ait i enV a hil { an lhe > Vou. XIX NOVEMBER, 1949 : Ne] THE CONTRIBUTIONS OF THE WOODS HOLE OCEANOGRAPHIC INSTITUTION C. O’D. IsELIN Director, Woods Hole Oceanographic Institution The dislocations of the war years have finally subsided at this Laboratory and it is hoped that the research program has become stabilized for a few years at least. The great majority of in- vestigations having direct practical applications have been terminated so that, of a total budget of about $750,000, which is now virtually assured through 1950, only about one-fifth comes under the heading of applied research or development. Thus, what we consider basic oceanography has grown at Woods Hole from a pre-war budget of about $110,000 to a post-war budget of about $600,000. This is not an exceptional increase. For the country as a whole, it has been estimated that the oceanographic budget has increased more nearly by a factor of ten and that it will double again during the next ten years. This is only to say that interest in the physies, chemistry, ge- ology and biology of the oceans is growing rap- idly. The Federal Government is especially aware of the need for increased knowledge of (Continued on page 3) THE MARINE BIOLOGICAL LABORATORY IN 1949 Dr. CHARLES PACKARD Director, Marine Biological Laboratory The 62nd session of the Marine Biological Lab- oratory formally opened with the first Friday evening lecture by Dr. Gilbert Tyler. But long before this time investigators had been arriving, some in May and many more before the middle of June. Others will continue to come until late August. By the middle of September most of them will have left, but the workers in the Insti- tute of Muscle Research, under the direction of Dr. Szent-Gyorgyi will remain throughout the year. The laboratory welcomes these newcomers, and wishes that more investigators would find it possible to carry on their research here after the summer season. As usual, more people applied for places in the laboratories and in the courses than we could accommodate. A few more investigators could have been accepted if there were more rooms available in our residences and in the village houses. The housing situation will not material- ly improve until a number of investigators build their own homes in the Devil’s Lane Tract or elsewhere. There is no need to call attention to Marine Biclogical Laboratory, Dr. C. Packard... 1 Contributions of the Woods Hole ccunoerep hie Institution, C. O’D. Iselin U. S. Fisheries Laboratory, D 5 Serological Aspects of Fertilization, Dr. A. Tyler. 6 Mechanism of Color Changes in Crustaceans, Dr. iraniaweAe SS TO WI) ise eee eee Labile P in Nueleie Acids, Abel Lajtha Structure of Fibrin Clots, Dr. E. Mihalyi Investigations on Muscle Fibers, Dr. A. G. Szent- Gyorgyi alhs) Evidence for Activity of DNAse i in Mitosis by Use of d-usnie Acid, Dr. Alfred Marshak... 16 Cold as a Means of Combatting Asphyxia in New- born Guinea Pigs, James A. Miller, Jr. 17 TABLE OF CONTENTS A Simple Method for Inducing Spawning of Sea Urchins and Sand-Dollars, Dr. A. Tyler 19 Biological Specificity and Protein Structure, Dr. IDYONROH Me Seba CN 2 ee 20 Reactions of Cells in Frog Tadpoles to Implants of Tantalum, Dr. Carl C. Speidel.__...___________ 21 Growth and Metamorphosis of the Pluteus of Arbacia punctulata, Ethel B. Harvey_ 22 Reversible Enzymie Reduction of Retinene to ‘Vita- min A, Dr, Alfred F. Bliss _ 2 Effects of Ultra-violet Light on the Catalase Ac- tivity and on Photosystheses, Dr. A. Frankel _ 24 Synthesis of Acetylcholine, Dr. D. Nachmansohn, S. Hestrin and H. Voripaieff : Pee), Directory of the Marine Biological Laboratory. pero 27 Pie lele ‘SIPLIpUIFT GUT[IO}S “puvjsvpy sepsnod ‘swing “We rT ‘SweENeMA “OM ‘ueLg ‘fp “yw OULU, “MH ‘PPypEYy DO “y Gaeduvg “yp oy ‘Yale pavuoory “WAT, WoqTy “400g es100H—MoL yuo ‘puvpsvoy, uwospuy OYsty Ypouusy “uUvwmozeg ‘poyruapray) ‘Koarvypy “eq pouyoy ‘Sqooer “HW ‘wang Wy “A ‘paep “POD “A “C “nvyteysO “A “fF “M “UUMAqTeR “A YT ‘atag “A “g “nqity [ey MOL OTPPUN = WOTFVH “TT “uuvuIyL, “A sy “s80yg PULA MOLOYIT AA OUTED ‘fT ‘poomdvyy oyjopreyp “lassotg TT “9 ‘uosaaugE qaoqoxp Spangy wraq ‘OSBYD “WV “UMOrg, “y yuRIyE ‘syoorg py pW Song UYOP “WOsTRUpPV NT "q—MoL yor HIOH SGOOM LV SLSIDOTOISAHd TVYANAD JO ALAIOOS AHL JO SLSAND AUNV SudgWa PSS November, 1949] THE COLLECTING NET 3 the crowded condition of the Mess. Sixty years ago the laboratory acquired a small steam launch at a cost of $1,350. Later the classes were carried to the collecting areas in the Vigilant, a sailing vessel which was towed by the launch. Now we have a fleet of five power boats —Dolphin, Limulus, Nereis, Sagitta and Tern. The first two are new; the others have been in our service for many years. The Dolphin, purchased last year, and used by the classes and the collect- ing crew is now provided with a Diesel motor. Thus the danger of explosion or fire, has been reduced to a minimum. This summer there are five labor fellows and five Atomic Energy Commission fellows in resi- dence. The former are investigators who already have their doctorate and are competent to carry on independent research in the fields of biophys- ies, biochemistry or biological chemistry. The latter are in general a younger group. Each has a definite problem involving the use of radio- active isotopes. One purpose of these fellowships is to train investigators in the new and exacting techniques required in this type of research. On the third floor of the Brick Building is a ‘‘hot laboratory’’ where transfers of highly ac- tive material can be made. Under Dr. Failla’s direction it has been equipped with the safe- guards necessary to protect the workers from radiations. Funds for the equipment were fur- nished by the American Cancer Society. The renovation of the Old Main Building, be- gun this Spring, is the first major change to be made in the oldest building of the Marine Bio- logical Laboratory. It was in 1888 that the south wing, now used by the Embryology class and some investigators, was erected. The next The Contributions of the Woods year the middle portion was added to serve as a lecture room and a library. Then in 1892 the north wing was built. Many notable biologists have occupied the little rooms on the first and second floors. Among these were Whitman, the first director; Loeb, physiologist; F. R. Lillie, embryologist; T. H. Morgan, geneticist; E. B. Wilson, cytologist ; and G. N. Calkins, protozoolo- sist. Some of the early pioneers—Conklin, R. S. Lillie, Mathews and Osterhout, have continued to return to the M.B.L., their places of summer work for more than fifty years. The renovation of the building was made pos- sible through the generosity of the Rockefeller Foundation, which has on previous occasions contributed to this laboratory. The basement, originally almost unexcavated, is now provided with well equipped laboratories used by the Physiology Class and by instructors in other courses. The upper floors will be remodeled next Fall. In the Embryology Laboratory the water table is to be rebuilt and moved toward the back of the room. On the Physiology side, the present stairway will be removed, and a new one will open from the street with stairs both to the basement and to the second floor. The present arrangement of rooms will be much al- tered. Upstairs the rooms will be enlarged, and some will be provided with salt water tables. It is hoped that insulation of the roof will keep room temperatures within reasonable limits. With all these changes, the building still re- tains much of its original character. It will al- ways be called ‘‘Old Main’’ to remind us of our debt to those who first worked in it and laid the foundations of American Biology. Hole Oceanographic Institution (Continued from page 1) the oceans and it is from this source that most of the money is coming to oceanography. How- ever, it may be significant to point out that our sister laboratory, the Scripps Institution of Oceanography, at present is receiving roughly $400,000 per year from the State of California. Many other coastal states also are making sub- stantial contributions for studies of the ecology of inshore forms, including the State of Massa- ehusetts which has given a contract to this In- stitution for the study of shellfish. There are, at present, 264 persons actively en- gaged in work at the Woods Hole Oceanographic Institution. These can be classified as follows: Full-time scientific and technical staff. 89 Part-time scientific ; i.e., summers only — 44 Fellowship holders So Visiting investigators 118 Secretaries and clerks 19 General maintenance and services ——_— 40 Crews of vessels JA Ghoawbangyiee yan ee The total summer increase, including fellowship holders and visiting investigators, is 68 which is roughly the pre-war figure. Of what does this boom in oceanography con- sist? It will only be possible here to describe very briefly some of the main lines of investiga- tion in which especially rapid progress is being made. The most active group in oceanography today are those interested in the geology and_ geo- physies of the ocean basins. The recording echo sounder when combined with new, radio-naviga- tional techniques makes it practical to examine in detail the topography of the ocean bottom. During the last few years, the Atlantis has ac- eumulated about 70,000 miles of bottom records 4 THE COLLECTING NET [Vol. XIX, No. 1 and gradually it is being revealed that the bot- tom of the ocean is as varied and complex as the surface of the land. Recent soundings in the neighborhood of Bermuda show that the island is situated on a ridge of low hills, trending northeast-southwest. Several submerged sea mounts, nearly as high, have been located in the general area. The Hudson River Canyon has re- cently been traced nearly halfway to Bermuda and at present our newest research vessel Caryn is tracing the continuation of this remarkable feature towards Bermuda. It is evident that the course and character of the ocean currents may be very much influenced in passing through regions of pronounced bot- tom topography. The Atlantis left two weeks ago to study these relationships in the area east of the Grand Banks. Another development in submarine geology has come about as the result of great improve- ments in coring tubes. The piston-type coring tube, first used by Swedish oceanographers, now permits cores thirty to fifty feet in length to be obtained from the deepest waters. Since the rate of sedimentation in deep water is relatively slow, there is great hope that much of the recent his- tory of the earth can be worked out rather quick- ly through studies of such cores. They form an undisturbed record of the changes in depth and climate extending over a period of several 1il- lion years. The seismic techniques developed in oil ge- ology have been adapted for use at sea and are allowing the examination of the rock structures underlying the ocean down to ten or fifteen miles below the bottom of the sea. Of special interest is the location of the edge of the granite on which the continents are built and to learn about the character of this edge. Turning to subjects more usually identified with oceanography, the study of the heat and water vapor exchange between the sea surface and the atmosphere is receiving special empha- sis. During the past twenty years it has been the fashion more or less to neglect heating and cool- ing as a cause of the general circulation, both in the atmosphere and in the hydrosphere. Recent studies of the Director of the Royal Netherlands Meteorological Institute are indicating that even on a day to day basis sea surface temperatures can exert a major effect on the development of the weather. The role of salt nuclei picked up by the air passing over the ocean also is proving to be a fascinating and important study connect- ing meteorology and oceanography. It will, of course, be a long time before the dis- tribution of temperature, salinity, oxygen and nutrient chemicals can be described in satisfac- tory detail. The broad seasonal and geographi- cal aspects of physical oceanography is a study requiring patience and a certain amount of sus- tained organization. If the collection of data was left entirely to the interests of individual investigators, it would proceed much more slow- ly than is desirable. In this sense, an oceano- graphic laboratory has somewhat the role of an astronomical observatory. Vast quantities of routine data must be collected and digested be- fore even the basic problems can be clearly de- fined. Improved instrumentation, both at sea and in the laboratory, is greatly accelerating the descriptive aspects of physical oceanography. It is not enough that the ships take in large quan- tities of, for example, routine temperature data. The process of correcting, sorting, averaging and digesting must, if possible, also be facilitated through machines of one kind or another. Al- though the instrumentation of oceanography has developed rapidly, it is clear that there is still much more to be done. The market for oceanographic instruments will probably always remain small. It is for these reasons that instru- ment design and construction remains an impor- tant activity at our laboratory. It would be nice to be able to report that bio- logical oceanography is going ahead with as much vigor as the physics and geology of the sea, but unfortunately in this case money is a serious limiting factor. Although it is clear that man will soon have to turn more and more to the sea as a source of protein, and this is already the case in several countries bordering rather bar- ren seas, there is little financial support in this country for basic studies of the produetivity of the oceans. We know about how many haddock are to be found on George’s Bank and about how much sustained yield can be expected from this area, but when it comes to the productivity of the oceans as a whole we know very little. Quan- titative studies of the smaller forms have been made, for they cannot easily escape a net, but as we advance up the food chain in the sea, the quantitative and geographical aspects beeome very vague indeed. Here again considerable instrumental develop- ment will be required. Two possible quantitative tools for marine biology are suggested by recent refinements in underwater acoustics and under- water photography. However, it also seems like- ly that marked improvements in the effective- ness of nets of various kinds can be made. Once really good sampling techniques have been de- vised the marine biologist will be face to face with the same problem that the physical ocean- ographer already has had to deal with. That is, he will soon be swamped with data, unless means are devised in advance to facilitate the analysis phase of a given investigation. November, 1949 | THE COLLECTING NET oO THE WORK OF THE UNITED STATES FISHERIES LABORATORY AT WOODS HOLE (fees = Lia e Dr. Paut 8S. GALTSOFP ce ; Director, United States Fisheries Laboratory \G la se ce a During the years following the end of the World War II, the United States Fisheries Lab- oratory at Woods Hole was gradually rehabili- tated and adapted for year-round operation. Investigators coming regularly to Woods Hole for the last ten years may recall that the build- ings and grounds of the Laboratory were seri- ously damaged by the hurricanes of 1938 and 1942. Although the most serious defects have been repaired, the signs of the ravages caused by the wind and sea and are still noticeable; the sea wall along the southeast side of the small boat basin is still in ruins, and the pool in which the sharks and seals were formerly displayed has not been restored. The Laboratory was able, however, to rebuild the sea wall around the grounds and to rehabilitate the laboratory build- ing and the residence which have been made suit- able for all-year occupancy. The sea water ta- bles, chemical benches and other laboratory equipment which were removed when the sta- tion was occupied by the U. 8. Navy during the war have been completely restored. The water pipes were repaired and the buildings rewired and reconditioned. The hatching of marine fish (cod, flounder and mackerel), which for many years had been carried out by the old Bureau of Fisheries, has been discontinued and the hatchery equipment adapted for biological research. In 1947, ar- rangements were made to transfer the headquar- ters of the section of the North Atlantic Fish- eries Investigations from Cambridge, Mass., to the Woods Hole Laboratory. The necessary re- arrangements to provide additional office and laboratory space for investigators and docking facilities for the research vessel Albatross III were completed that year. A comprehensive program of fishery research in the North Atlan- tic and its progress will be discussed in a sepa- rate article by Dr. William F. Royce, in charge of the project. Besides the studies carried on by the North Atlantic Section, the Woods Hole fisheries labo- ratory is engaged in shellfishery research con- ducted by Dr. Paul 8. Galtsoff and serves as a temporary headquarters for the clam investiga- tions carried on by John B. Glud. Following a well-established old tradition of working together with other scientific institutions at Woods Hole, the Service made a cooperative agreement in 1947 with the Marine Biological Laboratory for an exchange of services and facil- ities. The plan has proved mutually pleasant and profitable. Full cooperation with the Woods Hole Ocean- ographic Institute is likewise a very important factor in carrying on the research program of the Laboratory. Close association with the offi- cers and personnel of both institutions, mutual assistance in case of emergencies and free ex- change of ideas, creates a favorable environment which stimulates researeh work. The Aquarium of the Laboratory was re- opened to the public in 1947. Thanks to the cooperation of the Supply Department of the Marine Biological Laboratory, it was possible to assemble and display to the public from 55 to 65 different species of fish and invertebrates which occur in local waters. As an innovation, part of the former hatchery room on the first floor was set aside for special exhibits showing the various techniques used in marine biology. Of special interest are the exhibits arranged by the Woods Hole Oceanographic Institute, which show ocean- ographic instruments, automatic recording de- vices, underwater photography, sounds recorded in the depths of the sea, and various methods em- ployed by modern science in the study of the ocean. The Aquarium is open to the public every day, including holidays, from 8:00 A.M. to 8:00 P.M. The number of visitors, particularly on Sundays and holidays is surprisingly large, fre- quently exceeding 1,000 persons a day. Thus far, the Service has not been able to ob- tain sufficient appropriation for the complete re- habilitation of buildings and grounds and for the modernizing of scientific equipment and the Aquarium. Every year, however, the work of reconstruction and rehabilitation continues with the limited funds available for this purpose. The investigators of the Laboratory are confident that with this increased scope of scientific activi- ties the Laboratory will become an important center of research and training in fishery biology. 6 THE COLLECTING NET [Vol. XIX, No. 1 SEROLOGICAL ASPECTS OF FERTILIZATION Dr. ALBERT TYLER California Institute of Technology, Pasadena It seems quite appropriate that a talk on ferti- lizin and related substances should be presented here at Woods Hole since it was here that the subject was first developed by the late Professor Frank R. Lillie, who was director of this labora- tory for many years. It was here, too, that most of the early work in this field was done by Jac- ques Loeb, Otto Glaser, Alvalyn Woodward, C. R. Moore, E. E. Just, Myra Sampson and G. H. A. Clowes. After the highly interesting early work investigations along this line practically ceased from alout 1922 until 1939 when Max Hartmann and his colleagues, working at Naples, and we, in Pasadena, undertook a series of inves- tigations which have continued, with some inter- ruption, during the war years. More recently John Runnstrém and his co-workers in Stock- holm have entered this field. In general the re- sults of the latter investigators agree very well with our own while those of Hartmann and his co-workers differ in several points. Recent de- tailed reviews of the subject have been written by Bielig and Medem (1949) and by Tyler (1948, 1949). The present summary is based primarily on the work of the author’s laboratory. This work has been concerned with four kinds of substances that have been isolated from eges and sperm of marine animals; namely, fertilizins from eggs, antifertilizins from sperm, antifertilizins from egos and egg-membranes lysins from sperm. Early in this work it was shown that the ferti- lizin of eges of the sea-urchin and other animals is the macromolecular material of the gelatinous coat, and this has been confirmed by Hartmann, Runnstr6m and others. The gelatinous coat slowly dissolves as the eges stand in sea-water, yielding the so-called ege-water that has the property of ageglutinating homologous sperm. The gelatinous coat can be rapidly dissolved in slightly acidified sea-water, without injury to the rest of the egg, and concentrated solutions of fertilizin thereby obtained. Various tests, in- eluding the action of purified proteinases, showed the fertilizins of the sea-urchin and the keyhole limpet (as defined by their agelutinating action) to be of protein nature. By relatively simple extractions and precipitation procedures we have been able to prepare sea-urchin ferti- lizin in electrophoretically and ultracentrifu- gally homogeneous form. The purified material contains both amino-acids and sugars and may, therefore, be termed a glycoprotein or mucopoly- saccharide (depending upon the terminology adopted). It is of highly acidie character, show- ing little change in electrophoretic mobility be- tween pH 8.6 and 2.0 This is evidently due to the fact, discovered by Vasseur (1947) and con- firmed by us, that it contains over 25 per cent sulphate, most probably linked in the manner of a sulphuric ester. Some of the analytical data obtained on purified fertilizin is given in Table I. The values for the amino-acid and reducing sugar content are minimum due to the fact that a fair amount of not readily analyzable humin residue forms upon acid-hydrolysis. Galactose has been identified as the osazone. Paper chroma- tography has shown the presence of at least seven different amino-acids which are, most probably, aspartic acid, elutamic acid, threonine, lysine, arginine, phenylalanine and isoleucine in addi- tion to tryptophane in the humin residue. TABLE I Analysis of electrophoretically homogeneous preparations of fertilizin of Strongylocentrotus purpuratus IND trogen! eae - 9.6-5.8% Carbon 33.3% - Hydrogen cual eee 5.5% Sulphate asa omen 23% Phosphate 0.06% Reducing sugar >25% Amino acids —__ >20% Glucosamine (?) 1.6% Galactose pos. Glucuronie acid neg. neg. Molecular weight (eale. as sphere) — 82,000 On the basis of the results of attempts to sepa- rate protein and polysaccharide fractions of fer- tilizin it is concluded that these do not exist as loosely bound distinct entities in the molecule but rather that amino-acid and sugar residues are firmly inter-linked. In this connection it may be noted that other glycoproteins of somewhat similar composition, in particular those exhibit- ing the human ABO blood-group activity, have likewise proven refractory to attempts to disso- ciate protein and polysaccharide constituents (see Morgan, 1947). The antifertilizin from sea-urchin sperm has also been prepared in electrophoretically homo- geneous form. It is an acidic protein, isoelectric at pH 3. Investigations of its amino-acid com- position are in progress. One of the workers in November, 1949] Runnstrém’s laboratory (Hultin, 1947) has sue- gested that it may be a basic protein, but this is refuted by results published recently by Metz (1949) as well as by Runnstrém’s (1942) and our own data concerning its electrophoretic mo- bility. Evidence from electron microscopy of extracted sperm shows the antifertilizin to be located on the surface of the nuclear region of the head. The antifertilizin from eges and the egg-mem- brane lysin have likewise been shown to be of protein nature. Dr. Max Krauss, of our labora- tory, has obtained e@ood evidence showing that the action of the lysin of keyhole hmpet sperm is dependent upon the presence of sulfhydryl eroups. Electron microscopy of extracted sperm indicates that the acrosome may be the source of the lysin. Lillie considered the interaction of fertilizin and sperm to be analogous to that of serological agelutination and evidence has since accumu- lated that the kind of interaction exhibited by the various substances extracted from the eggs and sperm is essentially that of antigen and an- tibody. The finding of an antifertilizin within the ege alone with fertilizin in the coat, means, then, that in one and the same cell there are a pair of substances that are capable of interacting in antigen-antibody manner. This along with consideration of certain information from the literature of immunology has led to the develop- ment of a so-called auto-antibody concept of cell structure, growth and differentiation that has been presented recently in some detail (Tyler, 1947). Briefly this view states that the macro- molecular substances of which cells are con- structed bear the same relationship to one an- other as do antigen and antibody and that they are formed in essentially the same manner as are antibodies. In addition to various experiments of others that can be interpreted on the basis of the occurrence of such natural auto-antibodies the author has been able to demonstrate the pres- ence of an auto-antivenin in a venomous reptile, the Gila monster. The view also offers interpre- tation for certain serological anomalies, such as the Wassermann reaction, auto-agelutination phenomena, specific interaction of virus with cell surface, ete., and it offers the possibility of ob- taining protective agents against pathogenic or- vanisms by extraction of the organisms them- selves. Experiments; relating to the spontaneous re- versal of sperm agelutination by fertilizin in the sea-urchin (a phenomenon now known to occur also in the so-called Hirst reaction of hemagelut- ination by viruses) led to the finding that ferti- lizin could be converted. by simple treatments. into a non-agelutinating, ‘‘univalent’’ form. Evi- THE COLLECTING NET 7 id dence was also accumulated that fertilizin occurs normally in such ‘‘univalent’’? form in many species of animals and C. B. Metz (1945) dis- covered that specific agglutination of sperm could be obtained with such non-agelutinating fertilizins by the addition of a non-specific ad- juvant obtained from hen’s ege-white, serum- albumin or other sources. This latter result is paralleled by recent experiments of Wiener (1948) on ‘‘univalent’’ Rh antibodies which like- wise can cause specific agglutination in the pres- ence of certain non-specific proteins. Metz’s re- sults provide strong support for Lillie’s view that the fertilizins are of general distribution throughout the animal kingdom. As a side-line of some practical, as well as theoretical, interest a series of experiments were undertaken in which immune antibodies against manunalian blood cells, pathogenic bacteria and toxins were converted into the ‘“‘univalent’’ form by photo-oxidation and various properties of such antisera examined. The anaphylactic prop- erties of such treated sera were found to be greatly reduced. The evidence also showed that the ‘‘univalent’’ antidiptherial antibodies were capable of neutralizing the toxin but that ‘‘uni- valent’’ antipneumococcal antibodies were in- capable of acting as protective antibodies. With respect to serum-sickness factors there is, then, considerable improvement in the antisera in the former case but not in the latter. It was found, too, that the ‘‘univalent’’ anti-blood cell anti- bodies were incapable of acting as hemolytic sensitizer, or of fixing complement, and this offers some support of Heidelberger’s views concerning the mechanism of complement fixation. Investigations of the role of fertilizin and anti- fertilizin in fertilization have shown that, when present on the surface of the respective gametes, they facilitate the process. When present in solu- tion. however, they block fertilization, presuma- bly because the interaction of the sperm with fertilizin, or of the eges with antifertilizin, is completed before contact is made between the effective surfaces of the gametes. It has not, as yet, been possible to determine with any cer- tainty whether or not fertilizin-antifertilizin in- teraction is also essential for fertilization since, in the experiments on removal of fertilizin by methods that do not injure the rest of the eee, a minute laver of this substance evidently re- mains firmly bound to the surface. However, results of experiments employing immune anti- bodies against antifertilizin favor the view that the interaction is essential for fertilization. The role of the ege-membrane lysin, in species in which this agent has been demonstrated 1s mani- festly to enable the sperm to penetrate the mem- brane barriers that surround the unfertilved 8 THE COLLECTING NET [Vol. XIX, No. 1 ege. For the antifertilizin within the egg, Lillie had proposed a role in activation and establish- ment of the block to polyspermy but evidence concerning this is still lacking. Lillie’s demon- stration that fertilizin is obtainable from no other tissue than the gametes has been amply confirmed and this serves as a basis for under- standing the tissue-specificity of fertilization. An extensive investigation has been made, and is in progress, concerning the problem of species-speci- ficity. In general, the results show that the degree of cross-reaction of fertilizin and anti- fertilizin of various species is greater than the degree of cross-fertilization. Thus the specificity of fertilizin-antifertilizin interaction is not in itself sufficient to account for that of fertiliza- tion. Similar results are obtained with the lytic agent of sperm. Also, the specificity of these sub- stances as antigens in rabbits is not as great as that of fertilization. It appears then that other specific factors must be involved and this is not too surprising since it is quite likely that many other substances besides those discussed here are concerned in various steps in the process of ferti- lization. On the other hand, it should be noted that where cross-reaction between fertilizin and antifertilizin is lacking fertilization also fails to occur. A scheme has been proposed (Tyler, 1948) for the manner in which fertilizin-antifertilizin in- teraction may account for the approach and specific attachment of sperm to egg surface. As noted above Lillie also suggested that activation of the egg might involve these substances. At present the best available hypothesis concerning activation is that proposed by Heilbrunn (1943), which involves a protoplasmic gelation or clotting reaction initiated by a release of calcium. It is of interest to note, then, that the fertilizin-anti- fertilizin reaction is largely dependent upon the presence of calcium, as Loeb first showed and as Vasseur (1949) has recently demonstrated in some detail and, that fertilizin shows (see Im- mers, 1949) some heparin-like activity. References Bielig, H. J. and Medem, F. 1949 Experientia, 5: 11. Heilbrunn, L. V. 1943 An Outline of General Physi- ology, 2nd ed. Saunders, Philadelphia. Hultin, T. 1947 Arkiv Kemi, Mineral., Geol., 24B: No. 12. Immers, J. 1949 Arkiv Zool., 42A: No. 6. Metz, C. B. 1949 Proc. Soc. Exper. Biol. Med., 70: Morgan, W. T. J. 1947 Experientia, 3: 257. Runnstrom, J., Tiselius, A. and Vasseur, E. 1942 Arkiv Kemi, Mineral., Geol., 15A: No. 16. Tyler, A. 1947 Growth, 10 (suppl.) : 7. Tyler, A. 1948 Physiol. Revs., 28: 180. Tyler, A. 1948 Anat. Record, 101: 658. Tyler, A. 1949 Amer. Nat. (in press). Vasseur, E. 1947 Arkiv Kemi, Mineral., Geol., 25B: No. 6. Vasseur, E. 1949 Arkiv Kemi, 1: 105. Wiener, A. and Gordon, E. B. 1948 J. Lab. Clin. Med., 33: 181. 422. Nore: An abstract of a Friday Evening Lecture de- livered July 1, 1949, at the Marine Biological Laboratory. THE MECHANISM OF COLOR CHANGES IN CRUSTACEANS Dr. FRANK A. Brown, Jr. Professor and Chairman of the Department of Biological Sciences, Northwestern University The capacity for exhibiting changes in body coloration is widely distributed among higher animals. It is usually found only in species which possess well-developed eyes and ‘central nervous systems and the color changes are ordinarily complexly controlled through these organs. Of all the organisms showing color changes no group shows more striking examples of this capacity than the decapod crustaceans. Within this group the character of the changes spread through a wide range. Some species, as for example the fiddler crab, Uca, has as its major natural re- sponse simply a darkening of the body by day and a blanching by night. The sand shrimp, Crago, and the common prawn, Palaemonetes, on the other hand, are able to darken upon dark- colored backgrounds and lighten upon light-col- ored ones. In addition, both of these latter ani- mals are able to a good extent to become even the color of colored backgrounds upon which they come to rest. This is especially true of Palae- monetes. Still other crustaceans as, for example, the gulf-weed crab, Planes, and the shrimps, Latreutes and Hippolyte, appear able to imitate not only the color but also the pattern of the coloration of the algae upon which they live. These color changes in the crustaceans are ac- complished by means of special effector organs, the chromatophores, which are widely distributed over the surface of the body. These chromato- phores are highly branched, multinucleate cells, each containing one (monochromatic), two (di- chromatic) or more (polychromatic) pigments. The chromatophores normally function in both of two ways in the production of color changes. One of these two activities is referred to as physi- ological color change and involves the mechanical movement of the pigment within the chromato- phore. Any given pigment may concentrate into a spherical mass in the chromatophore center and thus not contribute to the gross coloration of the animal, or it may disperse out into the chromato- November, 1949] THE COLLECTING NET 9 phore branches and contribute to the coloration. By an appropriate differential activity of the chromatophoral pigments in physiological color changes, a prawn like Palaemonetes, possessing red, yellow, blue, and white pigments can readily assume the color of any background upon which it is placed. The rates at which physiological color changes are accomplished are relatively rapid, the time required for a pigment to pass from a maximally concentrated to a maximally dispersed state, or vice-versa, ranging from about five minutes to a few hours depending upon the chromatophore type. As would be ex- pected, the accuracy with which the background color can be matched by physiological color changes alone, depends upon the amounts of the required pigments present within the chromato- phores. The second of the two primary activities of chromatophores is termed morphological color change. This activity involves a differential syn- thesis or destruction of the pigments within the chromatophore. In Palaemonetes, for example, the red and blue pigments gradually disappear from the chromatophores of specimens kept upon a white background. Animals kept upon a black background show, on the contrary, a gradual gain in the amount of these dark pigments in the chromatophores. These morphological color changes are much slower than the physiological ones and require days or even weeks before the maximum adaptive change is completed. Extrac- tion ofsthe total pigment in the bodies of animals undergoing extensive morphological changes shows clearly that these changes involve chemi- eal alteration in the pigments and not simply a translocation of the pigments within the body. It was for many years assumed that the chro- matophores of crustaceans were directly under the control of nerves. Numerous attempts to dem- onstrate histologically the presence of nerves passing to the chromatophores all resulted, how- ever, in failure. Furthermore, when all the known nerve supply to a region of the body was transected, there seemed to be no disturbance, whatsoever, of the capacity of that region for color change. Even the accurately detailed tint- adaptation of a shrimp like Palaemonetes occurs in such denervated regions of the body. About twenty years ago, two investigators, working independently, demonstrated an action of blood-borne principles in the control of crus- tacean chromatophores. Koller, in Germany, utilizing the technique of blood transfusion, showed that blood from a black donor darkened a pale recipient and that blood from a yellow donor caused a pale recipient to become yellow. Perkins, in this country, observed that occlusion of a blood vessel resulted in an immediate cessa- tion of the color response of that region of the body supplied by that vessel, and that the abil- ity to respond reappeared at once following res- toration of the normal blood supply. Perkins demonstrated, in addition, that an aqueous ex- tract of the eyestalks of the shrimp, Palaemon- etes, hghtened dark specimens into which it was injected, and, conversely, removal of the eye- stalks left them permanently darkened. On the basis of these observations Perkins postulated the eyestalks to contain sources of a lightening hor- mone. At about the same time Koller reported that aqueous extracts of the rostral region of the sand-shrimp, Crago, darkened pale specimens and destruction of the region resulted in their remaining permanently pale. Koller therefore postulated a source of a darkening hormone to lie in the rostral region. These last observations of Koller have not yet been confirmed despite a number of attempts to do so. It appears clear at the present time that hor- mones conveyed by the blood constitute the only chromatophore- activating agents for the crusta- eceans. The great complexity of the endocrine mechanism which must be involved becomes ap- parent when, one recalls the independent activi- ties of the several pigments in single individuals in the.gourse of adaptation to different colored backgrounds. Palaemonetes possesses four kinds of pigments each of which is capable of activity independent of each of the other three. It is clearly evident that a minimum»of three hor- mones must be present in this shrimp to account for this complex differential activity of the pig- ments. Investigations during the past few years have provided us with considerable information as to sources, numbers and the activities of hormones influencing the various chromatophore types. The two major sources appear to be the sinus glands in the eyestalks and various regions of the cen- tral vervous system. The sinus glands are minute organs which are richly charged with secretory granules; these granules render the gland readily visible in the freshly dissected eyestalk. The glands are com- plexly innervated from the brain and optic gang- lia. Sinus glands may be easily dissected free of the remaining eyestalk tissue, extracted, and tested for activity by injection into other speci- mens. It seems quite clear at present that sinus glands of crustaceans possess at least three chro- matophorotropic principles. Evidence for this conclusion has come from two types of experi- ments: comparative physiological studies and chemical fractionation. Physiological studies of the actions of extracts of sinus glands of various species of crustaceans as tested by simultaneous assay of their actions upon two widely different 10 THE COLLECTING NET [Vol. XIX, No. 1 responding chromatophore types, Uca black pig- ment and Palaemonetes red pigment, have dem- onstrated that the activities of the extracts with respect to these two types do not vary in any correlated manner. This is what would be ex- pected were the activity upon one chromatophore type the result of one substance and the action upon the other, of another. Compelling the same conclusion are experiments upon chemical frac- tionation. An alcohol-soluble fraction of sinus glands from a wide variety of species of crusta- ceans exhibits a strong action upon Palaemonetes red pigment and little upon Uca black; the aleo- hol-insoluble fraction, on the contrary, has a strong action upon Uea black pigment and rela- tively little upon the red of Palaemonetes. There seem, therefore, definitely to be at least two prin- ciples in all sinus glands studied. Further comparative studies of the effects of sinus gland extracts lead to the conclusion that still an additional chromatophorotropie princi- ple is present in some. Extracts of the elands of Palaemonetes and other shrimp, but not of Uea or other true crabs, will, upon injection, lighten the telson and uropods of black Crago. It is still unknown as to whether the shrimp sinus elands possess three hormones and the crab, only two, or whether the glands of both groups possess only two, with one of the two differing in properties between the two groups. The presence of chromatophorotropins within the central nervous system is of general occur- rence among the crustaceans. The actions of these principles may supplement or may antagonize those of the sinus glands, depending upon the chromatophore type. The general roles of these substances in color change may perhaps be most lucidly illustrated by a description of a few types of results obtained with the shrimp, Crago. When one removes the eyestalks with their included sinus glands from this sand shrimp, a very char- acteristic color change ensues. First, the whole shrimp becomes very pale, except for the telson and uropods which become intensely black. After about an hour the shrimp gradually as- sumes a typical coloration of eyestalkless speci- mens, a mottled body and a completely pale tail; this coloration is then retained indefinitely. One can reproduce the transitory color ¢hange just described, by strong eleétrical or other stimula- tion of the cut ends of the optic stalks in the eve- stalkless animals. The same transitory response can be obtained by injection of an aqueous ex- tract of the central nervous System of another specimen. Incidentally, this peculiar coloration involving a light body and a dark tail is so often observed in normal individuals of the genus, that one common Pacific coast species bears the spe- cific name, nigricauda. If instead of applying a strong stimulus to the cut ends of the optic stalks, a mild electrical stimulus is used, the response is quite different ; the whole animal now undergoes a transitory blackening. It can easily be seen that at least two active principles from somewhere within the central nervous svstem are here involved. A study of the various parts of the central nervous system reveals that whereas an extract of any major portion of the system will lighten the body of darkened Crago, only extracts of the minute tritocerebral commissure, connecting the two cir- cumoesophageal connectives just posterior to the oesophagus, will both hghten the body and black- en the telson and uropods. If one goes further and now extracts the tritocerebral commissure with alcohol, this fraction, like the other parts of the nervous system, will lighten the body but not darken the tail. The alcohol-insoluble residue, now freed from its body-lightenine activity, blackens not only the tail of Crago, but the whole body as well. The central nervous system of Crago, therefore, clearly appears to have two principles, one of whose activities is to lighten the body but not the tail (sinus gland extract lightens both), and the other darkens the whole body. The mild stimulation of the optic stalk therefore caused a selective liberation of only one of the principles; the strong stimulation pro- duced extensive liberation of both. A histological examination of the tritocerebral commissures has disclosed that the neurilemma of this region shows a greatly thickened area whose cells are filled with secretory granules. When one examines the nervous systems of other crustaceans for the presence of this Crago- hghtenine and this Crago-darkening activity, it is found that all of the decapod crustaceans ex- cept the true crabs possessed both of these prin- ciples though their distributions within the ner- vous systems differ from genus to genus. True crabs, such as Uca, do not possess the Crago- darkening activity in any part of their nervous system. It can readily be demonstrated, how- ever, that the nervous system of Uca possesses two chomatophorotropins. One of the latter is a white pigment concentrating principle, the other is a black pigment dispersing one, and these two show quite different distributions through the system. It is seen from the foregoing that sources dis- tributed within the central nervous system and the sinus glands are important in the control of the complex chromatophore systems of crusta- ceans. There is no reason to believe that these initial demonstrations of a few principles from these sources have provided a complete picture. Undoubtedly, more will be shown to exist. Fur- thermore, little or nothing is known of the nature November, 1949 | of joint actions of the various principles. There are three general kinds of responses of crustacean chromatophore systems to environ- mental factors. The first general type is a re- sponse to total illumination. In this response it seems to be rather venerally true that the greater the illumination the ereater the degree of dis- persion of all the pigments, both dark and heht. This is apparently the primitive response of the system and is probably quite comparable to the primary or embryonic one of the vertebrate. It can be demonstrated by such a technique as shielding a limited portion‘of the integument that this response is at least in good measure an ‘““ndependent-effector’’ one in the crustaceans. On the basis of this response; crustaceans tend, other factors being equal, to become opaque in bright light and transparent in dim heht. A second general kind of response is one to the albedo, or in other words, a response to the ratio of incident to reflected light strikine the eye. This type of response is obviously depend- ent upon the possession of a complex eye. A eood white background diffusely reflects about 1/3 of the ineident light and a good black back- eround diffusely reflects about 1/200 of the inci- dent light, hence the ratios in the two instances are 3 and 200 respectively. The albedo responses of the chromatuphore system are ones resulting in a mimicking of the shade of the background upon which the animal lies. By this response the dark pigments typically disperse and the white pigment concentrates when the animal is upon a black background. The pigments assume the opposite condition upon a white background. It is clear from what has been said that the al- bedo response and total illumination response may supplement one another (e.g. the white pig- ment on black and white backgrounds) or oppose one another (e.g. dark pigment upon black and white backgrounds). The imitating of the colors of backgrounds similarly is an albedo type of response, but here obviously there is involved a capacity for color perception as well. The third major type of response of the chro- matophore system to environmental factors in- volves temperature. Elevation of the tempera- ture above the normal range tends to disperse white pigment and to concentrate dark pigment. The result is that at these higher temperatures the body reffects more, and absorbs less, radiant enerey. This would appear to serve to some de- eree as a body-temperature regulating mechan- ism. All three of the kinds of responses are normal- ly operating simultaneously upon any given chromatophore system, but the relative influences of the three vary with the species, the chromato- phore type, and the magnitude of the intensity THE COLLECTING NET 11 factor for each in any given situation. It can be seen that the characteristics of the responses of the system that have just been described are all compatible with all of the four commonly postulated adaptive significances of color change in animals in general. A function of obliterative coloration appears to be fulfilled by the albedo response.