Zooplankton fixation and preservation Edited by H. F. Steedman The Unesco Press Paris 1976 --.. .-_-___

Published by The Unesco Press, 7 Place de Fontenoy, 75700 Paris Printed by Les Imprimeries Rknies de Chambky ISBN 92-3-101272-X 0 Unesco 1976 Printed in France

Monographs on oceanographic methodology 4

In this series: 1. Determination ofphotosynthetic pigments in sea-water 2. Zooplankton sampling 3. A guide to the measurement of marine primary production under some special conditions 4. Zooplanktonjixation andpreservation

Preface Publication by Unesco of the series of ‘Monographs on Oceanographic Method- ology’ follows a recommendation adopted by the Scientific Committee on Oceanic Research (SCOR) at its meeting in Halifax in 1963. As a forerunner to the series, Unesco undertook to distribute to oceanographic laboratories of the world copies of the second and revised edition of A Manual of Sea-water Analysis by Strickland and Parsons (Fisheries Research Board of Canada, 1964). The monograph series was finally established by the compilation and printing of Volume 1, Determination of Photosynthetic Pigments in Sea-water, 1966. A second volume was published in 1968, entitled Zooplankton Sampling, followed by a third, A Guide to the Measurement of Marine Primary Production under some Special Conditions, in 1973. Further volumes in the series will reflect the results of current revision of various oceanographic methods being undertaken by several institutions and international bodies. The present publication results from the activities of SCOR Working Group 23 on Zooplankton Laboratory Methods, established in 1967 to ‘suggest methods for preserving zooplankton samples for taxonomic study and biomass determination’. To a certain extent the tasks of this group evolved from the studies conducted by SCOR Working Group 13, Zooplankton Sampling Methods, which had begun its work in 1964. During the intervening three years, it had become evident that whatever methods were established to assure standardization of the sampling process would have to be reinforced by standard techniques for preserv- ing such samples both on board ship and in the laboratory. Since the material covered in this volume is, in essence, a continuation and elaboration of the techniques set forth by Working Group 13 in Zooplankton Sampling, the two volumes may be used as companion texts. One explains the procedures used on board ship during the sampling and initial preservation stages, while the other follows in more detail the work carried out in the latter stages of laboratory fixation and preservation. Following its establishment in 1967, the first meeting of Working Group 23 was held in March 1968 at the Smithsonian Institution in Washington, D.C., with the following members : V. Kr. Hansen, Chairman (Denmark) H. J. Fltigel (Federal Republic of Germany) B. Kimor (Israel) H. F. Steedman (United Kingdom) T. Tokioka (Japan) M. E. Vinogradov (U.S.S.R.).

At that meeting a set of interim recommendations was developed to serve as the basis for further study by the group through laboratory experimentation and discussion. Three main subject areas-fixation, biomass determination and preservation and storage-were included among the twenty-four basic recom- mendations which were used as a point of departure for the present manual. Between 1968 and 1972 members of the group worked in collaboration with scientists and institutions to finalize the techniques set forth in this monograph. In order to allow the contributors and other specialists the opportunity to discuss and criticize the various chapters, a Symposium on the Fixation and Preservation of Marine Zooplankton was held at Bath University (United Kingdom), 13-20 July 1972, under the auspices of Unesco and SCOR. At this time the working group held its final meeting and approved the report. Discussions held during the symposium and the SCOR Working Group23 meeting included not only material that was to form the basis of the monograph but also what areas required further study. While some of these points are included within the final text, other related techniques were felt to require addi- tional investigation and have been passed to the Scientific Committee on Oceanic Research for consideration as to how such studies could best be undertaken. The Unesco Division of Marine Sciences will continue to maintain close collaboration with SCOR on this matter and will be pleased to receive additional information regarding new zooplankton preservation techniques, as they are developed. Unesco expresses its thanks and appreciation to the editor, H. F. Steedman, and his collaborators, J. R. Beers and V. Kr. Hansen, for their expertise and guidance in bringing this text to print. The scientific opinions expressed in this work are those of the authors and should not be interpreted as the views of Unesco. Equipment and materials noted in the text are given as examples of those most currently used during the experi- mental phase of Working Group 23 and their inclusion does not imply that they should be considered as preferable to others available at that time or developed since.

Acknowledgements It is a pleasure to acknowledge the additional substantial grants which were given in support of the SCOR Working Group 23 programme by the Natural Environ- ment Research Council (United Kingdom) the Smithsonian Institution’s Ocean- ography and Limnology Programme, Washington, D.C. (United States) and the Royal Society (United Kingdom) (for the provision of a research microscope). That part of the project which took place at the Smithsonian Oceanographic Sorting Center was supported through I. E. Wallen, and his successors, W. I. Aron and R. P. Higgins. Throughout the period 1968-72, H. A. Fehlmann was largely responsible for the continuous running of the programme at the centre, with the able technical assistance of A. Flerchinger. H. R. Adams. B. J. Landrum pro- vided further assistance, and D. Damkaer provided advice on crustacean matters. At Bath University (United Kingdom), M. Stribley and A. Greenaway maintained the technical efforts with the help of N. Storey. They also handled most of the local arrangements for the Bath symposium. The support of the university Vice-Chancellor, L. Rotherham, is gratefully acknowledged. During 1967-70, the Danish Institute for Fisheries and Marine Research provided much support to V. Kr. Hansen, Chairman of SCOR Working Group 23. E. Bertelsen, the institute’s Director, provided much advice as well. Assistance was provided by K. Albrechtsen, E. Rosendahl and A. M. Bresta. In Japan, experiments were carried out by courtesy of the Faculty of Fisheries at Hokkaido University, the Ocean Research Institute of Tokyo University, and at the Seto Marine Biological Laboratory of Kyoto University. R. Marumo and S. Motoda and M. Omori and T. Tokioka participated actively. Tn India, the project was supported by N. K. Pannikkar, Director of the Indian National Institute of Oceanography and by T. S. Rao, Officer in Charge, Indian Ocean Biological Centre where T. Balachandran carried out most of the experimental work. At Singapore, SCOR Working Group 23 research was conducted at the Regional Marine Biological Centre under the Director, Tham Ah Kow. Advice was always available from P. M. David of the Institute of Oceano- graphic Sciences, Godalming (England) and the frequent collection of plankton for the project by M. V. Angel and B. K. Rowbury helped greatly. Similar assistance was also available from R. I. Currie, Director of the Dunstaffnage Marine

Laboratory, Oban (Scotland), and also from R. F. Harden-Jones, D. Harding and J. H. Nichols of the Fisheries Laboratory, Lowestoft (England). Plankton catches were provided by K. F. Wiborg of the Institute of Marine Research, Directorate of Fisheries, Bergen (Norway) and by L. Eugene Cronin, Director of the Biological Laboratory, Solomon’s Island, Maryland (United States). L. Dove1 of the latter institution provided further field assistance. S. D. Ripley, Secretary of the Smithsonian Institution and R. Serene provided encouragement and guidance. K. W. Petersen edited the section on taxonomic data. The list of those who attended the SCOR Working Group 23 symposium, held at Bath University under the chairmanship of V. Kr. Hansen, 13-18 July 1972, is as follows: Adams, J. A. (United Kingdom) Angel, M. V. (United Kingdom) Balanchandran, T. (India) Be, A. W. H. (United States) Beers,* J. R. (United States) Burdon-Jones, C. (Australia) Dean, J. R. (Portugal) Faber, D. J. (Canada) Fawell, J. D. (United Kingdom) Fehlmann, H. A. (United States) Fenaux, R. (France) Flugel,* H. J. (Federal Republic of Germany) Griffiths, B. (Australia) Harding, D. (United Kingdom) Harris, R. H. (United Kingdom) Hasle, G. (Norway) Hunt, M. G. (United Kingdom) * Members of SCOR Working Group 23. Kimor,* B. (Israel) Langhelt, P. St. J. F. (United Kingdom) Millot, N. (United Kingdom) Nichols, J. H. (United Kingdom) Omori, M. (Japan) Ong Jin Eong (Republic of Korea) Petersen, K. W. (Denmark) Petersen, M. E. (Denmark) Raymont, J. E. C. (United Kingdom) Rowbury, B. K. (United Kingdom) Serene, R. (France) Steedman,” H. F. (United Kingdom) Taylor, F. J. R. (Canada) Tokioka, * T. (Japan) Turner, R. (United States) Vannucci, M. (Mexico) Williams, R. (United Kingdom) Williamson, D. L. (United Kingdom) It was at this meeting that SCOR Working Group 23 concluded its work. It had been in existence for five years.

Contents Introduction 13 I Shipboard and curating techniques F. B. GrifJiths, A. FIeminger, B. Kimor and M. Vannucci Appendix: Treatment of micro- and nanoplankton samples F. J. R. Taylor II Determination of zooplankton biomass J. R. Beers Introduction 1. Selecting a biomass measure and the use of ‘equivalents’ 2. Sample preparation 3. Gravimetric methods 4. Volumetric methods 5. Chemical and biochemical methods 6. Calorific content 7. Biomass estimates based on size measurements 8. Particle discriminators in studies of zooplankton biomass Appendix: Summary and suggested recommendations III Narcotizing agents and methods H. F. Steedman IV Freeze-drying Freeze-drying of marine zooplankton R. H. Harris 17 32 35 37 39 42 49 54 61 64 67 69 74 87 97 V Aldehydes 1. General and applied data on formaldehyde fixation and preservation of marine zooplankton H. F. Steedman 103 2. Chemistry of fixation and preservation with aldehydes D. Jones 155

VI Miscellaneous methods 1. Miscellaneous preservation techniques H. F. Steedman 2. Examination, sorting and observation fluids H. F. Steedman 3. Refractive index H. F. Steedman 4. Osmotic pressures in fixation and preservation H. F. Steedman 5. Permanent mounting media H. F. Steedman 6. Tissue sections of crustacean eyes F. M. Sweeney 7. Electronic measuring devices in the sorting of marine zooplankton J. K. Fawell W Cell products: calcium and oil 1. Cell products: calcium salts H. F. Steedman and M. Omori 2. Cell products: oil in plankters H, F. Steedman VIII Fixation and preservation of various marine taxa 1. 2. 3. 4. 5. 6. 7. 8. 9. The fixation and preservation of marine Protozoa: some problems and general considerations B. Kimor Methods for preserving Tintinnida K. Gold Preservation and laboratory study of actinopods in plankton samples J. R. Beers Preservation of planktonic Foraminifera and other calcareous plankton A. W. H. Bi and 0. R. Anderson 175 182 184 186 189 196 201 209 222 Flagellates F. J. R. Taylor Appendix: Preparation for examination by scanning electron microscopy (SEM) F. J. R. Taylor Fixation and preservation of planktonic Coelenterates K. W. Petersen Ctenophora fixation and preservation H. R. Adams, A. P. Flerchinger and H. F. Steedman Fixation and preservation of chaetognatha M.-L. Furnestin Fixation and preservation of planktonic polychaeta P. E. Gibbs 10. Laboratory methods for processing crustacean zooplankton M. Omori and A. Fleminger 231 236 240 250 259 265 268 270 272 279 281

Appendix 1: Bulk staining of small marine zooplankton using borax carmine J. H. Nichols Appendix 2: A stain for morphological study of copepods T. S. English and G. A. Heron Il. Fixation and preservation of molluscan zooplankton R. D. Turner 12. Propylene phenoxetol/magnesium chloride method for narcotizing gastropod veligers D. H. Leibowitz 13. Fixation, preservation and taxonomic aspects of Thaliacea T. Tokioka 14. The appendicularians R. Fenaux 15. Maintenance of quality in fish eggs and larvae collected during plankton hauls E. H. Ahlstrom 16. Criteria of satisfactory fixation and preservation of eel larvae (leptocephali) P. H. J. Castle Appendix: Some technical aspects of leptocephalus fixation and preservation H. F. Steedman 287 288 290 301 305 309 313 319 321 IX Fixation and preservation experiments on marine zooplankton 1. Fixation and preservation experiments on marine zooplankton in Japan: outline of experiments and results S. Motoda, R. Marumo and T. Tokioka 325 2. Fixation and preservation experiments on marine zooplankton at Cochin: outline of experiments and results T. Balachandran 333 Index 341

Introduction This monograph presents the results of an investigation by SCOR Working Group 23 into the best techniques of fixation and preservation of marine zoo- plankton for taxonomic and morphological study. In addition the book includes a discussion of the treatment of zooplankton samples on board ship from the time the net comes out of the water (Part I), and considerations of procedures requiring the handling of zooplankton material in the laboratory, such as biomass measurement (Part II). Separate sections have been devoted to narcotizing agents and to freeze- drying methods (Parts III and IV), and old and new reagents and methods were examined for their value as zooplankton fixatives and preservatives (Parts V and VI). Some attention was also given to celloidin work and electronic measuring devices (Part VI) and to cell products such as calcium and oil (Part VII). Because of the diversity of zooplankton forms in many natural communities and the specific requirements necessary to achieve optimal results with many taxa, a variety of associated methods have evolved which specialists use and advocate for dealing with a wide range of marine plankters (Part VIII). The SCOR Working Group 23 programme was international in character and scope and was made possible through the generous help of many institutions and individuals (see ‘Acknowledgements’). It is the pleasure of the editor to thank those who gave this aid so freely at all times, and it is the hope of the contributors that usage of the techniques cited may bring to marine biologists, whether new- comers or old hands, some measure of added success and satisfaction. In the hope that the publication of this monograph will encourage a continu- ation of efforts to improve the techniques and methods at present used in the laboratory study of marine zooplankton and to explore new processes, Working Group 23, before disbanding, recommended that the research should be continued whenever and wherever possible, and that international as well as national funds should be devoted to this end. H. F. STEEDMAN

Part I Shipboard and curating techniques l__l- __.- -.-

Shipboard and curating techniques F. B. Griffiths, A. Fleminger, B. Kimor and M. Vannuccir Introduction Well prepared and properly curated plankton samples may serve as useful sources of data on the nature and occurrence of planktonic organisms. Each sample is unique in providing an irreplaceable record of biological conditions at a particular time and place. For studies on systematics and natural history they have usually been found to be relatively durable their reputation for apparent fragility is primarily the result of poor initial preparation or subsequent neglect. In addition, offshore samples require the use of expensive equipment and personnel for considerable periods of time. When the sample is damaged by poor fixation, poor preservation, inappropriate labelling or by careless handling, its durability and usefulness are seriously jeopardized. Records of the sampling procedures and environmental conditions prevailing at the time of sampling are an inherent part of the sample and together with the sample require secure storage and convenient retrieval. In this chapter, methods are discussed for the satisfactory fixation, preser- vation and storage of zooplankton samples taken for taxonomic examination. The first section presents a step-by-step summary of procedures that will provide durable samples. These procedures start as the net is pulled out of the water and end where the samples are ready for examination in the sorting centre or labor- atory. In the second section shipboard and curatorial techniques are discussed in greater detail, including care of nets, choice of containers, labels, formulae for fixatives and preservatives, data recording, and transport, storage and mainte- nance of samples. A discussion of Gxation and preservation will be found in Part V. Special methods for the fixation of particular groups of animals are presented in Part VIII. Microplankton and phytoplankton present special problems in preservation. 1. Addresses: F. B. Griffiths, CSIRO, Division of Fisheries and Oceanography, P.O. Box 21, Cronulla, N.S.W. 2230 (Australia) A. Fleminger, Scripps Institution of Oceanography, P.O. Box 1529, La Jolla, California 92037 (United States) B. Kimor, Israel Oceanographic and Limnological Research Ltd, P.O. Box 1793, Haifa 31 000 (Israel) Marta Vannucci, Regional Science Officer, Unesco House, 40-B Lodi Estate, New Delhi 3 (India). 17

Zooplankton fixation and preservation Techniques for microplankton are fully discussed by Kimor, Beers, Taylor, BC and Anderson and Gold in Part VIII. Summary of recommended procedures 1. Secure the flow-meter propeller against spinning by the wind when the net is lifted from the water. 2. Without delay, wash the net from the outside with a sea-water hose to concentrate all the plankton into the bucket at the cod end. If no sea-water hose is available, dipping the net up and down in the surface water without submerging the mouth is a convenient substitute. 3. Remove the bucket from the end and drain excess water from the catch. If the bucket has no draining windows use a concentrating sock or similar device this must be done as quickly as possible to avoid deterioration of the catch. 4. After the catch has been concentraied, wash the plankton into the sample bottle with sea-water and fill to about three-quarters with sea-water. The volume of the plankton should not exceed IO per cent of the volume of the jar. If the catch is larger, use a larger jar or divide the catch among the number of jals required to maintain this 1: 9 ratio. 5. Fix the sample within 5 min. 6. Formaldehyde is a suitable fixative for general taxonomic work and for bulk material. Several formulations are available. The most common method is to dilute commercial formaldehyde (40 per cent) so that it makes either 2 per cent or 4 per cent with sea-water: 4 per cent formaldehyde: 40 per cent formaldehyde (borax-buffered), IO ml sea-water, 90 ml. 2 per cent formaldehyde: 40 per cent formaldehyde (borax-buffered), 5 ml sea-water, 95 ml. Before diluting the strong, commercial formaldehyde, add 2 g of borax to every 98 ml of 40 per cent formaldehyde. This will raise the pH to around 8 to 8.2. If a lower pH is needed use sodium glycerophosphate 4 g to 96 ml of 40 per cent formaldehyde. Additives which bring anti-oxidant properties, improved resistance to bacteria and moulds, and which give flexibility to tissues, may be employed with formaldehyde. A concentrated solution of these additives with formaldehyde is as follows: propylene phenoxetol, 50 ml propylene glycol, 450 ml 40 per cent formaldehyde (buffered with borax) 500 ml. Ten ml of this strong solu- tion with 90 ml of sea-water will produce a good, mild fixative suitable for most plankters. To ti the plankters add to the sea-water and specimens as given in 4 above sufficient strong fixative (either 40 per cent formaldehyde plus borax, or formaldehyde plus additives) to make the required strength of fixative. Thus if 4 per cent formaldehyde was the desired strength, add to a 500-m] container, filled as indicated in 4 above, 50 ml of strong formal- 18