BRARY HOLE * .. ruo tr 1! Ji. (O- 5? ? R 1960 METHODS FOR OBTAINING AND HANDLING MARINE EGGS AND EMBRYOS Methods for Obtaining and Handling Marine Eggs and Embryos D. P. Costello, M. E. Davidson A. Eggers, M. H. Fox and C. Henley MARINE BIOLOGICAL LABORATORY WOODS HOLE, MASSACHUSETTS 1957 Copyright 1957 by Donald P. Costello All rights reserved First edition Q, PRINTED IN UNITED STATES OF AMERICA BY LANCASTER PRESS, INC., LANCASTER, PENNA. CONTENTS PAGE Introduction viii General Methods xi Porifera Mycale fibrexilis 1 Coelenterata (Introduction) 3 Hydrozoa : Bougainvillia sp 4 Campanularia flexuosa 6 Clava leptostyla 8 Eudendrium ramosum 10 Eutima mira 12 Gonionemus murbachii 14 Gonothyrea loveni 16 Hydractinia echinata 18 Obelia sp 21 Pennaria tiarella 23 Podocoryne carnea 26 Tubularia crocea 28 Turritopsis nutricula 30 Scyphozoa : Aurelia aurita 32 Cyanea capillata 34 Anthozoa : Metridium dianthus 36 Ctenophora Mnemiopsis leidyi 38 Platyhelminthes Hoploplana inquilina 40 Polychoerus caudatus 42 Nemertea Cerebratulus lacteus 44 Bryozoa Entoprocta : Barentsia laxa 48 Pedicellina cernua 49 Ectoprocta : Bugula sp 51 Crisia eburnea 53 Electra pilosa 55 Sipunculida Phascolosoma gouldi 57 vi CONTENTS Annelida (Polychaeta) Amphitrite ornata 59 Arenicola cristata 61 Chaetopterus pergamentaceus 63 Cirratulis grandis 67 Cistenides gouldi 69 Clymenella torquata 71 Diopatra cuprea 73 Harmothoe imbricata 76 Hydroides hexagonus 77 Lepidonotus squamatus 81 Nereis limbata 83 Platynereis megalops 89 Podarke obscura 91 Sabellaria vulgaris 93 Sthenelais leidyi 98 Mollusca Amphineura : Chaetopleura apiculata 100 Pelecypoda : Callocardia convexa 103 Crassostrea virginica 105 Cumingia tellinoides 108 Ensis directus Ill Mactra solidissima 113 Mya arenaria 117 Mytilus edulis 119 Nucula proxima truncula 122 Pecten irradians 124 Teredo navalis 127 Venus mercenaria 130 Yoldia limatula 133 Gastropoda : Busycon sp 135 Crepidula sp 137 Haminea solitaria 141 Ilyanassa obsoleta 143 Lacuna vincta 147 Littorina obtusata 149 Thais lapillus 151 Urosalpinx cinerea 153 Cephalopoda : Loligo pealii 155 Arthropoda Balanus eburneus 160 Emerita talpoida 163 CONTENTS vii PAGE Lepas anatifera 165 Libinia emarginata 167 Echinodermata Asteroidea : Asterias sp 170 Henricia sanguinolenta 175 Ophiuroidea : Amphipholis squamata 177 Ophioderma brevispina 179 Ophiopholis aculeata 181 Echinoidea : Arbacia punctulata 184 Echinarachnius parma 191 Holothuroidea : Leptosynapta sp 194 Thyone briareus 196 Protochordata Enteropneusta : Saccoglossus kowalevskii 199 Tunicata : Amaroucium constellatum 202 Botryllus schlosseri 207 Ciona intestinalis 211 Molgula citrina 214 Molgula manhattensis 216 Perophora viridis 218 Styela partita 221 Chordata (Vertebrata, Teleostei) Fundulus sp 224 Menidia sp 228 Tautogolabrus adspersus 231 Other teleosts of the Woods Hole region: Cyprinodon variegatus 233 Opsanus tau 233 Scomber scombrus 233 Stenotomus chrysops 233 Strongylura marinus 234 Syngnathus fuscus 234 Table : Summary of egg characteristics 236 Index . 239 INTRODUCTION This manual represents a compilation of the methods for handling embryo- logical materials available at Woods Hole, Massachusetts. The material outlined was assembled from personal communications, from the laboratory guides prepared by members of the Embryology Course staff of the Marine Biological Laboratory at Woods Hole, from the observations made by Marjorie Hopkins Fox, Annette Eggers and Margaret E. Davidson as Embryology Course research assistants, and from the work on marine forms done by the senior author and Catherine Henley. In the past, much valuable time-saving information has been lost because it passed from investigator to investigator merely by word of mouth. E. E. Just's articles in the Collecting Net (1928) and his book, "Basic Methods for Experi- ments on Eggs of Marine Animals" (1939), were the first attempts to preserve this type of knowledge for the younger generations of investigators. This manual was started as an extension of Just's book, giving more details about the forms he described, and including data on additional species. Wherever possible, we have avoided descriptions of standard technical laboratory procedures which are avail- able in other books. Although our account is based upon those animals available at Woods Hole, it is apparent that the methods may be used to advantage elsewhere. Many of the forms described here are available along the entire Atlantic coast, and closely related forms (with similar breeding-habits and similar embryonic development) are to be found on the European, Pacific and southern Atlantic coasts. The breed- ing seasons listed in this account apply in most cases only to the Woods Hole region. Many workers at Woods Hole have been limited in the scope of their work by lack of information concerning the local fauna. Given a concise source of infor- mation, an investigator should be able to select the egg best suited for any purpose : large or small; transparent and colorless, or opaque and pigmented; with a tough membrane, or with a delicate one; with external jelly, or free of it. It is possible to utilize eggs at each of the four recognized stages of attainment of the fertilizable condition; eggs with a pronounced cortical change at activation, or with little or no change; eggs with or without micropyles; eggs with slow or rapid sperm pene- tration, and with slow or rapid development; eggs with any of the various types of etc. cleavage ; The present volume is admittedly incomplete, and better methods for handling certain of the materials may be forthcoming, or even already in use. We hope that workers will report errors, and make suggestions concerning improvements; gaps in the data should be filled in, and information for additional forms added. Each genus of animal at Woods Hole probably provides embryological materials worthy of investigation, and it is hoped that eventually all the most useful forms will be included in a later revision of this work. The listing of the names of the co-authors is on an alphabetical basis. Mrs. Fox served as research assistant in the Embryology Course at the M. B. L. during the summers of 1945, 1946, and 1947, obtaining information for this manual. She was succeeded by Miss Eggers for the summer of 1948, and by Miss Davidson for the summers of 1949 and 1950. After a lapse of several years, the work was resumed INTRODUCTION ix in 1956 under a National Science Foundation grant (NSF-G2477) with the aid of Miss Henley, who edited the sections previously prepared and added several new ones. In general, the nomenclature which we have adopted for the forms considered is that used in the classic earlier papers in the field of embryology, since we feel that these are the names by which the animals are best known to workers at Woods Hole. Thus, we have retained the generic names "Nereis" and "Mactra," for example, even though recent taxonomic revisions have assigned other names to both these forms. All the common synonyms known to us for the names of animals considered are included in the index to this volume, and in the heading of each description. The arrangement of sections is based on a phylogenetic sequence from "lowest" to "highest," and within each sub-division, the genera are described in alphabetical sequence. A bibliography has been compiled for each section, listing (1) those papers and books on which our description is based, and (2) some of the reports in the litera- ture which have utilized the form under consideration. We have made no effort to include all the papers describing work done on a given animal, but only those which were most useful to us in our compilation. There were many early investigators at Woods Hole who worked out the details of obtaining and handling one or more types of embryological materials, as well as the fundamental aspects of the embryology of such forms. Sometimes they incor- porated this information into their papers, but more often it was omitted. About 1940, when the older generation of embryologists at Woods Hole (including E. B. Wilson, E. G. Conklin, F. R. Lillie, A. D. Mead, A. L. Treadwell, C. M. Child, H. E. Crampton and others) was beginning to dwindle in numbers, Dr. Viktor Hamburger suggested that plans be made for a manual which would put on record some of the methods utilized those the of marine animals by studying development ; he obtained funds from the Marine Biological Laboratory for a summer research assistant to help with the project, beginning in 1945. Another factor contributing to the initiation of the work was the almost total disappearance of the sea urchin, Arbacia, from the usual collecting grounds near the Laboratory; this occurred about 1945. Earlier, when sea urchins were abun- dant at Woods Hole and thousands were often collected at a time, the Arbacia egg was the "standard living cell" upon which much of the physiological, biochemical and cytological work was done. With the marked decrease in numbers of Arbacia, however, it became impossible for many investigators to utilize this form, and there were frequent inquiries as to the availability of other egg-species. A collector-curator, George M. Gray (head of the Supply Department for many years), had very extensive and readily available knowledge of the Cape Cod marine organisms. He was succeeded in 1933 by James Mclnnis, and the staff of the Supply Department was trained under the able direction of Mr. Gray, Mr. Mclnnis and the older, experienced men. It would have been impossible to produce this guide without the willing help given by members of the Department. Although it is impossible for us to mention the names of all those who directly or indirectly contributed to the M. B. L. Embryology Course outlines (which served as a basis for some of the sections in this book), we should like to acknowl- x INTRODUCTION edge their contributions, as well as the assistance given us by investigators at the Laboratory, course instructors, teaching assistants, students and others. Some of these persons are mentioned in the list which follows : M. Jean Allen, P. B. Arm- strong, W. W. Ballard, C.