THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board JOHN M. ANDERSON, Cornell University JOHN B. BUCK, National Institutes of Health SALLY HUGHES-SCHRADER, Duke University LIBBIE H. HYMAN, American Museum of Natural History SHINYA INOUE, Dartmouth College J. LOGAN IRVIN, University of North Carolina L. H. KLEINHOLZ, Reed College JOHN H. LOCHHEAD, University of Vermont ROBERTS RUGH, Columbia University WM. RANDOLPH TAYLOR, University of Michigan ANNA R. WHITING, Oak Ridge National Laboratory CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 128 FEBRUARY TO JUNE, 1965 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. :. Tin: BIOLOGICAL Bn.i.Knx is issued six times a year at the Lancaster Press, Inc.. Prince and Lemon Streets, Lancaster. Penn- sylvania. Subscriptions and .similar matter should he addressed to The I'.iological Bulletin. Marine Biological Lahoratory, Woods Hole. Massachusetts. Agent for Great Britain : \Yheldon and \Yesle\ . Limited. 2, 3 and 4 Arthur Street. Xe\v Oxford Street. London, \V. C. 2. Single numhers, $3.75. Suhscription per volume (three issues). ^).00. Communications relative to manuscripts should he sent to Dr. Monald 1'. Costello, Marine Biological Lahoratory. \Yoods Hole. Massachusetts, hetwi-en June 1 and Septemher 1, and to Dr. Donald P. Costello, P.O. Box 429, Chapel Hill. North Carolina, during the remainder of the year. Second -class postage paid at Lancaster, Pa 1 \Vr\. COLYIX AND DO\\LD !•;. S\\TNIO\ Photoperiodic control of a physiological rhythm 177 I'll M'MAN, ( '.. The eg^ cocoons ot Sroloplos armiger ( ). I' . M tiller 189 i i NO, s. y. Heart rale and leucocyte circulation in ( Yassostrea virginica ((imelin). . . 198 FKINCS, Hi ni.KT. AND CARL FKIM,^ Chemosensory bases of food-finding and feeding in Aplysia Juliana (Mollusca, ( Ipisthobranchia) 211 ( '.OKDON, MALCOLM S. IntiMcellular osmoregnl.it ion in skeletal muscle during salinity adapta- tion in two species of toads 218 HARRIMIN, MARGARET X. The role of creatine and its phosphate in amphibian development 230 HOLLAND, NICHOLAS 1)., AND ARTHUR C. GIESE An autoradiographic investigation of the gonads of the purple sea urchin (Strongylocentrotus purpuratus) 241 HOLLAND, NICHOLAS D., JOHN II. PHILLIPS, JR. AND ARTHUR C. GIESE An autoradiographic investigation of coelomocyte production in the purple sea urchin (Strongylocentrotus purpuratus) 259 KUEN/LER, KinVAKD J., AND JAMES P. PlCRRAS Phosphatases of marine algae 271 MlLNE, LORUS J., AND M.VR^ERY MlLNE Stabilization of the vistial field 285 XOVICK, ALVIN Echolocation of Hying insects by the bat, Chilonycteris psilotis 297 ROPES, JOHN \Y., AND AI.DI-N P. STICKNEY Ke])roductive c> cle of Mya arenaria in New l^ngland 315 SHIVI^RS, C. AI.I'.X The relationship of antigenic components in egg-jellies of various am- j)hil)ian species 328 SHRIVASTAVA, Si ui ^11 C., AND A. GLICNN RICHARDS An autoradiographic siudy ot the relation between hemocytes and con- nective tissue in tin- wax molh, ( ialleria nu'lloiu-lla I ^37 No. 3. JiNi'., 1965 BROWN, I-"R\\K A., JR., AND YOUNG II. PARK Duration ot an at ter-elTect in planarians following a reversed horizontal magnet ic \-eclor 347 I I All li ID, PIIYI t is A. I'olxdoi.i commensalis Andrews larval development and observations on adults 356 HI.NI.I Y, CATHERINE, AND 1). I'. COSTH.I.O The cylological effects of podophyllin and podopln llotoxin on the fertilized eggs of ( 'haetoptcrus 369 KONIJN, THIO M., AND KENNETH 15. KATI-.R The iiillueni e of light on the time of cell aggregation in the 1 )ict\ osteli- ai <-ae. 392 CONTENTS v LOHAVANIJAYA, PRASERT Variation in linear dimensions, test weight and ambulacral pores in the sand dollar, Echinarachnius parma (Lamarck) 401 MUSCATINE, LEONARD, AND HOWARD M. LENHOFF Symbiosis of hydra and algae. I. Effects of some environmental cations on growth of symbiotic and aposymbiotic hydra 415 NASH, DONALD J., AND JOHN W. GOWEN Effects of x-irradiation of mice exposed in utero during different stages of embryological development on duration of mature life 425 PILSON, MICHAEL E. Q. Variation of hemocyanin concentration in the blood of four species of Haliotis 459 POST, CHARLES T., JR., AND TIMOTHY H. GOLDSMITH Pigment migration and light-adaptation in the eye of the moth, Galleria mellonella 473 STUNKARD, HORACE W. A digenetic trematode, Botulus cablei, n. sp., from the stomach of the lancetfish, Alepisaurus borealis Gill, taken in the South Pacific 488 SUGIURA, YASUO On the life-history of rhizostome medusae. III. On the effects of tempera- ture on the strobilation of Mastigias papua 493 WILLIAMS, CARROLL M., PERRY L. ADKISSON AND CHARLES WALCOTT Physiology of insect diapause. XV. The transmission of photoperiod signals to the brain of the oak silkworm, Antheraea pernyi 497 HUNTER, W. RUSSELL, AND STEPHEN C. BROWN Ctenidial number in relation to size in certain chitons, with a discussion of its phyletic significance 508 Vol. 128, No. 1 February, 1965 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY STUDIES ON VISCERAL REGENERATION IN SEA-STARS. IL REGENERATION OF PYLORIC CAECA IN ASTERIIDAE, WITH NOTES ON THE SOURCE OF CELLS IN REGENERATING ORGANS 1 JOHN MAXWELL ANDERSON Department of Zoology, Cornell University, Ithaca, Neiv York In a previous article (Anderson, 1962) it was shown that sea-stars from which pyloric caeca had been operatively removed were capable of making good progress towards the eventual replacement of these organs within the relatively brief period of time (8 weeks) covered by the experiments reported. These earlier studies demonstrated that in Hcnrlcla Icrinscnla (Family Echinasteridae) the preliminary steps in regeneration of the caecum produced a cylindrical, tubular outgrowth advancing between suspending mesenteries from the proximal stumps of the severed pyloric ducts. Presumably the Tiedemann's pouches, flagellary pumping organs which are normal appendages of the oral sides of the caeca, are subsequently elaborated by modification of the floor of the tubular outgrowth. Histological study of the regenerating organs showed a clear distal-proximal gradient of differentia- tion, with no trace of the precociously differentiated tip characteristic of the parietal parts of a regenerating ray. As no zone of cellular proliferation could be identified in the Hcnrlcla material, it was suggested that outgrowth of the regen- erating caecum might involve the mobilization and incorporation of amoebocytes wandering in the connective tissues and fluid spaces of the body, reaching the regenerate by way of the mesenteric sheets always reconstituted in advance of the outgrowing tube. Experiments similar to those already reported for Henricia have also been per- formed on three species (Asterias forbcsi, Pisaster ochraccns, and Leptasterias pusilla) belonging to the Family Asteriidae. In these sea-stars the pyloric caeca are relatively simple, lacking Tiedemann's pouches, and the replacement of a functional organ after extirpation might be expected to proceed more rapidly and in less complicated fashion than in Henricia. It is my intention to present here a relatively brief, composite account of the results of my studies on these three species, 1 These studies have received support from the Sarah Manning Sage and Dr. Solon . Sackett Research Funds of the Department of Zoology, and from NSF Grants G-6007 and G-20744 to Cornell University. 1 Copyright © 1965, by the Marine Biological Laboratory JOHN MAXWELL ANDERSON . '.* ~* >\? *^*T - • 8 FlGUHES 1-9. VISCERAL REGENERATION IN ASTERIIDAE 3 with particular attention to points of comparative interest in relation to the earlier studies on HenHcia and to points not brought out in those Indies A further series of experiments involved the administration of tritiated thymidine to specimens of Astcrias jorbcsi at a known stage in the regeneration of pyloric caeca only, or in the replacement of entire autotomized rays. Autoracliographs prepared from tissues of these animals have provided new information on the production of cells making up the regenerating parts, and the results of these experiments will be presented. The work on the West Coast species was carried on at the Hopkins Marine Station of Stanford University, Pacific Grove, California. Once again, I record my sincere appreciation of the many kindnesses extended by the Director and staff of this station. It is a pleasure to acknowledge also the competent technical assistance of Rebecca Folsom Ferguson in experiments carried out at the Marine Bioloical Laborator, \Yoods Hole. MATERIALS AND METHODS Small specimens of Lcptasterias pusilla, radius about 3 cm. or less, were collected intertidally at Mussel Point and Point Piiios, in Monterey Bay ; Pisastcr FIGURE 1. Astcrias: single pyloric duct leading from the pyloric stomach (upper left) and branching distally to form the paired pyloric caeca, characteristic of members of the Family Asteriidae. In operative removal of the pyloric caeca this duct is severed near its origin from the pyloric stomach. (Approx. 6 X.) FIGURE 2. Astcrias: stump of severed pyloric duct (arrow) one week after removal of the pyloric caeca. The diffuse dark area below marks the site of the incision in the aboral body wall and shows the extent of post-operative healing in 7 days. (Approx. 6 X.) FIGURE 3. Astcrias: pyloric duct (arrow) 10 days after removal of the pyloric caeca. (Approx. 8 X.) FIGURE 4. Astcrias: appearance of the regenerating duct two weeks after removal of the pyloric caeca ; this shows the incipient bifurcation of the regenerate. Arrow indicates the approximate level of the cross-section shown in Figure 26. (Approx. 12 X.) FIGURE 5. Astcrias: progress in regeneration three weeks after removal of the pyloric caeca. Note that one of the branches is longer and broader than the other and shows developing folds in its wall. The sections shown in Figures 7 through 11 were made from this specimen. (Approx. 3 X.) FIGURE 6. Astcrias: regenerated pyloric caeca 5 weeks after removal of the original pair. Note the extensive folding and branching of the walls of both replacement organs ; from all indications the regenerate is now fully functional. ( Approx. 3 X.) Figures 7 through 11 represent cross-sections of regenerating pyloric caeca in the three-\\eeK specimen shown in Figure 5. Tissue fixed in Helly's fluid, sections stained in phosphotungstic acid hematoxylin. FIGURE 7. Astcrias: section just distal to the end of the shorter, smaller regenerating tub. The mesenteric sheets hanging from the aboral body wall have fused to form a continuous tunnel : the summit of the tunnel at this level contains an accumulation of mesenchyme cells ( arrow i. (Scale = 25 /t.) FIGURE 8. Astcrias: similar section of the same regenerate just proximal to the l;i-i The dark mass of cells occupying the summit of the tunnel represents the closed end of tin- tubular outgrowth advancing between the mesenteric sheets. (Scale = 25 /j.. ) FIGURE 9. Astcrias: the same regenerate sectioned somewhat more proximally. '1 he lumen is well developed even at this far distal level, and its lining epithelium consists of tall cells with flagella and a brush border, with nuclei crowded toward the base. ( Scale = 25 .u.) 4 JOHN MAXWELL ANDERSON of about the same size were obtained at Mussel I'oiut and along the shore -rth of Santa Cruz, in an area where small individuals are locally abundant. • animals were kept in running sea-water at the Hopkins Station and were . asionully on cracked snails and pieces of mussel flesh. Similarly small speci- . is of .istcrias jorhcsi were provided by the Supply Department at Woods Hole and maintained in the circulating sea-water of the Laboratory ; these animals were also fed on small snails and bivalves. In most cases, operative technics for this series of experiments were as previ- ously described ( Anderson, 1962 ) for the studies on Henricia. Animals were immobilized by soaking in MgCl2 solution and the paired pyloric caeca removed from one ray through a longitudinal incision in the aboral body wall. Removal of the caeca involved transecting the pyloric duct (which in these species, unlike Henricia. is single as it leaves the pyloric stomach; see Figure 1) and cutting or tearing the mesenteries attaching the caeca to the body wall. In a few instances, to determine the effects of partial extirpation of the organs, only one member of the pair was removed from a ray, or only intermediate or terminal portions of a member. In other cases, to check the animals' ability to repair more massive defects, the pyloric caeca were removed from all the rays. Returned to running sea-water, the animals usually recovered rapidly, and the operative incisions healed well without the use of medication or sutures. To follow the course of regeneration, individuals were sacrificed and examined at approximately weekly intervals, beginning at the close of the first post-operative week. Observations were continued for 12 weeks on Pisustcr. for 10^ weeks on Leptastt'rius, and for (> weeks on Astcrias; the extended duration of the experiments with the \Yest Coast forms reflects the relatively slower rate of regeneration found to be characteristic of these species. Some individuals from which all of the pyloric caeca had been removed were followed for longer periods; in the case of one /'/\(/\/(T, observations were continued for 16 weeks. These animals were checked periodically with respect to feeding behavior and abilitv to digest material that had been ingested. Procedures for determining the progress of regeneration were as previously outlined and will not be described in detail here. As before, fixation of tissues was in Ilelly's fluid, chosen particularly in order to preserve cytoplasmic elements of the secretory cells in the regenerating organs. Decalcification involved soaking the tissues for about a week in 5', anneons disodium EDTA, a chelating agent. Paraffin sections of the aboral body wall with attached regenerating viscera were cut at ',' to 10 /. and Gained in .Mallow's phosphotungstic acid hemato.xylin. For the experiment.^ on Asterias utili/ing tritiated thymidine, groups of animals were prepared b\ removing one pair of pyloric caeca from each by the standard teclinic. After these animal- bad regenerated for two weeks each received, in tin- body cavity ot the operated ray or an adjacent ray, an injection of either 0.05 or 0.10 ml. ot tritiated ihymidine solution. The solutions were made up in sterile sea- water in concentrations calculated to provide 1.0. 10.0. or 100.0 /uc./ml. ; the total activity administered to each animal \\m~~ varied between 0.05 and 10.0 fie. Injected animals were kept separately in small bowls of cool sea-water for 3, 6, 12. or 24 hours For comparison, four additional individuals were used which had been regenerating entire rays, not just excised pyloric caeca, for two weeks following VISCERAL REGENERATION IX ASTERIIDAE 5 induced autotomy. These received comparable injections of the sa solutions and were maintained for 6 or 12 hours. Following the indicated period of incubation the animals regenerating pyloric caeca were relaxed in AlgCU and carefully dissected and the extent of regeneration noted. The aboral body wall of the operated ray, bearing the regenerating part^ was pinned out on wax and flooded with Bouin's fluid. After a brief hardening period the tissue was trimmed and transferred to a vial of the same fixative and placed under partial vacuum in a desiccator. Bouin's fluid was chosen because, in addition to its efficacy in preserving nuclear details, its acidity brings about rapid decalcification of the body-wall ossicles and obviates the necessity of soaking the tissue for long periods in the customary EDTA solutions, with attendant loss of radioactive compounds. The partial vacuum promoted the escape of gas bubbles resulting from decalcification and minimized tissue distortion. After one to two days' fixation and decalcification the tissues were washed in 80% alcohol, dehy- drated, embedded in paraffin by standard technics, and sectioned at 10 /u,. Animals regenerating entire rays were handled similarly except that they were not dissected before fixation. Alternate slides from the sets bearing sections of these tissues were used in the preparation of autoradiographs. They were dipped in Kodak nuclear track emulsion (Type NTB-3), air-dried, sealed in light-tight boxes with a desiccant, and kept in a freezer for 7 days. They were then developed in D-19 and stained in dilute Harris' hematoxylin. The remaining slides were routinely stained in Mallory's phosphotungstic acid hematoxylin, for purposes of comparison and to reveal cytological details obscured by the stained emulsion overlying the sections in the autoradiograph slides. RESULTS For any one of the species studied here, the sequence of events in caecal regen- eration, while not entirely uniform, is consistent and predictable within limit>. For example, Asterias forbcsi, under the conditions of these experiments, can replace a set of excised pyloric caeca with a reasonably complete regenerate within 5 to 6 weeks. The first postoperative week brings little grossly observable progress (Fig. 2) beyond closure and partial healing of the body-wall incision. By 10 days (Fig. 3) a blind tubular outgrowth appears at the end of the severed pyloric duct, and usually within two weeks this outgrowth has at least begun the process of bifurcation which is responsible for the pairing of the replacement organ. The two branches thus formed continue to elongate during the next couple of weeks, one usually extending somewhat farther than the other. At the same time (Fig. 5 I they become broader and deeper and develop folds and incipient sacculations in their side walls. By 5 weeks (Fig. 6) this process has produced the comple.\ branching side pockets characteristic of the complete organ ; the caeca at this time are still somewhat smaller than those in adjacent intact rays, but another week or two of continued growth will undoubtedly bring them to their normal size. Pisaster and Leptastcrias evidently replace extirpated caeca through the same processes observed in Asterias, as structurally similar stages can be found in all three species. In the Californian forms, however, the rate of regeneration i- considerably slower. Figure 12, for instance, shows a Pisaster regenerate typical 6 JOHN MAXNYKLL AXDERSON is of S-wcek specimens examined; comparison with Figure 5 makes it : that Astcrias has made more progress in three weeks than Pisastcr made The nearly complete pair of replacement caeca shown in Figure 14 for ister is structurally about equivalent to the .Istcrias regenerate pictured in Figure 6; it will he noted, however, that this represents a 5-week regenerate in Astcrias hut a 12-week growth in Pisaster. The rate in Leptasterias is closer to that in Pisastcr than to .Istcrias; the beginning of bifurcation is not seen until four weeks. The 5- and (>-week regenerates shown in Figures 16 and 17, while in some respects better developed than an 8-week Pisastcr (Fig. 12), are much simpler than the three-week .Istcrias caeca, with their walls already somewhat folded, seen in Figure 5. The oldest Leptasterias regenerate in the series, the 104-week specimen in Figure IS, does not approach the structural complexity of the 12-week Pisaster ( Fig. 14 ) or the 5-week .Istcrias ( Fig. 6). Rate differences aside, however, histological studies show that at equivalent morphological stages, irrespective of age, regenerates of the three species under investigation are very similar to one another and are obviously formed through similar processes. Therefore, in a general description of the events in regeneration and differentiation, illustrative examples drawn from any of the species should be broadly applicable to all. Further, in any regenerate, of whatever age, the youngest, simplest, most recently added part lies at the distal tip and is followed in a proximal gradient by regions successively older, more mature, and more complex. It should be borne in mind that the growing tip of an advanced regenerate is histologically equivalent to the corresponding region in any younger regenerate, and that follow- ing serial sections ot a single specimen from tip to base affords an opportunity of studying, in what amounts to a time-sequence, the processes of growth and differentiation in the developing organ. After the repair ot the operative incision, the earliest internal events in caecum regeneration involve fusion of the torn edges of the mesenteries to form a continuous tunnel. This consists of double mesothelial sheets separated by a thin connective- tissue and mesenchymal layer. There is no general hypertrophy of the peritoneum, as previously observed in Iloiricia (Anderson, 1962); in Leptasterias the meso- thelial cells immediately overlying the regenerate appear unusually tall and basophilic ( Figs. \(>. 20 ), but this is not at all comparable to the widespread involve- ment of the peritoneum seen in I/curicia. Under ideal circumstances the course of the mesenteric tunnel corresponds precisely to the lines of attachment of the original caeca, and thus the tunnel forks at the point of bifurcation of the original organ and continues into the ray as a pair of parallel structures, one on either side of the healing incision. I )i.stally these are low. Hat ridges, but proximal to the bifurcation they heroine more elevated and join the mesenteries supporting the healed-over stump of the pyloric duct. A.S in 1 1 cnricia, the preliminary formation of these tunnels provides ;i guide for the subsequent outgrowth of the regenerating caecum. Occasional cases of deranged regeneration can often be explained as result- ing from unu.sualK severe damage to the original mesenteries, and to the peritoneum with which they are continuous, during the operation in which the pyloric caeca were excised. With at least the proximal portions of the guiding mesenteries reconstituted, replacement of the caecum itself begins. Sections show that the outgrowth observed VISCERAL REGENERATION IX ASTERIIDAE in the dissections consists of a cellular tube, continuous with the lining of the pyloric duct, advancing through the mesenchymal layer between the me<>othelial sheets at the summit of the tunnel. As this outgrowth reaches the bifurcation of the tunnel, it broadens, and then from its two outer corners a pair of smaller tubr> appears and invades the parallel tunnels leading distally into the ray. Since the distal portions of regenerates of any age present the same histological feature , the nature of these very young outgrowths can be understood by reference to sections of older ones. Immediately in advance of the growing tip there appears an accumu- lation of mesenchyme cells, as shown in Figure 7. The outgrowth itself comprises a solid mass of cells only at its extreme end (Figs. 3, 19) ; within a very short distance proximally a lumen appears, continuous with that of the pyloric duct (Figs. 9, 13, 20). Very close behind the tip the cells lining the lumen show many of the characteristics of those found in the epithelium of the mature organ ; they are' relatively tall, with nuclei crowded toward the basal end and with flagella and a brush border at the free end, as seen in Figure 9. Further, even very near the end of the tube, and in regenerates of only 10 days' growth, the epithelium contains secretory elements like those found in the normal caecum; zymogen cells (Fig. 13) and mucous goblets (Fig. 20) appear in the epithelium almost as far out as the lumen extends. As the tip of the tube progresses distally, it thus establishes and leaves behind at more proximal levels the basic cell groups out of which the mature regenerate develops. Figures 10 and 11. representing sections some distance back from the tips of the two regenerating tubes seen in Figure 5, show the characteristics and relationships of the cell layers making up the forming organ. In the tall columnar epithelium lining the lumen, both zymogen and mucous gland cells are present in increased numbers as compared with more distal areas. The mesothelia covering the outer surfaces of the regenerate have become stretched and flattened as the diameters of the epithelial tubes have increased and their proportions have altered. Between the lining epithelium and the covering peritoneum, what appear to be remnants of the mesenchymal accumulations in the tunnel are spread in a thin layer, forming the probable antecedents of the subepithelial connective-tissue and muscle components of the developing organ. Figures 21 and 22 show, respectively, sections of distal and proximal levels of the Leptasterias regenerate seen in Figure 16. The thin- walled, cavernous nature of the tubes at these levels is evident, but it wrill be noted that in Figure 22, at the point of bifurcation of the regenerate, localized thickenings have produced inward bulges in the lining, representing the beginnings of the folds and pouches charac- teristic of the mature organ. Figure 23, showing sections through the branches of a much older and better-developed regenerate, illustrates a further stage in the pouching process as well as a second very significant change. This involves locali- zation of differentiated cell types in specific areas of the lining, as a result of which the side-walls, forming the precursors of the glandular pouches of the caecum, come to contain practically all of the secretory elements of the epithelium. In contrast, the roof and floor of the regenerate, which will constitute the median duel of the caecum, are lined with relatively unspecialized flagellated cells engaged in current production and responsible for maintenance of the characteristic pattern of circulation in the contents of the organ. That this circulation has already been set 8 JOHN MAXWELL ANDERSON i<;rki-:s Id 17. VISCERAL REGENERATION IN ASTERIIDAE ii]) even in very immature distal regions of a regenerate is indicated li\ Figure 13, which shows that debris characteristic of the external environment has been carried to the extreme end of the lumen of this regenerating tube. There can be little doubt that the functional capacities of the regenerate are established early and progressively develop towards normalcy as the size and structural complexity of the organ continue to increase, as secretory element > dif- ferentiate in increasing abundance and become localized in the glandular pockets, as the normal patterns of movement and circulation of the fluid contents assert themselves, and as the subepithelial muscular, connective-tissue, and nervous ele- ments develop in the wall of the caecum. It is evident that from relatively early stages the regenerate functions as an organ, even though in terms of structural and functional differentiation its immature distal extremities lag far behind the older proximal portions. For comparison with the general process of regeneration which can now be taken as established, a smaller series of experiments involving either partial or total caecal extirpation provides instructive material. Figure 24, for example, illustrates the result of an experiment in which a single member of the pair of caeca in one ray was excised, 6 weeks previously, in Lcptastcrias. Comparison with Figure 17 makes it apparent that the presence of the intact caecum in the ray has had no demonstrable effect on the rate of replacement of the excised member. The caecal tube has clearly grown out from the point of amputation of the original, following the path established by the mesenteric attachments of the missing portion. It is probably only coincidental that in the 6-week period provided, the regenerate has extended to a point almost exactly level with the tip of the normal member. Histologically, the tubular organ at this stage is in all respects comparable with FIGURE 10. Astcrias: the three-week regenerate at a still more proximal level. The tube here is taller than broad, and in addition to the conspicuous mucous goblet, the epithelium contains numerous zymogen cells lying in the region indicated by the arrow. Note also the large nuclei scattered high in the epithelium. (Scale = 25 /u.) FIGURE 11. Astcrias: cross-section of the larger of the two regenerating tubes shown in Figure 5. The epithelium contains numbers of zymogen cells and mucous goblets ; except for the presence of the unusually numerous large nuclei in the clear areas bordering on the lumen, it appears very similar to the normal caecal epithelium. (Scale = 25 /*.} FIGURE 12. Pisaster: specimen dissected after 8 weeks' regeneration following removal of pyloric caeca. The forked regenerate extends distally from the single pyloric duct at top center. Each fork is a deep tube with walls only weakly folded. (Approx. 6 X.) FIGURE 13. Pisaster: cross-section of an 11-week regenerate, far distally. As in Asterias (cf. Fig. 9), the regenerate advances between mesenteric sheets in a guiding tunnel. The lining epithelium shows differentiated secretory cells, as well as flagella and brush border, and the lumen contains detritus from the external environment. PTA hematoxylin. (Scale = 25 M.) FIGURE 14. Pisaster: well-developed regenerate 12 weeks after removal of the original pair of caeca. Structurally, this is perhaps slightly more advanced than the 5-week Astcrias regenerate shown in Figure 6. (Approx. 4 X.) FIGURE 15. Pisaster: one pair of regenerating caeca in an animal from which all 5 pairs had been removed almost 12 weeks previously. One member of this pair shows developing folds and wrinkles, but comparison with Figure 14 shows that the replacement of '.'t pairs of caeca is slower than the regeneration of a single pair in the same species. (Approx. FIGURE 16. Leptastcrias: dissection of a 5-week regenerate. The sections shown in Figures 19 through 22 were made from this specimen. (Approx. 8 X.) FIGURE 17. Lcptasteri,i\: progress in regeneration 6 weeks after removal of the pyloric caeca. As in most cases, one branch extends beyond the other. The size and relationship- of the intact pyloric caeca can be seen in the adjacent unoperated rays. (Approx. 4 X.) 10 JOHN MAXWELL ANDERSON i* v\."* ^fc. 'f*;\ ' > VISCERAL REGENERATION IN ASTKRIIDAE 11 either of the paired regenerates of the same age shown in Figure Conversely, the presence of the regenerating caecum has exerted no demonstral cl on the normal member of the pair. Although in Figure 24 the bulk of this <>rgan appear. s somewhat less than that of a normal caecum in the adjacent ray, this probably results from a difference in the way in which the organ lies in the oj.if-ni-.-d cnelomic cavity; approximately the same number of glandular pouches is present in all. Obviously, removal of one member of the pair has provided the remaining nu-iul-er additional space in which to lie. As Figure 25 shows, the extreme distal portion of the intact organ j^resents normal histological characteristics and shows no indications of stimulated growth or of de-differentiation, as might perhajDS have been expected. A different sort of experiment, not illustrated in the figures, provides addi- tional information bearing on questions of growth-gradients in the regenerating system. In Lcptastcrias, one ray was opened and the proximal portion of one member of its pair of caeca was excised, leaving intact a 1-cm. length of its distal end. At the same time, the proximal and distal portions of the other member were removed, leaving the middle part in place. Opened again after a 5-week period, the animal showed significant changes. Both of the isolated portions of the two caeca, which had been left attached only by their resj^ective mesenteries, had undergone considerable reduction and resorption. although remaining identifiable and histologically recognizable. Neither had produced regenerative outgrowths in either direction. But while these isolated distal segments had not themselves contributed any new growth, neither had they exerted any inhibitory effects on normal events in regeneration ; the usual mesenteric tunnels had formed proximally FIGURE 18. Lcptastcrias: well-developed regenerate, with simple sacculate foldings in its walls, lOi weeks post-operative. The section shown in Figure 23 was made from this specimen. (Approx. 6X.) FIGURE 19. Lcptastcrias: cross-section at the extreme end of the smaller branch of the five-week regenerate shown in Figure 16. The crowded cells occupying the summit of the tunnel mark the closed end of the advancing tube. This section is comparable in all respects with that shown in Figure 8, for Astcrias, with the exception of the characteristic hypertrophy of the peritoneum over the regenerate in Lcptastcrias. Helly-fixed ; PTA hematoxylin. ( Scale = 500.-) FIGURE 20. Lcptastcrias: section just proximal to the last. The lumen extends almost to the distal end of the regenerate, and even at this level the epithelium is well differentiated, containing conspicuous mucous goblets. PTA hematoxylin. ( Scale = 25 fi.) FIGURE 21. Lcptastcrias: section of the same regenerate about midway to its base. The wall is thin and consists almost entirely of the epithelium lining the tube, with abundant secretory cells. PTA hematoxylin. (Scale = 50 /u.) FIGURE 22. Lcptastcrias: the same 5-week regenerate sectioned at its point of bifurcation. The arrow indicates the region in which the floor of the broad proximal duct rises to meet its roof and separate the two distal branches. Note thickenings of the lateral walls of the wider tube, shown here, marking incipient folds. PTA hematoxylin. ( Scale = 100 /*.) FIGURE 23. Leptasterias : cross-section of the 10i-week regenerate shown in Figure 18, at about midlength. Note the vertical depth of the tubes, and the histological differentiation between the side walls and the oral gutter. PTA hematoxylin. (Scale = 100 ^.) FIGURE 24. Lcptastcrias: dissection of a specimen 6 weeks after removal of a single member of the pair of pyloric caeca in one ray. Compare the growth of this single regenerate with that shown in Figure 17, where both branches have been regenerating for 6 weeks. The arr<>\\ indicates the level of the section shown in Figure 25. (Approx. 6 X.) FIGURE 25. Lcptastcrias: cross-section of the distal end of the intact caecum shown in Figure 24 (arrow). The histology of this organ appears completely normal. PTA hematoxylin. ( Scale = 50 /*.) 12 JOHN MAXWELL AXDKRSOX , 26 27 28 29 FIGURI s 26-29. VISCERAL REGENERATION IN ASTERIIDAE 13 and extended to join with those of the distal fragments, and tubular caecal regen- erates had proceeded to grow from the pyloric duct, reaching a si comparable with the 5-week organ shown in Figure 16 (hut not attaining ! d of the resorbing fragment ) . Other experiments, involving simultaneous removal of all 5 pairs of pyloric caeca from specimens of Pisastcr and Asterias, demonstrate that these animals rapidly recover from the effects of such radical surgery on the digestive tract and are capable of making good progress, within the time limits of the experiments, towards eventual replacement of all the missing organs. The events in regenera- tion of individual caeca after multiple excision are the same as those described for replacement of single pairs, but the rate of regeneration is considerably slower. Figure 15 shows one of the 5 pairs of regenerating caeca in a Pisaster specimen just short of 12 weeks following removal of all its caeca; regeneration in the remaining rays had proceeded to an equivalent condition. Regenerative progress here obviously is slower than that shown in the 12- week specimen illustrated in Figure 14 and appears about comparable with that of the 8-week single-pair regen- erate shown in Figure 12. The rate of replacement in similarly operated specimens of Asterias is also reduced although characteristically more rapid than in Pisaster, as previously noted. Sea-stars deprived of their pyloric caeca are incapable of digesting food until the caeca are replaced. This fact makes it possible to chart the return of function after total caecal extirpation, by observing the performance of specimens in repeated feeding trials. The following observations describe the feeding behavior of an operated Pisaster at the indicated post-operative intervals : 1 1 days : offered food, ignores it 18 days : offered food, ignores it 19 days : attacks small mussel, later releases it unopened 3 weeks : attacks and opens small mussel, remains humped over it several hours but produces no evidence of digestion 4 weeks : repeats 3-week behavior, with same result 7 weeks : opens small mussel, partially digests soft parts during several hours of feeding 1 1 weeks : opens mussel, completely digests soft parts FIGURE 26. Asterias, autoradiograph : cross-section of a two-week regenerate (see arrow in Figure 4) fixed three hours after injection of 0.5 /JLC. of thymidine-H3, sectioned and prepared as described in the text. This is a normally growing regenerate, but it is evident that nuclear labeling is minimal or practically non-existent. Bouin, Harris hematoxylin. (Scale = 25 /u.) FIGURE 27. Asterias, autoradiograph : two-week regenerate, section showing mesenteric tunnel distal to advancing tube. The specimen was fixed 6 hours after injection of 1.0 /JL.C. of thymidine-H3. Note the localization of labeled nuclei in the mesenchyme and peritoneum involved in tunnel formation. Bouin, Harris hematoxylin. (Scale = 50 ^.) FIGURE 28. Asterias, autoradiograph : two-week regenerate, cross-section in proximal region of advancing tube; specimen fixed 12 hours after injection of 1.0 AIC. of thymidine-H3. Labeled nuclei appear in all cell layers of the regenerate but are particularly abundant in the lining epithelium where folds are developing. Bouin, Harris hematoxylin. (Scale : : 50 /u.) FIGURE 29. Asterias: oral aspect of a replacement ray known to have been formed within two weeks, following induced autotomy of the original. Approximately 15 pairs of tube feet have been produced, in addition to the terminal tentacle. This is the stage of regeneration represented in subsequent figures. (Approx. 10 X.) 14 JOHN MAXWKI.L ANDERSON When this specimen was dissected within a few days of the last recorded observa- tion, it proved to contain forked, tnlmlar. somewhat folded regenerates in all rays, similar to those found in the specimen shown in Figure 15. In the 5 similar experiments performed, Pisastcr specimens showed consistently that digestive functions were effectively re-estahlished within 11 to 12 weeks. For comparison, the same experiments on .Isfcrias indicated return of digestive function sometime between the fourth and sixth post-operative week. It may he noted as of passing interest that after a variable period of recovery all specimens acted "hungry" in the presence of food, and exhibited normal feeding behavior, for some time prior to the return of their ability actually to digest food enveloped by, or brought into, tin- cardiac stomach. Turning now to the thymidine-H3 experiments with .-Istcrias. we may first note the technical point that 0.5 ^.c. total activity, available to the tissues for three hours, produces practically no unclear labeling in the organism (Fig. 26). Doubling the total activity administered, and increasing the incubation time to 6 or 12 hours, brings about the abundant labeling shown in Figures 27 and 28. These figures illustrate clearly the very significant fact that thymidine incorporation, marking sites of impending or recent cell division, occurs at all levels of the two-week regenerate, from the elements of the far distal tunnel ( Fig. 27) to the proximal outgrowth of the pyloric duct (Fig. 28). As these figures show, background activity is very low. and labeling of nuclei appears almost entirely limited to tissues involved in the regeneration of the caecum. The results of the entire series of experiments are consistent and demonstrate that mitotic proliferation proceeds very actively in the growing regenerate. These observations are confirmed by the results of parallel experiments involving regeneration of pyloric caeca within regenerating rays. Figure 29 is an oral view of a two-week regenerate replacing an autotomized ray. The gradient of growth and differentiation in this young ray is clearly shown. Structures of the distal tip, while not vet of full size, are well differentiated; the most proximal features, such as tube feet, spines, and ossicles, are largest and oldest, and a gradient can be followed from the base of the new rav to a region just behind its tip, which contains the youngest structures and the zone of growth where they are formed. Sections of such a regenerating ray show that without question its visceral compo- nents are replaced through a sequence of events precisely comparable to those observed in the regeneration of excised pyloric caeca. Far distally, one encounters a low ridge-like structure ( Fig. 30) which is continuous proximally with a typical mesenteric tunnel (Fig. 31 ). More proximallv still, the summit of this tunnel is occupied by a tubular caecal outgrowth from the pyloric duct, and sections of such an outgrowth ( Fig. 32) show that it is quite comparable to similar levels of previ- ously observed replacement organs. ( Figs. (>. 13, for example). Further, a.s demon- strated in Figures 30. 31. and 32. the components of regenerating pyloric caeca within regenerating rays show the .same autoradiographic evidence for mitotic activitv a.s that found in material troin the excised-caecum experiments. I '.ut sections of regenerating caeca prepared for autoradiographs present not only the indirect evidence of thymidine uptake indicative of mitotic activity, but also conspicuous and unmistakable mitotic figures themselves. It will be recalled that this material was fixed in ISouin's fluid, while in earlier experiments all fixation VISCERAL REGENERATION IN ASTERIIDAE 15 was in Helly's fluid. Restudy of sections of the Helly-fixed tissues reveals struc- tures in the regenerating epithelium that can be interpreted as poorlv-fixed mitotic nuclei; some of these may he identified in Figures 9, 11, 13, and 20. ;~nr example. In both the excised-caecum and whole-ray regenerates, particularly in .Istcrias, the advancing epithelial tube of the developing organ displays numbers of large, clear, often somewhat granular nuclei. These are especially numerous in the extreme distal regions of the tube, where most of the nuclei present appear to be of this type ; proximal to the growing tip, in areas marked by differentiation of the epithelium into its specialized components, the cells with large nuclei gradually decrease in abundance, although remaining numerous and conspicuous. Here they lie at all levels in the epithelium, interspersed among the typical small, dense, basally crowded nuclei of the columnar cells lining the lumen ; they are especially noticeable in the clear cytoplasmic zone above the nuclear band. It is apparently with the large nuclei that thymidine uptake in the epithelium is most frequently associated, particularly in the distal region of the regenerate, and the localization of unmistakable mitotic nuclei, as shown in Figures 32, 33, and 34, indicates that it is these cells, high in the epithelium, that are dividing most actively. It is evident that the majority of mitotic figures here are oriented parallel with the surface of the epithelium, and as a consequence new cells will be added in such a way as to contribute to the increasing diameter of the caecal tube behind the growing point. Radioactivity in the epithelium is not altogether limited to the vesicular nuclei but is often found also in association with the small, dense nuclei of the differentiated cells. This is particularly evident in the older regions of the regenerate ; note, for instance, the distribution of grains among the basal nuclei in the proximal section >hown in Figure 35. There appears to be no zone in the epithelial lining of the two-week excised-caecum regenerate where mitotic activity can be said to occur with maximum intensity ; the concentration of heavily labeled nuclei and of mitotic figures is greater a millimeter or two behind the solid tip of the outgrowth than in the tip itself, but it is noteworthy that cell division continues throughout the length of the tube, occurring even in the older, more proximal levels where differentiation has produced an essentially mature epithelium. As seen in Figures 28 and 35, evidence of mitotic activity is concentrated in the regions of the expanding caecum involved in the production of outfolding glandular pockets. The replacement caecum within a two-week whole-ray regenerate has not reached the stage of development characterized by wall folding, and as Figure 36 shows, mitotic activity in this tube seems fairly uniformly distributed in both older and younger regions of the lining. Although directly observable mitotic figures are most conspicuous in the lining epithelium, cellular proliferation in the regenerate is by no means confined to this layer. As most clearly shown in Figures 35 and 36, thymidine uptake has occurred very actively in the peritoneal layer covering the outgrowing tube, and labeled nuclei are found also in the mesothelial constituents of the mesenteries, tunnels, and distal ridges. Here, as in the lining layer of the caecal tube, activity is most abundantly associated with large, clear nuclei and is less frequently found in the smaller, more irregularly shaped nuclei of typical cells of the peritoneum. Tin >ccurrence of labeling in the mesenchyme is less obvious ; some activity is apparent among the mesenchyme cells forming the core of the far distal ridge in Figure 30, and perhaps 16 JOHN MAXWELL ANDERSON 30 - - 34 ^ * * •' ' .^« • .- 35 **> 36 FIGURES 30-36. VISCERAL REGENERATION IN ASTERIIDAE 17 in the tunnel shown in Figure 31. The resolution of the radiographs heing what it is, however, it is impossible to state with confidence whether the developed grains scattered in the region hetween the lining epithelium and the covering peritoneum, at more proximal levels, actually pertain to nuclei of the mesenchymal cells, which are spread very thinly here. As the figures indicate, these grains almost always appear near heavily laheled nuclei of the peritoneum. DISCUSSION No significant differences appear when events in caecal regeneration are com- pared among the three asteriids studied. The rate differences encountered in the different experimental series are almost certainly expressions of temperature effects. According to station records (D. P. Abbott, personal communication), mean monthly sea-water temperatures at the Hopkins Marine Station for the period occupied by the regeneration experiments on Lcptasterias and Pisaster ranged between 14 and 16° C. In contrast, mean surface water temperatures at Woods Hole during the months covering the Asterias experiments averaged about 20° C. (D. Bumpus, WHOI sheets). It is thus not surprising that regeneration in Asterias should be found to proceed so much more rapidly than in the other asteriids studied. The rate in Henricia (Anderson, 1962) is comparable with that in the other West Coast forms, producing nothing more advanced than simple tubular regenerates in the 8-week period covered by the experiments. The peculiar involvement of the peritoneum in Henricia, lacking in asteriids, has already been mentioned. Beyond this, however, one may conclude that the asteriid experi- ments, carried to the point at which essentially complete, normal, and functional organs have been produced, reveal the kinds of changes that could be expected if the brief series of experiments on Henricia were extended to cover the later stages of regeneration. One would expect, of course, that some particular development in These figures are autoradiographs of sections showing regeneration of pyloric caeca within regenerating rays like that in Figure 29. Specimens were fixed 12 hours after injection of 1.0 MC. of thymidine-H3 and prepared as described in the text. FIGURE 30. Asterias: cross-section, far distally in the ray, showing localization of labeled nuclei in mesenchyme and peritoneum of a ridge forming in advance of the mesenteric tunnel. Bouin, Harris hematoxylin. (Scale = 25 /JL.) FIGURE 31. Asterias: same specimen, more proximally. The mesenteric tunnel has formed at this level and shows abundant labeled nuclei and mitotic figures, particularly in the peritoneum. Bouin, Harris hematoxylin. (Scale = 25 /JL.) FIGURE 32. Asterias: same specimen, more proximally still ; cross-section of tubular regenerate. Labeled nuclei and mitotic figures are apparent in the peritoneum as well as in the lining epithelium. Compare Figures 9, 13. Bouin, Harris hematoxylin. ( Scale = 25 ^,.) FIGURES 33 AND 34. Asterias: same specimen, sections similar to the last, in detail. The epithelium lining the regenerate is well differentiated ; large nuclei high in the epithelium are mitotically active ; labeled nuclei are numerous also in the peritoneum. Bouin, Harris hematoxylin. (Scale = 10 /j, in both figures.) FIGURE 35. Asterias: same specimen, sectioned more proximally where the tubular regener- ate has developed folded walls. Note that labeled nuclei are scattered in all layers, apparently including the subepithelial mesenchyme. Bouin, Harris hematoxylin. (Scale = 25 /JL.) FIGURE 36. Asterias: longitudinal section of the tubular regenerate in a similar specimen to the last, similarly prepared. Heavily labeled nuclei in all layers, relatively uniformly distributed through the length of the tube, indicate that cellular proliferation is not limited to the advancing tip. Bouin, Harris hematoxylin. (Scale — 25 /*.) 18 JOHN MAXWELL ANDERSON the floor of the regenerate would eventually lead to the formation of Tiedemann's pouch, with its specifically oriented flagellary channels — a structure not produced in the other forms. Such changes could be interpreted as simply an extension of the process of cell localization observed in all the other species, as a result of which the secretory cells become segregated in the incipient glandular pouches and the more generalized current-producing cells come to be concentrated in the roof and floor of the median duct. In Hcnricia the final disposition of glandular cells is somewhat different (Anderson, 1960). Studying the regenerative process in all the species observed, whether the new pyloric caecum is replacing an operatively-removed organ or is growing in connec- tion with the regeneration of an autotomized ray, one can only be struck by the aptness of Hyman's simple statement (1955, p. 314): "The pyloric caeca are replaced by outgrowth from the old ones. . . ." In all cases, after a period of reorganization, whatever remains as a stump of the original organ produces an outgrowth that always appears as a simple epithelial tube, continuous with its own lining layer, which secondarily bifurcates and forms pockets. Hyman's state- ment continues to the effect that regeneration occurs in the same manner as post- larval growth ; with this in mind, some descriptions of the events involved in the original formation of the caeca will be of interest, for purposes of comparison. Cuenot (1887), for example, refers briefly (p. 33) to the development of the pyloric caeca, pointing out that morphologically they are extensions of the gastric sac (sac stomacal) ; he gives a drawing of a developing caecum from a young Astropecten (Plate III, fig. 5), describing this as a tubular projection from the gastric sac without definite folds but with the same histology as that of its parent organ. Gemmill (1912), describing the development of Solastcr endcca, gives a full account of the origins and relationships of the pyloric caeca (p. 40) : "The paired radial diverticula appear as folds on the roof of the stomach which converge towards a central point. . . . The outer extremities of the folds . . . become elevated from the surface and grow out as blind pouches into the rays. . . . During the fourth month the radial diverticula begin to broaden at their outer ends. This is followed by bifurcation at these ends, with accompanying division of their suspensory mesenteries and of the pockets of epigastric coelom contained therein." Other accounts, notably those of MacBride (1896, 1914), provide generally comparable descriptions. The similarity between embryonic or post-larval development, as outlined in the foregoing passages, and the events involved in regeneration of the organs is clearly evident in the account by Yamazi (1950) of regeneration after autotomy in the fissiparous, multirayed sea-star Coscinastcrias acutispina. In this species the normal number of rays is 8; full-grown individuals divide across the disk, and each daughter individual then grows four small replacement rays. The pyloric stomach repairs itself, gives off a rectal sac, grows to the edge of the disk, and produces a small, tubular outgrowth leading into each of the four new rays. As Yamazi describes further changes (p. 182) : "The pyloric caecum soon furcates into two hollow canals at the position of the mesenteries placed on the median line of the ray cavity. The stalk of these branches remains as a common duct. . . . The branches which are originally simple canals, issue laterally into two series of short lobular branchlets. Meanwhile, the median canal becomes flattened laterally." VISCERAL REGENERATION IN ASTERIIDAE 19 The events thus described, and the structural features and changes illustrated in Yamazi's drawings, are obviously very closely similar to post-larval development as described by others ; they also resemble very markedly the kinds of regenerative changes found in the present studies of asteriids. It will be noted that Yamazi's statement refers briefly to a relationship between caecal outgrowths and the positions of the mesenteries in regeneration, and Gem- mill's description of post-larval growth, quoted above, indicates that such a relation- ship characterizes the original formation of the organs also. On this subject Gemmill has more to say (p. 34) : "As the paired diverticula of the enteron grow out, pockets from the epigastric coelom extend outwards along their aboral aspect, and these pockets bifurcate as the diverticula themselves become divided into two . . . the paired radial extensions of the mesentery ... do not become broken up into fibers, but remain as the boundary-walls of the epigastric coelomic pockets, which in the adult lie above the paired radial diverticula of the stomach." As a result of the present studies, and those reported earlier on Henricia, we are now in a position to understand that in caecal regeneration the paired mesenteries do not simply accompany the outgrowing regenerate. Rather, the positioning and bifurcation of the mesenteric tunnels (and of the pockets of epigastric coelom which they enclose) are events that occur in advance of the outgrowth of the caecal tube in each case. In regeneration of excised caeca, it is easy to visualize how the mesenteric tunnels form through the zippering-up, so to speak, of the remnants of the pre-existent paired mesenteries. What is perhaps more intriguing is the fact that in regeneration of an entire ray the mesenteric tunnels must form anew, traversing the newly formed aboral body wall, as in the original growth of the ray. The future mesenteric tunnel is represented far distally in the new ray by the low ridge which will later become elevated and contain a coelomic extension. Aris- ing in this way, the tunnel must bifurcate at the appropriate level so as to guide the caecal tube in its outgrowth, and in its subsequent broadening and bifurcation. What it is that guides the formation of the tunnel is not apparent. The partial-extirpation experiments on Leptasterias contribute to a demonstra- tion that caecal outgrowth, even with completely re-formed guiding mesenteries in place, occurs only in an outward direction, and only from centrally connected remnants or stumps of excised organs. They show also that remnants of an intact caecum, so long as they are centrally attached, are not noticeably utilized as a source of materials for the replacement of missing portions. Distally isolated frag- ments are resorbed without exerting any demonstrable effect, inhibitory or other- wise, on the progress of regeneration from the central stump. The limited series of experiments does not, however, provide an illustration of what interaction might occur when a centrally attached caecal outgrowth, advancing in its guiding mesentery, encounters a resorbing, distally isolated fragment. The ability of Pisaster and Astcrias (and presumably other species as well) to replace all pyloric caeca simultaneously after total extirpation is noteworthy. Ex- cision of all the caeca removes the only recognized source of digestive enzymes and thus precludes the possibility of effective feeding until functional organs can be regenerated. The observations on post-operative feeding behavior of caecumless specimens confirm the supposition (previously advanced for Patiria; see Anderson, 1959) that the caeca are indispensable for the digestive process. At the same time. 20 JOHN MAXWELL ANDERSON removal of the caeca eliminates the major storage organs for nutritional reserves, against which the animal would normally draw when for any reason feeding is not taking place. The energy sources for maintenance of metabolism during the period of enforced starvation pending caecum regeneration have not been identified. One can only assume that, in view of the invariably successful and relatively rapid replacement of the excised organs, subsidiary stores in the body wall and elsewhere must provide adequate reserves. As previously noted (Anderson, 1962), growth patterns in regenerating pyloric caeca are different from those in the surrounding body wall. Yamazi's work (1950) on regeneration in Coscinastcrias provides a graphic illustration (see his Figure 4, page 183) of the precociously developed distal structures in the body wall, advancing ahead of the zone of growth which establishes the age-gradient between itself and the base of the ray. Yamazi shows, and the present study of asteriids confirms with additional details, that there is no comparable differentiated region at the tip of the outgrowing caecum. Histologically, the tip of the caecum is the simplest and youngest region ; it is noteworthy, however, that differentiation of the lining epi- thelium begins almost immediately behind the advancing tip, with the production of secretory cells and the introduction of specialized characteristics of the epithelial cells. In view of the conclusive evidence, both direct and indirect, revealing the intensity of mitotic activity in the regenerating pyloric caecum of Astcrias, it is no longer necessary or valid to invoke mobilization of amoebocytes as a primary source of new cells for the replacement organ (cf. Anderson, 1962). Failure to recognize mitotic figures in the regenerating caecum of Hcnricia can be attributed chiefly to a technical deficiency, involving consistent use of a fixative (Kelly's) chosen as ideal for cytoplasmic features but providing poor preservation of nuclear details. Also relevant is the fact that regeneration in Hcnricia is relatively slow7, with mitotic activity much less prevalent than in the rapidly growing regenerates in Asterias. There can be little question, however, that many scattered structures in sections of Hcnricia material, originally interpreted as pycnotic nuclei, actually rep- resent collapsed mitotic figures. Having demonstrated the widespread occurrence of cellular proliferation in Astcrias, one has no reason to doubt that regeneration in Hcnricia. so closely similar in all other respects, must involve the same source of cells for incorporation into the developing organ. With a heretofore puzzling question disposed of, visceral regeneration in the asteroids studied can now be compared more meaningfully with the only other case of gut-replacement in echinoderms that has been similarly investigated — that of regeneration following evisceration in holothuroids. Dawbin (1949) has shown that in Stichopus, beginning about 40 days after loss of the gut, mitotic activity becomes widespread in the layer of cells lining the newly developed lumen of the regenerating alimentary tract. These cells, it will be noted, form a solid cord of mesenchyme at the thickened mesentery edge1, and it is in this cord that an irregular lumen gradually forms. The cells dispose themselves so as to constitute a lining for the tube-, and through continuing division provide for the growth of the gut until it eventually reaches normal size. As Dawbin points out, in order to accommodate the ever-increasing diameter of the tube the covering peritoneum must, and does, add cells also; but most of the mitotic activity observed is in the VISCERAL REGENERATION IN ASTERIIDAE 21 cells of the gut lining. Interestingly, cell division in the regenerating caecum of Asterias is observed in these same two layers, and its occurrence in the intervening thin layer of mesenchyme is questionable at best on present evidence. One point of contrast deserves emphasis : in asteroids, the cell layer lining the lumen of the regenerating caecum is always continuous with the epithelium of the pyloric duct and, as we have seen, extends outward by proliferation of cells belonging to this layer. In Stichopits, the lining proliferates and differentiates in place, from mesenchymal accumulations, without reference to or connection with the corre- sponding layer of the esophagus remnant lying anteriorly ( = orally) in the sup- porting mesentery. In this respect, of course (see discussion in Anderson, 1962), gut regeneration in S tic Ji opus differs even from that in other sea-cucumbers studied (Holothnria and Th\onc~], where the continuity of layers is similar to that found in asteroids. The existence of these differences makes it appear that a re-study of events in visceral regeneration in holothuroids, with particular attention to histo- logical details, localization of mitotic activity and cellular differentiation, and related matters, would provide a valuable contribution for comparative purposes. The thymidine-H3 experiments on Asterias were originally designed, in the absence of recognizable mitotic figures in material that had been studied, simply to provide evidence for the occurrence or non-occurrence of cell division in the regenerating caecum. They were not precise enough or extensive enough to serve as a basis for detailed analysis of cell-population dynamics in this system, and such an analysis has not been attempted. However, with the original objective now attained, with the added bonus of confirmation from directly observed mitotic activ- ity in the same sections, we may note briefly some additional information provided by study of the autoradiographic preparations. The appearance of the radioactive label in such profusion throughout the tissues of the regenerating caecum, after administration simply by injection into the coelomic fluid, provides a further indi- cation of the activity of metabolic exchange between the circulating fluid and the pyloric caeca, as already demonstrated for various nutrients by Ferguson (1964a, 1964b). Considering the difference in general metabolic levels, the duration of the mitotic cycle in Asterias is undoubtedly much greater than the 40 or so hours given by Lajtha (1957) for human bone-marrow cells in culture, or the approximately 20 hours suggested as a reasonable average by Quastler and Sherman (1959) for proliferating cells in the duodenal lining of the mouse. Thus, given the 6- to 12-hour incubation time in the present experiments, it is highly unlikely that any cell in the system can have proceeded through more than one mitotic cycle in the presence of the radioactive label. Heavily-labeled nuclei must have passed through a significant fraction of their premitotic synthetic phase during the period of incuba- tion ; more lightly labeled nuclei must represent cells exposed hardly at all to thymidine-H3, or immediately post-mitotic daughter cells carrying only a part of the label taken up by the parent nucleus. In some cases, particularly those involving 12-hour incubation periods, grain-density is so extreme over many nuclei that the mitotic status of the cells cannot be determined (cf. Fig. 36). Where labeled nuclei can be identified as premitotic, it has already been pointed out that they are most frequently of the large, vesicular variety with which mitotic activity seems usually to be associated, in both lining epithelium and peritoneum. These probably JOHN MAXWELL ANDERSON represent undifferentiated cells; their progeny, it would appear, either remain undifferentiated and furnish the stock for continuing divisions ; or else, in the lining layer, elongate, establish contact with the basement membrane, and transform into the columnar cells typical of the developing epithelium. The fact that some activity is associated with small, dense nuclei characteristic of these differentiated cells may identify them as post-mitotic types retaining label incorporated by a parent nucleus; alternatively, it may indicate that even differentiated cells can synthesize DNA and continue to proliferate. Leblond and Messier (1958) have reported encountering copious labeling of the nuclei of goblet cells in the intestinal epithelium of mice, under experimental conditions indicating that this is a premitotic phenomenon rather than a post-mitotic inheritance from a parent cell. It seems useless to attempt further analysis of these results until they can be extended and refined by additional experiments. It is suggested, however, that the regenerating pyloric caecum of asteroids, like the regenerating digestive system of holothuroids, provides excellent material for the study of cell proliferation and differentiation. The events in regeneration of the parietal parts of an autotomized ray, and the distribution of mitotic activity in regions other than those directly involved in caecal regeneration, are related subjects best reserved for discussion elsewhere. SUMMARY 1. The regenerative replacement of excised pyloric caeca has been studied in three species of sea-stars belonging to the Family Asteriidae. Regenerating speci- mens have been observed, and the histological events of regeneration followed by serial sections, to the point at which essentially normal organs are again in place and functioning. The process of regeneration is practically identical in all, but it occurs at a much slower rate in Lcf>tasterias and Pisastcr than in Astcrias. Rate differences are attributable to differences in environmental temperature. 2. Caecal regeneration involves a sequence of changes resembling those pre- viously observed in Henricia. Mesenteric tunnels guide the advance of simple tubular outgrowths from the pyloric duct ; bifurcation of the caecum occurs at the point where the tunnel forks, giving rise to paired tubes growing distally parallel with each other. Differentiation of epithelial elements, with functional secretory cells, proceeds close behind the advancing tip of each tube. More proximally, lateral expansions produce pockets in which gland cells become localized, while less highly- specialized current-producing cells line the roof and floor of the median duct. The gradient of differentiation extends from the young, undifferentiated growing tip tn the oldest region at the base of the regenerate. The pyloric caecum growing inside a regenerating ray following induced autotomy forms through the same sequence of events found in replacement of an excised caecum. Comparison with descriptions of post-larval formation of the pyloric caeca in the developing sea-star reveals that regeneration follows the same course as original formation. 3. Isolated segments of partially extirpated organs are resorbecl without pro- ducing regenerative growth ; their presence in the mesenteric tunnel does not affect normal outgrowth from the central stump. Pisastcr and Astcrias are capable of regenerating all 5 pairs of pyloric caeca at once, at rates somewhat slower than those involved in single-pair replacement. The return of function in these caecum- VISCERAL REGENERATION IN ASTERIIDAE 23 less animals has been charted by observation of feeding behavior and digestive capability. 4. The suggestion that growth of the replacement caecum occurs through mobilization and incorporation of amoebocytes, based on earlier failure to identify mitotic activity in Henricia, is now shown to be invalid. Autoradiographs made from sections of two- week regenerates in Asterias after injection of thymidine-H3 reveal large numbers of cells in some phase of mitotic activity. Direct observation of mitotic figures in these same preparations confirms the fact that growth of the regenerating caecum involves mitotic proliferation. Cell division is most common among apparently undifferentiated cells with large, vesicular nuclei ; it occurs where such cells occur, in both lining epithelium and covering peritoneum, at all levels of the advancing regenerate. In proximal regions, dividing cells are most numerous where the caecal walls are outfolding to form glandular pockets. LITERATURE CITED ANDERSON, J. M., 1959. Studies on the cardiac stomach of a starfish, Patiria miniata (Brandt). Biol. Bull, 117: 185-201. ANDERSON, J. M., 1960. Histological studies on the digestive system of a starfish, Henricia, with notes on Tiedemann's pouches in starfishes. Biol. Bull., 119: 371-398. ANDERSON, J. M., 1962. Studies on visceral regeneration in sea-stars. I. Regeneration of pyloric caeca in Henricia Icvinscula (Stimpson). Biol. Bull., 122: 321-342. CUENOT, L., 1887. Contribution a 1'etude anatomique des Asterides. Arch. Zool. cxp. ct gen., Scr. 2, T. 5, bis, Supp. Mem. 2: 1-144. DAWBIN, W. H., 1949. Auto-evisceration and the regeneration of viscera in the holothurian Stichopus mollis (Hutton). Trans. Roy. Soc. Neiv Zealand, 77: 497-523. FERGUSON, J. C., 1964a. Nutrient transport in starfish. I. Properties of the coelomic fluid. Biol. Bull, 126: 33-53. FERGUSON, J. C., 1964b. Nutrient transport in starfish. II. Uptake of nutrients by isolated organs. Biol. Bull., 126: 391-406. GEMMILL, J. F., 1912. The development of the starfish Solaster cndcca Forbes. Trans. Zool. Soc. London, 20: 1-71. HYMAN, L. H., 1955. The Invertebrates. Volume IV, Echinodermata. New York : McGraw- Hill Book Co., Inc. LAJTHA, L. G., 1957. Bone marrow cell metabolism. Physiol. Rev., 37: 50-65. LEBLOND, C. P., AND B. MESSIER, 1958. Renewal of chief cells and goblet cells in the small intestine as shown by radioautography after injection of thymidine-H3 into mice. Anat.Rec., 132: 247-259. MACBRIDE, E. W., 1896. The development of Asterina gibbosa. Quart. J. Micr. Sci., 38: 339-411. MACBRIDE, E. W., 1914. Text-book of Embryology. Vol. I. Invertebrata. London: Macmillan and Co. QUASTLER, H., AND F. G. SHERMAN, 1959. Cell population kinetics in the intestinal epithelium of the mouse. Exp. Cell Res., 17 : 420-438. YAMAZI, L, 1950. Autotomy and regeneration in Japanese sea-stars and ophiurans. I. Obser- vations on a sea-star, Coscinastcrias acntispina Stimpson and four species of ophiurans. Annot. Zool. Jap., 23: 175-186. NORMAL EMBRYONIC STAGES OF THE SQUID, LOLIGO PEALII (LESUEUR)1'2 JOHN M. ARNOLD a Marine Biological Laboratory, IVoods Hole, Massachusetts 02543, and the Department of Zoology, University of Minnesota, Minneapolis, Minnesota 55455 The need for a series of normal developmental standards for any experimental embryo is obvious, but some justification for further burdening the literature with another species might be in order. Recently, Loligo pealii embryos have been shown to be quite useful experimental organisms (Arnold, 1961a, 1961b, 1963a, 1963b). The only other developmental series for any of the cephalopods known to the author was done by Naef (1928) in his extensive monograph on this class. While the quality of Naef's work is indisputable his stages do not include cleavage in the numbered series and in some cases experimentally significant developmental events are not separated by his 18 stages for Loligo vnlgaris. Apparently, the major criterion used by Naef was chronological age by days of development rather than morphological events. This places some rather major events within the same stage (e.g., all early cleavage stage I) and places abnormal significance on develop- mental rate. Furthermore, slight differences in the sequence of events occur when the development of L. vulgaris is compared with that of L. pealii. For these reasons it was felt that a comprehensive staging of L. pealii would be useful. Hope- fully, the publication of such a staging would encourage further work on this embryo and serve as a teaching aid. MATERIALS AND METHODS The drawings which make up these stages were made under standardized conditions, to eliminate as much variation as possible. The embryos which were used were all of known chronological age and were kept in running sea water at 20 to 21° C. All of these eggs were laid in the laboratory under conditions pre- viously described (Arnold, 1962). An egg string with embryos of uniform age was selected, carefully examined, and a representative embryo drawn with the use of a 32-po\ver microscope and camera lucida. Details were added at a magnification of 100 power. After several such drawings had been made and compared, a preliminary staging was constructed and checked against living material. Two revisions of the preliminary staging were made with additions and corrections based on concurrent observations. 1 This work is a portion of a thesis submitted in partial fulfillment of the Ph.D. require- ments of the Department of Zoology, University of Minnesota. 2 Part of this work was done while the author was the recipient of an N.S.F. Predoctoral Fellowship. 3 Current address: Department of Zoology and Entomology, Iowa State University, Ames, Iowa 50010. 24 NORMAL STAGES OF L. PEALII 25 All of the drawings were made from living embryos, with the exception of stages 17 and 18. In these cases the outlines of the embryos were drawn from life and mercuric chloride was added to the sea water containing the embryos to make the cells more opaque. In this way it was possible to more easily observe the organ primordia and it was even possible to distinguish regions of columnar cells from those of cuboidal cells. However, with practice it is possible to make these same observations on living material. The later stages were immobilized with chloretone or by chilling. In order to have these drawings comparable in size, 1800 embryos were meas- ured in length and width. In the older embryos the distance across the eyes was used for the width measurement, and from stage 27 to stage 30 the distance from the posterior edge of the mantle to the base of the yolk sac was measured for the STAGE vs. TIME ISO 200 250 300 hours FIGURE 1. Chronology of development, based on 142 embryos observed from time of laying until stage indicated (20-21° C.). length measurement. These embryos were from at least three different egg strings which were laid by different females. The averages of these measurements were used to adjust photographically the relative sizes of the drawings so that the figures- have the proper size relationship. A total of 142 observations was made on embryos laid at known times so that a chronology could be constructed (Fig. 1). The variability shown here obviates the use of chronological age alone for a criterion of development age. However, these data are presented because they should prove useful in approximation. NORMAL STAGES OF DEVELOPMENT The following developmental stages are solely based on morphological features, and a certain amount of heterochrony should be expected when these drawings are compared with living material. These stages have been successfully used with 26 JOHN M. ARNOLD other genera (Sepiotcntliis. Octopus] by the author although some of the criteria used here must be ignored. The identifying characteristics mentioned below are listed more or less in order of prominence and/or importance. A detailed descrip- tion of morphological features will not be made here and the reader is referred to the general descriptions by Korschelt and Heider (1910, 1936) and MacBride (1914), as well as the more specific papers by Sacarrao (1945, 1952, 1953, 1954, 1956), Fioroni (1963), and von Orelli (1959) for an introduction to this literature. However, Figure 2 is included for the sake of convenience. FIGURE 2. Organ Anlai/en of embryo at about stage 21. Gl., gill ; an., anal papilla ; ot., otocyst ; ff., funnel folds ; yk., yolk sac ; sh., shell gland ; opt., optic ganglion ; mo., mouth ; sal, salivary pit. STAGE 1. STAGE 2. STAGE 3. STAGE 4. STAGE 5. STAGE 6. STAGE 7. STAGE 8. STAGE 9. STAGE 10. STAGE 11. STAGE 12. STAGE 13. STAGE 14. STAGE 15. Fertilized but lacking polar bodies. Ooplasmic streaming occur- ring to form the blastodisc. First maturation division. Blastodisc quite evident. Second maturation division. Slight increase in the size of the blastodisc. First cleavage along the plane of symmetry. Second cleavage occurs asymmetrically. Embryonic anterior and posterior are evident. Third cleavage. Division is unequal and asynchronous. Fourth cleavage. Division is unequal and asynchronous. Fifth cleavage. Blastoderm often asymmetrical and somewhat irregular. About 32 cells. Sixth cleavage. About 64 cells. Embryonic anterior and posterior no longer distinguishable. Formation of a second layer of cells to form a papilla of yolk in the center of the blastoderm. Yolk papilla flattened. Blastoderm spreading by marginal division. Blastocones can be distinguished but not easily in this species. Mlastodcrm covers about one-third of the egg. Blastocones no longer evident. About two-fifths of the egg cellulated. About three-fifths of the egg cellulated. NORMAL STAGES OF L. PEALII 27 STAGE 1 STAGE 5 STAGE 2 STAGE 6 STAGE 3 STAGE 7 STAGE 4 STAGE 8 STAGE 9 STAGE 10 STAGE 11 STAGE 12 STAGE 13 STAGE 14 STAGE 15 FIGURE 3. Stages 1 to 16. See text (ca. 24 X). STAGE 16 JOHN M. ARNOLD STAGE 16. STAGE 17. STAGE 18. STAGE 19. STAGE 20. STAGE 21. STAGE 22. STAGE 23. STAGE 24. STAGE 25. STAGE 26. STAGE 27. STAGE 28. STAGE 29. STAGE 30. Al>calii. Biol. Bull., 121: 380-381. ARNOLD, T. M., 1962. Mating behavior and social structure in Loligo pealii. Biol. Bull 123: 53-57. ARNOLD, J. M., 1963a. Developmental analysis of the cephalopod embryo. AT/ Int. Cong. Zool. 1: 61. ARNOLD, J. M., 1963b. Techniques for the in vitro culture of Loligo pcalii embryos. Assoc. Island Alar. Lab. Carib., 2: 19. FIORONI, P., 1963. Zur embryonalen und postembryonalen Entwicklung der Epidermis bei Zehnarmigen Tintenfischen. Verhandl. Naturf. Gcs. Basel, 74: 149-160. KORSCHELT, E., AND K. HEiDER, 1900. (Bernard translation, revised by Woodward.) Textbook of the Embryology of Invertebrates. Macmillan, New York. Vol. 4, pp. 235-310. , E., AND K. HEIDER, 1936. Verglcichendc Entwicklungsgeschichte der Tiere. Gustav Fischer, Jena. , E. W., 1914. Textbook of Embryology. Macmillan, London. Vol. 1. NAEK, A., 1928. Die Cephalopoden. Monographic 35. Fauna e Flora del Golfo di Napoli, Vol. 1. SACARRAO, G. F., 1945. fitudes embryologiques sur les Cephalopodes. Arq. Mits. Bocage, 16: 33-70. SACARRAO, G. F., 1952. Remarks on gastrulation in Cephalopoda. Arq. Mus. Bocage, 23: 43-47. SACARRAO, G. F., 1953. On the formation of germinative layers in Cephalopods. Arq. Mus. Bocage, 24: 21-64. SACARRAO, G. F., 1954. Quelques aspects sur 1'origin et le developpement clu type d'oeil des Cephalopodes. Arq. Mits. Bocage, 25: 1-29. SACARRAO, G. F., 1956. Contribution a 1'etude clu developpement embryonnaire clu ganglion stellaire et de la gland epistellaire endocrine des Cephalopodes. Arq. Mits. Bocage, 27: 137-152. VON ORELLI, M., 1959. "Ober das Schlupien von (.)c to pits vnlgaris. Sepia offieinalis, und Loligo vulgaris. Rev. Suissc Zool., 66: 330-343. AN ANGLE SENSE IN THE ORIENTATION OF A MILLIPEDE1 FRANKLIN H. BARNWELL Department of Biological Sciences, Nortlra-estcrn University, Evanston, Illinois, and the Muscii Paraensc "Emilia Gocldi," Belem-Pard, Brazil The literature of experimental psychology contains numerous studies of a maze- running phenomenon which has been called "reverse turning." This term refers to the subsequent reaction of an animal which has been forced to make a right-angle turn in a maze immediately before encountering a free choice point where it can turn to right or left. At the choice point an animal exhibiting reverse turning, turns to the side opposite that of the preceding forced turn ; for instance, a forced left turn is followed by choice of a right turn. This alternation of turning direction has been reported for a variety of organisms throughout the animal kingdom. The reasons for studying the phenomenon have been diverse and have led to several different interpretations. It seems worthwhile, since it has not been done before, to review the different approaches to the problem. The first extensive examination of reverse turning in a maze was made by Schneirla (1929) in his studies of learning in ants. He found a strong tendency for two species of Formica to alternate turning directions at a choice point immediately following a forced right-angle turn. Schneirla was interested in the phenomenon from the standpoint of maze design for the study of learning. The choice at a junction within the maze could be strongly influenced by the pattern of the preceding turns. If the reverse turning tendency were operating and the choice it favored were a blind alley, turning into this alley would be eliminated only with great difficulty during learning of the correct maze pathway. Schneirla explained reverse turning by "centrifugal swing," in which he distinguished two components, the first being the effect of momentum and the second, a thigmotactic response. According to his interpretation, the running ant, upon entering a right-angle turn, would be carried by its momentum into contact with the outside wall of the turn ; subsequently the ant would follow this wall and, at the free choice point, would tend to turn toward the side of antennal contact. Soon afterward, reverse turning was reported in the maze running of white rats by Dashiell and Bayroff (1931). During maze running in rats, these authors did not observe any obvious bodily displacements which corresponded to those upon which the centrifugal swing theory was based. To account for the reverse turning they proposed that "the factor most responsible is a forward-going tendency in animal locomotion that leads not only to maintenance for short distances of a direc- tion already set but also to a compensatory sort of correction when forced out of line by an obstruction" (p. 94). 1 This research was aided by a contract between the Office of Naval Research and North- western University, 1228-03, a grant from the National Institutes of Health, RG-7405, and funds of the Museu Paraense "Emilio Goeldi" and the Institute Nacional de Pesquisas da Amazonia. 33 34 FRANKLIN H. BARNWELL Following the report of Dashiell and Bayroff (1931) there appeared a number of papers (Schneirla. 1933; Ballachey and Buell, 1934; Witkin and Schneirla, 1937) which attempted to demonstrate that the reverse turning of rats could be accounted for by the centrifugal swing principle and that it was unnecessary to invoke any innate "forward-going tendency." Apparently these arguments for centrifugal swing were not wholly convincing. While acknowledging the important role of mechanical inertia and displacement in maze performance, Warden, Jenkins and Warner (1940, p. 805) wrote, "A more likely interpretation of the 'reverse turn' subsequently displayed at the junction, however, is that the phenomenon is primarily the direct outcome of behavioral inertia (postural activity) based in part on stimulation of tension receptors through inertial changes precipitated at the previous turn and in part on the set of the organism antecedent to the previous turn." In 1948 Hullo observed reverse turning in the cockroach Blatclla gcrmanlca run on an elevated maze. The turning was attributed to centrifugal swing. In the 1950s there was a renewal of interest in reverse turning. Demonstra- tions of its occurrence in several organisms were presented as evidence for Hull's principle of reactive inhibition. Hull stated the principle as follows (1943, p. 300) : "Whenever a reaction is evoked in an organism there is created as a result a primary negative drive ; this has an innate capacity to inhibit the reaction potentiality to that response ; the amount of net inhibition generated by a sequence of reaction evoca- tions is a simple linear increasing function of the number of evocations ; and it is a positively accelerated increasing function of the work involved in the execution of the response ; reactive inhibition spontaneously dissipates as a simple negative growth function of time." Presumably, then, in the maze situation there would occur, following a forced turn to the right, a temporary inhibition of the right- turning tendency, and at the next choice point the animal would turn left. As support for this concept Lepley and Rice (1952) reported that Paramecium inulti- micronucleatum in a microscopic maze tended to alternate its turning direction following a forced turn. However, Jensen (1959) pointed out that centrifugal swing had not been controlled in these experiments, and that this latter alternative seemed the more plausible explanation. Furthermore, using Paramecium caudatmn, Lachman and Havlena (1962) were unable to reproduce the results of Lepley and Rice. Perhaps it should be noted that this experiment differed from the experiment of Lepley and Rice in that the choice occurred at a Y rather than a T junction. This point is mentioned because it has been suggested that "invertebrates" may not alternate turning directions at a choice point in a Y-maze (Hayes and Warren, 1963). Apparently this generalization was based on the report that the beetle, Alcochara bilineata, did not exhibit spontaneous alternation (see below) in a Y-maze (Putnam, 1962). Even in this case, however, turning of the beetle in the maze was not random. Instead of alternating, the beetle showed a highly significant tendency to repeat its preceding choice. Mealworms, larvae of Tcuchrio niolilor. were found to alternate turning directions after a forced turn (Grosslight and Ticknor, 1953). The tendency to alternate was greater following two consecutive turns in the same direction and decreased when the distance between forced turn and choice point was increased. Following Jensen's (1959) criticism that centrifugal swing, in particular the ANGLE SENSE IN A MILLIPEDE 35 thigmotactic component, had not been controlled, the experiment was repeated with a narrow maze in which the mealworm was presumably equally stimulated on both sides of its body at the choice point (Grosslight and Harrison, 1961). The authors found that even with thigmotaxis controlled, the reverse turning tendency persisted with the same intensity found in the earlier study. They interpreted their results in terms of reactive inhibition. Dingle (1964b) has confirmed the occurrence of reverse turning in Tenebrio larvae, but he has made additional observations which he believes are incompatible with the principles of reactive inhibition, as originally formulated by Hull (1943). First, using a maze which presented the three alternatives of turning right or left or continuing straight ahead, he found that following a forced turn to the right, no animals chose to turn right and the number turning left was slightly greater than the number continuing straight ahead. Control animals not subjected to forced turning chose to continue straight ahead. Thus, the strong tendency for left turning following forced right turning reflected not simply inhibition of right turning, but rather a tendency to counteract specifically the preceding forced right turn. Second, reverse turning increased as the distance between starting point and forced turn was increased. Thus, the intensity of reverse turning in Tenebrio was dependent upon events occurring prior to the forced turn. Third, when the distance between forced turn and choice point was increased from four to eight centimeters, reverse turning disappeared and most larvae chose to continue straight ahead. However, when larvae were retained under cotton wool immediately following the forced turn, for a period of time necessary to crawl eight centimeters, and then were permitted to crawl four centimeters to the choice point, they evinced strong reverse turning. Thus, within the limits studied the decay of reverse turning tendency was not a function of time but rather of distance crawled. For these reasons Dingle rejects reactive inhibition as an explanation of reverse turning in Tenebrio. The terrestrial isopod, ArmadiUidium vnlgare, was reported to alternate its turning directions with high significance at successive choice points in a maze (Watanabe and Iwata, 1956). Reverse turning tendency was greater following two consecutive turns toward the same side and decreased as distance between the choice point and preceding turn increased. Results were interpreted in terms of reactive inhibition. The authors did not consider the possibility that their results could be explained by centrifugal swing. Rice and Lawless (1957) found no evidence for reverse turning in planarians at a choice point following a forced turn. The possible influence of thigmotaxis was eliminated by discarding all worms which followed the wall of the maze after making the forced turn. Of course, this criterion would eliminate all worms which had attempted to alternate their turning direction following the forced turn but before reaching the choice point and, as a consequence, had come into contact with the outside wall. Dingle (1961a, 1962) has reported that the boxelder bug, Lcpiocoris trivittatus, and several other insects, when forced to make a right-angle turn on an elevated maze, tended to turn in the opposite direction rather than continue straight ahead. In Leptocoris the tendency to alternate turning directions increased both when the distance between starting point and forced turn was increased and when the distance between forced turn and choice point was decreased. Dingle suggested 36 FRANKLIN H. BARNWELL that reverse turning is an adaptive behavior peculiar to insects living on the outer leaves of bushes and branches. He interpreted the response essentially as a mani- festation of a forward-going tendency. Like Warden, Jenkins and Warner (1940), Dingle believed that reverse turning was an attempt by the organism to re-establish a "set" which had been created by sensory inflow before encountering the forced turn. In these first experiments with boxelder bugs an open window served as the light source. Dingle performed control experiments in order to eliminate the possibility that phototaxis was the basis for reverse turning. Another possibility not discussed by him was that rather than being a simple phototaxis, the response was a menotaxis, or compass reaction, in which the bug moved at a fixed angle to the light source. This explanation would lie compatible with Dingle's analysis of the response. Thus, the "set" established prior to the forced turn would be the bug's orientation relative to the asymmetrical light field. Following the forced turn the bug would temporarily attempt to regain its original orientation or "set" until it assumed its newly imposed one. A concurrent report by Dingle (1961b) that the reverse turning tendency was reduced as the intensity of illumination was reduced indicated that vision indeed was involved in the response. In a recent paper Dingle (1964a) has reported additional characteristics of the reverse turning tendency in boxelder bugs. The tendency was present to a small but statistically significant extent in blinded bugs. Both normal and blinded bugs exhibited the tendency following forced runs on curved causeways ; the degree of reverse turning was increased as the distance run was increased and as the radius of curvature of the causeway was decreased. Dingle was able to quantify the turning tendency by substituting a level platform for the choice point ; upon the platform and about the point of emergence from maze pathway onto platform were inscribed arcs divided into 10° sectors. With this apparatus he was able to describe in terms of angular turning tendency the effects both of forced runs on curved causeways and of different starting point to forced turn distances. Also, he indicated that Akre (1962), using mazes similar to his own, had found reverse turning in the red milkweed bug, Tetraopes tetraopJitJialinus. A different approach to turning reactions has employed the simple T-maze. The turning phenomenon observed in the T-maze is referred to as "spontaneous alternation." In this maze the animal is required to make right-angle turns at the junction of the T on successive trials. On the initial trial the animal may be either permitted a free choice or forced to enter one of the arms. It is then returned to the starting arm. and its choice on the second trial is recorded as a repeat or alternation of the initial choice. White rats, which have been studied most extensively in this maze, display a strong tendency to alternate. Use of the T-maze eliminates the mechanical components of centrifugal swing, momentum and the subsequent contact response. However, a large body of work, reviewed by Dember and Fowler (1958), has demonstrated that the turning re- sponse in such a maze is in fact very complex. When a rat is subjected to a second trial in the same maze it is exposed to the same places and stimuli already en- countered on the preceding trial. With the introduction of these variables, it is possible that the rat may alternate its turning with respect to one or a combination of factors. The following are two examples of interpretations which have been suggested at various times for spontaneous alternation. First, alternation may be ANGLE SENSE IN A MILLIPEDE 37 the result of a persisting negative reaction to the turning response of the preceding trial, as the proponents of reactive inhibition have suggested. Thus, spontaneous alternation and reverse turning would have the same basis, that is, alternation of turning with respect to an immediately preceding response. This theory, however, has been found to be inadequate for explaining a number of aspects of spontaneous alternation. Second, alternation may occur with respect to the place and stimuli encountered on the preceding run. In line with this second viewpoint, spontaneous alternation has been interpreted in terms of exploratory drive, curiosity, and stimulus satiation, and it is upon these factors that recent psychological research has focused. A third explanation for spontaneous alternation, which has not been considered by alternation theorists, is that under certain circumstances spontaneous alterna- tion may reflect the forward-going tendency proposed for rats by Dashiell and Bayroff (1931). Alternation on the second trial would result from a persisting tendency to correct for the forced turn experienced on the initial trial. This explanation is similar to reactive inhibition because it states that spontaneous alternation and reverse turning are each manifestations of the same behavioral tendency and both are reactions to the preceding forced response. Although it is now established that psychological factors, such as curiosity, may be important determinants of spontaneous alternation, this third explanation is advanced because it may be significant in maze situations, and in organisms other than white rats, where the psychological factors are not dominant or even involved. One of the rat experiments possibly germane to the experiments to be described herein is the report of a spatial gradient in alternation tendency (Zeaman and Angell, 1953). Rats were run on a fan-shaped elevated maze consisting of choice alleys located at 90° to the right. 30° to the right, 30° to the left, and 90° to the left of the starting arm. The rats were first forced for either two or ten runs onto one of the 90° alleys and were then given a free-choice trial in which all alleys were open. There was a tendency to choose with increasing frequency the alleys farther removed from the original forced alley. The gradient of responses was more pronounced after ten forced trials than after two forced trials. Spontaneous alternation has been reported in earthworms. Lumbricus terrestris was found to alternate on successive trials in a T-maze (Wayner and Zellner, 1958). When the suprapharyngeal ganglion was removed, the tendency to alternate was reduced and in some worms was replaced by a tendency to repeat the preceding choice. In a recent review Jacobson (1963) describes the following additional unpublished experiments on alternation in annelids. Arbit and McLean (1959) employed the same technique used with rats by Zeaman and Angell (1953) but failed to find a spatial gradient in alternation tendency in Lumbricus. Fraser (1958) found that the earthworm, Allolobophora terrestris longa, alternated turns in a T-maze significantly more often than chance expectation, and the earthworm, Lumbricus rubelhts, alternated significantly less often. Kasper (1961) has reported that an earthworm can exhibit reverse turning in a maze pathway consisting of a forced turn followed by a free choice point. Iwahara (1956) found no evidence for spontaneous alternation in cockroaches run in a Y-maze with inter-trial intervals of either 20 or 120 seconds. From the foregoing review it is evident that in a number of experiments there FRANKLIN H. BARNWELL was failure to control for such well established behavioral responses as thigmotaxis, phototaxis, menotaxis, and in some cases, possibly, the following of chemical trails. Perhaps part of the reason for this failure is that many authors have regarded reverse turning and spontaneous alternation primarily as maze phenomena and have not recognized their possible biological significance as important components of animal orientation reactions. The further possibility that reverse turning represents a kinesthetic response was suggested more than twenty years ago, and good evidence for it has been provided by recent experiments. It is notable that two reviewers of animal orientation, Lindauer (1963) and Jander (1963), have considered the experiments on reverse turning in mealworms (Grosslight and Harrison, 1961) and boxelder bugs (Dingle, 1961a) as examples of kinesthetic orientation. For boxelder bugs it has been pointed out that more convincing evidence for a kinesthetic contribution was actually provided by later experiments in which visual cues were eliminated by blinding the bugs (Dingle, 1964a). The following experiments demonstrate a reverse turning tendency in another group, the Diplopoda. It has been possible to show that a millipede species is capable of precise quantitative compensation for forced turning through a large range of angles from an initial path. MATERIALS AND METHODS Tropical millipedes, Trigoniulus lumbricinus (Gerstaecker 1873), were ideal for experimental use. They were quite tractable, crawled about actively when handled, and did not appear to fatigue during an experiment. Generally they were collected on the day of the experiment from leaf litter beneath a breadfruit tree in the garden of the Museu Goekli in Belem, Brazil, where all experiments were performed. At least one-half hour before being tested, each animal was placed in the dimly illumi- nated orientation chamber for a period of light adaptation. The animals used were approximately 4 mm. in width and ranged from 43 to 54 mm. in length. The object of experimental manipulation was to force the millipede to turn from an initial path through a specified angle and to measure any subsequent orientative response to the forced turning. This was accomplished by causing the animal to crawl through a narrow corridor, in the middle of which was an abrupt turn. When the animal emerged from the corridor, its amount of right or left turning was measured in terms of degrees of angular deviation from a straight-ahead path. One of the corridors used is illustrated in Figure 1. A series of such corridors was constructed of smooth wooden blocks glued to sheets of graph paper. The dimen- sions of all corridors were the same : total length, 14 cm. ; height, 7 mm. ; and width, 6 mm. Only the angle of the forced turn was varied. For measuring the angle of emergence an arc was circumscribed on the graph paper at a distance of 6.35 cm. from the exit and divided into 5° sectors. The animal's orientation was recorded as the sector in which its head first reached the arc. A transparent glass plate served to cover the corridor. Dingle (1964a) has described a similar method for quantifying the reverse turning tendency in boxelder bugs. The length of each of the three components of the experimental path, corridor entrance to turn (7 cm.), turn to corridor exit (7 cm.), and corridor exit to arc (6.35 cm,), exceeded the length of any animal used. Consequently, each animal was ANGLE SENSE IN A MILLIPEDE 39 required to enter the corridor completely before encountering the turn, to straighten out completely between forced turn and exit, and to crawl a distance greater than its length from corridor exit to arc. Also, the corridor was sufficiently wide to permit the animals to emerge without touching either wall. The visual environment of the millipedes was controlled by placing the corridor in use within a large chamber, which was 49 cm. high, 79 cm. wide, and 62 cm. long. An opening 30 cm. wide in the back of the chamber behind the corridor permitted observation and access for handling the animals. The interior of the chamber was painted mat black. It was dimly illuminated through a small hole, in FIGURE 1. Orientation apparatus for quantifying reverse turning in millipedes. A millipede is depicted as responding to a 45° forced turn. the center of the top of the chamber, which was 1.5 cm. in diameter and covered with a white diffusing transmitter. The light source was a 25-watt frosted incandescent bulb centered 1.5 cm. above the small opening. During all experiments the exit of the corridor was directed southward and was centered directly under the light spot. Thus, at the instant of the animal's emergence from the corridor, the overhead light source itself could not serve as a horizontal directional reference cue. The experiments were always performed in a darkened room. The experiments consisted of measuring the response of individuals to each of a series of angular turns. Two different series were used. The first, which will be referred to as Series I, consisted of forced right-hand turns and comprised the 40 FRANKLIN H. BARNWELL following angles: 0° (no turn), 15°, 30°, 45°, 60°, 75°, 90°, 105°. and 120°. Between Nov. 15 and Dec. 2, 1962, 45 animals were run through this series at various times of day from 7 AM to 11 PM. The average time required to complete an entire experimental series with a single animal was 29 minutes. The second series, designated Series II, comprised the following seven angles, ranging from 30° to the right to 30° to the left : 30°, 20°, 10° to the right ; 0° ; 10°, 20°, and 30° to the left. Between Nov. 29 and Dec. 11, 1962, 50 animals were run through this series. The average time required for a single animal was 17 minutes. In both series the response to each angle for each animal was taken as the average of five consecutive trials. 120'- 40' 2 6.02 .06 15° R 15.51 279.53 > 13.33 <.001 30° R 28.84 259.23 >13.69 <.001 45° R 42.53 295.85 >11.60 <.001 60° R 54.13 232.06 > 6.87 <.005 75° R 61.00 301.11 > 15.90 <.001 90° R 76.90 267.01 > 6.53 <.005 105° R 83.43 280.16 >20.20 <.001 120° R 103.63 326.40 Series 1 1 30° R 30.48 302.95 >13.82 <.001 20° R 16.66 200.56 > 6.82 <.005 10° R 9.84 346.74 > 5.28 .02 0° 4.56 293.90 >20.12 <.001 10° L -15.56 296.82 > 7.22 <.02 20° L -22.78 394.71 > 4.90 .07 30° L -27.68 251.24 larity of variances for all angles (Table I). Comparison of mean values for the two independent series, I and II, revealed that the means for responses to the same angle, 30° to the right, were similar. Means for responses to intermediate angles in Series II, 20° and 10° right, were intermediate, although each of these two angles was not significantly different from means of responses to both of the forced 42 FRANKLIN H. BARNWELL angles in Series I which bracketed each of them. In both series the 0° response was asymmetrical, being twice as far to the left in Series I as in Series II. In Series I the relationship between stimulus intensity and response intensity was not linear throughout the entire range of forced angles. From 15° to about 60° the angle of emergence was equal to the angle of the forced turn ; above 60° the average angular turning upon emergence for all experiments was significantly less than the angle of the forced turn. For Series II the average values for all seven angles fell close to the regression line described by the equation Y = X, which indicates equivalence of stimulus and response. The computed least squares regres- sion equation, Y - 0.6° + Q.9SX, is scarcely distinguishable from this theoretical relationship. TABLE II Analysis of variance for comparing the turning responses of individuals to a series of angular forced turns Series I Source of variation Sum of squares Degrees of freedom Mean square F Total group Between means of indi- 473,799.2 404 dividuals 43,704.1 44 993.28 5.427* Between means of forced turns Error 365,666.8 64,428.3 8 352 45,708.35 183.03 249.731* Series 1 1 Total group lift ween me; ins of indi- 238,664.6 349 viduals 49,733.7 49 1,015.0 6.416* Between me; ins of forced turns Error 142,428.1 46,502.8 6 294 23,738.0 158.2 150.051* *P<0.001. Measurements for all angles in a series were made on every individual. There- fore, it was possible to test with an analysis of variance for the significance of dif- ferences both in response to forced angles and in the response of individuals. In each series both responses to forced turns, as was evident from inspection, and the individual responses were highly significantly different from chance expectation (Table II). A more detailed examination of the differences in response to forced angles was made by testing the significance of the difference between the means of responses to adjacent forced angles in each series. Comparisons were made with a t test in which the data were treated as non-independent samples in order to eliminate varia- tion due to individual differences. For each pair of forced angles the significance of the mean of a population of differences between two measures on each individual ANGLE SENSE IN A MILLIPEDE 43 was tested. Hence, the probabilities given in Table I for differences between means were not based on the variances listed in the neighboring column. In Series I, where 15° intervals separated adjacent forced turns, the means for all adjacent angles were highly significantly different with the exception of the difference between 15° and the asymmetrical 0° response (Table I). In Series II, where 10° intervals separated the forced angles, the probabilities were not so uniformly high as in Series I. Nevertheless, it appeared that on a statistical basis the millipedes were capable of distinguishing 10° differences in forced angles, even when the forced angle itself was only slightly larger than 0°. •z a. 120' 90° LJ in u. LJ LJ 60' O 30' ff A i i ! 60' Zuj40' 20" 0' ,20' 60' B - c o i 0' 15° 30° 45' 60° 75° TO RIGHT ANGLE OF FORCED TURN 90* 105" 120' 3Cf 20' 10* 0* 10° 20' 30° TO LEFT* 5»TO RIGHT ANGLE OF FORCED TURN FIGURE 3. A. Mean angular turning response to forced angles of Series I for three millipedes selected from six run on Nov. 19, 1962. B. Same as A, for three millipedes selected from seven run on Dec. 1, 1962, in Series II. The extent of the highly significant individual variation is indicated in Figure 3 by selected examples from each of the experimental series. Figure 3A represents the values for three individuals out of six which were run through Series I on Nov. 19. Figure 3B represents three out of seven individuals run through Series II on Dec. 1. There is a slight suggestion that the regression equation describing responses to a series of forced angles may differ in slope as well as intercept among individuals. The range of variation illustrated here is typical of that encountered among individuals on other days. There is an additional large component of the variance in Series I which was not accounted for in the preceding analysis of variance. This variance is associated with the order in which the nine different forced angles were presented within individual series. Since this order was shuffled in every case, each of the forced angles happened by chance to be presented at least one in each of the positions 44 FRANKLIN H. BARNWELL 120'- 15' 30' 45° 60° 75' TO RIGHT ANGLE OF FORCED TURN • 105° 120° 40" 20' 20 52 B I 30' 20' 10° 0° 10° 20* 30" TO LEFT « > TO RIGHT ANGLE OF FORCED TURN FIGURE 4. A. Mean angular turning response for all millipedes in Series I run first in sequence (open circles, dashed line) and ninth in sequence (closed circles, solid line). Mean values for each of the two series are indicated by horizontal lines to the right in the figure. Diagonal line has a slope of 1. B. Same as A, but for Series II. from first through ninth. There was, however, one exception to this statement. The 15° forced turn never occurred fifth in any series. For the analysis to be presented in Figure 5A a value was interpolated for the response to this forced angle by taking the average of the mean responses to the 0° and 30° forced turns occurring fifth in sequence. The extent to which the response was affected by sequence position is shown in Figure 4A where the mean value for each forced y sr a. a. P 54' Is" '•: 48" A I 2345 6 POSITION OF SERIES IN 789 SEQUENCE cr LJ a. . P ' < Ld - o i- er I 2345 67 POSITION OF SERIES IN SEQUENCE 5. A. Mean angular turning for scries occurring in each position in sequence in Series I. B. Same as A, but for Series II. ANGLE SENSE IN A MILLIPEDE 45 angle presented first in sequence is compared with the mean value when each angle was presented ninth, or last, in sequence. For all forced angles the response to the angle when it occurred ninth in order was greater than, or to the left of, the response to the same angle when it occurred first. The difference was largest at 15° and 30°, where it appeared that the animals did not distinguish among forced turns of 0°, 15°, and 30° on the first trial. To the right in Figure 4A two lines indicate the average difference between means for all nine angular forced turns in the two positions, first and ninth. The difference is 15°. The progressive character of the increase in turning tendency is shown in Figure 5A where the means for 15° 45° 60° 75 TO RIGHT ANGLE OF FORCED TURN 90° 105° 120' FIGURE 6. Mean angular turning response to forced angles for the last seven millipedes in Series I measured at two distances from the corridor exit, 6.35 cm. (open circles) and 10.2 cm. (closed circles). the angles of Series I occurring in each of the orders of first through ninth are graphed. These averages include the responses to the 0° corridor, although for this one angle there was no obvious trend toward displacement of the response to the left as one progressed through the nine orders of sequence. Values initially were low, increased rapidly between fourth and fifth positions and tended to level off during later positions. When the data for Series II were subjected to the same analysis, no consistent differences were found (Figs. 4B and 5B). It is evident, however, that animals were responding on the first trial to forced angles of 30° and less. As it emerged from the corridor the millipede oriented sharply and crawled off 46 FRANKLIN H. BARNWELL on a fairly straight course ; the animal showed no obvious tendency to circle in either direction. In order to determine the directness and stability of the orientation, a second, concentric arc was traced on the orienting surface at a distance of 10.2 cm. from the exit. Comparison of the orientation angles of each millipede at two distances from the corridor exit, 6.35 and 10.2 cm., would disclose any tendency in the animal for continued turning after it had crossed the first arc. This additional measurement was made on the last seven millipedes run in Series I. The results are shown in Figure 6. It is evident that essentially all turning had been completed before the animal reached the first arc at 6.35 cm. from the exit. Dingle (1964a) has reported a slight but not statistically significant tendency for boxelder bugs to continue turning after making the initial reverse turning response. In millipedes the tendency to continue turning is much less than it is in boxelder bugs for turning responses of comparable size, 55° and less. DISCUSSION In this study the millipede, Trigoniulus Imnbricinus, was found capable of executing a reverse turn which was, on the average, precisely related to the angle of a preceding forced turn. The animal, therefore, appeared to possess an accurate angle sense. Furthermore, since the reverse turning response was delayed while the animal was forced to straighten out completely in a narrow corridor, the animal necessarily possessed a memory for the forced angle. With the type of orientative apparatus used, the possibility that thigmotaxis, or contact response, was involved in the reverse turning reaction was unavoidable. However, it is doubtful that a simple thigmotaxis could account for quantitative compensatory turning. The 0° corridor served as a control for the thigmotactic component of reverse turning. In both experimental series the mean response to this corridor was closer to 0° than for any other forced angle. Of course, a mean response of 0° could result if the millipedes turned strongly with equal frequency to right and left. On the other hand, for the responses to the 0° corridor the variance, which would indicate the degree of strong right and left turning, was not signifi- cantly larger than the variances for other forced turns. Thus, a large thigmotactic response was not expressed on runs through the 0° corridor. Furthermore, it is presumable that thigmotaxis should influence responses for all forced turns, equally, since all such turns would tend, in the narrow corridor, to bring every animal into contact with the outside wall of the turn. It was observed that some- times after making a forced turn the millipede would not follow the outside wall of the turn. Instead, it would remain close to the inside wall, but would constantly tap the outside wall with its antenna. Upon emergence from the corridor the animal would alternate its turning in the expected direction. The tendency of the animal to follow by means of olfactory cues the trail of its own or another animal's earlier run did not appear to be an important factor. This is indicated, for instance, by the highly significant individual differences presented in Table II and Figure 3. Further evidence was provided by a brief preliminary experiment in which the same sheet of paper was used as the orienting surface for a series of forced turns. Although each animal had the opportunity of following its own or another millipede's trail for the same or different forced angles, the ANGLE SENSE IN A MILLIPEDE 47 results were the same as for the experiments reported here, namely, significant differences among individuals and angles. The effect upon reverse turning of varying the angle of the forced turn has been examined in boxelder bugs by Dingle (1964a). He measured the response to a range of forced angles by determining at the choice point the percentage of bugs that made a right-angle turn in the opposite direction rather than continuing straight ahead. He found that the percentages exhibiting reverse turning after 0°, 30°, 45°, and 60° forced turns were approximately the same, ranging from 19% to 23%, while the percentages after 75° and 90° forced turns were significantly greater, being 44% and 47%, respectively. He concluded (p. 119), "Rather than a steady increment in correcting as the amount of turn increased, there was a certain critical angle at which a marked and significant increase occurred." On this point the behavior of boxelder bugs apparently contrasts with that of the millipedes, which showed significantly different responses to all the angles used by Dingle. On the other hand, it is possible that the boxelder bug, too, possesses a capacity to respond to smaller forced angles but did not manifest this capacity under the conditions of Dingle's experiments. Two possible explanations for the apparent absence of this capacity are presented here. First, it was suggested in Series I (Fig. 3 A) that millipedes did not respond to forced angles of 15° and 30° when these were presented first in sequence. Only after several forced runs through the corridor did a response to these smaller forced angles become evident. Possibly boxelder bugs require several forced runs before responding to forced angles smaller than 75°. If Dingle gave the bugs only a single trial, they may not have been in a satisfactory physiological state for evincing a response to small angles. Second, the apparent absence of correcting for small forced angles may be due to the fact that the response was measured with a dichotomous scale, in which the bug was forced to chose between the alternatives of making a 90° -reverse turn or no turn at all. Although boxelder bugs may be capable of precise compensation for forced angles between 0° and 60°, even on the first run, this gradient of response could be masked if the correcting tendency were not strong enough to produce right-angle turning, and so led to a predominant choice of the straight path. Sup- porting this possibility would be the fact that no critical angle was found when Dingle used a continuous scale, that is, measurement of angular turning tendency over a 180° arc, to quantify the response of bugs to various curved causeways. It is of interest to compare the reverse turning tendency reported here with the homostrophic reflex described for millipedes by Crozier and Moore (1923). In this latter reaction a lateral displacement of the tail brings about a compensatory turning of the head so that its orientation is parallel and in the opposite direction to that of the tail. Jander (1963) has distinguished in any taxis mechanism two basic components, an afferent angle sensing and directing one and an efferent coordinating one. It may be that the two component mechanisms are similar in reverse turning and the homostrophic reflex, but the latter does not require a memory for the angle of bending of the body as does the reverse turning reaction. Possibly, however, Crozier and Moore were observing a memory for angle when they reported (1923, p. 600), "The fact that the reflex may be somewhat delayed increases the appearance of 'intelligent' pursuit of a straight path." Precautions were taken to reduce the significance of the light source as an 48 FRANKLIN H. BARNWELL external orientative reference cue for menotactic, or compass, reactions. This, plus the similarity of the response to the homostrophic reflex, which is dependent upon proprioceptive cues, suggests that the reverse turning reported here is basically a kinesthetic response. Such an internally controlled kinesthetic response would, of course, nicely supplement in a functional manner any externally controlled responses, including menotaxis and astrotaxis, which serve to maintain the organism on a directed course. It was not determined if any visual input at all was necessary for quantitative reverse turning in Trigoninliis. Apparently it plays a role in the boxelder bug, since blinding the bug diminishes its turning tendency (Dingle, 1964a). For a tropical millipede which normally inhabits the gloomy floor of the rain forest it is conceivable that photoreception would be relatively less important than propriocep- tion for the response. It should be noted, however, that the experiments as performed did not eliminate the possibility that pervasive extra-maze factors served as spatial references for a compass reaction. The recent demonstrations that animal orientation may be affected by very weak magnetic, electrostatic, and gamma radiation fields (Brown, 1962a, 1962b, 1963; Schneider, 1963) indicate that at least such a possibility should be considered. I am very grateful to Prof. Frank A. Brown, Jr., for discussions of this work and for his critical reading of the manuscript; to Dr. Nell B. Causey who kindly identified the millipede ; and to Mrs. Adrienne Barnwell for her assistance in Belem. To Mr. James A. Williams of the Office of Naval Research in Chicago we express our thanks for his help and advice on the matters of transporting personnel and equipment to and from Belem. We are deeply indebted to Dr. Eduardo Galvao for his interest in our research and to him and the rest of the staff for so generously placing at our disposal all the facilities of the Museu Goeldi. SUMMARY 1. The millipede, Trigoninliis himbricinns, when forced to crawl through a corridor containing an abrupt turn, tended to turn upon emergence from the corridor at an angle which was opposite and approximately equal to the angle of the forced turn. 2. The response held for forced angles ranging in size up to at least 120°. For forced turns greater than 60° the amount of reverse turning was less than the amount of forced turning. On a statistical basis the millipedes appeared to be capable of distinguishing 10° differences in the angle of the forced turn. 3. There were highly significant differences in the response patterns of individual millipedes to a graded series of forced angles. 4. The turning tendency \vus significantly increased following a series of turns in the same direction. 5. The precision and sensitivity of the reverse turning response suggest that it is an important orientation reaction for millipedes. A brief survey of the literature on certain maze-running phenomena, reverse turning and spontaneous ANGLE SENSE IN A MILLIPEDE 49 alternation, further suggests that the response is a type of kinesthetic orientation which may prove to lie widespread in animals. LITERATURE CITED AKRE, R., 1962. Correcting behavior by insects on vertical and horizontal mazes. Master's thesis, Kansas State University. ARBIT, J., AND J. P. McLEAN, 1959. The spatial gradient of alternation and reactive inhibition in the earthworm. Paper read at the annual meeting of the Illinois Academy of Science, Chicago, April, 1959. BALLACHEY, E. L., AND J. BUELL, 1934. Centrifugal swing as a determinant of choice-point behavior in the maze running of the white rat. /. Comp. 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THE RELATION OF SOROCARP SIZE TO PHOTOTAXIS IN THE CELLULAR SLIME MOLD, DICTYOSTELIUM PURPUREUM * JOHN TYLER BONNER AND FRANCES ELIZABETH WHITFIELD Department of Biology, Princeton University, Princeton, Neu' Jersey One of the most striking features of the cellular slime molds is orientation towards light during the migrating and culminating phases. This has been known since the work of the early workers, and more recently we were able to show that the sensitivity of this reaction is truly remarkable, although we suggested incor- rectly that the light and heat response might be explained by a similar mechanism (Bonner, Clarke, Neely and Slifkin, 1950). Gamble (1953) was the first to show that the light response was so sensitive that it was necessary to postulate a separate photoreceptor and this has been confirmed in detail by Francis (1964), who was even able to obtain a rough action spectrum of the photosensitive pigment. For some years in our laboratory we have attempted to demonstrate phototaxis in vegetative or aggregation amoebae with no success. Samuel (1961), in particu- lar, paid attention to this problem as has Francis (1964), and we have repeated a number of the obvious experiments during the course of this study. But in no case was any photo orientation demonstrated as it was in Davenport's (1897) early experiments on large soil amoebae. The purpose of this study was to determine whether or not small cell masses, which would be intermediate in size between single cells and the usual large pseudo- plasmodia, would respond to undirectional light with the same effectiveness as the larger masses, or whether they would show intermediate responses. As will be seen, for a given light intensity, the effectiveness of the response definitely decreases with size. The phototactic response is correlated with sorocarp size. METHODS The majority of these experiments were done on Dictyostelhiin purpurcum (strain No. 2), although a few comparative studies were made with Poly- sphondyliwn violacewn (strain No. 6), P. pallidimi (strain No. 4), Acytosteliuin leptosoinnin, and Protosteliuni mycophaga. In all cases they were grown on Eschcrichia coli which had been painted on the surface of 2% non-nutrient agar. When aggregation was largely completed and well formed centers were evident, these were punched out with a small glass tube and placed on the surface of a large block of agar within a Incite box 3x3x9 cm. in size (Fig. 1). These were in turn put in a darkroom so that each individual center resided at the same given distance from the light source. There was no temperature regulation of the dark 1 This study was supported in part by a grant from the National Science Foundation and in part by the funds of the Eugene Higgins Trust allocated to Princeton University. 51 52 JOHN TYLER BONNER AND FRANCES ELIZABETH WH1TFIELD blackened box door agar distance from light source. FIGURE 1. room and while the temperature was relatively steady during any one experiment, the temperature at which the experiments were run varied from 24° C. to 27° C. The light source was a 6.5-volt G.E. headlight lamp No. 1493. It was set with a transformer so that it received 1.14 amps, and 0.90 volts. The intensity of the light was checked hefore and after an experiment with a photocell and a galvanometer. After the pseudoplasmodia had fruited, the plastic boxes were placed upon their sides under a dissecting microscope and camera lucida drawings were made of the sorocarps. The angles were determined by drawing a line from the base through the upper end of the stalk at the point where it enters the sorus (Fig. 1). The vertical position was considered 0°, and any deviation towards light was given a positive angle value, while orientation away from the light was given a negative value. TABLE I Mean angles of deviation from the perpendicular for different size sorocarps at three different distances from a light source. The standard deviations are green fur each mean and the number of cases in parentheses (These are plotted in Figure 2) Ilciuht of sorocarp in mm. Angle of deviation from the perpendicular with the li.nlit at different distances iijioupeu in size classes of ±0.2 mm.) i foot 6 feet 18 feet 0.2 10° ± 23°(9) 3° ± 2()°(7) 1° ± 1<)°(10) 0.6 10° ± 30°n li -4° ± 10°(8) 7° ± 28°(12) 1.0 42° ± 26°U4) 3° ± 14°(14) -3° ± 140(16) 1.4 37° ± 31°(13) 21° ± 28°(12) 2° ± 22°(22) 1.8 48° ± 36°(10) 33° ± 17°((>) -4° ± 14°(22) 2.2 53° ± 15°(5) 38° ± 23°((>) 8° ± 20°(20) 2.6 52° ± 18°((» 35° ± 190(9) 2° ± 50°(15) SIZE AND PHOTOTAXIS IN DICTYOSTELIUM RESULTS 53 As can be seen from Table I and Figure 2, the experiment was run at three different distances, 1, 6, and 18 feet. Although the standard deviations show considerable variability in the response, it is clear that at one foot the larger sorocarps responded while the smaller ones did not. At 18 feet there was no 3.0 . 0.2 . + 80 +60 +40 +20 0 -20 -40 -60 Angle of orientation FIGURE 2. response, even of the largest sorocarp. In fact this agrees with a control run in the total darkness, involving 60 sorocarps. At 6 feet there was an intermediate response. For instance if one looks at the sorocarps that are 1 mm. long, then only those at 1 foot are oriented while those at 6 and 18 feet are not. However, at 1.4-mm. the ones at 6 feet from the light source are definitely oriented, although the 54 TOHN TYLER BONNER AND FRANCES ELIZABETH WHITFIELD atlgle of orientation is less than those at one foot. It should be added that this same result was obtained in a preliminary experiment involving 59 cases in which the intensity of the light was changed rather than changing the distance. This experi- ment, however, has the serious objection that the spectrum of the tungsten filament changes with intensity and therefore the data have not been included here. These results were repeated in a brief preliminary experiment comparing five species. In Protostelium, which has no aggregative phase and consists of a single cell raised on a minute stalk (Olive and Stoianovitch, 1960), and in Acytosteliuin, which is a very small species with an acellular stalk (Raper and Quinlan, 1958) , there was no orientation at all, even with strong light intensities. The remaining three were tested at the same light intensity and Polysphondyliitin pallidum (21 cases) was the least sensitive, Dictyostcliuin pnrpnrcnin (37 cases) next, and Poly- sphondylium I'iolaccuni (18 cases) the most sensitive and showed the lowest TABLE II Mean angles of deviation from the perpendicular for different-size sorocarps of Polys phondylinni pallidum. In the first column of angles two sorocarps are repelling each other, and in the second column a single sorocarp is orienting away from a vertical wall of agar. In both instances the distance between the sorocarp base and the other sorocarp or agar varies from 0 to .53 mm. The number of cases is indicated in parentheses Height of sorocarp in mm. (Grouped in size classes of ±.1 mm.) Anjjle of deviation from perpendicular Two sorocarps Sorocarp and agar wall .2 35°(1) 7°(1) .4 31°(8) 16°(2) .6 19°(12) 5°(S) .8 20°(8) 13°(0) 1.0 9°(2) 8°(S) 1.2 20°( 1 ) 17°(3) threshold. It is of interest that only the main stalk in Polysphondyliuin showed orientation ; the small side whorls never leaned towards the light, which might be expected on the basis of their small size. During the course of these experiments the question arose as to whether light might directly affect the rate of movement within the cell mass. This was tested by subjecting migrating masses of Dictyostelium discoideuni to alternating periods of light and dark (with the microscope lamp in the darkroom) and measuring the rate of movements. In IS separate experiments, most of them run for at least four hours, the light was alternately turned on and off at hourly intervals. The overall average rate during the dark periods is almost identical to that during the light period (Light— 1.6 mm./hr. ; Dark-: 1.7 mm./hr. ) and if one scores whether light has produced an increase or decrease in speed (or conversely the dark), both occur with equal frequency. A few further experiments were run with the light and dark alternating at 10-minute intervals and again there was no difference in the rate. These results are consistent with those of Francis (1964), who showed amoebae separated from a slug and subjected to strong increases in light intensity did not exhibit any change in speed. SIZE AND PHOTOTAXIS IN DICTYOSTELIUM 55 Besides the effect of light it should be recorded that two other types of orienta- tion were tested. By putting the plastic boxes on end in the total darkness it was possible to test the effect of gravity. The results do not show, as might be expected, that the large sorocarps droop. In 54 cases there is no clear downward trend, but the variance appears greater than the right-side-up controls in the dark and the right-side-up sorocarps 18 feet distant from light (Fig. 2). It would be interesting to know if the mechanism which controls orientation in the dark has difficulties in operating with precision when the sorocarp arises on a vertical wall. Presumably the method of orientation in the dark is entirely by gas gradients (Bonner and Dodd, 1962a). The old data from those experiments were re- examined and it is possible to compare the amount of repulsion between sorocarps and the size of the sorocarp. It is obvious from the results shown in Table II that the small sorocarps are as effective in orienting to gas gradients as large ones, and that this is true for a number of species. DISCUSSION The discussion of these results will be divided into two sections : the mechanism of orientation, and the question of adaptation. Unfortunately, these experiments tell us very little about the mechanism of orientation, but then perhaps this is not too surprising because despite all the detailed work on phototropism in Phycouiyccs, it is still far from understood. Orientation in the slime molds is similar to Phycouiyccs in that Francis (1964) showed that a small beam of light hitting one side of slug will cause the slug to bend away from that side, thus showing a parallel to Buder's (1920) experiment in Phycomyces. During the course of our work we repeated another experiment of Buder and placed the slime molds in mineral oil. As in Phvcoinyccs they oriented away from the light and all of this substantiates Francis' (1964) conclusion that the lens effect is operative in the slime molds. Unlike Phycomyces, there is no evidence that light affects the speed of move- ment, even for brief periods. From this Francis (1964) makes the reasonable suggestion that perhaps the light might be affecting the extensibility of the slime sheath which in turn directs the movement. Another difference is that in Phycoin\ccs the smaller sporangiophores are more sensitive to a given unilateral light than large ones, as Castle (1964) has shown. This is exactly contrary to the results here. From this we must conclude that the limiting factors are different for the two systems, but what they might be for the slime molds is difficult to surmise. It is not just light intensity, for if the lens effect operates, it should if anything be more effective in the small fruiting bodies at a given light intensity. Therefore, there must also be some limitation within the sorocarp itself. Something within the cell mass is quantitatively below threshold, but the threshold can be raised to some extent if the undirectional light intensity is raised. It is interesting that this should be in marked contrast to the orienting effects of gas gradients. Here the smallest sorocarps are as responsive as the large ones. The fact that single amoebae can also orient very effectively in chemical gradients, 56 JOIIX TYLER BONNER AND FRANCES ELIZABETH WHITFIELD such as the acrasin gradient, food gradients (Samuel, 1961) and mutual repulsion gradients (Samuel, 1961), is perhaps consistent with the notion that there is no size threshold for these chemical effects. To turn now to the question of adaptation, if orientation to light has adaptive value, then clearly there will he a selection pressure for large slime molds. Increase in size, however, may result in other features which are inadaptive, and some sort of balance must he reached. Size is to some degree fixed for a particular species or a particular strain and this is in part due to the spacing mechanism (Bonner and Dodd, 1962b; Bonner and Hoffman, 1963) and in part due to another mechanism which operates at higher amoeba densities (Hohl and Raper, 1964). Since there are different-sized species in nature we must presume that there are a number of adaptive advantages and disadvantages in size. This same question has been examined for different-sized Hydra by Slobodkin (1964), where he considers the whole question in terms of strategy, balancing the advantages and disadvantages in different ways. Here we can say that the ability to orient towards light improves with increased size, and if for any one species, in a particular environment, this is advantageous, there will be selection for size increase. The authors are indebted to Dr. L. S. Olive for cultures, and to Dr. D. W. Francis for his critical reading of the manuscript. SUMMARY Large fruiting bodies of the cellular slime mold, Dictyostelium piirpureum, orient more effectively towards a source of light of low intensity than do small ones. The threshold of sensitivity can lie changed either by changes in size of the sorocarp or by changes in the light intensity. However, in chemical gradients small cell masses are as sensitive as large ones. Therefore, if orientation to light is of adaptive value, selection pressure for size increase would be expected. LITERATURE CITED Box NEK, J. T., AND M. DODD, 1962a. Evidence for gas induced orientation in the cellular slime molds. Dn: Bio!., 5: 344-361. BOXXKK, |. T., AND M. DODD, 1962b. Aggregation territories in the cellular slime molds. Biol. Hull., 122: 13-24. BOXXER, J. T., AND M. E. HOFFMAXX, 1963. Evidence for a substance responsible for the spacing pattern of aggregation and fruiting in the cellular molds. /. Einhr\nl. Exp. M,»^h..2: 571-589. BONNKK, J. T., YV. \Y. CI.AKK, C. L. NEK.I.Y. AXD M. K. SLIFKIX, 1950. The orientation to light and the extremely sensitive orientation to temperature gradients in the slime mold Dictyostelium (liseoiileitnt. J. Cell. Ctnnf>. I'hysiol., 36: 149-158. BrDKK, J., 1920. Neue phototropische Fundamentalversuche. Her. Dcittsch. Hot. Ges., 38: 10-19. CASTLE, E. S., 1964. Differential growth and phototropic bending in Pliycouiyccs. J. Gen. riiysiol.. (in press). DAVKXPOKT, C. B., 1897. Experimental Morphology. Vol. 1. New York-London, The Macmillan Company. SIZE AND PHOTOTAXIS IN DICTYOSTELIUM 57 FRANCIS, D., 1964. Some studies on phototaxis of Dictyostelium. J. Cell. Comp. Ph\siol., 64: 131-138. GAMBLE, W. J., 1953. Orientation of the slime mold Dictyostelium discoideum to light. Senior thesis, Princeton University. HOHL, H. R., AND K. B. RAPER, 1964. Control of sorocarp size in the cellular slime mold Dictyosteliiun discoidcittn. Dcv. Bio!., 9: 137-153. OLIVE, L. S., AND C. STOIANOVITCH, 1960. Two new members of the Acrasiales. Bull. Torre\ Bot. Club., 87: 1-20. RAPER, K. B., AND M. S. QUINLAN, 1958. Acytostclium Icptosomum: a unique cellular slime mold with an acellular stalk. /. Gen. Microbiol., 18: 16-32. SAMUEL, E. D., 1961. Orientation and rate of locomotion of individual amoebas in the life cycle of the cellular slime mold Dictyostelium mucoroides. Dei'. Biol., 3: 317-335. SLOBODKIN, L. B., 1964. The strategy of evolution. Amcr. Sci., 52: 342-357. VARIABILITY IN LARVAL STAGES OF THE BLUE CRAB, CALLINECTES SAPIDUS 1 JOHN D. COSTLOW, JR. Duke I'nh'crsity Marine Laboratory, Beaufort, North Carolina Renewed interest in crustacean development, coupled with techniques which permit the rearing of larvae representing a variety of groups, has resulted in an increase in descriptions of larval stages and comparisons of similar stages obtained from laboratory rearing and from the natural environment. Comparisons of laboratory-reared larvae with those from the plankton frequently include observa- tions on certain variations, either in morphological characters within stages thought to be comparable or variations in the total number of larval stages. Some variability has been noted in larvae representing many of the groups within the Crustacea. Larvae of the Brachyura, however, while receiving perhaps greater attention than larvae of other closely related groups, have not been found generally to exhibit variability in either the number of stages or in morphological details within any one particular stage. The two exceptions are larvae of the blue crab, Callinectcs sapidits (Costlow and Bookhout. 1959) and larvae of the stone crab, Mcnipf>e incrccnaria (Porter, 1960). In studies on larvae of these two species of crabs, an "extra" stage was described, intermediate between the zoeal stage generally con- sidered to be the final zoeal stage, and the megalops stage. The present study was undertaken to determine if variability in larvae of Callinectcs sapidus reared under controlled laboratory conditions was limited to the occasional occurrence of an "extra" stage late in development or if irregularities occurred in the molting pattern of earlier larval stages which could result in morphological differences which might be interpreted as separate or intermediate zoeal stages. The author is indebted to Miss Judith Payne and Mr. Charles Lynch for their assistance throughout the study. METHODS The larvae of the blue crab, Curcau of Commercial Fisheries and Duke University. 58 VARIABILITY IN CALLINECTES LARVAE 59 30 p.p.t, and fertilized Arbacia eggs and recently hatched Artemia nauplii were added. Before the larvae were changed the watch glasses were examined under a dissecting microscope. If molting had occurred, both the shed exoskeleton and the zoea were examined under a compound microscope to establish what morpho- logical changes had occurred. For purposes of comparison, the descriptions and figures of the seven zoeal stages of C. sapid us (Costlow and Bookhout, 1959) were used as a basis for differentiating between the sequence of zoeal stages. Any variability in appendages or abdominal segments was recorded throughout progres- sive molts. RESULTS During larval stages I-IV, the morphological characteristics previously de- scribed by Costlow and Bookhout (1959) were found to be consistent. Within some series of C. sapidus larvae, however, variability in molting was observed after stage IV. The variability can be grouped into three general categories: (1) a molt without any perceptible morphological changes; (2) a molt which eliminated or "skipped" one of the previously described larval stages; and (3) a molt which resulted in a larva with a combination of morphological characteristics previously described for two separate larval stages. Table I represents the stages of zoeae observed at the time of each molt for one series of 20 C. sapidus larvae segregated as stage IV zoeae. Forty-five per cent TABLE I Stage of larval development at the time of each molt for 20 zoeae of Callinectes sapidus isolated as stage IV larvae. Roman numerals indicate stage of development Zoea Number Molt number 3 4 5 6 7 8 1 IV V VI VII Meg 2 IV VI VIII Meg 3 IV V V-VI VI-VII VIII Meg 4 IV V VII Meg 5 IV V VI VII Meg 6 IV V V-VI VII VII-VIII Men 7 IV V VI VII Meg 8 IV V VI-VII Meg 9 IV V VI (dead) 10 IV V V-VI VI-VII Meg 11 IV V VI VII Meg 12 IV V VI VII Meg 13 IV V V-VI V-VI VII Meg 14 IV V VI VII Meg 15 IV V VI VII Meg 16 IV V VI VII-VII1 Meg 17 IV V VI VII Meg 18 IV V VI VII Meg 19 IV V VII Meg 20 IV V VI VII Meg 60 JOHN D. COSTLOW, JR. O.I 1. Side view of four zoeal slaves nf the blur oral), Callinectes sapidus. A, Stage IV zoea; 15, Stage IV-V zoea ; C, Stage V-VI /<>ea; and I), Stage \"1 x.oea. VARIABILITY IN CALLINECTES LARVAE 61 of these larvae exhibited some variability during subsequent molting. In the three series which were studied, variability was observed in from 40% to 63% of the larvae. As shown in Table I, the frequency of molts without any perceptible morpho- logical change was low (Zoea #13). The few larvae which were observed to molt without any morphological change usually exhibited the morphological character- istics previously described for the next stage (Costlow and Bookhout, 1959) at the time of the next ecdysis and proceeded to molt normally in remaining larval molts. The complete elimination of one of the larval stages by "skipping" occurred more frequently (Table I, Nos. 2, 4, 19). In rare instances this type of variability was observed over several consecutive molting periods for the same zoea. The third type of variability, a molt resulting in a larva which combined morphological characteristics from two stages, was the most common (Table I, Nos. 3, 6, 8, 10, 13, 16). The "combined stages" most frequently observed were stage "V-VI" and stage "VI-VII." The morphological characteristics which usually were combined in these two stages were the antennae and maxillipeds of stage VI combined with the abdominal segments of stage V (Fig. 1, B), or the antennae and maxillipeds of stage VII combined with the abdominal segments of stage VI (Fig. 1, C). It should be noted that in a "combined stage" the more advanced characteristics were invariably in the anterior portion of the zoea; i.e., in a stage "VI-VII" zoea the anterior portion resembled the stage VII larva, as described by Costlow and Bookhout (1959), while the abdominal segments and development of pleopod buds were those previously figured for a stage VI zoea. A greater range of variability between stages was not observed. In many instances "combined stages" did, at the next molt, display the charac- teristics described for a single stage, but some larvae continued to molt with more advanced anterior development until the megalops stage was reached (Table I). The variability which was observed was accompanied by differences in the actual number of larval stages or molts required for development to the megalops stage. Some larvae (Table I), by eliminating stages V or VII, attained the megalops stage in six zoeal stages. Other larvae, which did not eliminate spe- cific stages, required seven or eight zoeal stages to complete development to the megalops stage. DISCUSSION Variability has been observed within the developmental stages of Crustacea representing a number of specific groups. Within the Cirripedia there has been general agreement on the occurrence of 6 naupliar stages prior to the cyprid stage. Two authors have described a seventh naupliar stage in rearing studies on Balanus amphitrite albicostatus (Ishida and Yasugi, 1937) and Balanus amphitrite hawaiien- sis (Hudinaga and Kasahara, 1941). In both instances, however, the only dif- ference between the sixth and seventh naupliar stages was the presence of paired eyes, a feature which is known to develop late in the sixth naupliar stage of all other species of barnacles described to date. It should be noted that while the two studies mentioned dealt with laboratory-reared material, differences have also been reported in barnacle nauplii obtained from the plankton. Norris and Crisp (1953) described "developmental intermediates" in nauplii of Balanus pcrjoratiis, 62 JOHN D. COSTLOW, JR. and Jones and Crisp (1954) found minor differences in setation of nauplii of Balanus improi'isus. Within the euphausids, Fraser (1936) described some "skipping" in the num- ber of stages of reared larvae of Euphausia snpcrba. In studies on larvae of Xyctipliancs simplex obtained from the plankton, Boden (1951) described six "dominant" furcilia stages but suggested that as many as 19 may occur in development. Larvae of several species within the Natantia have also been shown to vary in number of developmental stages or in minor morphological features within any one specific stage. Renfro and Cook (1963) described minor differences in the setation of naupliar appendages of the penaeid shrimp, Xiphopcneus kroyeri, reared in the laboratory. Broad (1957a), rearing the larvae of Palacmonctcs pugio and P. rnlfjaris, found skipping of stages and commented that the rate of develop- ment of the larvae and the frequency of molting were independent. Within the Reptantia, studies on the planktonic phyllosoma larvae of the spiny lobster, Panulints argus (Lewis, 1951), disclosed considerable variation between individuals in the same stage during the later developmental stages. Accounts of variability among larvae of the Anomura are available from rearing studies, as well as from studies on larvae obtained from the plankton. Working with reared larvae, variability was described for Plcnroncodes planipes (Boyd and Johnson, 1963), Calcinns tibicen (Provenzano, 1962a), Cocnoblta cl\pcatus (Pro- venzano, 1962b) and Emcrita talpoida (Rees, 1959), while Johnson and Lewis (1942) describe variability in larval development of Emcrita analoga obtained from the plankton. Within the Brachyura, or true crabs, the references to variability in larval development are limited to the description of an "extra" stage, late in the zoeal development of Callincctcs sapid us (Costlow and Bookhout, 1959) and Menippe uicrccnaria (Porter, 1960) reared in the laboratory. A zoea. comparable to the "extra" eighth zoeal stage of C. sapidus, has also been found in the plankton (Nichols and Keney, 1963). The foregoing resume of variability in larvae of widely varying groups of Crustacea, while not intended to be complete, serves to illustrate that variability does exist in larvae which develop through a series of successive stages by molting, be they reared larvae or larvae obtained from the natural environment. A number of factors have been investigated as possible causes of variability in crustacean larvae. Several authors have noted the occurrence of a "pre-zoea" stage in the development of the blue crab, C. sapidus (Robertson, 1938; Churchill, 1942; Sandoz and Rogers, 1944; Costlow and Bookhout, 1959). The "pre-zoea" occurs most frequently when the eggs are hatched in a salinity lower than 20 p.p.t. but this example of variability early in larval development is more closely related to abnormal hatching than to variations in rate of larval development or an actual stage of development. Other studies on the effect of salinity and temperature on larval development have either noted that these two environmental factors did not affect the number of larval stages (Costlow, Bookhout and Monroe, 1960; 1962) or the authors did not comment on any variability which may have existed (Coffin, 1958; Templeman, 1936a). A limited number of studies on the possible effects of light, or photoperiod, on VARIABILITY IN CALLINECTES LARVAE 63 larval development have failed to show any positive relationship (Templeman, 1936a; Costlow and Bookhout, 1962), although earlier work by Huntsman (1924) suggested that strong sunlight did reduce the per cent survival of young larvae of Homarns aincricanus. A third factor, diet, has been suggested as the cause of variability in larval development of several species of Crustacea. Templeman (1936b) attributed an occasional "extra" stage in the development of Homarns aincricanus to a reduction in available food. Broad (1957b) found that the rate of development of P. pugio and P. t'lilgaris larvae, as well as the number of larval stages, was associated with the amount of available food as well as the nature of the diet. Chamberlain (1961), working with the larvae of crabs belonging to the Xanthidae, found that although different diets affected the rate of development as well as the survival, the number of zoeal stages was not changed. In studies on the larvae of C '. sapid its, the diet was adequate in quantity and has, during the past five years, contributed to the successful and normal development of many thousands of zoeae of 30 species of crabs, including the zoeae of C. sapid us. The abundance of food, however, does not necessarily indicate that it is ingested or effectively utilized by all larvae. Previous studies on the larval development of other species of crabs have emphasized the regularity in molting frequency of the zoeae when environmental factors are optimal (Costlow, Bookhout and Monroe, 1960; 1962). While the mechanisms which control the regular sequence of molts in larval forms are not known, suggestions have been made on the basis of recent studies (Costlow, 1961, 1963a, 1963b). Earlier work by a number of investigators (Orlamunder, 1942; Pyle, 1943; Dahl, 1957), as well as the recent study by Hubschman (1963), described groups of cells in developmental stages of a number of Crustacea which are similar to the X-organ of the adult eyestalks. The stage of development when these cells first become functional, producing the molt-inhibiting hormone which, with the molt accelerating-hormone of the Y-organ, regulates the cyclic molting pattern of the adult crab, is not known for any larval form. Costlow (1963a), working with megalops of C. sapidns, has suggested that early in larval develop- ment only the accelerating hormone of the Y-organ is present. Late in larval development, perhaps at the beginning of the megalops stage, the X-organ sinus gland complex of the eyestalk becomes functional, prolonging the intermolt period of the megalops and eliminating the established frequency of molting of the zoeae. Results of studies on the effect of eyestalk extirpation on metamorphosis, molting frequency, and growth of C. sapidns megalops definitely established that molting and growth were independent and apparently controlled by separate mechanisms (Costlow, 1963a). If, as suggested by a study of molting and regeneration of chela in megalops of C. sapid us (Costlow, 1963b), molting has priority over regeneration, the regular pattern of molting which appears to be superimposed on the development of larval morphological features may also have priority over the normal pattern of morphological development. Under these circumstances one might expect that any factor which altered the rate of morphological development, but did not affect the mechanisms which controlled molting, would be observed as "skipping" of larval stages or "extra" larval stages. The three general types of variability which were observed in the present study of C. sapidus larvae could serve as examples. In the first type, molting without 64 JOHN D. COSTLOW, JR. any perceptible morphological changes, the frequency of molting was maintained while the mechanisms responsible for continued development were completely inhibited. In the second type of variability, the elimination or "skipping" of a larval stage, the molting frequency was again constant while the mechanisms con- trolling development were accelerated. The third type of variability was a molt which resulted in a larval stage which combined morphological characters nor- mally attributed to two zoeal stages. The molting pattern, still constant, would appear to have been preceded by normal or accelerated development in the anterior portion of the larvae, while in the posterior portion, development was inhibited or normal. It should be noted that in virtually all accounts of variability in number of larval stages, "skipping" or "extra" stages first become apparent late in larval development. The two cases described for the Brachyura involved a zoeal stage beyond the stage which is normally considered the final zoeal stage. Morpho- logically the "extra" zoeal stage had certain features, usually in the anterior por- tion of the animal, which closely resembled those features described for the mega- lops stage. In the present study, variability in larval stages was never observed before the fifth zoeal stage. As the majority of the larvae of C. sapidus pass through seven zoeal stages, the fifth stage may be considered "late" in development. If this variability is caused by the malfunction of an endocrine mechanism, the fact that the variability is limited to the later stages may be due to a gradual decline in secretory activity of the endocrine mechanisms rather than an abrupt cessation of activity. Another possible explanation should also be considered. Within the Brachyura the number of zoeal stages within any one species ranges from two (Libinia, unpublished) to seven (Callincctcs: Costlow and Bookhout, 1959), with a number of species having either 3, 4, or 5. Only within the Portu- nidae, the family to which Callincctes belongs, has the number of zoeal stages been shown to exceed five, and this family is considered to be the most primitive group within the Brachyura (Lebour, 192S). Thus, it is possible that this variability, so extremely rare in the larvae of other species of Brachyura with a relatively small number of zoeal stages, is associated with the primitive nature of the Portunidae. Experiments in our laboratory have shown that "extra" or supernumerary zoeal stages could be induced in the development of other species of crab larvae by the removal of both eyestalks prior to a critical period in larval development (Costlow, 1963c). Variability in these experimental larvae, similar in general external appearance to those described for extra stages of C. sapid us (Costlow and Book- hout, 1959) and M. mercenaries (Porter, 1960), occurred late in larval develop- ment and were directly associated with the absence of, or the reduced titer of, some substance which is elaborated within the neurosecretory centers of the larval eyestalks. If the Y-organ is present and functional in larval stages, activating the regular sequence of molts through the secretion of the molt-accelerating hormone without the inhibiting effect of the X -organ, removal of the eyestalks during the zoeal stages should not alter the molting frequency of the zoeae. Recent experi- ments (unpublished) would indicate that this is the case and further substantiate the concept that the pattern of development of morphological features is inde- pendent of molting and controlled by a separate mechanism within the eyestalks. In the sequence of development of normal x.oeae, i.e., zoeae which have not had VARIABILITY IN CALLINECTES LARVAE 65 eyestalks removed, variability may also be linked to malfunction of endocrine systems which do not permit the development of morphological features in the pattern generally regarded as "normal," to keep pace with the frequency of larval molting which is controlled by separate endocrine systems. Under this dual sys- tem of control, insufficient food, dietary deficiencies, or the absence of certain organic or inorganic trace elements in sea water could prevent or delay the normal functioning of the endocrine mechanisms which control development. Externally these deficiencies, as well as the resultant malfunctioning of the endocrine system, would be manifest as variability in the number of larval stages or in minor differ- ences in morphological characters of the larvae. LITERATURE CITED BODEN, B. P., 1951. The egg and larval stages of Nyctipliancs simplex, a euphausiid crusta- cean from California. Proc. Zool. Soc. London, 121: 515-527. BOYD, C. M., AND M. W. JOHNSON, 1963. Variations in the larval stages of a decapod crusta- cean, Plcuroncodcs planipes Stimpson ( Galatheidae). Biol. Bull., 124: 141-152. BROAD, A. C., 1957a. Larval development of P alaemonetes pinjio Holthuis. Biol. Bull., 112: 144-161. BROAD, A. C., 1957b. The relationship between diet and larval development of Palacnwncfcs. Biol. Bull., 112: 162-170. CHAMBERLAIN, N. A., 1961. Studies on the larval development of Neopanope te.rana sayi (Smith) and other crabs of the family Xanthidae (Brachyura). Tech. Report XXII, Ches. Bay Inst., 35 pp. COFFIN, H. G., 1958. The laboratory culture of Pagunts saiuuelis (Stimpson) (Crustacea, Decapoda). Walla Walla College Publ. No. 22, 5 pp. COSTLOW, J. D., JR., 1961. Fluctuations in hormone activity in Brachyura larvae. Nature, 192: 183-184. COSTLOW, J. D., JR., 1963a. The effect of eyestalk extirpation on metamorphosis of megalops of the blue crab, Callincctcs sapidus Rathbun. Gen. Coin p. Endocrinology, 3: 120-130. COSTLOW, J. D., JR., 1963b. Regeneration and metamorphosis in larvae of the blue crab, Calli- ncctcs sapidus Rathbun. /. E.vp. Zool., 152: 219-228. COSTLOW, J. D., JR., 1963c. Challenging problems : Larval development. A.I.B.S. Bulletin, October : 63-65. COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1959. The larval development of Callincctcs sapidus Rathbun reared in the laboratory. Biol. Bull., 116: 373-396. COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1960. A method for developing Brachyuran eggs in ritro. Limnol. Occanog., 5: 212-215. COSTLOW, J. D., JR., AND C. G. BOOKHOUT, 1962. The larval development of Scsanna rcticu- latnin Say reared in the laboratory. Cntstaccana. 4: 281-294. COSTLOW, J. D., JR., C. G. BOOKHOUT AND R. MONROE, 1960. The effect of salinity and tem- perature on larval development of Scsanna cincreuni (Bosc) reared in the laboratory. Biol. Bull,, 118: 183-202. COSTLOW, J. D., JR., C. G. BOOKHOUT AND R. MONROE, 1962. Salinity-temperature effects on the larval development of the crab Panopcus hcrbstii Milne-Edwards reared in the laboratory. Physio!. Zool., 35: 79-93. DAHL, E., 1957. Embryology of x-organs in Crangon allinanni. Nature, 179: 482. ERASER, F. C., 1936. On the development and distribution of the young stages of the krill (Euphausia superba). Discovery Repts., 1-192. HUBSCHMAN, J. H., 1963. Development and function of neurosecretory sites in the eyestalks of larval Palacnionctcs (Decapoda: Natantia). Biol. Bull.. 125: 96-113. HUDINAGA, M., AND H. KASAHARA, 1941. Larval development of Balunits amphitritc luncaiicn- sis. Zool. Mag. (Japan), 54: 108-118. HUNTSMAN, A. G., 1924. Limiting factors for marine animals. I : The lethal effect of sun- light. Contr. Canad. Biol,, N.S., 2: 83-88. 66 JOHN D. COSTLOW, JR. ISHIDA, S., AM) R. YASIT.I, 1937. Free-swimming stage of B. amphitritc albicostatns. Botany and /<>»/.. Tokyo, V: 1659-1666. JOHNSON, M. \V., AND \\'. M. LEWIS, 1942. Pelagic larval stages of the sand crabs Emcrita analoiia (Stimpson), Blcpharipoda occidcntalis Randall, and Lepidope m\ops Stimpson. Biol. Hull., 83: 67-87. JONKS, L. \\". G., AND D. J. CRISP, 1954. The larval stages of the barnacle Balanus improrisits Darwin. Proc. Zool. Soc. London, 123: 765-780. LEBOTK, M. Y., 1928. The larval stages of the Plymouth Brachyura. Proc. Zool. Soc. Lon- don, 1928: 473-560. LEWIS, J. B., 1951. The phyllosoma larvae of the spiny lobster Pannlints an/us. Bull. Mar. Set. C.nlj <"- Caribbean. 1: 89-103. NICHOLS, P. R., AND P. M. KENEY, 1963. Crab larvae (Callinectcs) in plankton collections from cruises of M/V Theodore N. Gill South Atlantic Coast of the U. S., 1953-1954. U. S. Fish and Wildlife Service, Special Scientific Report — Fisheries No. 448, 14 pp. NOKKIS, K., AND D. J. CRISP, 1953. The distribution and planktonic stages of the Cirripede Balanus pcrforatns Bruguiere. Proc. Zool. Soc. London, 123: 393-409. ORLAMUNDER, J., 1942. Zur Entwicklung und Formbildung des Birt/us lairo (L.) mit be- sonderer Berucksichtigung des x-organs. Zeitschr. u'iss. Zoo!., Abt. A, 155: 280-316. PORTER, H. J., 1960. Zoeal stages of the stone crab, Mcnippe incrccnaria Say. Chesapeake Sci., 1: 168-177. PROVENZANO, A. J., 1962a. The larval development of C ale huts tibiccn (Herbst) (Crustacea, Anomura) in the laboratory. Biol. Bull., 123: 179-202. PUOVKN/ANO, A. J., 1962b. The larval development of the tropical land hermit Cocnobita clypeatits (Herbst) in the laboratory. Crttstaccana, 4: 207-228. PYLE, R. W., 1943. Tlie histogenesis and cyclic phenomena of the sinus gland and x-organ in Crustacea. Biol. Bull., 85: 87-102. REES, G. H., 1959. Larval development of the sand crab Emcrita talpnida ( Say ) in the labora- tory. Biol. Bull., 117: 356-370. REN FRO, W. C., AND H. L. COOK, 1963. Early larval stages of the seabob, Xiphopcncns kro\eri (Heller). U. S. Fish U'ildlifc Service, Fishery Bull., 63: 165-177. ROBERTSON, R. L., 1938. Observations on the growth stages in the common blue crab, Calli- nectcs sapidns Rathbun, with special reference to post-larval development. Unpub- lished Thesis, Univ. of Maryland, 46 pp. SANDOZ, M., AND R. ROGERS, 1944. The effect of environmental factors on hatching, moulting, and survival of zoea larvae of the blue crab, Callinectcs sapidns Rathbun. Ecolo\ an 11-M/tiger larva. Sensory bristles omitted. STREBLOSPIO LARVAL DEVELOPMENT 75 when they were placed in rearing vessels containing sediment. When similar iso- lates were maintained without sediment, the larvae would usually reach the 13- setiger stage before disintegrating. Only two specimens metamorphosed success- fully in the absence of sediment. In both cases, metamorphosis coincided with the addition of setiger 14. DISCUSSION Both larvae at the time of hatching and eggs within females showed the char- acteristic blue-green coloration reported by Campbell (1957). Shortly after hatch- ing, all coloration is lost except a small area at the bases of the swimming setae on setiger 1. Several differences exist between the results of the present study and those reported by Campbell (1957). She noted that larvae, when released from the female, had three to four segments ; had well developed prototrochs and telotrochs ; and had serrated swimming setae, two pairs of red-brown eyes, and four anal cirri. In the present study, however, larvae first attained a length approximating that of a 7-setiger larva before a telotroch was formed or before setation or seg- mentation took place. It was also found that, unless disturbed, a female would retain larvae until the 9-setiger stage. These observations are supported by the size of Streblospio larvae found in the plankton of the Mystic River Estuary. Of the thousands of Streblospio larvae observed in plankton samples taken during two summers of study, none were found to have less than 7 setigers. The liberation of 3- and 4-segment stages reported by Campbell (1957) could be explained by rough handling of adults or by differences in reproductive behavior of this species in the two areas. The spawning season of Streblospio bencdlctl in the Mystic River Estuary is from June to October. During this period the sexes can be distinguished if the females are ovigerous or if the pale, milky posterior region of ripe males is dis- cernible. The sperm is of the aberrant type, a characteristic in keeping with its unusual mode of reproduction (Franzen, 1956). SUMMARY 1. The larval development of the larviparous spionid polychaete. Streblospio benedlctl, is described. Specimens were obtained from adults maintained in the laboratory and from plankton isolates. Early and late trochophores, 7-, 9- and 11 -setiger stages are illustrated. 2. Undisturbed adult females retain larvae until the 9-setiger stage. If stimu- lated, however, the female will release younger stages. Under laboratory condi- tions, late trochophores and all later stages can be reared through metamorphosis. Larvae can metamorphose once they acquire 9 setigers, but they can delay meta- morphosis until the 13-setiger stage in the absence of a suitable substratum. Rarely will larvae metamorphose in the absence of sediment. Pelagic life is usually three days or less but can be prolonged for at least two weeks. 3. Seven- to 13-setiger S. benedlctl larvae occur in the plankton of the Mystic River Estuary from June to October. Nine- and 10-setiger larvae are encountered most frequently. 76 DAVID DEAN LITERATURE CITED CAMPBELL, M. A., 1957. Larval development of Strcblospio bcncdicti. Biol. Bull., 113: 336- 337. DEAN, D., AND P. A. HATFIELD, 1963a. Pelagic larvae of Ncrinidcs af/ilis (Verrill). Biol. Bull.. 124: 163-169. DEAN, D., AND P. A. HATFIELD, 1963b. A method for holding small aquatic invertebrates for observation. Turtox Ncu's, 41 : 43. FRANZEX, A., 1956. On spermiogenesis, morphology of the spermatozoon, and biology of fertilization among invertebrates. Zool. Bidr. Uppsala, 31: 355-482. HARTMAN, O., 1945. The marine annelids of North Carolina. Duke Unir. Mar. Stat., Bull. No. 2: 1-54. HOWIE, D. I. D., 1958. Dried organic substances as food for larval annelids. Nature, 181 : 1486-1487. JONES, M. L., 1961. A quantitative evaluation of the benthic fauna off Point Richmond, Cali- fornia. Univ. California Publ. Zool., 67: 219-320. THE REACTION OF TWO STARFISHES, PATIRIA MINIATA AND ASTERIAS FORBESI, TO FOREIGN TISSUE IN THE COELOM 1 HELEN T. GHIRADELLA 2 Department of Zoology, Cornell University, Ithaca, New York The reactions of invertebrate animals to invasion of the body by foreign mate- rials are not clearly understood, largely because relatively little attention has been focused on this aspect of invertebrate biology. Phagocytosis appears to be the most common defense (Cantacuzene, 1923; Huff, 1940) and has been described by many workers, among them Durham (1888, 1891), Metchnikoff (1891), Kin- dred (1921), Lison (1930), and Boolootian and Giese (1958). These authors and others note that large objects, such as clumps of carmine which cannot be ingested by individual phagocytes, are generally encapsulated or surrounded by layers of amebocytes. Very little direct work has been done on the specific reactions of invertebrates to large implants, however; the results of Labbe (1929) working on molluscs, Cameron (1932) working on earthworms, Salt (1957) working on insects, and Triplett, Gushing and Durall (1958) working on sipunculid worms, are perhaps typical and suggest that encapsulation may be a general invertebrate reaction toward all foreign objects too large to be phagocytized. Triplett, Gushing and Durall note that the sipunculid worm, Dcndrostomuin zostericolum, discriminates between pieces of sipunculid and sea anemone tentacles introduced into the coelom, for although both are encapsulated, the sea anemone tissue is killed whereas the sipunculid tentacles are recovered from their capsules alive. The worms are evidently incapable, however, of distinguishing between pieces of sipunculid tentacle of homologous and heterologous origin. The data of dishing (1957), who made autologous and homologous grafts of eyes to the female portions of the gonads in the scallop, Aequlpecten irradians, suggest that the hosts are slightly more tolerant of autologous than of homologous grafts, but Gushing points out that his results are preliminary and need extension and confirmation. Anderson (unpublished) performed a brief series of exploratory studies in which he introduced pieces of pyloric caecum from the starfishes, Pisostcr ochraceus (Order Forcipulata) and Henricia levinsciila (Order Spinulosa) into the coelom of another starfish, Patiria ininiata (Order Spinulosa). He was able to recover the Pisastcr caecum in apparently healthy condition after ten days in the foreign environment. The Henricia caecum, on the other hand, was found to have under- gone almost complete disintegration. These results are only preliminary, but they 1 This paper represents an abridgement of a thesis submitted to the Graduate School of Cornell University in partial fulfillment of the requirements for the degree of Master of Science. The studies on which it is based were supported in part by the Sarah Manning Sage and Dr. Solon P. Sackett Research Funds of the Department of Zoology, and by funds from ! . S. F. Grant G-20744 to Cornell University (administered by Dr. J. M. Anderson). The studies were completed during tenure of a NSF Summer Fellowship. 2 Present address : Department of Biology, University of California, Santa Barbara. 77 78 HELEN GHIRADELLA present a contrast to the general pattern of encapsulation described above. They also serve to illustrate the inconsistency of reactions in invertebrate groups, for the I'isastcr tissue, which was tolerated, is less closely related to the host (in a different order) than the Hcnricia caecum, which was rejected. The present study was initiated as a further preliminary exploration of the nature of the reactions of starfishes to foreign tissue in the coelom, in the hope of determining whether the host distinguishes between the tissues of donors of different degrees of relationship, and how the foreign tissues are treated. For many reasons, the asteroids serve as excellent experimental material in transplant studies. The coelom is large and easily reached by knife and hypodermic needle. The coelomic fluid is similar to sea water in composition and tonicity, and transplants may be handled or held in ordinary sea water for considerable periods of time without being damaged. For the same reason, leakage of sea water through the implantation incision in the host body wall does not greatly alter the internal environment of the animal. The animals are generally hardy and regenerate easily. They can with- stand the loss of pieces of body wall or of viscera, and they heal experimental incisions well. The pyloric caeca, which are suspended in the coelom by mesenteries, provide excellent tissue for transfer. Caecum tissue is relatively active meta- bolically and shows a high rate of molecular turnover (Ferguson, 1964a, 1964b) so that its presence has a considerable effect on the composition of the coelomic fluid. It is easily removed from the donor. Above all, it is quite distinctive histologically ; in addition to classical accounts, more recent descriptions of normal histology have been provided for Astcrias forbcsi (Anderson, 1953) and Hcnricia Icrinscula (Anderson, 1960, 1962). Hence, it is possible to determine by histological study whether a sample seems to be normal and functioning, or to detect changes that may have taken place as a result of sojourn in a host animal. The general questions toward which this study was directed were the following : ( 1 ) Does a host starfish distinguish between implanted pieces of pyloric caecum from donors of different degrees of relationship? (2) What is the nature of the animal's reaction to foreign tissue, and by what means (if any) are large pieces removed from the coelom? (3 ) What effect, if any, does sojourn in a strange environment have upon tissue of different donors ? The author would like to express her sincere gratitude to Dr. John M. Anderson for his invaluable aid and advice during the course of the study, and for his help in the preparation of the manuscript and the photographs. MATERIALS AND METHODS The problem involved introducing pieces of pyloric caecum of one animal (donor) into the coelom of another (host) and determining the fate of such transplants. The host species used were Patina uiiniuta, obtained from the central California coast, and .Istcrias forhcsi, obtained from Woods Hole, Massachusetts. Fairly small specimens, ranging from one to three inches in diameter, were used because they required relatively little aquarium space and their body walls were STARFISH REACTION TO FOREIGN TISSUE 79 thin and easy to cut. Donors included members of the host species and of two others, Astcrias vulgaris and Hcnricia sanc/ninolcnta, both obtained from Woods Hole. The animals were kept in running sea water at the Marine Biological Laboratory at Woods Hole, where most of the experiments on Astcrias were done, and in tanks of circulating refrigerated sea water at Cornell University, where the balance of the experiments on Astcrias and all those on Patina were performed. Before being subjected to any experimental treatment, both hosts and donors were first relaxed in a solution of 7.5-8% MgCL in tap water. Pyloric caecum was then removed from the donors and, in order to distinguish it from the caecum of the host, was stained for at least two minutes in 0.01% tnethylene blue in sea water, to which had been added 0.03% egg albumin to reduce the toxicity of the dye (Chalkley and Park, 1947). The fact that homologous tissues stained in this way were not rejected by the host indicates that this treatment does not significantly alter the chemistry of the transplants. Implanted tissues retained the blue color for more than six weeks and thus were easy to identify in gross dissection ; the stain was completely removed, however, by subsequent histological procedures. The staining step was omitted when Hcnricia was the donor, for the caecum of this species is brilliant red-orange in color and is easy to identify without any special treatment. The tissue was trimmed and injected by means of a large-bore hypodermic needle (#16), which was inserted into the ray opposite the host ray. In this way, the body wall of the host ray was left intact and the tissue was not likely to slip out im- mediately upon implantation. The cardiac stomach, and perhaps the pyloric as well, suffered some trauma when the needle passed through it, but its many folds and great elasticity served to occlude any openings through which the tissue might escape. With the needle in position, the trimmed donor caecum was drawn into the syringe with a small amount of sea water, the syringe was connected to the posi- tioned needle and the tissue expelled into the coelom of the host. Care was taken not to introduce air bubbles into the coelom during this process. The advantage in first positioning the needle and then attaching the syringe lay primarily in the fact that there was less chance of macerating the tissue if it had to pass through the needle only once. Two rays of an individual were generally used as host rays, and each was con- sidered a separate experimental case. When an autologous transplant was being made, one of the rays opposite that intended to be the host ray was chosen as the donor ray, and the tissue was removed from it through a slit made in the aboral wall. The needle could later be inserted into the same incision. In some of the smaller specimens of Astcrias, where it was difficult to slit the body wall accurately because of the small area of the aboral surface of the rays, the tip of the donor ray was cut off and the caecum removed through the open end. Thereafter, the procedure for the autologous transplants was identical to that for the others. The hosts were examined daily for at least a week after implantation. Pres- ence of edema, autotomy of host rays, and such signs of morbidity as shedding of patches of body wall or development of excessive flaccidity were noted. In addi- tion, the dermal branchiae and oral and anal areas were checked for indications of the elimination of implanted tissue. At the end of a week (or longer in some cases), the hosts were dissected after relaxation in MgCL, and the disposition of the trans- 80 HELEN GHIRADELLA plants noted. If the tissue was recovered, it was fixed in Bouin's solution for at least 24 hours (48, if it included calcareous elements), and after washing in 80c/c alcohol, was dehydrated, cleared, and embedded in paraffin. Sections were cut at 10 /JL and stained with Mallory's phosphotungstic acid hematoxylin, which differentiates muscle, connective tissue, cell membranes, and secretion granules, and were examined for evidence of growth, necrosis, and other changes. Control pieces of caecum that had been passed through the needle without being implanted were similarly fixed, sectioned, and stained, in order to estimate the damage that might be caused to the tissue by the injection procedure. RESULTS a. Implantation experiments When the animals were examined and dissected, it was found that the disposi- tion of the donor tissue tended to fall into one of four categories, scored as follows : (a) "Leaking"- -The transplant caecum began to pile up in the dermal branchiae of the hosts, usually appearing here within one to three days after implantation. The distal halves of the branchiae, which had become swollen with donor tissue, often rounded up and pinched off from the liases, in effect undergoing autotomy. In other cases, the branchiae ruptured without autotomizing, so that the tissue simply passed out through the distal openings. In a few instances, where several adjacent branchiae had ruptured simultaneously, actual rifts in the body wall were formed, through which masses of implanted tissue could be seen emerging. In any case, when the hosts were dissected at the end of the week, the donor tissue was recovered, if at all, as a few small pieces projecting into the coelom from the bases of the branchiae. (b) "Not recovered"- -In many cases, the transplant was nowhere to be seen when the host was dissected. Parts of host caecum or body wall were stained with methylene blue, however, indicating that the donor tissue had been successfully injected into the coelom and had remained there long enough to impart some of its blue coloring to the surrounding host tissue. Asterias transplants tended to trans- fer more of the stain than Patiria transplants. It seems reasonable to assume that the tissue had in fact been eliminated, but by some route other than through the dermal branchiae. (c) "Recovered"- -The donor tissue was found apparently intact, either floating free in the coelom, or attached to the host body wall or pyloric caeca. (d) "Lost"- -When it was impossible to trace the disposition of the tissue, i.e., when there was no recovery or evidence of leakage, and no staining of any host tissues could be noted, it was assumed that the tissue had not been successfully introduced into the coelom, and the data for that case were discarded. Into this category were also placed animals that had become moribund or had autotomized the host ray(s). The first series of experiments involved transplanting pieces of homologous or heterologous pyloric caecum to determine the reaction of the hosts to transplants in general, and to see whether there were differences in the treatment of homologous and heterologous tissues. Table I presents a synopsis of the results of this series. STARFISH REACTION TO FOREIGN TISSUE 81 The figures for donor Asterias include both A. forbcsi and ./. ruli/aris. hetween which no distinction was apparently made by either Asterias or i'aiina hosts. These data indicate that both host species tend to tolerate their own tissue and to eliminate that of the other species. In order to confirm these observations, several double implantations were made, with each host receiving one homologous and one heterologous piece of caecum. Table II summarizes the results of these experi- ments. In the case of the single piece of Henricia caecum which was not recovered, the initial injection was probably unsuccessful. TABLE I Results of experiments demonstrating host reaction to single implants of pyloric caecum Host Donor Leaking Recovered Not recovered Total Patiria Patiria 3 29 0 32 Asterias 0 1 13 14 Hen rid a 9 1 (in frags.) f) 10 Asterias Asterias 0 11 0 11 Henricia 7 0 0 7 The results of both sets of experiments are mutually consistent and present evidence of what seems to be definite discrimination between homologous and heterologous transplants. These figures also demonstrate that the same animal can retain one transplant while eliminating another. There are evident contrasts between the modes of elimination of Asterias and Henricia caecum by both Patiria and Asterias. Patiria, though it eliminates Henricia caecum through the branchiae, may dispose of Asterias tissue by pushing TABLE II Results of experiments determining whether a single host discriminates between simultaneously implanted homologous and heterologous caecum Host Donor Leaking Recovered Not recovered Total Patiria Patiria f) 4 0 4 Asterias 0 0 4 4 Asterias Asterias 0 11 0 11 Henricia 9 1 1 11 it back into the cardiac stomach, for on several occasions, transplanted tissue was recovered projecting from the mouth. Asterias also eliminates Henricia caecum through the branchiae. Unfortunately, transportation difficulties made it impossible to obtain an adequate supply of Asterias in Ithaca, and I was unable to determine how this species reacts to Patiria implants. To determine whether tolerance of homologous material in the coeloni extended to tissue taken from other parts of the body, observations were made on homologous transplants of tissue other than pyloric caecum. Eight Patiria host rays were im- 82 I !!• I I \ CM IK KDELL \ "» » ' "*# ' 1 ".%-, ' '^j Kldl'KKS 1-4. STARFISH REACTION TO FOREIGN TISSUE planted with homologous rectal caecum, an organ normally found in the aboral region of the central disc. All eight transplants were recovered in apparently healthy condition, and in one case, the implanted rectal caecum was joined to an adjacent pyloric caecum of the host. On other occasions, recovered pyloric caecum transplants, especially older ones which had spent three or more weeks in the host coelom, had fragments of ossicle; embedded within them. The sources of these fragments are uncertain ; perhaps they had been picked up and carried in by the tip of the needle during the implantation procedure. This retention of both rectal caecum and ossicle fragments, despite their considerable physical differences from pyloric caecum, supports the view that elimination cannot be based on physical differences alone. It also suggests that the animal is not sensitive to the presence of tissue elements in the wrong places in the body. /'. Histological observations Normal pyloric caecum is composed of an outer peritoneum of cuboidal cells, layers of muscular, connective and nervous tissue, and an inner epithelium of tall flagellated columnar cells, this layer containing mucous gland cells and secretory cells producing vesicles and granules (Anderson, 1953 ; see also Fig. 1 ). Examina- tion of pieces of control caecum which had been passed through the needle before fixation revealed areas of tissue normal in every respect, interspersed with places where the tissue had been broken and abraded by the injection process. Both Patiria and Astcrias pyloric caecum transplants recovered after one to five weeks in homologous hosts generally showed the same mixed appearance as the injected and fixed controls, healthy areas alternating with damaged ones. The sound areas (Fig. 2) were generally indistinguishable from normal tissue. The presence of prominent secretion granules in the recovered caecum (Fig. 2) indicates that metabolic activities were continuing at the time of fixation. There were few amebocytes associated with the intact areas. Damaged areas, on the other hand, showed changes, particularly evidence of necrosis and disorganization associated with amebocytes. The amebocytes were often rounded and filled with what ap- peared to be cellular debris, including recognizable secretion granules. Whether these phagocytes came from the donor, the host, or both could not be determined. FIGURE 1. Patiria. normal pyloric caecum. Many of the tall columnar epithelial cells contain secretion granules and vesicles. X 260. 1, lumen of caecum ; p, peritoneum ; s. secre- tory cell with granules. FIGURE 2. Patiria, pyloric caecum recovered after four weeks in a homologous host. There is no evidence of damage or necrosis ; the secretory cells show evidence of continuing normal metabolic activity. X 520. 1, lumen of caecum ; s, secretory cell with granules ; v, vesicle. FIGURE 3. Patiria, pyloric caecum with attached mass of amebocytes and connective tissue recovered from a homologous host after four weeks. The mass of new cells is associated with damaged areas of caecum (not shown) but has extended to join the healthy caecum at left. The connective tissue fibers in the new tissue are continuous with those in the donor caecum. Peritoneum, also continuous with that of the donor caecum, covers the amebocyte mass a, amebocyte mass ; C, donor caecum ; c, connective tissue fibers ; 1, lumen of caecum ; p, peritoneum. FIGURE 4. Patiria, normal rectal caecum. The inner epithelium is thrown into folds supported by connective tissue partitions. >< 260. c, connective tissue fibers ; e, epithelium ; p, peritoneum. IIKLEN r.HIRADKLLA r/ * I ; . * B f e B w \ f FIGURES 5-8. '. V li e P ., •"• 8 STARFISH REACTION TO FOREIGN TISSUE 85 Recovered transplants were found joined to the host caecum or \n><\\ wall on several occasions. In some specimens, connections were made at several points. The more tenuous of these links appeared to he established by anu-bocvu^ alone, while more substantial bridges showed connective tissue fibers in addition, h seems probable that the amebocytes established the first bridges, with connective ti^ fibers invading later. Some of the older transplants (four or five weeks) had masses of amebocvU's and connective tissue filling in the gaps or folds in the implant (Fig. 3). Thi> growth was often quite extensive and gave the impression of rounding out the contours of the transplant. In some cases, peritoneal epithelium could be seen around the mass of new cells (Fig. 3). The development of the amebocyte masses usually began on the borders of the damaged sections, occasionally extending to the healthy areas as well. Where ossicles were embedded in the transplant, they were also associated with amebocytes and connective tissue, and appeared to be firmly joined to the surrounding caecum. In no case, however, did the recovered homologous tissue give the impression of undergoing encapsulation, for areas not obviously damaged were often free of amebocyte activity. Normal rectal caecum shows the same layers as normal pyloric caecum, but the inner epithelium is greatly folded, each fold being supported by inward extensions of the connective tissue layer (Fig. 4). Rectal caecum recovered after a week (Fig. 5) was for the most part indistinguishable from the normal caecum, although it did show some local damage associated with amebocyte activity and connective tissue formation similar to that described in the recovered pyloric caecum. One case of encapsulation was observed, involving a small fragment of Asterias transplant recovered from a host Patina. Sections showed a small core of ex- tremely disorganized donor tissue with a few scattered secretion granules remaining, surrounded by amebocytes. There was no evidence of connective tissue formation. Examination of sections of body wall in which the dermal branchiae were eliminating Hcnricia caecum showed interesting details. The branchiae were rounded and swollen at their tips, which either ruptured (Fig. 6), or pinched off (Figs. 7, 8). The walls of the swollen parts were stretched quite thin compared with the more typical branchiae (Fig. 8) ; the columnar epidermis appeared almost cuboiclal, the connective tissue layer was greatly attenuated, and the peritoneum was vague and indistinct. It was impossible to distinguish the muscle layers at all. FIGURE 5. Patiria, rectal caecum recovered from homologous host after one week (slightly oblique section). This tissue is virtually indistinguishable from the normal rectal caecum (Fig. 4). < 260. c, connective tissue fibers; e, epithelium. FIGURE 6. Hetiricia caecum being eliminated through ruptured Astcrias branchia. ('The section is not quite parallel to the long axis of the branchia.) This branchia and the ones on either side are packed with Hcnricia caecum. Note the expansion of the tissue already out of the branchia. X 90. B, body wall ; b, branchial wall ; C, Hcnricia caecum. FIGURE 7. Astcrias branchia eliminating Hcnricia caecum. (Section is slightly tangential to the long axis of the branchia.) The tip of the branchia containing the Hcnricia caecum is separated from the rest by a prominent constriction. Note the wrinkling of the base of the branchia. X 90. B, body wall ; b, branchiae ; e, epidermis ; f, coelom. FIGURE 8. Detail of Figure 7. Note how the wall of the autotomizing portion of the branchia has been stretched. (Compare with the more typical branchia at left.) < 260. C, Hcnricia caecum; e, epidermis of branchia; f, coelom (cavity of branchia); p, peritoneum. 86 HELEN GHIRADELLA In contrast, the bases of the branchiae, below the point of constriction, appeared quite normal histologically, except that they were slightly wrinkled transversely (Fig. 7) as if either circular or longitudinal muscles or both were somewhat con- tracted. "Where the branchiae had ruptured, Henricia caecum could be seen squeez- ing through the channel provided by the branchial walls and escaping to the outside (Fig. 6). The caecum in the branchiae was not in fragments, but rather gave the impression of being folded and packed into the lumen of the organ (Fig. 6). None of the Henricia caecum appeared to have unusual numbers of amebocytes n.vsociated with it. although it was clearly being actively eliminated. DISCUSSION The results of these experiments suggest that there are at least three different means of elimination of foreign materials from the asteroid coelom. The first, amehncytic attack, was expected from accounts in the literature. The second, elimination of tissue through rupturing or autotomizing branchiae, was somewhat of a surprise, for although Lison (1930) reported branchial autotomy in response to the presence of large clumps of amebocytes in the branchiae, I am aware of no other report of branchial elimination of pieces of tissue. The third method, that still undetermined means by which Patiria eliminated Asterias pyloric caecum (possibly through the cardiac stomach, although I have no histological evidence that this was the case), was also not predictable on the basis of previous reports of elimination of material. Phagocytosis of participate matter may be demonstrated easily in both Patiria and Asterias by injection of a carmine suspension into the coelom ; the branchiae shortly become filled with amebocytes bearing ingested carmine particles. However, amebocytic attack and invasion cannot be responsible for the branchial elimination of Henricia caecum, for the tissue in the branchiae remains in a relatively intact state and has few amebocytes associated with it. Nor does encapsulation by amebocytes seem to be a common response to the presence of large foreign bodies in the coelom, for it was observed only once during the whole study. It was not possible to determine at this time just why Henricia caecum was selectively eliminated through the branchiae by both hosts. The mechanisms in- volved might depend upon physical or chemical differences, or both, between this caecum and those of Asterias and Patiria. In any case, this means of elimination seems to be fairly important to both hosts; at least it was consistently employed. The tilling and rupturing of the branchiae must require a considerable amount of force, perhaps more than can be accounted for on the basis of ciliary currents alone. The musculature of the branchiae, consisting of both longitudinal and circular libers, might be aiding the movement of the tissue to the tip, perhaps by peristaltic action. The longitudinal muscles normally retract the branchiae in response to external disturbances, and the circular muscles are antagonistic to the longitudinal muscles. However, the fact that an autotomizing branchia may be pinching in strongly at one level alone and nowhere else along its length implies rather specific control over local contraction, which in turn suggests that peristaltic movement is at least theoretically possible. The wrinkled appearance of the autotomizing branchiae below the point of constriction, as seen in Figure 7, might indicate that such contraction wa> going on at the time the tissue was fixed. STARFISH REACTION TO FOREIGN TISSUE 87 In addition to eliminating material through the branchiae, Asl< can quickly and easily autotomize whole rays. Patiria cannot autotomize easilv, hut the effi- ciency with which it evidently eliminated the Astcrias trans] >lant> suggvMs tliat it is well protected despite its inability to shed rays. To determine whether Aslcrias can also eliminate material through the cardiac stomach (if this is in fact how Patiria eliminates the Astcrias tissue) or whether this species relies entirely on use of the branchiae and on autotomy requires further experimentation. The question of the level (i.e., tissue, organ, or higher) at which these elimina- tion mechanisms are operating is experimentally beyond the scope of the present study, but it presents possibilities for future investigations. Elimination through the branchiae, for example, may be a complex process ; it remains to be determined whether it is an automatic local reaction to the presence of anything in the branchiae, or whether the process is selective and under the regulation of the animal as a whole. If the latter is the case, one might well consider it an integrated response. It is also important to remember that local and general effects may be occurring simul- taneously and that demonstration of the one does not preclude the existence of the other. The role of the amebocytes associated with the damaged areas of the recovered caecum may lie a multiple one. It is unlikely that they are encapsulating the implant, walling it off from the coelom by forming a shell around it, as this process was found only once in this series of experiments. They are almost certainly engaged in cleaning up cellular debris in these damaged areas. They might also be participating in regeneration of fragmented caecum. Anderson (1962), studying the regeneration of pyloric caeca in Hcnricia lcrinscula, discusses the possibility that the amebocytes might actually be differentiating and supplying cells for the new caecum. Here, also, more work must be done to understand what is taking place. The results of the present preliminary study may or may not indicate that responses of an immunological nature are evoked by tissue implants. The response of the host does seem to vary in relation to the source of the implanted tissue. No distinction was made (within the framework of present experiments) between autologous and homologous tissue, or between tissue of two closely related species, Asterias forbesi and Asterias vulgar-is. On the other hand, there was discrimination between members of two different families in the same order (Henricia and Patiria}, and between members of two different orders (Astcrias and the other two). These latter results are in agreement with those of Anderson (unpublished) on the rejection of Hcnricia caecum by Patiria. They are contrary, however, to his findings on the Pisastcr implants, which were evidently tolerated by Patiria. This discrepancy is unexplained and might conceivably be another example of the "disconcerting inconsistency" mentioned by Cantacuzene (1923) as characteristic of invertebrate reactions. The mechanism of distinction is obscure, but as metabolizing caecum is un- doubtedly exchanging molecules with the coelomic fluid (Ferguson, 1964a. 1964b), the host may be reacting to the presence of atypical molecules in the coelomic fluid. On this basis, also, the distinction between "tissue" and "general" levels of reaction becomes a pertinent question. Much more work remains to confirm and extend these results. Xo attempt HELEN GHIRADELLA was made to follow the progress of any of the transplants for longer than five weeks. It may he that all foreign tissue is eventually rejected or perhaps en- capsulated if enough time is provided, although the experimental data suggest that this i> prohahly not the case. Other donor-host combinations should he tried to determine how specific and how consistent these reactions actually are and where the lines are drawn between tolerance and rejection. The role of the amebocytes needs clarification with respect to such questions as whether they are only removing debris, or are also taking part in regeneration of tissue in the damaged areas of caecum. Until these questions are answered, the precise bases of these reactions toward foreign implants will remain unknown. S I ' M M ARV 1. Two starfishes, Patiria miniata and Asterias jorhesi, were found to discrimi- nate between coelomic implants of homologous and heterologous pyloric caecum obtained from donors of these species and of two others, Asterias vulgaris and Henricia sanguinolenta. 2. Homologous transplants were recovered from the hosts one to five weeks after implantation and were found to be generally normal in histological appear- ance, although there was some growth of connective tissue and amebocyte masses in areas which had probably been damaged during the implantation procedure. Heterologous transplants were eliminated by the hosts within a week of implantation. 3. Henricia caecum was eliminated through the dermal branchiae by both hosts. Patiria, in contrast, eliminated Asterias caecum in a still undetermined way; the tissue may have been transferred from the host ray to the cardiac stomach, and either digested there or passed out through the mouth. 4. Contrary to expectation, amebocytic attack, phagocytosis, and encapsulation were not seen to play an important role in elimination of heterologous transplants, although amebocytes were associated with damaged areas in the homologous trans- plants. Such amebocytic activity is probably concerned with removal of cellular debris. 5. The results of these experiments may express a general tendency of starfishes to distinguish between implants of donors of different degrees of relationship, or they may represent special cases of discrimination. More work remains to deter- mine how specific these tolerance-rejection thresholds are and how consistently they vary with different host-donor combinations. UTKKATl'kE CITED \\HKKSO.V. ]. M., 195.1. Structure and function in the pyloric caeca of . Istcrins jorhcxi. Hii>l. Bull., 105: 47 61. A XMKKSOX, J. M., 1%0. Histological studies on the digestive system of a starfish, llcnrlcla, with notes on Ticdcinann's pouches in starfishes. Mini. Hull.. 119: 371-398. A xm-.ksox, |. M., 1°62. Studies on visceral regeneration in sea stars. I. Regeneration of pyloric caeca in 1 1 cnric'ut Icrutsculu ( Stimpson). Hn>l. Hull.. 122: 321-342. BOOLOOTIAX, R., AXD A. C. (liKSK, 1(>58. The coelomic corpuscles of cchinoderms. Hml. Hull., 115: 53-63. CAMKKOX, (i. 1\., 1M32. Inflammation in earthworms. ./. /'nth. Hue/.. 35: 933-977. CAXT.V i XKXK, I., 1923. I.e problem de 1'imniunite die/ les Invertehres. ( . A'. Soc. />'/«/. ( 7Se aim.) : 48-119. STARFISH REACTION TO FOREIGN TISSU 89 CHALKLEY, H., AND H. PARK, 1947. Methods for increasing the valiu- of hyd is material in teaching and research. Science, 105: 553. GUSHING, J., 1957. Tissue transplantation in Pcctcn irrudidiis. Biol. Hull., 113: 327. DURHAM, F., 1888. On the emigration of ameboid corpuscles in the starfish. / *\. Soc. London, Scr. B, 43: 328-330. DURHAM, F., 1891. On wandering cells in echinoderms, more especially with to excretory functions. Quart. J. Micr. Sci.. 33: 81-121. FERGUSON, J., 1964a. Nutrient transport in starfish. I. Properties of the coclomic fluid. Biol. Bull.. 126: 33-53. FERGUSON, J., 1964b. Nutrient transport in starfish. II. Uptake of nutrients by isolated organs. Biol. Bull., 126: 391-406. HUFF, C. G., 1940. Immunity in invertebrates. Physiol. Rcr.. 20: 68-88. KINDRED, J. E., 1921. Phagocytosis and clotting in the perivisceral fluid of Arbucia. Biol. Bull., 41: 144-151. LABBE, A., 1929. Reactions experimentales des molluscs a 1'introduction de stylets de celloidine. C. R. Soc. Biol., 100: 166-168. LISON, L., 1930. Recherches histophysiologiques sur les amebocytes des echinodermes. Arch. Biol., 40: 175-204. METCHNIKOFF, E., 1891. Lectures on the comparative pathology of inflammation. Translated by F. A. Starling and E. H. Starling. London : Keagan Paul, Trench, Trubner and Co. SALT, G., 1957. Experimental studies in insect parasitism. Proc. Ro\. Soc. London, Scr. B, 147: 167-184. TRIPLETT, E. L., J. GUSHING AND G. DURALL, 1958. Observations on immune reactions of the sipunculid worm, Dendrostomutn sostericolum. Amer. Nut., 92: 287-293. THK KKFKCTS OF TEMPERATURE AND SALIXITY CHANGE ON SWIMMING KATE IX THE I >I \( )F I. .V il-.LLATES, GOXVAL'LAX AND ( iVKODIXIL'M ' WII.LIAM (i. HAND, PATRICIA A. COU.ARI) AND DEMOREST DAVENPORT The L'nii'crxity <>/ (.\tHfoniui, Stintu liarhara, Sniita Hurhiira, Calif. 93018 Because of technical difficulties there have been few definitive studies in which swimming rate of the individual motile microorganism has been measured with accuracy. Wingo and Hrowning ( 1951 ) determined the velocity of Tctra- hynicthi. Dryl (1961) has studied the effects of pH change on linear velocity in Paramecium. These studies were conducted using conventional photomicroscope techniques. A more sophisticated approach to the investigation of swimming be- havior has been taken by Rothschild (1953) and van Duijn and Rikmenspoel (1960) in their studies on sperm; here photoelectronic methods yielded accurate data on velocitv, etc. Davenport, Wright and Causlev (1(^>2) have described an electronic tracking technique with which it is possible, with great accuracv and rapidity, to obtain data on linear velocity and rate of change of direction in the individual microorganism under a variety of conditions. The studies to be described were initiated because of the obvious importance of understanding the effects of changes in environmental parameters on the activity of motile phytoplankters. To this end we chose as experimental organisms two dinoflagellates, the littoral (ionyaula.v polycdra Stein and a to-date unidentified Gyrodinium. The former is the well-known causative agent of "red-tides" (Ilonnot and Phillips, 1^3S; Allen, lc>4(>; Council and Cross, 1950; etc.). Its vertical migration has been studied by llasle (1950. 1954) and the cyclic nature of its luminescent activity by llaxo and Sweeney (1955, etc.). Our Gyrodinium sp. was apparently "selected tor" by the particular environmental conditions of a shallow salt lagoon on the I'niversity campus which receives the outflow of the Marine Laboratory; it "bloomed" so richly as to contribute to the death of numbers of (iillichlhys and l: nndnlns. Since certain species of (', \rod\ni\iiu are known to be characteristically deni/ens of lagoons and salt marshes, we hoped that our form would show different tolerances than the littoral Gonyaulax. As shall be seen, this proved to be true. It was the purpose of these initial studies to establish the effects of temperature and salinity change on the behavior of the two organisms. 'These investigation^ uere conducted under Contract \'< )\ K- 4222-03 \\ith the Office of N'aval kc-rurch. 90 SWIMMING RATE IN GONYAULAX AND GYROD1NIUM 01 [MATERIAL, METHODS The apparatus - A gated square sweep (scan) of uniform low intensity is generated on a Dumont Type 304H oscilloscope mounted vertically (Fig. 1). The gated sweep is ac- complished by a generating circuit which is capable of producing several sweep frequencies. The scan frequency is further controllable by emphasizing a certain section of the generated sweep with accurate regularity. This is accomplished by the incorporation of a blanking circuit which can also be adjusted. By adjusting MICROSCOPE for visual observation HALF MIRROR PHOTOMULTIPLIER VIDEO AMPLIFIER field covered: 4x4mm raster reduced: 25:1 DISPLAY TUBE CAMERA ADJUSTABLE TIME INTERVAL GATE SCANNING TUBE FIGURE 1. Diagram of the scanning apparatus. the interplay between these two controls, as well as the oscilloscope intensity, it is possible to obtain gated scans with intervals of one scan per ten minutes to fifty scans per second. The gated scan is transmitted optically to a microscope stage above the oscillo- scope via a 1.0-mm. camera lens mounted below the stage. The position of this lens can be adjusted by a fine-adjustment so that the scan may be focussed on a prepared slide of the organism to be observed. The area of the test preparation scanned is a 4-mm. square, while the depth of field is less than 0.5 mm. The magnification of the subject may be changed by manipulating the horizontal and vertical sweep controls of the vertically mounted oscilloscope. The field scanned remains the same. L> The authors wish to express their appreciation to Dr. James Mover and Messrs. Robert King and Eugene Evinger, who cooperated in the construction of the apparatus by Servomechanisms Inc. of Santa Barbara. \Y. G. HAND, P. A. COLLAR!) A XI) I). DAVEXPORT 92 Tin- scan is transferred by a mirror to a type 1 P21 photomultiplier tube and associated preamplifier unit. Changes in light intensity within the scan, caused by objects (such as moving organisms) between the scan and the photomultiplier tube, are registered by that tube as changes in electrical potential. The potential change* are amplified and transmitted to a second Dumoiit 30411 oscilloscope mounted horizontally. This scope in turn displays a "negative" image of the intensity dif- ferences in the generated scan, i.e., the discrete areas of reduced illumination in the original scan are finally displayed as light "blips" on a dark field, each blip recording the organism's position at the moment it is scanned. Resolution of the system is fine enough for it to perceive relatively opaque organisms larger than 15-20 fj., and to distinguish the external morphological features of many forms in the 50-300 ^ range. By coupling a Dumoiit type 450A oscilloscope camera fitted Fi<;rif (/«»vi\ linear velocity Gonyaulax Approximately 0.4 cc. of Gonyaiila.r culture was pipetted into a Incite well-slide. The well was circular, with a flat bottom and vertical sides, its dimension being S.O mm. in diameter by 1.5 mm. deep. A coverslip was placed over the well. It was then scanned, photographed and discarded. Fifty individual pathways were recorded from such preparations and measured as described above. Krom these preparations a mean linear velocity of 250 ^/sec. ( with a range from 175 /u/sec. to 325 ju/sec. ) was obtained for the Gonyauhi.v cell at 20° C. Since in the above experiment it was possible to measure linear velocity in the hori/ontal plane only, a series was conducted to determine velocity in the vertical plane. This was accomplished simply by rotating the entire scanning elements of the apparatus, including the preparation under observation, through 90°. Velocities for the two planes were- com] tared statistically, using a one-way analysis of variance, and found not to be signilicantlv different ( I'" := 0.9d n.s. ). Gyrodinium Mean linear velocity for this form was found to be 319 /i/sec. at 20° C. 1 Tere again plane of movement was not a significant factor. The range was similar, being between 220 /A/sec, and 360 /A/sec. 77/c relation <>\ linear i'eli>cit\ to sa/nnlv Gonyaulax In order to determine the gross reactions ot these organisms to variations in -alinity, a series of subjective preliminary observations was conducted. SWIMMING RATE IN GONYAULAX AND GYRODIMl M 95 A series of culture samples was prepared, ranging at 10% inte :'vuls from 100% culture medium (28 /M to 40% (10 /{<•)• These samples were prepared from a single stock culture by diluting the appropriate amount of culture medium with twice-distilled water to make a 10-ml. sample of the desired salinity. The desired amount of distilled water was slowly added to the cell-containing medium ami stirred gently to insure proper mixing. The beakers containing the series of dilu- tions were covered with Parafilm, placed under culture conditions and left undis- turbed for a period of not less than four hours. In the examination of activity under hypersaline conditions, similar procedure was employed with the substitution of 200% sea water for twice-distilled water. Observations were then conducted on each series over a period of twelve hours. Immediately following sample preparation, the majority of the cells were observed to cease normal activity and settle to the bottom of the container. Controls differed from experimental samples in that the time necessary for recovery when one transferred cells from a normal culture into "cell-free" culture medium was a matter of a few minutes only. In such controls the proportion of cells which settled out, even for a brief period, was less than when salinity differed from the medium. At the end of the four-hour "acclimation period" motility was observed in the samples from 40% to 150% medium. In the 150% samples there were more motile cells observed at the end of 12 hours than after four hours. In all other samples, the total number of motile cells did not increase after the four-hour period. \Yith the information at hand from the above subjective observations, a test series was prepared in a similar manner and scanned after the four-hour acclima- tion period. The results of these experiments may be seen in Figure 3. The plot in the figure represents the best fitting (F - - 1201.73) mathematical relationship between linear velocity and salinity for these data. To compare the behavior of the Gonyaulax cell in varying concentrations of culture medium with its behavior in varying concentrations of sea water, the following test was conducted. One hundred ml. sea water were added to 25 ml. culture in a filter funnel lined with Whatman #5 filter paper. The addition was made slowly, the sea water being poured down the side of the filter funnel with care. Cells were not permitted to be drawn against the surface of the filter paper during filtering; by the use of a plastic squeeze bottle of sea water they were washed away from the filter paper surface. Additional sea water was added as needed to maintain original volume. Samples of the solution passing through the filter funnel were collected and tested with the conductivity bridge against sea water at the same temperature. When the conductivities of the two solutions were found to be equal it was assumed that the medium present in the funnel was pure sea water and the transfer of cells was complete. The transferred cells were observed under the microscope for damage. If noticeable damage was present, the transfer was discarded and the process repeated. Satisfactory transfers were placed under culture conditions for six hours and periodically observed for changes in behavior. At the end of this period salinity samples were prepared as described and scanned. It was observed that at corresponding salinities, mean velocity was consistently greater (by about 20 ^,/sec.) in medium than in sea water. A two-factor analysis of variance indicated that this difference was significant (F -- 6.85**). The inter- \V. G. HAND, I'. A. COLLARD AND D. DAVENPORT 400- 300- m r~ o o -< 200- 3 c. 100- 150 130 110 90 70 (42) (36) (31) (25) (20) CONCENTRATION % (%0) I;K, i KK ,•!. Relation of velocity to medium concentration in Gonyaulax 50 (14) 500-1 400- m i— o o 3 c O) o 300- 200- 100- 160 (45) 140 (39) 120 (34) 100 (28) 80 (22) 60 (17) 40 (10) 20 (5) l-h, i i'i 4. l\'i l.ition CONCENTRATION % (%<>) velocity to medium concentration in Gyrodinium -~ SWIMMING RATE IN GONYAULAX AND GYRODIXITM ()7 action between media used and salinities tested was not significant (P 0.96 n.s.). demonstrating that salinity change has the same effect in both media. The "absolute" hyposalinity tolerance for motility was U-> ; in sea water than that in medium, since cells were observed to remain motile down to 50 determined that the cells lost motility at 28°. Cells maintained at this temperature would recover motility in about an hour. If cells were allowed to undergo a gradual increase in temperature (approximately 2°/30 min.j, they could be raised to 34° before motility ceased. Cells maintained at 34° for twelve hours never recovered motility. Cells returned immediately to 20° after being raised to 34° likewi.M- never recovered. The scanned and measured series was conducted by placing a single culture in a temperature-controllable water bath. This bath could maintain a given tempera- ture within ±0.5° C. The slides used were placed in the bath in a dry, covered container. Samples were scanned at two-degree intervals from 20° C. to 34° C. Each two-degree increase was accomplished slowly over a period of not less than 30 minutes. The relationship of linear velocity to temperature in Gonyaulax is shown in Figure 5. Gyrodinium Strictly parallel experiments with Gyrodinium provided the data in the curve shown in Figure 6. DISCUSSION As Ryther (1955) has pointed out, independent movement may be of value to dinoflagellates in competition, under conditions of changing temperature, salinity, nutrient concentration and light, particularly in forms with a phototactically- controlled diurnal migration such as Gonyaulax polyedra (vide Hasle, 1950; Halldal, 1958). We have investigated the effects of variation in the first two parameters on the swimming speed of the single cell in two species. Simple calculation reveals that the demonstrated plateau for swimming rate (Fig. 3) in Gonyaula.r of approximately 250 /A/sec. (0.9 M./hr.) is well within the order of magnitude necessary to allow a single cell to accomplish the diurnal migration estab- lished by Hasle for Gonyaula.r in a Norwegian fjord (approximately 10 M./12hrs.). The littoral Gonyaulax and the lagoon-inhabiting Gyrodinium were both found to maintain a relatively constant plateau of velocity through a wide range of salinities. Gonyaulax maintained a velocity plateau above 225 /x/sec. from 120( culture medium (34 '/« ) through 70% culture medium (20 %o), while Gyrodinium maintained a similar plateau above 250 /x/sec. from 130% culture medium (36 %o) to 20^0 culture medium (5 %c). It is clear that an adaptation of this sort to variations in salinity is a definite asset to life in estuarine and coastal waters ; the wider tolerance in the lagoon-inhabiting Gyrodinium is to be expected. The ability of the two organisms to maintain swimming speeds within the range of their maxima means that salinity per sc is not an important limiting factor in the distribution of cells throughout their environment, unless it can be shown that a 100 \Y. (i. HAND, P. A. COU.AKI) AND M. DAVENPORT change in direction or a change in rate-of-change of direction (klinokinetic response) occurs when the cell encounters a steep gradient. The former possibility cannot he investigated without a change in our technique. A hriet analysis of the ratio of distance travelled over linear displacement ( Surtees, I'M), to give evidence of a change in klinokinetic behavior with change in salinity, indicated no difference in the deviosity of pathways in both forms within the salinity ranges in which a constant plateau of velocity was maintained. But it must be remembered that our technique in no way parallels what might occur when a cell encounters a steep gradient, because our pathways were- only recorded from cells which had had considerable time (four hours) to become adapted to the changed medium. The apparent difference in both species in swimming speed and salinity tolerance (at low salinities) when in culture medium and when in sea water war- rants brief consideration. Culture studies indicate that dinoflagellates can also reproduce normally under a considerable salinity and temperature range (Braarud, 11'51; Barker. 1935) but that an essential factor for their development is the presence of certain organic chemical constituents. In our media these are supplied by soil extract. Soil extract can be supplemented or replaced by vitamin B,._, (Provasoli and I'intner. 1953; Sweeney. 1955). In our transfer of cells from culture medium to sea water, these essential factors may have been removed from the environment, which possibly may account for the observed differences in the range of tolerance and swimming speed in our studies. In both species velocity was similarly maintained through a wide temperature range, Gvrudiniuni again having the widest tolerance, as one might expect in a species of its habit. What is more interesting is the response of the two species I" a rapid temperature decline. The encounter of such a phenomenon under natural conditions could result in marked effects on the distribution of cells in the surrounding medium, particularly in forms which undergo a marked vertical migra- tion such as Gon\'(nila.\-. It is possible that cells encountering a steep drop in temperature may become inactivated and sink to a different level. They may thus become concentrated by the action of thermoclines. Many workers (Braarud. personal communication) have reported great concentrations of dinoflagellates within an area of 3-4 meters, where in the same region the non-motile diatoms were randomly distributed. [. S. Kittredge (personal communication), working at Scripps Institution of Oceanography, has shown that "microthermocliues are nor- mally present throughout the summer in local (Southern California) near-shore waters. They vary in magnitude from a few hundredths of a degree to several tenths of a degree and. exceptionally, exceed a degree or two. Usually they are quite sharp, with a gradient of 0.1° to 1.0°/cm. depth. They vary in depth from a few centimeters to 10 M. (the greatest depth investigated). There may be several microthermoclines present in any record." Clearly it is imperative to combine the results of such valuable microphysical occanographic studies with observations on the behavior of the individual motile phytoplankter when it encounters a steep salinity gradient or thermocline. We know nothing about the behavior of the single cell under these conditions. \Ye do not even know whether as a discrete and independently motile unit such a cell ever crosses an interface under natural conditions. We have not as yet directed our attention to the technical problem of "facing" the single cell with such a condition. SWIMMING RATK IX GONYAULAX AND GYRODIXIUM 101 \Ye believe that when we have done this and investigated the correlation of such behavior with responses effected by changes in light intensity and wave length, we may be better able to understand the so-frequently reported "spotty" distribution of these phytoplankters. SUMMARY 1. Apparatus is described with which it is possible to make rapid measurements of linear velocity and rate of change of direction in the motile microorganism. 2. Observations on the relation between linear velocity and salinity have been made in the dinoflagellates, Gonyaulax and Gyrodinium. 3. Data are also presented on the relation of linear velocity to temperature in the two forms. 4. Results are discussed in relation to the ecology of the organisms. LITERATURE CITED ALLEN, W. E., 1946. Red water in La Jolla Bay in 1945. Trans. Amcr. Micr. Soc.. 65: 149-153. BARKER, H. A., 1935. The culture and physiology of marine dinoflagellates. Arch. f. Mikrobiol.. 6: 157-181. BONNOT, P., AND J. B. PHILLIPS, 1938. Red water, its causes and occurrences. Calif. Fish and Game. 24: 55-59. BRAARUD, T., 1951. Salinity as an ecological factor in marine phytoplankton. Physiol. Plant.. 4: 28-34. COXXELL, C. H., AXD J. B. CROSS, 1950. Mass mortality of fish associated with the protozoan Gonyaula.r in the Gulf of Mexico. Science, 112: 359-363. DAVEXPORT, D., C. WRIGHT AXD D. CAUSLEY, 1962. Technique for the study of the hehavior of motile microorganisms. Science. 135: 1059-1060. DRYL, S., 1961. The velocity of forward movement of Parainecinin caiuiufuin in relation to pH of medium. Bull, de L'Acad. Polonaise des Sci., 9: 71-74. HALLDAL, P., 1958. Action spectra of phototaxis and related problems in Volvocales, Ulva gametes, and Dinophyceae. Physiol. Plant., 11: 118-153. HASLE, G. R., 1950. Phototactic vertical migration in marine dinoflagellates. Oikos. 2: 162-175. HASLE, G. R., 1954. More on the phototactic diurnal migration of marine dinoflagellates. Nytt. Mag. F. Bot.. 2: 139-146. HAXO, F. T., AXD B. M. SWEENEY, 1955. Bioluminescence in Gonyaula.r polycdra. The Luminescence of Biological Systems. A.A.A.S. Washington. D. C. PROVASOLI, L., AXD I. J. PIXTNER, 1953. Ecological implications of in vitro nutritional requirements of algal flagellates. Ann. N. Y. Acad. Sci., 56: 839-851. ROTHSCHILD, LORD, 1953. A new method of measuring sperm speeds. Nature, 171: 512-513. RYTHER, J. H., 1955. Ecology of autotrophic marine dinoflagellates with respect to red water conditions. Luminescence of Biological Systems, A.A.A.S. Washington, D. C. SURTEES, G., 1964. Laboratory studies in dispersion behavior of adult beetles in grain. VIII. Spontaneous activity in three species and a new approach to the analysis of kinesis mechanisms. Animal Behavior, 12: 374—377. SWEEXEY, B. M.. 1951. Culture of the marine dinoflagellate Gymnodinium with soil extract. Amer. J. Bot., 38: 669-677. SWEEXEY, B. M., 1955. A marine dinoflagellate Gymnodinium splendens requiring vitamin Bu>. Amer. J. Bot., 41 : 821-824. VAX DUIJN, C., AXD R. RIKMENSPOEL, 1960. The mean velocity and velocity distributions of normal bull spermatozoa at different hydrogen ion concentrations, derived from photo- electric measurements. /. Agr. Sci., 54: 300-308. WIXGO, W. J., AXD J. BROWNING, 1951. Measurements of swimming speed of Tetrahymena geleii by stroboscopic photomicrography. /. E.vp. ZooL. 117: 441-445. STfDIES ON HOLOTHURIAN COELOMOCYTES. I!. THE ORIGIN OF COELOMOCYTES AND THE FORMATION OF BRO\YX BODIES HOWARD K. HKTZEL1 Department />f Zoolo!' I'.iolojiical Srirnrrx Illinois Stair I'niviTsily, Normal, I llinois. 102 HOLOTHURIAN COELOMOCYTES 1<>3 (PAS) technique, the Alcian blue method, and the ribonucleic acid extraction tech- nique, as outlined in Pearse (1960) on page 832, 838, and 916, respectively, were used. Some smears were stained with the mercuric chloride bromphenol blue technique of Mazia, Brewster and Alfert (1953 ), or with hematoxylin and eosin. Both experimental and control animals were sacrificed on the eighth day. Samples of the body wall and all major organs were fixed with Bourn's fixative, dehydrated in ethyl alcohol, embedded in paraffin, and sectioned at 10 microns. Sections were stained with hematoxylin and eosin. All sections and smears were searched for sites of coelomocyte production. Brown body formation was studied by means of injections of a 2% (W/V) suspension of lamp black (carbon) in filtered sea water into the perivisceral coelom of holothurians. Thirty specimens of Citcitinaria niiniata were injected with 2 to 4 ml. of the lamp black suspension, depending on the size of the organism ; five specimens of Eiipentacta quinquesemita and five of Psolns chitonoides were each injected with 1 ml. of the suspension. Each of five specimens of Leptosynapta chirki was injected with 0.5 ml. The injected animals were placed in individual fingerbowls or aquaria filled with sea water. One-mi, samples of coelomic fluid were withdrawn for examination after 30 minutes, 60 minutes, 2, 3, and 8 hours, and, thereafter, once daily for 30 days. One Cnciiuiaria was sacrificed every other day for a period of one month and after that at irregular intervals for up to six months. One Psolns was sacrificed each wreek for four weeks. The animals were dissected and searched for sites of carbon deposition. The entire digestive systems and respiratory trees were removed, and bleached and hardened in 95% ethyl alcohol for 12 hours before they were dissected and their contents and walls examined for the presence of black carbon deposits. No specimens of Enpcntacta or Leptosynapta were sacrificed during the course of the experiment. RESULTS No coelomocyte free in the coelomic fluid of experimental or control animals was ever observed in any stage of cell division. Samples of coelomic fluid taken from experimental animals showed no obvious differences from samples taken from control animals. Among the coelomocytes present in the samples taken from control and experimental animals were cells which could be arranged in graded series between lymphocytes and hemocytes, and between lymphocytes and amebocytes. I. Series of cells intergrading between lymphocytes and hemocytes. 1. Larger than average oval to spherical lymphocytes, with an increased number of Janus green B-positive granules. 2. Cells as described above with one or more of the Janus green B-positive granules containing a clear vacuole. 3. Still larger lymphocyte-like cells, similar to those of type 2 above, but with hyaline cytoplasm tinged straw-yellow. 4. Cells slightly smaller than hemocytes and with straw-yellow cytoplasm, but without the typical yellow refractile granules of hemocytes. 104 HOWARD R. HKTZKI. l;K,ri 1. Unusually large lymphocytes with more than three filiform pseudopndia. 2. Larger cells with still more complicated pseudopodial systems and increased granulation. 3. Cells that approach amebocytes both in size and in degree of cytoplasmic granulation. Cells belonging to Series II were found in all four species of holothurians examined. No histological differences were noted between tissues taken from experimental animals and tissues taken from control animals. I found no evidence of the origin of coelomocytes from the peritoneum covering the inner surfaces of the longitudinal retractor muscles. No observations suggested that coelomocytes originate from the walls of the respiratory trees, nor from the body wall, the wall of the digestive tract proper, the polian vesicles, the water vascular system in general, nor from any part of the reproductive system. Sections of the hemal vessels showed numerous lymplmcvtes within the lumina of the vessels (Fig. 1 ). The connective tissue layers of the hemal vessels and their associated mesenteries contained numerous mesenchymal cells with flattened, in- tensely staining nuclei. Mesenchymal cells located nearer the lumen of the hemal vessels contained oval, less intensely staining nuclei. Lymphocyte-like cells with oval nuclei and hyaline cytoplasm bulged from the connective tissue into the lumen of the hemal vessels (Fig. 2). Many of the latter cells formed a series exhibiting increasing size and granulation. These granulated cells can be arranged in a series intergrading with basophilic morula-like cells contained in the connective tissue layer of the mesenteries associated with the hemal vessels and the digestive tract (Fig. 3). III. Series of cells intergrading between lymphocytes in the hemal vessels and baso- philic morula-like cells. 1. Enlarged lymphocytes with homogeneous eosinophilic cytoplasm. 2. Enlarged cells as above, with finely granular eosinophilic cytoplasm ; these cells were also observed in the connective tissue of the mesenteries associated with the hemal vessels. 3. Cells with amphiphilic staining properties and with granular cytoplasm located in the mesenteric connective tissue. 4. Amphiphilic cells in the connective tissue of the mesenteries with morpho- logical features identical to those of morula cells. 5. Basophilic morula cells contained in the connective tissue layer of the mesenteries and free in the coelomic fluid. FIGURE 2. Cross-section of hemal vessel of Psolns chitonoidcs (1292X). Arrows indicate oval nuclei of mesenchymal cells believed to be differentiating into lymphocytes. C, connective tissue layer ; L, lumen of vessel ; h, lymphocyte with hyaline cytoplasm. FIGURE 3. Section of dorsal mesentery of Psolns chitonoidcs (970 X). Arrows indicate basophilic morula cells. 106 HOWARD K. HKT/KI. Numerous eosinophilic morula cells were present throughout the connective tissues of tin- hotly other than the connective tissues of the gut, henial vessels, and mesenteries adjacent to the heinal vessels and gut. Spherules of niorula cells fixed and stained at the moment of their release from cvtolyzing cells were composed of an outer hasophilic and metachromatic shell, and an inner core which was eosinophilic, hut not metachromatic. The outer shell was I1. \S-positive and stained green with Alcian hlue; the inner core stained blue when exposed to Maxia's hromphenol hlue techniijue. Morula cells lost their basophilia following treatment with rihonuclease. The injection of lamp black suspension into the coelomic cavity of holothurians apparently had little effect on the animals. Holothurians left undisturbed following the injection were observed to feed normally after one hour. Injected specimens were still normal and healthy six months after the injection. Samples of the coelomic fluid withdrawn 30 minutes following the injection contained very few coelomocytes when compared to the number normally present. A sample taken one hour after the injection contained many more cells than did the previous sample, but the number was still clearly below normal. A few petaloid amebocytes contained engulfed carbon particles at this time, but these represented only a small per cent of the amebocytes present in the sample. In Cncniuaria, carbon particles were occasionally observed within hemocytes. In many instances several amebocytes were observed clustered around masses of carbon particles. The number of amebocytes containing engulfed carbon particles gradually increased up to the tenth to the fourteenth day following injection. By that time, almost every amebocyte contained carbon particles. Lymphocytes, morula cells, and crystal cells were never observed containing engulfed carbon. Between the third and fourth weeks following injection the number of amebo- cytes containing carbon particles gradually decreased, and the number of amebocytes free of carbon particles increased. After six months an occasional amebocyte still contained particles of engulfed carbon. Amebocytes heavily laden with engulfed carbon particles did not produce large networks of filiform pseudopodia and. there- fore, did not contribute actively to the formation of clots as did amebocytes Iree of carbon. Most amebocytes with engulfed carbon particles also contained yellow granular material similar to the granular material composing brown bodies. Many spherical cells, completely filled with yellow granules and/or carbon particles, were observed. Large masses of carbon were still found in the perivisceral coelomic cavity of Cucinuaria after six months. As early as two days following the injection, notice- able accumulations of carbon were observed in the anterior portions of the water vascular system, especially in the polian vesicle, and in the ampullae of the anterior tube feet. Large masses of carbon were also located among the suspensor muscles of the cloaca, and along the junction of the dorsal mesentery and the gut. Carbon was accumulated in the same locations in /'solus chitonoidcs. After two weeks, carbon was also noted among the deposits of yellow granular material between the peritoneum and the wall of the sole of ['solus. The walls of the excised gut and respiratory trees of Cncuniaria and /'solus were searched for carbon deposits. Specks of carbon were occasionally found in the anterior and mid-gut regions two days following the injection, but no carbon was HOLOTHURIAN COELOMOCYTES 107 detected in the lumina or walls of the respiratory trees. Occasionally, small amounts of carbon were found in the feces of Cucumaria and I:npcntacta. After the second day. brown bodies composed partly of carbon were found in the aquaria containing injected Citcnmaria and Eupentacta. Masses of amebocytes laden with carbon emerged from the bases of the tube feet of Citciuuaria. Several specimens of Cucumaria were covered with carbon-laden mucus. After a period of three weeks the noticeable emission of carbon from the holothurians decreased and finalh stopped. Following the injection of lamp black, the normally clear coelomic fluid visible through the body wall of Leptosynapta appeared cloudy. Within two hours, how- ever, the coelomic fluid was again clear and the carbon was concentrated in the ciliated funnels that line the junction of the mesenteries and the body wall of the animal. The elimination of the carbon deposits from the ciliated urns of Leptosynapta occurred more rapidly than the elimination of carbon from bodies of the other holothurians studied. Within three days most of the carbon was gone from the ciliated funnels of Leptosynapta. Masses of carbon-laden amebocytes migrated from the ciliated urns through the body wall of Leptosynapta to the exterior. DISCUSSION On the basis of observations made in the present study, one is led to support Ohuye's (1938) conclusion that the various coelomocyte types are possibly derived from a common source or stock of stem cells, and that all types of coelomocytes may be produced by direct transformation of the stem cells. Lymphocytes are probably the stem -cells which differentiate into hemocytes, amebocytes, and morula cells. The source of crystal-bearing eoelomocytes is unknown at the present time, but Theel (1920) believed that they differentiated from amebocytes. The cells in Series I may be interpreted as lymphocytes undergoing differentiation into hemocytes. This view is further supported by the finding that these inter- mediate stages are found in Cucumaria and Eupentacta which possess hemocytes, but not in P arastjfkopus and Psolns which lack hemocytes (Hetzel, 1963). The course of differentiation is presumed to be the following : lymphocytes free in the coelomic fluid enlarge and produce increased numbers of Janus green-stainable granules ; as hemoglobin synthesis occurs, the cytoplasm becomes straw-yellow in color and the large yellow refractile granules typical of holothurian hemocytes appear; the refractile granules appear to be derived from the Janus green-positive cytoplasmic granules. Cells in Series If may be interpreted as lymphocytes differentiating into amebocytes. Lymphocytes are presumed to increase in size and produce more complex systems of pseudopodia as they differentiate into amebocytes. No evidence was found in the present study to indicate that morula cells dif- ferentiated from lymphocytes free in the coelomic fluid as Endean (1958) proposed. Observations indicate thatUymphocytes in the hemal vessels and closely associated gut mesenteries may form) morula cells. The course of this differentiation is be- lieved to be as follows : the hyaline cytoplasm of these lymphocytes gradually becomes granular; the granules gradually increase in size and are transformed 108 1 1 (WARD K. I1KTZEL into the spherules typical of monilu cells; during the transformation of the granules into >pherulcs. the cytoplasm of the lymphocytes changes from eosinophilic to amphiphilic to the hasophilic condition typical of morula cells located in the con- nective tissue framework of the mesenteries and morula cells free in the coelomic fluid. The haso|)hilia of morula cells was removed hy ribonuclease digestion, and the cores of the .spherules of the morula cells appear to he protein, as indicated by their p<»itive staining reaction to Mazia's bromphenol blue technique. These observa- tions indicate that morula cells may play an important role in amino acid uptake from the digestive tract and in protein synthesis and storage. If the morula cells indeed do function in amino acid uptake and in protein synthesis, as available evidence suggests, their possible .site of origin in the hemal vessels, which are so closely related to the digestive tract, would be especially significant. Eosinophilic morula cells found throughout the connective tissue layers of holothurians possibly represent morula cells that have completed their synthetic activities and await an as yet undescrihed function. Endean (1^5S) reported that spherules of morula cells of Holothuria Icucospilota also consisted of an inner proteinaceous core and an outer metachro- matic shell believed to be, at least in part, mncopolysaccharide. The present study confirms his observations of the structure of the spherules of morula cells, and supports Endean's theory that they differentiate from lymphocytes. In species studied in the present report, however, morula cells appear to differentiate in close association with the hemal vessels, and not in the perivisceral coelom, as Endean described. None of the observations made in the present study contribute to an understand- ing of the formation of crystal cells in holothurians. The crystals are not able to withstand the effects of fixation and preparation of tissue sections. Possible stages in the formation of crystal cells were likely destroyed in the prepared tissues, and only fully differentiated crystal cells were observed in living material. Theel ( 1920 ) claimed that crystal cells were derived from ameboid cells. The fact that crvstal cells, hemocytes, and morula cells are able to produce pseudopodia of some form (1 let/el. 19n.Si lends credibility to the hypothesis that these cells mav all be derived from a common form that also gives rise to amebocvtes. The site of origin of lymphocytes proved to be a problem not easily resolved, llausmann ( 1(>31 ) maintained they originate from cells of the peritoneal lining of the coelom. This mav be so. but would he difficult to prove conclusively. Ohuye i 1'MS) does not rule out the possibility that mesenchymal connective tissue cells may contribute to the formation of l\mphoc\tes in holothurians. Observations made in the present studv indicate that at least some of the lympho- cytes mav differentiate from mesenchymal connective tissue cells in the walls of the hemal vessels. | believe that Kmphocvtes more likelv are derived from mesenchyme than from epithelial cells such as those that make up the peritoneum. Mv observations suggest that lymphocyte-like cells in the hemal vessels possibly differentiate into morula cells. Other lymphocytes conceivably could migrate from their site of origin into the coelomic cavities, and there' differentiate into hemocytes and amebocvtes. HOLOTHUR1AX COELOMOCYTES 109 Schinke (1950) studied the origin and differentiation of coelotn. ; of the echinoid, Psammechinus iniliaris. Schinke reported that white ameboc.) mpho- cytes?) were formed by direct transformation of cells in the connective tissue matrix of the calcareous tests. Schinke stated that the formation of the coelomocytes never involved mitosis, nor did the coelomocytes themselves ever undergo cell division. Holothurian coelomocytes have not been observed in cell division. In light of Schinke's study, and the observations made in the present investiga- tion, it seems reasonable to assume that lymphocytes are produced from mesen- chymal connective tissue. The observations of Hatanaka (1939) of cells in the hemal vessels of holothurians also support this hypothesis. Cuenot ( 1897) defined a lymphoid organ in invertebrate animals as an organ closely related to the blood cells and coelomocytes, and one that is composed of amoeboid cells or produces them. Cuenot described a lymphoid organ as a con- nective tissue framework in which are found many corpuscles identical to those that occur free in the body fluid. On the basis of Cuenot's definition, and on suggestions proposed in the present study, the hemal system of holothurians may be regarded as lymphoid tissue that functions in part to produce cells from which the coelomo- cytes are derived. Cuenot, himself, considered the hemal ring of holothurians as a lymphoid organ. The rete mirabile, an extension of the hemal system, may also function in part in the formation of coelomocytes. On the basis of a survey of the coelomocytes of various invertebrate animals, in- cluding two species of holothurians, Ohuye (1938) derived lymphocytes from mesenchymaU and/or peritoneal cells. Ohuye derived erythrocytes (hemocytes), amphiphilic, ''edsinophilic, and basophilic granulocytes (morula cells), and pig- mented leucocytes from lymphocytes. The existence of holothurian coelomocytes possessing characteristics intermedi- ate between lymphocytes and hemocytes, morula cells, and amebocytes, lends support to Ohuye's views of coelomocyte lineages in holothurians. Amebocytes appear to have at least two functions in holothurians, as phagocytes and as active agents in the clotting reaction. These functions appear to compete against one another; two weeks after the injection of carbon- into Cucumaria, when the amebocytes were at>peak activity in disposing of injected carbon, the efficiency of the clotting mechanism was low. Later, when more amebocytes were again free from the chore of carbon elimination, the efficiency of the clotting reaction returned. The inability of carbon-laden amebocytes actively to contribute to the formation of a clot may easily be explained. As the amebocytes became filled with carbon particles, they became less ameboid and were eventually transformed into spherical cells laden with carbon; some also contained yellow granular material. Amebocytes partially filled with carbon granules were impaired in their ability to produce the extensive networks of filiform pseudopodia necessary for clot formation. Observations of hemocytes containing small quantities of engulfed carbon were especially interesting. This is the first report of such an observation, weakly phagocytic nature of hemocytes could indicate some relationship between hemocytes and amebocytes and lends further support to Ohuye'f scheme of coelomocyte lineages. ' The inclusion of carbon within brown bodies supports the hypothesis that brown 110 HOWARD K. IIKTZKL lutdy material must be considered a product, it" not truly excretory, at least of no to the holothurian. The fact that carbon is engulfed by amebocytes that often contain yellow granules, and the fact that the carbon-laden cells are accumulated in the same region of the body where masses of yellow granules were found, would seem to indicate that the injected carbon particles and yellow granular material are both disposed of by phagocytic amebocytes. The additional fact that yellow granules and carbon appear together in brown bodies would also indicate that brown bodies are indeed a material that is actively accumulated by amebocytes and eliminated by the holothurian. The presence of brown bodies in holothurians which both possess and lack hemocytes would seem to indicate that these granules are not necessarily related to hemoglobin synthesis or degradation. The lack of carbon in any of the morula cells or crystal cells would seem to indicate that these cells are very specialized and have lost all powers of phagocytosis. One other speculation may he made regarding the phagocytic nature of amebo- cytes and their role in removing unwanted materials from the body of holothurians. Hetzel (1963) reported amebocytes and brown bodies containing one or more hemocytes. morula cells, or crystal cells. These observations suggest that the elimination of old or damaged coelomocytes from the bodies of holothurians might be performed in a manner similar to the elimination of carbon or yellow granular material. If this were true, the constant loss or elimination of these coelomocytes in brown body material would require the continued replacement of these coelomo- cytes and, therefore, the continued production of new coelomocytes throughout the life of the holothurian. Sr\i MARY 1. Coelomocyte and brown body production were studied in four species of holothurians. Ciicitiinirni niininta. /iitpoitacta quinquesemita, Parastichopus cali- fornicus. and Psohis chitonoides. 2. Available evidence suggests that at least some, if not all. lymphocytes possibly originate from mesenchymal cells in the hemal vessels of holothurians. and possibly later differentiate directly into hemocytes. amebocytes. and morula cells. 3. The coelomic fluid of Cnciunaria and liupcntacta contains coelomocytes which can be arranged in a series intergrading between lymphocytes and hemocytes. These cells possibly represent stages in the transformation of lymphocytes into hemocytes. 4. Parastichopus and /'solus, which lack hemocytes. do not possess the series of cells interpreted as stages in hemocyte differentiation. 5. All four species of holothurians studied possess coelomocytes which can be arranged in a series intergrading between lymphocytes and amebocytes. These cells possibly represent stages in the differentiation of lymphocytes into amebocytes. 6. (iranular lymphocyte-like cells located in the hemal vessels of the four species studied possiblv represent stages in the differentiation ot lymphocytes into morula cells. 7. Ciicnnidi-id ininidtd, liupcntdctd quinquesemita, I 'solus chitonoides, and Leptosynapta clurki were injected with suspensions ot lamp black in sea water. 8. The carbon particles were engulfed by amebocytes which often also contained grannies similar to the granules found in brown bodies. HOLOTHURIAN COELOMOCYTES 1 1 1 9. Carbon particles were accumulated in the same regions of the body as brown body material. 10. Brown bodies composed partly of carbon particles were eliminate^ i by the holothurians. 11. Amebocytes are believed to play a role in the accumulation of brown body material and in the production of brown bodies. 12. Carbon particles were also concentrated in the ciliated funnels or urns of Lcptosynapta clarki. LITERATURE CITED CUENOT, L., 1897. Les globules sanguins et les organes lymphoides des invertebres. Arch. d' Anat. Microsc., 1: 153-192. ENDEAN, R., 1958. The coelomocytes of Holothnria Icncospilota. Oiuirt. J. Micr. Sci., 99: 47-60. HATANAKA, M., 1939. A study of the caudate holothurian, Molpadia rorctzii (v. Marenzeller). Sci. Rcpts. Tohoku Univ., so: 4, Biol., 14: 155-190. HAUSMANN, M., 1931. Entstehung und Funktion von Gefassystem und Blut auf Zellular- physiologischer Grundlage. Actu Znol., 12: 265-483. HEROUARD, E., 1889. Recherches sur les holothuries des cotes de France. Arch, dc Zool. E.vp. Gen., 2e sir., 7 : 535-704. HETZEL, H., 1963. Studies on holothurian coelomocytes. I. A survey of coelomocyte types. Biol. Bull., 125:289-301. MAZIA, D., P. A. BREWSTER AND M. ALFERT, 1953. The cytochemical staining and measure- ment of protein with mercuric bromphenol blue. Biol. Bull.. 104: 57-67. OHUYE, T., 1938. On the coelomic corpuscles in the body fluid of some invertebrates XII. General considerations on the results obtained by the preceding investigations. Sci. Rcpts. Tohoku Univ., scr. 4, Biol., 13: 359-379. PEARSE, A., 1960. Histochemistry Theoretical and Applied. Little, Brown and Company, Boston. PROSSER, C. L., AND C. JUDSON, 1952. Pharmacology of the hemal vessels of Stichopus calif ornicus. Biol. Bull., 102: 249-251. SCHINKE, H., 1950. Bildung und Ersatz der Zellelemente der Leibesholenflussigkeit von Psammcchimis miliaris (Echinoidea). Zcitschi: Zcllforsch. mikr. Anut.. 35: 311-331. THEEL, H., 1920. On amoebocytes and other coelomic corpuscles in the perivisceral cavity of echinoderms. III. Holothurids. Arkiv. f. Zool., 13: 1-40. DESCRIPTION OF HEMOCYTES AND THE COAGULATION- PROCESS IN THE BOLL WEEVIL, ANTHONOMUS GRAXDIS BOHEMAX ( CT'RCULIONIDAE)1 ROY E. McLAUGHLIX- AM) (1EORGE ALLEX :: Hull ll'ce-i'il Research Luhnnitory, State (.'nllci/c. Miss. This report presents the results of a study of the hemocytes of the boll weevil, Antliononnts (/rnndis Boheman. A knowledge of the hemocytes of healthy boll weevils was considered necessary before detailed study of the effect of various pathological conditions upon hemocytes could be conducted. Wittig (1962), in reviewing the pathology of insect blood cells, observed that diseases may affect hemocytes directly or indirectly. Knowledge of these effects may be useful not only in studying a microbial disease which attacks the hemocytes. but also as an aid to bioassaying a population for pathological conditions. Knowledge of the hemocytes of healthy insects is necessary for such comparisons. TERMINOLOGY Classification of hemocytes provides a means of reference for further studies concerning their function, changes due to normal physiological processes or to abnormal conditions, and for comparison with hemocytes in other species. Classi- fication systems have classically depended upon morphological characters. As \Viggle.s\vorth (1('5(^> stated, determination of function should be most important. Nevertheless, recognition of hemocyte morphology is necessary before detailed functional studies can be conducted. Classification on a purely functional basis would result in much confusion, since a distinct cell type often has more than one function. Moreover, hemocytes of dissimilar morphology from different species have similar functions. Accordingly, classification of boll weevil hemocytes has been established on the basis of morphological similarities, following to a large extent the system used b\ [ones ( lctt»2i. When a distinct hemocyte type was observed to transform in I'itro, the variations in form were described: but these were not considered to constitute a distinct type of hemocyte, especially since in ritro changes may not have been a true representation of in 7-/7v> activity. This factor was further emphasi/ed by the conditions which created artifacts during the investigation. The types of hemocytes present in the boll weevil correspond to previously recognized hemocyte groups of other insects. We feel that this system of nomen- clature mav serve as a basis for common usage, by insect pathologists at least. 1 In cooperation \\itli Mississippi Agricultural Experiment Station. Accepted for pub- lication October X, 1964. -Entomology Research Division, Agricultural Research Service, U.S.D.A., State College, Mississippi. 3 Present address : Department of Entomology, Mississippi State I'liiversity, State College, HEMOCYTES AND COAGULATION IN A WEEVIL 113 until further data regarding origin and function warrant a clmn. Each insect species apparently has its own characteristic hemocyte picture. Basic hernocyte types exist, but morphological adaptations to varying physiological requirements and conditions occur. The reader is referred to the excellent reviews In \Vigglcs\vorth (1959) and Jones (1962) for a more extensive coverage. The literature which served as a basis for classification is reviewed briefly in the following paragraphs to indicate the reasons for usage of the terms and to relate the terms to those which have been considered synonyms. Prohemocytes : Prohemocytes are generally characterized by having a small amount of cytoplasm, the nucleus comprising the greater amount of the cell volume. These cells develop into other types which presumably are then capable of perform- ing functions of mature hemocytes. The term "prohemocytes" was used by [ones (1950) and Arnold (1952). Jones (1959) stated that these are the same a> Yeager's (1945) proleucocytes. The term "prohemocyte" is proposed as the standard. Plasmatocytes: Hemocytes usually characterized by their pleomorphic capability and varied functions, as well as being the most abundant in the host, have been termed "plasmatocyte" by Yeager (1945). Yeager recognized a large number of cell types which differed mainly in their morphology. Arnold (1952) used the term in a report in which he included many of Yeager's types under that term. Jones (1959) retained Yeager's classification of plasmatocyte but also retained "podocytes" and "vermiform cells." Jones (personal communication) reported that podocytes or vermiform cells had not been observed to alter their form in t'ltro. The term "plasmatocyte" was adopted in this study to designate the hemo- cytes which were pleomorphic, exhibited cytoplasmic variations, were capable of altering their shape in vitro and were obtained in corresponding shapes in fresh preparations of blood, were capable of phagocytosis and took part in a coagulation process. Until further study can determine separate, distinct cell types for these functions, it is proposed that this term be used to designate such hemocytes. . Idipohemocytes : Hemocytes with lipoid or other refractive inclusions were termed "spheroidocytes" by Yeager (1945) and Jones (1959). Later, Jones (1962) preferred the term "adipohetnocytes," since "spheroidocytes" could be con- fused with a distinctly different type— -"spherule cells." The term "adipohemocyte" is proposed as the standard. The reader is referred to this review for further discussion of adipohetnocytes. Sp/ienile cells: Yeager (1945) described "eruptive cells." which Jones (1959) regarded as "spherule cells." Jones (1962) further reviewed the usage of this term. These cells contain one to several large, distinct, non-refringent. amorphous inclusions which often distend the cell membrane. These inclusions are often expelled into the hemolymph in ritro but are not associated with coagulation. They are a distinct and easily recognized type of cell. The term "spherule cell" is preferred because it adequately describes the distinctive appearance of the cell and does not relate to an activity which may or may not be an artifact, but which certainly can be affected bv conditions attendant to the technique employed to study the cells. Oenocytoids: Yeager (1945) described oenocyte-like cells free in the hemo- lymph. Arnold (1952) used the term oenocytoids. These have a small nucleus, a 114 ROY !•:. MCLAUGHLIN AND <;KOK<;K ALLEN compact cytoplasm which is usually opaque, arc lightly basophilic or eosinophilic and frequently contain a small number of granules or crvstals. The cell membrane may In- lightly creased, have elaborate canaliculi, or he complexly folded ( Hollande, 1'Ml; Jones, ]()5(); Yeager. 11H5). Oenocytoids are distinct from the non- circulating specialized cells known as oeiiocytcs which are enormous acidophilic cells of ectodermal origin, usually segmentally arranged in the abdomen. The term "oenocytoids" is ]ireferre(l to "oenocyte-like" cells for these reasons. MATERIALS AND METHODS Larvae were used for the initial descriptive work and an attempt was made to select those of uniform size and phvsiological condition to eliminate possible variations due to growth or developmental stages. Later, hemocytes were examined from all larval stages and from adults. Larvae were utilized soon after collection from local cottontields, to reduce artifacts which might have resulted from abnormal laboratory conditions for the insects. Larvae were placed ventral side up in model- ling clay and fastened securely. A sealed #30 hypodermic needle was used to make a puncture at the midventral line, which was free of adipose tissue formation. The- primarv method of observation was of wet mounts with a Leitz Ortholux 4 microscope by phase contrast, bright-field or ultraviolet illumination. A Metrimpex (Budapest) 3-D Condenser1 ( Xational Instrument Laboratories, Inc., 12300 Parklawn Drive, Rockville, Maryland, U. S. A.) was also used. Fixed and Gained preparations were utilized for biochemical and comparative purposes. Two methods were used for obtaining hemolvmph and placing it on a coverslip. The direct method consisted of touching a coverslip to the exuding hemolvmph. The capillarv method involved collection in a capillary tube made from a 1-nim. capillarv tube linelv drawn and fire-polished. The hemolymph was expelled onto a coverslip. Approximately 1 to 3 f»\. of hemolvmph were easily obtained from larvae. The coverslip was then inverted over a slide and .supported by a thin ring of white petroleum jelly so that a deep layer of hemolymph, not compressed by the weight of the coverslip, was formed. A similar preparation was obtained by use of a Leitz culture trough slide ( 1 M K )LI ) >.' Drying of the hemolymph was negligible and often a preparation remained satisfactory for 12 to 17 hours. Some preparations were diluted about 1:1. Dilution of hemolymph was accom- plished prior to ] dacing the cover-dip over the slide. The diluting solutions were: water, pi 1 7.3; O.S.V, X'aCl solution; 1.0' r potassium oxalate solution; distilled water, pi I (}.\ ; 0.5'; trehalose solution in distilled \\ater, final pi I 5.S ; phosphate buffer, pi I (>.4. Fluorescent dvcs were also added. Acridine orange, auramine 0, and rhodamine I! were prepared at 1 10 ; concentration in phosphate buffers of pi I (>.4 and 7.2. The phosphate buffers were prepared bv mixing the1 proper amounts of 0.07 M XaJll't), and KILI'O, solutions. \o autofluorescence occurred. Stained preparations were made by rapidly air-drying the fresh, unfixed liemo- Ivmph. Some preparations were then subjected to 40' /< formaldehyde or absolute 'The use ni' truth- or proprietary names does not necessarily imply tlie endorsement of tbese products by tbe U. S. Department of Agriculture. HEMOCYTES AND COAGULATION IN A WEEVIL 115 methanol fixation for several minutes. The stains used were Sudan 1Y, saturated iodine (Lugol's solution), Delafield's or Heidenhain's iron hematoxylin, fast green FCF, Giemsa, and Wright's blood stain, HEMOCYTE DESCRIPTION Prohemocytes. These were disc-shaped, pale gray, and nearly homogeneous by phase contrast. The nucleus occupied nearly the entire cell and only a thin band of peripheral cytoplasm was present. Cells averaged 7.0 /j. ±1.3 p., range (R) 6.3-9.9 /A, number (N) = : 11 (Plate I, Fig. 1). When stained with acridine orange, the large nucleus and small band of cytoplasm were very prominent. These cells were relatively rare and only in young first instars were they fairly numerous. \ ariations of this cell were observed which formed a complete range of develop- mental stages. The cytoplasm increased in quantity and progressively became more optically dense to phase contrast. A small number of granular inclusions were often present and large granules or vacuoles were occasionally seen in cells near the end of maturation. The final product was indistinguishable from disc-shaped plasmatocytes. Plasmatocytes. Plasmatocytes were characterized mainly by their morpho- logical variability. The cytoplasm was finely granular, dense, and uniform; or it contained various larger (about 1 p.) dark granules, exhibited areas of different density, and displayed small vacuoles or inclusions which were either gray or highly refractive by phase contrast. The nucleus was usually quite large and finely punctate or granulated. Plasmatocytes can become hyaline or highly re- fractive at their periphery. Disc-shaped plasmatocytes (Plate I, Fig. 2) measured 9.7 ju, ±1.9 ju, (R == 6.4-15.3 ^, N -= 75). The same data showed nuclei to measure 5.3/*±l.05/* (R ==3.4-8.5 /*). A common variant form of plasmatocyte is shown in Plate I, Figures 5 and 6. These cells had a granular cytoplasm and sometimes contained small vacuoles. granular inclusions, or dark, gray, small inclusions. They were also seen with one or two highly refractile lipoid globules. These were termed adipoid plasmatocytes. The frequency of cells with these inclusions was higher when free lipoid droplets were present in the hemolymph. Intentional disruption of adipose tissue, by rolling the larvae with pressure or by use of a needle to disrupt the tissue, produced a similar effect. The plasmatocytes were never observed ingesting lipoid droplets. The natural occurrence of free lipoid droplets was observed during the prepupal stage of larval development. Another form was characterized by possession of one to several hyaloplasmic extensions (Plate I, Figs. 4 and 5). When movement of the fluid caused the cell-- to be carried along, they would often temporarily attach to the surface by these extensions. This form was observed to change into a disc-like shape or to attach at one point and elongate into a fusiform, or spindle-shaped, cell. This type \vas highly unstable in vitro. The fusiform cell (Plate I, Fig. 3) was also seen upon immediate examination of a preparation, but was not as frequent us disc-shnped cells or cells with cyto- plasmic extensions. The fusiform cell could also be formed directly from a disc- 116 ROY K. MCLAUGHLIN AXD GEORGE ALLEN 12 13 All preparations \\nv of frt-sli. unfixed, undiluted i Plate I FlGURl 1 Prohemocyte. I'hasi' contrast. 875 X. FiGURE2. Disc-shaped plasmatocyte. I 'hase contrast. 1090 X. IP %l^ HEMOCYTES AND COAGULATION IX A WEEVIL 117 shaped plasmatocyte. A teardrop-shaped cell \vas also formed from disc-shaped plasmatocytes by elongation after attachment to the glass at the other end of the cell (Plate I, Fig. 8). The cytoplasm varied from dense and uniform hut finely granulated to a differentiated cytoplasmic area of slight refractivity by phase contrast and containing small vacuoles, or dark granules. Extreme elongation resulted in a thinner cell in which these contents were more easily observed. The spindle-shaped cell often had tenuous, hyaline, filiform, cytoplasmic extension- from either end which exhibited a searching and probing motion. This activity was fairly slow and not whiplike. The incidence of these cells increased as the preparation remained undisturbed on the microscope stage. Disc-shaped plasmatocytes were also capable of transformation into forms which played a major role in coagulation. They were observed to transform into cells which occasionally were flattened and expanded. The cytoplasm became hyaline at the periphery, accompanied by an extreme flattening of the cell. The cell membrane spread out quite thinly as the cytoplasm expanded onto the glass surface. Granules became very apparent. The edges of the expanded, hyaline cytoplasm ranged from scalloped to slightly pointed extrusions or to extremely long, filiform hyaline extensions, which were frequently very active (Plate I, Figs. 12 and 13). These morphological variations occurred only after the preparation wa> several minutes old. Rapid protoplasmic streaming was observed, with granules being carried along in the internal currents. Many hemocytes engaged in extension, retraction, prob- ing, and anastomosis with similar strands. This process may have originated from the form with several cytoplasmic extensions or the fusiform-shaped cell as well. Further description of this process is given under "Coagulation." in the section following this one. Adipohemocytes. A very distinct hemocyte was often observed, replete with lipoid droplets to the extent that the cell membrane was distended and the cell had a bubbly appearance (Plate I, Figs. 10B and 11). These were classed as adipohemo- cytes as denned by Jones (1962). They measured 10.9 p ±1.1 p.. R = 5.6-17.4 /*. FIGURE 3. Plasmatocytes of various shapes. Phase contrast. 700 X. FIGURE 4. Plasmatocytes with cytoplasmic extensions. Phase contrast. N75 X. FIGURE 5. Adipoid plasmatocyte with pseudopod-like extensions. Phase contrast. 875 X. FIGURE 6. Adipoid plasmatocyte with two lipoid inclusions. Phase contrast. 875 X. FIGURE 7. Small plasmatocyte with Sarcina lit tea packets. Phase contrast. 1050 X. FIGURE 8. Disc-shaped and teardrop-shaped plasmatocytes. 3-D condenser-phase-objectives. 875 X. FIGURE 9. Spherule cells. A. Normal. B. Dead. Hyaline form with eccentric nucleus and "dancing" cytoplasmic particles. 3-D phase contrast. 875 X. FIGURE 10. A. Spherule cell in abnormal, elongated shape. P>. Adipohemocytr. 3-D phase contrast. 875 X. FIGURE 11. Adipohemocyte. Phase contrast. 875 X. FIGURE 12. Flattening and expansion of a plasmatocyte on coversh'p, shnxving uranulcs in the cytoplasm. Phase contrast. 583 X. FIGURE 13. Plasmatocyte flattening onto coverslip and extending protoplasmic arms; cell membrane has become very thin and barely visible. Phase contrast. 583 X. FIGURE 14. Protoplasmic arm with well developed central body. Active streaming of protoplasm and movement of the body occurred. Phase contrast. 700 X. FIGURE 15. Same as Figure 14, taken about 10 seconds later. Bending of arms with central body as a fulcrum caused clump of cells to move in the preparation. 118 ROY !•:. Mel. U'UILIX AM) < iK( >K< ,K ALLEN -22. Rhodamine B and Sudan IV were used to confirm the lipoid nature of these droplets. The frequency of these cells was always low, hut increased during tin- prepupal stage and was correlated with the presence of free lipoids in the hemolymph. Complete transitional stages between an adipoid variant of a plasmatocyte with lipoid droplets and the hemocyte replete with lipoid droplets ( adipohemocytes ) were not ohserved. Spherule cells. These were very large and highly retractile cells, round, oval, or distended by lobes of gray, homogeneous inclusions which appeared as dark- clouds hv phase contrast. Cells contained one to several of these inclusions, which distended the cell into irregular shapes (Plate I, Figs. 9A and 10A). Cells usually were unstable after about one hour In I'ltro and tended to rupture, ejecting amorphous material which rapidly dissipated. Some .spherule cells never ruptured In ritro. After rupture, the nucleus, often previously obscured by the large in- clusion, was very prominent, granular, and remained in the cell ( Plate I, Fig. 9B). After intracellular disintegration the cell membrane was usually lined with granular debris. Measurements of 55 of these cells showed the longest axis to be 19.5 //, ±9.8 /* (R = : 11.9-34.7 ^) and the narrow axis measured 16.4 /* ±4.8 p. ( R - - 11.9- 29.6 p). Twenty nuclei measured 6.0 ± 1.0 /* (R = : 4.1-8.1 /*). The nature of the spherules is unknown. They did not fluoresce with any of the fluorescent dyes. Of the other stains used, only fast green FCF and Wright's stain were accepted. The spherules stained red with the latter stain. I n 1'itro changes of spherule cells occurred in direct preparations. Cells which had one or two medium-sized homogeneous gray inclusions transformed into large "mature" spherule cells. The cells lost their granular appearance, and the diameter increased as the typical gray refractile lobes appeared. The transformation was rapid or required up to 30 minutes in some preparations. Cells which looked like plasmatocytes were observed to develop inclusions which resembled spherules in 1'itro. However, plasmatocytes were not observed to develop into typical spherule cells. Oenocytoids. Xo cells resembling descriptions for oenocytoids (Arnold, 1952; Jones. l("ij; Y eager, 1(>45) were observed in wet-mount preparations, and all hemocytes could be placed into the above categories. Fixed, stained smears of boll weevil hemolymph did produce distorted cells which could have been mistakn for oenocytoids because of their uniformlv staining cvtoplasm, very small (shrunken) nucleus, and apparent creases or Striations in the cell membrane. However, prior observation of the slides in wet-mount preparations revealed that these were artifacts. A tew such cells were also seen in wet mounts diluted with phosphate butter, pi I Ci.4, or in partially dried wet mounts, not ringed with white petroleum jellv. Manv cells in these mounts were distorted and could not be recognized. Cells resembling oenocvtoids were never observed in preparations free from observable artifacts. F r \ ( "i i ( > N A i . I 'ROCESSKS Phagocytosis. The plasmatocytes actively ingested foreign bodies. Larvae were injected into the hemocoel at the ventral side of abdomen with India ink, living I'.sclicricliia coll (Migula 1895) Castellani and Chalmers I'M1', living Sarcina Intca hroeter lS8n), or .S". Intca stained with carmine. Memolvmph was drawn at HEMOCYTES AND COAGULATION IN A WEE\ 119 intervals up to one hour after injection. All forms of plasmatocytes \vere seen with ingested particles. Living S. lutca was used most frequently because- tin- packets were easily discerned in hemocytes (Plate I, Fig. 7). Hemocytes with spherule bodies were also observed with phagocytosed bacteria. The typical mature .spherule cells never exhibited phagocytosis. Coagulation. A phenomenon occurred in vitro which resulted in formation of extensive networks of protoplasmic strands interconnecting with hemocytes The process, seen in ?'itro, was first observed 15 minutes to an hour after a prepara- tion was made and proceeded slowly for several hours. Usually about an hour after activity was first seen the networks became quite extensive. One 17-hour- old preparation was a dense mass of closely interwoven plasma fibers and nbrobla>t- like structures connecting hemocytes, many of which still retained their normal shape. The process was first detected by the occurrence of filaments which often had a filiform extension. (The term "filaments" was used by Jones, 1962. See also Yeager, 1945, "plastids.") They were homogeneous except for one or two small black granules in the bulb-shaped body. These appeared suddenly in great numbers in preparations which later became very active in production of networks. These bulb-shaped filaments originated from plasmatocytes. Disc-shaped plasmatocytes underwent transformations, observed most fre- quently in capillary-obtained hemolymph diluted about 1 : 1 with pH 6.4 phosphate buffer, in which they flattened and expanded, or proceeded to produce filaments or cytoplasmic extensions. The most frequent event was production of one to four protoplasmic strands. After a long strand had been formed with a bulb-shaped thickening, further exten- sion distally occurred. The entire strand exhibited active protoplasmic streaming and movement of the granules. The distal portion engaged in a rapid extension- retraction movement, along with a probing motion in all dimensions. Whiplike movement of the strands was common. Two more events occurred simultaneously and represented the major phase of network formation (Plate I, Figs. 14 and 15). The proximal portion of the strand became thickened as cellular contents flowed into it. The thickened proximal strand changed frequently from a plastic to a turgid state and back again. However, the distal portion remained hyaline and continued to move rapidly. The amorphous bulb-like region was retained and a second such region often was formed in the proximal area of the strand. The flow7 of cellular content resulted in formation of a fusiform body in the strand, with granular inclusions and an additional body which by phase contrast resembled a nucleus. The second concurrent event was anastomosis of the distal portions t<> form the network. The extremely active tips often extended 10-50 /i in one direction to form a terminal bulb-like bodv, which increased great Iv in si/.e but remained j ' o - hyaline, and also sent out a second pseudopodial extension from the thickened body. Then both strands could be rapidly withdrawn while a third originated from a neu location on the thickened body (Plate II, Fig. 16). When the two strands touched each other they recurved along their entire length and joined into a single larger strand. The pseudopodia could become quite rigid and were occasionally observed to force their way between cells clumped together and to separate a cell from the clump. Plate I, Figures 14 and 15 show such activity. The photographs were 120 ROY i-:. MCLAUGHLIN AND <;KOK<;K AI.LKX taken ahout 10 seconds apart, The result of the entire process was an extensive network in all dimensions, with newlv formed thickened areas hardly distinguishable from fusiform plasmatocx tes, and manv areas of e\i)anded protoplasm containing dehris from parent cells. Kxtensive networks, formed one to two hours after removal of hemolympli and preparation of the slide, are shown in Plate II, Figures 17-1^. .More extensive formation frequently occurred after (> to 17 hours. 18 19 Plate If |-'u,rKK 16. Kapid recurving (if tun -trand> which have' coalesced at A, and subsequent extension of a new strand al II. Phase contrast. 700 X. I'K.rki.s 17, IN, 1°. Kxtcnsive network formations hO-120 minute- alter hemolymph with- drawal. Phase contrast. 350 X Rupture of spherule cells occurred during this process and frequently was seen prior to the earl\ stages of stranding and network formation. A definite relation- ship to initiation of the process hy the erupted spherule cells was not estahlished. However, protoplasmic stranding occurred in immediate proximity ot the cells and even appeared to he attached to intact large spherule cells on several preparations. Their si/e and high refractivity prevented ohservance of the tenuous strands at the cell memhrane, and no proof of actual connection was seen. Krom our ohserva- tioiis, spherule cells do not initiate coagulation, nor do thev play a role in coagulation HEMOCYTES AND COAGULATION IX A WEEVIL 121 process, with the possible exception of serving as a physical body to which the strands may attach. One important factor directly influencing the extent of network formation was the proximity of the plasmatocytes to each other. Strands were frequently seen which had extended apparently to the maximum extent possible. If no other strands were encountered, further activity ceased. Anastomosis with other strands appeared to rejuvenate or increase the vigor of the strands and promote continued formation. EFFECT OF PHYSICAL AND CHEMICAL TREATMENT ON HEMOCYTES Hemocyte morphology and in ritro transformations were altered by the physical action of hemolymph withdrawal and placement on the slide and by dilution with pH 6.4 phosphate buffer, 0.85^0 NaCl solution, 0.5% (w/v) trehalose in distilled water (final pH 5.8), 1% potassium oxalate (w/v) in distilled water (final pH 6.75), or tap water (pH 7.3). Although quantitative data regarding the extent of the effect on hemocytes were not obtained, comparisons were made between hemocytes observed in direct preparations and those in capillary prepara- tions or hemolymph diluted about 1 : 1 with the above diluents. Study of hemocyte morphology and in vitro transformations by several techniques of sample preparation provided an indication of pleomorphic variability and recognition of artifacts imposed by technique. The physical action of drawing hemolymph into a capillary tube and then dispensing it onto a coverslip involves tremendous rates of flow at the orifice. The direct method for allowing an exuding drop of hemolymph to flow onto a coverslip is much less rigorous. This latter, direct, method was considered to result in hemocytes most like those in natural in 1'lvo conditions. However, inter- pretation of observations from this method must also be tempered when one is attempting to draw conclusions as to the in 1'h'o hemocyte conditions. Plasmat- ocytes and spherule cells were affected by the physical forces involved in physical withdrawal of the hemolymph. Changes in appearance of adipohemocytes and prohemocytes clue to technique were never detected. The capillary method resulted in great changes in plasmatocyte morphology. Very few plasmatocytes observed immediately after sample preparation by this method exhibited cytoplasmic extensions or were spindle-shaped. Disc-shaped forms were dominant and the flattened and expanded forms were frequent. On the other hand, preparation by the direct method resulted in immediate observation of all pleomorphic forms of plasmatocytes. Dilution of hemolymph caused variations in hemocyte morphology. Although quantitative tests were not conducted, comparison of hemocytes in preparations diluted with the above materials with those in direct, undiluted preparations gave clear indication of artifacts. In the trehalose or potassium oxalate solutions, disc- shaped plasmatocytes were inflated and internal structural details more distinct. When the pH 6.4 phosphate buffer was used, disc-shaped plasmatocytes underwent a very rapid transformation to spindle-shaped and pseudopodial forms. However, dilution with water drawn directly from the tap (pH 7.3) did not result in this increased activity. Surprisingly, water from the central distillation source of the i- kov !•:. MCLAUGHLIN AND GEORGK ALLEN Laboratory was found to be ]>l I 6.1. Attention to the ]>1T of diluents may he quite important in a study of liemocyte morpholog\ . DISCUSSION The cells referred to as prohemocvte> were not observed hi vitro to change into mature cells. However, all intermediate stages from the typical prohemocyte described above to a spherical plasmatocyte were easily observed. As with all hemocUes. proof of origin may have to await application of tissue culture methods. Kmbryological study was not conducted. The reviews of AYigglesworth (1959) and Jones (1962) covered the origin of insect hemocytes. Imaginal discs, mitosis of hemocytes. and hemopoietic tissues have been reported as sources of post-embryonic replacement of hemocytes. Plasmatocytes were the most abundant forms in all preparations. They were capable of extreme pleomorphism and varied considerably with regard to cyto- plasmic inclusions. Close attention to nuclear size and placement, observation of in 1'itro changes, and intentional production of artifacts by various methods left no doubt as to the identity of all the observed forms. These hemocytes can carry on phagocytic activity, produce networks in a process which could be interpreted as coagulation, and possess different quantities and types of cytoplasmic inclusions. Because of transformation into many pleomorphic forms and variations of inclusion material, plasmatocytes are tentatively classed as a single category until further studv proves other types to be valid end products of plasmatocyte differentiation. The spherule cells are designated as a tvpe of hemocyte because of their dis- tinctive character. The slight phagocytic capabilitv of spherule cells does not imply they are plasmatocytes because phagocytosis bv pericardial cells and certain cells of adipose tissue has been reported (Cameron, 1^34). Moreover, the function of phagocytosis has not been shown to be restricted to onlv one type of cell. Spherule cells were not demonstrated to plav an active role in the coagulation proce». Adipohemocytes are considered a distinct type of cell because of their unique appearance and apparently unique function connected with lipid material. They were stable in vitro bv all techniques employed. Although complete transitional stages from any other type of hemocyte, especially the adipoid plasmatocyte, were not observed, this does not establish the independent lineage of adipohemocytes. Further research mav clarifv the relationship between these hemocvtes. j tf - The process of coagulation was reviewed by Jones ( 1('(>2) and \Yigglesworth (1959). (iregoire (1951, 1955, 195(>a, 1<)5%') studied the process in more than 400 species of insects. Four categories were described ((iregoire, 1951) and reviewed by Wigglesworth (195(>). Type IV (no coagulation) was reported from two species of curculios (Gregoire. 1955). In a further study ( l('5('b) he recorded Type IV from six additional species and observed the pre.seiice of hemocytes which exhibited the elongation, expansion, and retraction ot flexuous protoplasmic arms, as well as formation of loose mesh-works ot hemocyte protoplasm. In other studies ( l('5('a ) he reported network formation in many additional species of curculios. I le also described "plastic hemocytes" which produced activitv similar to that M-en among plasmatocvtes in . /. grandis. Gregoire also reported prohemo- cytes, which probably correspond to those in the boll weevil, as well as other cells HEMOCYTES AND COAGULATION IX A \YEF.YIL 123 similar to boll weevil plasmatocytes. Filaments with thickened regions, were also reported to take part in network formation. In the boll weevil, similar filamentous bodies originated from plasmatocytes and were the terminal portions of protoplasmic strands which frequently dissociated from the cell during the proces ; of network formation. The process of network formation did not occur in every preparation. It wa> most frequent when the direct method was utilized and the preparation appeared to be normal, or lacking in artifacts. The proximity of cells undergoing stranding \\a> important to the extent of network formation finally attained. When strands failed to contact others, the process was not as extensive as when cells were close enough to allow extensive contact and fusion, which appeared to rejuvenate the strands involved. The importance of the number of cells to coagulation was also observed by Yeager and Knight ( 1933 ). A very graphic example of the importance of recognition of artifacts and study by several techniques occurred when a hemolymph sample was diluted with pH 6.4 phosphate buffer. We observed thin plasma strands with adherent particles and granular plasma islands identical to those pictured by Gregoire (1951, Plate V, Figure 25 ) . We saw this condition only once, and our attempts to repeat it were unsuccessful. Although Gregoire did not dilute the hemolymph, he allowed it to flow by capillary action between an unsupported coverslip and the slide. This incident serves merely to illustrate the necessity for studying hemocytes by several methods and attempting to recognize the possible alterations caused by technique and physical or chemical conditions. The authors gratefully acknowledge the suggestions and comments of J. C. Jones in reading the manuscript. SUMMARY 1. Hemocytes of larval boll weevils, Anthonounis gnuidis Boheman, were classi- fied on a morphological basis into four types. Prohemocytes had a large nucleus and a thin band of peripheral cytoplasm, which increased in quantity and became more optically dense to phase contrast as the prohemocytes matured. All gradations to spherical plasniatoc\tcs were observed. PIasniatoc\tcs were characterized by their great pleomorphic capability. The cytoplasm varied in that it was granular with fine or large granules, dense and uniform to phase contrast or heterogeneous with areas of different optical density as well as possessing vacuoles or various inclusions, in addition to being hyaline or refractive at the cellular periphery. Plasmatocytes assumed spherical, pseudopodial or fusiform, as well as irregular, shapes during a process of stranding or when flattening onto a glass surface. Plasmatocytes were phagocytic. Cells filled with lipoid globules were tentatively classed as adipohemocytes. A fourth type of cell, spherule cells, was characterized by the presence of one to several large globules which were amorphous and non- refractive to phase contrast. These cells were large and often distended by the inclusions. 2. A slow process of network formation was the only observed indication of coagulation. Plasmatocytes underwent extension stranding by extension, retraction, KOY F. MCLAUGHLIN AND GEORGE probing, and annMomosis with protoplasmic arms from other cells to produce networks. The extent of these networks depended partial! v upon the proximitv of stranding cells. LITERATl'KK UTFD ARNOLD, J. \\'., 1952. The luiemocytes of the Mediterranean flour ninth, Eplicstld kuhniella Zell. (Lepidoptera: I'yralididae ). Cainui. J. Zool., 30: 352-364. CAMEROX. G. 1\., 1934. Inflaniniation in the caterpillars of Lepidoptera. J. Patliol. Bacterial.. 38: 441-466. GREC.OIRK. Cn.. 1951. Blood coagulation in Arthropods. II. Phase contrast microscopic observations on hemolymph coagulation in sixty-one species of insects. Blood. 6: 1173-1 IMS. GREGOIRE, Cn., 1955. Blood coagulation in Arthropods. V. Studies on hemolymph coagulation in 420 species of insects. . Irch. Biol., 66: 103-148. GREGOIRE. Cn.. 1959a. Hemolymph of Curculionidae and of Diptera. £.r/>. Pare .\\itl. Albert: I >cit.\-iciuc Ser. (Brussels), 10: 1-17. GREGOIRE, CH., l''5nl>. Further observations on distribution of patterns of coagulation of the hemolymph of neotropical insects. Smithsonian Misc. Ci>//., 139: 1-23. HOLLA XDE, A., 1911. Etude histologique comparee du sang des insectes a hemorrhee et des insectes sans hemorrhee. Arch. Zool. Ex p. cf Gen. (scr. 5), 6: 283-323. JONES, J. C., 1950. The normal hemocyte picture of the yellow mealworm, Tencbrio ninlitor Linnaeus. I»:cn State L'ollcf/c J. Sci.. 24: 355-361. JONKS, J. C., 1959. A phase contrast study of the hlood cells in Prodenia larvae (Order Lepidoptera). Quart. J. Mier. Sci., 100: 17-23. JONES, J. C., 1962. Current concepts concerning insect hemocytes. .liner. Zonl., 2: 209-246. WIGGLES WORTH, V. B., 1959. Insect hlood cells. Ann. Rev. Entoniol., 4: 1-16. WITTIG, G., 1962. The pathology of insect hlood cells: A review. Amcr. Zool., 2: 257-274. YKAC.ER, J. F., 1945. The hlood picture of the southern armyuorm Prodenia eridania. J . Atjr. Res., 71: 1-40. YEAGER, J. 1;., AND H. H. KNIGHT, 1933. Microscopic observations on hlood coagulation in several different species of insects. ./;;;;. I:.nlomol. Soc. Amer., 26: 591-602. AET AS A RADIOPROTECTIYE AGENT AT THE CELLULAR LEVEL1 ROBERTS RUGH AND KAREN FLT Radiological Research Laboratory, Department of Radinlnf/y, Collci/c of Physicians and Snrtictnis, Colninluti I'nii'crsity, A>;v York 27, N. Y. Many agents have been tested for their possible protective effect against the damaging and lethal action of ionizing radiations (Rugh, 1953a, 1958; Hollaender and Doudney, 1954; Thomson. 1962). Among the most effective agents for the mature mammal are AET (S,2-aminoethylisothiourea-Di-HBr), MEG (2-mercap- toethylguanadine) , MEA (B-mercaptoethylamine) and cysteine HC1 (Doherty and Burnett, 1955. 1961 ; Stern. 1956; Doherty ct al., 1957; Doherty and Shapira, 1958; Maisin and Doherty. 1960; Maisin and Popp. 1960; Maisin, 1961; Mundy et al., 1961 ; Dacquisto et al., 1961 ; Blouin and Overman, 1962; Melville and Leffingwell, 1962; Ehling and Doherty, 1962; Hanna and Colclough, 1963; Mittler. 1963). AET has also been shown to be protective against the effects of the so-called radiomimetic agents (Asano ct al., 1962. 1963). In every case the drug used, to be effective, had to be administered prior to exposure to the ionizing radiations. Usually such agents, injected after irradiation, were deleterious. Only spleen and bone marrow homogenates appear to have any protective value when adminis- tered after exposure (Bacq and Alexander, 1961). The usual explanation for the so-called protective action of any of these agents is that they somehow aid in hematopoietic recovery (Dickens and Shapiro, 1961; Colclough and Hanna, 1963) or cause hypoxia (Hanna and Colclough, 1963). How protection is actually accomplished is not at all clear. In order to understand better the mechanism of protection, it seemed wise to test the most effective agent, AET, at the cellular level. For this study the un- fertilized egg of the sea urchin, A r abac la pitnctnlata, was used. Thus, any irradia- tion and possible radioprotective action of AET on the haploid cells would be reflected in the early cleavage stages, as well as in early organogenesis. This is a report of the findings. MATERIALS AND METHOD The eggs of Arbacla punctulata were obtained from 5 or 6 mature females In cutting away the Aristotle's lantern from the oral region and inverting them in stender dishes of filtered sea water, and allowing them to shed naturally through the five gonopores. Such eggs were washed in two changes of filtered sea water, 15 minutes apart, and allowed to settle. When the eggs were to be subjected to AET, two times the desired concentration of the agent was used, added to an equal volume of egg suspension, resulting in the proper concentration of AET. 1 Based upon work performed under Contract AT- (30-1) -2740 for the Atomic Energy Commission, and aided by Grant RH-81 from Division of Radiological Health, Bureau State Services, Public Health Service. 125 126 KOI5KKTS Kn.II AND KAKF.X FT All eggs were fertilized simultaneously l>v a .sperm suspension made from a single sea urchin. The radiation facilities consisted of the ccsium-137 paired sources available at the Marine Biological Laboratory, Woods Hole. Mass. The eggs were suspended midway between the two sources of these gamma rays at a distance of 5.9 cm. from each source and a dose rate of 5000 r/min. After some exploratory tests a total exposure of 50.000 r was chosen, delivered in 10 minutes. The AKT was obtained from 1 )r. D. G. Doherty of Oak Ridge National Labora- tory, and was identical with that used by him so successfully with mammals (Doherty ct a!., 1957), and was kept in a desiccator. In order to convert the AET into the effective MEG, it was first dissolved in 10 cc. of phosphate buffer at pH 7.4, before' diluting with 490 cc. of filtered sea water, resulting in pH range from 7.3 to 7.(>. This is known to be optimum for the normal development of Arbacia (Harvey. 1957). The concentration of AET to be used was empirically deter- mined, being the threshold level just below toxicity (see below). The solution thus provided was largely MEG which had to be used within 30+ minutes, before its oxidation degradation to the disulfide. All >tudies were made at the laboratory temperature, which ranged from 21.5° to 23.0° C.. but was uniform for any single set of experimental variables. Arbacia samples were fixed in 10% formalin in filtered sea water at 20-minute intervals, beginning one hour after fertilization when the controls normally show a high percentage of cleavage. Percentages were based upon a minimum of 200 cell counts, the data presented here (Table I) being on 400 cell counts. When eggs were subjected to AET this was done for a minimum of 10 minutes prior to irradiation in order to bring this agent into equilibrium with the egg substance. "When Arbacia eggs were returned to sea water from the AKT solution they were diluted with approximately 100 times the volume of filtered sea water. I '" X I ' ]•: R 1 M K N T A L DATA Exploratory experiments indicated that exposures of the unfertilized eggs of . \rbacia to 5000 r or more resulted in cleavage delay and 50,000 r still allowed 1 1 ' < to cleave, after considerable delay ov( r the controls. Some 18rv showed anomalies during the earlv cleavages. Xoiie achieved the plnteus stage, nor even gastrulation. Thus, if AKT were able to "protect" such irradiated eggs this would be revealed in a shortening of the time from fertilization to the first cleavage, an increase in the percentage ot cleavage within a given period, reduction in the incidence of anomalies and/or effects on development past gastrulation. This exposure of 50,000 r did not render the eggs untertili/.able by normal sperm and thev readily formed fertili- sation membranes. There was some evidence that these membranes were not fullv elevated, and that the perivitelline .space was not as wide as in the controls, and the eggs were often clustered as if they were stick) ( Kugb. l('53b). Lower exposures did allow a higher percentage of cleavage, and further development, but the 50.000 r level ot exposure was chosen .since the low level of 13'< cleavage could be improved if there were any protection. In 13 separate .sets of data, involving thousands of eggs, only 5 or (> exposed to 50,000 r were able to achieve the pluteus RADIOPROTECTION AT CELLULAR LKVEL 127 stage, and not many more survived the process of gastrulation. This marie the end point of development, following 50,000 r gamma rays, clear-cut. The AET was neutralized to pH 7.3-7.6 but was found to be to ; . Irhucui eggs in concentrations comparable to those used for mammals. Membranes \vere elevated, asters were formed, and cleavages did occur in concentrations up to 60 mgm.c/c (2% cleavages). However, since as little as 5 mgm.% AET in sea water destroyed the developing Arbacla eggs some time after fertilization and early cleavage, if left in the solution, it was decided to use 3 mgm.%, which allowed 97% of the unirradiated eggs to be fertilized and cleave normally, both with respect to time and form. A lower concentration of 1 mgm.% allowed Arbacla eggs to be fertilized and to develop, similarly with the controls, through the pluteus stage. There appeared to be no evidence that this concentration was in any way toxic. The concentration of 3 mgm.% was therefore chosen for use prior to and during irradiation since, in a few experiments, this concentration did appear to have a very slight delaying effect on the time of the first cleavage if the eggs were left in the AET solution. This concentration was therefore considered to be at the threshold level of toxicity. The crucial experiment, based upon these empirical findings with irradiation and AET concentrations, was exposure of the eggs for 10 minutes prior to and 10 minutes during gamma irradiation (50,000 r) to the 3 mgm.% solution of AET, and then transferring them to filtered sea water for fertilization and development. Continuing exposure of the irradiated eggs to AET allowed slight improvement in the time and percentage of cleavage after fertilization ; only 20% became ciliated blastulae and all were dead by 48 hours. Thus, it seemed that the optimum conditions involved return of the irradiated eggs from the AET to filtered sea water for fertilization and development. The data of the final experiment alone will be given since they followed the general pattern of the prior experiments and had higher counts for each of the variables, of 400 cells per sample. The data of the table above substantiate Henshaw (1932. 1940) and Henshaw and Francis (1936), who report that a delay in fertilization of irradiated and un- fertilized eggs of Arbacia allows some of them to "recover"' so that the retardation in the initial cleavage normally caused by irradiation is somewhat nullified. The percentage difference is not great, after 50,000 r exposure, but the eggs irradiated at the beginning of the series started to cleave at a shorter elapsed time interval than those irradiated at the end of the series, due to the recovery phenomenon of Henshaw. Eggs in 12 mgm.% AET showed reduced percentage of cleavage throughout, and by 24 hours virtually all were dead. Likewise, those irradiated in 12 mgm.% but returned immediately to sea water for fertilization and development showed no "protection" by this agent, and all were dead by 24 hours. In 3 mgm.% AET the results were comparable to the controls, indicating no significant delav or deleterious effect on cleavage or development. When eggs were irradiated in 3 mgm.% AET and immediately returned to sea water for fertilization, there was no improvement in cleavage time in relation to those irradiated without the drug. By 24 hours only 30% became ciliated, and were retarded in development. As expected, 1 mgm.% AET had no effect on either fertilization or development. Thus, while AET in all concentrations used allowed fertilization of the treated eggs, and 3 mgm.% was definitely non-toxic, no concentration used prior to and during 128 KOI'.KKTS Kl (ill \XD KARKX FU irradiation had any "protective'" effect, either on the time of the initial cleavage, gastrulation or later development. "While 3 mgm.'/t' AET allowed unirradiated eggs to he fertilized and develop (by 4S hours) to motile plutei, no eggs irradiated in AKT to 50,000 r gamma rays ever reached the pluteus stage, or even gastrulation. TABUC I Cleavage ' , (-HM) eggs) at intervals after fertilization Conditions 60' 80' 100' 120' 24 hrs. 48 hrs. ( "mi irols 71', 92% 94', 999? Cil. gastrula .Norm, plutei 50, 000 r gamma rays 0 0 2 1 1 M)' , ell. hlaslula 0.1% cil. hlastula, no plutei 12 mgm. ' , AKT 25 32 36 49 .>()' ,' alive, 20' ,', alive, abnor- only retarded mal, retarded 12 inj- m. ', AKT + 0 0 1 4 99' ; dead 0.1' ;, cil. hlastula 50,000 r + sea \vate 3 mgm. ' , AKT only 72 90 95 99 Cil. gastrula Xorm. plutei .} mgm. ' , AKT + 0 0 1 7 M)1 ,' cil. hlastula 0.1' ,' cil. bias- 50,000 r + sea tula, no plutei water /; mgm. ', AI-:T + 0 0 9 21 M)< ,' cil. hlastula 100' , dead SO, 000 r + AKT 1 infill. ' , AKT only 69 90 96 99 99' ,' cil. gaslrula Norm, plutei 50,000 r Camilla rays* 0 0 0 2 10' , cil. hlaslula 100' , dead abnormal *l 'uleriili/cd c^^~. \\cre irradiated at the beijimimi; of the series and another group at the end niiimtes lalerj but all were ferlili/ed simultaneously so that the first group had M) minute- "recovery" time, due to delay in fert ilixation. The effect of the delay is reflected in the slight ditlerence in cleavage data. DISCUSSION "I'rotection" iii radiohiology is really a misnomer. Its limitations should always be clearly defined hy the user. Kven among mammals there are only a few agent. s which, if present in the body at the time of exposure, will provide greater tolerance of whole body exposure to ioni/.ing radiations, resulting in a higher LD/50/30 ( i.e., lethal dose to 50r/ of exposed animals in 30 days). Hut this is surt'h'al. and, to that extent, protection against death. The tendency is to assume that all animals that survive a 30-day post-irradiation period have "recovered" from anv and all ill-effects of the exposure and are as normal as they were prior to the exposure. This is simplv not true. Whole bodv exposure takes a toll which can be expressed in a variety o! sequelae, and some effects are irrevocable, irreparable. This is not to belittle the possible clinical importance of devising means to extend survival RADIOPROTECTION AT CELLULAR LEVEL 129 to a larger population of exposed individuals, no matter how tiirv fare in their survival. But we must not he hlinded to the sequelae of such exposure and survival. The mechanism of "protection" hy AET solutions has been elusive to mam investigators. It has been thought to function through aiding in hematopoietic recovery, but the same agents which seem to do this are deleterious if given after irradiation. It has been suggested that AET might help to remove toxic radicals before they can injure normal cells, or in some way to actually stimulate cell proliferation in specific tissues. While all theories have been vague, it has been presumed that any "protection" should ultimately be demonstrable at the cellular level. Tied in with protection of the adult mammal (Khym ct a!., 1957) is the demonstrated fact that genes and chromosomes are particularly radiosensitive. Recently, Mittler (1963) found that neither AET nor MEA had any protective effect against induced mutations or translocations in chromosomes of Drosophila spermatocytes or spermatids. But AET has been the most effective agent with mammals so that this present study seemed imperative. There is a wide species variation in the toxicity levels and the responses to AET (Colclough and Hanna, 1963), ranging from 10-20 mg./kg. given orally to man, to 640 mg./kg. injected into mice (Doherty, personal communication). Its chem- istry and degradation products are well documented by Doherty and his co-workers. Since it is not a highly stable compound, except under rather rigid laborator\ conditions, it may not prove to be as universally useful as might be desired. Nevertheless, among the hundreds of chemical agents tried it now appears to be the most effective for most mammals. The Arbacla egg is so well known, so dependable in its reactions to almost all environmental variables, that it proves to be an ideal haploid cell on which to test the efficacy of any possible radioprotective agent. The several criteria of effect are : fertilizability, membrane elevation, aster formation, cleavage time, cleavage form, blastula-gastrula transition, and plutei formation. Each successive criterion is the more sensitive, as development proceeds, and if the pluteus stage is attained one may consider survival to be quite complete, at least as complete as 30-day survival for the mammal. Ionizing radiations (x-rays) have been shown to have a direct effect on the cleavage mechanism of Arbacla so that, with increasing exposures (within limits) there is increasing delay in the time of the first cleavage (Henshaw, 1940). Re- cently Rao (1963) demonstrated that this was due to chromosome condensation interfering with the mechanism of mitosis. Time appears to heal, somewhat. irradiation damage because a delay in the fertilization of irradiated eggs rediuv- the effect of that irradiation on the initiation of the first cleavage. This was originally referred to as "recovery" (Henshaw, 1940). but was more recently shown not to be full recovery but rather a return toward the normal cleavage times (Rugh and Wolff, 1956; Rugh, 1958), with the ultimate embryonic death unaltered. This recovery of cleavage time was nevertheless of interest in view of the finding (Rugh, 1950) that ionizing radiations so affect (meiotic) chromosomes that they become sticky and are permanently clumped. Delay in fertilization could hardly dis- entangle fused chromosomes, although such delay could allow time for the re-fusion of fragmented chromosomes. Henshaw (1938) long ago showed that the delay in U(> ROBERTS RUGH AND KARF.X FU cleavage time .seen in irradiated eggs \vas not seen in the enucleated fragments of Arbacia eggs, so that this phenomenon is directly related to chromosome effects. \pparently it is the early prophase stage which is so affected, and such delay is not carried over into subsequent cleavages ( Yamashita ct . Thus, the maximum "recovery" of cleavage time would be demonstrated bv the first control-irradiated eggs ( which had maximum delay in fertilization) while the maximum damage from irradiation would be shown in the cleavage percentages of the final group, fertilized immediately after irradiation. Since the temperature was uniform for any set of variables of any single experiment, this factor could be ruled out. AKT was positively not protective to the egg of Arbacia, using any of the criteria listed above. This does not rule out the possibility that some other agent(s) might be "protective" in one or another sense. Certainly the mechanism of the so-called "protection" need not be identical for all cells, tissues, organs or organisms, but this agent, so useful with the mammal, is totally without benefit to the gamma- irradiated haploid egg cell of Arbacia, prior to fertilization. SUMMARY AND CONCLUSIONS 1. AKT (S,2-aminoethylisothiourea-Di-HBr) was used in a concentration just below the threshold toxicity level, to determine whether it might afford any radio- protection for the haploid Arbacia egg exposed to 50.000 r gamma radiation prior to fertilization with normal sperm. 2. ArhiH'ia eggs could be fertilized in 3 mgm.'/t AKT and would cleave and develop, both in time and manner comparable to the controls in pure sea water. Toxicity was indicated above 5 mgm.%, particularly after irradiation. 3. . Irhacia eggs exposed to 50,000 r gamma rays showed a delay in the initiation of the first cleavage with ultimate cleavage reaching only \\% and abnormalities reaching IX/v. Not a single egg so exposed ever reached the pluteus stage. The delay in the initiation of the first cleavage was also reduced by a delay in fertiliza- tion, and the percentage of ultimate cleavage was improved. 4. The optimum conditions provided were: Exposure to 3 mgm.% AET in >ea water for 10 minutes prior to and 10 minutes during gamma irradiation to 50.000 r, and yet thi> allowed no improvement in cleavage time, degree of membrane eleva- tion, or development. Xot a single egg thus treated reached either the pluteus or gastrula stages. 5. It is concluded that while AKT has proven to be radioprotective for the adult mammal, this protection (survival) may not be effected through individual cells but through tissue or organ regeneration. However, extrapolation is always hazardous and AKT may be cell- or species-specific. The haploid Arbacia cell (cvtoplasm and nucleus I is not subject to anv protective action from AKT. UTFk VITKF CITKI) AsANO, M., T. I'. M< I >OXAI,I> AND T. T. ( )m u., 1°62. FftVcts of nitmgi-ii mustard on mouse tissue and modification l»y AET. /;;/. ./. l\'inl. Hi, 'I., 4: 5('l 600. RADIOPROTECTION AT CELLULAR LEVEL 131 ASANO, M., T. T. ODELL, T. P. MCDONALD AND A. C. UPTON, 1963. Radiomimetic agents and x-rays in mice and AET protectiveness. Comparative pathological effect^. Arch. Path.. 75: 250-263. BACQ, Z. M., AND P. ALEXANDER, 1961. Fundamentals of Radiobiolo.uy. Academic Press, New York. BLOUIN, L. T., AND R. R. OVERMAN, 1962. Protection of the irradiated dog by aminoethyliso- thiouronium (AET) and p-aminopropriophenone (PAPP). Rail. Res., 16: 699-711. COLCLOUGH, N. V., AND C. HANNA, 1963. The effect of AET on the peripheral blood cells of the rabbit following gamma irradiation. Arch. Int. Phaniiacodyn., 143: 8-18. DACQUISTO, M. P., W. E. ROTHE AND E. W. BLACKBURN, 1961. Mechanism of the protective action of 2-mercaptoethylguanadine (MEG) against whole-body radiation in mice. Int. J. Rad. Biol.,4: 33-42. DICKENS, E. A., AND B. SHAPIRO, 1961. The mechanism of action of AET. Rad. Res., 14: 308-322. DOHERTY, D. G., 1961. Chemical protection to specific systems by AET and related compounds. Symposium on Radiation Effects and Milieu. Montreuz, Switzerland. DOHERTY, D. G., AND W. T. BURNETT, 1955. Protective effects of S, B-aminoethyliso- thiouronium-Br-HBr and related compounds against x-radiation death in mice. Proc. Soc. Ex p. Bio I. Mcd.. 89: 312-314. DOHERTY, D. G., AND R. SHAPIRA, 1958. Chemical structure and protection against radiation. Rad. Res.. 9: 107. DOHERTY, D. G., W. T. BURNETT AND R. SHAPIRA, 1957. Chemical protection against ionizing radiation. II. Mercaptoalkalamines and related compounds with protective activity. Rad. Res.. 7: 13-21. EHLING, U. H., AND D. G. DOHERTY, 1962. AET protection of reproductive capacity of irradiated mice. Proc. Soc. £.r/>. Biol. Mcd., 110: 493-494. HANNA, C., AND N. V. COLCLOUGH, 1963. Toxicity and tolerance studies on AET. Arch. Int. rhannacudyn., 142: 510-515. HARVEY, E. B., 1957. The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, N. J. HENSHAW, P. S., 1932. Studies of the effect of roentgen rays on the time of the first cleavage in some marine invertebrate eggs. I. Recovery from roentgen ray effects in Arbacia eggs. Amer. J. Rocnt. Rad. Thcr. Nucl. Mcd., 27: 890-898. HENSHAW, P. S., 1938. The action of x-rays on nucleated and non-nucleated egg fragments. Amer. J. Cancer. 33: 258-264. HENSHAW, P. S., 1940. Further studies on the action of roentgen rays on the gametes of Arbacia piiuctnlata. (Six parts.) Amer. J. Rocnt. Rad. Thcr. Nucl. Mcd.. 42: 899-933. HENSHAW, P. S., AND D. S. FRANCIS, 1936. The effect of x-rays on cleavage in Arbacia eggs. Evidence of nuclear control of division rate. Biol. Bull., 70: 28-35. HOLLAENDER, A., AND C. O. DouoNEY, 1954. Studies on the mechanism of radiation protection and recovery in the cysteamine and beta-mercaptoethanol. Symposium on Radiobiology, Liege, pp. 112-212; Butterworth, London. KHYM, J. X., R. SHAPIRA AND D. DOHERTY, 1957. Ion exchange studies of transguanylation reactions. I. Rearrangement of AET to MEG and 2-aminothiazoline. Cliein. Soc., 79: 5663-5667. MAISIN, J. R., 1961. Chemical protection of mammals against radiation. Rcr. Franc, d'htndes ' Clin. Biol.. 6: 378-393. MAISIN, J. R., AND D. G. DOHERTY, 1960. Chemical protection of mammalian tissues. Proc.. 19: 564-572. MAISIN, J. R., AND R. A. POPP, 1960. Effect of AET on sodium, potassium, and esterases of the alimentary tract of irradiated mice. Amer. J . I'hysiol., 199: 251-1 MELVILLE, G. S., AND T. P. LEFFINGWELL, 1962. Toxic and protective effects of AET upon normal and irradiated female rats. Brit. J. Radial., 35: 563-571. MITTLER, S., 1963. AET and MEA as protection against radiation induced chromosome aberrations in Drosophila. Genetics. 48: 902. ROBERTS RUGH AND K AKFX FU , R. L., M. H. HKIHH; \\n I!. MKIII.MAX, 1961. The pharmacology of radioprotectant chemicals. Biochemical changes in the dog following the administration of beta- mercaptoethylamine (MEA). Arch. Intern, rintniuicndyn., 130: 354-367. 1\ vo, B., 1963. AiuK sis of x-ray induced mitotic delay in sea urchin eggs. UCRL-10979. RUGH. R., 1950. The immediate and delayed effects of x-radiation on meiotic chromosomes. /. Cell. Comp. Physiol, 36: 185-203. RUGII, R., 1953. Radiobiology : Irradiation lethality and protection. Milit. Snn/cnn, 112: 395-413. RUGH, R., 1953. The x-irradiation of marine gametes : A study of the effect of x-irradiation at different levels on the germ cells of the clam, Spisula. Biol. Bull., 104: 197-209. RUGH, R., 1958. The so-called "recovery" phenomenon and "protection" against x-irradiation at the cellular level. Biol. Bull.. 114: 385-393. RUGH, R., 1958. Biological effects of ionizing radiations. /. Neuropath. Exp. Xeurnl., 17: 1-11. RUGH, R., AND T. WOLFF, 1956. Recovery from x-irradiation effects at the cellular level. Biol. Bull., Ill: 311-312. STERN, H., 1956. Sulfhydryl groups and cell division. Science, 124: 1292-1293. THOMSON, J. F., 1962. Radiation Protection in Mammals. Reinhold Publ. Corp., New York. YAMASHITA, H., K. MORI AND M. MINVA, 1939. The action of ionizing radiation on the sea urchin. Gann, 33: 117-121. HEAT TOLERANCE AND TEMPERATURE RELATIONSHIPS OF THE FIDDLER CRAB, UCA PUGILATOR, WITH REFERENCE TO BODY COLORATION JERREL L. WILKENS1 AND MILTON FINGERMAN Department of Zoology, Ncwcoiiil' College, Titian c University, Ncic Orleans. Louisiana, and Marine biological Laboratory. U'oods Hole, Massachusetts Fiddler crabs, Uca, being inhabitants of the littoral zone, offer a ready oppor- tunity for study of physiological adaptations associated with the intermediate step in the migration of crustaceans from an aquatic to a terrestrial habitat. Poikilo- therms, in general, that have left the aquatic environment require, among others, adequate mechanisms to survive greater extremes of temperature than they had experienced previously. Fiddler crabs are generally active and feeding about the time of low tide. Consequently, when low tide occurs in the middle of the day during the warmer months of the year fiddler crabs on open beaches are exposed to temperatures that are near the lethal level because of the direct solar radiation (Teal. 1958). The fiddler crab, /.>') the influence of body coloration on body temperature, and (c I the rates of transpiration at high temperatures. M. \TKKI.\I.S AND METHODS Adult male and female s])ecimens of I'ca pit Z LJ u rr LJ Q. 20 -O. 'O-o, 37 39 41 43 TEMPERATURE 45 47 FIGURE 1. Relationships between the temperature to which tiddler crabs were exposed for one hour in saturated air ( circles ) and in dry air ( dots ) and the percentage surviving. In Figure 3 are .shown (ai the mean rates of transpiration of water from male crabs in dry air from 28° to 48° C., and (b) the saturation deficit of dry air at each of the experimental temperatures. At each temperature the rate of transpiration was determined for each of 20 crabs. The saturation deficit values were taken from the Handbook of Chemistrv and I'hvsics, Fourth Edition. The increase in rate of evaporation with increase in temperature is proportional to the saturation deficit <>i the air. The similarity of the curves shows that transpiration of water from these fiddler crabs is a physical process completely dependent on the saturation deficit. The crabs apparently have no control over the rate at any temperature. I'.lject <>j coloration on body tempera/lire This experiment was devised to test the hypothesis that the blanching of fiddler • Tabs at high temperatures, first described by lirown and Sandeen (1948), has thermoregulatory significance. Dark crabs having maximally dispersed melanin and pale ones with maximally concentrated melanin were taken from the laboratory HEAT TOLERANCE OF A FIDDLER CRAM 137 37.5 •-•-•-•-•-•-«-•-• -•-•-• -•-• '•~Q~ft"fl ft-ft /•'*'* ! ox 35.0 ^o-o-o-o-o \J 0 UJ 32.5 / 1 tr _^O— o~o~o~o D o-o^ 1 £ 30.0 - 0'° 1 1 Q. z UJ 1 h27.S f/ A B i C 1 i / i 0 I 1 25.0 "l 1 1 i i 0 30 60 90 120 MINUTES FIGURE 2. Relationship between mean body temperature of crabs and time of exposure to slowly moving air of (A) 0%, (B) 50%, and (C) 100% relative humidity (circles). Ail- temperature is indicated by the dots. 17 Z o or to z cr h u. O UJ I- cr 13 II 28 33 38 TEMPERATURE 43 80 70 60 50 40 Z! o 30 20 48 FIGURE 3. Relationships between (a) rate of transpiration from fiddler crabs ( mg./cm.2/hr.) and temperature (circles) and (b) saturation deficit (mm. Hg) of dry air and temperature (dots). 138 1KKKKL L. VYILKKNS AX1) MILTON FINGERMAN 40 3 8 u o: D Q. 2 LJ 36 34 30 - - O— o— O- .0—0—0- 10 I 5 MINUTES FIGURE 4. Relationships In-t \vccn hody temperature of pale (circles) and dark (dots) fiddler crabs and time of exposure to sunlight. Substrate temperature is indicated by the half-filled circles. and exposed to sunlight on a bright, almost windless day. Their body temperatures were recorded over an interval of 15 minutes while they were clamped in position. The data are presented in Figure 4 where each point represents the mean of 10 . VuTininations. The effect of color ifferences in absorbing and reflecting radiant 10 u ui z LU u o: UJ Q. 8 400 500 WAVE 600 700 LENGTH I;K;UKK 5. Relationships between percentage of light rellected from the dorsal Mirlace ot the ceplialothorax of ]iale I upper curve) and dark (lower curve) crabs and the wave-length Of light. HEAT TOLERANCE OF A FIDDLER CRAB 139 energy and converting it to heat is evident in the difference between the rates of heat gain of the pale and dark crabs. Within five minutes after the crabs had been set in sunlight the dark crabs had a body temperature 2° C. higher than that of t he- pale crabs. At each of several wave-lengths of visible light from 400 to 700 m/A, the quantity of light reflected from the dorsal surface of the cephalothorax was greater with pale crabs than dark ones (Fig. 5). The difference at the longer wave-lengths was as much as 5%. Furthermore, for both the pale and dark crabs the percentage of light reflected was greater at the longer wave-lengths than at the shorter ones although the contrast between the shorter and longer wave-lengths was more readily apparent with pale crabs than with dark ones. Observations of field behavior of specimens of Uca pugilator Field observations of the activity and behavior of natural populations of I rca piKjilator were made throughout July and August, I960, at Chapoquoit Beach and Great Sippewissett Creek. Observations of individual crabs were made from a distance through binoculars. The days chosen were clear and hot with a minimum of wind ( 1 on the Beaufort scale : direction of wind shown by smoke drift only. Meteorological Observer's Handbook, 1942). The air temperature averaged 27.5° C. at a height of 2 cm. above the ground. The surface, dry sand, averaged 42.5° C. The air temperature in burrows in this dry sand 2 cm. below the surface averaged 30° C. and 15 cm. below the surface was 26° C. Excavation revealed that all burrows ended in very damp or wet sand. The average relative humidity on the days of observation was 60%, determined with a sling psychrometer held 60 cm. above the surface. On hot days crabs were observed actively feeding predominantly in any- where the surface of the sand was still moist ; however, occasionally they were also active on the drier parts of the beaches. In their normal course of activity the crabs would feed for a while and then retreat into their burrows. It was observed that each crab would feed for 15 to 20 minutes at a time, then disappear into a burrow for three or four minutes and reappear to resume feeding. Therefore, the frequency of this "feeding-retreat" rhythm is 18-24 minutes. The crabs did not enter their burrows en masse as they do when startled, but each individual adhered closely to its own cycle. DISCUSSION The lethal temperatures indicated in Figure 1 at which 50% of the crabs died are more meaningful statistics than are the temperatures at which 100% of the crab- died. The latter temperatures must necessarily include even the most resistant individuals. Teal (1958) found that 50% of the fiddler crabs, Uca pit.5 3 C. "by placing the animals in enough sea water of the desired temperature so that each animal was slightly more than half covered." The lowest corresponding value found in the present set of experiments was 40.7° C. with crabs in saturated air. This difference is probably a reflection of the difference in experimental technique because in the present experiments 10 crabs and only 20 ml. of sea water were placed 140 JKKKKI, ].. \Y11.KENS \\l> MILTON FJXGERMAN into a 500-nil tlask. \vith tin- result that very little submergence of the crabs occurred. It will be recalled these flasks were immersed completely in a constant temperature \vatrr bath but Teal did nut state how he kept the temperature of his flasks constant. The data presented herein confirm and extend the observations of Edney i 1('(>1 i on five species of African fiddler crabs. He found, as herein, that (a) the temperature within the burrows during the warmer months of the year is con- .siderahly cooler than the sand at the surface, and (hi an appreciable reduction in body temperature occurs as a result of transpiration. The ability to withstand high surface temperatures by transpiration undoubtedly has a significant survival value. The upper lethal temperatures at which all of Edney's crabs died ranged from 42° to 45° C. for the live species after immersion in water of the appropriate tempera- ture for 15 minutes. With one hour of exposure in saturated air the temperature required to kill 100', of the Uca pitijilator herein was 42° C. which is the same temperature that was lethal to all of Edney's specimens of Uca iiiarionis, U. urrillci. and U. chlorophthalmus. Edney obtained these particular data in January, 195^. Inhaca Island, where he worked, is south of the Equator, rendering Janu- arv one of the warmer months of the vear for these crabs. It was shown herein j for the first time with a fiddler crab that (a) the amount of cooling resulting from transpiration is proportional to the decrease in relative humidity (Fig. 2), and (b) the "saturation deficit law" as stated by Edney (1957) is obeyed (Fig. 3). The results shown in Figure 4 support the hypothesis of Brown and Sandeen (194S) that the blanching of fiddler crabs at high temperatures has a thermo- regulatory role. Pale crabs maintained themselves about 2° C. cooler than dark crabs. The measurements of quantities of visible light reflected from pale and dark crabs ( Fig. 5 ) are qualitatively similar to the findings of Deanin and Steggerda (194S) with pale and dark frogs. However, Deanin and Steggerda did not measure the body temperatures of their frogs but they did point out that the greater reflection of light at the red end of the visible spectrum than at the violet end may also have adaptive significance in view of the greater heating capacity of the rays having the longer wave-lengths. The observation that fiddler crabs cease feeding and enter their burrows every 18-24 minutes can be interpreted as a behavioral mechanism for control of bod\ temperature. Every time a crab descends into its burrow the escape from sun- light to the cooler air would allow flu- body temperature to decrease .several degrees. I 'aimer (1(^»2), working with a different species of fiddler crab, Uca piti/na.r, found a dailv phototactic rhythm; the crabs spent a greater proportion of lime in the illuminated end of the apparatus during the morning hours than at other times of dav. An attempt at thennoregulation may in part underlie- the change in strength of phololaxis because between 5 and S AM, when the crabs show a strong positive response to light, the air is usually cool, but during the wanner portion of ihe day the attraction to light, hence the tendencv to leave the burrows, is not so great. SUMMARY AND CONCLUSIONS 1. Upper thermal death points were determined for the- fiddler crab. I ca In saturated air the lethal temperature for 50', of the crabs, determined HEAT TOLERANCE OF A FIDDLER CRAB 141 graphically from the experimental data, after an exposure of one hour, wr ; 40.7° C. All of the crabs died after one hour at 42° C. In dry air the corresponding tem- peratures were 45.1° C. and 47° C. for the same time of exposure. 2. Five minutes after having been placed in sunlight the body temperature <>f dark crabs was 2° C. higher than that of pale crabs. More visible light is re- flected from the dorsal surface of the cephalothorax of a pale crab than fr< a dark crab. The difference is more striking with the rays of longer wave-length which have a greater heating capacity than the rays at the violet end of the visible spectrum. These observations support the hypothesis that the blanching that occurs at high temperatures has a thermoregulatory role. 3. The body temperatures of crabs maintained either in dry air or in air having a relative humidity of 50% were lower than the air temperature, un- doubtedly clue to transpiration of water. The body temperatures of crabs in satu- rated air were the same as the air temperature. The cooling that resulted from transpiration was proportional to the decrease in relative humidity. 4. Transpiration from this crab is a passive process. The rate is proportional to the saturation deficit of the air. 5. In their habitat specimens of Uca f>ntjUator exhibit a "feeding-retreat" rhythm that has a frequency of 18-24 minutes. There is no phase interaction between individuals in a population. On hot clays the frequent periodic return to the cooler burrows can serve to lower the body temperature. 6. These findings were discussed in relation to the observations of other investigators. LITERATURE CITED ABRAMOWITZ, A. A., 1937. The chromatophorotropic hormone of the Crustacea ; standardization, properties, and physiology of the eye-stalk glands. BioL Bui!., 72: 344-365. BROWN, F. A., JR., AND M. I. SANDEEN, 1948. Responses of the chromatophores of the fiddler crab, Uca, to light and temperature. Physiol. Zoo/.. 21: 361-371. DEANIN, G. G., AND F. R. STEGGERDA, 1948. Use of the spectrophotometer for measuring melanin dispersion in the frog. Proc. Soc. Expcr. BioL Mcd., 67: 101-104. EDNEY, E. B., 195 la. The evaporation of water from woodlice and the millipede Glomeris. J.Exf. BioL, 2S: 91-115. EDNEY, E. B., 195 Ib. The body temperature of woodlice. /. Ex p. Bio!.. 28: 271-280. EDNEY, E. B., 1957. The Water Relations of Terrestrial Arthropods. Cambridge University Press, Cambridge. EDNEY, E. B., 1961. The water and heat relationships of fiddler crabs (Uca spp.). Trans. Roy. Soc. S. Africa, 36: 71-91. ORR, P. R., 1955. Heat death. I. Time-temperature relationships in marine animals, riiysiol. Zoo/., 28: 290-294. PALMER, J. D., 1962. A persistent diurnal phototactic rhythm in the fiddler crab, Uca putina.v. BioL Bull, 123: 507-508. SOLOMON, M. E., 1951. Control of humidity with potassium hydroxide, sulphuric acid, or other solutions. Bull. Entoin. Res., 42: 543-554. TEAL, J. M., 1958. Distribution of fiddler crabs in Georgia salt matches. VERNBERG, F. J., AND R. E. TASIIIAN, 1959. Studies on the physiological variation between tropical and temperate zone fiddler crabs of the genus Uca. I. Thermal death limits. Ecology, 40: 589-593. WIGGLESWORTH, V. B., 1945. Transpiration through the cuticle of insects. 97-114. Vol. 128, No. 2 THE BIOLOGICAL BULLETIN PUBLISHED-'BY THE MARINE BIOLOGICAL LABORATORY STAGES IN THE NORMAL DEVELOPMENT OF FUNDULUS HETEROCLITUS a PHILIP B. ARMSTRONG AND JULIA SWOPE CHILI) Marine Bioloi/ical Laboratory, U'oods Hole, Mass. 02513, and State University of New York. Collcijc of Medicine. Syracuse, A"<"u' York 1321/1 Fundulus heteroclitus (Walbaum), the inuniinichog, is found along the Atlantic and Gulf coasts from the Gulf of St. Lawrence to Texas (Bigelovv and Schroeder, 1953 ). It is particularly abundant in the northern part of its range and, according to Nichols and Breder (1927), is replaced on the Gulf Coast by a closely related species. The typical habitats of F. heteroclitus include the inshore bays and inlets as well as the shallow tidal creeks, ditches and pools. According to Bigelow and Schroeder, practically all of the mummichogs of the Gulf of Maine will be included within a line drawn 100 yards out from the strand. The adults commonly measure 2 to 4 inches in length with occasional individuals measuring as much as 6 inches. The adults show a striking sexual dimorphism during the breeding season. The males are blue-black dorsally with a black spot on the caudal aspect of the dorsal fin. Ventrally they are predominantly yellowish. Pale vertical stripes may be present on the trunk and tail. The colors are less intense during the non-breeding season. The females are somber in appearance, olivaceous above, grading to pearly white on the ventral surface. Faint darker vertical stripes are sometimes present, most prominent on the tail. These stripes are usually lacking on the larger females. Both sexes show adaptive changes in color intensity to changes in background. I'luidnlus heteroclitus has been used extensively for embryological research due, in large measure, to several advantages. The adults are readily collected in minnow traps or bv seining, the eggs may be stripped from the females and fertilized in the laboratory. The chorionic membrane is transparent, affording a clear view of the developing embryo, the embryos are hardy and of adequate size for operative procedures, and the pace of development, closely resembling that of Amblystoma punctittiini, is convenient for a variety of experimental problems. The ovarian eggs are small in the fall, ranging from 0.16 to 0.4 mm. in diameter (Eigenmann, 1890). There is little change in size between October and April. 1 This investigation was supported by Public Health Service Research Grant No. 00936, from the Institute of Child Health and Human Development. 143 Copyright © 1965, by the Marine Biological Laboratory 144 PHILIP B. ARMSTRONG AND JULIA SWOPE CHILD The eggs increase slowly in size (luring April and by May 1, the largest eggs measure 0.8 nun. in diameter. According to Kigcnmann there is rapid increase in the size of the eggs during May so that thev reach their full size by June 1. However, on Cape Cod some fish do mature their eggs as early as the middle of May. Such adults are found in the ^mailer creeks, the pools and the ditches where the water warms up early in the season. The spawning season ends about the middle of July or shortly thereafter. Gentle pressure on the abdomen directed posteriorly will express the eggs from a ripe female. The milt can be obtained in a similar manner from the males. Fertilization is carried out conveniently in a glass fingerbowl containing enough water to wet the bottom of the dish. Kocking the bowl gently after the addition of the milt will usually assure the fertilization of the eggs. Difficulty is experienced in stripping if the eggs are immature. Such that are obtained will be white and opaque, in contrast to the mature eggs which are yellow and transparent. Diffi- cult}' in expressing the milt trom the males can be circumvented bv removing and mincing the testes in sea water and then decanting the sperm suspension onto the eggs. Eggs remain fertili/.able for 15-20 minutes after stripping (Kagan, ll)35). Adults collected in the larger inlets early in the breeding season may not have fully matured their eggs. Such fish, if held in the laboratory, will frequently mature their eggs over a period of days, permitting the collecting of a limited number of eggs daily for as long as eight or ten days. At the height of the season a fully ripened large female may yield several hundred eggs at a single stripping and. under fortunate circumstances, better than (ASr; of the eggs can be fertilized. The embryos are hardy. According to Solberg (1938), normal development proceeds at temperatures ranging from 12° to 26° or 27° F. They also develop in a wide range of salinities, even in distilled water, with some minor deviations from the normal. Developing in sea water, the embryos show an increase in the perivitelline space as the yolk is utilized in growth and differentiation. In distilled water the yolk sac, instead of decreasing, increases in size. Eventually the perivitelline space is obliterated, the embryo and yolk sac completely filling the chorion. Contraction of the somitic muscle is lost and the heart shows certain abnormalities of beat conduction with dropped beats in the ventricle. These physiological changes may be related to shifts in the calcium-potassium ratios incident to the onset of kidney function (Armstrong, 1932). The oxygen requirements of developing Fundulus embryos initially are low l Amberson and Armstrong. 1'M.V). The rate of development appears unaffected if the eggs are crowded (hiring this period, llowcver, on the second day the oxygen consumption starts rising sharply so it is essential to avoid crowding or clumping of the eggs in a culture if uniform parallel development of the embrvos is desired. It is neccssan to u^e a variei \ of lighting methods, at times in different combina- tions, to reveal the various morphological features of the embryos. The pigment cells show iiji to best advantage with light from a white substage reflector, the circulation with a substage mirror. Higher intensity of illumination from above, as from a Xirconarc lamp, is required to reveal finer de-tails of structure. There are minor time variations in which structures differentiate in relation to each other so that pinpointing any stage in time can be difficult and, on occasion, may appear somewhat arbitrary. Consecutively numbered stages are figured starting with the ovum, then proceeding \\itli the fertilized egg. NORMAL DEVELOPMENT OF FUXDULUS I1ETKK"- 145 Additional information on techniques and experimental methods for handling Fnndulus eggs are described by Costello ct al. (1957). There are references to technical procedures in work done by Nicholas (1927), Oppenheimer (1959) and Trinkaus (1951). Supplementary information on Fundnlus staging is available in Oppenheimer (1937), and Solberg (1938). Stage 1 - The fully matured unfertilized ovum is about 2 mm. in diameter. Eggs from the smaller females are commonly somewhat smaller than those from the larger fish. The egg membrane or chorion is 10-12 p. thick (f).5 p.; Eigenmann) and is composed of two layers (Shanklin and Armstrong, 1952), an inner compact meshwork of protein fibrils and an outer more homogeneous layer from which many fine fibrils arise to form a loose matting on the external surface of the membrane. When the ova are shed these fibrils are sticky and adhesive, a property soon lost on exposure to sea water. This membrane with its fibrils has been described by Eigenmann as a product of the follicle cells of the ovary. There are fine pores, 1.0-1.5 p. in diameter, through the external layer of the chorion) numbering 10,000-20,000 mm.-. The micropyle is most readily seen immediately after stripping the eggs from the fish. It shows up best when viewed on the horizon of the egg and appears as a small funnel-shaped indentation on the external surface of the chorion. The cortical granules (yolk platelets) are uniformly distributed over the surface of the yolk, just deep to the chorion, together with an aggregation of oil droplets in various sizes. There are a few small oil droplets scattered through the yolk which otherwise is a clear yellow viscous fluid, not granular as in some teleost eggs. FERTILIZATION Dramatic changes occur following fertilization. The cortical granules disappear. the perivitelline space forms, and the protoplasm heaps up to form a single cell. continuous at its margins with the plasma membrane surrounding the yolk. In addition, Huver (1960) has described the formation of the polar bodies. based on time-lapse microcinematography. He observed the first polar body approximately three minutes after fertilization. It was closely compressed to the egg surface in freshly stripped eggs. The second polar body appeared -to to 5.0 minutes after fertilization, adjacent to the first. Immediately following fertilization the cortical granules disappear in a progres- sive wave spreading out from the micropyle. the process bcini; completed in . minutes. The "lifting" of the chorionic membrane occurs first in the area encircling the micropyle. developing slowly immediately after fertilization. There is a brief delay in actual separation at the micropyle, this taking place quite regularly at approximately three minutes. It is noted that immediately after complete sepa- ration, the subjacent surface area of the egg assumes a dusky complexion charac- teristic of protoplasmic aggregation. This culminates in the formation of the one-cell stage through further protoplasmic accumulation at this >ite. In Fitininhts the chorionic membrane, which function.s as a fertilization membrane. doe> not '-The stage numbers used throughout this paper are illustrated by ti.mires with CHITC-IM Hiding numbers, in the plates at the end of the paper. 146 PHILIP i:. UIM STRONG AND JULIA SWOPE CHILD actually litt hut rather the egg shrinks a\vay from the membrane as the perivitelline space forms. This progressive formation of the perivitelline space closely parallels the disappearance of the cortical granules. Mature ova can he activated merely by stripping them into sea water. They go through similar changes as described above for fertilized eggs but not identical in all respects. The perivitelline space forms rather slowly, the cortical granules disappearing in a mosaic progression rather than in a continuous wave. .Also, the aggregation of the protoplasm to form the protoplasmic cap is slower. It is usually stated that the one-cell stage forms as a result of the "streaming" of the protoplasm from its general distribution over the surface of the egg to the submicropylar area. This "streaming" is not visible as such and, in the large egg of I'nndiilits. is a slow process. As noted above, there is some protoplasmic condensation at the submicropylar area when the perivitelline space first forms. At the end of an hour the one-cell stage appears as a biconvex lens-shaped structure. At the end of an hour and a half the protoplasmic cap bulges above the curve of the egg and at one and three-quarters hours the single cell is fully formed as illustrated. Stage 3 The first division is meroblastic and meridional, resulting in two cells of equal size. Shujc 4 As the second division approaches, the two primarv blastomeres flatten down. reducing the depth of the furrow between them. The second division is meridional and at right angles to the first. resulting in four blastomeres of approximately equal sixe. Static 5 The S-cell stage results bv a vertical division of the cells of the four-cell stage, the lines of cleavage paralleling that of the first cleavage. Two parallel rows of four cells each are commonly seen but minor irregularities in this arrangement are not uncommon. Stat'/c 6 Another vertical cleavage produces In cells in a single layer of four parallel rows of four cells each, though again minor variations in cell alignment may occur. Sttnje 7 The cells of stage 6 appear columnar as thev approach cleavage, particularly the central cells. The cleavage spindles are perpendicular to the egg surface, this cleavage being latitudinal. The central cells continue as a two-celled layer. There i- some rearrangement of the peripheral cells as deaxage proceeds, with irregu- larities in the layering of the cells. In some specimens the yolk appears to jut up into a depression on the dee]) surface of the hlasfodisc. NORMAL DEVELOPMENT OF FUXDULUS 1 1 KTEROCLITUS 147 s 8-9-10 \Yith successive cleavages there is a progressive increase in the number of cells with a reduction in cell size. However, there is little apparent increase in the size of the hlastodisc, marking this early period as one of cell multiplication rather than one of rowth. Sta(/c 11 The blastodisc is flattening out over the yolk (Trinkaus, 1963). The peripheral margin is serrated as if the periblast is about to migrate out. If the blastocoele has formed, it is not visible in the living embrvo. Static 12 The blastodisc is circular as seen from above and still has a well denned border. However, with proper illumination, the peripheral or marginal periblast nuclei can be visualized on the surface of the yolk at the margin of the blastodisc, forming a circumferential band about one-fourth the diameter of the blastodisc. The nuclei are disposed in four to five irregular concentric rows without cell boundaries. The density of the associated protoplasm can be demonstrated by staining with neutral red or by fixing the material in Stockard's solution (substitute 0.7% NaCl for distilled water to avoid shrinkage). This protoplasmic condensation and the periblast nuclei are not depicted in the illustrations. The former is seen only after fixation or vital staining. The periblast nuclei are not visible at the magnifications used for the illustrations, but are visible at higher magnifications, if the egg is illuminated with daylight both from above with direct light and from below with a substage mirror. The blastodisc appears as a biconvex disc when viewed on the horizon of the egg, the outer or free surface being of greater convexity than the surface in contact with the yolk. The blastocoele cannot yet be discerned in the intact egg. Static /.-? There has been some additional flattening and expansion of the blastoderm over the yolk. The blastocoele may be seen indistinctly in the transilluminated egg if the blastodisc is viewed on the horizon of the egg. The peripheral periblast is still relatively wide, the nuclei being somewhat irregularly distributed, three to four nuclei in width. Fine droplets are aggregating at the juJ&wuI pole, marking the future site of the closure of the blastopore. Stage 14 There has been a further extension of the blastoderm over the yolk and an elevation of the central area of the blastoderm, with an increase in the blastocoele resulting in a wide separation of the epiblast from the hypoblast. The peripheral periblast is somewhat reduced in width as the blastoderm has expanded. This stage may mark the onset of the transition from the blastula to the gastrula. Stage 15 There is a definitive germ ring in this stage which is narrow but complete around the margin of the blastoderm. The embryonic shield is rudimentary, slightly wider I'llll.ir B. ARMSTRONG AND JULIA S\\ < >I'K il f I IJ> than the rest of the germ ring and slightly more elevated when viewed on the horizon of the egg. The early embryonic shield frequently overlies the oil droplets. The peripheral periblast is narrow, reduced to a few nuclei at the immediate margin of the germ ring. The hlastocoele is extensive under the extra-embryonic ectoderm, separating the epihlast rather widely from the hypoblast, especially just anterior to the embryonic shield. The hlastocoele is not necessarily bilaterally symmetrical in its form. Stuyc l(i As epiboly proceeds, the blastoderm extends over the surface of the yolk, the germ ring advancing ahead of the extra-embryonic membrane. Concurrently the embryonic shield increases in size, the embryonic axis in length. There is a further accumulation of fine droplets at the vegetal pole at the future site of the closure of the blastopore. S I a ye 17 The gastrula has expanded to cover one-half of the yolk. The germ ring is little if any wider but the embryonic shield and axis have extended with epiboly. The latter is about one-sixth the circumference of the egg in length. It is best seen when viewed on the horizon of the egg. The fine droplets on the vegetal pole are converging to form a closer aggregation. Siaijc /.V The extra-embryonic ectoderm now covers three-fourths of the surface of the yolk. The embryonic shield lias extended in length and the embryonic axis can be clearly discerned not onlv when viewed on the horizon of the egg but also in a direct dorsal view. The blastopore is reduced to a small opening through which the volk may bulge. The embryonic axis is well defined with some condensation of tissue along its lateral margins. The optic vesicle.s are present but rudimentary. The embryonic keel is prominent in this stage. Stage 20 The closure of the blastopore is complete at thi.s stage. The main divisions of the brain, the forebrain, midbrain, and hindbrain, arc distinguishable with a well defined keel, ventral to the brain, indenting the yolk sac. There is an increased but variable condensation of cells lateral to the embryonic axis in the location of the future anterior somites. Kupffer's vesicle appears as the embryos advance toward the next stage. The ectoderm lifts to form a large vesicle anterior to the head, the forerunner of the pericardia! caxity. Stage 21 There are three to four pairs of somites at this stage. The embryo has increased in size, particularly toward the caudal end. NORMAL DEVELOPMENT OF FUNDULUS HETEROCLITUS Stage 22 The main divisions of the brain are now more sharply differentiated. There is an impression of segmentation of the hind brain into neuromeres, but the brain ventricles are still unformed. The optic lobes are more prominent than in the preceding stage. The optic cup can be seen best in a lateral view but the lens is not discernible. The pericardial cavity is present anterior to the head but has not yd extended posteriorly under or lateral to the head which is flush with the yolk. There is some condensation of tissue at the future site of the pectoral fin. There art- no pigment cells either on the embryo or yolk sac but the blood islands are forming as fine strands on the surface of the yolk. The Kupffer vesicle is deep and anterior to the tip of the tail, which is flush with the surface of the yolk. Under the tip of the tail is a collection of fine droplets. Stage 23 With additional growth and differentiation, the main divisions of the brain are better defined as are also the otic vesicles. The lens appears as a thickening but does not bulge out of the optic cup. The earliest indication of the olfactory placode can be noted. The outlines of the pericardial cavity, bilateral to the midbrain and hindbrain, are well defined. This cavity extends under the head, which has lifted off of the yolk sac beneath the optic lobes and anterior hindbrain. The anterior extremity of the forebrain is still flush with the yolk sac. In most specimens, the heart rudiment can be seen. There has also been a further elaboration of blood islands on the yolk sac. Pigment cells are present, scattered sparsely over the yolk sac with occasional cells on the dorsolateral aspect of the hindbrain. These cells are small and contain a small amount of densely aggregated pigment. The tip of the tail is rounded but not free of the yolk sac. Stage 24 The brain ventricles are forming as the embryos approach this stage, initially in the optic lobes with rapid extension into the other divisions of the brain. Some of the neural derivatives also are more prominent, including the olfactory placode and the otic vesicle. The lens of the eye fills the optic cup. There has appeared a condensation of tissue on the lateral sides of the hindbrain both anterior and posterior to the otic vesicles. Also, there is an increased number of melanophores and, as the embryos develop toward the next step, erythrophores appear on the deep side of the embryos under the hindbrain and trunk. Most of the melanophores are still unexpanded. The tip of the tail is rounded but bound down to the yolk sac. The earliest cardiac contractions were observed in embryos in the 14-somite stage and, as the heart elongates, there appears a regular initiation and conduction of pulsations from the venous to the arterial end. The blood islands of the yolk are linking up with each other, forming a syncitium, but there is, as yet, no circula- tion. Also, the melanophores on the yolk sac. have increased in numbers, Stage 25 This period is marked by the onset of the circulation. The pericardial cavity extends forward, lifting the head off the yolk ; the venous end of the heart likewise extends forward to form an elongate tube. Also, there develops a concentration PHILIP B. ARMSTRONG AND JULIA SYVOPK CHILD ni" blood cells in tin- lilddd islands on the yolk at the venous end (it the heart and near the root of the tail. The circulation is established first through the posterior vitelline arteries and \vas ohserved in \(i of 20 embryos in the 1' '-somite stage. Initially the circulation is sluggish, with red hlood cells in .scattered clumps moving slowly through the vessels. Late in this stage the circulation is established through the anterior vitelline arteries. The first contractions of somitic muscle are seen in enihryos in this stage, involving only a few of the most anterior somites. These contractions and the accompanying relaxations are slow and produce a slight bending of the body axis. The tip of the tail is now free of any yolk sac attachment. There has been some overall increase in the sizes of various embryonic structures but only partial expansion of some of the melanophores which also have increased in number and extended onto the optic lobes. There has been an increased extension of the pericardia! cavitv and an elongation of the heart anterior to the head, fully exposing the sinus venosus region of the heart to observation from above. The heart at its arterial end is curved, foreshadow- ing the development of the ventricle as a defined chamber. Although the heart chambers are not morphologically differentiated they are physiologically differen- tiated, showing stepuise depression of contractility progressively involving the presumptive ventricle, atrium and sinus venosus if the embryos are immersed in KC1 isosmotic with sea water. The brain ventricles have increased in size, especially the fourth ventricle which has a thin roof and is bounded anteriorly by a well denned rhombic lip. The paired otoliths are first observed in this stage as aggregations of very fine dark granules. The tail has increased in length, has lifted off the yolk sac at its root, and the fine droplets at the root of the tail are scattered along the posterior vitelline vessels. At the root of the tail the aorta arches ventrally onto the yolk sac, there branching into the posterior vitellinc vessels, \\ithin this arch is ^i niie condensation of cells from which the bilobed urinary bladder will form. There are also a few scattered contracted erythmphores on the yolk sac. Somitic movements are more frequent than in the preceding sta^e but not much more- vigorous. Stage 27 This and the subset |uent stages are in general characterized by a continuing increase in the si/e of the emhrvo, a gradual decrease in the si/e ol the yolk sac and active organodiflcreiiliation. The ventricle of the heart forms a definite chamber but not as we'll defined as later on. The otnliths are dense concrete bodies. There is a bilateral condensation of cells just posterior to the emerging anterior vitclline arteries, the rudiments ol the pectoral tins. They may jut up slightly above the \olk sac when viewed on the hori/on of the egg along the embryonic axis as the embryo advances through this stage. The body cavity is forming bilaterally but the gut rests ventrally on the yolk sac bisecting the body cavity longitudinally. The tail has increased in length and is still further elevated off the yolk. The melanophores on the surface of the yolk sac are expanding. i'.odv movements have increased in frc<|ucncy. Also in this Mage the prone])! iros actively eliminates dyes, though the urinary bladder is not yet formed (Armstrong, NORMAL DEVELOPMENT OF FUXDULUS HETEROCLITUS 151 Stage 28 This stage is marked by the earliest development of retinal pigment imparting a dusky tone to the eye. The body cavity has extended under the trunk, usually lifting the gut off the yolk sac. There is a blood vessel cour udally along the gut. The urinary bladder is small, bilobed but contains no prccipit: The tail has grown in length and lifted further off the yolk sac but contractility of the somites is limited to those anterior to the ventral flexure of the tail. The pert' fin juts up above the surface of the yolk sac parallel to the. bodv axis. Stage 29 There has been an increase in the pigmentation of the eye, which has also increased in size so that the lens does not completely fill the optic cup. The pectoral fin is a small acuminate projection on the horizon of the egg as viewed along the body axis. The urinary bladder is a small bilobed organ grooved above and anteriorly by a ventrally coursing blood vessel. There is no precipitate in the urinary bladder but selected dyes injected into the embryos in this stage are eliminated by the pronephros and color the bladder contents. The ventricle is well defined as a definitive chamber but the demarcation between the atrium and sinus venosus is vague. Stage 30 Although the retinal pigment has markedly increased, the outline of the lens can still be discerned on transillumination of the eye. The pectoral fin extends slightly above the lateral line. The tail is completely straightened out. On lateral flexion, the tip of the tail passes over the hindbrain. The aorta terminates in a small glomus in the base of the rudimentary caudal fin which is just beginning to flatten out. Stage 31 The heart chambers are all differentiated. The atrium is to the left and anterior to the ventricle, not directly to the left as it will be later, nor is it yet completely filled out. The tail fin shows additional flattening with a bifurcation of the aorta, one branch extending dorsally into the caudal fin, the other ventrally. The body cavity is well formed with the gut and liver in view. The liver is to the left of the body axis when viewed from above and blood can be seen circulating through the liver sinusoids. A small vessel is forming parallel to the pectoral fin margin. Circula- tion in this vessel usually appears late in this stage. There is a finely divided precipitate in the urinary bladder, which is bilobed. the two lobes communicating with each other through a large opening. Stage 32 The retina is more heavily pigmented, so much so that the outline of the lens can be seen only with strong transillumination. The atrium has increased in size. The posterior margin of the operculum is forming below the anterior margin of the otocyst, with a shallow depression extending forward deep to this margin. Branchial arches may be seen. There is a single blood vessel arching in the pectoral fin parallel to its border but as yet no motility is observed in the fin. There is a 152 PHILIP B. ARMSTRONG AND JULIA SWOPE CHILD tripartite branching of the aorta into the caudal fin with additional radial vessels appearing- later in this stage. Reflex vagal inhibition of the heart is elicited on mechanical stimulation of the embryo in this stage or shortly thereafter (Armstrong, 1931). During the past several stages there has been a developing flexure of the head at the level of the hindbrain, \vhich is most marked at this stage. In the succeeding stages the head gradual!} straightens out. The ectoderm of the yolk sac comes off the embryo anteriorly at the lower level of the forebrain. The rudiment of the lower jaw is inferior to this, under the head. The retina is heavily pigmented. masking the deep part of the lens. There may be sporadic movements of the fins but no coordinated rhythmic movements. Kays in the caudal tin are faintly seen, with some unexpandcd pigment cells along the rays. I hree to five radial vessels originate from an arterial arch in the base' of the caudal I'm and pass distally parallel to the tin rays. Stage 31 The lower jaw is well developed, the mouth is open and occasionally lower jaw movements are observed but these movements are not coordinated with movements of the gills, which are still immobile. The yolk sac ectoderm comes off the lower jaw just below the mouth. The pectoral tins mav occasionally show fluttering move- ments between long periods of quiescence P>od\ movements evoked by gross stimulation are spasmodic and uncoordinated and are frequently accompanied by fin movements (Coghill, 1933). The ravs in the caudal tin are well developed, with blood vessels radiating out between the ravs. There is a ventral fin along the entire length of the tail. The swim bladder is small and inconspicuous. Hatching takes place shortly after the mouth has come open, due in part to an enzyme liberated by cells in the mouth and gill cavities (Armstrong, 1936). Stage 35 Some extension of the head has taken place. The ectoderm of the yolk sac now comes off the ventral aspect of the head just in front of the continuity of the conns arteriosus with the ventral aorta. The margins of the operculum are well defined; no movements were noted but there were movements of the eyes. Un- dulatory swimming movements occur but are transitory and are not sustained enough to propel the embryo; however, there are well sustained alternating move- ments of the pectoral fins with intervals of quiescence. A dorsal fin is forming but is not as extensive as the ventral tin. Static 36 There has been some turther extension of the head and an apparent decrease in the size of the yolk sac. There is little if any significant change in the functional features of these embryos as compared with the preceding stage. Stage 37 The reduced size of the yolk sac, the extent of the development of the dorsal and ventral tins and the- serrations of ibe caudal fin are aids in differentiating this NORMAL DEVELOPMENT OF 1-1 XDULUS HETEROCLITUS 1 -^ stage. This stage is also marked by the maturation of motor activity. There is coordination of the lower jaw and opercular movements, and of the undulating swimming and pectoral fin movements. Staf/ c 38 There is a further reduction in the yolk and a strengthening of swimming activity. Stage 39 This is a transition stage from the embryonic to the larval state with the complete absorption of the yolk. The operculum and pectoral fins are almost continuously rhythmically active. The swim bladder has increased in size and the embryos commonly swim at the surface of the water but will swim to the bottom of the aquarium if disturbed, the pectoral tins being in constant motion. ]f the fish are anesthetized, the pectoral iin movements cease and the embryos float to the surface. LITERATURE CITED AMBERSON, WILLIAM R., AND PHILIP B. ARMSTRONG, 1933. The respiratory metabolism of Fundulus hctcroclitits during embryonic development. /. Cell. Coinp. Physio!., 2: 381-397. ARMSTRONG, PHILIP B., 1931. Functional reactions in the embryonic heart accompanying the ingrowth and development of the vagus innervation. /. £.r/>. Zoo!., SB: 43-67. ARMSTRONG, PHILIP B., 1932. The embryonic origin of function in the pronephros through differentiation and parenchyma-vascular association. Amcr. J. Anat., 51: 157-188. ARMSTRONG, P. B., 1936. Mechanism of hatching in Funduhis hcteroclitiis. Biol. Bull., 71: 407. BIGELOW, HENRY B., AND WILLIAM C. SCHROEDER, 1953. Fishes of the (iulf of Maine. Fish. Bull., US.. 53: 1-577. COGHILL, G. E., 1933. Somatic myogenic action in embryos of Fundulus heteroclitiis. Prnc. Sac. Exp. Biol. Mcd., 31: 62-64. COSTELLO, D. P., M. E. DAVIDSON, A. EGGERS, M. H. Fox AND C. HENLEY, 1957. Methods for Obtaining and Handling Marine Eggs and Embryos. Marine Biological Laboratory, Woods Hole, Mass. EIGENMANN, CARL H., 1890. On the egg membranes and micropyle of some osseous fishes. Bull. Mus. Comp. Zool. Harvard. 19: 129-154. HUVER, CHARLES W., 1960. The stage at fertilization of the egg of Fundulus hcteroclitiis. Biol. Bull.. 119: 320. KAGAN, BENJAMIN M., 1935. The fertilizable period of the eggs of Fundulus hctcroclitits and some associated phenomena. Biol. Bull., 69: 185-201. NICHOLAS, J. S., 1927. The application of experimental methods to the study of developing Fundulus embryos. Proc. Nat. Acad. Sci., 13: 695-698. NICHOLS, J. T., AND C. M. BREDER, JR., 1927. The marine fishes of New York and Southern New England. Zoologica, N. I'., 9: 1-192. OPPENHEIMER, JANE M., 1937. The normal stages of Fundulus hcteroclitiis. 68: 1-15. OPPENHEIMER, JANE M., 1959. Extraembryonic transplantation of sections of the Funduhis embryonic shield. /. £.r/>. Zool., 140: 247-267. SHANKLIN, D. R., AND PHILIP B. ARMSTRONG, 1952. The osmotic behavior and anatomy of the Funduhis chorion. Biol. Bull., 103 : 295. SOLBERG, ARC-HIE NORMAN, 1938. The development of a bony fish. Progr. Fish Cult., 1-19. TRINKAUS, J. P., 1951. A study of the mechanism of epiboly in the egg of Funduhis heterochtus. J. Exp. Zool.. 118: 269-319. TRINKAUS, J. P., 1963. The cellular basis of Fundulus epiboly. Adhesivity of blastula and gastrula cells in culture. Dev. Biol., 7: 513-532. 154 PHILIP B. \KMSTRONG AND JULIA SXVOI'K CHILD CHARACTERIZATION <>i STAGES Si.itii- \ Unfertilized ovum Stages 2-7 Clc.i \.iur Stages 8- 10 Cell nuilliplic.itioii Stages 11-14 Hl.iMiil.ie Stages 15-18 Gasirulae Siages 1() 20 Xeunil.ie Stages 21 .>•> Growth and organodifferentiation (Magnification, 22 X > TIME Si. (.'I ENCE OF DKYKI.ni'MKVI IN lldl KS AT 20° C. ± 0.2 Stage Time 1 0 2 1:75 3 2 :5() 4 3 :25 5 4:25 6 5 :0() 7 6:00 8 7 :50 9 0 :00 10 10:00 11 11 :()() 12 15:00 L3 20:00 1-1 24:00 IS 27:00 16 30:00 17 M :()() 18 37:00 19 40:00 20 46 :00 Stage Time 21 52:00 22 56:00 23 66:00 24 74:00 25 84 :00 26 ()2 :()() 27 112 28 12S 2(> 144 30 156 31 168 32 192 33 216 34 228 35 252 36 288 37 336 38 360 39 384 NORMAL DEVELOPMENT OF FUNDULUS HKTKROCLITUS 155 _/-* ' >^-irV !• -~ tr- ' ' - ' jiXi. : •rn::%?p3_ ;-5l^f^ u\^^"^V^---jV"C"'J-'; ^-'-i '-* l^r , ~^..'' ,l-i "v.*-^rf '"^0^ Wj..Xy(^. 'i-^'i ! "• J '\ ',r -• . -"--.. 'CVrtvv*v-i-^i'N ^SSsi$^S^^^'SO^Sc^f^'i:^ai:S^i^ 156 PHILIP r.. \KMSTROXG AXI) JULIA S\V()I'K CHILD 8 NORMAL DEVELOPMENT OF FUXDULUS HETEROCLITUS 157 . 15 158 PHILIP B. ARMS! RONG AND JULIA SWOPE CHILD 16 NORMAL DEVELOPMENT OF FUXDULUS HETEROCLITUS 15') 19 21 oil ~ 161) PHILIP H. ARMSTRONG AND JULIA S\\OI'K CHILD . ^~ ^^, ^ ., •-'< o -,* W . ,*: O ••- r-P ° / • O * - o • O / • ^•S^H!kN° o m f V ;/' 24 -o - NORMAL DKYKLOI'MHXT OF Fl'XDULUS HETEROCLITUS 161 ' s—\ ^ CP O • .^<5 , 'O,-? ; if y ' • vtW . ' O 25 *^M^ 26 V7. •teQ? 's~\ • — ' t >«-5«B ' *">?», - * O 7*v '•» H( !$ ^ B^A% fo\ Bi;a i.x-. -^ x'v- i-^ y^.:.^r 27 27 162 PHILIP I1,. ARMSTRONG AND IU.IA SWOPE CHILD * & . * ..--''.•' ^ «l : i ij@n * 28 JvmiSess «V^«5 '/ Ai f' ^ 7"\; r ' ' -,' tV. • -' •'fiut ' ' "• £ -^~ i yj l ff * ^f ' t * ' T WlH¥;/ -^ w I 29 I -a 1 29 :i.-~-'r-""»,^. ^, ^| '.'•/ f \ «it -t '?<> ;; rf .<( 1 ^y v l'mijjf f -rx \\%m?y -p - ^ ° * ^ i 30 30 \( (KM \\. DK\ KL< >PM] \ : ' , i ' : : • >• 163 31 ^^^^fr^. ''Jf A. ' i If.* // I ^7^7pt'J^***'?*7 -V^f-'jr1 -i 5v-2 , -x- -<^^:^ft^C'*t*;i''---^-*(-^Xi*r-^-*J^-1'-1'I1^i — Vy/,- ' ' >feJL\^.- ,*""1-"*2"''1 ^''V^* -Ji ^•' •to / il'~5 .: i! 38 168 PHILIP B. ARMSTRONG \\l> JUT.TA SWOPE CHILD m I h 1 39 K • THE CHROMOSOMES OF SEA URCHINS, ESPECIALLY ARHACIA PUNCTULATA; A METHOD FOR STUDYING UXSECTIOXED EGGS AT FIRST CLEAVAGE W \LTER AUCLAIR /V/if. uf Biological Sciences, University of Cincinnati, Cincinnati, Ohio 15221 anil Marine Biological Laboratory, U'oods Hole, Muss. 02543 Previously, there has been no truly adequate method for studying the chromo- somes of sea urchin eggs, even when sectioning procedures were utilized. With classical methods, such as acetic orcein, carmine and the Feulgen techniques, the cytoplasm stains rather intensely, which tends to obscure the chromosomes. More- over, the chromosomes are very small and numerous. Consequently, the chromo- some number and morphology have never been determined with certainty in Arbacia punctulata and in many other marine invertebrates that have large, yolky eggs. The present paper describes a simple and relatively rapid procedure that has been developed to examine the chromosomes and nuclear events of the early cleavage divisions of unsectioned eggs by a selective staining of the chromatin. With this method the chromosome number of A. piinctitlata has been established and that of the sand dollar, Echinarachnius panna. closely approximated. Also included is a description of the morphology of the chromosomes of the former species. A preliminary report has been published previously (Auclair, 1964). THE METHOD Shedding of the eggs of both A. punctulata and E. /><;;•//;BSI i- By this method the sequence of nuclear events from fertili/ation through < first two cleavage divisions has been examined in unsectiectioned egg of Arbacia pitnctulata. The smaller, more condensed male pronucleus is coining into contact with the larger female pronucleus. pronucleus then flattens over the female pronucleus surface and gradual fusion occurs, fi cation, approximately 2000 X. FIGURE 4. A partially squashed prophase preparation of an Arbacia egg, demonst the coiling of the elongated chromatid threads. Oil immersion-phase contrast approximately 2000 X. FIGURE 5. Metaphase chromosome spread of a colchicine-treated first cleavage division. The chromosomes do not split in synchrony and there is no attachment at the centromere position. Oil immersion-phase contrast ; magnification, approximately 2000 > 174 \\ \LTER AUCLAIR 1 8 10 11 12 13 14 15 16 17 IS 19 20 21 22 6 FIGURE 6. Karyotype obtained from the pre-metaphase chromosome spread preparation of Figure 2. Total magnification of the chromosomes, approximately 4000 X. Echinarachnius pan/ut. The chromosomes of the egg of the sand dollar also were examined by this method. These1 much larger eggs are more difficult to study because of the wide area ot dispersion of the chromosomes in the cytoplasm in spread preparations, ('omits gave either 44 or 46 chromosomes for the diploid number. The chromosomes of the sand dollar are somewhat larger than those of the sea urchin, and the centromere position tends to he more medial. An unequivocal karyotype still cannot be determined with any accuracy. Karyotype oj - "- >•' Arbacia punctulata during llirjirsl cleavage division ( liromosome 1 •i I 5 '' 10 1 t 15 H> 17 1 ') 20 1 \ 11 I i< > -up Large chromosomes, m-.irl\ terminal cehtromeres Chromosomes not as l.ir^c, IHHIT medial eenlroimTes Medium size chromosomes, approximately mi-di.m rent nmu-rr- \li-dmm size chromosomes, sub-median centromeres Mcdmni >i/c chromosomes, nearh' terminal cenlroinci'o Smaller chromosomes, nearly terminal centromeres Shmi chromosomes, --nli-mcnnchtlata. These vary from as low as 32 (Harvey, 1940) to as high as 40 (Tennent, 1912). Jn a discussion of these results, Harvey (1956, p. 102) concludes that most sea urchins, including A. pitnclulata, have 3o t<> .>X chromosomes, in agreement with the number reported by Matsui (1924). Halt/rr (1910), however, found 40 chromosomes in a related species, A. H.vnla. It is ; ])ortant to realize that in all these early studies the chromosome counts were made on sectioned material, which introduces many difficulties and uncertainties. More recent studies indicate a higher chromosome number, namely 42 to 44. Nishikawa (1961), on the basis of examining the meiotic divisions of spermatogonia, found a haploicl number of 21, or a diploid value of 42, for two species of Japanese sea urchins, Hcmicentrotus pulcherrimus and Anthocidaris crassispina. The num- ber. 44, reported in the present study, has also been confirmed recently by German (1964), who examined the chromosomes of A. pitnclitlata in the blastula stage blastomeres by the standard acetic orcein squash technique. German described attenuation at the kinetochore region of the metaphase chromosomes, so that it is likely that the lack of centromere attachment in the present work is due to the new technique and is not characteristic of sea urchin chromosomes. It may be that most echinoderms have a similar chromosome number and do not vary as widely as shown in the summary compiled by Makino ( 1951 ). One reason for this assumption is the chromosome number of E. panna, found in the present work to be 44 or 46, which is more in line with that found in Arbacia, and varies considerably from the earlier value of 52 determined by Matsui (1924). The method here reported should prove useful to workers dealing with physical studies on marine eggs, especially Arbacia, during the early cleavage divisions. It makes it possible to determine, without sectioning the eggs, the degree of synchrony and the exact stage of mitosis (Fig. 1 ) at the time of an experimental treatment. SUMMARY A method has been developed to stain the chromosomes and nuclei of unsectioned sea urchin and sand dollar eggs during the early cleavage divisions. The chromo- some number of Arbacia f'nnctulata is 44. and a karyotype of the pre-metaphase chromosomes of the first cleavage division has been made. The chromosome number of Echinarachnius panna is 44-46 and no karyotypc has as yet been made. LITERATURE CITED AUCLAIK, YV., 1964. On tin- chromosome number of Arbacia. Biol. Bull., 127: J BALTZER, F., 1910. Uber die Beziehung zwischcn dem Chromatin und der Entwicklung und Vererbungsrichtung bei Echinodermenbastarden. Arch. f. Zellfor., 5: 497-621. FRY, H. ]., 1936. Studies of the mitotic figure. V. The time schedule of mitotic changes in developing Arbacia eggs. Biol. Bull.. 70: 89-99. GERMAN, ]., 1964. The chromosome complement of the blastomeres of Arbacia punctulata. Biol. Bull., 127: 370-371. HARRIS, P., 1961. Electron microscope study of mitosis in sea urchin blastomeres. . Bioph. Bioch. Cytol, 11:419-431. 176 \V \LTER AUCLAik HARVEY, E. B., 1940. A comparison of the development of nucleate and non-nucleate eggs of Arbacia punctithita. Bio/. Bull., 79: 166-187. HARVKY, E. B., 1956. The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, X. J. M \KI.\O, S., 1951. An \tlas of tlie Chromosome Number in Animals. Iowa State College I *re>s, Ames. lo\\ a. M. vrsri, K., In24. Studie> on the hybridization amont; the Echinoderms with special reference to the l)ehavior of the chromosomes. J. Cull. .l;i>'. Imp. L'uit'. Tokyo. 7: 211-236. XISIITKAWA. S.. l''()l. Xotes on the chromosomes of lu'liinoderms, Haniccntrntiis pulcherrimits (A. Aiiassix ) and . Intliociduris crtissispina (A. Agassiz). Do/u/ts. /.usshl, 70: 425-428. 'I "i \\K.\T, 1 ). II.. 1912. Studies in cytology. I. A further study of the chromosomes of r,>.\-ot>n,-itstcs z-iirici/iitiis. 11. The behavior of the chromosomes in .•Irhiicui- Toxopncustes crosses. /. E.\-p. Zool.. 12: 391-411. PHOTOPERIODIC CONTROL OF A PHYSIOLOGICAL RHYTHM ' STANLEY D. BECK, IRENE B. COLVIN AND DONALD E. SWINTON Department of Entomology, University of Wisconsin. .Mtidismi (,. H'/.svi>,';\i» A hormone, proctodone, was recently discovered to be produced 1>\ epithelial cells located in the anterior portion of the proctodeum of the larval European corn borer. Ostrinin nnhiUilis (Hiibner) (Beck and Alexander, 19()4a). Proctodone appeared to be responsible for the activation of the neurosecretory processes leading to the production of the prothoracotropic hormone, thereby constituting a major endocrine component of the physiological processes underlying diapause develop- ment and prepupal morphogenesis (Beck and Alexander, 1964b). Cytological evi- dence of daily secretory cycles in the proctodone-producing epithelial cells was obtained, and some of the possible relationships between rhythmic physiological functions and extrinsic photoperiodic signals have been discussed (Beck, 1964). In the earlier publications, cited above, it was postulated that the rate of diapause development may be determined by the degree of synchronization between two or more interacting or interdependent physiological rhythms, such that when the rhythms were physiologically "in phase," development was rapid and diapause was soon terminated. Conversely, diapause development was slowr and the diapause state greatly prolonged when the participating rhythmic functions were physio- logically "out of phase." In support of this "phase theory" of developmental control, evidence was presented of the existence of secretory rhythms in both proctodone-producing epithelium of the proctodeum and lateral neurosecretory cells of the larval brain (Beck, 1964). The rhythms were found to be influenced by the environmental photoperiod, with the neurosecretory rhythm being sensitive to the onset of light and the proctodeal rhythm being sensitive to the onset of darkness. Both rhythms were found to be characterized by the possession of an 8-hour period, rather than the expected 24-hour period. Since the rhythms ran through three full cycles per day, they cannot be classified as circadian rhythms, although they cer- tainly utilized circadian photoperiodic signals as "Zeitgeberen." The present study was undertaken in an effort to clarify some of the relation- ships between photoperiodic signals and the temporal characteristics of the proctodeal secretory rhythm. Emphasis has been on the cytological changes involved in the rhythmic function, rather than on the role or fate of the physiologically active sub- stances produced during the secretory process. Although a number of physiologi- cal processes, including some endocrine functions, have been shown to display rhythmicity in plants, invertebrates, and vertebrates, there has been a marked paucity of cytological evidence in support of the hypothesis that secretory processes may show rhythmic characteristics (Halberg, 1960; Harker. 1960; Biinning, 1963 ; Beck, 1963 ; Wolf son, 1964) . 1 Approved for publication by the director of the Wisconsin Agricultural Experiment Station. This study was supported in part by a research grant (GM 07557) from the National Institutes of Health of the U. S. Public Health Service. 177 178 s. D. m-XK. i is. COLVIX AND D. i-:. s\vi. \TONI METHODS AND MATKRIAI.S The experimental insects employed in this investigation were larvae of the European corn horer. The larvae were reared on meridic diets as previously de- scribed (Heck, 1962). with diapause being induced by means of short-day photo- periods ( 12 hours of light and 12 hours of dark) during the larval growth period. Some experiments involved exposure of larvae to long-day conditions, which was accomplished bv means of incubators programmed for 16 hours of light and S hours of dark per day. All of the shorl-dav and long-dav incubators were synchro- nixed so that the clock times of the onset of light were identical. The photo- periodic conditions differed, therefore, only in the clock times at which the lights-ofT signal occurred. With onlv a few exceptions, noted specifically below, the tem- perature used for rearing, treatment, and post-treatment was 30° C. Tissue samples for permanent slide preparation were fixed in Houin's fixative, imbedded in paraffin, sectioned at 7 microns, and stained. Staining was by either iron hematoxylin and eosin or the paraldehyde-fuchsin technique of Cameron and Steele (1959). Nearly all of the experiments involved the examination of fresh proctodeal tissue for the presence of intracellular secretory granules. This was accomplished by dark-field fluorescence microscopy; a Leitz ultraviolet accessory was used, employing a U(i-l 2-mm. exciter filter and luiphos barrier filters. Living larval proctodeums were dissected out, and excess fat and tracheae removed. They were then split laterally, mounted flat in water on a microscope slide, and covered with a thin coverslip. The secretory granules occurring in the proctodeal epithelium were previously observed to display autofluorescence, leading to the detection of cyclic cell activity (Heck, 1964). In the present study, the secretory cycle was followed by the examination of tissue samples collected at different times during the photoperiodic cycle. Permanent records of the relative intensity of the fluorescence and the cellular disposition of the fiuorescing granules were obtained by photomicrography. For this purpose. 35 mm. color film was used (High-Speed F.ktachronie, ASA 160) with a standard shutter speed of 120 seconds. The pictures were developed and mounted as 2x2 inch transparencies. All subsequent considerations o! the secretory state of the samples were on the- basis of the colored photomicrographs, as the tissue samples themselves could not be preserved. In order to study tin- .secretory cvcle and the effects of variables on the cycle, it was necessary to devise a method for comparing the secretory state of different tissue samples. For this purpo.se. the secretorv cycle was divided into a sequence ot 5 arbitrary stages based on the characteristics of the fluorescence recorded in tin- colored photomicrographs. Hlack and white prints of the photomicrographs con- stituting the standard examples of each stage are presented as Figures 1-5. The general distinguishing characteristics of each of the several stages are given with the figures. Although .subject to the limitations invariably accompanying an atomistic consideration of a continuum, the use of this arbitrary scale of relative .secretorv activity has facilitated the studv and has yielded consistent and re- producible results. \\ ith only tew exceptions, all ol the epithelial cells of a given proctodeal sample were virtually identical in respect to the observable fluorescent inclusions, making PHOTOPERIODN Rm i HM roXTROL 179 FIGURE 1. Stage 0 of proctodeal secretory cycle. Cells are devoid of visible fluorescent cytoplasmic granules. FIGURE 2. Stage 1 of proctodeal secretory cycle. Cells contain scattered fluorescent granules; cell outlines are just discernible. FIGURE 3. Stage 2 of proctodeal secretory cycle. Cells contain numerous large fluorescent granules; cell and nuclear outlines easily discernible. FIGURE 4. Stage 3 of proctodeal secretory cycle. Fluorescent cytoplasmic granules vary numerous ; nuclear outlines well-defined. FIGURE 5. Stage 4 of proctodeal secretory cycle. Cells packed with finely divided fluores- cent inclusions ; nuclei partially obscured by inclusions. ISO s. 1). lll'A'K, I. I1,. COLVIN AM) I). !•'.. SWINTON it relatively easy to assign each preparation to one or another of the stage classifications. The procedure for assigning secretory stage values to the photo- micrographs was as objective as practicable. The pictures obtained in an experi- ment or series of tissue samples were marked with code numbers, randomized, and compared individually with the series of standards. On the basis of such com- parison, each was assigned to one of the arbitrary stages. Following this evaluation, the photomicrographs were decoded, and the stage values were recorded according to the experimental series to which they belonged. Statistical analyses of the data so obtained were limited to frequencv distribution considerations. The use of statistical tests, such as analyses of variance, was avoided because of the mathe- matical artificiality of numerical data describing arbitrary classifications. RESULTS AND DISCUSSION Rliytlnii cliaract eristics Stained sections of the anterior portions of the proctodeums of mature diapaus- ing European corn borer larvae usually disclosed that the epithelial cells contained granular cytoplasmic inclusions that stained dark purple with paraldehyde-fuchsin. The presence of such granules, irregular multilobate nuclei, and numerous small cytoplasmic vacuoles was interpreted as being indicative of much synthetic activity. In some specimens, the cells were devoid of the stainable granules, although showing the typical vacuoles and multilobate nuclei. Whether or not the epithelial cells contained stainable secretory granules appeared to depend upon the time of day that the larvae had been fixed. These observations suggested that secretory activity was on a cyclic basis. ( )ur interpretation has been that the paraldehyde-positive products accumulate in the small cytoplasmic vacuoles, and are then secreted into the hemolymph through the basal cytoplasmic membrane and the basement mem- brane underlying the cell layer. In the immature, feeding stages of the insect, these cells are known to absorb water and digested substances from the gut lumen and to secrete them into the hemolvmph (Wiggles worth, 1^50). During diapause and postdiapause, the larvae do not feed and the gut tract is quite empty. Our observations indicate, however, that the cells retain the capability of secretion into the hemolymph, and have an endocrine function. Study of the secretory cycle has been greatly facilitated by the finding that the secretory granules display a pale green autofluorescence in the fresh state. The cycle of elaboration and secretion could be followed by the examination of successive timed tissue samples without the laborious delay involved in fixation, embedding, sectioning, and staining. It was found that at the stage of the secretory cycle in which no paraldehyde-fuchsin-staining granules were present, no lluorescent gran- ules were detectable. This observation led us to the tentative interpretation that the stainable particle^ and lluorescent particles were identical. The absence of lluorescent inclusions in the epithelial cells was characterized as Stage 0 of the secretory cycle ( Fig. 1 i, and was interpreted as being the result of the cells' having liberated their accumulated secretory products. For reasons to be discussed below, this stage of the secretory cycle is thought to be of relatively short duration. Alter Stage 0, the cells again begin to accumulate secretory products, as shown bv progressive increase in the amount ol lluorescent granules PHOTOPERIODIC RHYTHM COXTROL 181 Stages 1, 2, 3, and 4 (Figs. 2-5 I. Our observation- have led us to believe that the cells are capable of liberating their products after they reach either Stage 3 or 4. For the purposes of studying the effects of photoperiod on the secretory cycle, Stages 0 and 1 were of the greatest interest, and their occurrence was contrasted to the occurrence of the other secretory stages. Proctodeal tissue samples were taken hourly from group* of diapau.Miig borer larvae that were maintained under either long-day or short-day pho results of typical 24-hour series are shown in Figures 6 and 7, in \vhkh each tissue sample examined is symbolized by a small circle, with the occurrence of Stage.-- 0 and 1 being depicted bv M>lid black circles and the other stages by open circles. 8 0 HOUR 16 20 FIGURE 6. Temporal distribution of proctodeal secretory cycle Stages 0 and 1 (solid circles) among diapausing larvae of the European corn borer held in a short-day photoperiod. It was apparent that Stages 0 and 1 did not occur at random during the 24 hours. but tended to be most frequent at the beginning of the scotophase (dark phase of the photoperiod) and at S-hour intervals thereafter. The borer larvae used in the series depicted in Figure 7 had been transferred from a short-day photoperiod to a long-day photoperiod 10 days prior to the experiment. The results indicate, therefore, that the insects were capable of adjusting the secretory cycle in accord with the photoperiod and that the beginning of the scotophase determines the time of occurrence of the subsequent secretion times. We have confirmed this conclusion by the results of a number of experiments in which the borer larvae were required to adjust to changes in the time of the beginning of the scotophase; the secretory rhythm always shifted so that one of the times of secretion (Stages 0 and 1) approximately coincided with the onset of the scotophase. The proctodeal 182 S. D. BECK, I. I'.. COLVIN \\l> I). !•.. SWINTON secretory rliythni is teui] » >rallv adjusted through tlic insect's response to tlie lights- ot'l stimulus provided bv the extrinsic photoperiod. The proctodeal secretory rhythm is not. in itself, a cireadian rhythin. because il displa\s a period approximating S rather ti.an 24 liours. It is phase-set (temporally adjusted) bv tin- 24-hour rhythm of daylight and darkness, and is most certainly a component of the in>ect's photoperiodism. The ])ossible significance of the effects of lights-on and lights-off stimuli on S-hour physiological rhythms in the control of growth processes was discussed in an earlier paper ( P>eck. Q HOUR 16 20 FIGURE 7. Temporal distribution of proctodeal secretory cycle Stages 0 and 1 (solid circles) among diapan>ing larvae of the European corn borer held in a long-day photoperiod. Data such as those of Figures 0 and / gave rise to some serious questions concerning the actual time-course of tin- physiological rhythm. One such question pertained to an explanation of the relatively low fre»|uency of occurrence ot Stages 0 and 1 at the be^inuin^ of the scotopha^e. \ot all (or even most) of the larvae sacrificed at that time showed proctodeal tissue that was nearly devoid ot thioresceiit granules. One might expect that the incidence of Stages 0 and 1 would he much higher than was actually observed, if the population was to be considered reasonably homogeneous and if all of the larvae were responsive to the' photoperiodic stimuli. These considerations prompted us to undertake a more detailed study of the distri- bution of secretory stages during the hours immediately before and after the beginning ot the scotophase. The percentage incidence of the several arbitrary stages of secretion for some different time periods is shown in l-'i^ure S. "Midcvcle" is the 60-minute period between 4.5 and 3.5 hours prior to the lights-off time; " 1 hr." identifies samples PHOTOPERIODIC RHYTHM CONTROL 183 examined between 90 and 30 minutes prior to the lights-ofi" time; "0 hr." designates samples taken from 30 minutes before to 30 minutes after the lights-off time; and ' + 1 hr." designates tissue samples collected from 30 to 90 minutes after the begin- ning of the scotophase. About 40 larvae were dissect < \amined to obtain the data for each of the different time periods. The "midcyde" distribution shows Stage 3 to be the most fn -:< merit, with Stages 3 and 4 constituting the condition of over 80% of the insect.- i \amined. At tl.U time there were no Stage 0, and Stage 1 was rare (one instance out of a total sample of 40 larvae). At " — 1 hr." the distribution changed only in that Sta^'e 0 and o ^ o 60 50 • z UJ 0 40 - QC UJ a Z 30 - UJ O z u 20 Q — ' u * i 10 o n in 1 n 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1234 MIDCYCLE -1 HOUR 0 HOUR -H HOUR (-1.5 to -0.5 hr) (-0.5 to +0.5 hr) b-0.5 to +1.5 hr. ) FIGURE 8. Incidence of different arbitrary proctodeal secretory stages (0-4) among European corn borer larvae examined at different times during the secretory cycle. Stage 1 were each present to the extent of about 5(/( . The distribution at "0 hour" showed two pronounced changes : (a) the incidence of Stage 1 was greatly im reused, and (b) the incidence of Stage 3 was much lower than in previous samplings. The distribution at " + 1 hour" showed a decline in the incidence of Stage 1 and an increase in Stage 3. Although not included in Figure 8, histograms of the incidence of the different secretory stages at +2 and +3 hours showed a return to the "midcycle" distribution. The cell state observed in Stage 0 and Stage 1 proctodeal tissue samples was interpreted as an indication that the cells had liberated their secretory products shortly before the larvae had been dissected. For this reason, the incidences of these two secretory stages were considered together, and their incidence compared 184 S. I>. BECK i B. r<)l.\ IN VXD I). K. SWIXTnX to the incidence of the other arbitrary stages in subsequent experiments. Combining the data from a number of series in which the larvae examined were from the standard short-day photoperiod. the temporal distribution characteristics of Stages 0 and 1 were determined for the period from two hours before to two hours after the beginning of the scotophase (Fig. 9). Of the total number of Stages 0 and 1 observed, 43',' occurred within 30 minutes of the beginning of darkness. The data form an obviouslv normal frequency distribution, with the mean falling very close to the lights-otT time of the photoperiodic cycle. From these data, we can conclude that the insect'- proctodeal secretorv rlivthm is, indeed, temporally adjusted to LJ O cr UJ CL o z O UJ cr 50 40 30 20 0 0 -2 -I 0 -H TIME IN HOURS + 2 LIGHT FIGURE 9. iTcqiu-ncy distribution of combined Stages 0 and 1 during the hours surrounding the beginning of the scotophase. the beginning <>f the scotophasc. .such that the proctodeal cells tend to release their secretory products in approximate synchrony with the lights-off signal. Because the secretory rhythm appears to possess an eight-hour period, it is evident that pliotoperiod can directly influence only one of the three daily cycles; the others must occur endogenously, but timed in accord with the cycle that responded to the photoperiod. Sampling characteristics If the proctodeal cells liberate their secretory products in approximate synchrony with the lights off signal, why is the incidence of Stages 0 and 1 often relatively PHOTOPKRIODK' RHYTHM CONTROL 185 low, even at the "0 hour" as shown in Figure This effect can be explained on the basis of the unfavorable probabilities attending the tissue sampling procedure. The measured response (Stages 0 and 1 ) is assumed to form a normal distribution such that two-thirds of the larvae may be expected to respond within 60 minutes on either side of the lights-off time. A second assumption is ,ide : the duration of Stage 0 is about 15 minutes and that of Stage 1 is about 30 minutes. When a larva is chosen at random for dissection at about the beginning of the scotophase, the probability that it will be among those that respond within the two-hour 60r Z UJ O cr LJ CL UJ O z Q U Z 50 - 40 - 30 - 20 - 10 - 0 -2 - - PHOTOPERIOD + THERMOPERIOD ^ PHOTOPERIOD ONLY 1 30' 10° I 0 +1 TIME IN HOURS + 2 TEMPERATURE FIGURE 10. Effect of a combined thermoperiod and photoperiod on the incidence of Stages 0 and 1 of the proctodeal secretory cycle in diapausing larvae of the European corn borer. period designated above is 0.67. The probability that it will be at Stage 0 at thr time of dissection is 0.67 X 0.125 (because there are eight 15-minute periods in two hours). This means that the probability of observing a Stage 0 tissue prepara- tion at even so favorable a time is but 0.083 ; on the average, one larva out of a sample of twelve should be found to be at Stage 0. If the duration of Stage 1 is twice that of Stage 0, about one larva out of six should be found to be at this stage when the population is sampled during the hour before and the hour after the lights-off time. The expected incidence of Stages 0 and 1 combined would be about 25% of the larvae examined; the "0 hour" distribution data in Figure 8 show a combined Stages 0 and 1 incidence of 28%. S. I). BECK, I. I', COLVIN \\l> D. E. SWINTON These probability considerations have some important implications that reach beyond the present limited study. In am instance where a physiological proofs cannot be tollowed continuously in each individual specimen, the investigator is limited to making' observations on a succession of specimens, each of which has been fixed at some point of time. Under such conditions, especially those in which successive physiological stages art' relatively fleeting, the existence of highly sig- nificant rhythmic functions may be overlooked or, if suspected, difficult to demon- -irate convincingly. In the present investigation, the demonstration of a photo- periodically sensitive secretory rhythm could not have been accomplished in the absence of the moderately large numbers of samples made feasible by the simple autofluorescence technique of examination. Jiffccls 0} thermoperiod An attempt was made to increase the1 incidence of Stages 0 and 1 among the borer larvae at the time of the lights-off signal. A thermoperiod was superimposed on the photoperiod, so that the incubator temperature fell from 30° C. during the photophase to 10° C. during the scotophase. Previous results (Beck, 1962) had shown that low temperatures during the scotophase increased the incidence of diapause, whereas high temperatures at that time tended to prevent diapause. As shown in Figure 10, combining a thermoperiod with the short-day photoperiod had the expected result of greatly increasing the incidence of observed Stages 0 and 1. The large increase in response that occurred at one hour before the light-off signal indicated that the effect was not a simple prolongation of the duration of Stages 0 and 1 because of low ambient temperature. It seems more likely that the effect ot the thermoperiod was one of decreasing the time range of the response, and thereby improving the probability of observing specimens in Stages 0 and 1 during the two-hour period of interest. Sliort-dtiv In l compared to Figure 7). Attempts to trace the detailed course of such a transition have not been wholly successful, however, because of the limitations of our sampling methods and the inherent variability among the individuals of the experimental populations used. A large group of borer larvae that had been reared under a short-day schedule in which the lights-off signal came at 12 noon was transferred to a long-day schedule in which the lights-off signal came at 4 P.M. The incidence' of Stages 0 and 1 was determined for three time periods: (a) 11 AM to 1 I'M, which was one hour on each side of the lights-off time of the original short-day photoperiod ; (b) 1 :30 to 2:30 PM, which was midway between the old and new scheduled lights-off time; and (c) 3 to 5 I'M. which was one hour on each side of the lights-off .signal of the newly imposed photoperiod. The data of Figure 11 are typical of the results obtained trom experiments of this tvpe. The incidence- of larvae responding in accord with the original noontime lights-off signal declined during the S da\s of the experiment. Responses during the 1 :30 to 2:30 interval were observed throughout PHOTOPERIODIC RHYTHM CONTROL 1ST the 8 days, with the incidence about constant from, the second day on. As expected, no larvae responded between 3 and 5 PM on day 0, but the incidence of response increased from day 1 until at least day 6. The results indicate that there was con- siderable individual variation in the larvae's ability to adjust to a four-hour change in the photoperiod. Some larvae were responsive to the new photoperiod by the second day, whereas others were still in the process of making the transition on the 8th day, as evidenced by the incidence of responses occurring in the middle hour of 1:30 to 2:30 PM. Although it was apparent that transient phases occurred during the process of becoming synchronized with a changed photoperiod, we were unable to determine the number of transient responses involved. Nonetheless, these results are in very good agreement with those of earlier work (McLeod and Beck, 1963), in which the effect of photoperiod on diapause development was 40-i ' am~ 1 pm l:30pm — 2;30pm 3pm — 5pm Z LJ — U ac LJ30- Q. Z 20- LJ U ^ LJ Z " D 024 6 8 0 2468 02468 DAYS DAYS DAYS FIGURE 11. Incidence of secretory Stages 0 and 1 during 8 days following a four-hour change in the time of the lights-off signal (European corn borer larvae in diapause). studied. In that work, transfer of diapausing borer larvae from short-day to long- day photoperiods and from long-days into continuous darkness disclosed that some of the larvae could adopt and maintain a long-day developmental schedule within two days, but at least 10 days were required for all of the population to make the adjustment. SUMMARY 1. In mature diapausing larvae of the European corn borer, Ostrinia nubllall (Hiibner), the epithelial cells of the anterior portion of the proctodeum were observed to show evidence of a regular cycle of synthesis and secretion. The cycle involved progressive accumulation and subsequent disappearance of paraldehyde- positive, autofluorescent cytoplasmic inclusions. For the purposes of this study, the secretory cycle was divided into a series of 5 arbitrary secretory stages. 2. The proctodeal secretory activity was shown to constitute a rhythmic func- tion. The rhythm was found to be non-circadian. in that it displayed an approxi- 188 S. D. BECK, T. R. COLV I X \\D D. E. SWINTON mately 8-hour period. .Mich that three complete secretory cycles occurred during each 24-hour day. 3. The proctodeal secretory rhythm was shown to he phase-set by photoperiod. The release of secretions ( loss of intracellular granules ) occurred in response to the beginning of the scotophase ( lights-off stimulus) and endogenously every 8 hours thereafter. 4. A thermoperiod that was superimposed on the photoperiod, so that a low temperature \vas concurrent with the scotophase. had the effect of intensifying the physiological response to the photoperiod. 5. From two to ten days were required for the secretory rhythm to re-syn- chronize with a four-hour change in the clock time of the lights-off signal. Because of great individual variability, the number of transient phase responses could not be determined. LITERATURE CITED BECK, S. D., 1962. Photoperiodic induction of diapause in an insect, Biol. /)'////., 122: 1-12. BECK, S. D., 1963. Physiology and ecology <>t insect photoperiodism. Bull. Rut. Soc. Aincr., 9: 8-16. BECK, S. D., 1964. Time-measurement in insect photoperiodism. .liner. Xatnrnlist, 98: 329-346. BECK, S. D., AND N. ALEXANDER, 1964a. Hormonal activation of the insect brain. Sciciii-c. 143: 478-479. BECK, S. D., AND N. ALEXANDER, l%4h. Proctodone, an insect developmental hormone. Biol. Bull., 126: 195-198. BUNNING, E., 1963. The Physiological Clock. Academic Press, New York. CAMEKOX, M. L., AND J. E. STEKLE, 1959. Simplified aldehyde-fuchsin staining of neurosecretory cells. Stain 7 Vc//.. 34: 265-266. HALBERG, H., 1960. Temporal coordination of physiologic function. Cold Sprhnj Harh. Symp. Qitani. BwL. 25: 289-308. II \kKER, J. E. I960. Endocrine and nervous factors in insect circadian rhythms. Cold Spriini Harb. Symp. Quant. Biol., 25: 279-286. Melj-.oD, 1). G. R., AND S. D. BECK, 1963. Photoperiodic termination of diapause in an insect. Biol. Bull., 124: 84-%. \YH.GI.ES\\OKTH, V. B., 1950. Principles of Insect Physiology. Methuen Publishing Company, I .ondon. \YOLI- so\. \., 1()64. Animal photoperiodism. F'hotopliysiolony, 2: 1-49 THE EGG COCOONS OF SCOLOPLOS ER O. F. MULLER G. CHAPMAN Department of Biology, Queen Elisabeth College, ( r/mrr.w/v «/ Lmidon), London, W .8. England In his review entitled "La ponte et I'incubation die/, les annelides polychelc-." Gravier (1923) listed 12 species which are known to lay their eggs in an agglomera- tion but which do not incubate them. Several more species have since been shown to spawn similarly (Herpin, 1925 ; Aiyar, 1931 ; Wilson, 1932 ; Day, 1934; Thorson, 1946; Bookhout and Horn, 1949; Rullier, 1954; Filial. 1958) but no study of the chemical and physical nature or ecological role of the egg cocoons seems to have been published. The present paper is concerned chiefly with the spawning of Scoloplos annigcr and the chemical nature of the cocoon jelly. S. annigcr has the same general distribution over the shore as Arcnicola marina (Thamdrup, 1935; Linke, 1939). The egg cocoons are found where the worms normally live, on the gently sloping muddy sand of the foreshore which does not dry out at low tide. They have often been described, for example by Cunning- ham and Ramage (1888), Ehlers (1892), de Groot (1907), Thamdrup (1935), Linke (1939), Thorson (1946) and Smidt (1951). At the time of formation the cocoon is a pear-shaped or globular mass of firm jelly, about 1 to 1.5 cm. in diameter and containing from 500 to 1200 eggs, and is inserted into the sand by a stalk about 6 cm. long. Since a mature female may contain between 3000 and 5000 eggs, it looks as if a worm may form more than one cocoon (Smidt, 1951). SPAWNING Numerous authors, for example Mau (1882), Thorson (1946) and Anderson (1959), state that spawning occurs in the spring and lasts for about a month, its exact dates varying with the particular district. At Whitstable spawning takes place in February or March and lasts little more than a week. The method of forecasting the spawning period bv the measurement of oocytes showed, during the winter 1949-50, that spawning might be expected in February, 1950. Its onset occurred on February 18, 1950, with a suddenness which is shown by the following observations. During a careful scrutiny of a wide area of the shore at Whitstable, lasting from 08.00 to 10.00 hr. on 18 February, only 5 (total) cocoons were SITU. After tin- shore had been covered by the midday high tide an average of two to three cocoons per ss. private communi- cation i while at Southend-on-Sea, on the opposite .side of the Thames Estuary, a tew worms spawned in February but the massive spawning did not occur until about 24-25 .March. l-oniittfioH of tJic cocoon Cocoon formation lias not been achieved in the laboratory and is difficult to observe on the shore, as it occurs at high tide. It is not known how many worms take part in the formation of a cocoon but indirect evidence suggests that male and female worm- come together in a burrow during spawning so that the eggs are fertilized as they leave the terminal glandular portions of the nephridia of the female. It is these organs which secrete the jelly which envelops the worms and their eggs while they are still within the burrow. On one occasion two worms were found within the stalk of a new cocoon and a search in the sand immediately below newly-formed cocoons usuallv revealed male and female worms occurring close together. The sex ratio, male to female, is approximately 1.3:1. The shape of the cocoon, that of a single large drop of a viscous substance emerging in mi a tube, is clearlv due to its method of formation. Rarely are mal- lornied cocoons found although occasionally two smaller drops are attached to a single stalk and more rarelv an additional stalk is found, as if one of the worms entered or left the cocoon by a different route. On one occasion male and female worms spawned in the laboratory soon after collection and before they had estab- lished their proper burrows. The cocoon jelly was released below the surface of the sand in irregular masses, emphasizing that the form of the cocoon normally results from ;ts method of formation as a drop emerging from a tube. De Groot (1909) stated that copulation occurs, but Anderson (1(>59) was unable to tmd sperm or fertilized eggs in the coelorn of the female. Observations made on stained sections of fixed ripe females confirm this; neither were living -permato/oa seen in the jelly of freshly-laid cocoons although the eggs which they contained were fertilized. It looks, therefore, as if the eggs are fertilized im- mediately they leave the body cavity of tin- female' before the jelly has had time to swell. THE COCOON JELLY Sniircc <>l the material The jelly of which the cocoon is formed is provided by the female and comes From the -penalized end portions of the nephridia. of which about 100 pairs in the middle region of the body develop conspicuous white swellings at the bases of the notopodia ( Fi.sig, 1914). These glandular portions surround the nephridiopores and consist of very larg • . 11-. each of which is greatly distended in the breeding season with aggregation- i measuring 20-30 //, in diameter) of rod-like bodies, each ? 10 IL long ( Fig. 1 I. Formation <>i jcllv jnun nxllcls When temporary preparations of the glandular nephridial cells are made and -<|iia.-hed or teased in sea water, some aggregations of rodlels may be forced out EGG COCOONS OF SCOLOPLOS 191 intact hut many are broken up into their co ; rodlets In such a preparation some of the rodlets appear to explode suddenly and at random but a rodlet can sometimes he seen to swell a little before suddenly bursting !er phase-contrast illumination the rodlets are sufficiently dense to appear quite black but the diffuse body formed after bursting can mily just be made out. \To ghosts or envelopes of rodlets are left, so that they appear to be simply a concentrated form of the jelly material which is able to absorb water and greatly to im rea in volume. The effect on the rodlets of various reagents was watched, using phase-contrast illumination, during irrigation under a cover glass or in hanging drop preparations. The tests were made about two weeks before spawning took place. FIGURE 1. Aggregation of rodlets from glandular portion of nephridium of female. Rodlets mounted in hanging drops of 100%, 80% and 60% S.W. were mostly intact after 5 hours but burst in that time in -KKv SAY. In 20% S.W. all burst within 13 minutes and in distilled water within 3 minutes. In S.W. made alkaline to pH 1 1 with XaOH, in N H2SO4 and in S.W./N H,SO4:1/1. all burst quickly but in S.W./N H,SO4:4/1 most remained intact. In Bouin's fluid all the rodlets burst quickly but in 5% formalin in S.W. only some burst. Subsequent irrigation with distilled water caused them all to burst. Surface-active agents in sea water, both ionized (0.1 % Teepol) and un-ionized (0.1% Triton X 100), caused the rodlets to burst. Teased male Scoloplos in S.W. had no visible effect. The conversion of the rodlets into jelly can thus be accelerated by various treatments but none of the tests throws much light on the natural process, which is rapid and once started goes to completion. Even in the tests with dilute S.W. there was never any suggestion of the rodlets taking up an intermediate size in equilibrium medium of osmotic pressure different from that of their normal environment. Microscopical appearance The cocoon jelly can be seen under the microscope (especially with phase- contrast) not to lie perfectly homogeneous but somewhat fibrous in appearance. This also shows up in the fixed material stained with Heidenhain's Azan stain (blue). Mallory's stain (blue), the P.A.S. stain (red), muci-carmine (red) and G. CHAPMAN Alcian blue. Metachromasy is shown with toluidine blue. The fibrous appearance is in no way dm- to the presence of clearly defined libers but rather to what are best described as local condensations of the jelly. Chemical properties The general chemical nature of tin- jelly is indicated by its staining properties. All these point to its being (or at aiiY rate containing) a mucoprotein. Further tots Yvere made in an attempt to find out details of its chemical composition and. it" possible, to relate this to its physical properties and biological role. Some 2000 cocoons were collected, teased up by hand, using needles, centrifuged at 5° C. for 20 minutes at 20,000 g and the jelly retained, the eggs and debris in the lower fraction being discarded. The process was repeated after further teasing ot the jellv and a clean, egg-free jelly was obtained. Examined under the microscope it was seen to contain only scattered specks of unidentifiable material probably derived from the substratum. Any jelly that was not used fresh was stored in 50rr> isopropanol in a refrigerator until required. l\ough determinations of the organic content of the centrifuged native jelly were made by weighing a sample, washing it at least 8 times in distilled water until demonstrably chloride-free when tested with silver nitrate, and then drying to constant weight in air on a hot-plate. Values of the salt-free dry weight were \.S% and 0.5'; of the wet weight, the difference probably being clue to the different amounts of the watery fluid which had been squeezed out by centrifugation before weighing, as well as to possible inequalities in drying. The jelly does not dissolve in sea water, distilled water or dilute acids. It dissolves in 0.5 X NaOH with gentle warming but it .seems likely that this process brings about considerable degradation. Nitrogen content The nitrogen content of the jelly was estimated as follows. A sample was taken, washed in repeated changes of distilled water until the washings gave no trace ot chloride when tested with silver nitrate solution. This was evaporated to dryness. washed again with distilled water and again evaporated to dryness. It was heated at 105° C. in an oven until of constant weight and two samples were used for total nitrogen estimation by the Kjeldahl method of Yuen and Pollard ( l('5,vi. The average of two estimations gave a figure of 12. 4' , X (12.3% and 12.5rr i. This figure is be-low that normally accepted for protein (16%) and sonieuhat above that for an amino sugar such as glucosamine (7.S',''). It looks, therefore-, as if the material is a combination of protein and carbohydrate moieties. . linino-acid composition \ sample of the dried material \\eighing 12.2 ing. uas hydrolyzed by heating for 7 hours at 105° C. with 10 ml. of n \ hxdrochloric acid in a sealed tube. The solution \\as evaporated in a rotary film evaporator and was twice redissolved and evaporated almost to dryness. The residue was taken up in distilled water and left in an evacuated desiccator over calcium chloride and sodium hydroxide to remove the last traces of hydrochloric acid. Two-dimensional paper chromatog- EGG COCOOXS OF SCOLOPLOS 193 raphy on Whatman Xo. 1. using butanol acetic acid wau-r (12:3:5 by volume) and phenol/water (4:1 by weight) as solvents, showed that some 15 amino acids were present. They were mostly revealed by ninhydrin and identified by com- parison with the positions taken up by known amino and also by the method of enhancement of each spot in turn by a known ami; ID acid applied in addition to the hydrolysate. Runs were also made using as solvents ethanol/ \vat- ammonia (12:1:1 by volume) and methanol/water pyridim- (20:5:1 by volume' The presence of aspartic acid, proline, hydroxyproline and alanine was the use of isatin. The following amino acids were present : alanine, argininc, aspartic acid, cystine, glutamic acid, glycine, hydroxyproline, leucine (or isoleucine), lysine, methionine (or valine), ornithine, proline, serine, threonine, tyrosine. A feature of interest is the presence of hydroxyproline which is usually found only in collagenous tissue proteins. These frequently have mucopolysaccharides a>Mid;it<-d with them. Carbohydrate components Sugars were separated on Whatman Xo. 54 paper, in the first place by descend- ing chromatography, using isopropanol/water (4:1 by volume). Runs were continued for 17 hours, which allowed the solvent to drip off the paper and increased the resolution of the spots with low Rf values. The sugars were revealed by silver nitrate in acetone, followed by sodium hydroxide in ethanol. The papers were bleached in ammonia and the spots blackened by hydrogen sulphide. The Elson-Morgan reagent for amino sugars was also used. These tests clearly showed the presence of glucosamine and or galactosamine and traces of two other sugars. Additional runs in ethyl acetate pyridine water (12:5:4 by volume), followed by revelation with silver nitrate, showed one of the simple sugars to be glucose. The other corresponded in position to fucose (a hexose ) and xylose (a pentose) which have similar Rf values in most solvents. Since the sugar was in low concentration in the hydrolysate the negative result of the phloroglucinol test for pentoses applied to the paper could not be relied upon to prove the absence of xylose. Accordingly the hydrolysate was tested for fucose. using the sulphuric acid/thioglycolic acid test for 6-cleoxyhexoses (Dische. 1947), to which group of sugars fucose belongs. A positive result was obtained. Testing the hydrolysate for pentoses by Bials' orcinol reaction and the phloroglucinol test gave a negative result. From this it was concluded that the trace of sugar present in addition to glucose and revealed as having an Rf value indicating either fucose or xylose was, in fact, fucose. The presence of both glucosamine and galactosamine was also confirmed ninhydrin degradation method of Stoffyn and Jeanloz (1954). followed by identifi tion'of the pentoses, arabinose and lyxose. which are produced by the reaction, This was done by paper chromatography, using ethyl acetate/pyridine solvent, followed by revelation with silver nitrate and comparison with th of known samples. The size and intensity of the spots obtained indicated two amino sugars were present in about equal amounts and in greater quanti glucose and fucose. Tests for uronic acids were made on the hydrolysate, using naph They were negative and confirmed the absence of any spot on the paper chroma- G. CHAPMAN terrain with an Rr corresponding to tliat of uroiiic acids. The presence of sulphate Croups \\-as tested for by adding hariuin chloride solution to the hydrolysate. No precipitate was obtained and the sugars are therefore not sul])hated. Attempts were made to separate any possible constituents of the jelly by electrophoresis on paper and cellulose acetate membrane at pH 8.5 and pH 4.0 and <) volts per cm., tolloued bv detection with SchitT's reduced fuchsin sugar reagent and naphthalene black for proteins. All the tests failed to demonstrate any mobile component whether they were made on small blobs of dialyzed native jellv or on washings of the jelly concentrated by ultrafiltration through Yisking tubing. All that can be concluded from this is that the molecular weight is verv high or that the molecules have little net charge, but the results lit in with the other properties (it the material, namely its insolubility and its extremely high viscosity at low concentration, which is suggestive of a cross-linked molecular structure. An estimate by the anthrone method ( P.angle and Alford, 1954) of the total quantity ot simple sugars present in the organic component of the jellv was made, using a weighed, salt-free, dry sample. (This method gives no color with amino sugars in the concentrations present. ) The intensity of color obtained was com- pared with that of standards from known concentrations of glucose in an EEL Spectra spectrophotometer at 625 nip.. The simple hexose component amounted to approximately 7% of the dry weight ;>f the jelly. From all these analytical procedures there is no doubt that the jelly can be called a mucoprotein having the carbohydrate moiety composed of galactosamiue and glucosamine, glucose and fuco.se. ( )ne of the chief properties of such materials is their ability to bind large amounts of water to a very small amount of organic matter. The jelly of Seoloplos is no exception to this and was easilv shown to be capable of imbibing water when dry and regaining its original volume or there- abouts. This process of drying and imbibition was repeated several times and was not affected by storage of the jelly in 50' r isopropanol. /'li\'sica! properties The bull< of the mucoprotein jellv does not dissolve in water but remains as a "bio])" even after teasing and centrifugation. Moreover it cannot readily be dissolved by chemical methods which do not degrade it considerably. These properties are obviously ot great importance m its role of egg container. ( )n the other hand its structure can be altered by certain purely physical means suHiciently to precipitate it or at least a component of it. \Yhen treated in aground- glass testtube homogenizer, in which the jellv is subjected to considerable shearing forces, it vields a white precipitate which on drving becomes powderv and is unable to imbibe water and regain the original volume of the jellv. The liquid left after spinning off the precipitate was tested with the anthrone reagent and with the Elson- Morgan reagenl for amino sugars. The anthrone test was positive but the Elson-Morgan gave only a verv faint coloration. The anthrone method ( Rangle and Alford. l''5-( i was then used to estimate the amount of hexose liberated from a sample ot jellv by homogenization. 1'v treatment in the homogenizer tor various lengths of time up to IP minutes, it was possible to split ot'f about 5'v ot the dry weight of the jelly, corresponding to about 70', of the .simple carbohydrate present. From these tests it can be concluded that the gel-forming property of the EGG COCOONS OF SCOLOPLOS 1(>5 material depends upon the presence of the carboln drale component of the complex, and that this is only loosely attached and can he removed hy physical means. The tensile strength of the stalk is such that, when rapidly loaded in sea water at room temperature, it extends and breaks within seconds when a weight of about X g. is applied. \Yhen loaded with 1 g. or even 0.5 . it stretche> quickly at first and then more slowly but continues to extend slightly for lx hours. Very slow extension was continuing when the experiment was stopped. When the tension was released there was an immediate shortening although not to the original length. These measurements were made using a smoked drum and light aluminum lever, and while it is clear that the jelly can he described in general terms as a viscous- elastic system, recording methods with less backlash and of greater sensitivity are needed to say how its properties may best be represented in a conventional wav. In nature the cocoons may be expected to be under a slight stress during most of their life because, although the direction of the tide changes at ebb and flow, the cocoon stalk is flexible and so ahvavs takes up the same position with respect to the current. This stress is presumably less than the 0.5 g. which was shown to cause extension, but the sudden stresses to which the cocoons may be subjected by wave action would be expected to be much greater. For overcoming the effects of these the elastic element in the behavior would be effective since the forces act for only a short time. A sample of the jelly was prepared for x-ray analysis by drying a film of the material on a glass plate. This film was peeled off and cut into rectangles 2x5 mm. which were stuck together by slight moistening to form a block 2x2x5 mm. The x-ray pattern perpendicular to the plane of the dried films of jelly showed disorientated protein but the x-ray pattern parallel to the dried films showed an indication of structure at the molecular level, composed of sheets separated by about 10 A. It is tempting to suppose that these correspond to proteins which are cross-linked by the carbohydrates but further experiments are required before this can be established. THE FUNCTION OF THE COCOONS The cocoon-forming habit occurs sporadically in the order Polychaeta and has thus presumably evolved many times. It is clearly based on the secretion of some part of the glandular epidermis which most annelids possess. Few authors have recorded the exact source of the cocoon jelly but the stage of hatching of the developing eggs is generally noted in the literature. It varies from the free-living trochophore, as in Phyllodocc nuicnlata, or the metatrochophore, as in Marphysa sp.. to chaetigerous larvae in .-Iricia spp. and Scoloplos spp. There is no positive evidence that the laying of eggs in cocoons confers any nutritive, thermal or osmotic advantage, although the opinion has been expressed that because algae and Infusoria are frequently seen to flourish in the cocoon they provide a food supply for the larvae. Bookhout and Horn (1949) stated that the embryos of Axiothella unicosa fed on the diatom Xitzscliia which multiplied in the cocoons, and Pillai (1958) is of the opinion that the embryos of Marphysa borra- dailci are nourished either by the jelly substance or the protozoans that flourish in it. It is possible that the larvae gain some nutriment from the jelly but it should be remembered that the eggs are generally packed with reserve food materials while 1()(> G. CHAPMAN the jelly contains only about \c/c of organic material, and in Scoloplos and 1'hyllodocc, at any rate, the cocoons seem to he little altered when the larvae have left. Also, no evidence has been put forward to prove that the concentration of food organisms is greater in the jelly than in the substratum. In the locality in which Scoloplos and Phyllodoce occur at Southend-on-Sea, the surface of the sand is frequently much richer in diatoms than the interior of the cocoon jelly. The chief function of the cocoon appears to be to keep the young in their proper station but even this advantage is partly lost if trochophores are liberated and especially if they are positively phototactic like those of Phyllodoce. Further ecological studies are required to determine more precisely the role of the cocoons in the biology of their producers and what advantages, if any, the cocoon-laying habit confers. I am grateful to Professor G. E. Newell, with whom many of the observations at \\ liitstable were made, to Dr. J. F. Forrest for information on the structure of the nephridia. and to Mr. A. (i. Tavlor who did much of the chemical analysis. SUMMARY 1. The egg cocoons of Scoloplos antii//cr are laid at Whitstable during the tew days of high spring tides in February or March. 2. The cocoon jelly is derived from secretion of cells forming the end portions of the nephridia of the female and contains only about 1 9r of organic matter which is mucoprotein. Fifteen amino acids, including hydroxyproline, were identified in the protein moiety and glucosamine. galactosamine, glucose and fucose in the carbohydrate portion. 3. The jelly can be dried but will swell again in water. This property can be destroyed by homogeni/.ation when 70'/r of the hexoses are split off. 4. It is unlikely that the cocoons play any significant part in the nutrition of the larvae, their chief function probably being to ensure that the larvae remain near a suitable substratum. LITERATURE CITKD AIYAK, R. ( i., 1('31. Aii account of the development and breeding-habits <>t a brackish-water polyehaete worm of the jiemi.s Mtirpltysa. J. Linn. Sue., 37: 387-403. AMIKKSOX, I). T.. 105''. Tin- embryology of the Polychaetc Scoloplos unui^/rr. (Jmirt. J. Mn r. Sci., 100: X<) lnf>. BANOI.K. l\., AND \V. C. \LFORD, 1(>54. The chemical basis of the periodic acid-Schiff reaction of collagen fibres with reference to the periodate consumption by collagen and by insulin. J. I listochcin. Cytochem.,2'. 62-76. lirioK norr, (_'. ()., AND K. C. llok\, 1949. The development of .-/.n<>//;<7/34. I )<-\ rlopnu-nt oi Scolecolepis juliginosa (Cl|id.). ./. Mitr. ttiol. Assoc., 19: 633-654. DE GROOT, G. J., 1('07. \aiiti-cl« ninuen over de ontwikkeling van Scoloplns unnincr. Disser- tation s'( iraveiilia^e. jip. 74. Disc1 1 1 1-., 7.., 1947. A vpccitic coloi n a< 'ion for ^Ineuronie acid. /. ttiol. (.'linn., 171: 725-730. Em.i-.io, I'".., 1892. 7.\\v Kenntnis von Arcmcolu uniriihi I.. lies. \\'i>s. ( .ottin«;en Nachr. pp. 413-418. EGG COCOOXS OF SCOLOPLOS t97 EISIG, H., 1914. Zur Systematik, Anatomic und Morphologic der Arididen nebst Beitragen zur generallen Systematik. Zoo!. Stat. Xcapd. Mitt., 21: 153-600. GRAVIER, C., 1923. La ponte et 1'incuhation chez les Annelides polychetcs. Ann. Sc. Xat. Zoo/. Paris, Ser. 10, 6: 153-247. HEREIN, R., 1925. La ponte et la developpement chez une Annelide polychete ^dt-ntaire Xiculcci sostcricola Mgn. C. R. A cad. Sci. Pans. 180: 864-866. LINKE, O., 1939. Die Biota des Jadesbusenwattes. Hclgoliindcr ivi rsnntcrsncluiiu/cu. 1: 201-348. MAU, W., 1882. Ueber Scoloplos annigcr O. F. Mnller. Beitrag zur Kenntm's (U-r . \natm und Histologie der Anneliden. Zeif. u'iss. Zoo/. Leipzig, 36: 389-432. PILLAI, T. G., 1958. Studies on a brackish-water polychaetous aunt-lid .l/o///. >rradailei sp. n. from Ceylon. Ccyluii. J. Sci ( Iliol. Sci.), 1: 94-106. RULLIER, F., 1954. Recherches sur la morphologic et la reproduction du nereidien Micruncrris varicgata Claparede. Arch. Zi/ol. E.vp. Gen., 91: 195-233. SMIDT, E. L. B., 1951. Animal production in the Danish Waddensea. Mcdd. K/niun. Havunder- sog. Kbn. Scr. Fisk.. 11 (6) : 1-151. STOFFYN, P. J., AND R. \V. JEANLOZ, 1954. Identification of amino-sugars by paper chromatog- raphy. Arch. Biochcin. Biophys., 52: 373-379. THAMDRUP, H. AL, 1935. Beitrage zur Oekologie der Wattenfauna auf Experimenteller Grundlage. Mcdd. Kiuniii. Havundersog. Kbn. Scr. Fisk., 10: 1-125. THORSON, G., 1946. Reproduction and larval development of Danish marine bottom invertebrates. with special reference to the planktonic larvae in the sound (0resund). Mcdd. Koiinu. Havundersog. Kbn. Scr. Plankton, 4: 1-523. WILSON, D. P., 1932. The development of Xcrcis pclagica L. /. Mar. Biol. Assoc., 18: 203-217. YUEN, S. H., AND A. G. POLLARD, 1953. Determination of nitrogen in soil and plant materials : use of boric acid in the micro-Kjeldahl method. /. Sci. Fd. Agric., 4: 490. HEART RATE AND LEUCOCYTE CIRCULATION IX CRASSnSTREA VIRGINICA (GMELIN)1- 2-2 S. Y. FENG nepartuient »l Zoology, l\iit in the blood is. therefore, of prime importance. M ATKRTAT.S AND METHODS 1. O\stcrs, m/iiarta, and sea 7U//na1 Institute of Allergy and Infectious Diseases of the National Institute- of I lealtli. 3 Present addres ;ter Research Laboratory, k'utuers, the State University, New Brunswick, X. J. 1 In tbe oyster tin t.ii - amebocyte, /eiii'neyie, and f>lniiineyie are used interchangeably, as by Stanbcr Mn50). 198 OYSTER HEART RATE MD LEUCOCYTE COUNT The water used to hold oysters in the laboratory was collected from the Shrewsbury River at Atlantic Highlands, New Jersey, where the salinity varied from 20 to 27/vc. Since ambient salinity change ( Keng. I'ViOi has also hern shown to influence the heart rate, all experiments listed below wen ir.ctcd in the sea water of 20^V which was obtained by diluting the stock water with appropriate amounts of aged tap water. The diluted sea water was stored in a oui-trmt tem- perature room (5° C.) before being used. Water was changed at least twice daily and vigorous aeration in each aquarium was maintained constantly. 2. Preparation of oysters for injection, bleeding and hear/ rale studies Oysters were prepared for intracardial injection and bleeding by tiling a window directly over the heart on the left valve of the shell with a power drill equipped with a circular grinding wheel. After a small hole was made in the nacremis layer, the opening was enlarged by subsequent fragmentation of the shell. For heart rate studies, however, the left valve anterior to the adductor muscles was completely removed, exposing the entire visceral mass, since according to Stauber (1940) valve closure also influences the heart rate. Oysters thus prepared were kept at 5° C. overnight to allow recovery from the shock of the treatment. Prior to the injection, the general physiological condition of each oyster was determined 1>\ noting (1) the strength of heart beat, (2) the response of adductor muscle to a mechanical stimulus such as a gentle tap on the aquarium wall, (3 ) the presence or absence of \vater pumping and (4) the ejection of fecal strings. Conditions (2) and (4) are not applicable to partially denuded oysters for heart rate studies, since their hinges are broken and consequently they are unable to snap their shells shut. Animals which did not meet the above standards and those which were infected with Bucephalus were discarded. 3. Preparation of inocula Seitz-filtered sea water (20/£o) was used as one of the inocula. One pound of fresh spinach was ground in a solution of 0.40 M sucrose, 0.05 M Tris, pH 7.8, and 0.01 M NaCl for 30 minutes at 5° C. (Jagendorf and Avron, 1958). The spinach pulp was filtered through several layers of gauze to eliminate large coarse fibers. The filtrate was then centrifuged at 500 g for 30 minutes at 5° C. The supernatant was discarded and the sediment, consisting of almost pure chloroplasts, was reconstituted in 20%o sea water to desired concentrations. 4. Injection and sampling procedures Seitz-filtered sea water and spinach chloroplast suspension were injected via the ventricular route. Injections were accomplished with a 1.0-ml. tuberculin syringe equipped with a 30-gauge (H") needle. Each oyster received 0.2 ml. of inoculum. There was some leakage immediately following the withdrawal of the needle, but in most cases the wound closed quickly. After injection the oysters were returned to the aquaria until the predetermined sampling intervals had passed. At the appropriate interval, 0.05 ml. of blood was withdrawn from the ventricle by the same type of syringe and needle used for inoculation. The samples were used for estimating the number of leucocytes and chloroplasts. 200 S. Y. FENG 5. Enumeration procedures a. Heart rate The heart was exposed for observation by the method described above. For temperatures abmr 15° C., the heart rates were calculated on the basis of the time required for 10 brats, while below 15° C.. only 3 to 5 beats wen- used to calculate ihe number of beats per minute. b. Unfixed ovster leucocytes During each sampling 0.05 ml. of blood was withdrawn from the ventricle. Samples were not diluted in most cases; but where dilution was warranted, the sample was diluted with filtered sea water in the syringe and the appropriate dilu- tion factor was noted in calculating the final number of unfixed leucocytes in a given volume of blood. The sample was shaken gently to keep the cells in sus- pension before the counting chamber was filled. The first two drops of blood ex- pelled from the syringe were discarded. The remaining blood in the syringe was used to fill the improved Neubauer counting chamber bv allowing the drops to flow under the cover glass. c. Fixed oyster leucocytes The procedure described above did not enumerate all the leucocytes in the sample, for in spite of the agitation of the blood sample, there was still a considerable num- ber of leucocytes adhering to the wall of the syringe. An experiment was de- signed to compare the fixed and unfixed leucocyte counts from the same blood sample. From each oyster 0.25 ml. of blood was obtained by the method described above. The blood was first used to fill 5 Xeubauer counting chambers and the remaining blood was fixed with a 5f/< acetic acid solution in 20',, sea water ( 1 part of blood and 4 parts of fixative). The diluted fixed blood sample was shaken gently to dislodge the adhered leucocytes from the wall of the syringe, from which 5 replicates of the leucocytes were obtained as above. d. Spinach chloroplasts In estimation of the concentration of chloroplasts in an inoculum, the suspen- Mon was first diluted serially in filtered sea water as follows: 1:10. 1:100, 1:1000 and 1 : 10,000. The counting chambers were filled with aliquots from the last three dilutions. ( 'ounting procedures similar to those for enumeration of oyster leucocytes were used to determine the total number of chloroplasts per ml. ot inoculum, l-or estimating ilie removal of chloroplasts from the blood stream, the tree chloroplasts present were counted simultaneously with the leucocvtes in the sample. e. Statistical treatment of the data A graphic representation of heart rates and leucocvtc counts was derived by calculating the symmetrical confidence limits of these numerical characteristics at the 99% level of probability. Such confidence limits were calculated for a group of control figures bv substituting the available information in the following expression s : OYSTER HEART KATE AND LEUCOCYTE COUNT 201 x — ts/n, where x == the arithmetic mean. t = "Student's" distribution value at the 99'.', level of significance, s — standard deviation and n = the number of observations. The 99% confidence limits of the control group are plotted. Any point of the experimental groups which falls outside the control limits is considered to be significantly different from the x at the 99% level of probability. This method of analysis is the standard procedure used in the study of fluctuations of oyster leucocyte counts, heart rates and chloroplast counts; hence it obviated the re- quirement of performing many tests of significance between groups of variables. I4H 12' 10 8 Y . 6- 4- 2 O X= Y= o A A L: Fixed or total leucocyte counts x 10 / ml blood Unfixed leucocyte Counts x 10 /ml blood Y plotted against X Y/X x 100 plotted against X Calculated value of Y/X x 100 Y = 0.26 + 0.41 X -100 -90 80 -70 -60 50 -40 o o X 30 4 8 12 16 20 24 28 32 X FIGURE 1. Relationship between unfixed and fixed leucocyte counts. RESULTS 1. Unfi.red leucocyte counts rs. fi.vcd leucocyte counts When 20 pairs of unfixed and fixed leucocyte counts are plotted, the points exhibit a linear relationship (Fig. 1 ). The diagram suggests that an increase in the unfixed leucocyte counts is accompanied by an increase in the fixed leucocyte counts (or the total number of leucocytes). The formula for this regression line is Y = 0.26 + 0.41 X, where Y is the unfixed leucocyte counts, X is the fixed (or total) leucocyte counts, 0.26 is the intercept of the line with the Y axis, and 0.41 is the slope of the line. The number of unfixed leucocytes may be expressed as a percentage of the total number of leucocytes (Y/X X 100). When these percentages are plotted against 202 S. Y. FENG TAHI.I I \'analnltt\ af leiii/ii vte (t>nnts hi a vron[> of 88 apparently normal oysters at 18° C. I.rucocyte X 10" nil. of lie.u t Moo, 1 0.5 2.0 3.5 5.0 6.5 8.0 9.5 1 l.ii 12.5 14.0 15.5 17.0 1 !c(|iieiir> 15 20 24 14 3 4 1 2 1 3 0 1 Mean 4.1(1 s(x — x)2 1027.83 Standiinl i >i-\ iaiion 3.43 Si.nidanl Krror 0.37 n 88 the total mimlicT of leucocytes (X), an exponential curve is obtained (dash line in Fig. 1). The curve suggests that there is little difference between unfixed and fixed leucocyte counts when the total number of leucocytes is in the order of 0.2 X lO'Vinl. blood. Presumably this is due to the fact that the chances of collision between leucocytes and the syringe wall are not great, hence most of the leucocytes remain in suspension. The greatest decrease of the Y/X X 100 term occurs at X values between 0.5 to 4.0 X 10':/ml. of blood. The adhesion of leucocytes to the syringe wall is probably the greatest in this range, since the chances of contact are enhanced and also the space on the syringe wall does not appear to be restricted. However, further increase in the total number of leucocytes from 4.0 to 30.0 X 10';/ml. of blood does not seem to affect the percentage term; this is reflected in the flatness of the curve between the above range of leucocyte concentrations. It fur- ther suggests that the space of the syringe wall is probably a limiting factor in de- termining the magnitude of the percentage term. Hased on the above analysis, it is concluded that the unfixed oyster leucocyte count, although not representing the total number of leucocytes is. nevertheless, a valid index of the total number of leucocytes. The data presented in this study are all in terms of unfixed number of leucocytes unless otherwise stated. 2. } 'aria hilit y <>j lcucoc\lc counts in 1.02 0.27 0.26 53 0.5 OYSTER HEART KATE \\!> LEUCOCYTE COUNT 203 3. Temperature-heart rate relationship Ten partially denuded oysters were placed in sea water at room temperature (23.5° C. ), followed by chilling in the cold room. While the \\ater was being chilled, the heart rates" were recorded for 23.5° 21.0°. 14.5 . 11.: and 10.0° C., respectively. After the observation was completed, tin oysters were placed in fresh sea water and kept in the cold room overnight. The next morning, the; were allowed to warm up to room temperature in fresh sea water. 1 hiring the .3- O O 7.0- 0 0 0 0 o o JD 1.5- 0 o 9"?>o o 00 g» ° ° 0 0 V) 0) 0 u ° ° 0° ° 0 %>« 0 ° s 0 ,000 ie.o- 0 o o o ° o - 1 0 oo 5- 0 S9 0 0 05 15 25 35 Wet weight in gms. FIGURE 2. A scatter graph of oyster body weights vs. leucocyte concentrations. The plot is based on observations made on 59 oystn>. warming process, heart rates were recorded at the above temperature gradient. Three sets of such data were obtained, which were combined to construct the temperature-heart rate curve of Figure 3. The result indicates that the heart rate changes from approximately 5 beats per minute at 10° C. to ^ beats at . and confirms the general conclusion reached by earlier workers (Roughley, 1926; Takatsuki, 1927; Federighi, 1929; and Stanber. 1940). 4. Temperature-leucocyte number relationship Twenty-one oysters were used in this experiment. At 6°, 12 5 and blood samples were taken from each oyster for the counting of leucocytes Oysters 204 S. Y. FENG 5- o _g .0 3- o> ?' o o o Q> O 0> 0 Leucocyte count Heart rate 35 -30 -25 -20 -15 CD O O a> o 0.10 <0.01 <0.01 6°C. us. 22° C. d.f. 40 40 ±t — — 1.44 2.33 12° C. vs. 18° C. P — — >0.10 >0.02 12° C. vs. 22° C. d.f. — — 40 ±t — — — 0.80 P — — — >0.50 18° C. vs. 22 °C. heart is probably quiescent at this temperature. It is possibly true (depending on the length of time) that in vivo at 0° C. leucocytes might not be expected to settle out completely due to the increased viscosity of "plasma." However, no leucocytes were found in the supernatant of a blood sample contained in a test tube which was allowed to stand overnight at 25° C. Extrapolation of the temperature- leucocyte relation beyond 22° C. is uncertain; the curve may become asymptotic if the mean of 4.85 X 10° leucocytes per ml. blood at 22° C. is the maximum for oysters, or it may rise further if all leucocytic clumping is not yet dispersed or if small foci of leucocytic accumulation in the tissues are resolved. 5. Effects of mechanical stimuli- (repeated bleedings) and of injection of sea ivatcr This experiment was attempted in order to discover the effects of repeated bleed- ings in small amounts of 0.05 ml. per bleeding on the number of leucocytes, and to TABLK IV Changes in the level of oyster leucocytes during a 20-hour period at lr> ( . Time (hr.) 0 l 1 i 1 i 3 5 10 20 ("•I: total Mean 3.20 4.15 4.00 3.15 3.45 3.65 3.10 2.55 3.40 3.57 s(x - x)2 29.10 42.03 62.00 73.03 59.73 38.53 24.90 64.23 61.40 463.10 cr 1.71 2.05 2.49 2.70 2.44 1.96 1.58 2 53 2.48 2.27 Om 0.57 0.69 0.83 0.90 0.82 0.65 0.53 0.84 0.83 0.24 n 10 10 10 10 10 10 10 10 10 90 d.f. 98 98 98 98 98 98 98 98 98 ±t 0.49 0.76 0.56 0.41 0.16 0.11 0.64 1.32 0.22 P* >0.50 >0.40 >0.50 >0.50 >0.50 >0.50 >0.50 0.20 >0.50 * Test for significance of the grand total mean and the means obtained at various time intervals. 206 S. Y. FENG establish a base line as the control for certain of the studies cited below. Ten oysters held at 1(> C. were bled nine times: 0, j. 7. 1. -\ 5, 10 and 20 hours after ihe start of the experiment. The grand total mean of leucocytes per ml. of blood based on (H) observations was 3.57 X 10" with a standard error of 0.24 and si \ - x) 4co.l. The statistical constants of leucoc\u> obtained at each time interval are summari/.ed in Table IV. Kach mean is com] tared with the grand total mean to ascertain whether the difference between them is significant. The large /' value, in each case, suggests that the mean number of leucocytes at any time interval dur- ing the 20-hour period does not differ significantly from the population mean. Tin- data are also presented in graphic form ( Kig. 4). Concurrently with the above experiment, five oysters were each injected with 0.2 ml. of sea water. Heart blood samples were obtained at the following intervals: ',. L :l. 2, 4, 8 and 96 hours after the injection. Significant changes in leucocyte numbers during the first H hours are observed (Fig. 4). Immediately after the injection, the number of circulating leucocyte's drops abruptly; it reaches the lowest point in .', hour and slowlv regains its normal number in about 1 \ hours. The sudden drop in leucocvte numbers after the injection of sea water is ascribed to the dis- placement of oyster blood in the ventricle by the inoculum or to dilution or to disturbance of heart rate or all three together. The gradual mixing of the inoculum with the influx of oyster blood containing many leucocytes results in the subsequent rise of leucocyte numbers. After 2 hours the number of leucocytes in this group • Repeated bleedings A Injected with sea water followed by repeated bleedings 99 % confidence limits 0 HOURS I;K,I KK 4. Tin- effect of injection of sea \\ater and repeated bleedings on the number of leucocyte- in sample of oyster heart blood at 16° ('. The cross-hatched area represents the 99% confidence limit-, of normal leucocyte number in control animals. OYSTER HEART RATE AXD LEUCOCYTE COUNT 207 > / 2 4 HOURS FIGURE 5. The effect of various experimental manipulations on the heart rate (a) and corresponding leucocyte number (b) of oysters. Group A is the control while Groups B, C, and D represent (1) repeated bleedings only, (2) injection of spinach chloroplasts, followed by repeated bleedings, and (3) injection of sea water, followed by repeated bleedings, respectively. The cross-hatched areas represent the 99% confidence limits of heart rates and leucocyte numlu-t - presumed to be present in unhandled oysters and in oysters subject only to repeated bleedings, respectively. is no longer considered to be significantly different from the- control group ( Fig. 4), even though one point does fall slightly outside the limits. 6. Effects of injection of spinach chloroplasts Nineteen oysters with hearts exposed were divided into four groups: A, B, C and D. Group A served as control. The remaining three groups were designed to determine the effects of repeated bleedings alone (Group B), injection of sea water followed by bleedings (Group C) and injection of spinach chloroplasts followed by bleedings (Group D) on the heart rate and number of leucocytes. Counts were 208 S. Y. FKNV, made on Groups A. 1'. and C at 15°-17° C. with 5 oysters in each group, and on ' in»up I) at 14 1(> C. with 4 oysters in the group. All experiments lasted for 4 hours. Heart rates were recorded for all groups hut leucocyte numhers we're sampled regularly for Groups B, C and I) only. Samples were taken at the following intervals: 0, j, i, f, 1. 2 and 4 hours. Leucocyte counts were not made for Groups C' and D at 0-hour, since 0-hour was also the time when injections were performed. Therefore, it was assumed that the 0-hour leucocyte counts for Groups C and D were prohahly similar to that of Group B. Each oyster in Group D was given 0.2 ml. of an inoculum containing 6.0 X 10;1 chloroplasts per ml., while in Group C each oyster was injected with 0.2 ml. of sea water. Ninety-nine per cent confidence limits of heart rates for Group A were calculated and plotted in the manner similar to that for the leucocyte counts. Repeated bleedings apparently have little effect on the heart rate during the 4-hour period ( Fig. 5). Even when the mean heart rate slows down to 8 beats per minute in Group C', which is a significant drop as compared with that of Group A, the leucocyte counts do not show appreciable difference from that of Group B one hour post-injection (Fig. 5). Injections of chloroplasts definitely suppress the heart rates which in turn prohahly contribute to the delay of the mixing process, among other things. However, the leucopenia immediately following the injection of chloroplasts could he due to incomplete mixing or other unknown factors which are independent of the heart rale. DISCUSSION Leucopenia and leucocystosis which are associated with infections in mammals are frequently used as indices of host response; the securing of such information becomes essential in clinical diagnosis. Two parameters may be employed as in- dices of host responses: ( 1 ) changes in ratio of various cells, and (2) quantitative change.-, in the total number of leucocytes. Although changes in the various typo of blood cells after injection of foreign materials or even after hemorrhage were ob- served by Cameron (l(M4i for the larvae of the wax moth caterpillar, and also pn-sumably normal leucocyte counts are available for some fishes, amphibians and reptiles (Gemeroy, 1938), the possibility of using this method in detecting host response in poikilothermic vertebrates and invertebrates has yet to be explored. It is probably true that one of the most serious hindrances to the progress is the lack of detailed leucocvte histology with respect to these animals. As a result of the present studv, however, it is evident that even the quantitative aspect of this problem is complicated bv the fact that the numbers of circulating leucocvtes are strongly influenced by the heart rate, which is in turn dictated by temperature and/or me- chanical stimuli, e.g., injections ()f participate and soluble materials. Thus, the mobilization of leucocvtes to the site of invasion in oysters at lower temperatures ma\ be greatly handicapped by the settling out of leucocytes at these low tempera- tures. The role of the two accessory hearts in circulation has been well established (Hopkins. 1(M4; Fble. I'Xoi A comparison of the two accessor) hearts and systemic heart with respect to iheir position, si/c and mechanical output suggests that the contribution of the former in this context is relatively minor ( Tr,ble, 1963; Stauber, personal communication). The available evidence also indicates that they are as temperature-dependent as the systemic heart (Stauber, personal communica- tion). Since leucocytes are also known to participate in the nutritive process, it OYSTER HEART RATE AND LEUCOCYTE COUNT 209 is possible that the number of circulating leucocytes may fluctuate with stages <>f feeding-, although experimental evidence is still lacking on this point. The transient disappearance of circulating granulocytes following intravenous injection of staphy- lococci and pneumococci in small mammals (Rogers, 1958) is attributed to ad- hesion of circulating granulocytes to the capillary endothelium (Wood. Smith. Perry, and Berry, 1951). A similar phenomenon in the oyster, which occurs immediate! \ following intracardial injection of sea water and chloroplasts, is thought to be as- sociated with the incomplete mixing of the inoculum and the influx of blood from the auricles. Bang (1961) describes the development of "intravascular clotting or thrombosis" in the circumpallial artery of the oyster following intracardiac injection of tissue extracts. He further reports that the "clotting" disappeared spontaneously within two hours after the injection. Based on the findings of the present study, an alterna- tive interpretation of Bang's observations might be offered. It is clearly demon- strated in the present study that the formation and dispersal of leucocyte aggregates or "clotting" can be manipulated by placing oysters at 5° C. followed by gradual warming to 23° C. This observation is further correlated with circulating leucocyte numbers, temperature and heart rate (Fig. 3). The spontaneous disappearance of "clots" within 2 hours after the injection, as reported by Bang, could, therefore, lie ascribed to restoration of normal heart rate following recovery of the heart from trauma, since it is the pulsation of the heart which keeps the leucocytes in suspen- sion. It was also not surprising to find that "clotting" did not occur in his control oysters injected with sea water, since in the studies reported here the reduction in heart rate from 13 to 9 beats per minute following injection of sea water (Fig. 5a) did not sufficiently reduce the velocity of blood flow to affect sludging of circulating leucocytes (Fig. 5b). Therefore, what Bang observed in his experimental animals could be a transient sludging of leucocytes induced by temporary heart failure rather than "clotting," which implies the involvement of biochemical processes. Such settling or sludging of blood corpuscles has also been observed in traumatized or sick experimental animals, sick domestic animals and in human patients ( Knisely and Hloch. 1942; Bloch. 1956; Harding and Knisely, 1958; Knisely, Warner and Harding, 1960 ) . These authors have clearly demonstrated that the cause of sludging is largely physical, namely, reduction in the velocity of blood (low, created by ex- perimental manipulations, pathological conditions or other factors. The physical aspect of this problem is reflected by the use, in their analysis of the phenomenon, of formulae originally designed for civil engineers in studying the sedimentation rates of various particles in a fluid medium and the transportation of silt by moving water. The author is deeply indebted to Dr. L. A. Stauber for his many suggestions, helpful criticism and generous support of this work. Without the technical as- sistance of Mr. Martin Horowitz this study would not have been possible. SUMMARY It was demonstrated that the number of circulating leucocytes from the heart blood samples of 21 oysters and the heart rate of oysters increase linearly with the rising ambient temperature at which the oysters were held. At ambient tempera- tures of 6°. 12°. 18° and 22° C., the mean leucocyte number per ml. heart blood -MO S. Y. I-KXG was. t"i uiiid to he l.d. 2.7. 4.1 and 4.(> million, respectively. The corresponding heart rates were 5 (10° C.), 9, 19 and 26 beats per minute. These findings indicate that the fluctuation of leucocyte counts in the blood of experimental oysters is probably associated with the intensity of agitation exerted by the heart beat, which is in turn influenced by the temperature. Effects of repeated bleedings and of injections of sea water and spinach chloroplasts on heart rate and number of leucocytes were also studied. Repeated bleedings do not affect the heart rate and the number of leucocytes, while injection of sea water and spinach chloroplasts does reduce the heart rate and the leucocyte number significantly for a period of two to at least four hours, depending on the type of inoculum used. These effects, therefore, do influence (1) the settling or sludging of leucocytes, (2) the mixing time of the inoculum and (3) the probable accumulation of leucocytes to the site of invasion, especially in oysters at lower temperatures. LITERATURE CITED BANG, F. B., 1961. Reaction to injury in the oyster (Crassostrca virqinica} . Blol. Bui!., 121: 57-68. BLOCK, E. H., 1956. Microscopic observations of the circulating blood in the bulbar conjunctiva in man in health and disease. Ergchn. Anat. Entu'., 35: 1-98. CAMERON, G. R., 1934. Inflammation in the caterpillars of Lepidoptera. /. Path. Bact., 38: 441-466. EBLE. A. F., 1963. The circulatory system of the American oyster, Crassostrca I'lrginica (Gmelin). Ph.D. Thesis, Dept. of Zoology, Rutgers, The State University, New Brunswick, N. J. FEDERIGHI, H., 1929. Temperature characteristics for the frequency of heart rate in Ostrea I'irg'mica Gmelin. /. E.vf>. Zool., 54: 89 04. FENG, S. Y., 1960. Unpublished report. Dept. of Zoology, Rutgers, The State University New Brunswick, N. J. liKMEKOv, D. G., 1938. Comparative studies in vertebrate hematology. ALA. Thesis, University of Western ( hitario, Canada. HAKDIXG, F., AMI M. II. KMSELY, 1958. Settling of sludge in human patients, a contribution to the biophysics of disease. Ain/iolni/y. 9: 317-341. HOPKINS. A. E., 1934. Accessory heart in the oyster, Ostrea ;<»/. Bull.. 67: 346-355. JAGEXIIOKK, A. T., AND M. AVRON, 1958. Co-factors and rates of photosynthetic phosphorylation by spinach chloroplasts. /. Biol. Cliem.. 231 : 277-290. KMSEI.Y, M. H., AND E. II. I'I.OCH, 1942. Microscopic observations of intravascular agglutina- tion of red cells and consequent sludging of the blood in human diseases. Aunt. Kee.. 82 : 426. KNISELY, M. H., L. WARM i> \\n \:. HAKIM. \G, 1960. Antemortem settling. Ainiioloi/y, 11 (6, part 2) : 535-58S. ROGERS, I). I'".., 1958. Cellular management of bacterial parasites. In: Pasteur Fermentation ( < nteiinial, 1857-1957, published by Chas. Pfizer and Co., Inc. ROI-GIII.EV, T. C., 1926. An investigation of the cause of an oyster mortality on the George's River, Xew South Wales, 1^24-1925. Proc. Limn. Soc. N. S. Wales, Sydney. 51: STAI m:k. I.. A., 1940. Relation of valve closure to heart beat in the American oyster. Natl. Shellfish. Assoc. Convention Addresses. \Y\\ Haven, Conn. July 31-August 2, 1940. STAIUSEK, L. A., 1950. The fate of India ink injected intracardially into the oyster, O.v//v<; virginica Gmelin. Hi«l. Hull.. 98: 227-241. TAKATSUKI, S., 1927. Physiological studies on the rhythmical heart pulsation in the oyster, listrcn circum picta PiKhury. Sci. Tohuku Imperial Vmv., Scmlai, Japan, 4th Ser.. 2: 301-324. \\'ooi). \V. 1!., JR., M. R. SMITH, \V. D. PERRY AND J. \Y. I'.KRRV, 1951. Studies on the cellular immunology of acute bacteremia. 1. Intravascular K'ukocytic rear) ion and surface phago< ytosis. J. Exp. Med.. 94: 521-534. CHEMOSENSORY BASES OF FOOD-FINDING VXD FEEDING IX APLYSIA JULIANA (MOLLUSCA, OPISTHO1 \TCHIA)1 HUBERT FRIXGS AND CARL FRINGS Department of Zoology, University of Hawaii, Honolulu, ffn-^'dii \Yhile many opisthobranchs have special feeding preferences, the sensory mechanisms involved in their food choices are mostly unstudied (Kohn, 1961 ; Paine, 1963). Aplysia Juliana, a common sea-hare of Hawaiian waters, is almost monophagous, feeding only on Ulva lactuca, if available, but taking Ulva fasciata. if given only this. On the latter, however, the animals do not grow normally. These sea-hares may nibble on other algae, but never eat enough to grow or survive for long. They live well in marine aquaria when fed Ulva lactuca, and make good subjects for research on sensory physiology. The animals we tested were maintained in the laboratory in ten-gallon aquaria or one-gallon jars fur- nished with sub-sand filters. Sea water was obtained locally and used unfiltered. If given food regularly, the animals usually lived for 2-4 months, and grew from a few millimeters to 12-18 cm. long. This report describes the responses of Aplysia Juliana (hereafter, aplysia) to its food plant, Ulva lactuca (hereafter, Ulva), with data on sensory processes involved. RESPONSES TO ULVA If aplysias are without food for a few days, they usually bury in the sand and remain hidden. One may, on looking into an aquarium, be quite unaware that any of these animals are present. If he drops a small piece of Viva into the water, within 10-15 seconds the oral tentacles of the animals appear, followed by the heads, as the sand seems to come alive, and the aplysias crawl out, with tentacles spread (Fig. 1). They climb the sides of the aquarium, holding fast with the posterior sucker that is characteristic of this species, and extend their tentacles as if sniffing the water, as a dog sniffs the air. If an aerator is in action, the animals may seem not to orient well, but if the water is relatively quiet or moving in a specific direction, they go fairly directly toward the Ulva. Upon touching it. they immediately seize it with the mouth and commence feeding. The response to food occurs, therefore, in three major steps — arousal, orienta- tion, and feeding, as in many predatory and carrion-feeding gastropods (Kohn. 1961). One is impressed, on first seeing the arousal and orientation, by the rapidity and precision of motion for these animals, and the obvious use of the oral tentacles. On contact with the Ulva, the aplysias react even more rapidly, as if they were using a different sensory modality. Certain questions immediately come to mind: (1) What is given off by the Viva that attracts the animals? (2) 1 These studies were aided by a contract between the Office of Xaval Research, Departmrnl of the Navy, and the University of Hawaii, NR 301-7-' 4. 211 212 [UBERT FRINGS AND CAKI. KUIXGS With what organ or organs is the stimulus received: ( 3 ) 1 low is this behavior related to the general feeding behavior of the animals? X.VIVKK OF T1IF. ATTRAITIVF. Sl.'KSTAXCE It was fairly obvious that only the chemical senses were involved in these reactions. Dropping pieces of other algae or other objects into the water did not arouse quiet animals, thus eliminating mechanical effects. The eyes of these animals would hardly seem to be involved, but to eliminate this possibility the following test was made. A few blades of I 'Ira were put in fresh sea water for about ten minutes, and only the water was then added dropwise to an aquarium holding the apK.sias. This proved just as stimulating as the I 'Ira itself. As few I II.URE 1. Younii Af>l\s'm julinnn ( 12 mm. l1) has noted, the relevance of these studies to normal behavior may be questionable. CHEMORECEPTION IN APLYSIA 215 DISCUSSION OF RESULTS It is obvious that a water-soluble material given off by ( Jlva into sea water acts as a powerful attractant, enabling the aplysias to find f. Either the same or another water-soluble material acts as a strong phagostimulant, inducing feeding even on unnatural foods. These act at remarkably low concentrations, dilutions at least 1 to 15,000.000. The chemical nature of the material or these materials remains to be determined. No attempt can be made, therefore, to designate it or them as contact or distance stimulants (Kohn, 1961 ). The receptors for the material acting in arousal and orientation are on the tentacles, and may be confined to these, although receptors in the mouth could be active. The rhinophores are not sensitive to this material, strengthening the con- clusions of Arey (1918), Crozier and Arey (1919), and Agersborg (1922) that the name, rhinophore, must not be thought to denote an olfactory function. All critical experimental evidence shows that the rhinophores are primarily receptors for mechanical stimuli, with no more sensitivity to inorganic materials than other parts of the body surface. Stimulation of the oral chemoreceptors causes feeding behavior. The change in reaction from that elicited by stimulation of the tentacles suggests that a different stimulating chemical or some change in sensory modality may occur. No direct evidence for this was found, however, and it may be that the same material both attracts the animal and stimulates the oral receptors to elicit feeding. The exact receptors were not discovered, but they are near or in the mouth, for stimulation of the mouth alone, without stimulation of the tentacles, elicits feeding reactions. The induction of feeding on filter paper and other unusual materials by stimula- tion of the animals with t//t'fl-water suggests that feeding is determined mainly by chemical stimuli. In a few cases, however, some of the animals refuse to feed on filter paper or other cellulose materials, even when flooded with Ulra-waier, sug- gesting that mechanical factors also determine food selection. OBSERVATIONS ON NORMAL FEEDING In captivity, the aplysias generally consume all of the Ulva fed to them. Yet, in nature, no matter how many of the animals bro\vse upon a bed of Ulva, they do not exterminate it. This observation led us to make some studies on feeding by these animals that indicate why this is so. Ordinarily, when feeding captive aplysias. we tear Ulra from the rocks on which it grows to bring it to the laboratory. If, instead, we bring in rocks with the Ulva attached, the aplysias do not completely devour the Ulva. Instead the}' eat the succulent-appearing outer portions of the blades and leave the heavier bases. It is the former that are usually collected when the Ulva is merely pulled from the rocks. The basal parts are noticeably coarser (Fig. 3). On examining Ulva in regions with high populations of aplysias, we found that the plants that are large enough for feeding obviously have two parts — a basal, darker green, thicker, coarser sec- tion, and a distal, succulent, leaf-like portion. Ordinarily, the aplysias eat only the succulent growth; the basal, heavier portions are left. The bases produce new succulent blades within a week or two. In the aquarium, the same thing is true. The aplysias eat the plants down to 216 III BERT FRINGS AND CARL FRINGS the heavy bases, hut they do nut cat these unless driven to it by long-continued starvation. In nianv cases, even this docs not cause them to eat the bases. The aplvsias can lie induced to eat the bases bv flooding their mouths with /7?'hore sea water in which ['Ira has stood is dropped onto the month of an aplysia. the animal starts feeding activity. The stimulating material acts at very low concentra- tions and is strongly excitatory. Normally these aplysias eat only the terminal thin parts of Ulra and leave the coarser bases which produce new blades. This may be due to the absence of phagostimulant in the bases. The animals therefore do not eliminate the Ulra. even when they are very numerous, and their food supply is thus maintained. LITERATURE CITED AGERSBORG, H. P. K., 1922. Some observations on qualitative chemical and physical stimulations in nudibranchiate mollusks, with special reference to the role of the "rhinophores." J.Exp. Zoo!.. 36: 423-444. AREY, L. B., 1918. The multiple sensory activities of the so-called rhinophores of nudibranchs. . liner. J. PhyswI.. 46: 526-532. BERGH, R., 1864. Anatomiske Bidrag til Kundskab oni Aeolidierne. I'idrnsk. Sclsk. Skr., NatnrridcHsk. og ma them. Ajd.. 7: 141-313. CROZIER, W. J., AND L. B. AREY, 1919. Sensory reactions of Chrninodnris zebra. J. E.rp. Zoo!., 29:261-310. KOHN, A. J., 1961. Chemoreception in gastropod molluscs. Amcr. Zool., 1: 291-308. PAINE, R. T., 1963. Food recognition and predation on Opisthobranchs by Xanana* inermis (Gastropoda: Opisthobranchia). Veliger,6' 1-9. INTRACELLULAR OSMOREGULATION IN SKELETAL MUSCLE DTK I XG SALINITY ADAPTATION IN TWO SPECIES OF TOADS * MALCOLM S. CORDON 2 Department <>/ Zmihniy, University of ('(ilifornid. Los Anijclcx 24, California The European green toad (Bnjo rirhiis} is a surprisingly euryhaline amphibian, some populations living in environmental salinities as high as 29%c (Stoicovici and I'ora. 1()51 I. This survival appears to he largely based upon a tolerance by the tissues of body fluid osmotic concentrations somewhat higher than those of the environment. Changes in osmotic concentrations of body fluids are due primarily (up to 84%) to changes in NaCl content (Gordon, 1962 ; Tercafs and Schoffeniels, L962). The occurrence of large changes in osmotic and salt concentrations in extra- cellular fluids immediately raises the question of the nature of possible simultaneous changes in intracellular fluid composition. The present paper describes the results of a study of the changes in the principal intracellular solutes in the skeletal muscles of adult green toads adapted to various environmental salinities. For comparison, similar data are presented for the less euryhaline western American toad, Bitfo f'orcas. A preliminary account of some of the results of this work was given by Gordon (1963). MATERIALS AND METHODS Adult green toads were collected on the island of Saltholm, near Copenhagen, Denmark, in May, 1962, and \vere shipped by air to Los Angeles. In Los Angeles they were maintained at 1(> ±2° C. in tap water dechlorinated with a charcoal filter. They were fed twice weekly with larval and adult Tencbrio. Survival was excellent. Some toads not used in experiments remained alive and in good condi- tion for more than two years. No attempt was made to separate the sexes. Adult western toads (Bn\o horcus) were obtained as needed from local suppliers in southern California. Maintenance conditions were as for green toads. Toads were adapted for two or more days to dilutions of natural sea water in groups of six or more in an air-conditioned room maintained at 19 ±2° C. Sea water dilutions (100% sea water '- ~ 32(/,( salinity " 950 mOsm/1. osmotic concen- tration) were made with dechlorinated tap water. Higher salinities were ap- 1 These studies have heeii .supported by research grants from the National Science Founda- tion ( ( 18802 ; G23855) and the U.C.L.A. Committee on Research. Basic arrangements for the study were greatly facilitated by a fellowship from the John Simon ( iuggenheim Memorial Foundation. -My thanks for aid, advice, and cooperation are due the following people: Dr. K. Allen, Department of Zoology, University of Maine; Dr. C. Barker J^rgensen, Institute of Zoophysiol- ogy A, I'niversity of Copenhagen; Dr. John I'ierce, Department of Biological Chemistry, and Dr. Maria Seraydarian, Department of /.oology, both of U.C.L.A. Valuable technical assistance was piovided by Maria I'olak, Dorothy McXall. and Dean Rickctts. 218 OSMOREGULATIOX IX Ml S< LES OF TOADS 210 TABLK 1 A nalytical procedures Analysis Procedure Plasma sample vol. (A.!.) Average pre- cision of dupl. analyses Muscle sample \vt. (ins.) Average pre- lupl. anal Reference Osmolarity Freezing point 0.0(11 ±20 mOsm/1. — — Ramsav & Brown depression (1955) Total solids \\Vt-dry weights — — 100-20(1 ±2 gm./kg. Gordon (1962) Chloride Ag+ titration 50 ± 3mEq/l. 100-200 ±1 mkq/ky. Cotlovt- (1 '.'fi.ii Sodium Flame photometer 100 ±3 mEq/1. 100-200 ±0.5 IllKq/kg. Gordon (1962) ; for muscle, v. homogenates of Potassium Flame photometer 100 ±0.2 mEq/1. 100-200 ±2 inEci/'kK- Gordon (1962); for muscle, watci homogenates of fresh samples. Non-protein N Nesslerization 50 ±20 mg./l. 100-200 ±0.03 Kin./kg. Natelson (1961) (NPN) a-NH2N Na 0-naphthoqui- 100-200 ±2 mg./l. 1(1(1 .MM) ±0.02 gm./kg. Frame el al. none-4-sulfonate (1943); Russell (1944) Urea Urease, micro- 25 ± 10 mg./l. 100-200 ±0.01 gm./kg. Conway (1957a); diffusion tor muscle as for sodium above Iiiulin Resorcinol too ±30 mg./l. 100-200 ±0.001 gm./kg. Roe et al. (1949); see text Total sugar Anthrone — — . 50-70 ±0.2 gm./kg. Seifter & Dayton (1950) Glycogen Anthrone — — 50-70 ±0.2 gm./kg. Seifter & Dayton (1950) Free amino acids 2-dimensional paper 100 — Allen & Awapara (qualitative) chroma tography (1960); Scher- baum et al. (1959); see text Free amino acids Spinco Model 120 — — 1000-1500 see text Spackman el al. (quantitative) amino acid (1958) analyzer preached in graded steps (e.g., successive three-day periods in fresh water, 20%, 40% and 50% sea water). Toads were not fed during salinity adaptation. Blood samples were ohtained by exposing and opening the heart, then collecting the blood in lightly heparinized glass capillary tubes. These tubes were closed with TABLI-: 1 1 I nul in spaces in toads adapted to various salinities [X ±S.E.(N)] btate ot adaptation Total extracellular fluid (l./kg. body wt.) Muscle extracellular fluid (l./kg. wet wt.) Bufo viridis FW 0.418 ±0.019 (5) 0.128 ± 0.036 (5) 20' ; S\Y. 7-15 days 0.398 ± 0.019 (4) 0.139 ± 0.035 (3> 40' , S\Y, 5-13 davs 0.364 ± 0.014 (5) 0.132 ± 0.060 (4) 50% S\Y, 2-6 days 0.338 ± 0.022 (5) 0.175 ± 0.011 (5) Bufo boreas FW, start 0.332 ± 0.026 (4) 0.116 ± 0.035 (5) F\\", starved 1 mon. 0.331 ± 0.019 (3) 0.120 ± 0.072 (3 20' ; S\Y, 7-15 davs 0.365 ± 0.020 (5) 0.139 ± 0.038 (3) 40' ; S\Y, 2-8 days 0.288 ± 0.037 (5) 0.164 ± 0.034 • 5 50' , S\Y, 3, days 0.270 (1) 0.159 ( 1 i 220 MALCOLM S. GORDON sealing; wax. centrifuged and resealed after the- ] lacked red cells were cut away. Plasma samples were stored frozen at —20° C. for later analysis. Samples of skeletal muscle ranging in weight, as required by the experiment, from 50 to more than 1000 mg. were taken from the thighs. Muscle samples were subdivided and the pieces weighed to ±0.2 mg. on a torsion balance. Prepared muscle extracts were stored frozen at -20° C. for later analysis. Procedures for sample preparation and analysis are summarized in Table I. Some additional information is necessary for two procedures. Iniilin spaces: Imilin spaces ("extracellular spaces") were determined both for the whole animal and for thigh musculature. Toads were weighed to ±0.1 g., then injected intraperitoneally with 0.5 ml./lOO g. of 10V' inulin in O.Sc/o Nad solution. Pilot studies on both species showed that plasma inulin concentrations reached a plateau stable for at least four hours within two hours ot injection. TAHI.K I I I I'liisniu concentration* in toads adapted to various salinities [x±S.E. (X)] State of adaptation Osmolaritv (mOsiu 1.) (1 (mH 1150 ±40(3) 88 ±8 (10) 850 ±130i 4i 2-8 days 50', SW — — — — — 84 ±6 (3) — 3 6 d.iv- Plasma and muscle samples were taken from injected toads 2-0 hours after injection. Paired control samples were taken from similarly treated uninjected animals. Following analysis, mean background values for inuloid substances in the muscles were subtracted from the measured inulin values in injected loads. This background was usually <|uite constant within groups treated in a given way. \'o background correction \\as necessary for plasma. There was always close agreement between the muscle inulin spaces measured in the two thighs ot individual animals. Muscle \rcc 0-.Y//...V ami jrcc ainhio acids: Muscle samples for these analyses were taken immediately after sacrifice of the experimental animal. They were weighed on a torsion balance and homogenized in ice-cold \(Y/c trichloroacetic acid i for a-NH2N), SO',' ethanol (for paper chromatography) or \(/< picric acid (for the amino acid analyzer). Checks on possible rapid production of ammo acids hv hvdrolvsis of tissue proteins were made for the first two methods by doing OSMOREGULATION IN MUSCLES OF TOADS 221 .^ ' -~~ s ~ -H ^ *f a LO LO LO" £> -t" C 1 Tl 1 Tl n TI TI TI £ o* c : 1*5 - h r^ t~ j if j r^j ^\ 0 - — •- •4 ^^ C ; c -^ — ' LO LO LO I/ ^. ^"x > LO LO «* §E o c o c" c d c 23 -ti -H -H -H i\ -H -H f^ r< : o o OO O 00 ^ ~ : -f -H H ^|' T ^ ^ LO LO LO LO LO LO ^ = u M _M _n _M Tl Tl Tl it Tl -H ^ ^" ; >tj ; o i- r^ oo IO W 5 LO LO 'O '*' •? + "3 ^ ps. T ^? i? cs ro OO 2 £ •H -H -H -H -H ji [ l Tl Tl ^-' Tf Ol OO OC f- '^ rr, ro 0 t- CM ^ VO ^^ IT) IT} IO LO IO LO ^t1 Og ^4] H 4J 1\ -H -H " — ' -+ t- t^ C > o r^- c* r^i r» i LO r~ CN 01 -i- 2 OC 5~ r-a 0s ^ o" o" 1? Tj< IO LO 'f LO " r CM O 00^ 1 M [I M ,23 "H T o — T 11 1 1 i OO C 111 Tl H ^- — -r oo o^-C-r^i^ o i*-. O '^; ^J f o *^ ,^J 'J* t/5 a 'f. £3 'S °^ „ >^ ^» >> ^ . X ^ "* ^ ^* ^ w rf» X rt O, . ^" **• — ^^* 'ti ••* ' ^ xi — 1^** ~ ^** ^". ^^ ^* . t/2-2 fo C/ 5 -3 CA> -0 C/ 1 -5 "* lJ C/^ "C C/^ ^t CD ~ rt ^>& ?i2o?2^ ? vo *^> ^ ^" '•- "** -^ IO • O v.O • *~ ^ ^— ^. • oo c.^ ^o ^^s : ! C 1 C: ^r T* - "T \ r~- -f LO LO ^i •*-. ~ r. ^ ' ^ 0 10 1 O i - f. ^ t- f C-l LO <^ 777 M \LCOLM S. GORDON parallel analyses on duplicate aliquots of tissue which were either allowed to remain at room temperature for several minutes before being homogenized or were homogenized in the appropriate solutions at room, rather than ice, temperatures. Xo statistically significant differences were found. These results agree with those of Roberts and Simouseii i 1962). Mean intracellular concentrations for substances in the muscles were calculated using the following equation: [H20]m -- Ve [A]; =: mean intracellular concentration of A [A]m == mean wet muscle concentration of A [AJP == mean plasma concentration of A ve =- mean muscle inulin space [H203m == mean wet muscle water content The most variable quantity involved in these calculations is v,,. Estimates <>t the variability in \-\\\ produced by variation in v,. were obtained by recalculating [ A]i using v,, ±2 S.E.'s. The variability figures given in Table A* are the average deviations about the means, calculated by this procedure. These calculations were made on the assumption that there are no large dif- ferences between plasma concentrations and concentrations in muscle extracellular fluid. The data of Conway (19571)) and Maurer (1938) support this assumption TABLE V Intracellular concentrations in muscles of loads adapted to various salinities (a/1 amounts per kg. cell icater; cf. text for method of calculation) State of adaptation ci- (mEq) Na+ (mEq) K+ (mEq) NPN (gm.) Free a-NHsN (gm.) Urea X (gm.) Bujo viridix FW 17 ±8 30 ± 10 95 ± 10 5.4 ± 0.5 1.42 ± 0.01 1.07 ± 0.01 20', S\\ 17 ± 10 22 ± 11 98 d= 10 4.9 ± 0.4 1.52 ± 0.01 1.18 ± 0.04 7-15 da\ - 40' , S\\ 58 ± 20 73 ± 20 105 ± 20 7.0 ± 1.0 2.10 ± 0.02 2.54 ± 0.1 1 5 13 cia\ > 5(1' , SVY M, ± 6 77 ± 5 130 ± 0 8.4 ± 0.3 2.68 ± 0.16 2.61 ± 0.02 2-6 da\ - Hufa liorens RV 19 ± 4 30 ± 4 110 ± 10 4.0 ± 0.4 1.04 ± 0.20 0.90 ± 0.02 start 1 \\ — — — — 1.03 ± 0.02 — starved 1 mon. 20<% SW 19 ± 10 34 ± 8 114 ± 12 4.3 ± 0.4 1.26 ± 0.01 0.75 ± 0.04 7-15 davs III', S\\ 37 ± 16 57 ± 15 150 ± 18 6.4 ± 0.6 2.14 ± 0.03 1.78 ± 0.12 1 x days SO' , S\\ 2.44 i 0.05 3 6 days OSMOREGULATION IN MUSCLES OF TOADS 223 600 '. I'iridis and /->'. horcas are presented in Tallies III and IV. These basic data have been combined with the muscle inulin space data of Table II to yield otimates of intracellular concentrations in the skeletal muscle fibers of the two toads. These latter estimates are presented in Table V. Figures 1 and 2 compare the major features of these sets of data. Both forms maintain osmotic concentrations of the plasma at levels higher than medium concentrations until maximum salinity tolerances are reached. Increases in plasma concentrations above those characteristic of fresh water are due primarily to XaCl (S6rv of the increase in A'. I'iridis between fresh water and 50% sea water: lOy/ of the increase in B. borcas between fresh water and 40% sea water— 500 in Q) O E c. O c Ki.uurr 1 hut for />';//" /'<''>vorcus appear to be the first vertebrates known to use large quantities of free amino acids in intracellular osmoregulation. The hagfishes ( Mvxini) also have high blood salt concentrations, but no more than 10-15% of intracellular osmotic concentrations in these forms are due to free amino acids. Intracellular free amino acid concentrations in My.vinc (/Ittt'niosu in 100% sea water are only 60-70 m,l//kg. liber water ( Bellamy and Jones, 1961 ). This level is about one-third that found in both species of toad adapted to only 50r,' sea water. Other species of toads apparently normally maintain about 100 m,l//kg. fiber water of intracellular free amino acids, even in the absence of livdration stresses ( Briner, l'"d). QSMOREGULATION IN MUSCLES OF TOADS 227 Free amino acids have long been known to be of importance in intracellular osmoregulation in many groups of invertebrates (recent reviews include Awapara, 1962; Florkin, 1963; Kittredge et al., 1962; Lockwood, 1962). There is as yet no experimentally defined relationship between the changes in intracellular solute composition described and the changes in intracellular osmotic concentration which occurred. Intracellular osmotic concentrations probably change with and are equal to simultaneous extracellular osmotic concentrate While this situation has not yet been conclusively proven (Robinson, 1960, for review), data such as those of Buckley ct al. (1958) and Bloch et al. (1963) seem con- vincing. However, all cell water may or may not be available to solutes (Dydynska and YVilkie, 1963 ; Lindenberg and Gary-Bobo, 1960). Intracellular sodium activity may be quite low (Lev, 1964). Very little is known of the degree to which "free amino acids" are actually free in solution, or whether other substances, such as urea, may not actually be partially bound (Bozler, 1961 ; Heinz, 1962) . The differences in the nature of the compounds changing in the two species presumably are reflections of metabolic differences between the two forms. It is probable that equally large metabolic differences exist among the various tissues within each species (Roberts and Simonsen, 1962). The chemical origins of the substances changing in concentration are unknown. Changes in external ionic environment have been shown to produce important metabolic shifts in isolated muscles of Bufo inarinits (Muller, 1962). The change in carnosine content in B. borcas may be accounted for in several ways. Severin ct al. ( 1962) indicate that carnosine may play a role in neuro- muscular transmission in frogs. They found that the carnosine content of pieces of muscle containing many motor end plates was significantly higher than the carnosine content of pieces containing few or no end plates. The present results may thus : ( 1 ) be related to the changing needs of intracellular osmoregulation ; (2) indicate an important change in neuromuscular function in B. borcas asso- ciated with salinity stress; or (3) be simply a sampling error due to differences in numbers of motor end plates included in the muscle samples analyzed. The present data on muscle inulin spaces agree well with some previous deter- minations of this quantity in amphibian muscle (Boyle ct al., 1941; Dee and Kernan, 1963; Simon ct al., 1957). They are significantly lower and less variable than other results (Steinbach, 1961; Tasker ct al.. 1959). These latter groups of higher and more variable results may be due to handling procedures used in experi- ments involving soaking of isolated muscles in Ringer solutions of various types (Dydynska and Wilkie, 1963). The present data on plasma composition in B. riridis agree with previous results (Gordon, 1962). Both sets of data are quite different from those of Tercafs and Schoffeniels (1962). The reasons for the disagreement are not apparent. SUMMARY 1. A detailed study of intracellular osmoregulation in skeletal muscles has been carried out in two species of toads, Bufo viridis and Bufo borcas, adapted to various external salinities between fresh water and 50% sea water (salinity 16%c). 2. Both species are osmoconformers, changes in plasma osmotic concentrations being due almost entirely to changes in NaCl concentrations. Muscle dry weight, M \iroi.M s. GORDON however, is more stable, increasing by less than ,S5rv in both forms in the face of osmotic concentration increases of 80-1.^5'f in the plasma. Muscle extracellular volume (innlin .space) is constant, independent of changes in plasma concentration. 3. Relative stability of muscle hydration is due to accumulation of intracellular solutes. Changes in intracellular osmotic concentration are broadly partitioned: 47 '/r inorganic ions ( Cl, Xa, K), 33 % free amino acids and related compounds, 20*7f urea in B. riridis: 43% inorganic ions, 409r free amino acids and related compounds. \7r/< urea in B. boreas. Free carbohydrates appear to be virtually absent. 4. Increases in intracellular free amino acids and related compounds involve different substances in the two species. B. t'iridis accumulates primarily taurine, glycine and alanine. B. boreas accumulates primarily taurine, glutamic acid and carnosine. Intracellular urea concentrations seem alwavs to be significantly higher than plasma concentrations. 5. Uncertainties in the data, related primarily to lack of knowledge of the physi- cal state of intracellular solutes, are discussed. Various implications of the results are considered. LITERATURE CITED ALLEN, K., AXD J. A \VAPARA, 1960. Metabolism of sulfur amino acids in M \tilns cdnlis and Ran«ui cuneata. Biol. Hull.. 118: 173-182. A \\AI-AKA. J., 1962. Free amino acids in invertebrates: a comparative study of tbeir distribution and metabolism. In: Amino Acid Pools (J. T. Holclen, ed. ) : 158-175. BELLAMY, D., AND I. C. JONES, 1961. Studies on M \.\-inc i/liitinnsn. 1. Tlie cliemical composi- tion of tlie tissues. Co/up, liiochctn. Phyxiol.. 3: 175-183. BLOCH, R., D. H. WALTERS AND W. KUHN, 1963. Structurally caused freezing point depression of biological tissues. /. Gen. I'hysiol.. 46: 605-615. BOYLE, P. J., E. J. CONWAY, F. KANE AND H. L. O'REILLY, 1941. Volume of interfibre spaces in frog muscle and tlie calculation of concentrations in tbe fibre water. J. Plivsiol., 99: 401-414. BOZLER, E., 1961. Distribution of nonelectrolytes in muscle, .liner. J. I'hysiol., 200: 651-655. BRINER, G. P., 1961. Tbe constituents of tbe tricbloroacetic acid extract of toad muscle and tbeir relationship to "ion balance." Biochiin. Biophys. Ada, 50: 353-356. BUCKLEY, K. A., E. J. CONXVAY AND II. C. RYAN, 1958. Concerning the determination of total intracellular concentrations by the cryoscopic method. /. I'hysiol., 143: 236-245. CON WAY, E. J., 1957a. Microdiffusion Analysis and Volumetric Error. Lockwood, London, 4th cd. CONWAY, E. J., 1957b. Nature and significance of concentration relations of potassium and sodium ions in skeletal muscle. I'hysiol. AYr.. 37: 84-132. CONWAY, E. J., AND M. J. CAREY, 1955. Muscle sodium, \\ititrc, 175: 773. COTLOVE, E., 1963. Determination of tbe true chloride content of biological fluids and tissues. II. Analysis by simple nonisotopir methods. Anal. L'hcin., 35: 101-105. hi.i, E.. AND R. P. I\KI:\A\, 1963. Energetics of sodium transport in /\•> IIYNSKA, M., AXD I >. l\. \\'n.KiK, 1963. The osmotic properties of striated musdc fibers in hypcrtonic solutions. /. I'liysiol.. 169: 312 32('. I;LoRKix, M., 1%3. Effectors and mechanisms of the intracellular iso-osmotic regulation in euryhaline invertebrates. Hidchciii. J., 89: 107P. 1'i'AME, E. (}., J. A. RCSSKI.I. AND A. E. W 1 1. 1 1 ELM i, 1(>43. The colorimetric estimation of amino nitrogen in blood. ./. li'nn. Chcin.. 149: 255-270. GORDON, M. S., 1%2. Osmotic regulation in the green toad (Huh' ;'iri<]is). J. E.\-f>. Biol., 39: 261-270. (,(ii.'Do\, M. S.. 1%3. [ntracellular osmoregulation in the muscles nf t\\o species of euryhaline toads. Fed. Proc., 22: 67<». OSMOREGULATION IN MCSCLKS OF TOADS 229 HEINZ, E. (ed.), 1962. State of the intracellular amino acids. In: Amino Acid Pools (J. T. Holden, ed.) : 762-777. KITTREDGE, J., D. G. SmoNSEN, E. RonKKTs AND B. Jr.LixEK, 1962. Free amino acids of marine invertebrates. /;;: Amino Acid Pools (J. T. Holden, ed.) : 176-18'.. LEV, A. A., 1964. Determination of activity and activity coc't'fidi!it potassium and sodium ions in frog muscle fibers. Nature, 201: 1132-1134. LINDENBERG, A. B., AND C. GARY-Bouo, 1960. Eau lion solvante et eau d'hyfinaaiion protei' de muscle de grenouille. Arch. Sci. Physiol., 14: 303-320. LOCKWOOD, A. P. M., 1962. The osmoregulation of Crustacea. Bin!. Rev., 37: 25 MAURER, F. \V., 1938. Isolation and analysis of extracellular muscle fluid from the frog. Amcr. J. Physiol.. 124: 546-557. MULLER, M. H., 1962. Metabolic aspects of ionic shifts in toad muscl<\ Ilinchiiu. Binpl. Acta, 57: 475-494. NATELSON, S., 1961. Microtechniques of Clinical Chemistry for the Routine Laboratory. Thomas, Springfield, 2nd ed. RAMSAY, J. A., AND R. H. J. BROWN, 1955. Simplified apparatus and procedure for freezing- point determinations upon small volumes of fluid. /. Sci. Instr., 32: 372-375. ROBERTS, E., AND D. G. SIMONSEN, 1962. Free amino acids in animal tissue. /;;: Amino Acid Pools (J. T. Holden, ed.) : 284-349. ROBINSON, J. R., I960. Body fluid dynamics. In: Mineral Metabolism ( C. L. Comar and F. Bronner, eds.), 1A: 205-248. " ROE, J. H., J. H. EPSTEIN AND N. P. GOLDSTEIN, 1949. A photometric method for the determi- nation of inulin in plasma and urine. /. Biol. Chcm., 178: 839-845. RUSSELL, J. A., 1944. Note on the colorimetric determination of amino nitrogen. /. Biol. Chcm.. 156: 467-468. SCHERBAUM, O. H., T. W. JAMES AM) T. L. JAHN, 1959. The amino acid composition in relation to growth and cell division in synchronized cultures of Tetrahymena pyriformis. J. Cell. Comp. Physiol.. 53: 119-138. SEIFTER, S., AND S. DAYTON, 1950. The estimation of glycogen with the anthrone reagent. Arch. Biochcm.. 25: 191-199. SEVERIN, S. E., P. L. VUL'FSON AMI L. L. TRANDAFILOVA, 1962. Carnosine content of different parts of frog muscles. Dokl. Akad. Nauk SSSR. 145: 663-665 (English translation). SIMON, S. E., F. H. SHAW, S. BENNETT AND M. MULLEK, 1957. The relationship between sodium, potassium and chloride in amphibian muscle. /. Gen. Physiol., 40: 753-777. SPACKMAN, D. H., W. H. STEIN AND S. MOORE, 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chcm.. 30: 1190-1206. STEINBACH, H. B., 1961. Na extrusion by the sartorius of Riinu pipicns. J. Gen. Physiol., 44: 1131-1142. STOICOVICI, F., AND E. A. PORA, 1951. Le comportement aux variations de salinite. Note XXX. Stud. C erect. Stiint., A cud. Kef. Pop. Romdiie, Fil. Clnj, 2: 159-219. TASKER, P., S. E. SIMON, B. M. JOHNSTONE, K. H. SHANKLY AND F. H. SHAW, 1959. The dimensions of the extracellular space in sartorius muscle. J. Gen. Physiol., 43: 39-53. TERCAFS, R. R., AND E. SCHOFFENIELS, 1962. Adaptation of amphibians to salt water. Life Sciences, 1 : 19-24. THE ROLE OF CKKATINE AND ITS PHOSPHATE IN AMPHIBIAN DEVELOPMENT1 M \RGARET N. HARRISON - / >i'fni>'fin<'iit of Zoi'!ni/\'. lattice Uiik'crsilv, Durham, North Carolina Kaldwin I 1(|57S) has summarized the chemical events that occur in a normal muscle working under anaerobic conditions : (1) Adenosine triphosphate (ATP) remains unchanged; (2) Phosphocreatine (PC) disappears; ( .1 i Free creatine (CF ') appears ; (4) Free inorganic phosphate (Pi) appears; (5) Glycogen disappears; (6) Lactic acid appears. The classical theory of muscle contraction accounts for these occurrences by means of assumptions schematized in Figure 1. As long as it is active, the muscle under- goes endergonic changes of state, as shown on the right. The necessary input of energy is provided by the dephosphorylation of ATP, converting it into ADP (adenosine diphosphate ) and liberating free PT. Part of the ADP thus formed is immediately phosphorylated by PC, thus regenerating some ATP as fast as it is utilized and elevating somewhat the level of Cp. The remainder, together with free P[, stimulates glycolysis, which in the absence of oxygen terminates in lactic acid, and which helps to relieve the drain on stored PC by generating further ATP. The imbalance finally reached is a measure of the discrepancy between the rate at which energy is demanded by the contractile process and the rate at which it can be supplied by glycolysis. An energy transfer and storage system similar to that operative in muscle is believed to occur in amphibian embryos, and in the sequel some of the evidence will be reviewed. However, the role of creatine and phosphocreatine has never been thoroughly investigated, although it has been hinted at by the data of Zielinski (1937) and of P.arth and Jaeger ( 1CH7). Thus, there has been a considerable hiatus in our knowledge of the major features of tin- energetics of amphibian development. The present investigation is. in part, a study of energy transfer and storage in Rana pipicns embryos, with special attention to the role of phosphocreatine under both aerobic and anaerobic conditions. Also, advantage has been taken of the opportunity to conduct a parallel study of gastrula-arrested hybrid embryos ob- tained by fertilizing R. pipiens eggs with R. sylvatica sperm (Moore, 1941, 1946; Gregg, 1957). Direct estimation of free and total creatine has made possible the calculation of the amounts of phosphocreatine phosphorus ( IVP) present at vari- 1 Tins paper is based on ;i thesis submitted to Duke I'niviTsity in partial fulfillment of the 1 1 .|uirementN for tin- decree of Master of Arts. 2 Present address: Fort Johnson Marine I'.iolo.m'ral Laboratory, Charleston, South Carolina. 230 ROLE OF CREATINE AND ITS PHOSPHATE 231 PI GLYCOGEN STATE B LACTIC ACID >* ATP -"^ STATE A FIGURE 1. Chemical events occurring in muscle contracting anaerobically. cms stages, aerobically and anaerobically, and this has been compared with directly estimated free inorganic phosphorus. Total phosphorus (P-r) and total acid- soluble phosphorus (PA) have also been estimated as checks against the possibility of artifacts arising from phosphorus loss to the surrounding medium; and further guarantee that the embryos have been maintained within physiological bounds has been sought in a study of the ability of R. f>if>iais embryos to develop normally after anaerobic treatment. I thank Dr. John R. Gregg for suggesting this problem, for his encouragement and helpful criticisms throughout the work, and for his assistance with the manuscript. METHODS Evnbryological Embryos were obtained by stripping eggs from pituitary-treated R. pipicns females into suspensions of R. pi[>icns or R. sylratica sperm. After 30 minutes, the sperm suspensions were poured off and fresh 10' '< amphibian Ringer's solution (without phosphate or bicarbonate) was added. After an additional 30 minutes. the embryos were separated into small groups and allowed to develop, as con- venient, at 10° C., 18° C. or at room temperature (21° C.). Before subjecting the embryos to anaerobiosis, their jelly coats were removed with jewelers' forceps. Aerobic embryos were de jellied just prior to chemical analysis. Developmental stages, and the corresponding standard ages at 18° C.. were ascertained by reference to Shumway ( 1CHO). Anaerobiosis The apparatus used to provide an anaerobic environment is represented in Figure 2. Dejellied embryos (125) were placed in the bottom of the chamber, along with enough lO^r Ringer's solution to immerse the tip of the gas inlet tube. After tipping the chamber so that the inlet tube cleared the surface of the medium. pre-purified nitrogen (oxygen content less than 45 p. p.m.) was flushed rapidly through the chamber for one minute. The chamber was then clamped vertically 232 M \.RG \KKT X. II ARRISOX in a rack, and lowered partly into a water bath maintained at 24 ('. Xitrogen uas allowed to bubble slowlv through the inediuni for 10 minutes, after which all stopcocks were closed. Aerobic control embryos were placed in nnstoppered Krlenmeyer flasks in the same water hath. /'riitchi-frcc c.rtracls After a prescribed time in the chamber, the embryos were quickly staged, trans- ferred into chilled graduated centrifuge tubes, washed with ice-cold deionized dis- tilled water, made up to a volume of 5 ml. with water, decanted into a chilled! GAS INLET GAS OUTLET -GROUND GLASS JOINT I'H.rkK 2. Diagram chamber. glass homogenizer containing 1.25 ml. of 24/4 trichloroacetic acid and homogenized. After taking an aliquot for the determination of total phosphorus, the homogenate was cenlrifuged at 0° C. for 10 minutes at 5000 acidified with 0.25 ml. of 0.3 N HC1, hydrolyzecl for 30 minutes in a water bath at 56° C, cooled and re-neutralized with 0.25 ml. of 0.3 N NaOH. Two ml. of freshly prepared diacctyl reagi-nt were then added to each tube. The tubes were placed in the dark, to prevent a 12 HOURS IN NITROGEN FIGURE 3. Changes in creatine and phosphorus fractions in Rmui pipiens embryos at Stage 10-, after various anaerobic periods. photoreaction between trichloroacetate and alpha naphthol, and incubated for 35 minutes at room temperature. Optical densities at 525 m/< were determined with a Bausch and Lomb Spectronic 20 colorimeter. A blank and two standards (creatine hydrate) were run simultaneously. The amount of phosphocreatine phosphorus was calculated, once the value of CK was known, from the equation PCP — 0.2362 CR, where the numerical constant is the value of the ratio of the molecular weight of phosphorus to the molecular weight of creatine. Inorganic phosphorus was determined by the method of Berenblum and Chain (1938), as modified by Martin and Doty (1949). A 2-ml. aliquot of protein-free extract was placed in a test tube, followed by 5 ml. of 1 :1 benzene :isobutanol and 1 ml. of molybdate reagent. The tube was stoppered with a ground-glass stopper 234 MARGARKT \. HARRISON1 and shaken vigorously for 15 seconds. \Yhen it liad formed, 2.6 ml. of the upper phase were transferred with a syringe into a centrifuge tube and made up to 5 ml. with acid alcohol. Stannous chloride reagent (0.5 ml.) was then added, and after 20 minutes the optical density at 625 mp. was determined with the colorimeter mentioned above. A blank and two standards ( KH._.PO4) were run simultaneously. The method of Martin and Doty (1949) was also used for the determination of i.'tal phosphorus and total acid-soluble phosphorus, but with the modifications sug- gested by Ernster ct al. (1950). Two test tubes were prepared, one (total HOURS IN NITROGEN K 4. Changes in ereatine and phosphorus fractions in Kana pipicns embryos at Stage 11 after short exposure to anaerobiosis. phosphorus) containing 0.2 ml. of homogenate, the other (total acid-soluble phos- phorus) containing 1 nil. of protein-free extract. After the addition of 1 ml. of 10 X 1I,S( ), to each tube, they were placed in an oven at 130-160° C. After two to three hours, and after partial cooling, one drop of 30^ H.XX was added to each tube, which was then heated over a bunsen burner until the contents became colorless or very pale yellow in color. The tubes were then replaced in the oven for 15 minutes. After partial cooling, 1 ml. of water was added to each tube, which was then returned to the oven, now at 100° C., for 10 minutes. The tubes were then cooled, and the contents made up to 7 ml. with water. Inorganic phosphorus present was then determined as described above, with the exception that H,SO, was omitted from the molybdate reagent. ROLE OF CRKAT1XK AND ITS 1'HOSl'lIATE 235 RESULTS AND Discussiox Preliminary experiments In order to avoid working with irreversibly damaged material, an attempt was made to ascertain how long embryos (R. pip ions) at various stages could be kepi anaerobic at 24° C. and still develop normally when aerobiusis was resumed. It turned out that late-cleavage embryos and gastrulae are less susceptible to pern nent damage by anaerobiosis than embryos of other stages, and will survive a sojourn of 12 hours in nitrogen; but two hours of anaerobiosis is the maximum that will permit subsequent normal development of embryos in all stages from fertilization to hatching. TABU'. I Creatiuc and phosphorus moieties of R. pipiens and R. pipiens 9 X R. svli-atica c? embryos after 2 hours of anaerobiosis at 24° C., -in micrograms per 100 embryos Treatment Range of stages R. pipiens Hybrids No. expts. CT PCP Pi PA PT CT PCP Pi PA PT Aerobic 3-4 94.4 14.0 2.2 90.0 2071 90.6 12.6 2.4 89.0 1929 5 Anaerobic 93.8 7.0 5.3 91.2 2068 91.6 6.7 6.1 94.0 1911 Aerobic 9-11 94.8 14.4 3.7 87.6 1998 91.7 13.8 3.3 88.8 1926 6 Anaerobic 95.8 2.9 11.8 90.0 2081 92.0 2.3 11.3 88.8 1944 Aerobic 12-14 93.8 12.1 5.1 85.2 2002 93.2 12.0 2.6 88.2 1936 5 Anaerobic 102.4 3.7 15.6 91.0 2062 95.2 2.8 12.1 87.4 2139 Aerobic 15-16 94.7 13.4 5.1 84.3 2055 86.7 11.4 3.1 80.7 1987 3 Anaerobic 91.7 2.4 18.5 89.3 2029 87.3 3.1 12.4 83.3 1947 Aerobic 17-18 109.0 15.4 7.4 100.5 1905 94.3 11.4 4.2 93.7 1914 4 Anaerobic 108.0 1.7 21.4 107.0 1931 91.8 1.4 13.8 90.7 2104 Aerobic 19-20 145.0 19.3 10.2 128.3 1946 86.0 9.9 6.6 96.0 1851 3 Anaerobic- 145.0 2.5 35.5 134.3 1922 83.0 2.1 17.5 92.0 1888 Having established this result, two experiments were made to determine the rapidity with which creatine and phosphorus imbalances are established in A'. pipiens embryos (gastrulae) under anaerobiosis. The results (Figs. 3 and 4) in- dicate that the major changes occur during the first hour in nitrogen, although some further change is evident throughout the 12-hour period examined. As a consequence of the foregoing, two-hour periods of anaerobiosis were adopted as standard for further experimentation ; and it is believed that this guarantees comparability of the results obtained with embryos of different ages— and of the results obtained with normal and hybrid embryos, although tests of the nitrogen-tolerance of hybrid embryos were not made. Results obtained by other investigators indicate that temperature, length of anaerobiosis and embryological type all affect the extent of the phosphorus im- balance induced by oxygen-deprivation. Barth and Jaeger (1947), in a study of 236 M. \UC.\kKT N. HARRISON R. pip/cits and J\. pi[>icns J X l\. syk'aliea ^ gastrulae, found that free inorganic phosphorus reached a maximum level between 4 to S hours after the onset of anaerobiosis and that this level was essentially maintained up to 22 hours ( 18° C.). Little change in the level of ATP was observed in either type of embryo; and this agrees with the results of Brachet (1954). who examined Bit jo I'lthjans and B. nil but increasing thereafter. As regards total phosphorus, free inorganic phosphorus and total acid-soluble phosphorus, aerobic hybrids exhibit roughly the same developmental changes, though to a lesser ex- tent in respect of the latter two fractions; while phosphocreatine phosphorus declines in amount after stage H>, and total creatine is constant throughout. Kor R. f>ipiens. it may be noted that the aerobic levels of inorganic phosphorus, phosphocreatine phosphorus, and total creatine approximate those reported for R. tempoi-m-id by Zielinski (1(M7), although his methods have been questioned on (unstated, technical grounds by Harth and Jaeger (l('47i. A comparison of the two groups of results is set out in Table 1 1. In general, the values for inorganic phosphorus in aerobic I\. pi^iens embryos agree more closeK with those obtained by Kutsky (1950) than with the higher values obtained by I'.arth and (aeger (1(H7) and by Gregg and Kahlbrock ( l'»57). Among other things, this may be the result of using a chemical method that reduces the breakdown of labile phosphates by shortening their exposure to acid molybdate reagent. As compared to hybrid embryos, the higher levels of free inorganic phosphorus developed by normal embryos suggested to Gregg, Maclsaac ROLE OF CREATINE AND ITS PHOSPHATE 237 and Parker (1964) that they operate at a relative < nerg) -deficit ; hut in view of the fact that the increases are more than halanced by incn-.-iM'n^ total acid-sohihle phos- phorus, it seems hetter to look upon them mereh as an n of hio-her steady- 20 W ? .8 K ID 5 16 U 0 |4 g O 12 - 10 Q. 2 Id c u u o o NORMAL O 2 4 6 B tO 12 14 IS 18 2O 22 INCREASE IN P IN pg/IOO EMBRYOS 8 o: ffi 2 Ul 0 0 a. o a. Ill U) IT _ Q O2 A 6 8 (O 12 14 16 18 2Q 22 INCREASE IN T? IN pg/lOO EMBRYOS FIGURE 5 (above). Ratio of change in phosphocreatine phosphorus to change in inorganic phosphorus in Rana pipicns embryos during a two-hour anaerobic period. The solid line is the theoretical regression line and the dotted line the calculated regression line. FIGURE 6 (below). Ratio of change in phosphocreatine phosphorus to change in inorganic phosphorus in Rana pipicns $ X Rana syhatica <$ hybrid embryos during a two-hour anaerobic period. The solid line is the theoretical regression line and the dotted line is the calculated regression line. 238 MARGARET X. HARRISON state levels. This interpretation is reinforced by the subnormal total creatine levels of post stage 16 hybrids, which may place some limitation on the amount of energy that can be stored as phosphocreatine. Under anaerobiosis (two hours), total phosphorus, total creatine and total acid- soluble phosphorus remain relatively stable in both types of embryos. Phospho- creatine phosphorus, however, undergoes a marked depletion, which is almost exactly balanced by the appearance of free inorganic phosphorus (Figs. 5 and 6). This may be taken to indicate that no change of ATP-level occurs, as in muscle. Under longer periods of anaerobiosis (12 hours), more free inorganic phos- phorus appears than can be accounted for by the disappearance of phosphocreatine 12 HOUR RUNG 2 HOUR RUN A 8 10 12 1-4 16 18 2O 22 24 26 INCREASE IN Fj> IN pg/lOO EMBRYOS FIGURE 7. Ratio of change in phosphocreatine phosphorus to change in inorganic phos- phorus during anaerohiosis in R'niia pipiais emhryos. The solid line represents the theoretical line on which points should fall if the ratio is 1:1. The dotted line is the calculated regression line for the two-hour runs and the dotted-dashed line is the calculated regression line for the 12-hour runs. phosphorus, at least in normal embryos (Fig. 7), perhaps owing to the breakdown of ATI". These observations are in agreement with the results of Carlson and Siger (1957), who found that net phosphocreatine breakdown in iodoacetate- poisoned frog sartorius muscle is a linear function ot work done, and that no net breakdown of ATI' occurs until the phosphocreatine concentration drops by 60% or more. They also account for the findings of liarth and Jaeger ( 1947) that dur- ing long periods of anaerobiosis (10-22 hours), more inorganic phosphorus appears than can be attributed to ATP breakdown, in both normal and hybrid embryos. It should be noted that the phosphocreatine phosphorus-inorganic phosphorus imbalance induced by anaerobiosis is much more pronounced in normal embryos than in hybrids, at least in older embryos. This is readily understandable in terms of the relatively greater energy-demands imposed by the complexity of the de- ROLE OF CREATINE AND ITS PHOSPHATE 239 velopmental changes occurring in normal embryos, in comparison with those of lesser complexity occurring in blocked hybrids. Taken all together, the foregoing results suggest that the role of the creatine moieties in the energetics of amphibian development is the same as in that of contracting muscle: the phosphorylation of creatine provides a store of phosphate bond energy that may be drawn upon in periods of anaerobic stress. Indeed, in view of the demonstration of a phosphorylating anaerobic glycolysis in amphibian embryos (Cohen, 1954), the parallel between muscle and embryos appears to be nearly complete. We may now add a few remarks about the role of energy metabolism in the development of blocked hybrids. Post-blockage hybrids have subnormal respirator v and glycolytic rates (Earth, 1946; Gregg, 1960, 1962), and utilize stored glycogen at a rate lower than normal (Gregg, 1948). Some ultimate limitation upon their capacity to generate and store energy is indicated by their subnormal respiratory response to the uncoupling agent 2,4-dinitrophenol (Gregg, 1960) and by their failure to synthesize creatine after stage 16, as mentioned in the discussion above. Nevertheless, homogenates of hybrid embryos exhibit respiratory and glycolytic rates that are quantitatively similar to those of normal embryos (Gregg and Ray, 1957; Gregg, Maclsaac and Parker, 1964), and the general picture of energy production, transfer and storage that emerges is one of essential normality, in which actually occurring energy-demand is met without undue difficulty. Thus, the lowered rates of energy metabolism may be regarded as a reflection of some inde- pendent block, internal or imposed by the embryonic environment, to developmental transformations that would normally require a greater expenditure of energy. SUMMARY 1. A study has been made of the role of phosphorylated creatine in the energy metabolism of Rana pipicns and gastrula-blocked K. pipiens J X /?. sylvatica ^ hybrid embryos. 2. It is shown that the longest period of anaerobiosis that can be survived by R. piplcns embryos of all pre-hatching stages, with subsequent normal aerobic development, is of two hours duration, at 24° C. 3. In hybrid embryos, creatine synthesis is deficient after stage 16, and the levels of phosphocreatine phosphorus and inorganic phosphorus are subnormal. 4. In both types of embryos, two hours of anaerobiosis result in a decrease of phosphocreatine phosphorus, accompanied by a quantitatively similar increase of free inorganic phosphorus. Both changes are greater in normal embryos than in hybrids. In R. pipiens embryos, prolonged anaerobiosis (12 hours) results in the appearance of excess inorganic phosphorus, perhaps at the expense of ATP. 5. A comparison is made of energy metabolism in muscle and embryos, and the role of energy metabolism in the development of hybrids is briefly discussed. LITERATURE CITED BALDWIN, E., 1957. Dynamic Aspects of Biochemistry. The University Press, Cambridge. BARTH, L. G., 1946. Studies on the metabolism of development. /. Exp. Zoo/., 103: 463-489. BARTH, L. G., AND L. JAEGER, 1947. Phosphorylation in the frog's egg. Ph\siol. Zoo!., 20: 133-146. 240 ,10, VRET X. HARRISON iSk.u'iiKT. J., 1954. (.'oiistitnli' in .inorinale du noyau et tenenr en adde adenosine-triphosphorique de la cellule. /f.r/v.vc/m, 10: 492. I'.KKi-.Nui.i'M, I., AND E. CHAIN, l'>38. An improved nietliod for tlu- colorimetric determination of phosphate. Bioch •»«. ./.. 32: 205-298. CARLSON, F. D.? AND A. SH.KR, 1957. The dependence of creatine phosphate and adenosine triphosphatc breakdown on work in iodoacetate poisoned muscles, Biol. Bull., 113: 324. C'OIIKN, A. I., 1^54. Studies on glycolysis during the early development of h'tnni pi^iens. Physiol. Zool, 27: 128-141.' EN NOR, A. H., AMI II. ROSENBERG, 1952. The determination and distrilnition of phosphocreatine in animal ti»ue>. Bioclient. J., 51: 606-618. ERNSTEK. L., R. ZKTTERSTROM AND O. LINDBEKG, 1950. Method for the determination of tracer phosphate in biological material. Acta Client. Scaml.. 4: 942-947. GREGG, JOHN K.. l'M8. Carbohydrate metabolism of normal and of hybrid amphibian embryos. /. Exf. Zool, 109: 119-134. GREGG, JOHN R., 1957. Morphogenesis and metabolism of gastrula-arrested embryos in the hybrid Kami pipicns $ X Rana sylratica c?. In: The Beginnings of Embryonic Develop- ment, edited by Albert Tyler, R. C. von Borstel and Charles B. Metz, Publication ,\n. 48 of the American Association for the Advancement of Science. Washington, D. C. GKKGG. Joii.v R., I960. Respiratory regulation in amphibian development. Riol. Bull., 119: 428 439. (ikhi.i,. IOHN R., 1962. Anaerobic glycolysis in amphibian development. Hit>l. l'KV, PHYLLIS B., 1950. Phosphate metabolism in the early development of I\ann pif>icns. J. Exp. Zool.. 115: 424-460. MARTIN, J. B., AND D. M. DOTY, 1949. Determination of inorganic phosphate — modification of i-.o-hntyl alcohol procedure. Anal. Client., 21: 965-967. MOORE, J. A., 1941. Developmental rate of hybrid frogs. ./. E.\-[>. Zool., 86: 405-422. Mi. in.' K J. A., 1946. Studies in the development of frog hybrids. I. Embryonic development in the cross [\niia f>ipiens $ • l\'ana sylvatica <$. J. E.\-f. Zool., 101: 173-220. Sni'M\\A'i, \\ ., 1940. Stages in the normal development of Ktina r>ipi<-n.\. I. lr.\ternal form. Anat. Rec., 78: 139-147. ZIKLINSKI, M. A.. 1937. Phosphagen and creatine in frog's eggs. /. E.rf>. Biol.. 14: 48-55. AN AUTORADIOGRAPHIC INVESTIGATION OF THE GONADS OF THE PURPLE SEA URCHIN (STRONGYLOCEXTROTUS PURPURATUS)1 NICHOLAS D. HOLLAND - AND ARTHUR C. GIESE Hopkins Marine Station of Stanford University, Pacific Grove, California The duration of the gametogenic events following DNA synthesis by primary spermatocytes and primary oocytes may be determined autoradiographically follow- ing the administration of tritiated thymidine. Primary spermatocytes and primary oocytes in premeiotic DNA synthesis are the most advanced germ cells that can incorporate tritiated thymidine. Therefore, by labeling such cells with tritiated thymidine and preparing autoradiograms of gonad samples taken at increasing time intervals thereafter, the labeled cells may be followed as they differentiate into gametes. It is assumed that, before the appearance of labeled gametes, the most advanced labeled cells in any gonad sample have been derived from labeled primary oocytes or primary spermatocytes, and not from labeled gonial cells. By this method, the latter part of oogenesis has been timed in mice (Lima-de-Faria and Borum, 1962; Rudkin and Griech, 1962), while the latter part of spermato- genesis has been timed in rats ( Clermont ct al., 1959), fruit flies ( Chandley and Bateman, 1962), and man (Heller and Clermont, 1963). In the present investi- gation, the method has been used to study oogenesis and spermatogenesis in the purple sea urchin, Strongylocentrotus pnrpuratns, a marine invertebrate having separate sexes and an annual reproductive cycle. Gonads from urchins collected at monthly intervals throughout one annual reproductive cycle were treated with tritiated thymidine to determine which gonadal cells (both germinal and non- germinal) were synthesizing DNA at each monthly sample. At five different times during the annual cycle, long-term experiments were initiated to time the latter part of gametogenesis. MATERIALS AND MKTHODS All specimens of Strongylocentrotus pnrpuratus used were collected at low tide from tide pools near Yankee Point, California. Most of the urchins collected weighed between 20 g. and 40 g. and had apparently gone through at least one previous annual reproductive cycle. Samples of 12 urchins were collected at approximately monthly intervals from March, 1963 to February, 1964. On the day of collection, the gonads of the sampled urchins were exposed to tritiated 1 This work was supported in part by NSF National Predoctoral Fellowships to N. D. Holland and in part by USPHS Grant RG-4578 to A. C. Giese. We are deeply indebted to Professor L. R. Blinks, Dr. A. K. Christensen, Dr. J. H. Phillips and Dr. A. L. Lawrence for providing facilities and for invaluable advice and assistance during the studies reported in this paper. - Now a NSF Postdoctoral Fellow at the Stazione Zoologica di Napoli, Naples, Italy. 241 242 NICHOL VS D. HOLLAND AND ARTHUR C. C.IKSK thymidine for one hour in I'h'o or in ritro. To lahel gonads /;; i'h'u, each urchin was injected intracoelomically via the peristomial menihrane with 0.5 /AC. of tritiated thymidine per gram of fresh weight of urchin. Before injection, the aqueous solu- tion of tritiated thymidine ohtained from Xew Kngland Nuclear Corp., Boston (with a specific activity of 2000 me. per m.l/ ) was diluted with four parts of sea water. The injected urchin was returned to an aquarium for one hour. Then, one of the five gonads was dissected from the urchin and fixed in sea water-Bouin's fluid. It did not matter which of the five gonads was chosen, since preliminary ex- periments showed that all acini of all gonads in any individual urchin were 15- S10 -D C 1 • • • • M A M J J A 1963 i I I I l S O N D J F M 1964 l-H,i'KK 1. The average gonad indices of urchins sampled at approximately monthly intervals during the 1963-1964 annual reproductive cycle. ( ionad indices of male and female urchins were considered together for calculation of each point. Long-term experiments were initiated at each ot" the five points indicated with arrows. at the same stage of development, From the wet weight ot the other four gonads, the gonad index was calculated hv the following formula: gonad index weight of four gonads >: 100 : 1.Z5 ) — . 1 he average gonad index tor each monthly weight of urchin urchin sample for March, 1('(>.} to February. I{)(i4 is given in Figure 1. To lahel goiinds in I'itro, one gonad was removed from each urchin and placed at once in 5 ml. of 14° C. sea water containing 5 /',c. of tritiated thymidine. One hour later, the u.onad was fixed in sea water-Bouin's fluid. There was no detectable difference in the numher or distribution of radioactive nuclei hetween the in 1'il'o- and the in T'/'/ro-labcled gonads. //; 7'///v>-la1>eling used less tritinted thymidine and resulted in more uniform distrihution of tritiatcd thymidine to the gonadal cells than did in ?'/ro-labe1ing, hut at the height of the reproductive season, in ?'//ro-labeling resulted in considcraMr loss of mature gametes from the dissected gonads. \fter fixation, the laheled gonads were emhedded in paraffin and sectioned at 7 j>. ;\\\(\ 2 ii . The sections were covered with Kodak A l\ 10 si rip] ting film ; exposure SEA URUIIV COX ADS 243 was for 4, 11 or 25 weeks. The autoradiograms were stained through the emulsion with nuclear fast red (Montruil-Langlois, 1962) or with hematoxylin and eosin. Gonad sections adjacent to those prepared as autoradiograms were always stained with hematoxylin and eosin. In each monthly sample, the number of male and female urchins was approximately equal. The gonads from each month's sample usually showed a fairly wide range of developmental stages. Therefore, after the histology for the whole annual cycle had been studied, each month's gonads were rated as typical, retarded, or advanced. Only typical gonads were used in the description of each monthly sample. At five different times during the 1963-1964 annual reproductive cycle (indi- cated by arrows in Figure 1), urchins were collected, injected with 0.5 /JLC. of tritiated thymidine per gram, and kept in 150-gallon running sea water tanks in the laboratory until sacrificed. In the interval between injection and sacrifice, the urchins were fed as much of the brown alga, Macrocystis, as they would eat. At sacrifice, gonad indices were calculated and autoradiograms of labeled gonads were prepared. Of 63 urchins collected and injected on March 22, 1963, 13 were killed after 11 days, 10 after 25 days, 10 after 41 days, 10 after 73 days, 10 after 119 days and 10 after 193 days. Of 19 urchins collected and injected on August 8, 1963, four were killed after 3 days, four after 6 days, four after 9 days, four after 12 days and three after 20 days. Of 31 urchins collected and injected on September 3, 1963, five were killed after 3 days, five after 6 days, five after 12 days, five after 20 days, five after 40 days and six after 100 days. Of 17 urchins collected and injected on November 3, 1963, six were killed after 6 days, six after 20 days and five after 125 days. Of 19 urchins collected and injected on January 1, 1964, six were killed after 6 days, six after 20 days and seven after 65 days. In addition to the 20-g. and 40-g. adult urchins studied, one group of ten very small urchins, ranging from 0.3 g. to 3.2 g., was collected on June 15, 1963. The gonads of these urchins were too small to be used in calculating a gonad index and too small to dissect from the urchin. Therefore, a cut was made around the ambitus of each urchin to obtain the aboral half of the test with the gonads attached. The gonads were labeled /';; situ for one hour in 5 ml. of 14° C. sea water containing 5 juc. of tritiated thymidine. Fixation in sea water-Bouin's fluid decalci- fied the tests and permitted sectioning for autoradiography of the entire aboral half of the urchin, including the gonads. RESULTS A histological section through an acinus of a sea urchin ovary or testis reveals an outer visceral peritoneum, a middle connective tissue-muscle layer and a lining germinal epithelium. The visceral peritoneum and connective tissue-muscle layer of the gonads of Arbacla f^unctulata were described in detail by Wilson in 1940. In S. purpuratus, as in Arbacla, the connective tissue-muscle layer is easiest to study in the months after spawning since it becomes stretched very thin and is difficult to see during the ripe season. In the connective tissue-muscle layer of spawned-out gonads, occasional 5 /A > ' 1 ^ nuclei of fibroblasts were labeled by a one- hour exposure to tritiated thymidine. No labeled nuclei of the smooth muscles of this layer were ever detected. Throughout the year, some of the nuclei of the visceral peritoneum of the gonads become labeled by a one-hour exposure to 244 NICHOLAS I). HOLLAND AND AKTIIt'K C. CIKSK • -^.rr u J V • • . • -.• '•<$£' *.&T' "'*-'•'• \ v o- '';':*f2^tafcr-."- \ *f '•• - *&s ^•.•,,w> • -^%^4 m .... - W.- • ,.- -XLL--'.^ / ^v"^' .-J- XL.-/-. *** — ««^\-:. 4 //. which contains a single, poorly-defined nucleolus and little chromatin. The cytoplasmic globules survive fixation and stain strongly with eosin. It is probable that all such globules seen in squash preparations (Fig. 2) come from the cytoplasm of cells disrupted by squashing. Staining with hematoxylin and eosin demon- FIGURE 8. Part of an acinus of a testis from an urchin collected October 4, 1963. The sperm-filled lumen is at the top. The scale line is 150 /*. Hematoxylin and eosin. FIGURE 9. Part of an acinus of a testis from an urchin collected November 3, 1963. The sperm-filled lumen is at the top. The scale line is 150 /u. Hematoxylin and eosin. FIGURE 10. Parts of two acini of a testis from an urchin collected on January 1, 1964. The scale line is 150 /j.. Hematoxylin and COMH. FIGURE 11. The periphery of an acinus of a testis from an urchin collected on January 30, 1964. Several primitive spermatogonia with large nuclei are attaching clumps of maturing spermatogonia and spermatocytes to the base of the germinal epithelium. The scale line is 25 /*. Hematoxylin and eosin. FIGURE 12. An autoradiogram of a testicular acinus from an urchin collected on January 30, 1964, and exposed to tritiated thymidine for one hour. Many of the maturing spermato- gonia and spermatocytes are heavily labeled. The scale line is 150 fj.. Nuclear fast red. FIGURE 13. An acinus of a testis from an urchin collected on February 27, 1964. Clumps of germ cells, detached from the base of the germinal epithelium, are proceeding toward the center of the acinus. The scale line is 150 /j.. Hematoxylin and eosin. 246 NICHOLAS D. HOLLAND AND ARTHUR C. GIESE strates cells and cellular ris invested by tin- nutritive phagocytes; the- ingested objects, which cannot lie seen in s<|iiash preparations, are usually located at the center <>t an eosinophilic globule. The cell boundaries ot the nutritive phagocytes are usually inconspicuous in stained sections ot gonad. In Xoyeniher and December, as the gonad index was approaching its maximum value ( 1963—1964 annual reproductive cycle), the nutritive phagocytes as seen on the slides begin to change to their alternate morphological state. They shrink to 15 /' in diameter and contain fewer and smaller cytoplasmic globules. Most of the globules shrink to a diameter less than 1 // or disappear entirely from the testis by December 1, 1963, and from the ovary by January 1, 1964; a month's differ- ence in the time of disappearance of the globules is the only dissimilarity between the nutritive phagocytes of the male and female. Xo change in nuclear morphology accompanies the loss of globules from the cytoplasm, and the single vacuole is otteii retained. Deglobulated nutritive phagocytes which retain their vacuoles appear as hollow spheres (usually stuck to one another in clumps) in squash preparations. In stained sections, these vacuolated cells give the germinal epi- thelium a net-like appearance referred to collectively as the netzformigen Syncytium by Lindhal ( 1(M2, p. 382) ; however, it is likely that they are individual cells and are not organized into a syncytium. In some ripe urchins, especially those that became abnormally ripe due to their failure to spawn in the laboratory, the nutritive phagocytes lose their vacuoles and can only be detected as scattered nuclei in the attenuated germinal layer. Such devactiolated nutritive phagocytes cannot be detected at all in squash preparations. After spawning, the deglobulated nutritive phagocytes remain in their alternate morphological state until the gonad index begins to rise ; then cytoplasmic globules reappear. . \ntoradiograms of gonads treated for one hour with tritiated thymidine reveal that many of the deglobulated nutritive phagocytes synthesize DXA, presumably in preparation for mitosis. Labeled nuclei of nutritive phagocytes occur at all levels in the germinal layer. Therefore, the production of new nutritive phago- cytes is not limited to the base of the germinal layer (as Caullery believed in 1925 ). Nutritive phagocytes in their deglobulated phase labeled on March 22, 1963 (and their progeny), can still be detected when they have reverted to the globulated state several months after injection. As the gonad index begins to rise and the cytoplasm of each nutritive phagocyte as seen on the slides begins to till with globules, the number of nutritive phagocytes synthesizing DNA declines. At the height of the reproductive season, no labeled nutritive phagocytes are detected after a one-hour exposure to tritiated thymidine. Thus, the nutritive phagocytes apparently originate from pre-existing nutritive phagocytes dividing by mitosis in the germinal layer of unripe gonads. This conclusion does not agree \\ith earlier schemes deriving the nutritive phagocytes from cells of the visceral peritoneum covering the gonad ( Tennenl <•/ 31), or Irom coelomocytes (Liebman, 1'bO). Nutritive phagocytes were found in the gonads of all the' immature specimens of .V. fiiirpnratns investigated. In most of these immature gonads, the recognizable nutritive phagocytes observed are vacuolated, but lack eosinophilic globules. Xutritive phagocytes in the gonad of the largest immature urchin (3.2 g) investi- gated contained a feu- eosinophilic globules. Exposure of the immature gonads to SEA URCHIN GOXADS 247 tritiated thymidine for one hour labeled many of the nutritive phagocytes. At pres- ent, it is not known whether nutritive phagocytes can be found in the gonads of speci- mens of S. piirpuratus weighing less than 0.3 g. From the descriptions of Hamann (1888) and MacBride (1903), nutritive phagocytes arc apparently not found at the gonadal tube stage and earlier in the life history of the sea urchin. This raises the question of the ultimate source of the nutritive. phagocx to. Cuenot in 1892 supplied a possible answer: that germinal cells and nutritive phagocytes both arise from a common primitive cell type very early in the life history of the urchin. These primitive cells may possibly be the Urkeimzellen of Hamann ("1SSS) and the "large cells" of MacBride (1903). The testis. It is most convenient to begin a description of the annual cycle of the urchin testis with the urchins sampled on April 21 and May 26, 1963 (Fig. 3). At this stage of the annual reproductive cycle the germinal layer consists chiefly of vacuolated, deglobulated nutritive phagocytes and the acinar lumens may or may not contain masses of relict spermatozoa, that is, spermatozoa left over from a previous spermatogenesis. Such spermatozoa are usually scattered at all levels in the germinal layer ; it is probable that most of them lie within the cytoplasm of the nutritive phagocytes, although this cannot be rigorously demon- strated by light microscopy (Figs. 3 and 4). A few scattered spermatogonia lie at the base of the germinal layer. These spermatogonia, which are usually de- limited by a distinct cell membrane, have a small amount of clear or slightly baso- philic cytoplasm. Their nuclei are at most about 5 //. in diameter and usually have one or two nucleoli and little chromatin (Fig. 4). In autoradiograms of typical testes from the April 21 and May 26 urchin samples, some of the spermatogonia are labeled by exposure to tritiated thymidine for one hour. DNA synthesis by the spermatogonia is presumably followed by mitotic division. In places at the base of the germinal layer where the spermatogonia have divided several times, they are grouped in cell nests. Such spermatogonia will be referred to hereafter as primitive spermatogonia. The germinal layer of a typical testis from the urchins sampled July 8, 1964, consists mostly of nutritive phagocytes, now containing eosinophilic globules as well as relict spermatozoa ; masses of such spermatozoa are still found in a few of the acinar lumens. The germ cell nests at the base of the germinal epithelium contain primitive spermatogonia and also a second type of cell which has a baso- philic cytoplasm and a nucleus 3 ^ to 4 //, in diameter; the nucleus often lacks a nucleolus and contains more chromatin than the nucleus of a primitive sperma- togonium. It is likely that cells of this type are maturing spermatogonia (which may be compared to the intermediate and type B spermatogonia of mammals). A one-hour exposure to tritiated thymidine labels many of the primitive and maturing spermatogonia. In the typical testes sampled on August 8. 1963, the individual nests of spermatogonia have become confluent, creating a continuous band of germ cells at the base of the germinal layer (Fig. 5). The cells of this band are by now so crowded that individual cell boundaries can rarely be seen even in 2- p. sections. Therefore, it is necessary to describe cell types by nuclear morphology alone. In general, the primitive spermatognia are the most basal cells in the band, and the spermatocytes in the band are closest to the lumen (Fig. 6). The spermatocyte 24S NICHOLAS I). HOLLAND AND ARTHUE C. C1KSK nuclei arc 3 /». to 4 /< in diameter, and some arc filled with a thread-like1 chromatin which stains strongly. Other spermatocyte nuclei contain numerous, stubby chromosomes, which give them a lumpy appearance. There is no reliable criterion for setting apart primary and secondary spcrmatocvtes in S. f>nrpnrains, although Xishikawa ( l()(d ) reported that secondary spermatocytes contain thinner chromo- somes than primary spermatocvtes in two Japanese sea urchins. Also in the band of germ cells are numerous cells intermediate in nuclear morphology as well as position between the spermatocytes and basal primitive spermatogonia. Since mitotic figures are often found among these intermediate cells, it is likely that they constitute a population of maturing spermatogonia rather than early primary spermatocytcs. As these maturing spermatogonia intergrade structurally with both the primitive spermatogonia and the spermatocytes, it is impossible to estab- lish precise boundaries between these cell types. In the typical testes sampled in August, a few trails of spermatids arise from the spermatocytes and extend, in the spaces between adjacent nutritive phagocytes, a short distance toward the center of the acinus (Figs. 5 and (>). The more basal, younger spermatids have darkly staining, round nuclei about 2 //, in diameter, while the more mature spermatids have more elongated nuclei, which resemble blunt spermato/oa ; spermatid cytoplasm is not demonstrable. A few newly-formed spermatozoa arise from the spcrmatids in the spaces between adjacent nutritive phagocytes. These spermatozoa have pointed conical heads, 2.5 /JL long and 1.2 /JL in maximum diameter, and are morphologically indistinguishable from relict spermatozoa in the acinar lumen or in the nutritive phagocytes. Many primitive spermatogonia, maturing spermatogonia and spermatocvtes are labeled by a one-hour exposure to tritiated thymidine (Fig. 7). The labeled primitive and maturing spermatogonia are synthesizing DNA in preparation for mitotic division. It is likely that mitotic divisions of primitive spermatogonia produce both primitive spermatogonia and maturing spermatogonia, while all ma- turing spermatogonia go through a limited number of mitotic divisions and then differentiate into primary spcrmatocvtes. Thus, the primitive spermatogonia prob- ably constitute the self-sustaining, progenitor compartment and the maturing spermatogonia probably constitute an amplification compartment of germ cell pro- duction in the testes. The labeled spermatocytes are presumably pre-leptotene primary spcrmatoc\ tes (comparable to the resting primary spermatocytes of mam- mals) synthesizing l).\.\ in preparation for meiotic divisions. In testes from urchins injected with tritiated thymidine on .August S and killed three days later, the only labeled germinal cells are primitive spermatogonia. maturing spermatogonia, and >permatocytes. Six davs after August S injection, tin- most advanced labeled cells are the vounger spermatids. Twelve and 20 days after the August S injection, the most advanced labeled cells an- spermato/oa. Therefore, the minimum lime required in August for a pre-leptotciic primary spennatocvte to become a spermatid is between three and six davs. This time is probably closer to six clays than three davs. since only the younger spermatids arc' labeled at six clays. The minimum time required in August for a pre-leptotene primary spermatocyte to become a mature sperm is more than six and less than 12 days. This time is probably somewhat less than 12 days (probably about 10 days), judging from the large number »\ labeled spermatozoa present 12 days alter SEA URCHIN GONADS 249 injection. In several testes from the August sample, the cytoplasm of the nu- tritive phagocytes contains many ingested spermatozoa embedded, sometimes singly and sometimes in clumps, within the eosinophilic globules. Twelve and 20 days after injection of tritiated thymidine, some of these ingested spermatozoa are labeled. Therefore, the nutritive phagocytes ingest not only relict spermatozoa, but also some newly-formed spermatozoa, and sometimes spermatids and sperma- tocytes. A typical testis from the sample taken September 3, 1963, resembles a typical testis from the previous month, except the basal band of germ cells is thicker. As in the previous month, many primary spermatogonia, maturing spcrmatogonia, and spermatocytes are labeled by a one-hour exposure to tritiated thvmidine. In testes from urchins injected with tritiated thymidine on September 3 and killed three days later, only primary spermatogonia, maturing spermatogonia, and spermatocytes are labeled. Six days after injection, the label has reached the younger, spherical spermatids. In testes from urchins injected on September 3 and kept in the laboratory for 20 days, continuous trails of spermatids and spermatozoa lead, in the spaces between the nutritive phagocytes, from the basal band of germ cells to the lumen, which contains a small mass of spermatozoa (see Fig. 8). Thus, by this time, at least some of the spermatozoa in the lumen are newly-produced and not from a past reproductive cycle. This conclusion, based on anatomical evidence, is borne out by autoradiograms showing labeled spermat- ozoa in the central sperm mass 20 days after injection. In the same testes, some nutritive phagocytes contain ingested labeled spermatozoa. In testes sampled 40 days after the September 3 injection, the lumen contained labeled spermatozoa, while some ingested clumps of labeled spermatozoa are still seen in the nutritive phagocytes. In testes sampled 100 days after the September 3 injection, labeled spermatozoa are still present in the lumen. After 100 days, no labeled sperma- tozoa are found in the cytoplasm of the nutritive phagocytes, but labeled amorphous basophilic granules are sometimes present ; presumably these labeled granules are the remains of partially digested, labeled spermatozoa. In typical testes from the samples of October 4 and November 3, 1963, the acinar lumen contains a mass of newly-produced spermatozoa (Figs. 8 and 9). A broad band of primitive spermatogonia, maturing spermatogonia and sperma- tocytes occupies the basal part of the germinal layer. Streams of spermatids and spermatozoa are proceeding between adjacent nutritive phagocytes toward the lumen. A one-hour exposure to tritiated thymidine labels many primitive sperma- togonia, maturing spermatogonia, and spermatocytes. In testes from urchins in- jected with tritiated thymidine on November 3 and killed six days later, the label has reached the younger, spherical spermatids. Twenty days after the injection of November 3, large numbers of labeled spermatozoa are found in the central sperm mass. Twenty days after injection, no labeled spermatozoa can be detected in the cytoplasm of the nutritive phagocytes. Apparently the phagocytosis of spermatozoa by the nutritive phagocytes of the testes ceases between September and November. The cessation of phagocytosis at approximately the same time that spermatozoa begin to accumulate in the lumen suggests that the nutritive phagocytes may play an important part in regulating the rate of accumulation of spermatozoa in the lumen. Testes from urchins injected on November 3. 1963, 250 NICHOLAS J>. HOLLAND A N D ARTHUR C. (JILiSE still contain labeled spermato/oa on March 7, No'4 (135 clays later). Apparently these urchins, which had an average gonad index of 17.1 at sacrifice, failed to spawn in the laboratory during the first part of January, 1(>(>4, when the urchins in the field spawned. In typical testes from urchins sampled on December 1, 1963, and January 1, 1964, the acinar lumen is distended with a great mass of spermatozoa. The germinal layer is markedly thinner than in earlier months, partly due to shrinkage of the nutritive phagocytes (which have lost most or all of their eosinophilic globules), and partly due to a thinning of the basal band of germ cells. Streams of spermatids and spermato/oa are still proceeding between nutritive phagocytes toward the lumen. In the testes of some individuals (Fig. 10), deglobulated nutritive phagocytes occur among the spermatocytes. maturing spermatogonia, and primitive spermatogonia. Some of these nutritive phagocytes appear to detach parts of the basal hand of germ cells from the basement membrane which separates the germinal epithelium from the connective tissue-muscle layer. As in previous months, primitive spermatogonia, maturing spermatogonia, and spermatocytes are labeled by exposure to tritiated thymidine for one hour. In testes from urchins injected with tritiated thymidine on January 1, 1964, and killed six days later, the label has reached the spermatids. Twenty days after injection, there are numerous labeled spermato/oa in the acinar lumen. No testes were available from the urchins injected on January 1, 1964, and sacrificed on March 7, 1(>M. since all were females. In a typical testis from urchins sampled on January 30, 1964, several weeks after spawning (A. L. Lawrence, personal communication), the acinar lumen contains only a small number of spermatozoa. The germinal layer still consists of deglobulated nutritive phagocytes and a basal region of germ cells. Many nutritive phagocytes occur among the basal germ cells and divide them into separate clusters of cells, which remain attached by one or two cells to the basement membrane. In many clusters, the most basal cells are primitive spermatogonia (Fig. 11). It is most likely that these cells are the very spermatogonia from which will spring the germ cells of the next annual reproductive cycle. Treatment of such a testis for one hour with tritiated thymidine labels many of the maturing spermatogonia and spermatocytes in the cell clusters (Fig. 12). The primitive spermatogonia at the base of the cell clusters are labeled only rarely. Typical testes from urchins sampled on February 27, 1964, and on March 22, 1(>63. have deglobulated nutritive phagocytes and clumps of germ cells at all levels in the germinal layer ( Fig. 13). Most of these clumps of maturing spermatogonia and spermatocytes have become detached from the base of the germinal layer and appear to be moving toward the acinar lumen. The lumen contains spermatozoa, often mixed with clumps of spermatids and spermatocytes. A few scattered primitive spermatogonia remain near the base of the germinal layer. Kxposure of such a testis to tritiated thymidine for one hour labels many ot the maturing spermatogonia and spermatocytes, even as they arc- progressing toward the lumen in disorganized clumps few of the primitive spermatogonia are labeled. In testes from urchins injected with tritiated thymidine on March 22, 1963, and killed 11 days later, the acinar lumen contains some labeled spermatozoa. Therefore, -permatozoa are produced after spawning has occurred, and the term relict SEA URCHIN GONADS 251 spermatozoa should not necessarily connote spermatozoa produced before spawn- ing. Some labeled spermatozoa were detected in tbe testes of urchins injected on March 22, 1963. and killed at increasing time intervals (up to 193 days) thereafter. The ovary. During the 1963-1964 annual reproductive cycle, germ cells were synthesizing DNA in typical ovaries sampled from March through October. These germ cells, which include oogonia and primary oocytes, occur in small groups or nests near the base of the germinal layer. In most ovaries, these cell nests are inconspicuous, and several acinar cross-sections must usually be searched before a nest is located. The oogonia have scanty, clear cytoplasm and a nucleus 5 /A in diameter ; the oogonial nucleus contains one or two indistinct nucleoli and little chromatin. The pre-leptotene primary oocytes have a thin shell of clear cytoplasm and a nucleus 4 /A to 5 p. in diameter; a nucleolus is often present and the chromatin is organized into tine threads. The cell nests also contain primary oocytes from the leptotene through the diplotene stages of the first meiotic prophase ; the chromatin of these oocytes is organized into a spireme. After the primary oocytes complete the diplotene stage, they enter the interphase-like dictvotene stage. Tennent and Ito (1941) referred to the dictyotene stage as the diffusion stage plus the resting nucleus stage. The primary oocytes remain in the dictyotene stage until they are fully grown and ready to undergo maturation divisions ; only then does the first meiotic prophase terminate. The smallest primary oocytes in the dictyotene stage are scattered at the base of the germinal epithelium. Exposure to tritiated thymidine for one hour labels some of the oogonia and pre-leptotene primary oocytes in each cell nest3 (Fig. 14). As early as three days after injection of tritiated thymidine, some of the primary oocytes in the spireme stage have become labeled. In none of the long-term experiments can the label be traced into growing primary oocytes, secondary oocytes and mature ova. Therefore, it mav well be that the label remains in the smallest primary oocytes in the dictyotene stage until the following annual reproductive cycle when the labeled primary oocytes grow and undergo maturation divisions. Such an ex- tended dictyotene stage in the human primary oocyte has been reported to last many years (Ohno et al., 1962; Baker. 1963), while a dictyotene stage lasting many months apparently occurs in female mice (Rudkin and Griech, 1962) and in female chicks (Hugres, 1963). It is convenient to begin a description of the animal cycle of the ovary v\-ith specimens of S. pnrpuratns sampled on May 26. 1()63. A typical ovary for this stage in the annual reproductive cycle is shown in Figure 15. Most of the acinar lumens contain a few ova from a preceding reproductive cycle (relict ova), some but not all of which are being attacked and digested by deglobulated vacuo- lated nutritive phagocytes. The germinal layer, in addition to the nutritive phago- cytes and germ cell nests already described, has as its base a number of small primary oocytes, apparently produced during the preceding annual reproductive cycle. These elliptical oocytes have a weakly basophilic, fibrous cytoplasm and a 3 These results are in general agreement with the earlier findings of Nigon and Gillot (1963), who incubated fragments of Arbacia lixula ovary in the spring for one hour in sea water containing tritiated thymidine and found a few labeled cells which they interpreted as "jeunes ovocytes" (p. 249). Nigon and Gillot did not attempt to follow the fate of the labeled cells in long-term experiments in vivo. 252 NICHOLAS 1). 1IOLLAN1) AND \KTMl K C. GIESE spherical nucleus containing a strongly l>aso|>hilic, spherical nucleolus. The largest of these primary oocytes is 1 5 ^ ) : 12 ^ and has a nucleus 9 /t in diameter containing a nucleolus 3 /JL in diameter. In typical ovaries ( Fig. 16) from urchins collected July S and August 7, 1963, the nutritive phagocytes have eosinophilic £••'.:*»••," - ft •/ .'••••.T-'\ .17 •:• ™ ,^'',:mi«^ i ©J o^ iOk!ahaa3C 22 FiGiki-. 14. An autoradiogram prepared from an ovary of an urchin collected September 3, 1963. Above the large, growing primary oocyte, there is a nest of oogonia and pre-leptotene primary oocytes labeled after e.\])osure to tritiated thymidine for one hour. One of the cells "f the visceral peritoneum is also labeled (below the growing primary oocyte). The scale line is 25 fj.. Xuclear fast red. l-K.i'kK 15. Several acini of an ovary from an urchin collected May 26, 1063. The scale line is 150 ,u. I lematoxylin and eosin. I'M, i RE l'i. I 'art of an acinus of an ovary from an urchin collected July 8, 1064. The center of the acinus is at the top. The scale line is 150 /i. Hematoxylin and eosin. Fi(;n4. Tin' center of the acinus is at the upper right. The scale line is 150 n. I Icmatoxylin and eosin. FJGUKK 20. An acmns of an ovary from an urchin collected on January 30, 1064. The scale line is 150 /u. Hematoxylin and eosin. IMM'KK 21. Several acini of an ovary from an urchin collected on March 22, 1063. Many large oocytes and ova arc being digested by deglobulated nutritive phagocytes. The scale line is 150 fj.. \ Icmatoxylin and eosin. I;[<;ri'. 18 p. containing a nucleolus 6 p. in diameter. October is the last month in which the germ cell nests occur at the base of the ovarian germinal epithelium. Typical ovaries sampled November 2, 1963, contain many primary oocytes of maximum size, some at the base of the germinal epithelium and others pro- ceeding between the nutritive phagocytes toward the lumen (Fig. 18). A primary oocyte of maximum size (which many embryologists term an "immature egg" or an "egg in the germinal vesicle stage") is usually elliptical (65 p X 45 p} and has weakly basophilic cytoplasm; the nucleus (or germinal vesicle) is 28 p X 22 p. and contains a nucleolus 8 p. in diameter. Some of the oocytes of maximum size (secondary as well as primary) contain maturation spindles (Fig. 18) and are giving off polar bodies. Maturation can occur at any level in the germinal epithelium or in acinar lumen. The acinar lumen contains a few newly-produced ova, which are 60 p. in diameter and each contains a nucleus 9 p in diameter. Since the diameter of a living ovum of S. pnrpitratns is about 80 p, it is evident that fixation and embedding causes considerable shrinkage. At the base of the germinal epithelium of ovaries sampled in November, there are growing primary oocytes of assorted sizes and also a number of small cells resembling the oogonia described previously. It is not clear whether these cells are oogonia, very small primary oocytes in the dictyotene stage, or both ; they are not labeled by a one- hour exposure to tritiated thymidine. Typical ovaries from the urchins sampled on December 1. 1963, resemble the typical ovaries of the previous month, but have far more ova in the acinar lumens. The typical ovaries sampled on January 1, 1964, contain more ripe ova than ovaries sampled December 1. 1963, and the nutritive phagocytes have become deglobulated (Fig. 19). In a typical ovary from urchins sampled on January 30, 1964, several weeks after spawning, the acinar lumen contains only a small number of ova, while the germinal layer is much the same as it was on January 1, 1964. Some primary oocytes of maximum si/.e and some oocytes in the process of maturation are still present (Fig. 20). It is, therefore, very likely that large primary oocytes continue to mature into ova after spawning has occurred. The large oocytes and the mature ova show no signs of being digested by the nutritive phagocytes. The ovaries sampled on February 27, 1964, unlike ovaries of the preceding months, contain no medium-sized primary oocytes. At the base of the germinal epithelium are only small primary oocytes, the largest of which is 20 /a ) 15 p., each with a nucleus 12 p in diameter containing a nucleolus 4 p in diameter. The acinar lumens contain some ova, a number of primary oocytes of maximum 254 NICHOLAS D. HOLLAND A XI) ARTHUR C. GIESE si/o, ;uul ;i few oocytes in the process of maturation. A few of these ova and oocytes arc being attacked and digested by the nutritive phagocytes. The disap- pearance of the medium-sized oocytes may lie due to the completion of growth and maturation hy all oocytes larger than .some critical size (which is apparently 20 |U /! 15 /i I. while all oocvtes smaller than this remain, without growing, at tin- base of the germinal epithelium. Spawning in 1(>(»4 occurred as in 1903, during the month of January (A. L. Lawrence, personal communication). Therefore, gonads sampled in late March of 1<>(>4 and in late March of 1963 should be at a similar stage of development. In ovaries sampled March 22, 1963, many of the ova and oocytes of maximum size are being attacked and destroyed by the nutritive phagocytes (Fig. 21). Where the attacking nutritive phagocytes come into contact with an ovum or oocyte, the boundary of the attacked cell becomes indistinct and its cytoplasm appears to be eroded away. At the base of the germinal layer, the germ cell nests are again present. These nests are probably reconstituted by mitosis of reserve oogonia carried over from the preceding fall. As in the previous month, the largest primary oocytes are still 20 p. ) 15 // and each has a nucleus 12 p. in diameter containing a nucleolus 4 /JL in diameter. A typical ovary from urchins sampled on April 21, 1963, has only a few ova remaining from the preceding oogenesis and no primary oocytes of maximum si/.e left in the acinar lumens. The nutritive phagocytes continue to attack some of these ova and also appear to be destroying some of the larger (20 /*, >: 15 /i) primary oocytes at the base of the germinal layer. 1-5 y May 26, 1963, when tin- sample which began this description of the annual ovarian cycle was taken, the largest primary oocytes remaining at the base of the germinal layer are 15 /A > 12 /j.; each has a nucleus 9 //, in diameter containing a nucleolus 3 /j. in diameter. Immature (/omuls. In all specimens of .V. piirpuratus weighing less than 3.0 g. (16 mm. in test diameter), the gonads remain immature throughout the year, even at the height of the reproductive season for the majority of the population. Although Fuji (I960) reported that the gonads of small specimens of Stron- (•ciitrotits intcrmedius are neuter, the sex of small specimens of 5". f>urf>uratus can be determined by microscopic investigation of their gonads. The germ cells of immature ovaries include oogonia, pre-leptotene primary oocytes, primary oocytes in the spireme stages of the first meiotic prophase and primary oocytes in the dictyotene stage. The largest of these dictyotene primary oocytes is 15 /x )! 12 /x and has a nucleus 10 /«, in diameter containing a nucleolus 3 p. in diameter (Fig. 22). Exposure of the immature ovaries to tritiated thymidine for one hour labels many of the oogonia and pre-leptotene primary oocytes. The primary oocytes which were produced on June 15, 1963, were presumably those which would grow and mature during the 1( '64-1965 reproductive cycle. The only germ cells in the testes of immature urchins are apparently primitive spermatogonia. A one- hour exposure to tritiated thymidine labels some of the primitive spermatogonia in these testes. I JISCUSSION The present investigation has elucidated the time course of the later events of spcrmatogenesis in .S'. pitrpnralits. The interval from primary spermatocyte l)\.\ svnthesis to early spermatid is about six days. It is probable that each SEA URCHIN GOXADS 255 primary spermatocyte spends most of this six-day period in an extended first meiotic prophase, and then divides into two secondary spermatoc\ tes, each of which quickly divides into two early spermatids. Since the first labeled sperma- tozoa appear tbout 10 days after labeling, approximately four days are required for a spermatid to differentiate into a mature ^>ermatO7.oon (the process of spermiogenesis) . The duration of the later phases of spermatogenesis in the sea urchin is comparable with those in the fruit fly, where the interval from primary spermatocyte DNA synthesis to early spermatid is five days and where spermio- genesis takes three davs (Chandley and Bateman, 1962). These intervals in both the sea urchin and the fruit fly are considerably shorter than in the labora- tory rat. In the rat, the interval from primary spermatocyte DXA synthesis to early spermatid is 18 days, while the interval from early spermatid to ripe spermatozoan is approximately 17 days (Clermont ct al., 1959). In A". f>nr^uratus, the time-course for the later events of spermatogenesis was the same for the production of spermatozoa in August, September, November. January, and March of the 1963-64 annual reproductive cycle. This uniformity in the duration of the cellular transitions between primary spermatocyte DXA synthesis and the appearance of mature spermatozoa means that this part of spermatogenesis can exert no control over the rate of sperm production. There- fore, the events which control the rate of sperm production must occur prior to the termination of DXA synthesis by the primary spermatocytes. The nature of these controlling events was not elucidated in the present investigation. Several possible schemes may be proposed to explain the seasonal fluctuations in the rate of sperm production. In the simplest of these, sperm production could be con- trolled by seasonal fluctuations in the duration of the growth-duplication cycle of the primitive spermatogonia. For example, in the spring the average duration of the growth-duplication cycle could be several weeks or months, but during the late summer and fall it may shorten to about one day. In S. pitrpuratits. the rate of sperm production does not necessarily equal the rate of sperm accumulation in the lumen of the testis. Early in the ripe season, newly produced spermatozoa are often ingested by the nutritive phagocytes, and never reach the lumen. Therefore, the rate of sperm accumulation in the lumen is determined both by the rate of sperm production and by the phagocytic activity of the nutritive phagocytes. In the ovary of S. purpiiraius, it is probable that the rate of increase in size of the primary oocytes (and not the rate of their production the year before) determines the rate of production of ripe ova. The possibility that the nutritive phagocytes of the ovary exert a partial control over production of ova was neither proved nor disproved in the present investigation. Since, in any given year, there is no significant difference in the gonacl index curve of male and female specimens of S. piirpnratns (Bennett and Giese, 1955), it is possible that the same factor or factors which influence spermatogonial proliferation may influence oocyte growth in the female. For a number of years, the annual reproductive cycle of .S". pHrpitratiis has been studied by monthly determinations of the gonad index of a periodically- sampled population of urchins (Giese, 1959). The shape of the gonad index curve and the time of spawning are often strikingly different from one year to the next. Spawning during the 1963-1964 annual reproductive cycle studied was J5(> NICHOLAS 1). HOLLAND \N1> ARTHUR C. C1KSE in [anuarv (if 1(>(>4. Spawning of the Yankee Point population of .V. [> urp unit its has been as late as May ( P.ennett and (iiese. l'1.^ I. At present, it is not known \vhether the time-course of germ cell proliferation and growth in years of early spawning and years of late spawning is different. The nutritive phagocytes, in addition to tin- phagocytic activity by which they destroy relict gametes and control the accumulation of spermato/.oa, also have a nutritive function in .V. piirpunitits. The cytoplasmic globules probably arise in part from ingested gametes and in part from nutrients translocated to the gonads by the coelomic fluid and hemal system. The cytoplasmic globules probably have a complex composition; a conspicuous component is a neutral mucopolysaccharide- protein complex (Holland, 1964). In the gonads of .9. f>itr[>uratits, the globules of reserve abruptly disappear as the gonads approach maturity Hearing the height of the reproductive season. Even as the globules disappear, the gonad index continues its steady rise due to the rapid increase in cell number in the testis and in the cell size in the ovary. The direct phagocytosis of globules from nutritive phagocytes by growing oocytes, reported for .Irbacia pnnctitlata by Liebman (1950). was never observed in the ovaries of .V. f^nr^iinifiis. Therefore, the re- serves stored in the nutritive phagocytes are presumably translocated in a soluble form to the germ cells (and possibly, to some extent, to other tissues as well). The globules in the nutritive phagocytes apparently were not utilized until most of the reserves stored in the inner epithelium of all major gut regions had been depleted during October and November of 1963 (A. L. Lawrence and J. M. Lawrence, personal communication). Tt is probable that a large part of these gut reserves was translocated to the gonads to support germ cell growth and pro- liferation early in the ripe season. SUMMARY 1. The DNA-synthesizing cells in the gonads of the purple sea urchin were labeled with tritiatcd thymidine and detected with autoradiography. 2. Some libroblasts in the connective tissue-muscle layer and some cells of the visceral peritoneum covering the gonads synthesize DNA. 3. Some of the non-germinal cells (the nutritive phagocytes') of the germinal laver proliferate during the .spring and earlv summer, but proliferation ceases in the late summer as the nutritive phagocytes begin to accumulate cytoplasmic globules ot reserve. Xutritive phagocytes labeled with tritiated thymidine in their deglolmlated phase in the spring are still labeled up to several months later, after thev have acquired cytoplasmic globules. 4. In the testes ot male urchins at the beginning of the reproductive season, not only relict spermatozoa, but also some newly-formed spermatozoa are ingeMed by the nutritive phagocytes. The phagocytosis of newly-formed spermatozoa ceases later in the reproductive season, at approximately the same time that spermatozoa begin to accumulate in the lumen. 3. In the germinal epithelium of the testis, the germ cells which synthesize l).\.\ are the spermatogonia and primary spermatocvtes. Primary spermatocvtes labeled with tritiated thymidine differentiate into early spermatids in about six days, while spermiogenesis takes approximately four days. SEA URC111X (iOXADS 257 6. Throughout the portion of the annual reproductive cycle when spermatozoa are heing produced (August through March), the time course for the later events of spermatogenesis remains constant. Therefore, the events which control the rate of sperm production must occur early in spermatogenesis. 7. In the germinal layer of the ovary, the germ cells which synthesize DNA are the oogonia and the pre-leptotene primary oocytes. During the same annual reproductive cycle, long-term experiments failed to demonstrate the differentia- tion of pre-leptotene primary oocytes into growing primary oocytes, maturing oocytes or ova. Therefore, it is likely that labeled primary oocvtes remain small and inconspicuous until the following annual reproductive cycle when tln-v grow and mature into ova. 8. The sex of very small, immature urchins mav he determined l>v histological examination of their gonads. The ovaries of immature female urchins contain small primary oocytes which are presumably the source of ova developing during the first annual reproductive cycle. LITERATURE CITED BAKKK, T. G., 1963. A quantitative and cytological study of germ cells in human ovaries. Proc. Roy. Soc. London, So: B, 158: 417-433. BENNETT, J., AND A. C. GIESE, 1955. The annual reproductive and nutritional cycles in t\vo western sea urchins. Biol. Bull., 109: 226-237. CAULLERY, M., 1925. Sur la structure et le functionnement des gonades chez les echinidi-s. TraT. Sta. Zoo/. U'iincrcii.v. 9: 21-35. CHANDLEY, A. C., AND A. J. BATEMAN, 1962. Timing of spermatogenesis in Drosophila mctanogastcr using tritiated thymidine. Nature. 193: 290-300. CLERMONT, Y., C. P. LEBLOND AND B. MESSIER, 1959. Duree du cycle de 1'epithelium seminal du rat. Arch. Anat. Micr. Morph. E.vp. 48 bis: 37-55. CUENOT, L., 1892. Xotes sur les echinodermes. 1. Ovogenese et spermatogenese. Zoo/. Anz., 15: 121-125. FUJI, A., 1960. Studies on the biology of the sea urchin. I. Superficial and histological gonadal changes in gametogenic process of two sea urchins, Strongylocentrotus nadn* and Strongylocentrotus intcnncdius. Bull. Fac. Fish. Hokkaido I'nii'., 11: 1-14. GIESE, A. C., 1959. Reproductive cycles of some west coast invertebrates. Pp. 625-638, in: Conference on Photoperiodism and Related Phenomena in Plants and Animals, ed. by Withrow. Amer. Assoc. Adv. Sci., Washington, D. C. HAMANN, O., 1888. Die \\-andernden Urkeimzellen und ihre Reifungsstatten bei den Echino- dernien. Ein Beitrag zur Kenntnis des Baues der Geschlechtsorgane. Zeitschr. wiss. Zoo/., 46: 80-98. HELLER, C. G., AND Y. CLERMONT, 1963. Spermatogenesis in man: an estimate of its duration. Science. 140: 184-186. HOLLAND, N. D., 1964. Cell proliferation in post-embryonic specimens of the purple sea urchin (Strongylocentrotus purpuratus) : an autoradiographic study employing tritiated thymidine. Doctoral dissertation, Stanford University. HUGHES, G. C., 1963. The population of germ cells in the developing female chick. J . Einhryol. Exp. Morph., 11: 513-536. LIEBMAN, E., 1950. The leucocytes of Arabacia punctulata. Biol. Bull.. 98: 46-59. LIMA-DE-FARIA, A., AND K. BORUM, 1962. The period of DXA synthesis prior to meiosis in the mouse. /. Cell Biol.. 14: 381-388. LINDAHL, P. E., 1932. Zur Kenntnis des Ovarialeies bei dem Seeigel. Arch. f. Entw., 126: 373-390. MACBRIDE, E. W., 1903. The development of Echinus csculcntus together with some points in the development of E. iniliaris and E. acutus. Trans. Roy. Soc. London, Set: B, 195 : 285-327. NICHOLAS D. HOLLAND AND ARTHUK C. CIKSK Mil MI;, K. A.. AND 11. I'.. SMITH, 1931. Ohservatioiis on tin- formation ol" the egg of Ecliinoiiictrii hicuntcr. Pups. Tortuijos /.. Carnegie hist. Washington, 27 (^413): 47-52. MONIKI n.-l .AXCLOIS, M., 1962. Staining sections mated with radiographic emulsion: a nuclear fast red. indigo carmine sequence. Stuin Tcchnol., 37: 175-177. \K;O\, \'.. AND S. (in. LOT, 1963. fitude radiographique du metaholisme des acides nucleiques et des proteines au cours du developpmu nt de 1'oeuf chez Arbac'm H.rnla. Cfihicrs Bio/. Marine. 4: 277-298. NlSHlKAWA, S., 1961. Notes on the chroiix»,oinr- of two species of echinoderms, Hcmiccntrohts piilchcrriiiuis (A. Agassiz) and Anth^cidaris cnissispiua (A. Agassiz). Zool. Mm/. Tokyo. 70: 425-428. ( )nxo, S., H. P. KLI.XUKK AND X. I'.. A i KIN, 1%2. Human oogenesis. Cytoi/nirfics, 1: 42-51. RUDKIN, G. T., AND H. P. GKIKIII. Inn2. ( )n the persistence of oocyte nuclei from fetus to maturity in the lahoratory mouse. ./. Hiopliys. Hiochciii. Cytol., 12: 169-175. TKXXKXT, D. H., M. S. (JAKDINKK A\D D. E. SMITH, 1931. A cytological and hiochemical study of the ovaries of the sea urchin Echinometra luciiiitcr. Pups. T<>rtii;/us Lab. ( 'arnegle Ins/., iruslihn/ton. 27 i =413) : 1-46. TKNNKNT, I.). H., AND T. ITO, 1'Ml. A study of the oogenesis of Mcspilia 50> held that the vibratile cells, after originating from the peritoneum, transformed into bladder and filiform amebocytes. Liebman further proposed a possible interconversion between eleocytes and colorless spherule amebocytes. Schinke ( l'»50) reported that the colorless spherule amebo- cytes, alter arising from connective tissue cells of the dermis, possibly differ- entiated into bladder and filiform amebocytes. The diversity of opinions concerning the origin and interrelationships of echinoid coelomocyte types prompted the present autoradiographic investigation employing tritiated thymidine. Autoradiograms were prepared from tissues of sea urchins killed one hour after injection of tritiated thymidine, to determine the possible sites of coelomocyte (or coelomocyte precursor) proliferation. Auto- radiograms were prepared from coelornocytes withdrawn from urchins at increasing time intervals after injection to demonstrate the possible transformation of labeled precursor cells into initiallv-unlabeled coelomocyte types. The circulating coelomo- cytes and the cells of the parietal peritoneum were studied quantitatively, since they could be prepared without histological sectioning, and yielded more reliable tritium indices than cells in sections. Autoradiograms of tissues that required sectioning were studied qualitatively. Coelomocyte production was investigated in starved as well as in freshly collected urchins. MATERIALS AND METHODS All specimens of Strongylocentrotus pitrpnrutiis investigated were collected from tide pools near Yankee Point, California, and were sexuallv mature. The weight and sex of each freshly collected urchin are given in Table I. On the day of collection, these urchins were injected intracoelomically ria the peristomial membrane with 0.2 /j.c. of tritiated thymidine per gram of fresh weight. One part of the aqueous solution of methyl-tritiated thvmidine (obtained from New Eng- land Nuclear Corp., Hoston, and having a specific activity of 6700 me. per mM) was diluted with tour parts of sea water before injection. The injected urchins were returned to containers of sea water for 50 minutes. Then coverslip prepara- tions of their coelomocytes were made bv the following method: A 10% (vv/v) aqueous solution of disodium ethylene-diaminetetraacetic acid (KI)TA). adjusted to pi I S.I bv slow addition of solid NaOIl, was used as an anticoagulant. A 1.0-ml. syringe (fitted with a 0.5-inch, 27-gauge needle) was filled with 0.2 ml. ot the KI)TA anticoagulant. The needle was introduced into the perivisceral coelom of an urchin bv puncturing the peristomial membrane in a radial position, and a 0.2 ml. -sample of coelomic fluid was withdrawn. The contents of the syringe were then quickly ejected into a flat-bottomed shell vial (15 ml. capacity, inside diameter 22 mm., and height 5 mm.) containing 5 ml. of the KDTA anticoagulant and a circular coverslip IS mm. in diameter. The SEA URCHIN COELOMOCYTE 1'RODUCTJOX 261 shell vial was placed in a regular centrifuge head, and the coelomocytes were spun down onto the coverslip at 500 rpm for ten minutes. The coverslip was then removed from the vial, and the adhering coelomocytes were fixed in formalin vapor under a hell jar. After overnight fixation, the still-moist coverslip prepara- tion was placed in two successive 10-minute baths of distilled water. The prrp;ira- tion was subsequently placed in a hath of 5% acetic acid (aqueous) for 30 minutes to remove all traces of acid-soluble tritiated material adsorbed to the slide. Tin coverslip was rinsed in distilled water, dried, and finally glued, cell-side-up, to a microscope slide with Permount. TABLE I Tritium indices of coeloiuocvtes und peritoneal cells from urchins killed one hour after injection Blad.-fil. amebocyte Col. sph. amebocyte Eleocyte Vibratili- cell Peritoneal cell Urchin weight Sex Av. # Av. = Av. # Av. = Av. * Tritium grains Tritium grains Tritium grains Tritium grains Tritium grains index per labeled index per labeled index per labeled index per labeled index per labeled cell cell cell cell cell 17.5 g. M 3.3% 32 0.2% 18 1.4% 27 0.0% — 1.6', 22 15.1 g. F 5.5% 40 1.1% 30 3.6% 37 0.0', — 16.5 g. M 1.5% 40 0.7% 19 2.6% 34 0.0', — 1.2', 26 16.0 g. F 2.5', 18 i.r; 5 1.9% 13 0.0' , — 1.8% 17 17.0 g. M 1 .0% 24 1.0', 19 1-4% 21 ().()', — 0.4% 15 16.8 g. F 1.6% 27 0.5', 12 1-2% 15 0.0', — 0.6% 27 16.0 g. M 0.9% 36 o..v ; 16 0.2% 32 0.0% — • i.r, 17 18.1 g. M 0.5% 31 0.6' , 11 0.4% 30 0.0% — 0.5' , 33 18.1 g. F 3.7% 37 2.8', 17 5.1% 38 0.0% — 1.3% 27 27.2 g. M 3.0% 38 0.3', 14 2-7% 26 0.0% — 35.7 g. F 3.7% 31 0.0% — 3.7% 34 0.0', — 29.4 g. F 1.7% 24 0.0' , — 1.4% 27 ().()', Average- tri- tium index + standard deviation 2.4 ± 1.5% 0.7 ± 0.8% 2.1 ± 1.6% 0.0 ± 0.0% 1.1 ± 0.5% After the coelomocyte sample had been taken, each urchin was replaced in its container for 10 more minutes and then fixed in sea water-Bouin's fluid. The Bouin's fluid had completely decalcified the urchins within one week. After de- calcification, the urchins were washed in water and dehydrated as far as 70% ethanol. At this point, samples of the following organs were dissected out for histological sectioning at 20 n, 7 p and 2 p, : gonad, axial organ, pharynx, esophagus, stomach, intestine. Polian vesicle with tooth, interradial body wall and radial body wall with radial nerve and water vascular elements attached. At this time, the parietal peritoneum with its supporting basement membrane was stripped off one interradial sector of body wall simply by seizing it with forceps and pulling. After a rinse in distilled water, the stripped' peritoneum was placed on a micro- scope slide and teased with two pins until it was spread out in a flat sheet with 262 X. D. HOLLAND, J. H. PHILLIPS, JR. AND \. C. GIESE no wrinkles. The sheet \vas mounted with the basement membrane facing the surface of the slide and the coelom-bordering epithelial cells up. Mescnteric strands (anchoring the gut to the body wall in intact urchins) arose from the coelomic side of the peritoneum an developer at IS C.), the coelomocytes and peritoneal cells were stained through the lilm with a 0.1 % aqueous solution of toluidine blue for one minute. The emulsion was then destained for several minutes in 70% etbanol. Alter a rinse in tap water, the stained autoradiograms were dried, and immersion oil was placed directly on the film to observe them. Kor quantification, a labeled cell was delmed as a cell having four or more silver grains above its nucleus. For each urchin, a minimum of 1000 cells was counted 1or each cell type quantified. Kor each cell type, the Iritiiini imfc.v equalled number of labeled cells LOO. I he average number ol grams per labeled total number of cells counted cell was recorded with each tritium index. The autoradiograms of the sectioned SEA URCHIN COELOMOCYTE PRODUCTION 263 tissues were stained through the emulsion with toluidine blue or with nuclear fast red by the procedure of Montruil-Langlois < l'">2). omitting the counterstain. Circulating coelomocytes from three uninjected donor urchins were exposed to tritiated thymidine in vitro. Each 30-g, donor was treated in the follouin- way: By peristomial puncture, 0.2 ml. of perivisceral coelomic fluid was taken into a syringe containing 0.2 ml. of the EDTA anticoagulant at 14° C. The contents of the syringe was then ejected into a shell vial containing a circular coverslip and 5 ml. of the EDTA anticoagulant at 14° C. One ^c. of tritia. thymidine in 0.01 ml. of distilled \vater was then added to the shell vial, and its contents were kept at 14° C. for 20 minutes. At least some of the cells collected in this way and subjected to washing with the EDTA anticoagulant are capable TABLE III Tritium indices of coelomocytes from three urchins serially sampled following a single injection of tritiated thymidine: the average number of grains per labeled cell is given in parentheses Interval from injection to samp ing 1 hour 24 hours 2>> days 57 days Sea urchin A Blad.-lil. amebocyte Col. sph. amebocyte Eleocyte Yibratile cell 3.0% (38) 0.3 'V (14) 2.7% (26) 0.0% (— ) 5.2', (21) 4.9', (13) 4.8%. (19) 1.2', (17) 4.6% (17) 7.9% (13) 5.9' ( (21) 2.7% (16) Sea urchin B Blad.-ril. amebocyte Col. sph. amebocyte Eleocyte Yibratilr cell 3-7% (31) 0.0% (— ) 3.7% (34) 0.0% (-) 3.9% (31) O.I', (5) 1.9', (32) 0.0% (— ) 5.5', (19) 5.0% (13) 6.9% (22) 0.7% (15) Sea urchin C Blad.-fil. amebocyte Col. sph. aincbiicyte Eleocyte Yibratile cell 1.7% (24) 0.0% (-) 1.4', (27) 0.0% (— ) 5.9% (31) 1.0', (23) 5.7% (28) 0-0% (— ) 6.0% (26) 6.3% (11) 8.8% (21) 0.3% (20) of proliferation in tissue culture ( Phillips and Farmanfarmaian, unpublished data). Therefore, exposure to the EDTA anticoagulant does not appear to affect the viability of the cells. Autoradiograms of the in z'/fro-labeled coleomocytes were prepared by the methods already described, the only difference being that they were exposed for eleven days rather than three weeks. Each of ten urchins, which had been starved in the laboratory from April 19 to August 27, 1963, was injected with tritiated thymidine on August 27 and killed one hour later. The weight and sex of each starved urchin are given in Table II. The urchins were injected and sampled by the procedures already described for the freshly collected urchins. Coelomocytes, peritoneal cells, and other tissues were prepared for autoradiography by the procedures already given. The only difference was that the amount of perivisceral coelomic fluid withdrawn was increased from 0.2 ml. to O.S ml., since the concentration of coelomocytes in the coelomic fluid was lowered bv the 130-dav starvation. 204 X. D. HOLLAND, 1. II. PHILLIPS, JR. AND A. C. GIESE The JUT! visa-nil codomic fluid from three urchins (A. \\ and C of Table III) was serially sani]iled following a single injection of tritiated thymidine. The urchins were injected and sampled by the procedures already described for the fre.shly collected urchins. From each urchin, a 0.2-ml. sample of codomic fluid was withdrawn one hour, 24 hours, 29 days and 57 days after the initial injection of tritiated thymidine. Autoradiograms were then made from the coverslip preparations of coelomocytes Throughout the experiment, the three urchins were fed continuously on the alga. Macrocystis. Each of the 30-g. urchins gained about 5 g. during the 59 days of the experiment. After the final sampling of coelomocytes. the three urchins were killed, and their peritoneal cells and other tissues were prepared for autoradiography by the procedures already described. * '•9 FK;I;KE 1. A toluidine blue-stained coverslip preparation of coelomocytes from the peri- visceral cot-lom of .V. piirpurntits. In the field are seven despherulated eleocytes, one colorlcv. .split-rule amebocyte (with a cytoplasmic process extending toward the upper left), one swollen \iliratile cell and one bladder-filiform amebocyte (at lower right). The scale line is 5 n long. Fna KK 2. A stripped preparation of interradial parietal peritoneum showing the regions of sparselj distributed nuclei and the regions of closely packed cells in surface view. Toluidine blue. The scale line is 30 /JL long. FIGURE 3. A toluidine blue-stained autoradiogram of a stripped preparation of the parietal peritoneum from an urchin killed 57 days after injection of tritiated thymidine. The plane of the silver grains is in focus. The scale line is 5 /u long. It was important to correlate the fixed and stained coelomocyte types, seen in coverslip preparations, with the living coelomocyte types described by Boolootian and ( "iie.se ( 1('5S). To accomplish this, a cross was scratched on one side of a circular coverslip, and coelomocytes were spun down onto the cross-bearing surface by the method already described. Prior to fixation, the living coelomocytes in the field with the cross were photographed. After fixation, the coelomocytes in the field with the cross were relocated and photographed a second time. The preparation was then stained in toluidine blue, and the same coelomocytes were photographed a third time. RESULTS The photographic correlation of the living coelomocyte types with the fixed and stained coeloniocvtes demonstrated thai the vibratile cells, eleocytes, and SEA URCHIN COKI.OMCH YTE PRODUCTION 265 colorless spherule amebocytes could be reliably identified in autoradiograms. The vibratile cells swell when fixed and flatten into discs 6 ^ to 20 /A in diameter when dried onto the slide. Toluidine blue stains the vibratile cell nuclei light blue and the cytoplasm (which contains an acid mucopolysaccharide) violet to purple. The flagellum is rarely visible in stained vibratile cells (Fig. 1 I. Colorless spherule amebocytes retain their spherules when fixed i Fig. 1). Toluidine blue stains their nuclei dark blue and their spherules light green. When fixed in formalin vapor, most but not all of the circulating eleocyu-s lose their spherules. Eleocytes without spherules typically have round nuclei 4 /A in diameter which stain dark blue with toluidine blue; their cytoplasm docs noi stain (Fig. 1). The eleocytes which retain their spherules are often swollen to the size of the vibratile cell in Figure 1, and toluidine blue staining reveal > large blue spherules (up to 3 p. in diameter) in their cytoplasm. It is probable that the circulating eleocytes which retain their spherules when fixed are identical to the orthochromatic coelomocytes described wandering in the gut wall of ,V. pitrpurutns (Holland and Nimitz, 1964). The bladder amebocytes, filiform amebocytes, fusiform corpuscles and hyaline hemocytes of Boolootian and Giese (1958) could not be reliably distinguished from one another when fixed and stained. For the purpose of this investigation, these four cell types are con- sidered to be a single type, the bladder-filiform amebocyte (Fig. 1). The fixed and stained bladder-filiform amebocytes are variable in shape and size (5 p. to 10 /A in diameter exclusive of cytoplasmic processes). They typically have a lightly staining nucleus and a basophilic cytoplasm which is often, but not always, ex- tended into processes. The data gathered from autoradiograms of circulating coelomocytes withdrawn from freshly collected urchins 50 minutes after injection of tritiated thymidine are presented in Table I. The circulating bladder-filiform amebocytes, colorless spherule amebocytes and eleocytes synthesize DXA. while the circulating vibratile cells do not. The average tritium indices for the circulating bladder-filiform amebocytes, colorless spherule amebocytes, eleocytes and vibratile cells labeled in riro are 2Ac/f , 0.7%, 2.]%, and 0.07^, respectively. Average tritium indices for the bladder-filiform amebocytes, colorless spherule amebocytes, eleocytes and vibratile cells removed from three urchins and labeled in vitro are 2.8%, 0.2%, }.S% and O.O^f, respectively. Thus, the circulating coelomocytes labeled in vitro immediately after removal from the urchin have tritium indices similar to those of Table I. These results rule out the possibility that all the labeled coelomocytes of Table I incorporated tritiated thymidine outside the coelomic circulation as they were finishing DNA synthesis and then entered the coelomic fluid in the 50 minutes between injection and sampling. Table I shows a striking variability in coelomocyte tritium indices between individual urchins. Since all urchins listed were treated at approximately the same time of day. the variability is not due to a diurnal cycle of fluctuating tritium indices. Such fluctuations in tritium indices do occur in some cell types in mice (Pilgrim ct a!.. 1963). It also appears from Table I that, within the coelomic fluid of each individual, the tritium indices for the bladder-filiform amebo- cytes, colorless spherule amebocytes and eleocytes are correlated — when one is high, the other two tend to be high also. To test the validity of this impression, 266 N. 1). HOLLAND, J. II. PHILLIPS, JR. \NI> A. C. G1ESE a correlation coefficient analysis was performed according to instructions in Moroney ( 1('5S. pp. .U(>-.UO ) . The analysis, the details of which are given in Holland (19()4). demonstrates that the correlation is significant at the 5% level. This significant correlation suggests that 1)X.\ synthesis in the circulating bladder- liliform amehocytes. colorless spherule amebocytes and eleocytes is under the con- trol of one or more common factors present in the plasma of the coelomic fluid. Possible controlling factors might include' either hormonal suhstances or nutrients necessary for DXA synthesis. Presumahly. DXA synthesis is e\eutually followed hy mitosis of the cir- culating coelomocytes. In autoradiograms of coelomocytes withdrawn trom urchins 24 hours after injection of tritiated thymidine. a numher of laheled coelomocytes have two groups of silver grains ahove them, indicating that ana- phase or telophase is occurring in the cell beneath. However, no chromosomes can he seen beneath the grains. Unequivocal mitotic figures also can not be de- tected in coverslip preparations of coelomocytes fixed in sea water-Bouin's fluid or Carney's acetic alcohol and stained with hematoxylin. It is apparent that some, and perhaps all, of the circulating coelomocytes which synthesize DXA subsequently divide in the coelomic fluid, but the mitotic figures are very difficult ( if not impossible) to detect by ordinary histological methods. This undoubtedly explains the failure of earlier workers to detect mitosis in sea urchin coelomocytes ( Kollmann. 1008; Yeager and Tauber, 1935; Schinke. 1950). It is interesting to note that Phillips and Farmanfarmaian (unpublished data ) found that coelomo- cytes taken from the perivisceral coelom of X. purpitratus proliferated when cul- tured for several weeks /';; ritro. "When coverslip cultures were fixed in Kahle's alcoholic formolacetic acid and stained with hematoxylin, some of the cultured coelomocytes showed all stages of mitosis with distinct chromosomes. I'n addition to coelomocyte tritium indices. Table I presents tritium indices for the cells of the parietal peritoneum labeled for one hour in t'ii'o. The interradial parietal peritoneum (Fig. 2) consists of regions of closely packed cells with elongate, often reniform, nuclei and regions of sparsely distributed, oval nuclei; cells of both regions are flagellated. The regions of closely packed cells run in interconnecting tracts, which may be seen in living, as well as fixed, stripped preparations ('living peritoneum may be stripped from the dermis in small pieces). A one hour's exposure to tritiated thymidine labels some cells in both regions. For calculating the tritium index, cells of both regions were counted together. Some, and presumablv all. of the peritoneal cells which synthesize DNA subse- quently divide, since autoradiograms of peritoneum from urchins killed .V days after injection of tritiated thymidine show that labeled cells (in both regions) frequently occur in groups of two to four ( Fig. 3). Groups of more than lour labeled cells also occur, but are less easily identified as progeny of a single initially-labeled cell. » In addition to cells of the parietal peritoneum, cells of all other tissues named by earlier workers as sites of coelomocyte production synthesize DXA. Auto- radiograms of sectioned tissues from freshly collected urchins killed one hour after injection show some labeled cells in the radial parietal peritoneum, in the visceral peritoneum, in the epithelium lining all parts of the water vascular system. in the axial organ, in ihe Polian \esicles. in the hemal strands and in the dermal SEA URCHIN COELOMOCYTE PRODUCTION 267 connective tissues of the body wall. Furthermore, there is DXA >\ nthesis by some of the eleocytes and colorless spherule amebocytes wandering in the body wall and gut wall. Cells of other tissues also synthesize DXA. but are not listed here since they have not been implicated in coelomocyte production. All tissues named as sites of coelomocyte production still contain labeled cells in urchins killed 37 days after injection. The tritium indices of coelomocytes and peritoneal cells from the starved urchins killed one hour after injection are presented in Table II. The average tritium indices of bladder-filiform amebocytes, eleocytes and peritoneal cells are significantly lower in starved than in freshly collected urchins. Due to large standard deviations, the average tritium index of colorless spherule ameboc\u- is not significantly lower (at the 5% level) in starved than in freshly collected urchins. The circulating vibratile cells, in the starved as in the freshly collected urchins, never synthesize DNA. Autoradiograms of sectioned tissues from the starved urchins showed that a few cells of all regions implicated in coelomocyte production synthesize DNA. The average number of grains per labeled cell is higher in starved than in freshly collected urchins, perhaps because a smaller number of cells synthesizing DNA results in less competition for the injected tritiated thymidine, perhaps because of a lesser dilution of the tritiated thymidine by unlabeled endogenous thymidine. After four months' starvation, protein in the perivisceral fluid of .V. ^nrpuratux declined from .37 mg. per 100 ml. to .14 mg. (L. Holland, unpublished). After 41 days' starvation, lipid in the perivisceral fluid declined from 25 mg. per 100 ml. to 15 mg. and carbohydrate from 2 mg. per 100 ml. to 1 mg. (A. Lawrence and J. Lawrence, unpublished). The tritium indices for the coelomocytes of the serially sampled urchins are presented in Table III. In donors A and C. labeled vibratile cells appear in the circulation sometime between 24 hours and 29 days after injection of tritiated thymidine. The 29-day sample for donor B was not countable, but labeled vibratile cells had entered its circulation by 57 days after injection. Therefore, some proliferating cell type must be differentiating into vibratile cells. The time course for this differentiation is on the order of several days at the least and several weeks at the most. The serial results for the bladder-filiform amebocytes, colorless spherule amebocytes and eleocytes are not very consistent. In most cases, the tritium index rises with time, but sometimes it drops. The striking rise in the tritium index of all three coelomocyte types of urchin C between one hour and 24 hours is unexpected and will be discussed below. The average number of grains per labeled cell usually falls slowly with time but in several instances remains constant or rises. DISCUSSION The circulating vibratile cells of S. pitrpnnitns are mature, non -proliferating cells, which must necessarily differentiate from one or more proliferating cell populations somewhere in the sea urchin. Of the many possible sources of vibratile cells, the cells of the parietal peritoneum are the most likely. The cytoplasm of some cells of the parietal peritoneum is filled with granules which stain violet with toluidine blue (i.e., show B-metachromasia') and which may be a mucopolysac- JOS \. |). HOLLAND, J. II. IM 1 1 I.I.I 1'S, JK. AND \. C. GIKSE charidc .similar to the one found in mature vibratile cells. Such a mucopoly- saccharide does not occur in the other cell populations which have been implicated in coclomocyte production. Furthermore, as I irhnian (1()50) pointed out, each cell of the i)eritoneum hears a single flagellum, which could he a precursor of the longer rlagellum of a vihratile cell. Since the vihratile cell is a non-dividing, differentiated cell type, it is most unlikely that it transforms into other coelonio- cyte types as Liebman claimed in 1950. The lack of DXA synthesis in circulating vihratile cells, as well as the ap- pearance of labeled vibratile cells in the circulation in the weeks following in- jection, does not support the suggestion of Cuenot (194S, p. 149) that vibratile cells are not urchin cells at all. hut are parasitic flagellate protozoans of the genus Oikoinonas. The conclusion that the vibratile cells are sea urchin cells and not parasites is further supported by Boolootian's 1(>(>2 report that the perivisceral fluid in all individuals of twelve different species of regular echinoids contained vihratile cells. Such universal distribution and lack of specificity is not char- acteristic of a parasite. Some of the bladder-filiform amebocytes, colorless spherule amebocytes and eleocytes of .V. f^iir^iinitiis synthesize DNA and apparently divide. All new coe'omocytes could ari.se by division of pre-existing coelomocytes, which would thus constitute self-maintaining cell populations. Cuenot (1897. p. 1S5) proposed just such a system for replacement of circulating cells of some arthropods, some annelids and some molluscs. However, all circulating and tissue coelomocytes synthesizing DNA could go through only one or a few growth-duplication cycles and thus constitute an amplification, compartment of a cell system which must necessarily have a self-maintaining compartment somewhere in the urchin. This self-maintaining, ultimate source of coelomocytes could be one or more of the tissues which have been named as sites of coeloniocyte production, since cell pro- liferation occurs in all of them. There are many possible schemes between these extremes. For example, only one of the three coeloniocyte types could be a self- maintaining cell population. Furthermore, tissue coelomocytes might have a dif- ferent renewal scheme from circulating coelomocytes of the same type. There is also the possibility (which was neither proved nor disproved in the present in- vestigation) that one type of coeloniocyte may differentiate into other types. The present lack of information on these points makes an unambiguous interpretation of Table 1 1 1 impossible. Interpretation of the data in Table III is further hampered by the lack of in- formation on the possible inter-changes of circulating and tissue coelomocytes and on the related <|iieMion of whether or not the circulating coelomocytes are in a Steady-State. It is known that large numbers of coelomocytes enter and leave the coelomic circulation in stressed urchins which have had all or part of their coelomic fluid removed ( Schinke, 1950; Roolootian and Lasker, 1964). In the present investigation, urchins A. ?>. and C of Table III were probably stressed to some extent by withdrawal of 0.2 ml. ('.V; to 10%) of their coelomic fluid at each sampling. The sampling of urchin C one hour after injection could well have induced an immigration of tissue coelomocytes (having high tritium indices) into the coelom between one and 24 hours after injection. In adult mammals, a renewing cell population has an initially-high tritium index ; with time, the tritium index and the average number of grains per labeled SEA URCHIN COELOMOCYTE PRODUCTION" 269 cell fall rapidly (Messier and Leblond, l('(>()j. An expanding cell population in mammals has an initially-low tritium index ; with time, the tritium index and the average number of grains per labeled cell tend to persist The data in Table III demonstrate that sea urchin coelomocytes rc-M-mble expanding, rather than renewing cell populations, of mammals. Indeed, most of the cell populations of adult sea urchins have the characteristics of expanding cell populations. In the adult urchin, only the gametogenic cells of the male during the ripe season and the tooth cells have the characteristics of renewing cell populations (Holland, 1964). The starved urchins of Table II showed no weight increase during starvation, although they appeared healthy in every other way. Presumably, the number of cells in their constituent cell populations was not increasing at the time of labeling on the last day of starvation. In most of the cell populations of the starved urchins, the few cells synthesizing DNA (33% of normal in the bladder- filiform amebocytes and 27% of normal in the peritoneal cells) are probably pre- paring to divide for replacement of cells lost from the populations and not for growth. Probably, even under normal conditions of growth, a minor component of proliferation in each expanding cell population is for replacement and not for growth. The reduction of tritium indices by starvation might be due to a re- duction in the nutrients necessary for cell proliferation or to starvation-induced hormonal changes. Since it is unlikely that starvation shortens the DNA syn- thesis period, the lowering of the tritium indices is probably due to an increase in the duration of the growth-duplication cycle by a lengthening of the period of the interphase preceding the DXA synthesis period. SUMMARY 1. Brief exposure to tritiated thymidine in riro or in vitro labels some of the bladder-filiform amebocytes, colorless spherule amebocytes and eleocytes circulating in the perivisceral coelomic fluid of the purple sea urchin ; circulating vibratile cells are not labeled. Some colorless spherule amebocytes and eleocytes are also labeled while wandering in the body wall and gut wall. 2. Some, and presumably all, of the circulating coelomocytes which synthesize DNA subsequently divide in the circulation. It is not known if this proliferation constitutes all or only a part of production of new bladder-filiform amebocytes, colorless spherule amebocytes and eleocytes in the sea urchin. 3. The tritium indices of the circulating bladder-filiform amebocytes, colorless spherule amebocytes and eleocytes within each individual urchin are significantly correlated. This suggests that DNA synthesis in these circulating cells is under the control of one or more common factors. 4. Brief exposure to tritiated thymidine labels some cells in the visceral peritoneum, the parietal peritoneum, the epithelium lining all parts of the water vascular system, the axial organ, the Polian vesicles, the hemal strands and the dermal connective tissue of the body wall. These tissues have been named by previous authors as sites of coelomocyte production. The present investigation found no evidence for or against their participation in the production of bladder- filiform amebocytes, colorless spherule amebocytes or eleocytes. 270 \. I). ITOLLAND, J. H. PHILLIPS, JR. AND A. C. GTKSK 5. In the weeks following injection of tritiatcd thymidine. I;i1>ele]>ear in the coelomie fluid . Tlie source of the vihratile cells is prohahly the parietal peritoneum. The vihratile cells thus constitute a sea urchin cell tvpe and are not, as has heen claimed, parasitic flagellates. (>. Serial autoradiographic data demonstrate that the coelomocytes (and most of the other tissues) of the young adult sea urchin have the characteristics of ex- panding, and not renewing, cell populations. 7. Starvation for several months causes a significant reduction in the tritium indices of the hladder-iiliform amehocytes, eleocytes and cells of the parietal peritoneum. LITERATURE CITED BOOLOOTIAX, R. A., l"r>2. The perivisceral elements of echinoderm body fluids. Amcr. 2: 275-284. BOOLOOTIAN, R. A., A xii A. C. CiiESE, 1958. Coelomic corpuscles of echinoderms. Bio!. Bull., 115: 53-63. BOOLOOTIAN, R. A., AND A. C. GIESE, 1959. Clotting of echinoderm coelomic fluid. /. E.rp. Zoo!., 140: 207-_7_"'. BOOLOOTIAX, R. A., AMI R. LASKER, 1964. Digestion of brown algae and the distribution of nutrient^ in the purple sea urchin Strongylocentrotus f>urf>itrtitns. Coinf*. Biochem. Physiol, 11: 273-289. CUEXOT, L., 1897. Les globules sanguins et les organes lymphoides des invertebres. Arch. Anat. Micr., 1: 153-192. CUENOT, L., 1948. Ediinodermes, pp. 1-363. In: Traite de Zoologie, Vol. XI, ed. Grasse. Masson, Paris. FREXZEL, J., 1892. Beitrage zur vergleichende Physiologic und Histologie der Verdauung. I. Mitheilung der Dannkanal der Echinodermen. Arch. Anat. Physiol, Physiol. Al>t.. 16: 81-114. GEDDES, P., 1880. Observations sur le fluide perivisceral des oursins. Arch. Zool. E.vp. Gen. (Ser.l), 8: 483-496. HOLT. A \i>, X. D., 1%4. Cell proliferation in post-embryonic specimens of the purple sea urchin tStroiniyloci'iitrolits purpuratnx} : an autoradiographic investigation employing tritiated thymidine. Doctoral Dissertation, Stanford University. Hoi. i. A\n. X. I)., \MI S. A. XIMITZ, 1964. An autoradiographic and histochemical investigation of the gut mucopolysaccharides of the purple sea urchin (Strongylocentrotus purpuratiis} . Bi,>!. Bull, 127: 280-293. KIMH;I:II, i. \-... \(>2(>. A study of the genetic relationships of the "ameb.-.cytes with spherules" in Arbacia. Biol. Hull.. 50: 147-154. KOLLMANX, M., 1908. Recherches sur les leucocytes et le tissue lymphoide des invertebres. Ann. Sci. Nat. Zool. (Scr. 9), 8: 1-240. l.iHiMAX, I'".., 1('5D. The leucocytes of . lrh from lMgun-s. 1'eiignin I'.ooks, Baltimore. I'n.r, KIM, C.. \\ . I. ii: \\u \V. MAI ii'. l'"i.i. Diurnal fluctuations in the numbers of DXA synthesizing nuclei in various mouse tissue's, \iilnrc, 199: 863. SAINT-HILAIKK, C., lS'<7. I'eber die Wanderzellen in der Darnnvand der Seeigel, Resume. Trai: Soc. /»//-. Nat., St. Petersburg, 27(3) : 221-248. SCHINKE, H., 1950. ['.ildung und Ersatz der /cllclcmente der Leibeshohlenflussigkeit von Psammechinus nnliin-is i Echinoidea) . Zcitschr. Zellforsch. nnkr. Aunt., 35: 311-331. Y^EAGER, T., AX» (). TAUBER, 1('35. < )n the haemolymph cell counts of some marine invertebrates. Biol Bull, 69: 66-70. PHOSPHATASES OF MARINE ALGA] EDWARD J. KUENZLER AND JAMES I'. I'KKK \S II <>i>\\ AKI) j. Kl'FA'/I.KK \.\D J \MI-'.S I'. I'KkKAS become phosphorus-deficient. This en/yme hydroly/ed dissolved phosphate esters on or outside tlic cell; tin- cell then absorbed oiil\- tin- phosphate ion. leaving the moietv in the inediuiii. \Ve are very grateful to 1 )rs. R. R. L. ( iuillard. |. C. Ilellebust, and C. A. I 'rice for inanv stimulating discussions and suggestions. Dr. (iuillard also gener- ouslv furnished the pure cultures of marine algae; the Chlamydomonas rcmhanli culture was obtained from the Harvard University laboratory of Dr. R. P. Levine through the courtesy of Miss |. C. Krungard. We are also grateful to Drs. |. H. Ryther. R. R. 1,. (Jnillard. and A. Torriani for reading and criticizing the manuscript. METHODS Stock axenic algal cultures were maintained in half-strength Medium / ((iuillard and Ryther. 1962) : 75 mg. XaXO,, 5 mg. XaHL,PO, • H,O.\5 mg. FeEDTA. 15-30 mg. Xa..Si( ).-''! I .,( ), 100 p.g. thiamine-HG. 0.5 //.g. Riotin, 0.5 //g. vitamin 1',.., 10 fig. CuS( ), • 51 1 J ). 22 /,g. XnSO4 -7H..O. 10Mg. CoCL/6H20, ISO Mg. .MnCL-41 1,6, '>.3 fig. Na2MoO4-2H2O, 1 liter filtered sea water. Chlamydomonas spp. were furnished with XH,C1 instead of NaXO:. as the nitrogen source. Stock cultures were tested periodically for bacterial contamination by inoculating into sea water medium containing 0.1 VP tryptone. Phosphorus-deficient algae were produced by growing them in sea water medium containing double the concentration of all the above nutrients except phosphorus; it was reduced to 7 ^M or less. All cultures were grown at 20° C. except /)ctomtUi conjervacea and Thalassiosira nordenskioldii which were grown at 5 C. Cells were counted using a Spencer "Bright Line" or a Palmer counting chamber. Cell dimensions were obtained with a calibrated ocular micrometer and cell volumes were calculated by formulae for similar geo- metric solids (sphere, cylinder, etc.). Cellular protein was measured by the Koliu-Ciocalteu phenol reagent by a method that we modified from Lowrv ct h dailv ) wa> added and the volume wa> made ii|) to <* ml. \\-ilb distilled water. After 10 minutes 0.5 ml. 1 X Folin-Ciocalteu ])benol reagent was added and the absorbance at 750 m// uas read 30 minutes later 011 a Beckman Dl' spectrophotometer. Protein standardization \\a> performed on bovine >erum albumin bv the Lowrv (•/ til. ( 1('51 ) procedure. Kn/yme aili\n\ was assa\ed bv the rate at \\bich nitropbeiiol accumulated during hydrolysis of /'-nitrophenyl phosphate (XPP) at known hydrogen ion concentrations. The \ I'P stock consisted of 0.1 g. of XPP plus 2.5 g. MgSO, in 50 ml. distilled water. The pi 1 range 3.0 to (>.S was covered by 1 M citric acid-potassium citrate solutions and the range 7.2 to ('.S bv 1 M Tris ( hvdroxy- meib\ 1 lamino methane • I I Cl solutions. The actual pi I value of filtered sea water PHOSPHATASKS OF MARINE AI.(,AP: 273 containing these buffer solutions was measured with a lU-ckman Model 7(i hydrogen ion meter. For each analysis, the algal culture: X IT stork: citrate1 buffer stock :auto- claved-sea water diluent ratios were usually about 1:1:1:30. In order to obtain adequate alkaline buffering however, it was necessary to use twice as much Tris. giving 1:1:2:30 ratios. Unless otherwise noted, all measurements of enzyme activity were performed on whole, living cells in darknes.-, or dim light at 19° -21° C. After incubation, sufficient pH 10 Tris buffer was added to the low pi I samples to bring out nitrophenol color (pH 9) and the absorbance of nitrophenol was measured in a Heckman 1)1* spectrophotometer at 410 m/i. in 1-cm. or 10-cm. cells. Knzyme activity was calculated from the nitrophenol-time regression; one "I'nit" represents the amount of enzyme that causes a change in optical density of 0.001 per minute in a 1-cm. cell. The rate of assimilation of phosphorus by algae from glucose-6-phosphate (G-6-P). adenosine monophosphate (AMP), and a-glycerophosphate (a-GP) was measured by periodically determining the phosphorus content of the algal cells. Two ml. of cultures of phosphorus-deficient cells were added to 8 ml. of filtered, auto- claved sea water ( pH ^ 8 ) containing 0.1-0.2 //.mole of one of these three ester- in centrifuge tubes. Periodically, duplicate tubes were centrifuged for 5 minutes, the supernatant was discarded, and the cellular phosphorus was determined by measuring the orthophosphate after hydrolysis in 0.55 X H._,SO4 at 140° C. for 5 hours ( Ketchum ct al., 1955). Total phosphorus initially present in the sea water, the algal cultures, and the three substrates was measured the same way. Inorganic phosphate of sea water was measured by the method of \Yooster and Rakestraw (1951). Rate of uptake of the glucose from G-6-P was determined from the decrease in radioactivity of a C'Mabeled solution. Ten ,uc. of the Ba salt of D-glucose-1-C14- 6-phosphate were dissolved in 3 ml. distilled water and converted to the Na salt by passage through a 7 ( 30-mm. strong cation exchange column (I)owex 50) in the XV form; the column was rinsed with 2 ml. of distilled water. This was diluted 1:100 to obtain a suitable count-rate and 0.25 ml. was added to centrifuge tubes containing autoclaved sea water ( pH - - 8), G-6-P carrier, and phosphorus-deficient algae as described above. Subsamples of this suspension were periodically cen- trifuged and samples of the supernatant were dispensed (0.2-0.5 ml ) onto planchets, dried, and the C14 counted with a windowless gas flow detector. The same volumes of the original whole suspension were also dried, and the initial count rate thus obtained had geometry and self-absorption factors similar to the supernatant samples. RESULTS Phosphatase synthesis b\ deficient ahjac Alkaline phosphatase is produced abundantly when algae are grown in a phosphate-limited medium, or stated conversely, its production is repressed in phosphorus-sufficient algae (Table I). In each species the enzyme activity of P-deficient cells was at least 30 times greater than in cells limited by vitamins, iron and trace metals, nitrogen, or silicon. (Phaeodactylum has no requirement for vitamins or silicon, nor Coccolitlnis for silicon; hence these experiments were not performed.) 274 KI>\\ \KI> J. ECUENZLEE AM) I AMI-IS P. 1'KKK AS IAKI.I, I /•.//<•(/ ni nutrient limitation mi /'induction of alkaline phosphatase. Cultures were grown in Medium f deficient in one of the 5 types of nutrients until cell numbers increased less than 10' , per day. Enzyme activity ( ('nits/ JO1 cells) was measured at 20: 25° C. All measurements were made at pH 8.0, although later it was found that this was not at the pH optimum for the phosphatase of these species. Nutrient-; Omitted : Thiamin Biotin, 812 Fe, Cu, /n, Co, Mn. Mo N Si p Pliaeiidactvluin tricornitlitm 1.1 2.1 — 62 Cyclotella nann (clone M 1) (1 0.03 0.10 O.O.i 6.3 C". nanti (clour M 1 > Skeletmienni custnlnm 0 0.04 0.17 0.27 0.02 0.27 0.21 0.13 12 12 Coccol it h us hu xley i 0.05 0.04 0 — 59 The low en/yme activities in 1 '-sufficient cultures are not the result of inhibition of enzyme activity by phosphate in the medium. In one experiment we measured the phosphatase activity of Coccolitlnis hii.vlcyi in the presence of various ortho- phosphate concentrations and found a nearly linear inhibition of activity amounting to S09f at 1.0 ni.U. However, the initial phosphate concentration of Medium f is PHAEODACTYLUM TRICORNUTUM 1 I I I 1 i I ~ • • Ct~ It1" — ^ | L/t LLo •-- PRDTFIM x\ •o^ 5 r t\U 1 L-\\\ • • PHOSPHATASE 44 40 ^ ^ N^ / ^ PROTE, CELLS / 9/ 36 32 ISP HAT A. 14 21 / 28 ^ 12 1 8 / 24 § 10 1 5 X 20 5 8 12 ^LlT^'^T^\; 16 | 6 9 /fl 12 4 6 •'/ ' 8 2 3 3^" i i« i i i i i i i i 4 1 3456789 10 11 £/4K I'H.i KI: 1. Changes in cell numbers, protein content, and cn/ynie activity during growth ol a Phacodactylmn tricornutum I'lilturr : 15 liters stirred by constant aeration; 2.^ f.M I'; 25 ( .: .ttio f.c, illnmination ; enzyme measured at |)ll (>.7. PHOSPHATASES <>F M \RI\K ALGAE 275 only 72 /x.l/ and tin- inhibition of enzyme activity should have only been about 6% even if no phosphate had been assimilated by the cells. As discussed below, the amount of phosphatase in a culture increases with time; thus, comparisons of the values for the different species in Table I are unwarrantable .1 5 ^ <0 ~J 8 «> ^ IS I 8?| fc ^ § $ 5 33 44 22 27 36 18 2 1 28 14 15 20 10 9 12 6 0 COCCOLITHUS HUXLEYI INITIAL PLUS P04 CELLS • — • A— A PROTEIN • — • A— A ENZYME • • A A I I I I 7 8 DAY 10 12 13 14 15 16 FIGURE 2. Changes in a-11 numbers, protein content, and enzyme activity during growth of a Coccolithits Im.rlcyi culture: 1 liter shaken several times daily; 3.7 ,u.l/ P; 20° C. ; 300 f.c. illumination; enzyme measured at ]>H ^.7; phosphate added on day 7 increased the content of the medium by 25 nM P. 'I'nnc course of phosphatase production The production of alkaline phosphatase in Phaeodactylum bc^an when the average cell still had enough phosphorus to divide at least once more. I.e., about two days before cell division and protein synthesis stopped ( Fig. 1 ). I£nzyme produc- tion continued at a nearly linear rate at least 5 days after the apparent halting of net protein production. The same pattern of initiation of enzyme production just before growth stopped was repeated in two experiments with Coccolithus, the results of one being shown in Figure 2. Repression of enzyme synthesis by phosphate was confirmed when this nutrient was added to half of the culture on day 7, the remaining half representing 276 KDXYAKI) I. KUENZLEE \.\i) FAMES I'. I'HKUAS a control. Phosphate stopped en/vine production whereas cell numbers and cellular ]>n>U'in increased again at rates comparable to those on days 1 to 3. The cellular phosphorus per unit volume of I '-deficient marine algae ranged from 1 to 15 > 10 17 mole I* !>"• in species of different sizes (Table II I. Cellular protein per unit volume, however, differed by less than a factor of 5 among these same species. Some variability in both 1'- and protein-content might arise from calcula- tion of cell volume, due to irregularity of cell shape and uncertainty as to the thick- ness of the enveloping frustules, coccoliths, or cell walls. A large vacuole would also tend to lower the amount of 1* or protein per unit whole cell volume. The l':l'rotein ratios calculated from Table II were more nearlv constant, from 230 I \m,i II Cell volume, phosphorus, protein, and HKIM'IIIUIII observed rule ot production of phosphdt/ise in phosphorus-deficient murim- Cell volume M< cell) Cell P (10-i-mole/V) Cell protein (10-" •+ M;1> Enzyme production rate (10~s units, V -day) Chrysophyceae Isochrysis pallid IKI 115 1.0 4..? 6 Cricosphaera cart era r 1240 1.7 5.8 16 Coccolithus hitxleyi 73 2.2 6.2 26 Bacillariophyceae Phaeodactylum tricomutinn 53 3.2 5.7 10 Cliaetoceros simplex 120 1.6 5.9 6 'riitil/issiosini fluvial il is 1300 2.9 7.4 10 Cyclott'llti cuspid 210 3.5 7,0 6 C. mind (clone 1 .> 1 i 140 3.4 10 0.2 ('. iia mi (clone .vl 1 ' 85 2.7 7.8 7 (.'. cry print 640 4.3 15 1 1 \it-_scliia clusteriiini 84 15 19 3 Skeletonema costal tun 160 8.0 12 0.5 1 Mnophyrear .1 mphidinium carteri ooo 2.9 to 0.4 to 7'H) //.mole I'/'g. protein. Some algae synthesized alkaline- phosphatase much faster than others when compared on the basis of cellulav volume (Table II). Further work will undoubtedly show conditions under which higher rates occur in some of these algae, but repeated experiments indicate that, under our conditions. there arc ohvious differences. Coccolithus, Cricosphaera, Phaeodactylum, and T. 'is repeatedly showed rapid enzyme production when phosphorus-deficient. ( > ptiiiiniii pi I The rate at which phosphorus-deficient algae hydroly/ed XI'P was markedly pH-dependent. The graphs of five representative species ( Kig. 3) show that significant enzvme activity generallv extends one or more pi 1 units on either side of the peak. Some >pecie> have more than one peak. It should be remembered that the absolute height of the alkaline-phosphatase peak i> a function of both the length of time since the cells became I'-dcficicnt and specific physiological factors. For this PHOSPHATASES OF MARINE \I.(,AK 277 reason emphasis is not placed on tin- amount ot en/\me of one species compared to that of others. Twenty-nine clones (at least 25 species i of marine algae were tested to determine the pH at which their phosphatases were most active (Table III i. Two species had maximum rates less than 0.6 Unit/ml, of culture and are not included in the table. All chrys< >phytes and diatoms ( Bacillariophyceae ) had an abundant enzyme with activity at pH S.6 or higher. Detonula had a slightly higher peak at pH 6 than at pH (). but this might have only been the result of its lower metabolic rate I 5 A • — • CRICROSPHAERA CARTER I B -— • COCCOLITHUS HUXLEYI C . — . PHAEODACTYLUM TRICORNUTUM D 0—0 RHODOMONAS LENS E O O CHLORELLA SP. 0 00- pH FIGURE 3. Five example «>f phosphomonoesterase activity from pH 3 to 1(1. at 5° C. ; the alkaline phosphatase activity presumably would overtake that of the acid in another few days. 1'hahissiosini nordenskioldii, the only other species grown at 5° C., was also relatively low in alkaline phosphatase, whereas T. fluriatilis was similar to other diatoms. The three clones of Rhodomonas, although originally isolated from different places, had almost identical pH graphs (see example. Fig. 3) and showed clearly that large amounts of alkaline phosphatase are not produced by Rhodonwiuis under these conditions. Their enzymes are probably constitutive; a repeat experiment with clone F-5A showed essentially the same amount of alkaline phosphatase as the first. The blue-green alga, Oscillutoria. produced alkaline phosphatase, whereas Coccoclilaris sp. (clone Syn) 278 EDW \KI> !. KUENZLER \\l> 'KKK \S (tint >howii i did lint. ( i \'ii; nod nun 111 lu'ls/nn is interesting because both the acid and the alkaline phosphatases move than doubled during four days of 1 '-deficiency. This is our only example of a phosphorus-repressible acid phosphatase. The two •-pecies of Chlamydoinonas were the only green algae with significant amounts of alkaline phosphata-e C'lone 0-5 was extremely rich; furthermore it had two alkaline peaks, the second being 475 Units/ml. at pi I S.u. The chlorophyte, iclla tcrtiolccta (not shown), was tested twice without acid or alkaline phosphataM- being detected. Pyramimonas was also tested twice. Chlorella sp. TABLE III The t>H optima at a/yil phosphatases. 1 he clone designation <>\ each ni the al^ae is in parentheses after the name. I'll/' Inihittil ty/>e fnnn which clones were obtained and isolated are: E, estiiariiie or rock pool; N, neritic; mid (>, oceanic. One ['nit /nil. is the enzyme activity per nil. of culture that causes a change in O.P. of O.I in I per minute in a I -cm. cell. I'll C)]itima Habitat Acid Alkaline t'nits ml. PH 1 mt- ml. pH Chrysophyceae Isochrysis ^alhana ( Isoi 1.9 6.8 28 9.8 E Monochrysis liitheri (Mono) — — 8 9.7 E Cricosphaera caiierae iCocco II) 3.9 6.8 23 8.6 N C oi' eolith us h/ix/eyi 'B T-6> 1.8 6.8 15 9.7 0 1'.. n illariopln ccar Phaeodactylum tricornutum (I'hat-o) — • — 26 9.7 E Melosini sp. (T-5) 0.6 6.8 6 9.7 E ( 'yclntella na na (1\\ > — — 2.6 9.8 E C. cryptica (O-.v\i — • — 17 9.7 E l>eti»/ula confervacea 1 1 >. con.) 1.1 6.0 0.7 9.0 K Thalassiosira fluviatilis (Aciin i 0.6 6.8 18 9.8 7. nordenskioldii ('I', imrd.) — — 1.3 9.0 \ \it':schia closterium (N. closi.) — — 2.7 9.8 \ Skeletonenid costal n/n (Skcl) — . — 0.7 8.6 \ ( 'hitetoi ei -os sp. (simplex?) (BBsmi 2..-! 6.8 18 9.7 0 ( 'yclntella cuspid < 10-5) 1.5 6.8 12 9.8 0 C. ;/,;>/(/ (13-1) 1.5 9.8 O < 'i \ pi< ijih\ n-.ir Rhodomonas (?) (3C) 0.5 4.4 0.8 8.6 E Rhadiniintia <• ( ?) ( 1' -5 A I 0.5 4.4 0.6 8.6 E Rhudoillona - /r//s i Khodi i 0.6 4.4 0.8 8.1 0 1 .mophvi eae < iscilldturid 1! '<>,< •mnchinii 'Sm 21) 0.6 6.8 3.7 9.0 E 1 )inophyceae Amphidinium carte \mphi 1 9 9.0 K o \'inni>(Jininm <•• ' .SBI . ) 0.8 5.8 0.3 9.8 K ( !hlorophyceae Chlorella sp. (580 (> (, S — — E ,S'/;'( /.." 0( C«5 sp ( -SB Si iclioi 2.1 1.8 — E /'yramimonas -]>. ( I'yr 2 ) 0.9 1 S E Chlamydomonas sji. M ) ^ 650 7.6 E Chlamydomonai -p. (F-17 7.5 8.6 E i'HOSI'H. \T.\SKS OF M \K1XE AUiAK 279 had more acid phosphatase than any other alga tested, hut even thi.s was low relative to the alkaline phosphatases of many other specie^. Fxcept for (> \tuno- dlniHin, acid phosphatase production did not seem to begin when these algae hecame P-deficient as was reported in Iltttjlctia - phatases found in only small amounts in certain algae are also constitutive; further study of these species is necessary. The phenomenon of alkaline phosphatase production when phosphate-deficient was found in algae from estuarine. neritic, and oceanic habitats (Table III ). I Intake o\ phosphate esters When P-deficient cultures were exposed to three different phosphate esters, all esters were assimilated at the same rate by each algal species (Fig. 4). The intercept at time-zero is the amount of phosphorus in the algae at the beginning of each experiment ; the somewhat accentuated uptake during the first 5-8 minutes simply reflects the amount of inorganic phosphate immediately available in the diluting sea water. The curves from 5 to 125 minutes represent uptake of phosphorus from the esters. In Phaeodactylum the uptake rate remained almost constant during this time, even though at 124 minutes about 80 Ov of the G-6 I' had been assimilated. The initial levels of the other substrates were higher, and such a high proportion was not assimilated during this period. The amount of phosphorus per Phaeodactylum cell may be calculated from Figure 4. The final value, 1 1 X 10 -r' mole P/l. 57 x 10- cells/1. =KXW '"'* P/CeU' is about 14 times higher than the initial value, but three times less than maximum cellular-P levels reported by Kuenzler and Ketchum ( 1962 ) and indicates that these cells were still not saturated. In the other two species not more than half of each substrate was used. The uptake rate decreased noticeably with time in Cricosphaera, perhaps indicating that it is more quickly satiated. The above experiments indicate that, instead of assimilating the whole ester, the algae hydrolyzed the ester extracellularly and took in the phosphate. One might expect the tiptake of whole glycerol phosphate, sugar phosphate, or nucleoside phosphate molecules to proceed bv different mechanisms and at different rates. However, the remarkable similarity in rates of accumulation of phosphorus from these three esters suggests that the rates were limited by a common mechanism. Alkaline phosphatases of other organisms frequently hydrolyze more than one phosphomonoester (Stadtman, 1961 ), sometimes at similar rates ( /:. coli; Torriani, 1960). Thus, the enzymatic hydrolysis rate might have limited the rate of phos- phorus uptake from these three esters. On the other hand, if sufficient phosphatase were present, the phosphate assimilation rate would be the limiting factor. The phosphorus content of the Phaeodactylum inoculated into each of the three esters (Fig. 4) was 1.4: 10 i:' mole/cell. Kuen/.ler and Ketchum (1962) found the phosphate uptake rate of Phaeodactylum in an 8 /i.l/ solution to be of the order of 0.32 ~> 10 '"' mole/cell -min. for two hours. These two values give an expected 2, SO Kl>\\ \K1> I. Kl'KX/I.Kk AND IAMKS I'. I'KKKXs doubling time of cellular phosphorus of 4.4 minutes ; cellular I' actually doubled more quickly than this in the initial period (Fit;. 4) when the sea water phosphate was being assimilated. The- lower rate of P accumulation from the esters than from the sea water phosphate surest. s that insufficient phosphatase was present to release phosphate as rapidly as the cells could accumulate it and, therefore, that the ester hvdrolvsis rate was the limiting' factor. The experiments were done at about pi I S although the phosphatases of these three species are more active at higher pi I values (Table 111 ). Here, however, we were interested in the capa- bilities of algae under approximately normal conditions rather than under conditions optimal for one en/vine but possibly damaging to the whole, living cell. 0—0 GLUCOSE -6- PHOSPHATE A— -A ADENOSINE MONOPHOSPHATE x x a-GLYCEROPHOSPHATE PHAEODACTYLUM TRICORNUTUM (5.7xl08 CELLS/*) THALASSIOSIRA FLUVIATILIS (3.0x107 CELLS/*) CRICOSPHAERA CARTERAE (3.4x10y CELLS/-?) 0 40 100 120 140 60 80 MINUTES l-'iiiikK 4. ImTr;t-,r in ])lio>pliuni- rontmt of tlinv spi-cies of al.uac \\lu-n exposed to tlirer phosphate e^terv The initial eoneciitrations of (i-6-P. AMI1, and o (il' were 12-14, 22 and 14 16 /j.\f I'. respeeti\-ely. PHOSPHATASES OF M \KI\K ALGAE 281 I ^ CL i <0 K 120 100 80 60 40 20 0 COCCOLITHUS (24 UNiTS/m«) A CRICOSPHAERA (36 UNlTS/m!) x THALASSIOSIRA (34UNlTS/m£) • HAEODACTYLUM (40UNlTS/m.P) C14 0----0 PHOSPHORUS O- I 0 20 40 60 80 MINUTES 100 120 140 FIGURE 5. The hydrolysis of C'Mabeled glucose-6-phosphate by P-deficient Coccolitlnts Int.rlcyi, Cricosphaera cartcri, Tltulussinsit'ii fliti'iatilis and Phaeodactylum triconiutniti. The concentration of the glucose moiety in the medium was determined by measuring C14 (dashed lines); the concentration of phosphorus remaining in solution (solid lines) \vas determined by the difference between the original concentration and the amount meu.Mired chemically in the cells. Enzyme activity was measured with XPP at the same time in other portions of each culture; results are shown in parentheses (Units/ml, of culture). The following experiment showed that only the phosphate is assimilated and that the organic part of the ester remains in solution. \Yhen C"-laheled G-6-P was mixed with four cultures of I '-deficient algae, radioactive carhon remained in solution while the phosphate was assimilated hy the cells ( Fig. 5 ). (The variability in the C" counts was probably caused by self-absorption and geometry variability.) The simplest explanation is that the phosphate was hydrolyzed extracellularly by phosphatase and taken in. whereas the glucose never entered the cell at all. Thi> is substantiated by the general correspondence between the slopes representing the decline of phosphorus in the medium and the phosphatase activity as measured independently with ^-nitrophenyl phosphate. Location of the enzyme The alkaline phosphatase of the two species studied more carefully, Coccolithus liu.rlcyi and Phaeodactylum tricornntitin. is firmly attached rather than being in solution in the cell. Repeated attempts on one or the other species, using sonication. explosive decompression, osmotic shock, freezing and thawing, heating, or treatment with lipase, cellulase, iso-butanol, and various salt and buffer solutions, failed to solubilize the enzyme. 282 Kl>\\ AKI> I. KrKXZI.HK AND IAMKS I'. I'KK'K Kothstein (N54) discussed several criteria that might indicate en/\nic attach- ment In a cell surface rather than to an internal organelle, and we have ap])lied some of these criteria to our own data. If the enzyme is located at the cell surface. disrupted cells should show no more activity than whole cells and the pi I responses of disrupted and whole cells should he the same. When we sonicated, then centrifuged, cultures of Coccolithus hn.\'lc\i and Chla/mydomonas reinhardi, all the enzyme activity was found in the pellet; additionally, the activity of the pellet was no more than that of whole, living cells. Experiments with six other species ( Table IV) showed that little of the enzyme activitv was in solution either before or after sonication. Here. too. except for Cricosphaera, there was no increase in enzvme activitv after disruption of the cell structure. In Coccolithus the activiu of each pi 1 between 7.7 and 9.7 was approximately the same in sonicated as in intact cells. As alreadv discussed, identical rates of assimilation of P from G-6-P. TAHU-. l\ Alkaline plwsphatasc ( i'nits/iul. of culture) in cells and nitrate he/ore und after sonication. The cells were sonicated at 0° C. in 5 X XaCI Whole culture Sonicated culture Cells Cpll< Filtrate after Before Aftei Phaeodact \li(iii tricorniitiun 00.4 9.6 83 80 9.2 L'ycltitelln crypticn 50 0 52 38 0.1 Chaetoceros sinif>le\ 73 0 72 58 0. 7 'hdld ss ios ira fluviatil is (.4.4 1.6 74 57 0.24 Isochrysis t^i/lhan/i 130 0 103 70 0 Cricosphaera ctn-terac 141 0 11') 201 0 AMP, and «-(!!'. more likely result from hydrolysis at the cell surface than from identical rates of transport of the whole molecules into the cells. Finally, the nitrophenol resulting from hydrolysis of XIM' was found in solution, just as the C" from labeled (1-h-l* staved in solution (Fig. <) ) ; these facts are more readily explained In extracellular hvdrolysis of non-penetrating substrates than by im- mediate excretion of nitrophenol, labeled glucose, or labeled CO., following intra- cellular hydrolysis. Our evidence for cell-surface enzymatic activitv in algae agrees with that of I'randes and Flston (1^56), Tal])asayi (1(H>2), ( lalloway and Krause I 1 ' "o i . and ( ) verbeck ( 1*^)2 ) alread\' mentioned in our introduction. I ) isc ess ION It appeal's that the alkaline phosphatases ot marine a enable them to regenerate phosphate from organic source's and thus circumvent heterotrophic regeneration. In general, heterotrophs may be expected to hydroly/e phosphate esters in order to obtain the energv bound in the organic moietv, whereas autotrophs with abundant energy available from sunlight would onlv need the inorganic phos- phate radical. There arc' similarities, however, between phosphatases of yeasts and bacteria and those of algae. Suomalainen ct al. ( l()'i()i reported that both the acid PHOSPHATASES OF M ARIXE ALGAE 283 and alkaline phosphatases of baker's ist increased markedly with phosphorus starvation. From comparisons of enzynir ,-ictivity of whole cells to that of dried, freeze-thawed, or cytolyzed cells, they concluded that the acid phosphatase was mostly at the cell surface and the alkaline phosphatase was mostly internal. In the bacterium, Escherichia coll, Torriani (1960) found that the acid phosphatase was always present (i.e., constitutive), whereas alkaline phosphatase synthesis was repressed by phosphate. Malamy and Horecker (1961) removed the cell walls from E. coli with lysozyme and reported that these protoplasts lost most of their alkaline phosphatase activity. They suggested that the enzyme lies outside the cell membrane. Yeasts and bacteria, therefore, also have a means of hydrolyzing phosphate esters extracellularly, especially when P-deficient. The ability to produce phosphatases that act on extracellular substrates may give some algae a competitive advantage over other species when phosphate in sea water is less abundant than dissolved organic phosphorus. It is probable that these phosphatases not only hydro lyze but also immediately transfer the phosphate to an acceptor in the cell. Such a transfer would improve the recovery efficiency and increase the competitive advantage even more. The components of the dis- solved organic phosphorus pool in the sea are still completely unknown, but un- doubtedly some of the compounds are phosphomonoesters. Kuenzler et al. (1963) reported that P-deficient Phaeodactylum tricornutum could rapidly obtain more phosphorus from natural sea water than was present as orthophosphate, and con- cluded that the difference came from dissolved organic phosphorus compounds. We have tried to learn the characteristics of the phosphatases of whole algae and the resulting capabilities for the living cells. In this regard, experiments on enzyme activity at very high or very low pH (Fig. 3) are not very meaningful because such conditions are abnormal in the sea and often lethal in cultures. Sea wrater usually has a pH of 8 or higher where active photosynthesis is occurring, and it seems significant that the phosphatases synthesized can hydrolyze esters at sea water pH. Constitutive internal phosphatases can be expected to have optima suited to particular pH values of various sites within the cell. It is possible that the ratio of phosphatase to cell volume, protein, or chlorophyll in natural phytoplankton populations can give direct information about their nutrient status. The fact that phosphate is undetectable in sea water does not prove that the natural phytoplankton population is phosphorus-deficient. The limit of chemical detectability is not yet as IOWT as the limit of assimilability by algae (Kuenzler and Ketchum, 1962). From Tables II and III we would guess that samples of phytoplankton dominated by chrysophytes and diatoms are phosphorus- deficient if the alkaline phosphatase activity is greater than 10 >: 10~s Units/ju,3 of cells, i.e., two days' production of this enzyme at a rate of 5 X 10~s Units//*,3 • day. We have been able to detect alkaline phosphatase in the participate matter of some samples of natural sea water. More analyses must be done, however, before we can draw conclusions about the phosphate nutrition of the plankton from enzyme measurements. SUMMARY 1. Phosphate-repressible alkaline phosphatases were found in axenic cultures of marine algae, especially Chrysophyceae and Bacillariophyceae. Enzyme synthesis 2S4 EDWARD j. ECUENZLER AND JAMES P. TERRAS >tarted when tin- algae hecame phosphorus-deficient and stopped if phosphate was rotored to the medium. 2. Maximal enxyme activity wa> usually ahove pli (), hut significant activity- was also present at ordinary sea water pi I. approximately pH 8. 3. The phosphatases enahled algae to split glucose-6-phosphate ; the phosphate was quickly assimilated hut the glucose moiety remained in the medium. Algae took phosphorus from adenosiue monophosphate and a-glycerophosphate at the same rate as from glucose-6-phosphate. 4. The phospha;a>cs appear to he firmly hound near the cell surface. They may enahle deficient algae to regenerate phosphate from soluhle organic phosphorus com] founds present in natural sea water. LITERATURE CITED BKA\I>K>. l>.. AM) R. X. KLSTOX, 1956. An electron microscopical study of the histochemical localization of alkaline ])lios])hatase in the cell wall of Chlorclla vul^aris. Nature, 177: 274-275. KITI.EY, R. W., 1962. Hydrolysis of polyphosphates hy I'orphvru and other seaweeds. Pliysiol. riant., 15: 246-251. GALLOWAY, R. A., AM) R. W. KKAUSS, 1963. Utilization of phosphorus .source's hy Chlorclla. hi: Microalgae and Photosynthetic Bacteria; Plant and Cell Physiol. Spec. Issue, pp. 569-575. GUILLARD, R. R. L., AND J. H. RvTiiKK, 1962. Studies of marine planktonic diatoms. I. Cvclotclld iKiiia Hustedt. and Dctonula con f SITU ecu ( Cleve) Gran. Canad. J. Micro- bi'ol., 8: 229-239. KETCHUM, B. H., N. CORWIN AMI 1). J. KKKN, 1955. The significance of organic phosphorus determinations in ocean waters. Deep-Sea Res., 2: 172-181. KTEX/LER, E. J., R. R. L. GUILLARD AND N. CORWIN, 1963. Phosphate-free sea water for reagent blanks in chemical analyses. Deep-Sea Res., 10: 749-755. KCKX/LER, E. J., AND B. H. KETCH I'M, 1962. Rate of phosphorus uptake by Phaeodactyhtin tricorinttuni. Hinl. Bull., 123: 134-145. I.O\\KY, ( ). II., N. J. ROSEBROUGH, A. I.. E\KK AND R. J. RANDALL, 1951. Protein measurement with the Folin phenol reagent. /. Hiol. Cliein., 193: 265-275. MALAMV, M., AND B. L. HORECKER, 1961. The localization of phosphatase in E. eoli Ki«. Hioehem. Biopliys. Res. Coin.. 5: 104-108. OVERBECK, J., 1962. Untersuchungen zum Phosphathaushalt von Grunalgen. II. Die Ver- \vertung von Pyrophosi>hat und organisch gebundenen Phosphaten und ihre Beziehung zu den Phosphatasen von Sceiiedesiints quadricuitda ( Turp.) Brep. Arch. HvdrobioL, 58: 281-308. I 'KICK, C. A., 1962. Repression of acid pho.sphatase synthesis in Eitijlcmi i/nicilis. Science, 135: 46. PROVASOLI, L., 1958. Nutrition and ecology of protozoa and algae. ./;/;;. A'.-:1. MicrohioL, 12: 279-30S. RoTHSTEIN, A., 1954. Tlie eii/ynxilogy of the ci'll surface. In: 1'rotoplasmatologia II. E. 4. ' I .. V. lleilbrunn and 1-'. \YcluT, eds. ) Springer- Verlag, Wien ; pp. 1-86. STADTMAX. T., l''nl. Alkaline phosphatases. In: The En/ymes (P. D. Boyer, H. Lardy and K. Myrbiick, eds. ). Academic Press : \\-\\ York. Vol. 5, pp. 55-71. SUOMALAINEN, II., M. l.ixko AND I1". Oi'KA, I960. Changes in the phosphatase activity of baker's vrast during the growth phase and location of the phosphatases in the yeast cell. Biochim. Biophys. Ada, 37: 482 490. TALPASAYI, \\. K. S., 1('62. Acid ]ihos]iliatase activity of some algae and its inhibition by molybdenum. Hinclrini, Hiopliys. .Ida. 59: 710-712. TMKRIAXI, A., \'n^l I n flue-nee of inorganic |)bos])bate in the formation of phosphatases by lisclicricliiu coll Biochim. ttinphys. . Icta, 38: 460-469. \\'OOS-IKR, W. S., AND X. \Y. RAKESTRAW, 1''51. The estimation of dissolved phosphate in sea water. ./. Mar. Res., 10: 91 100. STABILIZATION OF THE VISUAL FIELD1 LORUS J. MILNE AND MARGERY Mil. XI-. Department of Zoology, University of New Hampshire, Durham, A Most animals can make no adjustment in the orientation of their principal visual organs in relation to the body. Those that can move their eyes separate!} are the vertebrates, virtually all of the cephalopod mollusks, many crustaceans, and the comparatively few kinds of insects that have a mobile head. A remarkable number of these animals use this ability in a paradoxical way. They move their eyes in relation to the bod\ to prevent their eyes from moving in space. Involuntary movements of their eyes tend to stabilize their visual field. The anatomical basis for these adjustment is well known among vertebrates, which uniformly possess a conical array of four rectus muscles, and two oblique muscles in the orbit of each eye. Involuntary oscillatory adjustments (nystagmus) of the eye, chiefly through contractions of the external and the internal rectus muscles, have been noted for many years in man and some other vertebrates, elicited by visual stimuli. The obliques provide a correcting system that smooths out the horizontal and vertical sweeps produced by the antagonistic rectus muscles. They also produce "wheel movements" (Raddrehungen, Augenrollungen, cyclorotations) that confer on the organ a third degree of freedom. With its obliques, an animal may also be able to correct for skewness in its visual field. Involuntary responses of this kind follow stimulation of sensory centers in the muscles of the human neck (Nagel, 1896; Zoth, 1905), and in the inner ears of several other vertebrates (Benjamins, 1918, 1920; Magnus and de Kleyn, 1921). These adjustments based upon proprioceptive and gravitational cues show a latency in milliseconds, and commonly depend upon the obliques acting alone. Voluntary use of the rectus muscles in opposing pairs is found in some but not all vertebrates, and serves to keep centered in the visual field any object that moves. This following is usually jerky, with momentary pauses for fixation. Only these voluntary movements are discussed under ocular motility in The Vertebrate Eye and Its Adaptive Radiation (1942) by the late Gordon L. Walls, and in Les Yeii.v et la I'ision des Vcricbres (1943) by the outstanding French ophthalmologist A.-J.-F. Rochon-Duvigneaud. The latter divided reptiles into a category with immobile eyes (the snakes, geckos, and crocodilians) and another with mobile eyes (most lizards and chelonians). The true rarity of voluntary movements presumably led Sir Stewart Duke-Elder in his comprehensive volume, The Eye in Evolution (1958), to comment that most birds have fixed eyes and compensate for this fixity by bending their necks to follow objects of interest. Benjamins (1918) rotated a live perch about a transverse axis and recorded the wheel movements of its eyes (Fig. 1A). When the anterior end of the fish was 1 Presented before the Fourth International Congress on Photobiology, at Oxford, England, July 27, 1964. 285 286 LORUS J. MILXK \ N '!> M ^RGERY MILNE raised, to a position matching that assumed hy the animal while feeding at the surface, compensatory rotation of its eye maintained the horizontally of the horixoii to aliout 10 degrees, and then failed progressively to maintain corre- spondence as the angle between the longitudinal axis of the body and the horizontal was increased i Fig. IB). A maximum rotation of 28 degrees in this direction was reached when the body reached a 70-degree angle with the horizontal. Stability of the visual field was never as good when the anterior end of the fish was depressed, as it would be while the animal feeds from the bottom. A maximum rotation of the eyes was reached at 35 degrees when the body made an 80-degree angle with the horizontal. Curiously, the cyclorotational correction vanished 40 30 20 10 0 -10 -20 -30 -40 70 GO 50 40 30 20 10 0 B 180 225 270 315 0 45 90 135 180 180 225 270 315 0 45 90 135 180 head down head up inverted head down head up KH.I PI-: 1. When a European perch is rotated slowly aliout a transverse axis, its eyes exhibit compensatory rotational adjustments with respect to the body (.1), hut fail to hold the visual field level i />' ) except within about 8 degrees with head raised, about 5 degrees with head lourred, and about 1 degree with the body completely inverted. ( Replotted from data of 1'ieiijamiiis. 1(|1S. ) when the fish was inverted and its longitudinal axis again lav in the horizontal plane. The.se involuntary responses appear to depend entirelv upon stimulation of the maculae of the inner ear. Magnus and de Kleyn (1921) found remarkable excursions of the eyes in Kuropeaii rabbits, according to the static position of the inner ears. It made- no difference whether the whole animal was tilted or only its head was turned. Compensatory rotations of its eyes tended to stabilize the visual field within a degree or less over a range of head positions nearly 100 degrees in any direction. Beyond this limit, the rabbit's eyes tended to remain in their position of maximum excursion, and the animal showed a lessened response to visual stimuli. [nvoluntary cvclorotalion of the eves in a reptile came to public notice through some observations ol the copperhead snake (a pit viper of America) made by Professor I). K. Munro of Kansas State College and communicated by him to Albert (i. Ingalls (Scientific . I incrictin, March, 1(>54). Muuro had noted that the vertical slit pupils of this snake remain vertical even when the reptile's head is STABILIZATION OF THE VISUAL FIELD 287 raised at an angle of 65 degrees, or lowered a like amount. He reported further that just before dozing, the snake let the anterior pole of each eye rotate ventrad by about 10 degrees. When the snake was actually asleep, its pupils again took on a vertical orientation, but with the whole eye rotated until only the top of the pupil was visible through the corneal scale (Fig. 2). Normal alert position Just before dozing Head lowered 'Complete relaxation' — Ancistrodon contortrix (data of D. F. Munro — quoted by A. G. Ingalls) FIGURE 2. Rotational movements of the eye in the copperhead snake. (Data of D. F. Munro, prepared by permission from an illustration. Copyright © 1954 by Scientific American, Inc. All rights reserved.) In a subsequent issue of the same magazine (November, 1954), Ingalls pub- lished further information on cyclorotation, with observations made by Henri Morgenroth, a consulting engineer of Santa Barbara, California. The new data offered by Morgenroth related to a young turtle and a goldfish, and drew forth an additional comment from Professor Munro to the effect that stabilization of the visual field by cyclorotation is found also in snakes that do not strike at prey. One with a vertical oval pupil (unidentified) was cited as showing stability of eye orientation with respect to the field over a range of head position from 27.5 degrees below to 27.5 degrees above the horizontal. I. OKI'S J. MII.NK AM) MARGERY MILNE For the e\e of the- pigeon. Whim-ridge (1950) found a rotational correction amounting to about 10 degrees for head positions with the beak raised or lowered beyond the normal orientation. He did not establish whether the adjustment was a response to stimuli detected in the inner ears or in neck muscles. In man, the neck receptors alone call forth the response, and no stabilization of the visual field is gained if the whole trunk and head are inclined together to right or left. Analogous mechanisms can be recognized among several types of invertebrates. The slit pupil of the European Octopus I'lilt/aris has long been seen to remain horizontal despite movements of the bodv over irregular obstacles (Magnus, 1902; Muskens. 1(M34; van \\eel and Thor, 1936). These responses are obliterated if the statocysts are destroyed or the nerves from them are cut (Young, I960). No measurements of the maximum rotational adjustments possible seem to have been published for this cephalopod. Compensatory movements of the eyes have been observed also in crustaceans. When the mantis shrimp, Sj tlie neek (in Homo) or for elevating ( + ') or depressing ( — ) the anterior end ttf the hody. Reference points for detectini/ cyclnrotii- tion ean be found in pif/ment flecks on the iris in Homo, ()ryctnla the circular pupil in I'hyllomedusa ; and in a horizontal slit pupil in Sepioteuthis Response by Organism De>, rees Previously- class — order genus and species + - reported by A. Rotation of the eye in its orbit Mammalia — Primates Mammalia — Lagomorpha Aves — Columbiformes Kept ilia — Crocodilia Homo sapiens Oryctolagus cnuiculns Columba liri/i Caiman sclerous 8.6 i on 10 10 8.6 100 10 45 Xagel, 1896; Zoth, 1905; Merton, 1956 Magnus & de Klejn, 1921 \Yhitteridge, 1956 Kept ilia — Squamata A ncistrodon i untortrix Con strict or con strict or 32.5 30 32.5 30 Ingalls & Munro, 1954 Henndact\lu s mabouid 15 10 Kept ilia — Chelonia Amphibia — I )iplasiocoehi Pseudcinys scripta Bafo americaniis 70 5 10 0 Ingalls & Morgenroth, 1954 Rana pipiens 0 o \mphibia — M u tabilm Phyllomedusa buniieis- teri (tadpole) Pseiidotriton ntber 62.5 20 62.5 15 Ouesnel, 1956 Amphibia — Proteida \ectnrus maculosus 15 5 Osteirhthves — Percomorphi Osteichthyes — Ostariophysi Chondrich thves — Selach 1 1 Perca fluvial it is Cypriniis curpio Carcharias taunt s 28 60 ?0 35 60 20 Benjamins, 1918 Ingalls & Morgenroth, 1954 [[estimated ] Cephalopoda — I )i branch m .^epiotenthi s sepioidcs ?0 20 B. Bending head with eyes at neck 1 tia v iitni u v 8 8 C. Bending eyestalks Crustacea — Decapoda — by lateral adjustments Ca rein idea maenas (h'vpottc albicans 20 30 20 30 Sell one. 1954 /'i>di>i>lith//»/f tin- eyestalk are used to stabilize the visual field in the tihost crabs, Ocvpodc ceratophthalma and Macrophthalmus pectinipes. In both, the proximal ^•gim-nt of the eyestalk is greatly reduced, and movements involve only the elongated distal segment. The ghost crabs, ( h'vpodc ceratophthalma and Macrophthalmus pectinipes (Fig. 3), sho special development of the distal segment. Like Ocypodc icuns, these terrestrial crabs tend to stabili/e their visual field over a wide range of body position-. To do so they use the same muscles that serve to raise and lower the eyestalks the protective groo\c across the anterior edge of the carapace. \Yhen a gho ' crab begins the downward slant into its burrow in the beach, it quickly low- vestalk on the advancing side and then the eye on the opposite side as it enters the hole. I'pon return to the surface the crab raises its eyestalks verticallv at once, then drops them part wav into the groove while quickly wiping them clear of sand with the flexible tips of the third maxillipeds. Orientation of the eyestalks appears to depend upon speciali/ed sensory hairs in the statocyst which is present in the proximal segment of the antennules ( Dijkgraaf, 1955. 1(>5(>; Schone, 1951. 1^54. 1959; Cohen. 1960). Statoliths are lacking (Bethe, 1897). STABILIZATION OF THE VISUAL FIELD 293 A similar mechanism appears to control the distal segment of the eyestalks in Podophtlialnnis and Enphyhi.v. which possess .statocysts with specialized hairs hut no statoliths. In these marine crabs of shallow coastal water, the proximal segment of the eyestalk is greatly elongated, whereas the distal segment is scarcely larger than the compound eye (Balss, 1940). According to Mr. Bryant Sather (personal communication). Podophthalmus I'iyil of Hawaii holds its proximal segments at 45 to 50 degrees above the horizontal by day, and about 10 degrees at night (Fig. 4). \Yhen feeding in silt that envelops the body completely, Podophthalmus may raise its proximal segments to a still greater angle, perhaps *"* •" v :..'• water above silt normal daytime position of distal segment ocular, groove Podophthalmus vigil ? normal nighttime position of distal segment FIGURE 4. The Hawaiian crab, Podophthalmus rif/il, maintains the vertically of the distal segment of each eyestalk while moving about with the proximal segments raised by day or partly lowered at night, and while moving sidewise over an inclined surface, (Data from Sather, 1962.) 65 degrees. Often this suffices to keep its compound eyes in clear water above the silt, where vision is possible. Both at night and when the proximal segments are elevated into daytime position, the distal segments are kept vertical. Only when the crab is lifted out of the water or its eyestalks are molested is the distal segment lowered into the protective groove across the anterior edge of the broad carapace. Thus, the elongated proximal segments serve to raise the eyes high above the body of this silt-dwelling crab, while muscular adjustments of the short distal segment stabilize the visual field. Sand grains, serving as statoliths in the antennular statocysts of crayfishes and lobsters, appear necessary in the sensory system that permits these animals to right themselves and make compensatory adjustments of their eyestalks to l(l\ LORDS J. MILNE A\l> M \.RGERY MILXE match rotational displacements of the body ( Knhn. 1919; Scheme, 1954). But when the lobster, at least, walks over an irregular bottom and raises its anterior end to surmount some obstacle, its eyestalks droop only slightly. No compensatory movements of the eyes are apparent \vhen the lobster descends a slope. Stonuitopod crustaceans lack statocysts altogether ( Denioll, 1909; Balss, 1938), and make most of their compensatory adjustments by lowering the eyestalks in relation to the ocular plate, which is a hinged forward portion of the head region. These adjustments, which are conspicuous while the animal crawls up over some obstacle, occur at the same time that the eyestalks are performing scanning move- ments ( Milne and Milne. 1961). Organization of the compound eye in the cladoceran, Daphnia, is ou an utterly different plan. The 22 ommatidia are radially arranged around a median mass that arises by fusion of a pair of embryonic rudiments. With so simple a com- pound eye, Daphnin can gain only the crudest resolution from its field of view. The structure is at least two orders of magnitude simpler than any of the other animals discussed above. Seemingly the compensatory movements of the eye, which the four oculomotor muscles control, are unrelated to any feature of the field of view other than the most intensely illuminated part, and the principal plane of polarization if this is detectable. We are particularly grateful to Mr. Bryant Sather, Department of Zoology, I'niversity of Hawaii, for his notes on Podophthalmus and specimens of this re- markable crab. Our own observations on Caiman and Constrictor were made at the University of the West Indies, Mona, St. Andrews. Jamaica, B.W.I., through the courtesy of Mr. Garth Underwood; on Hemidactylus and Scpiotcitthis at the Bellairs Marine Laboratory of McGill University, Barbados, B.W.I., with assistance from Dr. John Lewis; on Carcliarias at Marine Studios, Marineland. Florida; on Ocy[>oessing camera-style eves. Xew data are offered on Caiman, Constrictor. Hemidactylus, Hiiju. Rana. Pseudotriton, Ncclnnis, Car- cliarias. and Sepioteuthis. 2. Comparable adjustments in the movable stalked eyes of crustaceans have been noted in both brachviiran and macrnran decapods and in stomatopods. New data are offered on Ocypode, I'odoplillialiints. ffonmnis, and Gonodaclylns. The silt dwelling crab. Podophthahnus, represents an extreme, in which the elongated STABILIZATION OF THE VISUAL FIELD 295 proximal segment of the eyestalk raises ilic terminal segment high above the body, while the terminal segment is stabilized in relation to gravity and usually extends above the silt into clear water. 3. Movements of the head in insects that have a flexible neck appear to relate less to stabilization of the visual held than to feeding, cleaning the legs and body, or recovery of normal flight attitude after displacement by air currents. Orienta- tion of patterns in space seems to have less significance than other parameters of important patterns. Some compensatory movements of the head are reported for dragonflies (insect order Odonata) but not for mantises (order Orthoptera). 4. Oculomotor activity of the median compound eye in the cladoceran Ihiphnia shows no definite relationship to gravity. Its responses to light represent a much lower order of reaction to the environment than is noted in malacostracan crustaceans with mobile compound eyes. LITERATURE CITED BALSS, HEINRICH, 1938. Stomatopoda. In: Dr. H. G. Bronns Klassen und Ordnungen des Tierreichs. Ed. by A. Schellenberg and T.-E. Gruner. Akademische Verlagsgesell- schaft, Leipzig, vol. 5, pt. 1, hook 7, pp. 1-173. BALSS, HEINRICH, 1940. Decapoda. Ibid., vol. 5, p. 1, book 7, pp. 1-160. BENJAMINS, C. E., 1918. Contribution a la connaissance des reflexes toniques des muscles de 1'oeil. Arch. Nccrland. Physio!., 2: 536-544. BENJAMINS, C. E., 1920. Versuche iiber Otolithenentfernung. Rcr. acsainptc PhysioL, 2: 176. BETIIE, ALBRECHT, 1897. Vergleichende Untersuchungen iiber die Funktion des Centralnerven- systems der Arthropoden. Pfliu/crs Arch. i/cs. PhysioL. 68: 449-545. BOYCOTT, B. B., 1954. Learning in Octopus rulf/nris and other cephalopods. Pubbl. Staz. Zool. Napoli, 25: 67-93. COHEN, M. J., 1960. The response patterns of single receptors in the crustacean statocyst. Proc. Roy. Soc. London. Scr. B. 152: 30-49. DEMOLL, REINHARD, 1909. Uber die Augen und die Augenstielreflexe von Squilla mantis. Zool. Jahrh.. Abt. Anat. n. Onto,,.. 27: 171-212. DITKGRAAF, S., 1955. Rotationssinn nach dem Bogengansprinzip bei Crustaceen. Experientia, 11(10) : 407-409. DIJKGRAAF, S., 1956. Kompensatorische Augenstieldrehungen und ihre Auslosung bei der Languste (Paliiiunts I'lilf/aris). Zcitschr. rcryi. PhysioL, 38: 491-520. DIJKGRAAF, S., 1956. Uber der kompensatorische Augenstielbewegungen bei Brachyuren. Pubbl Staz. Zool. Napoli, 28: 341-358. DUKE- ELDER, SIR STEWART, 1958. The Eye in Evolution. /;;: System of Ophthalmology. Ed. by S. Duke-Elder. Henry Kimpton, London. Vol. 1, pp. 1-843. EWALD, W. F., 1914. Versuche zur Analyse der Licht- und Farbenreaktionen eines Wirbellosen (Daphnia pulcx). Zcitschr. Sinncsphysiol, 48: 285-324. HERTZ, MATHILDE, 1929-1934. Die Organisation des optischcn Feldes bei der Biene. Zeitschr. vergl. PhysioL. 8, 11, 14, 21. INGALLS, A. G., 1954. The Amateur Scientist. Set. Aincr., 190(3) : 100-102. INGALLS, A. G., 1954. The Amateur Scientist. Sci. Amcr., 191(5) : 116-119. KUHN, A., 1919. Die Orientierung der Tiere im Raum. Gustav Fischer, Jena. LINDAUER, M., AND J. O. NEDEL, 1959. Ein Scliweresiniiesorgan der Honigbiene. Zcitschr. vcrgl. PhysioL, 42: 334-364. MAGNUS, RUDOLF, 1902. Die Pupillarreaktion der Octopoden. P fingers Arch. ges. PhysioL. 92: 623-643. MAGNUS, R., AND A. DE KLEYN, 1921. Uber die Funktion der Otolithen, Otolithenstand bei den tonischen Labyrinthreflexen. P fingers Arch. gcs. PhysioL, 186: 6-38. MAGNUS, RUDOLF, 1924. Korperstellung. In: Monographien aus dem Gesarnptgebiet der Physiologic der Pflanzen und der Tiere. Ed. by M. Gildemeister. Julius Springer Verlag, Berlin. Vol. 6, pp. 1-740. 2()6 LORUS J. Mil. XI-: AND MAKGKKY MILNE MERTOX. P. A., 1956. 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YOUNG, J. H., 1959. Morphology of the white shrimp Penacus setiferus (Linnaeus 1758). Fishery Bull., 145 (U. S. Fish & Wildlife Service, Washington, D. C., vol. 59) : 1-168. YOUNG, T. Z., I960. The statocysts of Octopus vitlyaris. Proc. Roy. Soc. London, Scr. B, 152: 3-29. XOTH, O., 1905. Augenbewegungen und Gesichtswahrnehmungen. Nat/els Handb. Physiol., 3: 318-326. ECHOLOCATION OF FLYING INSECTS BY THE EA CHILONYCTERIS PSILOTIS ALVIN NOVICK Department of Biology, Yale University, New Haven, Conn. 06520 Among the many aspects of acoustic orientation in bats that have been analyzed, studies of sonar design as it varies during insect pursuits and obstacle avoidance have been limited to a few genera, principally of vespertilionid bats (Griffin, 1953, 1958; Grinnell and Griffin, 1958, Griffin, Webster, and Michael, 1960; Cahlander, McCue, and Webster, 1964; Webster, 1963; Griffin, Friend, and Webster, 1964). Griffin (1962) has also reported briefly on pursuit behavior in Noctilio (Noctilio- nidae) and Rhinolophus (Rhinolophidae). Recently we have observed echolocation during insect pursuits in Pteronotns ( Novick, 1936b) and in Chilonycteris parnellli (Novick and Vaisnys, 1964), closely related bats of the subfamily Chilonycterinae (Phyllostomatidae) . Systematic alterations in pulse duration and interpulse interval (or pulse repetition rate) have been described in Pteronotns and C. parnellii which imply a dependence, for detection of insect prey, on pulse-echo overlap at the bat's ear and which further imply the value to these two bats of adjusting pulse duration relative to target distance so as to give fairly constant pulse-echo overlap sequences during insect pursuits. Such apparent utility of pulse-echo overlap encourages consideration of the possibility of the bat's using a variety of analytical methods which have been briefly discussed by Pye (1960, 1961a, 1961b), Kay (1961, 1962), and Novick and Vaisnys (1964). The systematic variations in pulse dura- tion have also implied measurement of target distance since pulse duration has, during a part of each pursuit, varied directly proportionally with bat-target sepa- ration. Variations in interpulse interval may be related to reaction time in the central nervous system and/or conservation of output. The sonar design of C. parnellii differs strikingly from that of Pteronotns, as, indeed, it does from C. psilotis (Griffin and Novick, 1955; Novick, 1963a). These differences in design, principally pulse duration and intensity, increased our interest in comparing the behavior of the members of this closely related group of bats, in the hope of unveiling some of the functions of the sound parameters used. The orientation sounds of one individual Chilonycteris psilotis Dobson (Phyllo- stomatidae) (Hall and Kelson, 1959) were recorded while the bat flew around a laboratory flight room and pursued and apparently captured common fruit flies, Drosophila sp. The orientation of C. personata has previously been studied by Griffin and Novick (1955) and Novick (1963a). This bat is either the same as C. psilotis or a very close relative. These bats are delicate in captivity. Of several individuals captured in a cave at Lake Tequesquitengo, Morelos, Mexico, only one survived to pursue fruit flies in New Haven and this one did so for less 297 -)()8 ALVIN NOV1CK than a week. I am grateful t interpulse interval 7.5 5.0 2.5 125 250 375 milliseconds 500 625 FIGURE 2. An example of a fruit fly pursuit by C. psilotis. Pulse duration and interpulse interval are plotted against milliseconds before the end of the pursuit. One msec. — 1.73 mm. (see text). The arrow indicates the first calculated pulse-echo overlap. Note that in Figures 2 through 5, time reads from right to left (larger numbers represent earlier events). The approach phase begins, by definition, in these analyses, at the point at which we first recognize that the bat is committed to a complete pursuit. In Pteronotus (Novick, 1963b), this point was recognized by the first obvious short- ening of the interpulse interval. Systematic changes in pulse duration also oc- curred, but their initiation point was harder to pinpoint. In C. parnellii (Novick and Vaisnys, 1964), the beginning of the approach phase is signaled by an in- crease in pulse duration while changes in interpulse interval occur gradually and are initially hard to identify. In C. psilotis, the beginning of the approach phase is marked by shortening of both pulse duration and interpulse interval (Figs. 1 and 2). During the approach phase, the bat apparently clarifies the position, nature, and velocity of the target and sets its own flight path for interception. 500 ALVIN NOVICK \\ lu-n plotted against time, close to linear shortening of the pulse duration and interpulse interval characterize this phase. It terminates sharply in a transition to a scries of evenly spaced, short pulses of high repetition rate called the terminal phase ( Figs. 1 and 2). In the terminal phase, the bat presumably closes in on. captures, and begins to eat the insect. Following the terminal phase there is a long silent period and then a return to searching sound production. O X3 c O O o> l/> I 25 ****< X y X X x x x * * X X I X jj 1 * x * x x x x x x x x 125 250 375 500 625 milliseconds FIGURE 3. Xinc iruit fly pursuits by C. psilotis. The pursuits bave been synchronized by a^uming equality ot" Ml nation at tbe beginning of the terminal phase which is shown here as /(.TO. Pulse durations preceding tlu- beginning of the terminal phase are plotted against time during the pursuit. Intermediate interpolated values (between pulses) are plotted for equal time intervals. Note the change in the distribution of points at about 250 msec, before the beginning of the terminal phase. One msec. = 1.73 mm. The nine best pursuits recognizably occupy 1('() to more than 350 msec., measured trom the pulse preceding the first recognized decrease in pulse duration. If the pulse preceding the first recognized decrease in interpulse interval is used instead as the index, the same pursuits last 210 to 3(>() mesc. Durations, by both criteria, of about 300 msec, are the most common. The terminal phase, identified by short interpulse intervals, occupies 28 to (H msec, of this time. Excluding one terminal phase which i- so short that it seems possible that the target escaped, the range is 50 to CH msec. Thus, the approach phase per sc ranges trom about 150 to 290 msec. The measurements are objective except tor the need to choose inflection points — the beginnings of the approach and terminal phases. If one plots all of the pursuits ( puKe duration and interpulse interval vs. time) together b\ assuming that the beginning of the terminal phase, in each ECHOLOCATION OF FLYING INSECTS 301 case represented somewhat the same situation to the bat (perhaps a given range from the fly or perhaps a clear bearing) and that the bat's flight speed was uni- form and constant, then mean pulse duration or interpulse interval at any point in time from the fly can be calculated. The first discernible decrease in mean pulse duration occurs at about 225 msec, before the beginning of the terminal phase (about 300 msec, before the end of the pursuit i i Fig. I 'sing mean interpulse interval, the first discernible drop may be at about 240 msec, and is 50 c o o O) 25 xx x x xx • x x x x X XX x x x x x x xx x X XX xx x xxx X X XX XX X XXXxx* x „ xxx xx *„ XX xx x x xx x ' XX X X JL 125 250 375 milliseconds 500 625 FIGURE 4. Nine fruit fly pursuits by (". psilotis have been synchronized and plotted together by assuming equality of situation at the beginning of the terminal phase. The duration of interpulse intervals (represented as events occurring at the beginning of the pulse preceding the interval) are plotted against time during the pursuit, setting the beginning of the terminal phase as zero. Note the transition immediately to the left of 250 msec. One msec. = 1.73 mm. certain by 225 msec, before the beginning of the terminal phase (Fig. 4). By these various criteria, therefore, a common value for the approach phase would he about 240 msec., for the terminal phase about 55 msec. Note that the entire termi- nal phase transpires in an interval comparable to a single interpulse interval of the search phase! A complete analysis, knowing only the time separating the moving bat from its moving target, is impossible. Unfortunately, we did not have facilities for photographing these pursuits nor do we have an objective measure of the bat's flight speed. Time, however, can be converted to distance if several assumptions and approximations are allowed. First, an empirically chosen flight speed of 1.73 m./sec. has been assigned to this bat. Second, we assume the bat's velocity ALVIX NOYK'K relative to tin- !l\ to IK- constant throughout the pursuit. And, finally, we assume that the pursuit cuds with a capture or that the lasi pulse represents a zero point from which the distance separating the hat and il\ can he calculated hack for each previous pulse. \Ye know the hat's velocity is not constant. During pursuits, hats appear to change direction as well as speed. \Ye have no useful information ahout the flies hut their directions of flight are prohahly random relative to the I \HLE I Various parameters oj insect pursuits l>y three closely related species of hats. The data for Pteronotus are from Xorick ( l<>63b) ; those fur C. parnellii are from Xovick and ]'aisnys (1964). C. psilotis Pteronotus C. parnellii Search phase pulse duration (msec.) interpulse interval (msec. ) repetition rate i pul-1-s sec.) I Miration of pur-uit time (msec.) distance, calculated (mm.) Approach phase duration in time ( mse< duration in distance, calc. (mm.) number of pu Im- pulse durat ion ( ui-^ec. I interpulse interval ( msec, i tinal repetition rate (pulse se< pulse-ech o o\ erlap (msei I erminal pli.i-e duration in time (msec, < dura) ion in distain e, i . ill . ! 1 1 1 1 1 1 . I number of pulses pulse duration (msei interpulse interval msec. ) Imal repelit ion rate i pulses/sec. > pulse-eclio overlap i msei 'I'heoretical range > ba-c-d on calculated pnlse-ei lio ovcrla|> and Ilitiht -pen! - I li-lit speed i mm./msi-i i 2.0 4.8 H St 17-18 210 360 360-620 150-290 200 500 5-9 decreasing to 1 .5-2.1 decreasing Ironi 40-7 about 100 ca 1.2 50 <)1 90 160 1 1 IS dec reasing to 0.6-1 .0 4.5 5 ca. 17(i 0.8 0.<> 100 700 mm. 1.73 3.9-5.0 70-200 10-12 550-620 690-790 350 (mean) 440 (mean) 3-10 decreasing to 2.4-3.2 dei reasmi; Irom about 50 to about about 25 about 35 ca .1.5 150-214 190 270 27-39 decreasing t<> 1 .0 1 .25 1.5 ca. 200 or more ca. l.o 6X0 S60 mm. 1.25 14-26 20-60 ca. 14 560-740 ca. 3,300 380-560 2,400 (mean i 7-10 rising from 20-21 to 28-37 and then fall- ing to 23-27 fluctuating about 20- 50 and then de- creasing to 4-5 about 20 or slightly more ca. 20 180-190 800 000 9-12 decreasing In 6 2.5 ca. 80 100 decreasing from 1 8 to ca. 5 3,800 mm. L5 ECHOLOCAT10X OF FLYING INSECTS 303 bat (unless they take evasive action as do some moths; Koeder and Treat, 1961) and their speed is probably small compared \\-iih that of the bat. In any event, we have ignored the movement of the flies in these analvses. The flight speed of 1.73 mm. /msec, was initially arrived at by plotting pulse duration vs. time from the end of the pursuit and arbitrarily seeking a flight speed which would yield a regular pattern of calculated pulse-echo overlap when time was converted to dis- tance. The flight speed is consistent with known bat flight speeds and is inde- pendently confirmed by correlation (cited below) between first calculated pulse- echo overlap and first behavioral response as well as by its yielding regular patterns of pulse-echo overlap in all 9 pursuits. \Ye have discussed, elsewhere, these extrapolations which have previously proved useful ( Xovick, 19631) ; Novick and Yaisnys, 1964). Fixing the bat's position at zero, then, at the end of the terminal phase, we have calculated its position at the beginning of each pulse during each pursuit. We find that the approach phases begin at about 500 mm. (360 to 620 mm.) from the fly and that the terminal phases begin from 90 to 160 mm. from the fly. The approach phase alone therefore occupies about 400 mm. (260 to 500 mm.) (Table I). Having calculated the bat's position relative to the fruit fly, one can calculate the time taken for sound to cover the roundtrip distance (using 350 mm. /msec, as the speed of sound). Knowing the pulse durations being used, one finds that the first pulse-echo overlap would occur at 230 to about 440 msec, or about 400 to 700 mm. before capture. The first pulse-echo overlap would normally be of very short duration (about 0.3 msec.). If the bat is flying at 1.73 mm./msec. and if the searching repetition rate is such that each pulse and its subsequent silent interval occupy about 60 msec., then the bat will have moved about 100 mm. between pulses. Neglecting the fly's movement, the round trip distance is de- creased by 200 mm. Sound covers this distance in 0.6 msec. Thus, if the echo from a given fly just fails to overlap with one pulse, it will, on the average, overlap by 0.6 msec, with the next. Provided the bat did not respond to non- overlapping echoes by lengthening pulse duration (no apparent evidence in C. psilotis) the average first overlap would therefore be about 0.3 msec. The average second overlap would be about 0.9 msec., provided the bat had not already altered its behavior in response to the first overlap. If about 0.5 to 1 msec, of overlap is needed for the bat to perceive or take an interest in an echo, the first pulses providing such information occur at 340 to 670 mm. Thus, the first sub- stantial pulse-echo overlap occurs at about the same time that the last normal interpulse interval is being produced (360 to 620 mm.). The coincidence of these values — the interpulse interval which is completely objective and the calculated first significant pulse-echo overlap — tends to justify the assumptions made above which allowed these extrapolations. Similar relationships have been reported in Pteronotus and C. parnellii (Novick, 1963b; Novick and Vaisnys, 1964). Specifically, the first calculated pulse-echo overlap precedes the first behavioral response by 34 to 150 msec. This puts vague limits on apparent response time. The range of values may well reflect the extrapolation error. In C. parnellii. where flight speed was objectively recorded, a possible response time of 130 to 150 msec, was reported (Novick and Vaisnys, 1964). Grinnell's (1963a, 1963b) 304 A I.YIN XOYK k studies of brain responses to acoustic stimuli in anesthetized vespertilionid liats suggest that cortical decisions may he involved in these initial echo recognitions. In subsequent portions of pursuits, time seems to be lacking for higher level judgment. The first, second, or third pulse following the first calculated pulse-echo overlap is distingnishably shorter than the searching pulses. Thereafter, pulse duration decreases close to linearly when plotted t's. time or distance separating the bat and tly. The slope of this line varies in different pursuits from 1 msec, of shortening of pulse duration per 100 mm. of apparent reduced separation to 1 msec./350 mm. These different slopes may indicate different closing speeds of bat on insect (as. for example, if the insect is flying directly toward rather than directlv away from the bat). In all pursuits, however, the relationship is linear during the approach phase. Pulse duration drops from average values of about 4.0 to 4.2 msec., initially, to values of 1.5 to 2.1 msec, at the beginning of the terminal phase. 2.0 TD C O o 125 250 375 milliseconds FIGURE 5. Pulse-rein i overlap in the longest, recorded single fruit fly pursuit by C. psilotis. Xote the rapid rise of pulse-echo overlap to values of 1.0 to 1.4 msec, during the approach phase and the shift to values »\ 0.6 to O.S during the terminal phase. The arrow indicates the beginning of the terminal phase. The calculated pulse-echo overlap is shown for each pulse during the approach phase but a representative value is shown for each 12.5 msec, during the terminal pha-c to increase clarity. As has been the case in C. paniellii, pairing of pulses in the late approach phase sometimes occurs. That is. two pulses of close to the same duration are followed by two of shorter but almost equal duration and so on. Such pairs mav be the functional units. The governing feature, here, mav be reaction or decision time (that is, that decisions concerning revision of pulse duration take longer to make than the time available between single pulses — about 25 msec, or less), or pairs may actually be functionally desirable (as, for example, to assess direction by exploiting the movement of the head between the two pulses which, because of beaming and directionality tuned ears, would alter the echo intensity ). The speed of sound is constant, the bat's ilight speed has been assumed constant, and pulse duration VS. time or distance drops linearly. \Yhen one calcu- lates the successive pulse-echo overlaps, therefore, these prove to remain constant throughout the approach phase after an initial rapid rise. Thus, at 3(H) mm. from the flv. where all nine pursuits have calculated overlap values, the range ot over- ECHOLOCATIOX OF FLYING INSECTS 305 lap is 0.4 to 1.7 msec, with a mean of 1.0 msec. At 300 mm., the range is 0.6 to 1.5, mean 1.1 msec.; at 170 mm., range 0.9 to 1.6 msec., mean 1.2 msec. In individual pursuits, the value is remarkably constant. Variations of less than 0.5 to 0.6 msec, are the rule during the approach phase. The characteristic individual approach phase overlap levels of the respective pursuits art- 1.1. 0.9, 1.2, 1.6. 1.2. 0.9, 1.1. 1.2, and 1.2 msec. (Fig. 5). During the approach phase, then, pulse duration drops off linearly from about 4.0 to 4.2 msec, to about 1.4 to 2.1 msec, as the pulse-echo overlap levels out at about 1.2 msec. During this time, interpulse interval also drops sharply but not linearly. Search phase interpulse intervals of about 56 msec, yield to intervals averaging about 40 msec, between 200 and 270 msec, before the beginning of the terminal phase; to 31 msec, between 95 and 125 msec.; to 21 msec, betwen 30 and 60 msec.; and to about 12 msec, between 13 and 30 msec., respectively, before the beginning of the terminal phase. The last interpulse interval of the approach phase or, perhaps, the first of the terminal phase is about 7 msec, in duration with little variation from one pursuit to another (Fig. 4). The repetition rate, about 17-18 pulses/sec, during the search phase, has increased to about 110/sec. by the end of the approach phase. The approach phase in C. psilotis varies in these analyzed pursuits from 5 to 9 pulses in length. The terminal phase in C. psilotis is grossly distinguishable from the approach phase. The interpulse interval drops immediately to a minimal value of about 4.5 to 5 msec, and remains at that level throughout. The pulse duration also drops from initial values of about 1.3 to 1.9 msec, to terminal values of 0.6 to 1 msec., without a dramatic shift from the previous linear drop of the approach phase until values of 1.0 to 1.3 msec, are achieved, after which the pulse duration curve flattens out and appears to approach a limit. Excepting one example, there were from 11 to 18 pulses in the terminal phase. In the exceptional case only 7 pulses appeared. This might have represented a rapid catch or a miss. The terminal pulse repetition rate, on the average, is about 170 pulses/sec. The transition from approach phase to terminal phase is generally non- ambiguous and is most clearly defined by a break in the interpulse interval vs. time curve. The interval just before the transition one is from 2 to 4 times as long as the interval just after the transition one. During the terminal phase, calculated pulse-echo overlaps are shorter than during the approach phase but again are uniform and regular with values ranging from about 0.6 to 1.3 msec. At a distance of 90 mm., the overlap averages 0.9 msec. ; at 65 mm., it averages 0.8 msec.; at 45 mm., 0.8 msec.; and at 20 mm., 0.9 msec. (Fig. 5). Sound spectrographs of pulses selected from search, approach, and terminal phases universally show a fundamental initially of about 21 kcps accompanied by its second, third, and fourth harmonics of 42, 63, and 84 kcps. respectively. The fundamental frequently appears faint. Either the second or, more often, the third harmonic is at the highest amplitude, especially during the terminal phase (Fig. 1). The initial frequency varies slightly from pulse to pulse (perhaps by 1 kcps), but does not vary systematically during a pursuit. The last pulses are essentially like the first in frequency components. During the course of a pursuit, however, the frequency pattern changes. In every case, the frequency drops during a pulse. The fundamental in search phase ALVIN NOVICK pulses drops to 17 kcps at the end of the pulse while the harmonics drop to mul- tiples of 17. The same 21 to 17 kpcs fundamental frequency drop seems to characterize all of the pursuit pulses. The slope of the drop, however, changes. In the search phase, the initial 1.3 to 1.5 msec, of each pulse seem to he close to constant in frequency. The final 1.0 to 1.3 msec, or so seem also to he constant in frequency or, at least, of \ cry shallow slope. The frequency drop occurs during the central part of the pulse (Fig. 1). The first apparent change in the frequency pattern is a change in slope of the final portion of the pulse which shifts, from being constant in frequency, to fusing more and more with the central pulse portion, lint can still be distinguished by an inflection point in the early terminal phase, after which it can no longer be clearly identified. The initial constant frequency portion shortens during the approach phase and also starts to have a frequency drop, though at a different slope from the rest of the pulse. The drop becomes more marked and fuses with the rest of the pulse early in the terminal phase. The central, frequency modulated portion remains much the same throughout a pursuit. Thus, during the search phase, the frequency pattern has a lazy Z shape (Fig. 1"). During the approach phase, the curve becomes sigmoid, and during the terminal phase looks like a slash mark. The frequency rs. time curve even in the terminal phase may not be a straight line, but the resolution of our equipment does not reveal any clear and regular inflection points. DISCUSSION The constant or almost constant frequency portions of the search and early approach phase pulses may provide markers for distance identification. In any event, if our calculations are approximately correct, the usual pulse-echo overlap, here about 1.2 msec., would tend to consist of an echo of constant fundamental frequency of about 21 kpcs overlapping with an outgoing constant frequency portion of 17 kpcs. During the search phase, at least three kinds of echoes could easily be distinguished. Those just barely overlapping ("about 1 msec.) would give a simple situation of two separate but constant frequency components. Closer objects would give pulse-echo overlaps involving the frequency modulated parts of the pulse and echo as well as the constant frequency parts. More distant objects would give no pulse-echo overlap. Thus, "too much" and "too little" overlap could be identified easily. Tn the approach phase, the constant frequency portions are systematically changed. Soon they, too, show a dropping frequency and by the early part of the terminal phase are no longer identifiable. It should be possible for such differing frequency slopes to provide intra-pulse markers which could be used for distance measurement or for the assessment of closing speed relative to the pursued target. If these portions are. indeed, regular, then the bat may well use the pulsr echo overlap as a way of deciding whether it is at the expected distance from its target, or closing in on it faster or slower than anticipated, simply by noticing whether the overlap occurs during one portion of the pulse or another. During the terminal phase, the typical pulse-echo overlap shifts to 0.8 msec. There is no clear marker at this time, though a poorly resolved change in fre- ECHOLOCATION OF FLYING INSECTS 307 quency may be present. If not, what does the constant pulse-echo overlap here signify and how is it measured and regulated? \ , answer is presently available. Thus, the possibility presents itself that one purpose served by pulse-echo overlap is to measure distance and, perhaps, changing distance. Such a measure- ment would be independent of any knowledge of the speed of sound or the ability to discriminate short intervals of time. The initial detection in the search plniM- would simply give the information that there was a target at the standard specific detection range. Later information would confirm satisfactory closing speed. If, indeed, the search pulses are designed with initial and final constant fre- quency portions in order to facilitate the recognition of echoes from a given useful hunting range, we still have to account for the frequency modulated character of the central portion. If the beginning of the pulse is to be different from the end, a frequency change must occur somewhere. The frequency change here may represent only that. Its slope may, instead, be the natural slope resulting from the relaxation of the laryngeal muscles in this bat. Other ways in which this frequency change may be exploited remain obscure. The previous records of C. paniellii have been reexamined in a search for coincidence between pulse-echo overlap and frequencv inflection points. In C. parnclHi, almost the entire pulse is of constant frequency. Only the final 1 to 2 msec, show a frequency drop. Such a drop could well allow for initial detection during searching but would soon be only a small part of the typical 20-msec. overlap. In C. parncllii, note, however, that the typical pulse-echo overlap of 20 msec, equals the usual search phase pulse duration of 20 msec, and the 1-2 msec, frequency drop equals the frequency drop seen in C. psilotis. These may be co- incidental but seem of possible interest. In any event, there is only an incomplete parallel with C. psilotis. Reexamination of the old records of Pteronotus does not permit more than a statement that the frequency pattern changes during pursuits. New examples will have to be recorded and analyzed. In summary, all pulses seem to include about the same amount of frequency drop (perhaps, a result of the mode of sound production; Novick and Griffin, 1961). Search phase pulses are each, during an initial and a final 1.5-msec. portion, constant or almost constant in frequency. These constant frequency portions shorten during the pursuit and also incorporate, more and more, the standard frequency drop so that any clear separation between the initial and final pulse portions rs. the central pulse portion disappears by the early part of the terminal phase. These frequency patterns and their changes could provide target distance information during the pursuit by changing pulse-echo contrast. Such frequency changes would allow a three-quantity judgment of distance — just right, closer than expected, or further than expected. Such judgments could be translated into prediction of the target's relative flight path. The data so far accumulated on the parameters of echolocation during insect pursuits by chilonycterine bats are summarized in Table I. Some of the data from previous papers have been slightly recalculated or restated. The closeness of these species of bats is exemplified by the decision of Burt and Stirton (1961) to include Chilonycteris in the genus Pteronotus. These authors felt that morphological considerations did not justify generic status, but other authors have disagreed and retain the two genera as we have chosen to do ALV1X NOVICK he-re-. All of the-M- closelv related hats we-re- recorded pursuing a mixed group of Drosophila while living in the same laboraton room. Thus, the targets and the auxiliarv orientation ])rohlems. as \\cll as the hat species, were similar. Flight sperel, sound output design ( fre<|iienc\ , freijnency pattern, pulse duration, in- tensity, and intcrpulse interval) differed and presumably other parameters, not \et assessed, also varied. A comparison of the three performances, at this point, however, raises interesting hut not yet answerable questions. "While searching. ( '. psilotis produce- slightly shorter pulses than f'tcronotus. Those of C. parnellii are 5 times as long. It pulse-echo overlap is needed for detection. pulse duration sets maximum range. Presumably flight speed also affects maximum practical range as must, indeed, pulse intensity. \Ye can onlv ob- serve for the moment that C. panic/Hi flies fastest and produces the loudest and longest pulses. Seeming! \ as a result. C. panicl/ii shows a fruit fly detection range of over 3 meters. ('. psilotis produces the shortest pulses of these three species, giving it the smallest range of detection. This shortest pursuit distance, combined with faster flight than J'tcnniotiis. results in the shortest pursuit dura- tion (about ' o that of I'tcronotus in time as well as in the number of pulses). Such a short pursuit involves many fewer decisions about the position of the flv and the hat's own flight control. Fewer decisions, of course, would seem to have been adequate since all of these bats seemed successful in their pursuits. If the range is small, the fly moves over a shorter distance during the pursuit. If tin- range is smaller, perhaps there is greater certainty about the position of the fly- its direction principally. The greater the range, the more problems would arise from confusing or ambiguous echoes, lint probably, the greatest reason for the vastly greater number of pulses during Ptcronotus' pursuits than during those of C. f>silotis is that the bat flew more slowly and simply had to follow the fly for a longer time. One might reasonably ask why all these bats would not have- hit on the same formula of pulse duration, intensity, and range. Flight speed itself might be the important difference but the size and flight habits of the common prey species might be even more important. All of these bats commonly occupy the same caves in large numbers. They seem to have very similar climatic de- mands. One might guess that they would hunt different insect populations and thus avoid competition. Insufficient information on their feeding habits precludes further speculation on this now. The striking anatomical difference in the wings of I-'tcronotiis (which originate- from the midline of the back as opposed to lateral attachment in almost all other bats ) implies different flight habits. The differences in interpulse interval (and. hence, repetition rate) mav well reflect flight speed. In all three .species, the repetition rate is such as to provide an apparently significant pulse echo o\ erlap from objects just bcvond range1 ot overlap with the- previous pulse. Again flight spe-ed serins to be- a prime- variable but there is also an apparent diffe-re-nce in the amount of overlap which draws the- bat's attention. ( '. f>silt>tis mo\rs about 100 mm. betwern pulses, J'tcronotits about 120 mm. and ( '. punic/lii about .^00 mm. Thus, objects just beyond overlap range- from one- pulse- will yie-ld about O.C», 0.7, anel 1.7 msec, of ove-rlap, re- spectively, with the- ne-xt pulse-. The behavioral re-sponses of each of these spccie-s suggest that this amount of overlap is e-nough to attract attention. The spacing of search phase pulse-s produce-el 1>\ e-ach of these- species is. howe-ver, surprisingly ECHOLOCATIOX OF H.YIXG INSECTS similar, aside from the above considerations. Since the speed of sound is so great compared even with the swiftest of these three species, the common pulse spacing of about 50 to 75 msec, may simply reflect the length of the central nervous system path involved in searching decisions. (Irinnell's (1963a, 19631)) recordings from vespertilionid bat brains may cast M>me light on these time relations. The time consumed in an insect pursuit is remarkably similar in Pteronotits and C. parncllii but only -i as long in C. psilolis. In C. psilotis both the approach and terminal phases are shorter than in the other two bats. The approach phaso in Ptcronotits and C. parnclln are roughlv e(|ual in length as are the terminal phases. The savings in the approach phase for C. psilotis seem to come from the more immediate and consistent shortening of the interpulse intervals. The num- ber of pulses is not noticeably different from that of the other bats. In the terminal phase, C. psilotis uses far fewer pulses than Pteronotits (11-18 vs. 27- 39) and, of course, much shorter pulses than C. parncUii. Possibly the prompt- ness of change in interpulse interval during the approach phase in C. psilotis is a direct result of the short range at which it detects fruit flies and the resultingly more clearly defined target. The distance covered in the approach phase is similar in C. psilotis and Pteronotus since C. psilotis flies somewhat faster. A bat of such size may require this minimal distance in order to align itself properly to intercept a fruit fly. But in the terminal phase, the distance is markedly shorter in C. f>silotis (90-160 mm. vs. 190-270 mm.). This may reflect more accurate identification of the insect's position by the end of the approach phase in the case of C. psilotis or may imply more information per pulse or may simply result from the greater flight speed or shorter range. That is, the difficult problem may be to line up on the fly during the approach phase. The task of the terminal phase may simply be to keep the fly's position identified while one covers the intervening distance. It is possible that the differences which we see in length of pursuit may simply reflect the relative adaptedness of C. psilotis to detecting, pursuing, and/or catch- ing fruit flies. In both C. psilotis and Pteronotus. pulse duration decreases sharply during the approach phase. In both, the decrease is close to linear vs. time, implying that the bat is measuring distance from the target. If pulse duration were adjusted so as to give essentially equal amounts of pulse-echo overlap, then dis- tance would, in effect, be measured. \Ve have no direct evidence so far concern- ing how well a bat can discriminate time intervals or lengths of overlap. See discussion above. There has previously been verv little direct evidence of proportional measure- ment of distance in bats. Search phase pulse durations are presumably set so as to give pulse-echo overlap at a desirable hunting range. Unfortunately, our analyses are limited by having studied only one kind and size of target in the same room. In C. parncUii. the pulse duration during the approach phase rises and then falls, but in doing so accomplishes the same result as that in the other two bats — constant pulse-echo overlap. Here. too. a measurement of distance is implied. And in the terminal phase, pulse duration falls linearly vs. time. Apparently either fixed amounts of pulse-echo overlap are useful during the ^1»> ALVIN NOVICK approach phase or SOUK- quantity proportional to pulse-echo overlap is being held constant purposefully. The decree of overlap is comparable in C. psilotis and Pteronotus hut is some 15 times longer in ('. parnellii. This is a striking differ- ence in such closely related hats hut its significance eludes us. During the approach phase, the longest interpulse intervals in all of these hats are ahout 40-50 msec., followed by or alternating commonly with intervals of ahout 25 msec, hefore shortening shar])ly to values of ahout 5-7 msec, at the heginning of the terminal phase. Such regularity suggests that these interpulse intervals represent the reaction times for decisions of decreasing complexity as well as the need for increasinglv frequent information. These two features- more frequent information vs. more thorough digestion and judgment of the information may well he segregated during the pursuit. In a pursuit which in all consumes suhstaulially less than a second. Pteronotus may emit over 40 pulses. A tempting suggestion would he that the hat, detecting an interesting echo for the first time, might turn on an ordered and predetermined sequence of pulses of varying- parameters and run through such a sequence without having to make more than a few intermediate decisions. Examining the various pursuits, how- ever, we find that they are similar hut non-congruent in almost every respect. The different numher of pulses (total and in each phase), the different pulse durations, repetition rates, and total pursuit durations all implv a large numher of intermedi- ate decisions. At least two decisions, the duration of a later pulse and of a later interpulse interval, seem necessarily to he made after at least each of the early approach pulses. Later, the frequent pairing of pulses in terms of duration and the final steadiness of the interpulse intervals in the terminal phase may indicate a reduction in the numher of decisions heing made per pulse. The numher of intermediate decisions involved in arriving at the desirahle pulse duration and interpulse interval cannot now he assessed. But, in any event, ahout 50 to 60 msec, seem to he available in the early approach phase, ahout 10 msec, in the late approach phase, and ahout 5 msec, in the terminal phase in C. psilotis and Pteronotus. The times are slightly different in (". purnellii. Information is rapidly accumulating on the limes involved in acoustic pathways in the central nervous system of hats hut does not seem ready to permit useful speculations on the length of the paths imolved here (Grinnell, l(>. Henson's (1('C>4) recent studies of cochlear microphonics and stapedius muscle activity in Tinlarida ( Molossidae ) , a free-tailed bat, have suggested additional possible interpretations of some aspects of repetition rate in insect pursuits. In this hat, the stapedius muscle may contract some (> to 15 msec, he-fore pulse emis- ::. when the pulse repetition rate is 5< > see. or less. At higher repetition rates, ECHOLOCATIOX OF FLYING INSECTS 311 60 to SO/sec., stapedius muscle contractions occur 4 to 8 msec, before pulse emission. At even higher repetition rates, 100-140/sec., stapedius contraction is continual. Such contractions reduce cochlear microphonics to a degree that implies about 20 db attenuation of incoming sound. Stapedius relaxation appears to be initiated within 1-2 msec, and complete by 10 msec, after the beginning of pulse emission at low repetition rates. At the higher repetition rates, the stapedius may remain contracted for up to 40 msec, after the end of the pulse train. At low repetition rates, stapedius relaxation occurs rapidly enough for echoes from nearby objects to evoke larger cochlear microphonic potentials than do the outgoing pulses (Henson, 1964). Now, during the search phase, when echoe> would presumably be faint and would occur unpredictably, there might well be advantage to having full stapedius contraction during the bulk of the outgoing pulse, with considerable relaxation at the time of echo reception (for example, during the last millisecond in C. psilotis}. This design feature would interact with pulse duration since the pulse would have to last long enough to allow some stapedius relaxation. During the approach phase, the intensity and timing of the echo can be anticipated to some degree but may well have to be assessed carefully and, of course, the echo will still be relatively faint since the fly is from 100 to 500 nun. away. Thus the repetition rate may be kept low to allow maximum contraction of the stapedius during pulse output and relaxation during echo input, yielding protection and relative amplification alternately. By the end of the approach phase, at least two general features will have changed. The echo will have been sub- stantially identified — the target located — and the echo will be predictable in time. Such anticipation of echo timing can improve perception of the echo. In addition, the target will be close, less than 150 mm., so that the signal will be physically more intense. These advantages may well permit sacrifice of stapedius relaxation and substitution of high repetition rate. At such close range auxiliary factors reducing the apparent intensity of the outgoing sounds (acoustic isolation of the ear capsule, directional pinnae, etc.), central facilitation (Grinnell, 1963a, 1963b), and increasing echo intensity might all combine to give satisfactory signal: noise ratios. Note, too, that the intensity of pulses during the search and approach phases ought to be greater than during the terminal phase because of the greater range. There is little objective evidence of absolute intensities during these pursuits since the distance and orientation of the bat relative to the microphone have not been recorded. Bats which routinely must hunt at short target ranges, however (Nycteridae and others), use low intensity pulses (Novick, 1958) while bats which routinely hunt at long distances (Rhinoloplins and C. panieUii) use high intensity (Novick and Vaisnys, 1964). High intensity output would also favor maximum stapedius contraction and relaxation during the search and approach phases while less contrast would be required during the terminal phase. Lastly, one should consider whether the frequency pattern exemplified by C. psilotis reflects simply coordination of the cricothyroid muscle with the stapedius muscle. Novick and Griffin (1961) have shown that cricothyroid muscle action potentials can be recorded, from several genera of bats, which have timings relative to the emitted pulses very similar to those of Tadarlda stapedius muscles (Henson, 1964). If these two systems are designed to work together, the rapid responses required may have led to development of reflex association of the muscles involved 312 A [.YIN NOVICK SO that tlu' trequcncv pattern and 'or the duration of the pulses during the various portions ot tin- pursuits mav be set by the need.-- of stapedius contraction and relaxation. hi summary, repetition rate during search and approach phases might be set to allow time for stapedius contraction and relaxation, in order to clarify echo perception. In the terminal phase, when echoes would he more intense, stapedius relaxation appears to he surrendered in favor of rapid repetition rate. \\ hat determines the ultimate pulse duration in the terminal phase in these three species has not heen clarified. In C. psilolis and I'tcrouotns, pulses of 1 msec, and shorter characterize this phase. Shorter pulses occur in insect pursuits in vespertilionids (Griffin, 1(>(>2; AYebster, 1(J(>3) and in the general emissions of many other hats ( i\ovick, ll'5S, l(X>3a), hut the pulses seen here are, nevertheless, very short and may represent the best that the physical apparatus of these hats can deliver. On the other hand, in these three species, we have substantial circum- stantial evidence that pulse-echo overlaps are essential to insect pursuit and that the useful amount of pulse-echo overlap in C '. psilotis and Ptcronotus is about 1 msec. If, indeed, they depend on such overlap, the limits on pulse duration are set. The vespertilionids apparently avoid overlap by using pulses as short as 0.3 msec, or less. All insectivorous bats, vespertilionids as well as chilonycterines. must be very close to an insect ultimately in order to capture it. We do not yet have sufficient information on the actual range between the ear and the mouth and the insect in any species of bat at the moment of capture though there are well documented examples of bats capturing insects in their wing membranes or inter- femoral membranes (Webster and Griffin, 1962). Such cases involved large insects and usually unnatural situations, however, and did not include our chilonycterines. But if a vespertilionid can avoid pulse-echo overlap up to the last moment by using pulses of 0.3 msec, and a chilonycterine uses pulses of about 1 msec., we may conclude, independently of other calculations, that in the last moments of a pursuit, C. psilotis and I'tcronotits are still getting about 0.7 msec. • if pulx'-echo overla] i, l-.ven though the terminal phase occupies only a very short time, during which the bats travel only a short distance compared with the distance covered in the approach pha»e, pulse duration must still decrease (and, indeed, it does) if pulse- echo overla]) is to be held steady. At the very end of the terminal phase, pulse duration may flatten out or even fluctuate a bit. This may be evidence of the relatively great effect of the movement of the fly at close range and/or variation in the physical site of capture (wing, mouth, etc.). In C. panic/Hi, where there would seem to be greater leeway to shorten pulse duration without losing pulse-echo overlap or overtaxing the sound producing mechanism, the pulse duration seems to continue to decrease linearly (in pairs) throughout the terminal phase. In all, these comparative data cast some light on the design of pulse duration, pulse repetition rate, pulse number, detection range and pursuit duration in these three species ot hats during searching and during insect pursuits. \Ye need more information on frequency pattern, intensity, flight speed, and the echolocation of targets of different sixes and velocities in order to reach more definitive conclusions. \Ye would also benefit from more information about the natural prey and hunting habils of each of these hats. ECHOLOCATION OF FLYING INSECTS 313 SUM MARY 1. Elements of the acoustic orientation of Chilonycteris pliilotis during insect pursuits have been observed and analyzed. Such pursuits can he subdivided into search, approach, and terminal phases. The search phase is characterized by pulses of about 4 msec, duration repeated at a rate of about IX/sec. On detection of an insect, apparently by pulse-echo overlap (at a typical range of about 400-700 mm.), the approach phase begins, characterized by shortening (linear TS. time) pulse duration (to about 1.5 to 2.1 msec.) and shortening interpulse intervals. The ap- proach phase (which lasts about 150-290 msec. ; 5-9 pulses) ends in a transition to the terminal phase — a rapid sequence of short (1-msec.) pulses produced at a rate of about 170/sec. The terminal phase lasts about 50-94 msec, and includes 11-18 pulses. Constant pulse-echo overlap of about 1.2 msec, characterizes the approach phase, implying distance measurement and overlap utility. During the terminal phase, pulse-echo overlap appears to be set at about 0.8 msec. The pulses of C. psilotis consist of a fundamental frequency, initially of about 21 kcps, accompanied by its second, third, and fourth harmonics. During the search phase, the initial and final 1.5 msec, of each pulse are of constant frequency but the central portion shows a frequency drop (for the fundamental about 4 kcps). During a pursuit, the constant frequency portions are shortened and then fused into the frequency modulated portion so that they are no longer recognizable beyond the early part of the terminal phase. Such a frequency pattern suggests possible mechanisms for recognizing and using pulse-echo overlaps. 2. The parallel parameters of Ptcronotus and Chilonycteris parncllii pursuits, previously studied chilonycterine bats, are tabulated and compared with C. psilotis. LITERATURE CITED BURT, \V. H., AND R. A. STIRTON, 1961. Tin- mammals of El Salvador. Misc. Pit!*!.. Mns. Zool., Univ. Mich. No. 117: 1-69. CAHLANDER, D. A., J. J. G. McCuE AND F. A. WEBSTER, 1964. The determination of distance by echolocating hats. Nature, 201 : 544-546. GRIFFIN, D. R., 1953. Bat sounds under natural conditions, with evidence for echolocation of insect prey. /. E.\-p. Zool.. 123: 435-466. GRIFFIN, D. R., 1958. Listening in the Dark. Yale Univ. Press, New Haven, Conn. GRIFFIN, D. R., 1962. Comparative studies of the orientation sounds of hats. Svinp. Zool. Soc. Loud.. 7: 61-72. GRIFFIN, D. R., J. H. FRIEND AND F. A. WEBSTER, 1964. Target discrimination by the echo- location of bats. Science, 144: 563. GRIFFIN, D. R., AND A. NOVICK, 1955. Acoustic orientation in neotropical hats. /. Ex{>. Zool., 130: 251-300. GRIFFIN, D. R., F. A. WEBSTER AND C. R. MICHAEL, I960. The echolocation of flying insects by bats. Aninnil He heir.. 8: 141-151. GRINNELL, A., 1963a. The neurophysiology of audition in hats : intensity and frequency parameters. /. Physio/., 167: 38-6 with special reference to the iiinction of tin- middle ear muscles. A thcM\ deposited in tlie lihrary of Yale University, \i w 1 1 a veil, Conn. KAY, I... l"ol. Perception of distance in animal echolocation. \iitin-c. 190: 361. KAY, L., 1('<>2. A plausible explanation of the hat's echo-location acuity. Animal Behav. 10: 34-41. Xovu-K. A., 1°5S. Orientation in paleotropical bats. 1. Microchiroptera. /. /:.r/>. Zoo!.. 138: 81-154. Xovu'K, A.. l('63a. Orientation in neotropical bats. 11. Phyllostomatidae and Desmodontidae. J. Mtniniiiil.. 44: 44-56. NOVICK, \.. l%3h. Pulse duration in the echolocation of insects by the hat. /'Icfunotns. ! rgebnisse Bioi. 26: 21-26. NOVICK, A., AND D. R. GRIFFIN, 1961. Laryngeal mechanisms in bats for the production of orientation sounds. /. ]l.\-f>. Zool., 148: 125-145. Xovu K. \.. AND J. R. YAISXYS, 1(")4. Echolocation of flying insects by the hat, Cliilini\cteris farncUii. Bid. Bull.. 127: 478-488. PYE, D., 1960. A theory of echolocation by hats. /. Laryn. Otol, 74: 718-729. PYE, D., 1961a. Perception of distance in animal echolocation. Xaturc. 190: 362-363. PYE, D., 1961b. Echolocation by bats. Endeavour, 20: 101-111. REVEL, T. P., 1962. The sarcoplaMin'c reticulum of the bat cricothyroid muscle. J. Cell. Blol 12: 571-580. ROEDER, K. D., AND A. E. TREAT, 1961. The detection and evasion of hats bv moths. Aincr. Scientist. 29: 135-148. \YEBSTER, F. A., 1%3. Active energy radiating systems: the bat and ultrasonic principles. II. Acoustical control of airborne interceptions by bats. /;;: Proceedings of the Inter- national Congress on Technology and Blindness, New York: American Foundation for the Blind. \YKBSTER, F. A., AND D. R. GRIKFIN, 1962. The role of the flight membranes in insect capture by bats. Anim. Bchai'., 10: 332-340. REPRODUCTIVE CYCLE OF MYA ARENARIA IN NEW ENGLAND JOHN W. ROPES i AND ALDEN P. STICKNEY U. S. Bureau of Commercial Fisheries, Biological Laboratory, Bnothbay Harbor, .!/"(,"• Knowledge of the reproductive cycle in the soft-shell clam, Mya arcniina L., is essential to an understanding of larval production and ultimately of the abundance of this commercially important bivalve. By following the progress of gonad and gamete development in Mya throughout the year, the times and duration of spawning can be determined — information useful in the management of this fishery. Although the literature contains numerous references to the times of spawning, information about gonadal changes in M\*a is fragmentary or incomplete. A\ V found the smear technique, used by Battle (1932), unreliable for determining gamete development because important details seen in the histological preparations were obscure in fresh, unstained smears. Coe and Turner (1938) described the development of juvenile Mva and gametogenesis from histological preparations. Unfortunately, their study indicated only the beginning of_ spawning and gave no data on its duration. In a preliminary report, Rogers (1959) described gonad development in Chesapeake Bay, Maryland, during the summer and fall. He observed a fall spawning period and ventured an opinion that a spring spawning also occurred, but he had not completed a full year of observations. Pfitzenmeyer (1962) also reported preliminary histological observations to support a conclusion that Maryland clams have two separate maturations. Plankton studies by various investigators have revealed both annual and biannual periods of larval abundance. Both Stevenson (1907) and Stafford (1912) reported their observations of larvae as most numerous during a single period of time. Sullivan (1948) used the term "broods," possibly to describe successive large groups of larvae, but did not mention intervals between the groups when none were caught. Landers (1954) observed both an early-major and later- secondary larval occurrence. Between the two swarms, larvae disappeared for intervals of one to two months. Pfitzenmeyer (1962) observed an early-secondary and later-major larval occurrence. Larvae were not caught for intervals of three to four months between these biannual swarms. Inferences about the frequency of spawning have also been made from observa- tions of the first appearance of newly settled juveniles in the flats. Successive ripening and spawning periods during a single reproductive cycle were hypothe- sized by Belding (1930) to explain the "not uncommon" occurrence during any one year of "two distinct sets" in areas on the southern shores of Cape Cod. Pfitzen- meyer (1962) used special collectors in Chesapeake Bay and obtained two distinct sets of juvenile clams each year. A period of two to three months when none were caught separated the biannual settings. 1 The senior author's present address is the U. S. Bureau of Commercial Fisheries Biological Laboratory, Oxford, Maryland. 315 316 JOHN VV. ROPES AND ALDEX P. STICKNEY , •• ». -" .. r u-. • 6 •V* * • *« • V r f»^' V . \ ••IHHk V 1,1 RE 1. Section oi uoiKid tissue irom female clam, inactive phase of reproductive cycle. Xote the small, dark phagocytes in the center of the alveolus, nutritive inclusions scattered within the follicle cells, and a large, round, almost completely cytoly/cd ovocyte. To permit a comparison of cell si/e during maturation, all the photomicrographs were taken at the same magnification and a scale < 100 M) i-s provided. FIGURE 2. Same, from female clam in active phase, showing early stages of ovogenesis. I'M, i RE .1. Same, from female clam in active phase, showing later stages of ovogenesis. REPRODUCT I VE C Y C 1 . 1C O K M YA 3 1 7 Unfortunately, no certain conclusions about the nature of the reproductive cycle itself can be drawn from observations of either planktonic larvae, or newly metamorphosed set. since several seasonal peaks of abundance may appear from spawnings resulting from a single annual reproductive cycle. While observations of larval swarms can provide corroborative evidence, the annual frequency and duration of the reproductive cycle of clams can best be learned from periodic histological examinations of their gonad tissue. This paper presents the results of such studies made on clams from several Xew England areas. METHODS Collections of clams were obtained periodically from the following New England areas north of Cape Cod. Plum Island Sound clam flats in Newbury, Massachu- setts, were sampled twice each month from April, 1951, through February, 1953; flats near Boothbay Harbor, Maine, from February to November, 1961 (except April) ; and flats in Sorrento, Milbridge, Roque Bluffs, Cutler, Lubec, Pembroke, and Robbinston, Maine (collectively designated as eastern Maine in the text), each month from September, 1959, through December, 1960. The clams were dug from the middle of the intertidal zone in the eastern Maine and Boothbay Harbor areas, but throughout the whole intertidal zone in Plum Island Sound because the clams were not abundant. Miscellaneous collections were also obtained from Falmouth and Woods Hole, Massachusetts. These samples from areas on the south side of Cape Cod provided valuable supplementary data. Each sample included approximately 10 clams, H to 3 inches in shell length. Within a few hours after field collections were made, the anterior third of the visceral mass of each clam was removed and preserved in Benin's fixative. Standard techniques of dehydrating in alcohol, imbedding in paraffin, sectioning at 10 fji, and staining in Delafield's hematoxylin and eosin were used to prepare slides of the gonad tissues. A microscopic examination was made to assign each specimen to a category which represented the gonad condition. The proportion of clams in each category, regardless of sex, was recorded for the individual samples. Although minor differences were observed, data on the clams from the seven eastern Maine sample areas, as well as the twice-a-month samples from I Mum Island Sound, were combined each month to simplify presentation. Since the combined samples had no single collection date, a date midway between the first and last collection dates was chosen to represent each month's observation of gonad condition. CATEGORIES OF GONAD CONDITION Female gonads Except for certain pre-meiotic stages, ovocyte maturation occurs not in the ovaries, but after the eggs are shed (Raven, 1958). Therefore, tissues could not be classified according to the meiotic stages of the ovocytes. Instead, arbitrary FIGURE 4. Same, from female clam in ripe phase. Note the amphinucleoli in the centrally located ovocyte. FIGURE 5. Section of gonad tissue from female clam in partially spawned phase. FIGURE 6. Section of gonad tissue from spent female clam. Note the small ovocytes at the periphery of the alveoli. SIS JOHN \\ . ROPES AND \I.I>K\ P. STICKNEY categories of development were distinguished by dividing the more or less con- tinuous reproductive process into five phases. These are described below. 1. lihicti-rc phase At certain times of the year the rate of gonad activity becomes so low that changes in appearance of the gonad tissues in successive samples are imperceptible. Therefore, this .state of relative quiescence is hereafter termed the inactive phase of the reproductive cycle. The term, however, is used for convenience and is not meant to imply that no activity at all is taking place. Small ovocytes occur at the peripherv of alveoli ( Fig. 1). A round nucleus in the ovocyte contains a conspicu- ous basophilic nucleolns and is encircled by an irregularly shaped cytoplasm. Vacnolated follicle cells that completely imbed the ovocytes sometimes fill the lumina of alveoli. Inclusions, apparently nutritive in function (Coe and Turner, I1 MS i, frequency occur in the follicle cells. Small migratory cells sometimes appear between alveoli and within the lumina of alveoli. These migratory cells are probably phagocytic for they surround cytolyzecl, unspent ovocytes. 2. .-let he phase That part of ovogenesis which takes place within the gonad tissue and where definite quantitative and qualitative changes in the ovocytes are perceptible is designated as the active phase of the reproductive cycle. Enlarging ovocytes grow between the follicle cells toward the centers of alveoli during the early stages ( Fig. 2). These ovocytes, which may be sub-conical, hemispherical or cylindrical in shape, are characteristically rounded at their apices and have broad cytoplasmic bases attaching them to the wall of the alveolus. The nuclei measure from 8 to 16 /j. and average 13.3 JL in diameter. Subsequent growth produces large, round ovocytes with constricted cytoplasmic bases (Fig. 3). The free ends of ovocytes extend beyond the basal membrane of follicle cells into the lumina of alveoli. The nuclei measure from 15 to 23 ^ and average 1(>.2 /> iu diameter. These ovocytes occur late in the active phase. The onsel of ovogenesis i- difficult to determine with precision, as pronounced changes in staining reactions do not occur. However, the slight enlargement of many ovocytes, the more regular shape of their cytoplasm and protrusion toward the alveoli centers are visible indications of growth activities. Thereafter, the growth of ovocytes and tissue change.s arc more definite. 3. Ripe phase In ripe clams, a very slender stalk may connect many of the largest ovocytes to the basal membrane (Fig. 4). Other ripe ovocytes appear as round cells in the lumina of alveoli, as if free of attachment to the basal membrane. The nuclei of tbe.se largo! ovocytes contain amphinucleoli ( Allen, 1'>53'). each consisting of an almost transparent nucleolns and a small opaque nucleolinus. The ampbinucleolns resembles a signet ring in some sections because the smaller nucleolinus occurs at the periphery of the uucleolus. The nuclei of ripe ovocytes measure from 20 to 31 />. and average 2S.2 /< in diameter. These large ovocytes till the lumina of alveoli and are nsnallv more numerous than less developed ovocytes. REPRODUCTIVK CYCLE OF MYA 319 4. Partially spawned pliase In partially spawned clams, gonad tissues contain a few ripe ovocytes in some of the alveoli (Fig. 5). Small ovocytes are imbedded in follicle cells at the periphery of empty alveoli. Nutritive inclusions appear in many of the follicle cells. An absence of ripe ovocytes in many alveoli and the cessation of ovogenesis in all alveoli is indicative of the partially spawned condition. 5. Spent phase Follicle cells form a thin layer covering the basal membrane of alveoli in some spent tissues, while in others they fill the alveoli (Fig. 6). Spent individuals can be distinguished from those in which no gametogenesis has taken place at all 1>\ the presence of a few unspent ovocytes in the early stages of cytolysis, which appear in the hmiina of some alveoli as large, darkly stained bodies with obscure nuclei. Also, very numerous spherical droplets of lipoids and other products of cytolysis are characteristic of individuals which have completed a period of ovogenesis and have spawned (or, when conditions were not conducive for spawn- ing, have resorbed their gametes). Since the reproductive process is cyclic, the spent phase merges with and overlaps somewhat the inactive phase. Sharp distinction between the two is a convenience rather than a natural phenomenon. Male g on ads The criteria for the live corresponding categories of gonad condition in the male are presented below. 1 . Inactive phase During the inactive phase, the male tissues frequently contain the products of an aberrant meiotic activity, which Coe and Turner (1938) have called "atypical sperma- togenesis." Pycnotic cells (Fig. 7a) and multinucleated, non-pycnotic cysts (Fig. 7b) appear in the follicle cells. Many tissues contain both types although one usually predominates over the other. Both types of inclusions disappear during normal spermatogenesis, but reappear before it ceases, as do the nutritive granules seen in female tissues. Follicle cells fill the alveoli, surround the aberrant cells, and imbed the few spermatogonia and primary spermatocytes at the periphery of alveoli. 2. Active phase The active phase of the reproductive cycle in the male includes the entire process of spermatogenesis, unlike that in the female where only pre-meiotic stages are included. At the onset of the active phase, tissue sections contain proliferating primary spermatocytes at the basal membrane of the alveoli (Fig. 8). These small and uniformly sized cells, very similar in appearance to the earliest ovocytes, force their way between follicle cells and toward the centers of alveoli. Early stages of meiosis occur in the spermatogenic cells near the basal membrane, whereas the 320 .KHIX \V. kol'KS AND AI.IIKX I1. STHKNKY **v-- f' ' t ••• ••.**'/.V i :$&£&.' Wm$ t* -\vi->. a.to »•—.<» -A.-" ^* :rv^ ..^< ^»;:: «k * A7 * * « *? /•'i ' %^ H%^ ?^»»*^J ^{HM^iSEIv, $5 ^S5H^fcSKS| ^•- v ;ilvXx:->:*A«v:V.^ 7;i. Si-rtioii of ^on;id tissiuj from inak- clam in inactive phase of reproductive cycle. Inactive phase of spermatogenesis in male soft-shell clams. Note the round, pycnotic c< IU, products of atypical spermatogenesis. l;i(, i -KK 7b. Same, showing the cysts of multinucleated, non-pycnotic cells, also products of atypical spermatogenesis. l;H,riiu' from male clam in active phase of reproductive cycle, showing early stages of spermatogenesis. FIGURE 9. Same, showing later stages of spermatogenesis. Late, active phase of spermatogenesis. REPRODUCTIVE CYCLE OF MYA 321 later spermatids occur at the centers of alveoli (Fig. 9). A very thin-walled membrane surrounds each spermatid. Meiotically active cells between the periphery of the alveoli and their centers eventually obliterate the follicle cells. A large number of spermatids produce a distinct mass in the centers of alveoli. 3. Ripe pJiasc Ripe male tissues contain masses of spermatozoa, arranged in more or less radial columns with their tails oriented toward the center. In fully ripe specimens these spermatozoa may nearly fill the lumina of the alveoli ( Fig. 10). 4. Partially spawned phase Partially spawned male tissues contain relatively few spermatogonia at the basal membrane (Fig. 11). Follicle cells occur between the basal membrane and groups of cells undergoing spermatogenesis. A scattering of pycnotic cells occur within the follicle cells. Spermatozoa still occupy a substantial portion of the central alveolar area. 5. Spent phase Spent male tissues contain no or very few spermatozoa in the central alveolar area (Fig. 12). Numerous follicle cells with multinucleated, non-pycnotic cysts and pycnotic cells from atypical spermatogenic activities surround small groups of spermatozoa. Spent tissues lack cells in the active phase of spermatogenesis. GEOGRAPHIC VARIATIONS IN THE REPRODUCTIVE CYCLE Eastern Maine Clams in the inactive phase were encountered throughout the year in eastern Maine, but were least numerous during June (Fig. 13). All were in this condition from September through December in 1959 and 1960. By January, 1960, some had begun gametogenesis. Clams in the active phase became more numerous each month after January, until May, after which fewer individuals were in this condition, and were absent from the August samples. Clams in the active phase were obtained to some extent for a period of about seven months. Ripe clams were first observed in the middle of May, but none showed indica- tions of having spawned. In June about half of the clams were ripe and some were partially spawned. Partially spawned clams were more numerous in July and by August about 75% of the clams had completely spawned and had returned to the inactive condition. The spawning period, then, extended from early June to about the middle of August, a period of 2\ months. Minor exceptions to the reproductive cycle described above were observed in clams from two eastern Maine areas where gametogenesis began earlier than in the FIGURE 10. Section of gonad tissue from ripe male clam. Note the columns of spermatozoa. FIGURE 11. Section of gonad tissue from male clam in partially spawned phase. FIGURE 12. Section of gonad tissue from spent male clam. Note the occurrence of both types of atypical spermatogenesis. 322 100 JOHN \V. ROPES AND XI.DEX P. STICKNEY EASTERN MAINE - I960 30 - \ I ' 1 I s i i 1 • - % Y, 1 V ^ +. , ^ 1 L i i i i — r 100 BOOTHBAY HARBOR - 1961 50 PLUM ISLAND SOUND - 1952 100 30 - x .'_ ? /f. ^ ''. % 1 / ^ /. X X - 4 \ I •? ' !___ 1 1 1 JAM FEB MAR APR MAY JUN JUL AUG StP OCT NOV D€C MONTH LEGEND FOR GONAD CONDITION: E23 INACTIVE . AND ^2 ACTIVE OAMETOGENE8I3. •I RIPE. ES3 PARTIALY SPAWNED. AND I I SPENT. 13. Gonacl conclitimis of .17 vi/ uroiitrin from Nc\v luiglaiul. Tlic length of each shaded area represi nts th( percentagi frequency of clams in each »onad condition. five other an-.-i>. More than half of the l.tilxr and I'emhroke rlaius were in the active phase in January and In-hruary. After l;ehniarv, clams in this condition were almost equally as numerou.s t'roin all seven eastern Maine areas. An earlier in Luher and I'emhroke was in it definitely indicated hy the data. REPRODUCTIVE CYCLE OF MYA 323 Boothbay Harbor Gametogenesis had commenced in about 75% of Boothbay Harbor clams during February and March (Fig. 13). More ripe clams occurred during May in the Boothbay Harbor than eastern Maine samples. Some partially spawned clams were observed as early as June in Boothbay Harbor, but both arc-as yielded numer- ous partially spawned clams during July. Completely spawned clams did not appear until July in Boothbay Harbor, or nearly one month later than in eastern Maine. More Boothbay Harbor than eastern Maine clams were in the ripe, partially spawned or spent condition in late August. The three-month spawning period of Boothbay Harbor clams, extending from late June to August, was not only later, but longer by a half a month than that of eastern Maine clams. Plum Island Sound The spawning season was still later in clams from Plum Island Sound than in clams from all Maine study areas (Fig. 13). Relatively few clams began gameto- genesis during February and March. Ripe clams appeared first in June, a month later than clams from Maine. Correspondingly, the earliest occurrence of spawned clams was later. Spawning, indicated by the appearance of partially spawned clams from July through September, lasted for three months in Plum Island Sound. Many spent clams were seen as late as October. DISCUSSION The results of gonad examinations suggested several generalizations about the reproductive cycle of Mya arcnaria. Gametogenesis began during the late winter or early spring in all areas sampled. Thereafter a progressive development of the gametes was seen until ripe cells filled the tissues and spawning began in late spring and early summer. A spawning peak occurred during the middle and late summer, after which gametogenesis ceased. The inactive condition prevailed during the fall and most of the winter. No more than a single reproductive cycle occurred in the clams from nine sample areas north of Cape Cod. No failure to spawn was indicated in clams from any of the sample areas. Despite the more southern latitude, the reproductive period in Plum Island Sound was characterized by a later rather than an earlier spawning season than that in Maine (Fig. 13). Furthermore, a still later spawning season than that of 1952 was indicated by the occasional gonad samples taken in Plum Island Sound during the preceding year. A few- ripe clams first appeared during July, 1951. Both ripe and partially spawned clams were most numerous during late July and early August. All were spent by late October, 1951. Most reports of larval occurrence and sex cell development indicated that Mya begins spaw-ning at an earlier date in areas north of Plum Island Sound (Fig. 14). At Malpeque Bay. Canada, the earliest soft-shell clam larvae were found during early June by Stafford (1912) and during late May bv Sullivan (1948). Stafford (1912) found an abundance of Mya larvae during Julv and August at St. Andrews, Canada. Most of the clams in Battle's (1932) samples from St. Andrews, Canada, contained ripe gonads during early June. She found some spent gonads by late June, indicating the beginning of the spawning season. M4 JOHN W. ROPES AND ALDEN P. STICKNEY After finding an early abundance of larvae, \Yelch ( 1953, unpublished report) 2 deduced that some were probably in the plankton of Robinhood Cove, Maine, during early May, 1951. He also caught a few larvae during June of 1952 (oral communication), even though maximum numbers occurred during the middle of August to the middle of October that year. A late spawning season was reported for AJya in northern Massachusetts areas. Belding (1907) observed that Mya spawns from the middle of August until early October (Fig. 14). Stevenson ( 1CK)7 I caught no larvae at Ipswich., Massa- chusetts, before late August, and reported the last half of September as the period of peak spawning. He also caught large numbers of larvae at Plymouth, Massa- chusetts, during late July and early August. Later Belding (1930) reported July and August as the spawning season for soft-shell clams north of Boston. These reports agreed with our observations of a late active phase and spawning period of Mya in Plum Island Sound. A study of Figure 14, summarizing available information on the reproductive cycle of M\a. shows clearly two important phenomena: (1) the tendency for clams to develop gametes and spawn progressively earlier in the season northward and southward from northern Massachusetts, and (2) the himodal nature of spawning activity south of Cape Cod. It is not entirely certain from this in- formation that the bimodality results from two distinct reproductive cycles. It may result merely from an interruption in the spawning activity during a single reproductive cycle. Lacking observations on gametogenesis in the clams from their study areas. Rumpus (1898), Mead and Barnes (1904), Belding (1907; 1930), Stevenson (1907), Nelson and Perkins (1931), Deevey (1948), and Landers (1954) neither described nor implied more than a single annual repro- ductive cycle to Mya, even though Mead and Barnes (1904), Belding (1907; 1930), and Stevenson ( 1907) found two distinct groups of newly settled juveniles, and Landers (1954) observed two distinct swarms of larvae. In addition, Coe and Turner (1938) and Rogers (1959) believed that Mya probably spawned more than once each year, but did not mention the possibility of more than a single annual reproductive cycle. Nevertheless, some evidence is available from several studies which suggests two separate reproductive cycles per year in southern latitudes. The clams from southern areas were reported to begin spawning earlier in the year than those from northern Massachusetts areas (Fig. 14). Deevey (1948) found that Mya larvae were dominant in Tisbury Great Pond on Martha's Vineyard, Massachu- setts, from late April to June. Landers (1954) obtained larvae as early as the middle of April in samples from Wickford 11 arbor. Rhode Island, whereas Pfitzenmeyer nr>(>2) captured larvae in the plankton of Chesapeake Bay. Mary- land, throughout May. More important, however, both Landers (1954) and Pfitzenmeyer (1962) caught no larvae during the middle of the summer, after a late spring to early summer larval swarm. This period of larval absence in the plankton corresponded to observations of gonads by Rogers (1959) and Pfitzen- meyer (1962) which were apparently in the inactive phase during the middle of 2 Welch, W. R., 105,3. Seasonal abundance of bivalve larvae in Robinhood Cove, Maine. Kourth Annual ContYruuT on Clam Research, U. S. Fish and Wildlife Service, Clam s F'.oothhay Harbor, Maine. (Mimeographed report). REPRODUCTIVE CYCLE OF M Y \ 325 the summer. Hence, the clams were unable to spawn because ripe sex cells were not present in the gonads. Further evidence of biannual spawning can be drawn from our own histo- logical examinations of clams collected in the "\Yoods Hole, Massachusetts, area. S PAWNING SEASON M A MONTH M J J A S N — i 1 — — i 1 1 — — i — MALPEQUE BAY eamwcuK STAFFORD ^^^^^^^^^^M 1912 do cKsn^Bm^^^^n ^^^^^^^^^^^^^^ SULLIVAN 1948 ST. ANDREWS mg^^^^g^m. STAFFORD ^^^^^^^^ 1912 do «^^^^^™n^B BATTLE 1932 EASTERN MAINE mmmmm ROPES 8 STICKNEr BOOTHBAY ^^^^^^ do. HARBOR ^^^^^^^^^* ROBINHOOD COVE WELCH 1953 do. do. PLUM ISLAND ROPES 8 STICKNEY SOUND •^^•••H N. of CAPE COD MM™™» BELDING ^^^^^^^ 1907 N. of BOSTON M^H^MHI ^^^^^^^^ 1930 IPSWICH M^^™ STEVENSON P LY M 0 U T H 1907 do. SOUTHERN CAPE BELDING COD ^^^^^^^^^^B 1907 do K^^^nm ^^^^^^^^* 1930 CHATHAM ^^^^^^^^^^^^ STEVENSON ^^^^^^^^^^^™ 1907 MARTHA'S DEEVEY VINEYARD 1948 WOODS HOLE ^^^_ BUMPUS 1898 RHODE ISLAND MEAD a BARNES ^^^^^^^^^ 1904 WICKFORD •HUM • I -< LANDERS 1954 do do do. IM-1 ™« .935 do NEW HAVEN ^^^^^^^^^^ COE a TURNER ^^^^^^^^^^^ 1938 NEW JERSEY ^^^^_ BELDING ^^^^^^ 1930 do. ^^^ NELSON 8 PERKINS ^^^ 1931 CHESAPEAKE ^^^^^^_ ROGERS BAY ^^^^^^^^ 1959 do. Hi HIM "56 PFITZENMEYER 1962 do • •HHMHII 1957 do. do. mHai mm • - do. do. •n m^ ,959 do. i i i i i i 1 1 1 1 1— — c A N A D A M A I N E N. M A S S. S. M A S S. S 0 U T H E R N A R E A S FIGURE 14. The duration of the spawning season of Mya arenaria reported in the literature for areas along the Northwestern Atlantic coast. JOHN \\ . koi'KS AND ALDEN P. STICKNEY Samples of clams collected July 1(> and M. 1(>50. from Falmouth Inner Harbor contained none with ripe or developing gametes. Some live clams from these samples were held in outdoor tanks of sea water at seasonal temperatures until September 1, 1950, at which time most of them were fully ripe. The numerous nutritive granules, typically products of cytoly/ed, unspawned gametes in females and the products of aberrant spermati genesis in males, which were seen in July samples suggest that the clams from this population had also been ripe earlier in the year. This inference was supported by subsequent observations of ripe clams which were common in samples collected from Woods Hole both in late September, 1961. and in late May. 1(><>2. The occurrence of ripe gonads during the early spring and fall corresponded to the times when larval swarms were found by both Landers (1954) and Pfitzenmeyer (1962). When the gonads of clams in the southern latitude's were in the inactive condition the gonads of clams in the northern latitudes were either ripe or partially spawned. Therefore, the two periods of garnet* >genesis in clams from southern areas were completely out of phase with the single annual reproductive period of northern clams, and indicated a biannual reproductive cycle. We used clams with an apparent biannual reproductive cycle in spawning experiments at the Boothhay Harbor laboratory. Spawnings were induced during a period of a month or more after collecting clams from Woods Hole. Massachu- setts, in late September, 1'">1, and also in late May, 1962, as well as from Chesapeake Bay, Maryland, in the middle of October, 1961. The Maryland clams remained in the inactive condition during the winter, and ripened again the following April in the laboratory tanks. These clams produced larvae when local clams were neither ripe enough to spawn, nor were the gametes developed to the ripe condition by the methods we tried. We wish to thank Mr. Malcolm V.. Richards, Marine Resources Scientist of the Maine Department of Sea and Shore Fisheries, for providing us with gonad >amplcs from eastern Maine. SUMM AKY 1. Only a single reproductive cycle was observed in Myu arcnaria gonads collected from areas north of Cape Cod. 2. In eastern Maine, the spawning .season extended from early June to the middle of August, llootbbay 1 larbor clams spawned from late June to late August. Clams from Hum Island Sound began spawning in July, a month later than eastern Maine clams, and completed spawning by late September. 3. Observations of gonadal changes in .17 y<> from southern Cape Cod indicated a biannual cycle occurs. These observations are supported by previous reports of spawning activities, larval ocvunvnce, and gametogenesis in M va from the southern latitudes. LITER VTURE CITED \LI.I \, R. D., 1953. Fertilization and artificial activation of the c^g of the surf-clam, Spisuld solidissima. Biol. Bull, 105: 213-239. BATTLE, HELEN L., 1932. Rhythmic sexual maturity and spawning of certain hivalve mollusks. Contr. Can. fiinl. J;i\li.. New Series, 7: 255-276. REPRODUCTIVE CYCLE OF MY A 327 BELDING, D. L., 1907. Report on the shellfislierics of Massachusetts. In: Kept. Comm. Fish. and Game 1906, Commonwealth of MassacluiMiK I'ultlu Doc. 2r< : -46-67. BELDING, D. L., 1930. The soft-shelled clam fishery of Ma^achusettv Ala^. Dept. Conserv., Div. Fish and Game, Marine Fisheries Ser., No. 1, pp. 1-65. BUMPUS, H. C., 1898. The breeding of animals at Woods Hull during the months of June, July and August. Science, Neiv Ser., 8: 850-858. COE, W. R., AND H. J. TURNER, JR., 1938. Development of the f:unads and gametes in the soft- shell clam (Mya areiuina). J. Morpli., 62: 91-111. DEEVEY, GEORGIANA B., 1948. The zooplankton of Tisbury Great Pond. Bull. Bingham Oceanog. Collection, 12: 1-44. LANDERS, W. S., 1954. Seasonal abundance of clam larvae in Rhode Island waters, 1950-52. U. S. Fish and Wildlife Service, Spec. Sci. Rept., Fish. No. 117: pp. 1-29. MEAD, A. D., AND E. W. BARNES, 1904. Observations on the soft-shell clam (fifth paper). R. I. Comm. Inland Fisheries, 34th Ann. Rept. : 29-68. NELSON, T. C., AND E. B. PERKINS, 1931. Annual Report of the Department of Biology. July 1, 1929- June 30, 1930. N. J. Agric. Expt. Stat. Bull. No. 522: 1-47. PFITZENMEYER, H. T., 1962. Periods of spawning and setting of the soft-shelled clam, Myii arcnaria, at Solomons, Maryland. Chesapeake Sci., 3: 114-120. RAVEN, C. P., 1958. Morphogenesis: The Analysis of Molluscan Development. Pergamon Press, New York, pp. 1-270. ROGERS, W. E., 1959. Gonad development and spawning of the soft clam. Maryland Tidewater News, 15: 9-10. STAFFORD, J., 1912. On the recognition of bivalve larvae in plankton collections. Contr. Canad. Biol, 1906-1910: 221-242. STEVENSON, J. R., 1907. Report of J. R. Stevenson upon observations and experiments on mollusks in Essex County during 1906. In: Rept. Comm. Fish, and Game 1906, Commonwealth of Massachusetts, Public Doc. 25 : 68-96. SULLIVAN, CHARLOTTE M., 1948. Bivalve larvae of Malpeque Bay, P. E. I. Bull. Fish. Res. Bd. Canada, 72: 1-36. THE RELATIONSHIP OF AXTJCI.MIC COMPONENTS IX EGG-JELLIES OF VARIOUS AM I'HIBIAN SPECIES 1>! C. AI.KX SII1VKRS Department of Zoology and Entoinol55). and tissue-affinity studies (Townes and Holtfreter, 1955) are examples of the type of cellular interaction which might be interpreted on the basis of complementary surface configurations. Perhaps the most thoroughly investigated cells with reference to specific inter- acting substances are the spermatozoa and eggs of various marine invertebrates, particularly echinoderms. Studies of these substances have been given consider- able attention in the past few years (for recent reviews, see Tyler, 1959; Met/., 1957, 1961; Runnstrom ct ; Shaver and I'.arch. 1(W>(); Subtelny and Bradt. I'M ). Since jelly-less body cavity eggs of the frog are capable of normal cleavage after artificial activation, either by inoculation of a cellular element (Bataillon, 1919), or after transfer of a blastula nucleus (Subtelny and Bradt. 1('M), it is clear that the egg-jelly layer is no! essential for development. It is reasonable to assume, then, that the jelly layer is involved in one or more essential interactions with the sperm in the process of normal fertilization. This assumption is supported by the observation that jellyles^ egg-, can become1 fertilizable when artificially enrobed by jellv capsules taken from ovulated eggs (Subtelny and Bradt, 1961). Finally, Shaver and Barch ( 11'00) have shown that antiserum prepared against the jelly- coat material of h'uini /i//i/V;/.v will inhibit fertilizability of eggs of the same species. Since the jelly-coat material is immunologieally tissue-specific (Shaver, Barch and Shivers. 1962) it appears that such inhibition of fertilization results from action of antiserum upon the egg jelly material. In studies using nonprecipitating, univalent anti-egg-jelly sera, Shivers and Metz ( 1902) again obtained inhibition of fertilizing 'This studv \va- supportm l>\ jjranl No. ( • K-l l.U.i (I .S.I'. M.S.) to thr author uii'i granl No. C-.5124 (U.S.P.H.S.) to John R. Shaver. \ portion of this \\ork was submitted as partial fulfillment lor the (k^rcc of Doctor of Philosophy, Department of Zoology, M irhi.nan State I 'niversity. 328 ANTIGENS IN AMPHIBIAN EGG-JELLIES capacity in frog eggs. These results with univalent antibodies indicated an actual blocking of egg-jelly receptor sites that perform sonic t^scntial interaction with the sperm at fertilization. Tn view of this evident importance of the amphibian egg-jelly in fertilization, a more detailed description of this material seemed warranted. The present report shows that several antigenic components are present in amphibian egg-jellies. Some of these are common to several species, \vhen-a> others are restricted to a few or even a single species. MATERIALS AND METHODS Jelly-coat material was mechanically removed with watchmaker forceps, sub- sequent to hydration in distilled water, from mature unfertilized eggs of four species of frogs (R. pipiens, R. clainitons, R. syh'atica and R. catesbeiana) . The jelly was washed several times with distilled water and lyophilized until dry with a Virtis freeze-mobile. Standard antigen solutions for injection into rabbits were prepared by blending ten mg. of this lyophilate with one ml. of 0.85% sodium chloride, buffered at pH 7.4 with Sorenson's phosphate mixture, to which sodium ethyl mercurithio-salicylate (merthiolate, Lilly) was added in a proportion of one part per 10,000. Antigen solutions were also prepared, in the same manner, from egg-jelly capsules of species of Anura (Bnfo americanus and Bufo inarhnis). as well as from another order of Amphibia (AmbystoynQ wiaculatmn). An antigen preparation of fertilizin obtained by acid extraction (Tyler, 1956) from eggs of Arbacia pnnctnlata (Echinodermata), which was kindly supplied by Dr. C. B. Metz, was also available. Blood for control serum was drawn from the marginal ear vein of large albino rabbits (2.7 kilograms average weight) prior to the injection of antigen. No cross-reaction has been observed between normal rabbit serum and any of the amphibian egg-jelly solutions thus far tested. One and one half ml. of the standard antigen solution emulsified with an equal volume of Freund's complete adjuvant were injected via the subscapular route for the production of antisera. A booster injection consisting of the same amount of antigen and adjuvant was repeated 10 days later. A trial bleeding for the presence of antibodies was made three weeks after the second injection. If antibodies appeared at this time, bleedings from the ear vein were continued every other week for 6 to 8 weeks. Generally 3-5 rabbits were given injections with the same preparation and sera from these rabbits were pooled. The antigenic components of the various jellies were analyzed by the agar-gel diffusion technique (Ouchterlony, 1949). slightly modified (Shaver, 1961). After agar plates were prepared in the usual way, various arrangements of wells were made, into each of which 0.75 ml. of the reactants wras placed. The plates were developed for 5 days at room temperature (20-22° C.) and photographed for a permanent record. In order to remove or neutralize specific antibodies the antisera were absorbed by mixing them with various dilutions of inhibiting antigen in glass tubes for 24 hours at 4° C. The antigen-antiserum mixture was centrifuged at 10.000 g to remove any precipitate which formed. The supernatant was then used as a test antiserum. Preliminary tests showed that an equal volume of the standard antigen preparation was sufficient to render serum non-precipitating. Unabsorbed 330 C. ALEX SHIVERS antibody preparations were mixed with an equal volume of Sorenson's phosphate mixture before analyzing to inaiutaiii equal dilutions in the absorbed and un- antisera. RESULTS sis of antii/cns hi species of RCDHI: Agar-gel diffusion precipitin anal\>is of the antigenic components of egg-jellies .if the four Rumi species revealed similar patterns to the extent that the jelly of each species contained at least Five distinct antigens. One of these antigens was common to all four species. Each of the ^pecies had a unique combination of cross-reacting antigens and two or three species-specific antigens. The relationships for anti-jelly serum of R. pipicns are This Figure is a drawing of a fully-developed agar diffu- illustrated in Figure 1. /^-^ FIGURE 1. Diagram of double diffusion plate, showing precipitation bands formed by reacting anti-jelly serum of R. pipicns (PA) with the egg-jelly material of R. syk-atica (SJ), R. pipicns CPJ) and R. clainitaus (CJ). a, b and c •= precipitation bands representing compo- nents which are species-specific, shared among the three species and shared between only two of the species, respectively. sion plate in which unabsorbed anti-jelly serum of R. pipicns (Well PA) was reacted with antigens prepared from egg- jelly' material of R. syk'atica (Well SJ), 1\. pipicns ( \\V11 PI i and R. ckunitans (Well CJ). Those components which are present in the jelly of eggs of R. pipicns are represented by lines formed between the anliserum well and well 1 'J (e.g., lines a, b and c. Figure 1). The three lines labelled a are species-specific to R. pipicns. The curvature of the line near< si the aiitisermn well does not represent a cross-reaction between the antiserum and the heterologous jellies. The component common to the egg-jellies of all three species is indicated by the continuous line extending between the- antiscrum well and the three anti-en wells ("line b, Figure 1). Components present in the egg- jellies of k. pipicns and l\ . claiuitans. but not present in the jelly of eggs of l\ . syh'alica, are represented by a continuous line between the antiserum well and wells CJ and I 'J (e.g., line c. Figure 1 ), but not present opposite well SJ. The-- relationships of jelly antigens were confirmed by appropriate absorption ANTIGENS IN AMPHIBIAN EGG-JELLIES 331 FIGURE 2. Diagram of double diffusion plate showing precipitation bands formed by reacting anti-jelly serum of R. pipicus, which had previously been absorbed with jelly of R. clamitans (PA+CJ), with jelly antigens shown in Figure 1. a = precipitation bands represent- ing components which are species-specific. of anti-jelly sera prior to the testing of these sera on agar-gel plates. As expected, antiserum failed to produce any precipitin lines following absorption with egg- jelly of the homologous species. In addition, antiserum prepared against the egg-jelly of one species was absorbed with jelly material from eggs of other species. Such absorbed serum was then diffused against egg-jelly solutions of several species (Figs. 2 and 3). Absence of precipitin lines between the absorbing heterologous jelly and the absorbed serum (e.g., wells CJ and PA + CJ, Figure 2; wells SJ and PA + SJ, Figure 3) served as a check for complete absorption. The spectrum of lines between the homologous jelly and the absorbed serum represented those antigens not present in the absorbing heterologous jelly. Some of these repre- FIGURE 3. Diagram of double diffusion plate showing precipitation bands formed by reacting anti-jelly serum of R. pipiens, which had previously been absorbed with jelly of R. sylvatica (PA+SJ), with jelly antigens shown in Figure 1. a and c = precipitation bands representing components which are species-specific and shared between two of the species, respectively. 332 C. ALEX SHIVERS sented species-specific antigens since the lines appeared even after absorption and failed to join with lines of the other heterologous species (e.g., lines a, Figures 2 and 3). Finally, joining of precipitin lines of the homologous and additional heterologous jellies indicated sharing of antigens not present in the original absorbing heterologous species (e.g., line c, Figure 3). By means of similar analysis employing antisera made against the egg-jellies of R. clainitans. R. syh'atica and R. catesbciana. the relationships of the antigenic components in the egg- jellies of these species were ascertained. The results are Jelly R. pip. R. clam. R. syl. R. cates. An t i se r a R. pip. R. clam. R. syl. R. cates. FIGUKK 4. Interrelationships of antigens in egg-jellies of species of Rana as determined by agar-gel diffusion analysis. Antigens were determined by reacting the anti-jelly serum of rach sprout with the various jellies. Common antigens are represented by continuous lines. Lines for each species represent a minimum number of antigenic components. diagrammed in Figure. 4. The figure shows a component shared among all four species of Rana; a component shared among R. pipiens and R. claiuitans and R. catesbciana which is not present in the jelly of R. syh'atica; a component shared between R. cltui/itmis and R. catesbciana which is not present in jellies of R. pipiens or R. syh'atica; and a number of components which are species-specific in each case. A component common to R. pipiens and R. syh'atica could be de- tected by the anti-jelly serum of R. s\h'atica but not by the R. pipiens antiserum. In addition to the interrelationships among the egg-jelly antigens of the four species of Rana, jellies of some more distantly related species were examined. These were Bujo aiiicricainis. I!, niarinus, . \inh\slonia niaculaliini and the echino- derm, Arbacia punctiihita. The>e results are presented in Table I. ANTIGENS IN AMPHIBIAN EGG-JELLIES 333 A number of antigenic components were observed which were specific to the jelly of B. americanus, as well as other antigens which were shared between this species and B. uiarinus. Cross-reactions were also observed between antiserum against B. americanus egg-jelly and the jellies from the four species of Rana. When reciprocal combinations of the jelly antigens of B. americanus and the anti- jelly serum of A. inaculatitni, R. claniitans and R. calcsbeiana were made to react, no precipitin bands appeared. Diffusion of Bufo inarhuis jelly against homologous antiserum resulted in at least five precipitin bands. Some represented specific antigens, whereas others represented antigens shared with B. aincriccnnis. In contrast to B. amcricmins, when B. inarhuis egg-jelly antiserum was reacted with jelly antigens from tin- species of Rana, no cross-reactions were observed. TABLK I Antigenic components in egg-jellies of species of Rana and Bufo (Antini), Ambyslonia (Urodeln\ and Arbacia (Echinodennata; Echinoidea) as determined by agar-gel diffusion precipitin tests \ \Jelly Anti-eKK-jelly\ B. amer. R. mar. A. mac. Ai'b. puitc. R. syl. R. cates. R. pip. R. fill HI. serum of\ Bufo americanus 5 3 2* 0 1 2a 2 2a Bufo marinus 3 5 0 0 0 0 0 0 , A mbystoma maculatii in Ob 0 5 0 Ob 2a 0 o •' Arbacia punctulatn 0 0 0 2 0 0 0 0 Numbers represent precipitin bands which appeared by reacting the anti-jelly serum with the jelly, a. reciprocal reaction produced no bands, b. reciprocal reaction produced two precipitin bands. The antiserum prepared against the egg capsules of Ambystoma uiaculaiitiu produced five precipitin bands when made to react with the homologous jelly antigen. When the antiserum of this urodele species was reacted with the jelly antigens of the four .species of Rana, and of the two species of Bufo, only the jelly of R. catcsbciana produced bands. The reciprocal combination of jelly antigens of A. inaculatinn with the anti-jelly serum of B. amcricunns and R. sylvatica produced two precipitin bands. The antiserum prepared against a sample of "fertilizin" made from jelly material of eggs of Arbacia pnnctttlata produced two specific precipitin bands when reacted with the homologous jelly but gave no visible reactions with jelly antigens from any of the species of Amphibia. DISCUSSION The species-specificity of fertilization is well nigh an axiom of biology. This specificity, combined with the high correlation between cross-fertilization and cross-agglutination of spermatozoa by substances from eggs, especially in echino- derms (cf. Tyler, 1959, for earlier work on this topic), suggests that there are specific molecular patterns on the surface of gametes which interact during fertili- zation. In addition to the specificity factor of fertilization, other roles in fertiliza- tion have been attributed in part to the interaction of egg and sperm surface 334 C. ALEX SHIVERS substances. These include attachment of sperm to the egg, initiation of acrosomal reaction, sperm engulfment and activation of the egg. The egg-jelly material of both echinodenns and amphibians has been suggested as one of the complementary egg .surface substances which interacts with the sperm at fertilization. Additional e\ idence for the egg- jelly of amphibians being important in the initial steps in fertilization is the fact that antibodies prepared against these jelly antigens in- hibit the fertili/ability of eggs (Shaver and Barch, 19(>(); Shivers and Metz, 1962). These observations suggest that surface components in amphibian gametes may represent the same mechanism for the insurance of specificity of fertilization in this group that the "fertilizin-antifertilizin" system may represent in the echino- denns. The speciliciu of fertilization in amphibians is not absolute, but allows a certain degree of cross-fertilization between species (for a summary of the variety of crosses which have been made among amphibians, see Moore, 1955). Crosses between species of Ranct employed in the present study (R. pipiens, R. clainitans. 1\. s\h'atica and R. catcsbcidna ) are capable of some development with the ex- ception of crosses involving the ova of R. c/aniitans. It may be inferred that one of the factors in the successful union of gametes of different species would he the degree of similarity of configurations on the surface of gametes participating in cross-fertilization. Thus, if the jelly-coat material of the amphibian egg plays a role in the specificity of fertilization one would expect to find similar substances in the jelly of eggs of species capable of hybridization. It should be noted that where cross-fertilization has been reported to occur between species of Rana whose egg-jelly antigens were analyzed in this study, common antigenic com- ponents were found to be present. In addition, common antigenic components were found to be present in the egg-jelly of some species which have not been reported as being capable of cross-fertilization (e.g., an antigenic. component shared between the egg- jelly of R. clainitaus and the heterologous species of Rana). Jt is possible that the sperm of these species are capable of making contact with and penetrating the surface of the ova of R. clainitaus without the subsequent rotation or further development of the egg. The reasons for the differences observed between reciprocal reactions (e.g., an antigen common to R. syli'atica and R. pipicns which could be detected by R. v\7?'<;//VM stronglv bv antibodies against the species-specific components; and these are probably the last jelly components to be laid down on the egg during its sojourn in the oviduct (Barch and Shaver, 1963). As expected, the antibodies against the species-specific components of heterologous egg-jellies (R. clamitans and R. syh-afica ) had no effect on the fertilizability of eggs of R. pipicns. Although much remains to be done to elucidate the role of frog egg-jelly capsules in fertilization, these observations on the interrelationships of antigenic components in the jellies and the effect of antibodies against them on the fertiliza- tion reaction offer more evidence for an essential role(s) for the jelly-coat material. The author wishes to express his thanks to Drs. John R. Shaver and S. H. Barch for their many suggestions during the course of this work. Thanks are also due Dr. C. B. Metz for reading the manuscript. SUMMARY 1. Antisera were prepared against the jelly-coat material of eggs of several species of Rana (R. pipicns. R. clainituns. R. sylratica and R. catcshciana) and other species of Amphibia (Bufo americanus, Bufo inarinits and Ambystoina maculatiini). Serological characterizations as to species-specificity of antigenic components found in these jellies have been presented. 2. Analysis showed that the jellies of each species contained a number of species-specific components. 3. Common components were observed in the jelly of species belonging to the same genus (either Rana or Bufo}. 4. In certain cases common components were observed between species of different genera (Bufo americanus and each of the species of Rana). 5. The results of these studies on the specificity of antigenic components in egg- jellies are discussed in connection with the possible role of these components in the process of normal fertilization. LITERATURE CITED BARCH, S. H., AND J. R. SHAVER, 1963. Regional antigenic differences in frog oviduct in relation to fertilization. Amcr. Zoo!., 3: 157-165. BATAILLON, E., 1919. Analyse de 1'activation par la technique des oeufs mis et la polyspermie experimental chez les batrachiens. Ann. Sci. Xat. Zoo!., (10) 3: 1-38. KAMBARA, S., 1953. Role of jelly envelope of toad eggs in fertilization. 26: 78-85. METZ, C. B., 1957. Specific egg and sperm substances and activation of the egg. In: The Beginnings of Embryonic Development, A. Tyler, R. C. von Borstel, and C. R. Metz, eds., Amer. Assoc. Adv. Sci., Washington, D. C., pp. 23-69. METZ, C. B., 1961. Use of inhibiting agents in studies on fertilization mechanisms. Rev. Cytol, 11: 219-253. 336 C. ALEX SHIVERS MOOKK, J. V. 1U55. Abnormal combinations of nuclear and cytoplasmic systems in frogs and toads. Adv. in Genetics, 7: 139-182. ( M < HTERI.OXY, O., 1949. Antigen-antibody reactions in gels. Ada I'utli. Microhm!. ScantUnar., 26: 507-515. RrxxsToM, J., B. HAGSTKOM AND P. PERLMANN, 1959. Fertilization. In: The Cell, Vol. 1, J. Brachet and A. E. Mirsky, eds., Academic Press, Xe\v York, pp. 327-397. SHAVER, T. R., 1961. A simple demonstration of antigen-antibody reactions. Mdrupol. Detroit Science Rev., 22: 18-20. SHAVF.K. J. I\., AND S. H. BARCH, 1960. Experimental studies on the role of jelly coat material in fertilization in the frog. Ada Einhryol. Morfh. E.rf.. 3: 180-189. SHAVER, I. 1\., S. IT. BARCIL AND C. A. SHIVERS, l'»<>2. Tissue-specificity of frog egg-jelly antigens. ./. /:.r/>. Zool., 151: 95-103. SHUI-K-.. C. \.. I'Hil. Immunobiological studies of the species-specificity of egg jellies of the frog. Doctoral Thesis, Michigan State t'niv. SHIVERS, C. V. AXD C. B. METZ, 1962. Inhibition of fertilization in frog eggs by univalent fragments of rabbit antibody. Proc. See. E.vp. Bwl. MctL, 110: 385-387. SPIEGEL, M., 1(>55. The reaggregation of dissociated sponge cells. Ann. N. Y. A cad. Set., 60: 1056-1076. SriiTEi.xv, S., AND C. BKADT, 1961. Transplantations of blastula nuclei into activated eggs from the body cavity and from the uterus of Raua pi picas. Part II. Development of the recipient body cavity eggs. />'/<'/., 3: 96-114. Tcnou-Su, AND Yu-LAN WANG, 1956. Etudes experimentales sur le role du mucus des oviductes dans la fecondation chez le crapaud, et la consideration generale sur le mechanisme de la penetration spermatique. Ada E.vp. Binl. Sinica, 5: 75-122. TONVNES, P. L., AND J. HOLTFRETER, 1955. Directed movements and selective adhesion of embryonic amphibian cells. /. E.rf>. Zoo!., 128: 53-120. TYLER, A., 1955. Ontogeny of immunological properties. In: Analysis of Development, B. H. \Villier, P. A. Weiss and V. Hamburger, eds., W. B. Saunders, Philadelphia, Pennsylvania, pp. 556-573. TYLER, A., 1956. Physico-chemical properties of the fertilizins of the sea urchin Arbacia pitnctitlata and the sand dollar Ecluiuiraclniius farina. E.vf. Cell Res., 10: 377-386. TYLER, A., 1959. Some immunobiological experiments on fertilization and early development in sea urchins. E.vf. Cell Res,, Stippl., 7: 183-199. WOERDEMAN, M. W., 1955. Immunobiological approach to some problems of induction and differentiation. In: Biological Specificity and Growth, E. G. Butler, ed., Princeton I 'niversity Press, Princeton, N. J., pp. 33-53. AN AUTORADIOGRAPHIC STUDY OF THE RELATION BETWEEN HEMOCYTES AND CONNECTIVE TISSUE IX TIIM WAX MOTH, GALLERIA MELLONELLA L.1 SURESH C. SHRIVASTAVA AND A. GLENN RICHARDS Department of Entomology, Fisheries, and Jl'ildliie, I'ni-i'ersity of Miniicsutit, St. !'cd had specific activities of 1.9 c./mJ/ = 0.50 me. /ml. Injections of 5 ^\. ( -- 2.5 ^c.) were used with larvae and pupae of ap- proximately 200 mg. live weight. In some cases, two or even three injections were made at successive intervals to increase the percentage of labeled cells. Safety bulbv Mixing chamber (dilution-70times)x / Self-filling 2jul tip ndex mark FIGURE 1. Diagram* Allowing' the quick-diluting micropipette (2 /*!.) and method of using it. Blood transfusion.s from labeled prepupae and pupae to other prepupae and pupae of the >ame age were made in a cool room (15° C.) six hours after injection of H3 thymidine into the donor. The receiving prepupae and pupae were bled as much as feasible i Id 60 /»,!. ) and then gi\en a corresponding amount of blood from the labeled donor. The transfused animals were returned to a 35° cabinet l( i continue de\ el< i| pineiit. I'arabiotic twins were made usiiii.1 a labeled and a normal pupa of the same age. A feu crvstals of phenylthiourea, streptonucin sulfate and K ])enicillin were placed around the wound site before sealing the partners together with beeswax. After the operation, the pupae were pumped alternately with finger pressure to HEMOCYTES AND CONNECTIVE TISSUE 339 mix the two blood streams and to assure free How of blood with hemocytes from the donor to the pupa receiving labeled hcmoc\tes. The fate of labeled hemocyte.s was checked ]>\ examination of serial sections. At intervals of 18, 72 and 92 hours after pupation, pupae were killed by injection of Zenker's fluid into the thorax. The insects were then opened and fixed for 18-20 hours, washed overnight, dehydrated through an ethanol series, embedded in Tropical Ester Wax, and sectioned. Alternate sections from the ribbon were mounted on two sets of slides. One set was stained with Mallorv's triple stain, the other set was used for autoradiography. Autoradiographs were prepared by the method of Joftes (1959), using Nuclear Emulsion type NTB3 and exposing for 2-3 weeks. Unincorporated label, RNA, etc. were removed prior to autoradiography by treating the sections with 10% perchloric acid for two hours at 20° (Woods and Taylor, 1959). RESULTS Prior to using autoradiography to study the role of hemocytes in connective tissue formation it is necessary to obtain labeled hemocytes. Using tritiated thymidine it is possible to label nuclei permanently but only during mitosis. As a prelude, then, it is necessary to know when mitoses occur in hemocytes, to verify that mitoses can be stimulated to occur, and to learn something of the life cycle and life expectancy of hemocytes. Normal hemocyte counts and the effects of bleediiiii Stephens (1963) has recorded the mean hemocyte concentration or hemocyte count per mm.3 of blood (HC) in normal larvae of G. uieUonella as 33,200 ± 1200. In insects in general the HC can change with age and stage due to an actual change in total hemocyte number, or the amount of plasma, or the number of cells adhering to the surfaces of various organs (Jones. 1('(>2; Wheeler. 1963). Our values for HC in G. inellonclla were considerably lower than those given by Stephens but this may be due to the determinations having been made at a later time in development and utilizing the third drop of blood to exude from chilled individuals. At the end of larval development, when the larva has finished spin- ning its cocoon, the HC was found to average 15,000/mm3. The value fell to 4000 in the motionless pharate pupa which is about to shed the larval skin, then rose gradually to regain the 15,000/mm.3 value about 12 hours later. The value then again decreased to about half this after another 12 hours (Figs. 2-3). How much of these changes may be due to each of the possible factors listed in the preceding paragraph was not determined ; the values stated here represent only hemocyte concentrations in the circulating blood of chilled specimens. Many years ago Dakhnoff (1938) reported that bleeding a larva of G. uiellonclla resulted in the production of excessive numbers of mitoses. Since it is only at mitosis that H3 thymidine is incorporated as a permanent label in nuclei, the report suggests a method for obtaining a reasonable percentage of labeled hemocytes for use in transfusions and parabiotic twins. But we needed not only to verify DakhnofFs report but also to see if the same increase in hemocyte count occurred during prepupal and earl}- pupal stages. For this purpose late larvae 340 SI kl'.SH C. SHRIVASTAVA AND A. GLENN RICHARDS and early pupae were lilcil cither a .small drop (<•hernle cells and oenocytoids (photomicrographs given in Ashhurst and Richards, 1(>(>4b). It is generally thought that prohemocytes differentiate into the other types, or at least into plasmatocytes which may later develop fat droplets to become- adipohemocytes. In the present study the follow- ing data were obtained : I iine after injection Picture of labeling of H3 thymidine 6 hours ca. 10' ,. ol prohemocytes labeled 12 hours sonic of labeled prohemocytes beginning to differentiate into plasmatocytes 24 hours numerous plasmatocytes labeled, some beginning to develop fat droplets, few labeled prohemocytes 48 hours both labeled plasmatocytes and labeled adipohemocytes present 72 hour- only adipohemocytes labeled (Pupal ion at this time) '>(> hours only adipohemocytes labeled 120 hours only a l"e\v labeled adipohemocytes pn-snil 1 11 hour-- no labeled hemoeyles ol any type The above data are interpreted as showing that prohemocytes become labeled promptly, and that some of these soon differentiate into plasmatocytes which, with the development of numerous sudanophilic vacnoles, become adipohemocytes. In three days the .sequence prohemocyte — -* plasmatocyte —> adipohemocyte has been completed, and within six davs the labeled adipohemocytes have vanished. It follows that the life expectancy of this cell type, at least at this stage in the life cycle of (,'. uiclloncUa, is less than six days at 35° C. The picture of blood cell differentiation is complicated by changes in hemocyte concentration treated in the preceding section and by changes in differential cell counts. All of the above cell types are found in late larval and early pupal life, but adipoheniocytes become more numerous and more conspicuous after pupation. These changes, however, do not interfere with the use of labeled cells as presented in the next section. Two other less common lypes of bemocUes are found in the blood of (/. These are the spherule cells and the oenocytoids. These two 1\pes HEMOCYTES AND CONNECTIVE TISSUE 343 A B • » x FIGURE 4. A. Autoradiograph showing labeled hemocytes in contact with the outer surface of the neural lamella of the nerve cord of GaHeria melloneUa 18 hours after pupation. B. Auto- radiograph showing labeled hemocytes between laminae of the disintegrating neural lamella of the nerve cord of GaHeria melloneUa at about 18 hours after pupation. are extremely difficult to identify with surety in our autoradiographed smears; hence nothing will be said about their origin or fate. The relationship between hemocytes and connective tissue The neural lamella around the central nervous system breaks down during the period 12-24 hours after pupation (Ashhurst and Richards, 1964a). Sections were prepared from 18-hour pupae which had received transfusions of labeled blood 6-48 hours previously. Labeled hemocytes were found throughout the hemocoel ; some of these are appressed to the nerve cord prior to dissolution of /• ' - Vi»" ' * - 20>L FIGURE 5. Autoradiograph of the abdominal nerve cord of GaHeria melloneUa 72 hours after pupation. This individual had been transfused with labeled blood. It shows the forming adult neural lamella without any label or labeled cells. 344 SI KKSH C. SHKIY \ST\\ \ AND A. GLENN RICHARDS the neural lamella ( Fig. 4 A), and, at a somewhat later stage, are found within the disintegrating laminae of tin- neural lamella (Fig. 4 13). During the period 24 <>0 hours after pupation there is no neural lamella around the nerve cord (the central nervous system becomes reorganized to the adult condition during this time). A new neural lamella tor the adult then begins to he formed and is clearly distinct hv 72 hours ( Ashhurst and Richards. l('()4a). Sections prepared from 72-hour pupae which had heen transfused with labeled blood or joined in parabiosis with a labeled pupa showed some labeled hemocytes in the hemocoel or appressed to various organs (especially fat body and tracheae) but no label in the sheath cells which are thought to secrete the neural lamella and no label in the neural lamella or immediately adjacent tissues (Fig. 5). These data support the conclusion of Ashhurst and Richards that adipohemo- cytes have some intimate role in the destruction of the larval neural lamella during early pupal stages, but that neither these nor other hemocytes participate in the formation of the connective tissue around the adult nerve cord. SUMMARY 1. The hemocyte count of (Jallcrla incllonclla during the prepupal and early pupal stages varies from 4000/mm.3 to 15000/m3 as a function of age. The number of freelv circulating cells can be approximately doubled within 6-24 hours bv bleeding the insect. 2. I sing tritiated thymidine and autoradiography it was found that there is a hemocyte differentiation sequence of prohemocyte — * plasmatocyte —* adipohemo- cyte. This differentiation requires about three davs at 35°, and the labeled cells have disappeared in another three days. The life expectancy of these cells, at least in prepupal and earlv pupal stages, is then less than six days. 3. Transfusion of labeled hemocytes, followed by autoradiography of sections, confirmed the previous suggestion that adipohemocytes have some role in the de- struction of the larval neural lamella, but that no blood cells are involved in the formation of the connective tissue around the adult nervous system. LITERATURE CITED ASHHURST, I). E., AND A. G. RICHARDS, 1964a. A study of the changes occurring in the i nnncctiv •(• tissue associated with tin- central nervous system during the pupal stage of the wax moth, Callcria nicllnnellti L. /. Mnrph., 114: 225-236. Asn HURST, D. E., AND A. G. KK ii Ai'iis, 19641). Some histochemieal ohscrvations on the hlood '(•11s of the wax moth, (nillcria nn l/miclla L. /. /I/or/1/;.. 114: 247-253. I'.OMIAI,. 1'. ]•'., AMI \V. ]. ARNOLD, 1961. Histology, histochemistry and tracheation of the ovariole sheaths in the American cockroach, I'criphtiicta aincricana L. J. Morph.. 108: 107-12". I ) \ K ii MM K, A., 1'J.iX. l.a regeneration dn sang chez Caller in incllaiicllii apres unc prise de sang. C. A'. Soc. Biol, 128: 520-523. I I \YDAK, M. 11., 1940. The length of development of the greater wax moth. Science, 91: 525. JOFTES, D. L., 195';. l.i(|uid emulsion autoradiography \\itli tritium. Lab. hrrcstiij., 8: 131-136. TOXKS, J. C., 1962. Current concepts concerning insect heinocvtcs. .liner, '/.ool., 2: 209-246. LAX \RI \KO, T.. 1('25. I'eitriigt- /nr viTgleichi-ndrn Ilistologii' drs Blutes und des Hindegeuelu-s. Z( itschr. nnkr. anal. Forsch., 3: 409 4' HEMOCYTES AND CONNECTIVE TISSUE 345 SCHARKEK, B., 1939. The differentiation hrt WITH ncuroylia and connective tissue sheath in the cockroach (Periplaneta americana). J. Comp. Neural., 70: 77-88. SHRIVASTAVA, S. C., AND A. G. RICHARDS, 1964. The differentiation of blood cells in the \vax moth, (iallcna nicllonclla. Aincr. Zool., 4: 312-313. STEPHENS, J. M., 1963. Effect of active immunization on tola' hrmocyte counts of larvae of d'lillcriii nicllonclla L. /. Ins. Path., 5: 152-156. WEKMEL, E. M., 1938. Die Explanation des Blutes von Seidenspinnerraupen. Bid!. Biol. MctL Exp. I'h'SS.S: 6-9. WHEELER, R. E., 1963. Studies on the total hemocyte count in Pcriphuicta <;;;;, 'n'nnirt. /. Ins. Physio!., 9:223-236. Wu;r,i-ES\voRTii, V. B., 1956. The hemocytes and connective tissue formation in an insect, Rhociniits proli.rns ( Hemiptera). Quart. J. Mia: Sci.. 97: 89-98. WOODS. P. S., AND T. H. TAYLOR, 1959. Studies of rihonucleic acid metaholism \vitli tritium labeled cytidine. Lah. Inresti,/.. 8: 3U('-318. Vol. 128, No. 3 June, 1965 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY DURATION OF AN AFTER-EFFECT IN PLANARIANS FOLLOWING A REVERSED HORIZONTAL MAGNETIC VECTOR1 FRANK A. BROWN, JR. AND YOUNG H. PARK Department of Biological Sciences, Northivestern University, Eranston, Illinois Responsiveness of the living system to very weak magnetic fields is well estab- lished. Studies have involved organisms ranging from the unicellular Pararneciuni (Brown, 1962), through Volvox (Palmer, 1963), Ditgesia (Brown, 1962), Nas- sarius (Brown, Brett, Bennett and Barnwell, 1960; Brown, Webb and Brett, 1960; Brown, Bennett and Webb, 1960; Brown, Webb and Barnwell, 1964), Drosof>hila (Picton, 1964). and Cockchafers (Schneider, 1963), to birds (Eldarov and Kholodov, 1964). The nature and strength of the response have been found to vary as functions of such factors as orientation of the experimental horizontal magnetic vector, of geographic orientation of the organisms, and of phase angles of the natural solar and lunar cycles. It was recently demonstrated, furthermore, that the mud-snail, Nassarius, is able to distinguish strength differences of the horizontal magnetic vector at least within the range, 0.05 to 10.0 gauss, displaying a maximum in capacity to respond to an experimentally reversed horizontal magnetic vector when the directional change is effected with minimal simultaneous change in vector strength (Brown, Barnwell and Webb, 1964). Following subjection to horizontal vector fields differing from the earth's 0.17-gauss local one, the snails, upon return to the nat- ural field, continued to display an influence of the experimentally imposed field. The effect lasted for at least three to five minutes. An after-effect of experimental magnetic fields, with strikingly similar characteristics, was discovered also for the planarian, Dnycsia (Brown, 1965). An after-effect has also been reported for birds (Eldarov and Kholodov, 1964). The present study was directed toward learning how long the after-effect, which results from an experimentally reversed magnetic field, persists beyond the pre- viously demonstrated short period, and to learn something concerning the charac- teristics of its decay. More knowledge concerning these characteristics is of 1 This study was aided by grants from the National Institutes of Health (GM-07405) and the National Science Foundation (G- 15008) and by a contract with the Office of Naval Research (1228-30). 347 Copyright © 1965, by the Marine Biological Laboratory 348 FRANK A. BROWN. JR. AND YOUNG H. PARK fundamental importance for the ultimate formulation of a sound hypothesis for the nature of the magnetoreceptive mechanism. MATERIALS, METHODS AND EXPERIMENTS Planarians, Dut/csia dorotocephala, were observed as they moved initially northward over a polar-coordinate grid in an asymmetrical three-light field (Brown, 1962) during about a seven-month period from October 8, 1963, through April 30, 1964. The experimental conditions were designed to reveal the duration of the after-effect following response to a reversed magnetic field. Experimental series were usually run in pairs. Each of the two observers involved in a double series recorded planarian paths as follows. Five worms were placed in a 9-cm. Petri dish of water inside an orientation chamber where they remained for the duration of a 50-minute series. First, two 5-minute samples of worm-paths, to serve as controls, were recorded under the natural ambient environmental conditions. Thereupon, an 18-cm., cylindrical, alnico bar magnet was placed horizontally, centered directly beneath the point of initiation of the worm path, oriented to oppose the direction of the earth's horizontal vector, and located at such a distance below the orienting worms as to produce at the level of the worms a reversed horizontal field of the desired strength. Immediately thereafter the mean rate of turning of all the worms orienting during the first, the second, and the third five-minute interval was determined separately. The experimental magnetic field was then removed and the mean path for all the orienting worms was determined for each of the subsequent five-minute intervals during a 25-minute period. One observer dealt with a reversed magnetic field of 4.0 gauss ; another observer used a reversed field of 0.05 gauss. During the course of the 7-month experiment a total of five different observers participated and all observers dealt with both magnetic field strengths. The observations were made at various hours of the day between 9 AM and 5 PM. About 60% were made during the morning hours. The total number of series run for each of the two magnetic field strengths for each of the seven calendar months was as follows: 4.11 u.niss 0.05 gauss Octolu-r 57 61 Xovenibi-r 78 7<> December 47 15 January <>7 <>4 l-'c] >ruury 43 48 March 28 26 April M 37 Total S57 It was learned later that the actual average number of worm-paths that was obtained in a 5-minute interval was about 14. The number varied from day to day depending upon the degree of activity of the animals. It is evident from the foregoing that during the 7-month period of the investigation there were about 10,000 initial control paths, about 15.000 paths during the 1 5-minute period 01" the applied fields, and about 25,000 paths during the 25 minutes following removal of the experimental fields. DURATION OF A M.-UA'ETIC AFTER-EFFECT 349 The data were reduced in the following manner. For each of the single 50- minute series, the mean paths of the worms during each of the three 5-mimite intervals while in the experimental field and during each of the five 5-minute intervals after removal of the field were expressed as differences from the average path determined for the initial two control samples in the same series. Mean values obtained in this manner for the "responses" to each of the two field strengths were then determined for all the data for successive 5-calendar-day periods. In addition, the mean was computed for each of the eight temporally corresponding differences between responses to the two experimental magnetic field strengths. Analyses of this report were based upon the eight values for each of the 5-day periods for the two experimental field strengths. This yielded a total of 656 indi- vidual data, or 328 differences. It was quite conceivable that the worms would adapt to each of the two altered experimental fields in the course of time, and that the initial kind of turning behavior might gradually be restored despite continued presence of the experi- mental, reversed magnetic fields. To establish or eliminate this possibility, an additional experiment was performed. This second experiment was conducted between October 27 and December 22, 1964. It was designed just like the initial one except that the magnets were left in place during the whole 40 minutes instead of being removed after 15 minutes. It differed in addition, however, in the following ways ; ( 1 ) Only two months were involved, overlapping in time of year, one year later, only about one-third of the period of the earlier experiment. (2) Six observers contributed to the results; four of them had not participated in the earlier observations and were given no information about either the earlier results or even the objectives of the study. (3) A larger fraction of the observa- tions, about 65%, were made during afternoon hours. A total of 30 series was run at 0.05 gauss and 32 series at 4.0 gauss, distributed over the whole two-month period. The data of this second experiment were treated like the earlier data in that responses were determined separately for each of the 5-minute periods by obtaining the path differences from those of the initial controls. The differences between the mean responses to each of the two field strengths for the corresponding con- secutive time intervals were computed. The mean response to each strength for the first 15 minutes, the last 15 minutes and the intervening 10 minutes was calcu- lated, together with standard errors of their means. RESULTS A. Removing magnets ojtcr 15 minutes It is evident that if there were no influence of either of the two imposed reversed magnetic field strengths, no significant difference between the mean paths of the worms for the corresponding, successive pairs of 5-minute samples would be ex- pected between the results for the two fields. If responses did occur, absence of significant difference between the two would indicate that exactly the same response resulted for the two strengths. Other than in magnetic-strength difference, the worms for the two kinds of series had been treated in precisely the same general manner by the same observers over a sufficiently long period to have reduced to insignificance the differences between the two means due to random sampling. 350 FRANK A. BKOYYX, JR. AND YOUNG H. PARK This method would also compensate for any systematic change that might tend to occur in the orientation of the worms over the course ol the 50-mimite observa- tion period while in the orientation chamber. Such a systematic change could conceivably arise from a gradual photic adaptation, the repeated mechanical stimulation, or simply fatigue of the live worms. The procedure employed would UJ u .• u .! I Ll i I- .. +1 • • . .• :- MAGNET s one, during three consecutive 5-mimite intervals after application of the experimental fields, and iluriny five 5-ininute intervals following field removal. B, the responses to the two .strengths of magnetic field, expressed as differences Irom initial controls, during and following the experimental field reversals. be expected to eliminate, therefore, all changes which were independent ol the specific differences between the effects of the two experimental magnetic treatments. A previous study (I'.rown, 1965) had established two facts: ( 1 ) north-directed worms in a 4.0-gauss reversed field turned more strongly clockwise than the same worms when they were subjected to a 0.05-gau>s reversed field, and (2 ) during the DURATION OF A MAGNETIC AFTER-EFFECT 351 initial 5-minute period following removal of the reversed field the worms that had been exposed to 4- to 6-gauss fields turned more strongly clockwise than the same worms subsequent to removal of 0.05- to 0.1 -gauss fields. In Figure 1 A, the mean path of the worms for each 5-minute interval in the 4.0-gauss series is plotted as the difference from the mean path for the correspond- ing interval in the 0.05-gauss series. Confirming the results of the previous study (Brown, 1965), worms in the 4.0-gauss field turned clockwise relative to worms in the 0.05-gauss one. There is suggestively present an initial overshoot and a subsequent tendency toward stabilization at a slightly decreased difference during the 1 5-minute period of exposure. Upon removal of the experimental, rever-ed fields, an after-effect persists with the same sign and of about the same magnitude during the first 5-minute interval. This is followed by a gradual decay of this after-effect with the difference between the after-effects from the two imposed experimental magnetic fields disappearing completely by the end of about 25 minutes. It is seen from Figure 1 P>. in which the mean responses of the worms to each of the two experimental field strengths, together with the post-field changes, are shown separately, that the results illustrated in Figure 1 A appear to come chiefly from a mean clockwise response to the 4.0-gauss field relative to essentially no mean response to the 0.05-gauss field. B. Leaving magnets in place jar -In minutes In Figure 2 A are seen the differences between responses of worms while in the reversed fields of 4.0 and 0.05 gauss for each of the eight consecutive 5-minute samples. In Figure 2 B the response to each of the field-strengths is shown separately. Although this figure is entirely comparable to Figure 1, it will be noted that the scale has been altered by a factor of 4. Both the responses of the worms to each field strength and the difference between the responses were much greater than for the earlier experimental series. In addition, a clear mean response was evident not only for the 4.0-gauss field (clockwise turning) but for the 0.05- gauss one (counterclockwise turning) as well. One fact was immediately apparent from the results of this experiment, namely that there was no overall reduction in the influence of the experimental fields on the worms during the 40 minutes of exposure to these fields. First, considering grossly the differences between responses to the two field strengths, the difference was found for the first 15 minutes in the fields to be 3.87 ± 0.921° (N - - IS'.i and for the last 15 minutes of the 40-minute exposure to be 3.97 ± 0.822° (^ - 186.) Fxamining each field strength separately, for the 0.05-gauss field the first and last 1 5-minute groups of responses were —2.78 ±0.605° (X =90) and -2.79 ±0.582° (X =90), respectively. For the 4.0-gauss field, the comparable values were +1.09 ± 0.695° (X := 96) and +1.18 ± 0.581° (N := 96). The two mid-series values were +1.44 ±0.559° (N = 62) for the 4.0 gauss and -1.61 ±0.678° (X := (.Oi for 0.05-gauss. It had been suggested, from examination of Figure 1, that there was an initial over-shoot in the response to the reversed fields with a stabilization at a slightly lower level prior to the removal of the experimental fields at the end of 15 minutes. A very similar behavior occurred again in this second experiment and, indeed, a 352 I-RAXK A. BROWX, JR. AND YOUNG H. PARK partial apparent acclimation suggestively continued fur about 20 mimiles. Also as in the first series this behavior seemed to result principally from an alteration in response to the weaker, 0.05-ganss, field and only to a lesser extent to the 4.0-gauss one. But equally suggestive was an apparent complete loss of this par- tially "acclimated" state by the end of the 40-minute period of observation. + 4 u 344 Although Polydora couiincnsa/is is often present in the shells of hermit crabs, many aspects of its life-cycle and biology have been overlooked. The larval stages have been incompletely known and there also is confusion as to the mode of fertilization. The aims of this paper are (1) to describe the larval development of P. com- mensal is so that it might be recognized in waters where it has heretofore been overlooked or confused with other species, and (2) to investigate problems sur- rounding the annelid's habitat and method of reproduction. MATERIALS AND METHODS Since a major aim of this investigation was to describe morphologically the larval development of Polydora cowimensalis, large numbers of the hermit crab, Eupagnnis pollicaris Say, were obtained. All hermit crabs used in this study were collected in Noank Harbor ( U.S.C. & G.S. Chart No. 358). One hundred and two shells of hermit crabs were opened from November, 1962, to September, 1963, in search of /'. coimncnsalis egg strings. The four egg strings found provided fertilized eggs and young larvae for laboratory studies. Stender dishes containing sea water which had been filtered through a Millipore filter, using pads with a porosity of 47 p. ( Millipore Filter Corp., Bedford, Massa- chusetts), were used as rearing vessels. These vessels were immersed in a water bath of running sea water. Sea water in these vessels was changed three times a week. Liver powder was used as a source of food and a freshly prepared sus- pension was added at the time of each water change (Howie, 1958). Larvae were also obtained in qualitative plankton tows, using a No. 10 net. Plankton tows were taken on the average of once weekly in front of the laboratory from November, 1%2, through December, 1963. Larvae from this source were maintained in the same way as those reared from fertilized eggs. Descriptions of laboratory-reared larvae and larvae collected from the plankton were made. Larvae were examined in either a hanging drop or under Saran Wrap (Dean and I latfield, 1963). Larvae of various stages were photographed with a Polaroid camera mounted on a phase microscope. All drawings except 1 Contribution No. 34 of tlio Marino Research Laboratory, Noank, Conn. Supported in part by grant No. (i22?4f) of tbe National Science Foundation. 2 Appreciation is extended to Dr. David Dean for constructive editorial suggestions and to ('apt. L. H. Malloy for providing hermit crabs. :i A portion of a thesis submitted in partial fulfillment of the requirements for the M.S. degree from the University of Connecticut, Storrs, Connecticut. 356 POLYDORA COMMENSALIS 357 Figure 1 were made from the photographs with the aid of a camera lucida or projector. Figure 1 was drawn from life, using a camera lucida. Larvae were returned unharmed to rearing vessels, following examination. I. OBSERVATIONS ON ADULTS 1 ) suggested that the larvae leave the egg sacs at this stage. It was determined that this was the case by using a method similar to that em- ployed by Wilson (1928) with Polydora ciliala larvae. An adult Polydora com- iiiensalis was left in its calcareous tube with its unbroken egg sacs in a dish of filtered sea water. For the next five days larvae were continually liberated. Twice a day these larvae were collected, examined, and the adult worm put in a dish with fresh sea water. In all cases the collected larvae were at a typical 5- segment stage. Furthermore, the earliest larval stage collected in the plankton possessed five segments, which adds credence to the above results. /'. Development outside tlic egg sac The pelagic larvae of Pol \dora coniinensalis were found in the plankton of the Mystic River Estuary from July to November. Egg strings were found in shells of hermit crabs on four separate occasions during the months of July and August, 1963. The following descriptions characterizing planktonic larvae are based upon examination of many larvae from the above sources. The general description of the pelagic larvae which follows precedes a more detailed description. One of the most noticeable features of the pelagic larvae is their pigmentation. Planktonic larvae of all stages possess a median dorsal row of black ramified chro- matophores on all segments, except occasionally on segments 1 and 2. As the larvae become older and reach the 12- to 16-segment stage, lateral unramified black chromatophores appear on the anterior aspects of each segment. The pigmenta- tion of the vestibule deepens as the larvae develop. The pygidium also appears quite dark. The median row of chromatophores becomes more ramified and spreads across the dorsum in older larvae. The anterior rim of the rounded prostomium is also noticeably pigmented in older larvae. The above pigmentation results in an overall dark appearance of the larvae when viewed with the naked eye or under low power of a dissecting microscope. The only other pigmentation is the black of the eyes. The latter number from zero to six in various specimens, but six is the most prevalent number. On either side there is usually a black ramified chromatophore between the most lateral eye and the eye nearer the midline of the body. One specimen, reared from a fertilized egg to a 28-segment stage, lacked eyes and bore only a median prostomial chromatophore. The other most obvious characteristic of these larvae is the large size they attain. Specimens surpassing 2 mm. in length are not uncommon. These general characteristics easily separate the larvae of P. coinmensalis from those of all other spionids reported in the literature to date, except Polydora hermaphroditic a Hannerz (Hannerz, 1956) and "Polydora A" (Gravely, 1909). 362 PHYLLIS A. HATFIEIJ) The descriptions of these larvae show great .similarities to /'. commensalis. The more detailed examination which follows is necessary, therefore, to avoid confusion. In early pelagic stages all setae are capillary. Notosetae are much longer than neurosetae. The fifth setiger does not become modified with stout hooks, charac- acteristic of the genus, until the larva has reached a considerable size. The precise setiger stage at which this modification occurs is variable. These hooks have been observed as minute setae beneath the integument as early as the 15-segment stage. Conversely, they have not been seen in some specimens of 19-segments. However, presence of hooks often is noted between the 18- and 19-segment stage. Associated with the appearance of these modified setae is the reduction in number of capillary 0.1 m FIGUKE 4. Anterior end of a 28-segment P. cniiiiiicn.'nilix larva. Dorsal view; ciliation omitted. neurosetae. The modified setae break through the integument at the 21- and 23- segment stage, and the capillary notosetae are lost. Large glands, named "poches glanduleuses" by Claparede (Hannerz, 1956), become noticeable with the advent of the modified setae. These glands enlarge as the hooks increase in si/.e (Fig. 4). Shortly after the appearance of the modification of the fifth setiger, hooded bidentate crotchets appear in the neuropodia of the posterior segments. These are accompanied by bent capillary setae. On a few occasions the hooded crotchets were observed on larvae with as few as 18 segments. They were always present on larvae of 22 or more >eligers and began on the eleventh to fourteenth setigers. All POLYDORA COMMENSALIS 363 segments have both dorsal and ventral cirri with the exception of the fifth. The ventral cirri are hetter developed (Fig. 5). Ciliated swellings lateral to the vestibule become very apparent by the 12-setiger stage. These lateral lips continue to enlarge and in advanced larvae appear enor- mous when the mouth is wide open. A prototroch runs across the ventral side of the lateral lips to either edge of the vestibule. It reaches the dorso-lateral edge of the lateral lips, but does not extend across the dorsal side. A minute prototroch is still observed as late as the 28- segment stage. The telotroch extends across the ventral side in patches. All larval stages exhibit a large dorsal gap. Neither a neurotroch nor ciliated pit has O.lmm Posterior end of a 25-segment P. commensalis larva. Dorsal view ; nototrorhs omitted. been observed at any stage. Prominent gastrotrochs are present in six to eight patches on segments 3, 5, 7, 10, 13, 15, 17, 19, 21, 23, 25, etc. Nototrochs are present in eight patches across the dorsum. The most lateral patch on each side is composed of "grasping cilia" (Wilson, 1928). Usually, nototrochs begin on segment 3 and are present on every segment thereafter. Occasionally, however, a small nototroch is present on segment 2. Between the 9- and 10-setiger stage, small swellings, palpi anlage, are observed posterior to the prototroch and anterior to the first segment. These buds gradually increase in length, but remain rather small in relation to the body size, even in the oldest larval stages (Fig. 4). They are greatly reduced compared to other spionid larvae. PHYLLIS A. 11ATFIFI.1) 1 Branchial buds are never observed prior to the 22-segment stage. Kven at the JS-segment stage they appear (|iiitc small, being present on setiger (> and onward. c. Metamorphosis and loujth oj larval life Unfortunately, experiments designed to induce metamorphosis were unsuccess- ful. Therefore, the length of larval life can he approximated only. Records were kept on the period of time it took for four larvae to develop from fertilized eggs to 27- to 29-segment stages. The results of four separate observations were 42 days, 44 days, 45 days and 38 days. This is an average of 42 days or approximately one and one-half months. After the 27-segment stage is reached, further develop- ment is very slow. For example, approximately 15 days were required for a larva to develop from a 27-segment to a 29-segment stage (Table I, specimen 1), whereas observations showed that a specimen might develop from a trochophore to a 14-segment stage in about the same length of time (Table I, specimen 4). The TABU, I Estimatex of length of larval life of P. commcnsalis Specimen no. Date examined No. segments No. days aiter initial observation 1 Julv 9, 196.? Fertilized Kgg '|ulv 28, 196.? 14 19 Aug. (), 1963 22 to 23 31 Aug. 13, 196.? 24 to 25 35 Aug. 14, 196.? 25 36 Aug. 20, 196.? 27 42 Sept. 4, 196.? 29 57 2 July 9, 1963 Fertilized Egg July 11, 196.? Trochophore 2 July 12, 196.? 3 3 July 15, 196,? 5 6 July 17, 196.? 8 8 "July 29, 196.? 14 20 Aug. 1, 1963 18 23 Aug. 5, 196.? 19 27 Aug. 10, 1963 23 32 Aug. 15, 1963 25 37 Aug. 22, 1963 27 44 3 July 11, 19ft.? Fertilized Fgg Aug. 2, 1963 12 22 Aug. 25, 1963 28 45 4 July 15, 196.? Trochophore* July 19, 1963 5 4 lulv 22, 1063 6 to 7 7 Julv 30, 1'>6? 14 15 Aug. 20, 1963 27 36 * Approximately 48 hours are needed to advance from the fertilized egg to the trochophore stage. Therefore, 2 days should he added to the figures in the column "Xo. days after initial observation" in order for trn-r figures to correspond to the other 3 observations. POLYDORA COMMKXSAL1S 365 TABLE II Length-frequency of P. commensalis adults Length (mm.) Frequency of occurrence Cumulative 0-1 0 0 1.5-2 5 5 2.5-2 18 23 3.5-4 6 29 4.5-5 9 38 5.5-6 1 39 6.5-7 4 43 7.5-8 8 51 8.5-9 0 51 9.5-10 1 1 62 10.5-11 0 62 11.5-12 2 64 12.5-13 3 67 13.5-14 3 70 14.5-15 8 78 15.5-16 0 78 16.5-17 1 79 17.5-18 1 80 18.5-19 0 80 19.5-20 4 84 20.5-21 0 84 21.5-22 1 85 22.5-23 0 85 23.5-24 0 85 24.5-25 3 88 25.5-26 0 88 26.5-27 2 90 27.5-28 1 91 28.5-29 0 91 29.5-30 0 91 30.5-31 0 9] 31.5-32 0 91 32.5-33 1 92 33.5-34 0 92 34.5-35 0 92 35.5-36 0 92 36.5-37 0 92 37.5-38 0 92 38.5-39 0 92 39.5-40 2 94 40.5-89.5 0 94 90 -90.5 1 95 Notes : The total number of worms collected from shells was 109. The total number of worms where measurements were taken was 95. latest stage a larva attained in these experiments was 29-segments. After remain- ing at this stage unmetamorphosed for 21 days, it was preserved. The length of larval life is probably shorter in nature. By the 22-setiger stage the larvae have acquired all the features of a young benthic worm. The fifth setiger has been modified, hooded crotchets have appeared and branchial anlage are present. 366 PHYLLIS A. HATFIELD If one a>Mimes larvae are capable of metamorphosing when these features have developed, then the larval life would be reduced to approximately one month (Table 1, specimens 1 and 2). d. Comparison o\ larvae oj Pol\dora coinincusalis witli Pol\dora hermaphroditic a The larvae of rol\dora hermaphroditica (Hannerz, 1956) and "Polydora A" (Gravely, 1909) greatly resemble larvae of Polydora commensalis. "Polydora A" has been reported as identical to P. hermaphroditica (Hannerz, 1956). Larvae of Polydora hermaphroditica are very similar to advanced larvae of Polydora commensalis in regard to pigmentation, size, ciliation and time of occur- rence in the plankton. However, the latter has the following distinguishing char- acteristics. Branchial anlage are clearly evident from segment 6 onwards. The dorsal median row of ramified chromatophores is present from segment 1 to the posterior end, except on occasion when they are absent on segments 1 and 2. Small imramified chromatophores are present laterally on the anterior side of each seg- ment. They often continue to the posterior end, although at times are lacking in the last few segments. Bidentate hooded crotchets in the neuropods begin between segments 11 and 14. whereas they are found more anterior in P. hermaphroditica. The above characters seem to be the most outstanding features by which to separate the two species. Although the author knows of no reports of the presence of P. hermaphroditica in American waters nor P. commensalis in European waters, it is important that specimens collected in the plankton be examined critically. It will be interesting to learn whether these species are more widely distributed than reported. DISCUSSION Polydora commensalis larvae exhibit typical polydorid characters in regard to morphology, deposition of eggs within the adult tube and planktotrophic pelagic larvae. Inconclusive data indicate that copulation, also typical of the genus, occurs. Polydora cunimensalis has a short period of brood protection followed by a long pelagic life. This species can delay metamorphosis, as shown by one specimen which remained unmetamorphosed at the 29-segment stage for 21 days. Both a long pelagic life and the ability to delay metamorphosis would be advantageous to this species in finding its host. Adults of Pol \dora- commensalis are found in several types of shells and in asso- ciation with more than one species of hermit crab. This distribution indicates that the commensal relationship is not highly specific. However, one must hesitate in referring to Pol\dora commensalis as a facultative commensal, since there are no reports of adults living in a free state. Usually, when a number of different species may act as host for a commensal, the factors which cement the relationship are (juite general in nature (Dales, 1957). In the case presently being discussed, it would appear that the main factors are food and shelter. Furthermore, the host is active and, thus, the more passive annelid obtains benefit by the avoidance of MaiMiation. The inside of the hermit shell is kept well aerated with fresh sea water. This is accomplished by the current of water from the branchial chamber of the crab, aided by the beating of the pleopods (Jackson. 1('13). The general nature of the relationship was further evidenced by the ability of POLYDORA COMMENSALIS 367 P. commensalis to form tubes on various shells which could never be occupied by hermit crabs, i.e., lamellibranch shells. All of the above, plus the fact that this annelid is reported from both sides of the globe, lead to the speculation that P. commensalis may be more widely distributed than reported. During the studies on the reproduction of P. commensalis sufficient evidence was collected to speculate that fertilization is accomplished by copulation. In summary, the data tabulated below support this theory. 1. Both males and females are often present in the same shell with their tubes in close proximity. 2. The worms are capable of extending far out of their tubes, thus allowing the sexes to meet and copulation to take place. 3. On occasion, worms in the size range of females are found to contain sperm as well as eggs, indicating copulation had taken place. 4. All egg masses found contained fertilized eggs. Although the occurrence of several P. commensalis of both sexes in one shell seems to have been overlooked previously, the occasional presence of a minute individual approximately 4 mm. in length has been noted (Andrews, 1891 ; Berkeley and Berkeley, 1936). Andrews had speculated that these minute annelids were males, and hence there was an interesting case of sexual dimorphism in regard to size. However, neither Andrews nor Berkeley and Berkeley were successful in determining if this were the case. In this study, ripe males were found to average a length of 12 mm. On only one occasion were sperm found in worms of 1.5 to 5 mm. in length, though a total of 38 worms were found in this size range (Table II). SUMMARY 1. Polydora commensalis larvae were found in the plankton from July to November, 1963, and fertilized eggs were found in the tubes of adults in July and August. 2. Fertilized eggs develop into 5-segment larvae within 5 to 7 days, at which stage they are liberated to the plankton. 3. Ciliation, setation, pigmentation and other taxonomically important features are described for larvae from the trochophore to 29-segment stage. Planktonic larvae of all stages possess a dorsal median row of black ramified chromatophores. Advanced larvae may exceed 2 mm. in length. The pigmentation and large size attained are the most outstanding characteristics of P. commensalis larvae. 4. Due to the similarity between larvae of P. commensalis and P. herma- phroditic a, a comparison of the two is given. 5. The length of larval life of P. commensalis is estimated to be between one and one and a half months. 6. Data on distribution, habitat and reproduction are reported. It is stiggeMed that this annelid is more widely distributed than reported, and that fertili/atinn is accomplished by copulation. LITERATURE CITED ANDREWS, E. A., 1891. A commensal annelid. Amcr. Nat., 25: 25-35. ANNENKOVA, N., 1938. Polychaeta of the North Japan Sea and their horizontal and vertical distribution. Tnid. Hydrobiol. Expcd. USSR in 1934, to the Jap. Sea., 81-230. 368 PHYLLIS A. HATFIKLD BERKELEY, E., 1927. Polychaetous annelids from the Nanaimo District — Part 3. Leodicidae to Spionidae. Contr. Canad. Biol. Fish.. 3: 407-422. BERKELEY, E., AND C. BERKELEY, 1936. Notes on Polychaeta from the coast of western Canada. I. Spionidae. Ann. Ma;i. Xat. PI 1st., 18: 468-477. BERKELEY, E., AND C. BERKELEY, 1956. On a collection of polychaetous annelids from the South Beaufort Sea, and from Xnrt Invest Alaska; together with some new records from the east coast of Canada. /. Fish. Res. Bd. Can., 13: 233-246. DALES. R. P., 1957. Interrelations of organisms, A. Commensalisrn. In: Treatise on Marine Ecology and Paleoecology, I. Geol. Soc. Ainer., Man.. 67, 1: 391-412. DEAX, D., AND P. A. HATFIELD, 1963. A method for holding small aquatic invertebrates for observation. Titrtox News, 41 : 43. GRAVELY, F. H., 1909. Polychaete larvae of Port Erin. Prac. Trans. Biol. Soc. Liverpool. 23: 575-653. HAXXEKZ. L., 1956. Larval development of the polychaete families Spionidae Sars, Disomidae Mesnil, and Poecilochaetidae X. Fam. in the Gullmar Fjord (Sweden). Zool. Bidr. 1'ppsala, 31: 1-204. HARTMAX, O., 1941. Polychaetous annelids. Part 3. Spionidae. Some contributions to tin biology and life history of Spionidae from California. Allan Hancock Pacif. E.rpcil., 7 : 289-323. HOWIE, D. I. D., 1958. Dried organic substances as food for larval annelids. Nature. 181: 1486-1487. JACKSOX, H. G., 1913. En^if Zoology, University of North Carolina, Chapel Hill, N. C. 27515 One of the clues to the mechanism of mitosis is a study of the ways in which it can be blocked; this has been discussed by Wilson (1963). There have been many such studies, utilizing a wide variety of chemical and physical agents ; one of the best known of these agents is colchicine, which has been extensively employed by Eigsti and Dustin and their co-workers, and by many others. Colchicine appar- ently acts by blocking the mitotic division at metaphase, destroying the spindle in the process; chromosome replication is not affected and polyploidy may result. Podophyllin (derived from the mandrake root) is a similar mitotic poison which destroys the spindle (Sullivan and Wechsler, 1947) ; however, polyploidy has ap- parently not been reported to be a sequence of its use. Cornman has made extensive investigations into the action of this substance (reviewed by Cornman and Corn- man, 1951) and of related or derived compounds, such as podophyllotoxin and quercetin. The early interest in these substances derived from their apparent efficacy against certain forms of warts (condyloiuata acuminata) and, later, against cancerous cells (see Cornman and Cornman, 1951, and Biesele, 1958, 1962, for specific references). The latter application has not proved to be especially valuable, but podophyllin and its derivatives are nevertheless of considerable interest as antimitotic agents, because of their great potency at very low concentrations. Riesele (1958) has extensively reviewed the literature on the action of mitotic poisons, including podophyllin and its derivatives, and colchicine. He lists the following agents as among the poisoners of metaphase and later stages of mitosis : colchicine and its derivatives, a number of physical agents, many organic compounds. podophyllin and related compounds, certain sulfhydryl reagents, quinones and phenols, antifolic acids, etc. Of these, we elected to test podophyllin and podophyllo- toxin (the active principle of crude podophyllin resin), using fertilized eggs of the polychaete annelid, Chactopierus. With this biological material, it was possible to have a large population of relatively uniform cells, all of which were at the metaphase of the first meiotic division (Figs. 1 and 2) and therefore at a susceptible stage for an antimitotic agent of this type. Cornman and Cornman (1951) used the eggs of certain echinoids (Arbacia, Lyt echinus, Tripneustes and Echinarachnius) , of the asteroid, Asterias, and of the gastropod, Chromodoris. In none of these experi- ments, with the exception of some tests with Asterias, were the eggs at the first meiotic metaphase after fertilization when they were treated. 1 Aided by grants from the National Institutes of Health, RG-5328 and CA 06662. 369 370 CATHERIXE II EX LEY A XI) J). P. COSTELLO PLATE I All drawings in this and the succeeding plates are of fixed Chactoptents eggs (whole- mounts, slightly compressed), drawn with the aid of a camera lucida, using 20 X oculars and an oil immersion or a 4-mm. objective, giving total magnifications of 3200 or 1300, respectively. The eggs illustrated were fixed in Kahle's fluid and stained lightly in Harris' acid haematoxylin. FIGURE 1. Normal first maturation metaphase in Chaetoptcrus, seen in side view. The nine maturation chromosomes are easily countable. Magnification: 1600 X. FIGURE 2. Polar view of a normal first maturation metaphase. The aster at only one of the spindle poles is shown. The large black body at the lower left is the nucleolus (which is not shown in Figure 1). Magnification: 1600 X. We are indebted to Miss Marion Seiler for her assistance in preparing the final drawings for publication, and to Mr. Donald E. Kent for photography and for his help in other phases of the investigation. METHODS Eggs and sperm of Chaeioplerus pcrgamentaccits were obtained and inseminated in freshly filtered sea water by the methods outlined by Costello ct al. (1957). Exactly five minutes after insemination (at which time the egg is at metaphase, preparing to undergo the first meiotic division) measured amounts of podophyllin - or podophyllotoxin - solution were added to 200 ml. of egg suspension, using a volumetric pipette, while the dish contents were being thoroughly mixed. Both control and experimental dishes were kept on the sea water table ; air temperatures varied from approximately 20° C. to 25° C. for different experiments but were relatively constant during the course of any one experiment. At the conclusion of the desired period of treatment, the eggs were washed with large quantities of freshly filtered sea water, and allowed to continue development. Control eggs were cultured in freshly filtered sea water and, except for the absence of the antimitotic 2 We wish to express our appreciation tu Mr. George Motoasca, ol" S. l'>. Peniek & Company, for samples of these drugs used in the earlier portion of our studies. EFFECTS OF PODOPHYLLIN 371 agents, were handled in a manner similar to that used for the experimental ova. Treatments were, in most cases, one hour in duration, except where otherwise noted. Development of the living control and experimental eggs was observed for periods up to three hours, and at intervals after that. Egg-samples were fixed at various times, calculated so that the ova would be killed at the metaphases of first and second polar body formation, and of first and second cleavages. Samples were also fixed approximately 24 hours after insemination, to ascertain what degree of later development, if any, had occurred. Kahle's fixative was routinely used, and the eggs were killed directly on #1 coverslips, by the method described by Henley and Costello (1957). The preparations were stained with Harris' acid haema- toxylin, dehydrated in an ethanol series and mounted in damar or Permount. Camera lucida drawings (Figs. 1-17) were made of living and fixed eggs as records; approximately 2100 permanent preparations were studied, utilizing an oil immersion or a 4 mm. objective and 20 X oculars. The podophyllin and podophyllotoxin stock solutions were made up as follows. Ten mg. of the dry drug were diluted in 10 ml. of distilled water, in a volumetric flask. Appropriate amounts of these stock solutions were then further diluted in volumetric flasks with distilled water to the desired concentrations (0.1 to 0.00001 mg./ml., final concentration, when added to 200 ml. freshly filtered sea water-egg suspension). All solutions were kept refrigerated until shortly before use, when they were allowed to come to room temperature. Fifty-five series of experiments were carried out. RESULTS The sequence of early events in the cytological effects of podophyllin In fertilized Chaetopterus eggs treated with high to moderate levels of podo- phyllin (0.1 to 0.005 mg./ml.), a characteristic series of early events takes place (Figs. 3-11). In eggs fixed as soon as one minute after the beginning of treatment (with a dosage of 0.005 mg./ml.), there is a suggestion of fading of the asters of the egg maturation figure ; the chromosomes look normal in such eggs. At 1 \-2 minutes after the beginning of treatment, this process of fading continues (Figs. 3, 9), so that by two minutes after the initiation of treatment, some of the maturation figures appear almost normal, while in others one or both asters are faint or absent, or slightly smaller than normal (Fig. 8). At higher dosages (0.01 mg./ml.), the spindles and asters have disappeared by three minutes after the beginning of treatment (Fig. 5), although the chromosomes remain in essentially the normal first maturation metaphase configuration. By 5 minutes after the beginning of treatment, what appears to be a faint "membrane" begins to appear around the group of chromosomes (Fig. 6), which have now begun to take on a somewhat vesicular appearance, but which still usually remain oriented in a ring of 9 around the periphery of the former spindle area. This same process of "membrane" formation begins about a minute later in eggs treated with lower doses of podophyllin. The containment of the chromosomes within a common membrane (Fig. 7) remains more or less unchanged for a period of a little less than half an hour, after which the chromosome vesicles are released, to lie loose in the cytoplasm (Fig. 10). Cornman and Cornman (1951) described a somewhat similar series of events in 372 CATHERINE HENLEY AX I) I). P. COSTELLO PLATE II Illustrations in Plate 11 arc of ova from the same experiment, treated with 0.01 nig./nil. podophyllin, beginning five minutes after insemination. Magnification: 1600 X. FIGURE 3. One aster (the inner one) has completely disappeared, and part of the first maturation spindle (seen in side view) is likewise no longer visible, in this egg fixed 90 seconds after the initiation of treatment. The large round black body is the nucleolus. Only 8 of the haploid complement of 9 chromosomes could be definitely counted. FIGURE 4. In this egg, fixed three minutes after the beginning of treatment, both asters have entirely disappeared, and the maturation spindle (side view) is only faintly visible. The fertilization membrane is present at the surface of the egg. (The wrinklings of this membrane, described in the text as typical results of podophyllin treatment, are not evident in this fixed material.) It appeared that only 8 of the 9 chromosomes were present, hut it is likely that one of the larger masses of chromatin actually represents two chromosomes. FIGURE 5. All 9 chromosomes are easily countable, in this polar view of the characteristic first maturation metaphase ring configuration. Both asters and the spindle have completely disappeared, and in the preparation there is only a slight staining at the center of the ring to indicate where the spindle substance was formerly located (not indicated in the drawing). Sample fixed three minutes after the beginning of treatment. FIGURE 6. In this side view of a first maturation metaphase, the 9 maturation chromosomes are countable, and there is a suggestion (at top) of "membrane" formation. Sample fixed 5 minutes after the beginning of treatment. FIGURE 7. "Membrane" formation is now completed and the 9 egg maturation chromosomes are entirely enclosed therein, as is the nucleolus (the lighter ring structure). Sample fixed 31 minutes after the beginning of treatment; at this stage, the control eggs were at the metaphase of the first cleavage. Note the similarity in diameter of the chromosome group in this treated egg to that of the normal first maturation metaphase ( Fig. 2). EFFECTS OF PODOPHYLI.IX 373 10 PLATE III 11 The eggs shown in this plate are from four different experiments, and were treated with varying concentrations of podophyllin (noted for each case) ; all treatments were begun five minutes after insemination. Magnification: 1600 X. FIGURE 8. There is marked reduction in the diameter of the asters and in the length (but, apparently, not of the diameter) of the maturation spindle, in this side view in an egg from a sample fixed three minutes after the beginning of treatment with 0.0055 mg./ml. podophyllin. The nucleolus is visible near the lower aster. FIGURE 9. Side view of a first maturation metaphase. The spindle is still clearly demarcated but the asters have entirely disappeared. Sample fixed three minutes after the initiation of treatment with 0.01 mg./ml. podophyllin (from the same experiment as the eggs shown in Plate II). FIGURE 10. The egg maturation chromosomes are now lying free in the egg cytoplasm, with no vestige of asters or spindle. The remnant of the "membrane" which formerly enclosed them (see Figure 7) is lying loose in the cytoplasm nearby. Only 8 of the 9 chromosomes could be counted with certainty. Sample fixed 21 minutes after the beginning of treatment with 0.1 mg./ml. podophyllin. FIGURE 11. In this egg from a sample fixed 115 minutes after the initiation of treatment with 0.0055 mg./ml. podophyllin, the egg chromosomes have become segregated in a large exovate, simulating a polar body (although very much larger than a normal polar body). There has been some fragmentation and, perhaps, re-consolidation of the chromatin, judging from the variation in size of the chromatin bodies. ->74 CATHERINE HENLEY AND D. P. COSTELLO living wisterias eggs treated with poclophyllin at the fir.st maturation division. This process of release occurs first for the egg chromosomes, but somewhat later (ap- proximately an hour after the beginning of treatment with 0.005 mg./ml.) it is also evident in the sperm chromosomes which have also become surrounded by a membrane. (See, also, Cornman and Cornman, 1951.) The two groups of chromo- some vesicles often eventually come to be intermingled and indistinguishable from one another. The exact sequence of events here appears to depend upon the entrance point of the sperm, with reference to the maturation figure, inasmuch as the two groups of chromosomes sometimes remain discrete. The empty "mem- branes" remain visible for a brief period (Fig. 10) but eventually disappear. Occasionally, there is some fusion of the egg chromosome vesicles with one another, so that fewer but larger vesicles are visible (Fig. 4). Usually, however, nine (the haploid number for Chaetopterus) can easily be counted. Cornman (1949) has also reported this apparent fusion of chromosome vesicles in living Asterias eggs which had been treated with podophyllin. The disappearance of the asters and spindles is so complete that there remains no evidence of these structures, except for a slightly more homogeneous staining of the general area they formerly occupied. (By the methods used, there is almost always some staining of the egg cytoplasm with haematoxylin, although this is not sufficiently great to interfere with observations.) The general sequence of events, as described above, occurs after almost all except the low doses of podophyllin (0.00005-0.00001 mg./ml.), although the tempo is noticeably faster in eggs treated with the higher concentrations. There is no further visible change in the eggs, and no further development occurs at doses between 0.01 mg./ml. and 0.0025 mg./ml. There was no evidence of any con- spicuous cyclic growth, disappearance and reappearance of chromosome vesicles, such as were described by Cornman and Cornman (1951) for podophyllin-treated echinodenn eggs. There was no recovery of treated eggs in our experiments, in contrast to the results reported by Inoue (1952) for colchicine-treated Chaetopterus ova. The most striking feature of these results is the remarkable rapidity with which effects are evident, as soon as 60 seconds after the initiation of treatment. This must mean that podophyllin penetrates the Chaetopterus egg very rapidly indeed. Swann and Mitchison (1953) found a comparable rapidity of effect of colchicine in causing the achromatic figure to disappear in treated Psammechinus eggs, as did Inoue (1952) for colchicine-treated Chaetopterus eggs. The source of the "membrane" which comes to surround the chromosomes is an interesting problem ; we are inclined to suspect that it may be derived from the spindle substance, but there is no real evidence for or against this theory except for the suggestion afforded by Figure 6. Gaulden and Carlson (1951) described the formation of a hyaline globule from the spindle substance of colchicine-treated grasshopper neuroblasts, and Kobayashi (1962) reported the complete disappear- ance of the spindle in demecolcine-treated Mespilia eggs, leaving a hyaline zone, which stained poorly with haematoxylin, around the nucleus. In Kobayashi's experiments, there was recovery from the effects of the mitotic poison, and during the early phases of this recovery process, the hyaline material was associated with the reconstituted spindle, spread over the fibrous structures of the mitotic apparatus. EFFECTS OF PODOPHYLLIN 375 At each of the first three or four divisions after recovery, the hyaline material was distributed to each of the daughter cells, after which it disappeared. It is interesting that although there is a pronounced reduction in the length of the maturation spindle in podophyllin-treated eggs (compare Figures 1 and 8, for example), the diameter is apparently not affected (compare Figures 2 and 5). (See, also, Inoue, 1952.) Furthermore, the chromosomes usually retain the normal maturation metaphase configuration. This affords confirmation for the idea that the spindle fihers are oriented longitudinally (Inoue, 1952, 1953). As shown in Figures 3 and 4, the asters were almost invariably the first structures affected by podophyllin, the spindle persisting for as much as one and one-half to two minutes longer. It was observed, also, that the first aster to be affected was usually (but not always — see Figure 3) the one which was nearest the periphery of the egg. It seems reasonable to believe that this is because the antimitotic agent would penetrate through the egg surface and first encounter the more peripheral of the two maturation asters. A similar susceptibility of Arbacia egg asters to the effects of another antimitotic agent, colchicine, was reported by Nebel and Ruttle (1938) and by Beams and Evans (1940). Sauaia and Mazia (1961) have demonstrated that in isolated sea urchin egg spindles, previously treated with colcemide (which is very close in chemical structure to colchicine), the asters are likewise the first components of the achromatic figure to be affected. Colcemide applied to the spindles after isolation resulted in no changes, which is not surprising. Beams and Evans (1940) found that the spindle was also destroyed by colchicine, but somewhat later than the asters. They attribute the destruction of asters and spindles to a solation of the cytoplasm, which causes destruction of the spindle, leaving the chromosomes relatively unaffected. In addition to the rapidity of the effects of podophyllin, another very striking feature is the completeness of the effects, the chromosomes and nucleoli alone remaining more or less unaffected (except for the somewhat vesicular appearance the chromosomes assume). There is no remaining trace of the asters, not even as the "lakes" described by Beams and Evans (1940) as resulting from colchicine- treatment of Arbacia eggs. The only evidence of the spindle which persists, as noted above, is a somewhat more homogeneous staining of the egg in the region formerly occupied by this structure. We found no evidence of any fibrous struc- ture in this area ; however, its boundary, which we have described as resembling a membrane, is very sharp. It seems quite likely that the association of the chromo- somes and the spindle fibers or their remnants may remain in effect somewhat longer than would be apparent at first glance, since the chromosomes retain their character- istic ring-shaped metaphase arrangement for some time after the spindle has visibly disappeared (Fig. 5). The effects of low doses of podophyllin (0.00005 mg./ ml. -0.00001 mg./ml.) After treatments with concentrations of 0.00005 mg./ml., for periods of 40 to 50 minutes, there was some recovery in treated eggs, although even with these very low doses, the resulting trochophores were usually atypical, with marked ciliary defects and other abnormalities. There was some evidence, also, of fusion of several embryos to form a single "giant." By 24 minutes after the beginning of treatment, there was reduction in the size of the first cleavage asters in some 376 CATHERINE HENLEY AND I). I'. COSTELLO I'l.ATK IV EFFECTS OF PODOPHYLLIN 377 eggs, while in others, these structures remained essentially normal. At 47-57 minutes there were some multipolar cleavage figures, and in other eggs, two separate complex metaphases (perhaps male and female in origin?) were apparent. Again, the asters and spindles were fairly normal in a number of ova, but reduced in size in others (see, also, Gaulden and Carlson, 1951, and Kobayashi, 1962). Sixty- eight minutes after the beginning of treatment, much variation was apparent : some eggs had a single large complex metaphase, some had several multipolar figures, some had from one to six interphase nuclei. Usually it appeared that no cytokinesis whatever had taken place, or that it had been partially suppressed, at least. By 113 minutes after the beginning of treatment, the cytological picture was much like that observed at 68 minutes, except that more interphase nuclei (up to 8 in one egg) were observed. Occasionally, cytokinesis had taken place — often resulting in an unbalanced distribution of chromatin, so that one of the daughter "cells" had none. Treatment with a still lower concentration of podophyllin (0.00001 mg./ml.) ( Figs. 12-17) resulted in the appearance of a number of multipolar cleavage figures (Fig. 12) at 27 minutes after the beginning of treatment, and in occasional reduc- tion in size of the asters and spindles (Fig. 13) ; otherwise, the eggs appeared essentially normal. At 52 minutes after the beginning of treatment, some relatively normal two- (Fig. 14) and four-cell stages, with both polar bodies, were found; other eggs had multiple or multipolar figures in non-divided cytoplasm (Figs. 15, All the eggs shown in this plate are from the same experiment, treated for 60 minutes with a low concentration of podophyllin (0.00001 mg./ml.), beginning five minutes after insemination. Magnification: 650 X. FIGURE 12. Tripolar figure, from an egg-sample fixed 27 minutes after the beginning of treatment. The control eggs fixed at this time were in the prophase to metaphase of the first cleavage, so it seems likely that the mitotic figure shown here is likewise a cleavage spindle, since the development of experimental and control eggs in this series was quite closely synchron- ous at this point. The chromatin is scanty and thread-like. One polar body is visible at the top of the figure, overlying it on the surface of the egg. FIGURE 13. In this egg, fixed at the same time as the one shown in Figure 12, the aster^ are smaller in diameter than usual, but otherwise the figure is similar to the cleavage metaphases in the control eggs. Both polar bodies are present. FIGURE 14. Two-cell stage, at the anaphase of the second cleavage. The asters are nut visible at the ends of the spindle ; this second cleavage was synchronous with the same division in the control eggs. One polar body is visible on the larger CD blastomere, below. Fixed 52 minutes after the initiation of treatment. FIGURE 15. There are two entirely separate anaphase figures in this uncleaved egg fixed at the same time as that shown in Figure 14. No cytokinesis has taken place, but karyokine*i> is proceeding, although the two discrete figures are somewhat smaller than normal (compare with Figure 14). Both polar bodies are present (at the top). FIGURE 16. This complex anaphase configuration is from the same egg-sample as those ova shown in Figures 14 and 15 (fixed 52 minutes after the beginning of treatment). Control samples fixed at this time were proceeding from the two- to the four-cell stage. At least three spindles can be discerned, their relationship to one another being somewhat obscure. One polar body is present beneath the complex array of spindles, at the right. FIGURE 17. In this egg, fixed 115 minutes after the beginning of treatment (when the controls were in advanced cleavage stages with ±16 cells), abnormal cytokineses has occurred, with six smaller cells in association with one larger one. Two interphase nuclei can be seen in two of the small cells, and most of the mitotic figures appear to be abnormal, including one or more multipolar spindles in the larger cytoplasmic mass. Both polar bodies are present on the periphery of the egg. The larger of the associated cells is undergoing an atypical anaphase. CATHERINE HENLEY AND D. P. COSTELLO 16). By 115 minutes after the initiation of treatment, there were occasional normal advanced cleavage stages, and other ova with partially or wholly undivided cyto- plasm. Quite often, suppression of cytokinesis had occurred only to a degree, although karyokinesis in such cases was usually highly abnormal (Fig. 17). A few multipolar cleavage figures were also observed. The further development of all these eggs was variable ; some fairly normal trochophores resulted, while others had marked ciliary defects and there were many dead larvae. It is important to emphasize here the fact that although there was considerable recovery in the eggs treated with these very low doses, subsequent development was not entirely normal (Figs. 12-17) and the resulting trochophores were almost in- variably atypical to at least some degree. Thus, there must be quite profound effects on the egg which are not necessarily apparent at its early stages of devel- opment, except in the reduction in size of the spindles and asters. The multipolar figures observed in cleavage stages of the treated eggs are very similar to those which occur in Chaetopterus ova after a number of other experi- mental treatments, including x-rays (Henley and Costello, 1957) and low tempera- ture (Henley, 1959). They have also been described in the eggs of other forms after a wide variety of experimental treatments, and in rat ascites tumor cells treated with podophyllin (Makino and Tanaka, 1953). Similarly, the suppression of cytokinesis, but not of karyokinesis, also occurs after such treatments. The fact that the polar bodies usually appeared in a more or less normal fashion (temporally and spatially) after low doses of podophyllin suggests that since the achromatic figures were not usually affected as early after the beginning of treat- ment with podophyllin at low concentrations as at higher concentrations, polar body formation could proceed in an essentially normal manner. It is of interest that even in eggs treated with higher concentrations of podophyllin (see following section) there was a simulation of polar body formation (Fig. 11) ; such pseudo- polar bodies were often resorbed, however. Shape changes in podophyllin-treated cc/i/s; effects on the c{/// membranes After treatment with even moderate to high doses of podophyllin. Ch <-ggs exhibited remarkable simulation of the normal shape changes which charac- terize the development of this form, even though maturation and cleavage spindles and asters were completely inhibited. With medium concentrations of podophyllin (0.0055 mg./ml., for example), a semblance of polar body formation took place, in which the entire egg nuclear complex was segregated into a large exovate (con- siderably larger than a normal polar body) (Fig. 11); this exovate usually remained attached to the egg cytoplasm by a stalk. Such "pseudo-polar body formation" occurred at the same time after insemination as the formation of a normal first polar body in the controls. The pseudo-polar body was often resorbed into the egg cytoplasm within about 10 minutes after its initial appearance, and there was usually (but not always) no repetition of the event at the time of second polar body formation in the controls. In occasional ova. exovates occurred in which no chromatin was present, as ascertained by examination of cytological preparations fixed at the time of exovate formation. It is possible that such exovates represent a rupture where the egg cortex and membrane were interrupted at the point of EFFECTS OF PODOPHYLLIX 379 ovarian attachment. I none (1952) has described similar pseudo-polar body for- mation in colchicine-treated Chaetopterus eggs. Concurrent in time with the characteristic "pear" and polar lobe stages of the control eggs, there were very normal-appearing similar stages in the experimental eggs treated with moderate dosages of podophyllin. The polar lobes were subse- quently resorbed by the eggs, approximately 20 minutes after they had appeared, and no further development occurred. Cytological examination of such treated eggs showed that in many cases, at least, the shape changes could be correlated in time with the release of the chromosome vesicles from the membrane. In some instances, abortive cleavages were initiated in the experimental eggs, but except after low doses, such furrows were resorbed within 10-15 minutes. Similar shape changes in podophyllotoxin-treated eggs, simulating those of normal control eggs, were also observed. Particularly after treatment with the higher doses of podophyllin (0.1-0.005 mg./ml.) striking effects on the eggs' membranes were observed. Following a dose of 0.1 mg./ml., for example, the membranes wrinkled around the entire peripheries of the eggs, at 57 minutes after the beginning of treatment. Seven minutes later, this wrinkling became very pronounced, and by 157 minutes after the beginning of treatment with this dosage, the membranes became symmetrically exaggerated in their elevation from the surfaces of the eggs. Subsequently, the symmetrical exaggeration often became asymmetrical. The general course of events in this process of exaggerated membrane elevation was strikingly reminiscent of that observed in Chaetopterus eggs after cold treatment (Henley, 1959), except that the ova usually did not become denuded of their membranes after podophyllin treatment, as was often the case after cold treatment. There is a normal series of wrinklings in the Chaetopterus egg membrane after fertilization (Pasteels, 1950), but the exaggerated crenations observed after podo- phyllin treatment are very much more conspicuous. There is no demonstrable relation between the integrity of the mitotic apparatus and the occurrence of shape changes in the podophyllin-treated Chaetopterus egg. In the normal egg of this annelid, the pear-shaped stage coincides with early meta- phase of the first cleavage division and the polar lobe stage lasts from approxi- mately the metaphase to anaphase of the same division. Similarly, the appearance of pseudo-polar bodies without chromatin is apparently entirely divorced from the presence of the functional maturation spindle and chromosomes in the egg; this is shown by the fact that in eggs fixed at the time when pseudo-polar bodies were present, the asters and spindles had disappeared (probably about 8-10 minutes earlier judging from the evidence obtained concerning the rapidity of effect of higher concentrations of podophyllin). The ultimate resorption of pseudo-polar bodies and of polar lobes may mean that although the initiation of these morpho- logical changes can occur in the absence of the spindle and asters (but, it should be noted, in the continuing presence of the chromosomes), it cannot continue to the point where polar bodies are actually given off as a consequence of cytokinesis. The observed exaggerated membrane elevation may result from a separate effect of podophyllin on the cell surface, as distinguished from the effects on asters and spindles. 380 CATI1KK1NK HENLEY AX1) D. P. COSTKI.LO An attempt to reverse the antimitotic action of podophyllin The possible antagonistic action of reduced and/or oxidized glutathione was studied, in an attempt to reverse the antimitotic action of podophyllin (see Kawa- inura, 1960, and Zimmerman, 1960). Chaetopterus eggs were subjected to treat- ment with concentrations of podophyllin (0.012-0.00032 mg./ml.) known to be effective in destroying astral rays ; they were then treated, two to five minutes later, with various concentrations of oxidized (0.25-0.025 mg./ml.) or reduced (0.25-0.05 mg./ml.) glutathione. In another series of experiments, the oxidized or reduced glutathione was added to the eggs concurrently with the podophyllin. In no case (regardless of concentration) was there a clear-cut reversal or prevention of injury to the amphiastral system. In some cases (high dosage — -0.25 mg./ml.) the effects of oxidized or reduced glutathione plus podophyllin in inhibiting cleavage were greater than those after treatment with podophyllin alone. Such high doses of glutathione, administered to eggs in the absence of podophyllin, were injurious, also. There is thus no indication that glutathione, either in reduced or oxidized state, can reverse the antimitotic effects of podophyllin, under the conditions of these experiments. The effects of podophyllotoxin In general, the effects of podophyllotoxin are entirely comparable to those observed after podophyllin treatment, although the requisite concentrations of podophyllotoxin for a given effect are lower for the active principle than for the crude resin (Table I). A range of concentrations, from 0.01 mg./ml. to 0.00005 mg./ml., inclusive, was tested. In a typical experiment, utilizing podophyllotoxin at a concentration of 0.001 mg./ml., for a duration of approximately 83 minutes, the general cytological picture was like that seen in eggs treated with much higher dosages of podophyllin (0.01 mg./ml., for example) ; the podophyllotoxin-treated ova showed discrete chromosome vesicles within a membrane, occasional appear- ance of a large pseudo-polar body, and eventual release of t'.e chromosome vesicles, so that they wrere lying loose in the egg cytoplasm, by 55 minutes after the begin- ning of treatment. With a lower concentration of podophyllotoxin (0.00025 mg./ml.), a normal first maturation metaphase was observed in treated eggs as late as 7 minutes after the initiation of treatment, and a normal-appearing second maturation metaphase also appeared at the usual time. By 40 minutes after the beginning of treatment, the first cleavage spindles had disappeared; here it was observed that the spindles were reduced in length and perhaps in diameter. By 65 minutes after the beginning of treatment, only three of 30-40 eggs on a given slide had cleaved and were at a normal-appearing two-cell stage. In the others, no spindles, asters or chromosomes could be seen. Polar bodies were sometimes present on the treated ova, sometimes absent. By 115 minutes after the beginning of treatment, at least one cleavage had occurred in nearly every egg, with most developing ova being at 4-, 6- or S-cell stages. There was a pronounced delay in cleavage of the treated eggs, as compared with the controls. After a dose of 0.0005 mg./ml. podophyllotoxin, for a period of approximately one hour, some of the first maturation metaphase asters, spindles and chromo- somes looked normal, but in others, there was a range of abnormalities, from a EFFECTS OF PODOPHYLLIX 381 slight decrease in size of asters and spindles to complete disappearance of these structures. By 27 minutes after the beginning of treament loose chromosome vesicles were present in the cytoplasm of some eggs, and by 53 minutes after the initiation of treatment, this condition held for all the eggs. No further develop- ment occurred. The less concentrated treatment of 0.00005 mg./ml. resulted, at four minutes after the beginning of treatment, in a reduction in size of maturation spindles and asters in certain ova, while in others, these were essentially normal. By the time of the second maturation metaphase there was a slight reduction in size of the TABLE I The comparative effects of podophyllin and of podophyllotoxin on the fertilized eggs of Chaetopterus Cone, and Druy Effect 0.00001-0.00005 mg./ml. podophyllin 0.00005 mg./ml. podophyllotoxin 0.0005 mg./ml. podophyllin 0.0005 mg./ml. podophyllotoxin 0.001 nig. /ml. podophyllin 0.001 mg./ml. podophyllotoxin 0.01 mg./ml. podophyllin 0.01 mg./ml. podophyllotoxin Multipolar iigures; abnormal trocho- phores. Karyokinesis without cyto- kinesis Reduction in size of asters and spindles; delayed cleavage : 4-cells in exp. ; advanced clvgs. in controls Reduction in size of asters and spindles; no polar bodies given off ; Very abnormal cleavages at first; some recovery, but not beyond 2-cell stage One or two polar bodies present @ 35 abt* ; no fusion of pronuclei. Mostly dead by 39 mins. after end of treat- ment Chromosomes in "membrane"; no fur- ther development Chromosomes within "membrane" ; no further develop. Chromosomes within "membrane"; no further develop. All treatments begun 5 minutes after insemination. * "Abt" = minutes after the beginning of treatment. spindles and asters. Subsequently, there was considerable delay in development, as compared with the controls, so that in samples fixed 115 minutes after the beginning of treatment, the experimental eggs were in the 4-cell stage, while the controls were in advanced stages of cleavage (about 16 cells). The resulting ex- perimental trochophores were abnormal, with the characteristic atypical features described above as resulting from podophyllin treatment. Thus, at this low con- centration, podophyllotoxin has only slight effects on the spindle mechanism, includ- ing a decrease in size ; a delay in cleavage occurs, and subsequent development is atypical. CATHERINE HENLEY AND D. P. COSTELLO The effects of increasing duration of exposure, at the same dosage Ici'd, on Chae- loptents ci/iis treated with podophyllotoxin To test the effects of increasing tlie duration of exposure of Chaetopterus eggs to podophyllotoxin at a single given concentration, eggs were exposed by the usual techniques to a dosage of 0.0005 mg./ml. ; as noted in the preceding section, this treatment could be expected to produce a predictable and reasonably uniform set of results (containment of the chromosomes in a membrane, with subsequent re- lease) when treatment was continued for 60 minutes. Tn the present series, treat- ments ranged from 5 to 20 minutes in length. After the 5-minute treatment (which, like the 10-, 15- and 20-minnte treat- ments, was followed bv two washes with large amounts of freshly filtered sea water, to remove the mitotic poison), there was a good deal of recovery, but the eggs were not entirely normal. At the earlier stages after the initiation of treat- ment, reduction in the size of maturation spindles was noted, as well as some incidence of multipolar figures in the cleavage stages. The 10-minute treatment resulted in considerable variation in effects; the>e ranged from the presence of thread-like masses of chromatin (sometimes with a suggestion, only, of asters nearby in the eggs fixed 55 minutes after the initiation of treatment, at a stage when the controls were in ±4-cell stages, to fairly normal-looking ±12-cell stages at 175 minutes after the beginning of treatment. In general, it can be said that this treatment resulted in retardation of development and in more marked abnormalities than the 5-minute treatment, but some degree of recovery did occur. A 1 5-minute treatment resulted in surface excrescences, of various sizes, simu- lating cleavage blastomeres, in samples fixed 100 minutes after the beginning of treatment ; in some of these pseudo-blastomeres, small abnormal spindles and/or asters were present, in others they were absent. Similar surface excrescences have been reported by Onnsbee ct al. (1947) for podophyllin-treated mouse tumor cells in tissue culture, by Cornman and Cornman (1951) for podophyllin-treated echino- derm eggs, and by Makino and Cornman (1953) for mouse tissues treated in vitro with podophyllotoxin. Thus only a very slight degree of recovery occurred after a treatment of even this short duration, and similar results were observed in eggs treated for 20 minutes. In general, the effects of these shorter exposures to a moderate dilution of podophyllotoxin were comparable to those observed after treatment with lower doses for longer periods. Five-minute treatments with various concentrations of podophyllotoxin Three different concentrations of podophyllotoxin (0.001, 0.0003 and 0.0005 mg./ml.) were tested for their effects on Chaetopterus eggs after treatments only 5 minutes in duration. At the highest concentration, the cytological picture charac- teristic of podophyllin and podophyllotoxin treatments was evident at least as soon as 15 minutes after the onset of treatment (10 minutes after the end of treatment) ; asters and spindles had disappeared and the chromosomes had already taken on a somewhat vesiculated appearance. (The first samples were not fixed until 15 minutes after the beginning of treatment, so we are not able to state how much earlier the characteristic effects were apparent. However, this concentration of fiodophyllotoxin, jn tests over longer periods, resulted in the usual cytological EFFECTS OF PODOPHYLLIN picture very soon after the initiation of treatment.) No further development occurred. After treatment with 0.0003 mg./ml., maturation was completed normally, and a semblance of cleavage occurred, although it was slightly delayed, as compared with the controls. Other eggs from the same batch, which were left in the experimental solution for as long as 20 minutes as a second type of control (in addition to eggs in plain sea water), showed no signs of recovery, and the cytological effects of podophyllin and podophyllotoxin noted above were seen. With an intermediate concentration (0.0005 mg./ml.) the first indication of an effect was in samples fixed 36 minutes after the beginning of treatment, in the first cleavage, where the spindles and asters were smaller than normal. In samples fixed 60 minutes after the beginning of treatment, there were many uncleaved eggs, some with two fairly normal (but small) spindles, others with a single multipolar spindle. None was observed to have progressed beyond the two-cell stage. However, between that time and 175 minutes after the beginning of treatment, when another sample was fixed, some degree of recovery appeared to have taken place, because there were a good many fairly normal advanced cleavage stages ; they developed into swimming forms which exhibited various types of abnormality in ciliation, surface blebs, etc., as is characteristic of trochophores developing from podophyllin-treated eggs. Ova which were left in the podophyllotoxin solution for 175 minutes did not develop farther than the usual stage found after relatively pro- longed treatment with these antimitotic agents. It appears, then, that eggs can recover to some extent from a 5-minute treatment with this intermediate concen- tration of podophyllotoxin, but they are not entirely normal. Treatment of Chaetopterus eggs with niitmnycin C, N-dichloroacetyl DL serine and quercetin In another series of experiments, the possible antimitotic effects of several other agents were tested on fertilized Chaetopterus eggs ; these agents included mitomycin C,3 N-dichloroacetyl DL serine (sodium) and quercetin, the last-named substance being a pigment component of crude podophyllin resin which has been reported by Cornman and Cornman (1951) to retard division of echinoderm eggs, but to have no effect in destroying the achromatic figure. The general methods followed in these experiments were the same as those described above for podophyllin and podophyllotoxin treatments. Mitomycin C was shown by Merz (1961) to induce chromosome breaks and inhibition of mitosis in root tip chromosomes of Vlc'ia faba, and by Matsumoto and Lark (1963) to block DNA synthesis in bacteria. In concentrations of 0.002 mg./ml. or 0.0001 mg./ml. this substance did not affect the division of Chaetopterus ova, nor their subsequent development, even when the treatment was continued for an hour or longer. N-dichloroacetyl DL serine, reported by Levi et al. (1960) to cause regression of sarcoma 37 in mice, had no effects, either cytological or developmental, at any of the concentrations tested (1-0.005 mg./ml.), except that there were some ciliary defects in a few of the experimental larvae observed the day following treatment. Quercetin, even in a concentration as high as 1 mg./ml., resulted in essentially : \Ye are indebted to Dr. Joel Flaks for his assistance in these experiments. 384 CATHERINE HENLEY A XI) I). P. COSTELLO normal trochophores and in no observed cytological abnormalities in samples fixed 5. 32 and 55 minutes, and three hours after the beginning of treatment. There was, however, a slight retardation of cleavage-. DISCUSSION There are, as Biesele ( 1('5S i has pointed out, a number of different places in which interference with one process or another may lead to the production of mitotic abnormalities or complete cessation of cell division. However, the oppor- tunities for obtaining abnormalities as a result of an interference with mitosis in eggs or cleavage blastomeres may be considerably greater than in relatively un- differentiated tissue culture cells. For normal embryonic development, there has to be a correlation between mitotic events and events such as ooplasmic segregation which lead to differentia- tion. Within the mitotic events, there is, of course, a coordinated relationship between karyokinesis (chromosomal activities), cytokinesis, and normal centriole replication, plus the axial relations of spindle orientations which must be coor- dinated with the segregation of cytoplasmic constituents destined to become in- corporated into particular cleavage blastomeres. If some mitotic poisons affect the chromosome replication or behavior, others the furrowing, still others centriole replication, and still others cell respiration and metabolism, the possibility for the abnormal distribution of cytoplasmic stuffs becomes so great that the chance of obtaining normal development is practically non-existent. Cytological c I) eels <>j podophyllin and podophyllotoxin on oilier materials The following table is of interest as a comparison of the effective doses of podophyllin and podophyllotoxin, respectively, required to block cleavage in fertilized marine eggs, as reported in this paper and by Cornman and Cornman (1951): Chaetopterus Arbacla' Echlnarachnlus** Podophyllin: 10 ni£./L.* Podophyllotoxin: 1.0 mg./L. 0.6 mg./L. 0.5-1 mir./L. 0.02-0.01 mg./L * For purposes of comparison, our dosages have here been expressed as nig. /I .. rather than as nig. /ml., as \ve have done elsewhere in this paper. ** Data of Cornman and Cornman (1051). It is apparent that the Chaetopterus egg is somewhat more resistant to the effects of podophyllin than is the egg of Arbacla, and very much more resistant than the egg of Echinaracliniiis (which is notoriously sensitive to any adverse condition, such as a slight increase in temperature). Similarly, a podophyllotoxin concentra- tion of 1.0 mg./L. is required to block Chaetopterus eggs, while slightly more than half that amount suffices to block .-Irbacta eggs, and even smaller amounts block Echinarachnius eggs. This illustrates again the greater efficacy of the active principle of podophyllin as compared with the crude resin. EFFECTS OF PODOPHYLLIX MacCardle (1951) found that in vivo treatment of mouse sarcoma 37 with N-acetyliodocolchinol methyl ether produced anastral spindles. In living cells (teased preparations), the spindle fibers were not visible, but in Heidenhain iron haematoxylin-stained sections, fragments of fibers were present. There were many multipolar mitoses. Low doses of podophyllin (20 micrograms) produced effects on sarcoma 37 cells similar to those found after acetyliodocolchinol treatment. After microincineration of cells treated with either agent, the spindle area was marked by the presence of large masses of white ash of calcium or magnesium ; these were much larger than similar masses present in untreated microincinerated cells. Biesele (1958) points out that this white ash might represent divalent ions which had united with a lipoidal constituent of the spindle liberated by action of the mitotic poisons. He also suggests that if the white ash originated in calcium or magnesium from the chromosomes, this might indicate a damaging effect of the poisons on the chromosomes themselves. A comparison of the effects of podophyllin it'iih those of colchicine* Inoue (1952), using polarization optics, has studied the effects of colchicine, in concentrations from 1 X 1O5 to 1 X 10~2 M, on the first maturation spindle of the Chactoptcrus egg. He observed that the spindles in such treated eggs began to disappear within a very few moments after the initiation of treatment, and his Plate II shows that this effect is first apparent in a diminution in size of the asters at 3 minutes 15 seconds after the beginning of treatment with 5 X 10~4 M (0.2 gm./L.) colchicine. With higher concentrations, the effects are apparent sooner and are complete, with disappearance of the spindle, by 4 minutes 5 seconds after the beginning of treatment with 5 X 10~3 M. In general, there was a direct relation between the concentration of colchicine used and the time required for complete disappearance of the spindle. Soon after the beginning of treatment with colchicine in Inoue's experiments, the characteristic birefringence of the astral rays and continuous fibers of the spindles began to decrease, while the spindle length shortened. As this process of spindle shortening continued, loss of birefringence became more pronounced. The length of the spindles at the time when birefringence disappeared seemed to depend upon the concentration of colchicine used, but in all cases he noted, as did we in the present experiments with podophyllin, that the chromosomes remained oriented on the equatorial plate until the spindle had completely disappeared. He observed that the chromosomes then began to scatter in the egg cytoplasm ; this is in contrast to our findings in podophyllin-treated eggs, and he does not describe any process 4An important contribution on the mechanism of colchicine inhibition of mitosis by E. \Y. Taylor (/. Cell Biol., 25: No. 1, Part II, 145-160; 1965) appeared while this paper was in press. H3-colchicine was shown to have no direct effects on the duration of the cell cycle (of strain K. P.. cultured human cells) or on macromolecular (DNA, RNA, and protein) synthesis, at a concentration of colchicine which completely inhibited mitosis. An exposure of 6 to 8 hours at 10~7 717 was sufficient to block essentially all the cells in metaphase, thus indicating that colchicine is bouii'l to the majority of interphase cells. The data are in agree- ment with the idea of a mechanism involving reversible binding of colchicine to a set of cellular sites, and suggest that if a critical fraction (3% to 5%) of the sites is complexed, the cell is unable to form a functional mitotic spindle. Presumably, a higher concentration of colchicine would be required to disrupt the mitotic spindle than to prevent its assembly. 386 CATHERINE HENLEY AXI) D. P. COSTELLO of containment of the chromosomes within a membrane, such as we found. As the chromosomes began to move inward from the egg periphery in his studies, a characteristic bulge was observed at the periphery of the egg above them; he interprets this as an abortive polar body, presumably comparable to the similar phenomenon described above for podophyllin-treated eggs. With low concentra- tions of colchicine. Inoue found that the spindle contracted very slowly ; the polar regions began to "disintegrate" and as many as seven parallel "spindles" (his quotation marks) were present, each with a pair of chromosomes at its center. We found nothing comparable to this effect in our studies. The effects of colchicine at the concentrations tested were apparently reversible in Inoue's studies. Eggs treated for 5 and 10 minutes with 1O4 M mitotic poison were washed in three changes of fresh sea water ; at this time the spindle had disappeared and pseudo-polar body formation had taken place. This condition persisted for more than an hour, at which time the polar bulge receded and a small spindle and asters began to appear. In about 3.*> hours this spindle had grown to approximately the normal size, and the egg could be successfully inseminated with ensuing polar body formation. Inoue (1952) interprets his data to indicate that the action of colchicine is to disorganize the orientation of the micelles in astral rays and spindle fibers, most probably by breaking down some chemical bond in or between the micelles and simultaneously causing some of the remaining micelles to contract (as well as breaking down some of the remaining linkages between them). He suggests that colchicine antagonizes the action of some ceil component which keeps the spindle substance polymerizing, and which maintains the spindle micelles in their extended form so that they cannot dissociate from one another. A comparison of the effective concentration of podophyllotoxin (ca. 1.0 mg./L.— equal to 2.4 :: 10 G M) with the effective dosages of colchicine, used on marine eggs by various authors (see Biesele, 1958, for references), indicates that the former is between 100 and 1000 times as effective in preventing cleavage. The Merck Index (1960) gives the empirical formula of colchicine as C22H25NOG, with a molecular weight of 399.43. Podophyllin is, of course, a mixture of substances, of which podophyllotoxin is the active antimitotic principle. The formula of podophyllotoxin is given as C22H22O|S. with a molecular weight of 414.4. There would, then, be relatively little osmolar difference (less than A%} between milligram/liter solutions of the two substances, colchicine and podo- phyllotoxin. Another study utilizing colchicine was that of Gaulden and Carlson (1951), who de.M-ribed the formation of a hyaline globule in colchicine-treated grasshopper neuroblasts. This globule was seen to arise either from the karyolymph of late prophase or from the spindle of metaphase or anaphase cells. When it originated from the spindle, the first sign of an effect was a reduction in the size of the spindle, to about half its initial size; the globule appeared at one side of the chromosome group and was not surrounded by a membrane. Gaulden and Carlson made three points in summarizing their findings : ( 1 ) The greater the concentration of colchi- cine, the greater its effects in dcstnning or interfering with development of the spindle. (This is in complete agreement with our findings for podophyllin-treated Chactoptcrns eggs.) (2) The more completely the spindle was developed at the EFFECTS OF PODOPHYLLIX time of its exposure to colchicine, the greater the concentration required to destroy it or prevent its further development. (In our experiments, the spindle was in most cases at the first maturation metaphase. so that we are unable to draw any meaningful comparisons on this point.) (3) A series of changes of orientation in the chromosomes was seen to he directly related to changes in the spindle structure. (We have already commented on this finding above — see section on "The sequence of early events in the cytological effects of podophyllin." ) Gaulden and Carlson (1951) suggest that colchicine does not destroy the spindle material in grasshopper neuroblasts, but merely alters its molecular configuration, so that it becomes a spherical mass with no mitotic function. The role of the achromatic figure in mitosis Hiramoto (1956) reported that in fertilized Clypeaster eggs at the dumbbell stage, the mitotic spindle and asters could be completely removed, using a micro- pipette, and furrowing would continue so that most eggs divided in two. If the spindle were removed in the anaphase or early telophase, division continued in some eggs, while in others the furrow receded. In some cases, the initiation of furrowing was seen after both spindle and asters had been removed as early as the metaphase. The cleavage plane was not affected by the removal of the spindle in Hiramoto's studies, although the speed of furrowing was somewhat slower than normal. He demonstrated, also, that the cleavage plane was unmodified when the position of the mitotic figure was displaced during anaphase or later, by removal of a part of the protoplasm ; thus, the cleavage plane is already fixed in the egg cortex. The effects of colchicine on Psainuiechinits eggs were used by Swann and Mitchison (1953) as a tool to test their hypothesis that the asters are only passive guides for the advancing cleavage furrow. They used the poison to suppress the spindles and asters at a time when the chromosomes had already separated, to see if cleavage would ensue ; it did so, even in cases where the asters and spindles had completely disappeared, if the cell had reached mid-anaphase when treatment was initiated. This is in agreement with Hiramoto's findings. Rappaport (1961) compressed EchinaracJinius eggs into a torus (doughnut) shape, and found that they then divided only in the spindle region, producing a binucleated horseshoe-shaped cell. The second division following this resulted in the isolation of two uniuucleated cells from the ends of the horseshoe. The bend of the horseshoe was binucleated, but within about five minutes after completion of the two "normal" divisions, a furrow appeared between the polar regions of the two asters in the binucleated cell. Thus, a furrow was completed in a region which had never been in close proximity to a spindle or to chromosomes but which was marked by the presence of the two asters. Rappaport suggests, then, that in normal cells, the position of the cleavage furrow may be determined by the "zone of confluence" of the asters. Kobayashi (1962) has described cases in demecolcine-treated multinucleated Mcsf-ilia eggs where furrows entered from the egg surface between the asters of neighboring achromatic figures ; he states that such cases were not frequent, but were usually encountered when the nuclei were crowded. Although they did not originate the idea of chemical evocation of cytodieresis, CATHERINE HEN1.KY AND D. P. COSTELLO Cornnian and Coriinian (lc'51) have attempted (p. 1479) to explain their results by the assumption that a furrow-organizer is released from the nuclei's at the end of prophase and distributed to the equatorial region by the achromatic h jure. This furrow-orgaui/cr then causes the furrow to form and to progress through the egg. When podophyllin incapacitates the achromatic figure, the furrow-organ i/.er reaches the cortex late, and in an irregular pattern, causing delayed, irregular furrowing. However, the Cornmans ( 11'51, p. 147(>) state that furrow activity maintains a relationship with the chromatin and not with the asters. We question this, since furrowing can occur between cytasters in enucleate fragments of marine eggs (Wilson, 1925). We question, also, whether the furrow-organizing substance is released from the nucleus of the mature egg or from the cleavage nuclei at each successive division. It seems more reasonable to assume that the furrow-organizing substance is a cytoplasmic component, possibly derived from the neutral-red- staining granules described by Kojima (1959). Kojima (1959) has reported that neutral-red-stainable granules appear in the cytoplasm of eggs of three species of Japanese sea urchins (Hemicentrotus, feinnopleurus and Mcspilia] ; at first these granules are uniformly dispersed in the cytoplasm of fertilized ova, but soon they gather around the mitotic figure and are distributed to the two cleavage blastomeres. Subsequently, they appear around the mitotic figures at subsequent cleavages ; no change in the number of granules was found. Similarly, in parthenogenetically activated eggs, the granules (at first dispersed in the cytoplasm) gathered around the monaster or cytaster, and in centrifuged eggs in which the granules were segregated, cleavage occurred only in the blastomere containing the granules. If unfertilized Teumoplcnrns or Mcspilia eggs were centrifuged into two halves, only the centrifugal half, which contains the granules, could develop further after fertilization. Kojima also treated fertilized eggs of these forms before and after vital staining, testing a number of mitotic inhibitors including colchicine and dinitrophenol ; he found that especially under the influence of DNP-inhibition, the appearance of the granules after vital staining was inhibited until the eggs were returned to sea water, the granules then gathered around the aster. The effects of colchicine were less clear-cut, there being only a reduction in the observed number of stained granules in such treated eggs ; these granules remained dispersed through the cytoplasm. It is important to note here that colchicine and DNP act as inhibitors of mitosis in two different fashions, colchicine being a spindle-destroyer and DNP being a respiratory poison; the significance of Kojima's findings on this point is thus questionable. Rebhun ( 1959) has described methylene-blue- and toluidine-blue-stainable granules in the Spisula egg, which move to the asters and subsequently migrate in a fashion which suggests the possibility that they are distributed by the mitotic apparatus. Zimmerman and Marsland (1960) studied Arbacla eggs which had been sub- jected after fertilization to centrifugation at high force (40.000-50,000 g) and high pressure (8000-12,000 pounds per square inch) for periods of two to five minutes. They demonstrated that such treated eggs could furrow long before the normal time, often irreversibly, as a consequence of the action of the combination of the two physical agents (if treatment was begun before prophase) or of centrifugation alone (if treatment was begun later). It was necessary that rupture of two groups of EFFECTS OF PODOPHYLLIN 389 structures have taken place before successful furrowing would occur: (1) the nucleus, and (2) the metachromatic beta granules. Zimmerman and Marsland postulate that the pressure acts by solating the gel structures of the cell, permitting them to break clown more readily and facilitating the separation and stratification of the cell's components. They suggest that the furrowing reaction is normally induced by the transport of materials, both nuclear and cytoplasmic in origin, to the two polar regions (spindle poles) of the cell cortex and, perhaps, the mitotic appa- ratus constitutes the transporting agency. The experimentally-induced reaction, on the other hand, seems to involve only the centripetal pole, and the transport is achieved through the medium of high centrifugal force. Whatever the source of the furrow-organizer, it is reasonable to assume that in animal cells, there is an accumulation of substance near or in the centrosomal regions, which is transported by the asters to the periphery of the ovum. This streaming would create a fountain movement, of the type described by Spek (1918) for the egg of Rhabditis, and by Conklin (1902, 1938) for the egg of Crepidula, with streaming from each aster to the cortical equatorial region (equator of the spindle axis) and then back toward the middle of the egg. The furrow as a constricting ring is initiated at the region of convergence of the fountain streaming. With the reduction or destruction of the asters by podophyllin (or by podophyllotoxin), the furrowing (cytokinesis) is inhibited or prevented. Destruction of the spindle pre- vents separation, etc., of daughter chromosomes (i.e., karyokinesis). There is nothing, in its molecular structure, to account for the much greater efficacy of podophyllin, as compared with colchicine, but this probably reflects our lack of knowledge of the structure of the mitotic spindle and the astral radiations. We incline toward the view that podophyllin has a direct effect on these structures, and that interference with the passage of some furrow-forming substance through the asters to the furrow region of a dividing cell is a secondary, rather than a primary, effect. The evidence we have presented in this paper supports such a conclusion. SUMMARY 1. Fertilized eggs of the polychaete annelid, Cliactoptcrus pergamentaceus, were treated with various dilutions of podophyllin and podophyllotoxin, beginning five minutes after insemination. Observations were made on the living eggs and on whole-mount cytological preparations made from samples fixed at various intervals during development. Dosages ranging from 0.1 to 0.00001 mg./ml. were tested. 2. Beginning as soon as 60 seconds after the beginning of treatment with moderate to high dosages (0.1 to 0.005 mg./ml.) of podophyllin, the asters of the egg maturation figure began to fade, followed by disappearance of the spindle by about three minutes after the initiation of treatment. The nine egg chromosomes remained in the ring configuration (characteristic of the first maturation division in this form) for approximately two to five minutes longer (depending on the exact dosage), after which they were gradually enclosed in a membrane-surrounded area which appears to have been derived from the spindle substance. By this time they had taken on a somewhat vesicular appearance. The sperm chromosomes were often similarly affected by the podophyllin, although the onset of these effects was usually somewhat slower than for the eggs. Both egg and sperm chromosomes CATHERINE HEXLEY AND U. P. COSTELLO remained contained in this membrane for approximately an hour, after which they were released to lie loose in the egg cytoplasm. No further development occurred at these dosages. 3. After treatments with podophyllin concentrations of 0.00005 to 0.00001 mg./ml.. a semblance of normal development ensued, although the resulting larvae were usually abnormal. In egg-samples fixed 24 minutes after the beginning of treatment, there was occasional evidence of reduction in size of the asters: by 47-57 minutes after the initiation of treatment, multipolar figures were observed, as well as two entirely separate complex metaphases, often in uncleaved cytoplasm. There was frequently karyokinesis without accompanying cytokinesis. When cleavage continued to occur, it was often retarded and/or abnormal. 4. 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The cytological and genetical significance of colchicine. J. Hered.,29:3-9. ORMSBEE, RICHARD A., IVOR CORNMAN AND RUTH E. BERGER, 1947. Effect of podophyllin on tumor cells in tissue culture. Proc. Soc. Exp. Biol., Med., 66: 586-590. PASTEELS, J., 1950. Mouvements localises et rythmiques de la membrane de fecondation chez des oeufs lecondes ou actives (Chactoptcrus, Mactra, Nereis.) Arch. Biol., 61: 197-220. RAPPAPORT, R., 1961. Experiments concerning the cleavage stimulus in sand dollar eggs. J.'Exp. Zoo!., 148:81-89. REBHUN, LIONEL I., 1959. Studies of early cleavage in the surf clam, Spisula. solidissiina, using methylene blue and toluidine blue as vital stains. Biol. Bull., 117: 518-545. SAUAIA, H., AND D. MAZIA, 1961. Action of colchicine on the mitotic apparatus. Path. -Biologic, '9:473-476. SPEK, ]., 1918. Oberflachenspannungsdifferenzen als eine Ursache der Zellteilung. Arch. j. ' £»/«•.. 44: 5-113. SULLIVAN, B. L, AND H. I. WECHSLER, 1947. The cytological effects of podophyllin. Science, 105: 433. SWANN, M. M., AND J. M. MITCH ISDN, 1953. Cleavage of sea-urchin eggs in colchicine. J. Exp. Biol., 30: 506-514. WILSON, E. B., 1925. The Cell in Development and Heredity. The Macmillan Co., New York (third edition). WILSON, G. B.. 1963. Studies on the disruption of the mitotic cycle. In: The Cell in Mitosis, 'ed. by L. Levine. Academic Press, New York. ZIMMERMAN, A. M., 1960. Physico-chemical analysis of the isolated mitotic apparatus. Exp. Cell Res.. 20: 529-547. ZIMMERMAN, A. M., AND DOUGLAS MARSLAND, 1960. Experimental induction of the furrowing reaction in the eggs of Arhacia piinctnlata. Ann. Ar. Y. Acad. Sci., 90: 470-485. THE INFLUENCE UK LIGHT UX THE TIME UF CELL AGGREGATIUX IN THE DICTYOSTEL1ACEAE THEO M. KONIJX1 AND KKXXETH B. RAFHK Departments of Bactcrioloy\ and Botany, University of Wisconsin, Madison 6, Wisconsin Light has long been known to influence the form and dimensions of the fructifica- tions, or sorocarps, of the Dictyosteliaceae. Both Potts (1902) and Olive (1902) reported positive phototropism in the developing fruiting structures of Dictyostelium nnicoroides, and Potts further observed that fructifications developed in light were smaller than those produced in darkness. Harper (1932) and Raper (1940) reported the latter phenomenon for Polysphondylium I'iolaccnni and Dictyostelium discoidcum, respectively. The high sensitivity to light of the migrating pseudo- plasmodia of D. discoidcum was first reported by Raper (1940). and has been further investigated by Bonner ct al. (1950) and Francis (1964). The object of our study has been to investigate the effect of light on still earlier stages of development, namely, 'the vegetative and preaggregative stages, with special emphasis on the beginning of cell aggregation in D. discoidcnrn. As early as 1940. Raper noticed that the myxamoebae of Dictyostelium nnicoroides and D. discuidcnui aggregated earlier if grown in light than if grown in darkness. More recently, Shaffer (1958) found that cells of D. discoidcum ag- gregated in bursts after they were transferred from darkness to light ; the maximal number of aggregations developed three to four hours after the myxamoebae were illuminated. He further observed (1961) that in /'. rioloceum the rate of center development increased rapidly after plates containing preaggregative myxamoebae were exposed to light for J to 1 hour. In our attempts to elucidate the influence of light on cell aggregation, primary emphasis was given to the type strain of Dictyostelium discoideitin, but three other strains of this species were investigated as well after preliminary experiments revealed that not all isolates respond equally to identical patterns of dark and light incubation. Myxamoebae of four additional species of the Acrasieae were then grown and tested under different conditions of light and darkness. Finally, an effort was made to determine if cells of D. (liscoidcuin grown in the light could influence the time of aggregation of cells grown in darkness. MATERIALS AND METHODS Cultures examined included several strains of Didvostclimn discoideum, repre- senting haploid and diploid cultures of NC-4 (the type) and three other haploid strains (TO-9, WS-7 and Acr-12), D. mncoroides, F-15a, D. furpurcuw. WS-321, Polysphondylium I'iolacciim, P-6, and P. pallidum. WS-320. The myxamoebae were usually grown on a rich peptone-glucose agar medium of pH 6.0 (Bonner, 1 Present address: Huhrccht Laboratory, International Kmbryological Institute, Utrecht, the Netherlands. 392 EFFECT OF LIGHT OX DICTYOSTFUUM 393 1947) in association with Escherichia colt #281. E. coll B/r, or Aerobactcr aero- genes #900. They were then harvested at the preaggregative stage and suspended in eold distilled water or in Bonner's salt solution (Bonner, 1947). Excess bac- teria were removed by 3 to 5 centrifugations at a relative centrifugal force of 110 g. One hundred-//.!, drops of a reconstituted suspension adjusted to 4 X 10"' cells/ml, were deposited on non-nutrient agar in dim fluorescent light. Three aliquots of the suspension were implanted in each of two Petri dishes to yield six replicate populations with a cell density of ca. 200 myxamoebae/mm.2 (Sussman and Noel, 1952). The dishes were kept uncovered until the excess water had evaporated or had been absorbed by the agar. The test populations were incubated at 23 ±1° C. Myxamoebae were also grown in shaken cultures according to the technique of Gerish (1959) : aliquots of 3 ml. of a bacterial suspension in IS :: 150 mm. test- tubes were inoculated with spores, grown at 25° C. on a rotary shaker at 250 rpm, and harvested in the vegetative or early preaggregative stage at a density of 3 to 5 X 106 myxamoebae/ml. (Hohl and Raper, 1963). When populations of myxamoebae were grown in the light on solid media, or were moved to the light after deposition of the 100-//1 drops, the Petri dishes were placed under General Electric "cool white" fluorescent tubes with a spectrum from 400 to 700 m/j. and a maximal peak at 580 niju.. The light intensity was ca. 60 foot candles at the level of the agar plates in which the myxamoebae were grown, or in which they were incubated after their deposition on solid medium, and ca. 300 foot candles at the surface of the tubes in which cells were grown in shaken cultures. Observations were made at hourly intervals as the myxamoebae ap- proached the onset of aggregation. RESULTS 1. The effect <>l an initial dark period on the time oj aggregation The first experiments were centered upon Dictyosteliuvn discoidcnin, NC-4(H), a haploid strain derived from the diploid stock culture, NC-4. Myxamoebae were grown in darkness on the solid medium. The cells were harvested in the pre- aggregative stage, centrifuged to remove bacteria, and resuspended as described above. After the myxamoebae were deposited on non-nutrient agar, the plates were incubated for increasing intervals in darkness and then transferred to light. The time of aggregation was defined as the moment when streams directed to a continuing center were clearly visible. Figure 1 reveals that cell aggregation was delayed in both constant light and constant darkness ; and that an initial dark period of 7 to 9 hours, followed by an exposure of from 4 to 2 hours to light, resulted in the earliest aggregation, i.e., 11 to 12 hours after the myxamoebae were deposited on non-nutrient agar. In occasional experiments, not shown in the graph, aggregation occurred after 6 hours darkness followed by 4 hours light. If the initial dark period was extended beyond 7 to 8 hours, aggregation was gradually postponed until the cells reached an age where they would have aggregated if kept in constant darkness. Early aggregation could still occur if the initial dark period was subminimal, but the number of such aggregations per Petri dish was limited to one, or a very few, and the majority THEO. M. KONIJN AND KEXXKTH B. KAPKR of the myxamoebae did not come together until ;i1)out two hours later. Populations of myxamoebae were recorded as being in the aggregative stage only if the total of developing aggregations in the three populations in a single Tetri dish was ten or more. The division between a subminimal and an optimal initial dark period was often quite sharp, and aggregation normally took place at about the same time in all populations of the duplicate plates, or the appearance of denser areas of cells indicated that aggregation was imminent. In other experiments, however, the time of aggregation of cells incubated under similar light conditions varied bv as much II llll I lill ill I ii iii _u i i 11 i i IBM •ii •in ni 0 2 4 6 8 10 12 14 IG Time in Hours FIGUKK 1. The influence of an increasing initial dark period on the onset of aggregation in Dictyostelium discoideitvi, N'C-4(H). The graph represents five experiments in which a dark period of ca. 8 hours \vas optimal for early aggregation. The myxamoebae were grown in the dark on the solid medium. Five experiments. D Incubation in light; • Incubation in darkness; | Onset of aggregation in individual experiments; | Average time for the onset of aggregation. as 2 to 3 hours. This was particularly true when the initial dark period was long enough for early aggregation to occur in some cases but too short for others. The exceptional variation seen in the time of aggregation for populations exposed to a 7-hour dark period may have been due, in large part, to differences in the physio- logical states of the cells that were harvested from the solid medium and used in the separate experiments (Fig. 1). If the myxamoebae were pregrown in liquid shaken culture, the time at which aggregation took place after deposition on agar was similar to or slightly earlier than for populations of cells grown on the solid medium. EFFECT OF LIGHT ON DICTYOSTELIUM 395 I • I I • I I • I 0 6 8 10 Time in Hours 12 14 16 FIGURE 2. The effect of a decreasing dark period on the onset of aggregation in Dictyostelium discoideum, NC-4(H), when the transfer to light takes place 8 hours after the deposition of the myxamoebae on non-nutrient agar. The cells were grown in the dark on the solid medium. The graph represents two experiments. D Incubation in light; B Incubation in darkness; | Onset of aggregation in individual experiments; | Average time of aggregation. • 2. The effect of later, short dark periods on aggregation Myxamoebae were deposited on non-nutrient agar, incubated first in the light for varying periods of time, then transferred to darkness. After a total of 8 hours, all plates were returned to the light. A dark period of at least 4-6 hours had to precede i m i • I (II I nni -H— r-r nn i inn jin i i n n inn i ni i riL j ni L 6 8 Time in Hours 10 12 14 16 Fuii'Kii 3. The influence of an increasing initial dark period on the onset of aggregation in Dictyostelium discnidemii, XC-4(H). The myxamoebae were grown on the solid medium. D Incubation in light; • Incubation in darkness; | Onset of aggregation of myxamoebae that were grown in the light (three experiments) ; [] Average time of aggregation of cells grown in the light; | Average time of aggregation of myxamoebae that were grown in the dark (data of Fig. 1). 396 THEO. M. KONIJN AND KKXXETH B. RAPER the transfer to light in order to ohtain aggregation within a 12-hour period (Fig. 2). A gradual decrease of the dark interval resulted in a progressive delay in the onset of aggregation. Not only does light affect the time of aggregation alter myxamoebae are harvested and deposited on agar, hut it also exerts an influence when applied during their growth phase. The results of three representative experiments are shown in Figure 3. Myxamoebae were grown on agar under cool fluorescent light hut were exposed to increasing dark periods after being harvested and deposited. The optimal dark period for early aggregation was reduced to ca. 4 hours, whereas dark periods of 3 hours or less did not accelerate aggregation significantly. When the initial dark period was increased above 4 hours, the time of aggregation was gradually delayed, in constant dark, and more particularly in constant light, cell aggregation was postponed still further. Under all test conditions, the cells grown , — ay I 1 IIBI i !B!I 1 1 IB 1 I 1 IB 1 § 1 IB 1 g I IB 1 | 1 IBB i 1 Pill i 1 18 1! 02 4 6 8 10 12 14 16 Time in Hours KM, i KF. 4. The influence of an increasing initial chirk period on the onset of aggregation in Dictyostelium discoideum, Acr-12. The myxamoebae were grown in darkness on a solid medium. D Incubation in light ; • Incubation in darkness; | Onset of aggregation in D. discoideum, Acr-12 (three experiments) ; | Average time of aggregation in D. disnudritni, Acr-12; [=] Time of aggrega- tion in D. discoideum, XT-HID (data of Fig. 1). on the agar medium in the light aggregated earlier than myxamoebae that were similarly grown in darkness. Comparable results were obtained for cells grown in liquid cultures as for those grown on the solid medium. 3. Light requirements jar optima! aggregation in different strains oj the same species Conditions of dark and light incubation similar to those optimal for f). dis- coideum, XC-4(1I), were required also for early aggregation in strains XV -4 (Stock), NC-4(S1) and NC-4(S2), TO-9, and \VS-7. The times of aggrega- tion under various light conditions were analogous to those shown in Figure 1. Dictyostelium discoideum, strain Acr-12, however, deviated remarkably from this pattern ( Fig. 4). Contrary to its effect on other strains, constant light was optimal EFFECT OF LIGHT OX DICTYOSTELIUM 397 for early aggregation in this case ; an increased delay in aggregation paralleled an increase in the dark period, and the longest delay took place in constant darkness. 4. The effect of light on aggregation in other species of the Dictvosteliaccac The influence of different light conditions on the time het\veen deposition of the myxamoebae and the onset of aggregation in species other than Dictyosteliuin discoldcum was first studied in D. purpurciun, strain WS-321. Myxamoebae were grown in the dark both on solid medium and in liquid culture. Aggregation began after 3-4 hours in populations grown on the solid medium and kept in constant light after harvest and deposition on non-nutrient agar (Fig. 5). An increasing dark- period caused a progressive delay in aggregation until the cells would have aggregated without any light. The time of aggregation was dependent not only on light, but also on cultural and environmental conditions. For example, cells grown in shaken cultures usually 1 — m- r KOBB 1 IB 1 | • II 1 II 1 III 1 III 1 1 • || 1 I II 024 Time in 6 E Hours FIGURE 5. The eifect of an increasing initial dark period on the onset of aggregation in DictyosteUum pur pure um, WS-321. The myxamoebae were grown in darkness in shaken liquid culture. D Incubation in light; • Incubation in darkness; | Time of aggregation in WS-231 (three experiments) ; | Average time of aggregation in WS-321. aggregated earlier than cells grown on an agar medium ; and aggregation was delayed if the excess of water in the lOO-/*!. drops evaporated slowly following deposition. Like DictyosteUum purpurcuiu, the other species examined, i.e., D. mucoroidcs, F-15a, Polysphondylium wolaceum, P-6, and P. pallid-urn, WS-320, required constant light for the earliest possible aggregation, while an increasing dark period gradually delayed the onset of this process. 5. The effect of light-grown cells on tJie aggregative behavior of cells grown in the dark Myxamoebae of D. discoid-emu, NC-4(H), were grown both in light and in darkness in liquid shaken cultures. After harvest and centrifugation, the light - grown cells and the dark-grown cells were mixed in ratios of 1:1 and 1:10. Populations of mixed cells, and of light-grown and dark-grown cells alone, were 398 THEO. M. KONIJN AND KENNETH B. RAPER plated on non-nutrient agar and incubated in constant light, constant darkness, and with initial dark periods of 4, 6 or 8 hours followed by light. The myxamoebae grown in the light aggregated earlier than the cells grown in darkness (Fig. 6). It" the light- and dark-grown cells were mixed in a ratio of 1:1 the time of aggrega- tion was intermediate between that of the light and the dark controls, but closer to that of the light-grown myxamoebae. Even if only \Q% of the myxamoebae were L 4D-L 6D-L 8D-L D Light Conditions Preceding Aggregation FIGURE 6. The influence of different light conditions on the time of aggregation in Dictyo- stelium discoideum, NC-4(H), in light-grown and in dark-grown myxamoebae and in mixtures of the two; myxamoebae grown in liquid shaken culture. Data represent averages for 3 experiments in each case. Populations tested: O : Cells grown in the light ; (j: Light- and dark-grown cells in a ratio of 1:1; (>l\splwnd\liui!i zriolaceum and P. pallid um included in the tests, and one strain of D. discoidcinn (Acr-12), aggre- gated earliest if exposed to constant light. Other strains of D. discoidcnni, includ- ing the type, NC-4, and substrains derived from it, aggregated optimally after an initial dark period of 6 to 8 hours followed by about 4 hours of light. When these latter strains were grown in light rather than in darkness, the initial dark period required for early aggregation was reduced to 4 hours. When light-grown cells of strain NC-4(H) were mixed with cells grown in the dark, the time of 400 THEO. M. KOXI.IX AND KENNETH B. RAPER aggregation of the latter was accelerated, but the addition of such cells did not induce dark-grown cells to aggregate as quickly as populations of light-grown cells alone. UTERATl'RK CITED ROXXER, J. T., 1947. Evidence for the formation of cell aggregates by chemotaxis in the development of the slime mold Dictyostelium discoideum. J. E.rp. Zoo!., 106: 1-26. ROXXEK, J. T., W. \V. CLARK, JR., C. I.. XKKI.V, JK. AND M. K. SLIFKIN, 1950. The orientation to light and the extremely sensitive orientation to temperature gradients in the slime mold Dictyostcliinn iliscoidcuiu. J . Cell. Com/). Physiol., 36: 149-158. FRAXCIS. D. \V., 1964. Studies on phototaxis in Dictyostelium. J. Cell. Comp. Pliysiol., 64: 131-138. GERISCH, G., 1959. Ein Suhmerskulturverfaliren fur entwicklungsphysiologische Untersuchungen an Dictyostelium discoiiiciuii. Natunviss., 46: 654-656. HARPER, R. A., 1932. Organization and light relations in P olysphondylium. Bit!/. Torrcy Bot. Club, 59:49-84. HOHL, H.-R., AND K. B. RAPER, 1963. Nutrition of cellular slime molds. I. Growth on living and dead bacteria. /. Bacteria]., 85: 191-198. KONITX, T. M., AND K. B. RAPER, 1961. Cell aggregation in Dictyostelium discoiileum. Dcv. Biol, 3: 725-756. OLIVE, E. \V., 1902. Monograph of the Acrasieae. Proc. Boston Soc. Nat. Hist., 30: 451-510. I'OTTS, G., 1902. Zur Physiologic des Dictyostelium unicoroidcs. Flora, 91: 281-347. RAPER, K. B., 1940. Pseudoplasmodium formation and organization in Dictyostelium (liscindcmu. J. Hlisha Mitchell Sci. Soc., 56: 241-282. SHAFFER, B. M., 1958. Integration in aggregating cellular slime moulds. Quart. J. Micr. Sci., 99: 103-121. SHAFFER, B. M., 1961. The cells founding aggregation centres in the slime mould Poly- sphondylium violaccum. J. Exp. Biol., 38: 833-849. SUSSMAX. M., AND E. NOEL, 1952. An analysis of the aggregation stage in the development of the slime molds, Dictyosteliaceae. I. The population distribution of the capacity to initiate aggregation. Biol. Bull., 103: 259-268. VARIATION IX LINEAR DIMENSIONS, TEST WEIGHT AND AMBl'LACRAL PORES IN THE SAND DOLLAR, ECHINARACHNIUS PARMA (LAMARCK; ' PRASERT LOHAVANIJAYA2 Department of Zn.iloi/y. L'nircrsity of AVzc1 Hampshire, Durham, New Huinf>s!ii>'c 03824 That the regular sea urchins have considerable variation in respect to a number of meristic and mensural characteristics has long been recognized. In varying de- grees Jackson (1912, 1914, 1927), Kongiel (1938), Vasseur (1952), and Swan (1962) have studied this variation and suggested that at least some of it might be either environmentally induced or selected. Kongiel also applied his methods of analysis to fossil remains of heart urchins, and Kermack (1954) and Nichols (1959a, 1959b, 1962) have more recently extended this work. Durham (1955) in his monumental "Classification of Clypeasteroid Echinoids" has indicated that in the sand dollars there is a great deal of variation, and Raup (1958) has gathered evidence that in the Pacific Coast sand dollar, Dendrastcr c.rccntricus (Esch- scholtz), heavier tests are characteristic of populations from colder water. In the present paper findings and suggested possible causal relationships with environmental factors are presented for Echinarachnins panna (Lamarck), based on collections from several localities in New England on the Atlantic Coast. The characteristics studied are ( 1 ) the relationship between length and width of the test, (2) test weight at comparable sizes, and (3) the numbers of pore-pairs in the petaloid areas of the aboral surface of the test. This material extends some of Durham's (1955) findings and is suggestive of the need for carefully designed statistical studies of variation in this species and for experimental studies on the effects of differing environmental factors on its growth pattern. MATERIALS AND METHODS Collections Six series of sand dollars were collected intertidally, either by hand, or with the aid of a rake. A series of specimens was taken from each of the following places: Crow Neck, North Trescott, Washington County, Maine (44° 52' 37" N, (.7° 07' 38" ±10" W) ; Bailey's Mistake, South Lubec, Washington County, Maine (44° 46' 23" N, 67° 03' 16" W) ; Paradise Point, Boothbay, Lincoln" County, Maine (43° 51' N, 69° 35' W) ; Hampton Beach, Rockingham County, New Hampshire (42° 54' 07" N, 70° 48' 40" W) ; Hampton Harbor, Rockingham County, New Hampshire (42° 53' 59" N, 70° 49' 07" W) ; and Scusset Beach. Barnstable County, Cape Cod, Massachusetts (41° 47' 18" N, 70° 30' 30" W). 1 This work represents a portion of a dissertation submitted to the University of New Hampshire in partial fulfillment of the requirements for the Ph.D. degree. - Present address : New England College, Henniker, New Hampshire. 401 402 PRASERT LOHAVANIJAYA The total number of specimens collected from these six areas was 1696. Two ad- ditional series, from New Castle, Rockingham County, New Hampshire (43° 03' 22" N, 70° 44' 17" W) and Eastern Point, Gloucester, Essex County, Massachu- setts (42° 35' 00" X. 70° 40' 00" \Y ) were collected subtidally. At New Castle a skindiver made the collection from about 5 feet of water while at Gloucester the animals were obtained with a rake over the side of a rowboat. The total number of specimens collected from these two localities was 519. The animals were pre- served in 10% formalin in fresh water for 24-72 hours and then dried at room temperature before being measured. The rake was purchased as a steel garden rake 14 inches wide and about 4 inches high at the opening, with two-inch teeth spaced one inch apart along the lower side of the opening. The handle, 5 feet in length, was made of wood. Hardware cloth (|-inch mesh) was used to construct a basket, 14 inches deep, FIGURE 1. Diagrammatic oral surface of the sand dollar, Echinarachnius panna, showing length and width of the test. which was fastened to the opening between the bow and the rake proper. This basket was attached by means of wire. Method of measuring A vernier caliper calibrated on the main scale in millimeters and on the vernier to tenths was used for all the measurements. The spines were removed from those portions of the test where contact with the calipers was to be made. Length The distance from the edge where the anus is located to the opposite edge of the animal is considered the length of the test (Fig. 1 ). Width The width of the tesl is obtained by measuring the distance from side to side, perpendicular to that of the length (Fig. 1). VARIATION IX SAND DOLLARS 403 Average diameter •J (Length + Width), as used by Durham (1955), has been used as the basic criterion of the animals' size. VARIATION IN RELATIVE LINEAR DIMENSIONS The specimens of each series were measured to the nearest tenth of a millimeter for length and width. The resulting data were tabulated into groups for each 5- mm. interval of mean test diameter, ^ (Length + Width). The numbers of speci- mens longer than wide, having length equal to width, and wider than long have been tallied. Tallies for all eight series were prepared. A summary of these data for all the series, but without reference to size of individuals, is given in Table I. The data tabulated according to size group show no appreciable change in re- TABLE I Relative lengths and widths of specimens from different localities Numbers of specimens Longer Wider L = W Total Hampton Harbor, X. H. 117 (37.6%) 176 (56.6%) 18 (5.9%) 311 Hampton Beach, X. H. 1 1 (3.0%) 358 (96.5%) 2 (0.5%) 371 Bailey's Mistake, Maine 23 (7.5%) 282 (92.2%) 1 (0.3%) 306 Crow Xeck, Maine 250 (78.9%) 57 (18.0%) 10 (3.2%) 317 Scusset Beach, Mass. 11 (4-5%) 229 (93.1%) 6 (2.4%) 246 Xew Castle, X. H. 53 (29.4%) 121 (67.2%) 6 (3.3%) 180 Boothbay Harbor, Maine 81 (55.9%) 55 (37.9%) 9 (6.2%) 145 Gloucester, Mass. 112 (32.6%) 211 (61.3%) 21 (6.1%) 344 lationship with size. Mean dimensions for the four chief series divided into size groups are plotted in Figure 2. Examination of these data reveals that the populations from Hampton Beach, Xew Hampshire, and those from Bailey's Mistake, Maine, have more wider than longer individuals and average wider than long more markedly than is the case for any of the other collections. In distinct contrast to these collections is the one from Crow Neck, Maine, whose members tend to be longer than wide. The collections from the other five localities fall well between these extremes. Comparisons of the mean dimensions of these five collections (Hampton Harbor, New Hampshire ; Boothbay Harbor, Maine ; New Castle, New Hamp- shire; Gloucester, Massachusetts; and Scusset Beach, Massachusetts) reveal that at Scusset Beach the tests grow appreciably wider than long but not as markedly so as at Hampton Beach and Bailey's Mistake. The collection from New Castle averages slightly over 1 % wider than long. In none of the other three series (Hampton Harbor. Paradise Point, and Gloucester) is one dimension more than 1% greater than the other dimension. Likewise in none of these series does the ratio of longer to wider specimens deviate from equality beyond 2:1 or 1:2. 404 PRASKRT LOHAVANIJ \Y \ The habitat at Cro\v Neck is unusual. Because it is on the shore of a con- stricted channel through which large amounts of water pass to and from large inner hays, to the extent of changing their levels many feet on each cycle, it has an almost continuous hut reversing flow of water. This resembles a rather swift river which flows nearly six hours in one direction, followed by a brief period of nearly slack water, and then another period of rapid flow in the opposite direction, again for nearly six hours. One would thus expect only rocks and relatively coarse sediments, which are in fact found in the main channels. Most of the sand dollars were found living in eddies which occurred in the small wider parts of the channel. The rather soft sandy substratum under these eddies was generally finer than in the main channels and was often overlain by a layer of mud, silt, or detritus. Thus, the sand dollars living in this habitat live in contact with a 80 70 .60 E 50 40 Ml) 411 50 60 Length ram. 70 80 FIGTKE 2. Relationship of width to length. HB — Hampton Beach; I1H — Hampton Harbor; BM— Bailey's Mistake; CN— Crow Neck. nearly continuous current hut encounter almost no pounding by waves, and the current's direction changes only a few times a day. It appears that they tend to grow longer than wide. In contrast with the conditions at Crow Neck we find at Hampton Beach and Bailey's Mistake little continuous current and considerable exposure to surf. At Hampton Beach, particularly, the surf action is considerable almost all the time. Here the sand grains are line and hard-packed. The situation at Bailey's Mistake is similar to Hampton Beach but there appears to be some reduction in surf action by the headlands to either side of the entrance to the bay, as is made evident by the eel grass growing in the bay, which in turn appears to give the organisms living among it additional protection. The sand dollars that live in two places (see Fig. 2) appear to grow quite consistently wider than long. VARIATION" IX SAXD DOLLARS 405 A situation between the continuously running water at Crow Neck and surf at Hampton Beach can be found at Hampton Harbor. It is a protected harbor southwest of the village of Hampton Beach. Since it is protected, it has little surf action. The current, due to the flow in and out of the tide, is appreciable but much less than at Crow Neck. The area where the sand dollars were collected is covered by water most of the time. During extremely low tides it becomes a shallow quiet pool. The substratum is a mixture of sand and mud with much organic matter. From Table I it can be seen that the animals usually grow a little wider than long. However, they are not nearly as wide as those from Bailey's Mistake or from Hampton Beach. This is true both in terms of average dimensions and in terms of the number of individuals wider than long compared with the number longer than wide. Thus there are three kinds of habitats involved in this study, namely: (1) soft sand substratum and continuous current, (2) substratum with appreciable amounts of mud, probably high in organic matter, under relatively quiet water with little surf action, and (3) hard-packed fine sand, containing no evident mud and probably little organic matter, associated with heavy surf. These observations suggest significant correlation between the kind of habitat and the usual form of the sand dollars inhabiting these habitats. Whether the differences in habitat influence the growth pattern of the animals or bring about a differential selection from a heterogeneous gene pool has not been determined. VARIATION IN TEST WEIGHT Ten specimens were picked from each 5 -mm. size group from the four main series. The spines of these specimens were removed, and then the tests were weighed in grams to the nearest 0.01 gm. The average weights obtained and the average dimensions of the groups of specimens used from the four localities, Crow Neck. Hampton Harbor, Bailey's Mistake, and Hampton Beach, were tabulated. Obtaining the real test weight, excluding the spines, the soft parts, and the contents of the digestive tract, is a problem. In order to approach a close ap- proximation to the true test weight, one must remove the spines and then cut the test with a thin saw. Raup (1957) used a diamond saw. Then his specimens were cleaned, dried, and weighed to the nearest 0.1 gm. After he determined the loss of weight (amounting to about 5% for a specimen 35 mm. in diameter) for 24 of his specimens, he considered it insignificant and disregarded it in his computations. To determine the loss resulting from the cut and the weight of the soft parts and the gut contents, I selected 10 specimens of approximately the same size (ca. 50 mm. in mean diameter) from each of the main series. The spines were removed and the tests were weighed to the nearest 0.01 gm. They were then cut with a thin electric saw and weighed again. The soft parts and the gut contents, but not the lantern, were removed and the weights were determined once more. The total loss of weight, loss from the cut, and the weight of the soft parts and the gut contents were calculated as percentages in the following manner : 406 PRASKRT LOHAVANIJAYA The difference in weight before cutting and after cleaning inside The total loss of weight -;— — — - X 100 \\ eight before cutting The difference in weight before and after cutting The weight loss from the cut = - ..T . . — :— -7— X 100 Weight before cutting The difference in weight after cutting and The weight of the soft parts after cleaning inside and gut contents Weight before cutting The percentage figures are shown in Table II. It should be noted that the lantern is considered part of the test weight. This is in contrast with Raup's (1957) method. From Table II we can see that the greatest percentage of total loss in \veight was 8.7% from a specimen from Bailey's Mistake and the lowest one was 1.9% from a specimen from Hampton Beach, with a mean of 4.14% for all 40 specimens. The weight of the soft parts and the gut contents ranged from 6.1% down to 0.6%- among the 40 specimens. It appears that the weight of the soft parts and the gut contents is of little significance and thus has very little effect on the total weight of the dried animal. However, it is interesting to note that the average per- centages of the soft parts and gut contents appear to vary with locality and, rounded to the nearest -i%, amount to H%, 2\%, 5%, and H% from Crow Neck, Hampton Harbor, Bailey's Mistake and Hampton Beach, respectively. Therefore, in this part of the investigation, the weight of the test used, in each series, was obtained by subtracting this predetermined percentage of weight of the soft parts and the gut contents from the specimen's weight before cutting. The test weights in grams are tabulated and plotted against mean diameters in Table III and Figure 3. As expected, the weights increase slowly when the animals are small in size and then increase somewhat more rapidly as they become larger. In general it appears that at comparable sizes the specimens from Crow- Neck are heaviest and those from Hampton Beach are lightest. The series from Bailey's Mistake and Hampton Harbor are between the extremes, with those from Bailey's Mistake being slightly heavier. Raup (1958) discussed "The Relation between Water Temperature and Morphology in Dcndrastcr," and found the tests of the Pacific sand dollar, Dcndraster exccntricus (Eschscholtz), of a given size to be heavier in cold water than in warm water. He also stated that this correlation is interpreted as the result of phenotypic ( nonheritable) adaptation to water temperature. The find- ings here noted appear to correspond to Raup's ideas. The weights of specimens from Crow Neck and Bailey's Mistake, both from eastern Maine, are heavier than the specimens from Hampton Harbor and Hampton Beach, both from the coast of New Hampshire. According to the "Surface Temperatures at Tide Sta- tions, Atlantic Coast" (U. S. Department of Commerce, 1951), the annual average surface water temperature for Eastport, Maine, from 1944 to 1950 was 44.1° F. and for Portsmouth, New Hampshire, from 1944 to 1950 was 47.4° F. It can VARIATION IN SAND DOLLARS 407 TABLE II Weight losses incurred by cutting tests and cleaning tests. The deductions from total weights made in Table III are based on these figures Weight Weight loss <% > Ave. soft part &< M t 1 # Before cutting After cutting After cleaning inside Total Cut Soft part & gut contents gut contents rounded to nearest J% C\ 121 11.60 11.44 11.33 2.3 1.4 0.9 125 9.95 9.75 9.55 4.0 2.0 2.0 130 9.15 8.95 8.80 3.8 2.2 1.6 135 5.94 5.84 5.71 3.8 1.7 2.1 136 137 11.69 9.61 11.52 9.47 11.24 9.36 3.8 2.6 1.5 1.5 2.3 1.1 1.5% 140 9.21 9.10 8.97 2.6 1.2 1.4 146 152 10.10 7.58 9.97 7.52 9.81 7.38 2.9 2.6 1.3 0.8 1.6 1.8 163 8.54 8.43 8.28 3.0 .3 1.8 HH 89 7.50 7.30 7.10 5.3 2.7 2.6 94 6.90 6.70 6.45 6.5 2.9 3.6 us 8.92 8.81 8.60 3.6 2 2.4 121 123 136 7.57 8.16 8.04 7.48 8.08 7.92 7.23 7.91 7.73 4.5 3.1 3.6 .2 .0 .5 3.3 2.1 2.1 2.5% 137 8.17 8.08 7.82 4.3 .1 3.2 154 7.52 7.43 7.24 3.7 .2 2.5 158 7.63 7.54 7.41 2.9 2 1.7 177 7.64 7.52 7.32 4.2 .6 2.6 BM 4 7.45 7.35 6.90 7.4 .3 6.1 5 8.81 8.68 8.21 6.8 .5 5.3 7 9 7.72 8.01 7.64 7.89 7.16 7.65 7.3 4.5 .0 .5 6.3 3.0 10 12 7.45 7.27 7.30 7.16 6.80 6.78 8.7 6.7 2.6 1.5 6.1 5.2 5% 16 6.90 6.81 6.46 6.9 1.3 5.6 17 8.94 8.82 8.49 5.0 1.3 3.7 19 8.29 8.19 7.78 6.2 1.2 5.0 24 7.15 7.06 6.64 7.1 1.3 5.8 HB 328 7.00 6.80 6.70 4.3 2.9 1.4 329 6.22 6.16 6.05 2.7 1.0 1.7 334 7.20 7.13 7.06 1.9 1.0 0.9 337 6.56 6.50 6.43 2.0 0.9 1.1 339 341 7.07 5.89 6.99 5.83 6.90 5.76 2.4 2.2 1.1 .0 1.3 1.2 1.5% 345 5.08 5.01 4.98 2.0 .4 0.6 346 6.75 6.65 6.50 3.7 .5 2.2 348 4.98 4.92 4.85 2.6 ? 1.4 167 5.82 5.76 5.70 2.1 .0 1.1 408 I'K ASKKT I.OHAVAXIJAYA Mean weights and diameters of specimens. Each average is that of 10 individual* unless a smaller number is indicated in parentheses after the mean diameter figures Crow Xeck Hampton Harbor Bailey'-! Mistake Hampton Beach i i L - W) mm. ToUil wt. -1.5% (SOft P.llt) gm. -i (I- +\V) mm. Total wt. -2.5', (soft part) gm. i (L + w.i mm. Total wt. 59! l-ott part) gm. i (L + VO mm. Total wt. -1.5% (soft part) gm. 14. S5 (8 20.99 i7i 0 0.54 — — — 21.59 0.61 — — — — — 25.% 0.95 31. 24 (7) 1.98 — — — — 30.52 1.53 36.51 3.00 — — 35.40 2.08 40. 64 4.12 41.82 3.90 — 40.13 2.77 15.76 (,.S6 45.48 5.20 — 45.21 4.0] 51.55 9.20 51.24 7.54 52. Ofl 7.50 51.69 6.17 55.44 11.11 00.52 12.9f, 55.30 60.63 1(1. 01 55.56 60.34 8.67 1 1.34 56.68 7.39 65.37 15.07 — 65.73 13.70 — — 72.40 (. 17.93 — 70.72 16.62 — — — — — 75.27 18.97 — — — — — 82.52 23.58 — — he seen that the difference in water temperature for this period was 3.3° F. More work needs to he done hefore any causal relationship can he demonstrated, and tin- differences found between the two New Hampshire series and between the two Maine series suggest that other factors may he involved. 20 30 40 50 1/2 (LtW) mm. 70 80 FIGUKK 3. Relationship of test weight to size, L' (L I YV ) . VARIATION IX SAND DOLLARS VARIATION IN PORE-PAIRS IN PETALS 40') The live smallest specimens of each 5-mni. size group from each of the four main series were used for the counting of pore-pairs in the petaloid areas of the ambulacra. The numbers of pore-pairs in all five petals in each specimen were counted. In order to set up a standard for all specimens, it was necessary to determine the distal limits of the petals. Figure 4 shows that there are two rows of pore-pairs in each, starting at the ambulacral plates nearest the apical system. Near the apex these two rows of pores are close together. Gradually these rows FIGUKE 4. An ambulacral sector of the aboral surface of the sand dollar, Echinarachtiinx panna, showing the arrangement of ambulacral pores in the "petal" of ambulacral area II. The arrows indicate the last pore-pairs to be counted in determining the number of these included in the "petal." Specimen from Boothbay Harbor, Maine (3.1 X). of pore-pairs spread apart and then toward the periphery they come closer to- gether again, but remain separated. Thus, in the terminology of students of the irregular urchins (cj. Durham, 1955) Echinarachnius has "open petals." Beyond the point of this coming together of the rows of pore-pairs, they diverge, and these conspicuous pore-pairs become infrequent. In this investigation counting of pore- pairs has been stopped at the point where the rows start diverging from each other (Fig. 4). 410 PRASERT LOHAVANIJAYA The mean diameters and numbers of pore-pairs in each size group for each series are summarized in Table IV. There are only two common size groups among these four series. When \ve compare the four populations and limit our findings to these two common size groups, it is obvious that, in both size groups, the animals from Crow Neck have on the average the smallest number of pore-pairs in the petals. The specimens from Hampton Beach and Hampton Harbor are close together and com- pete for having the highest numbers of pore-pairs. The Bailey's Mistake specimens show numbers between these extremes. Because of the differences in the size ranges among these series, it is difficult to compare them directly. To minimize this difficulty, a graph showing the relationship of the number of pore- pairs to size, ^ (L + W), has been drawn as Figure 5. From this graph it is apparent that among these four series, the series from Crow Neck has the lowest number of pore-pairs whereas the Hampton Harbor series has the highest at comparable sizes. The Hampton Beach and Bailey's Mistake series are in between with Hampton Beach being somewhat higher and at some sizes nearly the same as Hampton Harbor. The lines drawn on the graph have been fitted by eye. With the amount of data presently available, it appears doubtful that more careful fitting would be justified. Durham (1955, p. 87) states that it appears probable that the number of plates inside the petals is a better indication of the age than is the absolute size. If this is true, the sand dollars that live at Crow Neck are growing faster than those 70 60 : D, 30 20 BM. CN III! 10 40 50 60 1/2 (L+W) mm. 70 80 '.10 FIGURE 5. Relationship of the number of pore-pairs to size, J (L + W), VARIATION IN SAND DOLLARS 411 TABLK IV The mean dimensions and pore- pairs. Each average is thai of 5 individuals unless a smaller number is indicated in parentheses Crow Neck Hampton Harbor Bailey's Mistake Hampton Beach J (L + W) mm. Pore-pairs 1 (L + W) mm. Pore-pairs * (L + W) mm. Pore-pairs i (L + W) mm. Pore-pairs 13.9 19.6 20.1 27.1 — — — — 20.9 27.6 28.9 (4) 34.4 (4) — — — — 25.4 33.7 — — — — — — 30.2 36.4 35.3 40.4 — — — — 35.2 43.3 40.3 43.9 40.6 46.9 — — 40.1 46.3 45.4 47.0 45.1 53.3 — — 45.1 49.5 51.3 51.6 51.2 55.8 51.7 53.9 51.4 55.5 55.3 55.5 55.2 58.8 55.2 56.1 55.5 59.3 60.2 55.0 60.2 66.9 60.2 61.8 — — 65.1 57.4 — — 65.3 63.9 — — 72.0 63.6 — — 70.4 67.2 . — — — — — — 75.1 69.1 — — — — — 81.4 74.0 — • — living in the other localities studied. Also it would appear that the animals from eastern Maine generally grow faster than those from southern New Hampshire. DISCUSSION In this study variations among populations of sand dollars (E. par ma) from different localities have been noted. These variations pertain to relative linear dimensions, test weight, and numbers of pore-pairs in the petals. The preponderance of relatively longer specimens from Crow Neck, Maine, where they live in what is essentially a reversible tidal river, and the strong tendency for specimens to be wider than long at Hampton Beach, New Hampshire, Bailey's Mistake, Maine, and Scusset Beach, Massachusetts, where they live on surf-swept beaches, suggest that in some way the nature of the water movement where these animals live may affect their shape or bring about a selection such that longer individuals are selected where there are fairly strong currents flowing in one direction for several hours continuously, and wider specimens are selected where there is a mixture of current, often frequently reversing, and with the pounding of breaking waves. The occurrence of populations intermediate in this respect in localities where there is little surf, and what appears to be less current, supports the idea that there is some sort of correlation between the relative linear dimensions of these animals and the nature of the environment in which they live. That there may be correlations between habitat and relative linear dimensions in other scutellinids is suggested by the literature discussing the status of Den- draster excentricus var. clungatiis H. L. Clark 1935. Clark clearly considers it a variety. Grant and Hertlein (1938) at least tentatively accept Clark's opinion. MacGinitie and MacGinitie (1949) think there are two species which differ in form and habitat requirements. Mortensen (1948) considers this variation not 412 PRASKRT LOHAVANIJAYA to justify even varietal status. Durham (1955), without mention of the taxonomic question, states (p. 159). "It seems that the excentricity of the apical system and the greater posterior development of the food grooves is correlated with this habit [living on the edge where not too strongly affected by wave action]. The non- excentric species [specimens?] probably lie flat on the sea floor." Thus, there appears to be a somewhat similar problem among populations of Dcndrastcr on the Pacific Coast. Because the varietal name "elongatus" applied to the populations living in quiet water suggests that the animals are longer than those typical for the species that live on the surf-swept beaches, it would appear that the correlation between relative linear dimensions and water movement in the habitat is the same in Dcndrastcr as in Echinarachnius. There is, however, a ques- tion concerning what Clark (1935) considers length. He describes the holotype as being 41.5 mm. long and 40 mm. wide, but the specimen the MacGinities (1949) illustrate (Figure 100, page 238) is distinctly wider than long. Thus, the question arises as to whether this discrepancy results from differences in terminology — i.e., what is meant bv "length"-— or from real differences between Clark's and the Mac- Ginities' concept of the variety. To settle this. Clark's holotype (M.C.Z., No. 6040, not 3343 as Clark says) has been examined and measured. It is in fact longer than wide (41.2 mm. long X 40.5 mm. wide by my measurement) as are the two paratypes (M.C.Z., No. 6041). Thus, one is tempted to suspect that Clark (1935) and the MacGinities (1949) had rather different ideas concerning the characteristics of the variety, and careful reading of Clark's (1935) description and the MacGinities' (1949) discussion tends to substantiate this, not only in terms of shape but also in habitat. Even so it would appear that there is need for careful quantitative work aimed at determining the nature of any causal relationship that may exist between linear dimensions and habitat. As shown in Table III and Figure 3, there appear to be rather distinct differ- ences in the relationships between test weight and mean diameter. The populations from Crow Neck, Maine, and Hampton Beach, New Hampshire, appear to be at the extremes of the four collections examined, with those from Crow Neck being relatively the heaviest and those from Hampton Beach being the lightest. Again the populations from Bailey's Mistake, Maine, and Hampton Harbor, New Hamp- shire, are intermediate, with those from Bailey's Mistake appearing to be slightly the heavier, but because of the small amount of overlap in size-range between these two populations, this is a questionable difference. It is possible that whatever tactors may be responsible for the differences in relative linear dimensions may also be operating here in respect to the relationship between test-weight and mean diameter. However, it should be recalled that Raup (195S) found the tests of the Pacific sand dollar, /Vm/n/.v/rr c.vcenlricits, to be heavier in relation to diameter in populations from cold water than those from warmer water and that the available data appear to indicate that the mean annual water temperatures for the Maine localities are probably some 3° F. lower than those for the New Hampshire locali- ties. That there may be other factors involved can hardly be doubted. H. L. Clark (194S) in an attempt to find a possible causal basis for explaining differences in numbers of coronal plates in tests of the regular sea urchin. Stroiigvhccntrotits franciscanus, of comparable >i/c> suggests (p. 27S) that "unusual features of their habitat, such as excessivelv strong surf or tidal currents" mav affect this relation- VARIATION' IN SAND DOLLARS 413 ship. Swan (1962) found that specimens of S. droebachiensis living off the Gaspe, from shallower and presumably more turbulent water, had more coronal plates than those from deeper and presumably quieter water. Although 1 have found no statements in the literature on the subject. Swan in conversation tells me he strongly suspects that the tests of the specimens with relatively more numerous plates are also heavier at comparable sizes. In bivalve mollusks there appears to be a significant amount of evidence ( Shih, 1937; Swan, 1952) that, more or less generally, factors that reduce the rate of growth tend to increase shell thickness at comparable sizes. Likewise there is a vast literature (cf. Barlow, 1961) indi- cating that fishes grown under conditions retarding their growth rate through critical stages in their development often have larger numbers of meristic structures, ft should, however, always be remembered that the skeletons of the mollusks and the vertebrates differ greatly from each other and from the skeletons of echinoderms. Thus, although similarities may appear great, caution should be used when speculat- ing concerning what might happen in one group on the basis of what has been observed in another. That oxygen content of the water could affect shell or test thickness through its effect on ease of precipitation of CaCO:! should be considered. Finally, the possibility of the selection of different genetic types by different sets of environmental factors cannot be ruled out. Under some conditions it is possible that heavier tests would have distinct survival value whereas under others lighter tests might be advantageous. Nichols' (1962) study of differences between popu- lations of heart urchins (Ecliinocardiuin cordatuui Pennant) should be instructive to anyone trying to determine whether morphological differences are selected or induced by environmental differences. The findings concerning the numbers of pore-pairs in the petals of specimens from the four populations studied in this respect are puzzling in view of Durham's (1955) idea that the older specimens of comparable size would have more plates in the petals and in view of the aforementioned idea that the older specimens would have heavier tests. The fact that the specimens from Crow Neck are heaviest would indicate that they are slow growing, and their having the smallest numbers of pore-pairs in the petals would indicate that they are growing rapidly. Thus, it appears that until studies have been made in which the ages or growth-rates char- acteristic of populations are definitely determined, test weight and numbers of pore-pairs in the petals must be considered as independent of each other and neither should be considered as having a proven relationship to growth rate. SUMMARY 1. The widespread occurrence of skeletal variation in echinoids is indicated. 2. Variation in respect to several characteristics of the test has been studied in the sand dollar Echinarachnius panna (Lamarck) as it occurs in a number of New England localities. 3. Evidence is presented that indicates a tendency for these animals to produce tests longer than wide when living in flowing water and wider than long when living on surf-swept beaches. 4. In this species, as Raup (1958) reported earlier for Dcndraster on our west coast, tests tend to be heavier at comparable sizes in populations living in colder water than those of warmer localities. 414 PRASERT LOH\\ \\IJAYA 5. 1 'opulations vary from one another in numbers of pore-pairs in the petaloid areas of the ambulacra of individuals at comparable sizes, but no consistent corre- lations with environmental factors have been delected. LITERATURE CITED BARLOW, G. W., 1961. Causes and significance of morphological variation in fishes. Sv.?- tcmatic Zool, 10: 105-117. CLARK, H. L., 1935. Some new Echinoderms from California. Ann. May. Nat. Hist., scr. 10. 15: 120-129. CLARK, H. L., 1948. A report on the Echini of the warmer Eastern Pacific, based on the collec- tions of the Velero III. Allan Hancock Pacific E.vpcd., 8(5) : 225-351 (plates 35-71). DURHAM, J. W., 1955. Classification of Clypeasteroid Echinoids. U. of Calif. Publ. C-col. Sci.. 31(4) : 73-198 + frontispiece and plates 3 and 4. GRANT, U. S., IV, AND L. G. HERTLEIN, 1938. The west American Cenozoic Echinoidea. U. Calif. Los Angeles Publ., Math. Phy. Sci., 225 pp. + 30 plates. JACKSON, R. T., 1912. Phylogeny of the Echini, with a revision of Palaeozoic species. Mem. Boston Soc. Nat. Hist., 7: 491 pp. + 76 plates. TACKSON, R. T., 1914. Studies of Jamaica Echini. /'<;/>. Toi-tni/as Lai'. (PubL No. 1S2 Cam. Inst.), 5: 139-162. JACKSON, R. T., 1927. Studies of Arbacia pitnctiilata and allies and of nonpentamcrous Echini. Mem. Boston Soc. Nat. Hist., 8(4) : 433-565. KERMACK, K. A., 1954. A biometrical study of Micrastcr coranyuinum and M. (Isoinicrastcr) scnoncnsis. Phil. Trans. Royal Soc. London. 237 (B) : 375-428. KONGIEL, R., 1938. Rozwazania nad zmiennoscif jezowcow. (Considerations on the variability of Echinoidea.) Annu. Soc. gcol. Polognc, 13: 194-250. (In Polish with French summary and legends for figures and tables. English translation OTS 60-21506 avail- able from the Office of Technical Services, U. S. Department of Commerce, Washington 25, D. C.) MACGINITIE, G. E., AND N. MAcGiNixiE, 1949. Natural History of Marine Animals. McGraw- Hill, New York, London, Toronto, 473 pp. MORTENSEN, T., 1948. A monograph of the Echinoidea (vol. 4, part 2) Clypeastroida. C. A. Reitzel, Copenhagen, 471 pp. + 72 plates. NICHOLS, D., 1959a. Changes in the chalk heart-urchin Micrastcr interpreted in relation to living forms. Phil. Trans. Roy. Soc. London, 242B: 347-437. NICHOLS, D., 19591). Mode of life and taxonomy in irregular sea-urchins. Systematics Assoc. Publ., No. 3, pp. 61-80, 7 figs. NICHOLS, D., 1962. Differential selection in populations of a heart-urchin. Systematics Assoc. Publ., No. 4. pp. 105-118,8 figs. RAUP, D. M., 1957. Classification and morphological variation in the genus Dcndrastcr. Doctoral thesis submitted to the Division of Geological Science, Harvard Univ., 159 pp. + 24 pis. I\.\rr, 1). M., 1958. The relation between water temperature and morphology in Dcndrastcr. J. Geol, 66(6) : 668-677. SHUT, C. V., 1937. Die Ahhangigkeit der Grosse und Schalendicke mariner Mollusken von der Temperutur und dun Salzgehalt des Wassers. Sitz. Ges. naturf. Fr. Berlin, pp. 238-287. SWAX, E. F., 1952. The growth of the clam M\a arcnaria as affected by the substratum. Ecology, 33(4) : 530-534. SWAN*, 1C. F., 1962. Evidence suggesting the existence of two species of Strongylocentrotus ( Echinoidea) in the Xorthue.st Atlantic. Canad. J. Zool., 40: 1211-1222. LI. S. DKI'.MM MKNT OK CO.MMKIU K, COAST AND GEODETIC SURVKV, 1951. Surface water tempera- tures at tide stations, Atlantic Coast North and South America. Special Publ. No. 278 Kr\i-ed (1('51) Edition. L'. S. Government Printing Office, Washington; 57 pp. VASSKUK, J''.., 1(>52. Geographic variation in the Norwegian sea urchins, Strongylocentrotus droebacliii'nsis and ,V. pallidus. I'.rohtfion, 6: 87-100. SYMBIOSIS OF HYDRA AND ALGAE. I. EFFECTS OF SOME ENVIRONMENTAL CATIONS ON GROWTH OF SYMBIOTIC AND APOSYMBIOTIC HYDRA LEONARD MUSCATINE* AND HOWARD M. LENHOFF Division of Marine Biology, Scripps Institution of Oceanography, University of California, San Diego, California, and Laboratory for Quantitative Hiulut/y, University of Miami, Coral Gables, Florida Unicellular algae inhabit a variety of aquatic invertebrates. Their primary function as symbionts is in most cases not clearly understood (see review of Droop, 1963). Interest in this problem has led us to an experimental analysis of the association of algae and hydra. Green hydra are particularly suited to an investi- gation of symbiosis involving a metazoan because (1) methods for mass culture of these hydroids provide a practically unlimited supply of animals of similar genetic, developmental, and nutritional histories, cultured in a fluid of known ionic composition. (2) Aposymbiotic (= algae-free) control hydra are easily obtained (Whitney, 1907) and cultured, and (3) the specific growth rate constant, k (cj. Loomis, 1954), provides a quantitative measure of the effect of various factors, such as ionic composition of the medium, or the presence of symbiotic algae, on growth of the host. In preparation for studies on the effect of symbiotic algae on growth of hydra, it was necessary first to attempt to control environmental variables affecting growth. Since the most critical of these for laboratory-grown hydroids appears to be the ionic composition of the culture medium (Loomis, 1954; Loomis and Lenhoff, 1956; Ham, Fitzgerald and Eakin, 1956; Lenhoff and Bovaird, 1959. 1960; Fulton, 1962), the experiments described here were undertaken to determine the effect of some environmental cations on growth of symbiotic and aposymbiotic hydra. MATERIALS AND METHODS Chlorohydra viridissuna, obtained from the Carolina Biological Supply Co.. Burlington, N. C, and designated by us "Carolina Strain 1960," was used in all experiments. The general morphology of green hydra and associated algae is described by Goetsch (1924), Haffner (1925), and Brien and Reniers-Decoen (1950). Present knowledge of symbiotic algae is summarized by Droop (1963). Carolina Strain 1960, with an average resting length of about 5 mm., is small compared to other green hydra. Usually 15-25 unicellular green algae, each 3-6 microns in diameter, are situated basally within most of the host's gastrodermal cells. Electron micrographs of C. viridissima (Wood, 1959) show the intra- cellular location of the algae. 1 Present address : Department of Zoology, University of California, Los Angeles, California. 415 410 LEONARD MUSCATIXK AX!) HOWARD M. LENIIOI-T The culture medium ("M" solution) for this species of hydra consisted of 10~3 M CaCL, 10 :i AI NaHCO3. 10 ' M MgCL, 10 4 M KC1, and 10'3 M tris-(hydroxy- methyl)-aminomethane buffer (Sigma "121"), pH 7.6, in water deionized by pass- ing distilled water through organic removal and ion exchange resin columns (Barnstead Black and Red Cap Cartridges). The experimental rationale for the selection of this medium is given in the Results section (Fig. 2). Penicillin G (sodium, U.S. P., 50 mg./L.) was occasionally added to stock cultures to retard growth of contaminating microorganisms, hut was omitted from cultures during experiments. 4000 £ 2000 00 1000 K = 0.47 234567 DAYS FIGTKK 1. (iiouth curvt s tor a >tock culture of ( '. 7v'nWm/w<; (preen) obtained by counting hydranth.s (upper curve) or individual hydra (lower curve). Sampling period during logarith- mic growth indicated by bracket. Aposymhiotic control animals (referred to hereafter as albinos) were obtained by growing green individuals for eight days in culture solution containing 0.068 M glycerine (Whitney, 1907, 1908). Following this treatment, the absence of algae was confirmed by microscopic examination of macerated tissues and of 2- micron sections of paraffin-embedded whole animals. Aposymbiosis was perma- nent. Occasionally, algae reappeared in some individuals soon after glycerine treatment, apparently as a result of incomplete removal of algae. Under con- ditions defined elsewhere in this paper, albinos grew normally when returned to glycerine-l'ree culture solution, and have continued to do so through three years SYMBIOSIS OF HYDRA AND ALGAE. I. 417 .5r 0 (a) UJ a: x h- B£ O or .5 Ncf 0 (c) MgH o (d) K* 0.00 io5 io4 MOLARITY io3 i62 FIGURE 2. Histogram showing the growth rates of green (diagonal lined bars) and albino (open bars) C. viridissiina in "M" solution in which the concentration of each of four cations is varied in turn while others held constant. 41 S LEONARD MUSCATINE AXI) HOWARD M. LENHOFF of subculture in our laboratory. Since the approximate life span of a C. viridissima cell is 2-3 weeks (Brien and Reniers-Decoen, 1949, 1950; Burnett and Garofalo, I960), anv undesirable effects of glycerine treatment would presumably be diluted or absent after a few months of subculture. Stocks of hydra were cultured in Pyrex trays (33 cm. X 22 cm. X 4.5 cm.) and were ted daily on freshly hatched Artemia nauplii (cj. Loomis and Lenhoff, 1956). The hydra were kept at ambient laboratory temperatures (21-24° C.) and illumination ; additional illumination was provided continuously from a 40- watt fluorescent light (Sylvania-Cool White) kept 15 cm. from the center of a stack of 2-Ae trays. Stock cultures attaining a maximum of 4000 hydra/ 1500 ml. of culture solution were thinned and cleaned weekly by pooling the animals, wash- ing them in deionized water and finally re-distributing 600-800 hydra (1200- 1500 hydranths) to a tray containing 1500 ml. of clean culture solution. Through- out the course of these experiments, C. viridissima strain 1960 reproduced ex- clusively by asexual budding. Hydra used in experiments were sampled from asexually-reproducing stock cultures during the logarithmic growth phase as illustrated in Figure 1. The maximum specific growth rate constant (A'm;ix) for the stock population compares favorably with those values obtained for the growth rates of small experi- mental cultures (compare Fig. 1, Table II). Each sample consisted of duplicate groups of five hydra, each with a bud in an early stage of development, that were starved one day prior to the experiment. These animals can be defined as five "uniform" hydra (Lenhoff and Bovaird, 1961) or ten hydranths. Strict adherence to these sampling criteria was essential for reproducible experimental results. Growth was measured by the procedure of Loomis (1954). Five uniform hydra were placed in 30 ml. of culture solution in a 10-cm. (diameter) Petri dish placed 10 cm. from a single 40-watt fluorescent light (Sylvania-Cool White). TABLE I Growth of green and albino C. viridissima in unmodified and modified culture solution Culture solution Number of hydranths on day Growth rate k 1 2 3 4 s 6 A. "M" solution Green 10 15 25 32 50 01 0.4.U Albino 10 16 26 .?8 5,? 87 0.4, M magnesium ions always enhanced growth rates. Although growth occurred without potassium (Fig. 2d) its presence in the medium markedly enhanced the general appearance of the polyps and the re- producibility of growth curves. Average length of tentacles increased about three- fold when potassium was present. High concentration of calcium and potassium inhibited growth (Fig. 2a, d), while similar high concentrations of sodium or magnesium were without effect (Fig. 2b, c). The general growth response of green and albino C. viridissima to all ions tested was nearly the same with two exceptions: (1) albinos appeared more sensi- tive to lack of environmental sodium, disintegrating several days earlier than green hydra, and (2) green hydra produced significantly fewer buds than did albinos at high calcium concentrations. .hiions and other jactors Growth increased slightly but not significantly when NaCl was substituted for NaHCO3 (Table Ib), but HCCV was retained in all subsequent experiments to insure an ample external carbon dioxide pool for the algae. \Yhen penicillin was added to stock cultures (Table Ic), the growth rate in- creased slightly. Glycerine (0.068 .17) decreased the growth rate considerably (Table Id), and was used only to eliminate symbiotic algae. Growth rate under standard conditions Table II summari/es the growth rates of C. t'iridissuna measured in out- laboratory prior to July, 1962. Standard conditions are defined as growth in lab- oratory culture solution, pll 7.6, 21-24° C., continuous illumination (see Meth- ods), daily feeding on freshly hatched live Artcinia nauplii, twice-daily changes of the culture solution, constant volume of culture solution per vessel and initiation of growth experiments with uniformly developed hydra of known nutritional history sampled during the logarithmic growth phase of stock populations. The 54 measurements include data for 42 groups of green and 12 of albino C. viridis- sima. Under these conditions, albinos grow at about the same logarithmic rate as green individuals. Using an analysis of hydroid growth measurements as set forth by Fulton (1962), the standard deviation of the mean growth rate of the whole group (54 measurements) is ±0.06; the range, 0.20. For each individual set of experiments (21 measurements) the standard delation of the mean is r^O.03 (calculated from SYMBIOSIS OF HYDRA AX I) ALGAE. I. 421 ranges), the range, 0.11. Thus, the variation encountered from one group of experiments to another is about twice that encountered among replicates within any single experiment, indicating that conditions within each set of experiments were relatively constant. In 20 of 21 experiments, the growth rate of replicates differed from each other by 0.08 or less. In one experiment a difference of 0.11 was observed. From these data we may estimate that 95 % of the time a growth rate difference of 0.08 or more between cultures is significant. DISCUSSION The results demonstrate that in a solution containing calcium, sodium, mag- nesium, potassium, chloride, and bicarbonate in the concentrations described, growth of C. I'iridissima, Carolina strain 1960, is exponential with a mean k of 0.42 ± 0.06. This represents a doubling time of 1.45-1.90 days. This is the TABLE III Comparison of ionic requirements for growth of several hydroids in laboratory culture. Table modified from Fulton (1962) H. littoralis* C. laciistris C. viridissima Growth curve exponential exponent ial exponential k 0.37 0.21 0.42 Cat ion-; required Ca++ HO-* .17) Na+ (10-6 M)** Ca++ (10-3 M) \a+ (1C)-3 .17) K+ (K)"3 .17 ) Ca++ dO-' .171 \a++ (10-« M) Cations \vhicli rnhanrv growth but arc not required K-*** Mg++ K+, Mg++ * Data of Loomis (1954). ** Data of Lenhoff and Bovaird (1960). *** Data of Lenhoff (unpub.). fastest mean growth rate thus far encountered among laboratory-grown hydroids for which there are comparable data (see Table III) and in part reflects the smaller size of C. viridissima. The observation that the mean growth rates of green and albino C. viridissima are nearly identical under the conditions described suggests that the algae are not essential for growth at £mnx. This observation is dealt with in the next paper in this series. Using data of Loomis (1954) and Fulton (1962) some growth features of H \drci littoralis (a stream-dwelling non-symbiotic species, about 3-4 times larger than C. viridissima} and Cordylophora lacustris (a colonial hydroid typically found in fresh to brackish situations) are compared with those of C. viridissima in Table III. All have been grown in the laboratory under controlled conditions. All three hydroids require calcium ions in the environment for growth, although the minimal required concentrations vary with species. In the absence of calcium, the animals disintegrate very quickly, reflecting the fundamental role of this ion in maintaining cell and tissue integrity. At low levels of calcium, other factors 422 LEONARD MUSCATINE AND HOWARD M. LENHOFF which may ad\ rrscly affect growth arc (1) loss of calcium-dependent contractility necessary for positioning body and tentacles in prey capture and feeding; (2) failure of nematocysts to discharge even when contacted by live Artemia nauplii ; (3) inability to carry out the feeding reflex for which environmental calcium is required (Lenhoff and Bovaird, 1959). The animals are thus effectively pre- vented from feeding and obtaining additional calcium from food. C. viridissima also shares with Hydra littoralis and Cordylophora an absolute requirement for sodium in the external environment. Only trace amounts of sodium appear necessary for near-optimum growth of the fresh-water hydrids (H. littoralis and C. viridissima ) , while Cordylophora, as expected from its habitat, tolerates a relatively higher environmental sodium concentration. Environmental potassium ions are not an absolute requirement for growth of C. viridissima or Hydra littoralis, but when present, even at low concentration (10~G M) enhance the general appearance of polyps and the level of reproducibility of growth rates. The dramatic increase in length of tentacles observed when potassium is introduced into the medium may reflect the role of this cation in nerve and muscle irritability. Since tentacle length: body length ratio is often used as a taxonomic character in identification of hydras (Hyman, 1929), standardized culture media may help eliminate uncertainty in this parameter. Although environmental magnesium definitely enhances growth of C. viridis- siina (cf. Muscatine, 1961), it is uncertain as to whether or not it is absolutely required since maximum growth was occasionally observed in the absence of this cation. Magnesium also appears to be "less critical" for Cordylophora (Fulton, 1962). Acquisition of magnesium from food may offset a deficiency in the medium and thus interfere with the reproducibility of the effects of absence of environmental magnesium on growth. A magnesium requirement for C. viridis- shna is apparently not primarily related to a requirement by the algae since the growth rate of albinos is also lower in the absence of magnesium. Inhibition of growth of C. viridissima at high concentrations (10~- M} of calcium and potassium was probably not an osmotic effect, but more likely related to competition with other ions since similar osmolar concentrations of sodium and magnesium did not inhibit growth. Sodium-potassium competition is a well- known phenomenon, and has been demonstrated in hydroids by Fulton (1962) in growth experiments using Cordylophora. There is evidence that symbiotic algae may affect mechanisms of salt and water balance of the host. For example, in these studies albino C. viridissima disintegrated several days sooner than green C. viridissima in the absence of sodium. The same effect was observed in a medium also lacking in magnesium (Lenhoff and Bovaird, 1(V>0). On the other hand, albinos were less sensitive to high calcium compared to green individuals. Karakashian (1963) noted that algae-free Parawccium bursaria crenated and died within a few hours after in- oculation into an inorganic salt medium in which normal green individuals could be maintained. Hood (1927) observed that specimens of Frontonia Icucas, another ciliate harboring symbiotic algae, lived indefinitely in a \% dextrose solu- tion changed daily, and for several days when adapted to 3% dextrose. In con- trast, algae-free individuals averaged two days' survival in \% dextrose and died within a few hours in more concentrated solutions. However, in distilled water SYMBIOSIS OF HYDRA AND ALGAE. I. 42,:» green specimens disintegrated immediately while those without symbionts sur- vived for several hours. Part of this investigation was carried out at the Laboratories of Biochemistry, Howard Hughes Medical Institute, Miami, Florida, during the tenure of a Post- doctoral Fellowship from the Division of General Medical Sciences, United States Public Health Service (9653) to Leonard Muscatine, and an Investigator Award of the Howard Hughes Medical Institute to Howard M. Lenhoff. We thank Mr. John A. Bovaird, Mr. Alfredo Lopez, and Mr. Enrique Nagid for technical assistance. SUMMARY 1. Under controlled conditions in the laboratory, with daily feeding, Chloro- hydra viridissima grew exponentially in a solution containing calcium, sodium, magnesium, potassium, chloride and bicarbonate ions. The optimum concentra- tion of cations was ascertained and the effect of their deficiencies noted. The results are compared with data for Hydra littoralis and Cordylophora lacustris. 2. Calcium and sodium ions were required for growth. Magnesium and po- tassium enhanced growth and reproducibility of growth rates. Bicarbonate was not essential. 3. Both symbiotic and aposymbiotic C. viridissima grew at nearly identical rates, doubling in number every 1.45-1.90 days. In the absence of environmental sodium aposymbiotic hydra disintegrated several days sooner than normal green individuals, while growth of the latter was inhibited by high concentrations of calcium in the environment. LITERATURE CITED BKIEN, P., AND M. RENIERS-DECOEN, 1949. La croissance, la blastogenese, 1'ovogenese chez Hydra fusca (Pallas). Bull. Biol. Prance et Bclg., 83: 293-386. BKIEN, P., AND M. RENIERS-DECOEN, 1959. Etude d' Hydra riridis (Linnaeus) (la blastogenese, la spermatogenese, 1'ovogenese). Ann. Soc. Roy. Zool. Belgiqne. 81: 33-110. BURNETT, A., AND M. GAROFALO, 1960. Growth pattern in the green hydra Chlorohydra viridissima. Science, 131: 160. DROOP, M., 1963. Algae and invertebrates in symbiosis. In: Symbiotic Associations. Soc. Gen. Microbiol. Symp. no. 13. B. Mosse and P. Nutman, Eds. Cambridge, pp. 171-199. FULTON, C. H., 1962. Environmental factors affecting growth of Cord\lof>hora. J. Exp. Zoo!., 151:61-78. GOETSCH, W., 1924. Die Symbiose der Siisswasser-Hydroiden und ihre kunstliche Beeinflussung. Zcitschr. Morph. Okol. Tierc, 1: 660-731. HAFFNER, K., 1925. Untersuchungen iiber die Symbiose von Dalycllia viridis und Chlorohydra viridissima mit Chlorellen. Zcitschr. /. iviss. Zool., 126: 1-69. HAM, R. G., D. C. FITZGERALD AND R. E. EAKIN, 1956. Effects of lithium ion on regeneration of hydra in a chemically defined environment. /. E.vp. Zool., 133: 559-572. HOOD, C. J., 1927. The zoochlorellae of Frontonia Icitcas. Biol. Bull, 52: 79-88. HYMAN, L. H., 1929. Taxonomic studies on the hydras of North America. I. General remarks and description of Hydra amcricana, new species. Trans. Amcr. Microsc. Soc., 48: 242-255. KARAKASHIAN, S., 1963. Growth of Paramccium hursaria as influenced by the presence of algal symbionts. Physiol. Zool., 36: 52-67. LENHOFF, H. M., AND J. BOVAIRD, 1959. The requirement of bound calcium for the action of surface chemoreceptors. Science, 130: 1474. 424 LEONARD MUSCATINE AND HOWARD M. LKNHOKI- I.KMIOKF, H. M., AND J. BovAiRD, 1960. The requirement of trace amounts of environmental sodium for the growth and development of Hydra. Exf. Cell Res., 20 : 384-394. I.KMIOFF, H. M., AND J. BovAiRU, 1961. A quantitative chemical approach to problems of nematocyst distribution and replacement in Hydra. Dci'el. Biol., 3: 227-240. I.OOMIS, W. F., 1954. Environmental factors controlling growth in hydra. /. Exp. Zool., 126: 223-234. LOOMIS, W. F., AND H. M. LENHOKF, 1956. Growth and sexual differentiation of hydra in mass culture. J. Ex p. Zool., 132: 555-574. MUSCATINE, L., 1961. Symbiosis in marine and fresh water coelenterates. In: The Biology of Hydra, H. M. Lenhoff and W. F. Loomis, Eds., University of Miami Press, Miami, Florida, pp. 255-268. WHITNEY. D. D., 1907. Artificial removal of the green bodies of Hydra viridis. Biol. Bull., 13:291-299. \VIIITXEY, D. D., 1908. Further studies on the elimination of the green bodies from the endoderm cells of Hydra riridis. Biol. Bull., 15: 241-246. WOOD, R. L., 1959. Intercellular attachment in the epithelium of Hydra as revealed by electron microscopy. /. Biophys. B Indian. Cytol., 6: 343-352. EFFECTS OF X-IRRADIATION OF MICE EXPOSED IN UTERO DURING DIFFERENT STAGES OF EMBRYOLOGICAL DEVELOPMENT ON DURATION OF MATURE LIFE ' lutXALD J. NASH- AND JOHN \Y. <;o\YKXs In an carlic-r paper (Nash and Go\ven, 1962) we have shown that postnatal growth, as measured by weight changes to 75 days, following x-ray irradiation in ntero, was dependent on both the levels of irradiation and the embryological ages at the time of irradiation. The embryological ages in order of increasing sensitivity were 64, 17.1, 1-H and 101 days. Growth was measured for the periods from mating to birth, and to 12, 26, 40, 60 and 75 days postnatal ages. Body weight response was found to be noticeably dependent upon the age at which observations were recorded. Treatments that produced significantly lowered body weights did not usually show maximum effects until 40 days after birth. Growth changes, expressed as continuing reductions of the body weights with days post-parturition, were most obvious at 75 days of age. The reductions were greatest for the 320 r- treated mice, the lowering of the growth rates being dose-dependent. The data on which these observations were made have one common property ; the mice were alive at the time when the observations were initiated. This results in the population under observation being biased toward greater viability. This limitation is intrinsic at whatever level of development the data on the irradiation effects are taken. To, in part, reduce this difficulty in evaluating the irradiation effects on the animal's subsequent well-being, several other characteristics, such as anatomical changes in young born dead, lifetime fecundity and fertility and various parts of the lifespan. have been utilized for measuring as yet unrecognized physio- logical changes in those exposed to irradiation. This paper deals with those changes, which may be immediate or long delayed, which affect lifespan after the beginning of the reproductive period. Lifespan is a unique instrument in that it integrates the effects of external as well as internal agents influencing life processes. It covers both the short-term and long-term effects as well as the constitutional characteristics of those exposed. It has more variation, both innate and accidental, than other measures so differences between agents believed to affect life become harder to demonstrate. Yet it is in this characteristic most of us are interested. Irradiation of the mammalian embryo exposes rapidly dividing cells during the intense process of organ formation and differentiation. It is a time when 1 Journal Paper of the Iowa Agricultural and Home Economics Experiment Station, Ame--, Iowa. Project Nos. 1180 and 1187. This work has received assistance from Contract AT( 11-1) -107 from the Atomic Energy Commission, and Grants RH328 and Al-06239 National Institutes of Health. - Department of Zoology, Rutgers University, New Brunswick, New Jersey. :; Department of Radiology and Radiation Biology, Colorado State University, Fort Collins Colorado. 425 426 DOXA1.1) J. XASH AX!) JOHX \V. COXYF.X gene and chromosome changes would most likely result from single acute irradiation. Mitotic changes in single cells should grow +o islands of tissue having functions different from those of the rest of the body complex or organ. The changes in the compatibility of the functioning units can influence those of the body as a whole and be expressed in changes in life's duration (Go wen, 1962). MATERIALS AND METHODS The embryos irradiated were obtained exclusively from first pregnancies in matings within and among three of our long-inbred strains of mice. After mating, the mothers marked for subsequent irradiation were examined daily for the presence of a vaginal plug. The appearance of this plug was considered the time of fertiliza- tion and the start of the events covered by gestation. Embryo ages were calculated from this point. All mice were considered to have mated during the same time at night, with 4 AM as the most likely point for the union of sperm and egg. Irradia- tions were begun at 4 PM ; embryological ages at irradiation were set at 6|, 1(B, 14^ and 17i days. In a later series, irradiations of newborn young were indicated as of 19^ days of age. These mice add breadth to the observations in that they alone are irradiated, whereas both mothers and young are treated at the earlier ages with the possibility that a maternal effect may be added to the direct effect of irradiation on the progeny. The inbred females irradiated were of strains long under study and differentia- tion at the Genetics Laboratory of Iowa State University. The major criteria of differentiation have been relative resistance to several diseases of bacterial or virus origin, chemical poisons and radiant energy (Schott, 1932; Gowen and Schott, 1933a, 1933b; Hetzer, 1937; Gowen and Zelle, 1945; Gowen, 1955a. 1955b; Gowen and Stadler, 1956; Stadler and Gowen, 1957 a, 1957b, 1957c ; Gowen, 1960; Stadler and Gowen, 1961, 1963). The Committee on Mouse Nomenclature has designated the strains as BALB/Gw, K and S. The three strains cover a wide range of susceptibility to radiation. The BALB/Gw (hereafter called Ba) and K strains are relatively susceptible to acute irradiation, whereas the S is relatively resistant. All progeny were weaned at 30 days of age and males separated from the females. At 75 days of age mice were individually mated to non-irradiated mice of the Z strain. This strain has an entirely different origin than the Ba, K or S. It is noted for regularity, frequency and size of litters. Previously unmated Z males or females were used in these matings. The treated animals were mated to Z mates throughout their lives. To have balanced numbers of mice tested for lifespan and reproductive performance, two males and two females were selected when possible from each treatment group. The after-mating lifespans of these treated animals start at the day of mating and run to the day of death. The full lifespan from birth of any mouse is determined by adding 75 days to the mating-to-death interval. If a litter did not contain two males and two females in each treatment group, an additional litter from another female, raided to 75 days, was used to obtain the addi- tional mice to complete the cell, two each of treated males or females. When a Z mate died before its treated partner it was replaced within a few days by another virgin Z mouse of an age between two and six months. All matings were retained for the lifetime of the treated mouse. X-RADIATIOX EFFECTS OX MATURE LIFE 427 All niatings were observed daily and the following information taken : (a) life- span of each treated mouse, (b) total number of litters, (c) number of progeny within litters, (d) birth weights of individuals, (e) viability of mice within litters from birth to 21 days, and (f) any external anomalies which the mouse might carry. Irradiation exposures were chosen to cover the range from none to 320 roentgens, the highest exposure that would allow survival of at least some of the embryos in the embryonic age series. There were five exposures, 0, 20, 80, 160 and 320 r. Five stages in embryological development were treated with the chosen dosages of irradiation. The embryos treated were either males or females of which samples of two each were chosen. The genotypes were divided into nine groups ; three represented the pure inbred strains, six their possible hybrids made recip- rocally. The design is a factorial in which the treatments are regarded as fixed points. Unfortunately, of the mice treated with 320 r at 6.1, with 160 r and 320 r at lOJ-, none survived for lifespan determinations, and of the 320 r at 144 embryos only three progeny remained for determining lifespans for this group. In consequence, the design as a whole loses its orthogonality. However, the greater portions of the data may be arranged in different designs to retain the orthogonal principle even though they require separate analyses. A General Electric Maxitron operated at 250 pkv, 30 ma with 0.25 mm. Cu + 1 mm. Al filtration at a distance of 50 cm. from anode to mid-mouse, dose rate 133 r/minute, furnished the x-rays for the treatments. Dose was measured in air by a rate meter at a position corresponding to that of the mid-mouse. Pregnant mice were exposed to single closes of whole-body irradiation in circular wooden con- tainers 6^ inches in diameter and 1 inch deep. The base of the container was -]- X $ inch hardware cloth. The top was covered with two layers of cellophane. Newborn mice of a whole litter were irradiated whole body in small plastic trays and then immediately returned to their mothers. Following custom, all roentgen dose measurements were taken in air at the mid-position the mouse would occupy- when irradiated. These dosages do not represent the energies actually reaching different organs of the exposed mouse. They are subject to different adjustments dependent on the particular tissue and absorptions in different body tissues having characteristic atomic densities. If, for instance, the roentgen dose of 250 pkv, 30 ma filtered through 0.25 mm. Cu and 1 mm. Al to an adult female mouse is measured as 1 r, the dose when absorbed in passing through bone and back muscles to the ovaries becomes about 0.85 r at the position of the ovaries. The reader should take these effects into account. They will be discussed at some later date. The mice throughout were kept in a well ventilated room, in which the environ- ment and management were relatively constant. Compressed food pellets and water were before the mice at all times. FREQUENCY DISTRIBUTIONS OF PosT-75-DAY LIFESPANS AS AFFECTED BY SEX TREATED, PREVIOUS EARLY IRRADIATION EXPOSURES WHILE IN UTERO OR AT BIRTH, EMBRYOLOGICAL AGES AT TREATMENTS, AND GENOTYPES OF STRAINS TREATED Frequency distributions, even though based on rather small numbers, assist in evaluating the effectiveness and mode of action of the different treatment agents on the adult lifespans of those exposed. These distributions, for treated males and for treated females, are shown at the top of Figure 1. 428 DONALD I. XASI1 AND lOIIX \Y. COYVF-X 15 10- 10 500 1000 I-'KICUK 1. Frc INBRED AND RECIPROCAL CROSSES P,a x Ba x Ba K :< K K x Y ' x K i S x S 500 1000 S x Y 0 500 1000 MATURE LIFESPAN IN DAYS 500 1000 FIGURE 4. Percentage frequencies of deaths plotted on the different 100-day age intervals after mating for combined inbred mice of three strains and their possible hybrids are shown at the top of the chart. The individual inbreds and their possible crosses are plotted in the lower nine histograms, inbreds on the left. Crosses for the males of the same inbred in the middle, i' representing the averages of the two crosses. On the right the constant parent is the female inbred of the strain at the left. X-RADIATION EFFECTS ON MATURE LIFE 433 TABLE I Constants for frequency distributions Mean Median Standard deviation 0, fa t for gi t for g» Sexes Males 540 ± 14 568 249 ± 8 0.0178 2.405 1.0 2.2 (1) Females 332 ± 12 299 224 ± 6 0.1627 2.095 3.0 (4) 3.3 ( li X-ray exposure1 0 r 179 ± 29 505 243 ± 15 0.0416 2.043 0.7 1.7 20 r 474 ± 19 487 250 ± 11 0.0000 2.308 0.0 1.8 80 r 465 ± 19 468 257 ± 10 0.0078 1.991 0.5 2.7 (3) 160 r 438 ± 21 424 257 ± 14 0.0942 2.621 1.5 0.9 320 r 223 ± 22 157 189 ± 19 1.6021 3.946 4.6 (4) 2.0 (1) Embryological age untreated 545 ± 40 601 242 ±23 0.4270 2.328 1.7 0.8 65 days 507 ± 23 519 240 ± 14 0.0086 2.512 0.4 1.0 101 ciavs 493 ±32 518 276 ± 18 0.0002 2.163 0.5 1.4 14 1 days 461 ± 24 493 254 ± 13 0.0046 2.213 0.3 1.7 17J days 404 ± 23 390 280 ± 12 0.1550 2.112 2.0 (1) 2.2 (1) Newborn 360 ± 16 337 220 ± 10 0.2391 2.487 2.7 (3) 1.4 Strains Ba X Ba 414 ± 29 421 246 ± 14 0.0017 1.922 0.1 1.9 Ba X K 448 ± 31 428 261 ± 15 0.0091 1.933 0.3 1.9 Ba X S 491 ± 29 551 245 ± 15 0.1864 2.131 1.5 1.5 K X Ba 456 ± 31 433 260 ± 18 0.0350 2.304 0.7 1.2 K X K 451 ± 32 404 274 ± 21 0.3833 2.718 2.2 (1) 0.4 K XS 397 ± 31 332 262 ± 15 0.2189 1.983 1.7 1.8 S X Ba 445 ± 28 435 238 ± 15 0.0112 2.211 0.4 1.4 S X K 464 ± 33 514 279 ± 17 0.0159 2.046 0.5 1.7 S XS 359 ±27 332 231 ± 12 0.0170 1.818 0.5 2.1 Inbreds 407 ± 17 398 254 ± 1 1 0.1146 2.492 2.1 (1) 1.5 Hybrids 450 ± 12 448 260 ± 6 0.0103 2.035 0.9 4.1 (4) Reciprocal cn^-i-s Ba X Y 470 ± 21 499 254 ± 10 0.0250 1 .958 0.8 2.5 (2) Y X Ba 451 ± 21 435 250 ± 12 0.0250 2.290 0.8 1.7 K X Y 427 ± 22 390 263 ± 1 1 0.1026 2.099 1.6 2.2 (2) Y X K 456 ± 23 448 270 ± 11 0.0140 2.011 0.6 2.4 (2) S X Y 154 ± 22 463 260 ± 12 0.0186 2.163 0.7 2.1 (2) Y X S i 444 ± 22 461 258 ± 10 0.0006 1.802 0.1 3.0 (4) Probability levels (1) = 0.05, (2) = 0.025, (3) = 0.01, (4) = 0.005 or less. spans is positively skewed, the kurtosis being evident. Both skewness and kurtosis are significantly different from those of the "normal" curve. The frequency curves for other subdivisions of the data show lowering of the mature lifespans with increasing dosages of the x-ray energy as well as differences between its effects in the different stages in the embryological life when the radiation was received. Inbred mice showed shorter durations of life than hybrids between the strains, and both inbreds and hybrids showed variations dependent on their origins. Negative and positive skewness was observed in the curve forms. Save 434 DONALD J. NASH AND JOHN W. GOWEN for one case for mice treated with 320 r, the kurtosis was uniformly negative, reach- ing significance in 1 1 out of the 30 distributions. The differences exerted by the different variables show that analyses of the effects on the mature lives of the preadok scent mice must IK- analyzed separatelv for within sex, dosages and embryological ages of treatment. INFLUENCE OF SEX, X-RAY DOSAGES AND EM J:RYOLO<;ICAL A<;ES \YHEN THE IRRADIATIONS WERE RECEIVED ON MATURE LIFESPAN Table II presents means and standard deviations for the lengths of the later lifespans distributed for embryological age at irradiation, dose of x-ray exposure and sex of the parent treated. The mean day survival is followed by its standard deviation. The standard deviations of the distributions of the raw data follow. In every instance the after-mating lifespans of the males are noticeably longer than those of the females whatever may have been the time at which the developing embryos were irradiated, or the amount of irradiation to which they were exposed. This major factor in these lifespan differences, in consequence, is not attributable to the irradiations or the embryological ages at which irradiation occurred. They seem to turn on innate biological differences in the risk of death to the parent in carrying on reproduction. This risk is much greater to the females than to the males. The results support our earlier work in showing that pregnancies spaced at short intervals result in large reductions in the lifespans of the females, whereas the lifespans of the males are nearly unaffected (Gowen, 1960). Irradiations with 20 and 80 r occurring at the different in iitero periods showed little effect on the difference in life of the sexes, the females for the same treatments being about 0.6 those of the similarly treated males. A lengthening of the female lifespan occurred when the female was treated with 80 r as a newborn. MATING-TO-DEATH SURVIVAL FOR MICE IRRADIATED IN UTERO IN RELATION TO DOSK RECEIVED Figure 5 plots the mean after-mating lifespans of male and female mice dis- tributed within the embryological ages at which the irradiations were given and by the dosage of irradiation they received. The curves for the intrauterine irradiated mice were similar in form. Sex differences in the lengths of the lifespans were marked. Doses to and including 160 r of acute irradiation caused a 72-day loss to males and a 1 19-day loss to females in their average durations of life, 1 1 % and 27%, respectively, of their expected survivals after mating. The declines in the adult lifespans were irregular but in general followed trends which approach linearity. The reductions in life duration occurred even though the animals had 75 days in which to recover before measurements of the irradiation effects were begun. The groups were selected groups which had eliminated the rapidly fatal irradiation effects. After the 160-roentgen dosages the effects on later life survival became more severe. Lifespans for mice exposed to 320 r intrauterine were reduced to 43% and 30% of those enjoyed by untreated male and female mice, respectively. Figure 5 separates the effect of irradiations at different periods in the uterine development. The durations of adult life of inalc^ exposed at 6\, lOf, 144 and X-RADIATION EFFECTS ON MATURE LIFE 435 TABU-: II Mean dnys and standard errors mating to death Embryological age at irradiation Irradiation dose Sex treated Mean Standard deviation I 'n treated 0 r Male (M) 646 ± 49 208 Female (F) 443 ± 57 242 65 days 20 r M 597 ± 55 233 F 373 ± 56 237 80 r M 637 ± 55 234 F 383 ± 54 228 160 r M 576 ± 58 246 F 472 ± 34 143 10* 20 r M 616 ± 47 201 F 368 ± 63 268 80 r M 586 ± 70 297 F 401 ± 63 269 14! days 20 r M 620 ± 33 141 F 414 ± 57 244 80 r M 590 ± 48 210 F 313 ± 49 206 160 r M 596 ± 60 256 F 234 ± 43 184 320 r M 254 F 35 17| days 20 r M 668 ±55 232 F 359 ± 49 210 80 r M 591 ± 63 266 F 363 ± 50 212 160 r M 551 ±65 275 F 267 ± 48 202 320 r AI 284 ± 61 259 F 1 50 ± 39 165 Newborn 20 r M 447 ±41 174 F 275 ± 51 216 80 r M 440 ± 50 211 F 350 ± 50 213 160 r M 466 ± 62 263 F 335 ± 49 209 320 r M 289 ± 35 151 F 169 ± 31 133 Newborn 0 r M 518 ± 52 220 F 309 ± 46 194 days of uterine development are quite comparable. The lifespans for males treated as newborns are shorter than expected for the 0, 20, 80 and 160 r treatments. Females irradiated at 144 days uterine development with 160 and 320 r had their durations of life noticeably reduced. Newborn females either untreated or exposed to 20 r showed noticeably shorter lifespans than expected. No explanation is known 436 DONALD .1. XASH AND JOHN W. GOWEN which will account for this difference. The newborn group was selected for study some time after the other treatment groups, but the mice in the group were, so far as known, similar to the others previously treated with irradiation when they were chosen. 650 550 450 350 250 400 300 200 100 \ Males Females 20 160 DOSE IN ROENTCENS 320 K . Mature life-spans of mice treated ;;/ utcro with 0, 20, 80, 160 or 320 roentgens plotted for the given unhryolugical ages when the irradiations were absorbed. Embryo- logical ages in days at treatment \\ere designated by the following lines: 6i ; 10J ; 14^ -; 17i ; newborn mice - — , and untreated - — . X-RADIATION EFFECTS OX MATURE LIFE 43; Q a H .3 Q ia s- H O M f-c .7 .6 .5 .3 Male Treated Female Treated 6 1/2 10 1/2 14 1/2 17 1/2 New- born FIGURE 6. Lapsed days of life between mating to death of mice exposed at different embryo- logical ages to irradiations of given dosages divided by the survivals of the corresponding unexposed mice. The dose of irradiation is indicated as follows: 20 r — ; 80 r ; 160 r ; 320 r . The lower lifespans of the females are evident in all dosages and embryological ages and strains. For the in utero treated mice the average unirradiated females survived for only 69% of the mean male lifespan. This male-to-female difference is attributed to the liaxards which differentially affect the physiological processes 43S DONALD J. NASH AND JOHN W. GOWEX which surround reproductive functions. If the average female life under the different types of irradiation is adjusted for this difference to what would be expected for the male, the female still retains more susceptibility to irradiation than the male. Considering the available data for the dosages taken separately, the 20 r females had 85% of the mature mean lifespan observed for the untreated females, the males 96.7% that of the untreated males. Corresponding comparisons for the other dosages yield for 80 r. 82 vs. 93; 160 r, 73 vs. 89; and 320 r, 30 vs. 43% for females vs. males, respectively. Comparisons of the sensitivity of the factors which make for longevity in embrvos at different growth stages when irradiated are made difficult because all j mice exposed to 320 r at 61, 101. as well as those in the 160 r, 10.1-day embryo age groups, failed to reach the mating age. Only two males and one female lived beyond the 75-day date for mating in the 320 r 141-day treatment group. These mice survived 31, 478 and 35 days following mating. While these data confirm the sensitivity of these mice to irradiation at these dosages and stages, they failed to give a group by which the effects of irradiation at these dosages and embryological stages may be determined in the reproductive and senescence periods of life. The two males and one female in the 320 r, 141 embryological age group indicate that the life shortening would be severe. Figure 6, as the companion graph to Figure 5, brings out the contribution of embryological age to sensitivity toward high energy irradiation. The results are determined as the ratios of mature lifespans of irradiated males or females to those of the unexposed mice of like sex. They are plotted on the embryological ages when the irradiations were received. Full data on the separate sexes were obtained on the 0 r, 20 r, and 80 r for each embryological age. Curves for the 160 and 320 r are broken for the reasons previously indicated. Unirradiated mice on the average had longer lifespans between mating and death than those which were irradiated. For mice irradiated with 20 r and 80 r the mature lifespans change but little over the span during which embryological x-ray exposures occurred. The female adult lifespans were depressed somewhat more than those of the males. Dosages of 160 r and above tended to further reduce the days the mice survived. When the irradiations reach 160 r at 10^ days embryo- logical development or 320 r at 6i, 10i days, the development of all mice was interrupted before they reached the mating age. That these early uterine irradia- tions would have caused severe damage to after-mating life was shown by the fact that the two males and one female which did survive this dose at 141 days died early, 31 and 478 days for the males and 35 days for the female. REPRODUCTIVE AND SENESCENT LIFESPANS OF MALE AND FEMALE MICE FOLLOWING IRRADIATIONS OF DIFFERENT DOSAGES RECEIVED AT DIFFERENT PERIODS OF EMBRYOLOGICAL DEVELOPMENT The mean reproductive lifespans of the males were noticeably longer than those of the females in these data. The comparative differences are brought out in Figure 7. Figure 7 shows a rather constant relation between the lifespans of female to male mice for those treated with x-rays of 20 and 80 r over the different embryo- logical periods. The severity of the effects at 160 and 320 r for the 61, 101 and 14J X-RADIATION EFFECTS ON MATURE LIFE 439 .6 a 3.7 a .6 a 2 .5 .3 6 1/2 10 1/2 14 1/2 17 1/2 New* born FIGURE 7. Ratios of the mature lifespans of females to those of males for the same irradia- tion exposures, plotted against the embryological ages when treatment took place. Radiation dosages : 20 r — ; 80 r ; 160 r ; and 320 r . periods of gestation indicate that for higher dosages of x-ray, differences in the lethal or semilethal capacities do appear as embryological development unfolds. The individuals which survive to adulthood bear these unseen effects. The effects were more severe on the female than on the male mice. ANALYSES OF THE VARIATIONS OBSERVED IN MATURE LIFESPANS In these data the mature lifespans arc regarded as dependent on measurable events occurring some 75 or more days earlier, in the interim conception-to-birth, than when the mature lives took their origin. These events are the characteristics of the basic mouse genotypes and sexes, which compose the mouse population, acted upon by the different dosages of irradiation occuring at specific embryological stages of development in the life cycle. In symbols the equation describing the relationship is of the familiar linear type. The plan of the study as indicated earlier 440 DOXAI.I) J. NASH AXD JOHN* W. GOWF.X TABI.I 1 1 1 Duration of mature life as related to exposure to high energy irradiations at different stages of embryonic development Kmbryologieal age (1T) in days at exposure to irradiation Source of variation 61 10i 14* 17} Newborn df M.S. P df M.S. P df M.S. P df M.S. P df M.S. P Dose (T) 3 230 2 3 .'5 3 1261 (3) 4 6134 (4) 4 2066 (4) Strain (G) 8 227 8 435 8 1375 (4) 8 895 (2) 8 1640 (4) T X G 24 624 10 995 (1) 24 564 32 846 W 32 808 W Sex treated (F) 1 13718 (4) 1 12171 (4) 1 24748 (4) 1 24098 (4) 1 9344 (4) T X F 3 398 2 96 3 506 4 430 4 194 G X F 8 551 8 797 8 292 8 986 (2) 8 226 GTF 24 547 16 514 24 444 32 403 32 274 I'naccounted for iK) 72 469 54 548 72 346 90 383 90 216 Dosage classes 0, 20, 80. 160 r 0, 20. 80 r 0, 20. 80, 160 r 0, 20, 80, 160, 0, 20. 80, 160, 320 r 320 r df = degrees of freedom. M.S. mean square -=-100. P probability, level of significance utilizing the unaccounted for variance (/i) as the error term for fixed variates. P probability levels, 1 = 0.05, 2 = 0.025, 3 = 0.01, 4 = 0.005 or less. was orthogonal. The biological nature of the results made the results lack ortho- gonality at several points. However, there are several desirable orthogonal arrange- ments of the data which give useful information on the effects of the different independent variables on lifespan. We shall present the two analyses which have proved most informative. The variance constants of Tables III and IV allow evaluations of the after- effects on mature life processes of mice irradiated some 75 days or one-eighth of their expected lifespan earlier. The mice in these studies have withstood the TABLE IV Duration of mature life as influenced by the embryological age when the mice were irradiated with different dosages of high energy x-rays Dose of irradiation In roentgens Source of variation 20 r 80 r 160 r 320 r df M.S. P df M.S. P df M.S. p df M.S. P HmbrvolM^ir.il aye (U) 4 1487 (4) 4 724 3 1255 (2) 1 25 Strain (G) 8 942 (3) 8 539 8 1110 (4) 8 883 (4) U XG 32 698 (4) 32 791 24 1044 (4) 8 497 (1) Sex treated (F) 1 24123 (4) 1 19245 (4) 1 17308 (4) 1 2892 (4) II 4 235 4 724 3 1400 (2) 1 7 GF 8 661 8 662 8 321 8 457 GUF 32 524 32 698 24 M)2 8 76 I Jnaccounted for t !•'. i 90 328 90 155 72 360 36 210 Embryological av.e 65, 1'H, 14-4, 62, 10J, 1-1 i, classes 171 I7i 6i, 143, 17J, I7i Newborn Newborn Newborn Newborn Symbols as in Table 1 1. X-RADIATION EFFECTS ON MATURE LIFE 441 physiological consequences over the earlier parts of their lives following the acute embryonic irradiation of different dosages. They are to that extent a group, selected by nature as being resistant. The irradiation effects observed are long- time effects. The first arrangement of the data (Table III) is analyzed within constant embryological ages, when the irradiations occurred, and the numbers of mice for the different dosages. These data form an orthogonal design. The x-ray dosages given the treated groups are shown at the bottom of Table III. In the 6| embryo- logical age treatments there were 4 dosages of x-rays, 0, 20, 80, and H>0 r, which were complete in having all cells filled in the factorial design. In the 10^-day embryological age group there were 3 dosages which had equal numbers of mice throughout, 0, 20 and 80 r, etc. Table III shows that differences in irradiation doses, in the strains of mice exposed to the irradiations and in sexes of the individuals treated, all influenced the duration of mature life when the irradiations came in the latter half of pregnancy or in the newborn. Throughout, the greatest influence on the lifespan by a wide margin was the sex of the mouse. However, none of the sex interactions with dose or strains showed significant deviations. The fact that the variances for dose of irradiation and for strains were com- parable to the random variance (£) in the 64 and 10i embryological age treatments is not to be taken as indicating these variables have no effect in causing life shorten- ing for the full possible range of the irradiation dose treatments. The 320 r dose so severely affected lifespan as to cause all those exposed to die early. The limitation of studying only after-mating lifespan prevented the irradiations from showing their full effects, as the severely injured mice all die early, in utcro or shortly after exposure. Similarly, dosages of 160 r at 104 days of embryonic age also resulted in the early deaths of all the progeny. Actually the irradiation treatments at the 10^-day age were probably the most significant to over-all life. The severity of the effects prevented them from showing in the mature aged group. The interesting fact which comes from these comparisons is that the mice can take the lower dosages at these embryological ages and yet not be affected significantly in their later life- spans, even though higher dosages are so toxic. The data on the mice treated at 104 days were self-curtailed as mice exposed to higher dosages than 80 r all died before they reached breeding age. Those treated with 80 r showed less than a 10% drop in lifespan over those which were untreated. The mice treated at 64 days showed little dose effect. For 80 r there was a 6% and for 160 r only a 4% drop in lifespan. These facts suggest that the dosage effects in utcro on life's duration after reaching adulthood would be small in the 20 and 80 r range. This point was tested by analyzing the data on the 14], 17A and new- born treated mice restricted to those exposed to 0, 20 and 80 r x-ray exposures. These analyses show that these dosages had only statistically insignificant effects although they each lowered the lifespans somewhat, 17%, 14% and 3%, respec- tively, for the 80 r treatments of the 144, 174 and newborn mice. The data may be arranged to quantitate the effects of treatments at the different embryological ages when the dosages of irradiation are fixed at 20, 80, 160 and 320 r. These data are presented in Table IV. The mean squares of Table IV show intrauterine treatments reduced sig- 442 DONALD J. NASH AND JOHN W. GOWEN nificantly the durations of mature life in two of the four dosage treatments, 20 r and 160 r. Examination of the differences from which these variances were calculated shows that the observed significance values depend on the fact that the newborn mice, both untreated and x-ray irradiated, had lower average lifespans than the mice treated in ntcro. Why this should be is not clear. The mothers of these newborn mice were bred and randomly chosen from the same strains and in the same way as those bearing the mice exposed in utero. However, omitting this group from the calculations the results show that there were no real differences among mice treated at the different developmental stages, 6^, 104, 144 and 17-J days with 20 roentgens. If anything, their mature lifespans were increased. Mice exposed to 160 r, if males, had their lives reduced in length 8% to 15%. If females, a gain in lifespan of 4% was noted if the mice were treated at 6.1, days. A loss of 18% of the control lifespan occurred in the mice treated at 14£ days of embryological age with 160 r. The effects of the x-ray exposure to 160 r at embryological ages 6-J, 14^ and 17£ days were significant even when the newborn mice were not considered in the analysis. CONTRIBUTIONS OF THE DIFFERENT COMPONENTS TO VARIANCE In the following interpretation it is assumed that the variations in lifespan are due to components acting additively. Each component is regarded as fixed, as contrasted with the assumption of randomness, as it is doubtful if any factor which is subdivided into less than 10 to 15 parts, even though selected at random, can really be considered as random in the sense of representing the whole biological population of a species or even the laboratory mouse. The considerations lead to the general equation portraying contributions of the variables influencing lifespan, Y, as Yijl;l -- u + fji + tj + (cjt)n + /,, + (/0 ),-,.. + (ft)j, + (/ (i IRRADIATION DOSE IN ROENTGENS FIGURE 8. Contributions to variance of mature lifespan in these studies, upper chart, by genotypic and sex differences in strains of mice following in utcro exposures to specific x-ray dosages received at different stages in embryological development. The effects of the different components are shown by the following lines; Genotype (G) - — ; x-ray dose (T) - — ; interaction (GT) - -; Sex (F) - -; combined sex interaction (/) ....; and unaccounted for variations generally of environmental origin (E) — . Lower chart, the variance evaluations are distributed on x-ray dose received ; G, F, I and E have like significance to those shown by the same lines in the upper chart. The effect of stage in embryological development when irradiation was received (U) is shown by - -; and the (GU) interaction effects by - — . X-RADIATION EFFECTS ON MATURE LIFE 445 Genotype also interacts with both irradiation dosage (GT} and with stage in embryological development (GU). These contributions to variance range from 10% to 30%. Individually the embryological stages when the irradiations took place have relatively small percentage effects on the mature lifespans. 0-5%. although they do have pronounced effects on the juvenile survival. 100.0 10.0 u H 100 450 800 1150 DAYS LIVED FIGVRE 9. Survival rates of male and female mature mice exposed to various dosages of irradiation during uterine development. Males — , Females - - -. There are three different interactions which include sex of the treated mouse in each of the different embryological or dosage treatment groups. Individually these effects are small and by statistical standards insignificant. These interactions are combined in the lines marked / on the charts. They approach uniform values throughout the different treatment groups. Their total contribution to variance range is between 0 and 446 DONALD J. NASH AND JOHN1 W. GOWEN SURVIVAL (YkVF.s OF MATI/RL MICF [roi. LOWING EMBRYONIC IRRADIATION The survival patti-rn> of these mice- may hi- hnnight out more fully in life curves showing the proportions of mice surviving at successive ages over their known life- spans. Figure 9 gives these data separately for the males and females in the populations. The numbers of males and females in each group are equal. Male survival rates are at all ages greater than those of the females. At the stage when the population has reached only \% of those which started as mature mice at 100 days, the males have lived 300 days more than the females. As pointed out in the earlier analyses the greater part of the survival rate differences of the sexes is attributable to physiological differences in the reproductive functions rather than to the irradiations. The form of the male survivals is like that of the females in showing noticeable curvature with age. indicating that no one agent is responsible for the life changes. 100.0 10.0 UJ 1 < i.o 0.1 100 450 800 1150 DAYS LIVED FIGURE 10. Survival rates of mature mice exposed I'M utcro to different dosages of irradiation. Zero roentgens — , 20 r - - -, 80 r — — , 160 r , and 320 r - — . X-RADIATION EFFECTS ON MATURE LIFE 447 100.0 0.1 100 450 800 1150 DAYS LIVED FIGURE 11. Survival rates of mice exposed to x-irradiation at different stages of uterine development Untreated — , 6i days gestation - - -, lOi days - — , 143 days - — , 17* days — , and newborn — . Figure 10 arranges the data to show the survival rates for the different x-ray dosages. For these data the sexes are balanced. Survival rates are shown for the 5 dosage treatments. The survival rates for the 0, 20, 80 and 160 r are all smoothly flowing curves convex to the age axis for the mature mice. Deaths are few at the beginning of maturity. They increase and become more frequent as age advances. When 50 of the cohort of 100 have died, the survival rate differences are not large but they are in order of the x-ray dosages the mice received in utero. At 850 days of age the unirradiated control mice show a decrease in survival rate of unknown cause. In general, for those mice reaching adult age, the survival rates are fairly comparable in the range of x-ray dosages of 0 through 160 r. Changes induced by 320 r irradiation while the mice are still in utero have permanent effects on survival even when the mice reach young adulthood. The 44S DONALD J. NASH AND JOHN W. GOWEX rate of survival takes the form of a simple exponential instead of the convex form of the lesser dosages. Each day of life is accompanied by a constant per- centage decrease in survival. The chart makes it obvious that above 160 r the i it Vets of x-radiation on adult lifespan increase rapidly. For 160 r, 10% of mice remain alive at 835 days while with 320 r, 10% survival is attained at 600 days or 72% of the lifespan reached by the lower irradiated mice. The detrimental actions of 320 r x-ray acute irradiations resulted in pronounced lowering of adult lifespans. These losses in expected lifespan occurred even though the mice pre- viously had passed through an earlier intense selection period eliminating those so susceptible to irradiation as to die in utcro as in the juvenile period of develop- ment. Figure 1 1 gives the survival curves of these mice distributed by the stage in embryological development when the x-raying took place. The curves cover a fair range although not as great as that observed for the extreme x-ray dosages. At the point where 50% of the mice in the original populations had died, the untreated mice had the longest survival ; the 6-J- and 10^-day treatments were next and about equal to each other; the 14^-day treatments showed slightly less survival, 17^-day noticeably less and the newborn least of all. The sequence of the sur- vivals again comes from the fact that the severely detrimental actions of 320 r and 160 r at some embryological ages have destroyed the original orthogonal character of the experiment. For the lOi-day period only, the mice treated with 0. 20 and 80 r reached the beginning of the adult stage. Similar selective action occurs for some of the 61- and 14.1 -day treated mice. Some irradiated mice can live for long periods. In each irradiated group 1% to 7% of the group lived longer than the un- irradiated controls of this study. All of the curves for the mature age lifespans for mice irradiated at the different embryological ages are convex to the age axis, indicating that the mouse characteristics operated on by the radiant energy- are numerous and interacting. DISCUSSION Lifespan is a variable made up of many composite parts, each of which must integrate perfectly with the other, as well as the environmental factors, to bring it to its full expression. It is the variable which is most likely to measure quanti- tatively on a single scale the embryological, physiological and morphological dam- age accumulated by the exposed animal. Somatic cells arising from the germ line have equal opportunities for genetic change. Without any wastage it takes some 43 cell generations (successive divisions) to develop a mouse. This number is beyond our grasp, 243 for each individual. During these divisions there are many opportunities for gene muta- tions and chromosome aberrations to occur in the same way as they appear in the germ line. Mutations notoriously require conditions of proper embryological develop- ment for their effects to receive phenotypic expression. Different thresholds of change appear throughout life but the right one must be reached for the appear- ance of characteristic effect of the given gene. Aberrant phenotypes appearing in populations of mice receiving irradiation X-RADIATION EFFECTS OX MATURE LIFK 440 during embryo development likewise follow this step-like pattern. The aberrant phenotypes observed for the same radiation dosages given at different in utero stages show changes characteristic for expected stage of development when irradia- tion took place. Seldom do they show changes related to those naturally appear- ing in later development. The threshold pattern points to necessary specific conditions in both host and treatment for the appearance of the effect. The ultimate causes of the changes in physiomorphic development affecting lifespan seem best sought from this viewpoint, rather than in hypotheses generated through indistinguishable lumping of causative components into single constants to form one of the many curves which may be theoretically generated, e.g., the Gompertz curve. The direct effects of the irradiations on the exposed cell may be on the genes, the chromosomes, the action links between the genes and chromosomes, the cyto- plasm, or cytoplasmic elements. With the large numbers of ion pairs released per roentgen it seems impossible to think that any cell often escapes receiving at least one and often many when irradiated with the large dosages usually employed. Yet the evidence shows that sometimes cells do perform their functions even when they have been exposed to high dosages of radiation. It appears that cells have escape mechanisms for reducing the customary irradiation damage which may be immediately effective, or effective after an interval of further development. There are several categories into which these radiation effects may be classified. The growth pattern of the cell may be so altered as to make its future cells function abnormally with regard to the organism as a whole. Other cells may stop cell division, restricting an organ system essential to the whole. Subsequent growth processes may be so ineffective that a new event must occur if the damage is to be repaired. In the latter category may be included some gene or chromosome aberration — generated phenotypes. Chromosome aberrations initiated by break- age, on the other hand, may rejoin and the cell recover its previous normal functions. Differential cell growth where the irradiation damage is "healed" through replacement of defective cells by others of normal constitution, as in wound healing, offers another means of arriving at a normal phenotype. How- ever useful this mechanism is, it is not recovery of the irradiation-damaged cell but should l)e labelled one of cell replacement. Cytoplasmic elements affected by radiation frequently are not carried through future mitoses or are incapable of reorganization. They offer recovery mechanisms for their repair. Data on all types of cells seem to indicate that the greater parts of their vol- umes are not occupied by radiation-sensitive structures. Rather they allow the radiation to pass through, or, when absorption occurs, are unaffected. Earlier studies on malformations following ionizing radiations were concerned primarily with effects that were observed in embryos some time between the time of irradiation and shortly after parturition (Bagg, 1922; Job, Leibold and Fitz- maurice, 1953: L. B. Russell. 1950; W. L. Russell, 1954; O'Brien, 1056; ami Rugh, 1953, 1959). The results generally agree that the type of malformation depends both on the level of irradiation and the embryological stage of develop- ment during which irradiation took place. Most exposures were to high dose acute irradiations causing cell damage far beyond the physiological limits the exposed animals might repair. 450 DONALD J. XASII A XI.) JOHX \\'. GOYVKX Additional studies in recent years have attempted to evaluate the long-term sequelae of embryonic or fetal irradiation. It has become evident from these investigations that although an animal exposed to embryonic irradiation may ap- pear normal at birth, effects on morphological and behavioral characteristics may have been produced that become noticeable in later life, as cataracts (Stadler, 1959; Gowen, 1963) or volitional activity (Huff and Gowen, 1960). In some instances changes following embryonic irradiation have been found both by histo- logical study and by functional tests of the organ system under study. Beaumont (1960, 1962), in quantitative histological studies of the ovary and testis in rats, demonstrated a deficiency in germ cells following embryonic irradiation. Several workers, including Rugh and Jackson (1958), Russell ct al. (1959), and Nash and Gowen (1961), observed in mice long-term effects on the reproductive system in that adult reproductive capacity had been altered following embryonic irradia- tion. Effects of prenatal irradiation upon postnatal growth have been reported for several species, including the mouse (Russell ct al., 1959; Nash and Gowen, 1962), the rat (Ershoff and Bavetta, 1958), and cattle (Parish et al, 1962) . In several instances effects of prenatal irradiation upon growth did not become evident until some time after treatment. Nash and Gowen (1962) noted, for example, that certain embryological treatments did not produce a noticeable effect upon body weight until some two months after treatment. Similar "delayed effects" also have been reported for certain behavioral effects. Rugh (1956) found that mouse embryos irradiated with doses of 5 to 300 r exhibited more nervous excitability than controls when tested at 15 days and two months of age. In some cases behavioral changes have been observed in later life although no distinct morphological malformation or lesion could be demonstrated. It is evi- dent from these examples that there may exist changes induced by embryonic irradiation which may not have a noticeable effect until late in life. Although ionizing radiations have been widely used as an experimental means to accelerate changes sometimes associated with aging in mammals (Gowen, 1962), the exact relationship between lifespan and other physical and biological variables is still not clear. Attention has been directed towards changes progressive with age, sex, genetic background, and radiation dose response. Abrams (1951) found that the effect of irradiation on survival decreased rapidly with increasing em- bryological age and increasing age after birth. Similar results were reported for the rat by Steamer and Christian (1951), who observed that radioresistance in- creased rapidly between one and 48 hours after birth so that by two days of age rats were only slightly less radioresistant than adults. Although some workers ( Sacher, 1947, 1957; Blair, 1956) have postulated that radioresistance should probably decrease in some uniform manner with advancing age, other workers, including those in our laboratory, have found periods of relative stability of radioresistance. Kohn and Kallman (1956), for example, found that the LD50/3o was a linear function of age from 37 to 105 days of age, but remained nearly constant from 115 to 709 days. Spalding and Trujillo (1962) noted in mice irradiated between two and 21 months that radioresistance was relatively stable through the first half of adult life but then declined quite rapidly. Sacher (1957) also found little dependence on age and mean accumulated dose to death of ani- mals irradiated between 100 and 600 davs. X-RADIATION EFFECTS ON MATURE LIFE 451 There have been few systematic studies concerned with the effects of in liter o irradiation upon adult lifespan. Zuikova ct al. (1959) reported that in dogs irradiated during the last trimester of pregnancy, the duration of lifespan was in direct relation to dose. Russell et al. (1959) found that postnatal viability to 36 days of age in mice was lowered in animals irradiated with 200 r on days 9£, 7£, or 1H. In long-term survival, of the data available at that time, lifespan ap- peared reduced in those mice that had been exposed to 200 r on days 7£, 1H, or 13J. Considering the four embryological stages used in this investigation, 6£, lOi, 144, and 17^ days, and the levels of irradiation, through 160 r, it is difficult to discern clear-cut "critical periods" for the induction of changes in adult lifespan such as have been described for several other characteristics. Instead, noticeable effects on lifespan appear to be a result of interaction of a certain radiation dose with a certain specified embryological stage. The embryological stage at which embryos were irradiated did not seem to influence adult lifespan as long as the dosage did not exceed 80 roentgens. At the higher dose level the exceptions occur in female mice treated at 14^ and 17^ days gestation. After 160 r exposures life shortening occurs at a more rapid rate than expected from the previous trend in lifespans observed in 0, 20, 80, 160 r treatments. These changes are indicative of a threshold effect coming in this region of dosage, initiated by the radiation absorptions occurring in a more resistant set of receptors as well as those already taking up the radiant energy. The consequences of acute irradiations have generally been studied as changes closely following irradiation as expressed in different morphological types, morbidity and rapidity of death in 30 days. The data herein show that as far as duration of adult lifespan is concerned, the effects may be carried quiescent through to ex- pression over a fairly wide age span in later life. In this sense the late effects become comparable with more limited characters such as cataracts following irradiation, but on a different time scale. The shortening of life is the conse- quence of the experience through which the mouse passed at some embryological stages of development. The cataract stimulation, although having a long quiescent period of 300+ days during which no changes were evident, was dose-dependent and results from acute irradiation dosages occurring at 46 days from the birth of the animal when most of the embryological pattern of development was complete (Gowen, 1962). The nature and mode of action of the changes induced by embryonic irradia- tion that result in a shortening of the lifespan are still speculative. There is in- creasing evidence that aging may be caused by the gradual accumulation of spon- taneous chromosomal rearrangements and mutations in the somatic cells of the body (Gowen, 1934, 1962; Curtis, 1963). Through several methods of con- trolling chromosome aberrations it has been shown that their frequency was in- versely proportional to the life expectancy. Teratogens are also capable of producing chromosomal anomalies in various tissues. Mustard gas acting on sperm of Drosophila furnishes one of the early examples (Crowe, 1961). Ingalls ct al. (1963) demonstrated that a teratogen, 6-amino nicotinamide, when administered to pregnant female mice about the 13th day of gestation, produced chromosomal anomalies. Cells exhibiting polyploidy 452 DONALD J. XASil AND JOHN* \V. GOWEN and fragmented chromosomes were observed not only from tissues adjacent to palatal defects but iu tissues remote from the palate as well. In the present study, embryonic x-irradiation offers a means of producing chromosomal aberrations in the developing embryos. Although some of the em- bryonic cells may harbor mutated chromosomes they may function in a normal or near-normal fashion. Others may cause marked changes in body maintenance with a subsequent curtailment of life. A wide range of change leading, however, to like as well as different effects, may be induced giving high variability even within progenies of given inbred litters. A major concern of the present investigation was to evaluate the contribution of genetic background on lifespan in response to in utero irradiation. Although mammals, and the mouse in particular, have been widely used in radiation and aging studies, there is a surprising lack of information on lifespans in mice born of different mating systems. Inbred and hybrid mice, such as were used in this investigation, provide insight into the basic radiation responses over the period when aging is occurring. Several studies in the past have demonstrated that genetic constitution is a major factor in adult lifespan. Govven and Stadler (1956) utilized 10 genetically differentiated strains of mice to study the effects of irradia- tion at 40 days of age upon lifespan. The study included the three strains re- ported in the present paper. Genetic differences were found to influence the effects of x-rays throughout the acute dose range up to 960 roentgens. Genotypic strain differences were observed by the expression of different radiation syn- dromes. Chai (1959) also observed genetic differences in lifespan. In general, hybrid mice had longer mean life and lower mortality at earlier ages than those of the parental inbred strains. This may be due to the hybrids having a greater reserve of cells unaffected by such environmental agents as radiation on which to draw for life maintenance (Haverland and Gowen, 1960). Differences among inbred strains considered to be primarily genetic in origin, have been noted by Henshaw (1944), Grahn (1954), Gowen and Stadler (1956), Haverland and Gowen (1960), Kohn and Kallman (1957), and Grahn and I familton (1957). Progress has been made towards understanding the nature of the genetic and physiological factors conditioning the radiation response. Kohn and Kallman (1956) reported that in the strains of mice employed by them, greater sensitivity was a result of recessive genetic factors. Grahn (1958), in crosses involving the least and most resistant strains from a series of lines, esti- mated the genetic mean of the LDr,0 to be about 50% of the total variance. Pre- sumed single-gene differences in radiation response have been observed by Doo- little (1961) and Bernstein (1962). Stadler and Gowen (1957a, 1957b, 1957c) studied the effect of three body regions on radiation response in five inbred strains of mice, as measured by per- centage survival and length of survival. Differences between responses of strains were noted at different levels of irradiation. The extent of the interactions among x-ray doses and strains was large, indicating that each strain had a distinctive pattern of radiation response. In the results of the effects of embryonic irradiation upon lifespan reported in the present paper, a quantitative examination of genetic influences was pro- vided by a variance analysis and a breakdown of the variance into its component X-RADIATIOX F.FFECTS OX MATURE LIFE 453 parts. The variance analysis revealed that strain differences influenced the length of adult life when embryos were exposed to irradiation during the latter part of gestation, from 14-i- days on. This would indicate that by this stage in develop- ment genetic differences have been established sufficiently well in the mouse so that irradiation produces differential responses, as measured by adult lifespan. There are indications from other studies (Rugh, 1958, 1963; Nash and Gowen, 1963) that genetical differentiation, as measured by radiation response, may be expressed even earlier in embryological development. The lack of significant strain differences in the dosage sequences following irradiation at 6J and 10^ days is a reflection of the toxicity of lower acute x-ray dosages received by these strains. When the embryos have passed these thresholds in development the influence of strain differences on subsequent life becomes less. Of particular interest in the present data is the presence of significant dose- by-strain interactions following irradiation late in pregnancy. This indicates that the different genotypes are responding through different physiological channels over the range of radiation levels. Interactions of this sort following embryonic irradiation were also reported for postnatal growth by Nash and Gowen (1962). In the present study genotype-by-treatment interaction accounted for up to 30% of the total variation in adult lifespan. The existence of this type of interaction furnishes an excellent tool for further understanding of the basic radiation response. The possible existence of genetically determined variation in the timing of embryological development, as indicated by this analysis, lends biological sig- nificance to the mechanisms of radiation-induced lifespan changes. The early work of Painter (1928) and Venge (1950) demonstrated that differences in size between Flemish-giant and small Polish rabbits could be traced back to early embryonic stages and differences in cleavage rates of fertilized ova between large- and small-sized breeds. Mechanisms having these qualities offer means of un- derstanding how they and other genetic differences may appear in radiation effects on later lifespan and other effects. The evidence from Nash and Gowen (1962) shows that these mice irradiated in utcro were reduced in size following irradiation. The growth reductions were dependent on both dose and stage in embryological differentiation. The weight changes to 75 days were small for the 20 and 80 r treated mice. They became more severe at 160 r, particularly for the lOA-day irradiations where at birth the mice were but § size and unable to survive to the 12-day age. The 75-day weights were consistently reduced following 320 r irradiation. These changes can be related to observations on like actions by other agents which result in the separation of blastomeres, or cutting of Planaria (Morgan, 1898) into small pieces which on regeneration result in separate animals of small size. The x-ray action seems to come from reducing the numbers of certain vital cells. It is as though there was, for each egg type, a limit to the total cells which may divide. In eggs where the blastomeres lose their totipotence, the loss be- comes that of specific cells, precursors of specific organs. In this case these organs may be affected, giving the appearance of specificity to the abnormalities observed in mice irradiated at fixed stages of development in the embryological sequence. Schaible (1959, 1963) has studied the sequence of melanocyte distribution in 454 DONALD .1. \.\SH AND JOHN \Y. GOYVF.X mice and chickens of different genotypes. These cells distribute to eight centers of the mouse coat. Whiteness can he the result of absence, malfunction or death of the pigment cells. The distribution of the pigment leaves a record of the events through which the cells have passed in developing their final products. In general, changes in pigment cell density within the normal sequence do not in- fluence organizing cells to promote cell death. The products are left to reveal patterns of organogenesis. The observations become more complete than for other cells as in the organization of the nervous system where damage or loss of neuroblasts (Rugh, 1962) may remain obscure for lack of visible signs. The outward spread of pigment from eight centers is revealed in three ways: pigment spots on unpigmented background ; absence of pigmented cells in a pig- mented background ; and the presence of a spot of different-colored hair in the coat, representing the descendant cells of a primordial melanoblast which had changed to a genotype different from that of the original zygote from which it started. The pattern of melanocyte descent offers possibilities of control of the end products of development by both genetic and environmental factors. The factor interactions furnish a significant model for the variations observed spon- taneously and under x-radiation in the lifespan of this study. The coat color model displays differences between inbred strains of different genotypes ranging from zero to 90%. Substitution of different genes gives a staircase type of ascent to the frequencies of change within the mouse populations. Genotypic species changes are marked by even broader differences in the timing of the periods when hair or feather follicles may attract melanocytes : in the mouse all follicles of the coat seem to mature at one time and exhaust the epidermal supply of melanoblasts ; in the chicken the epidermal supply of melanoblasts does not exhaust, at least in the growing animal. In this model, tests of the pigment mosaics show they are, in large majority, somatic rather than germinal, agreeing with the evidence on the lifespans of mice under continuing low rate irradiation (Stadler and Gowen, 1963; Gowen and Stadler, 1964). There were no whole-body reversions of coat color in 5165 offspring nor did wild type segregates on test have unexpected gametes. Changes appear to occur only after at least one primordial pigment cell has established. X-radiation with 150 r to the region of the gravid uterus from i to 1H days, following copulation, in day intervals, revealed two significant high points in mosaic frequencies of the progeny, 2.5 and 10.5 days in utero development. How- ever, the acute dose administered had only slight influence in the total mosaics observed over the full period of the irradiations. The suggestions of periods of embryological development of greater susceptibility to acute x-ray dosages, and the lack of pronounced changes in frequencies of mosaics from the unexposed controls, again model what was observed in our data on survival in after-life for the lower dosages of acute x-ray irradiations. SUMMARY 1. Mature lifespans, 75 days of age to death, when the mice were exposed to irradiations at different stages of uterine development, showed reductions in mean longevity which were dependent on both x-ray dose and period of embryological cycle. The design for this study was factorial. There were five irradiation X-RADIATIOX EFFECTS ON MATURE LIFE 455 treatments, 0, 20, 80, 160 and 320 r. six embryological stages: untreated, 6i, lOi, 14£, 17^ and newborn (19^) days of uterine development, two sexes and nine mouse strains. Three of the inheritance groups were inbred strains and six were the reciprocal crosses of these strains. Efforts were made to obtain two mice for each cell of the design. However, some treatments were so severe that this was impossible. X-ray dosages of 160 and 320 r at embryological ages 6i, 10^ and 14i days were too lethal to obtain the requisite number of progeny in these groups. 2. Six hundred and forty-seven completed mouse lives were collected. Mature lifespan ranged downward from 1194 days for the males and 921 days for the females. Both of these long-lived individuals had been irradiated, the male with 160 r at 14 J days embryological development and the female with 80 r at 10i days embryological development. The frequency distributions of lifespan were of several types for the different groups, depending on the treatments, strains or sexes of the individuals. 3. In terms of changes in mature lifespan, sex differences in the mice resulted in marked differences in the mean days they survived. Considering the in utcro treated mice, the average unirradiated females survived for only 69% of the mean male lifespan. This male-to-female difference cannot be attributed to irradiation effects but rather to the hazards which differentially affect the physiological processes which separate the lives of the sexes, chiefly those associated with re- production. Adjusting for these effects shows that the average female life under irradiation still exhibits greater sensitivities to irradiation in utero. Considering the available data for the dosages taken separately, the 20 r females had 85% of the mean lifespan observed for the untreated females, the males 96.7% that of the untreated males. Like comparisons for the 80 r were 82 vs. 93 ; for the 160 r, 73 vs. 89 and for the 320 r, 30 vs. 43. 4. Intrauterine irradiations, when limited to 20 to 80 r, reduced the female durations of life at all stages of embryological development more than they did those of the males. 5. Variance analyses emphasized the importance of sex as a controlling ele- ment in adult survival when the mice are under pressure of reproduction. 6. Genotypic differences, as measured by strain and as separate from sex, have little effect on subsequent mature lifespans when the irradiation is small in amount, 20 including 80 r, or when given at 6J or 10^ days of embryological de- velopment. The amount of radiant energy absorbed becomes more important to mature life's duration when the dosages of radiation are larger, 160 including 320 r, or when the exposures occur in the latter half of pregnancy. The variance analyses support the significance of the differences on which the above conclusions were based. 7. Genotype also interacts with both stage of embryological development when irradiation occurred and with the dosage of x-rays absorbed. 8. Individually the embryological stages when the irradiations took place have but small percentage effects on mature lifespan, although they do have pro- nounced effects on survival in the juvenile stage. All changes in lifespan come as direct irradiation effect to the soma of the embryos, as caused by acute ir- radiation. Lifespan lowering comes largely in the 320 r treated embryos in the 456 DONALD .1. XASH AXI) JOIIX \V. GOWF.X form of a threshold effect \\hich tlien carries through to the adult as cryptic damage to appear later in life. LITERATURE CITED ABRAMS, H. L., 1951. Influence of age, body weight and sex on susceptibility of mice to the lethal effects of X-radiation. Proc. Soc. Exp. Biol Mcd., 76: 729-732. BAGG, H. J., 1922. Disturbances in mammalian development produced by radium emanation. Amcr. J. Anat.. 30: 133-161. BEAUMONT, H. 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Burdette, Holden-Day, Inc., San Francisco ; pp. 382-404. GOWEN, J. W., AND R. G. SCHOTT, 1933a. Genetic predisposition to Bacillus filiformis infection among mice. /. //v. Biol. Med., 93: 523-524. RUGH, R., 1958. X-irradiation effects on human fetus. /. Pediatrics, 52: 531-538. RUGH, R., 1959. Ionizing radiations ; their relation to the etiology of some congenital abnormali- ties and human disorders. Mil. Med., 124: 401-416. RUGH, R., 1962. Low levels of X-irradiation and early mammalian embryo. Amer. J. Roentgcnol.. Rod. Therapy and Nuclear Med., 87: 559-566. RUGH, R., 1963. The impact of ionizing radiations on the embryo and fetus. Amer. J. Roentgcnol., Rad. Therapy and Nuclear Med., 89: 189-190. RUGH, R., AND S. JACKSON, 1958. Effect of fetal X-irradiation upon the subsequent fertility of the offspring. /. Ex/>. Zool., 138: 209-221. RUSSELL, L. B., 1950. X-ray induced developmental abnormalities in the mouse and their use in the analysis of embryological patterns. I. External and gross visceral changes. ./. /•:.!•/>. Zool, 114: 545-602. RUSSELL, L. B., S. K. BADGETT AND C. L. SAYLORS, 1959. Comparison of the effects of acute, continuous and fractionated irradiation during embryonic development. Int. J. Radiation Biol., (Suppl.), June, pp. 343-359. RUSSFLL, W. L., 1954. Genetic effects of radiation in mammals. Radiation Res., 1: 825-859. SACHER, G. A., 1947. After survival of mice treated with single doses of X-rays. Manhattan District Report CH-3782, pp. 25-30. SACHF.K, G. A., 1956. On the statistical nature of mortality, with especial reference to chronic radiation mortality. Radiobiology, 67: 250-257. SACHER, G. A., 1957. Dependence of acute radiosensitivity on aye in the adult female mouse. Science. 125: 1039-1040. SCHAIBLE, R., 1959. Delimitation of pigment areas in mosaic and selected piebald genotypes of the mouse. Unpublished M.S. thesis. Iowa State University. SCHAIBLE. R., 1963. Developmental genetics of spotting patterns in the mouse. Unpublished Ph.D. thesis. Iowa State University. DONAI.I) J. XASII AND JOHN W. GOWEX Si IIOTT, K. (.;.. 1932. The inheritance of resistance to Salmonella acrtrycke in various strains of mice. Genetics. 17: 203-229. SPALDING, J. F., AND T. I. TRU.ULLO, 1962. Radiosensitivity of mice as a function of age. Radiation Res.. 16: 125-129. STADLER, J., 1959. Cataract in mice as affected by radiant energy. Rcc. Genet. Soc, Amcr., 28: 98. STADLER, J., AND J. W. GOWEN, 1957a. Contributions to survival made by body cells of genetically differentiated strains of mice following x-irradiations. Biol. Bull., 112: 400-421. " STADLER, J., AND J. W. GOWEN, 1957b. Radiological effects on resistance mechanisms of genetically differentiated strains of mice exposed to Salmonella typhimurium. J. Infect. Dis., 100:" 284-299. STADLER, J., AND J. W. GOWEN, 1957c. Radiation effects on active acquired immunity to Salmonella typhimurium in mice. /. Inject. Dis., 100: 300-311. STADLER, J., AND J. W. GOWEN, 1961. Viability in the mouse as affected by Cobalt-60 irradiation "continuous" through ten generations. Rec. Genet. Soc. Amcr., 30: 111-112. STADLER, J., AND J. W. GOWEN, 1963. Influence of Cobalt-60 continuing irradiation on fertilities and life spans of different strains of mice. XI International Congr. Genetics. STEARNER, S. P., AND E. J. B. CHRISTIAN, 1951. Effects of X-irradiation in newborn rats. /. Ext*. £""/.. H8: 495-507. YENGE. O.. 1950. Studies of the maternal influence on the birth weight in rabbits. Ada Zool., 31: 1-148. ZUIKOVA. YE. Z., N. Y. CHAVKOVSKAYA AND S. L. PETROSYAN, 1959. The effects of ionizing radiation on progeny following irradiation of animals during the last days of pregnancy. Translated from I'estnik Rcntgcnol. 1 Radio!., 34: 34-41. VARIATION OF HEMOCYANIN CONCENTRATION IN THE BLOOD OF FOUR SPECIES OF HALIOTIS 1 MICHAEL E. Q. PILSOX - Division of Marine Biology, Scripps Institution of Oceanography, La Jolla, California Hemocyanin is a copper-containing protein found in solution in the blood of certain arthropods and molluscs. It combines reversibly with oxygen, becoming blue when oxygenated, and is commonly regarded as a respiratory pigment. Hemocyanin has been recognized for about 100 years, and numerous investiga- tions of its chemistry have been carried out (Mamvell, 1960; Prosser and Brown, 1961). In a consideration of the function of a body constituent it is important to have available data on the quantity or concentration of the substance in the body. Data on the amount of hemocyanin actually present in the blood are, however, scanty. Redfield (1934) collected, from several sources, data on the oxygen capacity of the blood of several molluscs, and from these calculated values for the copper content of the blood. From the copper content and the known composition of the hemocyanin from these species he calculated the hemocyanin concentration in the blood of four molluscs. The values obtained, expressed as per cent, were: Octopus vulgaris, 5.9-9.1; Loligo pealei, 7.2-8.8; Helix pomatia, 1.5-3.9; and Busycon canalicitlatitin, 3.7—6.6. The greatest range of variation found in any one species was about 2.6-fold. It may be calculated from data on the copper content of the blood of two of these species (Prosser and Brown, 1961) that the hemocyanin content of Helix pomatia blood varies from 2.70% to 4.79% and that of Octopus vulgaris blood from 9.40% to 11.40%. Stewart et al. (1952) mentioned that the range in total protein concentration in the blood of Cryptochiton stelleri was from \% to 3%. Woods et al. (1958) carried out electrophoretic experiments with blood from 6 to 12 individuals belonging to each of 18 species of invertebrates, including Loligo pealei and Ostrea virginica, and stated (p. 519) that "within a given species differences in sex, size, or stages of the molt cycle produced no significant differ- ence in the electrophoretic patterns." Horn and Kerr (1963) have presented the most extensive data on the serum protein and copper (and by inference the hemocyanin) concentrations in the blood of Callinectes sapidus, the blue crab. They found a 10-fold variation in the total serum protein concentration and an 18-fold range in the serum copper concentra- tion. There was no relationship of either serum protein or copper concentration to the size of the crab, but it was found that female crabs had significantly higher serum protein and serum copper concentrations. No similar data for any molluscs are available. 1 Contribution from the Scripps Institution of Oceanography. 2 Present address : Institute for Comparative Biology, Zoological Society of San Diego, San Diego, California. 459 AflCHAKI. I*,. O. PI I.SOX In this paper i will document the low average concentrations and the rather unexpected variability in the amount of hemocyanin found in the blood of four species of Haliotis. including data from a total of 172 individuals. MATF.RIALS AND MF.THODS Collection of animals Abalones of the species Haliotis jnlgcns, H. comtgata, H. rufcscens and H. cracherodii were collected at intervals during the period from 1960 to 1962, mostly from the area around La Jolla, California. The depth from which they were taken varied from the intertidal region down to about 60 feet. Most of these animals were bled on the day that they were collected, while some were kept in aquarium tanks provided with running sea water for periods up to nearly two years before being bled; food (Macrocystis pyrifera] was provided during this period. The numbers of animals included in this study were: PI. fulgois, 107; H. cornigata, 26 ; H. rufescens, 32 ; H. cracherodii. 7 . Bleeding oj animals The abalones were removed from the water, quickly shaken to empty water from the branchial cavity, the shell dried with a towel and each animal weighed. The foot was then wiped with a towel, and a short incision, about 2 cm. deep, was made in the midline of the foot. The blood which welled into the cut from the large pedal sinuses was sucked up quickly with a large pipette, centrifuged to remove the few blood cells (the volume of cells was always less than 0.5%), and stored at 0° C. Blood from which the cells have been removed will be called plasma. Gonad inde.r The gonad-hepatopancreas was cut at a point midway between the start and ihe distal end of the horn. The diameter of the whole organ was measured in two directions at right angles to each other, as was also the diameter of the inner hepatopancreas. The gonad index was calculated as follows : Avg. diam. of whole organ donad index = Avg. diam. of hepatopancreas The sex of the animal was determined at the same time. Total nitrogen and NPN Total nitrogen was determined on 1-ml. aliquot s of the plasma by the micro- Kjcldahl method (A.O.A.C., 1955). Non-protein nitrogen was determined by the micro Kjeldahl method on protein- free filtrates prepared by the tungstic acid method of Folin and Wu (1919). Protein was estimated by multiplying the protein nitrogen concentration by the conventional factor of 6.25. All determinations were done in duplicate. HEMOCYANIN IX HA LI OTIS 461 . Ibsorbance Spectral absorption curves of the blood showed a broad, rounded maximum at 560 in/*,, as has been found for hemocyanins of other species. The absorbance, at 560 m/i, of undiluted and thoroughly aerated plasma was determined as a measure of the hemocyanin concentration. The absorbance at 280 m/A, a measure of the total protein concentration, was determined after dilution of 1 ml. of the plasma to 25 ml. with 0.5 N NaCl solution. Dilution with distilled water caused precipitation of the hemocyanin. Copper Aliquots of plasma (usually 1.0 ml.) were digested with 5 ml. of a mixture of 100 ml. of 70% perchloric acid and 400 ml. of concentrated nitric acid. Copper in the digests was determined according to method B given in Diehl and Smith (1958). In this procedure copper is reduced to the cuprous form, extracted as the 2,2'-biquinolate into isoamyl alcohol, and the absorbance of the colored complex measured at 546 m/x. The addition of hydroquinone in a final concentration of 0.5% to prevent fading of the color, as recommended by Riley and Sinhaseni (1958), was found to give good color stability. All determinations were done in duplicate. Electroplwrcsis 1 lorixontal paper electrophoresis was carried out at room temperature. Barbi- tal buffer, pH 8.6, ionic strength 0.05, was used, and the voltage gradient was about 3 volts/cm. Plasma was applied to the strips without prior treatment ; 10, 20, or 30 microliters were applied to each 3-cm.-wide strip. After the separation, the strips were stained with bromphenol blue. RESULTS Of the various measurements made on the plasma samples, the one for which most data are available is the concentration of total organic nitrogen. This varied TABLE I Average and range of total nitrogen and of hemocyanin in 4 species of abalnnes. Nitrogen as mg./lOO ml., protein as gm./lOO ml. H. fulgens H. corrugata H. cracherodii H. rufescens No. of aniniaU 107 26 1 32 Total N Median 129.0 67.4 91.3 92.2 Low 33.0 28.8 60.5 47.1 High 358.0 275.5 342.5 214.5 Hemocyanin N, Median 86.5 23.8 60.1 — Hemocyanin Median 0.54 0.15 0.38 — Low 0.030 0.0017 0.210 • — High 1.89 1.53 2.03 — Maximum variation 63-fold 900-fold 10-fold — 462 MICHAEL E. Q. PI I.SOX from 33.9 to 358.0 mg./lOO nil. for H. fuhjens. \\'ide ranges of variation were also found for the other species (Table I). It will be shown first that the hemocyanin concentration is directly related to the total nitrogen concentration. Then the total nitrogen concentration, as a measure of the hemocyanin concen- tration, will be compared to the environmental and physiological parameters which have been measured. The tabulated raw data have been reported by Pilson (1963|. 600 500 E 0400 \ en E 300 o o cc h- 200 o 100 257. 5 x 0.5 1.0 1.5 ABSORBANCE, 280 I-'IMKI. 1. Total organic nitrogen concentration in plasma from 4 species of Halii/lis r.v. the ahsorhaiuc at 2X0 m/i after dilution of 1 nil. of plasma to 25 nil. with 0.5 N NaCl solution. The plotted points for each succeeding species are displaced upward by 100 units. Non-protein nitroyen The median XI'N found for //. jitlt/ens (14 determinations) was 8.8 mg./lOO ml. If the plasma does not contain appreciable quantities of non-protein sub- Maiices absorbing in the region of 280 m/x, then the NPN can be estimated also from the relation of total nitrogen to the absorbance at 280 m/z. Figure l(a) HEMOCYANIN IN HALIOTIS 463 shows this relationship for the 27 samples for which these data were available. This and all subsequent regression lines have been calculated by the method of least squares. The y-intercept at 9.9 is satisfactorily close to the determined median value of 8.8 mg. N/100 ml. There is very little scatter about this line, indicating that large variations in the non-protein nitrogen fraction are unlikely. Figure 1 (b and c) shows the same plots for H. cracker odii and for H. cornigata. The y-intercept for H. cracherodii is 9.6 mg. N/100 ml. and this is the only available estimate for NPN for this species. The y-intercept for H. corrugata is 4.9, close to the median value of 5.0 mg. N/100 ml. obtained by direct determi- nation on three samples. Figure l(d) shows the same relationship for H. ntfcsccns, and the estimated average value for the NPN is 7.0 mg. N/100 ml. These values for NPN are minimal values, for any non-protein substance in the plasma which absorbs at 280 m/* will tend to shift the curve to the right, thus depressing the value at the y-intercept. The slopes of the regression lines are similar for the first three species, but that for H. rufescens is about 23% greater, indicating that the plasma proteins of H. rufescens may contain less of the amino acids which absorb at 280 m//. than do those of the other species. Non-hemocyanin protein The degree to which the variation in total nitrogen in the plasma is due to non-hemocyanin protein was estimated by paper electrophoresis, the relation of total nitrogen to the absorbance at 560 m//,, and by the relation of the total nitrogen to the copper concentration. Paper electrophoresis patterns were obtained for samples of plasma from four individuals of H. fulgens and one from H. corrugata. Two faint bands were present on the strips prepared for all samples, and appeared to be approximately constant in intensity. A third band, of greater mobility than the others, with a mobility approximately that of human a-globulin (human serum was run at the same time as a control) was variable in intensity; by visual estimation this vari- ation in intensity was thought to be approximately proportional to the blueness of the blood and to the total nitrogen content. This band was presumably due to hemocyanin. All known hemocyanins have, in the oxygenated form, a broad absorption maximum in the region of 560 m/*. Plasma from H. fnlgens had an absorption spectrum very similar to that of known hemocyanins, with a peak very close to 560 m/x. The absorbance in this region, then, can be taken as a measure of the amount of hemocyanin present. In Figure 2 the total nitrogen in the plasma is plotted against the absorbance at 560 m//,. The intercept of the regression line on the y-axis gives a minimal value for the non-hemocyanin nitrogen present. This is minimal because any other source of absorbance in the plasma will tend to shift the curve to the right, lowering the value for the y-intercept. The value for non-hemocyanin nitrogen is 24.0 mg./lOO ml. for H. fnlgens. The estimated values for the non-hemocyanin nitrogen in plasma from H. corrugata and H. cracherodii were 21.8 and 20.1 mg./lOO ml., respectively. The plot of the values from H. rufescens, at the top of Figure 2, shows a different pattern. The 5 points 4(>4 MICHAEL E. Q. PILSON enclosed in triangles appear to constitute a separate group. It seems that in this species there may he considerahle variation in the concentration of the non- heiiKH-\anin protein. No attempt was made to fit a regression line to these points. Another way to estimate the non-hemocyanin protein is to assume that all the copper in the plasma is hound in the hemocyanin and then to examine the relation- ship between the total nitrogen present and the amount of copper in the plasma. In Figure 3 the total nitrogen in samples of plasma is plotted against the copper concentration. The regression line fitted to the points from //. jitlgcns has a y-intercept of 42.5. This is probably the hest measure of the average amount of 500 : en E 400 llJ o 300 i Huhohs i'S. tlie absorbance at 560 m//. The plotted points for each succeeding species are displaced upward liv ICO units. non-hemocyanin nitrogen present. It should he noted, however, that some sam- ples of plasma had less than 42.5 mg. total nitrogen per 100 ml. Similar values for //. cnrrtii/dld and for //. crachcrodn were 43.6 and 31.2 mg./lOO ml., re- spectively. The plotted data from //. nijcsccns again indicate that in this species the non-hemocyanin protein may he quite variable. I corrected for the non-protein nitrogen and used the conventional conversion factor of 6.25 to calculate the following average amounts of non-hemocyanin protein present: //. juli/cus. 204; II. cornit/ata, 242; and //. crachcrodii. 135 ing. /100 ml. HEMOCYAXIN IX HALIOTIS 465 Hemocyanin concentration Figure 4 shows the relationship between the ubsorbance at 560 niju and the copper concentration of the plasma from H. jidgcns, H. corrmjata and //. rnfesccns. Jt is clear that, for the first two species named above, the absorbance at 560 m/x. is well correlated with the copper concentration. This indicates that most of the copper is present in combination as hemocyanin. For both species the y-intercept 600 500 o 2400 o300 o mvnt ration in plasma from //. fitl/n'iis VS. the total \vciglit of tlir animal. [here was no ; gnificant difference in the total nitrogen concentration of the plasma between mal< le specimens of //. jitlt/ois. Xiilritional state ami total nitrogen \:]\c individuals of th //. cornnjata were collected from a depth of 35 feet off Point Loma in an area from 'Ahich the kelp had been absent for at least a year. These animals all appealed shrunken and the foot muscle was weak and HEMOCYAXIX IX HALIOTIS 469 flabby; the gonads were undeveloped. Another group of five individuals, col- lected also from a depth of 35 feet only 200 yards away, had been living in an area where kelp was abundantly available. These were firm, healthy in appear- ance and with well developed gonads. The average total nitrogen content of the plasma from the starved animals was 62.1 mg./lOO ml., and that from the well fed animals was 47.5 mg./lOO ml. This difference was not statistically significant. From this admittedly small sample, it appears that hemocyanin concentration is not correlated with nutritional state. The great majority, and perhaps all, of the other animals examined were healthy and firm in appearance. 400 300 o o E * "o o 200 100 H. fulgtm 1.0 2.0 3.0 Gonod Index 4.0 5.0 FIGURE 7. Total organic nitrogen concentration in plasma from H. fulycns rs. the gonad index. Depth and season \Yithin the species H. fulgcns there appeared to be no relationship between the concentration of total nitrogen in the plasma and the depth at which the animals had been collected. Thinking that the warming of the water during the summer months might put sufficient stress on the abalones to cause an increased synthesis of hemocyanin, I plotted in Figure 8 the mean total nitrogen in various collections against the time of year. There is no obvious seasonal trend demonstrated in this figure. Copper a fid hemocyanin If it is assumed that hemocyanin from H. jnlyens contains two atoms of copper for each molecule of oxygen bound, as is known to be the case for other 470 MICH \KI E. Q. I 'II. SON species, it is possible to calculate, from the relationship shown by Figure 3, the minimum combining weight of the heinocvanin. For H. fulgens this is 10,300 gm. of nitrogen. Multiplying by the conventional factor of 6.25 yields a minimum weight of 64,400 gm. of protein. Comparable figures obtained by direct analysis of the hemocyaiiin from other molluscs are 52,800 for Hcli.r poinatia, 51,800 for Busycon canalicnlatnin. and 50,800 for Octopus ntlt/aris (calculated from data in I'rosser and Brown, 1961). Severy (1923) reported the copper content of each of five whole individuals of //. cracherodii to be 0.8 mg./kg. of wet tissue. Marks (1938) reported the following whole-body copper concentrations in three species of abalone : H. fulgois. 2.5 to 10 mg./kg. (11 samples); //. cniclicrodii. 1 to 13 mg./kg. (6 samples); 300 • E o ° 200 \ • E 100 o i Jon F*b Mar Apr May Jun« July dug S«pt Oct Nov D«c KH,I i'K X. Average total organic nitrogen concentration in various groups of H. fitochiton stcllcri. 1-cd.Proc., 11: 155. \\'OODS, K. R., E. C. PAULSEX, R. L. EXGLE AXD J. H. PERT, 1958. Starch-gel electrophoresis of some invertebrate sera. Science, 127: 519-520. PIGMENT MIGRATION AND LIGHT-ADAPTATION IN THE EYK OF THE MOTH. GALLKRIA MELLONELLA1 CHARLES T. POST, JR.? AXD TIMOTHY H. GOLDSMITH Department of Bioloc/y. Yale University, New ffaren. Connecticut With few exceptions, the compound eyes of arthropods fall into two group>- those with relatively short rhabdoms and pronounced migrations of accesx>ry shielding pigment, and those with rhabdoms extending the length of the retinulae and little or no movement of pigment (Exner, 1891). The former occur in noc- turnal animals or species which are found where there is low illumination; the second type is characteristic of diurnal forms which are active where there is ample light. Because of this ecological correlation it has been widely assumed since the time of Exner that migratory pigment is an adaptation for vision in dim light ; however, physiological evidence in support of this view has been provided only recently. The eyes of moths dark-adapt in two steps, hut the second, slower phase does not, as in the human eye, represent a second population of receptors with lower thresholds. The increase in sensitivity of the eye during this slow phase runs parallel with the migration of shielding pigment as it moves distally to a compact mass between the crystalline cones (Bernhard and Ottoson, 1960a, 1960b. 1964; Bernhard, Hoglund and Ottoson, 1963). Eyes which have no migratory pigment lack the second component in the dark-adaptation curve (Bernhard and Ottoson, 1960a; Bernhard, Hoglund and Ottoson. 1963; Goldsmith, 1963), and in the fully dark-adapted state they are not as sensitive as eyes in which the pigment sleeves have withdrawn distally (Bernhard, Hoglund and Ottoson, 1963). Compound eyes with short rhabdoms and migratory pigment can appropriately be called scotopic eyes ; those with no migratory pigment, photo f>ic."~ 1 This work \vas supported in part by a grant (NB-03333) from the Institute of Neurological Diseases and Blindness, U. S. Public Health Service. 2 Supported in part through the N.S.F. Undergraduate Science Education Program at Yale LTniversity. 3 A brief consideration of terminology is necessary here. The traditional names for these types of compound eye are superposition and apposition, and were introduced by Exner (1891). These terms refer to supposed differences in refractive properties and mode of image formation which Exner described in great detail but which subsequently have been shown not to be general properties of the two groups (Kuiper, 1962; Goldsmith, 1964). The raison d'etre of these names is thus rooted in an erroneous analysis of the optics of compound eyes, and the only argument for perpetuating them is that they have existed now for almost three-quarters of a century. \Ye feel they should be replaced by terms that relate to the known properties of the eyes and that are established in the vocabulary of visual physiology. By scotopic eye we thus mean one which is of the morphological type which was supposed by Exner to form superposition images and which is adapted (in an evolutionary sense) for high sensitivity in dim light. Obviously the basis for this adaptation — migratory shielding pigment — is different from scotopic vision mediated by a rod-rich vertebrate retina. There remains a relatively small number of compound eyes with the photopic (apposition) morphology but which possess migratory pigment. AYhat their proper physiological place may be must await experimental analysis. 473 474 CHARLES T. POST, JK. A.ND TIMOTHY M. GOLDSMITH many I .cpidoptcra light controls migration-, cither by direct action on the •it cells or indirectK through the sense cells; the must recent work indicates that pigment migrations can he observed in isolated retinas and that connection witli the optic ganglion or the circulation is not necessary (Tunrala, 1954 .1 . In such moths, cold or narcosis causes the pigment to move to the light-adapted position i I >ay. 1()41 ). suggesting that the process which holds the pigment in the dark- adapted state requires metaholic energy and that possibly the effect of light is to inhibit this process. The first part of this paper is a quantitative study of the light energy and time necessary to effect pigment migration in the scotopic compound eye of the wax. moth, Gallcria incllonctla. The second part deals with the rate of light adaptation and how it is influenced by the presence of migratory pigment. T. THE CoN'iuoi. OF I 'U:\TENT MIGRATION BY LIGHT Methods Intact moths (Gallcria mellonella) were placed on their backs and secured to a small cork platform with soft wax ("Tackiwax"). After a minimum of one hour • lark-adaptation the animals were exposed to an adapting light for varying lengths • it time. The adapting light was obtained with a high-pressure xenon arc and a Bausch and Lonib grating monochrornator, and consisted of a narrow band of wave-lengths (10 \\\jj. half hand width) centered at 500 in//,. An image of the diffraction grating was focused on the animals' heads. Intensity was controlled with a pair of cali- brated optical wedges. Energy flux was measured with an Eppley bismuth-silver thermopile of known sensitivity and a Keithley No. 149 millimicrovoltmeter. Alter a specified exposure the animals were decapitated under dim red light, the heads bisected, and the halves placed in Duboscq-Brasil fixative in the dark for -16 hours. The tissue was dehydrated and imbedded in paraffin by standard techniques. Sections 10 p thick were cut parallel to the ommatidial axes, cleared ot paraffin, and examined without staining with a phase contrast microscope to . the extent of pigment migration. Some sections were depigmentecl with < irenacher'.- solution and stained with Harris's haematoxylin to aid in orientation. I'igment position was measured as the distance between the distal end of the crystalline and the proximal border of the secondary pigment (see Fig. 1); but in order to comp s of different sixes, this figure was expressed as a fraction oi the distance fron 'one to the basement membrane. For ease in understand- ing, this index has bei \ ;sed in Figures 2 and 3 as a percentage of the total possible migration observed in eyes exposed to direct sunlight. speriments med in midafternoon during the months of June, July, and August, with animals d in the laboratory by the method of Dutky. Thompson and ( !an1 v - i Results Figure 1 , the positii pigment in dark- and light-adapted eyes. The nuclei of the retinular cells, which are located in the distal processes and are not readily visible in Figure 1, alsc n proximally in the light. LIGHT-ADAPTATION IN A MOTH EYE 475 The extent of pigment migration after 3 and 27 minutes irradiation is shown in Figure 2 as a function of incident energy. Not only does the pigment move farther the higher the energy, hut for each flux the pigment migrates more in 27 minutes than in three. These conclusions are amplified by Figure 3, which shows the time course of migration for two intensities of light. In each case migration starts immediately at a relatively high rate, then slows as the pigment reaches a steady-state position characteristic of the incident energy. The time required to attain the final steady- state position is longer the farther the pigment moves and is of the order of half dark-adopted light- adapted • HM t . *4 8 £/ :« b.m. FIGURE 1. Sections of Gallcria eyes cut approximately parallel to the long axes of the retinulae and showing the positions of the migratory pigment in the fully light- and dark-adapted states. The nuclei of the retinular cells, which are in the distal processes but are not readily visible without staining, also migrate distally in the dark. Sections were unstained and were photographed with phase-contrast optics. As a scale reference, the cystalline cones are about 45 fj. in length. Cor., cornea; ex., crystalline cone; d.p., distal processes of the retinular cell-.; rhb., rhabdoms ; b.m., basement membrane; p., migratory pigment of the secondary pigment cells. an hour for the nearly complete migration produced by 5 X 103 ergs sec."1 cm.~- at 500 in/*. Discussion Eyes exposed to the same combinations of intensity and time frequently show wide variation in pigment position; consequently a great manv sections must be prepared to achieve a high precision of measurement, and this has discouraged us from measuring an action spectrum by this histological procedure. 476 CHARLES T. POST, JR. AXI) TIMOTHY H. (.(H.DSMITH 100 80 yt o E 60 c o ° 40 c 0) E o> 20 0 1 I I I A = 500 m/y I I I 27 min 1234 Energy Flux (log ergs sec"1 crrr2) FIGURE 2. Proximal pigment movement as a function of energy for two different times of exposure, as determined from histological sections similar to those in Figure 1. Each point represents the average of about a dozen measurements on several sections from at least two eyes, each from a different moth. Energy flux was measured with a thermopile at the surface of the cornea. Wave-length was 500 rou. I = 5xl03 ergs sec-' crrr2 20 30 Time in the Light (minutes) - Proximal pigment mi , •.••;: m" lime in the light. Details as in Figure 2. LIGHT-ADAPTATION IN A MOTH EYE 477 The controlling effect of light is not all-or-none, for the extent of migration is ;i function of intensity. This is consistent with the thought that the rate of a chemi- cal reaction necessary for the pigment to he held in the distal (dark) position is slowed by light, but a more precise kinetic model cannot be tested critically on the basis of the experimental observations presently available. The finding that the pigment assumes intermediate positions in the presence of dim lights is in agreement with recent work of Bernhard and Ottoson (1964) who observed in Ccraf>tcr\.v i/raininis a partial retraction of pigment towards the dark position when the intensity of a steady adapting light was decreased but not extinguished completely. Our observations on the time course of pigment move- ment are in semiquantitative agreement with Hoglund (1963a), who reports that the large area of glow characteristic of dark-adapted eyes of various nocturnal Lepidoptera becomes minute when the pigment occupies a position intermediate between the distal and proximal extremes; and further, that glow requires 10 minutes or more to disappear when the eye is exposed to light. In a later paper Hoglund (1963b) shows a case in which glow has vanished in less than 5 minutes. Both of these times are in reasonable agreement with our results, considering that different species were studied and that there is no precise information on Hoglund's adapting energies or the relation between pigment position and the point at which glow vanishes. II. THE RATE OF LIGHT-ADAPTATION Bernhard and Ottoson (1960a, 1960b, 1964) and Bernhard, H5glund and Otto- son (1963) have shown that dark-adaptation of moths has two components which, under some conditions, are completely separated in time. The second phase is caused by the distal migration of the shielding pigment and produces a fall in threshold of the eye of 1-3 log units. It does not indicate an intrinsic change in sensitivity of the retinular cells, but rather an increased probability that light cross- ing the cornea will reach the receptors (Hoglund, 1963b). The correlation between sensitivity change and pigment position during dark adaptation of scotopic eyes prompted us to examine the corresponding relationship during light-adaptation. A photopic compound eye lacks both pronounced pigment migration and the slow phase of dark adaptation (Bernhard and Ottoson, 1960a, 1960b; Bernhard, Hoglund and Ottoson, 1963). Moreover, the rate of light- adaptation — measured as the change in increment threshold in the presence of the adapting light — is very rapid ; a steady value of sensitivity is achieved within seconds after turning on the adapting light (Goldsmith, 1963). On the other hand, in a scotopic eye where pigment moves for minutes after exposing a dark-adapted eye to the light, one might, at least on first thought, suppose that light-adaptation should be correspondingly extended in time and increased in extent. The following experiments are in agreement with Hoglund (1963b) in showing that this expecta- tion is- — for good reason — usually not fulfilled, but they indicate a greater variety in the shapes of light-adaptation curves than is suggested by his short but excellent paper. Light adaptation curves of scotopic compound eyes have a multiple origin. There is a rapid fall in sensitivity as the receptors adjust their thresholds to the adapting light, but with the inward migration of pigment there are two additional effects which are opposed to one another. The test light reaching the receptors is ill \Ul.l-> T. POST, JR. AND TIMOTHY H. GOLDSMITH MiKited. and this leads to a further /.^b) by conducting the light stimulus to the receptors through a glass liher inserted below the pigment, but we have come to the same conclusion from different experimental evidence. Rapid light-adaptation in the presence of pigment migration therefore means that the effect of attenuation of the test Hash by the shielding pigment is approximately balanced bv dark-adaptation of the receptors. Methods Optical. The test and adapting lights were (>-v. tungsten filament microscope lamps placed in light-tight metal boxes fitted with camera shutters. For most experiment.- the light paths originated at right angles to each other; they were combined with a beam splitter and arrived at the cornea on the same optical path. In some experiments a second test light was arranged so as to stimulate the eye from another angle. Intensity of the test light was controlled with a circular optical wedge, and the adapting light with neutral density filters. In the experiments described below. intensity of the adapting light was sufficiently great to move the pigment to the proximal position. Infrared was attenuated with 1.25 cm. of a 10% (w/v) copper snlfate solution. Duration of the test exposures was monitored with a photocell. /\ec(»'(iin(/. Animals were immobilized in soft wax at the focus of the light beams. A shield of aluminum foil prevented light from reaching parts of the animal other than the illuminated eye. The retinal action potential was recorded with silver: si her chloride electrodes which made contact with the animal through a Ringer-filled capillary with a tip diameter of about 20 //. placed beneath the cornea oi the illuminated eye, and a saline-soaked wick on the dark side of the head. The Ringer was that of Ephrussi and Beadle (1936). The responses were recorded with a direct-con] 'led amplifier (Grass P-6) and photographed from the face of an oscilloscope. A -cries of responses to increasing intensities was recorded for the dark-adapted eye and whenever the animal was held for an extended period in a The test flashes were 0.5 sec. The height of the sustained negatr as a function of log I. Response-energy curves for the light- adapted rallel. or nearly so, to those obtained from the dark-adapted by Mernhard and Ottoson (1960a). Their displace- ment on the < es the change in sensitivity of the eye. During ligl laptation the eye was stimulated every ten or more -'""ids i intensity. The shifting position of the response- energy curve, and nging sensitivity of the eye, was determined by comparing tl • ttial with the response-energy curve for the dark- adapted McDonald, 1(H7; Goldsmith, 11W>3). For the measurement of dark ; flashes were chosen that could be presented to 'he complete!; LO second intervals without themselves pro- ducing lighl adaptation. Adaptation measure] sualh. presented in terms of threshold rather ^ threshold is used below synonymously with LIGHT-ADAPTATION IN A MOTH EYE 479 -log relative sensitivity. It corresponds to the log of the intensity required for a constant effect. Twenty-three animals were studied. Results and Discussion Figure 4 shows that dark-adaptation of Gallcria incllonclla sometimes splits into two components. As shown by the more typical recovery curve in Figure 5, however, this is a species in which the two limbs of the curve are usurJly not distinct. o> > I I 9 • A o I I I I I I I I I I L_J I I I I L 10 20 30 minutes in the dc~x FIGURE 4. Dark-adaptation of the compound eye of Gallcria mcllonclla in an animal showing a distinct latency before pigment migration started. Overlap of pigment migration with physiological recovery has also been observed in other species (Bernhard, Hoglund and Ottoson, 1963). It means that following the extinction of the adapting light the dark migration of pigment has commenced with little or no latency. The important point, however, is that dark-adaptation of Galleria is increased in extent and prolonged in time by the presence of migratory shielding pigment, just as in other species studied by the Swedish workers. Not so light-adaptation. At least usually not ; we shall consider exceptions in due course. Figure 6A (filled circles) shows the abrupt rise in threshold of an initially dark-adapted eye as a function of time after turning on a steady adapting light. \Yithin seconds the sensitivity has reached its final value, even though the 480 CHARLES T. POST, JR. AND TIMOTHY II. (JOI.DSMITH laical observations of pigment movement indicate that migration requires minutes for completion. Figure <>A also shows that the wave form of the retinal action potential changes with time. The significance of this ohservation can be clarified by examining how the same eve responded when dark -adapted and stimulated by a series of flashes i increasing intensity ( Fig. 6B). (ianglionic components are small and are most evident at low intensities and at "on" the response is dominated by a graded O .c W O) 2 0) ^ 01 C7> O r i i T~T • "'* 10 20 minutes in the dark 30 al dark-adaptation curve than Figure 4, showing that in this migration usually overlaps the earlier, faster components of recovery. potential ly and persists as long as the stimulus. \Yith increasing intensity the increases. At high intensities, instead of rising smoothly to :i plateau ik, about 100-200 msec, duration and graded with intensity, pn idy value. This initial peak is evident in the response to the brig 0). F.vidence of other kinds, including intracellular recording ihat both the initial peak and the plateau arise in the retinular cells (see Foi review) ; for present purposes, however, it LIGHT-ADAPTATION IN A MOTH EYE 481 is sufficient to note that the initial peak is ohserved only when the cells are illuminated strongly. The test flash used for the light-adaptation in Figure 6 was log / : -1, which produced a barely perceptible initial peak in the dark-adapted eye. On turning on the adapting light the response was decreased, but the initial peak became more prominent relative to the plateau (cf. responses at 0 and 24 sec.). The latter is o - 1 T J I I L_L J I L_L J I I I L_L -4 a o T3 o * -2 5 10 minutes in the light 15 FIGURE 6. Light-adaptation of a compound eye of GaUcria. Filled circles, based on the maintained plateau of the retinal action potential ; open circles, based on the greatest excursion of the response. Several of the oscillograms from which the experimental points were calculated are included with the curve to show the progressive change in wave form of the retinal action potential during light adaptation. To the right of the graph is a series of responses to different intensities of stimulation recorded from the same animal while the eye was dark-adapted, just prior to irradiating with the adapting light. Test flashes were 0.5 sec. duration ; a 10 mv. calil). ation mark is included with each trace. of having more light- beams — incident on the the expected result receptors. The change in shape of the electrical response poses a problem : should one calculate sensitivity on the basis of the plateau or the highest part of the wave form? It is not obvious which is most important at the first synapse, so the issue has been avoided in Figure 6 by plotting both results. The filled circles are determined from the plateaus ; the open circles are based on the greatest excursion of the response, which in the early minutes was the initial peak. By six minutes the peak had sub- :v CHARLES T. POST, JR. AND TIMOTHY H. GOLDSMITH .sided. however. and tin- two light-adaptation curves meet. Even between 9.5 and 15 minutes, though, tlie form of the response continued to change; the height of response remained constant, hut its rate of rise decreased. These progressive changes in the shape of the retinal action potential are characteri.stic of decreasing intensities of stimulation. They can be accounted for by a gradual diminution of the light reaching the receptors caused by the proximal migration of shielding pigment. The pigment is known to be moving during this time, even though the sensitivity of the eve is constant. That attenuation of the test and adapting lights should produce equal and • >pi ">site effects on the sensitivity of the eye is a consequence of two factors: (a) equal absorption by the screening pigment, and (b) the applicability of Weber's law at the level of the retinal action potential. Insofar as the test and adapting beams are coaxial, for each position of pigment, absorption of the two lights will be the same. When the intensity of the test light reaching the receptors is decreased by the pigment screen to 0.1 of a previous value, clearly the energy in the flash will have to be increased ten-fold to maintain a constant response from the receptors; that is, sensitivity of the eye will have fallen one log unit. But if A/// ( ratio of incremental threshold to adapting intensity) is constant, at least over a significant range, the fall in intensity of the adapting light will produce an exactly equivalent increase in sensitivity of the receptors. Writing Weber's law. I^-Ja , where It and /„ are the intensities of the test and adapting lights, one can see that /, = /«(*+ 1) log It =-- log/,, + const. In other words, the curve log /, rs. log /„ has a slope of unity (except close to where the simple form of Weber's law used here must be modified), and f one log unit in /„. will produce an increase of one log unit in the i big. X). rates of light adaptation as shown in Figure 6A are not always ~ compares the light adaptation of two other animals, one rapid, I'.oth curves come within 0.2 log unit of the same final on the plateau of the retinal action potential. Other animals hgl ' at interim diate rates. ihe time course of light-adaptation is usually rapid - relatively slow in some. A slow rate of light-adapta- """ means i! siiioii of pigment (a) the tesl Hash is attenuated more than the adapt i flu- decrease in energy of the adapting light fails to produce an equal of the receptors. \\'e shall consider each of these i«>-.,ibilities in in: \\hat might be <|iial absorption of the test and adapting \fter the initial shold observed during the first few seconds, lurther changes \\-\\\ dep riately weighted averages of the optical LIGHT-ADAPTATION IN A AIOTH EYE 483 o .c d> 0> ~ I 0< o *-•-• I I I 5 10 minutes in the light FIGURE 7. Light-adaptation of two eyes, illustrating the variation in rate which is observed in different animals. Both curves are based on the height of the plateau in the retinal action potential. densities of the light paths in front of the receptors : log recovery oc O.D. of adapting path log rise in threshold oc O.D. of test path Consequently, A log threshold =: (O.D. test path) - - (O.D. adapting path). This difference in optical densities will vary as a function of time until pigment migration is complete. With coaxial beams it is not obvious how either light could be selectively screened, for both beams entered the eye along the same path. Nevertheless, in the hope of accentuating a selective absorption we- have performed several experiments with one or the other of the beams impinging on the eye obliquely. When the test light strikes the pigment screen obliquely and the adapting light enters more or less axially, the O.D. of the test path will likely be greater than the O.D. of the adapting path, and A log threshold will continue to rise until the pig- ment has reached the proximal position. On the other hand, if the adapting light is oblique and the test light axial, the difference in O.D. will be negative and log threshold should fall somewhat. When the optical densities are equal (coaxial beams) there will be no further change in threshold irrespective of pigment position. Of three experiments with an oblique test light, adaptation was very slow in two but essentially instantaneous in the third. In three experiments with an oblique CHARLES T. POST, JR. AND TIMOTHY H. GOLDSMITH adapting light the threshold ro.se to a maximum but did not subsequently decrease. In two of these animals tin- time course was rapid, but in the tbird it was slow, lu these experiments there is therefore no clear demonstration of the effects that are expected as a result of differential absorption. The most convincing experiments, however, are two in which light-adaptation was measured with a pair of test flashes presented alternately at intervals of several seconds, one oblique and a second coaxial with the adapting beam. These are shown in Figure u. In each case the change in sensitivity to the axial test light was greater than to the oblique test light. This means that the populations of receptors responding to the two test lights were not identical and that the adapting light did not have an equal effect everywhere in the eye. In each experiment the time courses of adaptation were the same with the two test lights, and in one experiment they were very rapid. The difference in the shapes of the light-adapta- tion curves clearlv depends on something other than the geometric arrangement of stimulating and adapting lights. Thus, under the conditions of these experiments there is no evidence for preferential absorption of either beam. This brings us to the second possibility — that under some conditions there is an a-\mnietrv in the physiological effects of decreased test and adapting lights. For example, attenuation of the adapting light might be more rapid than the ensuing equivalent recovery of the receptors. This could be observed if in some individuals resyntbesis of visual pigment or some other physiological component of recovery were unable to keep pace with the proximal migration of screening pigment, either cn o the increment threshold function for ;i photorcceptor e in the adapting intensity leads to an equal recovery of the i1'1"". . aicliun for a receptor in which a drop of adapting intensity ot l.H ]r,u unit only a ().<>/ log unit fall in the increment threshold. See the text : ' LIGHT-ADAPTATION IN A MOTH EYE 485 o JZ QJ i_ ,C o> I I o-o-o-o-0-o-0_0.0_0.0_0_0_o_o.o-o - coaxial oblique I I I I I I I I I 0 minutes 10 in the 15 20 light FIGURE 9. Pairs of light-adaptation curves for two animals (circles and triangles). Each pair of curves was measured during the same adapting exposure by stimulating the eye alter- nately with test flashes coaxial with the adapting beam (filled symbols) and obliquely (about 70°) to the adapting beam (open symbols). One animal (triangles) light-adapted much more rapidly than the other, irrespective of the angle between the test and adapting lights. All curves were based on the plateau in the retinal action potential. because the former were unusually slow or the latter unusually rapid. If such were the case, slon.' light-adaptation would be observed in eyes with the fastest rates of pigment movement. Whether these rates vary sufficiently to account for the different rates of light-adaptation which have been observed is not known. Judging from the speed of dark-adaptation of photopic eyes, however, it seems unlikely that recovery of the receptors of scotopic eyes would lag behind movement of the pigment. Alternatively, attenuation of the adapting light might not be followed by an equal dark-adaptation. If the increment threshold function, plotted as log /test vs. log lariapt. had a slope less than 1.0, partial dark-adaptation of the receptors caused by screening of the adapting light would IK it completely offset the rise in threshold CHARLES T. POST, JR. AND TIMOTHY II. (JOLDSMITH caused hv attenuation of the test light (Fig. 8). Measurements of this slope in seven animals gave values ranging from 1.0 to (U>7. The mean was 0.88 with a standard deviation of ±0.16, and the distribution was skewed toward lower values. In each case the measurement^ were made over approximately the same range of intensities. In the animal which provided the smallest value, attenuation of the test and adapting light 1 log unit by screening pigment would permit the receptors to recover only 0.67 log unit, and a slow component of light-adaptation covering 0.33 log unit might be expected to occur as pigment moved inward. The maximum range over which pigment controls sensitivity during dark-adaptation is about three log units (Bernhard and Ottosoii. 1V(>4). Making the most generous allowance which is justified by present evidence, therefore, a slow component of light-adapta- tion based on a non-linear relation between A/ and / might be expected to cover as much as one log unit. With the possible exception of two animals, all of the moths studied did not exceed this value. In the two apparent exceptions, light adaptation was not followed by a control dark-adaptation, so that the possibility of physiological deterioration cannot be eliminated. The experiments reported here lend support to the view that the presence of migratory shielding pigment in the compound eyes of arthropods extends the dynamic range of these eyes and is an adaptation for vision under scotopic conditions. \Ve are grateful to Mrs. Wiliam Palumbo for her skillful assistance, particularly with the histology; to Dr. Raimon Beard for several gifts of Galleria stock; and to Xathan Mandell for aid in the construction of equipment. SUMMARY 1. An energy flux at the cornea of 5 X 103 ergs sec."1 cm.~- at 500 ITI/A causes almost complete migration of the accessory shielding pigment in the eye of the wax moth, (idllcria iticllonclla. Movement requires approximately 30 minutes. Lower bring the pigment to steady-state positions intermediate between the light- and dark-adapted positions. .iglit-adaptation. unlike dark-adaptation, runs a faster time course than migration and is frequently complete in several seconds. This confirms llogluncl. The theoretical reasons for a rapid rate of light- face of slow pigment migration are discussed and are shown (a) equal absorption of the test and adapting lights by the '".nt, and (b) a linear relation between A/ and I. \ that the terms, superposition and apposition eye, be replaced "I1" .is the latter indicate more accurately the physiological properties LITERATURE CITED I960a Comparative studies on dark adaptation in the diurnal 1 -epidoptrra. ./. C,cn. Pliysiol., 44: 1(^5-20.3. Studies on the relation between pigment uu.m-ation !, adaptation in diurnal and nocturnal Lepi- doptcra. /. Gen. , 44: 205 215. LIGHT-ADAPTATION IN A MOTH EYE 487 i, C. G., AND I). OTTOSON, 1964. Quantitative studios on pigment migration and light sensitivity in the compound eye at different light intensities. /. Gen. PhysioL, 47: 465-478. BERNHARD, C. G., G. HOGLUND AND D. OTTOSON, 1963. On the relation between pigment posi- tion and light sensitivity of the compound eye in different nocturnal insects. /. Ins. Physiol.,9: 573-586. DAY, M. F., 1941. Pigment migration in the eyes of the moth, Ephcsfia kiihnicUa Zeller. Biol. Bull. ,80:275-291. DUTKY, S. R., J. V. THOMPSON AND G. E. CANTWELL, 1962. A technique for mass rearing the greater wax moth. Proc. Entornol. Soc. Washington, 64: 56-58. EPHRUSSI, B., AND G. W. BEADLE, 1936. A technique of transplantation for Drosophila. Am. Naturalist, 70: 218-225. EXNER, S., 1891. Die Physiologic der facettierten Augen von Krebsen und Insecten. Franz Deuticke, Vienna. GOLDSMITH, T. H., 1963. The course of light and dark adaptation in the compound eye of the honey-bee. Comp. Biochem. Physio!., 10: 227-237. GOLDSMITH, T. H., 1964. The visual system of insects. In: The Physiology of Insecta (M. Rockstein, ed.), vol. 1, chap. 10, pp. 397-462. Academic Press, New York. HARTLINE, H. K., AND P. R. MCDONALD, 1947. Light and dark adaptation of single photo- receptor elements in the eye of Limnhts. J. Cell. Comp. PhysioL, 30: 225-253. HOGLUND, G., 1963a. Glow, sensitivity changes and pigment migration in the compound eye of nocturnal Lepidoptera. Life Sciences, No. 4: 275-280. HOGLUND, G., 1963b. Receptor sensitivity and pigment position in the compound eye of noc- turnal Lepidoptera. Life Sciences, No. 11: 862-865. KUIPER, J. W., 1962. The optics of the compound eye. In: Biological Receptor Mechanisms (J. W. L. Beament, ed.). Symp. Soc. E.rp. Biol.. 16: 58-71, Cambridge Univ. Press, London. RUCK, P., 1962. On photoreceptor mechanisms of retinula cells. Biol. Bull., 123: 618-634. TUURALA, O., 1954. Histologische und physiologische Untersuchungen iiber die photo- mechanischen Erscheinungen in den Augen der Lepidopteren. Ann. Acad. Sci. Fenn., Ser.A,24: 1-69. A DIGENETIC TREMATODE, BOTULUS CABLEI, N. SR, FROM THE STOMACH OF THE LAXCETFISH, ALEPISAURUS BOREALIS GILL, TAKEN IX THE SOUTH PACIFIC1 HORACE W. STUNKARD The American Museum of Xatiiral History, Central Park ll'est at 79th Street, Ne^< York (luiart (1938) described a new genus and species, Botnlits alcpldosauri, on the basis of two specimens from the intestine of Alcpisaunts jcro.v taken July 1, 1897, at a depth of 2480 meters in the Atlantic Ocean south of the Island of Madeira. One of the worms. 27 mm. long, 7 mm. wide and 6 mm. thick, was photographed and later lost through desiccation; when found it measured only 18 mm. in length, 5 mm. in widtb and 4 mm. in thickness. Attempts to restore it were fruitless but the contraction disclosed the uterine loops which occupied "toute la region moyenne du corps." The other specimen was "en assez mauvais etat de conservation et avait etc deteriore, sans doute lors de 1'ouverture du tube digestif de 1'hote." It was Mctioned for histnlogieal .study, but the two photomicrographs showing cross- sections, taken near the middle and near the posterior end of the body, disclose the disintegration of the tissues. Owing to the condition of the material, the description of the species is superficial and incomplete ; no measurements are given other than the external dimensions of one specimen ; and it is impossible to recognize specific characters. To receive the genus Botnlus, Guiart erected a new family, Botulidae, in. ting that it has the external characters of the Azygiidae and the internal charac- ters of the Hirudinellidae. Accordingly, (iuiart placed the new family between the Azygiidae and the Hirudinellidae. several trematodes were found in the stomachs of .llcpisaitnts borcalis taken lary 23, 1963, at Station #12 (26° 04.9' S. and 101° 47.0' W.) near Faster Island in the South Pacific by the Shoyo Maru Cruise of the Inter-American una Commission. Eleven specimens were submitted by the collector, Mr. . to the writer for study and identification. Subsequently, two addi- re received from Mr. Klawe. They were taken November 5, 1(>< £13 (30° 57.9' S. and 127° 0.2.5' W.) by Mr. Eric D. Forsbei-h. Grateful acki enl is made to Mr. Klawe for his kindness. These worms - of ilie genus Ilotitlits, and since they cannot be determined specii with R. Icpidosanri, they are identified as a new species. ' Professor Raymond M. Cable and in recognition of his contributions to kno1 the Trematoda. Two of the spei stained and mounted /;; tola, three were sectioned serially, two trans' he other frontally ; while another was dissected by the use ot line needles and pins. All are gravid and in each the vitellaria are visible as a broad longitudinal (lark 1 ventral .side of the body. 1 Tin** investigation ;>y XSF G-23561. 488 NEW DIGENETIC TREMATODE 489 FIGURE 1. Whole-mount, type specimen, 13 mm. long. FIGURE 2. Cross-section through the oral sucker at the level of the genital pore. The pharynx appears above the oral sucker. FIGURE 3. Cross-section through the anterior part of the acetahulum, showing the junctions of the cuticular lined limbs of the esophagus and the epithelial lined digestive ceca, and the genital sac with sections of the metraterm (ventral) and the ejaculatory duct. FIGURE 4. Frontal section through the junctions of the digestive ceca and the excretory vesicle. 490 HORACE W. STUNKARD The worms (Fig. 1 i arc ovate to pyrifonn to fusiform; cither end may be bluntly n mn. led or attenuated to a pointed tip. The body is only slightly flattened dorsoventrally and is broadly oval ( Figs. 2, 3) in cross-section. The largest worm is 33.5 mm. long and 12 mm. in greatest width. It is spindle-shaped, attenuated at both anterior and posterior ends. The next largest specimen is 23 mm. long and 11 mm. wide. The sides an- almost parallel: it is rounded posteriorly and tapers sharply at the anterior end. The smallest specimen is 11 mm. long and 6.5 mm. wide, broadly ovate with a pointed anterior tip. The acetabulum is situated about one fifth of the body length from the anterior end; it measures 1.44 to 2.25 mm. in diameter and the wall is 0.35 to 0.40 mm. in thickness. The cuticula is heavy, 0.15 to 0.25 mm. in thickness; it is unarmed, although rows of tine, often imbricated, transverse ridges may simulate spination. The surface often presents a granular or pebbled-leatber appearance, with refringent dots, but these points are apparently the openings of subcuticular glands. The excretory pore is terminal and the tip of the vesicle may protrude as a papilla. The vehicle extends anteriad. but the exceedingly vacuolated parenchyma makes it impossible to work out the excretory pattern. The body wall is strongly muscular, especially in the anterior portion of the worm : it is composed of external circular, medial longitudinal, and internal diagonal layers. The longitudinal layer is especially developed, sometimes measuring as much as 0.0(>4 mm. in thickness and composed of six to eight superimposed bundles of libers. The parenchyma is highly vacuolate, with large fluid-tilled cavities. The mouth is subterminal, the oral sucker is 0.09 to 1.5 mm. in diameter with strong muscular walls (Fig. 2). The lumen is triangular with the ventral apex directed toward the mouth. The sucker is partially overlapped and partially con- tinuous dorsally and posteriorly with the pharynx, which is almost as large as the oral ^ucker. The lumen of the pharynx is rhomboid to cruciform and the esophagus arises from the dorsal median crus. The esophagus is short and divides into two limbs that diverge laterally, expand to form the two heavy- walled "crops" or 'stomachs," and continue laterad and posteriad to open into the digestive ceca The esophagus is lined with cuticula and the ceca by a high columnar i'linin whose free ediM-s are filamentous. Posterior to the gonads, the ceca luminous, often with folded walls, but they are constricted posteriorly, communicate with the excretory vesicle (Figs. 1,4). tal pon is located anteriorly and ventrally, a short distance behind the ng genital atrium extends to the level of the posterior end terminates in a dorsally located protrusible and retractile papilla \ openings of the male and female genital ducts. The testes an- opposite or somewhat oblique with the left one slightly anterior and dors; right, overlapping medially, and situated immediately posterior to tl Sperm ducts arise from the dorsal, anterior, medial 3 of the '< forward and mediad and join above the posterior border of the acetabulun onn the seminal vesicle, an expanded, thin-walled tube, tilled with spermato vhich coils about above the acetabulnm. It loops posteriad and turns forv it is enclosed by a layer. 0.11 to 0.15 mm. in thickness, of deeply stail cells, and surrounded bv a fibrous covering. This prostatic SCCti if the male duct coils anteriad and dorsad, then loses its NEW DIGENETIC TREMATODE 491 glandular covering and as a coiled ejaculatory duct, together with the metraterm, enters a muscular-walled sac (Fig. 3), which extends forward and terminates in the papilla which protrudes into the genital atrium. The ovary is oval. 1.00 to 1.60 mm. long; 1.50 to 1.75 mm. wide; and 1.4 to I/) mm. deep, situated slightly left of the median plane. The oviduct arises at the median, posterior face of the ovary and winds ventrad where it receives a duct from the vitelline receptacle and then anteriad and dorsal to expand into the ootype, surrounded by the cells of Mehlis' gland, which measures about 0.50 mm. in lateral and 1 .00 mm. in anteroposterior and dorsoventral dimensions. As the duct reaches the dorsal portion of the gland, Laurer's canal diverges and coils dorsally, opening to the surface of the body in the midline above the posterior border of the ovary. The uterus begins at the emergence from Mehlis' gland and the initial coils are filled with masses of spermatozoa. The uterus winds posteriad and laterad, the loops become longer to extend from one side of the body to the other, dorsally and ventrally, occupying the middle third of the body and extending forward on either side of the gonads to the level of the acetabulum. Here the wall of the uterus, which previously was very thin and flexible, becomes thick and muscular, and as the metraterm, passes forward to enter the muscular sac which encloses it and the ejaculatory duct (Fig. 3). The vitellaria are exceedingly numerous and filamentous, extending in a tangled mass on the ventral side of the body, posteriad in the central or median field at least four-fifths of the distance from the ovary to the posterior end of the body. Anteriorly the vitellaria converge and form a vitelline receptacle from which a short and narrow duct opens into the oviduct. The eggs are oval, thick-shelled, operculate and without filaments. They are rmbryonated when passed and measure 0.030 to 0.034 by 0.023 to 0.025 mm. The description of Botuhis alcpidosauri by Guiart is so incomplete that it is difficult to recognize the species. The textual account deals with generic characters and the specific features must be derived from the figures. Figure 5 portrays a sagittal section of the anterior region of the body and is highly schematic ; Figures 26 and 27 give little precise information. According to Figure 5. the pharynx is much smaller than the oral sucker and quite distinct from it, and the testes are represented as one behind the other. Figure 26 suggests that the vitellaria are dorsal in position, but it is probable that the ventral side of the section is uppermost in the plate. Since the present specimens do not conform to the description of B. alepidosauri, they cannot be assigned to that species. The worms are clearly members of the genus Botuhis and are described as a new species, Botuhis cablei. Type, cotype, and other specimens are deposited in the U. S. National Museum, Helminthological Collection, No. 60934. DISCUSSION Guiart reported the specimens of B. alepidosauri were from the intestine of the host, but noted resemblance between them and members of the Azygiidae and the Hirudinellidae. The finding of the worms in the intestine, both dead and one partially disintegrated, indicates that the fish had been dead for some time and that the worms had migrated from the stomach. The members of both the Azygiidae and Hirudinellidae are stomach-parasites of fishes, but they are only distantly related and their similarity is clearly a result of convergence. Accordingly, the 4(>2 HORACE W. STl'XKAK I) allocation of the family llotulidae to a position intermediate bei ween the two families is irrelevant. Actually, the inorplmlo--;, of Jlohtlus agrees completely with the characti -r^ of the family 1 I inulinellidae l)ollfus, 1('32, as given by Yamaguti ( l*'5Si. and permits the inclusion of ^utitliis in that family. Meanwhile the family I'.otulidae is reduced to a subfamily, 1'oiulinae. According to a report in Helminthological Abstracts. 30: 355 (1961), A. S. Skriabin ( 1('5S) described a dinurid trematode from .Ucpisciunts acscnlapius taken in the I'acitic ( )cean as a new genus and species, Profundiella skrjahini. The specimens differed from members of the Prosorchiinae Yamaguti, 1934. family I lemiuridae, in the position of the testes and the ovary and in the presence of so-called "stomachs" in the dige>tive system. These features were predicated as the basis for a new subfamily, Profundiellinae. But where these specimens differed from the I 'rosorchinae, they agree with the genus Botnlus and there is a possibility that the two generic concepts are identical. In that event Projundiclla will disappear as a synonym. Morphological agreement appears sufficient to warrant inclusion of Profundiella in the subfamily Botulinae, with suppression of the subfamily 1 'ri ifundiellinae. LITERATURE CITED Gi CART, J. K.. 1'J.iS. Trematodes parasites provenant des Campagnes scientifiques du Prince \lbert KT de Monaco. Result. Camp. Scient. de Albert I, Prince de Monaco. 75 pp. Monaco. SKKIAMIX, \. S., 1958. Papers on Helminthology presented to Academician K. I. Skrjabin on his 80th Birthday. Moscow: Izdatelstvo Akademii Nauk SSSR, pp. 340-344. \' \\IAGUTI, S., 1958. Systcma Helminthum. Vol. I, part 1: 251. ON THE LIFE-HISTORY OF RHIZOSTOME MEDUSAE. III. ON THE EFFECTS OF TEMPERATURE ON THE STROBILATION OF MASTIGIAS PAPUA YASUO SUGIURAi Misaki Senior HiyJi School, Miura-Shi, Kanagawa-Ken, Japan The fact that water temperature has a great deal to do with the initiation of strobilation has been reported for some time. However, most of the cases known are on Aurella and Chrysaoni etc.. in which the onset of strobilation is correlated with low temperature (Berrill, 1949; Thiel, 1962). Contrariwise, it is known that Cassiopca and Cotylorhiza strobilate in summer (Berrill, 1949). Mastn/ias papita belongs to this group (Uchida, 1926: Sugiura, 1963). At the same time, the present author reported an indispensability of symbiosis of zooxanthellae for strobilation (1964). But in Alastiyias papiia, too, since temperature effect on the metamorphosis is evident, the case will be described in this report. MATERIALS AND METHODS In the present work, two sets of temperature measurements were performed. ( 1 ) By following change of the temperature of natural sea water, the temperature at which the ephyrae made the first appearance was determined. (2) In the laboratory, the efficacy of the induction of strobilation under different temperature was examined. As reported in a previous paper (Sugiura, 1964), in the scyphistomae of Mastigias papua which have been reared in a zooxanthella-free condition, strobilation can be induced by supplying the symbionts in the culture. Temperature effect on such an induction was studied. For approximate regulation of temperature, rearing boxes provided with electric bulbs were used. For finer control of temperature, incubators and a constant temperature room were utilized. Considering the necessity of illumination for maintaining a healthy condition of zooxanthellae, scyphistomae were continuously exposed to the light from a fluorescent lamp during the temperature experiments. RESULTS Sea water temperature and strobilation in nature Every year, the earliest appearance of ephyrae of Mastigias papua in the vicinity of the Misaki Laboratory is early summer. According to the survey of 1962, it was July 11. On July 25. abundant ephyrae. which seemed to have been released recently, were obtained (Sugiura, 1963). 1 Present address: Misaki Marine Biological Station, Misaki, Miura-Shi, Kanagawa-Ken, Japan. 493 YASUO SUGIURA Simultaneously \vith these observations, temperature measurements were made on the habitat. Notwithstanding the fact that the scyphistomae of Masti(/ias Capita have not been found in nature ><> far. it mav not be unjustifiable to con- >ider that the scyphistomae must be living on the bottom ot the spot where ephyrae begin to appear, particularly because the area is very limited in the inner- most part of the bay. Considering that sea water temperature to be related to strobilation must be that at the bottom of the bay, measurements were taken at 1.5-3.0 m. in depth in the bays of Aburatsuho and Moroiso. The sea water tem- perature during the first week of the appearance of the ephyrae turned out to be 23° C. (Table I). However, taking into account the results of indoor experiments to be given in a following section, and also the possibility that the beginning of strobilation TABLE I .SV(/ -milter temperature at the time of the first appearance of the ephyrae July 11 July 18 July 25 2.1.0° C. 2,vO° C. 26.5-27.0° C. 26.0-29.0° C. in nature could be at least a week before the sighting of the ephyrae, the bottom temperature for strobilation was judged to be about 22° C. Effects of reariiii/ temperature for the strobilation in scyphistomae u'itJi zooxanihellae Schyphistomae having zooxanthellae, with previous history of strobilation, were reared from late autumn of the year until the next spring in boxes kept several degrees warmer than room temperature by electric bulbs. Three grades •<\ temperature were tested. The procedure was as follows: Test organisms were put in three boxes of the same size and although temperatures in the boxes were wed to fluctuate with the room temperature, box A was kept about 10° C. ter ronstantly and B a little less higher and C still closer to the room tem- In reality the lowest temperature in A was 19° C., in H it was 17° C. ' C. when the room temperature was around 10° C. - temperature rose with the season, initiation of strobilation ' March 21), and a month later (April 15-24) in B in 6 group, while no strobilation had taken place in C by that /ater temperature at the beginning of strobilation was 20-22° C. in both A and B • the result will be. if the culturing temperature is never allowed to K<>r this purpose, the culture C was transferred to the constant temperature of 20° C. at the middle of April before the atmos- phere readied that rature level. This group had not strobilated at least until June point (June 25), C was further divided into two classes, one brinsj, C. while the other was returned to room M-mperatnrr. The former did . robilate all through the .Mimmer. In the latt«T ^ronp. however, th iir>t strobilation took place on August 7. TEMPERATURE AND MEDUSAN STROBILATION 495 In conclusion, 20° C. seems to lie just below a critical level and culturing temperature has to go beyond this threshold in order to stimulate strobilation. On the basis of the almost perfect agreement between the data obtained in nature and in the laboratory, it is possible to say that 22° C. is the lowest effective tem- perature for inducing strobilation. Needless to say, in zooxanthella-free scyphi- stomae, even much higher temperatures are quite ineffective for causing strobila- tion. Temperature higher than 22° C. For studying the effect of temperature higher than 22° C., it was unavoidable to collect individuals which have failed, for some reason or another, to react to 27 24 20 •< oc J 1 1 I I 10 J 1 1 1 L 15 27 24 10 15 DAYS FIGURE 1. The effect of water temperature on two steps of strobilation. Upper chart : Latency between symbiosis and the beginning of morphological change for strobilation. Lower chart : Latency between the beginning of metamorphosis and the release of the ephyrae. 22° C. with strobilation. Using these organisms, a part maintained at 28-29° C. for 20 clays and the remainder back to 20° C. for a month (Sept. 21-Oct. 21) and group a, no strobilation occurred in all five dishes, while tion began within 10 days after raising the temperature In order to confirm the above situation further, the b were mixed and scyphistomae were randomly divided two parts were equally subjected to 20° C. for a month at when one part was transferred to 25° C. and the other of them (group a) was (group b) was first set then to 28-29° C. In among group b, strobila- in 4 dishes out of 5. group a and the group into two parts and the first (Nov. 26-Dec. 30) part kept at 20° C. In 4'H> YASUO SUGIURA the part made warmer, strobilation took place in all 4 dishes within a month, while in the cooler part, strobilation was seen in none of 4 dishes. Judging from the above experiments, when scyphistomae fail once to react to 22° C.. a further increase of the culturing temperature has no effect for causing stimulation. Instead, the temperature should be lowered once more for nearly a month before raising it, during which time the organisms seem to be made receptive to a warm temperature. At any rate, the main point of this finding is that it is now possible to induce strobilation by regulating the temperature, re- gardless of the season. Tempera/ lire effects <>n separate steps oj the process oj strohilation When scyphistomae which have over-wintered in a zooxanthella-free condition are given the symbionts, they strobilate immediately (within several days) with a high degree of reliability. Moreover, they can sometimes strobilate at 20° C.. showing their sensitivitv has been heightened by a long sojourn in a low tem- perature. Using such materials, the effects of 27°, 24° and 20° C. were examined concerning the latencv between supplying the symbionts and the beginning of strobilation and that between the onset of strobilation and the release of ephyrae (Fig. 1). As is clear from the figure, the latter step is more sensitive to change of temperature. Since the former step mostly corresponds to the period of multiplication of symbionts. this may mean that zooxanthellae are less sensitive to temperature change than the medusa itself. The writer wishes to express his thanks to Prof. K. Dan for his constant guidance in the course of the work. SUMMARY 1. In the habitat, the first appearance of the ephyrae coincides with the sea water temperature of about 22° C. In the laboratory, the minimum effective range of temperature for induc- tion o! strobilation by giving zooxanthellae is found to be 20-22° C. Individuals with zooxanthellae. which have once failed to react by strobila- ' C. can no longer be induced for strobilation by a further rise of temperature. • individuals do react if they are cooled to 20° C. for about a month -ed to the higher temperature. LITER ATI' RE CITED .•elopmeutal analysis of Seyphomedusae. Hint. Rcr.. 24: 3<)3-410. '•Yirm and LM'outh in the development of a Scyphomedusa. Hint. But!., 96: . the 1; tory of rhi/oslome medusae. 1. M'usthiias pupiia. Annot. Zoo SUGI listory of rhizostome medusae. II. Indispensability of i \fastigias pnpua. Ili>df>tiu L. Vgassiz, with ol ihylogeny of I\hi/ostomae. /. I'nc. .SV;. /;/;/>. I'ni''. Tokyo, Sect. IV. Zool, 1: PHYSIOLOGY OF INSECT DIAPAUSE. XV. THE TRANSMISSION OF PHOTOPERIOD SIGNALS TO THE BRAIN OF THE OAK SILKWORM. ANTHERAEA PERNYI * CARROLL M. WILLIAMS, PERRY L. ADKISSOX - AND CHARLES WALCOTT The Biolof/ieal Laboratories and the Dirixion ol Engineering and Applied l'h\sics, Hari'iird I Hii'ersity, Cambridge, Massachusetts The pupa of the oak silkworm, Anthcraca pcrnyl, is enveloped in a stout- walled cocoon which gives every impression of being opaque. It seems incredible that an insect in this situation could be influenced by the length of the day and night. Yet such is indeed the case. As demonstrated in the previous paper of this series (Williams and Adkisson, 1964), diapause is persistent at 25° C. when cocoons are exposed to short-day conditions (daily photophases of 4 to 12 hours). By contrast, photophases of 15 to 18 hours provoke the termination of dormancy and the initiation of adult development. It was possible to show that the sensitivity to photoperiod depends on the direct action of light on the pupal brain. In some unexplained manner, light of appropriate wave-length is able to penetrate the opaque cocoon and the pupal cuticle to act on the brain itself. The phenomenon is examined in detail in the present investigation. All ex- periments were performed on A. pertiyi. The cocoons were the diapausing first- brood harvested in late July ; on August 27 they were shipped from Japan in a series of cardboard boxes which arrived at Harvard University on September 30. The cocoons were spread on tables and stored at 25 ± 0.5° C. in a room programmed for a daily illumination of 8 hours. EXPERIMENTAL RESULTS 1. Photoperiod responses of naked pupae versus pupae in cocoons Ninety-six diapausing pupae were removed from cocoons. Forty-eight were placed in an incubator programmed for a daily 8-hour photophase at 25° C. ; the other 48 were placed in a similar incubator programmed for a 16-hour photophase. Each incubator also received 92 unopened cocoons from the same lot of material. The cocoons were spread on the shelves and thereby exposed to fluorescent illumi- nation having an average intensity of 175 foot-candles (1883 lux). The naked pupae were inspected once a week to detect the initiation of adult development in terms of the retraction of the wing epithelium. In the case of the intact cocoons, the emergence of the moths was noted daily ; the initiation of 1 This study was supported, in part, by funds from the National Science Foundation, the National Institutes of Health, and the Office of Naval Research. - Special Postdoctoral Fellow of the U. S. Public Health Service during the term of the study. Permanent address : Department of Entomology, Texas A & M University, College Station, Texas. 497 498 WILLIAMS, ADKISSOX AND \\.\LCOTT adult development \va> computed as having occurred 20 davs prior to adult emergence i Williams and Adkisson. 1('M). The results arc- summari/ed in Figure 1. .During the X-week term of the experiment, diapause was persistent in all naked pupae and intact cocoons ex- posed to the short-day regimen (horizontal line in Figure 1). By contrast, the entire series of animals exposed to the long-day conditions initiated development. \s indicated In the upper pair of curves in Figure 1, pupae in cocoons responded just as promptly to the long-day stimulus as pupae removed from cocoons. This finding shows that, despite the relatively low intensity of the incident illumination, the presence of the cocoon did not curtail the sensitivity to photoperiod. 100 80 a. o a 60 40 20 0 In cocoons ( 16-hr-photophase) Naked pupae (16-hr-photophase) 1 2345678 WEEKS AT 25°C ' ects nf long- and short-day photoperiods on the termination of pupal I naked pupae and by pupae in cocoons. All individuals exposed to the long-day men initialed development U«f> I MI curves'). None initiated development when exposed to 'loin line). 2. Spi'dral . uj dia pausing Pcniyi pupae <• removed from cocoons and ten were placed head-up in each oi ons of an aluminum "muffin tin." Arrangements were made tor po^itionin : ters over the depressions. For this purpose each •> cm. filter with masking tape to a 2-cm. collar cut from a 7.5- cm. thin-wall l»ras> ;.ach filter was positioned over a specific group of pupae and sealed m place with a gasket of plasticene. LIGHT TRANSMISSION TO SILKWORM BRAIN 499 With the filters removed, the entire assembly was placed at 25° C. in an incuhator programmed for a dailv l()-hour photophase. After 8 hours of ex- posure to the unaltered light, the filters were sealed iu place so that the pupae received filtered-light during the final S hours of the U>-hour photophase. The filters were removed the following morning at the outset of the next photophase. This regimen was repeated daily for 8 weeks. The five filters, together with their peak transmissions, were as follows : Corning color specification No. 5-62 (398 HI/A), No. 5-74 (434 HI/A), No. 4-105 (508 mfji), No. 3-110 (580 m^), and No. 2-78 (640 100 640 Red PEAK TRANSMISSION OF FILTERS (my) FIGURE 2. The spectral sensitivity of diapausing pupae of A. pernyi. The wave-lengths effective in causing the termination of diapause include violet, blue, and hlue-green light. Each of the five groups of pupae was inspected daily in order to detect the initiation of adult development. As controls, two groups of ten pupae were ex- posed to white light at 25° C. in incubators programmed for 8- and 16-hour photophases, respectively. During the 8-week term of the experiment, these con- trols behaved as follows: all the animals exposed to white light for 16 hours per day initiated development, while none initiated development under the short- day regimen. 500 \Y1I | | VMS, ADKISSON AND \\ \l > '< >TT The results observed in the live experimental groups are summarized in Figure 2. Diapause was terminated by all pupae expired to \iolet. bine, or blue- green light |3(>8 m// to 508 m// i ; indeed, these animals initiated development as promptly as the control series that received Id hours of nnliltered light. I'.y contrast, diapause was persistent in all pupae exposed to yellow light (580 m//, ) and in all hut one of the pupae exposed to red light (040 HI/A). 3. fj-Carotene- filtered lit/lit liecause of the involvement of carotenoids in so many photic reactions, a series of filters was prepared containing graded concentrations of /^-carotene dissolved in 95% ethanol. The solution was placed in optical flats prepared hy clamping a 0.5-cm. l.ucite ring hetween pairs of glass plates. The filters contained /^-carotene in final concentrations of 1000, 100, and 4 parts per million; the densest filter was a deep amher-yellow ; the least dense was almost colorless. The experiment descrihed in the preceding section was repeated using these filters. All individuals initiated adult development at approximately the same rate a> controls exposed to a Id-hour photophase of unfiltered light. This shows that /^-carotene does not remove all wave-lengths effective in the photoperiod response. \t the conclusion of the experiment, the absorption spectrum of each carotene solution was measured. Kven the most concentrated solution showed substantial transmission at wave-lengths higher than 470 m//,. Therefore, the carotene-filtered light contained wave-lengths (470-508 m/.t ) which were fully effective in imple- menting the photoperiod response of naked pupae. 4. Spectro photometric measurements A series of nine Kodak neutral density filters (0.9 ND) was calibrated for light transmission at wave-lengths ranging from 400 m/< to 700 m^. A Zeiss M 4 (J I 1 1 spectrophotometer was utilized for this purpose, the filter being placed in the1 experimental beam and its transmission measured against air. Transmission by the filters proved to be a function of wave-length and varied systematically from to 11', \ stack of five XI) filters was placed in the experimental beam and its trans- ion measured against a stack of four filters placed in the reference beam. The • in light transmission at all wave-lengths corresponded to that of a single1 'his demonstrated that the /eiss instrument was sufficiently sensitive to missions by objects as dense as five 0.9 ND filters. flattened fragment of a cocoon was positioned in the experimental beam and its mipared at each wave-length with that of a stack of four XD filters place nee beam. The transmission of the cocoon was then calculated for e; ized in the lower curve in Kignre 3 confirm that the cocoon is an extren o the simple transmission of light ; thus, at 400 m// the transmi ; at 700 ni/A. 0.014f/. P.y this same procedure, the transmission -d for the following fragments of pupal cuticle: (1 ) the unpigmented l the- tan wing cuticle of a palely pigmented pupa; •'"id <3) the black if a heavily pigmeiiled pupa. In these measure- LIGHT TRANSMISSION TO SILKWORM BRAIN 501 ments one or two XD tillers were placed in the reference beam. The results recorded as the top three1 curves in Figure 3 show the pupal cuticle to he 5000-fold more transparent than the cocoon to the effective wave-lengths. Moreover, the transmission of blue light (4(>0 m/i.) by the facial cuticle was 2 to 5 times that of the wing cuticles. This fact is illustrated in Figure 4. The anterior third of a Pernyi pupa has here been eviscerated and illuminated from behind to show the transmission of light through the unpigmented facial cuticle and, to a lesser degree, through the tan cuticle of thorax, legs, and antennae. 10 - WAVELENGTH (mu) FIGURE 3. Spectrophotometric studies of light transmission by the cocoon and by three types of pupal cuticle (top cities). curve') By a combination of the Spectrophotometric measurements on the cocoon and the facial cuticle, we calculate that, of blue light (460 m/0 incident on the outside of the cocoon, only 0.0000003 9£ could reach the brain by simple transmission. 5. The coc/xm us a light-integrating sphere Since the cocoon is essentially an opaque object in terms of the transmission of unscattered light, the demonstrated light-sensitivity of the pupa must depend on the collection and integration of scattered light (Jacqmv. and Kuppenheim, 1955). The geometry of the cocoon proves to be little- short of optimal for this purpose. This fact is illustrated in Figure 5 where an empty cocoon has been illuminated 502 \\ II I.I A MS. ADKISSON AND WALC< >TT FIGURES 4 7. LIGHT TRANSMISSION TO SILKWORM BRAIX 503 from behind by the foctissed beam of a 100-\vatt zirconium arc lamp. By excising the proximal face of the cocoon, one can witness the generali/ed illumination of the cavity of the cocoon. This same phenomenon is also illustrated in Figure 6. 1 I ere. the light-inte- grating hemisphere of an incident -light exposure meter bus been removed and replaced by the upper half of a Pernyi cocoon. A significant deflection of the meter is observed when the cocoon is illuminated. In order to describe this phenomenon in quantitative terms, the following experiment was performed : A cocoon was opened by a circumferential razor-blade cut around its long axis. After the removal of the pupa and the old larval skin, a micro-photocell (Texas Instruments LS-222 ) was suspended in the cavity of the cocoon. The latter was reassembled and sealed with Duco cement and a narrow band of opaque tape ; the paired leads to the photocell passed to the outside through the incision. The cocoon was suspended in a dark room at a standard distance from a microscope lamp (Bausch and Lomb Xo. SVB-73). The light first traversed a water-filled filter. 3 cm. in depth, and then illuminated one entire ISO0 hemisphere of the cocoon. The output of the photocell was measured in millivolts. The measurements were repeated with the cocoon oriented at various attitudes with reference to the incident white light. The output of the photocell varied from 120 to 270 millivolts, depending on the orientation of the cocoon, the average being about 240. The photocell was then removed from the cocoon and illuminated directly by the same white light at the same standard distance. The output was now 400 millivolts. Neutral density filters were then placed in the light beam to lower its intensity. A sandwich of two filters, each having lQc/< transmission for white light. decreased the photocell output to 240 millivolts. So. in rough terms, we can say that about \% of the incident white light reached the photocell when the latter was inside the cocoon. 6. Light-integration as a function of wave-length The preceding experiment was repeated with interference filters placed in the beam to establish monochromatic light. A total of nine wave-lengths was examined in this manner. Then with the photocell removed from the cocoon, the latter's properties at each wave-length were equated to that of the calibrated ND filters. The results are recorded as the upper curve in Figure 8. The light-integrating properties of the cocoon prove to be substantial, especially in the range 440 to 5 1 0 FIGURE 4. The anterior third of a Pernyi pupa has been eviscerated and illuminated from behind by white light. The photograph illustrates the transmission of light by the unpigmented facial cuticle and, to a lesser degree, by the' tan cuticle of the thorax, legs, and antennae. FIGURE 5. An empty cocoon is here illuminated from behind by an intense, focussed spot of white light from a 100-watt zirconium arc lamp. By excising the proximal face of the cocoon, one can witness the collection of scattered light within the cavity of the cocoon. FIGURE 6. A hemisphere of Pernyi cocoon serves as a light-integrating sphere when substituted for the integrating hemisphere of an incident light exposure meter. FIGURE 7. A micro-photocell has been sealed into a Pernyi pupa with its light-sensitive element placed just beneath the unpigmented facial cuticle. The pupa was then sealed inside the cocoon to permit measurements of the light energy reaching the brain at each wave-length. 504 WILLIAMS, ADKISSOX AND WALCOTT /. I.ii/lil reaching I lie / The cuticle overlying the legs was excised t'ruin an anesthetized pupa. The micro-photocell was then implanted through this mid-ventral area so that its light-sensitive element was positioned precisely beneath the transparent facial cuticle, i.e., in the place normally occupied by the brain. The photocell was held 1.6 - 400 Violet 450 Blue 500 550 Green 600 650 Red WAVELENGTH (mu) ' iif wave-length on t'nc per cent of incident li^ht energy which rein ti d-line) and \\hich reached the brain of a nupa scaled within a cocoon '. tem ^ho\\s a conspicuous "window" centering around 4n() ni^t. in place and i by the a])plication of opaque cement (see Kig. 7). The pupa wa> tl , imi a cocoon as described above. The measure ' under Section (\ were re])ealed with the results Limmarized by the Figure S. The optical properties of the entire M-Mem prove to }"•• ma: ••, , for blue light. Thus, in the range 440 to I'H), more than 0.14V hi n rgj reached the brain; indeed, at 4o() m/«, no t ban 0.5' ; of the scattered litdit ^ • ei-ollected to act on the brain. LIGHT TRANSMISSION TO SILKWORM BRAIN 505 I >isrrssioN 1. Spectral sensitii'itv o\ the pupal brain In the previous paper of the series (Williams and Adkisson, 1964), light was found to act directly on the pupal hrain to control the secretion of brain hormone. According to the present study, the effective wave-lengths extend throughout the lower third of the visible spectrum to include the violet, blue, and blue-green (398 m/x, to somewhat above 508 m//,)- Yellow or red light (580 mp. to 640 m/t) was without significant effects. The lowest wave-length tested (398 m/j.} was fullv effective; consequently, we are unable to state how far the spectral sensitivity extends into the ultraviolet. In order to have any effect, the pertinent wave-lengths must be absorbed. And since the effective wave-lengths include the lower third of the visible spectrum, the absorption of light is evidently accomplished by a pink brain pigment. The spectral sensitivity of the Pernyi brain is in close agreement with that described for the mite, Metatetranychus iiliui (Lees, 1955), and for certain, but by no means all. insects that have been studied (for reviews, see Lees, 1955; Bunning, 1960, 1964; Farner, 1961 ; de Wilde, 1962; Danilevskii, 1965 ). The Pernyi pigment therefore differs from the phytochrome system of the higher plants in terms of the latter's sensitivity to red and far-red (Borthwick, 1959; Hendricks, 1959). However, the photoperiodic responses of fungi (Bunning, 1964) and the phototropic reactions of higher plants (Shropshire and Withrow, 1958; Withrow, 1959) show a spectral sensitivity which is essentially the same as that of the Pernyi brain. The phototropic reactions of plants are thought to depend on the absorption of blue light by a carotenoid or flavin pigment. In the present investigation, /3-carotene was tested and found to be inappropriate in the insect system because of its scant absorption of certain wave-lengths (470-508 m/j. ) which were fully effective in the photoperiod response. However, it may be noted that numerous other carotenoids, such as the arthropod pigment, astaxanthin, show absorption spectra which include these higher wave-lengths ( Karrer and Jucker, 1950). 2. Optical properties of the cocoon and the pupal cuticle In order to be absorbed by the pink brain pigment, light must first traverse a series of barriers before reaching the brain ; namelv, the wall of the cocoon, the pupal cuticle, the underlying epidermis, and a narrow zone of hemolymph. Due to its opacity the cocoon is the major obstacle to the simple transmission of light (F"ig. 3). The pupal cuticle is, by contrast, 5000 times as transparent as the cocoon. Moreover, the measurements performed on the several categories of pupal cuticle show that, at 460 m/j., the unpigmented facial cuticle is twice as transparent as tan wing cuticle and .five times as transparent as black wing cuticle (Fig. 3). 3. Light-integration />v the cocoon Despite its opacity, the cocoon proves to be an effective vehicle for the collection and integration of scattered light ("haze"). The blue haze collected within the cavity of the cocoon then penetrates the pupal cuticle to act on the brain. Analogous 50(> WILL! \MS. ADKISSOX AND WALCOTT l)ii >lt >gical systems have been discussed in detail 1>\ Shibata (1958) and French (1959). ( )ur measurements reveal the surprising fact that the entire system is remarkably effective in the collection, integration, and transmission of blue light ranging from 440 HI/A to 4('() m//. As noted in Figure 8, a particularly prominent "window" centers around 400 m/i. All these wave-lengths, as mentioned above, are fully effective in the photoperiod reaction. 4. Lit /lit intensity In the experiments reported here, no attempt was made to obtain a true action spectrum in terms of the energy threshold of the brain as a function of wave-length. The fluorescent lam] is in our incubators saturated the brain's photoreceptive mechanism even though the incident illumination on the outside of the cocoons was only 175 foot-candles ( 1883 lux) — i.e., about 2-5r/r the intensity of direct sunlight. Spectroscopic examination of the fluorescent lamps revealed three intense emission lines (435.8 m//. 540.1 m//, and 577.0 m//, > of which only the one at 435.8 m/j, falls within the spectrum shown to be effective in the photoperiod reaction. If we assume that, of the' total light acting on the outside of the cocoon ( 175 foot- candles), about 2Sr/f is contributed by effective wave-lengths, the intensity factor is reduced to 44 foot -candles. Of this incident intensity, not more than about 0.2% could reach the brain after traversing the several barriers (Fig. 8). Hence we mav sav that the brain's photoreceptive mechanism is fullv saturated by intensities not in excess of about 1 foot-candle (10.8 lux) of blue light. This value is in line with those reported for other insects where saturating intensities ranging from 0.01 to 10 foot-candles (0.11-108 lux ) have generally been encountered (see Lees, 1955; Earner, 1(>ol ; de Wilde, SUMMARY 1. The pupal diapause of Anthcraca pcrnyi is sustained by exposure of cocoons -hort-day conditions (daily photophases of 4 to 12 hours") and terminated after ex pi isure to dailv phot< (phases of 1 5 to 18 hours. The photoperiod signal is conveyed by the direct action of violet, blue, and green light (398 508 m/< j on the brain itself. This finding implicates a pink brai In the absorption of the effective wave-lengths. its opacity, the cocoon functions as a light-integrating sphere in Tittered light — especiallv of blue light ranging from 440 m// to 510 m^.. on within the cavity of the cocoon, the blue "haze" penetrates the pupal in the brain. The brain's photoperiod mechanism is 'saturated" by 1 > 1 foot-candle of blue light. I.I I I K' \Tt'kK CITED BORTHWH K, II. [i< control "t llouering-. In: Photoperiodism and Related Phenomena in I'i. timals. T\. P,. XVitlirmv, edit., Tuhl. No. 55 of A.A.A.S., Washin MM., K., ]'"i<>. ' ircadian . i the time measurement in photoperiodism. Cold //,/•• /;;/>., 25: '49 256. LIGHT TRANSMISSION TO SILKWORM BRAIN 507 BUNNING, E., 1964. The Physiological Clock. Acad. Press. X. Y. DANILEVSKII, A. S., 1965. Photoperiodism and the Seasonal Development of Insects. Oliver and Boyd, London. FARNER, D. S., 1961. Comparative physiology: photoperiodicily. Ann. AY?1. I'liysiol., 23: 71-96. FRENCH, C. S., 1959. Action spectra and optical properties of cellular pigments. /;;: Photo- periodism and Related Phenomena in Plants and Animals. R. B. \Yithro\v, edit., Publ. No. 55 of A.A.A.S., Washington, D. C. : 15-40. HENDRICKS, S. B., 1959. The photoreaction and associated changes of plant photomorphogenesis. /;/: Photoperiodism and Related Phenomena in Plants and Animals. R. B. Withrow, edit., Publ. No. 55 of A.A.A.S., Washington. D. C. : 423-438. JACQUEX, T. A., AND H. F. KUPPENHEIM, In55. Theory of the integrating sphere. /. Opt. Soc Am.. 45: 460-470. KAKRER, P., AND E. JUCKER, 1950. Carotenoids. Elsevier, N. Y. LEES, A. D., 1955. The Physiology of Diapause in Arthropods. Cambridge Univ. Prc>v SHIBATA, K., 1958. Spectrophotometry of intact biological materials : Absolute and relative measurements of their transmission, reflection, and absorption spectra. /. Biochcin. Japan, 45: 599-623. SHROPSHIRE, W., JR., AND R. B. WITH ROW, 1958. Action spectrum of phototropic tip-curvature in Arena. Plant Pliysial.. 33: 360-365. WILDE, J. DE, 1962. Photoperiodism in insects and mites. Ann. Rcr. Enl/nnol., 7: 1-26. WILLIAMS, C. M., AND P. L. ADKISSON, 1964. Physiology of insect diapause. XIV. An endocrine mechanism for the photoperiodic control of pupal diapause in the oak silk- worm, Anthcraca pcniyi. Biol. Bull.. 127: 511-525. \\ ITHROW, R. B., 1959. A kinetic analysis of photoperiodism. In: Photoperiodism and Related Phenomena in Plants and Animals. R. B. Withrow, edit., Publ. Xo. 55 of A.A.A.S., Washington, D. C. : 439-474. CTENIDIAL Xr.Ml'.KK IX RELATION TO SIZE IN CERTAIN CHITONS, WITH A DISCUSSION OF ITS PHY LKTIC SIG X IFICANCE * \V. RUSSELL HUNTER2 AND STEPHEN C. BROWN * I 't-ptirliiicnl nf Znnloi/y. Syracuse i'nk'crsity, Syracuse, A'ru1 York, and Department nf Zoology, ['iii'i'crsitv a! Michigan, .Inn .lrbi>i\ Micliii/an Hefore the disci >very of tlie living tnonoplacophoran genus, X'eopilina. in 1952, anv sumniarv diagnosis of the phylum Mollusca included such a phrase as "coelomate, totally lacking metameric segmentation." This was completely accept- ahle despite' the \vell-kno\vn replication of several sets of organs — including shell valves, pedal muscles and ctenidia — in the polyplacophoran Amphineura — the chitons. However, the detailed morphology of Neopllina i/alathcac, so carefully described by Lemche and Wingstrand (T959b, see also Lemche, 1('57), led these authors to ])ro])ose that several features in both XcopUina and chitons were primitive metameric characters providing undeniable evidence of the segmental origin of the phvlum Mollusca. Certain workers with extensive knowledge of the functional morphology of molluscs have not been able to accept these characters as evidence of metameric origins ( Yonge. 1957; Morton and Yonge, 19(4)., regard- ing the multiplicity of certain organ systems (of cephalopods and chitons as well as of monoplacophorans ) as being replication with functional, rather than segmenta- tion with phyletic. significance. Other authors with extensive molluscan back- ground ( Fretter and Craham, 1962) have partially accepted the features as indica- tions of metamerism and to some extent revived an older theory of molluscan origins. This older theory was developed as a result of studies of the genital and systems of chitons and cephalopods (see Pelseneer, 1891J, l('0f); Xaef. proposed a stem group of molluscs with short segmented bodies. For ears it seemed that this older theorv had been completely dismissed as a result o1 the work on the functional morphology of primitive gastropods which important survey by Yonge ( l'M-7). which established as the most llusc a totally unsegmented animal with a posterior mantle-cavity cieuidia). The description of Xcopilinci has reopened dis- ible metamerism in primitive molluscs, and. as outlined above, this has again [deration of the multiplied organ systems in chitons. ; data on the length-weight relationship and the ctenidial number- tonnd in leura apiculata from Cape Cod and in Lepidochitona cinereus fr< oasl of Scotland, with some brief notes on conditions in certain oilier 5pe The data form part of other work by the present authors on the functi( log) of post larval chitons ( S. C. 1>.) and on Supported in pai . National Institutes of Health. (;.M Instructor and I Jcpai-tnirnt of Invi-rtohratr Xoolo^y. Marine Laboratory, Woods H 508 GILL Xl'Ml'-Ek AM) Si/.K IX CHITONS variations in growth and fecundity in chitons ( \\ . K. H.l, but arc presented here because of their significance in relation to the alleged metamerism of primitive molluscs. MATKRIALS AND MKTIIOHS * Two species of chitons were studied in detail. Chuctoplcitra apicnlala is the commonest chiton of the Atlantic seaboard of the northeastern United States, and was collected from the Northwest Gutter of Hadley Harbor and from I'enzance, Buzzard's Bay, both near \\oods Hole, Massachusetts. Lepidochitona chicrctts is one of the four commoner intertidal chitons of British waters, and was collected from rock pools at Leckmoram Xess on the Firth of Forth, near Xorth Berwick. Scotland. Both chitons were collected from between low- water of neap tides and low-water of springs. The species and collection localities of the other chitons not studied in detail are given in the text where they are mentioned. Initial ctenidial counts were made on living chitons and on material fixed and preserved bv various standard procedures. The principal material of both Chaetopleura and Lepidochi- tona used to establish the length-weight relationship and to count gills was prepared by a method which produces maximum flexibility of the ctenidia. A similar method has been employed extensively by one of the present authors ( \Y. R. H. ) in work on slugs and other pulmonate gastropods, and an account of this use was given by Owen and Steedman (1958; see also Rosewater, 1963, for further references). In the case of the chitons, they were placed in fingerbowls of clean sea water and propylene phenoxetol 4 gently added to form globules on the bottom of each dish ( not touching the chitons). The amount of propylene phenoxetol added was about 0.1—0.2% of the volume of sea water. They were then left undisturbed, in dim light, for about eight hours. Fixation of these partially-narcotized chitons was then car- ried out in \2c/f formalin in sea water, each being held flattened against a glass surface when first immersed in the fixative. They required to be held for only about 10-15 seconds to prevent curling. Fixation was continued for four hours, and the chitons then washed for four hours in running water. Finally they were transferred to the storage fluid: an aqueous solution of \f/< propylene phenoxetol, and \c/c ethyl alcohol in 10rv glycerol. Animals prepared in this way have life-like tissues, soft, flexible and excellent for dissection. For the benefit of anyone applying this method to other material, it should be emphasized that propylene phenoxetol has been used only (1) as a relaxing agent, and (2) as a preserving fluid (actually as a bacteriostatic agent). It should be used in the latter capacity only after adequate fixation (since it does not prevent tissue autolysis. etc.). In this work, chiton length was taken as the maximum distance along the midline from the anterior to the posterior "girdle" edges of flattened specimens. Length measure- ments were made to the nearest 0.1 mm. with an engineer's dial caliper. "Weights were "wet tissue weights'' determined to the nearest milligram after removing excess external water with filter paper, on a precision micro-torsion balance (V.D.F.. Holland ) with a scale of 1 gm. in milligrams. Ctenidia were counted under a dissecting microscope at about 20 X, using high-intensity incident illumina- tion, by leafing over with a fine dissecting needle. All counts were checked, "asymmetrical" chitons re-checked and significant counts double-checked by both 4 U. S. distributor: Goldsclimidt Chemical Cor])., 153 YYaverly Place, New York 14, Nr\v York. 510 \\ . RUSSELL HUNTER AND STEPHEN C. I'.ROWN 2000 1000 500 ^ 300 5 200 y 100 50 30 400 200 100 ( Q • 20 10 A. CHAETOPLEURA • t i i I - 1 - 1 - 1 - 1 - 1 - 1 I I L 10 12 14 16 18 20 22 24 26 LENGTH IN MM B. LEPIDOCHITONA *. .' • *• • : - 'v • •••v. r- v • 10 LENGTH IN 12 14 MM. FIGURE 1. "\\\-t tissue \\i-i.ulil" n body length in diilnns (\vfifthts in milli.iirams on tin ordinati are plotted logaritl 1 or ( hdctoplcum STEPHEN C. BROWN iidial number. h»r L. cinereus in this particular population, there is rather better circumstantial evidence relating size to age. \Vlien the weight distribution of Figure II1, is plotted on pn.bahility paper , as used by Harding, 1949, and ap])lied by Hunter. l(' on each side. Xo immediately post-larval material of Lepidochitona was available. In summarv, numerical asymmetry in Lepidochitona involved 61 out of a simple of 12(> individuals or 4S.4% of the population, and qualitative asymmetry involved a still greater proportion of the population. C'ertain general observations can be made. Except for a few very small speci- mens of each species, most of the Chacto pleura and Lepidochitona examined were adult, (irowth continues in adult chitons, and apparently the development of TABI.K I Extent of clcuididl iisviiitnetry in livo species <H. <>l individuals With extra left gills Symmetric With extra right yills Totals 3 2 i 1 2 3 (. 'hiK'-ii/jli'iini n f>/c:i'(i '54). had lengths ranging from 3.3 to 7/i iniii.. had gill numbers from 8 to 11 per .side, and included three asymmetric GILL NUMBER AND SIZE IN CHITONS 515 specimens (11L:10R, 11L:10R, and 9L:10R). A fresh sample of the commoner Clyde species of this genus (L. ascllus} was not available but six specimens in a museum collection proved to be all symmetrical with from 10 to 13 gill.-, on each side. A sample of nine specimens of Tonicclhi inarinorca from near Shandon, on the eastern shore of Gareloch, ranged in size from 12.5 to 23.7 mm., had gill numbers from 17 to 28 per side, and included one asymmetric individual (23L:24R). Samples of Lepidochitona cincrcus from this last locality, and from two localities in the Firth of Forth showed that nearly half of each population was asymmetric in gill numbers. Since L. cincrcus is also the most abundant intertidal Scottish chiton, it was decided to use this species along with Clun to pleura apicnlata in the main series. Lastly, 15 specimens of Acanthochitona crinitns (formerly known as Acanthochitona jasciculans} , collected from near low water mark on a boulder beach just south of Port Appin, Argyllshire, ranged in length from o.l to 13.0 mm., had gill numbers from 14 to 17 per side, and included two asymmetric individuals (both 15L:16R). Thus, of five species of Scottish chitons examined, four proved asymmetric in ctenidial numbers. It is worth noting that these five species include representatives of both the major orders of living Polyplacophora. Three genera: Lepidochitona and Tonicclhi (both in family Lepidochitonidae), and Acanthochitona (family Cryptoplacidae) are placed in the Order Chitonida, the group containing the more typical "modern" chitons usually living on rockv surfaces in the intertidal zone. The genus Lepidopleurus (family Lepidopleuridae) is placed in the Order Lepidopleurida, which includes some of the most ancient of polyplacophoran genera (e.g., Helminthochiton, from Lower Ordovician ) as well as four surviving genera which live offshore and in deeper waters (extending to beyond 6000 m. ). Subsequently, commercially obtained samples totalling 65 specimens of a moder- ately large chiton, Katherina titnicata from the Pacific Coast, probably collected near Gladstone, Oregon, were examined. Of these, 24 specimens proved to have ctenidial or pallial damage, and weights and gill counts were determined on the remaining 41. \Yet weights ranged from 13. 8 to 55.8 g., and gill numbers from 46 to 61 per side. Only 22 out of 41 specimens of Kathcrina were symmetrical, and the asymmetries (46.3% of the population) in two crises involved a difference of three gills, in five cases a difference of two gills, and in 12 cases a difference of one gill. Few previous authors have studied ctenidial numbers in chitons. In a footnote, Pelseneer ( 1897 ) refers to his finding single specimens of six species of Atlantic chitons, each with an asymmetry of one gill. His species were Acanthochitona discrcpans, A. zelandicns. Lepidopleurus cajctanits, L. articits. "Borcocliiton inar- inorcits" ( = Tonicc!la nntniiorca, above), and I'lo.rip/iora coclata. Perhaps the only previous detailed examination of a population was by Snyder and Crozier ( 1922) on Chiton titbcrculatits at Bermuda. These authors examined 100 speci- mens, and in individuals ranging in size from one to ten cm. long, found ctenidial numbers from 32 to 54 on each side with 69% of the sample asymmetrical. Thus, in four species of chitons studied in detail, the percentages of asymmetrical specimens found have been 19.5% (Chaetopleura apicnlata}, 46.3% (Kathcrina titnicata), 48.4% (Lepidochitona cincrcus}, and 69% (Chiton titbcrculatus). In addition, asymmetries of ctenidial numbers occur in at least eight other species. 516 \V. RUSSELL Hl'NTKK AND STEPHEN C. I'.KOYVX DISCUSSION A> noted in the- introduction, the discoverv of Xcopilina has reopened discussion of possible metamerism in ancestral molluscs Chitons show replication in several organ systems — including shell valves, pedal musculature, nervous system, circula- tory system, and cteuidia. As a result of their anatomical studies of Xeopilhut, I.eniclie and \Yingstrand (l(>5('b: see also Lemche. 1957, 19591)) proposed that such features of chitons, as well as similar structures in X'copilitht. were primitive metameric characters. Lemche il''5(^a, L'.^'c) has gone so far as to detail s hetween a ]>rimitive arthro])od triramous appendage and the gill in and hetween this latter and the ctenidia in the rest of the Mollusca (Lemche. I959b). Of course, the hypothesis that the molluscan ancestor was a short segmented animal has been advanced before (see, e.g., Pelseneer, 1899, 1906; Naef, 1926). These older protagonists of a segmented ancestor drew their evidence largely from morphological studies of the coelomic derivatives, notably reno-pericardial and genital ducts, in chitons and cephalopods. The significance of Neopilina in relation to such theories was stressed bv Lemche and "\Yingstrand (1959a. 19591) ). They suggested that the ancestral mollusc must have shown relatively complete meta- merism, that this is present to a somewhat reduced extent in Neopilina, that this is still further reduced in chitons, and that this metamerism degenerates so com- pletclv as to be undetectable in the rest of the molluscs. For about 30 years the original metameric hypothesis, set up by Naef and IVlscneer among others, was abandoned. This resulted from the extensive and convincing work of the molluscan comparative morphologists, such as Yonge, ( iraham. and Fretter. whose studies of ciliary mechanisms, ctenidial blood-vessels, reno-pericardial and genital ducts in the more primitive gastropods (as summarized in Yonge. 1947) indicated a very different pattern of ancestral mollusc. Although primitively bilaterally symmetrical, this animal was totally unsegmented and possessed a posterior mantle-cavity enclosing a pallial com] ilex of paired structures which included two ctenidia. The setting up of such a hypothetical ancestor had, of course, required consideration of those cases of molluscs in which there are more than two ctenidia, i.e., the primitive cephalopod Nautilus (see Yonge, 1947) and all the chitons i Yonge, L'.VM. Such consideration led to the conclusion that these cases represent secondary replications of structures of the ancestral mollusc. The publication ot the beautifullv detailed anatomical account of Xcopilhm (jalatlica niche and \Yim;strand. l('5''b), has made review of concepts of molluscan evolution inevitable. Few students of the Mollusca would accept the more extreme homologies proposed by Lemche ( l('5(>a, l('5('b, l(>5(>c). which equate structures in the more primitive mollusc--, with corresponding annelid parts. Fretter and (iraham ( 1 ''d_' '. in their excellent and detailed synthesis of existing knowledge of functional morphology in 'branch gastropods, have accepted the concept that XcopUina shows metamerism and 'nave linked this to the older theory of a segmental ancestor. They al>o >npport the oiicept that the mantle-cavity Was most primitively a groove bounded b\ the mantle and surrounding the head-foot, rather than a posterior cavity. Yonge (see Alorto and Yonge, 1(>M) has denied that metamerism occurs Y(v;/>/7///(/ and, although allowing that the ancestral mantle-cavity might have been a pallial groove, adhere ssentially to the concept of the ancestral mollusc GILL NUMBER AND SIZE IN CHITONS 517 outlined above. Obviously, aspects of the.se tbeories are mutually exclusive. The pattern of molluscan evolution. involving successive reductions of metameric structures. proposed by Lemche and \Vingstrand (195(>a. 1^5'Jb) requires as a premise that the multiplied organ-systems of chitons represent a simplification of more extensive metamerism. Therefore, the question must be asked: do chitons show anv vestiges of true metamerism? A seemingly negative answer is provided by existing evidence on the development of shell plates, on variation in auriculoventricular connections in the Class, and by the data on ctenidial numbers presented in this paper. As regards shell plates, their development in several chitons is similar. The elongate trochophore develops a mantle rudiment, which about the time of settle- ment secretes six shell plates. After an interval, a larger plate is added anterior to the series. Still later, a small last plate is added at the posterior end of the series. At no stage in the development is there a "budding zone" for shell plates. This sequence of events, known for several chitons, has recently been confirmed for Chaetopleura apicitlata by one on the present authors (S. C. B.). As a Class, the chitons show significant variation in the afferent chambers of the heart. These variations were surveyed by Pelseneer (1897. 1906). The pericardium is always dorsal and posterior, and contains an elongate ventricle in the midline which receives blood from two symmetrical elongate auricles. In a minority of chiton genera, Pelseneer found that there are single auriculoventricular (A-V) openings on either side; in the majority of genera there are two A-Y openings on either side; while in Chiton sqnauiosns there are three pairs and in C. goodalli four. Pelseneer also remarks that numerical asymmetries of the A-V openings can occur, although they are apparently extremely rare. It would be difficult to relate the paired auriculoventricular openings to any external "segmenta- tion'' such as shell valves. Similarly, although the renal organs of chitons are lobulated, relating particular lobes to external "segments" or to the renal lobes and discrete "nephridiopores" of Neopilina would be equally difficult. Turning to the ctenidia of chitons, their numbers, though approximately fixed for each species, show variations which are germane to the question of metamerism. First, gills are added anteriorly and somewhat irregularly as growth proceeds. Second, there can be marked asymmetry between left and right ctenidial rows in individual specimens (from 19.5% to 69(/o in four species studied in detail). Third, actual asymmetry of development can be more marked than mere enumera- tion of ctenidia would indicate, since the degree of differentiation of the anterior- most gills often varies. Fourth, the "definitive" ctenidial number in different species of chitons ranges from 8 to 160. (No one has ever suggested a primitive mollusc with 80 segments.) Final! v. individual ctenidia cannot be related to any other replicated structure, still less allocated to specific segments. The broad phyletic significance of the above can now be summarized. If no true metamerism occurs in primitive molluscs, the closest connections and possible origins of the phylum lie in the turbellarian-rhynchocoele phyla (as is concluded by Yonge. 1947; Morton and Yonge, 1964). If metamerism was a feature of the primitive mollusc, connections should be sought with the annelid-arthropod phyla (as urged by Lemche and Wingstrand, and partially accepted by Fretter and Graham, 1962). No one has attempted to establish a connection between W. RUSSELL HUNTER AND STEPHEN C. I'.ROWN molluscs and either of the other nu-ta/oan Croups .showing segmentation: the C'estoda or the C'hordata. At this point, it i.s appropriate to try to define metameric srgmeiitation as it is found in the Annelida and Arthropoda. Mynian (1^51, pp. JS _N) offers .several cogent ])hrases in defining segmentation as "a serial succession of sections, each of which contains identical or similar representatives of all the organ svstenis of the' hod\" in which "all parts . . . are serially repeated at regular intervals" and "the segments form in an anteroposterior sequence so that the first Moment is the oldest, the last the youngest." Thus, the essence of metamerism is the serial succession of .segments, each containing unit-subdivisions of the several organ systems. The most detailed homologies between chitons and Neopilina proposed by I -eniche and \Vingstrand ( 1()5(M involved the alleged metamerism of the gills and circulatory organs of both (although clearer "segmentation" in Neopilina is found in the coelomic cavities and their genito-renal derivatives). A major implication of the results presented above is that "segmentation" in chitons is more apparent than real. It is difficult to imagine morphogenesis of any metameric sort which would allow the addition {Ulteriorly of the penultimate shell valve to the six already developed, and this followed finally by the laying down of the last (eighth) valve posteriorly. Similarly it is difficult to invoke metamerism with regard to the anterior addition, irregularly and independently on either side, of ctenidia as growth 1 >n iceeds. Finally, it is difficult to characterize as a metamerically segmented animal : a chiton with X shell plates, with two, three or four auriculoventricular openings, with 21 ctenidia on one side and 23 on the other (or 15 and 18). and with a ladder- like nervous system of irregular transverse connectives — all these arranged appar- ently independently of each other. There is little of the serial succession of seg- ments, each containing unit-subdivisions of the several organ systems, about such replications as are found in chitons. Thus, it seems most probable that the multiplied structures of chitons reflect functional replication rather than ancestral segmentation. I --veil if we can thus dismiss true metamerism as regards chitons, the question of 'segmentation," both in Neopilina and in a hypothetical molluscan ancestor, re- mains. Similar arguments to those presented above can be used to criticize the concept of metamerism applied to the described structures of Neopilina. Neopilina has five pairs of gills, two pairs of auricles, six pairs of nephridia, one or possibly two pairs of gonads. X pairs of pedal retractor muscles, 10 sets of lateropedal nerve connections, and a single shell with a coiled protoconch. As Yonge has pointed out (Yonge, 1''57, I960; Morton and Yonge, 1('M), final assessment of the phyletic position ot Neopilina must await embryological studies — particularly on the develop- ment ot ihe allegedly metameric structures, and functional studies on the organiza- ti"ii "t the gills in living Neopilina. which could establish whether or not these structures are homologous with the ctenidia of the other molluscs. The question of ihe ancestral stock can be re-examined. According to the classical picture, derived from studies on the functional morphology of the most primitixc living gasl Yonge, 1*47), the ancestral mollusc had a posterior mantle cavity containing one pair of ctenidia connected to one pair of auricles. However, even before the discovery of Neopi/ina. there were several difficulties in relating this model b conditions in the primitive cephalopod Nautilus — with two GILL NUMBER AND SIZE IN CHITONS 519 pairs of ctenidia and four auricle's, and in the chitons — with main1 ctenidia and two elongate auricles (with two, four, or six auriculoventricular openings). There is a striking resemblance in hearts (each with four auricles and a "single"' ventricle) between Neopilina and Xnittilus. There is a further, though less obvious, re- semblance of both to conditions as the}- exist in the majority of chitons — with four auriculoventricular connections. Perhaps surprisingly, the arrangement of heart chambers and of their interconnections seems to form a relatively conservative feature in the evolution of other molluscan groups. For example, within the Order Neritacea, evolution of fresh-water and terrestrial forms has occurred completely independently of — though occasionally parallel to — the rest of the gastropods (Morton and Yonge, 1964; Hunter, 1964). The course of this evolution has in- volved the usual gastropod reduction of symmetrically paired structures. However, comparative studies on certain Neritacea by one of the present authors (W. R. H.. unpublished ) have shown that even in many forms where this reduction is complete for almost all of the other structures, a small right auricle remains, in addition to the functionally important left one. In a footnote in the classic paper of Yonge (1947), Dr. C. F. A. Pantin inquired if it was necessary to postulate the possession of only a single pair of ctenidia in the primitive mollusc. In view of the heart morphology of Xeopilhnt, Nautilus and chitons discussed above, it would seem as justifiable to set up a model ancestral mollusc with four ctenidia and therefore four auricles. Subjectively, a four-fold basic organization would seem a more reasonable starting point for two sorts of morphogenesis : involving either reduction to one pair or replication to many. Both a line of organisms with one gill on either side, and a line with many could thus evolve from an ancestral stock with two gills on either side. While it is realized that the setting up of yet another hypothesis, with four as the ctenidial number in the stem-mollusc, is but tenuously based on the observed phenomena of replication of parts in chitons, it is emphasized that the conclusion that chitons are not metameric is much more firmlv established. We wish to thank Myra Russell Hunter for checking calculations and for help collecting the Scottish chitons, and Robert T. Meadows for assistance with some weighings. We are also glad to record our gratitude to Dr. Clark P. Read who. by bringing us both back to Woods Hole, enabled this and other work to In- completed. SUMMARY 1. The discovery and description of Neopilina has reopened discussion of pos- sible metameric segmentation in the primitive molluscs. This has involved reconsideration of the multiplied organ systems in chitons, which some authorities regard as metameric in origin. The length-weight relationship was determined and ctenidial numbers counted for populations of two species of chitons: Chacto- plcura apicnhita from Cape Cod, and Lepidochitona cincreus from the North Sea coast of Scotland. Less detailed observations were made on other species of chitons and on other organ systems. Growth continues in adult chitons, together with the addition of ctenidia anteriorly, so that there is a correlation between gill-number and adult size. Chacto pleura apicitlata adults, weighing from 38 to 1324 mg., had 520 \v. RUSSELL HUNTER AND STEPHEN C. I'.KOWX gill-numbers ranging fnun 17 to J5 per side. Lepidochitona cincrcits adults, weigh- in- from (> to ,UO n is^.. had gill-numbers ranging from 11 to 20 per side. Asym- metry in ctcnidial nnmhers between the left and right sides of single specimens occurred in l('.5f; of ('littctoplciira aflcnhita, and in 4$>A% of LepidocJtitona ciiicrciis. (Jualitative asvmmetrv was even more exten.sive in the populations studied. Since such marked ctenidial asymmetries as reported above occur, and since individual gills are added in< e iently on either side, the ctenidia of chitons cannot be paired structure's. The oi -ain/ation of certain other replicated structures (including shell-plates, auriculoventricular connections, and renal lobes) is con- sidered more briefly, drawing on earlier pertinent work, and leads to the obvious result that individual ctenidia cannot be related to any other replicated structure— still le>s allocated to specific "segments.'' 2. These findings are di.scu.vscd in relation to the alleged metamerism of chitons, and it is also concluded that such replicated organs cannot represent a simplifica- tion of a more extensive metamerism. Further, it is shown that it is possible to criticize the concept of metamerism as applied to the described structures of Xcopilina. The phyletic significance of this is explored, and it is concluded that evidences for a connection between the primitive molluscs and the turbellarian- rhynchocoele phyla arc- better than are those for metamerism and an annelid- arthropod connection. A model ancestral mollusc with a }our-\old basic organiza- tion (e.g., four ctenidia, four auricles, four renal organs, etc.) is proposed. It is stressed that, while any model of an ancestral mollusc is highly speculative, the evidences against metamerism in chitons (and probably in all primitive molluscs) are overwhelmingly strong. LITERATURE CITED AKKV. I.. I',.. AND \Y. }. CKO/IKI-:, 1919. The sensory responses of Chiton. J E r/> Zoo! 29' 157-260. COMFORT, A.. 1('57. The duration of life in molluscs. Proc. Malacol. Sue. London, 32: 21()-241. pRETTER, \ .. AND A. Cii<.\n.\M, 1962. British Prosobranch Molluscs Their Functional Anatomy and Ecology. Ray Society, London, 755 pp. I IAKIMXG, J. I'.. ](>4<>. The use of probability paper for the graphical analysis of polymodal frequency distributions. ./. Mar. ttiol. Assoc., 28: 141-1 5.1. W. RUSSELL, 1%1. l.itY cycles of four freshwater snails in limited populations in I.och Lomond, with a discussion of infraspecific variation. Proc. Zool. Sue. London 137: 135 171. \V. RUSSELL, 1964. Physiological aspects of ecology in non-marine molluscs. In: Physiology of Mollusca, vol. I, K. M. Wilbur and C. M. ^'onge, eds., Academic Press New ^ork, pp. 83- 126. ND 1). A. Mrik, 1()54. On the situation and geological structure of the rvelloch Islands. Glasg. \\iL.17: 12('-Ki'i. The [nvertebrates. \"ol. II: Platyhelminthes and Uhynchocoela. The McGra\A Hill Hook Company, Inc., Xew York, 550 pp. living deep-sea mollusc of the Camhro-l )evonian class Mono plai , 179: 413 416. / ( un mohisco actual, con caracteres de ti]io I'osil, y su sigmTicado ) . B: 411 442. E, II., ]<>': n phylogeny in the light of VeopiKna. Proc. Xl'tli. Intermit. ".'/. tion 4: .iS(l .iSl. CHE, M. L9 i interrelationships in the light of \copilhiti. Proc. Xl'tli. . Section 4: .iSl ,iS4 GILL NUMBER AXI) SIZE I\T CHITOXS 521 LEMCHE, H., AND K. G. WINGSTRAXD, 1959a. The comparative anatomy of Ncof>ilina galathcac Lemche, 1957 (Mollusca Monoplacophora). Proc. XVth. Internal. Cong. Zoo!. (London). Section 4: 378-380. LEMCHE, H., AND K. G. WIXGSTRAXD, 19591). The anatomy of AY"/1/////./ i/ulnthcac Lemche, 1957 (Mollusca Tryblidiacea). Galathca Reft.. 3: 9-72. MORTON, J. E., AND C. M. YONGF., 1964. Classification and structure of the Mollusca. In: Physiology of Mollusca, vol. I, K. M. \Yilbur and C. M. Yonge. eds.. Academic Press, New York, pp. 1-58. NAEF, A., 1926. Studien zur generellen Morphologic der Mollusken. III. Toil: Die typi-chen Beziehungen der Weichtiere untereinander und das \Yrhaltm\ ihrc-r Urfonncn /u anderen Colomaten. Erycbn. Z <><>!., 6: 27-124. OWEN, G., AND H. F. STEEDMAN, 1958. Preservation of molluscs. Proc. Mahicol. Sue. I.midmi, 33: 101-103. PAXTIX, C. F. A., 1947. Sec Yonge, C. M., 1947. PELSENEER, P., 1897. Sur la morphologic des branchies et des orifices renaux et genitaux des chitons. Bull. Sci. Fr. Bel,,.. 31 : 23-30. PELSENEER, P., 1899. Recherches morphologiques et phylogenetiques sur les mollusi|ur- archa'iques. Mcin. Sur. etr. Acad. Roy. Bely., 57: 1-112. PELSENEER, P., 1906. Mollusca. In: A Treatise on Zoology, vol. V, E. R. Lankester, ed., A. and C. Black, London, 355 pp. ROSEWATER, J., 1963. An effective anesthetic for giant clams and other mollusks. Titrtox AVtc-.y. 41 : 300-302. SNYDER, L. H., AND W. J. CROZIER, 1922. Xotes on some problems of adaptation : 9. Ctenidial variation in Chiton. Hiul. Bull.. 43: 246-252. YOXGE, C. M., 1939. On the mantle cavity and its contained organs in the Loricata (Placophora). Quart. J. Mici: Sci., 81: 367-390. YOXGE, C. M., 1947. The pallial organs in the aspidobranch Gastropoda and their evolution throughout the Mollusca. Phil. Trans. Roy. Soc. London. Scr. B. 232: 443-5 IS. YONGE, C. M., 1957. Reflexions on the Monoplacophoran, \\-opilimi i/uluthcac Lemche. Nature, 179: 672-673. YOXGE, C. M., 1960. General characters of Mollusca. In: Treatise on Invertebrate Paleonto! ogy, Part I, R. C. Moore, ed., Univ. of Kansas Press, Lawrence, Kansas, pp. 13-136. INDEX AFT as radioprotective agent at cellular level. 125. Abalone blood, hemocyanin concentration in, 45'). Achromatic figure of Chaetopterns egg, ef- fects of podophyllin on, 36°. Acoustic orientation of Chilonycteris, 297. Actyostelium, phototaxis in, 51. Adaptation to salinity changes in toads, 218. Adipobemocytes of boll ueevil, 112. ADKISSON, P. I.. See C. M. WILLIAMS, 4('7. After-effect, duration of, in planarian orienta- tion. 347. Aggregation of cells in Dictyosteliaceae, in- fluence of light on time of, 392. Alepisaurus, new digenetic trematode from stomach ot, 488. Algae, marine', phosphatases of, 271. Algae and hydra, symbiosis between, 415. ALLEN, G. See K. F. Mi F.U-GHLIX, 112. Ambulacra! pores of sand dollar, variations in, 401. Ambystoma egg-jellies, relationship of anti- genie components ot, 328. Amebocyte production in Strongylocentrotus, 259. \mebocytes in holothurian coelomic fluid, 102. .\meboc\tes of oyster, circulation of, 198. \mino acids, role of in intracellular osmo- regulatiou in toads, 218. Amino acids of Scoloplos egg-jelly, 189. Amphibian development, role of creatine and it- phosphate in. 230. •ihihian 'lies, relationship of anti- genie components of, 328. erobiosis, effect of on frog embryos, 230. \n .•'< b is for stabili/ation of visual fields, 285. \ii.itomy of developing Fundnhis embryos. 143.' A MH RSO J. M. lies i .n viscei al re- • neratii in in Si rs. II., 1. Angle sense in orieip. f millipede, 33. \nnelid '. Polydora), lai - , > lopment of, 356. Annelid (Streblospio) , development of, f>7. Annelid eggs, effects of pod' on, 369. Annelid worm, egg cocoons of, 189. Antheraea brain, transmission of photoperi- odic signals to, 497. Aiithonomns, hemocytes of, 112. Antigenic components of amphibian eggs, re- lationships of, 328. Antimitotic agent, effect of on Chaetopterns eggs, 369. Aplysia, chemosensory liases of food-finding "in, 211. Aposymbiotic hydra, effect of environmental agents on growth of, 415. Arbacia eggs, method for studying chromo- somes of, 169. Arbacia eggs, radioprotective action of AET for, 125. Arbacia fertilizin antigens, relationships of. 328. ARMSTRONG, P. P>., AND J. S. CHILD. Stages in the normal development of Fundulus, 143. ARXOLD, J. M. Normal embryonic stages of the squid, Loligo, 24. Arthropod eye, pigment migration and light adaptation in, 473. Asterias, reaction of to foreign tissue in coelom, 77. Asteriidae, visceral regeneration in. 1. ArcLAiR, W. The chromosomes of sea ur- chins, especially Arbacia ; a method for studying uiisectioned eggs at first cleav- age," 169. \ntoradiographic study of relation between hemocytes and connective tissues in Gal- leria, 337. Autoradiographic study of sea urchin coelo- mocyte production, 25(>. Autoradiography of Asterias and Patina with foreign tissues in coelom, 77. Autoradiography of regenerating starfish caeca, 1. Autoradiography of sea urchin gametogenesis, 241. B Hacillariophyccae. phosphatasc production by, 271. I'.AKX \\KI.L, I'". II. An angle sense in the orientation of a millipede', 33. INDEX 523 P>at, echolocation of flying insects by, 297. BECK, S. D., 1. B. COLVIN AMI I). 1C. S WIN- TON. Photoperiodic control of a physio- logical rhythm, 177. Behavior of Chilonycteris, during pursuit of flying insects, 297. Bleeding of oysters, effect of on leucocyte counts, 198. Blood of Haliotis, concentration of hemo- cyanin in, 459. Blue crab, variability in larval stages of, 58. filue light, as photoperiodic signal to An- theraea brain, 497. Body coloration, in relation to heat tolerance of Uca, 133. Boll weevil, hemocytes of, 112. BONNER, J. T., AND F. E. WHITFIELD. The relation of sorocarp size to phototaxis in the cellular slime mold, Dictyostelium, 51. Botulus, new trematode species of, from lancet- fish stomach, 488. Brachyuran crab, variability in larval stages of, 58. Brain of Antheraea, transmission of photo- periodic signals to, 497. Breeding season of Mya, 315. Breeding season of Scoloplos, 189. Breeding season of Streblospio, 67. Breeding season of Strongylocentrotus, 241. BROWN, F. A., JR., AND Y. H. PARK. Dura- tion of an after-effect in planarians fol- lowing a reversed horizontal magnetic vector, 347. BROWN, S. C. See W. R. HUNTER, 508. Brown bodies, formation of in holothurians, 102. Bufo, intracellular osmoregulation during salinity adaptation in, 218. Bufo egg-jellies, relationship of antigenic components of, 328. Calcium, role of in growth of symbiotic and aposymbiotic hydra, 415. Callinectes, variability in larval stages of, 58. Cell aggregation in Dictyosteliaceae, influence of light on, 392. Cell division, effects of podophyllin on, 369. Cell size in Dictyostelium, relation of to phototaxis, 51. Cells in coelomic fluid of holothurians, 102. Cellular slime mold, phototaxis in, 51. Cephalopod, embryology of, 24. Cephalopods, stabilization of visual field in, 285. Chaetopleura, relation of gill number to size in, 508. C haetopterus eggs, effects of podophyllin on, 369. (.. IIAI'.MAN, (i. The egg cocoons of Scoloplos, 189. Chemosensory bases of feeding in Aplysia, 211. CHILD, J. S. Sec I'. I!. AKMSTKONO. 143. Chilonycteris, echolocation of flying insects by. 297. Chitons, relation of gill number and size in, 508. Chlorohydra, symbiosis of \\ith algae, 415. Chromosomes of Arbacia, method for study- ing, 169. Chromosomes of Chaetopterus, effects of po- dophyllin on, 369. Chronology of development in Fundulus, 143. Chronology of development in Loligo, 24. Chrysophyceae, phosphatases in, 271. Circulation of leucocytes in oyster, 198. Clam, soft-shell, reproductive cycle of, 315. Cleavage in Chaetopterus eggs, effects of po- dophyllin on, 369. Cleavage in Fundulus eggs, 143. Cleavage in Loligo eggs, 24. Cleaving Arbacia eggs, method for studying, 169. "Clotting" of oyster blood, 198. Coagulation process in boll weevil, 112. Cocoons, egg, of Scoloplos, 189. Coelenterate, effect of temperature on strobi- lation of, 493. Coelenterate (Hydra), symbiosis of with algae, 415. Coelomocyte production in Strongylocentro- tus, 259. Coelomocytes of holothurians, origin of, 102. Colchicine-treated Arbacia eggs, method for studying, 169. Cold, effect of on velocity of dinoflagellates, 90. Cold, role of in circulation of oyster leuco- cytes, 198. Cold, role of in diapause development in Ostrinia, 177. COLLAR, P. A. See \V. G. HAND, 90. Coloration, body, with respect to heat toler- ance of Uca, 133. Commensal of hermit crab (Polydora), de- velopment of, 356. Concentration of hemocyanin in Haliotis blood, 45". Connective tissue and hemocytes of Galleria, relation between, 337. Control, photoperiodic, of physiological rhythm, 177. COLVIN, I. B. See S. D. BECK, 177. Compensatory cyclorotation of eye, 285. 524 1\I>K\ Copper-containing protein in Haliotis blood, 45". COSTELLO, D. I '. See C. HENLEY, 3o". COSTI.OW. J. D., JK-. Variability in larval stages of the blue crab, Callinecte-, 58. Crab, variability in larval stages ot. 58. I 'rab, tiddler, heat tolerance of. 133. Crab, larval development of commensal of, 356. < rassostrea, heart beat and circulation of leucocytes in. 1"8. Crealiue and crcaline phosphate, role of in amphibian development, 230. Crustacean, heat tolerance of, 133. Crustacean, variability in larval stages of, 58. Crustaceans, stabilization of visual held in. 285. Ctenidial number in chitons, 508. Cucumaria, origin of coelomocv tcs in. 102. Cycle, reproductive, of Mva in New England. 315. < ili (rotation of eye, 28s. Cytological effects of podophyllin and podo- phyllolovin on Chaeloptenis eggs. 369. Cytology of developing Arhacia eggs, 169. Cytology of gametogenesis in Strongylocen- trotus, 241. D DX'A synthesis in Strongylocentrotus coelo- mocytes. 25". D.\.\ synthesis in Strongylocentrotus gonads, 241. I), irk. orientation of slime molds in, 51. Dark, role of in diapause development in Os- irinia, 177. Dark -adapted < ialleria, eye, pigment migra- tion in, 473. Darkness, role of in cell aggregation of Dic- tyosteliaceae, 3"2. DAVENPORT, 1). \Y. G. HAXD, "0. Day-length, rok' ol in diapause development in Ostrinia. 177. Da; . role of in termination of diapause in Antlu-raea, 4"7. I 'i \\, D. On the reproduction and larval dc1. • a of MreMospio, 67. I ><••. • h iping Arbacia • method for study- ing cytology of. In". I )e\ i loping Cha • • ' of po do]ili\ l!in on, 3d1'. I >e\ eloping mio I >evelopmen1 oi amphibians, \ • • . .itine and its phosphate in. I )e\-elc i].' iMindtiln-, 143. I levelopmenl ol ' iallei ia hemi : . nee live tissue, 337. Development ot I'olydora, 356. I Jevclopmcnl of si|uid, 24. Development ot Streblospio, 67. l)ia]ianse pliysiolo.i;y of insect (X\ '), 497. I h. i\ osteliaceae, inllnence of li.ulit on lime of cell a.iiiire.nation in, 392. 1 Jietyostelinm, pbototaxis in, 51. Differentiation of hemocytes in Galleria, 337. Di.uenetic trematode from lancetfisb stomach, 488. Dimensions of sand dollars, variations in, 401. Dinoflagellates, effects of temperature and salinity on velocity of, 'Ml. Distribution of Polydora, 356. Drosophila, echolocation of by Chilonycteris, 297. Dugesia orientation, duration of after-effect in. 347. Duration of after-effect in planarian orienta- tion. 347. Duration of mature life of x-irradiated mice, 425. E Echinarachnius, variations in dimensions and weights of, 401. Echinoderm, coelomocyte production in, 259. Echinoderm. reaction of to foreign tissue in coelom, 77. Echinoderm coelomocytes, origin of, 102. Echinoderm e.ugs, method for studying, 169. Echinoderm eggs, radioprotective action of AET for, 125. Echinoderm fertilix.in antigens, relationships of, 328. Echinoderm gametogenesis, autoradiographic study of, 241. Echinoderms, visceral regeneration in, 1. Echolocation of (lying insects by bat, 297. Ecology of dinoflagellates, as affected by their velocity, 90. Effects of environmental cations on growth of symbiotic and aposymbiotic hydra, 415. Effects of podophvllin on Chaetoptcrns eggs, 369. Effects of temperature and salinity on ve- locity rates of dinotiagellates, 9(1. Effects of temperature on strohilation ol Mas- tigias, 4'*3. Effects of \-irradiatioii of mice on duration of mature lite, 425. Egg cocoons of Scoloplos, 18'). Egg-jellies of amphibian, relationships ol anti- genie components of, 328. Eggs of Arbacia. method for studying cbro- mosomes of, 16". IXDEX 525 Eggs of Arbacia, radioprotective action of AET on, 125. Eggs of Chaetopterus, effects of podophyllin "on, 369. Eggs of Fundulus, development of, 143. Eggs of Loligo, development of, 24. Eggs of Streblospio, development of, 67. Electrophysiology of Galleria eye, 473. Eleocyte production in Strongylocentrotus, 259. Embryological development, role of x-radia- tion damage to mice in, 425. Embryological development of Chaetopterus, effects of podophyllin on, 369. Embryology of amphibians, role of creatinc and its phosphate in, 230. Embryology of Fundulus, 143. Embryology of Polydora, 356. Embryology of squid, stages in, 24. Embryology of Streblospio, 67. Embryos of Arbacia, method for studying. 169. Endocrine functions in Antheraea, 497. Endocrine functions in Ostrinia. 177. Energy flux in Galleria eye, 473. Energy metabolism of Rana embryos, 230. Environmental agents, effect of on growth of symbiotic and aposymbiotic hydra, 415. Environmental factors, role of in dimensions and weights of sand dollars, 401. Enzyme activity of marine algae, 271. Ephyrae of Mastigias, effect of temperature on time of appearance of, 493. Eupagurus commensal, larval development of, 356. Eupentacta, origin of coelomocytes in, 102. Eye of Galleria, pigment migration and light- adaptation in, 473. Eye movements in relation to stabilization of visual field, 285. FENG, S. Y. Heart rate and leucocyte circu- lation in Crassostrea, 198. Fertilization membrane of Chaetopterus CUM-. effect of podophyllin on. 369. Fiddler crab, heat tolerance of, 133. Field of vision, stabilization of, 285. FIXGEKMAN, M. See J. L. WILKENS, 125. Fish, development of, 143. Flatworm orientation, duration of after-effect in. 347. Flight of Chilonycteris, during insect pursuit, " 297. Food-finding and feeding in Aplysia, 211. Foreign tissue, reaction of starfishes to, 77. Formation of brown bodies in holothurians, 102. FKI.VGS, H., AND C. FRIXGS. Chemosensory liases of food-finding and feeding in Aplv- sia, 211. Frog embryos, n>lr of creatine and its phos- phate in development of, 230. Fruit fly, echolocation of by Chilonycteris, 297. Fu, See R. RUGH, 125. Function, return of, in regenerating starfish caeca, 1. Fundulus, stages in development of, 143. Galleria, relation between hemocytes and con- nective tissue in, 337. Galleria eye, pigment migration and light- adaptation in. 473. Gametogenesis in Mya, 315. Gametogenesis in Strongylocentrotus, 241. Gamma irradiation of Arbacia eggs, 125. Genotype, role of in radiation sensitivity of mice, 425. Geographical differences in breeding activity of Mya, 315. GHIRADELLA, H. T. The reaction of t\\o starfishes, Patiria and Asterias, to foreign tissue in the coelom, 77. GIESE, A. C. See N. D. HOLLAND. 241. 25". Gill number, relation of to size in chitons, 508. Glutathione, effects of on podophyllin-treated Chaetopterus eggs, 369. GOLDSMITH, T. H. See C. T. POST, JR., 473. Gonads of Mya, histolo.u\ of, 315. Gonads of sea urchin, autoradiographic inves- tigation of, 241. Gonyaulax, effects of temperature and salinity on velocity of, 90. GORDON, M. S. Intracellular osmoregulation in skeletal muscle during salinity adapta- tion in two species of toads, 218. GOWEN, J. W. See D. J. NASH. 425. Graftin.u experiment in starfishes. 77. Growth of sNinhiotic and aposymbiotic hydra, effect of environmental agents on, 415. (.\rodinium, effects of salinity and tempera- ture on velocity of, 90. H 1 labitat of Polydora, 356. Haliotis blood, hemocyanin concentration of, 459. 526 INDEX HAND, \Y. G., I'. A. COLLARD AND I). DAVKN- PORT. The effects ill" temperature and salinity change on -\\iiuming rate in tlie dinotlagcllate-, Gonyaulax and Gyro- diniuni. 90. HAKKISOX. M. X. The role of crcatine and it- phosphate in aniphihian development. 2M>. 1 1 \TKIKI.I), 1". A. I'olydora— larval dev< ment and observations on adult-. 356. Head movement-, in relation to stabilization of viMial field, 285. I h art rate and leucocyte circulation in Cras- sostrea, 1(W. Heat, effect of on strohilixation of Ma-.ti.nias, 493. Heat, effect of on velocity of dinoflagellates, 90. Heat tolerance of I'ca, 133. 1 leniocyanin concentration of Haliotis blood. 459. licmocyte production in Strongylocentrotus, 259. Hemocytes of boll weevil, 112. Hemocytes in coeloinic iluid of holothurians. 102. liemocyte.- and connective tissue of Galleria, relation between, 337. HHXLF.V, C., AND D. P. COSTKI.LO. The cyto- lo.yical effects of podophyllin and podo- phyllotoxin on the fertilixed eggs of Chae- topterus, 369. I lenricia caecum, reaction ot Asterias and Patiria to, 77. Hermit crab commensal, larval development of, 356. HKT/KI., H. K. Studies on holothurian coelo- mocytes. II., 1<>2. High temperature, effect of on velocity of dinoflagellates, (>0. I iistoflieiuistry of Scolo])los egg cocoons, 189. Histology of (ialleria hcmocytes and connec- tive tissue, 337. 1 li-i i holothurians, 102. 1 ll-toloi;y of M ' . ids, 315. Hi-- • 'i regenerating starfish pyloric 1. Ili-toloyy of Strongylocentrotus viiad-, 241. Hi tolo.uy of transplanted starti-h ti--ues, 77. HUM ••. M). .^ MI A. C. GlESE, Aii anto- radiographii ii^ation of the ^onads of the purple ;ea urchin, Stronyyloccii- trotus. 241. Hoi. 1. AMI, X. I).. i. II. I'll Il.l.ll'S, Jk., AMI A. C. Gil i Vi lu igraphii inves ti.uation of coelomi oduction in the inirple sea urchin, Sn ntrotus, 259. Holothurian coelomocytes, origin of, 102. Humidity, role of in heat tolerance of I'ca. 133. Hr.xiKK, \Y. K., AMI S. C. BKOWX. Ctenidial number in relation to size in certain chitons, with a discussion of its phyletic significance, 508. Hydra and algae, symbiosis between, 415. Immunology of amphibian egg-jellies, 328. In vitro cultures of Strongylocentrotus coelo- mocytes, 259. Influence of light on time of cell aggregation in Dictyosteliaceae, 392. Insect, relation between hemocytes and con- nective tissue in, 337. Insect diapause, physiology of (XV), 497. Insects, echolocation of by hat, 297. Insects, stabilization of visual field in, 285. Intracellular osmoregulation during salinity adaptation in toads, 218. Ionizing radiation, "protective" effect of AET against, 125. Irradiated Arbacia eggs, effects of AET on, 125. Jelly of Scoloplos cocoon, chemistry of. IS1'. K Karyotype of Arbacia eggs, 169. Killifisb, development of. 143. Kinesthetic orientation of millipedes, 33. KOXIJX, T. M., A xi) K. B. RAPER. The in- fluence of light on the time of cell aggre- gation in the Dictyosteliaceae, 392. KUKX/LKK, E. J., AXII J. P. PEKRAS. 1'hos- phatases of marine algae, 271. Lancettish stomach, new digenetic trematode from, 4SS. Larvae of Mya. -easonal differences in popu- lations ot, 315. Larval brain of silkworm, transmission of photoperiodic signals to, 497. Larval development of I'olydora, 356. Larval development of Streblospio, 67. Larval stages of (. 'allinectes, variability in, 58. Lead nitrate, use of in staining Arbacia egg nuclei. Id1'. I.KXllohK, II. M. See L MUSCATINE, 415. INDEX 527 Lepidochitona, relation of gill number to size in. 508. Leptasterias, visceral regeneration in, 1. Leucocyte circulation in oyster. 198. Life-history of rhizostome medusae, 493. Light, attraction of migrating slime molds to, 51. Light, influence of on time of cell aggregation in Dictyosteliaceae, 392. Light, role of in diapause development in Ostrinia, 177. Light-adaptation in Galleria eye, 473. Linear dimensions of sand dollars, variations in. 401. Localization of phosphatases in marine algae, 271. LOHANIJAYA, P. Variation in linear dimen- sions, test weight and ambulacral pores in the sand dollar, Echinarachnius, 401. Loligo, embryology of, 24. Longevity of x-irradiated mice, 425. Low temperature, effect of on velocity of dinoflagellates, 90. Low temperature, role of in circulation of oyster leucocytes, 198. Low temperature, role of in diapause develop- ment in Ostrinia, 177. Lymphocytes in coelomic fluid of holothurians, 102. ' M Magnesium, possible role of in growth of symbiotic and aposymbiotic hydra, 415. Magnetic vector, role of in orientation of planarian, 347. Marine algae, phosphatases of. 271. Mastigias, effect of temperature on strobila- tion of, 493. Mature life of x-irradiated mice, 425. MCLAUGHLIN, R. E., AND G. ALLEN. De- scription of hemocytes and the coagula- tion process in the boll weevil, Anthono- mus, 112. Medusae, rhizostome, life-history of, 493. Metamorphosis of Mastigias, effect of tem- perature on, 493. Metamorphosis of Polydora, 356. Method for studying Arbacia egg chromo- somes, 169. Mice, x-irradiated, duration of life of, 425. Migration of pigment in Galleria eye, 473. Millipede, angle sense in orientation of, 33. MILXE, L. J., AND M. MILNE. Stabilization of the visual field, 285. Mitomycin, tests of with Chaetopterus eggs, 369. Mitosis in Arbacia eggs, method for studying, 169. Mitotic activity in regenerating starfish caeca, 1. Mitotic poison, effect of on Chaetopterus eggs, 369. Mollusc, chemosensory bases of food-finding in. 211. Mollusc, circulation of leucocytes in, 198. Mollusc, (squid), embryology of. 24. Mollusc, relation of gill number to size in, 508. Mollusc, reproductive cycle in, 315. Mollusc blood (Haliotis), hemocyanin con- centration in, 459. Molting stages of Callinectes larvae, 58. Morphogenesis of Fundulus, 143. Morphogenesis of squid, 24. Morphogenesis of Streblospio, 67. Morphology of Callinectes larvae, 58. Morphology of new digenetic trematode, 488. Morula cells in holothurian coelomic fluid, 102. Moth (Galleria), relation between hemocytes and connective tissues in. 337. Moth eye, pigment migration and light-adap- tation in, 473. Motility of dinoflagellates, effects of tempera- ture and salinity on, 90. Movements of head, in relation to stabilization of visual field, 285. Mummichog, development of, 143. MTSCATINE, L., AND H. M. LENHOKF. Sym- biosis of hydra and algae. L, 415. Muscle, osmoregulation in, during salinity adaptation in toads, 218. Mya, reproductive cycle of, 315. Myxamoebae, influence of light on time of aggregation of, 392. N NASH, D. J., AMI J. \V. Go\vi \. Effects of x-irradiation of mice exposed in utero during different stages of embryological development on duration of mature life, 425. Neurosecretory processr- in ( Ktrinia, 177. \e\v digenetic trematode from lancetfish stomach, 488. Normal development of Fundulus, 143. Xormal development of squid, 24. XOVICK, A. Echolocation of flying insects by the bat, Chilonycteris, 297. Nutritive phagocytes of Strongylocentrotus, 241. Oak silkworm brain, transmission of photo- periodic signals to, 497. 528 IXDKX ( )cnloniotor activity, in relation to stahiliza- tion of vi>ua1 field. 2X5. ( >C1H IC\ tolds I It |)( ill \\ eCVll, 1 \2. ( logeiiesis in M \ a. 31 5. Oogenesis in Strongylocentrotus, 241. < )ptical properties of Anlheraea cocoon and impal pigment. 473. ( tricntation, acoustic, of Chilonycteris, 2' '7 Orientation of planarians, duration oi .itter- ct'fcct iii, 347. ( trientation sense of millipede, 33. Orientation of slime molds touards light, 51. ( tricntation of visual organs. 2X5. Origin of holothurian coeloinocytes. 102. ( )smoregulation during salinity adaptation in toads, 218. Ova of amphibian, antigenic components of jellies Of, 328. Ova of Arhacia, method for studying chromo- somes of, ]6°. ( )va of Arhacia. radioprotective action of AKT on, 125. Ova of Chaetoiiterns, effects of podophyllin on. 369. Ova of l.oligo, development ot. 24. Ova of Streblospio, development of, 67. O\ster, In-art rate and circulation of leuco- cytes in, T'X. I'arahiotic twins of < ialleria, use of in study- ing relation between hemocytes and con- nective tissue, 337. Parastichopus, origin of coelomocytes in, 102. PARK, Y. IF. See F. A. BROW \, JR., 347. I'atiria, reaction of to foreign tissue in coclom, 77. I'crnyi silkworm larvae, transmission of pho- i lodic signals to hrain of, 4('7. I'l RRAS, J. 1'. See I''.. J. KuENZLER, 271. id I optima tor phosphatase production by marine algae, 271. I tes of oysters, circulation of, 19X. ;ocyte« of Strongylocentrotus, 241. tosis in boll weevil larvae, 112. I'n n i IPS, J. II.. JK. See X. I). HOLLAND, 259. Phos],h;r i marine algae, 271. I'liosphonis metabolism in amphibian em- brxos, 2 I Miotoprrjc idic contn ical rhythm 177. Photoperiodic stimuli, Iran , . ion of to brain of Antheraea larvae, 4''7. Photoreceptors oi Galleria eye, physio'. n 473. I 'hotota xis, relatii HI of soroca to, in Dictyostelium, 51. I'livlctic significance ot gill number in chitons, " 50X. Physiological rhythm, photoperiodic control ' of. 177. Physiology of insect diapause, XV., 407. 1'igment of Antheraea larval brain, role of in transmission of photoperiodic signal, 497. I'igment migration in Galleria eye, 473. PILSO.N. M. !•".. O. Variation in hemocyanin concentration in the blood of four species of Haliotis, 459. I'isaster, visceral regeneration in, 1. Plauarian orientation, duration of after-effect in, 347. Planktonic larvae of Polydora, 356. Plasma osmotic concentration in toads, 218. Plasmatocytes of boll weevil, 112. Podophyllin and podophyllotoxin, effects of on Chaetopterus eggs, 369. I'olychaete, egg cocoons of, 189. Polychaete (Streblospio), development of, 67. Polychaete annelid, larval development of, 356. Polydora, development of, 356. Polysphondylium, influence of light on time of cell aggregation in, 392. Polysphondylium, phototaxis in, 51. Populations of Mya, differences in breeding activity of, 315. POST, C. T., JR., AND T. H. GOLDSMITH. Pigment migration and light-adaptation in the eye of the wax moth, Galleria, 473. Potential, retinal action, of Galleria eye, 473. Proctodone, photoperiodic control of secretion of, in Ostrinia. 177. Prohemocyfes of boll weevil, 112. "Protective" effects of AET, 125. Protein in blood of Haliotis, 45°. Protein content of marine algae, 271. Protostelium, phototaxis in, 51. Psolus, origin of coeloinocytes in, 102. Pulse-echo relations in pursuit of flying in- sects by Chilonycteris, 297. Pyloric caeca of starfish, regeneration of, 1. Oucrcctin, tests of with Chaetoptcrus eggs, 369. Radioprotective effects of AKT, 125. Rana egg-jellies, relationships of antigenic components of, 32X. Rana embryos, role of creatine and its phos- phate in development ot, 230. R VPER, K. II. See T. M. KoNIJN, 3()2. Rati' of heartbeat in oysters, 1('X. INDKN 520 Rates of swimming of dinotlagellates. effects of temperature and saliniu on, 90. Reactions of starfishes to foreign tissues in coelom, 77. Regeneration, visceral, in sea stars, 1. Relation of gill number to size in chitons, 508. Relation between hemocytes and connective tissue in Galleria, 337. Relation of sorocarp size to phototaxis in Dictyostelium, 51. Relationship of antigenic components in egg- jellies of amphibians, 328. Reproduction and development of Streblospio, 67. Reproduction of Polydora, 356. Reproductive cycle of Mya in New England, 315. Reproductive cycle of Strongylocentrotus, 241. Responses of planarians to weak magnetic fields, 347. Retinal action potential of Galleria eye, 473. Reverse-turning in millipede, 33. Reversed horizontal magnetic vector, role of in orientation of planarians, 347. "Rhinophores" of Aplysia, role of in food- finding, 211. Rhizostome medusae, life-history of, 4°3. Rhythm, physiological, photoperiodic control of, 177. ' RICHARDS, A. G. See S. C. SHRIVASTAVA, 337. Roentgen irradiation, effect of on mature life- span of mice, 425. Role of creatine and its phosphate in am- phibian development, 230. ROPES, J. W., AND A. P. STICKNEY. Repro- ductive cycle of Mya in New England, 315. RUGH, R., AND K. Fu. AET as a radio- protective agent at the cellular level, 125. Salinity, effects of on velocity of dinoflagel- lates, 90. Salinity adaptation in toads, 218. Sand dollar, variations in dimensions and weights of, 401. Scoloplos, egg cocoons of, 189. Sea-hare, food-finding in, 211. Sea stars, visceral regeneration in, 1. Sea urchin, coelomocyte production in, 259. Sea urchin, gametogenesis in, 241. Sea urchin egg fertilizin antigens, relation- ship of, 328. Sea urchin eggs, method for studying, 169. Sea urchin eggs, radioprotective effects of AET on, 125. Seasonal changes in Strong loeentrofiis gon- ads, 241. Serology uf amphibian egg-jellies, 32S. Sex cells of Strongylocentrotus, autoradio- graphic investigation of, 241. Sex ditternii-es in radiation sensitivity of mice, 425. Shape-changes in podophyllin-treated Chac- toptern-. • •szi-'s. -lo1'. SIIIVKKS, C. A. The relationship of antigenic component^ in egg-jellies of various am- phibian species, 328. SHRIVASTAVA, S. C., AND A. G. RICIIAKDS. An autoradiographic study of the rela tion between hemoe\tes and connective tissues in the wax moth, Galleria, 337. Silkworm brain, transmission of photoperiodu signals to, 497. Size in relation to gill number in chitons, 508. Size of sorocarp, relation of to phototaxis, in Dictyostelium, 51. Skeletal muscle, osmoregulation in, during salinity adaptation in toads, 218. Slime mold, phototaxis in, 51. Slime mold cell aggregation, influence of light on time of cell aggregation of, 392. Sodium, role of in growth of symbiotic and aposymbiotic hydra, 415. "Sonar" behavior of Chilonycteris, 297. Sorocarp size, relation of to phototaxis in Dictyostelium, 51. Spawning of Mya, 315. Spawning of Scoloplos. 189. Spawning season for Streblospio, o". Specificity of egg-jellies of amphibians, 328. Spectral sensitivity of Antlieraea pupal brain, 497. Spermiogenesis in Mya, 315. Spermiogenesis in Strongylocentrotus, 241. Spherule cells of boll weevil, 112. Squash method for studying Arbacia eggs, 169. Squid, embryologv of. 24. Stabilization of visual fields, 285. Stages in normal development of Fundulus, 143. Stages in normal development of Loligo, 24. Starfish, visceral regeneration in, 1. Starfishes, reaction of to foreign tissue in coelom, 77. STICKNEY, A. 1'. See J. \Y. ROPES, 315. Stimuli, photoperiodic, transmission of to brain of Antlieraea, 4n7. Streblospio, reproduction and larval develop- ment of, 67. Strobilation of Mastiuias, effects of tempera- ture on, 493. 530 Strongyloccntrotus. coclomocytc production iii, 259. Strongylocentrotus, gametogenesis in, 241. Studies on holothurian coelomocytcs. 11.. 102. STI \ KAKII. H. \\". A digcnetic trematodc, r.otuhis. I'nuii the stomach of the lancet- fish, Alepisaiirns, taken in the South I 'aril ic. 488. SUGIURA, V. On the life-history of rhizo- stonie medusae. III.. 4' '3. Swimming rate of dinoflagcllates, effects of temperature and salinity on, 90. SWINTON, D. E. See S. D. BECK. 17; Symbiosis between hydra and algae, 415. S\mbiotic organisms, role of in strohilation of Mastigias. 4('3. Syngamy in Arhacia eggs, lf>9. Synthesis of phosphatase by marine algae, 271. U I , a heat tolerance of. 133. L'lva as food for Aplysia, 211. V Variability in larval stages of Callinectes, 58. Variability of leucocyte counts in oysters, 198. Variation in hemocyanin concentration in Haliotis blood, 459. Variations in dimensions of sand dollars, 401. Velocity of dinoflagellates, effects of tempera- ture and salinity on, 90. Vertebrates, stabilization of visual field in, 285. Visceral regeneration in sea-stars, 1. Vision, role of in orientation of millipede, 33. Visual field, stabilization of, 285. Taxonomy of Botulidae, 488. Temperature, effects of on strohilation of Mastigias, 4°3. Temperature, effects of on velocity of dino- flagellates, 1_\ d> »ra, 356. I roi hophore oi Strehl< >spio, <>7. Turning tendency in millipedes. 33. W WALCOTT, C. See C. M. WILLIAMS. 497. \Vax moth, pigment migration and light-adap- tation in eye of, 473. Wax moth, relation between hemocytes and connective tissue in, 337. Weevil, hemocytes of, 112. Weight of chitons, in relation to gill number, 508. Weight of sand dollar, variations in, 401 WHITFIELD, F. E. See J. T. BONNER. 51. W'lLKENS, J. L., AND M. FlNGERMAN. Heat tolerance and temperature relationships of the fiddler crab, Uca, with reference to body coloration, 133. WILLIAMS, C. M., P. D. ADKISSON AND C. WALC-OTT. Physiology of insect diapause. XV., 497. X-irradiation of mice, effects of on duration of life. 425. Zoeae of Callinectes, variability in, 58. Xooxanthellae. role of in strohilation of Mas- tigias, 493. Volume 128 Number 1 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board JOHN M. ANDERSON, Cornell University L. H. KLEINHOLZ, Reed College JOHN B. BUCK, National Institutes of Health JOHN H. 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