R2 value of 0.66, also based on Mediterranean and Atlantic and Indian Ocean material representing a wide variety of product- ivity regimes. They argued that the abundance of oxyphilic taxa is regulated by the volume of aerated sediment (i.e. living space) rather than bottom- water oxygen values as such. When applied to a core from the North Adriatic, this proxy yielded oxygenation estimates for the past 160 years that corresponded well with historical data. This method seems to produce plausible results when applied cautiously, although it may require further refinement.

8. BOTTOM-WATER HYDROGRAPHY

8.1. General considerations

Foraminiferal distributions on continental shelves are often related to major water masses (Phleger, 1960, 1964; Culver and Buzas, 2000). Correlations between modern foraminiferal distributions and deep-sea, bottom-water masses, such as North Atlantic Deep Water and Antarctic Bottom Water, were first reported based on samples from the North Atlantic Ocean (Streeter, 1973; Schnitker, 1974) and numerous other studies were conducted during the 1970s and 1980s (reviewed by Douglas and Woodruff, 1981; Schnitker, 1994). On continental margins, where the water column is highly stratified and strong environmental gradients impinge on the sea floor, there are often clear relationships between bottom-water hydrography and foraminiferal assemblages (e.g. Douglas, 1979, 1981; Denne and Sen Gupta, 1991; Schmiedl et al., 1997). Mackensen et al. (1995) and Mackensen (1997) suggest that three deep-sea foraminiferal associations in the Atlantic and Southern Oceans reflect hydrographic influences: (1) assemblages dominated by the epifaunal species Cibicidoides 40 ANDREW J. GOODAY wuellerstorfi are associated with young, well-oxygenated water masses, for example, North Atlantic Deep Water; (2) Nuttallides umbonifer is closely linked to carbonate corrosive bottom water; (3) Lobatula lobatula (Walker and Jacob) and Trifarina angulosa (Williamson) are associated with strong currents. Occasionally, temperature contrasts between adjacent basins seem to provide the best explanation for faunal differences (Rathburn et al., 1996). In one of the earliest deep-sea biological studies, Carpenter (1869) and Carpenter et al. (1870) invoked contrasting temperature regimes to explain differences between the foraminiferal assemblages to the north and south of the Wyville-Thomson Ridge on the Scottish continental margin. Below, I consider two of these characteristics, carbonate undersaturation and current flow, in more detail.

8.2. Carbonate undersaturation

An abyssal assemblage dominated by Nuttallides umbonifer (Figure 3H–I) has been recognised in areas overlain by Antarctic Bottom Water (AABW), or its equivalents, in various parts of the world (Murray, 1991); for example, deep basins in the southern part of the North Atlantic (Streeter, 1973; Schnitker, 1974, 1980; Weston and Murray, 1984), on the Rio Grande Rise in the SW Atlantic (Lohmann, 1978), the SE and SW Indian Oceans (Corliss, 1979, 1983), and the eastern and western Pacific Ocean (Burke, 1981; Douglas and Woodruff, 1981, Table II therein; Nienstedt and Arnold, 1988). In the South Atlantic, it is restricted to the deep Cape and Angola Basins (Schmiedl et al., 1997), at water depths below 3800 m north of 51S and east of the Mid-Atlantic Ridge (Mackensen et al., 1993), and 3500–4000 m in the eastern Weddell Sea (Mackensen et al., 1990). Species that occur with N. umbonifer in the South Atlantic include Cribrostomoides subglobosa (Cushman), Epistominella exigua, Adercotryma glomerata (Brady) and Ammobaculites agglutinans (d’Orbigny) (Schmiedl et al., 1997). Nuttallides umbonifer is most closely associated not with AABW as such but with carbonate corrosive bottom water (Bremer and Lohmann, 1982). In the SE Atlantic Ocean and the Southern Ocean, it is restricted to carbonate-corrosive environments between the lysocline and the CCD (Mackensen et al., 1990, 1993, 1995; Harloff and Mackensen, 1997; Schmiedl et al., 1997). Mackensen concludes that ‘‘U. umbonifer is a characteristic constituent of dead assemblage on most of the world ocean floor over which AABW flows, i.e. between carbonate lysocline and CCD’’ (Mackensen et al., 1990) and that ‘‘this species unequivocally indicates abyssal carbonate-aggressive bottom water masses’’ (Mackensen et al., 1995). Carbonate dissolution is known to have a deleterious effect on calcareous foraminifera. Green et al. (1998) incubated shallow-water BENTHIC FORAMINIFERA 41 sediments containing meiofaunal organisms under conditions of carbonate saturation and undersaturation. Foraminifera exhibited significantly higher mortality in the undersaturated treatments than in the saturated treatments. Other experiments indicate that N. umbonifer is as susceptible to dissolution as other hyaline calcareous species, suggesting that its distribution is probably not related directly to carbonate undersaturation (Corliss and Honjo, 1981; Kurbjeweit et al., 2000). In the NE Atlantic Ocean, the distribution of N. umbonifer lies to the south of temperate areas that experience strong seasonal phytodetrital pulses (Gooday, 1993). Schmiedl et al. (1997) report a negative correlation between the N. umbonifer assemblage and organic matter flux and sediment TOC values in the eastern South Atlantic. In the eastern Equatorial Pacific, an assemblage dominated by N. umbonifer occurs in low-productivity areas situated well above the CCD (Loubere, 1991). Hence this species may be adapted to the highly oligotrophic conditions that prevail in deep, abyssal basins (Gooday, 1993; Altenbach et al., 1999) and associated only incidentally with corrosive bottom water. However, the observation by Kurbjeweit et al. (2000) of a N. rugosa (¼N. umbonifer) assemblage associated with phytodetritus at a bathyal site (WAST-T; 1920 m water depth) in the western Arabian Sea make it clear that the ecology of this important species is still not fully understood.

8.3. Current flow

Although much of the ocean floor is relatively quiescent, near-bottom currents occur in certain areas, particularly those with sloped topography such as continental slopes and seamounts (Heezen and Hollister, 1971; Hollister et al., 1984; Hollister and Nowell, 1991). Such currents can modify the structure and composition of benthic communities (Hall, 1994), particularly if the flow is strong enough to transport sediment (Levin et al., 1994). For example, strong bottom flow often depresses species diversity but can also enhance it (reviewed in Levin et al., 2001). Sessile suspension-feeding megafauna, such as the sponge Pheronema carpenteri (Thomson) and the deep-water coral Lophelia pertusa (Linneaus), are common on the upper continental slope around the NW European continental margin in areas where bottom-water hydrography and topography interact to enhance current flow (Rice et al., 1990; Rogers, 1999). These large organisms provide an important substrate for attached organisms, including suspension-feeding foraminifera (Lutze and Thiel, 1989; Klitgaard, 1995; Rogers, 1999). Associations between certain epifaunal foraminiferal species and currents are well established, particularly in upper bathyal settings 42 ANDREW J. GOODAY

(Mackensen, 1997). Sessile epifaunal suspension feeders are often common where the flow is strong, suggesting that species benefit from food delivered by currents. Scho¨ nfeld (1997) analysed living and dead foraminiferal assemblages (>250-mm fraction) on the Portuguese con- tinental margin. This region is strongly impacted by the Mediterranean Outflow Water (MOW) which flows as a contour current along the slope between 600 m and 1500 m water depth with sufficient velocity to transport sediment and create mud ripples. Attached epibenthic species (Cibicides lobatulus, Discanomalina coronata (Parker & Jones), Epistominella exigua, Hanzawaia concentrica (Cushman), Planulina ariminensis d’Orbigny, Vulvulina pennatula (Batsch)) that are presumed to be suspension feeders were common within this depth interval. Past changes in MOW flow patterns during the late Glacial and Holocene periods can be inferred from downcore changes in the abundance of this ‘‘Epibenthos Group’’ (Scho¨ nfeld and Zahn, 2000). In later papers, Scho¨ nfeld (2002a, c) described similar assemblages from the Gulf of Cadiz off southern Spain, where current activity is more intense and elevated epibenthic species correspondingly more abundant. Very high current velocities (up to 50 cm sÀ1) were encountered in the eastern Gulf of Cadiz, close to the Straits of Gibraltar. Here, epibenthic suspension feeders such as Deuterammina ochracea, Discanomalina semipunctata (Bailey), C. lobatulus and Cibicides refulgens de Montfort constituted 60–90% of the living foraminiferal assemblage. They were attached to hydroid colonies and to the tops of relatively large, heavy, stable objects, giving them access to a high food flux. In the western part of the Gulf, where current velocities were lower (4–25 cm sÀ1), attached epibenthic species (e.g. Crithionina mamilla Goe¨ s, Trochammina squamata Parker and Jones, Saccammina sphaerica Brady, Hanzawaia concentrica, Rosalina anomala Terquem) were relatively less abundant (7–21% of live assemblage) and confined to lower substrates (<3 cm above sea floor). Agglutinated tubes, mainly Rhabdammina abyssorum M. Sars, constituted up to 60% of the assemblages in these areas. Scho¨ nfeld (2002a) suggested that these faunal and ecological differences were related to the optimisation of food acquisition. Only at higher current velocities will the concentration of advected food particles increase at elevations >3 cm above the sea floor. Dead assemblages in the Gulf of Cadiz reflected the faunal differences apparent in the live faunas, suggesting that the proportion of elevated epibenthic species may provide the basis for a current velocity proxy that is independent of sedimentary parameters (Scho¨ nfeld, 2002c). Epibenthic foraminiferal faunas also occur on other parts of the NW European continental margin. An assemblage associated with MOW at water depths of 900–1200 m in the Bay of Biscay (Pujos, 1970) includes species that are found elsewhere living on Pheronema carpenteri (Lutze and BENTHIC FORAMINIFERA 43

Thiel, 1989). On the Norwegian margin, the attached suspension-feeding calcareous foraminiferan Rupertina stabilis (Wallich) is abundant at 600–700 m water depth in a border zone between two water masses where current speeds are enhanced (Lutze and Altenbach, 1988). Another attached species, Cibicides lobatula, occurs with the free-living Trifarina angulosa (Figure 3E) in areas of enhanced flow on the upper slope off SW Norway (Mackensen et al., 1985) and with the agglutinated species Reophax guttifer in seasonally ice-free parts of the Arctic Ocean (<500 m water depth) (Wollenburg and Kuhnt, 2000). The tiny rotaliid Stetsonia hovarthi dominates in current-affected regions at much greater water depths (>2700 m) under permanent ice cover in the Arctic Ocean (Wollenburg and Kuhnt, 2000). This species may be associated with very low flux rates (Altenbach et al., 1999), rather than current activity as such. A Trifarina angulosa association is also typical of the shelf edge and upper slope areas affected by strong bottom currents in the Southern Ocean (Mackensen et al., 1995; Harloff and Mackensen, 1997). Finally, Globocassidulina subglobosa is associated with areas of elevated topography and sandy sediments on the Walvis Ridge in the SE Atlantic (Schmiedl et al., 1997). Current flow impacts foraminiferal assemblages in a variety of other ways; for example, by physically transporting small individuals (Alve, 1999; Weinelt et al., 2001), modifying sediment characteristics, increasing or decreasing sediment heterogeneity, or by transporting oxygen into areas that would otherwise be oxygen depleted (Gage, 1997). Thus, on the Norwegian margin and eastern Weddell Sea, Trifarina angulosa may be related to coarse sediment rather than, or in addition to, current flow itself (Mackensen et al., 1985, 1995). An association with terrigenous sand is reported on the edge of the continental shelf to the east of South Island, New Zealand (Hayward et al., 2002). Scho¨ nfeld (2002a) suggests that T. angulosa occupies interstitial microhabitats in coarse-grained sediments which provide shelter from turbulent water in high-energy environments.

9. WATER DEPTH

Early investigators of deep-sea foraminiferal ecology were preoccupied with the bathymetric distribution of species (Phleger, 1960; Culver, 1988). However, water depth itself has no influence on benthic faunas, although it is related directly to hydrostatic pressure, a parameter which must set ultimate limits to the bathymetric ranges of species through its control on cellular biochemistry (Bradshaw, 1961; Belanger and Streeter, 1980; Somero, 1991, 1992). Within these physiological boundaries, actual species distributions are likely to reflect a variety of other environmental factors, such as water- mass characteristics, temperature, carbonate dissolution, and substrate 44 ANDREW J. GOODAY characteristics, all of which are also to some extent related to water depth (Hayward et al., 2002). It seems likely that organic fluxes to the sea floor are particularly important in determining the bathymetric distributions of foraminiferal species (Haake et al., 1982; van der Zwaan et al., 1999). Fluxes decrease with increasing water depth at any particular locality, and also vary from region to region. Analysing the relation between the percentage abundance of species and flux intensity, Altenbach et al. (1999) concluded that, down to a water depth of about 1000 m, species ‘‘patterns are depth dependent with reduced influence from organic matter flux’’. Below this limit, patterns of species abundance follow flux values down to depths of 2000 m, below which species tend to be very widely distributed. In the Mediterranean Sea, there is a clear relation between flux and bathymetric distribution of species. De Rijk et al. (2000) described a shallowing of the lower water depth limit for many species from west to east, corresponding to a change from eutrophic to oligotrophic conditions. They conclude that ‘‘...the bathymetric distribution of the dominant foraminiferal taxa seems indeed to be controlled by the level of the organic flux to the sea floor.’’ De Stigter et al. (in De Stigter, 1996, Chapter 6 therein) found that foraminiferal species lived at greater water depths in the South Adriatic Basin during the late Pleistocene compared with their present-day distributions. They attributed this upward bathymetric shift to a decrease in primary productivity and POC flux to the sea floor since the late Pleistocene. These results imply that benthic foraminifera can provide only a broad indication of bathymetry (Murray, 1991). Although sucessions of species invariably occur with increasing water depth along continental margin transects (Pujos-Lamy, 1972; Pflumm and Frerichs, 1976; Haake, 1980; Lutze, 1980; Douglas and Woodruff, 1981), species depth ranges are not consistent between regions. The distributions of modern foraminiferal species and species assemblages can therefore be used to reconstruct palaeobathymetry only when applied to fossil faunas in the same study area (e.g. Culver, 1988; Hayward, 1990; Kamp et al., 1998; Akimoto et al., 2002). A more generalised approach to estimating palaeodepths exploits the relation between the abundance of planktonic and benthic foraminifera and organic fluxes. Building on the ideas of earlier authors (e.g. Berger and Diester Haas, 1988), van der Zwaan et al. (1990, 1999) suggested that the planktonic/benthic ratio (P/B ratio ¼ the percentage of planktonic forami- niferal tests in the total assemblage) reflects water depth. A better relation between the P/B ratio and water depth was obtained when infaunal species, which are less closely linked to the freshly settled flux, were excluded. This relationship can be used to determine approximate palaeodepths and works fairly well as long as the ratios are not distorted by selective dissolution or by high inputs of organic matter and oxygen depletion. It has been used, for BENTHIC FORAMINIFERA 45 example, to estimate bathymetric changes within the Palaeocene El Kef Formation in Tunisia (Kouwenhoven et al., 1997) and the Miocene Monte del Casino section in Italy (van der Meulen et al., 1999). Explanations of metazoan distributional patterns in relation to water depth often involve food availability. Billett (1991) concluded that food availability, and hydrographic factors such as temperature, control on the distribution of holothurians in the Porcupine Seabight (NE Atlantic). Cartes and Sarda` (1993) emphasised the influence of decreasing food resources with increasing depth on the bathymetric ranges of decapod crustacean species in the Western Mediterranean. Hecker (1990) likewise invoked food availability, together with parameters such as sediment type and current intensity, to explain the bathymetric occurrence of megafaunal species on the New England slope. Biological interactions, particularly predation and competition have also been mentioned as factors contributing to the depth distribution patterns of macro- and mega-faunal metazoans. Rex (1977) argues that competition between species operating at lower trophic levels will be reduced by predation, allowing them to have broader depth ranges than predators. Although Rex’s hypothesis is problematical (e.g. Carney et al., 1983), biological influences should not be overlooked when considering controls on the distribution of deep-sea foraminiferal species, many of which feed at low trophic levels.

10. SPECIES DIVERSITY PARAMETERS AS TOOLS IN PALAEOCEANOGRAPHY

Species diversity parameters typically exhibit trends in relation to environmental gradients. On a global scale, diversity appears to decrease from low to high latitudes in a number of macrofaunal taxa (Rex et al., 1997, 2000) and in foraminifera (Culver and Buzas, 2000). At regional scales, species richness, diversity and dominance are influenced by, among other factors, the organic flux to the sea floor, bottom-water oxygenation, current activity and sediment heterogeneity (Etter and Grassle, 1992; Gage, 1997; Levin and Gage, 1998; Lambshead et al., 2001; Levin et al., 2001). Food and oxygen availability are often the most important parameters. Based on their analysis of macrofaunal diversity patterns in relation to sediment organic matter content (considered a proxy for food availability), Levin and Gage (1998) concluded that reduced macrofaunal species richness in eutrophic/dysoxic settings is due largely to lack of oxygen whereas the corresponding increase in dominance reflects increased food availability. The overall result is a decrease in diversity, although the effects may not become evident until oxygen concentrations fall below 0.5 ml lÀ1 46

Table 4 Species richness data for foraminifera at localities characterised by differing oxygen regimes. L ¼ live populations; D ¼ dead populations. E(S100) ¼ expected number of species per 100 specimens; R1D ¼ rank 1 dominance (percentage abundance of top- ranked species). Sources of data: A ¼ Bernhard et al. (1997); B ¼ Gooday et al. (2000); C ¼ Jannink et al. (1998); D ¼ Rathburn and Corliss (1994); E ¼ Gooday et al. (1998); F ¼Fontanier et al. (2002); G ¼ Jorissen et al. (1998); H ¼ Hughes et al. (2001); I ¼ Gooday and Hughes (2002); J ¼ Schmiedl et al. (1997); K ¼ Wollenburg and Kuhnt (2000); L ¼ Gooday unpublished.

Locality, size Bottom- Oxygen Calcareous All hard-shelled4 Source fraction, depth of water penetration species species core fraction, oxygen of depth of sample ml l-1 sediment Specimens Species E(S100) R1D(%) Specimens Species E(S100) R1D(%)

L D L D L D L D L D L D L D L D

Santa Barbara Basin: >63 mm; 0–1 cm 339 m 0.50 28 5 – 28.6 31 7 – 25.8 431 m 0.34 238 8 7.5 40.3 255 11 9.7 37.6 522 m 0.08 355 7 6.1 29.0 470 10 8.4 25.5 537 m 0.04 218 8 7.7 48.6 323 12 10.3 32.8 A 578 m 0.02 756 7 5.8 35.8 828 10 7.8 32.3 591 m 0.06 360 7 5.4 74.4 450 9 6.7 59.6 Santa Barbara Basin m >63 m, 0–1 cm GOODAY J. ANDREW 590 m 0.05 796 10 8.1 53.6 854 11 7.2 50.0 610 m 0.15 239 16 12.3 36.0 328 31 19.8 26.2 B Oman margin: >125 mm, 0–1 cm 412 m 0.13 4414 28 37.9 5505 41 30.4 B 3350 mm 3.0 18 15 12.5 406 61 13.1 Pakistan margin FORAMINIFERA BENTHIC >63 mm, 0–1 cm 495 m 0.18 2200 15 998 m 0.75 250 15 1254 m 0.22 700 16 1555 m 1.00 500 23 C 2001 m 2.20 1150 24 556 m 0.36 6500 18 1000 m 0.18 5400 24 1226 m 0.45 1400 22 1472 m 1.00 800 13 Sulu Sea: >63 mm, 0–20 cm 510 m 1.74 2869 112 5.12 1980 m 1.28 780 49 24.4 1995 m 1.24 499 39 25.4 D 3000 m 1.20 194 32 17.6 3995 m 1.21 24 10 41.0 4000 m 1.20 87 13 41.0 4515 m 1.19 252 14 80.0 1Porcupine Seabight: >45 mm 1340 m April ‘83: Oxic 0–1 cm 95 19 19.4 36.8 0–5 cm 133 23 20.1 36.9 E 21340 m July ‘83: 0–1 cm Oxic 368 20 14.1 34.2 0–5 cm 368 23 14.9 41.6

(continued) 47 48

Table 4. Continued.

Locality, size Bottom- Oxygen Calcareous All hard-shelled4 Source fraction, depth of water penetration species species core fraction, oxygen of depth of sample ml l-1 sediment Specimens Species E(S100) R1D(%) Specimens Species E(S100) R1D(%) L D L D L D L D L D L D L D L D

BENBO Site A 0–1 cm, >125 mm 3569 m (Aug. 1997) 19 3830 11 62 – 27.3 15.8 38.5 230 4398 50 77 31.2 32.6 36.1 30.9 H,L 3576 m (May 1998) 59 20 23.0 20.3 391 62 31.4 24.3 3567 m (July 1998) 103 17 17.4 75.0 298 43 25.1 26.2 BENBO Site B 0–1 cm, >125 mm 136 326 17 39 17.8 21.5 16.2 27.3 274 360 49 47 32.3 25.1 19.7 24.7 H,L 1100 m (Aug. 1997) BENBO Site C 0–1 cm, >125 mm 6.0 68 247 19 44 19.0 30.4 38.2 18.2 430 315 56 61 28.2 37.0 27.4 14.2 H,L 1926 m (Aug. 1997) GOODAY J. ANDREW BENBO Site C 0–1 cm; >63 mm 6.0 1913 m (May 1998) 397 1247 32 42 19.9 18.9 31.6 27.8 2265 1306 158 78 48 22 13.0 26.2 1913 m (May 1998) 237 2013 23 41 16.4 20.0 38.4 19.1 2115 73 24 19.3 I 1963 m (July 1998) 981 1668 33 39 15.1 15.8 46.1 24.5 6547 1855 176 62 34 22 20.4 24.5 1980 m (July 1998) 2818 1265 38 44 12.8 17.7 44.0 29.5 1350 68 23 27.6 1,2

Porcupine Abyssal FORAMINIFERA BENTHIC Plain: >63 mm Oxic 4845 m: 0–1 cm 210 15 11.7 38.0 0–10 cm 181 28 21.7 40.9 E 11908#70: 0–1 cm 1386 64 16.6 35.0 1763 102 25.1 27.5 11908#5: 0–1 cm 813 53 16.8 36.2 1010 87 25.6 29.1 11908#32: 0–1 cm 1034 58 17.6 34.5 1234 87 26.0 28.9 Madeira Abyssal Plain >63 mm Oxic 4940 m: 0–1 cm 54 13 38.8 217 80 0–10 cm 61 16 36.1 240 83 12174#24: 0–1 cm 182 24 18.2 30.2 311 58 33.3 17.7 12174#15: 0–1 cm 167 29 21.8 28.7 298 59 33.5 16.1 E 12174#88: 0–1 cm 65 18 18.0 21.5 152 45 37.2 21.5 Bay of Biscay: >150 mm 140 m 4.9 8 mm 0–1 cm 925 22 13.0 37.9 1221 31 15.1 26.5 0–10 cm 1584 25 14.3 34.9 1989 36 16.8 30.2 553 m: 4.8 27 mm 0–1 cm 914 34 13.4 37.7 1040 45 18.1 38.8 0–10 cm 1205 37 13.6 44.2 1345 49 18.6 33.8 1012 m: 4.4 20 mm F 0–1 cm 150 18 15.4 35.6 208 28 22.2 19.7 0–10 cm 385 25 15.8 27.3 476 37 22.9 28.8 1264 m: 4.7 64 mm 0–1 cm 72 14 14 39.0 76 16 16 39.5 0–10 cm 105 18 17.7 41.7 122 25 23.5 33.6 1993 m: 5.8 63 mm 0–1 cm 107 13 12.8 36.5 125 22 20.6 40.8

0–10 cm 159 18 15.4 47.7 179 27 22.2 32.4 49

(continued) 50

Table 4. Continued.

Locality, size Bottom- Oxygen Calcareous All hard-shelled4 Source fraction, depth of water penetration species species core fraction, oxygen of depth of sample ml l-1 sediment Specimens Species E(S100) R1D(%) Specimens Species E(S100) R1D(%)

L D L D L D L D L D L D L D L D

Off NW Africa: >150 mm 1195 m 3.67 1.5 mm 0–1 cm 558 39 19.8 32.8 848 63 27.3 21.6 0–10 cm 891 44 19.4 24.8 1297 69 28.2 17.0 1525 m 4.29 1.0 mm 0–1 cm 132 26 24 26.5 223 40 30.7 15.7 0–10 cm 253 32 23 16.6 449 48 31.7 11.4 2005 m 4.44 2.2 mm G

0–1 cm 104 23 22.6 24.0 187 33 27.0 13.6 GOODAY J. ANDREW 0–10 cm 179 28 22.2 22.3 272 39 27.8 14.7 2530 m 4.48 3.8 mm 0–1 cm 65 16 16 27.7 214 33 24.3 20.6 0–10 cm 249 22 13.8 39.4 449 40 22.4 21.8 3010 m 4.88 2.5 mm 0–1 cm 76 17 17 42.1 139 27 25.6 23.0 0–10 cm 166 22 19.7 11.2 359 40 26.0 20.9 ETI FORAMINIFERA BENTHIC 3SE Atlantic: 0–1 cm; >125 mm 250–700 m 2–3 287 572 41 43 400–1000 m >3 225 602 35 40 1000–2000 m >3 235 426 52 53 I 2000–3000 m >3 230 343 50 64 3000–4000 m >3 264 334 53 53 4000–5000 m >3 300 543 52 59 >5000 m >3 330 360 44 38 3Arctic Ocean: 17–38 gC mÀ2 yÀ1 flux: 37–100 m (n ¼ 4) 27–33 mm 594 676 49 37 6–8 gC mÀ2 yÀ1 flux: 200–250 m (n ¼ 4) 25 mm 616 756 56 51 0–2 gC mÀ2 yÀ1 flux: K 500–1000 m (n ¼ 9) 3–30 mm 657 691 66 63 1000–2000 m (n ¼ 7) 4–35 mm 464 640 57 51 2000–3000 m (n ¼ 8) 2–13 mm 612 648 41 47 3000–4000 m (n ¼ 4) 3–10 mm 503 604 39 40

1Values are medians of replicates. 2Samples contain phytodetritus. 3Median values; only samples with >200 specimens considered. 4Includes all calcareous species, multilocular agglutinated species, unilocular agglutinated species with rigid tests; 5excludes soft-walled allogromiids and saccamminids. 51 52 ANDREW J. GOODAY

(Levin et al., 2001). Foraminifera exhibit similar trends (Table 4). The numbers of ‘‘live’’ (rose Bengal stained) calcareous and total hard-shelled species were very low (maximum 8 and 12, respectively) in the Santa À1 Barbara Basin (O2 <0.5 ml l ) (Bernhard et al., 1997). More species (28 calcareous, 41 total hard shelled) occurred in the core of the Oman margin À1 oxygen minimum zone (O2 ¼ 0.13 ml l ), but this reflects the large number of specimens examined (4414 and 5505, respectively) (Gooday et al., 2000). In general, 20 or more ‘‘live’’ calcareous species and 30 or more total live species, are present at oxic sites, except where the number of specimens examined is <200. One exception is Rathburn and Corliss’ (1994) 4000 m À1 site in the Sulu Sea (O2 ¼ 1.19 ml l ) where only 14 calcareous species were represented among 252 ‘‘live’’ specimens. Where comparative data are available, the number of dead species is often higher than the number of ‘live’ species. Relative levels of species diversity can be related to the occupancy of sediment microhabitats, as summarised in the TROX model (Figure 1). Diversity is believed to exhibit a parabolic relation to productivity and food supply in deep-sea and other ecosystems (Levin et al., 2001). One would therefore expect diversity to be lowest at the oligotrophic and eutrophic extremes of the model, and highest at intermediate levels of food input, i.e. in mesotrophic systems. This expectation is generally supported by field evidence. In the Arctic Ocean, where food supply is severely limited by permanent ice cover, foraminiferal diversity is positively correlated with the organic carbon flux; i.e. higher productivity ¼ higher diversity (Wollenburg and Mackensen, 1998; Wollenburg and Kuhnt, 2000; Wollenburg et al., 2001). This presumably represents the ascending, left-hand side of the parabolic curve shown in Figure 1. In ‘‘normal’’ oxic deep-sea settings, foraminiferal assemblages are typically highly diverse (Gooday et al., 1998; Gooday, 1999). As discussed in Section 7, where a high organic matter flux is combined with low oxygen concentrations, species richness and diversity decrease and dominance increases; i.e. higher productivity ¼ lower diversity. Such situations correspond to the right-hand, descending side of the curve. Diversity parameters can be helpful in palaeoceanographic reconstruc- tions (Thomas and Gooday, 1996; van der Zwaan et al., 1999), provided that significant dissolution has not occurred. In the northern Arabian Sea, changes in the quantity and quality of organic matter arriving at the sea floor, and corresponding variations in the thickness and intensity of the OMZ, have been inferred from shifts in foraminiferal diversity (den Dulk, 2000). These changes may have been linked to fluctuations in the intensity of monsoon- induced upwelling in response to orbital and sub-orbital (precessional) forcing mechanisms. Den Dulk et al. (1998) studied Quaternary cores spanning 120,000 years from the Pakistan margin in the Northern Arabian Sea. Two foraminiferal assemblages were recognised, one characterised by BENTHIC FORAMINIFERA 53 low diversity and high dominance, the other by high diversity and low dominance. The low diversity assemblages recurred every 23,000 years and possibly reflected enhanced summer surface productivity (and therefore intensified OMZ development) linked to the precessional component of orbital forcing. A more sustained period of low diversity occurred under glacial conditions, perhaps related to a strengthening of the NE monsoon which led to higher winter productivity and hence lower bottom-water oxygen concentrations (den Dulk, 1998). In a detailed multiproxy study of shorter cores (spanning the last 30,000 years) from the same margin, von Rad et al. (1999) reported a switch from low to high foraminiferal diversity on the Pakistan margin during brief, late Quaternary to early Holocene climatic oscillations (Younger Dryas, Heinrich Events 1 and 2) when surface productivity was believed to be unusually low. Jian et al. (1999) studied fluctuations in benthic foraminiferal assemblages in the South China Sea over the last 40,000 years. They observed that species diversity (Shannon-Wiener index) decreased as surface productivity (inferred from the proportion of infaunal species) increased. In the Ionian Sea (E. Mediterranean Sea), on the other hand, Schmiedl et al. (1998) reported very low Shannon-Wiener values associated with very sparse faunas during interglacial periods. Glacial deposits were characterised by high abundance, high diversity faunas. The low diversity faunas persist in the modern E. Mediterranean, and probably reflect the extremely food-poor nature of this basin. These low-diversity faunas correspond, respectively, to the descending (eutrophic) and ascending (oligotrophic) sides of the parabolic curve (Figure 1). Diversity parameters are particularly useful as indicators of changing environmental conditions in absence of extant species. Examples include the increasing eutrophication and dysoxia that apparently affected Mediterranean basins at the end of the Palaeogene (Kouwenhoven et al., 1997) and continental margin basins in the North and South Atlantic Oceans in the mid- and Late Cretaceous (Koutsoukos et al., 1990; Holbourn et al., 1999, 2001a, b). In contrast to modern dysoxic environments, however, foraminiferal abundance in some Cretaceous deposits was unusually low, probably because oxygen depletion was very intense (Holbourn et al., 2001a). There is little evidence to link deep-sea foraminiferal diversity to current activity. However, enhanced bottom flow strongly affects the structure and composition of benthic faunas (Hall, 1994) and leads to changes in metazoan macrofaunal diversity (Levin et al., 1994; Gage et al., 1995; Gage, 1996, 1997) and taxonomic composition (Levin and DiBacco, 1995). Differences have been observed in the taxonomic/functional composition of predominantly agglutinated foraminiferal assemblages between quiescent areas and those subject to strong currents (Kaminski, 1985; Kaminski and Schro¨ der, 1987; Murray and Alve, 1994; Kuhnt and Collins, 1995). Hydro- dynamically active regions are widespread in the deep sea (Hollister and 54 ANDREW J. GOODAY

Nowell, 1991) and are likely to influence foraminiferal diversity parameters, although the effects will probably be more subtle than those resulting from organic enrichment and dysoxia.

11. SUMMARY OF ENVIRONMENTAL INFLUENCES ON LIVE ASSEMBLAGES

A number of authors, including Altenbach, Bernhard, Corliss, Jorissen, Kuhnt, Loubere, Lutze, Mackensen, Murray, Scho¨ nfeld, Rathburn, and van der Zwaan, have contributed substantially to the greatly improved under- standing, developed over the last two decades, of how environmental factors (particularly food and oxygen) influence deep-sea benthic foraminiferal faunas. To a large extent, the following points reflect the effort of these researchers. 1) The ocean-floor environment is food limited and usually less complex than shallow-water settings, making it easier to isolate the effects of individual parameters on foraminiferal faunas. In general, foraminiferal assemblages are believed to reflect a combination of local (organic flux) and regional (bottom-water mass) influences. However, the deep sea embraces various different environmental settings and the parameters influencing benthic foraminiferal assemblages therefore tend to vary with geography and bathymetry. 2) Regional patterns of foraminiferal abundance, and the species composition of assemblages, can be related to organic fluxes to the sea floor. These faunal attributes can provide a general indication of flux rates and therefore of surface primary productivity. The calibration of proxies for organic fluxes is hampered by inaccuracies inherent in the estimation of modern flux rates and by spatial and temporal variations in their quality and magnitude. Nevertheless, some progress has been made in using benthic foraminiferal accumulation rates (BFAR) and multivariate analyses of species assemblages as quantitative proxies for organic fluxes and surface productivity respectively. 3) At small spatial scales, species distributions within the sediment profile (i.e. their microhabitats) are controlled by a combination of food availability, pore-water oxygen concentrations, species interactions (e.g. competition and predation) and bioturbation. Again, these parameters depend ultimately on the flux of organic matter to the sea floor. Species can be divided into categories depending on whether they live near the sediment surface (epifaunal), within the upper few centimetres of sediment (shallow infaunal), or in deeper sediment layers (deep infaunal). Broadly speaking, these microhabitat categories are characterised by distinct test morphotypes, BENTHIC FORAMINIFERA 55 although the relationship is not perfect and microhabitat preferences cannot always be predicted from test morphology. 4) Except for species living at the sediment surface or on elevated substrates, foraminiferal species are influenced directly by oxygen concen- trations in the sediment pore waters rather than in the overlying bottom water. The ecological effects of oxygen depletion are not well understood. Some studies indicate a relationship between species occurrences and oxygen concentrations up to 3mllÀ1 and higher. Other evidence suggests that the threshold at which oxygen begins to have a significant effect on community parameters is much lower, probably <0.5 ml lÀ1. However, oxygen depletion clearly acts as a filter eliminating oxyphilic (predominately epifaunal) species. The deep infaunal taxa that tolerate oxygen depletion flourish in dysoxic settings due to the high concentrations of food and reduced competition and predation. 5) In a general sense, the proportion of infaunal vs. epifaunal morphotypes reflects a combination of organic-matter fluxes to the sea floor and bottom-water oxygen concentrations. Deep infaunal species tolerate anoxic conditions and appear not to be limited by oxygen. Their abundance (rather than presence) probably depends on the availability of more refractory (i.e. degraded) material and bacteria capable of decompos- ing it; i.e. of food ‘‘stored’’ within the sediment. Epifaunal species, on the other hand, are oxyphilic and disappear when oxygen levels fall below a certain threshold. Their abundance probably reflects adequate pore-water oxygen concentrations, the thickness of the habitable oxygenated sediment layer, and the availability of labile food. 6) Seasonality in the flux of organic matter to the sea floor is expressed in the abundance of small opportunistic, epifaunal/shallow infaunal species that respond directly to pulses of labile organic matter (phytodetritus). These species are associated with different overall levels of organic flux and also occur in areas where a seasonal flux signal is not clearly developed. In general, calcareous foraminifera (particularly rotaliids and buliminids) are more responsive to inputs of fresh organic matter than those with agglutinated or organic walls. 7) The regional distributions of some species and species assemblages are clearly related to bottom-water hydrography. Two examples stand out. First, the association between Nuttallides umbonifer and carbonate under- saturated water at abyssal depths below the lysocline in many parts of the world (but see Kurbjeweit et al. (2002)). Second, the development of distinctive foraminiferal assemblages dominated by suspension-feeders living on elevated substrates in regions of strong current flow. 8) The bathymetric distribution of deep-sea foraminiferal species reflects factors that change with water depth, particularly organic fluxes to the sea floor, rather than water depth as such. However, water depth is directly 56 ANDREW J. GOODAY related to hydrostatic pressure and this parameter must set upper and lower limits to bathymetric ranges through its effect on cell biochemistry. 9) Assemblage parameters (abundance, diversity, species richness, dominance) provide palaeoceanographic indicators that are independent of the identity of species and morphotypes. High productivity/low oxygen regimes are characterised by high abundance, high dominance and relatively low species richness and diversity. More oligotrophic, well-oxygenated settings are characterised by lower abundance, low dominance and high levels of species richness and diversity. Highly oligotrophic environments are characterised by low abundance and low species richness and diversity.

12. RELATIONSHIP OF MODERN AND FOSSIL ASSEMBLAGES

An understanding of how the living assemblages discussed above are transformed into dead, and ultimately into fossil assemblages is funda- mental to the use of foraminifera as palaeoceanographic indicators. Live and dead assemblages are never identical. The former are ephemeral, change with time, and are finely tuned to the contemporary environmental conditions. The latter represent an averaged view of the fauna over a considerable period of time (often hundreds or thousands of years in the case of deep-sea sediments) and incorporate the effects of mixing by bioturbation, different test production rates, and taphonomic processes such as the microbial decomposition of organic-walled tests and agglutinated tests with organic cement, the dissolution of thin-walled calcareous tests, the destruction of tests by macrofaunal ingestion, fungal attack and other forms of biological activity, and post-mortem transport of small tests by currents and other processes (Murray, 1976, 1991; Douglas et al., 1980; Douglas and Woodruff, 1981; Schro¨ der, 1986; de Stigter, 1996; Martin, 1999). Below the lysocline, and in high productivity areas, dissolution of carbonate tests may be considerable (Berger, 1979) with vulnerability to solution varying to some extent between species (Corliss and Honjo, 1981). These processes lead to changes in the overall taxonomic composition of assemblages, the percentage abundance of different species, and in the distribution of species on the sea floor (Douglas et al., 1980). The composition of dead assemblages will also be influenced by different rates of test production (Murray, 1976; Douglas et al., 1980). De Stigter et al. (1999) suggested that species occupying deep-infaunal microhabitats grow more slowly than epifaunal and shallow infaunal species and therefore have lower production rates. Jorissen and Wittling (1999) reached a similar conclusion based on a comparison between live/dead ratios of species from off NW Africa. There is some direct evidence to support this idea. The BENTHIC FORAMINIFERA 57

Figure 7 Distribution of ‘‘live’’ (rose Bengal stained) individuals of Eponides pusillus, Nonionella iridea and other calcareous foraminifera in three different size fractions of a sample collected at BENBO Site C during July 1998. Both E. pusillus and N. iridea are small species. The presence of substantial numbers in the >150 mm fraction reflects their occurrence within phytodetrital aggregates that are retained on this coarse mesh. sudden and substantial increases in population density of some opportu- nistic, surface-dwelling species following phytodetritus deposition (Gooday and Lambshead, 1989; Gooday and Hughes, 2002) suggests rapid rates of test production (Figure 7). The less distinct responses of infaunal species (e.g. Globobulimina affinis) to phytodetrital inputs, and their apparently slow rates of growth (Ohga and Kitazato, 1997), indicate lower production rates. The differences between live and dead assemblages resulting from these complex taphonomic and biological processes can be very substantial, particularly in the deep sea where a large proportion of the fauna typically consists of species with delicate agglutinated tests that decompose rapidly after death and have very little preservation potential (Douglas et al., 1980; Schro¨ der, 1986). At all three BENBO sites, for example, the dead assemblage (0–1 cm sediment layer) was dominated by calcareous for- aminifera while the live assemblage included numerous fragile, sometimes soft-shelled species, for example, psammosphaerids, saccamminids, hormo- sinaceans and Lagenammina spp. (Figure 8). Dead foraminiferal tests eventually pass out of the bioturbated zone into the permanent sedimentary record to form the fossil assemblage. Additional changes in faunal composition may result from diagenetic processes during fossilisation (Mackensen and Douglas, 1989). Usually, it is the agglutinated species that disintegrate. For example, species with cement containing iron oxides may be weakened and destroyed in deeper sediment layers where conditions are reducing and sediment compaction is greater (Schro¨ der, 1986). Such processes result in fossil assemblages that are dominated by calcareous tests with some contribution from resistant agglutinated taxa 58 ANDREW J. GOODAY

Figure 8 Relative abundance of different foraminiferal taxa in live and dead assemblages from multicorer samples (0–1 cm layer, >125 mm fraction) collected at BENBO Sites A–C, NE Atlantic. MAF ¼ multilocular agglutinated taxa other than Hormosinacea and Trochamminacea. Note the switch from dominance by agglutinated taxa in the live assemblages to dominance by rotaliids in the dead assemblages. BENTHIC FORAMINIFERA 59 with calcitic cement. In order to bridge the gap between dead and fossil assemblages, Mackensen et al. (1990) introduced the concept of ‘‘potential fossil assemblages’’, derived from modern dead assemblages by subtracting the non-resistant agglutinated species. Potential fossil assemblages do not reflect the effects of dissolution and other destructive processes and must therefore be considered as ‘‘ideal’’ fossil assemblages. Calcareous tests may also disappear as a result of dissoluton by corrosive pore waters (Mackensen and Douglas, 1989), leaving a predominately or entirely agglutinated assemblage (Gradstein and Berggren, 1981; Murray and Alve, 1994). Loubere (1989) and Loubere and Gary (1990) presented computer models and evidence from box cores (Gulf of Mexico, 1020–1170 m water depth) suggesting that microhabitat preferences can also influence the susceptibility of a species to taphonomic destruction. They argue that much of the destruction of foraminiferal tests occurs in the surface 1–2 cm of sediments where disturbance by benthic animals is most intense (see also de Stigter, 1996). It therefore has a particular impact on epifaunal and shallow infaunal species. Deep infaunal species live below this surface zone and therefore largely escape these destructive processes. Loubere et al. (1993) developed these arguments further, emphasising the ways in which the organic flux and bottom-water oxygenation interact to influence the formation of fossil assemblages. They suggested that assemblage generation depends on: (1) the distribution of living foraminifera within the sediment profile, (2) the rates of test production in different sediment layers, (3) the rates of destructive taphonomic processes (the ‘taphonomic filter’) and the variation of these rates within the sediment profile, (4) the style of bioturbation and the depth to which it extends (among other things, this will influence the extent to which deep-infaunal tests are exposed to taphonomic processes in surface sediments). The final fossil assemblage reflects a combination of these four processes (Figure 9), the importance of which change along gradients of organic carbon flux, oxygen availability, and depth in the sediment profile. On the basis of these ideas, Loubere et al. (1993) and Loubere (1997) suggested that in well-oxygenated, low-flux settings (e.g. central oceanic regions), most test production will occur close to the sediment surface and be subject to intense and uniform taphonomic processes leading to a substantial loss of tests in the fossil assemblage. In these environments, low sedimentation rates will also promote the formation of a homogeneous dead assemblage (de Stigter, 1996). Where a moderate organic flux is combined with well-oxygenated bottom water (e.g. bathyal continental margins), the fossil assemblage will form over a thicker sediment layer, species will be distinctly stratified within this habitable zone, and deep infaunal foraminifera will be subject to less taphonomic loss than those originating in near-surface layers. In high flux, low-oxygen settings (e.g. oxygen 60 ANDREW J. GOODAY

Figure 9 Important factors influencing the generation of fossil assemblages. For standing stocks, the dashed lines indicate the range of likely profiles within the sediment. Production rate is assumed to be related to available oxygen and therefore lower for deeper infaunal populations. The rate of taphonomic test destruction is also assumed to be dependent on available oxygen. Sediment mixing (bioturbation) will become less effective (dotted profile) as organic carbon flux increases. The standing stock and production rate curves together generate an assemblage of dead tests that are modified by taphonomic processes which are most intense in the surface layers of sediment. The efficiency of these processes depends on the depth in the sediment at which the tests were produced and the degree to which bioturbation mixes deep-infaunal tests into the surface layers. Slightly modified from Marine Micropaleontology, Vol. 20, P. Loubere, A. Gary, M. Lagoe, Benthic foraminiferal microhabitats and the generation of a fossil assemblage: theory and preliminary data, p. 179, Figure 10, 1993, with permission from Elsevier Science. minimum zones), taphonomic reworking will be limited (assuming that anoxic biogeochemical processes do not cause significant test destruction) and bioturbation will be reduced. The resulting dead assemblage will resemble the living assemblage and exhibit considerable spatial variability. To the analysis of Loubere can be added the fact that the proportion of hard-shelled, preservable foraminifera is higher in eutrophic, oxygen- depleted than in oligotrophic environments (Gooday et al., 2000) and that alkaline pore waters develop in anoxic sediments because of the presence of sulphate-reducing bacteria (Berger, 1979; Walter and Burton, 1990). Both these factors help to preserve carbonate tests. The scarcity of macrofaunal predators in severely dysoxic settings will enhance the preservation potential of foraminiferal tests in general (Phleger and Soutar, 1973). These and other considerations (e.g. Martin, 1999) lead to a number of general conclusions regarding the deep-sea foraminiferal signal. . Compared with those deposited in shallow-water settings, deep-sea sediments are less strongly bioturbated and provide a fairly continuous record of deposition over much longer time periods. BENTHIC FORAMINIFERA 61

. The resolution of this record will depend largely on the sedimentation rate and the thickness of the bioturbated zone. In the deep sea, sedimentation rates will normally be slow (a few cm 1000 yrÀ1) and the bioturbated zone will span several thousands of years of accumulation. Thus, the resolution of the palaeoceanographic record will normally be less detailed than in shallow-water settings where sedimentation rates are generally higher. Exceptions are bathyal dysoxic basins and oxygen minimum zones, where higher sedimentation rates combined with reduced macrofaunal activity yield a high resolution record (e.g. Cannariato et al., 1999). . In oligotrophic settings, only a small proportion of the living foraminiferal assemblage, which consists largely of non-fossilisable agglutinated forms, will be preserved. The residual fossil assemblage will be dominated by the shells of epifaunal/shallow infaunal species that originate in the surface sediments where taphonomic processes operate intensively. . Rapidly growing, opportunistic, epifaunal/shallow infaunal species add dead tests to the sediment at a faster rate than slower growing, deep infaunal species. These opportunists also have small, thin-walled shells and are therefore susceptible to dissolution and destruction by macro- and mega-faunal activity in the surface sediments. . In eutrophic, oxygen-limited continental margin habitats, a greater proportion of the living assemblage will be preserved than in more oligotrophic, well-oxygenated environments. To a large extent, this reflects the high proportion of fossilisable calcareous forms combined with limited taphonomic reworking. Considering the range and complexity of the biological and physico- chemical processes that intervene between the live and fossil assemblages, it is surprising that any information at all passes into the permanent record. Infact, Murray’s (1976) ‘‘palaeoecological’’ analysis of dead assemblages from a variety of coastal and shelf environments yielded interpretations that were at least moderately accurate, even when sharp differences existed between the live and dead assemblages. Further encouraging evidence for the resilience of the foraminiferal signal is provided by a series of experiments in which dilute acids were used to remove calcareous tests and agglutinated tests with calcareous cement from the original dead assemblage (ODA), leaving only agglutinated species with organic cement (Murray and Alve, 1994, 1999, 2001; Alve and Murray, 1995). The samples originated from environments ranging from deep-sea (>4000 m water depth) to intertidal. Remarkably, the acid-treated assemblages (ATAs) still conveyed a substantial amount of environmental information (for example, in the form of diversity patterns) despite the fact that they represented <5% 62 ANDREW J. GOODAY of the ODA. ATAs from slope, rise and abyssal plain settings were interpreted in terms of organic inputs, current flow, and water mass properties (Murray and Alve, 1994). Nevetheless, some modification of the signal must occur in deep-sea settings. Harloff and Mackensen (1997) reported that live and dead assemblages in the Scotian Sea and Argentine Basin correspond fairly closely. However, the potential fossil assemblages (sensu Mackensen et al., 1990), defined on the basis of Principal Component Analysis and consisting of species likely to fossilise, were generally more extensive than the corresponding dead assemblages and therefore embraced a wider range of environmental conditions.

13. PROBLEMS AND FUTURE DIRECTIONS

13.1. Relationship between environmental factors and spatial scales

Murray (2001) emphasised the multifaceted nature of foraminiferal ecology, and the need to understand its complexities when attempting to develop reliable proxies for use in palaeoceanography. One of his central points is that proxies require a clear, simple relationship between foraminiferal species and the environmental factors of interest, whereas in reality, species will be influenced by different factors, singly or in combination, at different times and in different parts of their ranges. As a result, abundance will only be related directly to a particular factor at times and in places where that factor is limiting. Experimental and field studies suggest that foraminifera do not have rigid ecological requirements and species will live where they can, not only where conditions are optimum for them (Bradshaw, 1961; Altenbach et al., 1999). This leads to wide geographical ranges and overlap between species that have different environmental preferences. For example, the co-occurrence of Uvigerina spp. (a high productivity taxon) and Epistominella exigua (a phytodetritus species) (Corliss 1979; Nees and Struck, 1999) reflect their relatively broad tolerances to organic carbon flux rates, particularly at low percentage abundances (Altenbach et al., 1999). Figure 10 represents some of the main environmental factors, biotic as well as abiotic, that lead to the formation of living deep-sea foraminiferal assemblages. In addition to affecting species directly, these factors interact with each other, sometimes making it difficult to disentangle their separate effects. To take just one example, elevated current flow can lead to the winnowing of fine sediment and increased mean grain size, the increased availability of suspended food particles, and the transport of oxygen into dysoxic environments. It can thereby influence faunal abundance, diversity, BENTHIC FORAMINIFERA 63

Figure 10 Inter-relationships between environmental factors that potentially influence foraminiferal species abundances and assemblage characteristics. Factors that have a direct effect on faunas are shown within the dotted oval line. Those that have an indirect effect are outside the line. composition, and the relative abundance of different feeding types (e.g. suspension feeders) in a variety of ways. Most studies of deep-sea foraminiferal ecology address distribution patterns at either large (100–1000 km2) or small (e.g. sediment micro- habitats) spatial scales. The influence of regional environmental gradients in organic matter flux, bottom-water hydrography (e.g. current flow) and chemistry (e.g. carbonate undersaturation) and hydrostatic pressure is clear from studies spanning large geographical areas, such as ocean basins or parts of continental margins (e.g. Mackensen, et al. 1995; Mackensen, 1997; Hayward et al., 2002). However, many individual foraminifera are small, live entirely within the sediment, and are finely tuned to its geochemical and structural fabric (e.g. Bernhard and Sen Gupta, 1999; Pike et al., 2001; Fontanier et al., 2002). As a result, the influence of large-scale 64 ANDREW J. GOODAY environmental gradients, particularly organic fluxes, bottom-water oxygen concentrations and perhaps current flow, rather than being direct, is mediated through their effects on small-scale gradients within the sediment milieu (Loubere, 1997). Thus, changes in organic fluxes and oxygen concentrations extending over horizontal distances of 10s–100s of kilo- metres act on local foraminiferal assemblages by altering centimetre-scale vertical gradients in physical, geochemical and microbiological parameters within the sediment. It is these small-scale gradients that directly influence foraminiferal assemblage characteristics. Figure 11 attempts to summarise the various routes by which regional environmental gradients impinge on the local assemblages that form the basis for the fossil record (Loubere et al., 1993; Loubere, 1997). Not all large-scale environmental gradients act indirectly. Some, for example hydrostatic pressure, have a more immediate influence on local faunas. Geochemical gradients within the sediment, and hence foraminiferal species, also respond directly to processes that occur across large geographical areas but over short time periods, e.g., seasonally pulsed inputs of organic matter (reviewed by Beaulieu, 2002). In addition to physico-chemical parameters, biotic interactions such as competition, predation, facilitation, biological disturbance, and recruitment, undoubtedly help to structure sediment communities (Gooday, 1986; Jorissen, 1999; Levin et al., 2001), although their role is poorly understood and difficult to quantify. This web of direct and indirect effects frustrates efforts to establish straightforward relation- ships between species assemblages and environmental parameters.

13.2. Calibration of proxies

The quantification of proxies, particularly for organic flux to the sea floor and bottom-water oxygen concentrations, remains a central challenge for palaeoceanographers. Reliable, globally applicable, quantitative, proxies for these parameters based on benthic foraminifera may always remain elusive because foraminiferal biology is so complex. Nevertheless, considerable progress has been made recently in a number of areas. Studies based on samples collected over a wide geographical area (e.g. Mackensen et al., 1995; Wollenburg and Mackensen, 1998) reveal qualitative relationships between faunal patterns and environmental parameters. Large data sets relating species abundances to a single parameter along an environmental gradient provide the basis for a more quantitative approach. For example, the per- centage abundance of species in the NE Atlantic Ocean has been related to the organic carbon flux to the sea floor (Altenbach et al., 1999) and multi- variate approaches have been used to develop transfer functions linking species assemblages to surface primary productivity (e.g. Loubere, 1994; BENTHIC FORAMINIFERA 65

Figure 11 Environmental gradients that act over regional spatial scales (outer rectangle) and their effects on foraminiferal faunas at local scales (inner rectangle). Some of the regional gradients (carbonate undersaturation, current flow, oxygena- tion) are water mass attributes. Organic fluxes and bottom-water oxygen act to modify local geochemical gradients within the sediment and these, in turn, influence faunal characteristics (species, morphotype composition, relative abundance of epifaunal/shallow infaunal vs. deep infaunal species) by accelerating or decelerating rates of reproduction. Current flow, organic fluxes, hydrostatic pressure and carbonate undersaturation have a more direct effect on faunal characteristics. Biotic interactions involving metazoan meio- and macro-fauna (not shown) will also influence foraminiferal faunas.

Wollenburg and Kuhnt, 2000). However, the calibration of these proxies requires reliable values for modern surface productivity and fluxes to the sea floor (Berger et al., 1994), both of which involve substantial errors. The good correlations obtained in some studies are encouraging but the large data sets required for calibration are not often available (Morigi et al., 2001). 66 ANDREW J. GOODAY

Another problem is that ancient assemblages may represent conditions that have no analogue in the calibration dataset. These are difficult to recognise and interpret (Mekik and Loubere, 1999). Clearly, there is a need for more precise calibration methods, for example using sediment oxygen profiles as measures of the flux actually arriving at the sea floor (Jahnke, 1996). For the present, it may be more realistic to report changes in relative productivity or flux rather than attempt to estimate absolute values when applying these approaches in palaeoceanography (Herguera, 2000; Loubere, 2000). Oxygen is a problematic parameter. Being tightly coupled to organic flux, it is difficult to determine whether faunas are influenced by these two factors acting together or by one of them acting alone. Dysoxic conditions are clearly associated with particular foraminiferal assemblage characteristics (e.g. low species diversity and high dominance) but there is less agreement about whether more subtle, species-level effects occur at higher oxygen concentrations (>1 ml lÀ1). Experiments may provide one way to explore these issues. Epifaunal/shallow infaunal species are most sensitive to oxygen depletion and therefore probably offer the best basis for developing bottom- water oxygen proxies. Again, large data sets, in this case spanning a wide range of oxygenation regimes, are required in order to calibrate such proxies.

13.3. Microhabitat studies

Studies of the small-scale distribution patterns of benthic foraminifera are valuable because they provide detailed information on the environmental preferences of individual species that cannot be gained by examining large- scale distribution patterns. Examples include the colonisation of phyto- detrital layers (Gooday, 1988) and elevated substrates (Lutze and Thiel, 1989; Scho¨ nfeld, 2002a, c) by some epifaunal species, the association of species with particular ranges of pore-water oxygen values (Scho¨ nfeld, 2001), and the occurrence of deep infaunal species within anoxic sediment layers (Jorissen et al., 1998; Fontanier et al., 2002). Such investigations are leading to a better understanding of the ecological requirements of deep-sea foraminifera and the relation of species to organic flux rates, oxygen concentrations, and food sources. In addition to direct observations, 13C values (i.e. the deviation of the observed 13C:12C ratio from an arbitrary standard) obtained from calcareous foraminiferal tests yield insights into the depth in the sediment at which calcification occurs and the relative mobility of different infaunal species (Rathburn et al., 1996; McCorkle et al., 1997; Mackensen et al., 2000). Despite these advances, many questions remain. Are infaunal taxa really tolerant of anoxia over long time periods or are they able to obtain oxygen BENTHIC FORAMINIFERA 67 by deploying pseudopodia into overlying oxygenated layers, as suggested by Bernhard and Sen Gupta (1999)? More information about diets is particularly crucial for understanding the balance between inputs of labile and refractory organic carbon. What do epifaunal/shallow infaunal and deep infaunal species feed on? Do the deep infaunal species consume anaerobic bacteria (e.g. Jorissen et al., 1998; Scho¨ nfeld, 2001; Fontanier et al., 2002), degraded organic matter (Goldstein and Corliss, 1994) or fresh phytodetritus (Kitazato et al., 2000) or can they utilise different food sources according to their availability? New approaches may help to resolve such questions. Lipid biomarkers can provide insights into the diets of infaunal and epifaunal foraminifera (Gooday et al., 2002; Suhr pers. comm.). In situ experiments using 13C-labelled algal substrates have considerable potential for investigating the utilisation of labile carbon sources by foraminifera in both shallow-water (Moodley et al., 2000) and deep-water environments (Levin et al., 1999; Moodley et al., 2002; Nomaki, 2002; Kitazato et al., 2003). Laboratory-based experiments can also provide information about aspects of deep-sea foraminiferal biology which otherwise would be very difficult to obtain (e.g. Kitazato, 1989; Hemleben and Kitazato, 1995; Gross, 2000; Heinz et al., 2001, 2002; Nomaki, 2002; Nomaki, pers. comm.). Finally, sediment impregnation techniques, when combined with fluorescent probes (Bernhard and Bowser, 1996; Pike et al., 2001), can reveal sub-millimetre details of the relationship between individual foraminifera and the sedimentary fabric in which they reside.

13.4. Problems in taxonomy

The accurate and consistent recognition of species is of fundamental importance in ecological studies. Considerable confusion has arisen over the application of names to some deep-sea foraminiferal species. To take one example, a small rotaliid that is common in the NE Atlantic Ocean and elsewhere has been variously referred to Eponides pusillus Parr, Epistominella pusillus (Parr), Alabaminella weddellensis (Earland), Eilohedra nipponica (Kuwano), Eilohedra levicula (Resig), Epistominella levicula Resig and Eponides leviculus (Resig) (Gooday and Lambshead, 1989; Gooday and Hughes, 2002). These problems were emphasised in a number of papers by Boltovskoy (e.g. Boltovskoy, 1978, 1983) who suggested that illustrations of species accompanied by references to the original description and, if necessary, brief remarks would avoid some of the confusion. This approach has been adopted by journals such as Marine Micropaleontology which publish taxonomic appendices and extensive illustrations of foraminiferal species. 68 ANDREW J. GOODAY

Modern advances in molecular genetics are providing a new understanding of species and their biogeography. There is increasing evidence for genetic differentiation among deep-sea metazoan species (Creasey and Rogers, 1999), particularly on topographically complex continental margins (France and Kocher, 1996; Chase et al., 1998; Etter et al., 1999; Quattro et al., 2001). Almost all benthic foraminiferal species currently described are morphos- pecies, i.e. they are based on test morphology. It is possible that some, for example, those occurring across a broad bathymetric range, include a number of cryptic species rather than being single genetic entities. Intraspecific morphological changes sometimes occur along bathymetric gradients (e.g. Boltovskoy, 1991; Spencer, 1992) and may reflect genetic differentiation. Molecular studies have revealed widespread cryptic specia- tion among planktonic foraminifera (e.g. Huber et al., 1997; De Vargas et al., 1999, 2001, 2002; Darling et al., 2000) and in the shallow-water benthic genus Ammonia (Holzmann and Pawlowski, 1997). Cryptic speciation remains to be demonstrated among deep-sea benthic taxa, although slightly different morphotypes have been recognised in some species, for example, Uvigerina peregrina (Loubere et al., 1995). In planktonic foraminifera, the distribution of cryptic species appears to be related to water masses of different productivity and hence to mesoscale upper ocean hydrography (de Vargas et al., 2001, 2002). This suggests that cryptic speciation is more likely to occur among benthic foraminifera on environmentally complex continental margins than on the more uniform abyssal plains, where species geographical ranges are probably very broad. If cryptic benthic foraminiferal species do exist in the deep sea, they should exhibit subtle morphological differences that could be used to distinguish them in the fossil record. Tests of otherwise almost identical planktonic species can be separated on the basis of porosity (Huber et al., 1997; de Vargas et al., 1999) and morphometric characteristics (de Vargas et al., 2001).

13.5. Biological–geological synergy in foraminiferal research?

Research by biologists and geologists has contributed to our under- standing of deep-sea foraminiferal ecology. The two disciplines tend to have different scientific aims and approaches. Biologists are concerned with the principles that govern the structure and functioning of ecosystems and therefore examine the effects of biological processes such as dispersion, interactions such as competition, predation and facilitation, and as physico-chemical factors like oxygen, food availability and currents. For geologists, the overriding aim is often the development and refinement of proxies for measurable physical and chemical variables that are important for understanding how ancient oceans functioned. As a BENTHIC FORAMINIFERA 69 result, they usually look for relations between species and groups of species (rather than assemblage parameters) and particular physical and chemical variables. Despite these contrasting approaches, there is considerable potential for synergy between palaeoceanography and biology (Gooday, 1994; Nees and Struck, 1999). Biologists and geologists share a common interest in many basic issues in deep-sea ecology and have often addressed them in the same geographical settings. Biology underpins the accurate reading of palaeoenvironmental signals, both faunal and geochemical, carried by fossil foraminifera. Palaeoceanographic studies, in turn, provide a record of faunal responses to changes in the environment over time scales that are much longer than those available to biologists (Cronin and Raymo, 1997; Den Dulk et al., 1998). It has long been known that deep-sea foraminiferal assemblages have responded over geological time to environmental fluctuations and recent studies reveal just how sensitive they are to rapid climatic oscillations (Cannariato et al., 1999). The long temporal perspective (103 to 106 or more years) provided by the palaeoceanographic record offers unique insights into the historical and macroecological processes that have helped to shape modern communities (Lawton, 1999). These are only now beginning to be exploited by marine biologists, for example, in the interpretation of large-scale patterns of genetic differentiation and species diversity in the deep sea (Rex et al., 1997; Quattro et al., 2001; Stuart et al., 2002). A broad perspective that combines biological and geological approaches to the study of benthic foraminifera (e.g. Loubere and Fariduddin, 1999b; Levin et al., 2001) may ultimately lead to a more complete understanding of the biology of these remarkable and immensely successful organisms.

ACKNOWLEDGEMENTS

I thank Frans Jorissen, John Murray, Joachim Scho¨ nfeld and an anonymous referee for critiques of various drafts of this paper and Alexander Altenbach, Elisabeth Alve, Joan Bernhard, Kerry Howell, Hiroshi Kitazato and Richard Lampitt for their comments on particular sections. I’m grateful to Lisa Levin, Hiroshi Kitazato and John Murray for discussions that helped in the formulation of ideas. Andy Henderson took the light photographs (Figures 2–3) using the PalaeoVision system at the Natural History Museum, London. Kate Davis prepared most of the figures. Financial support was provided by NERC Research Grant GST021749. 70 ANDREW J. GOODAY

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T. Subramoniam and V. Gunamalai

Unit of Invertebrate Reproduction and Aquaculture, Department of Zoology,University ofMadras, GuindyCampus,Chennai–600 025,India E-mail: [email protected]

1. Introduction ...... 92 2. Distribution and Natural History ...... 93 3. Sex Ratio and Size at Sexual Maturity ...... 95 4. Neoteny ...... 96 5. Protandric Hermaphroditism in E. asiatica ...... 99 6. Mating Habits ...... 104 7. Spermatophores and Sperm Transfer ...... 106 7.1. Morphology of spermatophores ...... 106 7.2. Histochemistry of spermatophoric components ...... 107 7.3. Origin of spermatophores ...... 111 7.4. Spermatophore dehiscence ...... 111 7.5. Adaptive role of spermatophores in sperm transfer ...... 111 8. Moulting Pattern of E. asiatica—A Case Study ...... 112 8.1. Moult cycle stages ...... 113 8.2. Size-related moulting frequency in E. asiatica ...... 117 8.3. Endocrine regulation of moulting ...... 117 8.4. Nutritional control of moulting in Emerita ...... 121 9. Reproductive Cycle ...... 122 9.1. Method of estimating reproductive cycle ...... 123 9.2. Reproductive cycle in E. asiatica ...... 124 9.3. Reproductive cycle of E. asiatica in relation to size ...... 128 9.4. Egg production ...... 129 9.5. Effect of temperature on egg development on the pleopods ...... 131 10. Interrelationship Between Moulting and Reproduction ...... 135 10.1. Role of haemolymph lipoproteins in moulting and reproduction ...... 136 10.2. Endocrine regulation of moulting and reproduction ...... 138

ADVANCES IN MARINE BIOLOGY VOL 46 Copyright ß 2003 Academic Press 0-12-026146-4 All rights of reproduction in any form reserved 92 T. SUBRAMONIAM AND V. GUNAMALAI

11. Biochemistry of Eggs ...... 139 11.1. Emerita yolk protein ...... 139 11.2. Carotenoid pigments in the eggs and yolk proteins ...... 143 11.3. Metal content of the yolk protein ...... 144 11.4. Hormonal conjugation to yolk protein ...... 145 11.5. Mechanism of yolk formation ...... 145 12. Yolk Utilisation ...... 146 12.1. Enzyme activity during yolk utilisation ...... 148 12.2. Energy utilisation in Emerita eggs ...... 153 12.3. Carotenoid metabolism during embryogenesis ...... 155 12.4. Embryonic ecdysteroids ...... 158 12.5. Occurrence and utilisation of vertebrate steroids in Emerita eggs ...... 160 13. Larval Development ...... 161 13.1. Larval description in Emerita talpoida ...... 161 13.2. Larval dispersal and megalopa settlement ...... 165 14. Emerita as Indicator Species ...... 168 14.1. Parasitisation of egg mass and ovary ...... 170 15. Conclusions ...... 170 References ...... 172

Emerita is a burrowing mole crab or sand crab, adapted to life in wave-washed sandy beaches of temperate and tropical seas. The reproductive biology of this anomuran crab presents several peculiarities, all contributing to its adaptation to this harsh environmental niche. We discuss the following aspects: 1) sex ratio and size at sexual maturity, 2) neoteny and protandric hermaphroditism, 3) mating behaviour and sperm transfer strategy, 4) synchronisation of moulting and reproduction, 5) environmental impact on reproductive cycle and egg production, 6) biochemistry of yolk utilisation and energetics, 7) larval development, dispersal and settlement and 8) the value of Emerita as indicator species. These aspects are discussed in the light of the life history pattern, comprising a sedentary adult and pelagic larval phases. The successful colonisation of the physically challenging habitat of the sandy beach by Emerita is attributable largely to reproductive strategy and the larval developmental and recruitment pattern. Sensitivity to changing environmental conditions, including pollution, make this intertidal crab an indicator species for monitoring anthropogenic impact.

1. INTRODUCTION

Crabs belonging to the genus Emerita burrow into wave-washed sandy shores and exhibit a high degree of adaptation to this precarious environ- ment. The morphological and behavioural features include modification of the appendages for fast burrowing, and filter feeding by means of modified BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 93 antennules. These features and a distinctive breeding biology, coupled with fast body growth and high fecundity, have enabled this group to colonise long sandy beaches of both tropical and temperate seas. The species of Emerita Scopoli, 1777, are medium-sized benthic crustaceans of the family Hippidae (Anomura: Hippoidea). Three genera are included in the family: Emerita Scopoli, 1777; Hippa Fabricius, 1787; and Mastigochirus Miers, 1878. Very recently, Haye et al. (2002) reported on the molecular phylogenetics of the group (Hippidae: Emerita) using sequence data from Cytochrome Oxidase I and 16S rRNA mitochondrial genes. Interestingly, these analyses suggest that Emerita analoga is closer to the Old World taxa than to the other New World species; thus the New World Emerita species do not constitute a monophyletic group. The life cycle of Emerita consists of two major parts, one sedentary and the other pelagic. The sedentary phase in the life cycle includes the juveniles, derived from the megalopa stage that settles on to the beach, as well as the different growth stages leading to adulthood. Many of the reproductive features of this crab exemplify adaptation for inhabiting wave-washed beaches. The life cycle includes the pelagic larval stages which live in offshore and open sea regions, followed by metamorphosis of the swimming zoea larvae into the crab-like megalopa, and the settlement of the latter onto the beach.

2. DISTRIBUTION AND NATURAL HISTORY

Exposed sandy beaches look superficially barren, but can have an abundant invertebrate infaunal community. The mole or sand crabs, including various species of Emerita, are often dominant inhabitants. Being a suspension feeder, Emerita is well represented in beaches characterised by large waves, wide surf zones, fine sands and gentle slopes (Dugan et al., 1995). The crabs play an important role in the economy of a sandy coast, contributing in a major way to secondary benthic production. The distribution pattern of each species is characteristic in that it is generally limited to long coastlines, though occasionally extending to offshore islands. In North America, E. analoga has a long distribution on the west coast, whereas E. talpoida inhabits predominantly the east coast. Two New World species, E. portoricensis and Hippa pacifica, have island distributions (Efford, 1976). In peninsular South India there are two species, Emerita asiatica (¼ E. emeritus) and E. holthuisi, the former inhabiting the east coast and the latter, the west coast. Figure 1 depicts the geographical distribution of the nine species of the genus Emerita. 94 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 1 Geographic distribution of Emerita species: Emerita analoga (Stimpson, 1857); Emerita asiatica (H. Milne Edwards, 1837); (¼ Emerita emeritus Linnaeus, 1767); Emerita austroafricana Schmitt, 1937; Emerita benedicti Schmitt, 1935; Emerita brasiliensis Schmitt, 1935; Emerita holthuisi Sankolli, 1965; Emerita portoricensis Schmitt, 1935; Emerita rathbunae Schmitt, 1935; Emerita talpoida Say, 1817). Data from Efford, (1976), Tam et al. (1996) with contributions from various other sources.

Different size classes of Emerita have different zonal distribution patterns. Weymouth and Richardson (1912) and MacGinitie (1938) observed that the youngest post-megalopa individuals are at the top of the wash zone and the oldest mature females of Emerita analoga are further down the beach towards the sea. Alikunhi (1944) and Subramoniam (1979a) also observed similar zonal distribution of E. asiatica in the Madras coast; the smallest individuals being commonest in fine sand near high water mark and the largest in coarse sand near low water mark, between these two zones specimens of intermediate sizes are found. Such a distribution of Emerita also minimises the stress of pounding waves especially on the young ones of the burrowing Emerita. Evidently, the smaller crabs move towards the low water mark as they grow bigger. In addition, they migrate vertically up and down beaches, using the tidal system to optimise filter feeding conditions. They can bury rapidly into the loose sand, can swim fairly efficiently by means of the modified uropods, and the carapace is streamlined from the flexure of the abdomen towards the head. Emerita burrows backwards, the burrowing being facilitated by the movements of the anterior pairs of legs as well as by the uropods and they come to rest in the sand facing oceanward. BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 95

The filter-feeding behaviour of Emerita is unique among arthropods (Weymouth and Richardson, 1912). Snodgrass (1952) described the anatomical modifications of the antennae in E. talpoida for filter feeding. They are feather-like structures with four rows of diverging setae, armed with inwardly directed secondary setae. When they unfold, the water passing over the animal from behind is filtered through the fine mesh of the setae. The mandibles of Emerita are much reduced structures. Zobell and Feltham (1938) suggested that sand crabs fed by ingesting sand and digesting the organic material including the bacteria mixed with it. However, detailed studies by Efford (1966) confirmed antennal filter feeding and comparisons were made with filter feeding by barnacles.

3. SEX RATIO AND SIZE AT SEXUAL MATURITY

Wenner (1972) proposed a size-related sex ratio for crustaceans and classified the male–female size relationships into four patterns. These are (1) standard (male–female ratios equal), (2) reverse (smaller individuals are all males and the larger ones, all females or vice versa), (3) intermediate (sex- ratios are intermediate between standard and reverse patterns and (4) anomalous (male–female overlap in a narrow size range). The anomalous pattern may also arise from factors such as differential growth rate and mortality as well as migration. In E. analoga, sex ratios based on size classes fit well in the anomalous pattern. Barnes and Wenner (1968) suggested that this close overlap between males and females, especially in the mid-size classes, could be interpreted as protandric hermaphroditism, by which the males change sex to females. However, Diaz (1981), from a population analysis of E. talpoida in the north Carolina beaches, concluded that sex ratio, calculated on the basis of the relative frequency of females in the population, fluctuated with season as well as recruitment pulses of the megalopa stage. For the tropical species E. asiatica, Subramoniam (1977b) calculated the size-related sex ratio, following the method of Wenner (1972). As seen from Figure 2 the overlap in size range between males and mature females is too wide to suggest a possible sex reversal in this species. Whereas the increased percentage of small size-group males indicates differential body growth of males and females, the declining percentage of males above 8 mm carapace length (CL) might be due to their death, as reported for the American species E. analoga (Efford, 1967). Further, the observation that males with distinct genital papillae and juvenile females devoid of genital papillae are found in almost equal proportions among the post-larval stages during settlement in the beach indicates that males and females develop 96 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 2 Size distribution of males, immature females and ovigerous females of E. asiatica. Note the close overlapping of both males and immature females in the population. The ovigerous females are continued from the last size group of 18–19 mm CL immature females. From Subramoniam (1977b). independently from the megalopa (Subramoniam, 1977b). In E. asiatica, the males achieve sexual maturity soon after metamorphosis from the megalopa (3.5 mm CL), whereas the females attained maturity only after considerable body growth (19 mm CL). This kind of difference in the size or age at sexual maturity between the sexes has been recorded in many species of Emerita (Table 1). Furthermore, Subramoniam indicated that the weight increase of the male gonadal apparatus is directly related to the increase in the male carapace length and body weight. These observations apparently indicated the parallel development of males and females from the megalopa (Subramoniam, 1977b).

4. NEOTENY

One recurrent feature of the life history of the sand crab genus Emerita is that with few exceptions the males are always smaller than the females (Table 1). In at least five species, the males are known to become sexually BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 97

Table 1 Size at sexual maturity of male and female of Emerita species.

Species Reference Male Female

E. analoga Knox and Boolootian, 10.0–22.0 15.0–30.0 1963 E. analoga Efford, 1967 6.0–12.0 13.0–31.0 E. analoga Barnes and Wenner, 6.0–11.0 8.0–22.0 1968 E. asiatica Menon, 1933 3.5–7.5 22.0–30.0 E. asiatica Subramoniam, 1977a 3.5–15.0 19.0–33.0 E. austroafricanus Barnard, 1950 *–35.0 23.0–37.0 E. emeritus Murugan, 1985 4.0–11.0 20.0–34.0 E. emeritus Murugan, 1985 3.0–10.0 20.0–37.0 E. holthuisi Sankolli, 1965 11.0–17.0 12.0–18.0 E. holthuisi Achuthan Kutty and *–10.0 10.0–15.3 Wafar, 1976 E. holthuisi Nagabhushanam and 3.0–11.0 4.0–18.0 Kulkarni, 1977 E. holthuisi Murugan, 1985 2.5–8.0 7.0–13.0 E. holthuisi Murugan, 1985 2.5–8.0 8.0–14.0 E. portoricensis Goodbody, 1965 *–8.0 9.0–17.0 E. portoricensis Quesnel, 1975 – 16.0–25.5 E. rathbunae Efford, 1967 2.5–** 33.0–41.0 E. talpoida Wharton, 1942 3.8–14.0 *–26.0 E. talpoida Efford, 1967 2.5–12.0 14.0–29.0 E. talpoida Diaz, 1981 3.25–10.25 14.10–29.25

*Minimum size not given; ** Maximum size not given. mature soon after their arrival on the beach as megalopas. The smallest mature males vary among species from 2.5 to 6 mm CL, whereas the females are not usually mature until they exceed 12 mm CL, except in E. portoricensis which matures at 8 mm CL. Although female maturity is attained as juvenile adults, mature males retain several larval characters. Subramoniam (1977b) described the secondary sexual characters, along with other morphological characters of the neotenic males of E. asiatica. The males lack the pleopods on the abdominal segments that are characteristic of mature females. However, they possess short stumps of the natatory pleopods found in the megalopa (Menon, 1933). Small males of E. talpoida also retain the stumps of the pleopods (Efford, 1967). They also show a general simplicity of the appendages associated with their small size. For example, the antennae are simple and do not have the regularly arranged, closely packed setal net of the larger animals. On the fifth thoracic leg of E. talpoida, situated at the inner side of the base of the coxa, there is a triangular sac, called a sperm sac (Wharton, 1942) 98 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 3 Male secondary sexual morphology of Emerita asiatica. (A) External morphology of genital papilla (ventro-lateral view); (B) fifth leg of male <5 mm carapace length showing location of genital papilla and arrangement of teeth in chela (lateral view); (C) chela of fifth leg of mature female (lateral view). OVD: opening of vas deferens; VD: vas deferens; G: genital papilla; BB: barbed bristles. Redrawn from Subramoniam (1979b). or genital papilla (Snodgrass, 1952). The external morphology of the genital papilla in E. asiatica is shown in Figure 3A. It consists of a protruding muscular sac with a tapering end. In freshly moulted males, the continuation of the vas deferens into this organ and its opening at the tip of the genital papilla is clearly seen. The papilla is supported from below by a separate process emanating from the basal segment. The latter has a rigid stem ending in an expanded hood-like structure, guarding the genital papilla in a semicircular fashion. The margin of the expanded border is beset with thick, inwardly curved barbed bristles. In the mature males, the arrangement of the teeth in the fifth chelate leg also presents peculiarities. In the adult females, the tips of the chelae are toothed, whereas in the males, the chelae possess 3 to 4 protruberances in the form of inwardly curved teeth at the tips (Figure 3B,C). Interestingly, the megalopa larva also displays the same structure in the chela of the fifth leg. The sexual dimorphism in size at sexual maturity and the resultant neoteny in the males appears to be brought about by two factors, namely, slower growth rate and lower survival time. In E. analoga and E. talpoida, the males normally die in spring and early summer (Efford, 1967). The smaller size of the mature males has an obvious advantage in accomplishing BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 99 mating with larger females in a turbulent marine environment such as the intertidal region (see below). Efford (1970) considered the generalisation that neoteny in the male is characteristic of Emerita, after examining several sources of published information. De Beer (1951) defined neoteny as precocious sexual maturity in which there is a relative retardation of body growth compared with the reproductive growth and maturation.

5. PROTANDRIC HERMAPHRODITISM IN E. ASIATICA

The problem of sex reversal in the sand crab Emerita is a story in itself. The occurrence of neoteny in several species of Emerita and the fact that the males die before reaching the size at which the females become sexually mature, result in considerable deviation from the 1 : 1 sex ratio. By applying a size-related sex ratio method, Barnes and Wenner (1968) found a sigmoid curve, characteristic of protandry in other crustaceans such as the deep sea prawns (Yaldwyn, 1966), and proposed for the first time a sex reversal hypothesis for E. analoga. In support, Eickstaedt, (1969) and Knapp and Wenner (as reported in Barnes and Wenner, 1968) postulated that some males, kept under laboratory conditions, changed sex. However, a series of laboratory culture experiments as well as natural environment observations on E. analoga (Auyong, 1981; Wenner and Haley, 1981; Conan et al., 1975), E. asiatica (Subramoniam, 1977b), E. talpoida (Diaz, 1981), E. portoricensis (Sastre, 1991) and an Island species, Hippa pacifica (Haley, 1979) suggest that the apparent anomaly in the sex ratio results only from the differential growth of males and females. Wenner and Haley (1981) summarised the arguments in favour of and against the sex reversal hypothesis for the hippid mole crabs, basing them mainly on population and sex ratio studies and laboratory experiments on differential growth and moult increments of males and females in different size groups. In all the above studies, there was no direct observation of the male gonads during the period when they might change to females, and hence the possibility of sex-reversal in Emerita species was at that time not resolved. Unequivocal existence of functional protandric hermaphroditism was demonstrated in E. asiatica by Subramoniam (1979c, 1981). The main reproductive events enumerated in the Figure 4 indicate that neotenous males continue to grow after serving an active normal male life, deviate from normal sexual behaviour, gradually lose their secondary and primary sexual characters, and undergo sex reversal by acquiring female characters around 19 mm CL. The disappearance of genital papillae around 15 mm CL, is the first visible sign of sex reversal. Concurrently, spermatogonial activity in testes ceases but hyperactivity of the mesodermic cells ensues 100 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 4 Chronology of sexualisation in female and male Emerita asiatica. From Subramoniam (1981). A.G. ¼ androgeni gland.

(Figure 5A). Such hyperactivity of the mesodermal cells in the testis during periods of sexual inactivity has also been shown in the crayfish Pontastacus leptodactylus leptodactylus (Amato and Payen, 1978). In the size range of 19–22 mm CL, these males possess a gonad comprising inactive testicular and active ovarian portions. The ovarian anlagen spread along the mid- dorsal line of the paired testis. During sex reversal, following the formation of separate ovarian anlagen, the ovarian structure contains typical follicle cells that have either migrated from the testicular region or differentiated from a prefollicular mesodermic tissue of the gonad (Figure 5B). These cells attach themselves to the vitellogenic oocytes, probably mediating uptake of yolk proteins from the haemolymph (Charniaux-Cotton, 1975b). The newly BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 101

Figure 5 Histological appearance of the hermaphroditic gonad in Emerita asiatica. From Subramoniam (1981). (A) Cross section through the hermaphroditic gonad of Emerita asiatica (19 mm CL). The testicular acini (T) contain spermato- gonial cells, but its lumen is devoid of spermatocytes and spermatozoa. The ovarian part (OV) is separated by connective tissue (CT). The oogonial cells (OG) are in the centre surrounded by previtellogenic (PV) and vitellogenic (VO) oocytes. OD ¼ Oviduct. Scale bar ¼ 100 mm; (B) Testicular acini showing oocytes in previtellogenesis (arrow). Note the basophilic cytoplasm of the previtellogenic oocytes. Spermatogonial cells (SG) are in the resting stage. Follicular cells are absent and a few residual spermatozoa (SPZ) are absent. Scale bar ¼ 25 mm; (C) Cross section through the proximal region of posterior median cord of the ovary. Germinal zone in the centre (GZ) surrounded by vitellogenic oocytes (VO). The connective tissue basal lamina contain many follicle cells (arrow). Scale bar ¼ 100 mm; (D) Transverse section through the middle region of a hermaphroditic ovary of an intersexual (CL ¼ 28 mm) to show the hyperactive somatic mesodermal cells (MC). TC ¼ Testicular acini; MT ¼ Metacercaria; PV ¼ Previtellogenic oocytes. Scale bar ¼ 150 mm; (E) Delamination of the follicle cells (FC) into the vitellogenic oocytes (VO) of the hermaphroditic ovary of an intersexual (CL ¼ 28 mm). Scale bar ¼ 10 mm. 102 T. SUBRAMONIAM AND V. GUNAMALAI formed median limb beyond the fused posterior extremity of the testis, however, lacks testicular elements (Figure 5C). It is composed of a central germarium surrounded by previtellogenic and vitellogenic oocytes. The vas deferens in these intersexuals is intact but its opening is occluded. They also begin to possess three pairs of pleopods and a pair of functional oviducts formed at the base of the coxae of the third walking legs. The external morphology of the reproductive system in the intersexual animals as compared with the testis and ovary of the normal crabs is depicted in Figure 6. These intersexuals with a functional ovary could be easily identified by the possession of only a few eggs on the pleopods. The malacostracan crustaceans are generally gonochoristic with geneti- cally determined sex. The genes for male morphogenesis act in the presence of an androgenic hormone and for females in its absence (Charniaux- Cotton, 1960). Sex reversal via protandric hermaphroditism has also been reported in other crustaceans (Ghiselin, 1969; Policansky, 1982). Inversion of sexual phenotype is influenced by epigamous factors exerted during growth (Gallien, 1959). In malacostracan crustaceans, the hermaphroditic potentialities are governed by the androgenic gland hormone (Charniaux- Cotton, 1965a). The sequential disappearance of primary and secondary male characters during the changeover phase of E. asiatica reported above, and the concomitant assumption of female characters strongly suggest

Figure 6 Diagrammatic representation of the testis, ovary and the hermaphro- ditic ovary of Emerita asiatica. t-testis; a.v.d.-anterior vas deferens; m.v.d.-mid vas deferens; p.v.d.-posterior vas deferens; a.l.o.-anterior ovarian lobe; m.l.o.-middle ovarian lobe; o.d.-oviduct; p.m.l.o.-posterior median ovarian limb; o-ovary; v.d.-vas deferens. Redrawn from Subramoniam (1981). BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 103 similarities with protandric hermaphroditic natantians such as Pandalus borealis (Carlisle, 1959) and Lysmata seticaudata (Charniaux-Cotton, 1960b) with regard to androgenic gland control of sexual differentiation. In the reproductively active males of E. asiatica, the androgenic glands, which are attached to the subterminal portion of the distal vas deferens consist of simple cellular strands, packed with columnar cells, whose nuclei are ovoid, conspicuous and endowed with dense chromatin. Dense secretory materials with vacuoles of various sizes fill the cytoplasm. In contrast, the androgenic glands of a larger male (9 mm CL) showed many degenerative changes. As seen in Figure 7, most of the gland has degenerated and a fine granular central portion devoid of normal cellular structure is evident. Continued degeneration of glandular cells in the periphery is evidenced by the presence of picnotic nuclei with wheel-shaped chromatin clumps adhering to the nuclear membranes. At 15 mm CL and above, the androgenic gland was not detected in the males. As inferred from the above histological account, high androgenic gland activity, when the neotenous males are reproductively active, and the degeneration of the

Figure 7 Composite diagram of androgenic gland of a large non-functional male (CL ¼ 9 mm). Large portion of the gland shows cellular degeneration (stippling). Scar-like thickenings (single arrow) are evident in the distal region of the gland. New cordon of cells appears in the basal region (double arrow). V.D. vas deferens; P.N. pycnotic nuclei. Redrawn from Subramoniam (1981). 104 T. SUBRAMONIAM AND V. GUNAMALAI androgenic gland during the gradual disappearance of male secondary sexual characters and stoppage of spermatogonial activity in large males, clearly suggest that a fall in circulating androgenic gland hormone may be responsible for these changes in E. asiatica. Extirpation and grafting experiments on the androgenic gland in Lysmata seticaudata have yielded similar results (Charniaux-Cotton, 1959; Berrear-Bonnentant, 1963). From a series of experimental studies on the hermaphroditic prawn L. seticaudata, Touir (1977a, b, c) suggested a bihormonal control over the androgenic gland as well as the gonadal activity from the brain neurosecretory factors. Interestingly, Subramoniam (1981) also came across instances of incomplete transformation of sex, as found in large females of E. asiatica (28, 29 mm CL). In these crabs, histological examination indicates that a separate ovarian portion is never found above the non-functional testis. However, the anterior half of the paired tubular gonad is dominated by oocytic differentiation, whereas the posterior half possessed inactive testicular tissues (Figure 5E). The mature oocytes in the anterior portion undergo oosorption, accomplished by the infiltrating follicle cells, which on entry into the ooplasm, turn phagocytic (Figure 5D). Interestingly, these intersexuals retain the vas deferens, but the genital papilla is absent. Paired oviducts are also present, but are incompletely differentiated, compared to the functional oviduct of the secondary females arising from successful sex reversal of E. asiatica. The bihormonal control of brain neurosecretory factors over sex reversal, as suggested by Touir (Touir, 1977a,b,c) for L. seticaudata may also explain the incomplete gonadal transformation in E. asiatica (see Subramoniam, 1981, for discussion). Protandric hermaphroditism in E. asiatica is significant and points to the high probability of sex reversal in other species of Emerita in which the males attain precocious sexual maturity in the post-larval stage.

6. MATING HABITS

Mating behaviour has been described in many species of Emerita. MacGinitie (1938) detailed the mating habits of E. analoga from both field and laboratory observations. He found mating to occur mostly in late spring or early summer. Males apparently gather around the egg-laying females as much as two to five days before coupling and remain in contact with the female. The males attach to the female by the dactyls of their fourth legs, which according to MacGinitie (1938) are equipped with a sort of sucker pad surrounded by stiff hairs. As the female burrows in the sand, the males collect on her ventral side and remain there until they deposit their BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 105 sticky spermatophores. This author also observed that spermatophore deposition occurred on the just-moulted females. He also reported a laboratory observation that the soft-shelled female was found to lie on top of the sand with its abdomen unflexed, while two to four males deposit sperm on the ventral surface in the cervix between the third and fourth pairs of legs. An interesting observation by the same author was that the males were more commonly found in the upper part of the surf zone; but during the mating season (late spring and early summer) they occurred with the female lower down on the beach. The behaviour of the males in the ventral region of the females has also been reported for other species of Emerita. According to Wharton (1942), the small males were found in the gill chambers, clamped between the coxae of the thoracic appendages, or attached to egg masses, and some even seemed to roam about on the ventral surface of the larger females. The relationship between the clinging males and the female is such that some males were found to moult whilst associated with the females, as recorded in E. asiatica (Menon, 1933). This kind of mating behaviour in the Emerita species has been termed ‘‘incipient parasitism’’ by Wharton (1942). In the temperate species, the occurrence of the males has been reported to be seasonal, limiting the mating season to late spring and early summer, as in E. analoga (Efford, 1967); however, in the tropical species where the reproduction and embryonic development in the pleopods go on uninterruptedly throughout the year, small functional males in the size range of 3.75 and 5 mm CL, occur throughout the year (Subramoniam, 1977b). This observation is at variance with an earlier finding on E. asiatica, from another location on the east coast of peninsular India, viz., Visakapatnam, that males occur only during the summer months (Ganapathi and Lakshmana Rao, 1959). From the Madras coast on the east coast of India, Subramoniam (1977b) has not only observed the year round occurrence of small functional males, but also observed the continued growth of the males to a larger size of up to 11 mm CL. However, these larger males have not been found to take part in mating with larger egg- laying females, although they possessed well-developed spermatophores in the swollen vas deferens. This author suggested that the sexually active smaller males, once metamorphosed from the megalopa, not only attain precocious sexual maturity without body growth, but also undergo a certain degree of growth regression during subsequent moults. Understandably, since these males are inside the burrow, clinging on to the females, they have no chance of active feeding by antennal filtering, thus resulting in considerable growth retardation. Furthermore, the smaller size is advanta- geous for their hide out on the ventral region of the burrowing females. More importantly, the females accept these smaller males, rather than the bigger males, for mating since they will not disturb normal activities such as 106 T. SUBRAMONIAM AND V. GUNAMALAI burrowing and reburrowing when the tides are in and out, as well as filter feeding. Again, mating between equally sized partners would be a cumbersome process in the intertidal region, as the larger males clinging onto females could be brushed off by waves, or by sand when the female burrows. Coupled with aggregation behaviour (Efford, 1965; Cubit, 1969), the neotenous male of Emerita species has probably evolved as a means to increase the chance of fertilisation in the unstable habitat. The occurrence of large non-functional but sexually mature males in the population of E. asiatica merits further comments on their sexual behaviour. The size-increase of non-functional large males suggests that the specialisa- tion towards neoteny in this species is still incomplete (Subramoniam, 1977b). A peculiar mating behaviour of these larger males has also been reported by Subramoniam (1979b). A male of 8.5 mm CL was observed to deposit a spermatophore ribbon on the ventral side of a freshly moulted immature female. Deposition of spermatophores by larger males on immature, helpless freshly moulted females indicates indiscriminate copula- tion, amounting to raping, in E. asiatica. Incidentally, Kittredge et al. (1971) provided some experimental evidence for sex pheromonal activity of the moulting hormone, crustecdysone, in a number of decapods. Whether such pheromonal attraction to the moulting females could trigger spermato- phoral deposition by males on the freshly moulted female E. asiatica is conjectural and needs experimental support.

7. SPERMATOPHORES AND SPERM TRANSFER

One of the significant reproductive attributes of Emerita in the successful colonisation of the sandy beach is the mode of sperm transfer and the epizoic fertilisation of the eggs deposited externally on the pleopodal hairs. As with most marine crustaceans, excepting the free-spawning penaeid shrimps, spermatophore production in sand crabs is a specialisation to transfer semen in the marine environment (Subramoniam, 1993). Emerita lacks an intromittent organ and hence spermatophores can be considered to be the alternative vehicle to transfer sperm. As in other decapod crustaceans, the spermatozoa of Emerita are not motile. Hence, the production of complex spermatophores is imperative for effective sperm transfer by the neotenous male.

7.1. Morphology of spermatophores

Early workers on E. talpoida and E. analoga reported deposition of a sperma- tophoric ribbon on the ventral sternum of the females (MacGinitie, 1938; BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 107

Wharton, 1942). A full description of the spermatophore of E. asiatica was given by Subramoniam (1977b, 1984). E. asiatica produces pedunculate spermatophores, characteristic of anomuran crabs and certain macrurans (Mouchet, 1931; Bloch, 1935; Pochon-Masson, 1983). However, the morphology of E. asiatica spermatophores reveals certain peculiarities. As in pagurid anomurans, the spermatophore consists of three distinct parts: the sperm-containing ampoule, the peduncle or stalk and a glutinous pedestal to fix the spermatophore on the sternal region of the female. In this crab, spermatophores are dimorphic in nature; one in the form of a truncated cone and the other in the form of a tumbler. These two types of spermatophores are arranged almost alternately in a single file (Figure 8A). The lower ends of the spermatophores possess peduncles, which join with a continuous gelatinous ribbon. The whole spermatophoric mass is embedded in a protective jelly-like matrix. In this respect, E. asiatica differs from other anomuran crabs, such as Diogenes pugilator and Pagurus bernhardus, wherein spermatophores are attached to the gelatinous base singly or in groups of two or three (Bloch, 1935). The extruded spermatophore has a thick double-layered refractile covering. The spermatozoa are glued together by a viscous fluid and packed closely and irregularly inside the spermatophore.

7.2. Histochemistry of spermatophoric components

A detailed histochemical analysis revealed that mucopolysaccharides complexed with proteins form the main components of the spermato- phores of E. asiatica (Table 2). The sperm mass substance within the ampoule is composed of highly sulphated acidic mucopolysaccharides (AMP) whereas the inner layer of the spermatophore contains carboxy- lated AMP. In contrast, the ventral gelatinous cord, peduncles and the outer layer of spermatophore ampullae when inside the vas deferens, are periodic acid schiff (PAS)-positive. The entire protective gelatinous matrix stains blue in Alcian blue-PAS indicating its acidic nature. The gelatinous matrix also contains vicinyl hydroxyl groups as revealed by PAS positivity, when used alone. The gelatinous layer and the peduncle stain intensely with Millon’s reagent suggesting the presence of tyrosin. Strong phenolase activity was detected in the ventral gelatinous chord, but diphenols are absent. While such findings may suggest ‘‘self tanning’’ (Hackman, 1974) in the spermatophoric mass, the phenolic compounds may have other roles such as antimicrobial activity (Brunet, 1980) for the exposed spermatophores. The outer layer of the spermatophore in the freshly extruded condition is refractile to all stains. The spermatophoric 108 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 8 Morphology and dehiscence of Emerita asiatica spermatophores. (A) Side view of spermatophore ribbon; (B&C), Truncated cone-shaped spermatophore before and during sperm release; (D&E), Tumbler-shaped spermatophore before and during sperm release. A: ampoule; GC: gelatinous cord; S: stalk; SP: spermatozoa. Redrawn from Subramoniam (1977b, 1993)

mass of E. asiatica does not undergo ‘‘hardening’’ on exposure to sea water. The predominance of various muco-substances in the spermato- phoric components of E. asiatica is correlated to their protective as well as structural functions (Jeanloc, 1970; Montgomery, 1970). The presence of a significant quantity of glycogen in the sperm cells may suggest a nutritive role. REIGBOOYO H ADCRAB, SAND THE OF BIOLOGY BREEDING

Table 2 Histochemical characteristics of mucopolysaccharide substances of spermatophoric mass of Emerita asiatica. Data from Subramoniam (1993).

Reagent or test Sperm mass Spermatophore Peduncle/ Gelatinous To indicate substance inner/outer layer gelatinous cord matrix

Best’s carmine þR þ/þR/R þþ/þþR/R þR Glycogen and mucopolysaccharides Schiff alone ÀÀ/ÀÀ/ÀÀFree aldehydes Periodic acid þþþM þþþ/þM/M þþ/þþM/M þM Glycogen, 1,2 glycols Schiff (PAS) Sperm cells Alcian blue - PAS þþM þþ/þM/M þ/þM/M þþþÀB Mucopolysaccharide and Sperm cells unsaturated fatty acids þB Acid and neutral EMERITA Sperm mass mucopolysaccharides substance Aldehyde fuchsin þþBB þ/ÀP/ À/ÀþP Sulphated and non-sulphated acid mucosubstances Bracco - Curti þþBB À/ÀÀ/ÀÀSulphated groups Toluidine blue at different pH pH 1 þþV þ/ÀB/ þ/þþÀV Sulphated mucosubstances pH 3 þþV þþ/ÀB/ þ/þV/V þþV Sulphated mucosubstances pH 4 þþBV þþþ/ÀB/ þ/þV/V þþþV Phosphated and carboxylated mucosubstances pH 7 þþB þþþ/ÀV/ þ/þB/B þV Carboxylated mucosubstances

(continued) 109 110

Table 2 Continued.

Reagent or test Sperm mass Spermatophore Peduncle/ Gelatinous To indicate substance inner/outer layer gelatinous cord matrix

Alcian blue: critical electrolyte concentrations of MgCl2 0.2 M þB þ/þB/ þ/þB/B þþB Carboxylated mucosubstances 0.6 M þB þ/ÀB/ À/ÀþþB Phosphated mucosubstances 0.8 M þþ/þÀ/ÀþþB Strongly sulphated mucosubstances 1.0 M þB À/ÀÀ/ÀþþB Strongly sulphated mucosubstances .SBAOIMADV GUNAMALAI V. AND SUBRAMONIAM T. 1% Aqueous alcian blue þB þ/þB/ þ/þþþB Sulphated mucosubstances Chitosan ÀÀ/ÀÀ/ÀÀChitin

B ¼ blue; BB ¼ benzidine blue; BV ¼ bluish violet; M ¼ magenta; P ¼ pink; R ¼ red; V ¼ violet; À¼negative; Ƽdoubtful; þ¼moderately positive; þþ ¼ positive; þþþ ¼ intensely positive. Sperm mass refers to sperm mass substance as well as sperm cells. When reactions are distinct for sperm cells and sperm mass substance they are indicated accordingly. BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 111

7.3. Origin of spermatophores

The spermatophore originates in the anterior region of the vas deferens. In the proximal region, the spermatozoa, as released from the testis, are agglutinated into many clusters, which are enveloped by a gelatinous membrane emanating from the columnar epithelial cells. The peduncle as well as the ventral gelatinous cord is secreted from the ventral epithelium of the distal vas deferens. In the dorsal region of the distal vas deferens the inner epithelial cells produce a typhlosole-like projection, which secretes a frothy substance, constituting the protective gelatinous matrix of the spermatophoric ribbon.

7.4. Spermatophore dehiscence

The mechanism of sperm release from the pedunculate spermatophores of anomuran crabs is controversial. In E. asiatica, sperm release in the deposited spermatophores does not occur until egg release from the oviduct. Spermatophores in different stages of sperm release have been recovered from the egg masses of freshly ovulated E. asiatica (Subramoniam, 1977b). Sperm release occurs only through a definite spermatophore opening. In the truncated cone-like spermatophores, the opening is made through the nipple-like projection found at the opposite end of the peduncle (Figure 8B,C). In the other larger type, the wider region is rimmed by a well-defined lip which is firmly closed before sperm release. During dehiscence, streaming of spermatozoa was first observed in the gaps formed at the corners of the wider end and then in several sites of the centre, resulting in the complete opening of the lips. After extrusion of all spermatozoa, the lips remain completely apart (Figure 8D,E). The fact that the spermatozoa release occurs only after contact with the eggs suggests that an oviductal secretion may be responsible for the digestion of the cementing material closing the lip of the spermatophore.

7.5. Adaptive role of spermatophores in sperm transfer

Spermatophore extrusion occurs through the muscular genital papillae situated at the inner side of the base of the fifth thoracic leg. In E. asiatica, the spermatophore deposition occurs only in the fresh moult condition and the sticky nature of the mucoid spermatophoric ribbon enables fast and firm attachment to the sternum of the females. Further, in E. asiatica, spawning 112 T. SUBRAMONIAM AND V. GUNAMALAI rapidly follows spermatophore deposition (Subramoniam, 1977b) and hence spermatophore ribbon remains as a jelly, enabling the dehiscence of the spermatophores by the oviductal secretion, as mentioned earlier. As the interim between spermatophore deposition and ovulation is brief, the spermatophoric ribbon does not undergo hardening, as in the lobster. Incidentally, mating via spermatophore deposition may also provide a stimulus for ovulation in Emerita. The highly adaptive nature of Emerita spermatophores in effecting epizoic fertilisation in a turbulent environment is also reflected in their possible evolution from the macruran type of spermatophoric mass. In the Macrura, the tubular spermatophores are enveloped in several acellular accessory mucoid secretions which protect the enclosed sperm cells during their prolonged epizoic storage on the female body (Radha and Subramoniam, 1985). Although anomuran crabs possess a pedunculate type of spermatophore, a sand crab species, Albunea symnista, also belonging to the family Hippidae, and co-existing with E. asiatica in the sandy beach possesses a macruran type of spermatophoric ribbon (Subramoniam, 1984). The spermatophoric tube of A. symnista, however, shows node-like constrictions giving rise to internal discontinuities. Such a breaking up of a continuous spermatophoric tube by constrictions (Albunea) and distinct spermatophoric ampullae with drawn-out peduncles set on a basal filamentous pedestal (Emerita) suggests that these anomuran sand crabs may be mid-way forms in the evolution of discrete pedunculate spermatophores of the anomurans from the tubular spermatophores of Macrura (Subramoniam, 1993).

8. MOULTING PATTERN OF E. ASIATICA—A CASE STUDY

Moulting facilitates continued body growth by periodic shedding of the old cuticle and secretion of a new cuticle. A characteristic feature, which is uncommon among other arthropod groups, is the continuation of moulting even after attaining sexual maturity in many crustacean species. In general, moulting and reproductive activities are temporally separated in large-bodied crustaceans such as lobsters and brachyuran crabs. On the contrary, crustaceans with high fecundity and faster body growth exhibit closeness in their moulting and reproductive cycles. In E. asiatica, there exists a close synchronisation between moulting and the female reproductive cycle (Gunamalai and Subramoniam, 2002). Hence, in order to evaluate the interrelationship between moulting and reproduction, a detailed delineation of different moult cycle stages is required. BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 113

8.1. Moult cycle stages

The moult cycle stages have been determined in E. asiatica using the criteria of changes in the cuticular morphology, epidermal retraction and setagenic events occurring in the pleopod. Furthermore, an aggregation of hemocytes characteristic of moulting stages was also evaluated throughout the moult cycle stages in E. asiatica, using microscopic observation on the pleopodal lumen (Gunamalai and Subramoniam, 2002). Four major stages, namely postmoult, intermoult, premoult and ecdysis have been distinguished. The defining features of different moult cycle stages are given in Table 3.

8.1.1. Postmoult (Stages A and B)

Postmoult stage refers to the crab immediately after ecdysis. During this period, the soft and pliable new cuticle undergoes hardening. The moulted animal is inactive during this phase, which lasts for 30 min; thereafter, it regains activity and burrows in the sand. The pleopods are soft and transparent. The setae are thin-walled, and their lumen is wide and prominent with a granular matrix filling up the space (Figure 9). This stage is further divided into A1,A2 and B.

8.1.2. Intermoult (Stage C)

As in many malacostracan crustaceans, the intermoult stage is the longest of all moult cycle stages. The exoskeleton has become progressively hard and calcified, making further subdivision of this stage difficult. The character- istic feature of the intermoult stage is that the setal development on the pleopods has been completed (Figure 9). Nevertheless, the intermoult stage can be divided into three substages (C1,C2 and C3) based on the hardness as well as the rigidity of the exoskeleton both on the dorsal and lateral sides.

8.1.3. Premoult (Stage D)

This is the preparatory stage to ecdysis and includes several substages (D0–D4). This stage starts with apolysis, the retraction of epidermis from the cuticle, creating a moulting space for the formation of new cuticle. The epidermal retraction followed by the secretion of new cuticle is easily seen in the pleopods. Hence, several substages characterising the extent of the epidermal retraction and the formation of new cuticle within the pleopodal tip can be examined microscopically. It may be seen from the Figure 9D–I that a setal groove originates as a deep depression in the retracted epidermis 114 T. SUBRAMONIAM AND V. GUNAMALAI

Table 3 Moult cycle characteristics of Emerita asiatica. From Gunamalai and Subramoniam (2002).

Moult Duration Characteristics of Microscopical observations stages of stage exoskeleton of the pleopodal changes

Post moult A1 5–6 h Freshly moulted crabs; Pleopods soft and transparent; cuticle soft and pliable; setal shaft thin walled; setal crab not active; after lumen wide and filled with 15–30 min becomes granular matrix; setal base active and burrows into evenly arranged on pleopods sand, if moulting is outside the burrow A2 24 h Exoskeleton pliable and No change in pleopods soft but begins to harden B 4 d Carapace continues to Pleopods hard and rigid harden Intermoult C1 5 d Exoskeleton remains Setal lumen becomes narrow; hard; lateral side of the setal wall thickened; setal carapace depressed by cone visible finger pressure C2 4–5 d Exoskeleton evenly hard Setal cone prominent, a throughout body surface tube-like structure observed under setal articulation; epidermis condensed with setal articulation (node) C3 3–4 d Carapace attains rigidity No changes in pleopods on dorso-lateral sides Premoult D0 3–4 d No changes in Appearance of setal groove exoskeleton at base of pleopod; no epidermal retraction D00 Same as stage D0 Apolysis starts; narrow gap between old cuticle and epidermis evident; setal groove extends up to tip of pleopods D1 2–3 d Exoskeleton becomes Retracted zone between old brittle cuticle and epidermis widens; tip of new setae still within setal groove; new cuticle appears wavy D10 No further changes in New setae protrude into exoskeleton. retracted zone. D100 Same as above. New cuticle clearly seen as a layer

(continued) BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 115

Table 3 Continued.

Moult Duration Characteristics of Microscopical observations stages of stage exoskeleton of the pleopodal changes

D2 3 d Exoskeleton becomes Epidermal retraction more brittle; epidermis continues; new setae clearly and secreted new cuticle visible and thin walled; appear as thick black appearance of setal layer on removal of old articulation cuticle at base of new seta D3 12–24 h Carapace becomes thin Setal articulation more and soft, cracks under prominent; new setae have pressure; exoskeletal extruded almost completely colour changes to pale in the retracted area; grey from white setal lumen clearly seen within new setae D4 3–6 h Animal inactive; Old setal exoskeleton appearance of suture completely separated from at intersegmental new setae membrane of carapace; ecdysis commences

in the pleopod. As the epidermal retraction continues with the formation of new cuticle, the new setae begin appearing from the base of setal grooves. In the following stages of premoult, the setal grooves get elevated pushing the internal setae to the outside of the groove. When the setal groove reaches the periphery of the epidermis, the new setae will be completely protruded out into the retracted zone. The raised epidermis and the cuticle surrounding the new setae form the basis for setal articulation. Concurrent with the internal changes in the setal development, there is resorption of old cuticle. When the process is complete, the old cuticle becomes brittle, and at stage D3 a gentle depression will result in the cracking of old cuticle. As the crack widens exposing the inner soft cuticle, water absorption begins through the soft cuticle, resulting in the swelling up of the body cavity. Figure 9A–I shows all the above described changes in the pleopods during premoult.

8.1.4. Ecdysis (Stage E)

This stage represents the emergence of the crab through the ecdysial sutures of the old cuticle. As a result of endocuticular resorption, the old cuticle is thin and friable. The first ecdysial suture appears in the intersegmental membrane connecting the cephalothorax and the abdomen. When flexed 116 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 9 Diagrammatic representation of epidermal and setagenic changes in the pleopods of Emerita asiatica during different moult stages. (A) Postmoult stage AB; (B) Intermoult stage C; (C) Early premoult stage D0; (D–I) Premoult stages. gm ¼ granular matrix; sl ¼ setal lumen; sa ¼ setal articulation; sc ¼ setal cone; sg ¼ setal grooves; er ¼ epidermal retraction; re ¼ retracted epidermis; erz ¼ epidermal retracted zone; ns ¼ new setae. Scale bars: A – 110 mm, B – 75 mm, C–80mm, D – 70 mm, E – 115 mm, F – 90 mm, G – 65 mm, H – 90 mm, I – 100 mm. Redrawn from Gunamalai and Subramoniam (2002). BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 117 ventrally the suture ruptures in a transverse direction allowing the animal to escape. When the crab emerges, the old exoskeleton along with all appendages is intact. After emergence, the animal remains inactive for about 5–10 min. On no occasion was the moulted crab found to consume the exuvium. The new cuticle continues to expand by water absorption, thereby increasing the body volume. Figure 10 depicts the sequences in the ecdysis of E. asiatica.

8.2. Size-related moulting frequency in E. asiatica

Growth in decapod crustaceans is facilitated by periodic moulting. As a rule, the frequency of moulting is high in the immature animals and, after the onset of reproduction, it declines considerably in order to facilitate the reproductive activities which are normally completed within an extended period of intermoult. In E. asiatica moulting continues even beyond sexual maturity facilitating simultaneous body growth and reproduction. We determined the size-related moulting frequency in both immature and mature females. The results are shown in Figure 11 which clearly demonstrates differences in the moulting frequency between three major size classes, namely immature (10–17 mm CL), actively reproducing females (18–22 mm CL) and large size females (23–33 mm CL). The percentage occurrence of moulting animals (as represented by premoult) is higher (52–72%) than that of non-moulting females (intermoult) in the first size class representing immature females. However, the percentage occurrence of premoult animals declines (30–40%) from 21 mm CL onwards with slight increase at 22 and 25 mm CL. In the actively reproducing females, the intermoult frequency is considerably higher (40–60%) in between 21–29 mm CL. Similarly, the large size group females ranging from 30–33 mm CL showed a high frequency of non-moulting females. Thus, the ratio of moulting and non-moulting forms is always higher in the smaller size group of females, whereas in the actively reproducing females and large size females, the percentage of intermoult animals slightly exceeds the percentage of premoult females (Figure 11).

8.3. Endocrine regulation of moulting

In decapod crustaceans moulting is usually controlled by a bihormonal system consisting of moulting gland (Y-organ, ecdysteroids) and eyestalk X-organ/sinus gland complex (moult inhibiting hormone) (Subramoniam, 2000). Whereas the ecdysteroids promote moulting, the neuropeptides from the eyestalk neurosecretory centres inhibit the synthesis of ecdysteroids by Y-organ. For Emerita species, although the moulting physiology has been 118 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 10 Moulting sequence in Emerita asiatica. (A) first phase of ecdysis in which the ecdysial suture is visible; (B and C) exposed part of its cephalothorax and abdominal region seen through the ecdysial suture (dorsal view); (D) fully moulted animal with soft exoskeleton. From Gunamalai and Subramoniam (2002). BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 119

Figure 11 Percentage occurrence of the premoult stage among females of Emerita asiatica, size class 10–17 mm CL (immature females); 18–22 mm CL (females maturing for the first time); 23–33 mm CL (repetitively reproducing females) during the period of one year (1998–99). understood well in E. asiatica, the control of moulting by endocrine means has not been adequately investigated. We have recently investigated the role of haemolymph ecdysteroids in moulting of E. asiatica, using radio- immunoassay techniques (RIA). This study has been made in three size classes of E. asiatica namely, immature (10–17 mm CL), maturing for the first time (18–22 mm CL) and repetitively reproducing females (23–33 mm CL). This study indicates a characteristic premoult peak as shown already in other crustaceans (Chang, 1991). In all the three size classes used, there is a gradual buildup of haemolymph ecdysteroids from early intermoult stages reaching a major peak in D2 stage of premoult, following which the ecdysteroids precipitously fall to a minimal level before ecdysis at D3–D4 stage (Figure 12). The premoult peak of haemolymph ecdysteroids, coinciding with the apolysis and the new cuticle synthesis, suggests a direct role for ecdysteroids in the moulting activity. Interestingly, the percentage value of ecdysteroids is found to be always highest during all moulting stages in the immature females in comparison with first maturing and repetitively reproducing females (Figure 12). The relatively higher concentration of haemolymph ecdysteroids in the immature females may indicate its profound effect in bringing about quick, repetitive moulting, uninterrupted by reproductive activity, thus achieving faster body growth. Our experimental studies with 20-hydroxyecdysone (20E) have adduced further evidence towards its positive influence on moulting (Gunamalai, 2001). The crabs receiving 20E at C3 stage of intermoult hastened premoult 120 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 12 Ecdysteroid level in the haemolymph of females of Emerita asiatica in different size groups. Immature females (10–17 mm CL); females maturing for the first time (18–22 mm CL); repetitively reproducing females (23–33 mm CL) during moult cycle stages. From Gunamalai (2001).

Table 4 Percentage of precocious premoult changes observed at various time intervals after 20 endysone injection at moult cycle stage C3 of vitellogenic females of Emerita asiatica. From Gunamalai (2001).

Group II C3 Stage Moulting Stages (%)

0 00 C3 D0 D1 D1 D1 D2 D3–4 E 0 day Control 100 – – – – – – – Experiment 100 – – – – – – – 1st day Control 100 – – – – – – – Experiment 83.33 16.66 – – – – – – 2nd day Control 33.33 66.66 – – – – – – Experiment – 16.66 16.66 66.6 – – – – 3rd day Control – 83.33 16.66 – – – – – Experiment ––––66.66 33.33 – – 4th day Control – – 50 50 – – – – Experiment –––––5050– 5th day Control – – – 66.66 33.33 – – – Experiment –––––––100

activities, culminating in precocious ecdysis (Table 4). Understandably, increased haemolymph ecdysteroid titre would bring about early onset of premoult changes, thus establishing the moult-inducing effect of ecdyster- oids in E. asiatica. Such experimental evidence on moult induction in BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 121

E. asiatica is in agreement with earlier results on other decapod crustaceans such as Homarus americanus (Rao et al., 1973). Although the role of ecdysteroids in moulting of E. asiatica is well illustrated in the preceding account, not much is known on the negative control of moulting by the neurosecretary centres of X-organ/sinus gland complex. Emerita is a burrowing crab and hence the paired eyestalks are secondarily reduced. Our unpublished observation indicates the absence of ganglionic structures such as the medulla externa, medulla interna and medulla terminalis in the eyestalk of E. asiatica. In the stalk-eyed decapods, the X-organ, the seat of major neuropeptide synthesis is found in the medulla terminalis and the neuronal ends of their neurosecretory cells establish connection with the neurohaemal storage organ, the sinus gland, found in between the medulla externa and medulla interna (Subramoniam et al., 1998). In the absence of any neurosecretory centres in the eyestalk of E. asiatica it is possible that the X-organ/sinus gland complex is embedded in the brain, as in the isopods, which also lack stalked eyes (Hanstrom, 1939). Indirect evidence to this effect is provided by Vasantha (1995) who found that the brain extract of E. asiatica contained the crustacean hyperglycemic hormone, which forms the principal neuropeptide (as much as 60% of all eyestalk neuropeptides) (Keller, 1992). The abbreviation of neurosecretory centres within the eyestalk has reached an extreme stage in Albunia symnista, another species of mole crab (family Albunidae) coexisting with E. asiatica in the Madras Coast. This species is totally blind and hence lacks eyestalks altogether (personal observation). Understandably, the moult-inhibiting neuropeptides are produced from the abbreviated X-organ/sinus gland complex embedded in the brain of E. asiatica and exert their inhibitory effects on ecdysteroid synthesis in the Y-organ, as in other malacostracan crustaceans.

8.4. Nutritional control of moulting in Emerita

Despite the fact that moulting is under hormonal control, environment may also play a significant role in determining the seasonality of moulting frequency at population level. For marine invertebrates in general, temperature, photoperiod, salinity and availability of food are known to exert influence on the vital physiological processes relating to growth and reproduction (Giese and Pearse, 1974). In Emerita species, the evidence indicates that abundance of food materials and the accumulation of nutrients have an influence on the seasonality and intensity of moulting (Siegel, 1984). As a filter feeder, Emerita might thus depend on the seasonal abundance of plankton to control the moulting process. While studying the 122 T. SUBRAMONIAM AND V. GUNAMALAI size-specific moult synchrony in E. analoga on the coast of California, Siegel (1984) concluded that physical factors such as temperature and lunar phase, and biological factors like reproductive seasonality or pheromones did not play any role in maintaining the intensity and synchrony of moulting in this crab. The moult frequency of the female crabs reared in the tank showed a peak during June–August; and a corresponding field observation also indicated high frequency of moulting during these months when food availability was high. In the same species of Emerita, Eickstaedt (1969) reported that the intensity of moulting coincided with the period of peak reproductive activity, suggesting that both reproduction and moulting at population level are controlled by a common environmental factor such as availability of food. Siegel (1984) also concluded that egg production did not affect moult synchrony, under laboratory conditions. In an elegant experiment of altering feeding regimen, using fresh and plankton-filtered sea water, he showed that synchronisation and desynchronisation of moulting could be achieved in the laboratory conditions. In a recent study on the size-related frequency of moulting in E. asiatica from the Madras Coast, India, Gunamalai (2001) observed continuous moulting all through the year. She found that during the months of September–December (1998–99) the frequency of moulting in all the three size classes studied was high (see Figure 11). Higher moulting rate during these months may be explained in terms of meteorological factors, including upwelling, influencing the availability of phytoplankton nutrients, with an overall increased production of plankton (Muthu, 1956).

9. REPRODUCTIVE CYCLE

The worldwide distribution of Emerita in tropical and subtropical sandy beaches has resulted in the acquisition of different reproductive periodicities for the different species. There are two major types of reproductive cycles; those inhabiting the tropical beaches show continuous reproductive cycles and the species occurring in temperate regions have an annual breeding cycle (Table 5). According to Semper (1881) all reproductive periodicities ought to be obliterated in tropical marine invertebrates, since in the tropics annual changes in temperature are minimal. Orton (1920) also supported the idea that all tropical marine animals breed continuously irrespective of the seasons. In agreement with Orton’s rule, two tropical species of Emerita, one from Jamaica (E. portoricensis) and the other from the east coast of India (E. asiatica) have been shown to breed continuously (Goodbody, 1965; Subramoniam, 1977a). Conversely, all the temperate species inhabiting the BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 123

Table 5 Summary of the breeding season of different species of Emerita.

Species Location Duration Reference

E. talpoida Beaufort, N.C. June–September Wharton, 1942 (U.S.A.) E. talpoida Bogue Banks January–August Diaz, 1980 (U.S.A.) E. analoga California (U.S.A.) April–October Boolootian et al., 1959 E. analoga San Diego, February–September Cox and Dudley, California (U.S.A.) 1968 E. analoga El Tabo, Chile March–November Osorio et al., 1967 E. analoga California (U.S.A.) March–November Eickstaedt, 1969 E. analoga California (U.S.A.) April–November Perry, 1980 E. analoga Caleta Abarea August–December Conan, 1978 (Chile) E. portoricensis Jamaica January–December Goodbody, 1965 E. portoricensis Trinidad (West January–December Quesnel, 1975 Indies) E. asiatica Madras, India January–December Menon, 1933 E. asiatica Madras, India January–December Subramoniam, 1977a E. emeritus Trivandrum, W. Coast of India: Site 1 Sangumughom February–January Murugan, 1985 Site 2 - Vizhinjam February–January Murugan, 1985 E. holthuisi Rathnagiri January–December Nagabushanam and Kulkarni, 1977 E. holthuisi Sangumughom September–December Murugan, 1985 E. holthuisi Vizhinjam July–December Murugan, 1985

east and west coast of America tend to concentrate their reproductive activities towards the summer months.

9.1. Method of estimating reproductive cycle

Like many other decapod crustaceans, Emerita carries the eggs on the pleopods of the abdominal segments where they are hatched and released as zoea larvae. The breeding season of these crabs can therefore be determined by plotting the percentage of ovigerous females against time (Boolootian et al., 1959; Knudsen, 1960). Although several workers on Emerita have used incidence of ovigerous forms to determine the reproductive cycle, this method has inherent difficulties in the estimation of gonad changes inside the animal. For example, in the species inhabiting the temperate seas, egg 124 T. SUBRAMONIAM AND V. GUNAMALAI masses remain on the pleopods long after cessation of gonadal activities for the particular reproductive season. Reproductive activities such as the formation and maturation of gametes may start well ahead of the spawning season, thereby obscuring the correct commencement point of the reproductive cycle. Therefore, more accurate quantitative methods such as gonad index and histological examination are needed to assess the cyclic seasonal reproduction. The gonad index can be calculated in several ways, but usually it is the ratio of the gonad wet weight to the wet weight of the whole animal expressed as a percentage (Giese and Pearse, 1974). It rests upon the assumption that the ratio of body parts varies little with changes in size of the animal. While several studies have employed the gonad index method to delineate the reproductive cycle of Emerita species, a study on the egg development in the pleopod may also be taken into consideration, especially when the crab breeds continuously throughout the year (Boolootian et al., 1959). Egg mass index is calculated as a percentage of the weight of the whole animal (Eickstaedt, 1969). As a corollary to egg mass index, seasonality in the pleopodal egg development can also be assessed by studying the mean developmental stages of eggs on the berried females in various months of the year. This method obviously necessitates a classification of the stages in egg development leading to the hatching of zoea larvae (Subramoniam, 1979a).

9.2. Reproductive cycle in E. asiatica

Emerita asiatica breeds continuously in the east coast of peninsular India. A detailed study of the breeding cycle was made by Subramoniam (1977a and 1979a) at Marina beach on the Madras Coast in 1974 and 1975. From the incidence of ovigerous females and the gonad index, the population appeared to be breeding continuously. A similar result obtained on this species by observing the year-round occurrence of zoea larvae in plankton collected from the near shore waters of the Madras Coast led to the same conclusion (Menon, 1933). Giese (1959) defined such continuous breeding of marine invertebrates as ‘‘an extended breeding season’’, meaning that the individuals of a species are producing several successive broods during the year or that they are breeding asynchronously. That is, ‘‘some are in the earlier stages of maturation, some are spawning and still others are already spent.’’ For E. asiatica, the population not only breeds continuously but also the individuals in the population breed repetitively. This is evidenced by the percentage of ovigerous females, which varies from 73% to 100% in size classes between 22 and 33 mm carapace length (Subramoniam, 1977a). Data collected on the gonad index during 1975 and 1976 showed high values, also suggestive of continuous breeding, though there was some BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 125

Figure 13 Annual fluctuations in the gonad, egg mass and hepatic indices of Emerita asiatica; range of carapace length given above bottom axis. From Subramoniam (1979a).

seasonal fluctuation (Figure 13; Subramoniam, 1979a). Breeding intensity throughout this study was also revealed by the egg mass index, which shows a pattern similar to that of the gonad index. As inferred from Figure 13, there is a steady rise in the gonad and egg mass indices from January to May followed by a fall in June and August and then a steep fall in November and December. As a whole, reproductive activity is steady between January and May while in the remainder of the year breeding is irregular with three declines in June, August and Nov–Dec. It is interesting that the dip in reproductive activity occurs during the monsoon rainy months, whereas high reproduction takes place during the premonsoon summer months. Comparison of the reproductive cycle of Emerita species from the west and east coast of India is instructive in relation to difference in hydrobiological features such as the rainfall and the consequent salinity changes in the inshore waters of these coasts. On the west coast E. holthuisi is the dominant species (Sankolli, 1965) whereas E. asiatica (¼ E. emeritus) is the only species recorded from the east coast of India. Rare occurrences of E. asiatica have been recorded from a few localities on the west coast. However, Murugan (1985) has described the co-occurrence of E. asiatica 126 T. SUBRAMONIAM AND V. GUNAMALAI and E. holthuisi in equal proportions on the south-west tip of the Indian peninsula. On the north-west coast of India, at Rathnagiri, E. holthuisi was shown to be a continuous breeder, but with two peaks of berried females occurring in the months of March and September (Nagabushanam and Kulkarni, 1977). These two peaks coincide with the pre- and postmonsoon seasons on the west coast. Murugan (1985) made extensive studies on the reproductive cycles of both E. asiatica and E. holthuisi, coexisting in two locations, Thiruvanandapuram and Vizhinjam on the south-west coast of India. For E. asiatica he found continuous breeding with three distinct peak periods at April–May, July and September–October, as determined by the percentage of ovigerous females in the population. However, the gonadoso- matic index indicated that the same population showed major peaks in July and March. Interestingly, E. holthuisi, which breeds more or less continuously on the north-west coast of India, shows only an extended breeding period from July–December on the south east coast of Thiruvanandapuram (Murugan, 1985). This season coincided with the postmonsoonal months. In Indian waters a major factor that influences intertidal as well as offshore life is the monsoon rain that differs in time and intensity on the two coasts (Panikkar and Jeyaraman, 1966). On the west coast, the southwest monsoon brings abundant rain during May and August. This results in the lowering of salinity in coastal waters, especially in the brackish water lagoons that also receive fresh water from many large rivers. On the east coast of India, the slow, retreating monsoon normally brings rain around October in places from 19 to15N, but in places south of 15Nit rains later, in November (Hu-Cheng, 1967). Varadarajan and Subramoniam (1982) made an estimate of breeding intensities of 78 marine invertebrates from both east and west coasts. Figure 14 indicates that each month between 60–80% of the east coast species are breeding continuously and there is no seasonal pattern of peak activity. In contrast, most breeding activity on the west coast occurs between September and March. While the deterrent action of the heavy summer rain checks reproduction on the west coast, its milder intensity than the retreating monsoon on the east coast, especially near Madras, without any swift flowing rivers, may enhance reproduction, as in E. asiatica. An interesting difference from the continuous reproduction found in E. asiatica from Madras occurs in another sand crab, Albunea symnista, belonging to the family Hippidae, coexisting with E. asiatica. Although this species does breed continuously on the Madras coast, there are two distinct reproductive peaks, one in January and another in July, which indicate a semiannual breeding pattern (Subramoniam and Panneerselvam, 1985). Examination of the ovary of this species during the rainy months of October to December showed cessation of reproductive activity in the BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 127

Figure 14 Percentage of marine invertebrate species breeding each month on the east and west coasts of India. Solid circles: percentage on west coast (N ¼ 15); open circles: those on east coast (N ¼ 63). From Varadarajan and Subramoniam (1982).

majority of females. Giese and Pearse (1974) thought this type of semiannual breeding pattern was characteristic of tropical seas influenced by monsoon rains. However, E. asiatica living on both the east and west coasts of India, breeds throughout the year, utilising the equable environmental conditions. Unlike the gonad index, the hepatic index of E. asiatica does not show any significant fluctuation throughout the year (Figure 13). The hepato- pancreas, as in other decapod crustaceans, constitutes the only central organ for mobilisation of precursor molecules both for reproduction and moulting (Parvathy, 1970; Gunamalai, 2001). In view of the year round reproduction and moulting, the hepatopancreas is expected to supply organic raw materials for these two physiologically energy demanding processes. Considering the steady synthesis and release of the protein materials from the hepatopancreas, a low hepatic index maintained all through the year is not unexpected. 128 T. SUBRAMONIAM AND V. GUNAMALAI

9.3. Reproductive cycle of E. asiatica in relation to size

A reinvestigation on the reproductive cycle of E. asiatica on the Marina beach at Madras, using a combination of gonad index (GI) and direct microscopic observation of the maturing ovary has yielded a better understanding of size-related breeding pattern in this locality. For this, we have classified the ovarian maturation stages into four categories on the basis of the colour changes and direct microscopic measurement of the oocyte diameter in the maturing ovary. Increase in the oocyte diameter is a function of ovarian maturation as indicated in Table 6. In this study, we have examined up to 1000 females with carapace length ranging from 16–33 mm. The frequency occurrence of four ovarian stages has been plotted against different size classes as a percentage value at each ovarian stage (Figure 15). The first stage of ovarian maturity has been obtained from 16 mm CL onwards with two peaks, one centred at 18 mm CL and a second at 25 mm CL. The first peak coincides with the maximum number of animals maturing for the first time at 18 mm CL and the second large peak coincides with the peak reproductive size class of 25 mm CL. Ovarian stage II appears to start from 19 mm CL onwards with a small peak at 20 mm CL and a larger peak at 25 mm CL. Similarly, stage III also appears to start from 20 mm CL with a gradual rise resulting in a flattened peak in the size range of 24–25 mm CL. The stage IV ovary appears to start from 21 mm CL with a broader peak reaching over 75% at 25 mm CL. This peak gradually declined to reach a minimum value at 32–33 mm CL. The overall data comprising the percentage frequency of four ovarian stages against the size classes indicates that the peak ovarian activity in Emerita occurs between 21–29 mm CL. Thereafter, the frequency of all the stages declined. It may be further inferred that the animals start the ovarian maturation at 16 mm CL onwards but continue maturation up to 21 mm, when the first ovulation is witnessed. From 22 mm CL onwards the frequency of all the four stages increases very steeply to reach the maximum at 25 mm CL, thereby

Table 6 Classification of the ovarian stages of Emerita asiatica. From Gunamalai (2001).

Ovarian Colour Oocyte diameter Gonadosomatic stages (mm) index

Stage I Whitish yellow 242.50 Æ 12.99 1.05 Æ 0.27 Stage II Yellow 248.00 Æ 19.39 1.06 Æ 0.28 Stage III Orange 257.50 Æ 42.64 2.12 Æ 0.58 Stage IV Bright orange 361.66 Æ 27.93 3.65 Æ 0.89 BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 129

Figure 15 Relationship between the different ovarian developmental stages and the size classes of Emerita asiatica. indicating that the intermediate size group ranging in carapace length between 24–25 mm CL has the maximum reproductive activity. From then onwards the frequency of all the four stages declines very gradually to reach minimum in 32–33 mm CL. The animals found in the range of 30–33 mm are not only rare but also most of them are in the reproductively senile condition without showing any gonadal recrudescence after the hatching of the larvae from the pleopods. Similar size-related breeding peaks have also been reported for west coast species, E. asiatica and E. holthuisi (Murugan, 1985).

9.4. Egg production

Emerita produces a large number of yolky eggs and attaches them to the setae of the endopodite of the pleopods. The abdomen, with the egg- carrying pleopods, is tightly flexed beneath the thorax. This gives protection to the developing embryos on the pleopods while the crab is inside the burrow. In Emerita, age of maturity, breeding frequency, and clutch size have a bearing on fecundity. As pointed out by Wenner et al. (1974), the maturation age of the female crab could vary in different populations of the same species, owing to differences in food availability. Thus, the E. analoga population from Santa Cruz Island attained sexual maturity at a lower carapace length due to poor food availability and slower growth rate. Conversely, with abundant food availability on the Santa Barbara coast, the 130 T. SUBRAMONIAM AND V. GUNAMALAI growth rate was not only high but there was also an increase in size of the females at sexual maturity. A season-dependent size at sexual maturity is also suggested for E. analoga by Eickstaedt (1969). Wenner et al. (1987) studied egg production in E. analoga in reference to the size and the year class at three California locations. They found that the overall pattern of egg number as a function of size was similar for the first two year classes, but egg production by the few third year crabs was highly variable at the San Clemente site. Interestingly, the slope of the regressions of size and egg number for each year class showed significant variation. The slope was quite steep for first year and less so for the second year, but in the third year the slope decreased considerably. This may suggest that the egg laying intensity is inversely proportional to the size of the crab. However, the number of eggs per spawning by the individual crabs always increased linearly with the size of the laying female. Several authors who worked on E. analoga at different beaches in north and south America also provided data on the number of eggs produced as a function of size, although the number per brood varied with season and locality (Osorio et al., 1967; Efford, 1969; Eickstaedt, 1969). The size-related fecundity has also been determined for the tropical species E. asiatica (Figure 16) indicating again that the egg- laying capacity increases in direct relation to its body mass (Subramoniam, 1977a). In another population of E. asiatica from the east coast of India,

Figure 16 Relationship between carapace length and the number of eggs carried in the pleopods of Emerita asiatica. From Subramoniam (1977a). BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 131 some 50 km south of Madras, the egg-laying adults reach a maximum size of 39 mm CL. Correspondingly, the number of eggs laid by these bigger females are also more than that of the females inhabiting Marina beach at Madras (unpublished observation). Table 7 summarises data on the fecundity of Emerita species inhabiting tropical and temperate beaches. In general, the number of eggs produced by the female per body size in the tropics far exceeds that of the temperate species. For example E. asiatica inhabiting the Madras coast produces 4000 eggs at a body size of 25 mm CL, whereas, the same sized temperate species E. analoga, in California produces a maximum of 2500 eggs. This is indicative of a temperature-dependent egg production in the Emerita species. Apart from temperature effect, other local environmental factors such as salinity and availability of food may also influence the rate of egg production. This is evident in E. analoga with a wide distribution on the west coast of America, ranging from Canada in the North to Mexico in the South. A major difference between the north and south living species is that in the south, the crabs grow to a larger size than those in the north. A corresponding difference in the number of eggs per female is also noticeable. In addition, on the east coast of southern peninsular India at Madras, E. asiatica produces a higher number of eggs when compared with its counterpart on the southwest coast of Trivandrum. Again, E. holthuisi inhabiting the west coast, not only grows to a lesser body size but also produces fewer eggs than E. asiatica coexisting in the same beach.

9.5. Effect of temperature on egg development on the pleopods

Using several sets of published and unpublished data on the sand crab E. analoga, which is widely distributed along the west coast of the Americas, Wenner et al. (1991) plotted the egg development time as a function of temperature. They found that the egg development time varied from 40 days at 25C to 100 days at 12C. Furthermore, the duration of embryonic development on the pleopod may also have a direct relation to the frequency of spawning in these crabs. Fusaro (1980) provided experimental evidence that decreased egg development time resulting from increased seawater temperature has a positive effect on the number of egg batches produced per female. Understandably, egg production by populations of E. analoga living in cooler waters may be depressed relative to those populations experiencing warmer water conditions. Eickstaedt (1969) calculated the monthly mean egg development of the berried females in the natural population to estimate the variation in the time of egg development, as influenced by environmental factors such as 132

Table 7 Fecundity profiles (number of eggs produced per female) of Emerita species in relation to body size and geographical occurrence.

Carapace E. analoga (Efford, 1969) E. analoga E. asiatica E. emeritus E. holthuisi length (Eickstaedt, (Subramoniam, (Murugan, 1985) (Murugan, 1985) (mm) 1969) 1977)

Northern Southern California Madras Sangumughom Vizhinjam Sangumughom Vizhinjam end of end of (East GUNAMALAI V. AND SUBRAMONIAM T. North America* North America** coast of India)

8 50 220 9 365 550 10 680 710 11 25/- 1115 1125 12 70/- 1285 1370 13 110/- 1795 14 225/- 15 -/125 16 -/425 17 -/425 REIGBOOYO H ADCRAB, SAND THE OF BIOLOGY BREEDING 18 -/550 19 700/225 20 1000/525 21 22 2800 23 7000 3100 1000 2000 24 1150/2300 3700 25 14000 4000 2800 3525 26 1750/3000 4600 27 17000 5000 5750 5350 28 1850/3750 5500 29 6100 8050 7800 30 2300/- 6250 31 6500 11500 9850

32 7300 EMERITA 33 7900 13100 11800 34 35 16300

*La Jolla to Tofino; **Garibaldi to Playa De La Mission. 133 134 T. SUBRAMONIAM AND V. GUNAMALAI temperature and salinity. His results agreed well with those of Fusaro in that a reduction in the mean egg development time coincides with high breeding activity in the summer month of August. While the monthly mean egg development time shows wide variation in temperate species such as E. analoga, in the tropical species, E. asiatica, egg development time is almost the same in all months of the year (Subramoniam, 1979a). The proportion of various stages in the egg development of berried females in different months of the year 1975–76 is given in Figure 17. It is clear from the figure that almost all stages, except stage X (hatching stage) are obtainable at any time in different individuals of the population, suggesting that egg development leading to the release of zoea larvae may be a

Figure 17 Egg development in Emerita asiatica: n – number of ovigerous crabs examined; MED – monthly mean egg development. From Subramoniam (1979a). BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 135 continuous process in accordance with the year-round breeding activity in the population (Subramoniam, 1977a). Obviously, on the east coast of India at Madras, the ambient seawater temperature as well as the other conditions such as optimum salinity and the availability of food both for the adult and the released larvae are conducive to promote and maintain both egg production and egg development on the pleopods at a high profile throughout the year (Panikkar and Jayaraman, 1966). By contrast, on the west coast of India, where the monsoonal rains as well as the swift flowing rivers lower the salinity of the coastal waters significantly, the intensity of breeding declines during June–August.

10. INTERRELATIONSHIP BETWEEN MOULTING AND REPRODUCTION

In the majority of the crustaceans, reproductive physiology is greatly influenced by somatic growth, permitted by periodic moulting in the adults. As evident from the preceding account, E. asiatica is not only a continuous breeder but also exhibits year-round moulting. A detailed analysis of the ovarian and moult cycle stages in the adult crab has not only indicated a close correlation between moulting and reproduction, but also provides evidence that some of the processes are closely linked and overlapping. In female E. asiatica, the reproductive cycle is repetitive; when the pleopodal embryos undergo development, there is a concurrent maturation of oocytes within the ovary, making it ready for the next spawning. Moulting invariably occurs after hatching of the larvae from the pleopods and before spawning. It was believed earlier that the presence of pleopodal embryos exerts an inhibitory effect on the onset of moulting in embryo- carrying malacostracan crustaceans (Adiyodi, 1988). However, in E. asiatica, initiation of the moulting process, such as the apolysis, invariably starts almost midway through the development of the brood. The premoult changes advance further up to D1, at the time when the pleopodal embryos hatch out as zoea larvae. No females examined at the time of embryo hatching are found in the intermoult stage. Tirumalai (1996) and Gunamalai and Subramoniam (2002) have also observed the occurrence of the yolk precursor protein vitellogenin in the haemolymph throughout intermoult and premoult stages in E. asiatica suggesting that the process of vitellogenesis continues well into the premoult stage. Evidently, there is a perfect synchronisation of moulting and ovarian cycles, thus allowing body growth and reproduction to occur simultaneously. The overlapping of moulting and reproductive activities in E. asiatica is further reflected in the haemolymph and ovarian total protein levels 136 T. SUBRAMONIAM AND V. GUNAMALAI

Table 8 Haemolymph protein levels during the moult cycle stages versus different size classes (10–17 mm CL immature females; 18–22 mm CL, first maturing females; 23–33 mm CL, continuously reproducing females). Numbers within parentheses indicate the total number of crabs analysed within each stage. From Gunamalai and Subramoniam (2002).

Haemolymph total protein (mg mlÀ1)

Moult stages 10–17 mm CL 18–22 mm CL 23–33 mm CL Immature First maturation Continuously females of the females reproducing females

AB 0.5418±0.2783 (3) 2.9865±0.8726 (11) 5.2388±1.5801 (5) C1 14.2266±5.2020 (35) CC2 1.1451±0.246 (3) 8.9593±4.1198 (11) 16.2619±7.0891 (14) C3 19.6398±6.9638 (18) D0 2.0705±0.4949 (4) 16.6802±1.1542 (19) 22.4768±5.3111 (42) D1 2.2545±0.6899 (6) 17.8527±3.0275 (8) 16.8872±4.8338 (13) D2 3.0418±1.0626 (6) 18.7403±4.0847 (16) 25.4245±3.2675 (14) D3–4 0.7438±0.1425 (4) 3.0367±2.0376 (6) 9.1816±0.3221 (3)

(Gunamalai and Subramoniam, 2002). Haemolymph protein is low soon after spawning in the postmoult stage, then gradually increases from intermoult stage C1 to C3 to reach a peak value at the onset of premoult stage D0 (Table 8). This period of increasing trend in haemolymph protein has been correlated with the intense vitellogenic activity that occurs in the ovary, while the eggs on the pleopods undergo embryonic development. However, the haemolymph protein exhibits a statistically significant decline during stage D0–D1, which coincides with the last stage of pleopodal embryonic development, leading to the hatching of the larvae. Following this decline, the haemolymph protein level reaches a peak at D2 stage, then once again drops very sharply in stage D3–4, suggesting a role in vitellogenesis and new cuticular synthesis respectively occurring in the intermoult and premoult.

10.1. Role of haemolymph lipoproteins in moulting and reproduction

As in other crustaceans, haemolymph plays a major role in the transport of precursor materials to the sites of egg formation and cuticle synthesis in Emerita. Lipoproteins are the major means of transporting lipid materials from the site of synthesis to the target tissues. Using electrophoretic techniques, Gunamalai (2001) isolated three slow moving lipoprotein BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 137

Figure 18 Comparison of haemolymph lipoproteins from male, immature and mature (Vitellogenic) females of Emerita asiatica. Msp ¼ male specific protein; LpI ¼ lipoprotein I; LpII ¼ lipoprotein II; LpIII ¼ lipoprotein III; Hcy ¼ hemocyanin; Sp ¼ simple protein. From Gunamalai (2001). fractions from the haemolymph of E. asiatica (Figure 18). Among them, lipoprotein I is the dominant one found in both males and females in all stages of development; but showed intensity differences in accordance with moulting and female reproductive cycles. There is increased intensity during vitellogenesis and new cuticle synthesis. Lipoprotein II is sex- specific and appears in the female during vitellogenesis, but is absent in males. This lipoprotein corresponds to the primary yolk precursor protein, viz. vitellogenin, as determined by similarities in electrophoretic mobility and immunological identity (Tirumalai and Subramoniam, 1992; Tirumalai, 1996). On the other hand, lipoprotein III is stage-specific in its appearance and is found only during the premoult stage of both males and females, suggesting a specific role in cuticle synthesis by the epidermal cells. Quantitative analysis of total haemolymph proteins also adduces evidence to support the role of haemolymph in the supply of raw materials to the vitellogenic process and new cuticle formation (Gunamalai and Subramoniam, 2002). In the immature female and first maturing females, the blood protein rises steeply in the premoult stage, corresponding to new cuticle formation, followed by a sharp decline in the late premoult stage when the cuticle synthesis is over. However, in egg laying adult females, the haemolymph protein level rises steadily during the progression of the intermoult stage when almost all vitellogenic 138 T. SUBRAMONIAM AND V. GUNAMALAI activities occur and, from the onset of premoult stage the protein registers yet another peak followed by a sharp decline in the late premoult stages (Table 8).

10.2. Endocrine regulation of moulting and reproduction

The close relationship between moulting and female reproduction in E. asiatica discussed above could imply common influencing endocrine factors. In several decapod species the moulting hormones, ecdysteroids, also subserve functions in the control of reproduction and embryogenesis (see Subramoniam, 2000, for references). As shown in Figure 12, the level of haemolymph ecdysteroids in the postmoult stage is minimal but it begins to rise gradually throughout the intermoult stage. From D0 onwards the titre demonstrates a sharp increase in the haemolymph to reach a maximum level in D2. From then onwards, the ecdysteroid level declines again to reach the basal level at D3–4 of the premoult stage. Analysis of ovarian ecdysteroids during the moult cycle stages also shows accumulation of ecdysteroids within the ovary (Gunamalai unpublished observations). Again, the titre of ecdysteroids in the haemolymph and the ovary exhibits a reciprocal relationship during the moulting stages, thus suggesting that the haemo- lymph ecdysteroids, when present in excess, could be sequestered in the ovary. The rising trend in the haemolymph ecdysteroids during the premoult stages also implies that vitellogenin synthesis and uptake by the ovary could occur under a high titre of ecdysteroids. In this context, it is of interest to note the studies by Okumura et al. (1992) in the fresh water prawn Macrobrachium nipponense and by Wilder et al. (1991) in Macrobrachium rosenbergii, where there is a close correlation between moulting and reproduction. However, in M. rosenbergii, two types of moulting namely, reproductive and non-reproductive moulting occur. The non-reproductive moult signifies repetitive moulting outside the ovarian cycle. During the reproductive moult, the ovarian cycle is completed within the intermoult stage and spawning occurs soon after ecdysis, as in the case of E. asiatica. The overlapping of moulting and ovarian cycle in the Macrobrachium species is further reflected in a parallel rise of vitellogenin and haemolymph ecdysteroids right up to the D1 stage of the premoult (Okumura et al., 1992). This study supports our observation in E. asiatica that vitellogenesis and the new cuticle synthesis during the premoult stage occur under high titre of haemolymph ecdysteroids. In other decapods also, such as Penaeus monodon, active vitellogenesis has been shown to occur during the extended premoult period (Crocos, 1991). Although the independent role of ecdysteroids in stimulating and maintaining vitellogenesis cannot be confirmed from these data on E. asiatica, it is evident that the ovary BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 139 accumulates large quantities of ecdysteroids both in free and bound forms (Subramoniam et al., 1999). Obviously, the stored ecdysteroids in the egg, derived maternally, may have a role during embryogenesis (see below). Experimental studies involving the exogenous injection of 20 hydro- xyecdysone (20E) in E. asiatica show the common influence of this hormone on moulting and reproduction (Gunamalai unpublished observations). Injection of ecdysteroids at the C3 stage resulted in precocious commence- ment of premoult changes as evidenced by new cuticular synthesis and pleopodal setagenesis (Table 4). More interestingly, in the ecdysteroid injected crabs, embryonic development on the pleopods was also enhanced. Injection of 20E stimulated protein synthesis in tissues such as ovary, hepatopancreas and integumentary tissue together with an increase in haemolymph total proteins (Table 9). This may again suggest that, in addition to their controlling effect on moult induction, 20E also acts as a metabolic hormone by inducing protein synthesis related to vitellogenesis and new cuticle synthesis, obviously under different titres.

11. BIOCHEMISTRY OF EGGS

11.1. Emerita yolk protein

Emerita asiatica lays a large number of yolky eggs at each spawning. As in other decapod crustaceans, the yolk proteins, also called lipovitellins, are high-density lipoproteins with carbohydrate as the major covalently linked prosthetic group. They are invariably conjugated to a carotenoid pigment. Tirumalai and Subramoniam (1992 and 2001) have characterised E. asiatica yolk proteins. These comprise two lipovitellins (Lv I and Lv II) constituting as much as 90% of the total egg proteins. In SDS-PAGE analysis, Lv I yielded two subunits with molecular weights of approximately 109,000 and 105,000 Daltons respectively; whereas, Lv II resolved into six subunits with molecular weights of 65,000, 54,000, 50,000, 47,000, 44,000 and 42,000 Daltons, respectively. The carbohydrate component of the yolk exists in three forms, namely free carbohydrate, protein- and lipid-bound carbohydrates (Table 10). The protein-bound carbohydrates are dominated by hexose, hexosamine and galactosamine. The Lv II contains the higher amount of N-linked oligosaccharides than the O-linked oligosaccharides. Sialic acid is absent. It is assumed that the abundant O-linked oligosaccharides of Emerita lipovitellin may play a role in the secretion of yolk precursor protein during yolk synthesis and recognition of its receptors on the oocyte membrane during yolk accumulation. In addition, the O-glycosylation may also render 140

Table 9 Quantification of protein in different tissues (hemolymph, ovary, hepatopancreas, integumentary tissues) of Emerita asiatica. Effect of exogenous 20 E (0.05 mg per crab) on moult cycle of C3 stage (Mean Æ SD). Data from Gunamalai (2001). Days Haemolymph Ovary Hepatopancreas Integumentary tissue (mg/ml) (mg/mg) (mg/mg) (mg/mg) Mean Æ SD Mean Æ SD Mean Æ SD Mean Æ SD

Control Experiment Control Experiment Control Experiment Control Experiment

0 3.77 Æ 0.76 3.74 Æ 0.44 18.81 Æ 1.98 19.22 Æ 2.33 11.69 Æ 1.94 11.08 Æ 0.71 5.68 Æ 0.86 6.09 Æ 0.18 GUNAMALAI V. AND SUBRAMONIAM T. 1 3.90 Æ 0.52 4.05 Æ 0.23 18.97 Æ 4.39 35.58 Æ 2.09 11.94 Æ 0.67 9.26 Æ 0.61 4.49 Æ 0.25 8.75 Æ 0.92 2 5.35 Æ 0.75 6.24 Æ 0.70 21.02 Æ 1.72 38.77 Æ 2.34 10.38 Æ 1.09 16.07 Æ 1.78 7.15 Æ 0.18 8.95 Æ 0.24 3 5.45 Æ 0.31 8.55 Æ 1.87 26.66 Æ 5.82 37.46 Æ 0.86 9.36 Æ 1.11 16.8 Æ 2.04 8.67 Æ 1.48 11.86 Æ 2.03 4 5.58 Æ 0.25 9.27 Æ 1.57 26.58 Æ 2.94 37.54 Æ 1.36 10.75 Æ 1.6 20.53 Æ 1.55 9.2 Æ 0.8 9.69 Æ 0.21 BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 141

Table 10 Sugar composition of delipidated Lv II of Emerita asiatica. Data from Tirumalai (1996).

Carbohydrates mg per 100 mg delipidated Lv II

Hexose 1.375 Æ 0.32 Hexosamine 1.460 Æ 0.14 Galactosamine 1.020 Æ 0.09 Mannose 0.730 Æ 0.11 Fucose 0.120 Æ 0.07 Glucose ND Sialic acid ND N-linked oligosaccharides 1.690 Æ 0.11 Mannose in N-linked oligosaccharides 0.680 Æ 0.04 O-linked oligosaccharides 1.045 Æ 0.16 O-linked oligosaccharides with N-acetyl 0.192 Æ 0.03 hexosamine as the terminal residue

ND ¼ Not Detected. the major yolk protein resistant to proteolytic cleavage (Berman and Lasky, 1985) during yolk degradation. Glycolipids of the major yolk protein have been reported for the first time in E. asiatica (Tirumalai and Subramoniam, 1992). Glycolipid formed the minor lipid species and constituted 2% of the total lipid fraction of the Lv II. Furthermore, Tirumalai and Subramoniam (2001) have demonstrated the presence of both glucose (monoglycosylceramide) and galactose (diglycosylceramide) containing glycolipids in the lipovitellin of E. asiatica. The galactolipids of yolk/yolk precursor protein may be involved in the recognition of its receptors on the oocyte membrane (van Berkel et al., 1985). Amino acid composition of the major yolk protein Lv II is given in Table 11. A characteristic feature of E. asiatica yolk protein is the high content of acidic amino acids such as aspartic acid and glutamic acid, the latter alone constituting 18.9 mole percent. The Lv II contains three amino acids with potential glycosylation sites such as serine, threonine and asparagine for the glycosylation of O- and N-linked oligosaccharides. Lv II, however, contained less basic amino acids such as lysine and the sulphur containing amino acid, methionine (Tirumalai, 1996). High levels of lipids are a defining character of eggs of marine invertebrates, constituting the main source of metabolic energy during egg maturation and embryonic development (Holland, 1978). The percentage distribution of different lipid species, including phospolipids, neutral lipids, and glycolipids in the eggs and embryos of E. asiatica is presented in 142 T. SUBRAMONIAM AND V. GUNAMALAI

Table 11 Amino acid composition of delipidated Lv II of Emerita asiatica. Data from Tirumalai (1996).

Amino acids Mole percent

Aspartic acid 8.7925 Glutamic acid 18.9271 Asparagine 8.2362 Serine 10.9370 Histidine 3.7342 Glycine 3.6943 Threonine 5.4295 Arginine 4.2299 Alanine 7.5289 Tyrosine 6.1480 Methionine 0.3951 Valine 5.1130 Phenylalanine 4.3210 Isoleucine 5.3851 Leucine 6.7835 Lysine 0.3446

Table 12 Relative percentage composition of different lipids in the egg (Stage I) and Lv II of E. asiatica. From Tirumalai (1996).

Lipid Species Egg Lv II

Neutral lipids 35 33±0.21 Cholesterol 4 3±0.11 Glycolipids 3 2±0.22 Galactolipids ND 0.038±0.009 Phospholipids 58 62±0.41

ND ¼ Not done.

Table 12. Phospholipids formed by far the greatest fraction of the total lipids in both freshly laid eggs and Lv II, as has been reported for the ovary of many crustaceans (Teshima and Kanazawa, 1983; Lautier and Lagarrigue, 1988; Teshima et al., 1989). As many as seven phospholipid species have been separated from the lipovitellin and eggs of E. asiatica, using thin layer chromatography (Tirumalai and Subramoniam, 1992). They are: (1) lysophosphatidylecholine; (2) sphingomylin; (3) phosphatidyl- choline; (4) phosphatidylenositol; (5) phosphatidylserine; (6) phosphatidy- lethanolamine and (6) cardiolipin. However, phosphatidyl choline and phosphatidyl serine were the predominant phospholipid species. These phospholipid species, accumulated within the eggs, have an important role BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 143

Table 13 Relative percentage composition of fatty acids in the neutral lipid fraction of the Lv II of Emerita asiatica. From Tirumalai (1996).

Fatty acid species Percentage composition

C12 17.214 C14 5.164 C16 6.886 C18:0 3.442 C18:1 3.452 C18:2 8.610 C18:3 17.206 C20:4 13.827 C22 10.329 Unidentified fatty acids 13.916 to play during embryogenesis. Thin layer chromatographic analysis of the neutral lipid species from the lipovitellin II of E. asiatica yielded cholesterol, cholesterol methyl esters, 1,2-diglyceride, 1,3-diglyceride, fatty acid methyl esters and carotenoid pigments. The relative percentage distribution of the fatty acids is given in Table 13. Saturated fatty acids constituted 43.0% of neutral lipid fatty acids of the Lv II, whereas the unsaturated fatty acids accounted for 43%. Arachidonic acid is predominant in the neutral fatty acid fraction of Lv II, constituting 13.8%. The increased percentage of neutral lipids in the eggs may result from the presence of glycerol, free fatty acids and different carotenoids (Kour and Subramoniam, 1992; Tirumalai and Subramoniam, 1992).

11.2. Carotenoid pigments in the eggs and yolk proteins

Crustaceans do not synthesise carotenoid pigments but ingest them from their plant food. Emerita, being a filter feeder on plankton and detritus, can accumulate carotenoids in large quantities in the gonad and other body tissues. The pigments of crustacean eggs are known to be derived from the haemolymph as conjugates of the yolk precursor protein, vitellogenin which is then sequestered into the growing oocytes (Wallace et al., 1967). During the female reproductive cycle of E. analoga, Eickstaedt (1969) observed that the haemolymph changed to bright orange in contrast to its usual bluish colour, suggesting a transfer of pigments from the haemolymph to the ovary. Gilchrist and Lee (1972) analysed the carotenoids in the ovary and other body tissues of E. analoga to find out the possible role of these pigments in female reproduction. They identified -carotene, -carotene, 144 T. SUBRAMONIAM AND V. GUNAMALAI echinenone, canthaxanthin, zeaxanthin, diatoxanthin, alloxanthin and astaxanthin in carapace, ovary, eggs and haemolymph of this crab. In general, carotenoids exist in three forms in crustacean egg/ovaries: (1) free pigments, carotenes and unesterified xanthophylls, (2) esterified to long chain fatty acids, and (3) attached to protein in the form of carotenopro- teins. In E. analoga eggs and ovary, Gilchrist and Lee (1972) found a predominance of carotenes ( and carotene) over the other species such as xanthophylls, astaxanthin and ketocarotenoids (echinenone and canthax- anthin) in decreasing order of abundance. Furthermore, these authors provided evidence from radiolabelled isotope studies that 14C labelled carotenoids of the alga Ulva are used in the metabolism of ketocarotenoids within the ovary. This experiment also indicates the fact that the ovaries are a site of astaxanthin production in Emerita; the presence of two intermediates in the process, viz. echinenone and canthaxanthin adducing further evidence to this contention. Using polyacrylamide gel electrophoresis, these authors also separated two carotenoproteins, viz. a blue carotenoprotein found in epidermis and carapace and a bright orange carotenoprotein found both in ovaries and eggs and also in the blood. The orange carotenoprotein occurs in three distinct forms in the slow moving region of the electropherogram. Interestingly, these three proteins have common electrophoretic mobility in the eggs and blood, suggesting that the egg carotenoids are derived from the haemolymph. Using column chromatography in conjunction with thin layer chromatography and spectrophotometric analysis, Tirumalai (1996) observed the presence of canthaxanthin in the purified yolk protein, Lv II, of E. asiatica as the chief carotenoid pigment. The carotenoid pigment of lipovitellin might be required for the stabilisation of the protein backbone of the major yolk protein (Cheeseman et al., 1967).

11.3. Metal content of the yolk protein

Emerita asiatica yolk proteins also contain several metal ions such as copper, iron, sodium, and calcium, also phosphorus (Table 14; Tirumalai and Subramoniam, 1992). These ions constituted as much as 3.5% of the purified major yolk protein. The calcium and copper are bound to lipid in Lv II, whereas the iron, phosphorus and sodium are both lipid and protein bound. The metalloprotein nature of Emerita lipovitellin assumes develop- mental significance inasmuch as lipovitellins serve important functions during embryogenesis of oviparous eggs. A characteristic feature of vertebrate yolk protein is its high phosphate content, helping in skeletal formation during embryogenesis (Wahli, 1988). Crustaceans lack an internal skeleton, but secrete a calcareous exoskeleton as armour. Whereas in BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 145

Table 14 Analysis of metal and phophorus content of the Lv II of Emerita asiatica. From Tirumalai (1996).

Metal ions Concentration (mg/100 mg Lv II)

Native Relative Delipidated Relative Lv II percentage Lv II percentage

Copper 290±1.96 8.288 ND ND Iron 1400±2.87 40.010 388±1.33 31.673 Sodium 933±1.73 26.660 800±0.96 65.306 Calcium 140±0.56 4.000 ND ND Phosphorus 736±0.87 21.030 37±0.73 3.020

ND ¼ Not detected. vertebrate as well as insect vitellin phosphorus is bound to protein by way of phosphorylation, in E. asiatica a large amount of phosphorus is linked to the lipid component of the lipovitellin (Tirumalai, 1996). Lipid-bound phosphorus has also been reported in an annelidan vitellogenin (Taki et al., 1989). The presence of a meagre amount of protein-bound phosphorus in crustacean lipovitellin may result from the O-linked glycosylation of serine moieties prior to phosphorylation (Della-Ciopa and Engelman, 1987; Dhadialla and Raikhel, 1990).

11.4. Hormonal conjugation to yolk protein

Emerita yolk protein also contains several conjugates of steroidal hormones involved in moulting and reproduction. In E. asiatica, Subramoniam et al. (1999) reported that the purified yolk protein, lipovitellin, contains both free and conjugated ecdysteroids. It is assumed that these hormones conjugated to the lipovitellin are maternally derived. The egg yolk proteins isolated from E. asiatica contain both estrogen and progesterone in significant quantities in a conjugated condition (Warrier et al., 2001). Interestingly, the isolated vitellogenin of these crabs also contains these steroidal hormones, suggesting that they are transported to the ovary by conjugation with vitellogenin. The steroidogenic ability of crustacean ovary is yet to be demonstrated.

11.5. Mechanism of yolk formation

As in the majority of decapod crustaceans, vitellogenesis in E. asiatica is also accomplished by heterosynthetic means. Using immunodiffusion 146 T. SUBRAMONIAM AND V. GUNAMALAI techniques, Tirumalai (1996) showed that the anti-Vg antibodies prepared against the purified vitellogenin of E. asiatica produced two precipitin lines with the supernatants obtained from the mature ovary. Interestingly, these two precipitin lines correspond to column chromatographically purified lipovitellin I and II. However, the anti-Vg antibodies of E. asiatica did not cross-react with the supernatants of any somatic tissue such as hepatopancreas, muscle and subepidermal tissues. Thus in Emerita, the final site of vitellogenin synthesis is still elusive. Perhaps molecular techniques such as Northern blotting (Yang et al., 2000) and real-time RT-PCR (Jayasankar et al., 2002) could unravel the ultimate site of vitellogenin synthesis in this highly fecund sand crab. Interestingly, the lipovitellin of another anomuran sand crab, Albunea symnista also cross- reacted with anti-Vg antibodies of E. asiatica suggesting the immunological and molecular similarities of the lipovitellins of these conspecific anomuran crabs. Homology in the amino acid sequences of vitellogenin of different decapod crustaceans as well as immunological relatedness between the lipovitellin and mammalian serum low density lipoproteins have also been revealed in recent studies (See Wilder et al., 2002; Warrier and Subramoniam, 2003).

12. YOLK UTILISATION

Emerita species fasten the eggs to the pleopodal hairs where the eggs develop and hatch out as larvae. The biochemical composition of E. asiatica eggs shows them to be a rich source of nutrition. The yolk comprises a glycolipocarotenoprotein complex, free lipids and glycogen granules. During maturation in the ovary the eggs also acquire various other organic and inorganic components needed for embryogen- esis and also early larval development. The embryos also absorb water and salts from the environment during the course of their development. A special feature of Emerita eggs is the large proportion of lipid in the yolk, forming as much as 30% of the lipovitellin (Tirumalai and Subramoniam, 1992), in addition to a significant quantity of free lipids. Lipid accumulation is a strategy to decrease density and to reduce energy cost of egg carriage in pelagic crustaceans, which are characterised by abbreviated development coupled with an extended period of incubation (Herring, 1973), but this has little bearing on benthic species such as Emerita. Studies on yolk utilisation in Emerita are limited to only two species, namely E. holthuisi (Vijayaraghavan et al., 1976) and E. asiatica (Subramoniam, 1991). BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 147

Emerita embryos also contain hemocyanin (Gilchrist and Lee, 1972; Gunamalai, 2001). The functional importance of haemocyanin, accumulated during vitellogenesis, in embryogenesis is not clear, but in other crustaceans haemocyanins are known to provide a source of protein and copper during yolk utilisation (Terwilliger, 1991). In Emerita, as in other ovigerous crabs, embryonic development occurs within a mass consisting of several thousand eggs. Hence, the embryos in the centre of the egg mass may experience a lower partial pressure of oxygen than do embryos on the outer surface. Embryonic accumulation of haemocyanin may help in oxygen transport or diffusion (Terwilliger, 1991). In freshwater prawns, Pandian (1994) observed grooming and aeration of the egg mass by the legs of the female. The same behaviour may also occur in Emerita, but direct observations are lacking. Yolk utilisation has been studied in several crustaceans, with particular regard to energy transformation during embryogenesis and the ecophysiol- ogy of the organism (Pandian, 1970a, b). During vitellogenesis, besides the accumulation of the yolk components, other metabolically important substances such as RNA, and a host of hydrolytic enzymes, are also synthesised and stored within the eggs (Adiyodi and Subramoniam, 1983). Taking advantage of the year-round availability of the berried females with eggs in different stages of embryonic development, Subramoniam (1991) made a thorough investigation into the biochemcal changes in the egg components of E. asiatica during embryogenesis. The eggs in the brood exhibit changes in colouration during embryonic development, thus facilitating an easy classification of development stages (Table 15). This table also summarises other microscopic observations such as percentage yolk clearance, appearance of morphological characters such as the eye spots, beating heart and the development of appendages. The time taken for each stage during egg development was also estimated by maintaining the freshly ovulated females in the laboratory (Temp. 26C; Salinity 34%). Changes in percentage values of protein, lipid and carbohydrates, calculated on wet weight and dry weight basis are summarised in Table 16 and 17. Protein value steadily declined from stage I to IX, corresponding to increasing water content. On the other hand, lipid content remained almost unaltered up to stage V; thereupon, the value fell precipitously, reaching a minimum of 0.49 mg per 10 mg in stage IX. Compared to protein and lipid, the total carbohydrate content was low, but different carbohydrate compo- nents exhibited an interesting pattern of fluctuation during egg develop- ment. The total free carbohydrates increased from a low value of 0.12 mg per 10 mg in stage I to 0.72 mg per 10 mg in stage IX. The free glycogen also exhibited a similar increase during egg development. Conversely, the protein-bound polysaccharides decreased from an initial high value of 0.072 mg per 10 mg in stage I to 0.021 mg per 10 mg in stage IX. 148 T. SUBRAMONIAM AND V. GUNAMALAI

Table 15 Classification of egg development in Emerita asiatica. Adapted from Subramoniam (1991).

Stage Approximate Description number of days

I 5 Yellow yolk granules seen; egg mass bright orange in colour II 5 Cleavage has taken place and blastomeres are seen; egg mass bright orange in colour III 1 A yolk-free white streak makes its appearance at the animal pole IV 1 One quarter of the yolk cleared; the white band encircles the yolk material which is now in the centre; at the animal pole a periodic twitching is recognised; red pigments are seen at the edge of the yolk; colour of the egg mass is dull orange V 2 One third of the yolk is cleared; two eye spots appear; red spots prominent and seen at the end of the animal pole; colour of the egg mass dull orange VI 2 Egg mass brownish orange in colour; eyes well developed; yolk is found in the vegetal pole; two-thirds of the yolk is cleared; red pigments seen all over the white space VII 1 Egg mass greyish orange in colour; yellow yolk is found as two clusters in the centre; appendages of the embryo are developed; heart beat seen; eye spots are very well developed VIII 1 Egg mass pale grey in colour; heart beat more prominent; embryo almost developed IX 1 Embryo fully formed; egg mass white in colour; no yolk globules seen; colourless yolk in the form of oil globules seen just below the eyes; about to hatch

12.1. Enzyme activity during yolk utilisation

Commensurate with storage of complex yolk proteins in E. asiatica eggs, a host of hydrolytic enzymes are available to release the component substrates in utilisable form. Table 18 summarises the stage-specific enzyme activity of esterases, proteases and glycosidases during embryogenesis in E. asiatica. Interestingly, the activity of all the three enzymes peaks during stage V and VI, coinciding with break down of the major yolk proteins into simpler subunits. Esterase activity involved in the breakdown of various lipids exhibits an interesting pattern during yolk utilisation. It starts only in stage III and attains a peak value during stage V. Thereafter, activity slowly declines to a very low value in stage VIII. The esterase activity correlates with lipid utilisation during embryogenesis. Inactivity of the enzyme before stage III may be caused by the proenzyme nature of esterases or there may REIGBOOYO H ADCRAB, SAND THE OF BIOLOGY BREEDING

Table 16 Major organic composition of eggs during different stages of development in Emerita asiatica. Values expressed as mg/10 mg wet weight (mean Æ SD). Esterase activity expressed as nM naphthol/mg protein/min. Data from Subramoniam (1991).

Biochemical Stages of eggs constituents I II III IV V VI VII VIII IX EMERITA Protein 1.32±0.150 1.09±0.143 0.949±0.107 0.90±0.03 0.77±0.09 0.66±0.043 0.44±0.08 0.28±.0.021 0.16±0.029 Free 0.12±0.013 0.24±0.009 0.28±0.012 0.32±0.012 0.36±0.010 0.48±0.012 0.52±0.008 0.68±0.009 0.72±0.015 carbohydrates Glycogen 0.005±0.001 0.0057±0.0001 0.0119±0.0002 0.0168±0.003 0.0109±0.0002 0.026±0.0022 0.0231±0.0013 0.0197±0.002 0.0207±0.0017 Protein 0.0729±0.00 0.0849±0.0024 0.0467±0.0032 0.0457±0.0014 0.0205±0.0071 0.0339±0.0021 0.0261±0.0018 0.0250±0.007 0.0210±0.003 bound sugar Lipid 31 1.88±0.014 1.87±0.010 1.85±0.014 1.80±0.012 0.64±0.009 0.52±0.014 0.51±0.015 0.49±0.009 Non-specific 1.89±0.014 – – 0.670±0.123 1.08±0.214 1.73±0.530 0.89±0.090 0.62±0.035 0.29±0.055 esterases 149 150

Table 17 Major organic composition of eggs during different stages of development in Emerita asiatica after correction for water content (expressed as mg per 10 mg of dry tissue, mean values only). Data from Subramoniam (1991).

Biochemical Embryonic stages constituents I II III IV V VI VII VIII IX

Protein 3.219 2.224 2.433 2.727 2.484 2.357 1.692 1.272 0.889

Free carbohydrates 0.293 0.489 0.718 0.969 1.161 1.714 2.000 3.090 4.000 GUNAMALAI V. AND SUBRAMONIAM T. Glycogen 0.012 0.012 0.031 0.051 0.036 0.093 0.089 0.089 0.115 Protein-bound sugars 0.178 0.173 0.119 0.139 0.066 0.121 0.100 0.114 0.117 Lipid 4.609 3.837 4.795 5.606 5.806 2.286 2.000 2.318 2.722 REIGBOOYO H ADCRAB, SAND THE OF BIOLOGY BREEDING

Table 18 Fluctuation of enzymatic activity during embryonic development in the crab Emerita asiatica.

Embryonic aEsterase activity #Protease activity $Glycosidase activity (mM p-nitrophenol released/10 mg embryo) stage (nmol napthol/mg in enzyme units protein per min) (1 mg leucine -Glucosidase -Glucosidase -Galactosidase -Galactosidase equivalent/30 min)

I ND* 5.5 – – – – II ND ND – – – – EMERITA III 0.1198 8.69 – – – – IV 0.1983 ND 0.058 0.018 0.054 0.086 V 0.3086 12.6 0.079 0.069 0.096 0.153 VI 0.1585 ND 0.085 0.036 0.157 0.172 VII 0.115 10.1 0.116 0.031 0.287 0.197 VIII 0.0523 9.35 0.075 0.028 0.195 0.112 IX – – 0.037 0.025 0.165 0.062

*ND, not determined. aData from Subramoniam (1991): $Data from Pravalli (1990): #Data from Gunamalai (1993). 151 152 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 19 Zymogram of esterases from the ovary and different egg develop- mental stages in Emerita asiatica. (E1–E5 represent different isozymes). Redrawn from Subramoniam (1991).

be specific inhibitors present. Esterases in Emerita eggs can be resolved into 2 groups; one is a homogeneously thick proximal fraction (E1) occurring in stage IV through IX (Figure 19). The second consists of a moderately staining fraction (E2) and two other thin fractions (E3 and E4). In stage V and VI, the E2 fraction declined in intensity but yet another fraction (E5) appeared in the fast moving zone in stage VI. E5 persisted up to the VII stage but was absent in stages VIII and IX. In general, the E1 fraction did not change significantly in intensity but the others decreased in intensity and disappeared in the last stage of egg development (Figure 19). All five fractions can be characterised as isozymes of carboxyl esterase since they were inhibited by silver nitrate and malathion, and unaffected by pCMB, EDTA and eserine sulphate. It is possible that freshly laid eggs contained a significant quantity of esterases, but the gradual increase of esterases as well as the appearance of new isozymes midway during embryonic development would suggest embryonic synthesis of this enzyme. Doyle et al. (1959) found a similar increase in esterase activity during embryogenesis in an isopod. The peak of activity of several hydrolytic enzymes in E. asiatica eggs coincides with commencement of lipovitellin degradation. Lipovitellins in Emerita are degraded by specific serine proteases (Pravalli, 1990). The proteolytic products of the lipovitellins gradually lose their PAS staining BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 153 properties, suggestive of dissociation of the carbohydrate prosthetic groups from the vitellins (Tirumalai, 1996). Activity of two glycosidases, glucosidases and galactosidase, is increased in embryos at a time when the PAS staining subsides in the vitellin fractions (Gunamalai, 1993). These glycosidases may be required to release bound glucose and galactose from the glycolipid and oligosaccharide components of the major yolk proteins, as well as to hydrolyse stored glycogen during embryogenesis in E. asiatica. Another enzyme that is active at the time of vitellin degradation in E. asiatica is phospholipase C (Ramachandran, 1992). Yolk utilisation in Emerita eggs necessitates extensive reshuffling of substrates, especially during the early stages of embryonic development. Such changes in the metabolic pathways involving interconversion of already stored substrates within the closed system of egg development of decapod crustaceans increase our understanding of embryonic development in the non-cleidoic eggs of marine invertebrates. The initial high content of lipid in Emerita egg is characteristic of lecithotrophic eggs. However, lipid utilisation starts only from stage V onwards, suggesting that protein may be the chief source of energy for initial embryonic development. On the contrary, the eggs of a freshwater crab Paratelphusa hydrodromus expend enormous reserves of lipid continuously during embryogenesis with a concomitant increase in the protein level (Pillai and Subramoniam, 1985). Suppression of protein utilisation and enhanced lipid metabolism is characteristic of cleidoic eggs, a feature found also in many crustacean species (see Pandian, 1970a). Conversely, in the bony fishes, protein is preferentially used during the entire course of embryogenesis (Lasker, 1962). Egg development of E. asiatica represents a condition intermediate between cleidoic and non-cleidoic developmental extremes. In spite of the increased utilisation of lipids in the second half of embryogenesis, lipid is retained in the form of colourless yolk globules in the embryo at hatching. These lipid reserves not only increase the buoyancy of the pelagic larvae on their release, but also are useful in delaying starvation during the rather protracted larval life of E. asiatica. In general, the protein/ lipid ratio is high in typically planktotrophic larvae such as cirripedes (Achituv and Wortzlavski, 1983), whereas lipids form the major reserves in lecithotrophic eggs. Although E. asiatica releases planktotrophic zoea with a long pelagic life, it produces many yolky eggs in which the lipid/protein ratio is much higher.

12.2. Energy utilisation in Emerita eggs

Conventionally, yolk utilisation in crustaceans and other animals has been expressed in terms of energy value (Pandian, 1994). Therefore, we have 154 T. SUBRAMONIAM AND V. GUNAMALAI

Table 19 Mobilisation of energy during egg development in Emerita asiatica: energy values (J per10 mg dry tissue) calculated from the organic composition values given in Table 16, 17 and by applying energy equivalents suggested by Brody (1968).

Egg stages Protein Total Lipid Total carbohydrates*

I 75.6 8.4 182.1 266.1 II 52.3 11.7 151.6 215.6 III 57.2 15.0 189.4 261.6 IV 64.1 20.1 221.4 305.6 V 58.4 21.9 229.3 309.6 VI 55.4 33.4 90.3 179.1 VII 39.8 37.9 79.0 156.7 VIII 29.9 57.0 91.6 178.5 IX 20.9 73.2 107.5 201.6

*Free carbohydrates þ glycogen þ protein bound sugars. converted the biochemical value given above (Table 16, 17) into energy value by considering the energy equivalents for total carbohydrates as 17.3 kJ gÀ1 dry weight, protein as 23.5 kJ gÀ1 and lipid as 39.5 kJ gÀ1 (Brody, 1968). The energy equivalent of total carbohydrates was calculated by pooling free carbohydrates, glycogen and protein-bound sugars. It is evident from Table 19 that the energy derived from the proteins is continuously utilised from stage I of egg development (75.6 J per 10 mg dry tissue) to the last stage (20.9 J per 10 mg dry tissue). On the other hand, the carbohydrate-based energy is continuously built- up from 8.4 J per 10 mg (stage I) to 73.2 J per 10 mg (stage IX). There is also an apparent increase in the lipid energy from stage II to stage V. There is then considerable utilisation of lipid energy in stage VI and VII. These stages correspond to the maximum yolk clearance coupled with faster organogenesis (eye and appendages development). However, there is a considerable retention of lipid energy in the last two stages of embryonic development, which may facilitate utilisation of stored energy in the absence of adequate food for the free-swimming zoea larva. These data suggest that the mobilisation of energy sources especially in the first phase of embryogenesis has changed the energy profile during egg development in E. asiatica. In the freshly laid eggs, the major energy source is lipid (68.4%), followed by protein (28.4%). Carbohydrate is a very poor source of energy (3.3%) in the beginning of the embryonic development (Table 20). However, prior to hatching, the energy profile changes dramatically. Protein contributes only 10.2% and lipid 53.3% at the end of embryonic development. Interestingly, the carbohydrate-based energy source has substantially increased to 36.3%. Furthermore, from the above BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 155

Table 20 Contribution (%) of protein, carbohydrate and lipid to the total energy in the stages of egg development in Emerita asiatica: data calculated from Table 19.

Egg stage Protein Total Lipid carbohydrate

I 28.4 3.2 68.4 II 24.3 5.4 70.3 III 21.9 5.7 72.4 IV 20.9 6.6 72.4 V 18.9 7.1 74.1 VI 30.9 18.7 50.4 VII 25.4 24.2 50.4 VIII 16.8 32.0 51.3 IX 10.4 36.3 53.3

data on the energetics of developing eggs of E. asiatica, the cumulative utilisation efficiency was found to be very high for protein (72.4%), followed by lipid (40%). On the contrary, the carbohydrate energy was built up by a factor of 8.7 times. From the above values the total energy utilisation efficiency for the entire embryonic period has been calculated as 24.2% in E. asiatica. It will be of interest to compare the value thus obtained for Emerita with values available in the literature on other crustacean forms. As given in Table 21 the energy utilisation efficiency found in E. asiatica conforms to the majority of crustaceans in respect of total energy utilised during embryogenesis. Summing up the above observations on energy utilisation by E. asiatica in comparison with other crustacean species, it may be said that E. asiatica not only efficiently utilises the energy stored in the egg but also metabolically converts them to readily usable substrates such as carbohydrate for the benefit of the free swimming larvae. Again considerable retention of lipid energy to the extent of 53% is advantageous for the newly released larvae to tide over possible adverse conditions.

12.3. Carotenoid metabolism during embryogenesis

Kour and Subramoniam (1992) reported on the qualitative and quantitative changes in the carotenoids during egg development of E. asiatica, using spectrophotometry in conjunction with column and thin layer chromatog- raphy. Table 22 shows the variation in the occurrence of different carotenoids in the embryonic stages analysed. By far, the most abundant form of carotenoid deposited in the developing eggs of E. asiatica is 156 T. SUBRAMONIAM AND V. GUNAMALAI

Table 21 Energy utilisation efficiency of eggs of crustaceans.

Species Energy content Energy References of eggs utilisation (kJ gÀ1 dry weight) efficiency (%)

ISOPODA Ligia oceanica 24.93 30.0 Pandian (1972) Probopyrus 32.90 5.0 Anderson (1977) pandalicola DECAPODA Macrobrachium 29.39 18.0 Balasundaram (1980) nobilli M. lamarrei 26.48 4.0 Katre (1977) M. idella 26.08 29.0 Vijayaraghavan and Easterson (1974) Crangon crangon 24.76 23.0 Pandian (1967) Homarus americanus 27.78 35.0 Pandian (1970b) H. gammarus 25.84 26.0 Pandian (1970a) Pagurus bernhardus 25.34 21.0 Pandian and Schumann (1967) Caridina nilotica 62.67 60.0 Ponnuchamy et al. (1979) Emerita holthuisi 17.95 77.0 Vijayaraghavan et al. (1976) Emerita asiatica 26.61 24.2 Subramoniam (1991)

-carotene, with its concentration varying between 15.4 mggÀ1 wet weight and 16.1 mggÀ1 wet weight in the early stages of embryonic development. After maintaining almost the same level up to stage V, -carotene started declining gradually to reach a low level of 3.7 mggÀ1 wet weight in the newly hatched out larvae. Alpha carotene also showed a declining trend during embryogenesis of E. asiatica. Obviously, these two parent carotenoids of dietary origin undergo bioconversion into more oxidised forms such as hydroxy and ketocarotenoids. The involvement of -carotene in the production of ketocarotenoids such as echinenone, canthoxanthin and astaxanthin is also evidenced in other crustacean species (Herring, 1968; Hsu et al., 1970). That oxidation of -carotene takes place via isocrypto- xanthin is revealed by the occurrence of this intermediate compound in all stages analysed, with the level declining as development proceeds. Kour and Subramoniam (1992) suggested a possible biosynthetic pathway of carotenoids during embryogenesis in E. asiatica (Figure 20). It can be seen from the figure that astaxanthin is the final product of -carotenoid metabolism. Free astaxanthin is found in all stages of embryonic development. However, esterified astaxanthin is found only in the last REIGBOOYO H ADCRAB, SAND THE OF BIOLOGY BREEDING

Table 22 Carotenoid content in different egg developmental stages of Emerita asiatica (mg/g wet weight). Data from Kour and Subramoniam (1992).

Carotenoids Stage

I III V VII VIII IX X

-carotene 0.853±0.056 0.921±0.189 1.490±0.026 0.833±0.013 0.960±0.012 0.031±0.002 - -carotene 15.560±0.122 16.072±0.141 15.445±0.087 14.320±0.097 12.220±0.034 7.220±0.034 3.700±0.069 EMERITA Lutein 2.080±0.067 –––––– Echinenone 0.846±0.031 1.960±0.036 3.540±0.036 – – – – Isozeaxanthin 4.373±0.068 1.500±0.019 3.540±0.039 3.380±0.048 0.031±0.010 – – Zeaxanthin 4.034±0.045 – 4.510±0.058 4.093±0.248 – 5.971±0.372 – Canthaxanthin – – – 2.972±0.323 5.806±0.528 4.613±0.264 2.606±0.264 -doradexanthin –––––– 0.666±0.117 Isocryptoxanthin 6.712±0.198 5.100±0.197 3.910±0.153 3.630±0.161 2.540±0.236 2.136±0.142 2.104±0.173 Free astaxanthin 0.600±0.022 0.216±0.016 0.686±0.034 0.608±0.016 1.192±0.055 2.440±0.100 0.848±0.044 Esterified astaxanthin –––––– 4.280±0.018 157 158 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 20 Biosynthetic pathway of - and -carotene metabolism, taking place in the developing eggs of Emerita asiatica. From Kour and Subramoniam (1992). stage of egg development. Herring (1974) correlated the appearance of esterified astaxanthin with the origin of chromatophores during the development of the decapod, Acanthephyra. In the light of this observation, it is suggestive that the astaxanthin, after esterification may give rise to the larval complement of chromatophores in addition to the possible biosynth- esis of visual pigments. Accumulation of large amounts of carotenoids, especially in the form of astaxanthin in the eggs is significant in the sense that they function as a heat or light shield to the developing embryos on the pleopods. Furthermore, as suggested by Gilchrist and Lee (1972), carotenoproteins may also be utilised by the developing young in the stabilisation and protection of food reserves.

12.4. Embryonic ecdysteroids

As shown in a preceding section, the vitellogenic ovary of E. asiatica accumulates significant amounts of ecdysteroids during the premoult stage when the haemolymph also contains a high titre of these hormones. The ovarian ecdysteroids are passed on to the eggs for possible elimination and to function as morphogenetic hormones partaking in the control of embryogenesis and early development. Using radioimmunoassay and high performance liquid chromatography, Subramoniam et al. (1999) reported the occurrence of a complex mixture of free and conjugated ecdysteroids in the developing eggs of E. asiatica. These hormonal complexes also exhibited multiphasic fluctuation during the course of BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 159

Table 23 Fluctuation of hormonal activity during embryonic development in the crab Emerita asiatica. Data from Subramoniam et al. (1999) and Warrier and Subramoniam (2001).

Embryonic Total Free Conjugated Estradiol Progesterone stage ecdysteroids ecdysteroids ecdysteroids 17 (pg per (ng gÀ1 (pg mgÀ1 lipid) (pg/mg lipid) (pg per 100 mg) egg wet weight) 100 mg) Polar Apolar

I 6.5 80.7 8.33 8.33 200 150 II ND ND ND ND 250 160 III 15.2 146.7 613.8 25.0 400 240 IV 4.62 ND ND ND 625 430 V 6.92 83.33 20.33 25.0 750 550 VI 15.0 125.0 18.33 20.83 650 500 VII 6.15 50.0 12.5 16.67 630 350 VIII 36.20 291.66 83.33 233.33 550 250 IX 450 175 embryonic development (Table 23). Such fluctuations are common to the free and conjugated forms, reflecting interconversions between them. However, the concentration of free ecdysteroids always predominated over the conjugated ones in all the developmental stages. Both 20-hydroxyecdysone (20E) and ecdysone are the prominent free ecdysteroids in the embryos. Furthermore, HPLC analysis indicated that the ratio of 20E to ecdysone is always higher during the entire period of embryogenesis. This study on E. asiatica reveals that the lipovitellin also contains significant quantities of both free and conjugated ecdysteroids, bound to it. As a result of intense esterase and protease activities, digesting the complex lipovitellins, there is a release of free ecdysteroids such as 20E and ecdysone from the conjugated polar compounds. A similar release of free functional ecdysteroids from the yolk protein ecdysteroid complexes as a result of esterase activity during embryonic development in insects has been reported by Bownes et al. (1988) and Hoffmann et al. (1986). Interestingly, in E. asiatica, both the conjugated and free ecdysteroid titres reach the maximum at stage VIII representing an almost fully formed embryo within the hatching envelope. The highest amount of ecdysteroid accumulation in the prehatching stage could also be due to combined contribution from maternally derived as well as endogenously synthesised ecdysteroids from embryonic Y-organ, as reported in the caridean shrimp, Palaemon serratus (Spindler et al., 1987). By comparison with other crustacean embryos (Chaix and De Reggi, 1982; Spindler et al., 1987), the first minor peak at stage III may be correlated with blastoderm extension and the second peak at stage VI, when 160 T. SUBRAMONIAM AND V. GUNAMALAI the eye and appendages are well developed (Subramoniam et al., 1999). The third major peak at prehatching stage (VIII) is correlated with the deposition of the embryonic cuticle of the zoea larva of E. asiatica, as suggested by Goudeau et al. (1990) for the control of secretory activities related to the synthesis of embryonic envelopes in European lobsters. Apart from acting as a morphogenetic hormone that controls several developmental events, including the secretion of embryonic cuticle and moulting, the accumulation of significant quantities of polar and apolar conjugates and their possible catabolism to other products such as 20, 26-dihydroxyecdysone (McCarthy and Skinner, 1979) and ecdysonic acids (Lachaise and Lafont, 1984) would suggest their elimination through storage excretion.

12.5. Occurrence and utilisation of vertebrate steroids in Emerita eggs

Accumulation of vertebrate steroids such as estradiol 17 and progesterone in the ovaries of several crustaceans has been reported (Fairs et al., 1989; Quinitio et al., 1991). Recently, Warrier et al. (2001) have reported the accumulation of these steroid hormones in the ovary of the crab Scylla serrata and E. asiatica. These hormones are possibly synthesised in the hepatopancreas and transported to the ovary bound to haemolymph yolk precursor protein, vitellogenin. In E. asiatica, the level of estradiol 17 and progesterone has been estimated in different embryonic stages using radioimmunoassay and microparticle enzyme immunoassay. The results, summarised in Figure 21, reveal that the levels of these two hormones are low in the first and second stages, but rise to a peak in stage V of embryonic development. After this, the level declines to a low value in the IX stage. Such a pattern in the fluctuation of these two steroidal hormones during embryogenesis of E. asiatica is very similar to that of the embryonic ecdysteroids, described earlier. Incidentally, the peak activity in stage V corresponds to the stage in which the stored lipovitellins undergo enzymatic degradation to release the bound hormones. Thus, the upsurge of these two hormones in stage V eggs may be due to the release of the protein-bound steroids (by protease action) into the general pool of free steroids. Unlike the ecdysteroids, the role of vertebrate steroids in the crab embryogenesis is not clear. However, accumulation of steroids, such as the thyroid hormones in the eggs of birds, has been suggested to have a controlling role in morphogenesis (Wilson and McNabb, 1997). Whether these steroids have any such role as morphogenetic hormones in the embryogenesis of Emerita remains to be seen. BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 161

Figure 21 Levels of estradiol 17 and progesterone in different embryonic stages of the eggs of Emerita asiatica (mean Æ S.D.; n ¼ 5, P < 0.001). Modified from Warrier et al. (2001).

13. LARVAL DEVELOPMENT

Like many other littoral benthic invertebrates, Emerita has a pelagic larval phase. Some Emerita species can have as many as seven zoeal stages that are spent in the open oceanic waters before metamorphosis to the megalopa stage, which then migrates back to the sandy seashore for settlement. The description of Emerita larvae dates back to 1877 when Smith described three zoeal stages namely second, third and last zoea and a megalopa collected from the plankton for a species described under the generic name Hippa (¼ Emerita talpoida). Subsequently, Faxon (1879) described the first zoeal stage hatched from the eggs in the laboratory. Much later, Menon (1933), Johnson and Lewis (1942) and Sankolli (1967) described larval development in three other species, E. asiatica, E. analoga and E. holthuisi respectively. Menon described five zoeal stages from the plankton. Similarly, Johnson and Lewis also described five zoeal stages from the plankton, and the first stage from the laboratory-hatched larvae.

13.1. Larval description in Emerita talpoida

The complete description of Emerita larvae was made possible only by laboratory hatching of the larvae and rearing them to the megalopa stage, 162 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 22 Zoeal stages of Emerita talpoida: (A) First zoea; (B) Second zoea; (C) Third zoea; (D) Fourth zoea. Redrawn from Rees (1959). first achieved by Rees (1959) with E. talpoida. This study described up to six zoeal stages before the megalopa stage. The number of zoeal stages could also extend to a seventh stage in certain individuals in laboratory culture. The details of different zoeal stages as well as the megalopa larva are given in Figures 22–24. In general, there is a uniformity of morphological structures in the first zoea of E. talpoida as compared with other Emerita species such as E. analoga and E. asiatica. The stage I zoea is characterised by a smoothly rounded carapace that is translucent and colourless. The shape of the carapace changes somewhat in stage III into a more or less pear-shaped structure. The lateral spines which are not present in stage I zoea are characteristic of the subsequent stages. BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 163

Figure 23 Zoeal stages of Emerita talpoida: (E) Fifth zoea; (F) Sixth zoea. Redrawn from Rees (1959).

The rostrum is short and broad in stage I zoea and continues to elongate and reaches about one and a half times the length of the carapace. The eyestalks are short and thick and lie close against the carapace, directed somewhat posteriorly. In the subsequent zoeal stages, the eyestalks increase in length and the eyes are carried somewhat farther forward than in the first stage. In the megalopa stage, which resembles the adult, the eyes are still relatively large as compared to the adult. The antennules in stage I zoea are short unjointed appendages which are thick at the base and taper to a blunt point where three setae of about equal 164 T. SUBRAMONIAM AND V. GUNAMALAI

Figure 24 Megalopa stage of Emerita talpoida. Redrawn from Rees (1959).

length are borne. These setae increase to four in number in stage IV, six in stage V and eleven in stage VI. The antennae in stage I zoea are rather stubby appendages, produced on the outer side into a spine-like process. From the base of the outer spine, there arises a somewhat slender dentiform process of about the same length. At the base of this inner process, there is a much smaller spine. The form of the antennae remains relatively unchanged through the first four zoeal stages, the first indication of a flagellum not appearing until the fifth zoeal stage. At this stage, the rudiment of the flagellum is visible as a conspicuous knob, which lengthens enormously in the VI stage zoea. In the megalopa stage, the antenna possesses all the important features of the adult form. BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 165

The mandible in the stage I zoea consists of an armed crown on its ventral edge followed by sharp triangular teeth. These appendages change very little, except for a general increase in size, throughout the zoeal stages. In the megalopa stage, the mandible has undergone a complete change in structure and function. It is no longer an organ of mastication but is adapted, as in the adult, for the purpose of scraping the antennae and passing food to the mouth. In the case of the maxillae and the maxillipeds, the structures remain more or less unchanged in the zoeal stages except for the increase in the number of setae in the second maxilla and the maxillipeds. In the megalopa stage, these structures possess all the parts of the adult appendage. The abdomen in the stage I zoea is composed of five segments projecting almost straight downward from the carapace, and is flexed so that the telson is carried beneath and parallel to the carapace. At this stage, no rudiments of the abdominal appendages are visible. The sixth segment is consolidated with the telson; this becomes apparent when the uropods appear in the III stage zoea. The uropod consists of a short basal segment with a long, flattened lobe extending from it. In stage IV zoea, the four free segments of the abdomen bears two small round thickenings on its inner side, the evidence of future pleopods, which eventually appear in the stage VI zoea. The pleopods are uniramous, unsegmented and appear on the second through fifth abdominal segments. The abdomen in the megalopa stage is composed of six segments, which are similar in form and proportion to those of the adult. In contrast to the uniramous pleopods of the zoeal stages, the megalopa stage bears four pairs of biramous pleopods.

13.2. Larval dispersal and megalopa settlement

In the temperate species, such as E. analoga, eggs are laid in the summer months and after incubation on the pleopods for about a month, give rise to zoeae, which are released into the plankton. Johnson (1940) estimated the time spent as zoea in the plankton to be about four and a half months, after analysing planktonic materials collected from tows off the coast of California. Following this, Johnson and Lewis (1942) described the zoeal stages of E. analoga from the plankton and suggested that they passed through at least five stages before moulting to the megalopa. The duration of larval development is also variable; for E. talpoida the laboratory rearing took 30 days (Rees, 1959) whereas, E. rathbunae took about 90 days (Knight, 1967). From laboratory rearing, Efford (1970) observed that the zoea larvae of E. analoga passed through as many as 9 moults in a total time duration of 130 days, before metamorphosing into megalopa. He also 166 T. SUBRAMONIAM AND V. GUNAMALAI observed that moulting into megalopa could occur at 8th or 9th zoeal moult. Although Hanson (1969) contended that the larval development in the Hippidae was temperature dependent, such variations in the larval duration, especially from the estimates of laboratory rearing and plankton analysis could not be explained in terms of temperature difference. The duration of the later stages is also so variable that there is a possibility that one or two stages from metamorphosis to megalopa are skipped if conditions are ideal for settlement on the beach. Similarly, the larvae may have the ability to delay metamorphosis if settlement conditions are unfavourable. This feature increases the chances for selection of a suitable substratum, thus contributing to the success of the population (Thorson, 1950; Wilson, 1952). Along the ocean coasts where the shelf is rather narrow and the deep sea is not far off, strong currents may carry the larvae away from their littoral and shallow water habitat. Johnson (1939), correlated water movements and the dispersal of pelagic zoea larvae of E. analoga along the southern Californian coast. He observed that the fourth zoeal stage of this sand crab is taken in plankton hauls at a distance of 125–130 miles from the mainland shores. However, the first zoeal stage, with a 4 week larval life in laboratory rearing, was found in plankton taken less than 20 miles from the shore. Evidently, such a long journey offshore for development and metamor- phosis into megalopa would cause a large wastage of larvae as suggested for oyster larvae by Korringa (1947). After spending a varying period of time in the plankton, zoea larvae of E. analoga metamorphose into megalopae and start arriving in large numbers in early April with a peak influx in early June at the Scripps beach, La Jolla (Efford, 1965). However, Wenner (personal communication quoted by Efford, 1970) observed the arrival of megalopae in the winter months of 1965–1966 on the beaches at Goleta, just south of Point Conception. Such differences in the recruitment period on different beaches along the west coast of North America may suggest that the timing of maximum recruitment perhaps depends largely on the distribution of the later zoeal stages in relation to local hydrographic conditions (Johnson, 1940; Efford, 1965; Barnes and Wenner, 1968; Cox and Dudley, 1968). Seasonality of the megalopa settlement in temperate species can be related to the seasonal reproductive cycle. With tropical species, such as the E. asiatica, that breed all through the year, we would expect to have a continuous or near continuous settlement pattern of megalopae. Year- round egg laying, coupled with continuous embryonic development of pleopodal eggs results in uninterrupted release of zoea larvae into the plankton. Hence, larval availability for metamorphosis to the megalopa stage and settlement occurs throughout the year. Yet, even on tropical beaches, seasonality in the megalopa settlement has been reported, BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 167 probably influenced by factors other than temperature. Menon (1933), by studying the occurrence of planktonic larvae of E. asiatica from the offshore waters of the Madras coast found the early stage zoea larvae abundant in September to November while in the succeeding months only the advanced stage larvae were present. The settlement of the megalopa larvae on this beach subsequently took place in April. This author apparently assumed that the planktonic larvae released from the same beach undergo development in the offshore waters and that they return to the same beach to restock the parent population. This would suggest an annual growth pattern for E. asiatica. However, later work by Subramoniam (1979a) on E. asiatica from the same locality has shown a year-round egg production coupled with uninterrupted zoea larval release. Consequently, their larval stock is more or less equally distributed in the plankton all year round. The megalopa settlement of E. asiatica on the Marina beach at Madras takes place first in June, corresponding to the onset of southwest monsoon rain and the second one in October and November, when the northeast monsoon brings heavy rain to this region (Subramoniam, 1979a). Similarly, Ansell et al., (1972) have documented the seasonal recruitment pattern during the premonsoon and monsoon months for E. holthuisi from the west coast of south India, suggesting a relationship between the monsoon rains and the megalopa settlement on the Indian coasts. Evidently, hydrographical conditions prevailing on the sandy beach determine the settlement time of the megalopa larvae. It is clear from the above account that the development, dispersion and settlement of the Emerita larvae depends mostly on hydrographic factors. Different species of Emerita inhabit long coastlines and hence, the dispersive power of their larvae not only augments the existing population, but also establishes new colonies in the beach. High exchange of genetic characters between populations is predictable as a result of this larval dispersal. Furthermore, gene flow may be enhanced by the possibility of multiple fertilisation of the females. Gene flow could offset the expected Hardy- Weinberg equilibrium in genotype frequencies in different populations. Despite these factors, Corbin (1977) found distinct allelic groups of E. talpoida colonising locations in North and South Carolina, based on polymorphisms of the phosphoglucoisomerase enzymes. Distinct allelic groups of local populations could be traced back to the Florida coast. This indicates that, besides a certain degree of horizontal gene flow amongst different regions, local selection pressures might favour different allelic groups in different local populations. An interesting observation in this connection is that the female population of E. asiatica at Marina beach in Madras grows to a maximum size of 33 mm CL, whereas the population of the same species at Kovalam and Kalpakkam coasts, just 40 km to the south, invariably reaches sizes of up to 40 mm CL. 168 T. SUBRAMONIAM AND V. GUNAMALAI

Beckwitt’s (1985) studies on E. analoga populations of southern California revealed lack of genetic differentiation among six samples of the study area indicating free horizontal gene flow within the species related to their dispersal abilities. The greatest difference in allelic frequencies was observed within two regions separated by only 7 km, indicating that local selection pressures might be involved.

14. EMERITA AS INDICATOR SPECIES

Extensive data on the biology of Emerita may contribute to their suitability as ‘‘indicator species’’ to investigate pollution on sandy beaches. Ever since Burnett (1971) reported on the bioaccumulation of DDT residues in E. analoga, this intertidal crab has served to indicate high pollution levels of DDT in Santa Monica Bay, California. Interestingly, the bioaccumulated DDT in the body tissues is transferred to the eggs, which after spawning and attachment to the pleopodal hairs remain undeveloped. That pollution on the sandy beach could cause abnormal reproduction in the sand crab has been clearly indicated by Siegel and Wenner (1984). These authors, studying the fecundity potentials of E. analoga in the vicinity of a nuclear generating station in Southern California (SONGS), found that a reduction in reproduction was not related to thermal enhancement associated with the operation of nuclear generating facilities. Instead it seemed to result from multifarious factors such as runoff of agricultural pesticides from a creek 3 km north of the nuclear generating plant, and the release of metals into nearshore waters. High levels of copper and zinc were reported in the body tissues of E. analoga in the vicinity of SONGS, near Santa Barbara, implying specifically that harmful metal contaminants in the environment might affect egg production and egg quality in these crabs (Siegel and Wenner, 1984). In the impacted area, egg membranes of the egg masses were found to be ruptured soon after egg extrusion. A similar incidence of egg disruption was reported by Wenner (1982) for a crab population at San Clemente Beach. When the sand crabs from the impacted area were brought back to the laboratory, they produced normal eggs, which underwent normal development to hatch into healthy zoeae. Apparently, some factors emanating from metal pollution in the beach could be responsible for the disruption of egg membranes of the sand crabs. In addition, Wenner et al. (1985) found that the size at onset of egg production was reduced in the crabs living in a stressed habitat, in comparison with those living in nonimpacted areas. Furthermore, Auyong (1981) found that the egg production season in the impacted area was shorter. Evidently, both organic and inorganic pollutants BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 169 in the beach could not only affect filter feeding, but also the channelling of metabolic energy to egg production, in addition to causing direct damage to the exposed egg masses. However, thermal effluents from an atomic power station at Kalpakkam, located south of Madras on the east coast of India, directly affect the distribution of E. asiatica in the vicinity. Figure 25 shows the distribution pattern of Emerita in the MAPS (Madras Atomic Power Station) region. Significantly, in the impact zone having an elevated seawater temperature of 35C, the crab is completely absent. However, as we move away from the impact zone, with normalisation of seawater temperature, the Emerita population gradually increases. No difference in reproductive activity was found between these populations and a population in the control region (Station 1). Another significant observation is that there was no megalopa settlement in the impact zone. This may suggest that both young and adult Emerita are sensitive to elevated temperatures caused by thermal effluents and they move to safer areas on either side of the impact zone.

Figure 25 Distribution pattern of Emerita asiatica in the vicinity of the Madras Atomic Power Station (MAPS) in relation to temperature: Arrow indicates the position of the mixing zone: Stations 1 to 4 are located south of the mixing zone at intervals of 500 metres, with Station 1 being the Control station; Station 5 to 9 are located at intervals of 500 m north of the mixing zone. –– sea temperature (C), –i– high water, –– mid water, –&– low water. Data from Subramoniam et al. (2002). 170 T. SUBRAMONIAM AND V. GUNAMALAI

14.1. Parasitisation of egg mass and ovary

Reproductive failure may also be caused by natural agents such as egg predators (Kuris, 1991) or other parasites. In E. asiatica, the eggs attached to the pleopods have been found to be occasionally parasitised by nemertean worms: these worms eat the eggs and assume their bright orange colour (Subramoniam, 1979a). Although the occurrence of nemertean worms has been recorded in the egg masses of the crabs Portumanus ocellatus and Carcinus maenas (MacGinitie and MacGinitie, 1949) and in the kelp crab Pugettia producta (Boolootian et al., 1959), only in E. asiatica have these worms been found to feed on the eggs. In addition, a vorticellid has also been associated with the egg-carrying pleopods (Krishnaswamy, 1954). The ovary of E. asiatica is invariably infested with numerous metacercaria of a larval trematode belonging to the genus Microphallus (Anantharaman and Subramoniam, 1976). The metacercariae are lodged only in the connective tissue epithelium surrounding the ovary and do not enter the ovary proper (Figure 5E); the midgut gland tubules are not infected except in extreme parasitisation, suggesting that the ovary is the primary tissue of infection. However, Young (1938) has observed that the midgut gland is the main site of metacercarial infection in E. analoga. Apparently, metacercarial association with the ovary of Emerita has no effect on oogenesis, but under heavy infestation, ovulation is incomplete, many ripe eggs remaining unspawned (Subramoniam, 1977a). Helminth infections on the ovary of the sand crabs also include the capsules of a tetraphyllidean larvae, belonging to the genus Phyllobothrium (Anantharaman and Subramoniam, 1980). Obviously, these crustaceans constitute the second intermediate host for these parasites that reach their final host in fishes and sea birds.

15. CONCLUSIONS

Mole crabs belonging to the genus Emerita are exclusively inhabitants of exposed sandy beaches in certain temperate and tropical seas. The main adaptive features for the sandy beach environment are the burrowing behaviour and the mode of filter feeding with a pair of long plumose antennules. While the morphology and behaviour of the mole crabs reflect the adaptive attributes of the species to the environment, peculiarities found in their sexual and reproductive biology imply a complex life history pattern. Filter feeding in Emerita species, coupled with the continuous availability of detrital food in the intertidal zone confers a favourable nutritional status to help ensure successful reproduction and moulting throughout the year, as shown in E. asiatica. BREEDING BIOLOGY OF THE SAND CRAB, EMERITA 171

A perfect endocrine coordination of these two energy-demanding processes is assumed to bring about the continued body growth even in egg laying adults. Detailed analysis of the egg components and of their efficient utilisation during embryogenesis, has unravelled the crab’s ability to produce healthy larvae, to be released into the open ocean for their subsequent development and metamorphosis. The protracted larval development, coupled with their dispersion, aided by ocean and nearshore water currents, enable them to spread far with the water masses, to settle in new areas and found new populations. Nevertheless, hydrographical conditions prevailing over the sandy beach intertidal zone may have a deciding role in the recruitment of the megalopa stage to the beach. The occurrence of neotenic males in the majority of Emerita species is again an adaptation to achieve easy sperm transfer via spermatophores deposited on the females, without affecting their normal activities.The sticky nature of the mucoid spermatophoric ribbon ensures fast and firm attachment to the ventral sternum of the females. Yet another feature of interest in the reproductive biology of a tropical species, E. asiatica is the occurrence of functional protandric hermaphroditism. This pattern of sexuality in the life history of this mole crab is of great adaptive significance because it vastly augments fecundity, by introducing the secondary females into the egg-laying female population. Obviously, natural selection has favoured a smaller size of males to accomplish mating in a turbulent environment, whereas the sex reversal of males at a larger size to enter the egg-laying population of female Emerita increases fecundity. Emerita is an ideal intertidal genus in which to investigate environmental influences on the growth and reproductive performance in an otherwise harsh substratum which provides habitation only to a few specialised invertebrate forms.

ACKNOWLEDGEMENTS

We thank the Council of Scientific and Industrial Research, New Delhi for financial support (Grant no. 21(0492)01/EMR-II/dt. 27.4.01). Grateful thanks are also due to Dr. E. Vivekanandan of the Central Marine Fisheries Institute substation at Chennai and Prof. Jeyaraman, Department of Genetics, IBMS, University of Madras and to former student Dr. R. Tirumalai for discussion during the preparation of this article. We also thank Mr. Sunil Israel and Ms. Santhoshi of the Unit of Invertebrate Reproduction and Aquaculture for their editorial assistance. 172 T. SUBRAMONIAM AND V. GUNAMALAI

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S. L. Coles1 and Barbara E. Brown2

1Department of Natural Sciences, Bishop Museum, 1525 Bernice St., Honolulu, HI 96734, USA 2School of Biology, University of Newcastle on Tyne, Newcastle on Tyne NE1 7RU, UK

1. Introduction ...... 184 2. Coral Upper Temperature Tolerance Thresholds ...... 186 3. The Coral Bleaching Process ...... 188 4. Coral Bleaching Protective Mechanisms ...... 190 5. Coral and Zooxanthellae Thermal Acclimation, Acclimatization, and Adaptation: Empirical Observations ...... 195 6. Coral Bleaching Recovery ...... 201 7. Bleaching and Coral Disease, Reproduction, and Recruitment ...... 204 8. Long-Term Ecological Implications of Coral Bleaching ...... 207 9. Conclusions ...... 209 Acknowledgments ...... 211 References ...... 212

Coral bleaching, i.e., loss of most of the symbiotic zooxanthellae normally found within coral tissue, has occurred with increasing frequency on coral reefs throughout the world in the last 20 years, mostly during periods of El Nino Southern Oscillation (ENSO). Experiments and observations indicate that coral bleaching results primarily from elevated seawater temperatures under high light conditions, which increases rates of biochemical reactions associated with zooxanthellar photosynthesis, producing toxic forms of oxygen that interfere with cellular processes. Published projections of a baseline of increasing ocean temperature resulting from global warming have suggested that annual temperature maxima within 30 years may be at levels that will

ADVANCES IN MARINE BIOLOGY VOL 46 Copyright ß 2003 Academic Press 0-12-026146-4 All rights of reproduction in any form reserved 184 S. L. COLES AND BARBARA E. BROWN cause frequent coral bleaching and widespread mortality leading to decline of corals as dominant organisms on reefs. However, these projections have not considered the high variability in bleaching response that occurs among corals both within and among species. There is information that corals and their symbionts may be capable of acclimatization and selective adaptation to elevated temperatures that have already resulted in bleaching resistant coral populations, both locally and regionally, in various areas of the world. There are possible mechanisms that might provide resistance and protection to increased temperature and light. These include inducible heat shock proteins that act in refolding denatured cellular and structural proteins, production of oxidative enzymes that inactivate harmful oxygen radicals, fluorescent coral pigments that both reflect and dissipate light energy, and phenotypic adaptations of zooxanthellae and adaptive shifts in their populations at higher temperatures. Such mechanisms, when considered in conjunction with experimental and observational evidence for coral recovery in areas that have undergone coral bleaching, suggest an as yet undefined capacity in corals and zooxanthellae to adapt to conditions that have induced coral bleaching. Clearly, there are limits to acclimatory processes that can counter coral bleaching resulting from elevated sea temperatures, but scientific models will not accurately predict the fate of reef corals until we have a better understanding of coral–algal acclimatization/adaptation potential. Research is particularly needed with respect to the molecular and physiological mechanisms that promote thermal tolerance in corals and zooxanthellae and identification of genetic characteristics responsible for the variety of responses that occur in a coral bleaching event. Only then will we have some idea of the nature of likely responses, the timescales involved and the role of ‘experience’ in modifying bleaching impact.

1. INTRODUCTION

‘‘Coral bleaching’’ was first described in detail by Yonge and Nicholls (1931a) as a reduction in cellular concentrations of symbiotic zooxanthellae in corals that had been exposed to elevated temperature at Low Islands, Great Barrier Reef, Australia. Their experiments also showed that bleaching could result from a variety of stresses acting on the coral–algal symbiotic association, such as exclusion of light or starvation. Earlier observations (Vaughan, 1914) had described loss of coral pigmentation as a result of reduced salinity and light exclusion. However, coral bleaching has been most frequently linked with elevated temperature, generally considered to be the primary stress causing coral bleaching worldwide and to be associated with global warming of the earth’s atmosphere and ocean CORAL BLEACHING 185 temperatures (see reviews by Brown, 1987, 1997b; Jokiel and Coles, 1990; Williams and Bunkley-Williams, 1990; Glynn, 1991, 1993; Goreau, 1992; Pittock, 1999; Boesch et al., 2000; Westmacott et al., 2000; Wilkinson, 2000; Fitt et al., 2001). The world’s mean ocean temperature has increased approximately 0.5C in the last century (Pittock, 1999), and various atmospheric models predict another 1–3C warming worldwide by the mid-21st century (Boesch et al., 2000). Reef corals have long been described as living at temperatures near their upper limits of thermal tolerance (Mayer, 1914; Edmondson, 1928). Maximum temperatures that have occurred in the tropics in the past two decades have coincided with episodes of coral bleaching that exceeded previous bleaching events in both frequency and magnitude. Coral bleaching reported in 1997–98 in the Indo-Pacific and the Caribbean was very widespread and was followed by extensive coral mortality in many areas (Cohen et al., 1997; Baird and Marshall, 1998; Spencer et al., 1998; Berkelmans and Oliver, 1999; Berkelmans and Willis, 1999; Fabricius, 1999; Hoegh-Guldberg, 1999; Mumby, 1999; Wilkinson et al., 1999; Aronson et al., 2000, Marshall and Baird, 2000; McClanahan, 2000; Mumby et al., 2000, 2001; Westmacott et al., 2000; Podesta and Glynn, 2001; Reyes Bonilla, 2001; 2002; Bruno et al., 2001; Carriquiry et al., 2001; Edwards et al., 2001; Feingold, 2001; Glynn et al., 2001; Guzman and Cortes, 2001; Jimenez et al., 2001; Lindahl et al., 2001; McClanahan et al., 2001; Vargas- Angel et al., 2001; Wellington et al., 2001). Periods of intense coral bleaching have often been preceded by ‘‘El Nino’’ episodes associated with the El Nino Southern Oscillation (ENSO) (Williams and Bunkley-Williams, 1990; Glynn, 1993; Spencer et al., 1998; Wilkinson et al., 1998; Hoegh- Guldberg, 1999; Mumby et al., 2001), when reduced mid-latitude high pressure systems result in weakened wind systems, less cloud cover, and lower evaporative cooling at the ocean’s surface both regionally and locally. However, there are equally as many recent bleaching phenomena that do not seem to follow ENSO signals (Brown, 1987). Bleaching episodes have apparently increased in their frequency and severity in the last 20 years (Glynn, 1993; Hoegh-Guldberg, 1999), initiating concern that, with maximum yearly temperatures increasing through the next century, thermal tolerance thresholds of corals throughout the world could be exceeded on an annual basis by 2030 (Hoegh-Guldberg, 1999). Corals and coral reefs therefore appear to be undergoing a historically unprecedented period of stress from elevated temperatures that may result in their ultimate decline as one of the major biotopes on the planet. Table 1 shows the range of dates projected by four global climate models for the dates when sea temperatures may increase to levels where coral bleaching temperature thresholds could be exceeded on an annual basis if acclimatization or adaptation does not occur (Hoegh-Guldberg, 1999). 186 S. L. COLES AND BARBARA E. BROWN

Table 1 Estimates of ranges and median dates when coral bleaching events may occur annually based on threshold temperatures proposed to induce coral bleaching locally and on projections of increasing ocean temperatures by four global climate models (derived from Hoegh-Guldberg, 1999).

Locality Threshold Projected dates for 10 bleaching temperature (C) events/decade

Range Median

Jamaica 29.2 2010–2030 2020 Phuket 30.2 2000–2040 2020 Tahiti 28.3 2035–2045 2040 Raratonga 28.3 2020–2040 2030 Southern GBR 28.3 2020–2060 2040 Central GBR 29.2 2025–2050 2037 Northern GBR 30.0 2020–2040 2030

Averaging the media values for the ranges derived from seven regions suggests that this could occur worldwide by about 2030. Various scenarios have been proposed to describe reef conditions resulting from continuing and repetitive bleaching events (Done, 1999). However, we should recognize that reef corals have been a subject of research for only a little over a hundred years, and that the last 30 years have produced the vast majority of observations and measurements on reef corals and their association with symbiotic zooxanthellae. Little is known regarding the capacity of corals or zooxanthellae to adapt or acclimatize to elevated temperatures, or the rates at which any such adjustment to stressful temperatures may occur. The purpose of this review is to summarize the information that is available on coral bleaching, focus on processes that may act as adaptive mechanisms and suggest needed research in this area.

2. CORAL UPPER TEMPERATURE TOLERANCE THRESHOLDS

The earliest observations on upper temperature limits to coral survival were made early in the 20th century (Mayer, 1914, 1917, 1918a,b, 1924) on corals in Florida, Australia, and Samoa. From this information, Mayer (1918b) concluded that upper and lower temperature death limits were similar for Florida and Great Barrier Reef corals for short exposures despite distinctly different temperature environments. He said that ‘‘the whole matter of temperature resistance is physiological and natural selection appears to have nothing to do with it’’ (Mayer, 1918b). Experimental measurements made CORAL BLEACHING 187 by Yonge and Nicholls (1931a) on the Great Barrier Reef and by Edmondson (1928) in Hawaii showed similar short-term upper temperature tolerances. However, these results were limited by the experiments being performed in static aquaria that allowed accumulation of toxic metabolites that limited the applicability of the results to the natural environment. Experiments using controlled temperatures in flowing systems were conducted in Hawaii (Coles, 1973; Jokiel and Coles, 1977; Coles and Jokiel, 1978; Jokiel and Guinther, 1978) and in Guam (Jones and Randall, 1973), and experimental results were compared to observations made in the vicinity of power station thermal discharges (Jokiel and Coles, 1974; Coles, 1975; Neudecker, 1981). Using these techniques, experimental comparisons were made between subtropical Hawaiian corals and tropical Pacific corals at Enewetak, leading to the conclusions that differences in coral thermal tolerances correspond to predictable differences in the ambient temperature patterns between geographic areas, and that ‘‘in both subtropical and tropical environments large populations of corals are exposed to tempera- tures precariously close (within 1–2C) to their upper lethal limit during the summer months’’ (Coles et al., 1976). Further experiments relating effects of temperature on energetic processes using measurements of oxygen flux suggested that there was a possibility of metabolic adaptation by corals to their ambient temperature regime (Coles, 1973; Coles and Jokiel, 1977). Comparisons of net photosynthesis and respiration across a temperature range of 18–31C for four coral species in Hawaii and Enewetak showed that P : R ratios differed between Hawaii and Enewetak specimens, resulting in linearly decreasing net photosynthesis with increasing temperature for Hawaiian specimens, compared with a response for Enewetak corals that suggested adaptation to the higher ambient temperature regime at Enewetak. P : R ratios throughout the tested temperature range also differed among species and corresponded to their different tolerances to increased temperature. Repeated coral bleaching episodes and additional experiments during the past 25 years have verified that upper temperature tolerances of corals are linked to geographic location and ambient temperature conditions. Increases of 1–3C above mean long-term annual maximum temperatures have consistently induced coral bleaching (Hudson, 1981; Glynn, 1984; Lasker et al., 1984; Harriott, 1985; Jaap, 1985; Brown and Suharsono, 1990; Cook et al., 1990; Gates, 1990; Glynn and D’Croz, 1990; Gleason, 1993; Brown et al., 1995; Cohen et al., 1997; Jones et al., 1997; Spencer et al., 1998; Berkelmans and Oliver, 1999; Berkelmans and Willis, 1999; Quinn and Kojis, 1999; Marshall and Baird, 2000; Berkelmans, 2001; Bruno et al., 2001; Edwards et al., 2001; Glynn et al., 2001; Podesta and Glynn, 2001; Vargas-Angel et al., 2001; Wellington et al., 2001). The threshold temperatures which induce coral bleaching and mortality range over 8C 188 S. L. COLES AND BARBARA E. BROWN worldwide, from 27C in Rapa Nui (Easter Island) during 2000 where summer ambient maximum is normally about 25C (Wellington et al., 2001) to 35–36C during 1998 in the Arabian Gulf (George and John, 1998; Wilkinson et al., 1998; Riegl, 1999, 2002), where normal ambient summer open water maxium usually ranges up to 34C (Coles, 1988). Clearly, maximum water temperatures normally occurring in particular geographic areas have principally determined the upper temperature tolerances of corals, indicating that the corals are adjusted to ambient conditions (Figure 1). This implies a capacity for reef corals and/or their algal symbionts to adapt to higher temperatures over as yet unknown periods of time. What is not clear is whether adjustment can occur through phenotypic acclimatization to acute stress conditions and the mechanisms involved, or require longer-term adaptation involving selection and breeding of eurythermal genotypes.

3. THE CORAL BLEACHING PROCESS

Various mechanisms of zooxanthellae loss from corals have been reported, including exocytosis, apoptosis (or programmed cell death), necrosis, and host detachment (Gates et al., 1992; Brown et al., 1995). The earliest description of zooxanthellae leaving a host coral’s cells was made by Boschma (1925, 1926), who concluded that this was a process of coral polyps digesting the algal symbionts at the mesenterial filaments where the zooxanthellae had aggregated. Yonge and Nicholls (1931a,b) reinterpreted this process to be an active removal of the zooxanthellae, or ‘‘bleaching’’ of the coral that could occur in response to a variety of stresses, including but not limited to increased temperature. Following earlier experiments on the effects of elevated temperature on Hawaiian corals (Edmondson, 1928), studies using a flowing seawater system with altered temperature and light regimes showed that high light levels interact with increased temperature in producing coral bleaching (Coles, 1973; Jokiel and Coles, 1977; Coles and Jokiel, 1978). A number of subsequent studies increased our understanding of the molecular processes which lead to zooxanthellar loss, coral bleaching, and the interaction of the effects of light and temperature (Iglesias-Prieto et al., 1992; Fitt and Warner, 1995; Jones et al., 1998, 2000; Warner et al., 1996, 1999; Brown, 1997b; Hoegh-Guldberg, 1999; Brown et al., 2000b; Fitt et al., 2001). The basis of the temperature–light interaction has recently been reviewed by Fitt et al. (2001). Briefly, under nonstressful temperatures and light, zoox- anthellar photosynthesis proceeds through a normal process of uptake of dissolved carbon dioxide and water and transfer of protons through the CORAL BLEACHING 189

Figure 1 Semilogarithmic plots of survival of Pocillopora corals with stress temperatures in Hawaii (thick line) and the tropical Pacific (thin line), from Figure 2 in Coles et al. (1976). Data from: Edmondson (1928) for Hawaii P. meandrina (solid circles) and P. caespitosa (syn. P. damicornis) (solid hexagons); Mayer (1918) for Great Barrier Reef P. bulbosa (syn. P. damicornis), open triangle; Mayer (1924) for American Samoa P. damicornis (open diamond); Jones and Randall (1973) for Guam P. damicornis (open circles); Jokiel and Coles (1977) for Hawaii P. damicornis (solid diamonds); Coles et al. (1976) for Enewetak P. elegans (open squares), Hawaii P. meandrina (solid square) and Hawaiian Podamicornis (solid triangle). photochemical systems of the light reaction, with the release of oxygen, and fixation of organic carbon in the dark reaction. At higher light intensities the rates of processes can become saturated, with photosaturation occurring as early as 09:00 h in shallow water corals (Brown, 1997b). With elevated temperatures, the rates of these processes increase to a level where more protons are produced in the light reaction than can be utilized to form organic carbon in the dark reaction. In the first studies of bleaching-related 190 S. L. COLES AND BARBARA E. BROWN molecular processes, Lesser (Lesser et al., 1990; Lesser, 1997) demonstrated that bleaching in corals and other cnidarians was preceded by the production of oxygen free radicals or other toxic forms of oxygen in the dinoflagellate symbionts and coral host tissues, subsequently causing cellular damage and expulsion of symbionts. Photoinhibition under the influence of increased temperature is therefore a primary factor influencing coral bleaching, and both internal and external processes that effectively reduce light levels or alter light quality to longer, less damaging wavelengths may reduce coral bleaching with increased temperatures. In experiments on Pocillopora damicornis at Heron Island, damage to zooxanthellae occurred with exposures of only 7 h in high light conditions (1000–1500 mmole quanta mÀ2 sÀ1) (Salih et al., 1998a; Salih, 2001), suggesting that the process of coral bleaching starts well before it manifests itself as actual zooxanthellae loss. A large proportion of algal cells showed greatly reduced chloroplasts, increased vacuolation and presence of lipid globules and increasing cell degradation along with coral bleaching two days after the high light exposure. Remarkably, these symptoms of cell damage occurred at both 26 and 32C, although bleaching was more pronounced in combined high illumination at 32C, and these corals continued to show progressive decline. These responses indicate that coral bleaching is not simply a direct result of increased temperature, but rather a result of combined stresses that include, but are not necessarily limited to, temperature and light conditions. Evidence from various studies has substantiated that high light levels are important in inducing coral bleaching (Hoegh-Guldberg and Smith, 1989; Fitt and Warner, 1995; Brown et al., 1999a). Individual corals usually show more pronounced bleaching and mortality on upper surfaces and on terminal branches than lower down on the colony. On a larger scale, corals at shallow depths are usually more sensitive to bleaching at a given temperature than those at greater depths (Marshall and Baird, 2000), and corals in offshore areas with high water clarity are usually more highly impacted during major bleaching events than corals in nearshore areas with higher turbidity (Phongsuwan, 1995).

4. CORAL BLEACHING PROTECTIVE MECHANISMS

Coral symbiotic algae must meet the challenge of all photosynthetic organisms in harvesting solar radiation efficiently while simultaneously safely disposing of dangerous excess excitation energy that would ultimately be harmful to both algae and coral host. Normally, when excess solar radiation is absorbed by the algae, an alternative dissipating pathway is activated that safely returns excited chlorophyll to ground state. In this CORAL BLEACHING 191 process the excitation energy is dissipated as heat via the xanthophyll cycle in a process termed nonphotochemical quenching (NPQ), which is well documented in both higher plants and algae (Demmig-Adams and Adams, 1993; Olaizola and Yamamoto, 1994; Olaizola et al., 1994; Owens, 1994; Wilkinson, 2000). In coral symbiotic zooxanthellae, heat dissipation is achieved by the reversible interconversion of the xanthophylls, diadino- xanthin, and diatoxanthin. These xanthophylls were first identified in coral zooxanthellae by Jeffrey and Haxo (1968), and an active xanthophyll cycle in corals was described by Ambarsari et al. (1997) and Brown et al. (1999b). Figure 2 shows a pronounced cycling of photoprotective xanthophylls in response to diurnal irradiance changes which induce photoinhibition in the shallow water coral Goniastrea aspera. When sea temperatures rise above the normal ambient maxima, corals become more susceptible to the effects of damaging solar radiation (Brown, 1997b; Hoegh-Guldberg, 1999); thus the xanthophyll cycle becomes a key photoprotective defense. Indeed it has been claimed that those corals more capable of dissipating excess excitation energy through NPQ are less prone to temperature bleaching (Warner et al., 1996). Another possible protective mechanism against stressful light levels may be fluorescent coral pigments, which have been indicated to reduce coral bleaching by reflecting and/or fluorescing absorbed light (Salih et al., 1998b, 2000; Dove et al., 2001). A total of 124 species of corals were found to have morphs containing fluorescent pigments on the Great Barrier Reef, often growing alongside of morphs of the same species without such pigments (Salih et al., 2000). Corals containing such fluorescent capacity were found to bleach significantly less than nonfluorescent colonies of the same species growing in the same area. Nonfluorescent corals were significantly more photoinhibited during peak irradiance periods, and bleaching resistance measured as tissue dinoflagellate biomass correlated significantly with fluorescent pigment concentrations in coral tissue. The protective capacity of these pigments may have important implications for long-term survival of corals exposed to thermal stress. At Phuket Thailand, Brown et al. (2002c) found abundant fluorescent pigment in cores from bleaching-resistant west- facing surfaces of G. aspera compared with low concentrations in bleaching- prone east-facing surfaces. Fluorescent pigments were most abundant in the endoderm surrounding the symbiotic algae, suggesting a photoprotective function. Such potential protective capacity has far-ranging implications for long-term survival of corals when additionally stressed by high temperature, although recent preliminary work by Dove (pers. comm. to BEB) suggests that some of these pigments are easily denatured by elevated sea temperature. An internal defense mechanism that may substantially influence coral tolerance to bleaching and mortality is change in heat shock proteins (Hsps) 192 S. L. COLES AND BARBARA E. BROWN

Figure 2 Underwater photosynthetically active radiation (PAR), effective quantum yield of photosystem II (iF/Fm0), and xanthophyll ratios of diatoxanthin to diadinoxanthin þ diatoxanthinin for the coral Goniastrea aspera in January 1998 at Phuket, Thailand. Points and bars represent means Æ one standard deviation (from Figure 2 in Brown et al., 1999b).

induced by increased temperature. The Hsps act as ‘‘molecular chaperones’’ (Hartl, 1996), preventing detrimental aggregation of structurally nonnative proteins, helping to refold reversibly heat damaged proteins and aiding in the insertion of proteins into organelles (Lindquist and Craig 1988; CORAL BLEACHING 193

Morimoto et al., 1994). Induction of a suite of Hsps is a well-characterized response to heat shock (and other stresses) in many marine organisms, e.g., Mytilus californianus (Roberts et al., 1997), the anemone Anemonia viridis, and various marine invertebrates, including scleractinian corals. In corals, Hsp 70 has been recognized during heat shock in Goniopora djiboutiensis and Goniopora pandoraensis (Sharp, 1995; Sharp et al., 1997), Montastraea annularis (Hayes and King, 1995), Montastraea franksi (Gates and Edmunds, 1999), Montastraea faveolata (Downs et al., 2000), Acropora grandis (Fang et al., 1997), and G. aspera (Brown et al., 2002c). Hsp 60 has been demonstrated in M. faveolata (Downs et al., 2000), A. grandis (Fang et al., 1997), and G. aspera (Brown et al., 2002c), while Hsps 27, 28, 33, 74, 78, 90, and 95 have been shown to occur in M. faveolata (Black et al., 1995). Fang et al. (1997) identified Hsp 35 in A. grandis as heme oxygenase, previously known to be induced by UV radiation and oxidative stress. In model organisms such as the fruit fly Drosophila spp., maximum rates of Hsp synthesis are achieved 1 h after initial heat shock; the rate of Hsp synthesis then declines, but if the high-temperature treatment is continued, Hsps accumulate since they have long half-lives. By 6–8 h they form up to 10% of the cell’s total proteins (Ashburner and Bonner, 1979). Similar temporal fluctuations have been observed in synthesis of Hsp 70 in the coral M. franksi (Gates and Edmunds, 1999), though detailed resolution of shifts in protein turnover in heat-stressed corals is lacking. In higher plant chloroplasts small Hsps are produced in response to many environmental stresses, with recent work showing that chloroplast small Hsps are important determinants of both photosynthetic and whole plant thermo- tolerance (Heckathorn et al., 1999). Chloroplast small Hsps are also present in coral symbiotic algae and in M. faveolata (Downs et al., 2000) are upregulated 3.5 fold compared to controls by an increase in temperature of 6C in dim light and as much as 50 fold in G. aspera by a temperature increase of 4C in bright light (Brown et al., 2002c). Important coral defenses against high light and elevated temperature also occur with oxidative enzymes which include copper/zinc superoxidase (SOD), manganese SOD, iron SOD, ascorbate peroxidase, and catalase, thereby preventing subsequent cellular damage from active species of oxygen. Both enzyme activity and concentration may be increased as a result of exposure to elevated temperature (Lesser et al., 1990; Fang et al., 1997; Downs et al., 2000). Changes in zooxanthellae symbionts may influence adaptive responses by the coral–algal association to thermal stress. Although all coral zooxanthellae were originally considered to be a single species described as Symbiodinium microadriaticum, numerous species and types of zoo- xanthellae are now recognized, and their environmental tolerances or composition may fluctuate with environmental conditions. This could 194 S. L. COLES AND BARBARA E. BROWN occur through changes in the phenotypic adaptation of the biochemical– physiological processes of the resident zooxanthellae (Brown, 1997b), or by rapid changes in their cladal composition (Coffroth et al., 2001). The latter possibility, first proposed over 25 years ago when Jokiel and Coles (1977) stated ‘‘a different strain of algal symbiont may inhabit the tropical representative of the various coral species,’’ was later formalized as the Adaptive Bleaching Hypothesis (ABH) (Buddemeier and Fautin, 1993; Ware et al., 1996). The ABH postulated that the loss of resident zooxanthellae in response to stress provides an opportunity for stress- adapted types to repopulate the coral, imparting greater resistance to the stress and competitive advantage for the coral–algal complex. A number of laboratory and field studies of the genetic diversity of symbiotic algae in corals and other cnidarians provide limited but inconclusive support for the ABH (Baker, 2001; Kinzie et al., 2001). These studies indicate consistent latitudinal differences that suggest the existence of thermo-tolerant zooxanthellae phylotypes (Loh et al., 2001; Rodriguez-Lanetty et al., 2001; Savage et al., 2002a). Glynn et al. (2001) found differences in bleaching resistance that corresponded to zooxanthellae symbiont genotypes in P. damicornis during the 1997–98 bleaching event. These examples suggest that inducible variations in genetic composition of zooxanthellae, as well as the capacity of the symbionts for phenotypic adaptation to stress events (Brown, 1997a,b), may contribute to adaptive selection of thermally resistant coral species and varieties. Some of these observations, however, should be viewed with caution. Baker (2001) argues that transplant experiments indicate that bleaching provides an opportunity for corals to rid themselves of suboptimal algae and acquire new partners. However, this work has been criticized by others (Hoegh-Guldberg et al., 2002) who believe that Baker’s data do not support the ABH. Although several criticisms were made, the main issue was that transplantation of corals to different depths confused interpretation of the results obtained. Most importantly, we are far from understanding the physiological traits of symbiotic algal genotypes. The photosynthetic characteristics of coral symbiotic algae cannot be deduced from the commonly used method of molecular typing of r-RNA genes (Savage et al., 2002b). Therefore, generalizations about photosynthetic traits of different algal genotypes based on these results are unconvincing. Indeed, Kinzie et al. (2001) showed that variability in physiological response to temperature (in this case growth rate) within a genotype might be as great or greater than between genotypes. Clearly, finer genetic differentiation will be required to understand not only the physiological tolerances of the algae but also their dynamics within the coral colony. Until this is achieved, the case for or against the ABH will not be determined. CORAL BLEACHING 195

5. CORAL AND ZOOXANTHELLAE THERMAL ACCLIMATION, ACCLIMATIZATION, AND ADAPTATION: EMPIRICAL OBSERVATIONS

The capacity of corals and reefs to adapt to elevated temperatures has been the subject of a number of reviews (Gates, 1990; Buddemeier and Fautin, 1993; Glynn, 1993; Brown, 1997a,b; Buddemeir and Smith, 1999; Done, 1999), and the mechanisms of phenotypic adaptation for corals have been discussed in Brown (1997a) and Gates and Edmunds (1999). As reviewed in Brown (1997a), adaptations by corals to elevated temperature or light regimes can occur under a range of time scales and conditions. Terms referring to these adjustments have been variously used, and we herein follow the terminology of Brown (1997a) and Gates and Edmunds (1999). Although acclimation has been used ambiguously to refer to adaptation over the long term, e.g., Ware (1997), acclimation more properly means changes in tolerances under laboratory or other experimental conditions, generally over the short term. Acclimatization refers to phenotypic changes by an organism to stresses in the natural environment that result in the readjustment of the organism’s tolerance levels. These phenotypic responses are usually reversible and are limited by the organism’s genotype, which determines the boundaries beyond which acclimatization cannot occur. Finally, selective adaptation occurs when the more stenotopic members of a population are eliminated by the environmental stress, leaving the more tolerant organisms to reproduce and recruit to available habitat. The primary evidence of long-term selection for temperature tolerant corals is based upon the linkage of thermal thresholds to maximum ambient temperature environments previously described, and reports of corals surviving temperatures well in excess of normally accepted limits. Gardiner (1903) observed abundant corals in a tidepool in the Laccadives at water temperatures up to 56C, and Kinsman (1964) noted massive Porites at over 40C near Abu Dhabi, Arabian Gulf. Motoda (1940), Orr and Moorhouse (1933), and Vaughan (1914) reported corals surviving temperatures up to 38–39C in Palau, Australia, and Florida, respectively. More recently Tomascik et al. (1997) reported a variety of corals living at 34–37Cneara thermal vent in Indonesia, with one species growing in the vent at 42C. On Ofu Island, American Samoa, Craig et al. (2001) found 52 coral species, including nine Acropora taxa, to survive daily temperatures as high as 34.5C for up to 3 h exposures daily for 35 days during the summer of 1998–99 with virtually no bleaching. Meesters and Bak (1993) found recovery of experimentally damaged bleached Porites asteroides in the thermal effluent of a power station in Curacao to be just as high as that of 196 S. L. COLES AND BARBARA E. BROWN normal colonies, and that the corals regained normal pigmentation at higher temperatures, suggesting acclimatization to have occurred at temperatures averaging 1.3C above ambient conditions. However, there have been few controlled experiments on reef corals that have attempted to determine the capacity of reef corals for even short-term acclimation to elevated temperatures. Experiments by Coles (1973) and Coles and Jokiel (1978) described in Brown (1997a) indicated that Hawaiian Montipora verrucosa acclimated for 56 days at 1–2C above summer maxima had higher survival for 5 days at stress temperatures of 30–32.5C than did ambient controls. Clausen and Roth (1975) showed shifts in coral calcification rates of Hawaiian P. damicornis corresponding to incubation temperature, suggesting a capacity for short-term acclimation. Glynn and D’Croz (1990) found corals from an upwelling area in the Gulf of Panama to undergo greater bleaching at 30C in controlled experiments than the same species from the nonupwelling Gulf of Chiriqui, where ambient temperatures were higher and more stable. Al-Sofyani and Davies (1992) found that respiration rates of Echinopora gemmacea in the Red Sea did not change with a 6C seasonal change in seawater temperature, suggesting acclimatization for this species, while respiration rates of Stylophora pistillata indicated no such acclimatization. Berkelmans and Willis (1999) found that the winter bleaching threshold of P. damicornis on the Great Barrier reef was 1C lower than the summer threshold for this species, and proposed that the winter temperature bleaching threshold of 31–32C was a reliable predictor of subsequent mortality observed when the stressed corals were returned to the field and observed for 84 days. This possibility of seasonal acclimatization, while intriguing, was not fully supported by these experiments, since postexposure observations were not made on corals during the summer trials, and lack of postexposure information on the fate of controls during the winter trial make the results subject to question. Also, these experiments did not find differences in thermal thresholds between corals from the reef flat compared to the reef slope, or from different reefs that had shown contrasting bleaching susceptibility. Such differences would be expected from Berkelmans’ (2002) conclusion that cross-shelf and latitudinal differences in coral bleaching thresholds correspond to tempera- ture regimes on the Great Barrier Reef, suggesting thermal adaptation at spatial scales of ca. 10–100 km. Observations comparing bleaching under field conditions during the 1997–98 periods of anomalous high temperatures at Ko Phuket Thailand (Dunne and Brown, 2001; Brown et al., 2002b) with previous episodes in 1991 and 1995 have indicated a complex interaction of light with temperature that may act to induce bleaching protection. Despite similar temperature elevations and durations in 1997 and even higher temperatures in 1998 than the two previous periods, bleaching was considerably less CORAL BLEACHING 197 in 1997 and 1998 than during previous episodes. High temperatures in 1997 and 1998 were preceded by periods of higher than normal light intensity that was indicated to stimulate photoprotective defenses in both coral host and algae when the sea temperature was lower than stress levels, and this tolerance then persisted through the periods of maximum temperature–light stress (Brown et al., 2002b). Anomalous low tides in 1997 and 1998 also accentuated the high light environment experienced by the corals in the area (Dunne and Brown, 2001). Complex interaction between sea temperature and light was also evident at the colony level at this Thailand site, where the west sides of colonies of G. aspera showed superior thermal tolerance to the east sides both in the field during major bleaching events as well as in laboratory experiments (Brown et al., 2000b, 2002a,c). In this example (Plate 1a) west sides of colonies are exposed to high irradiance in the dry season (November to May) and, as a result, may show solar bleaching. However, when anomalously high sea temperatures cause extensive bleaching on the reef in May, such bleaching is mainly restricted to the east sides of G. aspera colonies (Plate 1b). It appears that exposure of western surfaces of the coral to a high irradiance environment in the field subsequently conferred tolerance to high sea temperatures due to improved photoprotective defences on the west sides without alteration of the zooxanthellae genotype (Brown et al., 2002a). Recent experiments revealed increases of 10 to 50 fold for molecular biomarkers of stress and host stress proteins of G. aspera during elevated temperature (33C) exposures (Figure 3). Higher levels of oxidative stress occurred on east sides than west sides, concomitant with higher concentra- tions of defenses, such as Hsps and oxidative enzymes (Brown et al., 2002c). Interestingly, in this experiment the differences lie in the host defenses rather than those of the algae. This model is useful in showing that, in this shallow water coral, limited acclimatization to high temperature does occur in the field, that the timescale for acclimatization is relatively short (days–weeks– months) and that photoprotection in the host can be an important defense against elevated sea temperatures. Observations comparing the responses of corals in the eastern Pacific to elevated temperatures that occurred during the ENSO events of 1983–84 (Glynn, 1983, 1984; Glynn and D’Croz, 1990) and 1997–98 (Glynn et al., 2001; Jimenez et al., 2001) suggest that corals or coral assemblages may become more thermally resistant or tolerant of bleaching with repeated bleaching events. Elevations of sea surface temperatures (SSTs) and durations of elevations in the Gulfs of Panama and Chiriqui, the Galapagos Islands, and the coast of Ecuador were of similar magnitude during the 1987–88 and 1982–83 ENSO events (Glynn et al., 2001; Podesta and Glynn, 2001). However, coral bleaching and mortality from 1997–98 198 S. L. COLES AND BARBARA E. BROWN

Plate 1 Experience-mediated bleaching in Goniastrea aspera. (A) Solar bleaching evident on the west sides of a colony in February 1995. The arrow marker across the top of the colony points north-south. (B) Temperature-induced bleaching on the east sides of colonies in May 1995 when sea temperatures were anomalously high. The lesions caused by solar bleaching earlier in the year can clearly be seen on the west side of the colony. CORAL BLEACHING 199

Figure 3 Concentrations (pg mgÀ1 except Ubiquitin and Cu : Zn SOD in ng mgÀ1) of 12 molecular markers in soluble protein in Goniastrea aspera held at an elevated temperature of 33C for three days at Phuket, Thailand. Open bars represent west sides of colonies, shaded bars east sides. Markers included three indicators of oxidative stress: (4-hydroxynoneal [HNE], alondialdahyde [MDA] and ubiquitin) four coral host-specific biomarkers: (oxidative enzymes copper/zinc superoxide dismutase [Cu : Zn SOD] and manganese superoxide dismutase [MnSOD] and heat shock proteins Hsp60 and Hsp70, and five symbiotic algae host-specific biomarkers: Cu : Zn SOD, MnSOD, Hsp60, Hsp70, and chloroplast small heat shock protein (ChlsHsp). Bar represent means Æ one standard error. Significant differences: *< 0.05, **< 0.01, ***< 0.001 (from Figure 2 in Brown et al., 2002c). 200 S. L. COLES AND BARBARA E. BROWN was substantially less than in 1982–83 (Glynn et al., 2001; Podesta and Glynn, 2001). Coral mortality from the 1982–83 event was 97–99% in the Galapagos Islands, 85% in the Gulf of Panama, and 75% in the Gulf of Chiriqui. By contrast, mortality in 1987–88 was 26% in the Galapagos, 13% in the Gulf of Chiriqui, and undetectable in Gulf of Panama (Glynn et al., 2001). Although these comparisons are not unequivocal due to differences in seasonal timing of anomalies, duration of exposures (Podesta and Glynn, 2001) or upwelling (Glynn et al., 2001), the lower bleaching and mortality that occurred in 1997–98 suggest that selection for resistant species or genotypes of corals and zooxanthellae may have occurred during prior ENSO-related temperature events (Podesta and Glynn, 2001). Jimenez et al. (2001) also report higher bleaching and mortality to corals on Costa Rican reefs in 1982–83 than in 1997–98, despite the temperature stress from the later event having been as strong or stronger than in 1982–83. Unfortunately, no information is provided concerning the prevailing light climates in this region for the two major El Nino events that would clarify whether differences in solar radiation might have influenced the generally lower bleaching that occurred in 1997–98. Coral bleaching was also minimal in the Society Islands during the 1998 event, but the cause there was attributed to reduced light during the event. Mumby et al. (2001) found no coral bleaching in the Society Islands in 1998 despite high temperature anomalies, but attributed lack of bleaching to high cloud cover and reduced light levels during the period of elevated temperatures. Statistical analyses of bleaching occurrence based on cumulative temperature elevations, wind speed, and cloud cover predicted the correct scenario for the 1998 event only when high cloud cover was included in the analysis, indicating that the interactive effect of cloud cover can reverse bleaching predictions based solely on temperature elevation. Other findings suggest that coral populations can adapt to localized temperature conditions. Cook et al. (1990) found that Bermuda corals at lagoon and inshore sites, where they were subject to higher and more variable temperatures, were more resistant to bleaching in 1987 than the same species at offshore sites. Similar patterns have been observed on the Great Barrier Reef (Marshall and Baird, 2000) and the East Pacific (Guzman and Cortes, 2001). Berkelmans (2002) proposed that thermal adaptation had taken place over both local (10s of km) and regional (100s to 1000s of km) scales in the Great Barrier Reef, although Berkelmans and Oliver (1999) concluded that inshore reefs were more prone to bleaching than offshore reefs because of higher inshore temperatures and probably reduced circulation. An indication of localized thermal adaptation was found in the Colombian Pacific (Vargas-Angel et al., 2001), where coral responses to the 1997–98 elevated temperatures showed less bleaching and lower mortality in an area where long-term temperatures were consistently CORAL BLEACHING 201 higher by 0.5–1.0C. Although Bruno et al. (2001) did not find significant differences in bleaching between sites at 3–5 m compared with 10–12 m depths from the severe 1997–98 ENSO event in Palau, Coles (unpublished report) found high coral survival in nearshore compared with offshore areas in August 1999, one year after the event. Corals at various nearshore sites around the island of Babeldoab in 1999 were abundant, well pigmented, and in apparently healthy condition at temperatures up to 31.7C, equivalent to the temperatures that occurred during the bleaching event (Bruno et al., 2001). Coral coverage and species composition in these nearshore areas was indistinguishable from observations made on surveys in 1997. By con- trast, virtually all Acropora and many other species on offshore reefs were dead in 1999. These examples indicate a capacity for selective adaptation by various coral species to elevated temperatures. However, nothing is known about the conditions or time frame under which this capacity was acquired. The critical question pertaining to large-scale survival of corals and continued viability of coral reefs over the next century is whether the temperature tolerances of corals and their symbionts can adjust rapidly enough to a changing ocean temperature environment, and whether the maximum temperatures that ultimately occur will exceed adaptation capacity. Attempts to predictively model reef conditions that may result from rising sea temperatures have usually used fixed coral thermal tolerances (Hoegh- Guldberg, 1999) predicting coral declines and phase shifts to algal- dominated reefs over the next century. However, models comparing projected global seawater change with various estimates of acclimation (i.e., adaptation) times (Ware et al., 1996) suggest that, although probable bleaching events are likely to increase over the next century, development of higher temperature thresholds in 25–50 years may dramatically reduce bleaching probabilities and frequencies. This suggests that models projecting future conditions for reef corals and coral reefs could utilize specific information relative to thermal acclimatization and adaptation of corals and their symbionts. Especially needed are data on the timeframe required for selective adaptation to both gradually increasing temperature and to infrequent temperature increases in order to project the eventual impacts of both global warming and El Nino events.

6. CORAL BLEACHING RECOVERY

In contrast to the limited experimental evidence for corals adapting to higher temperatures, there are numerous instances of repeated coral recovery from bleaching events, and recolonization and substantial 202 S. L. COLES AND BARBARA E. BROWN regrowth of corals in areas denuded by coral bleaching can occur as rapidly as within two years (Plate 2). The most documented area is the central Great Barrier Reef, where bleaching occurred in 1979–80, 1981–82, 1986–87, 1992–93, 1993–94 (Harriott, 1985; Oliver, 1985; Jones et al., 1997), and 1998 (Berkelmans and Oliver, 1999; Marshall and Baird, 2000; Berkelmans, 2002). The bleaching episode that occurred in January–March 1982 resulted in an estimated 50% mortality (Oliver, 1985) or more (Harriott, 1985) by November 1983. Despite this and lesser impacts that occurred with bleaching episodes every 2–5 years, recolonization and recovery was sufficient to reestablish a coral community by 1998 prior to the most extensive bleaching that has occurred there to date. Berkelmans and Oliver (1999) reported 65% of inshore reefs and 7% of offshore reefs in the central GBR to have had bleaching levels of 30% or more, with subsequent mortality of up to 60–80% on the reef flats at Orpheus Island. Marshall and Baird (2000) reported 53% of all coral colonies on Magnetic and Orpheus Island to have been affected by the 1998 bleaching event, with a preliminary report of mortality up to 16% on replicate transects and substantial differences among species and spatial variation in bleaching resistance. On a larger scale, Berkelmans (2001) reported good recovery only 6–8 months following the severe 1998 bleaching event on most GBR inshore reefs, where bleaching had been heaviest. Mortality was greatest in the Palm Islands region with up to 73% on the reef flat at Rattlesnake Island, but coral cover in the Magnetic Island and Whitsunday Islands region was generally unchanged with no significant decreases on these reefs over time. Mortality was highly variable and generally less extensive on most offshore reefs, where maximum mortality was 50–55% at Otter and Little Kelso Reefs. On the Heron Island reef flat, where 80% of corals showed bleaching discoloration during the 1998 event (Jones et al., 2000), observations in July 2001 (SLC, pers. obs.) indicated a flourishing coral community with >50% total coverage. Similar recurrent bleaching and recovery occurred in Moorea, French Polynesia in 1984, 1987, 1991, and 1994 (Salvat, 1992; Fagerstrom and Rougerie, 1994; Hoegh-Guldberg and Salvat, 1995) with sharp reductions observed following the 1991 but not the 1994 event. Minimal bleaching occurred again in 1998 (Wilkinson, 2000). In the Andaman Sea off the coast of Thailand, bleaching occurred in 1988, 1991, and 1995 (Phongsuwan, 1995; Brown et al., 1996; Brown, 1997b). However, little mortality occurred that could be attributed directly to these bleaching events, although coral coverage decreased substantially on the outer reef flat due to high sediment loading from a deep water port development (Brown, 1997b; Brown et al., 2002b). Little bleaching or mortality occurred from similar to higher temperatures in this area in 1997–98 (Dunne and Brown, 2001; Brown et al., 2002b). Guzman and Cortes (2001) describe low-level coral recovery on CORAL BLEACHING 203

Plate 2 (a) Bleached corals near the entrance to Suva Harbor in March 2000. Extensive mortality and wave breakage of branching and arborescent colonies followed the bleaching event. (b) Coral recolonization near this reef in March 2002, showing competition between colonies for available habitat space was already underway. Settlement of new colonies was observed as early as three months following the end of the bleaching event in 2000. (Pictures and information provided by Ed Lovell.) 204 S. L. COLES AND BARBARA E. BROWN

Pacific reefs of Costa Rica following the 1982–83 ENSO. They attribute this recovery to corals more tolerant of thermal stress, and note that mortality of such corals was very limited during the 1997–98 ENSO warming. Kayanne et al. (2002) noted that recovery of Montipora to prebleaching conditions two years after the 1998 bleaching event had resulted in high mortality in the southern Ryukus, although Montipora patches with coverage of less than 10% did not recover in that time period. Mortality and recovery varied among the other genera surveyed, with low mortality and little overall change shown for Heliopora and massive Porites, high mortality and moderate recovery for branching Porites and Acropora, and high mortality with no recovery shown for Pavona.

7. BLEACHING AND CORAL DISEASE, REPRODUCTION, AND RECRUITMENT

A major consideration in recovery and maintenance of coral assemblages and coral reef integrity following bleaching events is the impact of thermal stress on coral resistance to disease, reproduction, and recruitment. Observations and experiments have suggested infectious disease to be both a cause and an effect of coral bleaching. A series of studies (Kushmaro et al., 1996, 1998, 2001; Toren et al., 1998; Banin et al., 2000; 2001; Israely et al., 2001; Fine et al., 2002a,b) in the Mediterranean have linked bleaching of an introduced coral, Oculina patagonica, at elevated temperature with the growth of the bacterium Vibrio shiloi. Recent experiments have indicated a similar relationship between Pocillipora damicornis and the bacterium Vibrio coralyticus in Zanzibar (Ben-Haim and Rosenberg, 2002). These pathogens can be isolated in culture, and are experimentally transferable between coral colonies. They cause lysis of coral host tissues, especially when temperatures are elevated above normal ambient maxima (Ben-Haim and Rosenberg, 2002). Vibrio coralyticus has been isolated from diseased P. damicornis in the Red Sea, and bacterial strains from bivalve larvae in the North and South Atlantic were found to be pathogenic to this coral species. These findings offer a new perspective that requires consideration for its implications regarding widespread coral bleaching events. However, it is unlikely that such bacterial processes are the primary cause for most of the coral bleaching events that have been reported worldwide, which have been found to be reversible if temperature–light stresses are not too extreme or long lasting. As indicated by results and figures in Ben-Haim and Rosenberg (2002), these bacterial infections lead to partial tissue lysis scattered throughout the coral colony within seven days after infection, followed by 100% lysis and mortality within three weeks. CORAL BLEACHING 205

In contrast, temperature–light induced bleaching produces aggregation of zooxanthellae to polyp mouths and zooxanthellae expulsion, which may be followed by recovery if the stress subsides. Even so, these findings on temperature-correlated bacterial infections suggest an additional factor to be considered within the general context of coral bleaching and its ramifications. Lower energy reserves caused by prolonged and repeated coral bleaching are probably related to the extensive outbreaks of coral diseases that have occurred in Florida and Caribbean waters in the last decade (Cervino et al., 1998; Richardson et al., 1998). Chronic decreases in energy reserves of bleached corals have also been indicated to reduce the long-term reproductive capability of corals on reefs. Experiments on hard corals in Florida (Szmant and Gassman, 1990) and Jamaica (Mendes and Woodley, 2002) and a soft coral on the Great Barrier Reef (Michalek-Wagner and Willis, 2001a,b) have shown reduced fecundity of bleached corals that resulted in reproductive failure or delay in spawning of one year and reduced ability to complete gametogenesis, long after the symptoms of bleaching had ended in the adults. Reduced fecundity appears to result from lower energy resources available to a coral that has survived and recovered from a bleaching episode (Szmant and Gassman, 1990). This potential impact of bleaching on coral vitality, reproduction, and planula release has serious long-term implications, especially if bleaching events increase in frequency. The limited information available also indicates that temperature increases are as stressful to coral planulae as to adult stages. Experimental exposure of P. asteroides planulae in Florida to 33C for 24 h (Edmunds et al., 2001) significantly increased mortality and shortened metamorphosis time compared with exposures at ambient temperatures (28C). Also P : R ratios decreased with short-term exposures to elevated temperatures in these experiments, similar to that found for adult Hawaiian corals (Coles, 1973; Coles and Jokiel, 1977). This suggests that overall coral recruitment may be reduced through lower energy availability, reduced lower planula survival, and restricted planktonic dispersal following premature metamorphosis. For postlarval stages, contrasting results have been reported for the impact of thermal stress on settlement and recruitment of coral larvae that have been exposed to temperatures that can induce coral bleaching. Experiments in Hawaii found coral settlement to be highly sensitive to long term temperature increases (Jokiel and Guinther, 1978), with 10-fold reductions at an increase of 1C above the annual temperature maximum. However, Edmondson (1946) and later Coles (1985) demonstrated that brief exposures to elevated temperatures significantly increased settlement and survival of coral recruits, with a temperature optimum for settlement of 206 S. L. COLES AND BARBARA E. BROWN

P. damicornis at approximately 34C for 10-min exposures, or about 7C above annual maximum ambient (Coles, 1985). Coral recruitment near a thermal outfall in Hawaii, where long-term mean temperature elevation was 0.63C above ambient, was 10 times greater than rates elsewhere in Hawaii (Coles, 1984), and coral abundance adjacent to the outfall remains the highest in the area (Coles, pers. obs.). Mumby (1999) determined that the 1998 3.5-month long bleaching event which caused 70–90% bleaching of adult corals in Belize produced only 25% bleaching of recruits 2–20 mm in diameter, with ‘‘no measurable effect on recruit density or community structure,’’ comparing conditions before and after the event. Similar reduced susceptibility to bleaching in juvenile corals was also noted by Loya et al. (2001) during extensive bleaching in Japan in 1997–98. Observations by Edwards et al. (2001) showed high recovery by recently settled juveniles compared with adults following the 1998 bleaching in the Maldives. They noted recruitment of 202 branching acroporid and pocilloporid corals within 10 months after bleaching had eliminated 98% of nearly 1500 corals counted on artificial structures in 1994. These findings suggest that, although elevated temperature may reduce planula survival and restrict planktonic dispersal, exposure to thermal stress may also increase coral settlement rates and perhaps select for more resistant surviving planulae. The apparently lower susceptibility of juvenile corals to bleaching at elevated sea temperatures compared with adults is interesting in terms of their molecular defence mechanisms. Preliminary experiments comparing the temperature tolerances of adult and juvenile corals reveal that juvenile corals show a greater defensive response (in terms of concentrations of Hsps and oxidative enzymes) than adults (Brown, unpublished). These differences may be the result of age-related energetic costs (i.e., reproduction and/or lesion healing processes) that reduce the defensive ability of adults and/or their capacity to maintain homeostasis in the face of stress as the organism ages (Beckman and Ames, 1988; Halliwell and Gutteridge, 1999). Whatever the mechanisms involved, these findings have important bearing on the recovery potential of some reef sites following bleaching events and the ultimate community structure that might result. The weight of the available evidence therefore suggests that the reproductive problems posed by coral bleaching are of greater concern than impacts on adult corals, but that survival of recruits is less affected. Given the importance of reproduction and recruitment to long-term reef viability and the contrasting results that have been obtained for some of the studies, it is clear that substantial further research is needed in this area. CORAL BLEACHING 207

8. LONG-TERM ECOLOGICAL IMPLICATIONS OF CORAL BLEACHING

A recent review (Fitt et al., 2001) has emphasized that reliable conclusions about coral bleaching and mortality should be based on measurements of a variety of environmental factors, such as duration of thermal stress, light intensity, and quality (Warner et al., 2002). It was considered that substantial reductions in algal symbiont concentrations, i.e., subliminal bleaching, can be normal annual events. Fitt et al. (2001) also question whether bleaching is a meaningful indicator for coral mortality, given the lack of information linking zooxanthellae loss to coral death. Going further, the available information is, in our view, insufficient to provide definitive conclusions about the long-term fate of corals and reefs impacted by coral bleaching. Uncertainties remain concerning the tropical seawater tempera- ture environment and frequency of thermal events in the next century. We are only beginning to acquire basic information on bleaching thresholds, and the capacity of corals and their symbionts to acclimatize or adapt to increasing temperatures or thermal events. Limited information is available concerning linkages between bleaching and mortality, reproduction, recruitment, and the capability of coral assemblages to recover and reestablish after a bleaching event. Even less information is available as to whether coral acclimatization and adaptation can occur sufficiently fast to adjust to temperature anomalies that may occur. Uncertainties also remain concerning the interaction of the stresses which induce coral bleaching with other sources of coral stress and reef alteration (Buddemeir and Smith, 1999), such as nitrification and eutrophication, increased macroalgal growth that may result from overfishing of herbivores and reduced coral growth rates that result from ocean pH changes related to increased atmospheric CO2 (Pittock, 1999). The combined effects of these and other important factors with temperature and light effects on coral survival and propagation may be additive, synergistic, or neutral, but not necessarily negative in all cases. Turbid environments, generally considered to inhibit coral growth and survival, may shield corals from high light intensities and act as refugia for corals during times of thermal stress, and contribute to acclimatization and adaptation (Meesters et al., 2002). This attests to the potential importance of nonreef communities containing resistant corals, both locally and globally, in providing recruits during periods of large-scale disturbance (Buddemeir and Smith, 1999). Various scenarios resulting from mass coral bleaching have been presented by Done (1999), which include coral tolerance and adaptation, shifting of coral populations to smaller size classes, changing of species, compositions toward more tolerant coral species with decreasing diversity, 208 S. L. COLES AND BARBARA E. BROWN

Figure 4 Model summarizing range of responses by reef corals to environmental stresses inducing bleaching and long-term changes in composition of the reef community. (Adapted from Done, 1999.) and phase shifts to reefs dominated by fleshy macroalgae instead of corals and coralline algae (Figure 4). Where coral bleaching has been severe, fast growing branching acroporids and pocilloporid species have often died. In contrast, slow growing massive poritid and favid species have usually recovered their zooxanthellae and survived (Brown and Suharsono, 1990; Gleason, 1993; McClanahan, 2000; Edwards et al., 2001; Baird and Marshall, 2002; Kayanne et al., 2002; Riegl, 2002). However, Mumby et al. (2000) reported high Porites mortality following extensive bleaching at Rangiroa Atoll in 1998. Species composition has generally been reduced in the short term after bleaching, but recruitment of Acropora and Pocillopora has often occurred within two years (Edwards et al., 2001; Guzman and Cortes, 2001), unless macroalgae came to dominate the benthic habitat space (McClanahan et al., 2001; Diaz-Pulido and McCook, 2002). These examples indicate that the character of dominant reef assemblages in years following extensive bleaching vary from location to location, both locally and globally. Even assuming a worst-case scenario of annual coral bleaching and widespread reductions in diversity and abundance of reef corals occurring worldwide in 30 years, it is unclear how such alteration of coral assemblages might impact other major components of the coral reef system. Fishes and macroinvertebrates that are symbionts or direct CORAL BLEACHING 209 consumers of living corals would be as diminished as their coral hosts, but for the majority of reef organisms not directly linked to corals, the total result of pervasive coral bleaching is difficult to foretell. Although all of these alternatives are likely to result in less aesthetically attractive reefs, we do not know that the reefs would be functionally diminished as biotic systems, at least in the short term. Species diversity and abundance of small invertebrates would be likely to increase initially as new habitat spaces opened up in recently dead corals (Coles, 1980), and benthic macroalgae would become more abundant. In the year after the 1998 bleaching event in the Indian Ocean, McClanahan et al. (2001) found a 75–85% decrease in hard and soft corals on Kenyan reefs and 88–220% increases in turf and fleshy algae. Diaz-Pulido and McCook (2002) found a similar shift in dominance to macroalgae on Porites that had undergone severe bleaching and mortality on the Great Barrier Reef. Regarding fish assemblages, studies of postcoral bleaching event conditions have sometimes found shifts in dominant feeding groups but no overall population decreases. Wellington and Victor (1985) found no significant change in a damselfish population in the Gulf of Panama after coral mortality from the 1982–83 El Nino caused massive increases in available algal food and nesting sites. Lindahl et al. (2001) found that fish community diversity was unchanged after the 1998 bleaching event that killed 88% of corals on Tanzanian reef plots, but fish abundance rose 39%, mostly due to increase in herbivores apparently responding to a greater availability of macroalgae. Halford (1997) reported herbivorous scarids to become the dominant fish taxa within a northwestern Australian bay where the dominant benthos had shifted from corals to macroalgae following large-scale coral and fish mortality due to hypoxia. Three years after a bleaching event on the southern Great Barrier Reef, which had reduced coral cover > 75%, Doherty et al. (1997) found fish recruitment to be indistinguishable in both numbers and diversity from when coral cover was high. Victor et al. (2001) found that fish populations on East Pacific reefs were not reduced by the 1997–97 ENSO event.

9. CONCLUSIONS

For the last 20 years corals and coral reefs have globally undergone repeated stress from periodic elevation of seawater temperatures that is unprece- dented in approximately one hundred years during which scientists have been studying corals and their environmental responses. If these stresses continue and seawater baseline temperature increases in the next century, the tolerances of corals and their symbiotic zooxanthellae will be severely 210 S. L. COLES AND BARBARA E. BROWN tested in many parts of the world where corals and coral reefs are the dominant biotope. There is ample evidence that global temperature, including SST, has risen substantially and that the rise is continuing (Wigley et al., 1997; National Research Council, 2002; Hansen, 2003). Responses to this warming have been shown by both terrestrial and aquatic ecosystems (Parmesan and Yohe, 2003). However, the rise has been most pronounced in the Atlantic and at higher latitudes in the northern hemisphere (Hansen, 2003), and changes have been less obvious in some tropical seas. Recent analyses of satellite SST and in situ seawater temperatures (Liu et al., 2002; Strong et al., pers. comm.) suggest that, with ENSO events excluded, the overall trend in SSTs in certain tropical waters, notably the western tropical Pacific, has been stable for the last two decades and in some regions temperature has fallen. There has also been some controversy about tropical temperatures during past ‘‘greenhouse’’ periods in the Eocene and Cretaceous (Zachos et al., 2002). Thus, projections of a steadily increasing baseline of SSTs underlying periodic ENSO events (Hoegh-Guldberg, 1999) may not apply to all tropical regions. Even if SST warming occurs generally in the tropics and temperature anomalies associated with ENSO periods continue, there is evidence that a degree of adaptability, not yet rigorously defined, exists for corals and their zooxanthellae, suggesting that these organisms could continue to dominate coral reefs. We base this conclusion on demonstrated differences in coral thermal thresholds linked to ambient temperatures, both locally and regionally, on experimentally demonstrated protective mecha- nisms such as HSPs, coral fluorescent pigments, and zooxanthellae adaptability, on limited experimental evidence for acclimatization and/or adaptation, and on the rapid recovery of corals and reefs that has been observed following bleaching events. Repeated bleaching events followed by various levels of coral mortality during the last two decades has led to the perception among many reef scientists and the general public that coral bleaching is likely to result in degradation and demise of coral reefs as a major tropical biotope within the next 50 years. Although most of the available information and projections are not encouraging in terms of the environmental stresses that are likely to occur, there are also indications that reef corals have ‘‘potential for greater physiological tolerance than might have been previously expected’’ (Done, 1999), and ‘‘possess effective mechanisms of adaptation and acclimation that have ensured their survival and recurrence over geologic time’’ (Buddemeir and Smith, 1999). Additional research is needed to clarify the potential for corals and zooxanthellae to adapt to increasing temperatures occurring in both brief events and over the long term. Since recruitment plays a major function in reef recovery after bleaching events, it will be critically important CORAL BLEACHING 211 to clarify the tolerance of coral larvae and newly settled juvenile corals versus adult stages, and determine the importance of habitat diversity in providing refuges for juveniles, both during and after bleaching events. Carefully managed, long-term monitoring programs with high statistical power need to be established or continued on reefs worldwide to clarify initial and long-term impacts of coral bleaching events, and to test whether certain environmental factors may provide resistance and resilience to coral bleaching (Done, 1999; West, 2001; West and Salm, in press). This information could then be used to establish criteria for protected areas to provide refugia as sources of recruitment for coral reef recovery after bleaching events (Salm and Coles, 2001; Salm et al., 2001). Only after considerably more basic research has been completed will we be able to make meaningful projections of the long-term impacts of coral bleaching. The biologist’s scope for understanding the complex interactions of environmental stresses on coral bleaching and the equally complex responses of the coral/algal symbiosis to these stresses may be significantly expanded in the future by the application of environmental genomics. Recent developments in DNA and protein-based technologies offer an enormous increase in the efficiency of gene discovery and characterization, placing focus specifically upon those genes that are upregulated as a result of stress. Attempts to understand just how well corals may adjust to rising seawater temperatures will need to focus increasingly on genetic variation, both in terms of selection and phenotypic plasticity for ecophysiological traits. Regarding phenotypic plasticity, Pigliucci (1996) comments ‘‘the old metaphor of genes as blueprints for the organism has to be abandoned in favor of a more complex view that sees organismal properties emerging from local and limited genetic effects.’’ Work on noncoral organisms has shown that there is considerable genetic variation for phenotypic plasticity in natural populations and that this variation is both character and environment specific (Via et al., 1993; Ackerly et al., 2000). Targeting those ecophysiological processes that appear to confer thermal tolerance in corals (e.g., xanthophyll cycling capability, HSPs, and oxidative enzyme production to name but a few) and identifying the genes responsible for plasticity in these traits in coral/algal symbioses from different environments would be major advances in our understanding of the scope of corals to survive an era of global warming.

ACKNOWLEDGEMENTS

These concepts expressed in this review have been influenced through many years of observations and discussions with researchers in the field of coral 212 S. L. COLES AND BARBARA E. BROWN biology, including those who may not totally agree with all of the conclusions. These include stimulating conversations on coral bleaching that occurred among participants in the workshop on Coral Bleaching and Marine Protected Areas. Mitigating Coral Bleaching Impact Through MPA Design, held at Bishop Museum in May 2001, namely R. Salm, B. Causey, T. Done, P. Glenn, W. Heyman, P. Jokiel, G. Llewellyn, D. Obura, J. Oliver, and J. West. Important input has also come from A. Salih, T. Nahaky, T. McCleod, and E. Lovell, who kindly provided the photos for Plate 2. Two anonymous reviewers and A.J. Southward provided very helpful comments and suggested changes that resulted in major improvements to the article. Figure 1 is reprinted by permission of University of Hawaii Press, and Figures 2 and 3 by permission of Inter-Research. Thanks to The Natural Environment Research Council, The Royal Society, and The Leverhulme Trust in the United Kingdom for supporting research conducted by BEB in Thailand over the last 23 years that has provided insight to some of the issues raised by this review. Contribution No. 2003- 001 to the Pacific Biological Survey.

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Johanne Dalsgaard,1 Michael St. John,2 Gerhard Kattner,3 Do¨ rthe Mu¨ ller-Navarra2 and Wilhelm Hagen4

1University of Copenhagen c/o Danish Institute for Fisheries Research, Charlottenlund Castle, DK-2920 Charlottenlund, Denmark 2University of Hamburg, Center for Marine and Climate Research, Institute for Hydrobiology and Fisheries Research, Olbersweg 24, D-22767 Hamburg, Germany 3Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, D-27570 Bremerhaven, Germany 4Marine Zoology, University of Bremen, P.O. Box 330440, D-28334 Bremen, Germany

1. Introduction ...... 227 1.1. Purpose and structure of the review ...... 227 1.2. The trophic marker concept ...... 230 1.3. Applications of fatty acid trophic markers in marine research ...... 231 1.4. Lipids and fatty acids in higher organisms ...... 235 1.5. Fatty acid biochemistry ...... 236 2. Fatty Acid Dynamics in Marine Primary Producers ...... 238 2.1. General aspects ...... 238 2.2. Biosynthesis of fatty acids ...... 239 2.3. Impact of growth, environmental and hydrodynamic factors ...... 240 2.4. Specific fatty acid markers of primary producers ...... 241 2.5. Specific fatty acid markers of heterotrophic bacteria and terrestrial matter ...... 251 3. Fatty Acid Dynamics in Crustaceous Zooplankton ...... 255 3.1. General aspects ...... 255

ADVANCES IN MARINE BIOLOGY VOL 46 Copyright ß 2003 Academic Press 0-12-026146-4 All rights of reproduction in any form reserved 226 JOHANNE DALSGAARD ET AL.

3.2. Uptake of dietary fatty acids and de novo biosynthesis of specific fatty acid markers ...... 256 3.3. Mobilization of fatty acids during starvation and reproduction ...... 264 3.4. Validation of the fatty acid trophic marker approach in crustaceous zooplankton ...... 266 4. Fatty Acid Dynamics in Fish ...... 269 4.1. General aspects ...... 269 4.2. Incorporation of dietary fatty acids ...... 269 4.3. Modifications and de novo biosynthesis of fatty acids ...... 271 4.4. Mobilization of fatty acids during starvation and reproduction ...... 273 4.5. Validation of the fatty acid trophic marker approach in fish ...... 275 5. Applications of Fatty Acid Trophic Markers in Major Food Webs ...... 278 5.1. General aspects ...... 278 5.2. The Arctic ...... 281 5.3. The Antarctic ...... 291 5.4. Northwest Atlantic ...... 298 5.5. The North Sea ...... 305 5.6. Gulf of Alaska ...... 308 5.7. Mediterranean Sea ...... 310 5.8. Upwelling and sub-tropical/tropical systems ...... 312 6. Summary and Conclusions ...... 313 6.1. State-of-the-art ...... 313 6.2. Future applications ...... 316 Acknowledgments ...... 318 References ...... 318

Fatty acids have been used as qualitative markers to trace or confirm predator–prey relationships in the marine environment for more than thirty years. More recently, they have also been used to identify key processes impacting the dynamics of some of the world’s major ecosystems. The fatty acid trophic marker (FATM) concept is based on the observation that marine primary producers lay down certain fatty acid patterns that may be transferred conservatively to, and hence can be recognized in, primary consumers. To identify these fatty acid patterns the literature was surveyed and a partial least squares (PLS) regression analysis of the data was performed, validating the specificity of particular microalgal FATM. Microalgal group specific FATM have been traced in various primary consumers, particularly in herbivorous calanoid copepods, which accumulate large lipid reserves, and which dominate the zooplankton biomass in high latitude ecosystems. At higher trophic levels these markers of herbivory are obscured as the degree of carnivory increases, and as the fatty acids originate from a variety of dietary sources. Such differences are highlighted in a PLS regression analysis of fatty acid and fatty alcohol compositional data (the components of wax esters accumulated by many marine organisms) of key Arctic and Antarctic herbivorous, omnivorous and carnivorous copepod species. The analysis emphasizes how calanoid copepods separate from other copepods not only by their content of microalgal group specific FATM, but FATTY ACID TROPHIC MARKERS 227 also by their large content of long-chain monounsaturated fatty acids and alcohols. These monounsaturates have been used to trace and resolve food web relationships in, for example, hyperiid amphipods, euphausiids and fish, which may consume large numbers of calanoid copepods. Results like these are extremely valuable for enabling the discrimination of specific prey species utilized by higher trophic level omnivores and carnivores without the employment of invasive techniques, and thereby for identifying the sources of energetic reserves. A conceptual model of the spatial and temporal dominance of group-specific primary producers, and hence the basic fatty acid patterns available to higher trophic levels is presented. The model is based on stratification, which acts on phytoplankton group dominance through the availability of light and nutrients. It predicts the seasonal and ecosystem specific contribution of diatom and flagellate/microbial loop FATM to food webs as a function of water column stability. Future prospects for the application of FATM in resolving dynamic ecosystem processes are assessed.

1. INTRODUCTION

1.1. Purpose and structure of the review

At present, one of the key issues for both marine and terrestrial ecologists as well as resource managers is to resolve and predict the impacts of global change on ecosystem dynamics. The objective of these activities is the development of ecosystem-based management strategies with the ultimate goal of preserving the structure and functioning of ecosystems and contributing to the sustainable management of natural resources. Contingent upon developing such strategies is a clear understanding of the bottleneck processes (both biotic and abiotic) that impact the population dynamics of key trophic level species, and which are influenced by global change. Resolution of these bottleneck processes has to date been determined primarily via an approach whereby the growth and overall condition as well as trophic links of individuals are related to in situ conditions and assumptions about the survival potential of the individual are scaled up to the population level. Such classical approaches to the resolution of key processes are limited, as they are reliant upon temporal snapshots of complex and highly variable (both spatially and temporally) interactions obtained from individuals that might never survive to become part of the reproductive population. In order to expand the temporal window of resolution of key processes, an approach termed ‘‘characteristics 228 JOHANNE DALSGAARD ET AL. of survivors’’ (Fritz et al., 1990; Taggart and Frank, 1990; St. John et al., 2000), has recently been used to identify processes leading to enhanced survival success. This approach is based upon the examination of phenotypic and genotypic characteristics of individuals before and after experiencing an event. If, after exposure to a specific process, a random subset of survivors exists from the initial population, no phenotypic or genotypic selective advantage exists with respect to that process. However, if a particular characteristic results in an increased survival success, this characteristic can be described as increasing the individual’s fitness. To date, the characteristics of survivors approach has primarily been used to identify survivors in terms of growth rates (Miller et al., 1988; Meekan and Fortier, 1996), food webs (St. John and Lund, 1996; Storr-Paulsen et al., 2003) and transport processes (St. John et al., 2000). All of these studies employ a biomarker approach to identify in situ processes contributing to enhanced growth, condition and survival success. Specific biomarkers included in these studies comprise otolith microstructure (e.g., Meekan and Fortier, 1996) and fatty acid trophic markers (e.g., St. John and Lund, 1996). The application of otolith microstructure for the study of larval, juvenile and adult fish is now a common tool in fish ecology (e.g., Campana, 1996), however, the application of fatty acid trophic markers (FATM) to address issues in marine science is so far relatively limited. Hence, the major objective of this review is to summarize applications of fatty acids (FA) as trophic markers in marine ecosystems and furthermore, to assess the future prospects for their application in resolving ecosystem dynamic processes. For three decades, detailed information on the FA composition of marine organisms has been generated under the assumption that, among other things, such data may provide valuable insight into predator–prey relationships. Studies employing FATM have taken place in both marine and freshwater pelagic systems as well as in demersal and deep-sea applications. In order to constrain this review and avoid duplication we will focus on applications in the pelagic marine system, and will not consider other lipid biomarkers such as sterols and hydrocarbons (but see, e.g., Sargent and Whittle, 1981; Volkman et al., 1998). Furthermore, for an introduction to FATM in freshwater ecosystems we refer readers to Desvilettes et al. (1997) and Napolitano (1999). In the first part of this review, we introduce the FATM concept and give a chronological synopsis of the development and application of FATM in marine food web research. General FA biochemistry is briefly presented and the distribution of lipids and FA in marine organisms is discussed. Subsequently, the dynamics of lipids and FA at the various trophic levels (i.e., primary producers, zooplankton and fish) are described in more detail. At the first trophic level microalgae are given most emphasis, as they are the principal primary producers in the marine environment, supporting both FATTY ACID TROPHIC MARKERS 229 pelagic and offshore benthic food webs (Parsons, 1963; Kayama et al., 1989). In order to summarize and compare the information on algal groups, the characteristic FA patterns of various marine microalgal classes are visualized through a partial least squares (PLS) regression analysis based on published laboratory culture studies, and the conclusions compared to natural plankton communities. We comment briefly on macroalgae, which are largely confined to shallow coastal regions. Here they may support local benthic food webs (Ackman et al., 1968 and references therein), while they generally have little importance in the pelagic marine environment. Finally, FATM of heterotrophic bacteria and terrestrially derived organic matter are summarized. In general, bacteria make important contributions in the marine environment, particularly in microbial loop food webs, which develop primarily in stratified and nutrient depleted areas (e.g., Cushing, 1989 and references therein). Terrestrial matter can be important in coastal and estuarine ecosystems, and differences in the FA pattern between the terrestrial and marine environment have been used to detect the entrainment of terrestrial organic matter into coastal food webs. We look therefore briefly at characteristic terrigenous FATM. At the next trophic level, zooplankton form an essential link between primary producers and higher order consumers (Sargent, 1976; Sargent and Henderson, 1986). We focus on herbivorous calanoid copepods, as they are the best studied group of zooplankton with respect to FATM. We describe the uptake, incorporation and modification of dietary FA during different life history stages, and give examples of studies that have verified the conservative incorporation of specific phytoplankton-derived FATM by copepods. Moreover, apart from incorporating and transferring dietary FA from primary producers to higher trophic levels, calanoid copepods are themselves important producers of specific FA and fatty alcohols (the moieties of wax esters). Hence, we discuss their capacity to biosynthesize such compounds de novo, focusing on those FA and fatty alcohols that can be used to elucidate predator–prey relationships at higher trophic levels. The FA characteristics of omnivorous and carnivorous copepods are subse- quently discussed, and FA that have been used as markers of carnivory are summarized. Lastly, the information on herbivorous, omnivorous and carnivorous copepods is summarized and compared in a PLS regression analysis based on FA and fatty alcohol compositional data of key Arctic and Antarctic copepod species. Next, we review the dynamics of FA in fish, primarily teleosts, which principally catabolize and transform dietary FA (Sargent and Henderson, 1986). We describe the processes of uptake, incorporation and modification of dietary FA, de novo biosynthesis, mobilization of FA during starvation and reproduction, and summarize studies that have validated the FATM approach in this group of organisms. 230 JOHANNE DALSGAARD ET AL.

Finally, in the last section of the paper, we review major marine food webs in which FA have been used to trace or confirm predator–prey relationships and key processes impacting on ecosystem dynamics, i.e. the Arctic, the Antarctic, northwest Atlantic Ocean, the North Sea, the Gulf of Alaska, Mediterranean Sea, upwelling and subtropical–tropical systems. The section is introduced by a comparison between these different systems based on the influence of stratification processes on phytoplankton group dominance.

1.2. The trophic marker concept

The perfect trophic marker is a compound whose origin can be uniquely and easily identified, that is inert and nonharmful to the organisms, that is not selectively processed during food uptake and incorporation, and that is metabolically stable and hence transferred from one trophic level to the next in both a qualitative and quantitative manner. Such a marker would provide essential insight into the dynamics of ecosystems by presenting unique information on pathways of energy flows, i.e., crucial information on which all ecosystem models are eventually built. However, such markers are unfortunately rare if nonexistent and instead we have to be content with less ideal components, a category to which FA belong. In the case of FATM, these lipid components are in many circumstances incorporated into consumers in a conservative manner, thereby providing information on predator–prey relations. Moreover, contrary to the more traditional gut content analyses, which provide information only on recent feeding, FA provide information on the dietary intake and the food constituents leading to the sequestering of lipid reserves over a longer period of time (e.g., Ha˚ kanson, 1984; St. John and Lund, 1996; Kirsch et al., 1998; Auel et al., 2002). This integrating effect helps to resolve the importance of specific prey items and can validate prey utilization strategies based on traditional stomach content analyses (Graeve et al., 1994b). Furthermore, traditional stomach analyses suffer from the fact that food items in the gut are frequently difficult to identify and are quantitatively biased due to differential digestion rates of soft and hard parts. For example, exoskeletons and otoliths may be retained in the stomachs whereas softer tissue parts are rapidly digested, and hence, seldom observed (e.g., Iverson et al., 1997a and references therein; Budge et al., 2002). These problems are partly circumvented by FA but unfortunately replaced by other constraints. For example, no single FA can be assigned uniquely to any one species and depending on the condition and metabolic strategy of the consumer, FA are not necessarily metabolically stable (e.g., Section 3.2 and 4.3). In addition, the temporal dynamics, i.e., turnover rate of individual FA, can be species- specific and are often linked to the metabolic condition or reproductive FATTY ACID TROPHIC MARKERS 231 status of the organism (Section 3.3 and 4.4), and have seldom been quantified (St. John and Lund, 1996; Kirsch et al., 1998). Consequently, FA have so far only been used as qualitative and ‘‘semi-quantitative’’ food web markers, the latter in concert with other tracers such as stable isotopes (Kiyashko et al., 1998; Kharlamenko et al., 2001). It still remains to be established whether they can be used for more than that. This is a serious challenge given the fact that whereas the FA composition may be used to elucidate the dietary source of lipid reserves, it is not possible to discern whether an individual is incorporating or depleting reserves in its current situation, using a marker which gives no indication of the temporal dynamics of growth or conditional status.

1.3. Applications of fatty acid trophic markers in marine research

The concept of FA being transferred conservatively through aquatic food webs was first suggested in 1935 by Lovern. This seminal work found that Calanus finmarchicus could be distinguished from three freshwater copepod species based on lower proportions of C16 and C18 unsaturated FA and higher concentrations of C20 and especially C22 unsaturated FA. Similar relationships had previously been observed in fish from the two habitats, and the author speculated that the ‘‘whole character of fish fats’’ was derived from the crustacean diet, suggesting further that these differences propagate all the way down to the algae. Almost 30 years later, Kayama et al. (1963) performed one of the first experiments demonstrating the transfer of FA through a linear, experimental food web consisting of Chaetoceros simplex (diatom) – Artemia salina (branchiopod) – Lebistes reticulatus (freshwater guppy). The FA profile of the branchiopods and guppies clearly showed the transfer as well as endogenous modifications of dietary FA. In particular, the branchiopods were able to elongate and 1 further desaturate C18 polyunsaturated fatty acids into 20:5 . In addition, the guppies contained both 22:5 and 22:6, with more of the latter when the water temperature had been lowered from 24Cto17C. These results were supported by Jezyk and Penicnak (1966), who examined a discontinuous,

1The IUPAC-IUB Commission on Biochemical Nomenclature (1967, 1977) short- hand notation of fatty acids z:y(n-x) is employed throughout the paper. Here, z ¼ number of carbon atoms in the acyl chain; y ¼ number of double bonds; n ¼ chain length; x ¼ number of carbon atoms from the last double bond to the terminal methyl group, i.e., (n-x) defines the position of the first double bond counting from the terminal methyl group of the acyl chain. In some, particularly older studies, FA isomers were not determined and are cited accordingly. 232 JOHANNE DALSGAARD ET AL. linear, experimental food web consisting of (i) algae - brine shrimp, and (ii) brine shrimp nauplii - Hydra at 10C and 20C. These authors found that the FA composition of the neutral lipid (NL) fraction resembled the diet more closely than did that of the polar lipid fraction, and that polyunsaturated fatty acids (PUFA) were predominantly concentrated within the polar lipid fraction. In a study involving the culturing of twelve species of unicellular marine algae from the phytoplankton classes Chrysophyceae, Cryptophyceae, Bacillariophyceae (diatoms), Dinophyceae (dinoflagellates), Chlorophyceae (green algae), Prasinophyceae, Rhodophyceae (red algae) and Xanthophyceae, Ackman et al. (1968) discovered that despite large variations of individual FA within the different taxonomic classes, common features could still be recognized. Subsequently, consistent with these findings, Jeffries (1970) performed the seminal work on the changes in the FA composition accompanying a succession of species within a natural plankton community (Figure 1). In this study, a succession from diatoms to flagellates in Narragansett Bay, Rhode Island, was found to be associated with a decrease in the 16:1/16:0 ratio from >2 to <0.3. This trend was, furthermore, partly mirrored in locally abundant Acartia sp., assumed to feed on the algae. Complementary but much less pronounced trends were also evident in the 18:4/18:1 ratio, highest when the 16:1/16:0 ratio was lowest and commensurate with the peak in flagellate dominance. Shortly thereafter, Lee et al. (1971b) demonstrated that dietary FA were incorporated conservatively into the wax ester (WE) fraction of marine copepods. In this study, Calanus helgolandicus fed on either diatoms (Lauderia borealis, Chaetoceros curvisetus, Skeletonema costatum)or dinoflagellates (Gymnodinium splendens) showed a WE fatty acid composi- tion very similar to its prey. Such similarities were, however, not observed between the diet and the WE fatty alcohol composition, which consisted primarily of saturated and monounsaturated fatty alcohols, purported to be biosynthesized de novo by the copepods. Subsequently, Sargent (1976) concluded that calanoid copepods differ from phytoplankton in containing high proportions of C20 and C22 monounsaturated FA and fatty alcohols biosynthesized de novo, and that these moieties can be recognized (as FA) in copepod predators. To verify the potential of phytoplankton FA as trophic markers, Graeve et al. (1994a) performed a feeding experiment with herbivorous, Arctic calanoid copepods (see also Section 3.4). After 42 days on a diet of Thalassiosira antarctica (diatom), the level of 16:1(n-7) in Calanus finmarchicus had strongly increased, whereas 18:4(n-3) was almost depleted. Opposite trends were observed in C. hyperboreus fed on Amphidinium FATTY ACID TROPHIC MARKERS 233

Figure 1 Seasonal distributions of particular FA within the phytoplankton and zooplankton community of Narragansett Bay, Rhode Island. Redrawn with permission after Jeffries (1970). carterae (dinoflagellate), i.e., the level of 16:1(n-7) had decreased, while the level of 18:4(n-3) had increased. Kharlamenko et al. (1995) demonstrated how a combination of FA identified in samples of pelagic diatoms, seston, microbial mats, sediments and macroalgae collected in an isolated shallow-water hydrothermal ecosystem in Kurile Islands, east Pacific, could be used to identify potential, major food sources of locally abundant macrozoobenthic species. Their ratio of 16:1(n-7)/16:0 and the 20:5(n-3) content indicated that diatoms were a major food source of all species. Furthermore, some of the deposit and 234 JOHANNE DALSGAARD ET AL. suspension feeders studied contained higher than average levels of bacterial markers (branched and odd-chain FA; see also Section 2.5) indicating a significant dietary input, whereas the gastropod Littorina kurila had a FA profile comparable to that of the locally abundant brown macroalgae Fucus evanescens. Despite several applications, the 16:1(n-7)/16:0 ratio was first validated as a specific food web tracer in 1996 by St. John and Lund, who performed a controlled laboratory experiment with first-feeding North Sea cod larvae (Gadus morhua; see also Section 4.5). The larvae were maintained on either a Heterocapsa triquetra (dinoflagellate) or Skeletonema costatum (diatom) based food web or a mixture of the two. Using Acartia tonsa nauplii as an intermediary, the larvae mirrored the tracer index of their respective diets within thirteen days of feeding (Figure 2). Recently, FA combined with stable isotope analyses have proven to be particularly helpful for identifying major sources of organic matter con- tributing to the diet of marine benthic invertebrates (Kiyashko et al., 1998;

Figure 2 Validation of the 16:1(n À 7)/16:0 specific food web tracer in larval North Sea cod (Gadus morhua) raised on food webs based on either Skeletonema costatum, Heterocapsa triquetra, a 50% mix of the two or starved. The algae were fed to Acartia tonsa and the resultant nauplii fed to the cod larvae. Each point represents an average of five cod larvae. Redrawn with permission after St. John and Lund (1996). FATTY ACID TROPHIC MARKERS 235

Kharlamenko et al., 2001), warranting a possible future direction of this approach.

1.4. Lipids and fatty acids in higher organisms

Fatty acids are ubiquitous components of all living organisms where they form an essential and integral part of neutral and polar lipids and constitute important precursors of ‘‘local’’ hormones (eicosanoids). One major role of polar lipids is to provide the basic matrix of the cellular membranes into which cholesterol, proteins and other membrane constituents are embedded (Spector and Yorek, 1985; Stubbs and Smith, 1990; Cook, 1996; Vance, 1996). The dual structural and functional role of polar lipids limits the type of FA that are incorporated, consisting principally of PUFA of the (n-3) and (n-6) series (reviewed by Vaskovsky, 1989). These particular FA provide special conformational properties to the biomembranes, and assist tissue specific cells in reacting to external stimuli such as, e.g., changing environmental temperatures and light regimes (Sargent et al., 1993; Cook, 1996). The principal role of neutral lipids (NL), which in marine systems consist predominantly of triacylglycerols (TAG) and WE, is as an energetic reserve of FA that are destined either for oxidation to provide energy (ATP) or for incorporation into phospholipids (PL) (Sargent and Whittle, 1981; Hølmer, 1989; Lee and Patton, 1989). The NL content and the constituent FA is linked to the physiological status of the organism, and is determined by the rate of turnover of the lipid depots, i.e., the coupled processes of anabolism and catabolism. An organism experiencing a dietary surplus of energy may accumulate lipids either directly, in which case the FA composition is similar to the diet (Ackman and McLachlan, 1977; Sargent and Whittle, 1981), or after modifying the FA to suit particular physiological needs, e.g., for the formation of reproductive tissue. The former situation underpins the belief that FA in many cases can be used to explore predator–prey relationships, i.e., that they can be used as trophic markers. In reviewing the literature and the use of FATM, it has become apparent that different approaches have been used for detecting dietary relationships. Either the total lipid (TL) composition has been analysed, or individual lipid classes have been evaluated separately. Neutral lipids are preferred for resolving dietary contributions in ‘‘end’’ predators, since the FA composi- tion of this lipid class usually reflects trophic influences much better than PL (e.g., Bell and Dick, 1990; Stubbs and Smith, 1990; Parrish et al., 1995). However, if the objective of the study is to characterize the FA signature of potential prey organisms, and as most predators consume their prey whole, 236 JOHANNE DALSGAARD ET AL. analyses of TL of these potential prey species are preferable (e.g., Iverson et al., 1997b; Kirsch et al., 1998).

1.5. Fatty acid biochemistry

The biochemistry of lipid classes and their FA components as well as WE fatty alcohols has been thoroughly described in the literature. For a general introduction to the biochemistry of lipids in living organisms consult for instance Gurr and Harwood (1991) and Vance and Vance (1996). In particular, Christie (1982, 1989, 1992, 1993, 1996, 1997) and Hamilton and Hamilton (1992) are excellent sources of information on methodologies applied in lipid research, while Ackman (1989a, b) contains an impressive compilation and summary of work relating to (i) marine lipid classes, (ii) the distribution of marine lipids in plants, invertebrates, fish, mammals and seabirds, and (iii) utilization of marine oils and lipids. Lastly, a comprehensive summary of the recent status of knowledge on the roles of lipids in aquatic ecosystems, with emphasis on freshwater systems, can be found in Arts and Wainman (1999). In the following, we limit the discussion to the basic processes of FA biosynthesis in primary producers and marine animals with emphasis on their potential as trophic markers. The de novo biosynthesis of FA generally follows the common lipid pathway, i.e., the Type I fatty acid synthetase. The major end product of this pathway is 16:0 but FA with 14, 18 and 20 carbon atoms may also be released or produced by further chain elongation (the latter referring to acyl chains with 18 and 20 carbon atoms). The most universal pathway for the formation of monounsaturated fatty acids (MUFA), which most organisms are capable of, is aerobic desaturation catalysed by the enzyme Á9 desaturase (the delta nomenclature, where carbon atoms are numbered from the carboxylic acid end of the acyl chain, is used for describing biochemical reactions; Figure 3). This leads to the introduction of a double bond between carbon atom 9 and 10 to form 16:1(n-7), 18:1(n-9) and 20:1(n-11) (Sargent and Henderson, 1986; Gurr and Harwood, 1991; Cook, 1996). In animals, these MUFA may also be biosynthesized from 14:0 and 16:0 precursors obtained from the diet and undergoing further chain elongation and desaturation, rather than by de novo biosynthesis. These basic patterns of FA biosynthesis and modification leave enough flexibility for different species to select specific pathways best suited for their metabolic requirements (Kattner and Hagen, 1995). The processes of chain elongation and desaturation lead to major FA end products, which have been widely used to infer trophic relationships. For example, the de novo biosynthesis of the long-chain MUFA, i.e., 20:1 and 22:1, is par- ticularly pronounced in herbivorous calanoid copepod species (Figure 4B) FATTY ACID TROPHIC MARKERS 237

Figure 3 Positions of fatty acyl desaturation by enzymes of certain insects, animals in general, plants in general and lower plants (most marine algal species). The delta-designation (numbering the carbon atoms from the carboxylic acid end of the acyl chain) replaces the n-designation when describing biochemical reactions. Reproduced with permission after Cook (1996).

Figure 4 Major pathways of FA biosynthesis in (A) marine algae, modified after Gurr and Harwood (1991) and Cook (1996), and (B) herbivorous calanoid copepods, modified after Sargent and Henderson (1986) and Kattner and Hagen (1995). 238 JOHANNE DALSGAARD ET AL. which, in the process of forming WE, reduce a considerable amount of the MUFA into the corresponding long-chain monounsaturated alcohols. Generally, only plants are capable of biosynthesizing (n-3) and (n-6) PUFA de novo (although a few invertebrates and protozoa may also be able to do so; Gurr and Harwood, 1991; Cook, 1996; Pond et al., 1997a, b, 2002). Oleic acid (18:1(n-9)) is the precursor of all (n-3) and (n-6) PUFA (Figure 4A), which are essential to heterotrophic organisms. Unlike animals, primary producers possess the enzymes Á12 and Á15 desaturase, which enables them to insert double bonds between the existing double bond in the Á9 position and the terminal methyl group (Figure 3). Thus, the next double bonds are introduced to form 18:2(n-6) and then 18:3(n-3). Through the combined actions of i6 and i5 desaturase and 2-carbon unit chain elongations, 18:2(n-6) may be converted further to 20:4(n-6) (AA) and 18:3(n-3) to 20:5(n-3) (EPA) and 22:6(n-3) (DHA). The final steps to produce DHA via C24 PUFA intermediates rather than direct chain elongation of EPA was discovered by Sprecher (1992). Typical FA of this biosynthetic scheme are found in dinoflagellates, in which 18:4(n-3) and DHA are often dominant. An alternative to this pathway is the desaturation of 16:0 to 16:1(n-7) and further desaturation to C16 PUFA with 16:4(n-1) constituting the final desaturation product. This biosynthetic pathway is very characteristic of diatoms, in which not only 16:1(n-7), but also C16 PUFA, in addition to EPA, are major FA, and often used as markers of this group (Section 2.4). More details concerning FA in marine primary producers and animals are presented in the following sections.

2. FATTY ACID DYNAMICS IN MARINE PRIMARY PRODUCERS

2.1. General aspects

The basic FA pattern in marine food webs is laid down by primary producers (Kelly et al., 1963; Jeffries, 1970) consisting of phytoplankton and macroalgae, with phytoplankton comprising both microalgae and photo- autotrophic bacteria (Raven et al., 1992). However, for the purposes of this review we assume that photoautotrophic bacteria have a minor impact on the dynamics of marine ecosystems (but see Paerl and Zehr, 2000), and hence, they will receive little attention. Phytoplankton communities in the pelagic, marine environment are represented predominantly by Bacillariophyceae (diatoms), Dinophyceae (dinoflagellates) and Prymnesiophyceae (e.g., Parsons, 1963; Le Fe` vre, 1986; Mann, 1993 and references therein; Thomsen et al., 1994), while other taxonomic classes FATTY ACID TROPHIC MARKERS 239

(see below) are much less abundant except in bloom conditions (e.g., Parrish et al., 1995; Cripps et al., 1999). Phytoplankton are the major providers of metabolic energy in pelagic food webs (Parsons, 1963), which is transferred to higher trophic levels via grazing by herbivorous and omnivorous planktivorous species including the larvae of fish and larger invertebrates. Similarly, phytoplankton support benthic food webs that, in addition, may receive considerable inputs from macroalgae (seaweeds), belonging to one of the three classes: Chlorophyceae (green algae), Rhodophyceae (red algae) or Phaeophyceae (brown algae) (Raven et al., 1992). While a few species of the brown algae Sargassum are free-floating (Raven et al., 1992), the dominant life phase in most macro- algal species is benthic, and because of limited light availability they are restricted to the shallower coastal areas (Levring, 1979; Kristiansen et al., 1981). Here, they constitute an important refuge for fish and invertebrates and are either grazed directly or, as is mostly the case, enter the detrital food webs via microheterotrophs (Dunstan et al., 1988; Sherr and Sheer, 2000 and references therein; Graeve et al., 2002 and references therein).

2.2. Biosynthesis of fatty acids

Autotrophic organisms biosynthesize all of their cellular constituents de novo including a great diversity of FA (Sargent and Henderson, 1995; Cook, 1996). A description of the structure of these lipids and FA in algae can be found in Pohl and Zurheide (1979) and in Wood (1988), while algal metabolism is discussed by Harwood and Jones (1989). In summary, FA are biosynthesized in the chloroplasts comprising the thylakoid membranes (Harwood and Russell, 1984; Raven et al., 1992). They consist predomi- nantly of even-numbered, straight-chain, saturated or cis-unsaturated compounds with 12 to 24 carbon atoms (Pohl and Zurheide, 1979, 1982; Wood, 1988; Cobelas and Lechado, 1989; Harwood and Jones, 1989; Kayama et al., 1989). Low amounts of more unusual FA with more than 24 carbon atoms, as well as some trans-unsaturated (particularly trans- 16:1(n-13)) and odd-chain FA of varying chain length, are also biosynthe- sized by some species (Pohl and Zurheide, 1979; Volkman et al., 1980a; Harwood and Jones, 1989; Mansour et al., 1999a). The FA are esterified chiefly to glycolipids (particularly rich in (n-3) PUFA and the major constituents of the thylakoid membranes), whereas PL and NL are comparatively minor lipid constituents of algae (Pohl and Zurheide, 1979; Sargent et al., 1987, 1989; Wood, 1988; Harwood and Jones, 1989). As mentioned in Section 1.5, plants are usually the only organism within the system that can biosynthesize 18:2(n-6) and 18:3(n-3) de novo. These particular PUFA and their derivatives (i.e., AA, EPA and DHA) are 240 JOHANNE DALSGAARD ET AL. essential constituents of heterotrophic organisms, stressing the central position of algae within marine food webs (Pohl and Zurheide, 1979; Gurr and Harwood, 1991; Cook, 1996; Smith and Fitzpatrick, 1996). Consistent with this, recent experimental evidence (particularly in freshwater research) has shown that the level of (n-3) PUFA is an important food quality indicator (Jo´ nasdo´ ttir et al., 1995; Mu¨ ller-Navarra, 1995; Mu¨ ller-Navarra et al., 2000; Wacker and von Elert, 2001), which may affect trophodynamic relationships (Mu¨ ller-Navarra and Lampert, 1996; Sterner and Schulz, 1998; Mu¨ ller-Navarra et al., 2000). Consequently, (n-3) PUFA may determine the rates at which carbon (Brett and Mu¨ ller-Navarra, 1997), and hence other marker FA, are channeled through the food web.

2.3. Impact of growth, environmental and hydrodynamic factors

The FA signature of microalgae is an expression of both genotypic (Alonso et al., 1994) and phenotypic characteristics. Large qualitative and particularly quantitative fluctuations, both within and between species, are observed that can be related to the combined effects of environmental conditions and the physiological state of the algae (see below). Variations in the biomass, distribution and species composition of microalgae, and hence the basic FA pattern in the marine environment, are ultimately driven by hydrodynamic processes. The reason for this is that hydrodynamic processes affect both the availability of nutrients and light, and influence the distribution of microalgae through horizontal and vertical circulation patterns coupled with behavioral or buoyancy characteristics (e.g., Franks, 1992). Nutrient and light availability are tightly coupled to water column stability, which can be simplified into two extreme hydrodynamic regimes in the pelagic environment: stratified and mixed water columns. Stratified water columns arise in areas characterized by low turbulent energy, and primary production in these areas is typically nutrient limited. The primary producer community is generally composed of small, autotrophic flagellates and cyanobacteria (< 10 mm) that form the basis of low biomass, microbial loop food webs. In contrast, areas of high turbulence result in mixed or weakly stratified water columns with a consistent influx of nutrients. As a consequence, primary producers in these areas are light limited rather than nutrient limited due to the increased depth of mixing. Primary production is usually carried out by relatively large diatoms (> 10 mm) giving rise to ‘‘simple’’ food webs with an efficient transfer of energy to higher trophic levels. Algal growth within these regimes is largely controlled by the local environmental conditions, with FATTY ACID TROPHIC MARKERS 241 temperature, light and nutrient availability being the three key factors affecting the FA pattern of the local community. The impact of these environmental factors has been studied primarily in laboratory cultures, and has been reviewed, e.g., by Pohl and Zurheide (1979), Cobelas (1989), Kayama et al. (1989), and Roessler (1990). Typically, lower water temperatures result in an increase in the level of unsaturation (e.g., Ackman et al., 1968; Pohl and Zurheide, 1982), whereas the impact of light is ambiguous and more species-specific. In general, however, the level of glycolipids, and hence (n-3) PUFA, increases under nonlimiting light conditions, whereas photo-inhibition and reduced light intensities reportedly lead to the accumulation of TAG (the major lipid storage product in algae), which is richer in saturated fatty acids (SFA) and MUFA (Cohen et al., 1988; Harrison et al., 1990; Mayzaud et al., 1990 and references therein; Thompson et al., 1990; Sukenik and Wahnon, 1991; Smith et al., 1993; Parrish et al., 1994). Algal growth, as previously mentioned, is influenced by the availability of limiting nutrients (principally nitrogen, phosphorus or silicate), which influence the transition from the exponential phase (non- nutrient limited) to the stationary growth phase (nutrient limited), the latter being characterized by the accumulation of TAG (see above for consequences on FA patterns; Kattner et al., 1983; Morris et al., 1983; Ben-Amotz et al., 1985; Harrison et al., 1990; Kattner and Brockmann, 1990; Mayzaud et al., 1990; Fahl and Kattner, 1993; Reitan et al., 1994; Falk-Petersen et al., 1998; Henderson et al., 1998). During the exponential growth phase of phytoplankton blooms, carbon fixed through photosynthesis is allocated to growth and cell division rather than lipid storage (e.g., Morris, 1981; Kattner et al., 1983; Parrish and Wangersky, 1990). As a consequence, the relative proportion of glycolipids is particularly high in this phase (Sargent and Henderson, 1986; Roessler, 1990), and the concentration of (n-3) PUFA may approach 50% of the TL content (e.g., Napolitano et al., 1997; Claustre et al., 1989; Sargent et al., 1989; Falk-Petersen et al., 1998; Henderson et al., 1998). This exponential algal growth phase occurs during spring bloom conditions and the FA pattern of the exponentially growing algae is particularly evident in field examinations of phytoplankton lipid dynamics (e.g., Kattner et al., 1983; Hama, 1991).

2.4. Specific fatty acid markers of primary producers

2.4.1. Microalgae

It is well established that whereas FA cannot be used as taxonomic indicators at the species-specific level, the presence and combinations of certain FA can be characteristic of particular algal classes and thus have potential as markers 242 JOHANNE DALSGAARD ET AL.

(e.g., Ackman et al., 1968; Chuecas and Riley, 1969; Pohl and Zurheide, 1979; Kattner et al., 1983; Sargent et al., 1987; Cobelas and Lechado, 1989; Mayzaud et al., 1990; Mourente et al., 1990; Fahl and Kattner, 1993; Viso and Marty, 1993; Napolitano, 1999; Volkman et al., 1998). Since the early 1960s, a large number of laboratory studies have examined the FA composition of marine microalgae (reviewed by Ackman et al., 1968; Pohl and Zurheide, 1979; Cobelas and Lechado, 1989; Kayama et al., 1989). In these studies, the algae have been cultured under a wide range of treatment conditions, and have been analyzed using standard, organic- solvent extraction and methylation procedures combined with thin layer chromatography (TLC) and gas chromatography (GC) later combined with mass spectrometry (GC-MS) (Ackman, 2002; Traitler, 1987). Many of the earliest studies were characterized by incomplete compound separation and loss of PUFA due to improper sample handling and storage protocols. Hence, the results from these studies should be interpreted with caution (discussed by Ackman et al., 1968; Chuecas and Riley, 1969; Conte et al., 1994). Subsequently, techniques have improved (especially column technol- ogy), resulting in a higher degree of sensitivity. As a consequence, more precise estimates of total FA contents may be obtained, and in addition, more FA have been identified. For example, trace amounts of the very-long- chain, highly-unsaturated-fatty-acids (VLC-HUFA) 28:7(n-6) and 28:8(n-3) have been identified in several species of dinoflagellates (Mansour et al., 1999a, b). Intriguingly, octacosaheptaenoic acid (28:7(n-6)) and other VLC- HUFA had previously been detected in Baltic herring where they were suspected to originate from the diet (Linko and Karinkanta, 1970). However, except for a few examples like this, these more unusual FA usually occur only in trace amounts in phytoplankton (e.g., Nichols et al., 1986), and are even more difficult to recognize in the consumers due to the low levels of occurrence, limiting their potential as trophic markers (see also Section 5.7.2; Ackman and Mclachlan, 1977; Mayzaud et al., 1999). Aside from sample treatment and identification procedures, another obstacle associated with the application of FATM has been the interpreta- tion of the large data sets routinely produced in these types of analyses (typically arrays of more than 30 FA determined simultaneously from one or more samples). With the development of computer power, easily accessible, multivariate statistical methods have advanced to become particularly useful for interpreting such large data sets (e.g., Wold et al., 1988; Frank, 1989; Kaufmann, 1992; Smith et al., 1997, 1999; Legendre and Legendre, 1998). Here, we present the results of such an analysis, indicating the patterns of FA similarities within and among eight classes of microalgae (Bacillariophyeae, Chlorophyceae, Cryptophyceae, Dinophyceae, Eustigmatophyceae, Prymnesiophyceae, Prasinophyceae and Raphidophyceae). The outcome of the analysis is visualized in Figure 5, FATTY ACID TROPHIC MARKERS 243

Figure 5 PLS regression analysis of logarithmically transformed FA composi- tional data of the eight classes of marine microalgae summarized in Table 1. Plots show (A) the scores of the first two of six principal components, and (B) the corresponding loading weight plot. Ellipses in (A) are drawn only to indicate the major grouping of the different microalgal classes relative to each other. 244 JOHANNE DALSGAARD ET AL. which was constructed by applying a PLS regression analysis2 to logarithmically transformed FA compositional data compiled from laboratory culture studies reported in the literature3. The analysis was performed on nineteen FA variables summarized in Table 1. The variables comprise both individual FA as well as combinations (sums) of FA selected based on the presence in the compiled data set, i.e., only FA that were identified in all studies were included. In this analysis, the first six PLS components (linear combinations of the variables) explained 89% of the variance of the FA compositional data (predictor variables) and 61% of the variance contributed to microalgal ‘‘class-affiliation’’ (response variables). Despite considerable overlap, particularly between dinophytes and prymnesiophytes and between bacil- lariophytes and eustigmatophytes, the eight classes of microalgae can still be recognized in the score plot of the first two principal components (Figure 5A). The corresponding loading weight plot4 (Figure 5B) shows the importance of the different FA variables for the two first PLS components. Fatty acids roughly in the same direction from the center as the microalgal classes are positively linked to and particularly important predictors of those classes, whereas FA in the opposite direction are negatively linked with the algal classes. Figure 5B shows that bacillariophytes clearly separate from the other classes along the first PLS component linking positively with 16:1(n-7), C16 FA, C16 PUFA, C20 FA and EPA and negatively with C18 FA (see also Mayzaud et al., 1990). Although not included in the analysis, another important FA is 16:4(n-1), which has been suggested as a specific marker of this microalgal class (Viso and Marty, 1993). It has been detected in most of the species of Bacillariophyceae studied to date, whereas it is more or less absent in

2This particular analysis models simultaneously the FA composition and microalgal ‘‘class-affiliation’’, and can be perceived as a PC-hyperplane tilted slightly so as to make microalgal ‘‘class-affiliation’’ better explained by the latent variables of the FA matrix (Wold et al., 1988). Analyses were performed using The UnscramblerÕ v7.6 SR-1 CAMO ASA software. 3The model is only preliminary and not adopted for predictive purposes by applying it on an independent test set. 4 ‘‘Loading weights are specific to PLS ... and express how the information in each X-variable [predictor variables] relates to the variation in Y [response variables] summarized by the u-scores. They are called loading weights because they also express, in the PLS algorithm, how the t-scores are to be computed from the X-matrix to obtain an orthogonal decomposition. The loading weights are normalized, so that their lengths can be interpreted as well as their directions. Variables with large loading weight values are important for the prediction of Y.’’ Copyright ß 1996-2000 CAMO ASA. All rights reserved. AT CDTOHCMARKERS TROPHIC ACID FATTY

Table 1 Summary of the FA composition (as % total FA) of marine microalgal classes used in the PLS regression analyses.

Bacillario- Chloro- Crypto- Dino- Eustigmato- Prasino- Prymnesio- Raphido- phyceae phyceae phyceae phyceae phyceae phyceae phyceae phyceae (n ¼ 31) (n ¼ 14) (n ¼ 4) (n ¼ 11) (n ¼ 4) (n ¼ 4) (n ¼ 21) (n ¼ 4)

Fatty acids 14:0 14.1 ± 6.9 1.1 ± 1.0 6.8 ± 1.9 6.9 ± 3.4 5.9 ± 0.9 2.8 ± 2.5 25.3 ± 14.0 6.5 ± 1.0 16:0 15.9 ± 8.4 21.1 ± 5.2 21.2 ± 8.4 26.2 ± 15.5 26.8 ± 6.5 25.2 ± 10.2 19.0 ± 9.3 28.8 ± 11.0 16:1(n-7) 23.6 ± 6.5 1.6 ± 2.1 2.0 ± 1.8 3.7 ± 5.4 26.6 ± 2.3 4.0 ± 4.4 4.6 ± 4.0 10.5 ± 3.8 18:0 1.2 ± 1.3 0.9 ± 0.6 1.1 ± 0.4 3.4 ± 4.9 1.0 ± 0.7 1.8 ± 1.4 3.3 ± 3.7 0.5 ± 0.3 18:1(n-7) 1.9 ± 1.9 4.8 ± 14.1 3.9 ± 0.7 1.8 ± 1.9 0.4 ± 0.2 2.7 ± 0.6 2.0 ± 2.1 0.9 ± 0.1 18:1(n-9) 1.4 ± 1.4 5.3 ± 3.5 9.5 ± 8.0 4.3 ± 4.7 6.3 ± 4.7 7.3 ± 2.9 12.7 ± 8.1 1.3 ± 0.8 18:2(n-6) 1.2 ± 0.9 11.0 ± 6.4 14.2 ± 3.6 2.3 ± 2.6 1.2 ± 0.6 4.0 ± 2.4 4.6 ± 3.5 3.0 ± 1.2 18:3(n-3) 0.6 ± 0.6 22.1 ± 12.9 13.1 ± 1.6 1.1 ± 1.3 0.1 ± 0.1 13.5 ± 2.4 4.5 ± 4.1 3.7 ± 0.6 18:4(n-3) 1.8 ± 1.7 2.2 ± v2.5 17.7 ± 3.5 4.1 ± 4.2 0.1 ± 0.1 11.2 ± 7.0 7.5 ± 6.4 15.5 ± 5.8 20:5(n-3) 16.2 ± 10.5 1.8 ± 2.1 7.2 ± 5.1 6.9 ± 7.3 14.9 ± 3.0 5.0 ± 1.1 2.6 ± 4.6 12.6 ± 3.4 22:6(n-3) 2.4 ± 1.8 0.2 ± 0.2 3.6 ± 2.2 17.5 ± 8.4 0.1 ± 0.2 0.4 ± 0.6 5.5 ± 5.6 2.0 ± 1.0 Sums of Fatty acids C16FA 54.4 ± 8.3 44.6 ± 5.0 25.3 ± 10.9 33.2 ± 15.5 59.5 ± 3.2 40.5 ± 9.2 26.0 ± 10.0 44.9 ± 14.0 C16PUFA 13.6 ± 9.2 15.3 ± 6.3 0.0 ± 0.0 3.0 ± 3.7 0.6 ± 0.7 4.6 ± 4.4 1.0 ± 2.0 0.1 ± 0.2 C18FA 8.3 ± 4.3 46.9 ± 9.0 54.4 ± 1.2 31.9 ± 12.0 9.1 ± 4.6 39.9 ± 9.3 36.5 ± 12.6 28.1 ± 7.2 C18PUFA 4.1 ± 2.0 35.9 ± 13.3 39.8 ± 9.0 22.3 ± 16.0 1.4 ± 0.6 29.3 ± 10.6 18.4 ± 13.3 25.4 ± 7.4 C20FA 18.0 ± 10.8 3.0 ± 3.2 8.2 ± 6.1 8.0 ± 7.4 18.4 ± 4.0 7.2 ± 1.7 2.3 ± 4.3 14.1 ± 4.0 C22PUFA 2.5 ± 2.1 0.2 ± 0.2 3.7 ± 2.2 17.9 ± 8.6 0.5 ± 0.2 0.3 ± 0.4 5.9 ± 6.3 2.5 ± 1.4 (n-3)PUFA 21.1 ± 12.1 37.7 ± 18.1 35.6 ± 18.8 46.2 ± 20.8 15.9 ± 3.6 34.5 ± 7.7 21.3 ± 16.2 37.3 ± 10.1 (n-6)PUFA 3.6 ± 2.5 16.1 ± 7.9 16.0 ± 1.6 3.2 ± 2.8 4.7 ± 1.6 5.4 ± 3.7 5.2 ± 3.8 4.7 ± 2.5

Data from Dustan et al. (1994), Mansour et al. (1999b), Mourente et al. (1990), Napolitano et al. (1990), Nichols et al. (1987, 1991), Parrish et al.

(1990, 1994), Servel et al. (1994), Viso and Marty (1993), Volkman et al. (1981, 1989). Values are mean ± one standard deviation. 245 246 JOHANNE DALSGAARD ET AL. other algal classes (e.g., Volkman et al., 1989; Napolitano et al., 1990; Parrish et al., 1990; Thompson et al., 1990; Dunstan et al., 1994). Trace amounts have, however, been detected in a few species of chlorophytes (Chuecas and Riley, 1969), dinophytes (Mansour et al., 1999b), prasino- phytes (Chuecas and Riley, 1969), prymnesiophytes (Ackman et al., 1968; Chuecas and Riley, 1969) and rhodophytes (macroalgae; Graeve et al., 2002). Prymnesiophytes (except for two species of Hymenomonas) and dino- phytes separate from the other classes by positive anomalies of 18:0, 18:1 (n-9), 18:4(n-3), C22 PUFA and DHA. Another important FA of these two classes, though not included in the analysis, is 18:5(n-3). This FA was identified for the first time by Joseph (1975) in several species of Dinophyceae. Later, it has been identified in species of prymnesiophytes (Volkman et al., 1981, 1989; Sargent et al., 1985; Claustre et al., 1990; Napolitano et al., 1990; Viso and Marty, 1993), raphidophytes (Nichols et al., 1987; Viso and Marty, 1993), prasinophytes (Viso and Marty, 1993) and bacillariophytes (Reitan et al., 1994). Chlorophytes (except for one species of Nannochloris) are discriminated by 18:3(n-3), 18:2(n-6) and other (n-6) PUFA. The close association of the chlorophytes with prasinophytes in Figure 5A is consistent with both classes belonging to the same division of Chlorophyta (Viso and Marty, 1993). A characteristic FA of both these classes is 16:4(n-3) (not included in the analysis; Ackman et al., 1968; Viso and Marty, 1993), whereas the presence of >C20 FA in prasinophytes distinguishes them from the chlorophytes (Viso and Marty, 1993). Cryptophytes, raphidophytes and eustigmatophytes can be distinguished as more or less separate groups. However, together with prasinophytes their variations are poorly explained by the model, clustering around the center on the loading weight plot (Figure 5B). It should be emphasized that limited FA compositional data were available for these four classes of microalgae. Hence, they were only represented by four observations each, which are really too few for ensuring stability of the model (Albano et al., 1981; Wold et al., 1988). This was taken into account in a second analysis, considering only Bacillariophyceae, Dinophyceae, Prymnesiophyceae and Chlorophyceae, which all contributed sufficient sample sizes and, as mentioned in Section 2.1, dominate the phytoplankton biomass in most marine ecosystems (except for Chlorophyceae). In this analysis, the first four PLS components now explain 83% of the variance of the FA compositional data and 76% of the variance contributed by microalgal ‘‘class-affiliation’’. A plot of the second vs. third principal component (Figure 6A) reveals that apart from two species of Hymenomonas, two large clusters of prymnesiophytes can be recognized consisting predominantly of Isochrysis spp. (upper ellipse), and FATTY ACID TROPHIC MARKERS 247

Figure 6 PLS regression analysis of logarithmically transformed FA composi- tional data of Bacillariophyceae, Dinophyceae, Prymnesiophyceae and Chlorophyceae (summarized in Table 1). Plots show (A) the scores of the second and third of four principal components, and (B) the corresponding loading weight plot. Ellipses in (A) are drawn only to indicate the major grouping of the different microalgal classes relative to each other. 248 JOHANNE DALSGAARD ET AL.

Phaeocystis spp. and Chrysotila spp. (lower ellipse). Consistent with their position in Figure 6A close to the Dinophyceae, species of Isochrysis are characterized by a low 16:1/16:0 ratio, and high concentrations of C18 PUFA and EPA (Conte et al., 1994). The loading weight plot (Figure 6B) shows that the prymnesiophytes in general separate from the dinophytes by higher, positive anomalies of 14:0, 16:1(n-7), 18:1(n-9) and 18:4(n-3) while the dinophytes link positively with DHA, C22PUFA and (n-3) PUFA. These analyses re-emphasize that individual FA cannot be used as taxonomic indicators of particular algal species or classes, whereas combinations of FA reveal certain patterns when microalgae are compared class-wise. This conclusion confirms the statement of Viso and Marty (1993) who identified the need to combine several FA criteria to distinguish natural assemblages of microalgae belonging to different taxonomic classes. To date, most of the criteria (ratios of FA) that have been developed have focused on bacillariophytes and dinophytes, reflecting the relative impor- tance of these two classes in the marine environment. Here, in particular, high values of 16:1(n-7)/16:0 (typically >1) and ÆC16/ÆC18 have been associated with a dominance of bacillariophytes (e.g., Miyazaki, 1983; Claustre et al., 1988, 1989; Mayzaud et al., 1990; Viso and Marty, 1993; Budge and Parrish, 1998; Budge et al., 2001; Reuss and Poulsen, 2002), whereas high values of 18:5(n-3)/18:3(n-3) and Æ (C18PUFA, C22PUFA) have been associated with a dominance of dinophytes (Nichols et al., 1984; Viso and Marty, 1993). Combining these criteria, i.e., high values of ÆC16/ÆC18 together with low values of 18:5(n-3)/18:3(n-3), has been proposed as a means whereby bacillariophytes can be distinguished from dinophytes (Viso and Marty, 1993). This could be further strengthened by examining the ratio of 22:6(n-3)/20:5(n-3) as suggested by Budge and Parrish (1998). Here, a value 1 signals a dominance in the contribution of dinophytes while conversely, a value <1 is suggestive of a greater contribution of bacillariophytes. A quantitative summary of the PLS analyses and the criteria discussed above is presented in Table 2 with mean values for the eight classes of microalgae included in the analyses. It must be recognised that the figures in the table should be perceived only as a very rough guideline of potentially useful FATM.

2.4.2. Macroalgae

Compared to microalgae, most of the information on macroalgae (belonging to one of the three classes: Chlorophyceae, Rhodophyceae and Phaeophyceae) originates from field studies (reviewed by Kayama et al., 1989), rather than laboratory experiments (e.g., Ahern et al., 1983; AT CDTOHCMARKERS TROPHIC ACID FATTY

Table 2 Specific FATM (as % of total FA) of the marine microalgal classes used in the PLS regression analyses.

FATM Bacillario- Chloro- Crypto- Dino- Eustigmato- Prasino- Prymnesio- Raphido- phyceae phyceae phyceae phyceae phyceae phyceae phyceae phyceae (n ¼ 31) (n ¼ 14) (n ¼ 4) (n ¼ 11) (n ¼ 4) (n ¼ 4) (n ¼ 21) (n ¼ 4)

16:4(n-1) 3.2 Æ 4.1 0 0 0.2 Æ 0.6 0 0 0 0 18:5(n-3) 0 0 0 14.1 Æ 13.4 0 0.2 Æ 0.4 1.6 Æ 2.8 2.9 Æ 2.5 ÆBaca 42.9 Æ 7.9 2.5 Æ 2.7 9.2 Æ 3.3 10.7 Æ 10.2 41.4 Æ 2.3 9.0 Æ 5.4 6.7 Æ 7.6 23.1 Æ 2.4 ÆDinb 2.1 Æ 1.9 0.1 Æ 0.2 3.6 Æ 2.2 31.6 Æ 17.5 0.1 Æ 0.2 0.4 Æ 0.8 7.1 Æ 7.3 4.8 Æ 3.1 ÆPryc 3.1 Æ 2.0 7.5 Æ 5.6 27.2 Æ 4.7 8.4 Æ 5.0 6.4 Æ 4.7 18.5 Æ 7.0 20.2 Æ 8.8 16.7 Æ 5.8 ÆChld 1.9 Æ 1.8 38.7 Æ 17.3 20.7 Æ 4.0 5.3 Æ 4.3 1.3 Æ 0.6 21.6 Æ 5.4 8.6 Æ 7.0 6.8 Æ 1.4

C18 þ C22PUFA 6.6 Æ 3.3 36.0 Æ 13.3 43.5 Æ 11.2 40.2 Æ 19.5 1.8 Æ 0.7 29.6 Æ 10.4 24.3 Æ 17.5 27.9 Æ 8.6 EPA þ DHA 18.3 Æ 11.4 1.3 Æ 1.9 10.8 Æ 7.2 24.3 Æ 13.2 15.0 Æ 3.1 5.2 Æ 1.0 7.6 Æ 7.6 14.6 Æ 4.3 16:1(n-7)/16:0 2.0 Æ 1.3 0.1 Æ 0.1 0.1 Æ 0 0.2 Æ 0.3 1.1 Æ 0.3 0.2 Æ 0.2 0.3 Æ 0.2 0.4 Æ 0.1 18:5(n-3)/18:3(n À 3) -e 0-36.8 Æ 61.4 0 0 0.3 Æ 0.5 0.9 Æ 0.8

C16FA/C18FA 8.5 Æ 4.8 1.0 Æ 0.3 0.5 Æ 0.2 1.2 Æ 0.7 8.7 Æ 6.3 1.1 Æ 0.3 0.9 Æ 0.7 1.8 Æ 1.3

C16PUFA/C18PUFA 4.7 Æ 4.7 0.4 Æ 0.1 0 0.1 Æ 0.1 0.6 Æ 0.7 0.2 Æ 0.2 0 Æ 0.1 0 EPA/DHA 11.9 Æ 10.7 - 1.8 Æ 0.6 0.4 Æ 0.4 - - 1.2 Æ 3.3 7.4 Æ 3.1

Boxed entries indicate that the particular FA criteria may be a useful tracer of the algal class. Based on data compiled from the literature; see Table 1 for references. a16:1(n-7) þ 16:4(n-1) þ EPA; b18:5(n-3) þ DHA; c18:1(n-9) þ 18:4(n-3); d16:4(n-3) þ 18:2(n-6) þ 18:3(n-3); eCould not be determined (dividing by zero). 249 250 JOHANNE DALSGAARD ET AL.

Honya et al., 1994). Some general trends distinguishing the three classes have been recognized, which are largely independent of geographical locations and morphological differences (e.g. Ackman and McLachlan, 1977 (Nova Scotia); Chuecas and Riley, 1966 (Isle of Man); Dembritsky et al., 1993 (Caspian Sea); Fleurence et al., 1994 (French Brittany coast); Graeve et al., 2002 (Arctic and Antarctic); Khotimchenko et al., 2002 (Pacific coast); Li et al., 2002 (Bohai Sea); Pohl and Zurheide, 1982 (Baltic Sea)). C18 and C20 FA constitute the principal PUFA in macroalgae whereas C22 PUFA are more abundant in microalgae (Chuecas and Riley, 1966; Graeve et al., 2002). Moreover, the (n-6) family (particularly AA) is more prevalent in macroalgae than in microalgae. High concentrations of AA combined with insignificant amounts of C18 PUFA distinguish rhodophytes from phaeophytes in which both C18 PUFA (particularly 18:4(n-3)) and C20 PUFA (principally EPA and AA) are major FA constituents. The FA pattern of chlorophyte macroalgae is similar to that of chlorophyte microalgae. Overall, the chlorophytes differ from the rest of the eukaryotic algae by a FA composition more similar to that of higher plants (Pohl and Zurheide, 1979; Wood, 1988; Kayama et al., 1989). For example, few of the constituent FA have more than three double bonds and most species have only modest amounts of >C20 PUFA, while the proportion of C16 and C18 PUFA is generally high (see Table 1; Wood, 1988; Lechevalier and Lechevalier, 1988; Volkman et al., 1998; Graeve et al., 2002). A particular trait of chlorophytes is the high content of 18:3(n-3) regarded as a characteristic of the phylum Chlorophyta (Li et al., 2002). Furthermore, several species exhibit a 18:1(n-7)/18:1(n-9) ratio >1 (Khotimchenko et al., 2002; Li et al., 2002), which in combination with 18:2(n-3) and 18:3(n-3) may potentially serve as a biomarker of this algal class.

2.4.3. Comparisons with natural plankton communities

Natural plankton communities consist of a mixture of species and dead organic matter that are exposed to concurrent fluctuations of different environmental factors. This makes comparisons and extrapolations of results obtained in the laboratory to the field situation extremely difficult. The proportion of PUFA is, for example, usually lower in natural phytoplankton communities than in algal cultures (e.g., Kattner et al., 1983; Morris, 1984; Morris et al., 1985; Kattner and Brockmann, 1990; Fahl and Kattner, 1993), and the FA signature of lipid-deficient algae is often masked by the signature of more abundant and lipid rich species such as diatoms (e.g., Skerratt et al., 1995; Budge et al., 2001). Despite these uncertainties, studies of natural phytoplankton communities generally confirm the characteristic FA patterns summarized above. Hence, FATTY ACID TROPHIC MARKERS 251 elevated concentrations of 14:0, 16:1(n-7), C16 PUFA (particularly 16:4(n-1)), and EPA are characteristically measured in diatom-dominated enclosure studies (Morris et al., 1985; Kattner and Brockmann, 1990; Mayzaud et al., 1990; Pond et al., 1998), during temporal spring blooms (Kattner et al., 1983; Claustre et al., 1988, 1989; Napolitano et al., 1997; Budge and Parrish, 1998; Budge et al., 2001), in open waters of polar and boreal systems (Lewis, 1969; Sargent et al., 1985; Kattner and Brockmann, 1990; Pond et al., 1993; Skerratt et al., 1995; Cripps et al., 1999; Cripps and Atkinson, 2000; Reuss and Poulsen, 2002) and in Arctic and Antarctic attached sea-ice algae (Fahl and Kattner, 1993; Nichols et al., 1993; Falk-Petersen et al., 1998; Henderson et al., 1998). Similarly, typical dinoflagellate markers, i.e., particularly high levels of 18:4(n-3), 18:5(n-3) and DHA are consistent within dinoflagellate dominated communities both at temperate (e.g., Kattner et al., 1983; Mayzaud et al., 1990; Napolitano et al., 1997; Budge and Parrish, 1998; Budge et al., 2001) and at high latitudes (Falk-Petersen et al., 1998). Consistent with the PLS analysis in Section 2.4.1, the FA composition of blooms dominated by prymnesiophytes is more variable. In several regions, such blooms have been associated with elevated levels of 14:0, 16:0, 18:0 and 18:1(n-9) and low levels of (n-3) PUFA (Al-Hasan et al., 1990 (Kuwait Bay); Claustre et al., 1990 (the Irish Sea); Skerratt et al., 1995 (Antarctic); Cotonnec et al., 2001 (the English Channel); Reuss and Poulsen, 2002 (west Greenland)). The low concentration of (n-3) PUFA makes Phaeocystis in these regions of low nutritional value for grazers. For example, Claustre et al. (1990) estimated that Phaeocystis constituted only a minor dietary component of Temora longicornis in the Irish Sea. Cotonnec et al. (2001), however, found that T. longicornis, Acartia clausi and Pseudocalanus elongatus, sampled in the English Channel during a Phaeocystis-dominated spring bloom, had all consumed large quantities. They argued that this was a result of low rejection of the algae due to its very high concentration in the field. In contrast, Phaeocystis blooms have in other regions been associated with high concentrations of 18:4(n-3), 18:5(n-3), EPA and DHA (Sargent et al., 1985; Hamm et al., 2001 (Balsfjord)), and are here heavily grazed (e.g., Sargent et al., 1987; Tande and Ba˚ mstedt, 1987; Sargent and Falk- Petersen, 1988).

2.5. Specific fatty acid markers of heterotrophic bacteria and terrestrial matter

2.5.1. Bacteria

Marine heterotrophic bacteria are particularly abundant in sediments (Sargent et al., 1987) and as colonizers of settling particulate matter 252 JOHANNE DALSGAARD ET AL. following major plankton blooms (e.g., Morris, 1984; Mayzaud et al., 1989; Skerratt et al., 1995; Najdek et al., 2002). As a consequence, the FA composition of marine bacteria has been studied predominantly by geochemists seeking to resolve the source and diagenetic state of POM and of sediments (e.g., Brooks et al., 1976; Haddad et al., 1992; Harvey, 1994; Colombo et al., 1997; Harvey and Macko, 1997; Volkman et al., 1980a; Wakeham and Beier, 1991). However, as mentioned in Section 1.1, heterotrophic bacteria are also very important in areas dominated by the microbial loop, where they occupy a critical position, recycling DOM and POM to higher trophic levels (Sherr and Sheer, 2000 and references therein). Unfortunately, very few studies have examined the FA dynamics of these systems (e.g., Claustre et al., 1988; Ederington et al., 1995). Bacteria do not store TAG but incorporate FA chiefly into PL (Fulco, 1983; DeLong and Yayanos, 1986; Parkes, 1987). Fatty acids commonly biosynthesized by bacteria are within the range C10–C20 and are dominated by SFA and MUFA, whereas PUFA, with a few exceptions including deep- sea bacteria and some bacterial strains isolated from fish intestines, are rarely detected (e.g., Johns and Perry, 1977; DeLong and Yayanos, 1986; Yazawa et al., 1988; Pond et al., 1997a, 2002; Nichols and McMeekin, 2002). Bacteria, moreover, differ from eukaryotes in biosynthesizing large amounts of odd-numbered, branched trans-unsaturated and cyclopropyl FA such as 15:0, 17:0, 15:1, 17:1, iso and anteiso-branched SFA and MUFA, 10-methylpalmitic acid, trans-16:1(n-7), cy17:0 and cy19:0 (Perry et al., 1979; Volkman et al., 1980a; Gillan et al., 1981; Parkes, 1987; Vestal and White, 1989 and references therein; Rajendran et al., 1994). In the same way as for microalgae, several combinations of FA have been used to detect the presence of bacteria (summarized in Table 3). Bacteria also biosynthesize large amounts of more common FA including 16:1(n-7) and 18:1(n-7) (e.g., Volkman and Johns, 1977 and references therein; Perry et al., 1979; Gillan et al., 1981; Parkes, 1987 and references therein; Vestal and White, 1989; Volkman et al., 1998). These particular FA are, however, also biosynthesized by and used as markers of eukaryotic organisms, principally diatoms and their entrainment into food webs. Therefore, unless elevated levels of some of the more specific bacterial marker FA summarized above are detected, and if PUFA are present in large amounts, it is in most cases presumably safe to assume that 16:1(n-7) and 18:1(n-7) derive from eukaryotic rather than bacterial production. The only controlled laboratory experiment so far to demonstrate the transfer of bacteria (and diatom) FA markers to higher trophic levels was carried out by Ederington et al. (1995). In this experiment, cultures of either bacterivorous ciliates or diatoms were fed to Acartia tonsa for 96 hours and FATTY ACID TROPHIC MARKERS 253

Table 3 Summary of particular bacterial and terrestrial FATM.

FATM Reference

Bacterial markers Æ Odd carbon numbered Budge and Parrish þ branched chain FA (1998), Budge et al. (2001) Æ Iso- and anteiso-C15 Viso and Marty (1993) and C17 18:1(n-7)/18:1(n-9) Volkman et al. (1980b) Iso þ anteiso 15:0/16:0 Mancuso et al. (1990) Iso þ anteiso 15:0/15:0 White et al. (1980) Æ 15:0, iso- and anteiso-C15 Najdek et al. (2002) and C17, 18:1(n-7) a brC15/15:0 Najdek et al. (2002)

Terrestrial markers 18:2(n-6) Napolitano et al. (1997) 18:2(n-6) þ 18:3(n-3) > 2.5 Budge and Parrish (1998), Budge et al. (2001) 22:0 þ 24:0 Budge et al. (2001) Æ C24:0–C32:0 Meziane et al. (1997)

aUsed as a measure of bacterial growth in mucilaginous aggregates, as bacteria experiencing favorable growth conditions yield higher

proportions of branched-chain C15 over straight-chain C15:0 (Najdek et al., 2002).

their FA composition subsequently examined. The bacterivorous ciliates were characterized by high concentrations of typical bacterial FA, accounting for 14.6% of total FA, suggesting the direct incorporation of these FA from the ingested bacteria. Elevated levels of bacterially derived FA, particularly 17:0, were likewise measured in the Acartia feeding on the bacterivorous ciliates when compared to starving and diatom-fed copepods (7.1%, 4.4% and 2.4%, respectively, of total FA). On the other hand, Acartia feeding on diatoms contained higher concentrations of characteristic diatom FATM, i.e., 16:1(n-7) and EPA. Moreover, the different dietary FA patterns were partly recognizable in the copepod eggs. These observations strongly support the hypothesis that bacterial and diatom FATM can be transferred to copepods and their eggs via protozoa, or in case of diatoms, directly from grazing on the microalgae. It is also notable that in this experiment, although not commented upon by the authors, the level of 18:1(n-7) was very high both in the bacterivorous ciliates, ciliate-fed copepods and their eggs (34.6%, 22.5% and 11.5%, 254 JOHANNE DALSGAARD ET AL. respectively, of total FA), whereas it was comparatively low in the diatoms, diatom-fed copepods and their eggs (1.4%, 2.2%, 2.9%, respectively, of total FA). This substantiates the hypothesis that 18:1(n-7) can be used as a bacterial indicator when present in combination with other, more typical bacterial FATM, although is should be emphasized that the level of 18:1(n-7) was also fairly high in starving copepods (11.2% of total FA).

2.5.2. Terrestrial markers

Differences in FA patterns between terrestrial and aquatic environments suggest that FA can be used as markers of terrestrial contributions to aquatic ecosystems. It is outside the scope of this review to provide a thorough overview of terrigenous biomarkers in aquatic ecosystems, and instead we refer readers to the papers by, for example Sargent et al. (1990), Yunker et al. (1995), Meyers (1997) and Naraoka and Ishiwatari (2000). Very briefly, PUFA in terrestrial (vascular) plants consist predominantly of 18:2(n-6) and 18:3(n-3) (Harwood and Russell, 1984). Hence, their FA composition is similar to that of green algae with which terrestrial plants have common ancestors (Raven et al., 1992), but different from the FA of the majority of marine primary producers which are characterized by higher levels of EPA and DHA (Section 2.4.1 and 2.4.2). Long-chain SFA (>C20), which are a component of cuticular waxes, may also make up a large share of FA in vascular plants (Sargent and Henderson, 1995; Sargent et al., 1995a). The presence of these FA has been used as a marker for terrestrial input into freshwater (e.g., Scribe and Bourdier, 1995 (>C26)) as well as marine sediments (e.g., Colombo et al., 1997 Budge et al., 2001; (Æ22:0, 24:0)). There are also several examples where inputs of terrigenous matter into marine food webs have been deduced from the detection of particular FA. For example, elevated concentrations of 18:2(n-6) in the particulate matter and in grazing calanoid copepods following a diatom bloom in the Bahı´ a Blanca estuary, Argentina, was attributed to agricultural products routinely being scattered into the bay (Napolitano et al., 1997). In other examples, elevated levels of typical bacterial markers, traces of C18 PUFA and long- chain SFA (C24–C32) in macrozoobenthos from coastal ecosystems were attributed to the ingestion of particulate matter derived from halophytes (Meziane et al., 1997), mangroves and macroalgae (Meziane and Tsuchiya, 2000; Meziane et al., 2002). Furthermore, using Æ(18:2(n-6), 18:3(n-3)) as specific markers, Budge et al. (2001) concluded that Barred Island Cove, Newfoundland, may receive considerable inputs of terrestrial matter from a neighbouring forest, corroborated by stable isotope analyses. Combining FATTY ACID TROPHIC MARKERS 255

FA and lipid class biomarkers, hydrocarbons, sterols and carbon stable isotope ratios, Canuel et al. (1997) concluded that the largest input of organic matter into Cape Lookout Bight, North Carolina, originated from phytoplankton and sedimentary bacteria, whereas vascular plants contributed a comparatively smaller fraction. These studies confirm that coastal and estuarine ecosystems can receive considerable inputs of terrestrial organic matter, which is characterized by the presence of particular terrestrial marker FA (summarized in Table 3). Terrigenous inputs can be traced as far out as to the continental slope using sterol rather than FA markers, as the latter are broken down or reworked rapidly (Harvey, 1994; Prahl et al., 1994). In order to estimate the fluxes of terrigenous matter between the water column and the sediment, an understanding of the processes leading to sediment production and diagenesis is crucial, especially regarding the incorporation and/or alteration of biomarker signatures. For example, Ahlgren et al. (1997) found a significantly lower content of PUFA (2–40% depending on season) in sediment trapped at just 15 m depth, 2 m above the bottom in Lake Erken, when compared to net plankton. Likewise, Fredrickson et al. (1986) showed that phytoplankton-derived FA were efficiently metabolized in the oxic part of the water column of Lake Vechten. In addition, a tremendous shift in the distribution of FA may take place across the oxic–anoxic interface. For example, whereas algal-derived FA (e.g., 16:3, 16:4, 18:3, 18:4) were abundant under oxic conditions in a coastal salt pond, they were completely replaced by bacterial FATM (e.g. 16:1(n-7), 18:1(n-7), anteiso-C15)inthe anoxic layers (Wakeham and Canuel, 1989).

3. FATTY ACID DYNAMICS IN CRUSTACEOUS ZOOPLANKTON

3.1. General aspects

The concept of FATM has been frequently applied to marine invertebrates, especially herbivorous zooplankton that represent a key link between primary producers and higher trophic levels (Lee et al., 1971b; Sargent et al., 1977; Falk-Petersen et al., 1987, 1990). There is a large body of information on the lipids of ‘‘juicy’’ larger calanoid copepods (reviewed by Sargent and Henderson, 1986), which dominate the zooplankton biomass in large parts of the world’s oceans (e.g., Geynrikh, 1986; Smith and Schnack-Schiel, 1990; Boysen-Ennen et al., 1991; Hirche et al., 1994), and which are particularly important in northern temperate and polar latitude pelagic food webs (Sargent and Henderson, 1986). More recently, lipid and FA research has also focused on euphausiids, especially from the Antarctic, where they 256 JOHANNE DALSGAARD ET AL. are very prevalent and often constitute the major prey of squids, fish, marine mammals and seabirds (Pond et al., 1993; Virtue et al., 1993; Hagen et al., 2001; Saito et al., 2002). In contrast, very little information is available on FA of cyclopoid and poeicilostomatoid ‘‘microcopepods’’, which usually dominate in terms of copepod abundance but not in terms of biomass (Paffenho¨ fer, 1993; Metz, 1998; Bo¨ ttger-Schnack et al., 2001). This holds true also for other invertebrate groups of noncommercial interest such as, e.g., pteropods and amphipods, which nevertheless are essential members of marine food webs (Joseph, 1989; Kattner et al. 1998; Hagen and Auel, 2001). However, there is a large body of literature on the general distribution and composition of lipids in marine invertebrates, and a comprehensive compilation was provided by Joseph (1982, 1989). In this next section, we deal predominantly with the dynamics of FA in calanoid copepods for which most information is available. A discussion of fatty alcohols is also included, since these are the constituents of WE accumulated in large amounts by some of the species. Some fatty alcohols are unique to certain copepods, and therefore, of potential biomarker value. The lipid and FA dynamics of other zooplankton groups are mentioned where pertinent, but otherwise confined to Section 5, where they are discussed in conjunction with major food webs.

3.2. Uptake of dietary fatty acids and de novo biosynthesis of specific fatty acid markers

3.2.1. Herbivorous calanoid copepods

Given their central position within the food web, a key aspect of FA dynamics in copepods and other zooplankton is whether they modify dietary FA, and if so, to what extent do these modifications take place, and how might this interfere with the interpretation of FATM? On the basis of controlled laboratory experiments (Section 3.4), it is generally accepted that phytoplankton FATM are incorporated largely unaltered by phytopha- geous species, allowing conclusions to be drawn on the major type of food ingested. Herbivorous calanoid copepods from higher latitudes are classical examples of this. They typically accumulate large lipid reserves as an adaptation to the pronounced seasonality and strongly pulsed supply of food in these regions (Lee et al., 1971a; Lee and Hirota, 1973). The lipid reserves consist predominantly of WE, and may contain considerable amounts of specific FA such as 16:1(n-7), 18:4(n-3) and EPA, presumably incorporated directly from the consumption of microalgae (e.g., Sargent and Henderson, 1986; Graeve et al., 1994a). Moreover, calanoid copepods are so far the only known organisms that biosynthesize de novo considerable FATTY ACID TROPHIC MARKERS 257 amounts of MUFA and monounsaturated fatty alcohols with 20 and 22 carbon atoms. Consequently, the latter may be used to resolve food web relationships at higher trophic levels, and have, for example, been detected in euphausiids and fish which typically consume large quantities of calanoid copepods (e.g., Sargent, 1978; Falk-Petersen et al., 1987). The lipid biochemistry of calanoid copepods was reviewed in detail by Sargent and Henderson (1986), who also discussed the possible pathways involved in WE biosynthesis. The long-chain MUFA are biosynthesized following the common pathway (section 1.5.). Strictly herbivorous copepods, such as species of the genus Calanus and Calanoides, biosynthe- size large amounts of 20:1(n-9) and 22:1(n-11), which are produced by one- step chain elongation of 18:1(n-9) and 20:1(n-11), respectively (Figure 4B; Sargent and Henderson, 1986; Kattner and Hagen, 1995). A large fraction of these long-chain MUFA are subsequently reduced to their fatty alcohol homologues. Clear species-specific differences in the type and ratios of these MUFA and monounsaturated fatty alcohols are observed. Hence, highest amounts of 22:1(n-11) and highest ratios of 22:1(n-11) to 20:1(n-9) have, for example, been detected in Calanus hyperboreus (Falk-Petersen et al., 1987; Kattner et al., 1989; Albers et al., 1996; Scott et al., 2002), whereas the 20:1(n-9) component comprises the largest fraction in Calanoides acutus and Calanus glacialis (Tande and Henderson, 1988; Albers et al., 1996; Scott et al., 2002). C. propinquus, which deviates from the other herbivorous Calanus species by storing TAG rather than WE (Hagen et al., 1993), has evolved a slightly modified biosynthetic pathway unique to this species, elongating 20:1(n-9) further into 22:1(n-9), (Kattner et al., 1994). The other major FA biosynthesized by C. propinquus, 22:1(n-11), is an end product of the common pathway. C. propinquus is known to switch to omnivorous feeding during winter (Bathmann et al., 1993; Hagen et al., 1993; Kattner et al., 1994), a strategy apparently evolved by this species to cope with the seasonal availability of primary production in lieu of accumulating large WE reserves. Moreover, contrary to other calanoid species, C. propinquus does not store large proportions of typical microalgal FATM, and it is hypothesized that it catabolizes such dietary FA to provide energy for the biosynthesis of long-chain MUFA, which are then incorporated into TAG (Kattner and Hagen, 1995). Another interesting biosynthetic pathway is followed by Neocalanus cristatus and N. flemingeri. These species, in addition to 22:1(n-11), produce considerable amounts of the 20:1(n-11) rather than the 20:1(n-9) isomer (Lee and Nevenzel, 1979; Saito and Kotani, 2000), resulting from the desaturation of 20:0 to 20:1(n-11). Fatty acids can be synthesized de novo from nonlipoidal dietary components such as monosaccharides and amino acids. In addition, it is also possible that shorter-chain saturated dietary FA such as 14:0 and 16:0 258 JOHANNE DALSGAARD ET AL. enter the biosynthetic pathway (Section 1.5) and are modified to longer- chain SFA and MUFA (Sargent and Henderson, 1986). The entrainment of such short-chain SFA probably varies with the dietary regime, e.g., throughout a phytoplankton bloom. This may account for some of the variation observed within the WE fatty alcohol composition of a given species as well as the differences observed between different developmental stages of copepods (Sargent and Falk-Petersen, 1988; Tande and Henderson, 1988). However, dietary 16:1(n-7), which is used as a specific diatom tracer, does probably not enter this internal biosynthetic pathway as it may only be elongated to longer-chain (n-7) isomers (Figure 4B), which are generally not detected in large amounts in calanoid copepods (Sargent and Falk-Petersen, 1981, 1988). The reduction of SFA and MUFA to fatty alcohols is presumably mediated by a NADPH-fatty acyl coenzyme A oxidoreductase specific to WE producing animals, and once formed they may subsequently be esterified to dietary FA by a nonspecific ester synthetase (reviewed by Sargent and Henderson, 1986). Through these processes, dietary carbohy- drates, proteins and FA may effectively be converted to WE even in periods of high intakes of dietary FA. In contrast, this situation usually causes a feedback inhibition of FA biosynthesis in other organisms such as fish (e.g., Sargent et al., 1989; Section 4.3). Hence, the possession of this specific biosynthetic pathway is presumably largely restricted to higher latitude herbivorous species. These species have both to accumulate enough energy reserves during the short feeding season to survive the prolonged periods of starvation, and to fuel reproductive processes starting prior to the onset of phytoplankton spring blooms (Sargent and Falk-Petersen, 1988; Hagen and Schnack-Schiel, 1996). Altogether, these processes sustain the hypothesis that the FA component of WE in herbivorous calanoid copepods is largely derived from the diet (i.e., phytoplankton), whereas the fatty alcohols are derived from the animal’s internal biosynthesis (Sargent and Henderson, 1986). The conservative incorporation of dietary FA into WE has been established through controlled laboratory experiments (Section 3.4), even though it has also been demonstrated that herbivorous marine invertebrates can modify dietary 18:3(n-3) to EPA and DHA at very slow rates (e.g., Moreno et al., 1979; Sargent and Whittle, 1981 and references therein). As the natural diet of herbivorous copepods is typically rich in EPA and DHA and relatively poor in C18 PUFA (e.g., Scott et al., 2002), they presumably do not need to undertake these modifications to sustain their growth requirements (Sargent and Henderson, 1986). In all circumstances, FATM are most ‘‘applicable’’ to herbivorous copepods sampled in mid- or late- summer (Sargent and Henderson, 1986) when they are actively accumulat- ing lipid reserves, whereas specimens sampled from mid-winter and onwards FATTY ACID TROPHIC MARKERS 259 will be mobilizing their energy reserves (for moulting and gonad development; see Section 3.3).

3.2.2. Omnivorous and carnivorous crustaceous zooplankton

Not all zooplankton are characterized by suitable FATM as are the calanoids. This is true of omnivorous and carnivorous copepods for which FA biosynthesis is rather simple. It typically ends with carbon chain elongation to 18:0, which is almost completely desaturated to 18:1(n-9). Moreover, considerable amounts of SFA, especially 16:0, are often produced. Omnivorous and carnivorous copepods accumulate lipids in the form of TAG but may also produce large amounts of WE. In contrast to herbivorous copepods, the fatty alcohols are composed largely of 14:0 and 16:0, reduced from the corresponding FA (Sargent and Henderson, 1986; Graeve et al., 1994b; Kattner and Hagen, 1995; Albers et al., 1996). Only the euphausiid Thysanoessa macrura is known to reduce large amounts of 18:1(n-7) and 18:1(n-9) to the corresponding 18:1 alcohols (Kattner et al., 1996). The reason why long-chain MUFA are not biosynthesized by omnivorous and carnivorous copepods is still under discussion. It has been hypothesized that these species are provided with a less efficient lipid ‘‘economy’’, and that they are less dependent on the seasonal pulse of phytoplankton production in high-latitude ecosystems (Graeve et al., 1994b). Carnivorous and omnivorous polar copepods may also take up large amounts of WE from their diet (Sargent et al., 1977). However, by comparing the lipid composition of Euchaeta antarctica with that of its potential prey, Hagen et al. (1995) concluded that the WE moieties are biosynthesized de novo rather than incorporated directly from the prey (see also Sargent, 1978). Substantiating this conclusion, gut tissue from Euchaeta has been shown to oxidize fatty alcohols to FA as well as to biosynthesize fatty alcohols de novo (reviewed by Sargent, 1978; Sargent and Henderson, 1986). Tracking trophodynamic relationships in omnivorous and carnivorous species in general, using FATM, is more complex than for herbivores. A major reason for this is that the lipid signatures may originate from a variety of different dietary sources. Hence, it generally applies that markers of herbivory become ‘‘blurred’’ and trophic relations become less clear with increasing trophic levels (Auel et al., 2002). However, other FATM may increase in importance, reflecting the changes in feeding behavior (see also Section 3.2.2). Typical algal FATM may be ingested either directly from phytoplankton or indirectly via herbivorous prey species, which themselves may exhibit very different lipid characteristics (e.g., calanoid copepods) that may be transferred to higher trophic levels as well. 260 JOHANNE DALSGAARD ET AL.

As mentioned earlier, high concentrations of C20 and C22 MUFA are presumably unique to, and used as tracers of herbivorous calanoid copepods in secondary and higher order consumers (Sargent and Falk- Petersen, 1981, 1988). Moreover, 18:1(n-9) is used as a general marker of carnivory taking into account that it is a major FA in most marine animals (Falk-Petersen et al., 1990; Sargent and Falk-Petersen, 1981, 1988). In addition, the 18:1 (n-7)/18:1(n-9) ratio has been used to distinguish carnivores from herbivores (e.g., Falk-Petersen et al., 1990, 2000; Graeve et al., 1997; Auel et al., 2002). Here it should be emphasized that microalgae such as Phaeocystis spp. may also contain elevated levels of 18:1(n-9). Hence, when fed to Euphausia superba, this resulted in a decrease in the 18:1(n-7)/18:1 (n-9) ratio (Virtue et al., 1993) as would usually only be expected of species feeding as carnivores. Lastly, the 18:1(n-7)/18:1(n-9) ratio may also increase during starvation (e.g., Ederington et al., 1995), and thus, this ratio is not an unambiguous indicator of herbivorous versus carnivorous feeding. Besides the use of EPA/DHA to distinguish between a diatom and a dinoflagellate-based diet in strictly herbivorous species (preferably along with other FA indices; Section 2.4.1 and Table 2; Graeve et al., 1994a; Nelson et al., 2001; Auel et al., 2002), this ratio may potentially also be used to determine the degree of carnivory. The reason for this is that DHA is highly conserved through the food web being preferentially incorporated into PL (Section 1.4; Scott et al., 2002). As a result, EPA/DHA should decrease toward higher trophic levels. Finally, Cripps and Atkinson (2000) showed that the PUFA/SFA ratio could be used to detect changes in the recent feeding history of Euphausia superba, which may resort to carnivory during nonbloom periods with a consecutive increase in this ratio (see also Section 3.4). The FA and fatty alcohol patterns of typical polar herbivorous, omnivorous and carnivorous copepods are summarized and compared in Figure 7. The figure was constructed by applying a PLS regression analysis to standardized FA and fatty alcohol compositional data summarized in Table 4. The analysis produced three distinct clusters of copepods on a plot of the first versus third of nine PLS components (Figure 7A), which altogether accounted for 80% of the variance of the FA and fatty alcohol compositional data, and explained 84% of the variance attributable to ‘‘species-affiliation’’. The first component separates carnivorous from herbivorous copepods, and Figure 7B shows that the type of alcohol, i.e., short-chain saturates versus long-chain monounsaturates is particularly important for this partitioning. The third component separates Calanus propinquus characterized by 22:1(n-9) from the WE accumulating calanoid copepods in which 20:1(n-9) and 22:1(n-11) FA and fatty alcohols are more important. Overall, the distribution of the variables is consistent with the FATTY ACID TROPHIC MARKERS 261

Figure 7 PLS regression analysis of standardized FA and fatty alcohol compositional data of eight key species of polar copepods summarized in Table 4. Plots show (A) the scores of the first vs. third of nine principal components, and (B) the corresponding loading weight plot. Ellipses in (A) are drawn to indicate the major clusters of zooplankton species. 262

Table 4 FA and fatty alcohol compositional data (as % of total FA and fatty alcohols, respectively) of polar marine copepods used in the PLS regression analysisa.

C. acutus C. propinquus C. finmarchicus C. glacialis C. hyperboreus Euchaeta M. gerlachi R. gigas (n ¼ 23) (n ¼ 21) (n ¼ 24) (n ¼ 12) (n ¼ 65) (n ¼ 8) (n ¼ 12) (n ¼ 7)

Fatty acids 14:0 4.4 ± 1.3 3.6 ± 0.7 16.9 ± 5.1 9.8 ± 4.0 3.7 ± 0.5 1.6 ± 0.4 4.4 ± 0.6 0.7 ± 0.1 15:0 0.2 ± 0.3 0.6 ± 0.6 0.7 ± 0.4 0 0 0.9 ± 1.3 0.6 ± 0.4 0 16:0 4.5 ± 2.1 13.0 ± 1.4 12.7 ± 2.4 6.9 ± 1.2 4.3 ± 0.8 2.4 ± 2.2 12.3 ± 2.1 3.3 ± 0.8 16:1(n-7) 7.7 ± 2.7 4.3 ± 1.2 6.2 ± 2.0 25.2 ± 6.3 10.6 ± 4.0 20.3 ± 4.6 5.6 ± 2.9 11.6 ± 1.9

16:1(n-5) 0.2 ± 0.2 0.2 ± 0.1 0.4 ± 0.3 0.7 ± 0.3 0 0.3 ± 0.5 0.2 ± 0.1 0.0 ± 0.1 DALSGAARD JOHANNE 16:2(n-6) 0.6 ± 0.1 0.4 ± 0.3 0.9 ± 0.3 1.0 ± 0.2 1.8 ± 0.6 0.9 ± 0.1 1.3 ± 0.7 2.7 ± 0.5 16:3(n-3) 0.2 ± 0.3 0.1 ± 0.2 0.3 ± 0.3 0.9 ± 0.4 0.5 ± 0.7 0.4 ± 0.3 0.2 ± 0.2 0.7 ± 0.3 16:4(n-3) 0.6 ± 1.3 0.2 ± 0.2 0.0 ± 0.1 2.0 ± 1.2 0 0.1 ± 0.2 1.0 ± 0.9 3.4 ± 1.2 18:0 0.1 ± 0.2 1.3 ± 0.1 1.5 ± 0.8 0.4 ± 0.3 0.4 ± 0.2 0.4 ± 0.3 1.4 ± 0.6 0.3 ± 0.5 18:1(n-9) 4.8 ± 1.0 2.9 ± 0.6 5.3 ± 1.2 3.7 ± 0.8 3.2 ± 0.7 37.9 ± 12.4 12.8 ± 3.1 18.2 ± 1.7 18:1(n-7) 1.5 ± 0.4 1.1 ± 0.3 0.4 ± 0.9 1.0 ± 0.2 0.9 ± 0.4 1.3 ± 0.4 3.4 ± 2.0 3.3 ± 0.2 18:2(n-6) 1.6 ± 0.5 1.2 ± 0.4 1.8 ± 0.6 0.9 ± 0.2 1.7 ± 0.7 1.5 ± 0.3 1.7 ± 0.2 1.7 ± 0.3 18:3(n-3) 0.5 ± 0.3 0.6 ± 0.2 1.1 ± 0.4 0.5 ± 0.4 0.7 ± 0.4 0.5 ± 0.1 0.8 ± 0.3 0.9 ± 0.2 TAL ET 18:4(n-3) 4.6 ± 5.2 2.8 ± 1.7 9.5 ± 6.5 3.2 ± 2.4 10.3 ± 7.3 2.8 ± 1.2 5.1 ± 1.9 14.6 ± 4.2 . AT CDTOHCMARKERS TROPHIC ACID FATTY 20:1(n-9) 23.1 ± 6.2 2.7 ± 0.5 7.7 ± 3.8 12.3 ± 3.4 19.8 ± 3.2 2.2 ± 1.1 1.3 ± 0.2 0.6 ± 0.3 20:1(n-7) 0.8 ± 0.2 0.6 ± 0.1 1.0 ± 0.5 1.0 ± 0.1 1.9 ± 0.9 0.1 ± 0.1 0.1 ± 0.1 0.0 ± 0.1 20:4(n-6) 1.4 ± 0.8 0.9 ± 0.4 0 0.2 ± 0.3 0 1.9 ± 1.2 0.9 ± 0.3 0.6 ± 0.6 20:5(n-3) 17.1 ± 4.7 12.4 ± 4.6 13.2 ± 5.8 16.0 ± 7.2 14.1 ± 4.5 10.5 ± 5.8 20.9 ± 2.9 27.4 ± 2.1 22:1(n-11) 9.8 ± 2.4 20.1 ± 6.4 8.0 ± 4.1 7.1 ± 1.7 15.0 ± 2.5 0.4 ± 0.6 0.7 ± 0.9 0.8 ± 1.9 22:1(n-9) 3.7 ± 0.8 19.2 ± 6.5 0.3 ± 0.3 1.1 ± 0.3 3.5 ± 1.6 0.4 ± 0.3 0.4 ± 1.1 0.1 ± 0.2 22:5(n-3) 0.8 ± 0.6 0.8 ± 0.1 0.3 ± 0.3 0.6 ± 0.7 1.0 ± 1.3 0.3 ± 0.3 0.9 ± 0.2 0.3 ± 0.2 22:6(n-3) 11.8 ± 4.5 10.9 ± 5.6 11.6 ± 6.3 5.2 ± 1.5 7.8 ± 1.7 12.8 ± 6.2 24.1 ± 3.8 15.5 ± 17.5 Alcohols 14:0 6.2 ± 1.7 0 1.7 ± 0.7 3.2 ± 1.3 2.8 ± 1.5 58.9 ± 5.7 50.0 ± 3.5 45.6 ± 2.0 16:0 8.1 ± 2.8 0 9.6 ± 4.3 11.2 ± 2.6 6.1 ± 2.5 37.3 ± 5.4 48.1 ± 4.1 48.1 ± 2.4 16:1(n-7) 2.4 ± 1.3 0 3.2 ± 2.5 7.1 ± 3.1 3.6 ± 1.7 3.8 ± 1.2 1.9 ± 2.4 4.3 ± 0.9 18:0 0 0 1.7 ± 1.9 0 0.4 ± 0.4 0 0 0 18:1(n-9) 1.2 ± 0.6 0 2.6 ± 1.3 2.1 ± 0.5 0.5 ± 0.6 0 0 1.9 ± 0.3 20:1(n-9) 55.0 ± 4.8 0 36.6 ± 4.3 43.4 ± 5.9 32.6 ± 3.9 0 0 0 22:1(n-11) 27.2 ± 4.8 0 44.6 ± 6.2 30.4 ± 4.7 55.0 ± 7.2 0 0 0 aBased on unpublished data compiled from field trips to the Arctic and Antarctic. Values are mean ± one standard deviation. 263 264 JOHANNE DALSGAARD ET AL. findings discussed above on the typical FA compositions of the herbivorous calanoid copepods C. glacialis, C. hyperboreus, Calanoides acutus and Calanus propinquus, revealing moreover that the 18:1(n-9) alcohol is particularly important in C. finmarchicus, which is known on occasions to feed as an omnivore (Levinsen et al., 2000).

3.3. Mobilization of fatty acids during starvation and reproduction

The applicability of FATM to higher trophic level organisms is constrained by the degree to which they alter their FA signature through de novo biosynthesis, metabolization and breakdown (oxidation) of dietary FA. The dynamics of these processes are coupled to factors such as life history stages, environmental conditions and lipid storage types. For example, most calanoid copepods store minor amounts of TAG that are readily mobilized during starvation (Ha˚ kanson, 1984; see also Sargent and Henderson, 1986; Sargent and Falk-Petersen, 1988 and references therein). These stores are hypothesized to derive ‘‘directly’’ from microalgae (for assimilation of lipids across gut epithelia, exemplified for fish, see Section 4.2.2), and to represent the recent feeding history of the animals (Ha˚ kanson, 1984; Sargent and Henderson, 1986). In contrast, a large fraction of the NL accumulated by herbivorous stage CV copepodites during summer is mobilized to provide energy for moulting into adults early the following year and subsequently, for the production of reproductive tissues (Sargent and Henderson, 1986; Sargent and Falk- Petersen, 1988). These are highly energy demanding processes, which are not understood in detail. Sargent and Henderson (1986) hypothesized that WE are mobilized by a hormone-sensitive lipase to form free fatty acids (FFA) and fatty alcohols. The alcohols are presumably oxidized to FA and added to the ‘‘fatty acid pool’’, before they are oxidized in the mitochondria by conventional beta-oxidation to yield ATP. Wax esters that are not catabolized during moulting are presumably transferred to the gonads. As in fish (Section 4.4.2), the eggs and larval stages are rich in EPA and DHA, while they are relatively deficient in long-chain MUFA (Sargent and Falk- Petersen, 1988). Copepod nauplii do not feed, and juvenile herbivorous copepods do not start to elaborate large lipid reserves until the later copepodite stages (Sargent, 1978; Sargent et al., 1989; Kattner et al., 1994). This is reflected in their content of long-chain monounsaturates and microalgal FATM, which typically increase according to the developmental stage as illustrated in Figure 8. This figure shows the ontogenetic development of selected MUFA in Calanus finmarchicus (CI - adult) sampled in the North Sea. Apart from FATTY ACID TROPHIC MARKERS 265

Figure 8 Ontogenetic changes of MUFA from copepodite stage I to adult Calanus finmarchicus. Based on data from Kattner and Krause (1987).

18:1(n-9), the levels of 16:1(n-7), 20:1(n-9) and 22:1(n-11) all increase in the older stages. Similar trends have been reported for the Antarctic C. propinquus and Calanoides acutus (Kattner et al., 1994), substantiating the hypothesis that de novo biosynthesis of FA and fatty alcohols is less developed in the younger copepodite stages, which presumably catabolize dietary FA to provide energy for rapid growth and development rather than accumulate lipids (Kattner et al., 1994). Lipids also play an important role in euphausiids, and FATM have been successfully applied in several species to identify dietary preferences. The ontogenetic changes in the TAG fatty acid composition of Euphausia superba are shown in Figure 9A. Here, the FA composition of calyptopis and furcilia larvae indicate a dietary input of phytoplankton more clearly than does that of the more advanced postlarval and adult stages, although there is nonetheless an algal signature throughout (Hagen et al., 2001). Figure 9B shows the ontogenetic changes in E. crystallorophias which, in contrast to E. superba, switches from a herbivorous to a more omnivorous diet as it grows (Kattner and Hagen, 1998). Hence, the FA composition of the calyptopis and furcilia suggests a dietary input of microalgae in these stages, but this tendency disappears in the older stages as the diet becomes less specialized. The increase in the level of 16:1(n-7) toward the older stages indicates an intake of diatoms either directly or through the ingestion of primary consumers. 266 JOHANNE DALSGAARD ET AL.

Figure 9 Ontogenetic changes of FATM in the (A) TAG of Euphausia superba, and (B)WEofEuphausia crystallorophias. Based on data from Hagen et al. (2001) and Kattner and Hagen (1998).

3.4. Validation of the fatty acid trophic marker approach in crustaceous zooplankton

The incorporation of dietary FA into crustaceous zooplankton has been established through a series of controlled studies. Hence, Lee et al. (1971b) demonstrated for the first time that the herbivorous copepod Calanus helgolandicus was able to biosynthesize WE from a microalgal diet deficient in fatty alcohols. Moreover, the FA composition resembled the diet closely, the similarities being more obvious in animals ingesting more algae. Thus, it FATTY ACID TROPHIC MARKERS 267 was deduced that C. helgolandicus incorporates dietary FA largely unaltered into WE, and that the fatty alcohols are biosynthesized de novo. Feeding three different concentrations of the dinoflagellate Scrippsiella trochoidea to Calanus helgolandicus, Harvey et al. (1987) later found that PUFA were almost completely retained from the diet at all food concentrations, which were designed to resemble a natural food range. The assimilation of SFA and MUFA was lower than PUFA but increased with higher food concentrations (from 60 to 80% during 1.5 days). The differences in the uptake dynamics of the different FA resulted in higher concentrations of PUFA, particularly 16:4 and 18:4, in the animal tissues. Similar to the FA, the major fatty alcohols 22:1, 20:1 and 16:0 also showed a consistent rise with increasing food levels. Hence, the results emphasize the findings by Lee et al. (1971b) that dietary FA are efficiently assimilated by C. helgolandicus, particularly at high food concentrations, and are incorporated more or less directly into WE. The applicability of diatom and Phaeocystis specific FATM for tracing food web relationships in Euphausia superba was demonstrated by Virtue et al. (1993). After five months feeding, specimens on a Phaeocystis diet contained significantly higher concentrations of 18:1(n-9) than specimens on a diatom diet. The latter, on the other hand, were significantly enriched in 16:1(n-7) and displayed a consistently and significantly higher 16:1(n-7)/16:0 ratio. E. superba is believed to resort to omnivorous-carnivorous feeding during nonbloom periods (Cripps et al., 1999), and the PUFA/SFA ratio has been suggested as an index to detect such changes in its recent feeding history (Cripps and Atkinson, 2000). This was based on a controlled laboratory experiment in which E. superba, caught in an area of high diatom abundance, and hence, believed to have been feeding as a herbivore, was fed exclusively on copepods for 16 days. As a result, the PUFA/SFA ratio increased from <1 to 2. Alternatively, this increase could have been due to starvation and thus a depletion of TAG comparatively rich in SFA. However, as the level of PUFA in the experimental animals increased not only in relative but also in absolute terms, this alternative was excluded. Finally, to verify the potential use of specific FA as trophic markers in calanoid copepods, Graeve et al. (1994a) fed unialgal cultures of either Thalassiosira antarctica (diatom) or Amphidinium carterae (dinoflagellate) to three species of Calanus. Using 16:1(n-7) and 18:4(n-3) as specific markers, clearest signals were observed in Calanus finmarchicus fed diatoms (Figure 10). Over a period of 42 days the proportion of 16:1(n-7) increased from 3% to 14%, whereas 18:4(n-3) declined from 22% to 4% of total FA. In comparison, the level of 16:1(n-7) and 18:4(n-3) in the diatoms was 36% and 4%, respectively. Complementary but less pronounced changes were observed in C. hyperboreus fed dinoflagellates for 47 days. The proportion of 16:1(n-7) decreased from 14% to 11% whereas 18:4(n-3) increased from 268 JOHANNE DALSGAARD ET AL.

Figure 10 Temporal development of selected FA in Calanus finmarchicus (CV stages) fed on the diatom Thalassiosira antarctica for 42 days (upper panels) and 24 days (lower panels). In each time period (A) is the total lipid fraction, and (B) the WE fraction. The inserts are linear regression curves derived from the original FA data to elucidate the trends. Redrawn with permission after Graeve et al. (1994a).

1% to 10% of total FA. The proportion of the two FA in the dinoflagellate was 1% and 30%, respectively. C. glacialis, on the other hand, deviated from the other Calanus species as a diet of dinoflagellates did not result in the expected increase in the proportion of 18:4(n-3). The few other published data on C. glacialis have also shown low to zero concentrations of 18:4(n-3), whereas the level of the diatom signature FA 16:1(n-7) has typically been high (Tande and Henderson, 1988; Graeve, 1993; Hirche and Kattner, 1993; Albers et al., 1996). An explanation of the differences in the distribution of FATTY ACID TROPHIC MARKERS 269

18:4(n-3) between species might be that C. glacialis readily converts 18:4(n- 3) to EPA, whereas C. finmarchicus incorporates 18:4(n-3) directly. It should, however, also be noted that all animals were losing weight (measured as wax ester content) during the experiment, in particular C. glacialis, and the results may simply be due to C. glacialis starving on the experimental diet.

4. FATTY ACID DYNAMICS IN FISH

4.1. General aspects

A review on the dynamics of lipids in fish, focusing on marine species, was first presented by Shul’man (1960), who pointed out that many of the major conclusions could have been drawn from data obtained by the end of the 18th century, and that little fundamentally new knowledge had been added from then until 1960. Subsequently, a large body of literature on the dynamics of lipid and FA metabolism in marine fish has been generated. This research has focused in particular on the optimization of artificial diets for meeting the nutritional requirements, and improving the growth and development of cultured species. However, as pointed out by Ackman (1980), much of this literature is of little relevance for natural systems because of the ‘‘designed’’ lipid composition of artificial diets, and the ‘‘unnatural’’ growth rates and fat levels achieved by cultured species. We therefore focus on the literature that is pertinent for the interpretation of FATM in fish, i.e., the uptake, incorporation and modification of dietary FA as well as mobilization of FA during periods of starvation and maturation. Finally, we summarize studies that have demonstrated the incorporation of FATM in fish.

4.2. Incorporation of dietary fatty acids

4.2.1. Lipids and enzyme specificity

Marine fish use lipids as a chief metabolic energy source (Shul’man, 1960), fulfilling their energetic requirements primarily through the oxidation of cellular lipids and proteins rather than carbohydrates (Cowey and Sargent, 1977; Jobling, 1994; Sargent et al., 1993). TAG is the primary mode of lipid storage in most species whereas WE are usually much less important (Shul’man, 1960; Love, 1970; Owen et al., 1972; Ackman, 1980; Navarro and Gutie´ rrez, 1995; Sargent and Henderson, 1995). Many meso- and 270 JOHANNE DALSGAARD ET AL. bathypelagic species, such as the lantern fish (Myctophidae) and bristle- mouths (Gonostomatidae), however, accumulate large amounts of WE reserves (>10%; reviewed by Lee and Patton, 1989), consisting of relatively simple FA and fatty alcohols, i.e., primarily 16:0 and 18:1 (Sargent, 1976; Sargent et al., 1977). The exact role of these WE is not known but they most probably serve either as long-term energy stores in species living in an environment characterized by irregular food supply, or as a means to provide buoyancy since WE have a lower specific gravity than TAG (Lee and Patton, 1989; Sargent, 1976). Laboratory experiments have established that the FA composition of fish can be highly affected by their diet (Section 4.5). On a biochemical basis, this may be due to the low enzyme–substrate specificity of the FA converting enzymes of the common lipid pathway, which rely on weaker ‘‘hydro- phobic’’ interactions contrary to, for example, amino acid and protein metabolism that depends on stronger ionic and hydrogen-bond interactions (Sargent et al., 1993). Hence, whereas the amino acid composition of proteins is controlled by highly specific transfer RNAs, Á6 desaturase (which is central to the common lipid pathway) may readily desaturate a number of dietary FA (Sargent et al., 1993). The introduction of polar groups, however, enhances slightly the specificity of the enzyme–substrate complex, as demonstrated by the selective incorporation of PUFA rather than SFA and MUFA into PL (Sargent et al., 1993). Still, the acylases and transacylases that esterify PUFA to PL do not have absolute specificity for any one FA in particular, and a dietary excess of, e.g., EPA may lead to elevated levels of this FA at the expense of DHA if the latter is present in lower concentrations (Sargent et al., 1999). These processes largely explain why storage lipids are generally more similar and respond more readily to the diet than specialized tissues such as the heart and brain, which are comparatively rich in polar lipids (Navarro et al., 1995; Grahl-Nielsen and Mjaavatten, 1992; Mjaavatten et al., 1998).

4.2.2. Uptake of dietary fatty acids

The digestion, absorption and deposition of lipids and FA in fish has been studied in detail and thoroughly reviewed (Cowey and Sargent, 1977, 1979; Sargent, 1978; Henderson and Tocher, 1987; Sheridan, 1988; Sargent et al., 1989, 1993). Briefly, upon consumption the dietary lipids are emulsified by bile salts and hydrolysed by pancreatic lipases in the gut to form FFA in addition to 2-monoacylglycerols and glycerol (from dietary TAG), alcohols (from dietary WE) and lysophospholipids (from dietary PL). Wax esters are more hydrophobic than TAG and PL and therefore more difficult to emulsify. Hence, fish consuming large quantities of WE generally exhibit a FATTY ACID TROPHIC MARKERS 271 longer retention time of food in their gut presumably to facilitate hydrolysis and absorption (e.g., Cowey and Sargent, 1979). The various lipid components are absorbed into intestinal epithelial cells where they are re-esterified into TAG and PL. Dietary fatty alcohols are oxidized to the corresponding FA by a NAD-dependent dehydrogenase prior to esterifica- tion (with glycerol) to form TAG. Shortage of preformed glycerol in the diet is compensated for by converting nonessential amino acids and glucose to triacylglycerol-glycerol (see also Sargent and Henderson, 1986). The lipids are concurrently assembled into lipoprotein particles and transported to the liver or extra-hepatic adipose tissues by the blood or lymphatic system. In most species the liver, rather than the adipose tissues, is the principal site of lipogenic activity including de novo biosynthesis and modification of dietary FA (see also Henderson and Sargent, 1985). This short summary explains how zooplanktivorous fish, which may consume large quantities of WE rich calanoid copepods, are able to accumulate TAG rich in 20:1(n-9) and 22:1(n-11) (see also Sargent, 1978; Sargent and Henderson, 1986). However, whereas the ratio of 20:1(n-9) and 22:1(n-11) is typically 1:2 in the copepods, it decreases to 2:3 in clupeids (Ackman and Eaton, 1966b) and is close to one, e.g., in capelin, indicating a preferential catabolism of 22:1(n-11) (Pascal and Ackman, 1976; see also Henderson et al., 1984). Moreover, both MUFA are essentially absent from fish PL suggesting that they are used preferably for the provision of metabolic energy rather than involved in biomembrane functioning (reviewed by Sargent and Whittle, 1981; Henderson and Sargent, 1985). These observations sustain that the FA composition of storage lipids resem- bles the diet more closely than does the FA composition of polar lipids. Larval fish may not be capable of biosynthesizing the glycerophospho- base backbone of phosphoglycerides de novo, but presumably obtain these moieties from their diet. They may, however, readily exchange FA between and within dietary-derived PL and TAG (reviewed by Sargent et al., 1999), consistent with the findings that larval fish consuming large amounts of microalgae and microzooplankton have a total FA composition very similar to their prey (e.g., Klungsøyr et al., 1989; St. John and Lund, 1996).

4.3. Modifications and de novo biosynthesis of fatty acids

Like most other organisms, fish can readily biosynthesize SFA with up to 18 carbon atoms de novo (Ackman, 1980; Henderson and Sargent, 1985) and desaturate them into monounsaturates following the common lipid pathway (Section 1.5). However, in contrast to calanoid copepods discussed in Section 3.2.1, a dietary excess of FA (>10%) apparently suppresses de novo 272 JOHANNE DALSGAARD ET AL. biosynthesis while the deposition of dietary FA continues (reviewed by Sargent et al., 1989, 1993). Fish incorporate dietary FA either directly or after modifying them slightly through further elongation and desaturation. To date, detailed research has been conducted mostly on the ability of fish to convert 18:2(n-6) and 18:3(n-3) to AA, EPA and DHA, which are essential for their normal growth and development (e.g., Bell et al., 1986; Sargent et al., 1995a, b, 1999). Early experimental evidence from rainbow trout (Castell et al., 1972a, b, c), and later from numerous other studies of freshwater species (reviewed by Cowey and Sargent, 1977; Watanabe, 1982; Henderson and Tocher, 1987; Sargent et al., 1989, 1993), has established that freshwater fish can generally carry out these modifications. In contrast, most marine species studied so far cannot undertake these conversions at any significant rates (e.g., juvenile gilthead sea bream, Mourente and Tocher, 1993a; juvenile golden grey mullet, Mourente and Tocher, 1993b; plaice, Owen et al., 1972; red sea bream, rockfish and globefish, Kanazawa et al., 1979; and turbot, Owen et al., 1975; Cowey et al., 1976; Scott and Middleton, 1979; Linares and Henderson, 1991). It has been hypothesized that since the diet of both larval and adult marine fish is naturally rich in (n-3) PUFA, a deficiency or impairment of the Á5 fatty acid desaturase necessary for converting C18 PUFA to EPA and DHA has evolved in these species (reviewed by Sargent et al., 1993, 1995a). However, it has also been argued that the ability to undertake these conversions is a matter of feeding habit rather than water salinity (Sargent, 1995; Sargent et al., 1995a). For example, similarly to marine piscivores, freshwater pike (Esox lucius) do not convert C18 PUFA to EPA and DHA at any significant rate (Henderson et al., 1995). Moreover, the capacity to undertake these conversions might be coupled to ontogenetic changes in the diet composi- tion (Sargent, 1995; Sargent et al., 1995a). Rapidly growing salmon fry in freshwater can, e.g., readily convert 18:3(n-3) ingested from aquatic insects to DHA, whereas slower growing juveniles entering the marine environment and turning into piscivores, do not need to undertake these conversions, as they have a ready dietary source of DHA (Sargent, 1995; see also Lovern, 1934 and Mjaavatten et al., 1998). In a comparative study of 56 fresh and brackish-water fish species, Ahlgren et al. (1994) found that differences in FA patterns were a matter of overall lipid content rather than water salinity. Hence, they found strong correlations between the total FA content and SFA, MUFA and (n-6) PUFA, respectively, in all species. In contrast, the concentration of (n-3) PUFA was independent of the total FA content after a breakpoint at about 100 mg FA gÀ1 dry mass (DM). PUFA are preferentially incorporated into polar lipids, and high concentrations of (n-3) PUFA in the biomembranes of fish have been FATTY ACID TROPHIC MARKERS 273 linked to the generally low temperature in the aquatic environment (e.g., Cowey and Sargent, 1977, 1979). The fluidity of biomembranes is largely determined by the degree of membrane FA unsaturation and by selectively incorporating (n-3) PUFA, fish may ensure the functional integrity of their biomembranes at lower water temperatures (reviewed by Cowey and Sargent, 1977, 1979; Henderson and Sargent, 1985; Bell et al., 1986; Sargent et al., 1989). More recently, the abundance of (n-3) PUFA in fish membranes has been related to their structural rather than fluidizing role (reviewed by Sargent and Henderson, 1995; Sargent et al., 1995b). High concentrations of di-22:6(n-3) phosphatidylethanolamine and di-22:6(n-3) phosphatidylserine in the retinal rod outer segment membranes and brain synaptosomal membranes of fish are believed to provide a unique and highly ordered bi-layer that remains relatively constant despite changing environ- mental temperatures and pressure, while facilitating fast conformational changes undergone by membrane signaling proteins (reviewed by Sargent and Henderson, 1995; Sargent et al., 1993, 1995a, b). Substantiating this hypothesis, Bell et al. (1995) showed that herring larvae (Clupea harengus) reared on a diet deficient in DHA fed less actively at different light intensities than larvae reared on a diet supplemented in DHA (see also Navarro and Sargent, 1992). In summary, the FA composition of fish lipids is a blend of endogenous and exogenous sources, determined by (i) de novo biosynthesis of short- chain SFA and MUFA, (ii) selective uptake and ‘‘direct’’ incorporation of dietary FA and fatty alcohols, and (iii) uptake and modification of dietary FA and fatty alcohols prior to incorporation.

4.4. Mobilization of fatty acids during starvation and reproduction

4.4.1. Starvation

The metabolism of lipids and FA in fish is strongly linked to physiological and behavioral traits such as size, age, sex, state of maturity, spawning, depth distribution and migration as well as to biotic and abiotic factors such as food abundance, water temperature, salinity, etc. (e.g., Shul’man, 1960, 1974; Friedrich and Hagen, 1994; Sargent and Henderson, 1995; Anthony et al. 2000). Prolonged periods of starvation are common in fish and have often evolved as part of their reproductive cycle, for example in spawning- migrating salmon (Henderson and Tocher, 1987). Starvation is accompa- nied by a reduction in FA biosynthesis (reviewed by Sargent et al., 1989), and increased mobilization of energy stores. TAG is mobilized either simultaneously or after carbohydrates, but usually before proteins and 274 JOHANNE DALSGAARD ET AL. always before PL (Takama et al., 1985; Ha˚ kanson, 1989; Sargent et al., 1989; Moyes and West, 1995; Navarro and Gutie´ rrez, 1995). If starvation is prolonged, skeletal muscles by virtue of their large mass and protein content may become the main energy source (Moyes and West, 1995; Navarro and Gutie´ rrez, 1995). The mobilization of lipid stores is effectuated by intracellular, hormone- sensitive lipase activity (reviewed by Sheridan, 1988; Sargent et al., 1989), and a list of agents known to enhance lipid mobilization in fish can be found in Sheridan (1988). The mobilization of lipid reserves results in the hydrolysis of TAG and the subsequent release of FFA. The time between initiation of starvation and an increase in the plasma FFA concentration is highly species-specific, varying from a few days in rainbow trout to 145 days in eels (reviewed by Sargent et al., 1989). Mobilized FA are transported to the liver where they are oxidized via microsomal beta-oxidation to provide energy (reviewed by Henderson and Sargent, 1985; Sargent et al., 1989). In fish undergoing ovarian development, mobilized FA are also used for biosynthesis of vitellogenin, which is transferred to the ovary (Sargent et al., 1989).

4.4.2. Reproduction

The mobilization of particular FA depends on whether they are required solely for provision of metabolic energy or destined for gonad development (Sargent et al., 1989). This was shown by Takama et al. (1985), monitoring the progressive depletion of particular FA in starving adult cod, half of which were maturing and half of which had been surgically gonadectomized. Cod accumulate lipids in their liver, and a reduction in the liver level of both DHA (significant) and 18:1 (insignificant) was detected in the maturing cod but not in the gonadectomized cod. As these two FA were among the major constituents of the gonads, it was hypothesized that they had been selectively mobilized from the liver for incorporation into the gonads. The mobilization of particular FA during gonadogenesis was also examined by Henderson et al. (1984) studying a natural population of endogenous capelin (Mallotus villosus) in Balsfjord, northern Norway. Contrary to cod, capelin accumulate lipids in their muscles, and moreover, presumably do not feed during gonadogenesis. Hence, an interesting comparison could be made between the FA composition of the muscles at the onset of gonadogenesis with muscle and ovary FA compositions imme- diately prior to spawning. In both sexes 14:0, 20:1(n-9) and 22:1(n-11) were selectively retained in the muscles, increasing in relative proportions during gonadogenesis. In contrast, all other major FA (>1% of total muscle lipid) were partly mobilized: 14:0, 16:1(n-7), 18:2(n-6), 18:3(n-3), 18:4(n-3) FATTY ACID TROPHIC MARKERS 275 and DHA were preferentially deposited in the ovaries, whereas 16:0, 18:0, 18:1(n-7), 18:1(n-9), 22:1(n-11), 22:5(n-3) and EPA were catabolized after mobilization. These results emphasize that the FA patterns of fish depleting their lipid reserves are highly distorted, reflecting internal metabolic processes rather than potential dietary signals. In fish roe, EPA and DHA typically constitute 50% of the TL, suggesting an essential need of the developing embryo for the formation of cellular membranes. Interestingly, the FA composition of fish roe is remarkably similar among species and presumably optimized nutritionally for the growth of the developing embryo and yolk- sac larvae until first-feeding (Kaitaranta and Linko, 1984; Tocher and Sargent, 1984; Klungsøyr et al., 1989). The dietary FA composition of the parent fish typically has little impact on the FA composition of the eggs. However, when comparing the roe of Atlantic and Baltic herring (Kaitaranta and Linko, 1984), relatively large proportions of 20:1(n-9) and 22:1(n-11) (i.e., 3.1% and 1.5% of total FA, respectively) were detected in the Atlantic herring roe, whereas these FA were absent in Baltic herring eggs. Calanoid copepods are much less common in the Baltic Sea compared to the Atlantic, presumably because the lower salinity in this system (Ackman, 1980), and this probably explains the absence of these tracers in Baltic herring roe. In another example, Lasker and Theilacker (1962) found a relatively close similarity between the FA composition of the ovary of Pacific sardine (Sardinops caerulea) and the diet of the adult fish, consisting mostly of Calanus. However, apart from a few such exceptions, it may be anticipated that FA add a limited amount of information useful for resolving the trophodynamic processes resulting ultimately in the production of offspring.

4.5. Validation of the fatty acid trophic marker approach in fish

Only a handful of studies have validated the FATM approach in fish, examining the FA composition of prey and predators under controlled experimental conditions either in the laboratory or in mesocosms. Such studies are nevertheless essential for the application of any trophic marker in studies of ecosystem dynamics. Two laboratory studies have been performed. In the first case, St. John and Lund (1996) examined the potential of 16:1(n-7)/16:0 as a specific food web tracer in a study with the overall objective of identifying the dominant microalgal class, and hence the hydrographic regime (Section 2.3), contributing to the condition of juvenile North Sea cod (Gadus morhua). In order to establish a relationship between lipid tracer content and food utilization in situ, the tracer was first validated in the laboratory. Using Acartia tonsa nauplii 276 JOHANNE DALSGAARD ET AL. as an intermediary, larval North Sea cod were reared on food webs based on monocultures of either the diatom Skeletonema costatum or the dinoflagellate Heterocapsa triquetra, i.e., algae dominating in the mixed and stratified regions of the North Sea, respectively. The cod larvae required 8 days on either food type before the tracer lipid signals started to change from their original values to those similar to the algae at the base of their respective food webs (Figure 2). After 13 days, the lipid tracer content in the larvae was no longer significantly different from that of the cultures of Skeletonema costatum or Heterocapsa triquetra. Subsequently, a sub-sample of 100 juvenile cod from stratified, mixed and frontal regimes in the northeastern North Sea was examined for the content of FA tracers and condition (as determined by the ratio of total lipid content to total length). Juvenile cod displaying a lipid tracer content indicating utilization of a diatom-based food web (found in proximity to regions of frontal mixing) were in significantly better condition (P > 0.05) than those containing a lipid signal indicative of a flagellate-based food web (found in stratified regions of the North Sea; Figure 11). In another laboratory study, Kirsch et al. (1998) examined how the FA signature of whole adult Atlantic cod changed when offered first a prepared diet of low-fat squid (Illex illecebrosus, 2% lipid DM) for six weeks, followed by a prepared diet of high-fat Atlantic mackerel (Scomber

Figure 11 Plot of condition, as determined by residuals of the total lipid content to total length relationship, against the specific food web tracer 16:1(n À 7)/16:0 for a random sample of 100 juvenile North Sea cod. Redrawn with permission after St. John and Lund (1996). FATTY ACID TROPHIC MARKERS 277 scombrus, 16% lipid DM) for another eight weeks. Intriguingly, after only 3 weeks on the squid diet, and despite the absence of any mass gain, the FA composition of the cod had changed significantly toward that of the squid, changing no further after 6 weeks of feeding. When switched to the mackerel diet, the overall tissue lipid content of the cod increased from 2% to 4%. Furthermore, the FA patterns had reversed toward that of the mackerel diet within 5 weeks of first feeding, with no further changes during the last three weeks. Applying a classification and regression tree analysis (CART) to the FA compositional data, the authors showed that the cod treatment groups, despite the influence of dietary FA, were still readily differentiated from each other and from their diet. The results of these two studies demonstrate the relevance of dietary FA as qualitative markers for resolving trophic interactions in both larval and adult fish. Moreover, the latter study supports the application of FATM for assessing the diet of yet higher trophic level predators such as marine mammals (e.g., Iverson et al., 1997b). A series of enclosure studies have been carried out in Loch Ewe, Scotland, demonstrating the impact of ontogeny and varying dietary regimes on the FA composition of herring larvae (Clupea harengus). In the first study, Gatten et al. (1983) observed that a switch in the diet of herring larvae from microalgae and nauplii (as determined from gut analyses) to WE rich stages of copepodites and adult calanoid copepods, was accompanied by a gradual replacement of typical dinoflagellate and flagellate FATM (18:4(n-3), EPA, DHA) by calanoid FATM. Considering the condition of the herring larvae, Fraser et al. (1987) later found that a dietary resemblance was much more pronounced in well-nourished larvae, which were accumulating TAG, than in under-nourished larvae. Finally, using 18:4(n-3) as a specific flagellate tracer, Fraser et al. (1989) were able to follow a natural succes- sion in the enclosed microalgal community from dinoflagellates and flagellates to diatoms, and furthermore, could detect the signal, presumably through zooplankton, to herring larvae (Figure 12). However, whereas the zooplankton community closely mirrored the temporal development in the phytoplankton, the peak in the tracer content was delayed by 23 days in herring larvae. This delay suggests that the fish larvae either continued feeding selectively on dinoflagellates and flagellates rather than on diatoms or zooplankton, or that the turnover rates of the tissue lipid pools decreased as the larvae grew (see also Section 5.2.5). The authors did not, however, discuss this. Apart from the studies summarized above, several studies of natural fish populations have been carried out, comparing the FA composition of fish and their potential prey, and assuming simply a conservative transfer of FA from prey to predators. These studies will be summarized in Section 5. 278 JOHANNE DALSGAARD ET AL.

Figure 12 Temporal changes in the level of 18:4(n À 3) in phytoplankton total lipid (circles), zooplankton total lipid (squares) and TAG (diamonds) of herring larvae from an enclosure study in Loch Ewe, Scotland. Reproduced with permission after Fraser et al. (1989).

5. APPLICATIONS OF FATTY ACID TROPHIC MARKERS IN MAJOR FOOD WEBS

5.1. General aspects

The application of FATM in ecosystem analyses falls under two broad categories of research, these being (i) identifying species and group interactions, and (ii) resolving the impact of hydrodynamically driven processes on population dynamics. The first approach conforms with the old adage ‘‘you are what you eat’’, and aids in the definition of trophic interactions and food webs thereby defining trophic exchanges (e.g., Kattner et al., 1994; Iverson et al., 1997b). The second approach goes a step further and identifies the key climatically driven processes that impact on ecosystem dynamics through bottom-up pathways (e.g., St. John and Lund, 1996). This is particularly important for resolving mechanisms by which climate change might modify the dynamics of key species, and thus marine ecosystem structure and functioning. The latter approach is based on the assumption that climate change impacts water column stability through fluctuations in surface temperature and freshwater inputs. These processes cause spatial and temporal variations in stratification, and in addition, contribute to variations in its intensity. As discussed in Section 2.3, stratification is one of the key mechanisms determining the structure of phytoplankton communities in pelagic ecosystems (e.g. Sverdrup, 1953; Kiørboe, 1993; St. John and Lund, FATTY ACID TROPHIC MARKERS 279

1996), and hence, the basic FATM patterns recognized in higher trophic levels. Seasonal patterns of phytoplankton group dominance, driven by stratification, are most pronounced in high latitude and temperate systems, and are used here as an example to outline the general processes, conceptualized in Figure 13. First, as light intensity increases in early spring, the phytoplankton community is dominated by small flagellates, typically Phaeocystis spp., with blooms occurring in some situations. Accompanying such blooms are typical FATM (Section 2.4.1), available for transfer to higher trophic levels. With the onset of stratification, the spring diatom bloom is initiated and flagellate FATM are largely replaced by diatom FATM. Continued and increased stratification results in a period of nutrient limitation. As a consequence, the phytoplankton community becomes dominated by flagellates, dino- flagellates and microbial loop production again with a characteristic FATM distribution. Variations in the content of these different group specific FATM in higher trophic levels during the succession of phytoplankton dominance are indicative of the importance of the various algal groups for the transfer of energy up the food webs. The importance of the different temporal components of this evolution of phytoplankton dominance, and hence FATM, varies dramatically between geographic regions (e.g., polar, temperate regions and tropics), and is in essence based on the dynamics of water column stratification as indicated in Figure 13A. A comparison of the dynamics of FATM in these different systems has not been made. However, based on the processes outlined above, a continuum of the importance of diatom versus flagellate, microbial loop and dinoflagellate production to higher trophic levels (dependent upon transfer efficiencies), coupled to the relative contribution of these different groups to the total phytoplankton biomass of the system, might be expected (Figure 13B). For example, in boreal and temperate systems the spring diatom bloom contributes a higher proportion to the overall phytoplankton biomass than in tropical systems. The reason for this is that tropical systems are generally stratified and dominated by flagellate phytoplankton and microbial loop production. The latter comprises also cyanobacteria, however, these are more difficult to categorize. They are N-fixers and may act like diatom blooms, but as they are not necessarily driven by stability, they are not included in Figure 13. Phytoplankton group dominance is also influenced by mesoscale features such as coastal upwelling and tidal mixing processes, which impact on water column stratification and nutrient availability. These systems in essence create localized ‘‘spring bloom’’ conditions for phytoplankton communities, 280 JOHANNE DALSGAARD ET AL.

Figure 13 A conceptual model of the dynamics of stratification, phytoplankton group dominance and FATM over a seasonal cycle in (A) polar, temperate and tropical ecosystems. (B) Predicted trends in the contribution of group-specific FATM as a function of water column stratification. and are also dominated by diatom production (e.g., St. John and Lund, 1996). The dynamics of phytoplankton group production in upwelling systems is well understood, but the dynamics of FATM has not received very much attention. On the other hand, in tidal mixing regions the FATTY ACID TROPHIC MARKERS 281 distribution of FATM relative to water column structure and phytoplank- ton group dominance has been studied, and links to higher trophic level condition have been identified (St. John and Lund, 1996). The following section describes the state-of-the-art of FATM in pelagic marine ecosystems.

5.2. The Arctic

5.2.1. Primary producers

Light, nutrients and stratification are the major driving forces in the Arctic, controlling the short but intensive period of primary production with 60–70% of the total annual primary production taking place between mid- March and early July (Falk-Petersen et al., 1990 and references therein). The pelagic spring bloom is initiated in fjords (where fresh water run-offs result in early stratification), followed by blooms in the open water of the marginal ice zone (MIZ) (Falk-Petersen et al., 1998 and references therein). Ice algae consist predominantly of diatoms, whereas open water phytoplankton communities are relatively richer in dinoflagellates and smaller flagellates (Falk-Petersen et al., 1998; Henderson et al., 1998). In particular, Phaeocystis spp. often dominate at the onset of the open water spring bloom (Sargent et al., 1985; Falk-Petersen et al., 1990, 2000 and references therein; Marchant and Thomsen, 1994; Hamm et al., 2001). The different phytoplankton communities are accompanied by typical FA signatures reflecting the dominant algal classes (Section 2.4.1). A notable exception is Phaeocystis pouchetii in Balsfjord (Sargent et al., 1985; Hamm et al., 2001), which contained a FA pattern quite different from that observed in other areas (Section 2.4.3), i.e., high proportions of 18:4(n-3), 18:5(n-3), EPA and DHA combined with relatively low levels of C16 PUFA. The FA signature of size-fractionated plankton samples collected during the spring and post-plankton bloom off the west coast of Greenland was recently combined with detailed microscopic analyses of biomass and species level composition of microalgae (Reuss and Poulsen, 2002). This study revealed that most of the spring bloom biomass was contained within the 11–300 mm size-fraction and was dominated by diatoms, while 80% of the biomass in the 6–11 mm size-fraction was composed of Phaeocystis pouchetii. The spring plankton bloom was succeeded by flagellates (Haptophyceae; < 11 mm) with the total biomass of FA being an order of magnitude lower and significantly different (r ¼ 0.95, P < 0.001) from the spring bloom. On this basis, specific FATM were coupled with the phytoplankton species composition. The biomass of diatoms correlated significantly and positively with 16:1(n-7)/16:0, ÆC16/ÆC18, 16:1(n-7) and 282 JOHANNE DALSGAARD ET AL.

Figure 14 Total FA (mglÀ1) and ratios of specific FA in plankton samples obtained off West Greenland in (A) May 2000 and (B) June 1999. Note separate and different scales. Redrawn with permission after Reuss and Poulsen (2002).

EPA and negatively with ÆC18 FA and 18:1(n-9). The temporal develop- ment in the diatom FATM composition of the particulate matter is shown in Figure 14. In contrast, the typical dinoflagellate FATM 18:4(n-3) and DHA did not correlate with the biomass of either flagellates or dinoflagellates. The authors emphasized that dinoflagellates are a complex group of organisms comprising auto-, hetero- and mixotrophs that contain chloroplasts of diverse endosymbiotic origin. This may explain some of the variation in specific FATM observed within this group (Table 2), and based on the FATTY ACID TROPHIC MARKERS 283 results in the study, the authors deduced that ÆC18 FA provide a better indicator of flagellate contribution than 18:4(n-3).

5.2.2. Copepods

High levels of phytoplankton FATM have typically been detected in the major species of predominantly herbivorous Arctic copepods including Calanus finmarchicus, C. hyperboreus and C. glacialis (Lee, 1974, 1975; Sargent and Henderson, 1986; Sargent and Falk-Petersen, 1988; Kattner et al., 1989; Scott et al., 2002). The distributions of FATM, however, vary. For example, the proportion of 16:1(n-7) and 18:4(n-3) in C. hyperboreus collected off the northeast coast of Greenland was found to change depending on the hydrographic regimes they were collected in (Figure 15A; Kattner and Hagen, 1995). Hence, a strong decline in the concentration of 16:1(n-7) was found from specimens collected in the ice-free and diatom dominated area (site A), to specimens from the unproductive pack-ice (site B), and to specimens sampled in the marginal ice zone (site C–E), where the phytoplankton community was dominated by dinoflagellates and Phaeocystis. At the same time, a complementary trend was evident for the specific dinoflagellate tracer, 18:4(n-3). FATM have also been useful for resolving temporal changes in the diet composition and lipid metabolism of these copepods. This is illustrated in Figure 16, which shows a marked increase in the concentration of 18:4(n-3) in spring and summer in Calanus finmarchicus sampled in Balsfjord (Sargent and Falk-Petersen, 1988), and which was consistent with a major dietary intake of Phaeocystis pouchetii as verified by visual examination of gut contents (Sargent et al., 1987), and laboratory feeding experiments (Tande and Ba˚ mstedt, 1987). A less pronounced increase in the sum of 16:1 and EPA suggested a ‘‘switch’’ in diet to include diatoms, and this was accompanied by the generation of WE reserves as indicated by the increase in the concentration of long-chain monounsaturated fatty alcohols. In contrast to calanoid copepods, the FA and fatty alcohol composition of another abundant polar copepod genus, Metridia, does not show the characteristics typical of species relying on highly efficient energy stores (Falk-Petersen et al., 1987, 1990; Graeve et al., 1994b; Saito and Kotani, 2000). In this genus, long-chain monounsaturated fatty alcohols are replaced by shorter-chain saturated alcohols (Section 3.2.2), consistent with a more omnivorous diet (Falk-Petersen et al., 1987, 1990). Supporting this hypothesis, FATM suggestive of both a phytoplankton (e.g., 16:1(n-7), 18:4(n-3)), and an animal (e.g., 18:1(n-9)) derived diet have been detected in Metridia spp. This is exemplified in Figure 17A, which shows an increase in the sum of 16:1 and EPA during the spring and summer period in M. longa, 284 JOHANNE DALSGAARD ET AL.

Figure 15 Spatial variation of the dietary FATM 16:1(n À 7) and 18:4(n À 3) in (A) Calanus hyperboreus (CV stages) collected in the Fram Strait (4Wto4E, 78N to 80N), July 1984 and (B) Calanoides acutus (CV stages) collected in the south eastern Weddell Sea (site I þ II: 36Wto42W, 77300Sto78S; site III–V: 18Wto 21W, 72Sto73300S), January–February 1985. Redrawn with permission after Kattner and Hagen (1995). sampled in Balsfjord (Sargent and Falk-Petersen, 1988, and references therein). This increase indicates a dietary intake of diatoms, whereas the less pronounced increase in the concentration of 18:4(n-3) later in summer indicates that diatoms were succeded by dinoflagellates and flagellates in the diet. Furthermore, a complementary trend in 18:1 suggests a higher degree of carnivory during winter. FATTY ACID TROPHIC MARKERS 285

Figure 16 Seasonal development in specific WE fatty acids and alcohols in Calanus finmarchicus from Balsfjord, northern Norway. Based on data from Falk-Petersen et al. (1988) cited and reproduced in Sargent and Falk- Petersen (1988).

Metridia longa and M. okhotensis may, however, deviate from the general fatty alcohol pattern summarized above, and their WE contain substantial amounts of C20 and C22 monounsaturates. It is not clear whether these long-chain monounsaturates are biosynthesized de novo or derived from feeding on calanoid copepods (Falk-Petersen et al., 1987; Albers et al., 1996). Assuming that the latter is true, Figure 17B indicates an uptake of calanoid copepods in late winter by M. longa. Similarly, the WE fatty acid composition of another carnivorous Arctic copepod, Pareuchaeta norvegica, indicated that this species also feeds on calanoid copepods (Sargent and McIntosh, 1974). Interestingly, the WE of the Antarctic congeners, Metridia gerlachei (Graeve et al., 1994b) and Euchaeta antarctica (Hagen et al., 1995), were characterized by the near absence of calanoid FATM. E. antarctica has been observed to prey on Calanus acutus (Øresland, 1991), and Metridia gerlachei is believed to show similar feeding behavior. Hence, it is not fully understood why long-chain monounsaturates are apparently entirely catabolized in these Antarctic species (Hagen et al., 1995), while retained in the Arctic congeners (Auel, 1999). However, it obviously weakens the potential of these long-chain monounsaturates as calanoid FATM, particularly in the southern hemisphere, due to the uncertainty of their dietary and biosynthetic origin. 286 JOHANNE DALSGAARD ET AL.

Figure 17 Seasonal development in specific WE (A) fatty acids, and (B) fatty alcohols in Metridia longa collected in Balsfjord, northern Norway. Based on data from Falk-Petersen et al. (1988) cited and reproduced in Sargent and Falk-Petersen (1988).

5.2.3. Euphausiids

High levels of 16:1(n-7), C18 PUFA and EPA have been detected in the two major Arctic euphausiids, Thysanoessa inermis and T. raschii (Ackman et al., 1970; Sargent and Falk-Petersen, 1981; Saether et al., 1986; Falk- Petersen et al., 2000; Hamm et al., 2001), indicating that these species feed as herbivores during the Arctic summer. Substantiating this hypothesis, the ingestion of Phaeocystis pouchetii by Thysanoessa spp. has been verified both in the field (Balsfjord) and in the laboratory (T. raschii; Hamm et al., 2001). In addition, analyses of Thysanoessa inermis sampled in autumn in Balsfjord and Ullsfjord revealed increasing proportions of calanoid FATM suggesting a switch in diet to include copepods in the Arctic dark period (Falk-Petersen et al., 2000). However, whereas a low 18:1(n-7)/18:1(n-9) ratio in T. inermis from Kongsfjord, Svalbard also suggested an animal FATTY ACID TROPHIC MARKERS 287 dietary input, low proportions of calanoid FATM indicated that these copepods did not make important contributions to the diet of T. inermis in this area (Falk-Petersen et al., 2000). The seasonal and spatial changes in the FATM pattern of Thysanoessa raschii generally resemble those of T. inermis, though this species stores TAG rather than WE, suggesting a slightly more omnivorous feeding behavior (Falk-Petersen et al., 1981, 2000).

5.2.4. Other zooplankters and nekton

Amphipods perform an essential role in the Arctic, linking sympagic (ice fauna) and pelagic production to higher trophic levels (Scott et al., 1999; Auel et al., 2002). Here, FA analyses have allowed the identification of both interspecific and regional differences in trophic interactions (Falk-Petersen et al., 1987; Scott et al., 1999; Auel et al., 2002). Amphipods are considered opportunistic feeders (Hagen, 1999), and have been observed to store both WE and TAG in varying amounts. Auel et al. (2002) suggested that WE in these animals are stored mainly to provide buoyancy. Intriguingly, the WE often contain considerable amounts of 20:1 and 22:1 MUFA and monounsaturated fatty alcohols. Hence, either amphipods have evolved a mechanism for depositing WE directly from preying on calanoid copepods, or they are capable of biosynthesizing these monounsaturates de novo (Falk- Petersen et al., 1987). If the latter is the case, it seriously undermines the case for the use of these compounds as calanoid FATM. Assuming that amphipods cannot biosynthesize 20:1 and 22:1 de novo, and considering the varying amounts of typical diatom or flagellate tracers, it has been deduced that many of these species, including Themisto libellula, T. abyssorum, Gammarus wilkitzkii, Onisimus nanseni and O. glacialis feed as omnivores (Scott et al., 1999; Auel et al., 2002). Furthermore, on the basis of lower 18:1(n-7)/18:1(n-9) and EPA/DHA ratios in the deeper-living Themisto abyssorum relative to the epipelagic and ice-associated T. libellula, it has been hypothesized that the latter is a secondary consumer whereas T. abyssorum is a tertiary consumer (Auel et al., 2002). Using similar reasoning, Scott et al. (1999), in a study of ice-fauna, suggested that Gammarus wilkitzkii is a secondary consumer while Onisimus spp. are tertiary consumers. FATM have also been applied in studies on the feeding preferences of Arctic ctenophores. Hence, in Raudfjord, Svalbard, Clarke et al. (1987) found a remarkably similar FA composition in the TAG of all three levels of a presumably simple food web comprising: Calanus glacialis - Bolinopsis infundibulum (ctenophore) - Beroe cucumis (ctenophore). However, the FA patterns were not consistent within the WE fraction, which constituted the 288 JOHANNE DALSGAARD ET AL. dominant lipid class in all three species. These observations conform to the hypothesis that TAG represents the recent feeding history of animals whereas WE integrate over a longer period of time (Ha˚ kanson, 1984; Sargent and Henderson, 1986). Alternatively, Bolinopsis infundibulum is not a ‘‘true’’ intermediate link between Calanus glacialis and Beroe cucumis. This hypothesis is proposed based on a closer examination of the data in the paper, revealing a quite similar WE fatty acid composition of Calanus glacialis and Beroe cucumis, i.e., suggesting that Bolinopsis infundibulum is not part of the food web. Support for this hypothesis may be found in the paper by Falk-Petersen et al. (2002), where a close coupling of the FA composition of the NL, mostly WE, between the dominant calanoid copepods Calanus hyperboreus, C. glacialis and C. finmarchicus, the ctenophores Mertensia ovum and Beroe cucumis was found. Based on the presence of calanoid FATM these authors suggested that WE moieties are transmitted unmodified from Calanus spp. via Mertensia ovum to Beroe cucumis. The chaetognath Sagitta elegans is another active carnivore in the Arctic, and high abundances have been observed, e.g., in Balsfjord. S. elegans stores moderate amounts of TAG with a low 18:1(n-7)/18:1(n-9) ratio and high proportions of calanoid FATM (Falk-Petersen et al., 1987), suggesting that it is an important predator of these copepods. Finally, stomach content analyses of different age-groups of the deep- water prawn Pandalus borealis, collected during spring and summer in Balsfjord, revealed very clear ontogenetic changes in diet composition (Hopkins et al., 1993). Age-groups 0–1 were found to consume mostly calanoid copepods whereas older prawns (II–IV) contained remains of euphausiids (Thysanoessa spp.) and scales from capelin (probably from fish discarded by prawn trawlers). These observations were substantiated by FATM showing that the concentration of calanoid FATM was highest in the youngest age-classes, whereas in the more mature prawns, higher proportions of 18:1(n-9), EPA and DHA were found.

5.2.5. Fish

Balsfjord has a large resident population of Thysanoessa inermis and T. raschii, which constitute the major prey of indigenous capelin (Mallotus villosus; Falk-Petersen et al., 1986b). As mentioned in Section 5.2.3, these euphausiids feed primarily as herbivores during the Arctic summer and are therefore relatively deficient in calanoid FATM (Sargent and Falk-Petersen, 1981; Falk-Petersen et al., 1982). This pattern was also reflected in the capelin (Henderson et al., 1984; Falk-Petersen et al., 1987), and was a trait that distinguishes them from offshore Norwegian (Falk-Petersen et al., FATTY ACID TROPHIC MARKERS 289

1986b) and eastern Canadian (Ackman et al., 1969; Eaton et al., 1975) capelin populations (see also summary table by Jangaard, 1974). Furthermore, in capelin caught off Novaya Zemlya, northern Norway, a similar FA signature to that seen in Balsfjord was observed (i.e., low levels of calanoid FATM and elevated proportions of 16:0 and 18:1), consistent with a mixed diet of calanoid copepods and locally abundant Thysanoessa inermis (Falk-Petersen et al., 1986b, 1990). In contrast, large proportions of calanoid FATM were detected in Maurolicus muelleri and Benthosema glaciale, important members of the pelagic fish community in Ullsfjord located adjacent to Balsfjord (Falk-Petersen and Sargent, 1986a; Falk-Petersen et al., 1987). Calanus finmarchicus, C. hyperboreus and the predatory amphipod Themisto abyssorum are common species in this fjord (Falk-Petersen et al., 1986a, 1987), and based on FA signatures are all hypothesized to contribute to the diet of these fish. In another study including FATM in fish, the 16:1(n-7)/16:0 ratio was applied as a food web tracer to clarify the impact of food quantity and quality on the condition of juvenile snail fish (Liparis sp.) off west Greenland (Pedersen et al., 1999). On the assumption that mesozooplankton >400 mm (consisting predominantly of Calanus) constituted the major prey, the 16:1(n-7)/16:0 tracer was observed to follow the same spatial pattern in the fish and in the mesozooplankton (Figure 18). Hence, the ratio of the diatom tracer in the fish increased significantly (T-test, P < 0.001) toward the northern part of the region, and in addition, correlated significantly (T-test, P < 0.001) with the condition of the fish. Concurrent analyses of size-fractionated plankton samples revealed a succession from a hetero- trophic dinoflagellate and nanoflagellate dominated plankton community in the south to a late spring bloom, diatom dominated community in the north, consistent with the withdrawal of sea ice in this area. Intriguingly, the 16:1(n-7)/16:0 ratio did not show a significant south–north trend in the phytoplankton, and it was therefore deduced that the tracer signal in the mesozooplankton in the north (high 16:1(n-7)/16:0, low DHA) originated from a recent diatom bloom (which was not sampled), reflecting a lower turnover rate of FATM with increasing body size (see also Section 4.5). The effect was carried over to the snail fish, whose FA composition suggested that they had been feeding on a flagellate-based food web in the south and a diatom-based food web in the north. The Arcto-Norwegian cod, Gadus morhua, utilizes the Lofoten area, northern Norway, as an important spawning site, and FATM of first- feeding cod larvae have been examined to ascertain the contribution of phytoplankton to their diet (Klungsøyr et al., 1989). Close similarities between the FA compositions of the phytoplankton community, composed primarily of diatoms and Phaeocystis pouchetii, and the cod larvae were 290 JOHANNE DALSGAARD ET AL.

Figure 18 C16:1(n À 7)/C16:0 ratio in (A) mesozooplankton > 400 mm in body size, and (B) for the snail fish Liparis spp. sampled along a transect (65N–72N) off West Greenland in 1993. Reproduced with permission after Pedersen et al. (1999).

found. As the larvae grew, changes in their content of 18:2(n-6) reflected largely that of the phytoplankton. On the other hand, this tracer was relatively absent in copepod nauplii considered an alternative prey, and thus it was concluded that phytoplankton initially constitute the major diet of first-feeding cod larvae. Polar cod (Boreogadus saida), which is typically found in association with sea ice (Scott et al., 1999), is another major predator in the Barents Sea as well as an important prey of marine mammals, birds and fish (Frost and Lowry, 1981, and references therein). Juveniles of this species caught in the marginal ice zone in the Barents Sea (Scott et al., 1999) and Isfjord, Svalbard (Dahl et al., 2000), contained high concentrations of calanoid FATM suggesting a diet containing significant amounts of these copepods, or alternatively, a secondary input through predation on amphipods. This observation was consistent with earlier stomach content analyses where both calanoid copepods and the amphipod Parathemisto libellula were found (Dahl et al., 2000, and references therein). FATTY ACID TROPHIC MARKERS 291

5.2.6. Marine mammals

FATM have also been used to assess the potential prey of white whales (Delphinapterus leucas; Dahl et al., 2000). Comparing blubber FA signatures derived from biopsies of white whales foraging close to Svalbard with FA compositional data of potential prey species by principal component analyses, Dahl et al. (2000) deduced that juvenile polar cod, capelin, Calanus hyperboreus and Pandalus borealis constituted the most likely prey. From observations of feeding behavior and stomach content analyses it was, however, suggested that copepods are not ingested directly but represent a secondary input via predation on polar cod and capelin.

5.3. The Antarctic

5.3.1. Primary producers

A large share of primary production in the Antarctic takes place in the sea- ice. Here, as in the Arctic, the microalgal communities are dominated by diatoms (Fahl and Kattner, 1993; Nichols et al., 1993), although a variety of autotrophic flagellates, particularly Phaeocystis, are also present (Marchant and Thomsen, 1994). Most of the pelagic primary production occurs in the marginal ice zone rather than in the open ocean (Marchant and Thomsen, 1994, and references therein). Pelagic phytoplankton is also composed largely of diatoms superimposed on a background of Phaeocystis and dinoflagellates. Moreover, Phaeocystis spp. typically bloom in the marginal ice zone in the spring prior to the increase in diatoms (Pond et al., 1993; Marchant and Thomsen, 1994; Skerratt et al., 1995; Cripps et al., 1999; Cripps and Atkinson, 2000, and references therein). As in other regions, the phytoplankton biomass in the Antarctic is typified by characteristic signature FA reflecting the prevailing class of microalgae (Section 2.4.3). However, in contrast to the Arctic and as discussed in Section 2.4.3, Phaeocystis spp. in this region are much less rich in (n-3) PUFA, which together with a low 16:1(n-7)/16:0 ratio and an elevated concentration of 18:1(n-9) distinguishes them from diatoms (Skerratt et al., 1995).

5.3.2. Copepods

Herbivorous and omnivorous copepods in the Antarctic deviate in several ways from their Arctic counterparts. Many species such as Calanus propinquus (Hagen et al., 1993; Kattner et al., 1994; Falk-Petersen et al., 1999), C. simillimus and Euchirella rostromagna (Hagen et al., 1995; Ward et al., 1996), store TAG rather than WE. This storage pattern suggests that 292 JOHANNE DALSGAARD ET AL. these copepods feed throughout the year and have evolved a more opportunistic feeding strategy than strictly herbivorous species. In WE- storing copepods, long-chain monounsaturated fatty alcohols are typically replaced either by short-chain saturated fatty alcohols (e.g., Rhincalanus gigas, Graeve et al., 1994b) or the concentration of 20:1(n-9) is higher than that of 22:1(n-11) (Calanoides acutus, Graeve et al., 1994b; Kattner and Hagen, 1995; Falk-Petersen et al., 1999; Section 3.2). FATM are typically less evident in Antarctic copepods as compared to Arctic copepods (e.g., Calanus propinquus, Kattner et al., 1994; Falk-Petersen et al., 1999; C. simillimus and Euchirella rostromagna, Hagen et al., 1995; Ward et al., 1996), though this does not apply to all species. High concentrations of 16:1(n-7) and 18:4(n-3) have, for example, been detected in the dominant circum-Antarctic species, Calanoides acutus (Graeve et al., 1994b). This is illustrated in Figure 15B, which was based on samples of C. acutus from the Weddell Sea. Here, specimens from site III–V contained very high concentrations of 18:4(n-3) probably as a result of the uptake of Phaeocystis, which was the dominant microalgae in these areas at the time of sampling (Kattner and Hagen, 1995, and references therein). Lower levels of 18:4(n-3) combined with elevated concentrations of 16:1(n-7) at site I suggested a higher uptake of diatoms there, whereas the resolution of group- specific phytoplankton contributing to the diet of specimens from site II was less easy to interpret, suggesting a more mixed diet. These observations were supported by data from the Lazarev Sea, where the FA signature of C. acutus indicated extensive feeding on a mixed but probably diatom- dominated phytoplankton diet, with seasonal differences in the uptake of dinoflagellates and Phaeocystis (Falk-Petersen et al., 1999). Fatty acid trophic markers have also been used to resolve the diet composition of the Antarctic copepod Rhincalanus gigas revealing char- acteristics of both a herbivorous and omnivorous feeding behavior (Graeve et al., 1994b; Ward et al., 1996). Hence, the WE fatty acid composition of specimens from the Weddell Sea revealed a mixture of 18:1(n-9), typical of a carnivorous diet, and 16:1(n-7), 18:4(n-3), EPA and DHA indicating additional uptake of phytoplankton (Graeve et al., 1994b). Omnivorous feeding behavior by R. gigas on phytoplankton, detritus and zooplankton has indeed been reported by Arashkevich (1978, in Bathmann et al., 1993). Hence, utilizing FATM, R. gigas has been suggested to be a facultative herbivore able to switch to nonphytoplankton food when algae are scarce. Similarly, the predominance of 16:1(n-7) and 18:1(n-9) in the WE of older, lipid-rich specimens of another Southern Ocean species, Pareuchaeta antarctica, collected in the southeastern Weddell Sea (Hagen et al., 1995), suggested omnivorous feeding behavior by this species as well. Older stages of P. antarctica are, however, known to feed as carnivores consuming only small amounts of phytoplankton (Hopkins, 1987). Hence, whereas the FATTY ACID TROPHIC MARKERS 293 predominance of 18:1(n-9) agreed with conventional feeding studies, the high concentrations of 16:1(n-7) may be explained by indirect ingestion via herbivorous copepods (Hagen et al., 1995). Moreover, FA and fatty alcohols can be subjected to intense restructuring processes and apparently, P. antarctica completely catabolizes any long-chain monounsaturated com- pounds ingested with, e.g., Calanoides acutus or Calanus propinquus (Øresland, 1991). Cripps and Hill (1998) examined the effect of different dietary regimes on the FA (and hydrocarbon) composition of five common Antarctic copepods in addition to the krill Euphausia superba, sampled along a transect from the MIZ to the open water. A principal component analysis of the FA data grouped the copepods into dinoflagellate-feeders, diatom-feeders and omnivores, whereas E. superba formed a group of its own. The dinoflagellate-feeding copepods consisted of Calanoides acutus, Calanus propinquus and Metridia gerlachei, sampled chiefly under the pack-ice. These specimens were all characterized by high levels of DHA and a low 16:1(n-7)/ 16:0 ratio. In the MIZ, Calanus propinquus and Metridia gerlachei had apparently switched to a more omnivorous feeding behavior, as specimens from this sampling location contained higher proportions of 16:0 and 18:1(n-9), while typical microalgal FATM were absent. This was also true of cyclopoid copepods (Oithona spp.), common in the MIZ as well. Diatom feeding copepods were confined to the open ocean and comprised specimens of Calanoides acutus, Metridia gerlachei and Rhincalanus gigas. Diatom FATM were most evident in Calanoides acutus and Rhincalanus gigas, which both contained a 16:1(n-7)/16:0 ratio >1 in addition to high concentrations of EPA. The FA composition of Metridia gerlachei, on the other hand, was quite similar to specimens of this species sampled in the pack-ice. Dinoflagellate markers were indeed present in all three species sampled in the open ocean, indicating that these microalgae, in addition to diatoms, contributed to the diet at this location. In contrast to the copepods, there was no spatial resolution in the FA pattern of Euphausia superba, suggesting a dietary regime and lipid metabolism distinct from the copepods.

5.3.3. Euphausiids

Euphausia superba is a key Antarctic species which predominantly accumulates TAG (but also phosphatidylcholine; Hagen et al., 1996; Mayzaud, 1997). Typical microalgal FATM (16:1(n-7), 18:4(n-3) and EPA) in specimens sampled in the Weddell Sea and Lazarev Sea indicated that E. superba feeds primarily on phytoplankton during the austral spring and summer (Mayzaud, 1997; Hagen et al., 2001; Phleger et al., 2002). 294 JOHANNE DALSGAARD ET AL.

Pronounced ontogenetic differences have, however, been observed in this species as discussed in Section 3.3 (Figure 9A). A comparative study of the FA (and sterol) composition of Euphausia superba, E. tricantha, E. frigida and Thysanoessa macrura collected near Elephant Island was carried out by Phleger et al. (2002). Euphausia superba separated from the other euphausiids containing higher concentrations of 18:4(n-3) as well as higher ratios of 16:1(n-7)/16:0, 18:1(n-7)/18:1(n-9) and EPA/DHA, consistent with a more herbivorous diet than suggested for the other species. However, as discussed in Section 3.4, E. superba is believed to resort to a more omnivorous feeding behavior during nonbloom situations. This hypothesis was reinforced by the absence of typical microalgal tracers in E. superba collected in the waters off South Georgia, accompanied by an increase in the PUFA/SFA ratio (under nonstarving situations; Cripps et al., 1999; Cripps and Atkinson, 2000). A near absence of 20:1 and 22:1 furthermore indicated that calanoid copepods were not an important prey (Price et al. 1988; Atkinson and Snyder, 1997; Cripps et al., 1999), or alternatively, that these monounsaturated compounds were selectively cata- bolized as has been suggested for other omnivorous Antarctic zooplankters. The detection of 20:1(n-9) fatty alcohol in another common Antarctic euphausiid, Thysanoessa macrura, collected in the southeastern Weddell Sea and in the open water off Dronning Maud Land (Hagen and Kattner, 1998; Falk-Petersen et al., 1999) indicated that this species had been feeding on Calanoides acutus. Supporting this hypothesis, Reinhardt and Van Vleet (1986) had observed Thysanoessa macrura to feed on Calanoides acutus. However, the significant concentration of 22:1(n-11) typically found in C. acutus was not reflected in the lipids of Thysanoessa macrura suggesting that it selectively catabolizes this fatty alcohol. Recent evidence suggests that the high-Antarctic ‘‘ice-krill’’, Euphausia crystallorophias, may have evolved an unusual lipid storage strategy. Hence, Falk-Petersen et al. (1999) observed that small specimens of E. crystallorophias collected in the Lazarev Sea contained TAG as their main storage lipid, whereas larger specimens from the same area contained WE as their main storage lipid. In contrast, WE was generally the major depot lipid detected in the whole size range of E. crystallorophias collected in the Weddell Sea (Hagen et al., 1996; Kattner and Hagen, 1998). The lipids of the smaller specimens from the Lazarev Sea were relatively deficient in PUFA whereas they were comparatively rich in SFA and MUFA, and this FA pattern was believed to have originated from the ingestion of decaying and detrital material (supported by the detection of phytol in their WE; Falk-Petersen et al., 1999). In contrast, the WE of the large specimens were composed largely of short-chain fatty alcohols and the FA 18:1(n-9), consistent with earlier findings (Kattner and Hagen, 1998). The high concentration of 18:1(n-9) (>70% of total FA) suggested a predominantly carnivorous feeding behavior. In addition, significant FATTY ACID TROPHIC MARKERS 295 proportions of 18:1(n-7) indicated a considerable uptake of either diatoms or bacteria, although the rather constant ratios between the two 18:1 isomers (between 3 and 4 to 1) may also suggest de novo biosynthesis of these FA (Falk-Petersen et al., 1999). Intriguingly, small amounts of 18:5(n-3) (0.2–1.2% of TL) and very-long- chain PUFA (C24–C28; trace – 0.1% of total FA) were detected in several species of Antarctic euphausiids sampled in 1998 but not in 1997 (Phleger et al., 2002). This was true also of other zooplankters including salps, cnidarians, ctenophores, pteropods and amphipods (up to 5.8% and 5.3%, respectively, of total FA; Phleger et al., 1999, 2000, 2001; Nelson et al., 2000, 2001). As mentioned in Section 2.4.1, trace amounts of 28:7(n-6) and 28:8(n-3) have recently been identified in several species of dinoflagellates (Mansour et al., 1999a, b). Hence, the observations from 1998 suggested that dinoflagellates presented a particularly high contribution to the pelagic Antarctic food web in that year. Unfortunately, no phytoplankton FA data were available for the period, and this hypothesis could not be tested (Phleger et al., 2000).

5.3.4. Other zooplankters

Analyses of the FA composition of several important but often neglected pelagic Antarctic zooplankters including salps, cnidarians, ctenophores, pteropods and amphipods have recently been carried out (Kattner et al., 1998; Phleger et al., 1998, 1999, 2000, 2001; Nelson et al., 2000, 2001). These animals generally do not accumulate large lipid reserves, and hence, FA may be expected to provide only limited information on trophic interactions (Clarke et al., 1987; Phleger et al., 1999, 2001). Fatty acid trophic markers (and sterols) were, however, applied in an attempt to verify the diet of the pteropod Clione limacina. This species is an extreme trophic specialist believed to feed exclusively on the herbivorous pteropod Limacina helicina in polar regions, or L. retroversa in temperate regions (Phleger et al., 1997b; Kattner et al., 1998, and references therein). Very low amounts of 16:1(n-7) in Antarctic Clione limacina suggested an indirect uptake of diatoms via Limacina helicina. In addition, high levels of the more unusual lipid, alkyldiacylglycerol ether (DAGE) comprising considerable amounts of odd-chain FA, were detected in Clione limacina (see Phleger et al., 2001, for review on DAGE in various organisms). These lipids were hypothesized to have been biosynthesized by C. limacina (from propionate derived from phytoplankton dimethyl-sulphoniopropionate (DMSP)), as they were not detected in Limacina helicina (Kattner et al., 1998, and references therein). However, Phleger et al. (2001) alternatively hypothesized that the odd-chain FA came from thraustochytrids, which are 296 JOHANNE DALSGAARD ET AL. common marine microheterotrophs that feed as saprobes or parasites, and which are reported to contain elevated levels of odd-chain FA (Phleger et al., 2001, and references therein). Fatty acids have also been used as more general markers in amphipods and gelatinous zooplankton from this region. The detection of calanoid FATM combined with relatively low 16:1/16:0, 18:1(n-7)/18:1(n-9) and EPA/DHA ratios in several of such species collected in the Elephant Island region of the Antarctic Peninsula suggested a predominantly omnivorous – carnivorous diet (supported also by sterol markers; Nelson et al., 2000, 2001). A single species of cnidarians (Stygiomedusa gigantea) was observed to contain relatively higher ratios of 16:1/16:0 and EPA/DHA than other gelatinous zooplankton (Nelson et al., 2000), indicating that it was feeding of a predominantly diatom-based food web. Finally, a near absence of long-chain monounsaturated compounds in the TAG of the common Antarctic hyperiid amphipod Themisto gaudi- chaudi was probably due to a commensalistic relationship with gelatinous zooplankton such as salps and jellyfish (Nelson et al., 2001). In contrast, and as discussed in Section 5.2.4, its Arctic congeners, T. abyssorum and T. libellula, often contain large amounts of calanoid FATM (Auel et al., 2002).

5.3.5. Fish

Research in the Antarctic has also employed FATM to examine feeding relationships in fish. Here, enhanced proportions of calanoid FATM (6–15% of the total FA) in two pelagic (Aethotaxis mitopteryx, Pleuragramma antarcticum) and one benthopelagic (Trematomus lepidorhi- nus), Antarctic notothenioid fish species suggested an intake of both Calanoides acutus and Calanus propinquus (Hagen et al., 2000). This was supported by the detection of the 22:1(n-9) isomer, unique to C. propinquus. Additionally, high concentration of 18:1(n-9) suggested that these fish potentially also feed on other important copepods (e.g., Rhincalanus gigas, Metridia gerlachei, Euchaeta antarctica) and euphausiids (e.g., Euphausia superba, E. crystallorophias and Thysanoessa macrura; Hagen et al., 2000). Bottom-dwelling notothenioid fish, such as Bathydraco marri and Dolloidraco longedorsalis, are known to feed primarily on benthic invertebrates (Hagen et al., 2000, and references therein). Consistent with this, these species were found to contain higher proportions of EPA, DHA and particularly AA in their PL as compared to pelagic species (Hagen et al., 2000; see also Graeve et al., 1997 for the FA composition of Arctic benthos). However, small amounts of calanoid FATM suggested that FATTY ACID TROPHIC MARKERS 297 copepods may also form part of the diet. Moreover, higher concentrations of 20:1 than 22:1 indicated that Calanoides acutus rather than Calanus propinquus forms part of the diet, conforming with the vertical distribution pattern of these copepods (Hagen et al., 2000). Remarkably high levels of monoenoic fatty alcohols (37–90% of total fatty alcohols) and FA (37–88% of total FA), comprising mainly 18:1(n-9), 22:1 and 20:1, were also found in lipid rich myctophids (lantern fish) caught in the northern sub-Arctic Pacific (Saito and Murata, 1996, 1998; Seo et al., 1996) and in the Antarctic (Phleger et al., 1997a). Consistent with these findings, remains of copepods and other crustaceans have been recognized in the stomachs of myctophids from the northern Pacific (Saito and Murata, 1998), whereas amphipods, copepods and euphausiids (Thysanoessa macrura) comprise the major prey of the Antarctic myctophid Electrona antarctica (Phleger et al., 1997a, and references therein). Interestingly, it has been suggested that myctophids in general, and in contrast to northern hemisphere zooplanktivorous species, incorporate dietary lipids directly, including zooplankton WE (Saito and Murata, 1996, 1998). If that is the case, FATM may prove a very valuable tool for resolving trophic interactions in these species.

5.3.6. Marine mammals

As will be discussed in Section 5.4.6, FATM have been employed to distinguish Antarctic and northern Atlantic finbacks (Borobia et al., 1995). In the Antarctic, FATM have also been applied to examine the feeding dynamics of Antarctic fur seals (Arctocephalus gazella) during nurturing. The females remain ashore suckling their pups for a short period (perinatal fasting period), before they start making intermittent foraging trips to the sea (Iverson et al., 1997a, and references therein). Hence, whereas the FA signature of milk secreted during the perinatal period is derived from blubber mobilization, the milk FA in the subsequent foraging period is derived largely from the diet (Iverson, 1993; Iverson et al., 1997a, and references therein). Consistent with this, large differences in the milk FA composition were observed when comparing the two periods in lactating fur seals from South Georgia (Iverson et al., 1997a). High levels of 18:1(n-9), 20:1(n-9) and 22:1(n-11) in milk secreted during the perinatal period indicated that the seals had been preying on fish in a different geographical location prior to returning to the breeding ground. In the initial foraging period, this pattern changed to suggest the consumption of Euphausia superba. A second shift in the FA pattern was observed later in the lactating period consisting of a large increase in the proportion of 20:1(n-9) and 22:1(n-11), indicating a switch in diet from euphausiids to 298 JOHANNE DALSGAARD ET AL. myctophids. These observations were supported by faecal analyses and other independent evidence showing that the availability of krill was greatly reduced within this particular period (austral summer 1990–1991; Iverson et al., 1997a). Similar observations were made by Brown et al. (1999). In this study the FA signatures of milk secreted by lactating Antarctic fur seals and Southern elephant seals (Mirounga leonina) were compared with potential prey species using CART and cluster analyses. The analyses generally confirmed the hypothesized switch in diet of fur seals in 1990–1991. The nature of the diet in the second half of the period could, however, not be established as the milk samples did not cluster with any of the potential prey species sampled and included in the analyses. On the other hand, samples from 1992 and 1993 clustered predominantly with krill and krill-eating fish, giving no indications of a switch in diet in these years. The FA signature of milk secreted by elephant seals indicated that they had been foraging on fish that do not prey on krill (e.g., larger notothenioids and myctophids), thereby resolving that the two species of seals utilize very different diets. Elephant seals, in contrast to fur seals, remain on land while suckling their pups and consequently, the milk FA during the whole nurturing period reflects the dietary intake during the previous fattening period (Brown et al., 1999).

5.4. Northwest Atlantic

5.4.1. Primary producers

Consistent temporal changes in the particulate FATM composition have been measured all over the northwestern Atlantic (Bedford Basin, Mayzaud et al., 1989; Georges Bank, Napolitano and Ackman, 1993; Newfoundland, Parrish et al., 1995; Napolitano et al., 1997; Budge and Parrish, 1998; Budge et al., 2001). In this system, the spring bloom is usually dominated by diatoms (confirmed by microscopic analyses; Parrish et al., 1995; Budge and Parrish, 1998; Budge et al., 2001) and an associated elevated level of diatom markers, i.e., 16:1(n-7)/16:0, ÆC16/ÆC18 and 16:4(n-1) (Figure 19A). Mayzaud et al. (1989) established that the spring bloom in Bedford Basin terminated on depletion of nutrients and was replaced by relatively larger detrital particles (64.0–101.6 mm), associated with a mixture of SFA, MUFA and typical bacterial FATM (iso and anteiso-FA). In addition, Parrish et al. (1995) found that this period was accompanied by a large increase in the abundance of ciliates and tintinnids and a smaller peak in nanoflagellates, establishing the potential for a microbial loop food web. However, except for 18:5(n-3) and 20:4(n-6) in the polar lipid fraction of the FATTY ACID TROPHIC MARKERS 299

Figure 19 Values of various FA indicators in net-tows collected during a spring bloom in Trinity Bay, Newfoundland in 1996 (mean Æ S.D., n ¼ 9). (A) diatom indicators, and (B) dinoflagellate and bacterial indicators (the latter equal to the sum of 15:0, 17:0 and all iso and anteiso-branched chain FA expressed as percent of total FA). Redrawn with permission after Budge and Parrish (1998). microzooplankton, no significant correlations with typical microalgal FATM within this period were detected. Later in the summer, a second bloom composed of small (2.0–6.4 mm) dinoflagellates and flagellates usually develops, characterized by increasing proportions of C18 FA and DHA (Mayzaud et al., 1989; Parrish et al., 1995). 300 JOHANNE DALSGAARD ET AL.

Consistent with the temporal development in the phytoplankton community summarized above, Budge and Parrish (1998) observed that the DHA/EPA ratio in Trinity Bay, Newfoundland, was at a minimum throughout the spring bloom (Figure 19B), further reflecting the changes in dominance, prior to and after the spring bloom when dinoflagellates and flagellates were more prevalent. Interestingly, in one year, the occurrence of a dinoflagellate maximum was completely masked by a coinciding diatom bloom (Budge et al., 2001). This observation demon- strated that FATM of plankton samples reflect the dominant microalgal group. To obtain higher resolution, e.g., if the objective of the study is to characterize the algal group composition of the phytoplankton community, or to identify potential prey preferences of various grazers, size-fractionated plankton samples must be obtained and analyzed separately (e.g., St. John and Lund, 1996).

5.4.2. Euphausiids

The euphausiids Meganyctiphanes norvegica and Thysanoessa inermis are very abundant off Nova Scotia (Ackman et al., 1970), where they constitute an important prey for fish and marine mammals (Ackman and Eaton, 1966a). Meganyctiphanes norvegica has a wide distribution, ranging from the Mediterranean Sea (Section 5.7) to the Arctic Ocean (Virtue et al., 2000). In boreal waters, the FA of this species contain lower levels of phytoplankton FATM as compared, e.g., to Thysanoessa inermis. In contrast, calanoid FATM are usually highly prevalent in Meganyctiphanes norvegica from this region (but see Section 5.7). These observations are consistent with data on the feeding ecology of this species, which is known to feed preferentially on calanoid copepods (Sargent and Falk-Petersen, 1981; Virtue et al., 2000). Thysanoessa inermis, on the other hand, is a boreal-Arctic species storing large amounts of WE with a FA and fatty alcohol composition suggestive of a more herbivorous diet as discussed in Section 5.2.3. The differences between Meganyctiphanes norvegica and Thysanoessa inermis were already established in the late 1960s when Ackman et al. (1970) reported on the FA composition of the two species collected from stomachs of finbacks (Balaenoptera physalus) captured off Nova Scotia.

5.4.3. Other zooplankters

A highly unusual FA pattern consisting of large concentrations of odd-chain FA (chiefly 15:1 and 17:1) were observed in smelt (Osmerus mordax)in Jeddore Harbour, Nova Scotia, and were coupled to the consumption of the FATTY ACID TROPHIC MARKERS 301 amphipod Pontoporeia femorata (Paradis and Ackman, 1976). This amphipod contains extremely high levels of these FA (50%), and stomach content analyses of smelt moving into the harbor prior to their spring spawning runs revealed that they had been preying heavily on P. femorata. The conservative propagation of the odd-chain FA through this short food web was substantiated by a nearly identical distribution of monoethylenic isomers in the amphipod and the fish, whereas the isomeric ratio of more common even-chain FA was quite different. Intriguingly, high levels of similar odd-chain FA (i.e., 15:0 and 17:1(n-8)) have later been reported for Clione limacina (pteropod; Kattner et al., 1998), which is an extreme trophic specialist as discussed in Section 5.3.4. Since the prey of C. limacina con- tained only traces of these odd-chain FA, and because of the close trophic coupling, it was deduced that C. limacina biosynthesize these FA de novo (Kattner et al., 1998), or alternatively, obtain them from thaustochytrids (Microheterotrophs; Phleger et al., 2001). The situation may be similar for Pontoporeia femorata, however, this remains to be examined. Another less well studied organism, which periodically occurs in very large abundances in the North Atlantic, is the tunicate Dolioletta gegenbauri. This species is known to graze on a wide variety of microplankton ranging from bacteria to diatoms, and is believed to contribute significantly to the diet of many larval fish (Pond and Sargent, 1998, and references therein). However, being gelatinous, this prey is difficult to detect in stomach content analyses and here, FA may provide additional information. Free-swimming, sexual stages of D. gegenbauri sampled in the western Atlantic off central America   (58 W, 20 N) contained high concentrations of EPA, DHA and C18 PUFA. This FA pattern was consistent with a primary producer community dominated by coccolithophores and smaller contributions of diatoms, dinoflagellates, flagellates and picoplankton (Pond and Sargent, 1998). On the basis of the high growth and mortality rates observed in D. gegenbauri,it was hypothesized that the tunicates sediment rapidly to the deep ocean, bringing with them large amounts of labile PUFA to the benefit of bathypelagic and deep-sea benthic ecosystems.

5.4.4. Macrobenthos

Measuring the organ-specific FA composition of the sea scallop Placopecten magellanicus, a major local primary consumer in Trinity Bay, Newfoundland, Napolitano et al. (1997) found that the digestive gland (which is the major NL storage site and is composed of 60% TAG) exhibited a series of compositional shifts, reflecting the temporal develop- ment in the phytoplankton. In this study, the digestive gland prior to the spring bloom was characterized by dinoflagellate- and flagellate-specific 302 JOHANNE DALSGAARD ET AL.

FATM (18:1(n-9), 18:4(n-3) and DHA), which were partly replaced during the spring and post-bloom period by diatom-specific FATM (16:1(n-7), 16:1(n-4) and EPA). Similar patterns were recognized in the digestive gland and in the gut content of the scallop Placopecten magellanicus from Georges Bank, Nova Scotia (Napolitano and Ackman, 1993). Here, maximum concentrations of C16 PUFA (mostly 16:4(n-1)) and EPA also coincided with the diatom dominated spring bloom, while a smaller increase in 18:4(n-3) in addition to a marked increase in the proportion of DHA occurred in the fall, coinciding with a dinoflagellate and flagellate-dominated fall bloom. These findings were supported by the trend in the polyunsaturation index (the summed products of PUFA weight percentages >1 multiplied by the number of double bonds) measured in the digestive gland. Hence, this index increased from summer to fall, consistent with an intensive feeding on particulate matter rich in AA, EPA and DHA. It was followed by a dramatic decrease from fall to winter reflecting the mobilization of TAG reserves from the digestive gland to the maturing gonads. Based on the presence of typical algal FATM, combined with an overall lack of typical bacterial FATM both in the gut content and in the digestive gland, it was deduced that the supply of photosynthetically produced organic matter on Georges Bank was sufficient to sustain the scallop population throughout the year (Napolitano and Ackman, 1993). Comparable temporal patterns in the FA composition were also observed in the tissue of the blue mussel, Mytilus edulis, from Notre Dame Bay, Newfoundland (Budge et al., 2001). Here, the level of AA was five-fold greater than in the phytoplankton, indicating a selective retention of this FA by the mussels. Moreover, 18:5(n-3) was not detected in mussel tissues despite significant concentrations in the phytoplankton presumed to comprise the bulk of their diet. On this basis it was hypothesized that 18:5(n-3) was chain-elongated to EPA, and the potential of employing 18:5(n-3) as a specific dinoflagellate tracer at higher trophic levels was dismissed. In contrast, Mayzaud (1976) had earlier applied 18:5(n-3) as a specific dinoflagellate tracer to a natural plankton community in Bedford Basin, Nova Scotia. In that study, the FA was observed to decrease by roughly a factor of 10 for each trophic level in a ‘‘linear food web’’ consisting of microalgae (9% of PL fatty acids) – copepods (2% of TAG fatty acids) – chaetognaths (0.1% of TAG and WE fatty acids).

5.4.5. Fish

The impact of frontal primary production on the condition of juvenile cod (Gadus morhua) and haddock (Melanogrammus aeglefinus) on Georges Bank FATTY ACID TROPHIC MARKERS 303 has recently been assessed using FATM (Storr-Paulsen et al., 2003). In this study, a significant positive correlation between larval condition and the specific diatom tracer 20:5(n-3)/18:4(n-3) suggested that utilization of a diatom-based food web contributed to enhanced larval condition. In contrast, a significant negative correlation between larval condition and the specific flagellate tracer C18 PUFA/total FA indicated that larvae trapped in areas of flagellate-dominated primary production experienced sub-optimal feeding conditions. These observations are consistent with earlier findings on juvenile cod and sandlance in the North Sea (St. John and Lund, 1996; Møller et al., 1998; Section 5.5.3) and juvenile snail fish off West Greenland (Pedersen et al., 1999; Section 5.2.5). The inter- and intraspecific variability in the FA signature of 28 species of fish and invertebrates from the Scotian Shelf, Georges Bank and the southern Gulf of St. Lawrence has also recently been assessed (Budge et al., 2002). In this study, a CART analysis successfully classified 89% of the samples, demonstrating that FA, besides containing information on diets, have the potential to resolve between species based on species-specific FA compositions. A discriminant analysis separated the 16 species with sufficient sample sizes into three distinct groups likely to share similar feeding strategies (Figure 20). The groups separated were the Pleuronectidae (American plaice, yellowtail flounder, winter flounder), small planktivorous fish (capelin, herring, northern sandlance) and a third group consisting mostly of Gadidae (cod, haddock, pollock, silver hake, white hake), but also including redfish, ocean pout, longhorn sculpin and shrimp. Shrimps were believed to cluster with Gadidae as they comprise a large fraction of the diet of this group, resulting in similar FA compositions. Capelin, herring and northern sandlance separated from the other two groups by the first discriminant function defined primarily by 22:1(n-11) and 20:1(n-9). These results are suggestive of a zooplanktivorous diet and are supported by previous FA analyses of these species from the same region (e.g., capelin, Ackman et al., 1969; sandlance, Ackman and Eaton, 1971; Jangaard, 1974; Eaton et al., 1975; Pascal and Ackman, 1976; capelin, herring and mackerel, Ratnayake, 1979; Ratnayake and Ackman, 1979). Significant size-related changes in the FA composition were also observed in several species from this region, and were consistent with reported stomach content analyses. Moreover, in all species with statistically significant sample sizes, there was a significant effect of the sampling location on the FA signature. As discussed by the authors, such findings may be attributed to broad-scale differences in prey assemblages in the northwest Atlantic and ultimately to subtle geographical differences in primary production (Budge et al., 2002). 304 JOHANNE DALSGAARD ET AL.

Figure 20 Discriminant analysis of FA compositional data of 16 common species of fish and invertebrates from the Scotian Shelf, Georges Bank and the Gulf of St. Lawrence. The plot shows the average scores of the first two of 15 discriminant functions that classified individuals to species with a 98% success rate. Ellipses surround the three major clusters of groups and are based on the data point clouds of individuals. Reproduced with permission after Budge et al. (2002).

5.4.6. Marine mammals

A comparative analysis on the blubber FA composition of sympatric populations of finbacks (Balaenoptera physalus) and humpbacks (Megaptera novaeangliae) from the Gulf of St. Lawrence was carried out by Borobia et al. (1995). Blubber FA data from earlier studies on finbacks from the Antarctic, Nova Scotia and a single sample from Spain were incorporated in the analysis as well, as was data on stable carbon isotope ratios. Calanoid FATM clearly separated the northern hemisphere baleens from Antarctic finbacks. Consistent with these finding, Antarctic finbacks are known to feed heavily on Euphausia superba, which are relatively deficient in long-chain MUFA (Section 5.3.3; Ackman and Eaton, 1966a). Furthermore, Gulf of St. Lawrence humpbacks separated from finbacks in the same area on the basis of higher than average levels of EPA and DHA. Based on this and a slightly more depleted stable carbon isotope ratio in humpbacks as compared to finbacks, it was deduced that the humpbacks fed slightly lower in the food web than finbacks. FATTY ACID TROPHIC MARKERS 305

5.5. The North Sea

5.5.1. Primary producers

The temporal dynamics of primary production in this area is similar to that in the northwest Atlantic (Section 5.4.1). In a study on Fladen Ground, northern North Sea, Kattner et al. (1983) performed one of the first systematic determinations of the particulate FA composition during the course of a natural spring plankton bloom (but see also Jeffries, 1970). A clear relationship between the species composition of microalgae and the FA profile of the particulate matter was found. The initial bloom was dominated by diatoms and was associated with peak proportions of 14:0, C16 FA, 18:4(n-3), EPA and DHA as illustrated in Figure 21. The bloom was terminated with the exhaustion of nutrients and was shortly followed by a second, smaller bloom consisting mostly of dinoflagellates, which was accompanied by a temporary increase in the proportions of C18 FA and 22:6 (Figure 21B). In another study on the coupling between FATM and larval and juvenile cod, St. John and Lund (1996) examined the distribution of phytoplankton and their associated FA composition across a frontal system in the northern North Sea. Here, in situ, size-fractionated plankton samples analyzed for phytoplankton species and concurrent FA composition, verified the co-occurrence of diatom and dinoflagellate species and their representative FATM across a tidal mixing region.

5.5.2. Copepods

In general, little information exists on the lipid and FA composition of small copepods either in this or other regions, although they can be important members of zooplankton communities (Schnack et al., 1985; Morales et al., 1991). The majority of small zooplankters, such as Acartia, Pseudocalanus, Temora and Centropages from temperate regions are omnivorous and their feeding behavior is tightly coupled to food availability. This was shown, e.g., by Cotonnec et al. (2001) who, using a combination of phytoplankton pigments and FA, found that Temora longicornis, Acartia clausi and Pseudocalanus elongatus all had consumed large quantities of low quality Phaeocystis during a Phaeocystis dominated spring bloom in the English Channel. In addition, the specific diatom marker 16:1(n-7) and the PUFA, EPA and DHA have been detected in specimens sampled in the southern North Sea and Wadden Sea, and may give some indication of seasonal variations in food availability (Kattner et al., 1981). 306 JOHANNE DALSGAARD ET AL.

Seasonal changes in the FA composition of the omnivorous copepod Calanus finmarchicus, sampled in the North Sea, generally followed the seasonal pattern in phytoplankton dominance, i.e., high levels of C16 FA and EPA were observed in the spring reflecting the dominance of diatoms, changing to higher concentrations of C18 FA during summer as indicative of increased flagellate production (Kattner and Krause, 1989). In conjunction with a Phaeocystis bloom in 1984, particularly high concentrations of 18:4(n-3) were detected in Calanus finmarchicus, suggesting that they were feeding of this bloom (Kattner and Krause, 1987). Similar FA patterns have

Figure 21 Temporal development in the mean concentration (filled circles) and percentage (open circles) of individual FA in particulate matter sampled above the thermocline during a plankton spring bloom in the Fladen Ground area, the North Sea, 1976. Redrawn with permission after Kattner et al. (1983). FATTY ACID TROPHIC MARKERS 307

Figure 21 Continued. also been observed in the closely related but more temperate species, C. helgolandicus, sampled in the eastern North Sea (Kattner and Krause, 1989). Together, these observations support the hypothesis that the foraging by C. finmarchicus and C. helgolandicus is closely coupled to the seasonal phytoplankton production. In comparison with Calanus finmarchicus and C. helgolandicus, Kattner and Krause (1989) found a significantly different FA composition in Pseudocalanus elongatus. These observations were attributed to a different 308 JOHANNE DALSGAARD ET AL. feeding strategy utilized by P. elongatus, as this species is known to consume large amount of detritus (Kattner and Krause, 1989, and references therein). A high concentration of 18:1(n-9) both in P. elongatus (see also Cotonnec et al., 2001) and in the particulate FA was observed in this study, and thereby proposed to confirm the utilization of detritus by this species (Kattner et al., 1983).

5.5.3. Fish

The links between phytoplankton class composition, copepod consumption and larval fish growth and condition in the North Sea have also been examined using FATM (St. John and Lund, 1996; Møller et al., 1998). Here, the enhanced condition of juvenile North Sea cod was linked to diatom production in frontal regimes, using the 16:1(n-7)/16:0 ratio as a food web tracer. Juvenile cod with a higher-than-average-tracer content suggestive of a diatom-based food web were found to be in significantly better condition than fish with a lower tracer content indicative of a flagellate-based food web (Figure 11). Similar findings have been made for larval and juvenile sandlance using 20:5(n-3)/18:4(n-3) as a specific diatom tracer (Møller et al., 1998). Larvae with a higher than average tracer content, indicative of a diatom-based food web and hence a frontal mixing regime, were larger and in better condition than predicted from the size-specific mean of the population.

5.6. Gulf of Alaska

5.6.1. Primary producers

Unfortunately, there is a lack of FATM related studies with focus on lower trophic levels in this region. Intriguingly, however, research on higher trophic levels (Iverson et al., 1997b, 2002) has revealed that in contrast to food webs in the northern Atlantic, 20:1(n-11) is more abundant than 20:1(n-9). This ‘‘unusual’’ isomer ratio has been observed in species of Neocalanus (Saito and Kotani, 2000), and has been recognized all the way up to harbor seals (Phoca vitulina), indicating that the FA composition at the base of the food web is very different in the two regions (Iverson et al., 1997b).

5.6.2. Fish

A few studies on the FA composition of secondary and higher order consumers in the Gulf of Alaska have recently been carried out (Iverson FATTY ACID TROPHIC MARKERS 309 et al., 1997b, 2002). In one study, 22 common species of forage fish and invertebrates were sampled within Prince William Sound (PWS) over a four year period (1994–1998). The species were readily distinguished by their total FA composition via a CART analysis (92% classified correctly; Iverson et al., 2002). Species with partly overlapping diets such as walleye pollock (Theragra chalcogramma), Pacific herring (Clupea harengus pallasi) and Pacific sandlance (Ammodytes hexapterus) were, however, less success- fully classified. These observations were supported by a discriminant analysis in which the three species tended to cluster together on a plot of the first two discriminant functions. Flatfishes, which presumably also share a similar diet and life history, constituted another cluster. Ontogenetic changes in specific dietary FATM (14:0, 20:1(n-11), 22:1(n-11), EPA, DHA) were also observed in this system. Hence, herring showed a shift in FA composition commensurate with a dietary switch from zooplankton in earlier life stages to a more piscivorous diet as the fish grew larger, an observation consistent with stomach content analyses. Similar changes have previously been reported for both herring and pollock in PWS (see below, Iverson et al., 1997b), and more lately for several species of fish in the northwest Atlantic (Section 5.4.5). Finally, unusually high levels of 20:1(n-11) and 22:1(n-11) were found in young herring, pollock and sandlance sampled in the spring and summer 1995/1996. These changes in FA composition were attributed to a more highly stratified ocean surface layer contributing to a reduced biomass of calanoid copepods in these two years, an occurrence which was hypothe- sized to have forced a dietary shift in the young fish (Iverson et al., 2002).

5.6.3. Marine mammals

In a study of harbor seals (Phoca vitulina) from this system, Iverson et al. (1997b) employed a CART analysis on blubber FA. The analysis readily classified the seals according to region (PWS, Kodiak Island, Southeast Alaska) and even specific haulout sites within PWS, suggesting site-specific diets (Iverson et al., 1997b). Moreover, herring and pollock were classified according to size (length) and sampling location in a CART analysis on the FA composition of potential prey, and the authors commented: ‘‘One result of these findings is that given a fatty acid composition of an unknown herring or pollock, one could essentially determine its size-class and location within the study area with reasonable certainty... This could provide an important tool for studying foraging ecology and stock structure of fish species’’. In a preliminary analysis, the FA data of the seals were subjected to the classification rules derived from the FA composition of their potential prey. Intriguingly, the seals separated into two groups suggesting possible prey 310 JOHANNE DALSGAARD ET AL. differences. Hence, seals from the southern PWS and Kodiak Island grouped with yellowfin sole and larger herring and pollock, whereas seals from the northern and eastern part of PWS and southeastern Alaska grouped with smaller herring and pollock, smelt, sandlance, cod, octopus, squid and shrimp (Iverson et al., 1997b).

5.7. Mediterranean

5.7.1. The microbial loop

Detailed research on the temporal and spatial FATM dynamics of primary production in this system is presently limited. However, the trophodynamics of an oligotrophic food web in the coastal Ligurian sea, Villefranche- sur-Mer Bay was examined by Claustre et al. (1988) using FATM. Characteristic seasonal patterns in FA distributions were observed within the 53–100 mm plankton size-fraction. Here, a bloom of the tintinnid (ciliate) Stenosemella ventricosa was observed in late March–April (Figure 22) and was associated with increasing proportions of 18:5(n-3), Br20:0, 18:1(n-7)/ 18:1(n-9) and (isoC15:0 þ anteisoC15:0)/C15:0 (Figure 23). These observations suggested that the tintinnids were feeding on small autotrophic flagellates

Figure 22 Temporal variations in the composition of major microplanktonic groups encountered at a standard oceanographic station at the entrance to the bay of Villefranche-sur-Mer (404101000N, 7190000E) from 11 March to 30 May 1986. Redrawn with permission after Claustre et al. (1988). FATTY ACID TROPHIC MARKERS 311

Figure 23 Temporal changes in selected FA and FA criteria of the micro- planktonic community illustrated in Figure 22. A:16:1/16:0. B: ÆC16/ÆC18. C:C18:5 (expressed as the percentage of total identified FA). D: BrC20:0 (expressed as the percentage of total identified FA). E: C18:1(n À 7)/18:1(n À 9). F:(iso- 15:0 þ anteiso-C15:0)/C15:0. Redrawn with permission after Claustre et al. (1988). and bacteria associated with detritus. The transfer of bacterial FATM through ciliates to copepods was later verified in a controlled laboratory experiment (Ederington et al., 1995), discussed in Section 2.5.1. The tintinnid bloom was temporally replaced by diatoms in late April–early May, and conforming to typical diatom FATM, the ratio of 16:1(n-7)/16:0 increased from <1 to >4 and the ratio of ÆC16/ÆC18 from <2 to >7 (see also Claustre et al., 1989).

5.7.2. Euphausiids

The euphausiid Meganyctiphanes norvegica is at its southern limit of distribution in the Mediterranean Sea and, as inferred from its FA composition, seems to be feeding more opportunistically than its higher latitude counterparts, presumably a trait evolved to cope with the oligotrophic conditions in the Ligurian Sea (Mayzaud et al., 1999; Virtue et al., 2000). Thus, higher flagellate-dinoflagellate signals (i.e., a low 16:1(n-7)/16:0 ratio and relatively high proportions of C18 PUFA and DHA) were detected in the Mediterranean species compared to those from the 312 JOHANNE DALSGAARD ET AL.

Clyde Sea and Kattegat, which contained higher diatom signals (a high 16:1(n-7)/16:0 ratio and high EPA). The latter also contained higher concentrations of calanoid FATM suggesting that they were relying heavily on copepods. In contrast, M. norvegica from the Ligurian Sea contained significantly lower concentrations of long-chain MUFA, although copepods from this area were also relatively deficient in these compounds. Hence, 20:1 and 22:1 cannot be used as copepod FATM in this area (Virtue et al., 2000). Mayzaud et al. (1999) emphasized that one should exercise caution when interpreting FATM in omnivorous species such as M. norvegica. They wrote: ‘‘To be of practical use under natural conditions, fatty acid tracers in omnivorous species should at least be present at concentrations higher than 1% of the total fatty acids (below that the tracer is likely to be a contaminant from ingested grazers) and display over time a pattern coherent with that of the food supply’’.

5.8. Upwelling and sub-tropical/tropical systems

There are comparatively few studies on the dynamics of lipids and FA in food webs from lower latitude temperate and tropical regions despite the fact that these areas comprise the world’s largest pelagic fisheries, centered on major upwelling systems (e.g., Cushing, 1989; Kiørboe, 1993). These systems are regions of highly turbulent mixing and are generally dominated by diatoms, which are consumed either directly by the major fish stocks in the region (i.e., Peruvian anchovy) or by meso- and macrozooplankton, which are then consumed by fish predators. The application of FATM has primarily focused on identifying the feeding ecology of zooplanktivorous fish. As a result, it has been determined that planktivorous fish from northwest African waters are typically rich in DHA and particularly EPA, whereas they contain only traces of calanoid FATM (e.g., Njinkoue´ et al., 2002), reflecting their closer association to the base of the food web (reviewed by Sargent et al., 1989; Sargent and Henderson, 1995). Low levels of fat (<2% of wet mass) combined with high levels of (n-3) PUFA, (n-6) PUFA (particularly AA) and trace amounts of calanoid FATM are common traits of low latitude fish species apart from upwelling systems, as shown for Malaysian and temperate – tropical Australian coastal species (Gibson, 1983; Gibson et al., 1984; Evans et al., 1986; Dunstan et al., 1988). Such observations are consistent with the more regular food supply experienced by these species, and therefore, the absence of need for them to accumulate large lipid reserves. Dunstan et al. (1988) noted that macroalgae from Australian waters are grazed directly by omnivorous and herbivorous fish species. This observa- tion was supported using FATM, as the authors found that omnivorous FATTY ACID TROPHIC MARKERS 313 teleosts could be differentiated from carnivorous teleosts by higher concentrations of AA, EPA and a lower (n-3)/(n-6) ratio, suggesting that they were feeding partly on macroalgae. Among the cartilaginous species examined in the same study, highest levels of AA were found in Port Jackson sharks (Heterodontus portusjacksoni), which presumably feeds on macroalgae via predation on sea urchins and snails. Lowest concentrations of AA were found in piked dogfish (Squalus megalops), which feeds primarily on cephalopods believed to rely on a microalgal-based (and hence low AA) food web. Subjecting FA compositional data of either black bream (Acanthopagrus australis) or red fish (Centroberyx affinis) to principal component analyses, Armstrong et al. (1994) found distinct seasonal clusters on the scatter plots of the first two principal components (explaining 59.5% and 64.6%, respectively, of the total variance). Corresponding bi-plots revealed that specimens caught in spring correlated positively with (n-3) and (n-6) PUFA, whereas those caught in autumn correlated positively with SFA and MUFA. These results were consistent with a higher concentration of storage lipids in specimens caught in autumn at the end of the feeding season, whereas specimens caught in spring had used up their lipid reserves, and as a consequence, contained relatively higher proportions of PL rich in PUFA. Supporting this hypothesis, the FA composition of John dory (Zeus faber) and ling (Genypterus blacodes) failed to reveal similar seasonal clusters, consistent with the lack of seasonal lipid accumulation in these species. Finally, when comparing the 22:1(n-11) to 20:1(n-9) fatty alcohol ratio in specimens of orange roughy (Hoplostethus atlanticus) and deep-sea oreo (Oreosomatidae) from Australian waters with that of their north Atlantic counterparts, significant differences were observed reflecting different FA compositions at the base of the food web in the two regions (Bakes et al., 1995). Hence, in specimens from the north Atlantic the ratio ranged from 1.4 to 2.2 indicating a significant dietary contribution from calanoid copepods. In contrast, the ratio was much lower in the Australian specimens, ranging from 0.2 to 0.9 and consistent with the relatively lower concentration of the two monounsaturates in copepods from the southern hemisphere (Section 5.3.2).

6. SUMMARY AND CONCLUSIONS

6.1. State-of-the-art

In a sense, the state-of-the-art in the field of FATM research remains at the level indicated by Sargent (1976) over 25 years ago: ‘‘At the present 314 JOHANNE DALSGAARD ET AL. state of our knowledge it would appear that fatty acid analyses represent a rather blunt tool in defining food chain inter-relationships. Until further knowledge is accumulated it would appear best to apply fatty acid analyses as a corroborative method to support prey–predator relationships already indicated on independent grounds, such as the analyses of stomach contents’’. Fraser et al. (1989) later added that: ‘‘to clarify the transfer of lipid biomarkers up the food web, the availability of tracer lipids in algae and the zooplankton prey of larval fish must be established before these tracers may be employed either quantitatively or qualitatively’’. St. John and Lund (1996), while recognizing the statement of Fraser et al. (1989), were more specific about problems concerning the quantitative application of FATM. They stated: ‘‘these biomarkers may be suitable as a qualitative index of utilization of a specific food source in field studies, however, quantitative estimates of transfer between trophic levels in the field may prove to be difficult for a number of reasons. It is evident that a better understanding of the dynamics of lipid incorporation and utilization with respect to environmental conditions such as temperature, light and nutrients in phytoplankton as well as during ontogeny in zooplankton and larval and juvenile fish is required before these biomarkers may be used quantitatively’’. With the recommendations of these authors in mind, it is clear that to quantify relationships using FATM, information would need to be available on a number of aspects of the dynamics of FA in marine animals, including not least, time scales for incorporation of new FA signatures into tissues. This has been examined in a few laboratory studies of copepods, larval and adult cod (Graeve et al., 1994a; St. John and Lund, 1996; Kirsch et al., 1998) and in one field experiment on mussels (Mytilus galloprovincialis; Freites et al., 2002), but there is still a long way to go, considering the physiological status of the organisms (i.e., adding or depleting lipid reserves), growth rates and ontogenetic state of development, the impact of mixed diets, etc. Given the resolution of FATM, we question whether turning FATM into a quantitative tool is worth the effort, although in some studies a quantitative approach has been considered (e.g., Desvilettes et al., 1997). Resolution of ecological niches is the strength of the FATM approach and a key to resolving complex trophic interactions. FATM are incorporated largely unaltered into the NL pool of primary consumers, especially in periods of low catabolic activity, as when the animals are accumulating lipid reserves. In particular, 16:1(n-7), C16 PUFA and EPA have been used as indicators of diatom-based diets, whereas 18:4(n-3), C18 PUFA and DHA are used as dietary tracers of dinoflagellates and prymnesiophytes. Secondary and higher order consumers may also incorporate dietary FA largely unaltered into their NL reserves, but the FATTY ACID TROPHIC MARKERS 315 signals of herbivory are obscured as the degree of carnivory increases and FA may derive from many different sources (Auel et al., 2002). Markers of herbivory may be replaced by markers of carnivory, reflecting changes in feeding behavior such as during ontogeny. This is most obvious at higher latitudes, where the mesozooplankton communities are dominated by herbivorous and omnivorous calanoid copepods. These copepods usually accumulate large WE reserves containing large amounts of C20 and C22 MUFA and monounsaturated fatty alcohols, which they are believed to biosynthesize de novo. These particular monounsaturates have been used to trace and resolve food web relationships at higher trophic levels, for example in hyperiid amphipods, euphausiids and zooplanktivorous fish that typically consume large quantities of calanoid copepods (e.g. Sargent, 1978; Falk-Petersen et al., 1987; Kattner and Hagen, 1998; Hagen et al., 2001; Auel et al., 2002). Additional information on the ecological niches occupied by various zooplankton species may be obtained by combining FATM and lipid class compositions. Hence, at higher latitudes, the largest concentrations of WE are typically found in strictly herbivorous zooplankton, which are directly and immediately linked to primary production in these regions. The level of WE generally decreases from herbivores through omnivores to carnivores (Sargent and Falk-Petersen, 1988), and is partly replaced by TAG. Ratios of particular FA have also been used to assess the extent to which various species occupy different ecological niches. The proportion of 18:1(n-7) to 18:1(n-9) (as a marker of primary or heterotrophic bacterial production vs. animal production) was, for example, found to decrease in Arctic benthic organisms when considering a ‘‘succession’’ from suspension feeders via predatory decapods to scavenging amphipods (Graeve et al., 1997). As an example of how to combine this criterion with the various FATM summarized above, in addition to lipid class compositions, Falk-Petersen et al. (2000) used all these indices to classify seven common species of Arctic and Antarctic krill into different ecological niches. Hence, based on the results they concluded that Thysanoessa inermis and Euphausia crystal- lorophias are true herbivores, whereas Thysanoessa raschii, T. macrura and Euphausia superba are omnivores, and Thysanoessa longicaudata and Meganyctiphanes norvegica are carnivores. At higher trophic levels, i.e., in fish and marine mammals, specific FATM are often less evident than in zooplankton and consequently more difficult to interpret. The advancement of multivariate statistical methods of pattern recognition has, however, proven particularly valuable for resolving trophic interactions in these organisms (Smith et al., 1997; Iverson et al., 1997b, 2002; Budge et al., 2002), and we urge that this becomes an integrated tool in future applications of FATM at all trophic levels. 316 JOHANNE DALSGAARD ET AL.

6.2. Future applications

FATM are obviously good tools for assessing trophic interactions in the marine environment, adding information that is at times difficult, and in some instances impossible, to derive from more traditional techniques, such as stomach content analyses. In particular, FA provide information on the origin of lipid reserves generated over a period of time. Primary producer communities in the marine environment are dominated by diatoms, dinoflagellates and prymnesiophytes, which can be distinguished based on the presence and combinations of particular FA (as summarized in Table 2; see also Mayzaud et al., 1990). The spatial and temporal resolution of the various phytoplankton groups, and hence the basic FA pattern in the marine environment, is largely determined by macro and mesoscale stratification processes acting on phytoplankton group dominance through affecting light and nutrient availability. Large concentrations of phytoplankton biomass, essentially dominated by diatoms, evolve under spring bloom type conditions and form the basis for an efficient transfer of energy to higher trophic levels. Flagellates, on the other hand, typically dominate the phytoplankton communities before and after diatom bloom events when either light or nutrients are limiting, establishing the potential for microbial loop food webs. This coupling between hydrodynamic processes and the transfer of group-specific phytoplankton production to higher trophic levels has been established using FATM. For example, St. John and Lund (1996) showed a coupling between the growth and condition of larval and juvenile fish to spatial variations in frontal primary production, linking ultimately with physical frontal mixing processes, using 16:1(n-7)/16:0 as a food web tracer. Hence, applied in this manner, in examination of the potential impact of mesoscale processes, FATM may provide a tool for resolving the impact of global change on marine ecosystem dynamics. To further develop this approach, we suggest that it could be combined with the analyses of larval fish otolith microstructures. These allow an indication of the growth history of the individuals, thereby contributing to the identification of the potential dynamics of FATM incorporation. Fatty acids have principally been used as qualitative markers of trophic interactions in shelf sea ecosystems with an emphasis on higher latitudes. In contrast, very few FATM studies have been carried out in upwelling and open ocean, oligotrophic areas, including tropical systems. Primary producers in oligotrophic systems are composed largely of small, autotrophic flagellates and cyanobacteria, forming the basis of low biomass, microbial loop food webs. As microorganisms usually do not accumulate large lipid reserves, FATM may be of less relevance here. However, despite FATTY ACID TROPHIC MARKERS 317 their lack of storage lipids, heterotrophic bacteria, which contribute significantly to these systems, are still recognizable by specific FA. Considering the fast turnover rates of microorganisms, we therefore hypothesize that FATM may help resolve trophic interactions in microbial loop food webs, and we support the strengthening of FA research in this area, as suggested also by Strom (2000), recognizing the importance of these systems in the global carbon budget. The combination of FATM and stable isotope analyses may provide additional information for resolving trophic interactions in marine ecosystems (e.g., Kiyashko et al., 1998; Kharlamenko et al., 2001). This approach has proven particularly helpful in identifying the contribution of major sources of organic matter contributing to detrital food webs, and hence, the diet of, e.g., detritivorous benthic invertebrates, which cannot be inferred from stomach content analyses. Using this approach Kiyashko et al. (1998), for example, established that in addition to bacteria, benthic rather than pelagic diatoms, which are characterized by similar FA signatures, constituted the major food source of sea urchins in Vostok Bay, Sea of Japan. Another interesting approach that can be used to characterize carbon fluxes between prey and predators as well as to validate the applicability of FATM, involves feeding experiments with 13C-enriched experimental diets. Such studies provide information on carbon accumulation, transfer and turnover rates as well as biosynthesis of lipids and individual FA. Hence, in a preliminary study involving 13C-enriched phytoplankton, it was shown that long-chain MUFA and monounsaturated fatty alcohols synthesized de novo by herbivorous copepods feeding on the 13C-enriched phytoplank- ton were also enriched in 13C isotopes (Albers, 1999). In general, studies applying FATM have been dominated by those correlating individual FA with the dynamics of individuals, for example growth or reproductive output. In many instances the biological relevance of the FA employed is not clear. This points to a key issue in the field, which is a general lack of validation, and at times an uncritical application of FATM. Many of the studies cited in Section 5 have, for example, applied FATM on the assumption that they derive from certain prey species or groups of species, without testing the validity of this assumption (e.g., often as simple as examining stomach contents). A second flaw within FATM research is that this technique has been applied in a fragmentary manner, i.e., employed in studies in which the FATM results have not been validated by other approaches. Hence, future applications of FATM should form part of integrated research programs, with FATM as an ecological tool to establish trophic interactions on an ecosystem level. It is here that their strength lies. 318 JOHANNE DALSGAARD ET AL.

ACKNOWLEDGEMENTS

The first author thanks Dr. B. Jørgensen for fruitful discussions on multivariate statistical analyses, Dr. H. A. Thomsen for advice on microalgal taxonomy and G. Møller, C. Anderberg and K. Prentow for excellent library service. We wish to thank Prof. A. J. Southward and an anonymous reviewer for very constructive comments on the manuscript. The authors would like in addition to thank the GLOBEC Focus Group 2 on Process Studies for convincing Gerhard Kattner and Michael St. John of the necessity of this review. The Danish Institute for Fisheries Research, the Danish Research Agency and the European Union Fifth Frame Work Programme, Quality and management of living resources, Q5RS-2000- 30183 (LIFECO) provided funding for the first author.

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Acanthopagrus australis 313 Bulimina marginata 20 Acanthephyra 158 Bulimina spp. 20, 35 Acartia 253, 305 Buliminida 5 Acartia clausi 251, 305 Bullimina spp. 19 Acartia tonsa 252, 275 Acropora 195, 201, 204, 208 Calanoides 257 Acropora grandis 193 Calanoides acutus 257, 264, 265, 284, Adercotryma glomerata 40 285, 292, 293, 294, 296, 297 Adercotryma glomeratum 23 Calanus 257, 267, 268, 275, 289 Aethotaxis mitopteryx 296 Calanus acutus 285 Alabaminella weddellensis 20, 21, Calanus finmarchicus 231, 232, 264, 29, 31, 67 265, 267, 268, 269, 283, 285, 288, Albunea symnista 112, 121, 126, 146 289, 306, 307 Allogromiida 5 Calanus glacialis 257, 264, 268, 269, Ammobaculites agglutinans 20, 21, 40 283, 287, 288 Ammodytes hexapterus 309 Calanus helgolandicus 232, 266, 267, 307 Ammonia 68 Calanus hyperboreus 232, 257, 264, 267, 283, Amphidinium carterae 233, 267 284, 288, 289, 291 Anemonia viridis 193 Calanus propinquus 257, 260, 264, 265, 291, Arctocephalus gazella 297 292, 293, 296, 297 Artemia salina 231 Calanus simillimus 291, 292 Astrorhizida 5 Calanus spp. 288 Carcinus maenus 170 Balaenoptera physalus 300, 304 Caridina nilotica 156 Bathydraco marri 296 Cassidulina reniforme 20 Bathysiphon spp. 35 Cassidulina spp. 19, 35 Benthosema glaciale 289 Centroberyx affinis 313 Beroe cucumis 287, 288 Chaetoceros curvisetus 232 Bolinopsis infundibulum 287, 288 Chaetoceros simplex 231 Bolivina albatrossi 23 Chilostomella oolina 13, 19, 20, 21, 22 Bolivina pacifica 30 Chilostomella ovoidea 22, 38 Bolivina spp. 19, 22, 34, 35 Chilostomella spp. 35 Boreogadus saida 290 Chrysotila spp. 248 Brizalina spp. 22 Cibicides lobatula 43 Bulimina aculeata 13, 21, 30 Cibicides lobatulus 42 Bulimina alazinensis 21 Cibicides refulgens 42 Bulimina exilis 22 Cibicidoides kullenbergi 23 342 TAXONOMIC INDEX

Cibicidoides pseudoungerianus 23 sex ratio 95–6 Cibicidoides wuellerstorfi 12, 22–4, 40 size at sexual maturity 95–6 Clione limacina 295, 301 of male and female species 96–7 Clupea harengus 277 stimulus for ovulation 112 Clupea harengus pallasi 309 yolk utilisation 146–60 Crangon crangon 156 zonal distribution patterns 94 Cribrostomoides subglobosum 23 Emerita analoga 93, 94, 97–9, 105, 122, 123, Crithionina mamilla 42 130, 161, 170 allelic frequencies 168 carotenoids in ovary and egg 143–4 Delphinapterus leucas 291 copper and zinc levels 168 Deuterammina ochracea 42 effect of temperature on egg development Diogenes pugilator, spermatophores 107 on pleopods 131–5 Discanomalina coronata 42 egg production 131 Discanomalina semipunctata 42 fecundity profiles 132–3 Dolioletta gegenbauri 301 larval dispersal and megalopa settlement Dolloidraco longedorsalis 296 165–8 Drosophila spp. 193 mating habits 104–6 morphology of spermatophores 106–7 Echinopora gemmacea 196 sex ratios based on size Eilohedra levicula 67 classes 95 Eilohedra nipponica 29, 67 Emerita antarctica 285 Electrona antarctica 297 Emerita asiatica 93, 94, 98–102, 105, 106, 113, Emerita 91–182 114, 121, 123, 126, 129, 130 as indicator species 168–71 amino acid composition astaxanthin production in 144 of Lv II 141–2 behavioural features 92–3 androgenic glands 103 biochemistry of eggs, annual fluctuations in gonad, egg mass and yolk protein 139–46 hepatic indices 125 breeding season 123 biosynthetic pathway of - and -carotene carotenoid pigments in eggs and yolk metabolism taking place in developing proteins of 143–4 eggs 158 distribution 93–5 carotenoid content in different egg production 129–31 egg developmental stages 157 energy utilisation in eggs 153–5 carotenoid metabolism during fecundity profiles 131 embryogenesis 155–8 filter-feeding behaviour 95 chronology of sexualisation in female life cycle 93 and male 100 male–female size relationship classification of egg development 148 patterns 95 classification of ovarian stages 128 morphological features 92–3 contribution (%) of protein, natural history 93–5 carbohydrated and lipid neotenic males 171 to total energy in stages neotony 96–9 of egg development 155 nutritional control of moulting 121–2 deposition of embryonic cuticle of occurrence and utilisation zoea larva 160 of vertebrate steroids in eggs 160 diagrammatic representation of testis, overview 92–3 ovary and hermaphroditic ovary 102 reproductive cycle 122–35 distribution pattern 169 role of haemolymph lipoproteins in ecdysteroid level in haemolymph moulting and reproduction 136–8 of females 120 TAXONOMIC INDEX 343

egg development 134 relationship between carapace length and egg production 131 number of eggs carried in pleopods 130 embryogenesis 147, 153, 156 relative composition of fatty acids in embryonic ecdysteroids in 158–60 neutral lipid fraction enzymatic activity during embryonic of Lv II 143 development 151 relative percentage composition of enzyme activity during yolk different lipids in egg 142 utilisation 148 reproductive cycle 124–7 epidermal and setagenic changes in relation to size 128 in pleopods during sex reversal 99–100, 104 moult stages 116 size distribution of males, esterases in 152 immature females and fecundity profiles 132–3 ovigerous females 96 fluctuation of hormonal activity during size-related moulting frequency 117, 122 embryonic size-related sex ratio 95 development 159 sperm release 111–12 functional protandric spermatophore 107 hermaphroditism 171 spermatophores 108 glycolipids 141 sugar composition of delipidated Lv II 141 haemolymph lipoproteins vitellogenin of 146 from male, immature year-round moulting 135 and mature 137 yolk protein 139–43 hepatic index 127 yolk utilisation 146 histochemical characteristics Emerita austroafricana 94 of mucopolysaccharide Emerita austroafricanus 97 substances of Emerita benedicti 94 spermatophoric mass 109–10 Emerita brasiliensis 94 histological appearance Emerita crystallorophias 265, 296 of hermaphodite gonad 101 Emerita emeritus 94, 97, 123, 125 hormonal conjugation to fecundity profiles 132–3 yolk protein 145 Emerita frigida 294 levels of estradiol 17 and Emerita holthuisi 93, 94, 97, 123, 125, 126, progesterone in different embryonic 129, 156, 161, 167 stages 161 fecundity profiles 132–3 major organic composition of eggs during yolk utilisation 146 different stages of Emerita portoricensis 93, 94, 97, development 149, 150 99, 122, 123 mechanism of yolk formation 145–6 Emerita rathbunae 94 metal content of yolk protein 144–5 larval dispersal and megalopa settlement mobilisation of energy during egg 165–8 development 154 Emerita Scopoli 1777 93 moult cycle stages 113–17 Emerita talpoida 93–5, 97, 98, 99, 123 moulting pattern 112–22 allelic groups 167 moulting sequence 118 larval description in 161–5 parasitisation of egg mass and ovary 170 larval dispersal and megalopa settlement percentage of precocious premoult 165–8 changes 120 megalopa stage 164 planktonic larvae 167 morphology of spermatophores 106–7 premoult stage 119 sperm sac or genital protandric hermaphroditism 99–104 papilla 97–8 quantification of protein 140 zoeal stages 162, 163 344 TAXONOMIC INDEX

Emerita tricantha 294 Lagenammina spp. 31, 57 Epistominella arctica 23, 29, 31 Lagenida 5 Epistominella exigua 12, 23, 29, 30, Lauderia borealis 232 31, 40, 42, 62 Lebistes reticulatus 231 Epistominella levicula 67 Lenticulina spp. 35 Epistominella pusillus 67 Ligia oceanica 155 Epistominella spp. 35 Limacina helicina 295 Eponides leviculus 67 Limacina retroversa 295 Eponides pusillus 29, 30, 57, 67 Liparis sp. 289 Eponides tumidulus 23 Littorina kurila 234 Euchaeta antarctica 259, 296 Lituolida 5 Euchirella rostromagna 291, 292 Lobatula lobatula 40 Euphausia crystallorophias 266, 294, 315 Lophelia pertusa 41 Euphausia superba 260, 265, 266, 267, 293, Lysmata seticaudata 103 294, 296, 297, 304, 315 androgenic gland 104 Euphausiids 311 Macrobrachium idella 156 Fucus evanescens 234 Macrobrachium lamarrei 156 Fursenkoina mexicana 20 Macrobrachium nipponense 138 Fursenkoina spp. 35 Macrobrachium nobilli 156 Macrobrachium rosenbergii 138 Gadus morhua 234, 275, 289, 302 Mallotus villosus 274, 288 Gammarus wilkitzkii 287 Mastigochirus Miers 1878 93 Genypterus blacodes 313 Maurolicus muelleri 289 Globobulimina 19 Meganyctiphanes norvegica Globobulimina affinis 22, 37, 38, 57 300, 311, 315 Globobulimina auriculata 13, 21 Megaptera novaeangliae 304 Globobulimina pyrula 20 Melanogrammus aeglefinus 302 Globobulimina turgida 20 Melonis barleeanum 13, 19, 20, 21, 22 Globobulimina spp. 16, 20, 21, 22, 23, 35 Melonis zaandami 19, 23 Globocassidulina subglobosa 22, 43 Melonis spp. 16, 20, 22 Goniastrea aspera 191, 192, 193, 197, 198, 199 Mertensia ovum 288 Goniopora djiboutiensis 193 Metridia gerlachei 285, 293, 296 Goniopora pandoraensis 193 Metridia longa 283, 285, 286 Metridia okhotensis 285 Metridia spp. 283 Hanzawaia concentrica 42 Microphallus 170 Heliopora 204 Miliolida 5 Heterocapsa triquetra 234, 276 Mirounga leonina 298 Heterodontus portusjacksoni 313 Montastraea annularis 193 Hippa 1787 93 Montastraea faveolata 193 Hippa pacifica 93, 99 Montastraea franksi 193 Hoeglundina elegans 12, 22, 23 Montipora verrucosa 196 Homarus americanus 121, 156 Montipora spp. 204 Homarus garmmarus 156 Mytilus edulis 302 Hoplostethus atlanticus 313 Mytilus californianus 193 Hormosina dentaliniformis 20, 21 Mytilus galloprovincialis 314 Hydra 232 Hymenomonas 246 Nannochloris 246 Illex illecebrosus 276 Neocalanus cristatus 257 Isochrysis spp. 246, 248 Neocalanus flemengeri 257 TAXONOMIC INDEX 345

Neocalanus spp. 308 Pontoporeia femorata 301 Nonion scaphum 22 Porites 195, 204, 208, 209 Nonion spp. 35 Porites asteroides 195, 205 Nonionella iridea 57 Portumanus ocellatus 170 Nonionella fragilis 30 Probopyrus pandalicola 156 Nonionella iridea 29 Pseudocalanus 305 Nonionella opima 20 Pseudocalanus elongatus 251, 305, 307, 308 Nonionella stella 22 Pyrgo murrhina 23 Nonionella spp. 35 Pyrgo murrhyna 23 Nuttallides rugosa 41 Pyrgo rotalaria 23 Nuttallides umbonifer 13, 17, 22, 23, 30, 40, 41, 55 Rectuvigerina cylindrica 13 Reophax guttifer 43 Oculina patagonica 204 Reophax spp. 35 Onisimus glacialis 287 Rhabdammina abyssorum 42 Onisimus nanseni 287 Rhincalanus gigas 292, 293, 296 Onisimus spp. 287 Robertinida 5 Oridorsalis umbonatus 22, 23 Rotaliida 5 Osmerus mordax 300 Rupertina stabilis 43

Saccammina sphaerica 42 Pagettia producta 170 Sagitta elegans 288 Pagurus bernhardus 156 Sardinops caerulea 275 spermatophores 107 Scomber scombrus 276 Palaemon serratus 159 Scrippsiella trochoidea 267 Pandalus borealis 103, 288, 291 Skeletonema costatum 232, 234, 276 Paratelphusa hydrodromus 153 Sphaeroidina bulloides 21–3 Parathemisto libellula 290 Squalus megalops 313 Pareuchaeta antarctica 292, 293 Stainforthia apertura 30 Pareuchaeta norvegica 285 Stainforthia fusiformis 31 Pavona 204 Stainforthia spp. 21, 35 Penaeus monodon 138 Stenosemella ventricosa 310 Phaeocystis pouchetii 281, 283, Stetsonia hovarthi 23, 43 286, 289 Stygiomedusa gigantea 296 Phaeocystis spp. 248, 251, 260, Stylophora pistillata 196 267, 291, 292, 305, 306 Symbiodinium Pheronema carpenteri 41, 42 microadriaticum 193 Phoca vitulina 308, 309 Phyllobothrium 170 Placopecten magellanicus 301, 302 Temora 305 Planulina ariminensis 42 Temora longicornis 251, 305 Pleuragramma antarcticum 296 Textularia kattegatensis 30 Pocillipora damicornis 204, 205 Textulariida 5 Pocillopora bulbosa 189 Thalassiosira antarctica 232, 267, 268 Pocillopora caespitosa 189 Themisto abyssorum 287, 289, 296 Pocillopora damicornis 189, 190, 194, 196 Themisto gaudichaudi 296 Pocillopora elegans 189 Themisto libellula 287, 296 Pocillopora meandrina 189 Theragra chalcogramma 309 Pocillopora spp. 189, 208 Thysanoessa inermis 286, 287, 288, Pontastuacus leptodactylus 289, 300, 315 leptodactylus 100 Thysanoessa longicaudata 315 346 TAXONOMIC INDEX

Thysanoessa macrura 259, 294, 296, Uvigerina auberiana 20 297, 315 Uvigerina mediterranea 23, 24 Thysanoessa raschii 286, 287, 315 Uvigerina peregrina 20, 23, 68 Thysanoessa spp. 286, 288 Uvigerina spp. 21, 28, 35, 62 Trematomus lepidorhinus 296 Trifarina angulosa 13, 40, 43 Valvulineria laevigata 20 Trifarina fornasinii 23 Vibrio coralyticus 204 Trochammina squamata 42 Vibrio shiloi 204 Trochammina spp. 35 Valvulina pennatula 42 Trochamminida 5 SUBJECT INDEX

abyssal environments 6 small-scale distribution patterns 66–7 acid-treated assemblages (ATAs) 61–2 see also foraminiferal species acidic mucopolysaccharides (AMP) 107 benthic foraminiferal accumulation rate Adaptive Bleaching Hypothesis (ABH) 194 (BFAR) 25–7, 54 agglutinated foraminifera 31 and differences in quality of deposited alkyldiacylglycerol ether (DAGE) 295 organic matter 26–7 allogromiid foraminifera 31 and organic matter flux to sea floor 25 amino acid composition of Lv II in benthic foraminiferal faunas used in E. asiatica 141–2 palaeoceanographic reconstructions 16–17 androgenic gland 103 benthic storms 15 androgenic gland hormone 104 bentho-pelagic coupling 18 annual flux rates, reconstruction 19–28 biological–geological synergy in foraminiferal Antarctic Bottom Water (AABW) 39, 40 research 68–9 Antarctic Circumpolar Current 30 bottom-water hydrography 39–43, 55 Arabian Sea 10, 29 current flow effects 41–3 Arctic Ocean 28, 43, 52 water depth effect 43–5 Argentine Basin 62 box corers 7 Asko¨ splitter 7 Buliminida 35 assemblage data, multivariate analysis 27–8 assemblage parameters as calcareous foraminifera 8 palaeoceanographic indicators 56 calcareous species 31–3 astaxanthin production in Emerita 144 calcareous test morphotypes 11 Atlantic Ocean 27, 31, 39–40 calibration dataset 28 calibration of proxies 64–6 California Borderland 34 bathyal continental margins 29 Carbonate Compensation Depth (CCD) 8, bathymetric distribution of deep-sea 30, 40, 41 foraminiferal species 55–6 carbonate undersaturation 40–1 Bay of Biscay 42 carnivorous crustaceous BENBO programme 4 zooplankton 259–64 BENBO Site A–C 58 carotenoid pigments in eggs and yolk proteins BENBO Site C 31, 32, 57 of Emerita 143–4 benthic foraminifera 1–90 carotenoids as proxies 4 in ovary and egg of E. analoga 143–4 faunal approaches based on 4 metabolism during embryogenesis in in palaeoceanography 8 E. asiatica 155–8 overview 7–8 Central Pacific 32 348 SUBJECT INDEX characteristics of survivors 227–8 correspondence factor analysis (AFC) 28 Chilostomellidae 35 crab see Emerita in Taxonomic Index classification and regression tree analysis crayfish 100 (CART) 277 Cross Seamount 32 climate/ocean system 3 current flow 15 Colombian Pacific 200 continental slopes 14 DDT pollution levels 168 copepods, FATM in 283–5, 291–3, 305–8 deep-infaunal species 34 copper : zinc SOD 199 deep-sea environments 5–6 coral–algal association 193 deep-sea faunas 3 coral–algal symbiotic association 184 deep-sea foraminiferal diversity and current coral bleaching 183–223 activity 53 and ENSO events 183, 185, 197, 200, 201, deep-sea foraminiferal ecology 7–15, 63 204, 210 deep-sea foraminiferal signal 60–1 and fish assemblages 209 deep-sea foraminiferal species, problems in and global warming 184–5 taxonomy 67–8 and photoinhibition under the influence of deep-sea sediments 3, 4 increased temperature 190 deep-water production 3 early studies 184 discriminant function analysis 27 future studies 211 dissolved oxygen index (BFOI) 37–8 internal defense mechanism 191 dysoxic conditions 66 link with elevated temperature 184–5 dysoxic foraminiferal assemblages 37 long-term ecological implications 207–9 observations under field conditions 196 ecdysis stage 115–17 present studies 185 ecdysteroids, moult-inducing process 188–90 effect of 120–1 protective mechanisms 190–4 ecosystem dynamics and global change 227 range estimates and projected median dates egg production, Emerita 129–31 186–7 El Kef Formation 45 recovery 201–4 El Nino Southern Oscillation scenarios resulting from 207 (ENSO) and coral bleaching 183, uncertainties concerning interaction of 185, 197, 200, 201, 204, 209, 210 stresses inducing 207 embryonic ecdysteroids in corals E. asiatica 158–60 acclimatization and adaptation to elevated endocrine regulation temperatures or light regimes 195–201 of moulting 117–21, 138–9 adjustment to ambient conditions 188 of reproduction 138–9 and metabolic adaptation to ambient environmental factors and temperature regime 187 spatial scales 62–4 defenses against high light and elevated environmental gradients 64 temperature 193 environmental influences on fluorescent pigments 210 live assemblages 54–6 long-term selection for temperature enzyme activity during yolk utilisation tolerance 195 in E. asiatica 148 mechanisms of zooxanthellae loss 188–9 enzyme specificity in fish 269–70 mortality 210 epibenthic foraminiferal faunas 42 resistance to disease, reproduction and epifaunal/shallow infaunal species 14 recruitment 204–6 epifaunal species 11, 14 symbiotic algae 190 esterases upper temperature tolerance activity 148 thresholds 186–8 in E. asiatica 152 SUBJECT INDEX 349 euphausiids, FATM in 286–7, impact of growth, environmental and 293–5, 300, 311–12 hydrodynamic factors 240–1 eutrophic systems 11 in higher organisms 235–6 mobilization during reproduction factor analysis 27 264–5, 274–5 fatty acid trophic markers (FATM) 225–340 mobilization during starvation 264–5, applications in major food webs 278–313 273–4 Antarctic 291–8 modifications 271–3 Arctic 281–98 seasonal distributions 233 Mediterranean 310–12 temporal development 268 North Sea 305–8 uptake of dietary 256–64, 270–1 Northwest Atlantic 298–304 fatty acyl desaturation 237 upwelling and sub-tropical/tropical fatty alcohols 264 systems 312–13 faunal approaches applications in marine research 231–5 based on benthic foraminifera 4 bacterial 251–4 to reconstructing palaeoceanography concept 230–1 15–18 crustaceous zooplankton 266–9 faunal indicators 15 de novo biosynthesis 256–64 fish future applications 316–17 enzyme specificity in 269–70 Gulf of Alaska 308–10 FATM in 288–90, 296–7, 302–3, 308–9 heterotrophic bacteria and terrestrial fatty acid dynamics in 269–77 matter 251–5 food availability 11, 14, 19, 25, 32, 45, 129 in copepods 283–5, 291–3, 305–8 foraminifera, characterisation 4 in euphausiids 286–7, 293–5, 300, 311–12 foraminiferal abundance, regional in fish 288–90, 296–7, 302–3, 308–9 patterns of 54 in macrobenthos 301–2 foraminiferal distributions 14 in primary producers 281–3 foraminiferal ecology 4 interpretation of large data sets 242 foraminiferal microhabitats 9 of marine microalgal classes used in PLS foraminiferal proxies 15 regression analyses 249 foraminiferal research, biological–geological primary producers 241–51 synergy in 68–9 state-of-the-art 313–15 foraminiferal species terrestrial 253–5 and assemblages associated with high validation in fish 275–7 productivity areas 20–1 fatty acids (FA) relationship with organic flux to the basic pattern in marine food webs 238 seafloor and surface primary biochemistry 236–8 production 23 biosynthesis 237, 239–40 see also benthic foraminifera in primary producers and marine foraminiferal standing stocks 19 animals 236 fossil assemblages combined with stable isotope analyses 234 factors influencing generation 60 composition of marine microalgal living assemblages relationship to 56–62 classes 245 free fatty acids (FFA) 264, 274 de novo biosynthesis 236, 257–8, 271–3 dietary 269–71 genotypic characteristics 228 dynamics in crustaceous zooplankton global change and ecosystem dynamics 227 255–69 global climate 3 dynamics in fish 269–77 global warming 3, 201 dynamics in marine primary producers and coral bleaching 184–5 238–55 glycolipids, E. asiatica 141 350 SUBJECT INDEX granuloreticulate pseudopodia 4 macroalgae 248–50 Great Barrier Reef 186–7, 209 macrobenthos, FATM in 301–2 Greenland-Norwegian Sea 24 Madras 135 Gulf of Alaska, FATM 308–10 malacostracan crustaceans, hermaphroditic Gulf of Cadiz 42 potentialities 102 Maldives 206 haemolymph 100–2 manganese superoxide dismutase haemolymph protein levels during moulting (MnSOD) 199 136–8 MAPS (Madras Atomic Power Station) 169 heat shock proteins (HSPs) 191–3, 210 mating habits in E. analoga 104–6 Heinrich Event, H1 and H4 37 Mediterranean Outflow Water (MOW) 42 herbivorous calanoid copepods 256–9 mesodermal cells 100 heterotrophic bacteria 251–4 mesotrophic settings 11 high performance liquid metazoan distributional patterns 45 chromatography 158 microalgae 241–8 high productivity areas, foraminiferal species microhabitat preferences 11 and assemblages associated with 20–1 microhabitat studies 66–7 high productivity assemblages 19 microparticle enzyme immunoassay 160 hormonal conjugation to yolk protein in monounsaturated fatty acids (MUFA) 236–8, E. asiatica 145 241, 252, 257–9, 267, 270, 271, 294, hyaline calcareous foraminifera 31 304, 315 hydrographic factors 14–15 Monte del Casino 45 hydroxyecdysone (20E) 139 morphotypes as flux indicators 24–5 moult-inducing effect of ecdysteroids 120–1 moulting 112 Iberian Peninsula 37 and reproduction interrelationship 135–9 INDAR (Individual Accumulation Rate) 26 cycle stages 113–17 India 127 endocrine regulation 117–21 Indian Ocean 27, 31 frequency 117 infaunal morphologies 14 haemolymph protein levels during 136–8 infaunal morphotypes 25 in decapod crustaceans 117 vs. epifaunal morphotypes 55 nutritional control 121–2 infaunal species 9, 11 postmoult stage 113 intermoult stage 113 premoult stage 113 Intertidal Sand Crab see Emerita in multicorers 7 Taxonomic Index multilocular agglutinated taxa 58 isopods 15 multivariate analysis of assemblage data 27–8 Italy 45

Narragansett Bay, Rhode Island 232 Kalpakkam 169 natural plankton communities 250–1 NE Alantic 58 larval description in E. talpoida 161–5 NE Atlantic 29, 31, 41, 64 lipid biomarkers 67 NE Pacific 29 lipids neutral lipids (NL) 235 in higher organisms 235–6 Nonionidae 35 in marine fish 269–70 nonphotochemical quenching (NPQ) 191 lipoproteins 136–8 North Atlantic 29 lipovitellins (Lv I and Lv II) 139 North Atlantic Deep Water living assemblages, relationship to fossil (NADW) 30, 39, 40 assemblages 56–62 Northern Arabian Sea 52 low-oxygen environments 33 Northern blotting 146 SUBJECT INDEX 351 ocean-floor environment 3, 54 POC flux 44 ocean surface productivity 27 Polar Front 30 ocean temperature increase 185 polar marine copepods 262–3 oligotrophic systems 9 polyacrylamide gel electrophoresis 144 omnivorous crustaceous zooplankton 259–64 polyunsaturated fatty acids (PUFA) 232, 235, oocytic differentiation 104 238–42, 248, 250–2, 255, 267, 270, 272–3, organic carbon flux rates 27–8 291, 294, 302, 312 organic-matter fluxes 18–33, 55 Porcupine Seabight 31, 32, 45 original dead assemblage (ODA) 61–2 postmoult stage 113 otolith microstructure 228 premoult stage 113 oxic species 37–8 principal components analysis 27 oxygen, as limiting factor for foraminifera 34 productivity signal 32 oxygen availability 11, 14 protandric hermaphroditism 99–104 oxygen concentrations 33–9, 55 E. asiatica 104 qualitative approaches 35–7 proxies quantitative approaches 37–9 calibration of 64–6 oxygen depletion 14, 33–5 quantification 64–6 oxygen fluxes across sediment–water pseudopodia 67 interface 18 pycnogonids 15 oxygen gradients 9 effect on foraminiferal species richness 36 Quaternary sediments 15 Oxygen Minimum Zones (OMZs) 33 oxygenation regimes 66 radioimmunoassay (RIA) 119, 158, 160 regional distributions of species and species Pacific Ocean 31 assemblages 55 Pakistan margin 52–3 regional patterns 14–15 palaeoceanography 3 foraminiferal abundance 54 benthic foraminiferal faunas used in replication 7 reconstructions 16–17 reproduction and moulting relationship 135–9 faunal approaches to reconstructing 15–18 Rotaliida 35 species diversity parameters as tools in RT-PCR 146 45–54 palaeoecological analysis of dead assemblages 61 Sagami Bay, Japan 9 PalaeoVision system 12, 13 sampling devices 6–7 particulate organic matter (POM) fluxes San Clemente Beach 168 18–19, 22 Santa Barbara 129, 168 pelagic ecosystem 19 Santa Barbara Basin 10, 37 periodic acid shift (PAS) 107 Santa Cruz Island 129 phenotypic characteristics 228 Santa Monica Bay 168 phospholipid species 142 saturated fatty acids (SFA) 241, 252, 258, 267, photosynthetic characteristics of coral sym- 270, 294 biotic algae 194 Scotia Sea 62 photosynthetically active radiation (PAR) 192 sea-surface temperatures 4 physico-chemical factors 68 seasonality in flux of organic matter to sea phytodetritus, pulsed fluxes 29 floor 55 phytodetritus deposition 19 seasonally varying fluxes 29–31 phytodetritus species 29–31 sediment characteristics 15 planktonic/benthic ratio (P/B ratio) 44 sediment community oxygen consumption planktonic foraminiferal assemblages 4 (SCOC) 18 PLS regression analysis 244–8, 261, 262–3 sediment fractions 6 352 SUBJECT INDEX sediment porewater oxygen profiles 18 Suva Harbor 203 sediment–water interface, oxygen fluxes SW Pacific 30, 34 across 18 sieve sizes 6–7 thermohaline circulation 3 small-scale patterns 8–11 triacylglycerols (TAG) 235, 241, 269 Society Islands 200 Trinity Bay, Newfoundland 299 South Atlantic 30 TROX model 9, 52 South China Sea 27–8, 53 Tunisia 45 Southern Californian Bight 30 turbidity currents 15 Southern Ocean 29, 40 spatial scales and environmental factors 62–4 species abundances 19 very-long-chain, as indicators of absolute flux rates 22 highly-unsaturated-fatty-acids species distributions within (VLC-HUFA) 242 sediment profile 54 vitellogenin of E. asiatica 146 species diversity parameters as tools in volcanic ash falls 15 palaeoceanography 45–54 species richness data for foraminifera at WAST-T 41 localities characterised by differing oxygen wax ester (WE) fraction 232 regimes 46–51 Western Mediterranean 45 sperm transfer 106–12 World Ocean 27 spermatophores in 111–12 spermatogonial cells (SG) 101 yolk proteins 100–2 spermatophores 106–12 dehiscence 111 yolk utilisation deposition 105 in E. asiatica, enzyme activity during 148 in Emerita 146–60 histochemistry of components 107–8 in sperm transfer 111–12 morphology 106–7 zooxanthellae origin 111 adaptability 210 SSTs 210 symbionts 193 sulphate-reducing bacteria 60 thermal acclimation 195 sulphides, toxic effects 34 zooxanthellar, photosynthesis in coral surficial sediments 27 bleaching 188–9