THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, Vanderbilt University JOHN B. BUCK, National Institutes of Health T. H. BULLOCK, University of California, Los Angeles E. G. BUTLER, Princeton University K. W. COOPER, University of Rochester M. E. KRAHL, University of Chicago J. H. LOCHHEAD, University of Vermont E. T. MOUL, Rutgers University ARTHUR W. POLLISTER, Columbia University MARY E. RAWLES, Johns Hopkins University A. R. WHITING, University of Pennsylvania CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 112 FEBRUARY TO JUNE, 1957 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. 11 THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Penn- sylvania. 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Vol. 112, No. 1 THE BIOLOGICAL BULLETI PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE DISTRIBUTION OF POLYSACCHARIDES AND BASOPHILIC SUBSTANCES DURING THE DEVELOPMENT OF THE MUSHROOM COPRINUS1 JOHN TYLER BONNER, ALLAN A. HOFFMAN, WILFRED T. MORIOKA AND A. DUNCAN CHIQUOINE Department of Biology, Princeton University, N. J . As Buller (1909 ct scq.) has pointed out, some species of the genus Copriniis sow their spores once during a short interval of time and the fruiting body disap- pears shortly thereafter by auto-digestion. Characteristically the small buds will, all in one clay, go through a period of rapid expansion and elongation, shed their spores, and deliquesce. This rapidity is no doubt related to their small size, for larger species of hymenomycetes will go through many days of continued production and shedding of spores. The origin of the gills and their individual lamellae has been described in detail by Atkinson (1916). They are formed at an early stage by the orientation of hyphae and the final result ( which is illustrated in Figure 1 ) has a number of distinguishable component parts : the outside is covered with the hymenium which consists of a mixture of basidia and sterile paraphyses ; below this there is a sub- hymenial layer of small hyphae ; and finally the central portion of the gill lamella, the tramal layer, which is composed of large hyphae. Borriss (1934) has shown that the development of Coprinus occurs in two distinct stages, one in which cell division and the initial cell orientation takes place, and it is during this stage that the gill primordia are formed. The second stage consists of rapid cell elongation and in the latter phase of this period the spores bud from the basidia. It may be inferred, from the recent results of Madelin (1956), that this period of rapid expansion involves the transfer of material from the vege- tative mycelia. Such a transfer is in keeping with the views of Buller as well as with the situation in Agaricns canipcstris ( Bonner, Kane and Levey, 1956). Using histochemical techniques, it has been possible in this study to follow the distribution of certain groups of substances in the fruiting body. It could be dem- onstrated that these substances accumulated at specific locations in the gills and that they were all transferred into the spores, so that by the advent of auto-digestion there were virtually no demonstrable substances left within the cells at the time of the final destruction. Not only does this indicate an efficiency, an economy in the 1 This research was supported in part by funds of the Eugene Higgins Trust allocated to Princeton University. 1 2 BONNER, HOFFMAN, MORIOKA AND CHIQUOINE fruiting process, hut also it is of some interest to find that prior to sporulation the different suhstances are stored in different parts of the gills. METHODS Two species of Coprinus were studied : C. lag opus Fr. and C. curt us Kalch. (The authors would like to thank Dr. Haig P. Papazian of Yale University for the culture of C. lagopus, and Dr. Alexander H. Smith of the University of Michigan for the identification of C. curt us.} They were grown in jars on moist, sterile horse dung and kept at room temperature (approximately 24° C.) The entire fruiting bodies were fixed for 12 hours in Rossman's fluid (9 parts absolute alcohol saturated with picric acid plus 1 part formaldehyde) at 4° C. The selection of this particular fixative, as well as the temperature, follows from the recommendations of Deane et al. (1946), from their studies on the preservation and localization of glycogen in mammalian liver. The suitability of the fixation for the mushroom material is evidenced by the absence of any false localization of gly- cogen, "glycogen flight," due to the pathways of penetration of the fixative. Follow- ing fixation the tissues were dehydrated, cleared with methyl salicylate (14 hours), and embedded in paraffin. Sections were cut at both 5 and 10 /A and mounted on slides in the usual manner. Polysaccharides were demonstrated by the periodic acid-Schiff reaction as de- scribed by Gomori (1952). This technique is based upon the oxidation with periodic acid of the vicinal hydroxyl groups of polysaccharides to aldehyde groups and the subsequent visualization of the .aldehyde groups by reaction with the Schiff reagent. The sections were deparaffinized, run to water, and oxidized for 10 min- utes at room temperature with 0.5% periodic acid. Schiff reagent was prepared according to the method of Lillie (1954) and the slides were treated with it for 20 minutes. Excess Schiff reagent was removed by three sodium metabisulphite rinses, the slides dehydrated, cleared and mounted. Control sections were treated exactly the same, although not oxidized with periodic acid. Any material which stained with the PAS technique and did not stain in the control slides is hereafter referred to as polysaccharide. It was possible to differentiate mucopolysaccharides from glycogen by digestion of the slides with salivary amylase prior to the periodic acid oxidation. The slides were run to water as before and digested for three hours at room temperature in saliva. Following digestion the slides were run through the same PAS procedure outlined above. Any material which was PAS-positive but removable by salivary digestion is hereafter referred to as glycogen. Any material not removable with salivary digestion is referred to as mucopolysaccharides. Additional slides placed in water for three hours, in place of saliva, served as control slides. Since the pattern of localization in those control slides differed in no way from the standard PAS pattern, one can conclude that the saliva removes glycogen by enzymatic hydrolysis specific for glycogen and not by a leaching-out of materials by simple dissolution in an aqueous solution. Regions of basophilia were demonstrated in the same material by staining with a 0.1% aqueous solution of toluidin blue. Cytoplasmic basophilia can be attributed to the presence of ribose nucleoproteins and/or acid mucopolysaccharides. Since all DEVELOPMENT OF MUSHROOMS 3 basophilic material observed in this stud}' was not PAS-positive, it is assumed that the basophilia was due to ribose nucleoproteins. RESULTS A series of stages of both species of Coprhuts was fixed and sectioned. Then duplicate slides of each fruiting body were stained by ( 1 ) the PAS method for all polysaccharides, (2) the PAS method with salivary digestion for the mucopoly- S H FIGURE 1. A section through a gill indicating the distribution of basophilic substances (above) and glycogen (below). T, tramal layer; S, sub-hymenium ; H, hymenium ; B, basidiv.m ; P, paraphysis. (Drawing by Miss Marcia J. Shaw.) saccharides, and (3) with toluidin blue for the basophilic substances. Therefore, it was possible to compare a specific fruiting body at a specific stage for three sub- stances : mucopolysaccharides, glycogen, and basophilic substances. First of all it should be said that both species, C. lagopits and C. ciirtus, showed the same staining characteristics, and therefore the description given below applies 4 BOXXKk, HOFFMAN, MORIOKA AND CHIQUOINE to both. The general staining properties of the cap and stipe could be seen more advantageously in the smaller C. ciirtns, while C. lagopus was especially suitable for examination of the gills. .An intense staining with SchifT reagent was observed in the globular cells of the rap in (". curt us which was not dependent upon periodic acid oxidation. There was also a faint staining of all cell walls in non-oxidized control sections, and character- glycogen basophilia FIGURE Photograph of sections through the gills showing the distribution of glycogen (left column) and basophilic substances (right column) at different stages of development; a. 1), c represent the three stages of before, during, and after spore formation. Each pair (e.g., a and a,) are from the same mushroom. DEVELOPMENT OF MUSHROOMS 5 istically the stain was more intense at the basal end of the mushroom. The basis for these two staining reactions is unknown. Following periodic acid oxidation the cell walls stained intensely, indicating the presence of mucopolysaccharides, pre- sumably chitin. The cell walls were the only regions where mucopolysaccharides were observed in a significant concentration. Exclusive of the gills, there was no general difference in the distribution of glycogen and the basophilic substances ; they both appeared to be present in small quantities all over the mushroom at early stages, although the growth zone just below the cap always showed a higher concentration. The intensity of staining reactions was inversely proportional to the cell length, that is, the short cells of the growth zone at early stages were darkly stained, but after complete expansion these cells were depleted of stainable material as were the cells of the rest of the mushroom. In the gills, prior to spore formation, there is a dark layer showing a concen- tration of basophilic substances, as well as a similar layer of glycogen. The inter- esting point is that, as shown in Figures 1 and 2, the positions of these layers do not correspond. The basophilic layer lies at the tips of the basidia, that is, the end at which the future spores will be formed. Furthermore, basophilia is observed only in the basidia and not in the paraphyses. The glycogen lies primarily in the sub- hymenium, although it extends to the base of the hymenium (in both the basidia and paraphyses) on one side and into the tramal layer on the other. This staining picture is present at the earliest stages that gills are discernible, and in subsequent stages there is no obvious increase in the intensity of the staining. The only significant change is the increase in the size and the extent of the gills with age. It was possible to show that once the spores are formed (Fig. 2, c) both the stainable layers disappear, showing a complete absence of glycogen and basophilic substances. Therefore, it is presumed that these materials enter into the spores, but the spores themselves become darkly pigmented with maturity, which masks any staining reaction they might show. In order to verify the hypothesis that the spores contain these substances, a new series of mushrooms of intermediate size was fixed to find ones in the middle of the process of spore formation. A particular set of sections of C, lag opus was for- tunately exactly at this stage (Fig. 2, b). The glycogen is now present in and around the hymenium, as well as concentrated in the young spores. The basophilic substances, which were at the basidial tips to begin with, appear to enter directly into the spores during their formation. DISCUSSION The presence of glycogen about the base of the basidia, and its outward move- ment during spore formation, suggest that it might supply energy for the process of spore formation. However, a considerable portion of the glycogen enters directly into the spore and becomes part of its reserves for future germination and growth. The basophilic substances are at the end of the basidium that forms the spores and therefore the material is transferred directly upon spore formation. Presum- ably the nuclear material is associated with this basophilic zone and both are trans- ferred together. It is of interest to note that the staining appears equally intense in the gills in 6 BONNER, HOFFMAN, MORIOKA AND CHIQUOIXK both small young mushrooms and large fully developed ones. The only difference is the extent of the gill material and from this we might presume that the gill is laid down with all its food material for sporulation right in the beginning, and during the period of growth the sole change is that the total amount of gill material is extended. From the work of Madelin (1956) it is likely that materials are constantly being drawn up from the vegetative hyphae into the mushroom during its expansion, and this material must be led directly to the newly forming gill. The observation that essentially all stainable glycogen and basophilic substances are gone after sporulation implies an economy, and one would expect this phenome- non to be correlated with the fact that in small species of Coprinus there is but one short period of spore formation and discharge. Mr. Anthony J. Schmidt of this laboratory has made some preliminary PAS and toluidin blue preparations of Agaricus campestris and here one finds large concentrations of glycogen and baso- philic substances in the cap with channels of conduction to the hymenium. There- fore in a large species which forms spores over a long period there is a large store of substances that can be continuously poured into the spores. SUMMARY Two species of Coprinus (C. lagopus and C. curtus) were examined, using histochemical techniques, and it was found that prior to sporulation there were two distinct zones in the gills, one containing glycogen and one containing basophilic substances. The glycogen zone is at the base of the hymenium, extending into the central tramal layer. The basophilic zone is at the outer tips of the basidia. Upon sporulation both these groups of substances entered the spores, leaving no demon- strable material within the cells of the gills. LITERATURE CITED ATKINSON, G. F., 1916. Origin and development of lamellae in Coprinus. Rot. Gas., 41 : 89- 130. BONNER, J. T., K. K. KANE AND R. H. LEVEY, 1956. Studies on the mechanics of growth in the common mushroom, Agaricus campestris. Mycologia, 48 : 13-19. BORRISS, H., 1934. Beitrage zur Wachstums- und Entwicklungsphysiologie der Fruchtkorper von Coprinus lagopus. Planta, 22 : 28-69. BULLER, A. R. H., 1909, et seq. Researches on fungi. Longmans, Green and Co., New York. DEANE, H. W., F. B. NESBETT AND A. B. HASTINGS, 1946. Improved fixation for histological demonstration of glycogen and comparison with chemical determination in liver. Proc. Soc. Exper. Biol. Med., 63 : 401-406. GOMORI, G., 1952. Microscopic histochemistry. University of Chicago Press, Chicago. LILLIE, R. D., 1954. Histopathologic technic. Second Edition. Blakiston Co., Philadelphia. MADELIN, M. F., 1956. Studies on the nutrition of Coprinus lagopus Fr., especially as affecting fruiting. Ann. Bot., 20 : 307-330. RELATION BETWEEN POSITION OF BURROWS AND TIDAL RHYTHM OF UCA 1 MILTON FINGERMAN Department of Zoology, .YVar<>;;f/> College, Tulanc Unii'crsity, Ncu> Orleans 18, Louisiana The literature concerning tidal and semilunar rhythms of color change has been reviewed by Fingerman (1957). These rhythms were first observed in the fiddler crab Uca pugnax collected in the region of Woods Hole, Massachusetts, where the tides are semidiurnal (Brown, Fingerman, Sandeen and Webb, 1953). The crabs darkened by day and lightened by night in accordance with their 24-hour rhythm of color change. Superimposed upon the latter was a tidal rhythm that progressed across the 24-hour rhythm at the average rate of 48.8 minutes per day, as evidenced by a supplementary dispersion of pigment in the melanophores about the time of low tide. Also evident was a semilunar rhythm of 14.8-day frequency, the average in- terval between days on which the 24-hour and tidal rhythms repeat similar time relations to one another. The relationship between the time of supplementary dispersion of the melanin and the time of low tide was determined directly by the local tidal situation where the crabs were collected, as evidenced by the phase differ- ence of the tidal rhythms of Uca pugnax collected from two localities where the times of low tide were different. Persistent tidal and semilunar rhythms of color change have been observed in the blue crab, Callinectcs sapidus, by Fingerman (1955). The rhythms were simi- lar to those described above for Uca pugnax in spite of the fact that the Callinectes were collected in the vicinity of New Orleans, Louisiana, a region of diurnal tides. Evidently, the center of tidal rhythmicity in Callinectcs operates on the basis of tides spaced 12.4 hours apart in spite of the fact that the crabs were collected in a region where 24.8 hours is the interval between two successive low tides. Tidal rhythms of color change have been observed in two species of fiddler crabs in addition to Uca pugnax, Uca pugilator and Uca speciosa by Fingerman (1956). The latter two species were collected at Ocean Springs, Mississippi, where the tides are diurnal. The tidal rhythms of both species were similar in nature to the tidal rhythms of Uca pugnax and Callinectes sapidus. Both the Uca pugilator and the Uca speciosa were collected from limited portions of the beach. Analysis of the tidal rhythm of both species revealed that the Uca speciosa behaved as if low tide occurred for them 7.5 hours earlier in the day than low tide for the Uca pugilator. Inspection of the beach at Ocean Springs revealed that the burrows of the Uca speciosa were closer to the high tide mark than were the burrows of the Uca pugilator. The phase difference of the tidal rhythms appeared to be due to the fact that when the water began to recede after high tide, a local low tide occurred earlier for the Uca speciosa than for the Uca pugilator. The Uca speciosa would be free, therefore, to leave their burrows and feed earlier than the Uca pugilator living 1 This investigation was supported by Grant No. B-838 from the National Institutes of Health. 8 MILTON FINGERMAN closer to the actual low tide mark. Measurements of the beach where both species were collected revealed that the water actually began to uncover the burrows of the Uca pugilator 4.9 hours after the burrows of the Uca speciosa began to be uncovered. The present study was undertaken to investigate further the nature of the tidal rhythm of color change of the fiddler crab Uca pugilator and to test the hy- pothesis presented above that the phases of the tidal rhythm are. set by the time of low tide at a limited portion of beach. The hypothesis was tested by comparing the phases of the tidal rhythm of color change of specimens of the same species col- lected from distinct sets of burrows different distances from the high tide mark, be- cause the possibility existed that the phase difference between the Uca speciosa and Uca pugilator was a species difference and not due to the tides as hypothesized above. MATERIALS AND METHODS Adult male and female specimens of the fiddler crab Uca pugilator were collected on the beach near the Gulf Coast Research Laboratory, Ocean Springs, Mississippi, for use in the observations reported below. The specific collection site consisted of two discrete sets of burrows. Crabs could be collected from each set of burrows with assurance that no mingling of the two groups occurred normally. Figure 1 is a diagrammatic representation of the section of the beach where the animals were col- lected. Included in this figure are the distances of the burrows from each other and from the high and low tide marks. A sub-surface drainage pipe, not shown on the diagram, emptied its contents between the two sets of burrows. The water flowing from this pipe at times of low tide was another factor that kept the crabs of the two sets of burrows isolated from one another. The animals were placed into stainless steel aquaria and transported to an air- conditioned laboratory in New Orleans. From the evening of the day of collection until the end of the period of observation, the animals were maintained in darkness except for the few minutes required to make observations of the chromatophores and to change the water in the aquaria on days observations were performed. The animals were kept in inclined stainless steel aquaria containing sufficient sea water to cover approximately one-half of the bottom of the aquaria. The crabs were, therefore, free to move into and out of the water. Chromatophores were staged in the manner described by Fingerman (1956). The average index was determined for the melanophores on the anterior aspect of a walking leg with the aid of a stereoscopic dissecting microscope and lamp. The chromatophore indices of Hogben and Slome (1931) were used in staging the chromatophores. The most concentrated state of the pigment is referred to as stage 1, the most dispersed stage 5, and the intermediate conditions stages 2, 3, and 4. RESULTS AND DISCUSSION The measurements of the portion of the beach at Ocean Springs, where the animals were collected (Fig. 1), were analyzed to determine the phase difference that would be expected between the tidal rhythms of the animals from the two sets of burrows if the hypothesis of Fingerman (1956) is correct. The distance from the high tide mark to the low tide mark was 215 feet (Fig. 1). Twelve and four- tenths hours is the average interval required for the water to recede from the high TIDAL RHYTHM OF UCA tide mark to the low tide mark. One set of burrows was 28 feet closer to the high tide mark than was the second set. If the water recedes 215 feet in 12.4 hours, then the water will recede 28 feet in 1.6 hours assuming, of course, that the slope is uniform. The set of burrows closer to the high tide mark would, therefore, be- gin to be uncovered by the receding water 1.6 hours earlier each day than would the set of burrows closer to the low tide mark. On the basis of the average tidal progression of 48.8 minutes per day, 1.6 hours in a tidal cycle are equivalent to two days. Therefore, a phase difference of two days was anticipated between the specimens from the two sets of burrows when they were examined in the laboratory. 215 FT (12.4 MRS.) 00 FT 5^(578 HRS/ 73 FT L_ 10 128 FT (739 MRS.} DIFFERENCE-LSI HOURS \ FIGURE 1. Diagrammatic representation of the positions of the burrows on the beach at Ocean Springs, Mississippi, where the specimens of Uca pugilator were collected. Figure 2 presents the times of high and low tide at Ocean Springs on days chro- matophores were observed in the laboratory. The diurnal nature of the tides is obvious. However, the tides did not follow the usual pattern of advancing 48.8 minutes daily across the 24-hour day. High tides occurred generally between mid- night and 2 P.M. and low tides from 2 P.M. to midnight. The data for Figure 2 were selected from the tide tables published by the Coast and Geodetic Survey, United States Department of Commerce. On July 3, 1956, several hundred crabs were collected from each set of burrows for the first series of observations that started at 8 A.M. on July 4. On days when observations were performed, 50 specimens from each group were observed hourly 10 MILTON FINGERMAN from 8 A.M. through 7 P.M. and the average chromatophore stage was calculated for each group. The crabs from the burrows closer to the high tide mark will be re- ferred to as Series A and the crabs from lower on the beach as Series B. In Series A a sufficient number of crabs survived throughout the period of observation so that 50 specimens were always available. Many of the Series B crabs died sud- denly between August 6 and 8 so that this group had to be discarded. The average hourly chromatophore stages for Series A and B on each day observations were per- formed are presented in Tables I and II, respectively. Observations were usually made every second day, but from July 12 through 17 observations were daily. Some of the data were taken from Tables I and II in order to prepare Figure 3. Use of this figure aids in discussion of the phase difference of the tidal rhythms I2P.M 6 P.M. I2M. 6AM. 12P.M. o HIGH TIDE • LOW TIDE 0° I j I I 4 14 24 3 1323 JULY AUGUST 2. Times of high and low tide at Ocean Springs on days observations of the crabs were made in the laboratory. of the two sets of animals. In Figure 3 the daily patterns of Series A and B crabs were arranged according to the expected two-day phase difference. According to the calculations low tide at any given hour of the day, e.g., noon, would occur two days earlier for Series B crabs than for Series A crabs. Therefore, the curve of Series A for any given day was placed beside the curve of Series B that had been obtained two days previously. As is evident from inspection of Figure 3, a two- day difference in the tidal rhythms was present as shown by the similarity of the curves of Series A with the curves of Series B that had been obtained two days earlier. The shapes of the daily curves obtained during the first three weeks of observa- TIDAL RHYTHM OF UCA 11 tion did not turn out as predicted on the basis of the observations of chromatophores of Uca pugilator performed during the summer of 1955 (Fingerman, 1956). The maxima of the curves did not progress across the day but were restricted to the left side of the curves, decidedly different from the results obtained with the same spe- cies collected in 1955 in the same area (Fingerman, 1956). The specimens of Uca pugilator observed during the summer of 1955 exhibited the typical pattern of tidal rhythm of color change just as was found in Uca pugna.v by Brown, Finger- man, Sandeen and Webb (1953) and in Callinectcs sapidus by Fingerman (1955). TABLE I The average melanophore index for each of the 12 daily observations of the Uca pugilator animals of Series A 8 A.M. 9 10 11 12 M. i P.M. 2 3 4 5 6 7 July 4 3.7 3.4 3.6 3.3 3.6 3.8 3.8 3.4 3.2 3.6 3.3 2.9 6 3.9 3.8 3.6 3.7 3.3 3.5 3.3 3.1 3.3 3.2 2.8 2.9 8 3.8 4.2 4.2 3.7 3.6 3.7 3.5 3.7 4.0 3.8 3.4 3.4 10 4.0 3.9 3.9 4.2 4.1 4.1 3.7 3.5 3.3 3.8 3.9 3.6 12 3.3 3.2 3.9 3.9 3.6 4.0 3.8 3.2 3.5 3.4 3.2 3.1 13 3.5 3.8 3.5 3.6 3.6 3.6 3.4 3.3 3.6 3.6 3.9 3.4 14 3.6 3.6 3.7 3.6 3.6 3.5 3.5 3.6 3.7 3.4 3.4 3.6 15 3.9 3.7 3.1 3.1 3.4 3.3 3.2 3.4 3.3 3.5 3.3 3.5 16 3.8 4.0 3.6 3.4 3.5 3.2 3.0 3.1 3.1 3.4 3.1 3.2 17 3.6 3.9 3.9 3.8 3.8 3.5 3.5 3.0 3.4 3.2 3.2 3.3 19 3.6 3.2 3.6 3.3 3.3 3.4 3.5 3.2 3.0 3.1 2.9 2.5 21 4.2 3.8 4.0 3.6 3.5 3.7 3.9 3.7 3.7 3.6 3.6 3.1 23 3.9 3.8 4.1 3.2 3.4 3.0 3.3 3.2 3.3 3.2 3.0 3.6 25 4.2 3.9 3.7 3.4 3.0 3.2 3.9 3.4 3.0 3.1 3.3 2.9 27 3.6 3.7 3.0 2.8 3.0 3.1 3.2 3.3 3.3 3.2 3.3 2.9 29 3.8 3.2 3.4 2.9 2.8 3.0 3.3 3.2 3.5 3.3 3.2 2.8 31 3.6 3.9 3.2 3.0 3.1 3.3 3.0 3.0 3.0 3.3 2.9 2.8 August 2 3.6 3.4 3.5 3.2 3.1 2.9 3.2 3.0 3.1 3.2 2.9 2.7 4 3.5 3.3 3.0 2.8 2.9 3.0 3.2 3.3 3.1 3.5 3.2 2.9 6 2.6 3.2 3.6 2.9 3.3 3.2 3.3 3.3 3.4 3.1 2.9 3.1 8 3.1 2.8 3.1 3.0 2.9 3.3 3.0 3.4 3.2 3.2 3.5 3.3 10 3.0 2.9 2.7 2.6 2.8 3.1 3.0 2.8 3.2 3.3 3.5 3.0 12 3.4 3.4 2.8 3.0 2.9 2.7 3.4 3.2 3.2 3.0 3.0 2.9 14 3.0 3.3 3.5 2.8 3.1 2.8 2.9 3.0 2.7 2.9 2.7 2.4 16 3.1 3.0 3.0 2.8 3.2 3.2 3.2 3.5 3.5 3.0 3.4 3.0 18 3.3 3.1 2.7 3.2 2.8 3.1 2.8 3.1 3.0 3.3 3.0 3.1 20 3.2 3.2 3.1 3.2 3.0 2.9 3.1 3.0 3.2 3.4 3.5 3.1 22 3.0 3.0 2.7 2.8 3.0 2.8 2.9 2.9 3.0 3.1 3.2 3.2 The tidal maximum of pigment dispersion of the crabs observed in 1955 progressed across the 24-hour day at the usual tidal rate of 48.8 minutes per day, with the result that maximum pigment dispersion occurred in the right half of approximately 50 per cent of the curves depicting the daily pattern of pigment dispersion. However, the maximum did not progress into the afternoon during the first three weeks of observation in 1956. More will be said of this phenomenon below. In order to reaffirm these observations specimens of Uca were collected on July 18, 1956, from the same sets of burrows as were the animals of Series A and B and 12 MILTON FINGERMAN were treated in the same fashion as the animals collected previously. Crabs col- lected on July 18 from burrows nearer the high tide mark will be referred to as Series C and crabs from burrows lower on the beach as Series D. The average chromatophore indices determined for Series C and D are presented in Tables III and IV, respectively. Fifty specimens were available from Series C throughout the observations. However, with Series D 50 were available only from July 19 through August 8. The number then gradually diminished from 49 on August 10 to 26 on August 22, the last day of observation. A two-day phase difference in the tidal rhythms of Series C and D crabs was evident during the first week of observa- tion. Maxima were restricted to the left half of the curves during this period as were the maxima of the curves of Series A and B. About July 26, however, unex- pected results that will be described below appeared. TABLE II The average melanophore index for each of the 12 daily observations of the Uca pugilator specimens of Series B 8 A.M. 9 10 11 12 M. i P.M. 2 3 4 5 6 7 July 4 3.8 3.8 3.6 3.4 3.3 3.5 3.5 3.3 3.3 3.1 2.5 2.6 6 3.7 3.8 4.0 3.6 3.2 3.5 2.9 3.2 3.4 3.3 3.3 2.6 8 3.6 3.8 4.1 3.9 3.9 3.6 3.7 3.8 3.2 3.6 3.6 3.2 10 3.3 3.7 3.4 3.8 4.1 4.0 3.8 3.7 3.2 2.8 3.5 3.1 12 3.4 3.4 3.8 3.2 3.5 3.3 3.3 3.6 3.5 3.1 3.3 3.2 13 3.2 4.0 3.5 3.3 3.4 3.5 3.3 3.8 3.3 2.8 3.2 3.2 14 4.0 3.7 3.7 3.3 3.2 3.5 3.0 3.3 3.3 3.3 3.2 3.1 15 3.9 4.0 3.9 3.7 3.8 3.2 3.4 3.0 3.5 3.3 3.3 3.2 16 3.4 3.6 3.6 3.4 4.0 3.7 3.2 3.6 3.1 3.4 3.0 3.4 17 3.9 4.0 3.7 3.9 3.5 3.3 3.5 3.2 3.3 2.9 3.1 2.7 19 3.4 3.9 3.7 3.5 3.4 3.3 3.3 2.9 3.2 3.1 2.8 2.4 21 3.5 3.7 3.8 3.1 3.3 3.0 3.1 2.8 2.7 2.5 2.7 2.5 23 3.4 3.5 3.5 3.0 2.7 2.9 2.8 3.1 2.8 2.8 2.7 2.3 25 3.9 3.6 3.3 3.1 3.2 3.1 3.0 2.9 2.9 2.9 2.7 2.4 27 3.2 3.2 2.7 2.8 2.5 2.6 2.7 2.6 2.5 2.5 2.6 2.3 29 3.1 3.1 2.5 2.4 2.5 2.6 2.3 2.6 2.6 2.4 2.5 2.1 31 3.7 2.9 2.8 2.7 2.6 2.6 2.4 2.5 2.6 2.7 2.4 2.2 August 2 3.0 3.0 2.6 2.7 2.7 2.5 2.4 2.4 2.6 2.2 2.6 2.1 4 3.0 2.9 2.9 2.4 2.5 2.4 2.4 2.5 2.4 2.3 2.4 2.3 6 3.2 3.0 2.9 3.0 2.8 2.8 3.1 2.8 2.9 2.6 2.3 2.2 To determine the precise rate at which the tidal rhythm progressed across the 24-hour rhythm, and to demonstrate clearly the phase difference between the tidal rhythms of the crabs from the two sets of burrows, Figures 4 and 5 were prepared as follows. The data of Series A and B were used for Figure 4 and Series C and D for Figure 5. The 12 periods of observation were divided into four periods of three hours each, 8-10 A.M., 11 A.M.-! P.M., 2—4 P.M., and 5-7 P.M. The 12 hourly averages were summed and 12 subtracted from the total because if there had been no daily excursion of the pigment the sum would have been 12. Likewise, the average chromatophore indices for each three-hour period were summed and three subtracted from the total. The percentage of the total that each three-hour period occupied was then calculated. TIDAL RHYTHM OF UCA 13 3 4 3 4 t- Q_ O cr x u 3 " 3 4 3 4 h HIGH GROUP _OW GROUP 10 8 I I 12 10 14 12 15 13 16 14 17 15 8A.M. 12 M. 7PM. 8A.M. I2M. 7P.M. FIGURE 3. Daily pattern of melanophores of Uca pugilator from Series A (high group) and B (low group) on selected days. Analysis of the data in the manner described above revealed a two-day difference, relative to the time of day, in the phases of the tidal rhythms of specimens of Series A and B and Series C and D through July 26 as indicated by the horizontal bars in Figures 4 and 5. For any given hour of the day the groups closer to the low tide mark exhibited corresponding maxima and minima two days earlier than the crabs from burrows closer to the high tide mark. Analysis in the same manner of the data obtained with Uca pugilator during the summer of 1955 revealed regularly recurring 14.8-day cycles (Fingerman, 1956). 14 MILTON FINGERMAX o HIGH GROUP(A) • LOW GROUP(B) 3 13 AUGUST 23 FIGURE 4. TIDAL RHYTHM OF UCA 15 However, because of the absence of afternoon maxima through August 26 in the data collected in 1956, the pattern of 14.8-day cycles was different. Furthermore, the phase difference evident through July 26, 1956, disappeared and the four groups of crabs became rhythmically similar. Inspection of Figure 4 reveals that the first 14.8-day cycle (shown by the first two solid diagonal lines) consisted of two 7.4-day rhythms as shown by the broken diagonal line. The diagonals were drawn midway between the two-day difference of Series A and B and C and D. The 7.4-day rhythms were due to the absence of late afternoon peaks. Instead of the tidal rhythm progressing across noon and into the afternoon the peak shifted back to early morning, thus producing a 7.4-day rhythm rather than the typical 14.8-day rhythm. Only two 7.4-day cycles were evi- dent in Series A and B and were followed by a cycle of different frequency. A 7.4- TABLE III The average inelanophore index for each of the 12 daily observations of the Uca pugilator animals of Series C 8 A.M. 9 10 11 12 M. i P.M. 2 3 4 5 6 7 July 19 3.7 4.0 3.5 3.8 4.0 4.0 3.5 3.9 3.4 3.4 3.4 2.6 21 4.0 3.9 3.7 3.6 4.0 3.6 3.9 3.8 3.4 3.5 3.8 3.1 23 3.4 4.3 4.2 3.6 4.0 3.8 4.1 3.7 3.7 3.3 3.8 3.6 25 3.7 4.0 4.1 3.6 4.2 4.2 3.8 3.7 4.3 4.0 3.6 3.4 27 3.7 4.1 3.7 4.1 3.7 3.4 4.0 3.8 3.6 4.1 3.8 3.7 29 4.1 4.1 3.8 3.8 3.8 3.2 3.5 3.8 3.5 3.9 3.6 3.6 31 4.1 4.0 4.0 4.0 3.2 3.1 3.3 3.0 3.2 2.9 3.2 2.5 August 2 4.0 3.4 4.1 3.4 4.0 3.2 3.1 2.8 3.0 3.1 3.3 3.2 4 3.3 3.0 3.3 3.6 3.2 2.7 3.0 3.2 3.1 2.7 2.6 2.7 0 2.8 3.1 3.4 3.6 3.5 3.4 3.4 3.5 3.7 3.3 3.1 2.6 x 2.6 3.0 3.1 3.0 3.2 2.9 3.1 3.4 3.5 3.2 3.6 2.8 10 2.9 2.9 2.7 2.6 3.1 2.5 2.5 2.6 2.5 3.3 3.0 3.5 12 3.2 3.0 3.7 2.9 2.8 2.8 2.9 3.2 2.8 3.0 2.8 2.8 14 2.9 2.7 3.4 2.7 2.6 2.7 2.4 2.4 2.1 2.5 2.8 2.7 16 2.9 3.2 3.2 2.9 2.8 2.9 2.5 2.9 3.0 3.1 3.0 2.4 IS 3.0 3.0 3.4 3.1 2.8 2.7 2.7 3.1 3.0 2.9 3.1 3.1 20 2.8 3.1 3.1 3.0 3.2 3.1 3.2 3.2 3.4 3.3 3.0 2.9 22 3.0 2.9 3.0 3.0 3.2 3.3 3.3 3.4 3.6 3.4 3.3 3.5 day cycle was not evident in Series C and D because when they were collected Series A and B crabs were completing their second and last 7.4-day cycle. Furthermore, the two-day phase difference between the tidal rhythms of the crabs of Series C and D was not evident for longer than a week because of the unexpected loss of the phase difference that occurred in the four series of crabs about July 26. When the phase difference disappeared a rhythm appeared with a frequency that had not been observed previously. Instead of a typical 14.8-day cycle, an 11.5-day rhythm appeared (Figs. 4 and 5). As evident from inspection of Tables I, III, and IV, maxima occurred in the afternoon on August 8. From the latter date until the FIGURE 4. Relationship between percentage of daily melanin dispersion of Uca pnyilator collected July 3 that occurred at each of four periods during the day and the day of the month. Circles represent fiddler crabs from the burrows closer to the high tide mark (Series A). Dots represent fiddler crabs from the burrows closer to the low tide mark (Series B). 16 MILTON FINGERMAN o HIGH GROUP CG) • LOW GROUP CD; 3 13 AUGUST 23 TIDAL RHYTHM OF UCA 17 observations of Series A, C, and D ended on August 22, the daily patterns ex- hibited a typical tidal progression of the peak across the day as observed with Uca pugilator during the summer of 1955. A typical 14.8-day cycle followed the 11.5- day cycle concomitant with the gradual progression of the tidal maximum across the day. The relationship between the times of high and low tide (Fig. 2) and the maxi- mum of the chromatophore readings determined each day of observation could not be determined with accuracy, since at the start of the observations the maxima of the chromatophore readings did not progress across the day but were confined to the morning hours. On some days the maximum would occur later than the time of TABLE IV The average melanophore index for each, of the 12 daily observations of the Uca pugilator crabs of Series D 8 A.M. 9 10 11 12 M. i P.M. 2 3 4 5 6 7 Julv 19 3.1 3.0 3.7 3.4 3.5 3.3 3.5 3.4 3.0 3.0 2.6 1.8 21 3.2 3.7 3.6 3.8 3.2 3.5 3.4 3.3 3.3 3.3 2.8 2.4 23 2.3 3.0 3.7 3.7 3.3 3.1 3.2 3.2 3.1 3.1 2.7 2.6 25 2.7 3.5 3.8 3.8 3.3 3.5 3.6 3.4 3.7 3.1 3.1 2.9 27 2.1 3.2 2.9 3.1 3.1 3.0 3.2 3.0 3.2 3.2 3.3 3.1 29 2.2 3.4 3.5 3.3 3.3 3.1 3.0 3.1 3.0 3.1 3.2 2.9 31 2.9 3.5 3.6 3.8 3.4 3.3 3.2 3.1 3.3 2.8 3.1 2.7 August 2 2.5 3.3 3.4 3.2 3.3 3.1 3.0 2.7 2.5 2.7 2.6 2.8 4 2.4 2.8 3.1 3.2 3.1 3.0 3.1 2.8 2.9 2.8 2.8 2.2 6 2.0 2.7 3.3 3.4 3.2 3.3 3.4 3.1 2.9 3.3 2.9 2.5 8 1.6 3.0 2.8 2.8 3.0 2.8 3.0 3.1 2.9 3.1 3.2 3.2 10 2.0 2.0 3.1 3.1 3.2 3.2 2.9 3.0 3.1 3.4 3.5 3.1 12 2.4 3.0 3.5 3.3 3.4 3.4 3.3 3.4 3.4 3.3 3.2 3.1 14 2.7 3.1 3.4 3.1 3.1 3.1 3.3 2.9 3.0 3.1 2.9 2.7 16 2.9 2.9 2.6 3.1 2.9 2.9 2.8 2.9 3.0 2.7 2.9 2.6 18 3.1 2.7 3.1 2.9 2.5 2.6 2.7 2.8 3.0 2.9 2.8 2.9 20 2.8 2.8 3.0 2.9 2.8 3.2 2.9 2.9 3.0 3.1 3.1 3.0 22 2.9 2.9 2.8 2.9 2.6 2.8 2.9 2.9 2.8 3.0 2.9 2.8 the tide and on other days earlier since the tides occurred at all hours of the 24-hour day but the maxima were restricted to the morning hours. The relationship between the maximum of pigment dispersion and the time of tide from day to day must have been different at the end of the period of observa- tion than at the beginning because of the 11.5-day cycle that intervened between the two 14.8-day cycles shown in Figure 4. The tides progressed at the typical semi- lunar rate of 14.8 days throughout the period of observation so that the phase rela- tionship must have changed by 3.3 days, equivalent to 2.7 hours in a tidal cycle, in addition to the complication imposed by the appearance of typical cycles and maxima in the afternoon. FIGURE 5. Relationship between percentage of daily melanin dispersion of specimens of Uca pugilator collected July 18 that occurred at each of four periods during the day and the day of the month. Circles represent fiddler crabs from the burrows closer to the high tide mark (Series C). Dots represent fiddler crabs from the burrows closer to the low tide mark (Series D). 18 MILTON FINGERMAN The disappearance of the phase difference between the groups of crabs was in all probability due to some exogenous factor and not to removal of the crabs from direct contact with tides. If the latter explanation were correct, then crabs col- lected July 3 (Series A and B) would have lost their phase difference and taken on an 11.5-day cycle two weeks prior to the animals of Series C and D collected July 18. Since the crabs were not exposed in the laboratory to any overt stimuli capable of evoking the 7.4- and 11.5-day cycles observed during the summer of 1956, some exogenous factor might be considered as the mediating agent although natural rhythms have neither 7.4- nor 11.5-day cycles. Brown, Bennett and Ralph (1955) obtained evidence for a reversible effect of cosmic ray showers upon the responses of the chromatophore system of the fiddler crab Uca pugnax. The re- sponse of crabs exposed to increased intensity of cosmic ray showers, as compared with controls, was decreased pigment dispersion during the start of the day phase of the endogenous 24-hour cycle and increased dispersion during most of the remain- ing hours of the day. Brown and Stephens (1951), in an investigation of the influence of length of photoperiod upon the amplitude of the daily pigmentary excursion of Uca pugnax, found that the longer the daily photoperiod the greater was the amplitude of the daily pigmentary excursion after the crabs were placed in constant darkness. Crabs with their burrows close to the low tide mark experience a shorter daily photoperiod than do animals whose burrows are close to the high tide mark. When the water covers the burrows the crabs are in darkness and emerge when the burrows are uncovered by the receding water. Burrows close to the low tide mark are uncovered later than burrows near the high tide mark and are covered earlier each day. Therefore, crabs living in burrows close to the low tide mark experience a shorter photoperiod each day than do crabs in burrows near the high tide mark. The averages of the daily totals obtained for the first 33 days the crabs of Series A and B were in darkness were 40.96 and 37.59, respectively ; averages for the first 34 days the crabs of Series C and D were in darkness were 39.65 and 36.62, re- spectively. The values for the crabs closer to the low tide mark (Series B and D) were less than the values for the crabs from the burrows close to the high tide mark (Series A and C). An influence of change in day-length is also evident from inspection of the aver- ages of the amplitude values. The time from sunrise to sunset in New Orleans was nine minutes less on July 18, 1956, than on July 3, 1956. The amplitude values for groups of crabs from the same set of burrows likewise decreased. Analysis of the daily amplitudes obtained with Uca pugilator during the summer of 1955 (Fingerman, 1956) also reveal an effect of change in photoperiod upon the ampli- tude of the daily pigmentary dispersion. On June 15, 1955, the time from sunrise to sunset was seven minutes more than on June 1, 1955. The average amplitude for Uca pugilator from the same set of burrows for the first 34 days in the laboratory was 37.75 for those collected on June 1, 1955, and 39.22 for those collected June 15, 1955. In this instance the day-length had increased between the first and second collection and the amplitude was consequently greater. GENERAL DISCUSSION The results presented above support the conclusions of Fingerman (1956) concerning the tidal rhythms of color change of the fiddler crab Uca pugilator, as TIDAL RHYTHM OF UCA 19 well as provide new information about the nature of the rhythm. The time the bur- rows are uncovered at a particular level of the beach, and not the time of actual low tide on the beach, appears to be the primary determinant of the phases of the tidal rhythm relative to the 24-hour rhythm of color change. Chromatophore rhythms with frequencies other than 12.4 and 24.0 hours and 14.8 days have not been observed previously. In the present investigation 7.4- and 11.5-day rhythms were described. The latter two, however, did not persist for more than one or two cycles. Evidently, the center or centers of rhythmicity of the fiddler crab can be set at one of several frequencies, some persistent and others not. Brown, Webb and Bennett (1955) have shown that Uca pugnax has the en- dogenous ability to mark off periods of solar and lunar day lengths in the absence of all possible rhythmic external signals. The persistent frequencies because of their correlation with frequencies of environmental events are probably of adaptive significance. On the other hand, no cosmic phenomenon has a 7.4- or 11.5-day frequency nor do fiddler crabs carry on activities correlated with these frequencies. The non-persistent rhythms of 7.4 and 11.5 days may have been imposed upon the crabs by exogenous factors and when these factors were no longer able to express themselves the crabs returned to the usual rhythm of 14.8-day frequency. SUMMARY AND CONCLUSIONS 1. The tidal and semilunar rhythms of color change of the fiddler crab Uca pugilator have been subjected to further analysis. 2. The phases of these rhythms appear to be set according to the time the bur- rows of the crabs begin to be uncovered by the receding water following a high tide and not the time of actual low tide on the beach. 3. The amplitude of the daily pigmentary dispersion is also influenced by the time when the burrows are covered and uncovered. Crabs in burrows close to the high tide mark experience a longer daily photoperiod and consequently exhibit a greater daily amplitude of pigment dispersion. 4. A two-day phase difference, equivalent to 1.6 hours in a tidal cycle, was found between two groups of crabs collected from two discrete sets of burrows different distances from the high tide mark. The tidal maximum of pigment dispersion at any given hour of the day occurred two days earlier in the crabs collected closer to the low tide mark. 5. Measurements of the beach where the crabs were collected revealed that the receding water begins to uncover the burrows close to the high tide mark 1.6 hours earlier than the burrows closer to the low tide mark begin to be uncovered, the same difference observed in the laboratory. 6. The daily patterns of pigment dispersion were different during the first two weeks of observation from the patterns observed in previous investigations. Max- ima of pigment dispersion did not occur in the late afternoon during the first three weeks of observation but were restricted to the morning hours. The absence of peaks in the afternoon resulted in cycles with a 7.4-day frequency. 7. An 11.5-day cycle also appeared and was followed by a typical 14.8-day cycle accompanied by maxima of the daily curves in the late afternoon. 8. The results are discussed in terms of possible exogenous causes of the 7.4- and 1 1.5-day cycles. 20 MILTON FINGERMAN LITERATURE CITED BROWN, F. A., JR., M. F. BENNETT AND C. L. RALPH, 1955. Apparent reversible influence of cosmic-ray-induced showers upon a biological system. Proc. Soc. Exper. Biol. and Med., 89 : 332-337. BROWN, F. A., JR., M. FINGERMAN, M. I. SANDEEN AND H. M. WEBB, 1953. Persistent diurnal and tidal rhythms of color change in the fiddler crab, Uca pugnax. J. Exp. Zoo!., 123: 29-60. BROWN, F. A., JR., AND G. C. STEPHENS, 1951. Studies of the daily rhythmicity of the fiddler crab, Uca. Modifications by photoperiod. Biol. Bull., 101 : 71-83. BROWN, F. A., JR., H. M. WEBB AND M. F. BENNETT, 1955. Proof for an endogenous com- ponent in persistent solar and lunar rhythmicity in organisms. Proc. Nat. Acad. Sci., 41 : 93-100. FINGERMAN, M., 1955. Persistent daily and tidal rhythms of color change in Callinectcs sapidus. Biol, Bull., 109 : 255-264. FINGERMAN, M., 1956. Phase difference in the tidal rhythms of color change of two species of fiddler crab. Biol. Bull., 110: 274-290. FINGERMAN, M., 1957. Lunar rhythmicity in marine organisms. Amer. Nat., in press. HOGBEN, L. T., AND D. Su)ME, 1931. The pigmentary effector system. VI. The dual character of endocrine coordination in amphibian colour change. Proc. Roy. Soc. Land., Ser. B., 108: 10-53. EVIDENCE FROM SEA URCHIN-SAND DOLLAR HYBRID EMBRYOS FOR A NUCLEAR CONTROL OF ALKALINE PHOSPHATASE ACTIVITY REED A. FLICKINGER 1 Dept. of Zoology, University of California, Los Angeles, California and Friday Harbor Laboratories, University of Washington, Friday Harbor, Washington Several investigators (Hultin, 1948a, 1948b; Bohus Jensen, 1953; Tyler and Metz, 1955) have shown that hybridization in echinoids can be facilitated by treat- ment of the eggs with trypsin. A recent review by Moore (1949) has covered the problem of inheritance in hybrid plutei of echinoids. It seemed of interest to deter- mine the activity of two enzymes (acid and alkaline phosphatase) in the developing embryos of two separate species and of the hybrids. In the hybrids it was hoped that it would be possible to assess the relative role of the nuclei and the cytoplasm in directing the synthesis and activity of these two enzymes. Alkaline phosphatase activity rises rapidly from gastrulation on to the pluteus stage ( Mazia ct al., 1948; Gustafson and Hasselberg, 1950), while acid phosphatase maintains a constant level of activity during sea urchin development according to Gustafson and Hasselberg (1951). However, the latter authors have noted a slight but definite rise in acid phosphatase activity in the sea urchin Paracentrotus lividus. Paternal antigens arising by the late blastula stage in sea urchin hybrids have been demonstrated serologically by Harding, Harding and Perlmann (1954), but little information exists dealing with quantitative chemical differences in hybrids. Brachet (1954) has expressed the belief that the nucleus exerts a very important control over enzymes of the microsomes, as evidenced from work upon nucleated and enucleated halves of amoebae. Utilizing the Gomori-Takamatsu cytochemical technique Krugelis (T947b) has noted that nuclear alkaline phosphatase activity increases from gastrulation to the pluteus stage while that of the cytoplasm declines. In the unfertilized egg the weak staining reaction for the enzyme is primarily located in the cytoplasm (Krugelis, 1947a) and this is borne out by Mazia ct al. (1948) who utilized biochemical meth- ods and found equal activity in nucleated and enucleated halves of unfertilized eggs separated by the Harvey method. The Gomori-Takamatsu technique has been criticized by Novikoff (1951) and Johansen and Linderstrom-Lang (1952) ; these authors believe that diffusion obscures the accurate localization of the calcium phos- phate precipitate and that nuclei may adsorb the precipitate. Novikoff ct al. (1950) isolated rat liver nuclei and found less alkaline phosphatase activity in that fraction as compared to the cytoplasm, and Stern ct al. (1952), utilizing a non-aqueous me- dium for isolating nuclei (Behren's technique), obtained similar results for nuclei isolated from the thymus gland. However, Dounce (1943) found a higher con- 1 Lalor Foundation Summer Research Fellow, 1956. 21 22 REED A. FLICKINGER centration of alkaline phosphatase in the isolated nuclei from rat liver as compared to the whole tissue. Danielli (1953) expresses the belief that the Gomori-Taka- matsu cytochemical technique can successfully localize alkaline phosphatase in the cell and this would mean that a number of workers who cite a nuclear localization of alkaline phosphatase (Danielli. 1946; Krugelis, 1947b; Brachet and Jeener, 1948; and Bradfield, 1950) are correct. The question of localization of this enzyme is an open one and it was hoped the method of hybridization might help to clarify it. It was originally planned to hybridize reciprocally Strongylocentrotus purpuratus and Strongylocentrotus jranciscanus (see Moore, 1943, for a discussion of maternal and paternal inheritance in this cross), but it was found that there was essentially no difference in acid and alkaline phosphatase activity between the two species. Also it is known that when S. jranciscanus eggs are fertilized by S. purpuratus sperm, development is blocked at the late blastula stage (Moore, 1943). Hybridi- zation of 5*. purpuratus and Dendraster excentricus is advantageous in that these genera are more distantly related ; they differ markedly in their speed of develop- ment, and the cross can be made reciprocally. MATERIALS AND METHODS Eggs and sperm of Dendraster and 6". purpuratus were obtained by injection of 0.5 M KC1 (Tyler, 1949) into the body cavity. The eggs of Dendraster could be fertilized by the usual dilute suspension of 6". purpuratus sperm used in the homolo- gous crosses. In order successfully to fertilize S. purpuratus eggs with Dendraster sperm (Tyler and Metz, 1955), the eggs were placed in a 0.05% trypsin (crystal- line-lyophilized preparation from Worthington Biochemical Sales Co.) solution for ten minutes and the eggs were then washed several times with sea water to remove the trypsin. They were then fertilized with an amount of \% sperm (1 drop of dry sperm/5 cc. sea water) which was in forty-fold excess of the volume of sea water containing the eggs. After the eggs were left in this concentrated sperm solution for 30-40 minutes (with frequent agitation of the solution), the eggs were then washed four or five times to remove the excess sperm and cultured in a slowly ro- tating four-liter flask which floated in a tank of running sea water. The tempera- ture of this running sea water was usually about 10° C. With this procedure about 40% of the eggs were successfully fertilized and hatched swimming blastulae could be separated from the unfertilized eggs. On the first, second, third, and fourth days after fertilization embryos were col- lected by centrifugation and were washed several times with the appropriate buffer. These washes were carried out rapidly so as to prevent any loss of the enzyme. For the acid phosphatase assays this was a 0.1 M sodium acetate-acetic acid buffer of pH 5.32 ; for the alkaline phosphatase assays a 0.1 M sodium veronal-HCl buffer (0.0015 M MgCL) of pH 9.0 was utilized. For a given assay the embryos were suspended in 4 cc. of the acid or alkaline buffer and disintegrated in an all-glass homogenizer which was kept immersed in an ice bath during homogenization. Then a two-cc. aliquot of the brei was added to two cc. of the substrate (0.1 M so- dium ft glycerophosphate) ; for the acid phosphatase assays the pH of the substrate solution was adjusted to 6.0. One of the mixtures of brei plus substrate was im- mediately inactivated by the addition of 0.8 cc. of 60% trichloracetic acid while the other was allowed to incubate at 25° C. for a two-hour period at which time a simi- NUCLEAR CONTROL OF PHOSPHATASE 23 lar amount of trichloracetic acid was added. The control and experimental samples were then centrifuged at approximately 10,000 G and the trichloracetic acid super- nates were collected. Phosphorous assays were made upon these samples utilizing the Fiske-Subbarow technique (1925) and the control values subtracted from the experimental ones. The trichloracetic acid precipitates were each suspended in one cc. of water and total nitrogen determinations (Umbreit et aL, 1948) were made upon aliquots from these samples. Acid or alkaline phosphatase activity is stated as the ratio of total micrograms of phosphate released in a two-hour period divided by the total micrograms of trichloracetic acid-insoluble nitrogen. Acid-insoluble nitrogen is a good standard for these measurements since it remains fairly constant during early development. RESULTS Preliminary assays of acid phosphatase in developing embryos of Strongylocen- trotus purpuratus and S. jranciscanus showed the activity of this enzyme to be es- sentially similar in the two species, and preliminary determinations of acid phospha- tase in Dendraster gave values within this same range. Since acid phosphatase could not be used quantitatively to distinguish S. purpuratus from Dendraster, this enzyme was not assayed in the hybrids nor were further determinations made in the homologous species. The series of determinations for any given batch of eggs seemed to indicate a slight increase in activity of acid phosphatase during develop- ment, but the average values only substantiated an increased activity from the bias- tula to the gastrula stage. The activity of alkaline phosphatase was quite different in Dendraster and 5". purpuratus, rising quite sharply for the sand dollar embryos and much more slowly for the sea urchin embryos (Fig. 1). The points in Figure 1 represent two series of experiments, all of which were conducted under similar conditions and gave excellent reproducibility. It was of interest to see if the activity of this enzyme in the hybrids was characteristic of the maternal or the paternal species, or if it might be intermediate between the two. If the latter alternative held true, this would be a quantative indication of a nuclear control of alkaline phosphatase ac- tivity, whereas a maternal rate of activity would indicate a cytoplasmic independence of this enzyme. It was not expected that a paternal activity rate would be found. Hybridization of Dendraster eggs by S\ purpuratus sperm results in normal de- velopment up through gastrulation but in the experiments reported here the em- bryos then became abnormal and gave rise to so-called spherical plutei. These "plutei" did not swim actively and died within a few days. It is believed that the low alkaline phosphatase activities which were found here are a reflection of the poor condition of the embryos. Even if these embryos had developed normally, this would not have been the best cross to ascertain nuclear or cytoplasmic de- pendence of the enzyme since the maternal species (Dendraster) normally has a high alkaline phosphatase activity by the pluteus stage. An intermediate value in this case would be a decrease from the normal activity of the maternal species and might be ascribed to lowered vitality of the hybrids. However, in the S. purpuratus 5 X Dendraster <$ hybrids an intermediate value would be an increase from the nor- mal activity of the maternal species and could clearly be ascribed to the effect of the paternal nuclear material during development. It was fortunate that these hybrids 24 REED A. FLICKINGER developed perfectly normally. Some were kept as long as 12 days after fertilization and they were still perfectly healthy plutei at that time. The S. purpuratus 5 X Dendraster <$ hybrids were strictly maternal in terms of rate of development both before gastrulation and up to the pluteus stage ; Den- .20 .15 y PHOSPHORUS *ACID INSOLUBLE NITROGEN .10 .05 O DAYS O I FIGURE 1. Alkaline phosphatase activity of Strongylocentrotus purpuratus, Dendraster excentricus and Strongylocentrotus purpuratus $ X Dendraster excentricus <$ hybrids. Expressed as the ratio of micrograms phosphorus released in a two-hour period per microgram of acid- insoluble nitrogen. Solid dots indicate determinations upon Dendraster, open circles are for S. purpuratus and X indicates determinations upon the hybrids. draster reaches the pluteus stage three days after fertilization whereas S. purpuratus takes four days. The day after fertilization (first day) the hybrids were early blastulae, the next day (second) they were hatched swimming blastulae or early gastrulae, the third day they had reached the prism stage and on the fourth day after fertilization they were plutei. In terms of morphological appearance the hybrid NUCLEAR CONTROL OF PHOSPHATASE 25 plutei look very much like those of S. purpuratus. They lack the extended oral and anal arms of the Dendraster plutei and the skeletal rods are located as they are in S. purpuratus plutei. Alkaline phosphatase determinations in the S. purpuratus $ X Dendraster <$ hybrids were routinely made on the third day after fertilization when the hybrids had reached the prism stage. Assays were made at this time (third day) since no estimations were made upon Dendraster plutei after this period, and also by this third day there was a marked disparity between the alkaline phosphatase activity of the two homologous species which is not true at the earlier stages when alkaline phosphatase activity of the two species is more similar. The enzyme estimations re-))) vealed values intermediate to those of either homologous species (Fig. 1) at a similar time after fertilization. These hybrid prism activities were not only greater than those of 6". purpuratus prisms but also distinctly higher than the values obtained for four-day S. purpuratus plutei. This indicates that alkaline phosphatase activity is not merely reaching an appropriate level for a given stage of development, as is the case with so many metabolic activities. These data are interpreted as an eleva- tion of alkaline phosphatase activity due to the presence of Dendraster nuclear ma- terial in the developing embryos. It is not known if this nuclear stimulation of alkaline phosphatase indicates a nuclear localization of the enzyme, but it certainly seems to demonstrate a quantitative nuclear control for this enzyme with the inter- mediate value resulting from the action of both maternal and paternal nuclear elements. Several attempts to induce higher acid and alkaline phosphatase activities in S. purpuratus embryos were carried out by adding sodium /3 glycerophosphate (final concentration 0.1 M) to several of the cultures at the mesenchyme blastula stage. The blastulae were allowed to develop for two days in the sea water contain- ing the substrate and then acid and alkaline phosphatase assays were made at the pluteus stage. However, in two sets of experiments involving such cultures and their controls (where no substrate was added) no increase in activity was noted. Usually the activity of the controls was slightly higher than that of the cultures to which glycerophosphate had been added. In order to determine if inadequate homogenization might account for the low alkaline phosphatase values, 6". purpuratus embryos were frozen and thawed several times before homogenization so as to facilitate rupture of the cells. This procedure had no effect upon alkaline phosphatase activity since similar values were obtained for fresh material, once frozen, and twice frozen samples. Microscopic examinations of the homogenates were made routinely in all experiments. In all cases no large clumps of cells were observed, but undoubtedly not all cells were broken by the homogenization. This does not affect the interpretation of the results since the em- bryos of 6". purpuratus, Dendraster and the hybrids were disintegrated in the same manner, and an equal degree of homogenization apparently was obtained in each case. It might also be expected that the hybrids would yield homogenates much like those of S. purpuratus since the only Dendraster component of the hybrids is nuclear and hence they are probably of equal fragility. The author wishes to express his gratitude to the staff of the Friday Harbor Laboratories for their many kindnesses during the summer. 26 REED A. FLICKINGER SUMMARY 1. Alkaline phosphatase estimations upon sea urchin embryos (S. purpuratus) showed a slow rate of increasing activity up to the pluteus stage while sand dollar embryos (Dcndr aster excentricus) showed a very rapid increase up to this same stage. 2. Hybridization between Strongylocentrotus purpuratus 5 and Dendraster ex- centricus <$ resulted in alkaline phosphatase activity at the prism stage which was intermediate between that of the two homologous species and higher than that of the maternal species. This is interpreted as indicating a nuclear control of alkaline phosphatase activity. 3. Addition of substrate (0.1 M sodium (3 glycerophosphate) to cultures of de- veloping S. purpuratus embryos did not affect an increase in acid or alkaline phos- phatase activity. LITERATURE CITED Bonus JENSEN, A., 1953. The effect of trypsin on the cross fertilizability of sea urchin eggs. Exp. Cell Res., 5 : 325-328. BRACKET, J., 1954. Nuclear control of enzymatic activities. Reprinted from Vol. VII of the Colston Papers, 91-102. BRACKET, J., AND R. JEENER, 1948. Recherches sur le role de la phosphatase alcaline des noyaux. Biochcm. ct Biophys. Acta, 2: 423-430. BRADFIELD, J. R. G., 1950. The localization of enzymes in cells. Biol. Rev., 25: 113-157. DANIELLI, J., 1946. A critical study of techniques for determining the cytological position of alkaline phosphatase. /. Exp. Biol., 22: 110-117. DANIELLI, J., 1953. Cytochemistry. John Wiley and Sons, Inc., New York. DOUNCE, A. L., 1943. Enzyme studies on isolated cell nuclei of rat liver. /. Biol. Chem., 147 : 685-698. FISKE, C. M., AND Y. SUBBAROW, 1925. The colorimetric determination of phosphorous. /. Biol. Chem., 66: 375-400. GUSTAFSON, T., AND I. HASSELBERG, 1950. Alkaline phosphatase activity in developing sea urchin eggs. Exp. Cell Res., 1 : 371- 375. GUSTAFSON, T., AND I. HASSELBERG, 1951. Studies on enzymes in the developing sea urchin egg. Exp. Cell Res., 2 : 642-672. HARDING, C. V., D. HARDING AND P. PERLMANN, 1954. Antigens in sea urchin hybrid embryos. Exp. Cell Res., 6: 202-210. HUT.TIN, T., 1948a. Species specificity in fertilization reaction. I. The role of the vitelline membrane of sea-urchin eggs in species specificity. Arkiv. Zool., 40A : No. 12, 1-9. HULTIN, T., 1948b. Species specificity in fertilization reaction. II. Influence of certain factors on the cross-fertilization capacity of Arbacia lixula. Arkiv. Zool., 40A : No. 20, 1-8. JOHANSEN, G., AND K. LiNDERSTROM-LANG, 1952. Liberation, diffusion, and precipitation of phosphate in the Gomori test. Acta Med. Scand., Suppl, 266: 601-613. KRUGELIS, E. J., 1947a. Alkaline phosphatase activity in oocytes of various marine invertebrates. Biol. Bull., 93: 209. KRUGELIS, E. J., 1947b. Alkaline phosphatase activity in developmental stages of Arbacia. Biol. Bull., 93: 209. MAZIA, D., G. BLUMENTHAL AND E. BENSON, 1948. The activity and distribution of desoxyribo- nuclease and phosphatases in the early development of Arbacia punctulata. Biol. Bull., 95: 250. MOORE, A. R., 1943. Maternal and paternal inheritance in the plutei of hybrids of the sea urchins Strongylocentrotus purpuratus and Strongylocentrotus franciscanus. J. Exp. Zool., 94: 211-228. MOORE, A. R., 1949. On the problem of inheritance in the hybrid plutei of echinoids— a per- spective. Portug. Acta Biol., (A), 258-272. NOVIKOFF, A. B., 1951. The validity of histochemical phosphatase methods on the intracellular level. Science, 113: 320-325. NUCLEAR CONTROL OF PHOSPHATASE 27 NOVIKOFF, A. B., E. PODBEK AND J. RVAX, 1950. Intracellular distribution of phosphatase ac- tivity in rat liver. Fed. Proc., 9: 210. STERN, H., V. ALLFREY, A. E. MIRSKY AND H. SAETREN, 1952. Some enzymes of isolated nuclei. /. Gen. Physiol., 35: 559-578. TYLER, A., 1949. A simple, non-injurious method for inducing repeated spawning of sea urchins and sand-dollars. Coll. Net, 19 : 19-20. TYLER, A., AND C. B. METZ, 1955. Effects of fertilizin-treatment of sperm and trypsin-treatment of eggs on homologous and cross-fertilization in sea urchins. Pubbl. Staz. Zool. Napoli, 27 : 128-145. UMBREIT, W. W., R. H. BURRIS AND J. F. STAUFFER, 1948. Manometric techniques and related methods for the study of tissue metabolism. Burgess Publishing Co., Minneapolis, Minnesota. OBSERVATIONS ON OSMOREGULATION IN THE ARCTIC CHAR (SALVELINUS ALPINUS L.)1 MALCOLM S. GORDON 2 0 shorn Zoological Laboratory, Yale University and Woods Hole Oceanographic Institution, Woods Hole, Mass. The different groups of euryhaline fishes have developed somewhat different mechanisms for maintaining the relative constancy of the concentration of their "milieu interieur" in the face of large changes in the external osmotic pressure. Following such an external change, many change their internal concentrations only transitorily, in the same direction as the external variation. A return to essentially the original conditions usually follows shortly (in adult female eels (Anguilla) : Boucher-Firly, 1935; Duval, 1925; in sticklebacks (Gasterosteus} ; Gueylard, 1924; Koch and Heuts, 1943; in killifish (Fundulus) : Burden, 1956). Anadromous salmonid fishes, however, have long been known to regulate their blood concentra- tions on two distinct levels (probably the end-points of an acclimation curve). In almost all such salmonids studied so far, the transition from salt to fresh water (or the reverse) is accompanied by a fall (or rise) in total blood concentration of about 25% (the Atlantic salmon, Salnw salar, and the Chinook salmon, Oncorhynchus tshawytscha, however, change by only 12% (Fontaine and Koch, 1950; Greene, 1926)). Plasma freezing point depressions vary from species to species, being 0.67-0.90° C. in salt water, 0.55-0.72° C. in fresh water (Benditt et al, 1941 ; Fontaine, 1943, 1948; Fontaine, Callamand and Vibert, 1950; Fontaine and Koch, 1950; Greene, 1904; Kubo, 1953). The Arctic char (Salvelinus alpinits L.) is an anadromous salmonid fish com- mon in fresh and coastal salt waters throughout most of the Arctic. As in other sea-going char, its migrations from fresh to salt water and back again are somewhat different from those of other salmonids in that its winter periods in fresh water lakes and rivers are long compared to its summer periods in the ocean, rather than vice versa (Andrews and Lear, in press; Backus, 1952; Sprules, 1953). Osmo- regulation in this form has not been studied, probably due to its inaccessibility. The present paper describes some observations made on the osmoregulatory abilities of adult Arctic char on their return (spawning) migration to fresh water after a .summer in the ocean. 1 These studies were aided by contracts between the Office of Naval Research, Department of the Navy, and the Arctic Institute of North America. Reproduction of this paper in whole or in part is permitted for any purpose of the United States Government. The author's ac- tivities were also supported in part by National Science Foundation Predoctoral Fellowships. Contribution Number 868 from the Woods Hole Oceanographic Institution. 2 The author's thanks for aid and cooperation are due the following people : In Labrador (1954 "Blue Dolphin" Expedition), Cmdr. D. C. Nutt, Drs. P. F. Scholander and L. van Dam, and P. Hay. At Churchill, Manitoba, Mr. P. Bussadore, Mr. and Mrs. A. Maclver, Mr. and Mrs. I. Smith, and Dr. D. E. Sergeant. Mr. C. L. Claff kindly permitted the use of his flame photometer. Dr. van Dam performed the plasma chloride determinations at Hebron. 28 \ OSMOREGULATION IN ARCTIC CHAR 29 The material is of a preliminary nature in many ways, but serves to show that the Arctic char is similar to other salmonid fishes in regulating its blood concentration on two levels. Agreement between experimental results and data obtained from fish living in fresh and salt waters provides a basis for further experimental study of osmotic phenomena during the migrations of these fish. Some data on regulation of muscle concentrations are also presented. Differences in results obtained from char in northern Labrador and Hudson Bay indicate the possibility of physiologi- cally different populations in this species. MATERIALS AND METHODS Regulation of total plasma concentration, chloride and potassium, and of total muscle solids, chloride, and potassium was studied in mature adult char of both sexes by means of observations on fish living in salt or fresh water, and by time series of observations immediately following direct transfers of fish from salt to fresh water. Conditions of temperature, stage in life cycle, etc., were kept fairly constant in these experiments. Char were taken by means of gill nets from Hebron Fjord, Labrador, and Hudson Bay near Churchill, Manitoba, during late July and early August of 1954 and 1955, respectively. Seven char were used to establish the normal salt water ranges for plasma freez- ing point and chloride concentration in fish in Hebron Fjord (water of 28.6 %o sa- linity, 9° C. temperature). Five Churchill char were used similarly, observations on these consisting of plasma chloride and potassium concentrations, and total muscle solids, chloride, and potassium concentrations (Hudson Bay water was of approxi- mately 26 %o salinity, 10-12° C. temperature). Fresh water ranges for plasma freezing point and chloride concentration were determined at Hebron in several small land-locked char from a small lake, in some pre-sea-run parr from a river, and in an adult char that had returned to fresh water by itself. No fresh water char were obtainable at Churchill. The osmotic stresses these fish undergo during the course of their migrations from the sea were approximated by transferring four Hebron fish and seven Church- ill fish directly from salt to fresh water. Temperatures at Hebron were : salt water, 9°, fresh water, 5-10°; at Churchill: salt water, 10-12°, fresh water 14-16°. Changes in plasma freezing point with time were followed in three of the Hebron fish via serial blood samples taken at intervals up to 77 hours following transfer. The fourth fish was sampled initially and after 77% hours. Two of the Hebron char were then returned to the Fjord and sampled again following death (after some fifteen hours in salt water ) . Only two of the Churchill char survived for more than one hour after transfer from salt to fresh water — these for two and six hours, respectively. Blood and muscle concentrations were determined in these, after the periods mentioned, as in the salt water fish. The approximately 5° C. thermal shock to which these fish were subjected, combined with a lack of running fresh water, hence a need for aeration by hand dipping, probably explains in great part the lowered survival as compared with the Hebron fish. The speed with which death followed transfer makes it seem unlikely that this is the complete explanation, however. Blood samples were taken via heart puncture from all fish. The samples were heparinized, centrifuged, and the plasma pipetted off. Plasma freezing points 30 MALCOLM S. GORDON were determined at Hebron using the method of Pounder and Masson (1934) modified for field use (Scholander et «/., in press). Plasma chloride determinations were made using the method of Schnohr (1934). Plasma potassium concentration was determined on the Churchill samples following dilution with Pyrex-distilled \vater on a Baird Associates internal standard flame photometer. Probably due to coagulation of the proteins in these samples, which may have interfered with acti- vation of the ions by the flame, only three of these analyses gave what appear to be reasonable values. Four samples of tissue from the dorsal muscle mass, averaging about 100 mg. wet weight, were also taken from each of the Churchill fish. Total solids were de- termined by weighing these before and after complete drying in an oven at 105° C. 1.00- o ' o o o e oe o e o 0 10 20 30 40 50 60 70 80 TIME AFTER TRANSFER ( HOURS) — FIGURE 1. Changes of plasma freezing point depression with time following direct transfer from salt to fresh water of Arctic char (Salvelinus alpinus L.) from Hebron Fjord, Labrador. Ranges for fish naturally acclimated to salt water (black bar) and fresh water (black and white bar) indicated on ordinate. Temperature 5-10° C. Following digestion with concentrated nitric acid and 30% hydrogen peroxide, du- plicate chloride and potassium analyses were carried out. Methods were as above. Precision of the freezing point determinations is ± 0.05° C. Plasma chloride analyses agreed within 1%, as did total muscle solids. Muscle chloride and potas- sium duplicates agreed within 5%. RESULTS AND DISCUSSION Figure 1 shows the behavior of total blood concentration in control and ex- perimental char from Hebron Fjord. The total plasma concentration in Hebron char living in fresh water is about 25% lower than the salt water concentration. The char is thus like most other anadromous salmonids in this regard. Beginning \ OSMOREGULATION IN ARCTIC CHAR 31 two hours after transfer from salt to fresh water, the experimental fish also de- creased their plasma concentrations by about 25% over a period of approximately twenty hours. They then became fairly stable. It therefore seems that sudden transfer experiments duplicate at least some of the physiological events occurring during the migrations of these fish. This last might have been expected, since individual salmonids frequently make the transition from salt to fresh water and back again as rapidly as they can swim through the estuaries involved (Benditt et a! 1941; Greene, 1910; Killick, 1955). The continuing decrease in plasma concentration below the normal fresh water level which occurred in the experimental fish after about 35 hours in fresh water could have been the result of several influences. First, the fish were not fed ; sec- ond, they may well have lost salt via their urine as a result of having been handled TABLE I Blood concentrations in arctic char Source of fish Plasma AF (°C.) Plasma [Cl~] (meq./l.) Equivalent Cl AF (°C.)* Plasma [K+] (meq./l.) Hebron Fjord, salt water J.80 0.80 148 191 0.28 0.36 0.81 191 0.36 0.82 213 0.40 0.82 214 0.40 0.83 0.76 Hebron Fjord, fresh water (land-locked) 0.59 0.61 0.63 144 144 161 0.27 0.27 0.30 Churchill, salt water 177 153 0.33 0.28 5 8 156 0.29 1 130 0.24 1 132 0.25 2 Churchill 2-hr, transfer 134 0.25 1 6-hr, transfer 124 0.23 8 * Equivalent Cl~Ap calculated from: AF = 1.86 [Cl ] (laboratory diuresis of Grafflin, 1931, 1935, and Forster, 1953) ; and third, their skin permeability, hence rate of water uptake in a hypotonic medium, may well have been increased as a result of loss of slime during handling. The two Hebron char transferred back to the Fjord after the experiment, after fifteen hours in salt water, had plasma freezing points of - 0.75° C. This is es- sentially the original salt water value. Table I summarizes the data on blood concentrations obtained from both Hebron and Churchill control fish and Churchill transfers. With the exception of the first salt water char from Hebron, the calculated equivalent Cl freezing point is essen- tially a constant fraction of the total freezing point (45-50%). More exact regu- lation of the concentrations of other plasma components is thus indicated. Similar behavior of chloride and total concentrations has been noted in brook and brown trout (unpublished data of the writer and van Dam). Fontaine, Callamand and 32 MALCOLM S. GORDON Vibert ( 1950), however, found a decrease of only 4-% in plasma chloride in Atlantic salmon (Salmo salar} when total concentration dropped 13%. The Churchill material generally supports the Hebron results. Plasma chloride concentration in the Churchill fish in fresh water for six hours is approximately 18% lower than the mean plasma chloride concentration for Churchill char in salt water. Plasma freezing point behaves similarly in the Hebron char. Note, how- ever, that plasma chloride concentrations in the Churchill fish are generally much lower than in the Hebron fish. The mean difference of 20% seems too large to be the result of acclimation to differing salinities (the salinities differing only by 10%.). The marked differences between Hebron and Churchill char with respect to sur- vival following transfer were noted earlier. Even allowing for the poor conditions encountered at Churchill it seems likely that real physiological differences exist between these populations. Marked differences in growth characteristics differ- entiating these two groups (Andrews and Lear, in press; Backus, 1952; Sprules, 1953; unpublished data of the author) also make this seem likely (though differ- ences in food supply might well account for this last). TABLE II Muscle concentrations in Churchill arctic char Source of fish Total muscle solids (gm./kg. wet weight) Muscle [C1-] (meq./kg. wet weight) Muscle [K+] (meq./kg. wet weight) Salt water 276 22 123 224 20 130 266 8 120 226 8 120 260 6 125 2-hr, transfer 242 4 127 6-hr, transfer 244 3 150 Table II indicates that the one Churchill fish surviving transfer for six hours regulated total muscle concentration very well. Muscle potassium, however, seem- ingly increased markedly. The concentrations of the same muscle components in brook and brown trout under similar conditions behave very differently, however (data of the author and van Dam) . In these other forms there is a close parallelism between changes in muscle concentrations and changes in the blood. Further work on the char is obviously needed. In closing it should be noted that the blood and muscle potassium concentrations reported by Jones (1956) for fresh water brown trout agree very well with the figures given in Tables I and II for the Churchill salt water char (low plasma potas- siums excepted). SUMMARY 1. Adult Arctic char (Salvelinus alpinus}, taken in summer from Hebron Fjord, Labrador, and Hudson Bay near Churchill, Manitoba, were transferred directly from salt to fresh water under fairly constant conditions. 2. Decreases in blood freezing point and chloride concentration of the order of 25% were found, the char thus being like most other anadromous salmonids in this OSMOREGULATION IN ARCTIC CHAR respect. The possibility of much better regulation of muscle concentrations is indicated. 3. Data are presented on plasma freezing point, chloride, and potassium, muscle solids, chloride, and potassium. 4. Physiological differences between populations of char are indicated. LITERATURE CITED ANDREWS, C. W., AND E. LEAR. The biology of Arctic Char (Sah'clinns alpinus L.) in north- ern Labrador. /. Fish. Res. Bd. Canada (in press). BACKUS, R. H., 1952. Growth in the Arctic Char (Salvdiniis alpinus L.) in the Nain region of Labrador. Unpublished thesis, Cornell University. BENDITT, E., P. MORRISON AND L. IRVING, 1941. The blood of the Atlantic salmon during mi- gration. Biol. Bull, 80 : 429-440. BouCHER-FiRLY, S., 1935. Recherches biochimiques sur les teleosteens apodes (Anguille, Congre, Murene). Ann. Inst. Oceanogr. (Monaco}, 15: 217-327. BURDEN, C. E., 1956. The failure of hypophysectomized Fundulus hetcroclitus to survive in fresh water. Biol. Bull, 110: 8-28. DUVAL, M., 1925. Recherches sur le milieu interieur des animaux aquatiques. Modifications sous 1'influence du milieu exterieur. Ann. Inst. Oceanogr. (Monaco}, 2: 233-407. FONTAINE, M., 1943. Des facteurs physiologiques determinant les migrations reproductrices des Cyclostomes et Poissons potamotoques. Bull. Inst. Oceanogr. (Monaco}, No. 848: 1-8. FONTAINE, M., 1948. Physiologic du saumon. Ann. Sta. Cent. Hydrobiol. Appliq., 2: 153-183. FONTAINE, M., O. CALLAMAND AND R. VIBERT, 1950. La physiologic du saumon. Ann. Sta. Cent. Hydrobiol. Appliq., 3 : 15-26. FONTAINE, M., AND H. J. KOCH, 1950. Les variations d'euryhalinite et d'osmoregulation chez les poissons. /. de Physiol., 42 : 287-318. FORSTER, R. P., 1953. A comparative study of renal function in marine teleosts. /. Cell. Comp. Physiol., 42 : 487-509. GRAFFLIN, A. L., 1931. Urine flow and diuresis in marine teleosts. Amer. J. Physiol., 97: 602- 610. GRAFFLIN, A. L., 1935. Renal function in marine teleosts. I. Urine flow and urinary chloride. Biol. Bull, 69 : 391-402. GREENE, C. W., 1904. Physiological studies of the chinook salmon. Bull. U. S. Bur. Fish., 24 : 429-456. GREENE, C. W., 1910. An experimental determination of the speed of migration of salmon in the Columbia River. /. Exp. Zool, 9: 579-592. GREENE, C. W., 1926. The physiology of the spawning migration. Physiol. Rev., 6: 201-241. GUEYLARD, F., 1924. De 1'adaptation aux changements de salinite. Recherches biologiques et physico-chimiques sur 1'fipinoche (Gastcrosteiis Iciurus C. et V.). Arch. Phys. Biol., 3 : 79-197. JONES, I. C., 1956. The role of the adrenal cortex in the control of water and salt-electrolyte metabolism in vertebrates. Mem. Endocrin., No. 5: 102-120. KILLICK, S. R., 1955. The chronological order of Fraser River Sockeye Salmon during migra- tion, spawning, and death. Intl. Pacific Salmon Fish. Comm. Bull., No. 7: 1-95. KOCH, H. J., AND M. J. HEUTS, 1943. Regulation osmotique. cycle sexuel et migration de re- production chez les epinoches. Arch. Intern. Physiol., 53 : 253-266. KUBO, T., 1953. On the blood of salmonid fishes of Japan during migration. I. Freezing point of blood. Bull. Fac. Fish. Hokkaido Unit'., 4: 138-148 (English summary). POUNDER, F. E., AND I. MASSON, 1934. Thermal analysis and its application to the dinitro- benzenes. /. Chcm. Soc., 1357-1360. SCHNOHR, E., 1934. A study of the cause of death in high intestinal obstruction. Coll. Pap. Univ. Zoofys. Lab. Kjobenhavn. 12A (No. 185) : 1-176. SCHOLANDER, P. F., L. VAN DAM, J. W. KANWISHER, H. T. HAMMEL AND M. S. GORDON. Supercooling and osmoregulation in Arctic fish. /. Cell. Comp. Physiol. (in press). SPRULES, W. M., 1953. Arctic Char of the west coast of Hudson Bay. /. Fish. Res. Bd. Canada, 9: 1-15. A COMPARATIVE STUDY OF THE GILL AREA OF CRABS I. E. GRAY Department of Zoology, Duke University, Durham, N. C. In a comparative study of the gill area of marine fishes (Gray, 1954), it was shown that a definite correlation exists between the size of the gill area, the degree of activity, and the habits of the fishes concerned. It was found that sluggish bot- tom-dwelling species have proportionately much less gill surface than do fast swim- ming pelagic fishes. In this paper an attempt is made to find out if similar corre- lations exist in crabs from different habitats. Several correlations pertinent to the present discussion have already been pointed out by others. Ayers (1938) indi- cated that intertidal and land crabs consumed oxygen at a higher rate than did the strictly aquatic species. Pearse (1929a, 1929b, 1950) in his study of the emi- gration of animals from the sea has reported that there is a lessening of gill volume as crabs emigrate toward land. Pearse determined only the gill volume, not the gill area. More recently Vernberg (1956) has shown in a series of crabs that oxygen consumption of the whole animal and of gill tissue is highest in terrestrial species and decreases progressively as the habitat approaches ocean depths. This paper presents the results of a study of the gill areas of sixteen species of brachyuran crabs from six taxonomic families, and representing both pelagic and benthic species, and those living below the low tide level, those of the intertidal zone, and those that live out of water most of the time. Grateful acknowledgment is made to the Duke University Research Council for partial support of this research and to Miss Darlene Connor and Miss Barbara Galloway for the technical assistance. MATERIALS AND METHODS The method used in the determination of gill area in crabs was similar to that employed in the determination of gill area in fishes (Gray, 1954). With crabs, however, since the gill platelets are so much larger, the procedure is somewhat less tedious. Each crab was weighed after first removing surplus water from the body and gill chambers with paper toweling. The gills were then removed from one side and each placed in a separate Petri dish. The total number of platelets for each gill was counted under a dissecting microscope or computed after measuring the length of each gill with vernier calipers and determining the average number of platelets per millimeter of length. After preliminary trials to observe the range of sizes, what appeared to be average size platelets from each gill were removed and mounted in sea water on slides. Using a dissecting microscope, camera lucida drawings were made of the selected platelets and the area of these determined by means of a planimeter. Knowing the magnification used and the total number of platelets, the total gill area could be readily calculated. A weak point in the pro- 34 GILL AREA OF CRABS 35 ceclure is that it calls for judgment on the part of the observer in selecting average size platelets for the camera lucida drawings. However, the method seems no less accurate and is far less cumbersome than the mathematical methods employed by Riess (1881), Putter (1909), and Price (1931) in estimating the gill areas of fishes. Gill areas were obtained for the following species, listed more or less in order from the most land-adapted to the strictly aquatic : the ghost crab, Ocypode albicans; the wharf crab, Sesarma cinerea; Sesarma reticulata; the fiddler crabs, Uca ininax, Uca pugna.v, and Uca pugilator ; Panopeus hcrbstii; the stone crab, Menippe mer- ccnaria; the spider crabs, Libinia dubio and L. emarginata; Hepatus epheliticus; and five portunids, the blue crab Callinectes sapidus, Arencus cribarus, Ovalipes occllatus occllatus, Portunus gibcsii, and Portunus spinimanus. The spider crab, Libinia emarginata, was obtained at the Marine Biological Laboratory, Woods Hole, Massachusetts and collected from the north side of Cape Cod. All other species were collected in the vicinity of the Duke University Marine Laboratory, Beaufort, North Carolina. HABITS AND HABITATS OF THE CRABS Of the crabs studied, the ghost crab, Ocypode albicans, is by far the most land- adapted. It may make its burrows in the dunes at considerable distances from the high tide mark. It spends very little time in the ocean and, in fact, cannot survive prolonged submergence. It can, however, withstand desiccation to only a very limited degree and in hot weather comes out mainly at night and feeds near the wa- ter's edge. The wharf crab, Sesarma cinerea, also spends most of its time on land, hiding out under the drift in the daytime and coming out to feed at the water's edge at night. Both Ocypode and .S. cinerea are very active and move at a rapid rate when disturbed. The fiddler crabs usually make their burrows near the high tide mark where they will be submerged for at least a portion of each day. The sand fiddler, Uca pugi- lator, is found in large numbers along the sandy protected beaches of the estuaries near the ocean. It appears from its burrows with the receding tide, migrates to the water's edge, and may be exposed on the moist sand of the beach for several hours. Of the fiddlers, Uca minax is farthest removed from the ocean, making its burrows in mud banks of the marshes and ditches, often quite far from the sea and where tidal effect is less than on the open beaches and where the salinity may be greatly reduced. It does not travel as far from its burrows as U. pugilator and is exposed for shorter periods of time. Uca pugna.v occupies a somewhat intermediate habitat between that of U. minax and U. pugilator and tends to occupy the salt marshes. Sesarma reticulata, more robust and less active than 5". cinerea, lives in mud banks with Uca minax. Panopeus herbstii may be easily captured at low water in the lower intertidal zone of exposed oyster reefs, in crevices and under stones of rock jetties. The stone crab, Menippe mercenaria, can be found in crevices of rock jetties at and be- low the low tide level. Panopeus and Menippe are not as active as the fiddler crabs, but are much more active than Libinia. Libinia dubia and L. emarginata are slow moving bottom species living well be- low the low tide level. These are the most sluggish of the crabs studied. In 36 I. E. GRAY marked contrast, another bottom-dwelling species, Hepatus epheliticus, is very active indeed and can dig rapidly in the bottom sand where it is prone to hide. The portunid crabs are all active swimmers, quick in movement, and capable of rapid burrowing in the sandy bottom. The blue crab, Callinectes sapidus, invades both the open sea and the estuaries. Areneus cribarus frequents the area of surf along the outer beaches. Ovalipes ocellatus, Portunus spiniinanus, and P. gibesii are small crabs commonly taken in shrimp trawls in 20 to 60 feet of water, but are also occasionally found in the estuaries. RESULTS AND DISCUSSION Table I presents the gill areas and number of gill platelets per gram of body weight for the sixteen species of crabs, arranged according to families. In addition TABLE I Gill area and gill platelet number in crabs No. of Body weight grams Platelet number Gill area inm.Vgrn. Body vol.* Oxygen** Species determi- Gill vol. ^l./gm./ nations ratio min. Min. Max. Aver. Max. Min. Aver. Max. Min. Aver. Ocypoclidae: Ocypode a'.bicans 31 11.2 77.3 45.8 93 13 31 446 197 325 67.4 2.35 Uca ininax 33 3.8 11.8 6.9 238 74 131 904 282 513 40.0 1.28 - 40 UJ —I UJ LIBINIA EMARGINATA O A A A A BODY WEIGHT-GRAMS 400 BODY WEIGHT-GRAMS FIGURE 1. Relationship between the number of gill platelets and body weight in Callincctes sapidiis and Libinia cinaryinata. Circles indicate females, triangles males. survive when it is submerged? The fact that it does survive is an indication that the gill area is adequate and suggests that the crabs not only have enough gill sur- face for their normal needs, but enough for emergencies, too. Also, it has been dem- onstrated (unpublished data) that the crabs, Callincctes and Panopeus at least, go into a state of suspended animation for several hours when the O2 tension is greatly reduced. This is long enough to carry them through a good part of a tidal cycle. In this connection, if one may be permitted to guess without supporting experi- mental evidence, it would be to predict that the oxygen consumption of fiddler crabs, while in their burrows with the tide covering them, is very low. Presenting the gill areas of the various crabs as average amounts per gram of body weight suggests that the gill area per unit of measurement is relatively con- stant throughout life. This is not the case, however. Putter (1909) determined GILL AREA OF CRABS 39 the gill areas of a few crabs and fishes and maintained that the young had propor- tionately greater gill surface than did older animals. Krogh (1941) stated that while this was undoubtedly true Putter's work did not prove it. Figure 1, showing in Callinectes and Libinia how the number of gill lamellae per gram varies inversely with the weight of the crabs, and Figure 2, showing the decrease in gill area per gram of body weight with increased body growth in Menippe and Libinia, appear to substantiate the claim of Putter. Gill platelet number per gram, relatively high in very young crabs, falls off rapidly as the crabs grow older. The curves tend to level off in the older crabs. Evidently the addition of new platelets does not keep pace with the growth of the crab. Gill area per unit of weight, though more diffi- cult to demonstrate as clearly, follows a somewhat similar pattern. Very young crabs have a greater gill surface per unit of weight than do the adults. Five small 900 * I < UJ o LIBINIA EMARGINATA A A f 1400 UJ < 800 J O 6OO 400 A MENIPPE MERCENARIA 00 0 200 400 600 BODY WEIGHT - GRAMS 100 200 300 BODY WEIGHT - GRAMS FIGURE 2. Relationship between gill area and body weight in Libinia eman/inata and Menippe mercenaria. Circles indicate females, triangles males. specimens of the genus Menippe, not included in Table I, weighing between 1.5 and 2.8 grams, had an average gill area of 1443 (range 1349-1496) sq. mm. per gram of body weight, whereas the gill area of individuals of the genus Menippe varying in weight from 10 to 600 grams averaged only about half this amount. Similarly, two crabs of the genus Ocypodc weighing 1.0 and 2.5 grams had gill areas of 713 and 472 sq. mm. per gram of body weight, both values greater than the maximum for 31 crabs varying between 10 and 77 grams which averaged but 325 sq. mm. per gram. Though still apparent, the falling off of gill area per unit of weight is not as pronounced in older crabs as they increase in weight as it is in the young. The relative decrease in gill area is more easily demonstrated in large species that have great differences in weight between young and old than in the small species where individual variations may obscure the pattern. It is quite possible that had Pearse (1929a) made enough determinations he 40 I. E. GRAY would have found that the body volume-gill volume ratio was not uniform for crabs of all sizes within the same species. A factor deserving of comment is the percentage of inert skeleton. This differs among various species but is least in the fast-moving active land crabs, Ocypode and 7000 6000 N 5000 2 o I <4000 Id CC 3000 O 2000 1000 O MENIPPE MERCENARIA O O O O o 1 I 20 40 60 60 100 120 140 CARAPACE WIDTH-™™ 160 FIGURE 3. Correlation between the growth of the carapace and increase in gill area in Menippe mercenaria. GILL AREA OF CRABS 41 S. cinerea, and greatest among the heavier bodied, slower moving crabs, Menippe and Panopcus. Among the aquatic species the exoskeleton of Callinectes accounts for approximately 16 per cent of the total weight and that of Libinia about 22 per cent (unpublished data). The differences in skeletal weight, however, do not alone account for the differences in gill areas among the different species. It may be argued that weight is not a satisfactory basis for comparing gill areas of crabs. This perhaps is true, but seems much more adequate than either body surface area or linear measurements which vary so greatly in different species. Within a species, however, linear measurements may be directly correlated with gill area. This is demonstrated in Figure 3, which shows the normal increase in total gill area of Menippe as the carapace increases in width. There appears to be little or no sexual dimorphism among crabs as far as gill area or platelet number is concerned, except in those species where a major skeletal difference exists between males and females. In fiddler crabs, as illustrated by Uca mina.r (Table I), males, with greater weight because of the large chela not possessed by females, have smaller gill areas per unit of body weight than do females. In the spider crab, Libinia cinarginata, males attain much larger size than females and may weigh several times as much. Per gram of weight the females have a greater number of gill platelets and larger gill area than do the males. It seems obvious from Figures 1 and 2 that these differences between males and females are not so much a matter of sex as of body weight. It has been found with other species, as well as with Libinia, that the relative number of gill platelets and the relative size of the gill surfaces decrease as the crabs grow larger and heavier. SUMMARY 1. A comparative study has been made of the size of the gill areas of 16 species of brachyuran crabs from six families and representing land, intertidal, and wholly aquatic habitats. 2. The size of the gill area is correlated with both habitat and metabolic activity. 3. There is a tendency toward reduction in gill area per unit of weight in going from wholly aquatic to intertidal to land species. 4. Among wholly aquatic species the active, fast moving crabs (portunids) have greater gill area than do the sluggish bottom-dwelling species (Libinia). 5. Both the gill area and the number of gill platelets per unit of weight, relatively high in very young crabs, decrease as the crabs grow older. 6. Apparent sexual dimorphism in gill area is a function of weight differences between the sexes. LITERATURE CITED AYERS, J. C., 1938. Relationship of habitat to oxygen consumption in certain estuarine crabs. Ecology, 19 : 523-527. GRAY, I. E., 1954. Comparative study of the gill area of marine fishes. Bio]. Bull., 107: 219- 225. KROGH, A., 1941. The comparative physiology of respiratory mechanisms. University of Penn. Press, 172 pp. PEARSE, A. S., 1929a. The ecology of certain estuarine crabs at Beaufort, N. C. J. Elisha Mitchell Sci. Soc., 44 : 230-237. 42 I. E. GRAY PEARSE, A. S., 1929b. Observations on certain littoral and terrestrial animals at Tortugas, Florida, with special reference to migrations from marine to terrestrial habitats. Pap. Tortugas Sta., Carnegie Inst. Washington, 391 : 205-223. PEARSE, A. S., 1950. The emigrations of animals from the sea. Sherwood Press, Washington, 210 pp. PRICE, J. W., 1931. Growth and gill development in the small-mouth black bass, Microptcrus dolomicii, Lacepede. Frans Theodore Stone Laboratory, Contribution. 4: 1-46. PUTTER, A., 1909. Die Ernahrung der Wassertierre. Gustav Fischer, Jena, 168 pp. RIESS, J. A., 1881. Der Bau der Keimenblatter bei den Knochenfischen. Arch. f. Naturcjesch., 47: 518-550. VERNBERG, F. JOHN, 1956. Study of the oxygen consumption of excised tissues of certain marine decapod Crustacea in relation to habitat. Physio}. Zoo/., 29 : 227-234. AN ANALYSIS OF RESPONSE TO OSMOTIC STRESS IN SELECTED DECAPOD CRUSTACEA WARREN J. GROSS 1 Department of Zoology, University of California, Los Angeles, California Krogh (1939) and Prosser ct al. (1950) have reviewed the subject of osmotic behavior in aquatic animals. Beadle (1943) has reviewed the importance of os- motic regulation in the evolutionary migration of marine animals to fresh water habitats. Pantin (1931) has discussed the origin of body fluids in animals. Robertson (1949, 1953) presents extensive information concerning ionic regulation among several groups of invertebrates. Hober et al. (1945) consider the physical chemistry involved in osmotic regulation. Jones (1941) showed that the crab Pachygrapsus crassipes regulates in dilute or concentrated sea water after 72 hours of immersion. However, osmotic regulation as a function of time for Pachygrapsus apparently has not been studied. This would seem to be an essential parameter in its ecologic importance, especially in the first few hours. Salt and water pools have been suggested several times (Hukuda, 1932 ; Scholles, 1933; Beadle and Shaw, 1950; Gross, 1954.) The present investigation demon- strates that in the crabs studied, osmotic changes in the blood are brought about mainly by salt exchanges and not water. The presence of functional salt and water pools is considered. Exoskeleton permeability is very unequal among decapods. Nagel (1934) found a correlation between regulating ability and permeability of the exoskeleton to applied sodium iodide in several crabs. The correlation is also established in the present study on the permeability for electrolytes and water, comparing six species of crabs and a crayfish. The gills as seats of salt and water exchange and organs of regulation in crabs have been implicated mainly by eliminating other probable structures (Margaria, 1931; Nagel, 1934; Krogh, 1938). Webb (1940) produced histological evidence of a correlation in crabs between the ability to regulate and the complexity of the gills. Pieh (1936) demonstrated that isolated gills of regulating crabs show in- creased respiratory rates when exposed to osmotic stresses, thus suggesting increased work for regulation in these tissues. Koch et al. (1954) have produced direct evi- dence that in vitro the gills of Eriocheir can remove salts from a medium against a gradient. The work presented here offers direct evidence that the gill chamber of Pachygrapsus is a locus of electrolyte and water exchange, and that an osmotic gradient can be held in the chamber during an osmotic stress. The energy expended for osmotic regulation has repeatedly been a subject of study by oxygen uptake determination. Schlieper (1929), Schwabe (1933) and Flemister and Flemister (1951) demonstrated that the metabolic rate increases when crabs are under osmotic stress. This they attributed to added osmotic work. 1 Present address : Division of Life Sciences, University of California, Riverside. 43 44 WARREN J. GROSS Krogh (1939), Wikgren (1953) and Potts (1954) throw doubt on this interpre- tation. The present study indicates that rates of oxygen consumption do not manifest increased osmotic work, but muscular activity. MATERIALS AND METHODS The principal subjects for this investigation were three species of decapod Crustacea: (1) Biryu latro Linnaeus, the anomuran coconut crab, native of the Indo-Pacific region and an inhabitant of land, was collected on the island of Guam and maintained in the laboratory in Los Angeles. (2) Emerita analoga Rathbun, the common anomuran sand crab, found on sandy beaches burrowed in the sand near the level of the washing waves, was collected at Santa Monica and Corona Del Mar, California. (3) Pachygrapsus crassipes Randall, the brachyuran shore crab, found in high intertidal zones and in semi-terrestrial situations, was collected at Ballona Creek and Flat Rock Point, California. The concentration of fluids was determined in two manners: (a) melting point method as described by Gross ( 1954) , which permitted determinations on volumes as small as one mm.3 ; (b) conductivity measurements using a 1000-cycle bridge. This allowed determinations on a two-mi, sample which was not necessarily expended but could be returned to the experimental vessel. Of course, this method measured only electrolytes. Units of resistance were converted to per cent of a standard sea water. Oxygen consumption was measured by means of the Scholander-Wennesland respirometer as described in Wennesland (1951). All determinations were made at 16° C., a temperature to which the experimental animals were accustomed. Particular care was taken to assure that immersed animals were completely covered. No readings were made until the animal remained in the chamber for at least one hour ; this was to allow them to become accustomed to the new environment. RESULTS 1. Osmotic Regulation as a Function of Time Measurements of blood concentration during immersion in water were made throughout the range of water salinity, in which life could be sustained. The be- havior of a species was determined partially from single readings on specimens ex- posed to certain stresses for given periods. However, regulation as a time function in individual specimens was followed over extended periods by two methods : ( 1 ) melting point determination on blood samples extracted periodically during immer- sion, and (2) periodic measurements of losses or gains of conductivity of the me- dium. A combination of these two methods was found best for studying individual responses over extended periods. Emerita, a non-regulator It was first established by melting point determinations that the body fluids of Emerita are isotonic to normal sea water (3.46% salt) and that this animal cannot sustain an osmotic gradient between its blood and external medium as a steady- OSMOTIC RESPONSES BY CRUSTACEA 45 state condition. When three specimens were immersed in each of the following concentrations of sea water: 50, 75, 90, 110, 125, and 150% (total of 18 animals), their body fluids were isotonic to their respective external media within two hours or less after immersion. Thus Emerita shows no ability to regulate osmotically. TABLE I Solute space calculated from the relationship between concentration changes in the medium and the blood of the animal immersed in 5 times its volume of water Specimen number Medium (% sea water) Change in blood (% sea water) Change in medium (% sea water) Change in blood Calculated solute space (water equivalent) % body weight Change in medium Emerita 1 50 46.3 3.7 12.5 36 2 50 45.6 4.4 10.4 44 3 60 36.5 3.5 10.4 44 4 75 23.0 2.0 11.5 40 5 75 23.1 1.9 12.2 37 6 75 22.8 2.2 10.4 44 7 125 23.2 1.8 12.9 38 8 125 23.1 1.9 12.2 37 9 150 45.3 4.7 9.6 47 10 150 46.3 3.7 12.5 36 Mean 11.5 40 Pachygrapsus 1 25 22.6 2.7 8.4 50 1 25 15.3 1.9 8.1 51 2 50 13.7 2.0 6.9 60 3 39 13.2 1.8 7.3 57 4 50 14.7 1.9 7.8 53 5 50 14.2 1.8 7.9 53 6 50 9.5 1.3 7.3 57 7 125 10.5 1.4 7.5 56 8 145 7.9 1.0 7.9 53 9 150 15.8 2.0 7.9 53 Mean 7.7* 54 5 = 5/7.7 1.2 X 100 = 54%. Individual specimens which had never been removed from normal sea water, were then immersed in dilute or concentrated media of 5 times the volume of the animal. Then the electrical resistance changes in the medium were followed until the resistance was stabilized and here the body fluids could be considered to be iso- tonic to the medium. The change in the blood concentration is therefore equal to the difference between the final concentration of the stress medium and the concen- tration of normal sea water. If, then, a constant ratio between osmotic changes in the blood and medium could be established, a conductivity variation in the medium 46 WARREN J. GROSS could be interpreted in terms of a change in the blood concentration, so that at any- time the osmotic pressure of the blood could be estimated by such conductivity readings. Data in Table I demonstrate that such a ratio is relatively constant over a wide range of osmotic stresses, the mean value showing a change in the body fluids equivalent to 11.5% sea water for each 1% sea water change in the medium. The rate of approaching equilibrium with the external environment suggests a physical, diffusion phenomenon. The curves are indicated in Figure 3. How- ever, there are individual variations which result particularly with size, the smaller animals reaching equilibrium first. Exploratory experiments suggested that Callianassa affinis, Upogebia sp., Cancer antennarius, C. gracilis, and Pugettia producta behave similarly. Regulating forms immersed in water Pachygrapsus in normal sea water is not necessarily isotonic to the medium. (Note the variation in initial blood concentration in Figure 1.) Specimens were immersed in stress media of five times their respective volumes and salinity changes in the medium were repeatedly noted by the conductivity bridge. After a significant change in the medium was observed, a melting point determination was made on the blood. As with Emerita, a relatively constant ratio between conductivity change of the external medium and osmotic pressure change in the body fluids of the animal was demonstrated (Table I) . On the average, a change in the external medium equiva- lent to \% sea water meant a change in the blood equivalent to 7.7% sea water. Therefore, if the initial or final blood concentration of Pachygrapsus were known, conductivity measurements of the medium could be converted to represent the ap- proximate osmotic pressure of the blood. The crabs were able to tolerate the small volumes of medium for long periods, if moved to a fresh medium of the same conductivity at least every three hours. Over extended periods the rate of regulation in an individual specimen of Pachygrapsus could be estimated by converting changes in the medium to body fluid concentrations assuming the above mean ratio of change in blood to change in medium. Occasional checks were made by melting point determinations of the blood. Subsequent deviations did not exceed 10%. Such an error could not obscure a trend. When readings were not needed at close intervals, the specimen was placed in a large volume of the desired salinity and after an appropriate period a melting point determination was made on the blood. This was done on most of the late readings (e.g., 72 hours). Figure 1 illustrates osmotic regulation in Pachygrapsus as a function of time. It should be pointed out that a small error is introduced by using the conductivity method, for when a change occurs in the medium, the osmotic gradient is consequently reduced. In the extreme case where a change in the blood was equivalent to 40% sea water, about a 5% error was effected. However, since the conductivity method was used for brief periods, not exceeding 12 hours, osmotic changes detected were small and the consequent er- rors caused by reducing the osmotic gradient were usually insignificant. As shown in Figure 1, Pachygrapsus can regulate osmotically in both hypotonic and hypertonic media. This confirms the work of Jones (1941). However, while Jones showed this to be true after 72 hours, the present investigation shows that OSMOTIC RESPONSES BY CRUSTACEA 47 regulation is established immediately, and is sustained perfectly by some specimens in moderate stresses for a few hours. Regulation then diminishes gradually until equilibrium is reached, usually within 24 hours. However, several plateaus and steps on the osmotic behavior curves may be produced before equilibrium is finally reached. Equilibrium in the case of Pachygrapsus does not mean that the body fluids are isotonic to the external medium. Rather, it means that the blood of the animal has reached a steady state with respect to osmotic pressure. 140 ISO I2O 110 100 150 I4O I3O 120 MO IOO ISO I4O ISO 120 no 100 ISO I4O 130 120 jt 110 ~- 100 IOO 9O 80 70 60 50 UJ o z o o Q O 3 CD IOO 9O 80 70 60 50 IOO 90 80 70 GO SO IOO 90 80 70 60 SO 160% R- 0 I = 0 130% R- 5 I • 19 125% R* 5 I - IO 100% -8- o — o — g- »— 8- R- 3 I • O 75% R= 4 I • 0 R = 5 I • 2O 50% 25% R' 5 I ' 31 12 IS 18 21 24 27 30 33 36 39 42 45 48 TIME IN HOURS 54 37 60 63 66 69 72 FIGURE 1. Osmotic regulation in Pachygrapsus as a function of time. "R" represents those specimens whose behavior was followed for extended periods by repeated determinations and is indicated by open points (O) ; "I" represents those specimens on which only one deter- mination was made and is indicated by solid points ( • ) . Solid line represents approximate median and is drawn through points representing the actual behavior of one specimen. All points are indicated unless coinciding with others. Concentration of the medium is indicated in per cent of normal sea water above the respective curve. 48 WARREN J. GROSS The plateaus and steps on the osmotic behavior curves reflect grading of the active regulatory processes or changes in accessory processes, e.g., water movement over the gills. Only occasionally do fluctuations or dips occur. Such behavior is shown in Figure 1 by a crab immersed in 25 % sea water. A high degree of individual difference is demonstrated by the wide spread of points and the varying slopes among individual histories. Such variations could be caused by differences in size, age, sex, metabolic rate variances caused by physio- logical periodicities, e.g., molting cycle, or external environment changes. Concurring with Jones (1941), it was demonstrated that Pachygrapsus regulates better in hypotonic media than in hypertonic media. This phenomenon becomes evident from (a) the period of perfect regulation, (b) the slope of the regulation curve, (c) the total change in the concentration of the blood at equilibrium and (d) the extreme osmotic stresses endured by the crabs. These lines of evidence become apparent with inspection of Figure 1. However, it is worth mentioning that no animal lived in 175% sea water for as long as 6 hours. Yet several crabs actively survived 25% sea water for 72 hours. Prosser et al. (1955) demonstrated rela- tively strong tolerance to 170% sea water by Pachygrapsus. However, their ex- perimental animals were gradually acclimated to lesser stresses before immersion in 170% sea water (personal communication). There seems to be variation in the blood concentration of Pachygrapsus living in normal sea water. Jones (1941) reports that the body fluids of Pachygrapsus are hypotonic to normal sea water. Pearse (1931) found the body fluids of this crab hypertonic to normal sea water. As can be seen from Figure 1 the crabs used in the present investigation were usually hypertonic to their external medium when they were immersed in normal sea water. Such variations possibly can be explained by the fact that the osmotic pressure of the blood is a function of the molt cycle (Baumberger and Olmsted, 1928). Token experiments suggested that Uca and Hemigrapsus regulate similarly to Pachygrapsus. Confirming the work of Jones (1941), Uca was found to regulate more strongly in hypertonic and hypotonic sea water than Pachygrapsus. In the salinity ranges from 50% sea water to 150% sea water, this form maintained almost perfect regulation for 36 hours. While Jones (1941) found that Hemigrapsus could regulate strongly in dilute sea water, he was unable to show regulation in concentrated sea water after 72 hours immersion. The present investigation revealed that this crab can regulate up to 33% perfectly for 20 hours in 150% sea water. Osmotic regulation in the land crab Birgus latro The implications of osmotic regulation in the land crab Birgus latro have been discussed by Gross (1955). As mentioned there, these anomurans will drown when completely immersed for a day or so. It was therefore necessary to allow all the animals to rise slightly out of the water for exposure of their respiratory mem- branes to air. By this operation they partially released themselves from the im- posed osmotic stresses. However, most of the external surface was immersed all of the time. Because of the limited number of specimens, only one specimen of Birgus could be studied in the representative stresses which were : 25, 50, 66 and 137% sea water, respectively (four specimens). In these cases the changes in the OSMOTIC RESPONSES BY CRUSTACKA 49 osmotic pressure of the blood were followed by repeated melting point determina- tions on blood samples extracted at chosen times. Results are illustrated in Figure 2. These data show at least that Birgus is a strong regulator in both dilute and concentrated sea water. The moribund condition of the crabs after prolonged im- mersion in 25% and 137% sea water is not believed to be the direct result of the osmotic changes, since the two blood concentrations, specifically 70 and 119% sea water, are readily tolerated by this species (Gross, 1955). Anoxia seems to be a more satisfactory explanation for the moribund condition of these two specimens; however, high oxygen tensions failed to revive them. It is perhaps pertinent that UJ CO z 110 o < 100 cr UJ o 90 80 o Q 70 O O CD 60 50 137% MORIBUND MORIBUND -L. 0 12 16 2O 24 28 TIME IN HOURS 32 36 40 44 48 FIGURE 2. Osmotic regulation in Birgus as a function of time. Behavior of four specimens demonstrated by repeated melting point determinations on the blood. Concentrations of the medium are indicated in per cent of normal sea water over the respective curves. the two animals exposed to the greatest stresses were most affected, but the fact that Birgus can regulate in concentrated sea water corroborates the observation that hypo-osmotic regulation is common among crabs showing some degree of terrestrial behavior (Jones, 1941 ; Gross, 1955). Salt exchanges When Emerita and Pachygrapsus are immersed in dilute or concentrated sea water, the osmotic pressure of their body fluids changes but corresponding weight changes under these conditions are small. In the case of Emerita a change in the body fluids equivalent to 25% sea water resulted in a weight change of less than 2% of the body weight. If pure water, this could effect a concentration change in the 50 WARREN J. GROSS blood of less than 6% on the assumption of 40% of the body weight being osmoti- cally active water. This of course means that changes in the concentration of the body fluids are effected mostly by net changes in the solute content rather than water ; 20% of the total concentration change of the blood was caused by water and SO'/r by solutes, in this case. 120 no 100 90 80 ro 60 so ,0 130 o 100 s 90 rr eo t- Z 70 UJ O 60 O 50 (J 8 '50 140 130 120 110 100 90 80 70 60 50 MORIBUND 150% 150% MORIBUND 125 % 100% 75 % kl .50 % MORIBUND 8 10 12 14 TIME IN HOURS IB 20 22 24 FIGURE 3. Comparative osmotic behavior of Emcrita, Pachyf/mpsits and Birgus. Solid line indicates behavior of individual specimen which, in the case of Emcrita and Pachygrapsus, represents the approximate average behavior of all individuals investigated for a given osmotic stress. All cases of Bir X C/j ^i III ^ ^o X Q 0. S 02 o ^ ^ to ^ ^ K ^ ^j ^ H Cfc v) ^^ O ^ ^ oe S Co ^ z >x CO -^r ^C _J; M ° 01 t x t l- _l 5 ^ ^ < v> ^ 5 !£ to ^ I;K;URE 4. The relative permeability of exoskeleton in several decapod Crustacea. Values represent the means of three determinations for each species with a 50% sea water gradient. Variation among the three determinations for each species was in no case more than 25%. dilute sea water if it were as permeable as Cancer? Much evidence is available demonstrating that animals which cannot regulate osmotically are strong ionic regu- lators (Prosser et al., 1950). It may be that in the case of crabs, the mechanism for active, osmotic regulation is present and functioning in the non-regulators, but the permeability of the exoskeleton is so great that an osmotic gradient cannot be sus- tained. No evidence has been obtained on this point with respect to crabs but it 54 WARREN J. GROSS has been shown in the case of poikilosmotic sipunculids that salts can be removed from the medium without benefit of a gradient (Gross, 1954). Osmotic regulation in the gill chamber It is generally believed that the gills of marine crabs function as osmotic regu- latory organs. Koch et al. (1954) have produced direct evidence in Eriocheir sup- porting this view. Gross (1955) demonstrated that the gill chamber of Pachy- grapsus is a site of salt exchange under conditions of desiccation. As one way of testing whether this is more generally true, the salinity changes which occur in the branchial cavity of crabs removed from water were measured after the cavity was filled with water of varying salt content. It was previously shown that no signifi- cant changes occur under similar conditions when the gill chambers are filled with normal sea water (Gross, 1955). First, the volume of fluid held in the chambers was estimated by a method re- ported previously (Gross, 1955). Crabs whose gill chambers had been pierced for flushing were immersed in tap water, 25% sea water or 150% sea water for a period of one hour ; each cavity was then flushed out with 10 ml. of distilled water, the flushings were caught and the salinity of this fluid was measured by means of a conductivity bridge. Assuming that the gill fluid is the same concentration as the medium in which the crab was immersed and knowing the volume and salinity of the flushings, the volume of the branchial fluid can be estimated as well as the ab- solute quantity of salt in that fluid. The animal was then immersed in normal sea water for about two hours to per- mit recovery. Again it was returned to its respective stress medium for an addi- tional period of one hour. The crab then was removed from the water and placed in a closed container which was kept at saturated humidity by a paper towel soaked in the same medium from which the animal had been removed. Such paper was placed where the crab could not reach it. After two to seven days the animal was removed from the container and its gill chamber flushed out with distilled water, the salinity of this fluid being measured. If the latter salinity determination differed from the former for the same crab, it was suggested that salt exchanges occurred in the branchial chamber. Such was the case, for all animals with dilute branchial fluid showed an absolute salt increase after the period in the humid container, while all animals with concentrated branchial fluid showed an absolute decrease. It should be said, however, that the crabs which had been immersed in 150% sea water showed some signs of desiccation. It is not surprising to find salt exchanges in the gill chamber, for as described above, even the chitinous exoskeleton is permeable to both salts and water. How- ever, if it could be demonstrated that the salinity of the branchial fluid were differ- ent from that of the blood when a steady-state had been reached, then it would seem that a dynamic mechanism is present in the gill chamber, permitting an ex- change of salt with the body fluids, but also being able to hold an osmotic gradient. Now, the volume of branchial fluid for each animal was determined with the first flushing operation described above. Thus, the salinity of the second flushing will yield an estimation of the branchial fluid concentration after the period in the humid container. Results suggest that a gradient is sustained in the gill chambers of all animals which had been immersed in dilute media. The mean salinity of such OSMOTIC RESPONSES BY CRUSTACEA 55 fluid from two animals which had been immersed in tap water was equivalent to 14% sea water while the blood was estimated to be greater than 75% sea water in concentration. The mean salinity in the gill chamber of four animals removed from 25% sea water was equivalent to 64% sea water while the blood concentrations were estimated to be greater than 85% sea water. Data from the specimens which had been immersed in 150% sea water were considered invalid for this aspect of the experiment. It might be argued that the apparent gradients described above are not real, but that a great amount of branchial fluid rests on impermeable tissues. In such a case, the fluid in contact with the permeable tissues could become isotonic with the blood and this would affect the total salt content in the gill chamber only enough to give the impression that an osmotic gradient was being held. However, the large ap- parent gradient sustained when the branchial fluid was initially tap water, and the large salt exchange under conditions at desiccation (Gross, 1955), do not support this argument. Since all animals remained out of the water for at least two days before the salinity of their branchial fluid was measured, there was plenty of op- portunity for salt exchanges to take place. It is probable, therefore, that the above described gradients are real, but the fact that more salt change occurred for animals from 25% sea water than for those from tap water raises doubt as to the quantita- tive value of the data, since the osmotic gradient between blood and gill chamber was greater for the tap water animals. One more point of interest arises from this experiment. It was previously esti- mated that the mean branchial fluid volume for 20 Pachygrapsus was 1.7% body weight (Gross, 1955). The mean branchial fluid volume of those animals im- mersed in 150% sea water was only 0.71% body weight. Yet, again, those im- mersed in dilute sea water averaged 1.7% body weight. This, of course, suggests that Pachygrapsus has some control over the water that enters the gill chamber, which would be an aid to osmotic regulation. If such an aiding mechanism for regulation exists, it seems that it functions only for hyper-regulation, but confirma- tion is needed for this finding. Metabolic work in increased osmotic stresses It has been reported that when certain crabs are exposed to osmotic stresses, they respire more rapidly ; this increase in metabolism has been interpreted as work accomplished by the regulatory mechanism (Schlieper, 1929; Schwabe, 1933; Flemister and Flemister, 1951). However, Krogh (1939) suggested that such metabolic increases are caused in part by activities of the organism other than os- motic regulation. I have observed that certain crabs, e.g., Pachygrapsus, show violent attempts to escape from a medium which departs much from normal sea water in concentra- tion. Obviously such activities would step up the entire metabolic rate of the crab, making it difficult to show the increased rate due to osmotic regulation alone. The necessary increase in the osmo-regulatory mechanism cannot be predicted simply by knowing the normal osmotic pressure of the body fluids of an animal and the osmotic stress imposed. Rather, a knowledge of the sustained osmotic gradient is needed to give the relative amount of regulation necessary to withstand a certain stress. It is conceivable that a given amount of osmotic work will main- 56 WARREN J. GROSS tain the body fluids at viable concentrations over a range of osmotic stresses. This could be possible if the animal holds a constant gradient between blood and external medium, although the actual blood concentration alters to permit the constant gradi- ent. Over such a range of stress, then, the constant rate of regulation would be 70 te. u 60 * < w m 8* 50 40 -I < Ul o o o 30 20 10 r. uj O K o 0 ISOTONICITY j 25 5O 75 100 125 150 MEDIUM CONCENTRATION ( % SEA WATER) FIGURE 5. Crabs sustain greater osmotic gradients at equilibrium as greater stresses are imposed. Points are approximate mean values and for Pachygrapsus are calculated from the present investigation ; values for Uca, H. oregoncnsis, and H . nudus, are calculated from Jones (1941). expected to cause no variations in the total metabolic rate assuming constant relative roles of the various regulatory organs. On the other hand, if an increasing gradient is established more work would be expected. It has been pointed out recently by Potts (1954) that semi-permeable animals inhabiting fresh water could maintain their blood hypertonic to the medium with less work by excretion of dilute urine than by regulating at the tissue surfaces which are exposed to the external medium. OSMOTIC RESPONSES BY CRUSTACEA 57 However, it was also demonstrated that such a mechanism would not be significantly advantageous over the other unless large osmotic gradients were sustained between the blood and external medium (greater than 50% sea water). The possibility should therefore be considered that at certain stresses the mechanism of regulation may shift from one process to another, and that the various processes are not neces- 350 _ 3OO 25O I 200 z o t- a. 2 150 ^ CD Z o o IOO CD > X o 50 2 4 5 6 100 5O TAP MEDIUM CONCENTRATION ( % SEA WATER) FIGURE 6. Oxygen consumption in Uca as a function of osmotic stress. Numerals repre- sent individual specimens. Solid line connects mean values for all cases. Measurements made at 16° C. on crabs of approximately two grams. These could not stand out of the solution. sarily equally efficient. Nevertheless, it does not seem sume that in general more metabolic work is required gradient than to sustain a small osmotic gradient. Figure 5 demonstrates that in Uca, Pochygrapsus, H. oregonensis, more osmotic work would be expected the sustained gradients increase with the larger stresses, of Hemigrapsus when they are immersed in concentrated not regulate. It can be observed in Pachygrapsus that to be unreasonable to as- to sustain a large osmotic Hemigrapsus nudus, and in greater stresses because except for the two species sea water where they can- the sustained gradient in- 58 WARREN J. GROSS creases almost linearly with the stress, from a medium of normal sea water up to one of 50% sea water; but in 25% sea water the gradient drops off from the linear relationship with stress. This indicates that the crab must work only slightly faster, and it can be seen that with the same metabolic output an equal gradient could be held in even more dilute water. Uca shows almost a linear increase in the gradient up to a medium of 25% sea water and only a slight fall off from this re- lationship to stress in 5% sea water. Hemigrapsus nudus and H. oregonensis show behavior in dilute sea water which is somewhat different from Pachygrapsus and Uca, for they regulate weakly in 75% sea water, sustaining less than a 10% sea water gradient. With like stresses Uca and Pachygrapsus hold 22% and 20% sea water gradients, respectively. However, as the stress increases both species of Hemigrapsus regulate strongly, as illustrated by the increased slopes for these crabs in Figure 5. If the additional osmotic work could manifest itself, the animals would be ex- pected to consume more oxygen as the medium departs farther from the osmotic pressure of the body fluids. The oxygen consumption of individual specimens of Uca was compared when the crabs were immersed in normal sea water, 50% sea water, and in tap water. Since this species may be found commonly in normal sea water, its respiratory rate was measured first in this medium, assuming that harmful effects would be at a minimum here. For the next determination, half the animals were tested in 50% sea water, the other half in tap water. For the third determina- tion the order was reversed so that each specimen was studied in three media. This was a control against permanent injury which might result from the heavy stress imposed by tap water. However, the order of immersion did not seem to make a difference. After determinations in 50% sea water and tap water, the crabs were placed in 100% sea water for 24 hours to allow recovery before the next determina- tion was made. All specimens whose behavior is recorded lived in sea water for at least 48 hours after completion of the experiment. Results in Figure 6 illustrate that the metabolic rate of an individual animal does not necessarily increase with osmotic stress. In fact, of 8 animals, only numbers 2 and 7 show such a behavior. With the exception of two extreme cases, however, number 3 in normal sea water and number 6 in tap water, the respiratory rates for the group of animals seem to be higher in higher stresses. The mean of the 8 specimens shows a value for oxygen consumption in 50% sea water of about 108% of what it is in normal sea water, while in tap water it is about 135% of the value for 100% sea water. Assuming that the normal osmotic pressure of Uca blood is equivalent to that of 85% sea water, there will be a 15% sea water gradient when the crab is immersed in normal sea water; in 50% sea water there will be a maximum gradient of about 35% sea water, and in tap water a maximum gradient of 85% sea water. When the animal is exposed to an osmotic stress, the amount of work should increase by the same factor as does the increased sustained gradient. When immersed in 50% sea water, regulation might increase by 35-15 or 20 units. The mean respiratory rate in 50%- sea water shows 8% increase over animals immersed in normal sea water ; thus, the fraction of total metabolism responsible for regulation in sea water might be calculated by simple proportion to be 6.0%. In tap water, this value is calculated to be 7.5% of the total metabolism. These results do not agree with those of Flemister and Flemister (1951) who OSMOTIC RESPONSES BY CRUSTACEA 59 studied another crab, Ocypodc. Flemister and Flemister regarded the increased respiratory rate in hypertonic and hypotonic sea water as a manifestation of greater chloride regulation, and probably osmotic regulation. If this is the case, then about the same amount of metabolic work serves to maintain almost perfect regulation in any medium which establishes a gradient of from 25% to 50% sea water, yet in an isotonic medium, the metabolic rate is at least 20% lower. Could it be that osmotic regulation in Ocypodc is an all-or-none process which is triggered by a salinity change in the medium? Uca is also a member of the family Ocypodidae, and its habitat is similar to that of Ocypode. It would seem questionable, that the osmo- regulatory mechanism of these closely related organisms could be essentially different. It is this author's opinion that the interpretation of increased respiratory rates of animals subjected to osmotic stresses as a manifestation of greater osmotic work is to be questioned. This opinion is based on the inconsistent results found in indi- vidual animals in the present investigation (Fig. 6) and on the knowledge from numerous observations on a number of species that crabs are sensitive to osmotic stress and attempt to escape. DISCUSSION The osmotic responses of the species studied in this investigation are not always obviously adaptive. The stenohaline forms represented by Emerita are presumably limited in their choice of habitat by their tissue tolerances, since they are unable to regulate. Yet, this species can tolerate salinities of from 75% sea water to 125% sea water for at least 24 hours, suggesting that the dilute waters of estuaries and the concentrated waters of tide pools might partially afford a refuge for them. It would seem to be the habits of this animal that dictate that it live near the level of the wash- ing waves, rather than its lack of osmotic regulation. The strong osmo-regulatory ability of Pachygrapsus and Birgus superficially seems superfluous in animals of their habits. Pachygrapsus generally is found in normal sea water where it does not have to regulate, or on land where it is sub- jected to desiccation, not direct osmotic forces. Birgus is found usually on dry land except for periods when it returns to the sea to reproduce. Gross (1955) discusses the significance of the ability to regulate among terrestrial and semi-ter- restrial crabs, and suggests that in nature where osmotic stresses in aqueous media are rarely encountered it is of little importance as such, but appears under the arti- ficial conditions of experimentation, i.e., osmotic stresses, as a secondary manifesta- tion of physiological processes important for life on land. It is interesting that the regulators Pachygrapsus and Birgus show large tolerances to variations in their blood concentrations. The immediate resistance to osmotic stresses demonstrated by regulating animals may be a matter of inertia permitted by relatively impermeable exoskeletons, for the present investigation has shown a correlation between osmo-regulatory ability and integumental impermeability. The latter significantly contributes to the time factor which is important to intertidal animals which are subjected to tidal rhythms. In the case of Hemigrapsus, hypo-osmotic regulation for 20 hours would be adequate, because it would be rare for this animal to be exposed to the air for such a period. Pantin (1931) has said that non-regulatory animals may posses the same media- 60 WARREN J. GROSS nisms permitting osmo-regulation as do regulators but to a lesser degree. It may be, in view of the above findings, that the difference between a regulator and a non- regulator could be merely a difference in permeability. Previous investigations have produced evidence that the gills are major osmo- regulatory organs among marine crabs. The present investigation has established that a dynamic flux of salts and water occurs in the gill chamber of Pachygrapsus. Although no direct evidence has been produced, it is probable that the gill tissues are capable of actively transporting salts. However, the regulating mechanism may not lie entirely at a tissue level, for there is evidence suggesting that stress media can be partially excluded from the branchial chamber, therefore preventing contact with the gill tissues in large volumes. The green glands of Pachygrapsus are probably not important organs of osmo- regulation, because the urine is close to the concentration of the blood in various concentrations of sea water. Prosser et al. (1955), however, have produced evi- dence that the green glands are important ion regulators. Potts ( 1954) has shown theoretically that a semi-permeable animal would not gain much regulatory effi- ciency in excreting a dilute urine, unless extreme stresses were encountered. How- ever, the crabs used in this study are not semi-permeable. In fact, net changes in the osmotic pressure of the blood were shown to be effected mainly by salts, not water. The relatively constant values for solute space which were calculated from the ratio of concentration changes in the blood to concentration changes in the external medium have interesting implications, namely, that if salt pools are contributing to the blood changes occurring under osmotic stress, they are doing so in a constant manner. The question remains open, whether or not a given alteration in the blood can be accounted for in the external medium. When regulating crabs are exposed to increasing osmotic stresses, they generally sustain greater osmotic gradients. This would necessitate greater osmotic work. However, increased rates of respiration which are registered by crabs when they endure an osmotic stress cannot be interpreted as the manifestation of that increased work, for other activities, stimulated by the stress, e.g., struggle to escape, cannot be isolated. It may be possible that greater osmotic work alone does not add appreci- ably to the total metabolic rate. This investigation was conducted under the direction of Professor Theodore Holmes Bullock, to whom I am grateful for his most helpful encouragement and guidance. I am also indebted to Professor C. L. Prosser and Dr. J. D. Robertson for reading parts of the manuscript and offering helpful suggestions. SUMMARY 1. The decapod Crustacea, Emerita, Callianassa, Upogebia, Cancer antennarius. C. gracilis and Pugettia cannot regulate osmotically and from lack of tolerance are generally stenohaline. 2. Pachygrapsus, Birgus, Hcnrigrapsus and Uca can regulate osmotically in con- centrated and dilute sea water. Hemigrapsus is the weakest hypo-osmotic regulator of the four species. OSMOTIC RESPONSES BY CRUSTACEA 61 3. Among species where osmotic regulation occurs, it is established immediately and may be long lasting or may grow weaker with time. Although blood concen- trations may fluctuate in a given stress, the phenomenon is not common. 4. Equilibrium of the blood concentration, where perfect regulation does not oc- cur, is usually established within 24 hours following immersion ; changes occasionally occur later when extreme stresses are imposed. 5. Estimates on the solute space volumes were calculated as 40% for Emerita, 54% for Pachygrapsus and about 50% for Birgus. 6. Concentration changes occurring in the blood of Pachygrapsus and Emerita are caused mostly by salt rather than water exchanges. 7. There is a dynamic flux of salt and water in the gill chamber of Pachygrapsus, thus furnishing further evidence that the gills are osmo-regulatory organs. 8. The osmotic regulating crustaceans Cambarus, Pachygrapsus, Hemigrapsus nudus and H. oregoncnsis have less permeable exoskeletons than the non-regulators, Cancer gracilis, C. antennarius and Pugettia by a factor of at least three. 9. Pachygrapsus, Uca, H. nudus and H. oregonensis sustain greater osmotic gradients when greater osmotic stresses are imposed. 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Ionic regulation in Carcinus maenas. Proc. Roy. Soc. London, Ser. B, 129: 107-136. WENNESLAND, R., 1951. A volumetric microrespirometer for studies of tissue metabolism. Science, 114: 100-103. WIKGREN, B., 1953. Osmotic regulation in some aquatic animals with special reference to the influence of temperature. Ada Zoo/. Fennica, 71 : 1-102. STUDIES ON FEEDING, DIGESTION, AND FOOD STORAGE IN FREE-LIVING FLATWORMS (PLATYHELMINTHES : TURBELLARIA) J. B. JENNINGS Department of Zoology, The University of Leeds, England Investigations upon turbellarian nutrition (summarized by Hyman, 1951 and Yonge, 1954) have so far dealt mainly with triclads, where intracellular digestion has been demonstrated but the possibility of some supplementary intraluminar di- gestion not fully explored. Triclad nutrition has therefore been re-examined, us- ing Polycelis cornuta, and the opportunity taken to make comparable investigations on representatives of the other turbellarian orders. In each case the nature of the food and the feeding mechanism, the structure of the gut and the course of digestion, and the nature and location of the food reserves have been studied. The various mucoid and rhabdoid secretions have also been examined and an assessment made of their value in feeding. MATERIALS AND METHODS The following Turbellaria, listed systematically with details of their habitat, were examined. Order Acoela. Convoluta parado.va. Oersted. From rock pools rich in Ulva and Entcromorpha in Cullercoats Bay, Northumberland. Order Rhabdocoela. Sub-order Notandropora. Macrostomum sp.. Oe. Sub-order Opisthandropora. Stenostomum sp., Duges. Both from ponds in the Leeds area. Sub-order Lecithophora. Mcsostonia tctragonum, O. F. Miiller. From Middlerigg Tarn, Troutbeck, Westmorland. Order Tricladida. Sub-order Paludicola. Polycelis cornuta, Schmanda. From under stones in small fast-running streams in the Leeds area. Order Polycladida. Sub-order Cotylea. Cycloporus papillosus, Lang. On colonial tunicates (Botryllus and Botrylloidcs) beneath weed-covered rocks at low- water mark on St. Mary's Island, Northumberland. Sub-order Acotylea. Lcptoplana tremellaris, O. F. M. Beneath stones on a predominantly sandy shore at Llanfairfechan, North Wales. The flatworms were starved to encourage a readiness to feed and to clear the gut lumen and the gut cells of any remnants of previous meals. The animals as- sociated with the flatworms under natural conditions were then presented to them so that the methods of capture and ingestion of the selected prey could be followed in detail. Flatworms were killed where possible in the act of feeding, Steinmann's fixative (equal parts concentrated nitric acid, distilled water and saturated mercuric chloride in 5% aqueous sodium chloride) giving the necessary instantaneous fixa- 63 64 J. B. JENNINGS tion. and after washing in running water the preparations were stained in borax carmine. The natural food was used, whenever possible, to determine the site and course of digestion, but in forms with a phagocytic gastrodermis (especially the triclad) both this and the soft foods used by previous investigators (Arnold, 1909; Willier, Hyman and Rifenburgh, 1925; Kelley, 1931) disintegrated during ingestion and it could not be ascertained whether any intraluminar break-up preceded the phagocyto- sis and intracellular digestion, nor whether the separate constituents of the food (proteins, carbohydrates and fats) were dealt with differentially. Hence the flat- worms were fed either on food which reached the gut in a visibly recognizable con- dition, or on homogeneous food substances whose digestion could be detected by simple chemical tests. Some of these were readily eaten, but with others the natural prey had to be used as a carrier. This particularly useful technique was applied by extracting the body contents of a normal prey with a hypodermic syringe and replacing them with the appropriate food substance. In all cases the flatworms were fixed at intervals after an observed feed, and examined histologically. All fixation (in Susa, saturated mercuric chloride or 10% formalin) was carried out at about 40° C. to prevent rupture or discharge of the gut contents. Sections cut at lOjtt were stained with Ehrlich's haematoxylin and eosin, Heidenhain's iron haematoxylin and Feulgen's stain. Squash preparations were also made, either directly in 0.5% saline or after preliminary maceration in saturated aqueous boric acid, and examined both fresh, and fixed and stained. In cases where ingested food was visible within the living flatworm its fate was also followed by direct observation. Fat reserves were studied from squashes fixed in osmium tetroxide vapour and from sections prepared after fixation in Flemming's fluid. Carbohydrate reserves were studied after fixation in 95% alcohol, the sections being stained with Best's carmine for glycogen or periodic acid-Schiff's reagent for carbohydrates generally. For protein reserves flatworms were fixed in neutral 10% formalin and the sections stained by the modified Millon method (Bensley and Gersh, 1933). Special methods for the study of pH changes during digestion and the examina- tion of the mucoid and rhabdoid secretions are described in the text. OBSERVATIONS TRICLADIDA. Polycelis cornuta The food and feeding mechanisms P. cornuta (1-1.5 cm. long and 2-3 mm. broad) feeds upon small annelids, crustaceans and insect larvae, particularly those of Ephemeroptera, Plecoptera and Diptera. It is also a scavenger and feeds upon injured or dead animals, provided they are not too decomposed — juices diffusing from such animals attract all the flat- worms in the vicinity and they can be collected in great numbers by anchoring a crushed earthworm in the stream as bait. The living prey may be seized directly but is often caught after it has become entangled in the mucus produced for locomotion and adhesion by the Polycelis popu- lation in general. The normal slow movement by ciliary gliding is facilitated by mucus secretions which are laid down in tracts comparable to the slime trail of a snail ; this mucus is not particularly sticky but small animals are occasionally trapped FEEDING IN FREE-LIVING FLATWORMS 65 in it. Often, however, ciliary locomotion is supplemented by direct muscular move- ment in which waves of contraction pass down the body and urge it forward. During such movement, and particularly when the flatworm is facing a current or changing levels upon a plant or stone, mucus of the type usually used for adhesion when at rest is produced from the edges of the body to provide temporary anchorage during the muscular contractions. This mucus is very sticky and as the flatworm moves it is drawn out into strands 2-3 cm. long which stretch between stones and leaves or along a level substratum. The many single strands produced in this way tend to cross or coalesce to form a complex tangle which becomes a most ef- fective trap for small animals. Such deposits occur all over the natural habitat, and can be easily demonstrated in laboratory culture dishes by flooding with weak eosin (Fig. 11). Poly cells is gregarious and this habit results in a large amount of mucus being deposited within a given area. Animals with many appendages, such as crustaceans and insect larvae, are easily trapped in these mucus "snares" and become still more entangled in their struggle to escape. The flatworm does not lie in wait near the "snares" but is rapidly attracted by any disturbance in the water created by the struggles of the prey. Several individuals are often attracted to the same prey and their concerted efforts enable them to feed upon animals too large for a single individual. The prey is seized by the flatworm wrapping itself about it and covering it with large amounts of the sticky mucus. In this way even relatively large ani- mals such as gammarids are rapidly immobilized. There is no evidence of any toxic effects of the mucus since prey rescued after complete immobilization show no ill effects. Inert food does not stimulate the formation of much mucus; only the amount necessary for adhesion is secreted when such food is being eaten. After the prey is seized the pharynx is protruded and makes exploratory movements over the body surface. With arthropods it is eventually forced through some weak point in the exoskeleton, as between sclerites or the area of articulation of a limb, and rapidly sucks out the body contents (Fig. 10). The pharynx is very extensible and moves about within the prey, entering the head and larger appendages to draw out all the organs and musculature, so that virtually only the empty exoskeleton is finally left. With annelids the cuticle is breached and the body of the worm extracted, whilst with suitable carrion minute pieces are sucked off by the pharynx. The disintegra- tion of the food on its way to the gut is so rapid that it appears to be due entirely to mechanical disruption during its passage through the pharynx ; there is no evidence that digestive juices assist the process since the waves of muscular contraction in the pharynx invariably pass backwards towards the gut, never forwards as would be expected if such juices were being regurgitated. The efficacy of the process is indicated by the homogenized condition of the food as it arrives in the gut lumen. The structure of the gut and course of digestion The large cylindrical plicate pharynx lies in the pharyngeal chamber in the pos- terior half of the body and is directed backwards so that it can be protruded through the posterior central "mouth" by simple muscular elongation (Fig. 1). It is very muscular and contains both eosinophilic and basophilic mucus glands whose se- cretions help ingestion. The gastrodermis (Figs. 12 and 14) consists of a single layer of cells resting upon a delicate basement membrane. There are two types of cells, of which the 66 J. B. JENNINGS larger and more numerous are columnar, 35-40 fj. in height with basal vesicular nuclei ; their distal ends often bulge freely into the gut lumen and their basophilic cytoplasm is finely granular and may contain various eosinophilic inclusions. These inclusions disappear progressively after feeding and hence appear to repre- sent phagocytosed food undergoing intracellular digestion. The second type of gut cell, later referred to as "sphere cells" on account of their appearance, is 20-30 /A high and normally contains intensely staining spheres which appear to constitute a protein reserve. The finely divided condition and mixed nature of the normal food after ingestion made it difficult to determine whether phagocytosis was preceded by any intra- luminar break-up, and experimental feeding by the carrier technique was applied. ANT. EPID SUB.EPMUS. POST. FIGURE 1. Diagrammatic longitudinal sections of Poly cells to show the cylindrical plicate pharynx. A, The normal condition with pharynx retracted. B, Pharynx protruded for feed- ing ; ant. : anterior ; a.g.b. : anterior gut branch ; ev.phar. : everted pharynx ; m.g.b. : median gut branch ; mes. : mesenchyme ; phar. : pharynx ; post. : posterior ; p.g.b. : origin of posterior gut branches ; prot.phar. : protruded pharynx ; sub.ep.mus. : subepidermal muscles. The carriers used in this case were gammarids which were boiled before injection to ensure that no active enzymatic juices of the crustacean remained to attack the test food. The series of test foods was chosen with three objectives: (a) to fol- low the separate fates of carbohydrates, proteins and fats; (b) to supply unequivo- cal evidence of the presence or absence of intraluminar digestion; and (c) to de- termine the maximum size of particle which can be phagocytosed by the gut cells. ( 1 ) Experimental feeding with carbohydrates Polycelis readily ingested boiled starch paste, and sections prepared immediately after feeding and stained with Lugol's iodine showed the blue and unchanged starch lying in the gut lumen. The paste was quickly phagocytosed and twelve hours after feeding only a small amount remained in the lumen, quite unaltered and still stained blue with Lugol, indicating the absence of any intraluminar digestion of carbohy- FEEDING IN FREE-LIVING FLATWORMS 67 drates. The intracellular digestion of the phagocytosed starch was followed in saline squashes of fed flatworms. Two hours after feeding the columnar gut cells contained large amounts (Fig. 13), which stained with Lugol in various shades of blue and brown. As time passed all stained brown and then eventually disappeared as digestion was completed. Starch was also found in mesenchymal cells so that a certain amount of phagocytosed material passes back into the mesenchyme for digestion. Indicators mixed with the starch paste were unchanged in the gut lumen, giving further evidence of the absence of intraluminar digestion, but as the pH value after phagocytosis was 6.8-7, intracellular digestion of carbohydrates occurs in a neutral to slightly acid medium. Raw starch grains were ingested and readily phagocytosed if sufficiently small, and could be seen very clearly in sections by polarized light in the columnar cells. They were unaffected by the intracellular diastatic enzymes and were eventually rejected by the cells and returned to the gut lumen. In this case, therefore, no capacity for the selection of suitable food material appears to be possessed by the gut cells themselves. On the other hand, no grains above about 30 p were taken up and apparently this is the critical size above which phagocytosis is impossible. Later all the starch grains were expelled in the usual manner by taking in water through the pharynx and flushing out the gut contents by violent contractions of the body. (2) Experimental feeding with proteins Polycelis fed directly upon clotted frog and chick blood. The erythrocytes were quickly phagocytosed and could be seen in the columnar gut cells in saline squashes made thirty minutes after feeding. The number phagocytosed increased with time and six hours later almost every cell was packed full. Occasionally erythrocytes were also seen in mesenchymal cells. The erythrocytes quickly changed in ap- pearance after phagocytosis and condensed into homogeneous deeply staining spheres which decreased in size and number as digestion and absorption progressed (Fig. 14). They showed no signs of digestion in the gut lumen prior to phagocytosis. Chopped frog muscle stained with various indicators was fed to the flatworms by means of gammarid carriers. The particles reached the gut in recognizable pieces still showing their characteristic striations and the larger remained unchanged in size, shape and pH value until ejected from the gut some 24 hours later. This indication of the absence of intraluminar proteolysis was confirmed by feeding coagu- lated albumen, gelatin and fibrinogen, all of which likewise remained unchanged throughout their stay in the gut lumen. The smaller particles of chopped muscle were phagocytosed and neutral saline squashes made one hour after feeding showed many present in the columnar gut cells. Particles stained with brom-cresol green were then a clear blue in color (pH 5.2) but changed with time until six hours after feeding when they had become a clear green (pH 4.6), so that intracellular protein digestion proceeds in a distinctly acid medium. This color faded as the particles themselves became rounded and less distinct. Stained inclusions passing through the same series of color changes were occasionally found in mesenchymal cells. This indicator incidentally proved to be most attractive to these flatworms and they ingested large amounts of food stained by it ; particles of the indicator dropped into culture dishes attracted the flatworms to them and stimulated protrusion of the pharynx. 68 J. B. JENNINGS (3) Experimental feeding with fat Large amounts of cod liver oil suspensions stained with Sudan IV were readily ingested from carrier gammarids, often increasing the body volume of the flatworms by 40-50%. Saline squashes and frozen sections prepared thirty minutes after feeding showed some stained oil globules within the columnar cells, and the num- ber increased with time. Irrigation of the preparations with Nile Blue (Smith, 1907; George, 1951) showed the globules changing from red to deep blue as the stain penetrated the cells showing that much of the phagocytosed oil was undergoing intracellular lipolysis with the production of free fatty acids. Nothing suggesting intraluminar lipolysis was seen. Stained globules appeared early in the mesen- chymal cells, and later became much more numerous, but only a few ever showed any color reaction with the Nile Blue. Since the Sudan IV stains the fatty acid radical of fat molecules and remains with it through lipolysis and resynthesis, it appears probable that the red globules in the mesenchyme cells had mainly been broken down and re-formed in the gut cells into the flatworm's own particular type of fat and then passed into the mesenchymal cells for storage. Flatworms with this stored fat had a diffuse pink color which persisted for several weeks and faded only gradually as the fat was metabolized and the Sudan IV excreted. Saline squashes made six weeks after feeding still showed occasional red globules in the mesenchymal and columnar gut cells. It was not possible to determine the pH conditions attending intracellular lipolysis since indicators dissolved in the water of the cod liver oil suspension were not taken up by the cells. Suet particles were also fed. The largest particles remained in the gut unchanged in size and shape until ejected 24 hours later, confirming the absence of intraluminar lipolysis. The smaller particles were phagocytosed and digested intracellularly. The nature and location of the food reserves Polycclis forms large reserves of protein and fat, supplemented by some carbo- hydrate reserve, and these enable the flatworm to survive for at least three months without food. The gonads, mesenchyme and general body tissues constitute a protein reserve which is drawn upon during starvation, with a consequent reduction in body size, but there are also specific protein reserves in the sphere cells of the gastrodermis. These contain 8-16 small homogeneous concentrations which normally stain deeply with modified Millon, eosin and iron haematoxylin, but their appearance and stain- ing affinity varies with the nutritive state of the animal. In well-fed laboratory individuals, and in those collected out of doors in late summer and well nourished in preparation for winter, the spheres are all compact and densely staining. During starvation they become increasingly indistinct, stain poorly and finally disappear. At the same time a progressive reduction in the volume of both columnar and sphere cells results after about three months in a flattened gastrodermis devoid of any inclu- sions. The significance of the sphere cells as protein reserve can be demonstrated by comparing the proportion of sphere and columnar cells under a variety of nutri- tive conditions. These are summarized in Table I which is compiled from cell counts of every third section of a number of histological series. The fat reserve is laid clown as globules in the inner mesenchyme and columnar FEEDING IN FREE-LIVING FLATWORMS 69 gut cells. It decreases only slowly during starvation, and significant amounts still remain after three months without food. The carhohydrate reserve occurs as small irregular granules of glycogen, scat- tered throughout the mesenchyme and columnar cells. It decreases rapidly on starvation and disappears from the mesenchyme after about a fortnight. Small amounts persist in the gut cells, however, for much longer than this, but these may possibly result from the conversion of other reserves. A purely carbohydrate diet results in an enormous increase in the amount of glycogen in the mesenchyme and the flatworms, provided they were mature, survived as well on this diet as upon purely protein or fat diets. The epidermal and subepidermal (/lands and their relation to feeding These secretions can be divided into mucoid, giving a positive reaction with P.A.S. and Alcian Blue (Steedman, 1950), and the non-mucoid or rhabdoid, giving a negative reaction. The mucoid can be further differentiated into eosinophilic and basophilic. The latter is produced by scattered gland cells in the epidermis and outer mesenchyme, TABLE I The proportion of cell types in relation to nutrition Nutritive condition Ratio of "sphere" to columnar cells High protein diet 930:3723 = 1:4 Normal diet 625:3957-1:6.3 Starvation: 14 days 435:2957 == 1:6.7 Starvation : 28 days 371:3545 == 1:9.5 Starvation: 2 months 176:2668 == 1 : 15.5 Starvation: 3 months "Sphere cells" absent and gastrodermis syncytial and is used in gliding and to a minor extent in trapping prey as already mentioned. There are no large aggregations of basophilic glands as described in some other triclads. The much more adhesive eosinophilic mucus is produced both from scattered gland cells and from concentrations of mesenchymal gland cells, the "marginal adhesive glands" of Hyman (1951), situated along the borders of the ventral surface. During its discharge the epidermis becomes elevated into small temporary papillae so that the secretion is laid down in lines of oval deposits, from which are formed the main elements of the "snares." The non-mucoid rhabdoids are transparent greenish rods, 15-20 p, in length, pro- duced by mesenchyme cells and passed out to the epidermis. Once outside the body the rhabdoids take up many times their own volume of water ("hydrate") and fuse to form a semi-fluid gelatinous layer which closely invests the body. This material can be collected for examination in two ways. It can be removed from the living flatworm by drawing a fine glass needle a number of times across the dorsal surface of the body. Alternatively, immersion of the worm in strong sodium chloride solu- tion causes the discharge of enormous numbers of rhabdoids and at the same time inhibits their hydrolysis, so that they can be fixed in 95c/c alcohol for histological examination. Irrigation of unfixed rhabdoids with water causes them to hydrate rapidly (Figs. 15 and 16), producing the same sort of gelatinous sheet as invests 70 J. B. JENNINGS the normal flatworm. The lowest critical concentration of salt to give discharge without hyclration is about 3.75'/£ ; possibly hydration within the epidermal cells may be inhibited by some comparable combined effect of the cytoplasmic salts. The rhabdoid material is basic (pH 8.0-8.2), soluble in acids and alkalies, and stains strongly with Alillon ; apparently it is of a purely protein nature, as all fat and carbohydrate tests were negative. The hydrated material is not toxic to other animals, annelids and crustaceans surviving for long periods in contact with it, and it plays no part in feeding. It is distasteful to fish, pieces of earthworm smeared in it being rejected by sticklebacks, but its main function is probably mechanical, acting as a "fluid cuticle" to protect the epidermis from abrasion and bacterial and fungal attack whilst still permitting ciliary activity, and in life it is probably in a continual state of loss and renewal. There is also a rapid discharge of rhabdoids if a flat- worm is injured, to give a protective clot-like effect over the region of the wound. ACOELA Convoluta parado.va (2-3 mm. long and pear-shaped with a pronounced ventral curling of the broader anterior end) feeds upon small marine crustaceans, protozoa, diatoms and similar organisms which are captured by various methods, depending upon their size. Minute prey such as protozoa are captured by the flatworm gliding slowly over algal fronds or stones with the solid syncytial gut partially extruded through the mouth like a large pseudopodium and the food engulfed amoeboid fash- ion and passed back into the syncytium for digestion. Alternatively the flatworm may spend long resting periods attached to the substratum by the adhesive papillae of the tail and with the rest of the body slightly raised in a "sitting-up" position. If larger prey, such as crustaceans or their larvae, come sufficiently close, the flatworm rapidly extends its body and grasps the prey with the curved anterior margin, which is covered with abundant sticky mucus produced by the frontal and other subepidermal glands. The flatworm then curls up ventrally, starting from the anterior end, so that the prey is drawn beneath it and pressed into the mid-ventral mouth. This is very distensible and the food is ingested whole and intact. More rarely Convoluta captures actively swimming creatures whilst it is itself either swimming freely or gliding over the algal fronds, but in all cases capture and in- gestion are extremely rapid and occupy only 5-10 seconds. The flatworm shows marked avoiding reactions when dead food is encountered ; it feeds only upon living- organisms and does not act as a scavenger. The simple pharynx is a very short ciliated tube leading directly to the solid "gut"-— a central compact or vacuolated syncytium, one-third to one-half of which can be protruded through the mouth (Fig. 5). Its cytoplasm is finely granular and contains many vesicular nuclei. All prey when ingested is enclosed in temporary vacuoles which follow no definite path, but merely move back inside the animal to come to rest in any part of the syncytium. Occasionally vacuoles containing di- gesting prey may be found in the mesenchyme, but the cytoplasm about these vacu- oles is always distinct from the surrounding tissue and is apparently a temporarily isolated part of the digestive syncytium. As digestion progresses the food becomes disorganized and breaks up and in Crustacea only fragments of the exoskeleton re- main when digestion and absorption are complete, some 18-24 hours after feeding (Fig. 6). These indigestible residues then pass to the mouth to be thrown out. FEEDING IN FREE-LIVING FLATWORMS 71 It was not possible to determine the pH conditions controlling digestion as Conrohtta refused crustaceans stained with indicator. Attempts to recover food in various stages of digestion for pH examination also failed, as it inevitably became contami- nated with mesenchyme, mucus and sea water. Fat forms the principal food reserve in Coni'olitta, occurring in large amounts as globules of 2-3 p diameter in the outer, more compact mesenchyme and in the digestive syncytium. Small amounts of glycogen also occur as irregular granules scattered throughout the mesenchyme and digestive tissue. There are no specific protein reserves. RHABDOCOELA (1) Macrostomnin sp. (2-3 mm. long and 0.5 mm. broad) feeds upon minute fresh water crustaceans, annelids, nematodes, rotifers and large ciliate protozoans. The mouth and pharynx are capable of great distension and any small creature moving near the flatworm is seized and ingested whole. Only living food is taken and mucus plays no part in its capture. EPID. ANT SUB.EPMUS. POST. PHAR. GUT MES. FIGURE 2. Diagrammatic longitudinal section of Macrostoiiniin to show the simple pharynx. Abbreviations as in Figure 1. The ventral slit-like mouth opens into a simple ciliated pharynx longer than that of Conrolnta but still with no thickening of the underlying muscular layers (Fig. 2). Eosinophilic glands in the mesenchyme open into the pharynx but they are not mucus-producing and their precise function remains unknown. The gut is a simple unbranched sac extending almost the whole length of the body and its wall is made up of two kinds of cells, standing upon a thin basement membrane (Fig. 7). The larger and more numerous are columnar, some 30-40 p. tall and 5-6 /A broad, with basal vesicular nuclei and finely granular cytoplasm which may have inclusions, giving the cell a phagocytic appearance. The second type of cell is smaller and bears a close resemblance to the "sphere-cell" of the triclad gastrodermis. It is club-shaped, 1 5-20 //, tall and 3-4 /* broad distally, with the nucleus in the narrower basal region where the cytoplasm is often strongly basophilic. These cells con- tain 10-15 eosinophilic spheres which also stain with iron haematoxylin and modi- fied Millon. Sections of Macrostomnin fixed 15 minutes after feeding on DapJmia showed the intact crustaceans lying free in the gut. In others fixed 10 hours later digestion was well advanced, with the muscles and organs greatly disorganized, whilst 24 hours after feeding only the exoskeleton remained, but as the latter retained its general shape there can be little movement or contraction of the gut during digestion. 72 J. B. JENNINGS This was confirmed by observation of living flatworms. During digestion the columnar gut cells became shortened, swollen and indistinct from one another, prob- ably as a result of secretory activity or absorption of digested food, or both. The "sphere cells" remained unchanged. These observations confirmed the presence of intraluminar digestion in Macrostomum but after feeding upon Paramecium or small annelids almost all the gut cells showed discrete inclusions which were clearly particles phagocytosed from the disintegrating food mass in the gut lumen (Fig. 7). Thus digestion in Macrostomum must be a combination of intraluminar and intra- cellular processes, the latter occurring when the food in the lumen is sufficiently disintegrated for the particles to be phagocytosed. The small amount of phagocyto- sis and intracellular digestion seen with crustaceans may be due to the exoskeleton retaining most of the disintegrating material until it is rendered completely soluble. The pH conditions of digestion could not be determined as the flatworms refused to ingest prey stained with indicator. EPIQ SUB.EPMUS. GUT PHAR. B MES. EV. PHAR. FIGURE 3. Diagrammatic longitudinal sections of Mesostoina to show the bulbous pharynx. A, The normal condition with the pharynx retracted. B, Pharynx everted for feeding. Ab- breviations as in Figure 1. Small amounts of glycogen and fat occur in the columnar gut cells and in the mesenchyme, whilst the "sphere-cells" of the gut epithelium appear to constitute a definite protein reserve, since they disappear after 10-14 days' starvation. (2) Stcnostomum sp. (1.5-3 mm. long and very slender) feeds mainly upon large ciliates and rotifers, which are seized in the anterior ventral mouth and en- gulfed whole. These animals are usually found by chance but the flatworm may .search actively for them, crawling slowly forwards with the anterior end slightly raised and the mouth wide open. The latter is capable of great distension and is fringed with large cilia which probably help to sweep the prey back into the pharynx, where it is retained a few seconds for examination. If unsuitable it is rejected, but if acceptable the mouth is closed and the pharynx contracts violently to force the food into the gut. The pharynx is a simple ciliated tube, similar to that of Macro- stomum, with some basophilic gland cells producing mucus to help ingestion. The FEEDING IX FREE-LIVING FLATWORMS 73 gut is a simple blind sac extending almost to the posterior end of the body, its anterior end is contracted and open communication with the pharynx exists only during actual ingestion. The gastroclermis is of unciliated club-shaped cells, 20—25 fj. tall and 6-8 p. broad distally with basal nuclei and granular inclusions. When ingestion is complete, and the pharynx again closed ofY, the gut contracts violently and the food is driven to and fro in the lumen (Fig. 9). Ciliates are broken up within fifteen minutes, and indeed are often disintegrated by the pharynx during ingestion. Rotifers resist longer but eventually their body contents are squeezed out through the mouth or some weak spot in the integument and the empty cuticle ejected. The resultant particles are phagocytosed by the gut cells and show for a time as eosinophilic inclusions until intracellular digestion is completed. Oc- casionally Paraincciiiin stained with indicators covering a pH range of 3.5—8.4 were accepted but during their disintegration the pH remained unaltered, showing the absence of intraluminar digestive activity. ANT EPID. SUB.EPMUS. M.G.B. PHAR. B MES. PRO! PHAR. FIGURE 4. Diagrammatic longitudinal section of Leptoplana to show the ruffled plicate pharynx. A, The normal condition with pharynx retracted. B, The pharynx protruded to envelop food. Ahbreviations as in Figure 1. Fat forms the only food reserve in Stenostoinuin, occurring as small globules in the gut cells. The amount decreases rapidly and little remains after a week, which is about the maximum period the flatworm can endure without food. (3) Mesostoina tctnn/onuin (5-8 mm. long and 1-2 mm. broad) feeds upon small oligochaetes, crustaceans and insect larvae, which are captured directly by the flatworm wrapping itself around them. The bulbous pharynx is then protruded through the median ventral mouth (Fig. 3) and applied to the prey's body wall. Strong sucking movements made by alternate contractions and expansions of the radial musculature of the pharynx draw small prey bodily into the gut (Fig. 8). Larger animals are held until the body wall ruptures and the pharynx can be thrust into the body to suck out the contents, but in a somewhat inefficient manner so that frequently only the body fluids are withdrawn. Mesostouia is also a 74 J. B. JENNINGS fr^KrefEBM *••#"-' •;.' * ' r** *» * I 1 1 1 FIGURES 5-10. FEEDING IN FREE-LIVING FLATWOKMS 75 scavenger and will feed upon any sort of dead prey, if not excessively decomposed. The gut is a simple sac and the gastrodermis is syncytial, 15-20 ^ thick, often with spherical inclusions. The ingestion of whole prey implied that digestion in Mesostoina is at least partially intraluminar and this was confirmed by experimental feeding with clotted frog blood. The erythrocytes are largely broken up within thirty minutes, though their freed nuclei are still distinct, but there is no evidence of phagocytosis by the gut cells during this initial period. After three hours the material in the gut lumen has become a homogeneous mass, though a light reaction with Feulgen indi- cates that the ruptured nuclei are not yet entirely decomposed, and as this stage is reached material of similar appearance begins to show as small spheres within the gut cells. After six hours the lumen was empty and the gut cells packed with spheres, which in turn decreased and disappeared within 24 hours. Intracellular digestion in Mesostoina is therefore preceded by some degree of intraluminar digestion, but details are unknown as no successful method of feeding with indi- cators could be found. POLYCLADIDA (1) Cycloporus papillosus (1-2 cm. long and broadly oval) is usually found attached by its single ventral sucker to the surface of the encrusting colonial tuni- cates Botryllus and Botrylloides, and it appears to feed exclusively upon these ani- mals. The anterior cylindrical plicate pharynx, shorter but otherwise resembling that of Polyeelis, is protruded forwards and thrust down into the colony to suck out individual zooids. The pharynx leads into the median gut branch which gives off 6-8 pairs of lateral diverticula, subdividing in turn to give the typical polyclad arrangement. The distal gut branches lead up to large "anal chambers," ranged along the margins and opening by pores through the epidermis. In histological preparations the branches and anal chambers always appear closed off from each other, and no actual passage outwards of gut material has ever been found ; possibly temporary openings may develop for the passage of excess water taken in with the food. The majority of the gastrodermal cells are columnar, 35-40 p. tall and 5-8 p. wide, with their free margins ciliated, the cilia being particularly strong in the median diverticulum (Fig. 17). There is, however, an anomalous region of columnar cells at the posterior end of this diverticulum where the cilia are replaced by what ap- pears to be a "brush border." Scattered amongst the columnar cells are smaller FIGURE 5. Longitudinal section of C oiii'oliitii showing the central vacuolated syncytium. Haematoxylin and eosin. Scale 0.25 mm. FIGURE 6. Transverse section of Convoluta showing a recently ingested intact crustacean and one completely digested except for the exoskeleton. Haematoxylin and eosin. Scale 0.1 mm. FIGURE 7. Longitudinal section of Macrostomum showing part of the simple pharynx (left) and the gastrodermis with small, club-shaped "sphere cells" between the long, projecting phagocytic cells. Haematoxylin and eosin. Scale 20 JJL. FIGURE 8. Mesostoina ingesting an annelid. From life. Scale 1 mm. FIGURE 9. Stcnostoiintin immediately after ingesting a Paraincciinn stained with neutral red. The gut is beginning to contract to break up the ciliate. From life. Scale 0.4 mm. FIGURE 10. Polyeelis killed whilst feeding on a large Dafhnia. Note the protruded pharynx. Steinmann's fixative and borax carmine. Scale 1 mm. 76 J. B. JENNINGS II *^ % * i «» *• TS; ' -ff * 4 *» * k* /* ^: * /J * S 13 t i^ ^ xf V . x/ \ ^ •a. FIGURES 11-17. FEEDING IN FREE-LIVING FLATWORMS numbers of spherical glandular cells, up to 45 /x, in diameter, their cytoplasm laden with tiny granules. These cells occur in various sizes, the smaller appearing be- tween the bases of the columnar cells and the larger nearer the lumen ; occasionally they were seen free in the lumen, and their disintegrated remains were found both here and between the bases of the columnar cells. Their phases of growth and disintegration, however, could not be related in any way to the condition or amount of food in the gut and it is not at all certain that they are concerned in digestion. The columnar cells themselves, on the other hand, do appear to secrete juices into the lumen, becoming swollen and vacuolated soon after feeding. In feeding, the tunicate zooids reach the gut almost undamaged ; histological examination at intervals after an observed feed shows a progressive homogenization of the food, leading to complete absorption after about twelve hours. At no time is there any indication of other than intraluminar digestion. Confirmation came from feeding starved Cycloporus on Botryllus colonies injected with starch paste; Lugol sections showed digestion confined within the lumen, with unchanged starch never ap- pearing within the gut cells. Food containing indicators, however, was consistently refused. Fat globules in the ciliated cells and mesenchyme constitute the only significant food reserve. Only minute amounts of glycogen occur in the mesenchyme and there are no specific protein reserves. The flat worm cannot withstand more than about seven days' starvation. Only basophilic mucus glands are present, scattered throughout the epidermis and ventral mesenchyme, and mucus plays no part in feeding. Rhabdoid-producing cells are common in the mesenchyme, particularly between the posterior gut diver- ticula, and their cycle of activity can be followed in its entirety. Immature cells contain small eosinophilic granules and rods which enlarge into the fully formed rhabdoids. As these are discharged the cells shrink to a fraction of their former size and migrate into the nearest gut cliverticulum ; there they disintegrate and are presumably expelled with the unwanted food material. The discharged rhabdoids themselves follow one of two courses. Some pass to the epidermal cells and so to the exterior in the usual way. The experimental removal and examination of these rhabdoids gave results identical to those reported for Polycelis. Others aggregate to form conspicuous tracts, greyish white in the living flatworm, which converge upon the gonopore. The rhabdoids pass through or between the cells of the gonopore wall and are discharged with the eggs. Sections of the egg masses show the individual eggs to be embedded in a gelatinous matrix which has properties similar to those of hydrated rhabdoids. The rhabdoids are not concerned in any FIGURE 11. Mucus "snares" produced by Polycelis. Alcian blue. Scale 0.5 mm . FIGURE 12. Polycelis. Transverse section of gut diverticulum showing columnar phago- cytic cells and smaller heavily staining "sphere cells." Iron haematoxylin. Scale 40 /u. FIGURE 13. Polycelis. An isolated columnar cell with phagocytosed starch. From a saline squash. Lugol. Scale 40 p. FIGURE 14. Polycelis. Transverse section of the gut diverticulum 24 hours after feeding on chick blood, showing phagocytosed erythrocytes undergoing intracellular digestion. Iron haematoxylin. Scale 40 /*. FIGURE 15. Polycelis. Rhabdoids discharged after immersion in 5% aqueous sodium chloride. Scale 50 n. FIGURE 16. Polycelis. Rhabdoids discharged as in Figure 15, then irrigated with tap water. Scale 50 /u. FIGURE 17. Cycloporus. Longitudinal section of the median gut branch showing ciliated gastrodermis. Haematoxylin and eosin. Scale 40 M- 78 J. B. JENNINGS way in the formation of the egg membranes since these stain strongly with P.A.S. and are already fully formed in the oviduct well above the point of entry of the rhabdoids. (2) Leptoplana tremellaris (2-2.5 cm. long and pear-shaped) feeds upon poly- chaetes, isopods and amphipods which are captured directly without any trapping devices. The prey is seized by the anterior part of the flatworm's body and wrapped round by the expanded body margins. The flat worm then curls up and passes the prey back along the ventral surface to the median mouth. The pharynx is of the ruffled plicate type (Fig. 4), consisting of a large thin convoluted oval curtain suspended from the roof of the peripharyngeal chamber, and is protruded through the mouth to envelop the prey. Small animals are ingested whole, either by simple peristalsis or by the pharynx being drawn back within the mouth, a process requiring five minutes. Larger organisms are enveloped as far as possible and the pharynx is retracted slightly so that the prey is partially ingested ; the flat- worm then presses itself down on the substratum and the pharynx makes strong sucking movements which are often supplemented by movements of the whole body. This process may continue for an hour or more, whilst the prey is disintegrated and ingested piece by piece or has its integument ruptured and the contents squeezed out. Leptoplana also feeds upon dead animal material provided it is not too decomposed. The gut is of the usual polyclad form but the gastrodermis here consists of un- ciliated vacuolar columnar cells. In well-fed individuals these are indistinct in out- line or even syncytial and the cytoplasm loaded with eosinophilic spheres which disappear on starvation. This appearance of phagocytic activity was confirmed by experimental feeding with frog blood and small pieces of Littorina muscle. The former were rapidly taken up by the gut cells and their intracellular digestion and disappearance easily followed. The small pieces of muscle entered the gut entire but subsequent changes in their histological appearance showed that they were partially broken down by enzymatic activity in the gut lumen before entering the gut cells, where their fate follows that of the usual eosinophilic inclusions. In this case, therefore, intraluminar digestion is incomplete and is succeeded by phagocytosis and intracellular digestion. The possibility that enzymes from the gut could be regurgitated to assist disin- tegration in the protruded pharynx was investigated by using pieces of Littorina foot muscle too large to be ingested whole. These pieces were removed from the flatworm's pharynx after a series of time intervals and checked against control muscle for pH and enzymatic changes. Control muscle was slightly acid (pH 6.6-6.8), but after ten minutes decreased to 5.4 and after twenty to 4.5. For pro- teolytic activity the muscle was laid on gelatin-coated slides and half an hour al- lowed for any enzyme to diffuse out. The gelatin below and around a piece of muscle which had been in the pharynx for a quarter of an hour was dissolved away and the holes formed in the gelatin film showed clearly after staining the slide with eosin. Control muscle had no such effect. Corresponding acidity and enzyme production in the gut lumen can therefore be reasonably inferred. Fat again forms the only significant food reserve, and what has been said of Cycloporus about mucus and rhabdoids applies also to Leptoplana. FEEDING IN FREE-LIVING FLATWORMS 79 DISCUSSION The general pattern of flatworm nutrition consists more of a series of intimate relationships between the nature of the food, the structure and function of the pharynx, and the course of digestion, than of a simple increase in complexity from primitive to more elaborate forms. Possibly the "amoeboid" method of feeding in the Acoelan Convoluta is primitive to the group (cf. Hyman, 1951), and no longer found in other forms. Certainly the alternative method in Convoluta of using the body as a whole to envelop the prey is still retained in one form or another by the majority of flatworms, with its effectiveness enhanced by elaboration of the pharynx. The limitation of the simple pharynx, found also in the rhabdocoels Macrostomum and Stenostomum, is that prey has to be engulfed whole and its size is thereby restricted. In contrast, the bulbous pharynx of the rhabdocoel Meso- stoma can not only ingest small animals but can be forced into larger animals to suck out their contents, though little triturating effect is possible. The cylindrical plicate pharynx of the triclad Polycclis is used in a similar but much more effective manner, the whole contents of the prey being withdrawn and discharged into the gut in a finely divided condition. The comparable pharynx of the polyclad Cycloporns, however, is used to deliver largely undisrupted food into the gut, the difference being reflected in the subsequent course of digestion. The ruffled plicate pharynx of the polyclad Leptoplana, though developmentally akin to the last, is used rather as an extension of the gut in which preliminary breakdown is enzymatic instead of mechanical. Thus the elaboration of the pharynx has made available to the flatworms an increasing range in the size and variety of their food. Mucus plays a minor part in the feeding of the majority, but does serve to hold the prey and facilitate ingestion. In Polycelis alone it is vised for the actual capture of prey. The course of digestion is linked with the feeding mechanism, since this deter- mines the condition of the food on entry to the gut. In Convoluta the position is anomalous in that digestion in its gut syncytium might be looked upon either as intra- cellular or as occurring within temporary lumina. In Polycclis the food is very finely divided by the pharynx and the particles taken in by phagocytosis for intra- cellular digestion. No evidence of intraluminar digestion was found in this species, either of mixed food or of the three food elements fed separately, in agreement with Willier, Hyman and Rifenburgh (1925) and Kelley (1931) ; the contrary opinion of Arnold (1910) was based on the erroneous supposition that the "sphere cells" of the gut were glandular and discharged into the lumen. Some evidence of intracellular digestion in mesenchyme cells was observed, of food apparently re- ceived indirectly from the endoderm cells. In striking contrast is the purely intra- luminar digestion of Cycloporus, which shows that the flatworms are capable of evolving this more advanced process. In all the other types examined some pre- liminary breakdown of the food, either by mechanical or enzymatic means, occurred in the pharynx or in the gut lumen, to be followed by phagocytosis and intracellular digestion ; this preliminary breakdown should perhaps be interpreted as an adapta- tion to allow the persistence of the more primitive form of digestion. Experimental feeding with proteins, carbohydrates and fats given separately showed that fully grown Polycelis can not only utilize and store all these food ele- ments but can survive upon any one indefinitely. Polycclis also forms normal food 80 J. B. JENNINGS reserves of all three types : specific protein reserves in the "sphere cells" of the gut, fat and glycogen in the gut and mesenchyme cells. Glycogen is the first to be used up and appears the least important. In the others fat forms the main food reserve, supplemented by protein in Macrostomum, but they are less prominent than in the triclad, and the capacity to withstand starvation is correspondingly reduced. I wish to thank Professor E. A. Spaul for suggesting this problem and for as- sistance, and Dr. T. Kerr for constant guidance and encouragement. SUMMARY 1. A study has been made of the food, feeding mechanisms, digestion and food storage in the triclad Polycelis cornuta, supplemented by observations on representa- tives of the other three flatworm orders. 2. Each form has a characteristic method of feeding, but in general the range of available prey has been greatly increased by elaborations in the structure and use of the pharynx. Mucus plays a minor part in feeding, except for the "snares" of the triclad. Rhabdoid material has no function in feeding. It forms a temporary cuticle and in polyclads a covering for the eggs. 3. In Polycelis mechanical breakdown of the food is followed by phagocytosis into the gut cells and intracellular digestion. Elsewhere the preliminary breakdown may be'wholly or in part enzymatic; only in Cycloporus is there purely intraluminar digestion with absorption in place of phagocytosis. 4. Polycelis can make use of all three food elements, and stores of each type normally occur. In the other forms food storage is less well developed though reserve fat at least is usually to be found. LITERATURE CITED ARNOLD, G., 1910. Intra-cellular and general digestive processes in Planariae. Quart J. Micro. Sci,, 54 : 207-220. BENSLEY, R. R., AND I. GERSH, 1933. Studies on cell structure by the freezing-drying method. Anat. Rec., 57 : 217-238. GEORGE, W. C., 1951. The recognition of lipolytic activity with the microscope. Stain Tech., 26: 175-176. HYMAN, L. H., 1951. The Invertebrates. Vol. II : Platyhelminthes and Rhynchocoela. McGraw-Hill Book Co. Inc., New York. KELLEY, E. G., 1931. The intracellular digestion of thymus nucleo-protein in triclad flatworms. Physiol Zool, 4: 515-541. SMITH, L., 1907. On the simultaneous staining of neutral fat and fatty acids by exazine dyes. /. Path. Bact., 12 : 1-4. STEEDMAN, H. F., 1950. Alcian Blue 8G.S. A new stain for mucin. Quart. J. Micro. Sci., 91 : 477-479. WILLIER, B. H., L. H. HYMAN AND S. A. RIFENBURGH, 1925. A histochemical study of in- tracellular digestion in triclad flatworms. /. Morph., 40: 299-340. YONGE, C. M., 1954. Tabulae Biologicae. Vol. XXI, parts 3 and 4. VARIATION OF NITROGEN AND CARBOHYDRATE CONSTITU- ENTS DURING THE DEVELOPMENT OF HIMANTHALIA ELONGATA (L.) S. F. GRAY RAYMOND F. JONES Botany Department, King's College, Nezvcastle upon Tyne, England Seasonal variations in the chemical composition of brown algae have been stud- ied by various workers (Lapicque, 1919; Lunde, 1940; Black, 194Sa, 1948b, 1949, 1950a, 1950b, 1954a, 1954b ; Channing and Young, 1952, 1953 ; 0y, 1951 ; Smith and Gordon Young. 1953; Wort, 1955). Black and Dewar (1949) correlated the physical and chemical properties of the sea water with the chemical constitution of algae. In all these investigations, however, no indication of the age, stage of de- velopment or sex of the plants is given. Moss (1950) found that marked changes in the chemical composition of Fitcns vesiculosus were associated with the development of the reproductive struc- tures. In later work Moss (1952) noted that during the Spring a variation in chemical constituents occurred in several developmental stages of Himanthalia donga to collected at the same time from the same habitat. Black (1954a) has shown that at certain times of the year gradients of carbohy- drates, proteins and mineral matter occurred along the frond of Laminaria sac- charina. Studying the variation of nitrogen in F. vesiculosus and L. saccharina, Jacobi (1954) observed horizontal and vertical gradients of nitrogen, water con- tent and fresh weight. During the time of sporulation the frond of L. saccharina showed a higher percentage of soluble nitrogen than during the time of vegetative growth. Jacobi suggested that ontogenetic factors were more decisive in bringing about variations in chemical composition of brown algae than the availability of nutrients in the sea water as postulated by Black and Dewar (1949). For Himanthalia elongata similar variations in chemical constituents occur along the length of the plant (Jones, 1956). The same author noted that the mature vege- tative tissues of the plant were lower in nitrogen content than the actively growing- reproductive tissues, and that the male receptacles were higher in nitrogen content than the female receptacles. No work appears to have been carried out to determine the variation of chemical constituents during the growth and development of brown algae from germination to maturity and senescence. For siich a study, therefore, the brown alga Hinian- tltalia elongata has been selected, for it enables an independent study to be made of the variation in chemical composition of both vegetative and reproductive tissues during the growth and development of the plant. MATERIALS AND METHODS Collection of material Samples of Himanthalia elongata were collected from St. Mary's Island, North- umberland, at low water as the tide receded. All samples were returned to the 81 82 RAYMOND F. JONES laboratory in large glass vessels, thus ensuring that the plants, prior to analysis, were fully turgid. During 1954, the following major stages of growth and develop- ment of the plant were investigated : 1) The development of the young vegetative buttons until they produced re- ceptacles at the end of the year. At St. Mary's Island, the young receptacles made their appearance during September to October. 2) The maturation of the plants in their second year of growth. During this period the receptacles grow rapidly in the summer months and develop conceptacles containing the gametes. For analysis, samples were sorted into their respective stages of development. Each sample contained at least 100 individuals. Wherever possible the vegetative buttons were separated from the receptacles and analyzed separately so that varia- tions in chemical composition throughout the development of both vegetative and reproductive tissues could be studied. Because of the difference in nitrogen con- tent of male and female receptacles of Himanthalia (Jones, 1956), for the mature plants only female ones were used. Analytical procedure Methods of analysis were those described previously (Jones, 1956). Fucoidin as combined L-fucose, however, was estimated by the improved method of Black, Cornhill, Dewar, Percival and Ross (1950). Expression of experimental results For the purpose of this study it was considered that the results would lend them- selves best for interpretation if they were expressed on a unit plant basis. RESULTS 1. Young developing plants During the development of the young vegetative button there is a gradual in- crease in fresh and dry weight until the young receptacles make their appearance in October (Table I). Thereafter the weight of the button remains comparatively steady. The receptacles, however, increase in both fresh and dry weight after their initial appearance. The total nitrogen of the young plants gradually increases throughout the year. In the button the protein content steadily increases, while the non-protein nitrogen increases with the growth of the button until October, when, at the time of receptacle initiation, it falls appreciably (Fig. 1). The relationship between the protein and non-protein is illustrated in Figure 2. During the period January to August the ratio rapidly decreases, suggesting a building-up of non-protein constituents. From August to the end of the year, the ratio increases indicating a synthesis of protein. This synthesis precedes the development of the young receptacles, which are themselves rich in protein and non-protein nitrogen. The build-up of a solu- ble non-protein reserve, of which peptide nitrogen is the chief constituent, is there- fore clearly indicated during the early stages of the development of the button. DEVELOPMENT OF HIMANTHALIA 83 TABLE I Variation of constituents during the development of young plants, 1954 Weight of plants Nitrogenous constituents Carbohydrate constituents gm./unit mg./unit plant mg./unit plant T* * 1 plant Total Month ash Fresh Dry Total N Protein N Non- protein Volatile base Free amino Pep- tide Alginic acid Man- nitol Lami- narin Fu- coidin mg./unit plant January B 0.54 0.07 1.02 0.87 0.15 0.04 0.04 0.07 13.9 3.9 1.4 5.1 28.0 February B 0.72 0.11 1.48 1.25 0.23 0.06 0.06 0.10 — 12.1 — — — March B — — — — — — — — — — — — — April B 0.78 0.14 2.02 1.68 0.34 0.08 0.09 0.15 26.1 13.6 2.7 10.0 56.0 May B — — — — — — — — — — — — — June B 1.69 0.24 3.71 2.79 0.92 0.31 0.15 0.35 43.8 32.4 5.1 18.6 91.0 July B 2.00 0.32 4.62 3.58 1.04 0.45 0.24 0.61 56.0 41.5 7.0 20.5 112.0 August 1.80 0.31 4.31 3.06 1.25 0.38 0.15 0.50 58.3 43.1 12.3 28.0 108.0 September B 1.96 0.38 4.67 3.67 1.00 0.36 0.19 0.50 64.8 47.8 34.5 31.4 110.0 October B 1.62 0.33 4.44 3.69 0.75 0.21 0.25 0.31 71.5 36.6 27.0 20.7 97.0 November B 1.76 0.36 5.16 4.24 0.92 0.17 0.14 0.33 82.0 25.1 21.5 22.5 120.0 November R 0.15 0.03 0.56 0.33 0.23 0.02 0.02 0.03 4.0 2.0 — 1.7 8.0 December B 2.00 0.36 5.32 4.58 0.74 — — — 83.0 24.4 11.1 26.0 127.0 December R 0.32 0.04 0.91 0.53 0.28 ' 6.6 2.4 0.5 2.6 13.0 B = Button. R = Receptacles. Throughout the growth of the young plant the alginic acid content increases even after the production of the receptacles. The other carbohydrate substances, namely mannitol, laminarin and fucoidin gradually increase during the early months of the year, then become more concentrated during the summer months, no doubt the result of active photosynthesis. Between August and October the vegetative but- Button o—o Receptacles O N M A O N 0 FIG.I J JY A FIG.Z FIGURE 1. Variation in nitrogen during development of young plants. FIGURE 2. Ratio protein : non-protein nitrogen during development of young plants. 84 RAYMOND F. JONES tons exhibit a fluctuation in carbohydrates associated with the production of the re- ceptacles. It is seen that the laminarin falls to a much lower level after receptacle formation in October. A similar fall in mannitol and fucoidin also occurs at this time. In the young receptacles the mannitol and fucoidin increase during the last two months of the year. These latter results indicate the building-up of a reserve of carbohydrates throughout the early months of the year, which, with the increased photosynthesis during the summer, reaches a high concentration in the button. However, with the advance of the winter months this reserve is utilized for the rapid development of 60_ o40- fact m 20- (— I O- i o. a 8o_ t 7O- ? 60_ MANNITO _ 3 _ 2 Button Receptacles m c z 5- 4 _ o h- < 3-1 or STAGES OF DEVELOPMENT ABC D M J JY FIG. 3. FIG.4. FIGURE 3. Variation in carbohydrates during development of young plants. FIGURE 4. Variation in the ratio protein : non-protein nitrogen during development of re- ceptacles in their second year of growth. A : Period of slow growth ; conceptacles absent. B : Period of steady growth and development of conceptacles. C : Period of rapid growth and development of gametes. D : Cessation of growth, extrusion of gametes, and breakdown of receptacles. the receptacle initials which, by repeated apical growth, give rise to the reproductive thongs. Concomitant with this there is, in the button, a fall in mannitol, fucoidin and laminarin (Fig. 3). The receptacles, after their appearance, carry on their own metabolic activity and synthesize their own carbohydrates. The ash content of Himanthalia, like that of most marine algae, is considerably higher than that found for land plants. With the growth of the young plants there is a relatively high rate of accumulation of minerals from the surrounding sea water, particularly during the early months of development. 2. The mature plants In the mature plants, during the second year of growth, the button remains rela- tively constant in fresh and dry weight (Table II). The receptacles, however, ex- DEVELOPMENT OF HIMANTHALIA 85 TABLE II Variation of constituents during development of mature plants, 1954 ( The vegetative button) Weight of plants Nitrogenous constituents Carbohydrate constituents gm./unit mg./unit plant mg./unit plant plant Total Month ash Fresh Dry Total N Protein N Non- protein N Volatile base N Free amino N Pep- tide N Alginic acid Man- nitol Lami- narin Fu- coidin mg./unit plant January 2.19 0.39 4.95 4.07 0.88 0.21 0.21 0.53 83.3 39.0 18.0 42.4 125.0 February 2.51 0.45 5.68 4.77 0.71 0.15 0.17 0.42 — 37.8 42.6 162.0 March 2.17 0.40 5.40 4.47 0.83 0.16 0.16 0.38 88.4 22.1 — 23.5 147.0 April 1.88 0.38 4.58 4.00 0.58 0.12 0.12 0.33 88.7 25.6 8.1 — 13S.O May 2.17 0.40 5.02 4.29 0.73 0.13 0.22 0.40 88.7 28.5 — 23.3 144.0 Tune 1.97 0.37 4.92 4.29 0.63 0.13 0.20 0.31 83.4 32.8 — 27.9 138.0 July 1.95 0.40 5.60 5.06 0.54 0.12 0.21 0.32 88.0 30.6 — — 1 16.0 August 1.95 0.38 5.41 4.85 0.56 0.10 0.19 0.25 84.0 29.1 9.5 33.9 105.0 September — — — — — — — — — — — — — October 1.81 0.35 4.74 4.23 0.51 0.11 0.26 0.20 77.5 21.5 — — 100.0 November 1.62 0.33 4.81 4.37 0.44 0.13 0.18 0.18 75.2 20.9 7.3 30.5 95.0 December 1.64 0.33 4.77 4.32 0.45 — hibit marked changes. During the early months of the year, growth of the re- ceptacles, as determined by the fresh and dry weights, is slow, but steady. In the spring and early summer there is an increase in both fresh and dry weights, reach- ing a climax during the month of July, when the fresh weight of the receptacles has increased some five-fold. This increase is not due to water uptake and turgor ex- tension of the cells alone because a similar five-fold increase in the dry weight also occurs (Table III). After the month of July, with the approach of the winter months, the fresh and dry weights of the receptacles begin to fall. From October to December this decrease is rapid. The receptacles undergo several anatomical changes during this period of growth. For the first two months of the year the re- ceptacle tissue is non-fertile. From March to May, conceptacles develop. From May to July there is a rapid development of conceptacles, terminating in the pro- TABLE III Variation of constituents during development of mature plants, 1954 (The receptacles] Weight of plants Nitrogenous constituents Carbohydrate constituents gm./unit plant mg./unit plant mg./unit plant Total Month ash Fresh Dry Total N Protein N Non- protein N Volatile base N Free amino N Pep- tide N Alginic acid Man- nitol Lami- narin Fu- coidin mg. /unit plant January 0.67 0.08 2.24 1.56 0.68 0.07 0.08 0.23 . February- 1.19 0.14 3.85 2.50 1.35 0.10 0.15 0.42 — 11.2 — — — March 1.58 0.19 5.32 3.73 1.59 0.14 0.22 0.60 30.6 18.7 2.2 6.3 81.0 April 2.14 0.27 7.63 5.65 1.98 0.17 0.32 0.60 34.6 23.2 2.6 10.8 134.0 May 9.76 0.87 24.57 19.11 5.46 1.04 1.16 1.91 143.9 59.4 6.9 28.5 446.0 June 26.24 2.81 78.51 55.38 23.13 7.64 3.85 8.46 477.4 152.5 31.8 82.9 1324.0 July 112.80 14.97 347.00 231.14 115.86 40.00 21.26 41.62 2776.5 938.5 308.4 473.0 6719.0 August 101.50 13.37 251.49 182.50 68.99 14.97 11.17 23.40 2535.0 1458.8 266.0 764.8 6000.0 September 95.80 13.89 243.91 189.46 54.45 12.83 10.69 21.81 2868.3 830.1 314.3 923.5 6376.0 October 89.50 12.28 203.47 169.21 34.26 7.00 9.70 11.79 2773.5 746.6 391.7 — 5511.0 November 58.62 7.60 126.38 105.81 21.20 3.57 3.80 8.44 2009.4 266.0 195.3 — 3405.0 December 16.00 2.05 34.60 29.86 4.74 — — — 540.8 51.3 43.3 — 906.0 86 RAYMOND F. JONES duction of antheridia and oogonia. By August the receptacles are "ripe" through- out their entire length, except for some 5 to 6 cm. above the button which remains sterile. Growth ceases and there is a gradual extrusion of the gametes in a basi- petal sequence. Disintegration from the tips of the receptacles then follows. This occurs comparatively slowly over the months of August to October, but thereafter very quickly, so that by January of the next year only a few plants remain, the majority having completely disintegrated. Very little change, if any, occurs in the nitrogenous constituents of the button in the second season after it has reached maturity. Most of the nitrogen in the button is in the form of protein ; very little non-protein nitrogen is present. The receptacles show great fluctuations in their nitrogen content (Table III) . The protein and non- protein nitrogen increases steadily during the early months of the year, but from March to July there is a rapid increase, particularly in protein. From August to the end of the year the nitrogen content decreases. The ratio protein : non-protein nitrogen (Fig. 4) shows a correlation between the ratio and the stage of develop- ment reached by the receptacles. During the early months of the year when growth of the receptacles is slow but steady, the ratio gradually rises in favor of protein synthesis within the receptacles. Between May and July the ratio falls sharply and this is coincident with the period of rapid growth and development of gametes within the conceptacles. This suggests that the early synthesis of protein is associated with extra conceptacle tissue, and that, at a later date, this protein is broken down to soluble constituents and transported to the conceptacles where re-synthesis takes place in the production of the gametes, resulting in the increase in the ratio from August to the end of the year. From October onwards the gradual extrusion of the gametes and the wearing away of receptacle tissue keep pace with one another and the ratio continues to rise. It is seen that the peptide nitrogen exceeds the free a-amino nitrogen in the soluble non-protein constituents of the mature receptacles. During the production of the receptacles there was a proportion of the soluble non-protein nitrogen which could not be accounted for. Nitrate nitrogen could not be detected in the plants at any stage of development. It was therefore consid- ered to be some nitrogenous substance associated with reproduction such as nu- cleic acid. In the button the carbohydrates remain relatively constant throughout the year. Like the young vegetative button the "old" tissue remains high in alginic acid con- tent. The mannitol is low, similarly laminarin and fucoidin, the latter being higher than the laminarin and similar in magnitude to the mannitol. Throughout their growth the receptacles are particularly low in fucoidin and laminarin. Alginic acid is the major constituent of the receptacle tissue, mannitol being slightly lower. During the early months of the year there is little change in the content of alginic acid and mannitol. However, with increased photosynthesis in the spring and sum- mer months both these constituents increase rapidly. After the extrusion of the gametes the alginic acid and mannitol are considerably reduced. With the increased growth the accumulation of inorganic ions ,by the developing receptacles occurs during the months of January to July and thereafter falls following the disintegration of the tissue. The mature button remains comparatively uniform in mineral ash content throughout the year, although there is a tendency toward a falling-off during the latter part of the plant's existence. DEVELOPMENT OF HIMANTHALIA 87 GENERAL DISCUSSION During the present investigation, variations in chemical constituents have been followed throughout the development of H. elongata. Other workers have generally selected their plants at certain times of the year, or have used well established and fully developed plants for their investigations, failing to include the young stages. This is particularly the case where the grapnel has been used for collection, for this method selects only the larger algae. The number of individuals analyzed from one habitat is also of great importance. The results obtained by Black (1949, 1950b) for the Laminariaceae were based on results of the chemical analysis of two plants per month. Baardseth and Haug (1952) have since shown that certain con- stituents of marine algae vary considerably from one plant to another in a uniform TABLE IV Expression of results obtained for the receptacles of mature plants during the months of April and August 1954 Month Protein X Ash Alginic acid Marmitol Laminarin Fucoidin a. Results expressed as mg. per gm. dry weight April August 21.04 13.65 500.0 445.7 128.0 189.6 86.6 109.6 13.4 19.9 39.9 32.2 Month Protein X Ash Alginic acid Mannitol Laminarin Fucoidin b. Results expressed as mg. per unit plant April August 5.63 182.50 134.0 5959.0 34.6 2535.0 23.2 1458.8 2.6 266.0 10.8 430.5 population, even when collected from the same habitat. The ash and alginic acid contents of 55 L. digitata plants which they collected at one time showed variations as great as those obtained by Black throughout the whole year. The majority of studies concerning the chemical composition of marine algae have been carried out by chemists concerned with the possible utilization of algae for industrial purposes. These studies, based on the variation of chemical constitu- ents expressed as a percentage of the dry weight, have lead to considerable mis- conceptions of the physiological problems of growth and development in marine algae. For example, Black (1948a, 194Sb, 1949, 1950b) found that the Laminari- ales and the Fucales exhibit a decrease in "crude protein" content expressed as a percentage of the dry weight, during the period of rapid growth. He attributed this decrease of protein to the lack of nitrate in the sea water at that time of the year. One can hardly accept the statement made by Black ( 1950b, p. 52) that "rapid growth of the plant results in a decrease in the crude protein content." Where an increase in growth occurs, increase in protoplasm must be the determining factor. An increase in protoplasm with a decrease in protein is difficult to imagine, particularly as protein is one of the major components of protoplasm. RAYMOND F. JONES n:he time of maximum growth in Laminaria and Fucus the carbohydrates and other substances, particularly ash, are high. An increase in these other substances will result in an apparent decrease in the protein content expressed on a dry weight basis. Had Black expressed his results on a unit plant basis a truer picture of growth and the variations accompanying growth and development would have been made, as the examples for the receptacles of Himanthalia illustrate (Table IV). A decrease in protein, ash and fucoidin content between April and August occurs when the results are expressed on a dry weight basis. In the actual plant, however, these constituents have greatly increased over this period (Table IVb). As a result of such an increased synthesis of metabolic products the plant has increased in size, form and reproductive development. Parke (1948), in her studies on the growth of L. saccharina, found that the plant reached its maximum weight during June to July after the period of rapid growth. Plants were found reproducing at all months of the year, but the greatest number were found during October and March. Black (1948b, 1949) found that for the fucoids the maximum dry weight of the plants occurred during the period May to June. For Himanthalia, the present author recorded maximum fresh and dry weight values during the months July and August. At all these times of maximum weight the plants commence their reproductive phase, producing either spores or gametes. The development of the reproductive tissues will therefore exert a considerable influence upon the chemical composition of the plants. Moss (1950, 1952) showed that for F. vcsiculosus, and later for Himanthalia elongata, reproduction definitely influenced the chemical composition of the plants. Jacobi (1954) found that when F. vcsiculosus and L. saccharina were grown under favorable conditions, seasonal variations in nitrogen similar to those published by Black occurred, and that prior to reproduction a mobilization of protein was evident. In L. saccharina a higher percentage of soluble nitrogen was recorded for the reproductive tissue than the adjacent vegetative tissue. This lead Jacobi to con- clude that the increase in nitrogen content of the plant was most probably due to the development of the reproductive tissues which are higher in nitrogen during spore production or gamete formation. The present work on the change in chemical composition during the growth and development of Himanthalia substantiates the findings of Moss (1952) and Jacobi (1954). During the first year of growth the young vegetative plants gradually build up a reserve of nitrogen and carbohydrate constituents which are utilized for receptacle production. The young receptacles, after their initiation in October, continued to grow steadily until the following year, when, during the months of April to July, they grew very rapidly and reached maturity. The fucoidin, mannitol and laminarin, accumulated during the summer months in the young vegetative buttons, on the appearance of the young receptacles, were greatly depreciated, sug- gesting they were utilized in the production of reproductive tissues. Associated with the receptacle formation there was a rapid synthesis of protein from the non- protein constituents. In the button and also in the developing receptacles the alginic acid content continued to increase. After the establishment of the receptacles the composition of the vegetative button throughout the second year of growth remained relatively constant. With the increase in the sea temperature and the increased photosynthesis DEVELOPMENT OF HIMANTHALIA during the early summer months active metabolism resulted in the extensive growth of the receptacles. The two polysaccharides, fucoidin and laminarin, were very low during this period. Alginic acid, mannitol and mineral ash, however, continued to increase. Utilizing the inorganic nitrogen sources of the surrounding sea water, the receptacles synthesized amino acids and peptides which were later utilized in protein synthesis during the formation of the gametes in the conceptacles. The plant by August reached maturity and the gametes were extruded in a basipetal sequence. This process was accompanied with one of disintegration of receptacle tissue, resulting in a reduction in the chemical composition of the re- ceptacle, until, by December, little remained other than the sterile bases attached to the senescent button. The whole plant then quickly decomposed. The high ash content of the tissues, particularly of the receptacles, is of interest for the ash content of land plants is usually only represented by about 5 per cent of the dry matter of the plant (Thomas, 1949). Unlike land plants, marine algae are immersed in their nutritive medium, the sea water. In many cases seaweeds concentrate elements several thousand times the concentration found in the sur- rounding sea water (Black and Mitchell, 1952). The mineral ash consists of in- organic salts, presumably in solution in the cell sap, and cations in combination with such organic substances as alginic acid and fucoidin. Again the reproductive tissues exhibit differences from the vegetative tissues of the plant, insofar as they were found to be considerably higher in ash content. The receptacles contained as much as 50 per cent of their dry wreight in mineral ash. Similar high results were found for the receptacles of F. vcsicidosns (Moss, 1950), particularly when the re- ceptacles were ripe. The effect of the onset of reproduction upon the chemical composition of plants is not only to be found in marine algae. Variations in nitrogen with development of reproductive tissue have been found to occur in land plants. McCalla (1933), growing wheat in water cultures, found a decrease in nitrogen in the vegetative parts of the plant after the ears had emerged. These findings were later con- firmed by Miller (1939) who showed that from the end of May onwards the inflorescences of winter wheat were the only aerial parts of the plants to gain in nitrogen, whereas the stems and leaves lost their nitrogen progressively. Blanck, Giesecke and Heukeshoven (1933) and Blanck and Giesecke (1934) found that maturation of the oat plant was accompanied by a redistribution of nitrogen, which first decreased in the roots and later in the stems and leaves while, on the other hand, it accumulated in the inflorescences. In the barley plant Richards and Templeman (1936) also indicated a pronounced movement of nitrogen from the vegetative parts to the developing grain. It appears, therefore, that in the life history of Hinwnthalia elongata from germination to maturity and senescence separate parts of the plant undergo their own ontogenetic changes which greatly affect the chemical composition of the plant. The author wishes to thank Dr. Betty L. Moss, under whose supervision this work was carried out, for helpful advice in the preparation of the manuscript, and to express his appreciation of a research grant from the Institute of Seaweed Re- search which enabled the work to be undertaken. The author is also indebted to the Director, Dr. F. N. Woodward, for permission to publish. 90 RAYMOND F. JONES SUMMARY 1. Monthly variations in chemical constituents have been followed throughout the development of Hirnanthalia elongata. 2. Nitrogen, laminarin, mannitol, alginic acid and fucoidin increase during the first year's growth of the vegetative button. 3. Associated with receptacle initiation, the carbohydrate and nitrogen reserve of the button is utilized and there is a synthesis of protein from the non-protein constituents. 4. The young receptacles build up a reserve of non-protein materials which are later utilized in protein synthesis during the formation of the gametes. 5. It was concluded that during the life history of Hitnanthalia elongata from germination to maturity and senescence, separate parts of the plant undergo their own ontogenetic changes which are correlated with changes in the chemical composition. LITERATURE CITED BAARDSETH, E., AND A. HAUG, 1952. Individual variation of some constituents in brown algae and reliability of analytical results. First Int. Seaweed Synip. (Edin.}., p. 36. BLACK, W. A. P., 1948a. The seasonal variation in chemical constitution of some of the sub- littoral seaweeds common to Scotland. Parts I. II and III. /. Soc. Chcin. Ind. Lond., 67: 165-176. BLACK, W. A. P., 1948b. The seasonal variation in chemical constitution of some of the littoral seaweeds common to Scotland. Part I. Ascophyllum nodosum. J. Soc. Chem. Ind. Lond., 67 : 355-357. BLACK, W. A. P., 1949. The seasonal variation in chemical constitution of the littoral sea- weeds common to Scotland. Part II. /. Soc. Chem. Ind. Lond., 68 : 183-189. BLACK, W. A. P., 1950a. The effect of the depth of immersion on the chemical constitution of some of the sub-littoral seaweeds common in Scotland. /. Soc. Chem. Ind. Lond., 69: 161-165. BLACK, W. A. P., 1950b. The seasonal variation in the weight and chemical composition of the common British Laminariaceae. /. Mar. Biol. Ass. U. K., 29 : 45-72. BLACK, W. A. P., 1954a. Concentration gradients and their significance in Laminaria sac- charina (L.) Lamour. /. Mar. Biol. Ass. U. K., 33: 49-60. BLACK, W. A. P., 1954b. The seasonal variation in the combined L-fucose content of the common British Laminariaceae and Fucaceae. /. Sci. Food Agric., 5 : 445-448. BLACK, W. A. P., W. J. CORNHILL, E. T. DEWAR, E. G. V. PERCIVAL AND A. G. Ross, 1950. An improved method for the estimation of combined fucose in seaweeds. /. Soc. Chem. Ind. Lond., 69: 317-320. BLACK, W. A. P., AND E. T. DEWAR, 1949. Correlation of some of the physical and chemical properties of the sea with the chemical composition of the algae. /. Mar. Biol. Ass. U. K.,27: 673-699. BLACK, W. A. P., AND R. L. MITCHELL, 1952. Trace elements in the common brown algae and in the sea water. /. Mar. Biol. Ass. U. K., 30 : 575-584. BLANCK, E., AND F. GIESECKE, 1934. Zweiter Beitrag zur Frage nach dem zeitlichen Verlauf der Nahrstoffaufnahme des Hafers. /. Landiv., 82 : 33-59. BLANCK, E., F. GIESECKE AND W. HEUKESHOVEN, 1933. Ein vorlaufiger Beitrag zur Frage nach dem Verlauf der Nahrstoffaufnahme des Hafers warhend seiner Vegatationszeit. /. Landw., 81 : 91-103. CHANNING, D. M., AND G. T. YOUNG, 1952. Peptides and proteins of brown seaweeds. Chem. and Ind., p. 519. CHANNING, D. M., AND G. T. YOUNG, 1953. Amino acids and peptides. Part X. The nitro- genous constituents of some marine algae. /. Chem,. Soc. Lond., pp. 2481-2491. DEVELOPMENT OF HIMANTHALIA 91 JACOBI, G., 1954. Die Verteilung des Stickstoffs in Fucus vcsicitlosits und Laminar ia sac- charina und deren Abhangigkeit vom Jahresrhythmus. Kicler Meeresforsch., 10: 37-57. JONES, R. F., 1956. On the chemical composition of the brown alga Himanthalia cloni/ata (L.) S. F. Gray. Biol. Bull., 110: 169-178. LAPICQUE, L., 1919. Variations saisonieres dans la composition chimique des algues marine. C. R. Acad. Sci Paris, 169 : 1426-1428. LUNDE, G., 1940. Seaweed as a source of raw materials. Zeitschr. anyeiv. C'hcin., 50: 731-742. McCALLA, A. G., 1933. The effect of nitrogen nutrition on the protein and non-protein nitrogen of wheat. Canad. J. Res., 9 : 542-570. MILLER, E. C., 1939. A physiological study of the winter wheat plant at different stages of its development. Kans. Agric. Exp. Sta. Tech. Bull., 47 : 1-167. Moss, B. L., 1950. Studies in the genus Fucus. II. The anatomical structure and chemical composition of receptacles of Fucus vesiculosus from three contrasting habitats. Ann. Bot. Land., N. S., 14: 396-410. Moss, B. L., 1952. Variations in chemical composition during the development of Himanthalia clongata (L.) S. F. Gray. J. Mar. Biol. Ass. U. K., 31: 29-34. 0v, E., 1951. The nitrogen compounds in seaweeds. Tijdschr. for Kjcini Bergresen og Metallurgy, pp. 82-84. PARKE, M., 1948. Studies on the British Laminariaceae. I. Growth in Laminaria saccharina (L.) Lamour. /. Mar. Biol. Ass. U. K., 27: 651-709. RICHARDS, F. J., AND W. G. TEMPLEMAN, 1936. Physiological studies in plant nutrition IV. Nitrogen metabolism in relation to nutrient deficiency and age in leaves of barley. Ann. Bot. Land. N. S., 1 : 367-402. SMITH, D. G., AND E. GORDON YOUNG, 1953. On the nitrogenous constituents of Fucus vesiculosus. J. Biol. Chem., 205 : 849-858. THOMAS, M., 1949. Plant physiology. J. and J. Churchill, London. WORT, D. J., 1955. The seasonal variation in chemical composition of Macrocystis integrifolia and Nereocvstis luetkcana in British Columbia coastal water. /. Canad. Bot.. 33: 323-340. STUDIES ON SHELL FORMATION. VI. THE EFFECTS OF DINITROPHENOL ON MANTLE RESPIRATION AND SHELL DEPOSITION *• 2 SAMUEL P. MARONEY, JR.,3 ALBERT A. BARBER AND KARL M. WILBUR Department of Zoology, Duke University, Durham, N. C. and Duke University Marine Laboratory, Beaufort, N. C. Shell formation in mollusks concerns the elaboration of an organic framework or matrix and the orderly growth of calcium carbonate crystals within this matrix. This matrix is composed of three fractions : a water-soluble protein, a scleroprotein, and a polypeptide (Gregoire, Duchateau and Florkin, 1955). Synthesis and secretion of matrix and any active transport of inorganic ions involved in shell deposition will require metabolic energy. Glycolysis (Humphrey, 1950) and the utilization of tricarboxylic acid cycle substrates (Humphrey, 1947; Humphrey and Jeffrey, 1954; Cleland, 1951 ; Jodrey and Wilbur, 1955) have been demonstrated in oyster tissues and would lead to the formation of high energy phosphate compounds. If these high energy phosphate compounds are utilized in shell deposition, then dinitrophenol (DNP) by preventing phosphorylation (Hunter, 1951; Lardy and Wellman, 1953; Shacter, 1955) or reducing the tissue concentration of the com- pounds already formed (Hunter, 1951; Shacter, 1955) may be expected to retard shell deposition. This problem has been examined in the oyster Crassostrea virginica. The action of dinitrophenol was studied with respect to ( 1 ) respiration of shell-forming tissue; (2) calcium deposition using radioactive calcium as an indicator; and (3) shell regeneration in the whole oyster. METHODS Measurements of the effect of DNP on respiration were carried out by the Warburg method using a strip of tissue about one cm. wide taken from the posterior portion of the mantle. Endogenous respiration was measured in sea water for 30-60 minutes. DNP was then added from the sidearm, and measurements were continued for periods of one to six hours. DNP solutions were buffered at pH 8 with 0.03 M glycine. The isolated mantle-shell preparation (Hirata, 1953) was utilized for measure- ment of Ca45 deposition as described by Jodrey (1953). These mantle-shell prepar- ations were placed in one liter of sea water containing DNP for one hour. Ca45 (one ml. of high specific activity) was then added and the preparations remained in this solution for an additional six hours. Reversibility of DNP action on calcium deposition was tested by first immersing mantle-shell preparations in DNP solu- 1 Supported by a grant from the Office of Naval Research under Contract No. N7onr- 45505 with Duke University. 2 We wish to express our appreciation to Mr. Tadashi Tsujii for microscopic observations in the regeneration experiments. 3 Present address : Department of Biology, University of Virginia, Charlottesville, Virginia. 92 DNP AND SHELL DEPOSITION 93 I o o H Q- 5 CO o o CVJ o 80 - 6CH 20- 0 - -20 10 -6 IO"5 IO'4 DNP - MOLAR CONC 10-3 FIGURE 1. Effect of DNP on mantle respiration. Tissue weight, 180-220 mg. \vet weight; temperature, 25.6° C. ; pH 8.0; DNP, 0.2 ml. in sideann ; total volume 2.2 ml.; gas phase, air; salinity, 33.6-35.4 r/,r ; mean oxygen consumption of control, 15.6 /ul O., per 100 mg. wet weight per hour. tions for seven hours and then washing them in running sea water for two hours. The preparations were then placed in sea water containing Ca45 for six hours and the deposition of Ca45 was measured. Control preparations were carried through the same procedures but without DNP. TABLE I DNP effect on Ca46 deposition DNP cone. 11 DNP added counts/min.* a No DNP counts/min. p 10~3 M 5 35 ± 24 5 323 ± 179 <0.01 1CT4 M 12 69 ± 37 12 193 ± 143 <0.01 10~s M 9 376 ± 171 9 481 ± 205 0.3 > P > 0.2 Figures in columns three and five 'show mean calcium deposition by isolated mantle-shell preparations with standard deviations ; n gives the number of cases. The concentration of Ca45 added per liter varied from 5.25 /xc to 12.75 juc. For uniformity of presentation calcium deposition is expressed as counts per minute per 10 juc added per liter sea water per 6.2 cm.2 of shell. Tem- perature 25-26° C. ; salinity 31.7-34.0 °/oo- The experiments were not all carried out at the same time which probably accounts for the differences between the three mean values in column five. * Shells without mantle were included along with the mantle-shell preparations in these experiments in order to obtain a measure of Ca45 exchange. However, it is not certain that the concentration of calcium for exchange is the same for both (Wilbur and Jodrey, 1952), and accord- ingly exchange corrections have not been made in the figures in Table I. When exchange values are subtracted from the deposition in the mantle-shell preparations the statistical significance is essentially that given. 94 MARONEY, BARBER AND WILBUR TABLE II Reversibility of DNP effect on Ca45 deposition n DNP added, then washed n No DNP, then washed p 12 129 ±93 12 87 ± 34 0.2 > P > 0.1 Details given in Methods and footnote of Table I. DNP concentration, 10 4 M. The action of DNP on shell regeneration was studied on intact oysters with shells notched at the posterior edge and placed in 200 ml. or 1000 ml. of sea water containing various concentrations of DNP. All solutions were aerated and were changed daily. The extent of regeneration was determined microscopically. Oysters were collected near Beaufort, N. C., and maintained for two weeks prior to use in natural waters or in tanks with running sea water supplied through hard rubher lines. RESULTS DNP caused a stimulation of mantle respiration at concentrations of 10~3 M and 10~4 M (Fig. 1). This increased respiration remained constant for at least six hours. The concentration of DNP which caused maximum respiratory stimulation (10~3 M, Fig. 1) gave essentially complete inhibition of Ca45 deposition by mantle- shell preparations (Table I). The same relationship of respiratory stimulation and inhibition of Ca45 deposition held true for 10'4 M DNP (Fig. 1 and Table I). In further experiments the inhibitory action of 10~4 M DNP on Ca45 deposition was found to be reversible (Table II). When the concentration of DNP was lowered to 10~5 M and 10~6 M, there was no statistically significant effect on mantle respira- tion (Fig. 1). Likewise, at 10~5 M, DNP had no significant effect on the deposi- tion of Ca45 by the mantle-shell preparations (Table I). Shell regeneration in the presence of DNP was inhibited as the concentration of the inhibitor was increased (Table III). The inhibitory concentrations of DNP were about the same for both calcium deposition by the mantle-shell preparation and for regeneration in the whole oyster (cf. Tables I and III). The last column in Table III indicates that DNP is toxic. However, of 20 oysters which were dead by the eleventh day of DNP treatment (all DNP concentrations), 10 showed re- generation. In those cases in which regeneration occurred, the structure of the TABLE III Effect of DNP on shell regeneration DNP cone. n Oysters showing regeneration in DNP Comments 0 10 10 No deaths in 14 days 10-5 M 10 8 1 dead after 14 days 5 X 10~5 M 10 7 9 dead after 11 days 10-" M 10 3 9 dead after 5 days Temperature 24.0-29.5° C. ; pH 7.7-8.1 ; salinity 18.0-35.99 °/oo. both valves in all but one case. Regeneration occurred in DNP AND SHELL DEPOSITION 95 shell as observed microscopically was the same in DNP-treated oysters and oysters in sea water. DISCUSSION The effects of DNP on the respiration of oyster mantle followed the general pattern as seen in many animal and plant tissues (Simon, 1953). Mantle respira- tion increased with increasing concentration of DNP, reaching a maximum 87% above the endogenous level. This degree of stimulation was considerably higher than that produced in the mantle by the addition of citric acid cycle intermediates (Jodrey and Wilbur, 1955). At concentrations of DNP which caused a respira- tory stimulation, Ca45 deposition by the oyster mantle was reversibly inhibited. The action of DNP may involve one or more of the following mechanisms con- cerned with shell deposition : ( 1 ) transport of calcium and carbonate ions across the mantle; (2) transfer of CaCCX crystals from the interior of mantle cells; (3) synthesis and secretion of the shell matrix; or (4) amoebocyte activities and shell regeneration. If the transport of the calcium and carbonate ions across the mantle requires the expenditure of energy, this active transport could well be inhibited by DNP (Taggart and Forster, 1950; Mudge, 1951; Levinsky and Sawyer, 1953). The transport of calcium carbonate crystals from the cell interior, as suggested by Bevelander (1953), might utilize an energy mechanism susceptible to the action of DNP. In the regeneration experiments failure of intact oysters to deposit matrix alone in the presence of DNP may be due to an effect on matrix synthesis since peptide bond and protein synthesis are known to be inhibited by DNP (Borsook, 1954; Tarver, 1954; Siekevitz, 1952). However, in view of the toxicity of DNP (Table III) one should not conclude that failure of regeneration reflects only a direct effect on matrix synthesis or secretion. Another possibility arises from the fact that in the snail Helix aspersa, amoebo- cytes play a part in shell regeneration and are thought to be responsible for the deposition of matrix and calcium carbonate crystals (Wagge, 1955). However, the role of amoebocytes in oyster shell regeneration has not been studied and accordingly we do not know their significance with respect to the inhibition of regeneration by DNP. SUMMARY 1. Dinitrophenol stimulated oyster mantle respiration at 10~3 M and 10'* M. Respiration was not significantly different from the endogenous rate at 10~2 M, 10~5 M and 10-6 M DNP. 2. Calcium deposition, as measured by Ca45, was inhibited in isolated mantle- shell preparations at 1O3 M and 10"* M DNP. the same DNP concentrations which stimulated respiration. Inhibition was found to be reversible. 3. Shell regeneration in whole oysters was inhibited by DNP. DNP concen- trations which inhibited regeneration were toxic. LITERATURE CITED BEVELANDER, G., 1953. 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WILBUR, K. M., AND L. H. JODREY, 1952. Studies on shell formation. I. Measurement of the rate of shell formation using Ca4n. Biol. Bull., 103: 269-276. TEMPERATURE DEPENDENCE OF BREATHING RATE IN CARP A. L. MEUWIS AND M. J. HEUTS Agricultural Institute, University of Loi/-i\iin (Belgium] Temperature is an essential factor in the aquatic environment ; it exerts a profound influence on the morphologic and physiologic characteristics of fishes. To infer from this the importance of temperature in adaptation, in its genetic meaning, seems to be logical. An adequate study of this adaptation presupposes easily recognizable adaptive variants within a population. Our original intention was to work out an easy method to detect such physiologic variants which would not involve necessarily the death of the experimental objects, for breeding purposes. For several reasons— among which figure the data of Fox (1939) concerning the frequencies of respira- tory movements with regard to the geographical distribution of poikilotherms — we have studied the same frequencies in Cyprinus carpio L. in relation to temperature. It appeared soon that the breathing rate in this fish is highly dependent on size. As a first contribution to the problem we intended to solve, this paper reports the influence of age and size on the mentioned physiologic trait. MATERIAL AND METHODS Our experiments have been performed on Cyprinus carpio, belonging to the race of glass-carps from the temperate warm waters. The best temperature for development for Cyprinus carpio would be situated, according to Huet (1953), between 20° and 25° C. ; according to Schaeperclaus (1949) the optimum is nearer to 27° C. but at 15° C. a good production is still obtained. As experimental objects we used fishes of three months, one year, two years and four years old, having live weights from 20 to 2000 grams. The fishes have been obtained from a single commercial stock. A starvation period of at least 48 hours preceded all experiments. Each individual of the different weight and age classes has been examined at several temperatures, from 4° C. up until death occurred, at about 38° C. Trans- fers always took place from lower to higher temperatures. Intervals can be read from the graphs to be discussed presently. During measurements each temperature was kept constant within 0.1°, and the water abundantly aerated to provide a constant saturation with oxygen. For practical purposes the fishes were put in small wire baskets. Measurements have been performed only on perfectly quiet animals. RESULTS The dependence of the frequency of respiration upon the temperature in a sinc/le individual 1. Temperature acclimation By temperature acclimation we mean the temporary adaptive alteration of the phenotype after an external temperature change.1 It is known from Wells (1935a) 1 We wish to reserve the term adaptation for a similar alteration of the genotype, which is included in the definition of acclimation or acclimatization by Prosser (1955) and by Bullock (1955). 97 98 A. L. MEUWIS AND M. J. HEUTS 0 12 24 36 48 60 72 HOURS FIGURE 1. Successive mean respiration frequencies after transfer from 32° to 36°, plotted against acclimation time (carp No. 16). Vertical bars added to the points cover the range M ± ff. TABLE I Frequency of respiration as a function of temperature (Carp No. II) Temperature (°C.) Duration of respiratory pauses Number of pauses per minute Breathing frequency 4 1-2 mins. 1 2 8 \—2 mins. 2-1 6 12 15 sees. 3 9 18 — — 24 15 16 sees. 3 13 20 10 sees. 5 24 26 10 sees. 5 25 29 10 sees. 5 24 34 10 sees. 5 24 35 — — 24 37.4 3 sees. 15 32 BREATHING FREQUENCY IN CARP 99 50 20 LU I— < cc o z LU CC CD d o 10 5- 2- 1 Fiy. „ N811 (20g) x N°10(21g) a N°16(24g) N°6 15 39 21 27 33 TEMPERATURE FIGURE 2. Rate-temperature curves for breathing frequency in carps of 20 to 24 grams. and Schlieper (1950), that, in fishes, this acclimation takes from 24 hours to a few days after the transfer from one temperature to another. Only after this lapse of time, constant and consistent results, on metabolism at least, have been obtained. Before acclimation is completed, the oxygen consumption is generally higher than normal. Figure 1 shows that similar phenomena characterize the accommodation of the frequency of respiratory movements. This frequency, after an initial rise of 100^, reaches a stable value after 48 hours following a transfer from 32° to 36° C. Usually a steady-state is reached within three to four days, although rarely it may last fourteen days. Acclimation is generally shortest between 16° and 30°. All fishes which maintained this steady-state for 48 hours were considered as com- pletely acclimated. Only measurements obtained on such fishes are considered further in this paper. 100 A. L. MEUWIS AND M. J. HEUTS At given high temperatures the fishes die hefore having attained complete acclimation as defined. These temperatures are indicated in this paper as the upper lethal temperatures. 2. Respiratory pauses and jreijueney of respiration at a given temperature Typical for the respiration of fishes, especially of small ones, are the so-called pauses, during which no respiratory movements occur. After such a pause, which may be variable in length, the fish breathes a few times at a rapid rate and then pauses again. The duration and the frequency of the pauses vary with the temperature (Table I). 20 LU I— < DC. 10 LU o: CD O O 2- 1 N°8(25g) N° 2 (39 g) N° 3 (39g) N°7 (67g) 15 33 39 21 27 TEN PERATURE FIGURE 3. Rate-temperature curves for breathing frequency in carps of 25 to 67 grams. BREATHING FREQUENCY IN CARP 101 50 20 10 UJ a: CD O O N° Vlll(318g) N° VI (320g) x N" VII (320 g) A N° I (1975g) a N° II (2050g) 15 21 27 TEMPERATURE 33 39 FIGURE 4. Rate-temperature curves for breathing frequency in carps of 318 to 2050 grams. In this paper breathing frequency is expressed as movements per minute, ignoring the pauses. The time intervals used for one count were chosen long enough to eliminate the disturbing influence of the pauses. As a rule these inter- vals were five minutes at low, and two minutes at high temperatures, the counts being repeated at each temperature as many times as needed for adequate statistical treatment. Immediately after transfer to a higher temperature no respiratory pauses may occur, making for lower standard deviations (Fig. 1). Otherwise no trends in standard deviations could be detected during acclimation, with respect to time, or after acclimation, with respect to temperature. 102 A. L. MEUWIS AND M. J. HEUTS 3. Respiration frequency as a function of temperature The variations of the mean frequencies of respiratory movements with respect to temperature, show, in the case of the smaller fishes, a typical pattern : first, at lower temperatures, a progressive increase of the frequency with a rise in tempera- ture ; further a less steep or even horizontal central part, where the frequency in- TABLE II Q lo-values ,* breathing rates at 24° and lethal temperatures for carps, arranged according to weight and age Serial number Weight in grams Age in years Qio Rate at 24° Lethal tempera- ture Low Intermediate High 11 20 1 2.10 (4-19) 1.00 (19-35.4) 2.82 (35.4-37) 24.6 38 10 21 i 3 2.13 (4-15.6) 1.10 (15.6-35.4) 3.84 (35.4-38.4) 18.0 38.5 6 24 J 1.98 (4-17) 1.22 (17-35.2) 3.36 (35.2-38) 18.2 39 16 24 * 1.69 (4-16.3) 1.30 (16.3-35.3) 2.28 (35.3-37.8) 15.9 38 8 25 * 1.84 (3-16.5) 1.31 (16.5-35.4) 2.31 (35.4-37.5) 14.2 38 2 39 k 2.07 (4.7-18.3) 1.26 (18.3-35) — 19.5 39 3 39 * 2.16 (4.7-17) 1.31 (17-35.5) 2.66 (35.5-37) 14.0 39 7 67 2 1.89 (4-18) 1.37 (18-35) 2.66 (35-37.7) 17.6 38 VIII 318 2 2.19 (4-15.5) 1.40 (15.5-34) — 17.4 36 VI 320 2 2.83 (3.5-12.3) 1.35 (12.3-32) — 20.2 36 VII 320 2 2.08 (4-16) 1.34 (16-35) 4.20 (35-36.5) 18.2 37 IX 325 2 — — — — 35.8 I 1975 4 — 1.39 (9-35) — 25.9 36 II 2050 4 — 1.51 (9-30) — • 33.0 36 The temperature limits, in degrees C., are indicated in brackets. BREATHING FREQUENCY IN CARP 103 creases much less with temperature, and finally a sudden rise of the respiratory rate at a point which is near to the lethal temperature. This pattern is particularly well recognizable when the data are plotted on semilog coordinates, showing the proportionate response of the breathing rate to the temperature (Figs. 2, 3 and 4). This proportionate response appears to show, over broad temperature ranges, practically constant values, which change abruptly to other such values, at given critical temperatures. From eye-fitted curves through the data for individual fishes Qin-values for each of these ranges can be readily estimated, while their intersections localize, with a fair approximation, the critical temperatures. The lower critical temperatures appear to be situated between 12.3° and 19°, the higher between 32° and 34.5° (Table II). The lower critical temperatures delimit a first zone of high Q1(, -values from 1.69 to 2.83. Within the range extending from the low to the high critical temperatures Qln- values vary from 1.00 to 1.51. Within the range of high temperatures Q10's reach values between 2.28 and 4.20. Because of the short interval separating the high critical and the lethal temperature, only a few data can be obtained in this range, making for a limited significance of these last Q10's for most fishes. Nevertheless it is perfectly clear that for the population of carps studied, typical regulation phenomena characterize the respiratory rates over given homeo- static zones between 12.3° and 34.5°. However, within these zones a considerable individual variation, as to the Q10's, as well as to the situation of the critical tempera- tures, is showing up. a. The influence of weight A closer inspection of the log R-T curves in connection with the weight variants, leads readily to the recognition of the following facts. First, all small fishes (39 grams and lower) exhibit more effective regulatory mechanisms than larger fishes. In the first category all QH,'s, within the homeo- static zone, fall between 1.00 and 1.31, and, moreover, the correlation between weight and the respective values is very close (Table II). All larger fishes, on the other hand, have Q](p's between 1.34 and 1.51 and, again, the largest fishes have very low regulator)7 capacities. Concomitant with the occurrence of lower Q,,,'s within the homeostatic zones of larger fishes, is the lowering of the upper lethal temperature threshold of the same fishes, with respect to the small fishes. Fishes of 67 grams and less have lethal temperatures at 38° and 39°, while those of higher weights have lethal temperatures between 35.5° and 37° On the other hand, the largest fishes (± 2000 grams) fall easily into a lethargic state at 4°, where the measurement of barely recognizable respiratory movements is no more possible. Therefore, a subdivision of the R-T curves of large fishes into three, with differential regulatory characteristics, seems to be impossible. Their narrowed viable range of temperatures can be characterized only by a single, high Q10-value. A last remarkable feature is the variation of the mean respiratory rate. The different individual R-T curves are clearly situated at unequal over-all rate levels. It is difficult to express this shift of the curves along the ordinates. We have tried to do this by indicating the estimated rate for all fishes at 24° (Table II). It appears, then, that the largest fishes have very high rates, smaller fishes attaining 104 A. L. MEUWIS AND M. J. HEUTS only about half of these rates. The smallest fish, however, shows again a high over-all rate. 1). The influence of age On the basis of our data it is not possible to differentiate clearly the effect of age from the influence of size. A certain indication is obtained from fish No. 7 (Table II). This fish was intentionally starved during one year in the laboratory. During that year its weight practically remained constant (67 grains), and was far below the weight of normally fed fishes of the same age (±320 grains). For as far as the lethal temperature is concerned (38°) this fish reacted according to its weight class. Its Q10 within the homeostatic zone, however, falls within the range of its age group (1.37). c. Residual variation The number of individuals studied within each weight and age class does, of course, not allow an estimation of the individual variation of breathing rate within a homogeneous group. From our data we can, however, infer its existence. Most interesting, in this respect, is the fact that the R-T curve of fish No. VI is, in its whole, shifted to lower temperatures, for a distance of about 3°, when com- pared to fish No. VII, belonging to the same weight and age group (Fig. 4 and Table II). This might indicate a differential adaptation to temperature of both fishes. Individual variation, with respect to the efficiency of the breathing regulation within homeostatic zones of equal extension, seems to exist as well. This is indi- cated by the detected differences in Qln's within age and weight classes. DISCUSSION Two main points of interest are revealed by our analysis. The first is the shift of the upper lethal temperatures from 38°-39° for small fishes, to 35-36° C. for large-sized individuals. The second is the gradual disappearance of homeo- stasis for breathing frequencies with increase in size. Decreased resistance to high temperature with size, if we restrict the survey to post-embryonic life, is not a general trend among fishes. The literature concerning the subject is reviewed by Hart (1952). All three types of fish species seem to exist : those with increasing, those with stationary, and those with decreasing resistance with increasing size. In most cases the separate effects of size and age have not been studied. In Salvelinus fontinalis no size effect was recorded for yearlings of the same age (Fry ct al., 1946), indicating seemingly that the age effect on temperature resistance would be preponderant. However, Spaas (un- published results) found a size effect in one-year-old trout and a mean increase in high temperature resistance in two- and three-year-old fishes of the same species, which, however was lower than expected on the basis of the size differences for temperature tolerance within yearlings. Both factors, age and size, seem to interact in some way. The fe\v data recorded in this study are not opposed to such a view. A second main point, disclosed by our analysis, is the progressive increase of dependence of the frequency of respiratory movements upon the temperature. It BREATHING FREQUENCY IN CARP 105 is illustrated by the gradual disappearance of a homeostatic zone of practical stability of this frequency at several temperatures in small fishes, and by a cor- related increase of the over-all Q10-value of this process in the course of life history. By reputation, poikilotherms are animals without homeostatic mechanisms regu- lating their most essential physiologic functions, such as oxygen consumption, heat production, activity and development. More specifically, for oxygen consumption, they have been generally assumed to follow Krogh's "Normalkurve." However, a certain number of workers have shown that the type of response to temperature of such functions is not only a specific trait among poikilotherms, but even that it varies intraspecifically as well. According to recent reviews of the data (Rao and Bullock, 1954; Bullock, 1955), it seems justified to admit that in most cases a higher dependence on temperature is characteristic for animals from warmer habitats. A survey of the data on speed of development leads to the same conclusion with regard to specific and to racial geographic variation (Heuts, 1956). More effective homeostatic mechanisms seem to be a distinctive trait of inhabitants of colder areas, especially when intraspecific comparisons are made. Poikilotherms are poikilostatic organisms, but they are such to varying, genetically determined, degrees. On the other hand, it is clear enough that the homeostatic mechanisms of the homoiotherms also are operating only within a given temperature range in a given individual (the thermoneutral zone), and, further, that the ranges of this zone are equally subject to specific variation in relation to the habitat, northern animals possessing more efficient homeostatic mechanisms operating at low temperatures (Scholander e t al., 1950). The conclusion is obvious, and has been drawn, that a homeotherm is clearly adapted to the temperature range where the Q1(l for biological processes appears to be equal to 1.00, and where, consequently, its behavior is most homeothermic. In poikilotherms such homeostatic zones, with O1(1's approaching 1.00, are gen- erally not found or not recognized. Only recently Bullock (1955) strongly called attention to the existence of homeostatic mechanisms for a number of rate processes among several poikilo- therms. In addition to the cases cited by this author a few other examples may be mentioned here. A clearly delimited homeostatic zone for oxygen consumption is indicated, al- though not recognized by the author, in Fitndnlus parripinnis between 18° and 22° for small fishes (Wells, 1935a, 1935b). Its existence shows up due to almost con- tinuously increasing experimental temperatures between 10° and 24°. A definite fall of QI(, values for oxygen consumption between 15° and 20° in Salvclinus jontinalis is demonstrated by Job's data (1955). Unfortunately no measurements have been made beyond 20°. The same author mentions the results of Edwards (1946) on the click beetle Mclanotns conunnnis, which, between 17° and 27°, shows a definite decrease of O,,,-values for oxygen uptake. Job, however, is inclined to regard this phenomenon as due to an experimental error. A number of indications seem thus to point to a possibly widespread phenome- non of homeostatic mechanisms among poikilotherms, which may be brought t< > light by more accurate experimental procedures. These zones being presumably narrow, their absence in current observations might be due to the experimental 106 A. L. MEUWIS AND M. J. HEUTS procedure making use of too discontinuous temperature gradients. Still another source of error might be an insufficiently long acclimation to the experimental temperatures. If, however, homeostatic zones can be demonstrated to exist in poikilotherms. then it seems logical to admit, as is done in homeotherms, that they delimit adaptive zones, and to conclude that a poikilotherm is adapted to such conditions, where it is least poikilostatic. Such a situation exists clearly in young carps. In the smallest fishes Q10-values approach 1.00 over a temperature range from 16° to 35°. Within this range they are homeostatic for the studied trait, and to this range they are prob- ably best adapted. How far the frequencies of respiratory movements reflect the oxygen consump- tions at different temperatures remains, of course, an open question. They will not truly reflect oxygen consumption, if, principally, the utilization coefficient of the oxygen present in the water, flowing over the gills during each breathing movement, would be different according to temperature. For trout and eel, at least, van Dam (1938) has shown that the utilization percentage is not affected by a temperature difference of 8°. For obvious reasons, the complete disappearance of homeostatic zones in carps, with increasing size, cannot be asserted. They certainly are narrowed in the course of life, though this progressive alteration is not, or not directly, dependent on age. A similar narrowing of homeostatic zones is obvious in Funduhis parvi- pinnis (Wells, 1935b), concomitant with the over-all higher dependence of oxygen uptake on temperature with increasing size. In other cases, however, the individual evolution is exactly the opposite. Job (1955) recognizes a flattened proportionate response to temperature, a higher tem- perature independence, in large-sized Salvelinus fontinalis. Similarly Spaas (un- published results) finds an increased dependence of oxygen consumption in large versus small yearlings of brook- and sea-trout. The click beetles already mentioned (Edwards, 1946) show, on the other hand, a response of oxygen uptake to temperature per unit wet weight, which is inde- pendent of size. The case is, however, not strictly comparable, because all click- beetles, whatever their dimensions, have reached final sizes. Between the first fact disclosed by our analysis (the shift of upper lethal tem- perature to lower values in the course of life) and the second (the decrease in homeostatic efficiency) there seems to be a close relation in our material, as one would logically expect. Lack of adequate data on post-embryonic development pre- vents the generalization of this observation for individual life cycles. Some data on embryonic development, however, indicate clearly that a similar relationship does not hold in racial and interspecific comparisons. Races or species with a higher temperature independence for speed of embryonic development can nevertheless have a narrower temperature tolerance range during development than another race or species with a more dependent development. If the relationship between temperature tolerance limits and regulation holds in indi- vidual cycles, then this cycle can be labelled as physiologically regressive (with re- spect to the general trend of the phylogenetic record) in Cyprinus carpio and in Fundulus parvipinnis. Several Salmonidae seem to follow a physiologically progres- sive ontogeny. A last point of interest is the fact that the methods used seem to be adequate to BREATHING FREQUENCY IN CARP 107 detect individual adaptive differences to temperature ranges, as well as individual differential degrees of homeostatic attributes, without necessarily killing the ex- perimental objects. This permits genetic studies of the mentioned characteristics, and creates thus the possibility of filling gradually the evolutionary gap between poikilotherms and homeotherms. SUMMARY 1 . The dependence of the frequencies of respiratory movements upon temperature has been studied in Cyprimis carpio L. 2. The degree of dependence of these frequencies upon temperatures is primarily determined by the size of the fishes. Large fishes are highly, small fishes only slightly, dependent. 3. Small fishes are characterized by a broad homeostatic zone of independence of the breathing frequency on temperatures. This homeostatic zone disappears in large fishes. The upper lethal temperatures decrease concomitantly with the disappearance of the homeostatic zones. 4. Individual variants, as to the degree of homeostasis and as to the adaptive temperature ranges, can be detected by the method presented. It can be used for studying genetic determination of these characteristics. 5. A survey of the data in the literature allows one to distinguish poikilotherms with progressive and with regressive ontogenies, with respect to homeostatic behavior. LITERATURE CITED BULLOCK. T. H., 1955. Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev., 30: 311-342. VAN DAM, L., 1938. On the utilization of oxygen and regulation of breathing in some aquatic animals. Doctoral Thesis, Groningen. EDWARDS, G. A., 1946. The influence of temperature upon the oxygen consumption of several arthropods. /. Cell Coinp. Physiol., 27: 53-64. Fox, H. M., 1939. The activity and metabolism of poikilothermal animals in different lati- tudes. V. Proc. Zool Soc. Land. Scr. A, 109: 141-157. FRY, F. E. J., J. S. HART AND K. F. WALKER, 1946. Lethal temperature relations for a sample of young speckled trout, Sah'cliniis fontinalis. Pnbl. Ontario Fish. Research Lab., 54 : 9-35. HART. S. P., 1952. Geographic variation in some physiological and morphological characters in certain freshwater fish. Pnbl. Ontario Fish. Research Lab., 72: 1-79. HEUTS, M. J., 1956. Temperature adaptation in Gastcrostcits acnlcatus. Ptibbl. Staz. Zool. Napoli. 28: 44-61. HUET, M., 1953. Traite de pisciculture. La Vie Rustique, Bruxelles. JOB, S. V., 1955. The oxygen consumption of Sak'dimis fontinalis. Pnbl. Ontario Fish. Re- search Lab., 73 : 1-39. PROSSER, C. L., 1955. Physiological variation in animals. Biol. Rci'., 30: 229-262. RAO, K. P., AND T. H. BULLOCK, 1954. Q1(1 as a function of size and habitat temperature in poikilotherms. Atncr. Nat., 88: 33^44. SCHAEPERCLAUS, W., 1949. Grundriss der Teichvvirtschaft. Paul Parey, Berlin und Hamburg. SCHLIEPER, C., 1950. Temperaturbezogene Regulationen des Grundumsatzes bei YVechsel- warmen Tieren. Biol. Zcntralbl., 69: 216-227. SCHOLANDER, P. F., R. HOCK, V. WALTERS, F. JOHNSON AND L. IRVING, 1950. Heat regulation in some arctic and tropical mammals and birds. Biol. Bull., 99 : 237-258. WELLS, A. N., 1935a. The influence of temperature upon the respiratory metabolism of the Pacific killifish, Fitndiilus parvipinnis. Physiol. Zool.. 8: 196-227. WELLS, A. N., 1935b. Variations in the respiratory metabolism of the Pacific killifish, l-'tindnliis pari'i/yinnis. due to size, season and continued constant temperature. Ph\siol. Zonl., 8: 318-336. OXYGEN UPTAKE IN INSECTS WITH CYCLIC CO2 RELEASE A. PUNT, W. J. PARSER AND J. KUCHLEIN Laboratory for Comparative Physiology, University of Amsterdam, Amsterdam, Holland The periodical release of carbon dioxide (CO2), as first found by one of us in bugs (Punt, 1944), is well established now in several insects in rest (Punt, 1950, 1956a) and in diapausing pupae of Lepidoptera (Punt, 1950; Schneiderman and Williams, 1953, 1955; Buck, Keister and Specht, 1953; Buck and Keister, 1955). As to the oxygen uptake, there is some controversy among the above mentioned authors. Punt described in Corobus nenioralis (Mull.), Locnsta migratoria ini- (jratorioidcs (Rch. and Frm.), Meloc proscarabaciis L. and Rhodnius proli.vus (Stal.) a diaferometrically recorded periodical oxygen uptake, which ran parallel to the periodical CO2 bursts. In diapausing pupae, on the other hand, a continuous oxygen uptake was found by Schneiderman and Williams and by Buck and Keister using the Warburg technique. The aim of this paper is to give more details about our investigations concerning the oxygen uptake in carabids and in the pupae of the cecropia silkworm. Some results of experiments on the influence of oxygen tension on the burst frequency will be mentioned. A possible explanation of the oxygen records will be given and the preliminary results of the estimation of the CO2-binding power of insect haemo- lymph will be discussed. MATERIAL AND METHODS The insects under investigation were Carabus nemoralis (Miill.), Hadrocarabus problematicus Hrbst., Pcriplancta americana L., pupae of the cecropia silkworm (Hyalophora cecropia (L.)) and pupae of Spliin.v ligustri L. The cecropia pupae were received from Dr. Schneiderman to whom we want to express our acknowledgment here. A single animal was put in a glass tube, through which a very constant current of CO2-free outdoor air was sucked. The gas exchange was continuously estimated by means of two diaferometers as described earlier (Punt, 1950, 1956a). In these instruments the gas which is to be analyzed passes through a copper tube, in the center of which an electrically heated platina wire is suspended. The temperature of the wire, and as a consequence the electric resistance, depends on the CO2 and O2 percentage, respectively, of the passing gas. The wire is connected in a Wheatstone bridge with a galvanometer. In all experiments two parallel diaferom- eters were used to record the CO2 production and the O2 uptake simultaneously. The gas coming from the animal container was dried and equally divided among both machines ; the portion for the O2 diaferometer was absolutely freed from CO2 by soda lime. As the O2 reading is slightly influenced by the removal of the CO,, a valid R. O. cannot be extracted directly from the curves. Both galvanometer 108 OXYGEN UPTAKE IN INSECTS 109 responses were recorded on one paper strip in a rotating-drum camera, the slit of which was perpendicular to the direction of rotation. Care was taken that the diaferometers had the same "latent period" in order to get synchronous points on the ordinate of the graphs. The galvanometers were connected in such a way that O2 uptake and CCX release were recorded in the same direction. In other experiments, for comparison, the direct Warhurg method was used to estimate the O., uptake. The animals were placed in standard Warhurg flasks (16 ml.) containing filter paper drenched in I0r/c KOH. The temperature was kept constant at 20° C. FIGURE 1. Photographically recorded CCX production and CX uptake of one specimen of Cai'tjbns ncuwralis. The index on the left side indicates approximately 10 /zL per hour (CX and C(X). The inclination of the CO, line is caused by galvanometer drift; 20° C. The CO2 dissociation curve of haemolymph was estimated by means of the Haldane method for blood gas analysis. In order to work with small quantities, a special apparatus was built with a 10-ml. reaction vessel, the thermo-barometer ves- sel being of the same size. Gas mixtures were analyzed in the Haldane gas analysis apparatus. RESULTS 1. Oxygen uptake hi carabids In a previous paper (Punt. 1956a) the discontinuous oxygen uptake in Carabits ncuwralis (Mull.), Locnsta inigratoria migratorioides ( Rch. and Frm.), Meloc proscarabaeus L. and Rhodnius prolixus (Stal.) was described. The oxygen uptake was found to be ( at any rate partly ) periodical ; the spikes in the photographic records were exactly synchronous with the CO2 bursts. But 110 A. PUNT, W. J. PARSER AND J. KUCHLEIN there could be noticed a marked difference between the forms of the CX and CO2 curves, which difference was most evident in Carabns (Figs. 1 and 2). Both the opening and the closing moments of the spiracles are rather distinct in the CO., line : a steep curving up of the galvanometer record indicates the mo- ment of opening and a more or less sharp notch in the line, followed by a decline to zero level (or so close to zero that the difference was within the range of experi- mental error), marks the moment of closing. Sometimes, in Carabus and Pcri- plancta, the line rises somewhat in the interburst period (Fig. 1), probably due to small interburst CO2 release (cf. Schneiderman and Williams, 1955). 1 HOUR FIGURE 2. As Figure 1, but from a beetle in which the ratio between the open period and the closed period is discrepant from the mean value (1:2) as is occasionally found. The O2 line, too, shows these peculiarities, but while the CO., spike shows a maximum just after opening of the spiracle and declines rather regularly till the moment of closing, the CX line goes down much steeper nearly to the interburst level. After the moment of closing of the spiracles (corresponding to the sharp bend in the CO, line) the O2 line declines still more to a minimum, to curve up- wards again after some time to come to a "steady-state" which is maintained till the next burst. This steady rate of oxygen consumption is slightly lower than the minimum O2 uptake in the burst period. As at that time we only meant to deter- mine the type of respiratory activity and not the total gas exchange, there were un- fortunately no zero readings recorded in the above mentioned paper. The respira- tion was recorded over periods of 6 hours continuously and it is rather inaccurate to interpolate a zero line over so long a period, particularly as there may have been some galvanometer drift due to external circumstances. Next we performed some experiments of much shorter duration, preceded and followed by zero readings in order to be able to interpolate the zero line in our OXYGEN UPTAKE IN INSECTS 111 records. Though there was considerable individual variation, the CX uptake in the interburst periods could be calculated from planimeter measurements to be approxi- mately 60% of the total CX consumption. So in Carabus the oxygen uptake is both continuous and periodical. In a great number of experiments with Pcriplaneta americana we came to the same conclusion. It has already been shown that these O., spikes could not be caused by CO., interference in the O., diaferometer (Punt, 1956a). As was mentioned, Schneiderman and Williams, and Buck and Keister reported the CX uptake in diapausing pupae to be continuous only. In order to determine whether this discrepancy between our results in Carabus and the results of Schneider- man and Buck was the consequence of the use of different techniques, the gas ex- O2 - O) -2 c O — — i £_ — 2 u <5 1 — ' JJ 4 I_ I — a. J 4 E _ , J" J ~L p •6 E h j 1 — i 6 L J |_ 20 4D 6O 8O 1OO minutes 120 FIGURE 3. Manometrically estimated O. uptake in Hadrocarabus problematicus (440 mg.). Warburg flasks provided with KOH-drenched paper ; 20° C. change of carabid beetles was estimated in a Warburg apparatus. There were performed a great number of experiments with specimens of Hadrocarabiis prob- Icuiaticits of a weight of 250-650 mg. The insects were placed in a normal side-arm flask with gas vent. The side-arm contained filter-paper, which protruded into the flask above the beetle. The paper was drenched in 10% KOH. After half an hour of temperature equilibration the stopcocks were closed and manometer readings made at intervals of three or five minutes. The change in pressure in this interval as compared with the foregoing reading was plotted against time (Fig. 3). In this way the oxygen consumption could be calculated from these figures, taking the flask constant into account. As the insect's volume was not exactly known, the index on the right of Figure 3 represents an arbitrary value only. The results show that CX consumption in Hadrocarabus is at any rate partly periodical, which is in ac- cordance with the diaferometer experiments. Sometimes the curve rises above the zero line, which would indicate that there is in this interval an increase in pressure in comparison with the foregoing reading. This can only mean that at these mo- 112 A. PUNT, W. J. PARSER AND J. KUCHLEIN ments the CCX bursts occur and the CCX absorption is not yet completed. But as the CO. 2 is readily absorbed, the pressure is considerably decreased in the following five minutes, accentuating the apparent CX uptake in this period. From a large number of experiments with the diaferometer we know that at this temperature (20° C.) in carabicls the open spiracle period can be sharph distinguished from the closed period, the interburst period being mostly twice as long as the burst. As this ratio cannot be clearly seen in the manometrical curves it mav be concluded that J * in Hadrocarabus CX uptake is partly continuous as well. When instead of containing KOH-drenched filter paper the side-arms were empty, the line represented in Figure 4 was found. Here, too, the moment of opening of the spiracles could be found in the curve as an increase in pressure, cn c o -82 y>o -4- Jl 2O 4O 6O 8O KX> minutes 120 FIGURE 4. Hadrocarabus problematicus (500 ing.). Variations in pressure in a "\Yarburg flask, without KOH ; 20° C. reaching a maximum in about ten minutes. This was followed by a slow decrease in pressure, lasting for about fifteen to twenty minutes by which the cycle was completed. The decrease in pressure in the closed period is interpreted as a continuous CX consumption, but Buck, Keister and Specht (1953) considered the possibility that telescoping of the insect was involved, which in this manometric method could not be distinguished from some other phenomenon causing pressure loss. Our results, however, in the "open circuit" with the diaferometer made this assumption doubt- ful. Still we thought that in this beetle the space between the abdominal tergites and the elytra, which can be rather well closed and into wrhich eight out of nine pairs of spiracles open, had something to do with the cyclic gas exchange. So we per- forated the elytra. This had not the slightest influence on the periodical respira- tion. The sealing of three pairs of the spiracles with melted bee's wax made the pe- riods more irregular and increased their frequency. It did not matter very much which pairs of spiracles were sealed. Introduction into the spiracles of very tiny glass capillaries, which were fixed with bee's wax, made the CX uptake more con- OXYGEN UPTAKE IN INSECTS 113 FIGURE 5. Oxygen uptake in Hadrocarabus, manometrically estimated at three-minute intervals ; 20° C. A : normal insect at rest, the elytra being removed. B : Some of the spiracles sealed with bee's wax. C : Some of the abdominal spiracles kept open with glass tubes. D : The same with the thoracic spiracles. tinuous. The clearest effect resulted here from placing the capillaries in the large thoracic spiracles (Fig. 5). 2. The oxygen uptake of Hyalophora cecropia pupae Two years ago, in the Physiological Laboratory, University of Utrecht, the fol- lowing experiments were performed with cecropia pnpae. The gas exchange was CO2 1 HOUR FIGURE 6. Photographically recorded CO, release and C), uptake in a pupa of the cecropia silkworm ; 20° C. 114 A. PUNT, W. J. PARSER AND J. KUCHLEIN estimated with two diaferometers simultaneously, one for recording the CO2 pro- duction, the other for the CX consumption. In Figure 6 the photographically re- corded galvanometer responses are shown. At 20° C. these diapausing pupae showed one CCX burst in about four hours. The form of the CCX line is exactly the same as described earlier in pupae of Sphinx lignstri. Just after the CO, spike the line falls to approximately zero level and is very smooth, but after about one hour small perturbations return in the line, which go on and increase until the next CO2 burst. Exactly synchronous with the C(X bursts small O, spikes were found. This CX line falls after some minutes but remains slightly above the interburst level. In the interburst period the CX line, too, is at first very smooth, but shows after some time the same small perturbations as the CO., line (in fact running exactly parallel to them). It looks as if the spiracles are no longer hermetically closed but are leaking or fluttering from time to time. Unfortunately we did not esti- 02 CO2 FIGURE 7. The same pupa as in Figure 6. A double burst is recorded. mate the exact value of the O2 uptake in these prolonged experiments, as was pointed out in the foregoing section. But as the O., diaferometer was adjusted to be approximately as sensitive as the CO., apparatus in order to get the same re- sponse for the same percentage of gas, it is clear from these graphs that O2 uptake must take place in the interburst periods as well. The area of the O.2 spike was much smaller than that of the CO., burst, whereas the interburst perturbations were of the same size in both lines. The impression which we have from some zero readings of the O., line and from calculations of the O, spike area is that there must be a relatively large interburst O., uptake which was considered to have a mean value of 92% of the total O., uptake. This confirms the findings of Schneider- man and Williams and of Buck ct al. But still a periodic O2 uptake is found as well. Perhaps of interest in this connection is what we find on page 147 in the paper of Buck and Keister (1955) where we can read: "Small but statistically significant perturbations were in fact seen in some of our O, uptake records. . . ." Occasionally a double burst was recorded as was already described for pupae of Sphinx and Papilla (Punt. 1950; Fig. 7). OXYGEN UPTAKE IN INSECTS 115 02 CO2 1 HOUR FIGURE 8. Gas exchange of a cecropia pupa, a few weeks before hatching (May, 1954). Drawn from photographic records. In Figure 8 we find a record of simultaneously estimated CO2 release and O2 uptake of the same cecropia pupae made a few weeks before hatching. The graph is on the same amplification and on the same time scale as Figure 6. The hurst frequency has increased to two per hour. The OL, uptake in the bursts is relatively larger. 3. Influence of o.vygen tension on burst frequency The influence of CO2 tension on the burst frequencies in Carabus has been previ- ously described (Punt, 1955, 1956b). Increasing pCO2 caused a prolonged open- 21% 02 A /V V_ " U^U _/y /y A A A 10% 'WwWxJU/v/ 5% FIGURE 9. Hadrocarabus problematicus ; COL, hurst frequency as influenced by different OL. tensions. 116 A. PUNT, W. J. PARSER AND J. KUCHLEIN ing of the spiracles, until at about \%c/< of CO, the closing of the spiracles was inhibited. In the present investigation experiments were performed on the in- fluence of decreasing oxygen tension on burst frequency in Hadrocarabits prob- leinaticus. In Figure 9 a summary is given of the results. A decrease of pO2 caused an increase in C( ).. burst frequency and consequently a decrease in burst volume. This is in accord with Wigglesworth's observation on spiracular move- ment in fleas (Wigglesworth, 1953). 4. The C02 dissociation curve of haemolymph For reasons given in the discussion, we thought it worthwhile to know some- thing about the CO, dissociation curve of insect haemolymph. Much work has been done on the composition of the body fluid of insects, but as to the possibility of buffering the CO, our knowledge is scanty (Reali, 1955; van Asperen and van Esch, 1956; Levenbook and Clark, 1950). We therefore tried to estimate the CO, TABLE I The COz-binding property of haemolymph of Sphinx ligustri pupae Quantity of haemolymph pCO? Total CO2 (mL) (mm.Hg) (volume1,) 0.50 0 2.2 ± 0.8 0.70 0 1.7 ±0.6 0.69 0 2.2 ± 0.6 0.49 19 5.1 ± 0.9 0.23 31 10.9 ± 1.0 0.50 38 9.2 ± 1.1 0.53 38 8.9 ± 0.9 0.38 73 18.2 ± 1.4 0.24 84 17.9 ± 2.2 0.33 105 20.0 ±1.5 saturation of haemolymph under different tensions of CO,. The haemolymph of diapausing pupae of Sphinx Uijustri was gathered by puncturing the pupae with a syringe which was previously moistened with tromboliquine (heparin), in order to prevent clotting. The exactly measured quantity of haemolymph was put in the small bulb-flask of the Haldane blood-gas analyzer and saturated with CO, under a certain tension by rotating the flask mechanically for one hour. After that time the analysis was performed in the normal way, tartaric acid being used to expel the CO, from the haemolymph. The results are to lie found in Table I and in Figure 10. The data are corrected for dissolved CO, at the different tensions and at the temperature used (20° C.) so that the total amount of CO, could be plotted against the pCO2. Though only a few experiments were performed (our stock of pupae being exhausted for this season ) , it proved that the CO ..-binding capacity exceeds the line which represents the dissolving of CO2 in water at the same temperature (the solid line in Figure 10; data from Umbreit ct al, 1949). Our data are too fewr in number to allow drawing a correct line through the points. Insect haemolymph probably contains some CO2-binding principle but in our pupae the capacity was rather disappointing as only twice as much CO2 could be extracted from the haemolymph as would dissolve in pure water. In certain other OXYGEN UPTAKE IN INSECTS 117 insects (Gastrophilns and Hydrophilus; cj. Florkin, 1937) the quantity of bound CO2 was found to be much larger, but these may perhaps represent special cases. More work on this subject is in progress. DISCUSSION We will try to give a possible interpretation of the recorded O2 uptake and CO2 release in Hadrocarabus. This interpretation is, of course, only hypothetical, but 20 o 016 1 12 O > 8 2O 4O 6O 8O pCO2 mmHg FIGURE 10. The CO,, capacity of haemolymph of Sfhin.v liijustri (20° C). Solid line: CO,. capacity of pure water. is in agreement with the results of our and other investigations and partly based upon assumptions from Wigglesworth (1953) and from Buck and Keister (1955, p. 162). Probably in other insects, including diapausing pupae, the same phe- nomena may occur. We may assume that oxygen is taken up continuously in some way or another. But the quantity which can penetrate into the insect body in the "closed spiracles" 118 A. PUNT, W. J. PARSER AND J. KUCHLEIN period is insufficient to cover the metabolic oxygen want. So an oxygen debt is incurred, oxidation being incomplete, and acid metabolites are formed and accumu- lated in the body fluid. This is in accordance with the theory of Wigglesworth, who attributes the withdrawal of fluid from the tracheal endings to increased osmotic value of the tissue fluids, due to the increasing amount of acid metabolites. Carbon dioxide is formed by metabolic processes and stored in the body fluid, not only as dissolved gaseous CO2 but probably bound in some buffer system or CO2-binding principle. But as soon as the pO2 in the tracheal system is diminished and an oxygen debt develops in the tissues, the acid metabolites drive the CO2 from the bound phase into the gas phase and as a consequence the pCO2 in the tracheae will increase. As soon as the pCO2 has reached a certain threshold the spiracles open and the CO2 burst takes place. In the open period the CO, diffuses out, not only from the tracheae but from the tissue fluid as well, until the moment when the pCO2 reaches a level at which the spiracles may close. In the meantime O, has entered the tracheae, which is seen as the initial spike in the O2 uptake line. But after this initial spike the O2 line falls down to a level slightly above the steady state of the interburst period. Probably this part of the O2 line represents the real oxygen consumption of the animal per unit of time. When the spiracles are closed at last, the amount of O2 in the tracheal system is sufficient to cover the metabolic want for some time (the O2 line going down to nearly zero), but as soon as the pO2 inside the trachea is low enough, a leakage of O2 starts (continuous O2 uptake). This leakage is not sufficient, however, to cover the real oxygen want and acid metabolites are formed again, by which the described respiratory cycle is completed. This hypothesis is in accordance with the assump- tion that CO2 controls the sudden opening of the spiracles (Buck and Keister, 1955 ; p. 161). The action of decreased pO2 on the triggering of the spiracular opening may be seen as an indirect one : decreased pO2 increases the degree of hypoxia and hence the amount of gaseous CO2 liberated into the tracheae. Similarly Wiggles- worth's assumption that in pure oxygen, opening is induced by a large amount of CO2, and in 5% O2 by a very small amount of CO2, could be interpreted to mean that in pure oxygen the CO2 remains bound in the body fluid and that in both cases (1007o of O2, and 5% of O2) the tension of free CO2, triggering the spiracles, may be the same. Probably this threshold CO2 tension is not very high, for there is evidence that in about 1%% of CO2 the spiracles do not close at all (Punt, 1956b). So it looks as if in all cases it is the pCO2 which controls spiracular movement. When oxygen tension is lowered, oxygen debt develops more readily and the pCO2 will reach the critical level sooner. As a consequence the burst frequency is in- creased. The burst volume is decreased, as no large quantities of CO2 can be ac- cumulated in the short interburst period. It would be worthwhile to determine the pH of the haemolymph under these circumstances. When oxygen tension is raised, acid metabolites are not formed so soon and more metabolic CO2 may be bound in the body fluid, the intertracheal pCO2 only reaching the critical value after a longer period; burst frequency is decreased, burst volume increased (Punt, 1956b). As to the experiments in pure oxygen, when the spiracles are opened the tracheae are filled with pure oxygen and as a consequence the oxygen leak in inter- burst period is much less. Schneiderman and Williams (1955; p. 134) stated that the "interburst CO2 output," too, may become undetectable in pure oxygen. OXYGEN UPTAKE IN INSECTS SUMMARY 1. The simultaneous determination of CCX production and O, uptake in Hadro- carabus problematicus Hrbst. is described. Both CO2 release and (X uptake are cyclic, but there is some interburst O2 uptake. 2. The diaferometrically recorded O2 uptake is discussed, and compared with the results as obtained with the Warburg technique. 3. In diapausing pupae of the cecropia silkworm (Hyalophora cecropia) the continuous O2 uptake forms a larger percentage of the total amount of O2 consump- tion than in Hadrocarabus. Still there was found an initial maximum of O2 uptake of short duration at every CO, burst. 4. Some experiments on the CO2 dissociation curve of haemolymph of pupae of SpJiin.v lignstri are mentioned. There is evidence that in haemolymph a CO2- binding principle is present. 5. The interaction of O2, haemolymph and CCX, resulting in a certain inter- tracheal pCO2 controlling spiracle movement, is discussed. Probably the pO2 is only indirectly involved in the triggering of spiracle opening. LITERATURE CITED VAN ASPEREN, K., AND I. VAN ESCH, 1956. The chemical composition of the haemolymph in Pcriplancta amcricana. Arch. Ncerl. dc Zool., 11: 342-360. BUCK, J. B., M. L. KEISTER AND H. SPECHT, 1953. Discontinuous respiration in diapausing Agapema pupae. Anat. Rec., 117: 541. BUCK, J. B., AND M. L. KEISTER, 1955. Cyclic CO., release in diapausing Agapema pupae. Biol. Bull., 109 : 144-163. FLORKIN, M., 1937. Sur la composition du plasma sanguin des insectes adultes. Arch. Intern. Physiol, 45 : 6-16. LEVENBOOK, L., AND A. M. CLARK, 1950. The physiology of carbon dioxide transport in insect blood. II. The effect of insect blood on the rate of hydration of CO-,. E.vp. Biol., 27: 175-183. PUNT, A., 1944. De gaswisseling van enkele bloedzuigencle parasieten van warmbloedige dieren (Cime.v, Rhodnhts, Triatoina). Ondcrz. Physiol. Lab. R. U. Utrecht, 8th Ser., 3: 122-141. PUNT, A., 1950. The respiration in insects. Physiol. Coinp. ct Occol., 2 : 59-74. PUNT, A., 1955. New diaferometric investigations on the respiration in insects. A eta Physiol. Pharmacol. Nccrl., 4: 114. PUNT, A., 1956a. Further investigations on the respiration in insects. Phvsiol. Coin p. ct Oecol.,4: 121-129. PUNT, A., 1956b. The influence of carbon dioxide on the respiration of Carabits ncinoralis Mull. Physiol. Coinp. ct Occol.. 4 : 130-139. REALI, G., 1955. Studi sull' emolinfa degli insetti. Boil. Zool. Agraria, 21 : 185-188. SCHNEIDERMAN, H. A., AND C. M. WILLIAMS, 1953. The physiology of insect diapause. VII. The respiratory metabolism of the cecropia silkworm during diapause and develop- ment. Biol. Bull., 105 : 320-334. SCHNEIDERMAN, H. A., AND C. M. WILLIAMS, 1955. An experimental analysis of the discon- tinuous respiration of the cecropia silkworm. Biol. Bull.. 109: 123-143. UMBREIT, W. W., R. H. BURRIS AND J. STAUFFER, 1949. Manometric techniques and related methods for the study of tissue metabolism. Burgess Pub. Co., Minneapolis. WIGGLES WORTH, V. B., 1953. Surface forces in the tracheal system of insects. Quart. J. Microsc. Sci., 94 : 507-522. STUDIES ON MARINE BRYOZOA. X. HIPPADENELLA CARSONAE, N. SP. MARY DORA ROGICK College of New Rochelle, New Rochelle, N. Y. The writer wishes to express her most sincere appreciation to the National Sci- ence Foundation for research grants which have greatly aided this and other stud- ies, and to the Smithsonian Institution, U. S. National Museum for the loan of Bryozoa collected by Comdr. David C. Nutt on the U. S. Navy's 1947-48 Antarctic Expedition. The purpose of this paper is to give a detailed description of the morphological features of Hippadenella carsonae, new species and to note other species which are ecologically associated with it. Hippadenella carsonae is a lepralioid Ectoproct, Order Cheilostomata, Sub- order Ascophora, of the Family Hippoporinidae of Bassler (1953). It is named in memory of Louisa Carson, a beloved teacher of my early school years. SPECIES DATA Hippadenella carsonae, n. sp. Diagnosis: Colony calcareous, twig-like ; branching open. Twigs slender, cylindrical, with usually nine to twelve, sometimes more, wedge-shaped zooecia in cross-section. Mural rims raised, crinkled. The smooth to tubercled frontal is a pleurocyst with three to five pairs of oblique, tubular pores. Orifice lepralioid, rounded. Lyrula absent but two slight cardelles are placed low. Operculum with lateral parenthesis-like sclerites. Very broadly oval median suboral avicularium placed at a varying angle to the frontal plane and mounted on a wide porous avicu- larial chamber that contains prominent avicularial glands, muscles and vestigial polypide. Hyperstomial ovicell very salient, globose, but slightly flattened, tubercled and non-porous. Its arched rim is a continuation of the zooecial mural rims. Two communication areas (a multiporous pore chamber and corresponding opening) in lateral wall. Distal wall a sieve plate of numerous pores. Measurements: The first figures are the minimum, the next the maximum and the last (in parentheses) the average of ten readings (or occasionally more) for each structure. Readings are in millimeters. L is for length, W for width, D for diameter, H for height. 1.073-1.406 (1.247) L Zooecia 0.407-0.555 (0.444) W Zooecia 0.115-0.173 (0.146) L Avicularial apertures (rostal and back areas) 120 HIPPADENELLA 121 0.101-0.173 (0.137) 0.058-0.086 (0.072) 0.094-0.173 (0.124) 0.144-0.202 (0.173) 0.144-0.216 (0.179) 0.158-0.187 (0.170) 0.173-0.202 (0.192) 0.432-0.475 (0.452) 0.389-0.490 (0.422) 0.675-0.705 (0.690) W Avicularial aperture 1 - Mandible W Mandible L Zooecial orifice \Y Zooecial orifice I . Operculum W Operculum L Ovicell W Ovicell L Tentacles (two readings only) The zooecial polypide measurements below are based on only one reading each : 0.147 D Tentacular bundle \vithin the tentacular sheath 0.264 L Esophagus 0.132 D Esophagus, just below the tentacles 0.073 D Esophagus, just above the cardia 0.029 H Esophageal epithelial cells near tentacles 0.014 H Esophageal epithelial cells near cardia 0.132 D Stomach caecum (empty) 0.250 L Rectum (empty) 0.088 D Rectum (empty) Zoarhtui: The zoarium or colony consists of openly branching twigs which some- times fuse or anastomose with other twigs at point of contact. The twigs are fragile and brittle, breaking into shorter twigs, so that no true picture of the extent of a colony, nor the ultimate pattern of branching, whether dichotomous or irregular (Figs. 11, 13) can be gotten from the mass of short fragments at hand. Some of the broken fragments were up to 51 mm. long and about 2 or 3 mm. in diameter. There was about a pint of material in the collection, but not one of the stalks was a complete, intact colony. To the naked eye the twigs appear smoothly cylindrical (Figs. 13, 21) except in the ovicelligerous region where they are covered with bumps (ovicells) as in Figure 11. The usual number of zoids around a branch is about nine to twelve, although occasional stalks may have almost twice that number just before a bifurca- tion (Figs. 14. 21). Zoids face outward around the usually cylindrical branches radially. Dead twigs are white, living twigs yellow. In practically all the living twigs, i.e., twigs collected when they were in the living state, the polypides were confined to the tips or distal part of the stalk while the basal, proximal part of the twig was dead, polypideless. The yellow color of live twigs is due to the presence of yellow polypides within the zooecia. Zooecia: The outside calcareous skeletal case in which the soft zoid parts (di- gestive tract, musculature, tentacles) are housed is the zooecium. Zooecia are wedge-shaped in cross section (Fig. 21). mostly rectangular in frontal or face view and quincuncially arranged (Figs. 6, 12) like bricks in a wall. In side view the end wall (Figs. 8, 10) slants a bit obliquely downward and backward. In some zooecia 122 MARY DORA ROGICK it is nearly horizontal. The frontal wall is of variable thickness, sometimes being twice as thick as the side wall. Zooecial boundaries are well defined by mural rims (Figs. 3, 12, 17, 18, 23). The height of these partitions varies with age and with exposure to molar forces. Sometimes these rims are high and very crinkled. Other times they are nearly level with the zooecial front. In early secondary calcification the distal mural rims send slender calcified trabeculae across the operculum (Figs. 18, 23). Later, the orifice may be incompletely obliterated by more extensive calcification over the operculum (Figs. 6, 17). The frontal wall is flat to convex, porcellanous to sparsely tubercled, non-porous centrally but completely perforated by about three to five pairs, usually three pairs (Fig. 10), of obliquely directed marginal tubular pores or tubes whose diameter varies (Figs. 30, 32). These tubes end in slight elliptical craters above the frontal (Fig. 32). Viewed from the front, the top pair of tubes slants diagonally, con- verging downward toward the zooecial mid front. The middle pair slants toward the zooecial mid line. The bottom pair slants diagonally upward toward the zooecial mid front, as in Figure 30. In other words, the marginal tubes lean toward the zooecial center front. The front may be perfectly smooth except for the raised pores or it may be roughened by a few tubercles (Figs. 6, 17). The side walls are straight and have two widely spaced, multiporous, blister-like interzooecial communication areas. These areas are variously known in bryozoan literature as rosette plates, septula, pore chambers, corresponding openings. The distal one is a pore chamber and the proximal one is a corresponding opening (Figs. 8, 10, 19, 26). The pore chambers have six to twelve small pores (Fig. 19). The corresponding opening has a single large hole rimmed about by an irregular annulus (Fig. 26). The quincuncial arrangement of zooecia brings the pore chambers of one vertical row of zooecia against the corresponding openings of the left or right vertical rows of neighboring zooecia, and vice versa. Silen (1944) has given an excellent and extensive account of pore chambers, corresponding open- ings, pore plates and various types of interzooecial communications for various species. Conditions in Hippadenella carsonac are in agreement with his findings for related species. The end wall is oval to pear-shaped (Figs. 10, 21 ). Its smaller, medial part is punctured by numerous (about forty, more or less) small, closely spaced pores and slants downward more than the broader peripheral non-porous part. Some- times the slant is rather steep, sometimes deviating little from the horizontal. The calcareous frontal walls are a bit too opaque usually for satisfactory study of soft internal parts as tentacles, gut, gonads, musculature and avicularial apparatus. Decalcification was not attempted because of the nature of the colony — a number of zooecia radially arranged, very close together. Crushing the stalk gently in a drop of Euparal on a slide usually gave well dispersed material quickly and in rea- sonably satisfactory condition for microscopic study. Attempts were made to clear the youngest, slenderest, polypide-containing tips in glycerine and others in dioxan. Some clearing was accomplished with the glycerine but none with the dioxan. So, the description of the soft internal parts is based on crushed, dispersed material and also on what could be seen through the walls of the younger, less calcified zooecia. HIPPADENELLA 123 For a study of the exoskeleton or zooecial walls calcining (burning off the chitinous and membranous coverings with a small blow pipe) is the most satisfactory method of preparation. However, calcining must be halted before the specimen becomes too fragile and disintegrates to powder. Autosooccial polypidc: The autozooecial polypide consists of the tentacles, gut and associated musculature (Figs. 2, 7, 16, 29). All polypides are in a retracted position, the polypides withdrawn into the body cavity, none with tentacles ex- truded through the orifice, so characteristics of the retractor muscles, parietal body wall muscles and tentacle number could not be studied. As far as can be deduced from retracted specimens there seem to be about twelve to fourteen tentacles. The tentacles are ciliated, rather stout and not of excessive length. They are withdrawn into a transparent membranous tentacle sheath which is closed at the top near the operculum (Fig. 16) by a sphincter or diaphragm. Two flask-shaped "oral" or "sub-oral" glands of unknown function are part of the sheath, just beneath the diaphragm. Attached to the diaphragm are two bundles of muscle fibers, the parieto-diaphragmatics, one on each side (Figs. 2, 16, 29). Their muscle fibers extend diagonally back and upward to attach to the zooecial wall. A short distance below the parieto-diaphragmatics are two membranous bands, the parieto-vaginals, which contain a few delicate fibers that are deviated from the tentacle sheath. The parieto-vaginals connect the tentacular sheath to the lateral zooecial walls. The gut consists of mouth, esophagus, stomach ( which has three divisions : cardiac, caecal and pyloric), rectum and anus. In some bryozoa there is a ciliated pharynx between mouth and esophagus but not in H. carsonac. The esophagus of H. carsonac is short and tapers slightly. Its epithelial mucosa cells are tall colum- nar, hyaline and not ciliated. Those near the mouth are twice as tall as those near the cardia, the diminution in height being gradual. A sphincter separates the esophagus from the cardia. Retractor muscles attach the lophophore (region at the base of the tentacles and around the mouth) to the body wall, so there originates a curtain of muscle fibers just at the beginning of the esophagus. The epithelial cells of the cardia are low, cuboidal. non-ciliated and not much different in diameter LIST OF ABBREVIATIONS USED ON THE PLATES A Abductor mandibuli muscle fibers B Adductor mandibuli muscles C Avicularial back area D Avicularial chamber E Avicularial gland F Avicularial polypide G Avicularial rostral area H Avicularium I Cardelle J Cardia K Esophagus L Mandible M Mural rim N "Oral" gland O Orifice or aperture P Ovicell Q Parieto-diaphragmaticus muscle R Parieto-vaginal band S Pore chamber or rosette plate T Pylorus U Rectum V Sclerite \Y Stomach X Tentacles Y Tentacular sheath All figures, except Figures 4, 8, 10, 11 and 13, were drawn with the aid of a camera lucida, and are of Hippadcnella carsonae, new species, from Antarctic type locality, Sta. 104. Figure 11 is from the holotype, the others from paratypes. Measurements for structures are given in text. 124 MARY DORA ROGICK PLATE I HIPPADENELLA 125 from those of the esophagus but their cytoplasm is granular rather than hyaline. The carclial wall is thin. The cardia is J-shaped, parallelling the esophagus. The stomach caecum originates at the upper side of the cardia, near the pylorus, and has a highly granular cytoplasm. The pylorus is ciliated, internally. The different parts of the stomach and also the rectum are all rather thin-walled. Diatoms must be a part of the H. carsonac diet because diatom shells are in some of the recta. The gut and tentacles amply fill the body cavity. The ovary is attached to the side wall, behind the tentacular sheath and below the operculum. It is present in non-ovicelled zoids and presumably also in ovi- celled ones as well as in zoids with or without an avicularium. If an avicularium ic EXPLANATION OF PLATE I FIGURE 1. Mandible, top surface view. Transparent oval in center is the lucida. Ad- ductor muscle fibers attach as individual dots in the two semicircular areas in front of lucida. Transparent rimming membrane (cf. Figs. 9, 25) here barely visible about rounded tip. FIGURE 2. Side view of upper third of polypide. Compare with Figures 7, 16, 29. FIGURE 3. Frontal view of three calcareous ovicelled zooecia. The upper two have avi- cularia whose chambers are small. From calcined specimen. FIGURE 4. Diagram showing contents of avicularial chamber, as seen from top and front. One of the avicularial glands contains a hardened or coagulated crescent-shaped secretion. The delicate abductor muscle fibers are shown bent upward, out of their natural position, for the sake of clarity. They should extend downward and backward toward the base of the avicularial chamber, some distance away from and back of the adductor tendons. FIGURE 5. Side view of "diaphragm" or sphincter and the two "oral" glands which con- tain some secretion. The sphincter is slightly relaxed, so is evident a narrow central passage- way that must widen considerably to permit extrusion of tentacles. FIGURE 6. Frontal view of three calcareous zooecia. The upper two have fully developed and functional orifices and avicularia. The bottom one has a nearly obliterated orifice and avicularium due to secondary calcification. Avicularial chambers are large as compared with those of Figure 3. FIGURE 7. Retracted polypide. The slender upper third corresponds to Figure 2 but is from a different side. The esophagus is partly hidden by the stomach caecum. The anus is at the uppermost tip of rectum. FIGURE 8. Diagrammatic side view of zooecia, front wall to the left, side wall facing observer. Ovicelled zooecium complete, the non-ovicelled one only partly shown. The end walls slant, sometimes less obliquely than shown, may even be nearly horizontal in some zooecia. Side walls are perforated by two communication areas. Upper, distal multiporous area (S) is like an internal blister (cf. Fig. 19) and is called a rosette plate or pore chamber. Lower, proximal single opening fits against a rosette plate of a neighboring zooecium which is not here shown. Single opening bears the inadequate name of "corresponding opening." FIGURE 9. Mandible, edge view. The delicate serrated membrane hangs vertically down from front edge. More heavily cuticularized parts are darkened. FIGURE 10. Diagram of a single, non-ovicelled, wedge-shaped zooecium. Its side wall (with two communication areas) is at left. Its frontal wall (with six frontal pores) is at right, A multiporous end wall is at bottom. Three pores are shown on the avicularial chamber. FIGURE 11. Colony fragment, drawn to the one-cm, scale at immediate left. Bumps along stalk are ovicells. FIGURE 12. Frontal view of four zooecia, two with avicularia and two without. From a calcined specimen. FIGURE 13. Another colony, with more branches. Drawn to same scale as Figure 11. FIGURE 14. Cross-section of a slightly flattened branch which has thirteen wedge-shaped zooecia at this level. FIGURE 15. Four eggs in ovary. Nucleoli prominent, excentric. Nuclei clear, vesicular. Cytoplasm homogeneous and denser. Drawn to the 0.06-mm. scale at left. 126 AlARY DORA ROGICK PLATE II HIPPADENELLA 127 present the ovary is about half-way between the operculum and the avicularial chamber, back of the tentacular sheath. It contains several eggs of different sizes. The ova have a large excentric nucleolus and an almost clear nucleus (Fig. 15). Whether an ovary is present in every zoid cannot be ascertained because of the opacity of zooecial walls. Whether this species is hermaphroditic or dioecious is unknown at present. It was not possible to determine the exact site of origin of spermatogenic tissue although some material, apparently spermatogenic, was found near the stomach in rather sizable patches in the crushed specimens. Orifice: The H. carsonac orifice resembles those of the genera Cryptosula and Hippodiplosia. It is rounded, lepralioid. Part of its distal wall is formed by the next distal zoid. A median tooth, lyrula, is absent. Two slight cardelles or ledges for operculum articulation are placed low at the sides, dividing the orifice into two areas of unequal size. The larger distal area is formed by the anter (distal orifice lip) while the smaller proximal area is bounded by the poster (proximal orifice EXPLANATION OF PLATE II FIGURE 16. Operculum, "diaphragm," "oral" glands, tentacle sheath, tentacle tips and anchoring musculature shown from the back surface. The channel for protrusion of tentacles is more contracted than in Figure 5. FIGURE 17. Frontal view of zooecium with completely sealed-over orifice in a more ad- vanced stage of calcification than that of Figure 6. FIGURE 18. Frontal view of the upper half of a zooecium in an early stage of secondary calcification where calcareous trabeculae extend from the mural rims across the orifice. A partly opened mandible articulates with the pivot. FIGURE 19. Multiporous rosette plate of side wall. FIGURE 20. Profile of zooecial orifice and avicularium with operculum and mandible, re- spectively, in place. The avicularial wall is left more transparent than it normally would ap- pear to show the position of the avicularial contents. Compare with Figures 4, 22, 27. FIGURE 21. Stereogram of typical cylindrical stalk. Orifice and avicularium on bottom zooecium. Porous end wall tops another. FIGURE 22. Oblique top view of avicularial apparatus. Glands curve around adductor tendons to fit the avicularial chamber. FIGURE 23. Front of a zooecium which has an ovicell and avicularium, and shows secondary calcification trabeculae spanning the orifice. Ovicell opening is completely plugged up by domed calcareous lamina which occupies about half the ovicell interior and which is here faintly outlined, cf. Figure 24. FIGURE 24. Ovicell whose front wall has been broken away to show the double outer wall and the third (innermost) lamina which has sealed off the ovicell cavity. The lamina resulted from secondary calcification. FIGURE 25. Edge view of another mandible with serrate membrane and adductor muscle tendons which attach to semicircular areas (cf. Fig. 1). FIGURE 26. Looking through a "corresponding opening" with its irregular annulus or ledge, into the multiporous blister-like rosette plate (cf. Fig. 19). FIGURE 27. Avicularial contents from the under side. Abductors are not shown. FIGURE 28. Detail of orifice, cardelles and avicularium. The bands leading from orifice to avicularium represent differences in thickness of frontal calcification and are also the distal limits of the avicularial chamber. FIGURE 29. Frontal view of "oral" glands, operculum and tentacle tips which are very close to extrusion. The "diaphragm" opening is wider than in Figures 5 and 16. FIGURE 30. Interior of frontal zooecial wall showing shape and direction of tubular frontal pores. The interior openings have been blackened. The exterior openings are faintly rimmed to show their raised nature (cf. Figs. 3, 6, 32). FIGURE 31. Inner face of operculum. Occlusor muscles attach to sclerites. FIGURE 32. Side view of frontal wall tubular pore and its raised external terminus. 128 MARY DORA ROGICK lip). The cardelles form the dividing line between the two areas, cj. Figure 28. In side view (Fig. 20), the orifice is not a flat plane. The poster curves outward in lepralioid fashion. There is no difference in size between orifices of the ovi- celled and non-ovicelled zooecia. The zooecial orifice is covered by an operculum shaped to fit ( Figs. 20, 31 ). It has been the universal and long-time practice for bryozoologists to use the term "chitinous" when describing opercula of those bryozoa which have a more or less horny, yellowish to brown, sometimes unevenly stiffened or reinforced oper- culum. Its use is based more on the visible physical appearance of the substance (its resemblance to the insect exoskeleton) rather than on its chemical composition. Dr. Libbie Hyman suggests that the term "cuticularized" would be more appropriate since little chemical evidence exists for the presence of chitin in the ectoprocts (bryozoa). So, where I have used the term "chitinized" in the present and in past articles in connection with bryozoa the term "cuticularized" would perhaps have been more suitable. The H. carsonae operculum is lightly cuticularized (Fig. 31). Near its lateral borders, internally, are parenthetical sclerites to which attach delicate occlusor muscle fibers and from which a flange may develop in some older zoids. The orifice in many old zoids is overgrown or secondarily calcified and may be entirely sealed over or obliterated (Figs. 17, 18). Sometimes the secondary calci- fication extends to the avicularium and ovicell, so that the avicularium too fuses over and the ovicell opening is blocked by a dome-shaped calcareous lamina (Figs. 6, 23, 24). Peristome and oral spines are absent. Ovicells: The ovicells were collected long past the breeding season because larvae are absent. The ovicells are either empty or partitioned off by the internal second- ary calcification lamina. Ovicells generally occur in groups at periodic intervals along the stalk. They are not immersed or covered over by the frontal of the next zoid but rest on a cushion formed by it (Figs. 3, 8). They are large, salient, non-porous. Their surface is beaded to tuberculate and faintly ridged. The thin raised rim about the highly arched opening is continuous with the raised mural rims. Avicularia: An avicularium occurs on some zoids, either ovicelled or non-ovi- celled. Its size varies from small to medium, some avicularia being twice as large as others. In position it is constant, always sub-oral, median, slanting obliquely forward-downward, away from the orifice. A pivot bar or hinge (Figs. 18, 28) for articulation with the mandible separates the avicularial surface into two slightly inclined regions : (a) the back area and (b) the mandibular, beak or rostral area. The rostral area is closed by the mandible. The membrane-covered back area is shorter and broader than the rostral area and in this species is always nearest the orifice, the rostral area being the farthest away, both in the mid line and sub-oral (Fig. 20). In face view the avicularium is broadly oval, wider than long, perched on a wide mound-like avicularial chamber of variable size. Usually three, occasionally more or fewer, small pores perforate the front of this chamber. There is a great difference in the degree of development or complexity of the soft structures inside the avicularia of various species but in H. carsonae they are well developed. HIPPADENELLA 129 Levinsen (1909) called the avicularia and their contents heterozooecia and heterozooids ; Borg (1926) heterozoids ; Silen (1938) heterozoids, avicularial polypides, polymorphic individuals. Earlier references exist to the avicularial contents (Waters, 1888, 1892, 1900) but Waters' 1892 account is very adequate and understandably figured, although Silen (1938) published an extensive and excellent study of avicularia of several species. The avicularial contents of Hippadenella carsonae are similar to those described by Waters (1892, pp. 272-274, and PI. 19, Figs. 1, 2, 4, 5) for Lepralia foliacea Ellis and Sollander. In H. carsonae the avicularial chamber contains the avicularial polypide, avicularial glands, abductor mandibuli and adductor mandibuli muscles (Figs. 4, 20, 22, 27). The function of the polypide and glands is unknown. Marcus (1939, PI. 19, Fig. 46) shows the avicularial contents of seven species and on p. 275 says of avicularial glands : "These glands can neither belong to the nervous, nor to the nutritive, or reproductive system and might perhaps have something to do with the stronger skeleton of the Ascophora . . . the function of the (se) organs still remains unknown ; they might be poisonous." The H . carsonae avicularial glands are sometimes large, hollow and alveolar, with a large lumen and thin wall. Other times they are partly filled with a homogeneous, hardened secretion. The two bilaterally placed glands are united in a saddle-shaped unit that curves about the two bundles of adductor tendons and follows the contours of the very wide avicu- larial chamber. The avicularial polypide is a small, dense cellular body pinched in the middle so it seems double. It is attached to the back of the avicularial gland isthmus, in the mid-line. A vestibulum connects the polypide to the rostral area below the mandible tip, although this is difficult to see because of the opacity of the calcareous wall, the infrequency of favorably oriented crushed specimens and the small size of the soft structures involved. The musculature of the avicularium is well developed. The adductor mandibuli muscles are more anterior, proximal and massive than the abductor mandibuli muscles. The adductor muscles have numerous short, thick, faintly striated muscle fibers. Their endings at origin and insertion differ in appearance. Their origin is over an extensive area on the walls and floor of the avicularial chamber. Their insertion is in two small pits, one at each side of the lucida on the inner mandibular surface. The muscle fibers attach bluntly and broadly at the origin while before the insertion is reached the muscle fibers have given way abruptly to delicate mem- branous and fine tendinous tissue (Figs. 4, 20). The tendon fibers attach by bead- like enlargements to the insertion site (Figs. 1, 25, 27). The abductors form a very diffuse, sparsely fibered curtain against the distal end of the avicularial chamber. Their fibers originate on the back avicularial chamber wall and insert on the circularized membrane very close to the pivotal bar against which the mandible articulates. They do not seem to be striated and are consider- ably more slender than the adductor fibers. Marcus (1939, p. 273) states that in species studied by him the avicularial adductors are striated but the abductors smooth. This is true of H. carsonae also. The avicularium is topped by a cuticularized mandible and a back area mem- brane (Figs. 20, 27). The mandible has a narrowly elliptical lucida flanked on each side by a somewhat semicircular pit into which the adductor tendons attach. A broad cuticularized band reinforces the mandible edges and base (Figs. 1, 22). A 130 MARY DORA ROGICK short, delicate, transparent and serrated membrane decorates its front border (Figs. 9, 25), hanging down vertically, so it is almost invisible from the top (Fig. 1). Distribution and ecology: Hippadenella carsonae turned up in only two dredg- ings of Jan. 29, 1948, Comdr. D. C. Nutt collector; one fragment from Sta. 101, and a pintful of twigs from Sta. 104. Both stations were from the Ross Sea area, Antarctica, off Cape Royds, Ross Island, from 58 fathoms. Most of the twigs were empty of polypides and relatively clean of extensive extraneous growths or encrustations. When other forms did grow on or in them these were relatively few in number and sparsely distributed over the branches, so apparently the twigs of H. carsonae did not present as hospitable a stratum for settling of other forms as does Phylactellipora lyrulata or some other species. The following organisms or their products are attached to the dead parts of H. carsonae twigs from Sta. 104: brown and green Folliculinids. Foraminifera, brown and white sponges, yellow egg cases (of flatworms?), calcareous tubes of annelids and scraps of various bryozoa. The bryozoa growing in small patches or attached to H. carsonae are several species of the Order Cyclostomata and the following of the Order Cheilo- stomata: Ccllaria inoniliorata, Hippothoa bongaimnllei, Hippothoa distans, Phylac- tellipora lyrulata and a number of other species awaiting fuller identification. The Cyclostomata are especially numerous and seem to favor this species. Some Sta. 104 H. carsonae twigs grow on or partly engulf alcyonarian and sponge spicules, Cyclostomata, Cellaria inoniliorata, Ccllaria vitritnuralis , Smittina ordinata and other cheilostomes yet to be identified. In a discussion of Smittina ordinata (Rogick, 1956, p. 300) reference was made to Smittina ordinata growing on "other Bryozoa at Sta. 104," the other bryozoa in this case being H. carsonae. Hippadenella carsonae specimens are deposited in the Smithsonian Institution, U. S. National Museum, USNM Cat. Nos. 11357 through 11364. SUMMARY 1. The morphology of Hippadenella carsonae, new ectoproct from the Antarctic, is described in detail and measurements made of many of its structures. 2. At the time of its collection, Jan. 29, ovicells were empty of embryos but de- veloping eggs were in the ovaries. Developing embryos were absent from the body cavity also. 3. Living polypides occurred at the tips of some twigs but most of the material was dead or empty of polypides, at the time of collection. 4. The species has an unusually well developed avicularial apparatus consisting of large glands, reduced polypide, abductor and adductor muscles. 5. So-called "oral" or "sub-oral" glands are present. They are near the operculum but have no actual connection with, or proximity to, the true polypide mouth. 6. The function of "oral" glands, avicularial glands and avicularial polypide is unknown in this species, and a matter for speculation in other species. 7. Other peculiarities of this species are the oblique tubular frontal wall channels (frontal pores) and the mode of secondary calcification where trabeculae span the orifice and a domed lamina seals the ovicell. HIPPADENELLA 131 LITERATURE CITED BASSLER, R. S., 1953. (G) Bryozoa, in R. C. Moore's Treatise on Invertebrate Paleontology. Geol. Soc. Amer. 253 pp. BORG, F., 1926. Studies on recent cyclostomatous Bryozoa. Zool. Bidrag frdn Uppsala, 10 : 181-507. LEVINSEN, G. M. R., 1909. Morphological and systematic studies on the cheilostomatous Bryozoa. 431 pp. MARCUS, E., 1939. Briozoarios marinhos Brasileiros, III. Univ. Sao Paulo Bol. Faculd. Filosof., Cienc. e Letras, XII, Zool., No. 3: 111-354. ROGICK, M. D., 1956. Bryozoa of the U. S. Navy's 1947-48 Antarctic Expedition, I-IV. Proc. U. S. Nat. Mus., 105 : 221-317. SILEN, L., 1938. Zur Kenntnis des Polymorphisms der Bryozoen. Die Avicularien der Cheilostomata Anasca. Zool. Bidrag frdn Uppsala, 17: 149-366. SILEN, L., 1944. On the formation of the interzoidal communications of the Bryozoa. Zool. Bidrag frdn Uppsala, 22 : 433-488. WATERS, A. W., 1888. Supplementary Report on the Polyzoa collected by H. M. S. Challenger, 1873-76. Repts. Voy. Challenger, Zool., 31 : (Part 79) : 1-41. WATERS, A. W., 1892. Observations on the gland-like bodies in the Bryozoa. /. Linn. Soc. Zool. London, 24 : 272-278. WATERS, A. W., 1900. Bryozoa from Franz-Joseph Land. Part 1. /. Linn Soc. Zool. London, 28 : 43-105. EXCYSTATION OF APOSTOME CILIATES IN RELATION TO MOLTING OF THEIR CRUSTACEAN HOSTS WILLIAM TRACER Rockefeller Institute, Netv York, and the Marine Biological Laboratory, Woods Hole, Massachusetts The dependence of certain aspects of the life cycle of a parasite on certain physiological activities of its host is a common phenomenon. One need only men- tion, as examples, the synchronicity of reproduction in malaria parasites, which is dependent on the diurnal rhythm of activity of the host (Stauber, 1939), and the appearance of sexual reproduction in the intestinal protozoa of the roach Crypto- ccrcits, which is provoked by the molting of the host (Cleveland and Nutting, 1955). In a similar way, some species of apostome ciliates exist as small cysts (phoronts) on the gills of various Crustacea and excyst only at the time when the host molts (Chatton and Lwoff, 1935). The excysted forms (trophonts) then engorge rapidly on the proteinaceous fluid in the shed skin of the host. The much larger organisms so produced form free-living cysts. Within these cysts a series of divisions occurs which results in the formation of a number of small daughter ciliates (tomites). These swim about, apparently without feeding, until they have been drawn into the gill chamber of a suitable crustacean host. Here they encyst on the gills and again remain quiescent until the new host molts. The entire cycle depends on a single meal obtained from the molting fluids of the host, and the molting of the host provides the only stimulus to excystation (Chatton and Lwoff, 1935). It seemed of interest to attempt to produce excystation in ritro. MATERIALS AND METHODS Two host-parasite combinations have been used : ( 1 ) the fiddler crab Uca pugna.r and a probably undescribed species of Gymnod'mioidcs; (2) the hermit crab Pagurus longicarpus and Gymnodinioides inkystans. The fiddler crabs were kept in dishes with a shallow layer of sea water which was changed daily. Isolated individuals were kept in paper cups with a little sea water and a screen cover. The hermit crabs were maintained in running sea water. Isolated crabs of this species were placed in 100-ml. beakers covered with a piece of gauze held on by a rubber band. The beakers were immersed in an aquarium of running sea water. Both species were fed pieces of clam once or twice a week. Individuals of Uca pugna.v which were near the molt were generally recognizable by a peculiar pale cast to the carapace and legs. Those in the process of molting were observed to have milky white blood. In order to identify crabs very near the molt, the tip of a leg was cut off to permit a drop of blood to exude. The blood had a clear appearance except just before molting, when it was milky. Crabs of the species Pag urns longicarpus near molting could be recognized by a marked gray color of the carapace and legs, as distinguished from the reddish cast 132 EXCYSTATION OF APOSTOME CILIATES 133 of individuals which had recently molted. In small groups of isolated "gray" crabs about 50% molted within three days after isolation, whereas none molted in corresponding groups of "red" crabs. For the in vitro experiments, sterile Petri dishes holding two discs of filter paper and one or two depression slides were used. The filter paper was moistened with sterile sea water. In the concavity of each depression slide was placed a droplet of sterile sea water containing, per ml., 500 or 1000 units of penicillin G and 0.5 or 1 mg. of streptomycin. The lo\ver concentrations wrere used in the experiments with Uca and the higher concentrations in the experiments with Pa-gurus. A crab selected to serve as a source of infected gill material was immersed briefly in 70% ethanol, rinsed in sterile sea water, and blotted on sterile filter paper. The legs were cut off close to the body with sterile scissors and the blood allowed to exude into the droplet of sea water with antibiotics. Two such blood-sea water mixtures could be prepared from one crab. In the experiments with Uca the blood w-as diluted by the sea water about 1:1, in those with Pa-gurus about 1 : 3. The carapace of the crab was then torn off. At this step, if the animal was close to molting, the old carapace cuticle would readily come loose revealing the soft newly formed skin beneath it. After exposure of the gill chamber, the gills were plucked off and placed in the mixtures of blood and sea water with antibiotics. The preparations were kept at room temperatures in dim light and observed with a dissecting microscope for the appearance of trophonts. The gills were later placed between slide and coverslip and examined for the presence of phoronts and for possible signs of excy station. RESULTS A. Observations 1. Gyninodinioides sp. of Uca pugna.r. About 80% of the crabs of this species examined showed a few to many phoronts on their gills. The incidence of trophonts was, however, much lower. Out of a series of 105 recently shed skins only 24 contained trophonts. This might have been the result of very rapid engorgement and early escape of the trophonts from the exuviae, especially since molting usually occurred during the night. On the other hand, it might be that Uca is a relatively unfavorable host. The phoronts were frequently surrounded by a cellular reaction, often containing considerable brown pigment. Such a host reaction was never seen in Pagurus. The living trophonts of this ciliate had a more pointed and twisted posterior end than those of Gyinnodinioidcs inkystans from Pagurus. A silver preparation sug- gested 10 or 11 rather than 9 ciliary bands. The developmental cycle was typical of the genus Gyinnodinioidcs (Chatton and Lwoff. 1935). More detailed morpho- logical study would be needed for the precise identification of this organism, which does not quite fit any of the described species of Gyinnodinioidcs. In gill cuticle material taken from crabs found in the act of molting, small trophonts which had already excysted were present, as well as cysts showing a motile trophont within, and also seemingly unchanged resting phoronts. Cysts containing a motile trophont did not show the conspicuous large refractile granules present in most of the other phoronts. This might indicate a utilization of reserve food granules during the encysted state, a suggestion already made by Miyashita 134 WILLIAM TRACER (1933). The following observation on re-infection, however, is not entirely in ac- cordance with this idea. One Uca isolated in a small dish with a little sea water molted during the night. In the morning engorged trophonts were found swimming about in the dish. The crab was removed to a separate dish. By the following day cysts undergoing tomite formation were observed in the first dish and the crab was returned to it. The next day active tomites were noted in the water of this dish. One day later (three days after the molt) the crab was killed and its gills examined. Eighteen phoronts were found. All of these had few reserve granules and most had a large vacuole near the posterior end. In several this vacuole was seen to collapse and re-form slowly. Had these cysts not been found on a host which was known to have just molted and just been re-infected, they might have been considered "ripe" phoronts ready to excyst. 2. Gymnodinioides inkystans of Pa gurus longicarpus. At least 80% of the molt skins of Pagurus examined during three summers contained moderate to large numbers of trophonts which were clearly G . inkystans. Phoronts were found on the plicae of the gills of most of the hermit crabs examined. B. Experiments in vitro 1. Gymnodinioides sp. of U. pngna.v. In an initial experiment gills from a crab in the act of molting were placed in a droplet of sea water and in sea water con- taining a segment of leg integument. Active small trophonts were already present in the gills, but no\vhere else, at the time of the preparation. Eight hours later only small trophonts were seen in the preparation with sea water alone, but in the other preparation numerous partially to fully engorged trophonts were swimming about. A few were already encysting. By the second day many active tomites had been formed. Thus, once excystation had occurred, the engorgement and subsequent development in vitro were essentially normal. In all the later experiments, with both host-parasite combinations, the methods detailed in the section on Materials and Methods were used. The general plan was to pair a crab judged to be not near the molt with one considered close to molting. The gills of each were divided into two portions. One portion was placed in a droplet of sea water with antibiotics plus blood of the same crab, and the second portion in a similar droplet with blood of the other crab. Out of five experiments of this type in which numerous phoronts were present on the gills of both crabs, fully formed trophonts ready to excyst and showing slight movements were found in three. In these three experiments the crab judged to be near molting had milky blood, whereas in the other two experiments the crab con- sidered near molting had clear or faintly turbid blood. In two of the three positive experiments the signs of excystation occurred only on gills of the crab near molting in its own blood. In the third, they occurred on gills of the crab near molting in the blood of the non-molting one. In no case did phoronts from the gills of the non- molting crab show signs of excysting. 2. Gymnodinioides inkystans of P. longicarpus. Seven experiments of the paired type were done in which phoronts were present on the gills of both the non-molting crab and the crab near molting. The following observations were made. EXCYSTATION OF APOSTOME CILIATES 135 Ex p. 1. Seven hours after placing of the gills in the blood-sea water-antibiotics mixture, four large trophonts were seen in the preparations containing gills from the crab near molting in its own blood, and one small trophont in the preparation of gills from this same crab in the blood of the non-molting crab. Exp. 2. No trophonts were seen after seven hours, but after one day one large trophont was present in the preparation of gills from the crab near molting in its own blood, and two large trophonts in the preparation of gills from the crab near molting in the blood of the non-molting crab. When the gills of the non-molting crab in its own blood were then examined under higher magnification, the unexpected observation was made of three phoronts which showed ciliary movement within the cyst (out of a total of 16 seen). Of 20 phoronts seen on the gills of the non-molting crab in blood of the one near molting, none showed movement although three had a large vacuole near the posterior end. Exp. 3. A large trophont was present after one day in the gills from the crab near molting held with its own blood. None of the other preparations showed any indication of excystation. Exp. 4. After one day four trophonts had developed from the gills of the crab near molting held with its own blood, and one from the gills of this crab with blood of the non-molting crab. Exp. 5. Within seven hours six trophonts had developed from the gills of the crab near molting in its own blood, and one trophont from the gills of this crab in blood of the non-molting one. By the next day two additional trophonts had ap- peared in the latter preparation. Exp. 6. Six trophonts developed within seven hours from the gills of the crab near molting in blood of the non-molting one. Partial drying had occurred in the preparation containing gills of the crab near molting in its own blood. Exp. 7 . No trophonts or signs of excystation could be found in any of the preparations. DISCUSSION Excystation of both species of Gymnodinioides can evidently take place in vitro from phoronts which have not yet begun to excyst in vivo. The host crustacean must, however, be very close to molting time for this to occur, so that the statement of Chatton and Lwoff (1935) that these phoronts excyst only in response to molting of their host still holds. Phoronts from the gills of crabs near molting excysted, on the whole, a little better in the presence of the homologous blood than in the pres- ence of blood from a non-molting crab. Miyashita (1933) noted that certain "ripe" cysts of G. caridinae from a fresh- water shrimp excysted within a few min- utes after being placed under a coverslip in body fluid of the host, whereas many "unripe" cysts did not. It might be that the successful excystation occurred only with cysts which happened to have been taken from a shrimp near molting. The results of the experiments described in the present paper indicate that the encysted phoronts of Gymnodinioides may require a series of stimuli from the host in order to prepare them for the final stimulus which produces excystation just be- fore the actual molt. Such a course of events would be somewhat analogous to that described by Cleveland and Nutting (1955) for the sexual phenomena of the protozoa of Cryptocercus. Protozoa transferred from a roach at one stage of the 136 WILLIAM TRACER molting cycle to another at a different stage invariably died, whereas those trans- ferred to another roach at the same stage continued their development. SUMMARY Observations and experiments have been made with the encysted phoronts of Gymnodinioides inkystans on the gills of the hermit crab (Pagurus longicarpus) and of Gymnodinioides sp. on the gills of the fiddler crab (Uca pugnax}. The phoronts of both species would excyst in vitro, in a mixture of crab blood with sea water and antibiotics, and give rise to engorged trophonts, only if the cysts were taken from gills of a crab which was near molting. This excystation occurred somewhat more readily in the presence of blood from the same crab, near the molt, than in the presence of blood from a crab not close to molting. It is concluded that the encysted phoronts probably require a series of stimuli from the host in order to prepare them for the final stimulus which produces excystation just before the actual molt. LITERATURE CITED CHATTON, E., AND A. LWOFF, 1935. Les cilies apostomes. I. Apergu historique et general ; etude mongraphique des genres et des especes. Arch, de Zool. Exptl. ct Gen., 77 (Fasc. 1) : 1-453. CLEVELAND, L. R., AND W. L. NUTTING, 1955. Suppression of sexual cycles and death of the protozoa of Cryptocercus resulting from change of hosts during molting period. /. Exp. Zool., 130: 485-513. MIYASHITA, Y., 1933. Studies on a freshwater foettingeriid ciliate, Hyalospira caridinac n. g., n. sp. Jap. J. Zool, 4 : 439-460. STAUBER, L. A., 1939. Factors influencing the asexual periodicity of avian malarias. /. Parasitol, 25: 95-116. BIOLOGY MATERIALS The Supply Department of the Marine Biological Labora- tory has a complete stock of excellent plain preserved and injected materials, and would be pleased to quote prices on school needs. PRESERVED SPECIMENS for Zoology, Botany, Embryology, and Comparative Anatomy LIVING SPECIMENS for Zoology and Botany including Protozoan and Drosophila Cultures, and Animals for Experimental and Laboratory Use. MICROSCOPE SLIDES for Zoology, Botany, Embryology, Histology, Bacteriology, and Parasitology. CATALOGUES SENT ON REQUEST Supply Department MARINE BIOLOGICAL LABORATORY Woods Hole, Massachusetts CONTENTS Page BONNER, JOHN TYLER, ALLAN A. HOFFMAN, WILFRED T. MORIOKA AND A. 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The effects of dinitrophenol on mantle respiration and shell deposition 92 MEUWIS, A. L., AND M. J. HEUTS Temperature dependence of breathing rate in carp 97 PUNT, A., W. J. PARSER AND J. KUCHLEIN Oxygen uptake in insects with cyclic CO2 release 108 ROGICK, MARY DORA Studies on marine bryozoa. X. Hippadenella carsonae, n. sp. 120 TRAGER, WILLIAM Excystation of apostome ciliates in relation to molting of their crustacean hosts . 132 Volume 112 Number 2 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, Vanderbilt University J. H. LOCHHEAD, University of Vermont JOHN B. BUCK, National Institutes of Health g. x. MOUL, Rutgers University T. H. BULLOCK, University of Californk^^ ^^ w POLLISTER> Columbia University E. G. BUTLER, Princeton University MARY E" ^w^8. Johns Hopkins University K. W. COOPER, University of Rochester A- R- WHITING, University of Pennsylvania M. E. KRAHL, University of Chicago CARROLL M. 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Vol. 112, No. 2 April, 1957 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY DIFFERENCES IN SUSCEPTIBILITY TO WHOLE-BODY GAMMA IRRADIATION IN THE LAYERS OF THE RETINA OF BUFO BENNET M. ALLEN AND MARION HUBBLE DEVICK Atomic Energy Project, School of Medicine, University of California at Los Aiu/clcs Differences in susceptibility to irradiation constitute a problem of biological significance heightened by the fact that they occur in comparable tissues in different groups of animals. We have found that in Bnfo the cells destroyed under the conditions of our experiments include certain small nerve cells of the brain, of the olfactory membrane, and of the retina. This paper deals with the latter because it offers an especially instructive example of these principles. MATERIALS AND METHODS Recently metamorphosed Bnfo borcas halophilits with trunk lengths of 10 to 12 mm. were firmly held in the zone of maximum irradiation while being exposed to total-body irradiation by a cobalt60 source. The average dosage rate was 950 r/minute. Fixation was by Benin's fluid. Serial paraffin sections were cut at a thickness of one to five microns. A thickness of three microns was the most suit- able. Slides were left over-night in commercial HoO? in order to expose the rods and cones by depigmenting the strands from the pigment layer. Some material was stained with Delafield's hematoxylin and eosin, but by far more useful was Heidenhain's iron-alum hematoxylin, slides being left one-half hour in each of the two solutions. This stain proved especially valuable not only because of its sharpness but because it stained the pyknotic nuclei most intensely. RESULTS Gamma irradiation of 10,000 r, 20,000 r, 30,000 r, and 60,000 r caused destruc- tion to the nerve cells of the inner nuclear and ganglionic layers. The few nuclei surviving after a dose of 60,000 r may be largely identified as those of the fibers of Mueller and the amacrine cells. On the other hand, even 60,000 r within the twenty-four hour survival period of the toads caused no destruction to the outer nuclear layer nor to the rods and cones belonging to them. At the lower irradiation levels the ganglionic layer appeared to be somewhat less susceptible than the inner nuclear layer, but with an irradiation of 60,000 r it, too, was completely destroyed (Fig. 2). The occurrence of structures interpreted as chromosomal vesicles in all 137 138 B. M. ALLEN AND M. H. DEVICK the retinal nuclei is considered to be normal because they are the same in experi- mental and control animals. Toads irradiated with 10,000 r or 20,000 r and killed at twenty-four hours (Fig. 4 and Fig. 6) showed considerable destruction of the inner nuclear layer but this was almost complete at 30,000 r and 60,000 r. Toads irradiated at 10.000 r $&m&i%ii3P* • FIGURE 1. The retina at twenty-four hours after being given 60,000 r ; X 60. FIGURE 2. A higher magnification of the area in Figure 1, showing a portion of the more edematous area. X 239. FIGURE 3. The retina six days after having been given 20,000 r. The thickness of the internal nuclear layer is reduced. The lowest part of the picture is the nearest to the ora serrata. X 329. FIGURE 4. The retina twenty-four hours after having been given 20,000 r. X 239. IRRADIATION OF THE RETINA OF BUFO 139 and 20,000 r and killed at an interval of six days after irradiation (Fig. 3 and Fig. 7) showed decided reduction in the thickness of the inner nuclear layer due to resorption of dead nuclei. With doses of 60,000 r, groups of toads were killed immediately and at short intervals of one hour, two hours, and three hours after irradiation. In general, the effect was a delayed one, pyknosis becoming more and more extensive up to 24 hours. This was observed both after x-ray and gamma irradiation, the results being closely similar. A few cases of pyknosis were seen by the time irradiation was completed. In these early stages pyknosis involved a darkening of the nuclei and only later was there destruction of the cytoplasm. These pyknotic cells occurred throughout the inner nuclear layer of the retina but were most numerous a short distance in from the margin. Not only do the nuclei become deeply pyknotic but decided edema of the retina results. This was localized in the case of lower irradiation dosages, but with 60,000 r the entire retina becomes very heavily pyknotic and edematous (Fig. 1). Figures 6, 7, 8 and 9 show portions of the retina under different degrees of irradiation. Pyknosis is roughly proportional to the amount of the dose. It is clear that even the highest dose does not affect the outer nuclear layer or the rods and cones, as observed at 24 hours or at six days after irradiation with 20,000 r. Experiments were performed to show the effect of divided doses on the basis of a 60,000 r total. Irradiation was given on consecutive days and at the following rates : 10,000 r, six times ; 20,000 r, three times ; and 30,000 r, two times. Destruc- tion of the inner nuclear layer was comparable to that caused by a single dose of 60,000 r. There was a marked reduction in thickness of the inner nuclear layer due to resorption during the course of the experiment (Fig. 8). Divided doses did not cause the amount of edema produced by a single dose. A very interesting condition is seen at the ora serrata (Fig. 10). The cells of this region show no visible differentiation and there is no sharp line of demarcation between prospective sensory and nerve cell layers, but the nuclei toward the cavity of the eyeball can be followed to the inner nuclear and ganglionic layers, and we consider them to be prospective nerve cell nuclei. It is significant that irradiation renders these highly pyknotic. On the other hand, the nuclei adjacent to the choroid coat, considered to be prospective sensory cells, are not pyknotic. In the region more peripheral to the ora serrata there is likewise no pyknosis in spite of the fact that it is the zone where mitosis had added to the retina, up to a stage shortly before this. It would seem clear that some change has taken place in the prospective nerve cells that renders them especially sensitive to irradiation. Following the differentiated nerve cell layers central to the ora serrata the dis- tribution of pyknotic nuclei shows that cells of the peripheral portion of the retina are far more readily destroyed than the ones nearer to the center of the retina. At a lower degree of irradiation, 10,000 r and 20,000 r, this is quite evident but at 60,000 r the entire retina is deeply affected. DISCUSSION The effects of irradiation upon the retina of amphibians have been chiefly studied in young stages. Brunst (1955) applied x-rays in doses of 1000 r to 8000 r to axolotl larvae from 9 to 65 days of age, the experiment being terminated in 18 to 140 B. M. ALLEN AND M. H. DEVICK FIGURE 5. Section from the retina of a normal control animal. (1) pigment layer, (2) the rod and cone layer, (3) outer limiting membrance, (4) outer nuclear layer, (5) outer plexiform layer, (6) inner nuclear layer, (7) inner plexiform layer, (8) ganglionic layer, (9) nerve fiber layer, (10) internal limiting membrane. X 1110. FIGURE 6. The retina twenty-four hours after having been given 20,000 r. X 1402. FIGURE 7. The retina six days after having been given 20,000 r. Pyknotic nuclei are shrunken and resorbed. The thickness of the inner nuclear layer is reduced as compared to Figure 6. < 1402. IRRADIATION OF THE RETINA OF BUFO 141 28 days after irradiation. He stated that in all cases the rod and cone layers dis- appeared first. Eventually the retina degenerated leaving only pigment cells. Brunst (1955) further states (p. 289) : "These observations justify the conclusion that the eyes of animals used for this investigation are organs in the process of differentiation and active growth and are therefore, according to the law of Burgonie and Tribondeau, sensitive to roentgen irradiation." Rugh (1954) finds that in Ambystoma larvae 22 mm. in length, x-irradiation of 15,000 r not only causes destruction of mitotic cells, but is equally destructive in regions lying quite apart from them. In a discussion following the reading of this paper, Rugh stated (p. 63), "the rods and cones are separated from the pigment layer but are not individually damaged as are the neuroblast cells. . . . The rods and cones are apparently not relatively sensitive or delicate." In our own work the rods and cones and the plexiform layers are completely developed (Fig. 5). At the same time this condition has been rather recently attained. The peripheral region of the retina is the youngest part, having been built up as a result of mitotic activity in the region of the ora serrata, as shown by Spear and Glucksman (1941). We have shown that irradiation with 10,000 r and 20,000 r produces very heavy destruction of the inner nuclear and ganglionic layers a short distance central to this region. This leads to the assumption that the sus- ceptibility of these cells is conditioned by their degree of maturity, together with the intensity of the irradiation. In our work we find no evidence that the outer nuclear layer or the rods and cones are affected even by 60,000 r in the 24 hours through which the toads survive. Edema is localized in the case of 10,000 r and 20,000 r irradiation but it is general when 60,000 r is given in a single exposure. It is of secondary importance because it does not appear when 60,000 r is given in divided doses. We have shown that the amount of pyknosis produced by divided doses appears to be roughly equal to that observed when irradiation is given in a single dose. This was shown in Tritunts by Brunst and Sheremetieva-Brunst (1949). In sharp contrast to our findings in Bufo, the investigators who have irradiated the eyes of mammals have found most extensive injury to the outer nuclear layer, notably to the nuclei of the rods and to the rods themselves. The cones with their nuclei were less affected. This was the finding of Lorenz and Dunn (1950), who exposed newborn mice to 400 r of x-irradiation and killed them at the end of twelve months. Noell et al. (1954) gave x-irradiation to the eyes of rabbits, resulting in the destruction of the visual cells while the inner retinal layers were spared. Similar findings were recorded by Brown ct al. (1955), and Cibis and Brown (1955), who used gamma and x-irradiation upon the monkey, Macaco, rhesus. With doses of 10,000 r the rods and their nuclei were affected as early as two hours after irradiation. Only when doses exceeded 30,000 r, was there considerable destruction of nerve cells of the inner nuclear layer. FIGURE 8. This animal was given 10,000 r on 6 consecutive days. The amount of pyknosis is comparable to that produced by 60,000 r in one dose. The thickness of the inner nuclear layer is much reduced by resorption. There is no edema. < 1402. FIGURE 9. The retina twenty-four hours after having been given 60,000 r. A large amount of edema and many pyknotic nuclei. X 1402. FIGURE 10. Undifferentiated margin of the retina near the ora serrata, twenty-four hours after 60,000 r was given. The pyknotic nuclei are continuous with the inner nuclear and ganglionic layers. The pigment epithelium is toward the bottom of the picture. X 1402. 142 B. M. ALLEN AND M. H. DEVICK Noell (1953, 1955) experimented upon rabbits by injecting sodium iodate, sodium iodacetate, and also by applying oxygen poisoning as well as x-irradiation. All of these were especially injurious to the rods and in a lesser degree to the cones. They all produced quite comparable effects. The nuclei of the outer nuclear layer were largely killed except those belonging to the cones. He found that with the sodium iodate-poisoning, the pigment epithelium was first affected and the injury to the sensory organelles came later, leading to the view that there is a causal relation. It is clear that in all of the above papers dealing with experiments upon mam- mals, it was shown that the rods and their cell bodies in the outer nuclear layer were most sensitive to irradiation, while the nerve cells were affected in far less degree. We have shown that just the reverse is true in Bufo, within the time limits used. In fact, we find no injury to the rods and cones and their cell bodies in the outer nuclear layer, while on the other hand, the inner nuclear and ganglionic layers show a very high degree of pyknosis. We shall not attempt in this paper to speculate upon the reasons for this difference but the facts are clear enough, and the point is most significant. Attention is called to the sensitivity of the prospective nerve cells observed at the ora serrata where structural differentiation has not yet taken place. It is evident that in very early stages these prospective nerve cells have already under- gone invisible changes that render them vulnerable to irradiation. Allen (1956) showed that irradiated toads used in this work not only suffered Heavy destruction of the nerve cell layers of the retina but also of the nerve cells of the brain. SUMMARY 1. Gamma irradiation doses of 10,000 r, 20,000 r, 30,000 r and 60,000 r were given to recently metamorphosed Bufo boreas halophilus. Toads receiving the two lower dosages were killed at the end of twenty-four hours and at six days. Those receiving the two higher dosages were all killed at twenty-four hours. Cell destruc- tion was found in the inner nuclear and ganglionic layer of the retina but none was found in the outer nuclear layer nor in the rods and cones. 2. Destruction increased as the dosage increased. At doses of 10,000 r and 20,000 r, pyknotic nuclei were most abundant near the marginal portion of the retina, where the cells had been formed more recently than in the central portion. In animals that were given 60,000 r, pyknosis of retinal nerve cells was general and almost complete. 3. Divided daily doses of 10,000 r given six times, 20,000 r three times, and 30,000 r two times produced as much destruction as a single dose of 60,000 r. The amount of edema in these cases was never as great as that which was caused by a single dose of 60,000 r. 4. At the ora serrata where structural differentiation was not yet evident, cells interpreted as prospective nerve cells were destroyed while there was no destruction of prospective sensory cells of the outer nuclear layer. LITERATURE CITED ALLEN, BENNET M., 1956. Cell destruction induced by heavy irradiation of recently metamor- phosed Bufo. Anat. Rec., 124 : 387. BROWN, DAVID V. L., PAUL A. CIBIS AND JOHN E. PICKERING, 1955. Radiation studies on the monkey eye. Arch. Ophthal, 54 : 249-256. IRRADIATION OF THE RETINA OF BUFO 143 BRUNST, V. V., 1954. The effect of local x-ray irradiation upon the development of the anterior part of the head of the axolotl. /. Morph., 95 : 373-391. BRUNST, V. V., 1955. The influence of roentgen irradiation on the development of the eye of the axolotl. Amer. J. Rocnt., Rod. Thcr. and Nuc. Med., 73 : 281-293. BRUNST, V. V., AND E. A. SHEREMETIEVA-BRUNST, 1949. The time factor in lethal effects of total roentgen irradiation. Amer. J. Roent. Rod. Thcr., 62: 550-554. CIBIS, PAUL A., AND DAVID V. L. BROWN, 1955. Retinal changes following ionizing radiation. Amer. J. Op thai, 40 : 84-88. LORENZ, E., AND EGON DUNN, 1950. Ocular lesions induced by acute exposure of the whole body of newborn mice to roentgen irradiation. Arch. Ophthal., 43: 743-749. NOELL, WERNER K., 1953. Experimentally induced toxic effects on structure and function of visual cells and pigment epithelium. Amer. J. Ophthal., 36: 103-114. NOELL, WERNER K., 1955. Metabolic injuries of the visual cell. Amer. J. Ophthal., 40: 60-68. NOELL, W. K., E. EICHEL AND P. A. CIBIS, 1954. Visual cells and pigment epithelium after high intensity x-irradiation. Fed. Proc., 13 : 106. RUGH, ROBERTS, 1954. The effect of ionizing radiations upon amphibian development. /. Cell. Comp. Physiol., 43 : suppl. 31-72. SPEAR, F. G., AND A. GLUCKSMAN, 1941. The effect of gamma radiation on cells in vivo. Brit. J. Radio!., Part 3, 14: 65-76. LARVAL DEVELOPMENT OF PALAEMONETES PUGIO HOLTHUIS1- 2 A. C. BROAD Duke University Marine Laboratory, Beaufort, N.C. Knowledge of the larval development of the marine species of Palaemonetes of the eastern United States is limited to Faxon's (1879) account of the development of P. vulgaris. This study was based almost wholly on planktonic larvae collected in an area from which two other closely related species, P. pngio and P. intermedius, have been described since (Holthuis, 1949). Holthuis (1952) feels that even mature adults of these species often have been confused. The larvae, presumably, may be quite similar, although those of the two latter species are unknown. The problem of distinguishing between decapod species during the larval phase has received relatively little attention. Lebour (1927 through 1943) has found generic, and in some instances specific, taxonomic characters for British decapod larvae, but sometimes only was able to state that larvae of certain species were alike. Gurney (1942) feels that similarity between larvae indicates the relationship be- tween the species. Often larvae of commercially valuable decapods have been studied without consideration of the larvae of related species. Churchill's (1942) account of the zoeae of the blue crab, Callinectes sapidus, is thought by Hopkins (1944) to include larvae of another species, possibly C. ornatus. Pearson's (1939) description of the development of the white shrimp, Pcnaens sctiferus, has been questioned by Burkenroad (1949) and Heegaard's (1953) descriptions of larvae of the same species are thought by Gunter (editorial comment in Heegaard, 1953) to include larvae of other penaeids. Among species most studied there has been relatively little agreement on either the form or the number of larval stages. Churchill (1942) found five blue crab zoeae. Hopkins (1943, 1944) found four, but feels that a fifth may exist which has never been found. Sandoz and Rogers (1944) obtained a pre-zoeal blue crab in the laboratory which has not been found in nature. Heegaard (19S3) questions the number of white shrimp stages found by Pearson (1939). The number of naupliar stages reported for other species of Penacus varies still more (Hudinaga, 1942; Heldt, 1938). Many of these accounts were based on larvae caught in plankton and have been questioned. The validity of a reconstruction depends upon the ability of the author to recognize species during the larval phase. The difficulty is apparent, especially when another closely related species may exist in the same region. Intraspecific variation in development among euphausids has been fairly well established (MacDonald, 1927; Fraser, 1936; Boden, 1950, 1951), but variation 1 Supported in part by a contract with the Office of Naval Research, Nonr-1232(00) , and a National Science Foundation grant, NSF-G-1214. 2 Part of a thesis submitted to the graduate faculty of Duke University in partial fulfill- ment of the requirements for the degree of Doctor of Philosophy. 144 LARVAL DEVELOPMENT OF PALAEMONETES 145 in decapod larvae has been denied (Gurney, 1942). Heegaard (1953), however, feels that, at least among penaeids, variation in the rate of larval development may result in differences in the number and form of larval intermolts for each species. Some doubt may have been cast on this hypothesis by the paucity and questionable identity of the material on which it was based. The pre-zoeal stage of Callinectes reported by Sandoz and Rogers (1944) has usually been dismissed as abnormal. The present paper is first a description of the larval development of Palae- monetes pngio Holthuis based on observations of larvae reared in the laboratory. The larvae and the development are compared to that of Palaemonetes vulyaris (Say) reared concurrently. Finally, the considerable variation in the number and structure of larval intermolts, found in the development of both species, is described. The author wishes to express his sincere appreciation to Professor C. G. Book- hout, under whose guidance and direction this work was done and who read and suggested improvements in this manuscript. Thanks are also clue to Dr. T. R. Rice, who supplied stocks of all unicellular algae used, and to Dr. Fenner A. Chace, Jr., who provided working space and specimens of Palaemonetes from the collection of the U. S. National Museum. METHODS Mature adult P. pnyio and P. vulgaris are abundant in the vicinity of the Duke University Marine Laboratory at Beaufort, N. C., from late April until mid-Octo- ber. Egg-bearing females were caught in dip nets and held individually in dishes of sea water in the laboratory until the eggs hatched, after which they were pre- served for later identification and reference. Groups of 10 newly-hatched larvae, all from the same clutch of eggs, were placed in clean four-inch finger bowls of sea water. The water was changed only if evidence of cloudiness appeared. The bowls of larvae were placed near north windows but not in direct sunlight. Because of other experiments which required constant illumination in an adjacent part of the laboratory, the room was never com- pletely dark. No method of controlling the temperature of the building was avail- able during the summer months. Most of the larvae were reared at temperatures which ranged from about 25 to 27° C. and none at lower than 20° C. The diet of each larva was the same during its lifetime, but a variety of different foods or combinations of foods was offered to various larvae. Some were not fed. Other individuals received daily rations of either a species of unicellular algae, or a combination of species. Algae used included two species of Nitzschia and one species of each of the following genera : Chlamydomonas, Thorocomonas, Nanno- chloris, Porphyridium and Pyramimonas. Some larvae were fed species of algae combined with zooplankton which had been obtained by net and first killed by immersion in distilled water to prevent the fortuitous inclusion of living larvae similar to those being studied. The diet of some other larvae consisted of freshly killed zooplankton alone. A few larvae were fed on chaetognaths removed from the plankton and some others were fed tiny bits of the visceral mass of the mud snail, Nassarins obsoletiis. The remaining larvae were fed living Artcuiia nauplii. Food was added to the bowls and uneaten food was removed daily. Each larva was inspected daily under the low power of a stereoscopic binocular microscope. A record of molting by each larva was kept. Only the presence of 146 A. C. BROAD all or a large part of an exuvium or cast exoskeleton was accepted as evidence that a larva had molted. Morphological changes in larvae established which individuals had undergone the molt. Camera lucida drawings were made of entire living larvae which had been anesthetized with ethyl carbamate and placed on slides beneath supported cover slips. These preparations were then sealed with oil. Larvae lived for as long as two hours under observation. Individual appendages from alcoholic specimens were studied and drawn. Finally, adult females from which larvae were obtained were identified after comparison with type and other material at the U. S. National Museum. RESULTS The number of individuals of each species which survived each of the several molts is given in Table I. Only those larvae which were fed diets containing some animal tissue were able to survive. The number of ecdyses and, conversely, the number of intermolts or so-called larval stages were not constant among individuals of the same species. In general, the differences were slight and the similarities great between the larvae of P. pugio and those of P. vnlgaris. In some instances, preserved larvae were identical. Among living larvae, howrever, a difference in the distribution of chromatophores on the ventral surface of the abdomen proved to be an invariable index to species. Larvae of P. pugio bear, on the sternites of abdominal somites TABLE I Number of larvae of Palaemonetes pugio and Palaemonetes mdgaris which survived each molt in the laboratory and the number of postlarvae obtained by metamorphosis. Column headings indicate diet of larvae Molt number Palaemonetes pugio Palaemnnetes vulgaris No food Uni- cellular algae Algae plus animal tissue Animal tissue Artemia nauplii No food Uni- cellular algae Algae plus animal tissue Arlemia nauplii 0 60 280 ' 608 732 100 80 100 667 390 1 42 162 491 464 92 0 1 30 324 2 0 0 143 177 87 0 15 282 3 77 84 87 13 261 4 54 70 85 11 227 5 47 50 82 11 191 6 37 36 82 9 149 7 29 28 69 8 131 8 24 25 8 9 18 14 8 10 17 11 17 Number postlarvae 0 0 16 6 65 0 0 6 122 LARVAL DEVELOPMENT OF PALAEMONETES 147 2 and 3, pairs of chromatophores. The larvae of P. vulgar is bear chromatophores on the sternite of abdominal somite 3, but lack pigment spots on abdominal sternite 2. Although the individual larvae and the general pattern of development of P. pugio are nearly identical to that of P. vulgaris, differences between individuals of the same age and molting history and differences in the duration and tempo of larval development were observed in each species. Larvae which passed through the greatest number of molts during development showed the least morphological change after each molt. The structural characteristics of the individual larva were more readily associated with its total length than with its age or the number of molts completed. Although larvae which were of the same age often differed from one another in extent of development completed, those of about the same size were quite similar in structure regardless of the age or previous molting history. All larvae \vere alike upon hatching. This similarity began to disappear after the second molt. Considerable variation was found after the third molt, but. at the end of larval development, all larvae again resembled one another. The sequence or order of developmental events was the same for all larvae. The number of steps or stages passed through varied. Differences in the form of larvae were accompanied by variation in the actual rate of development. Larvae fed living Artemia nauplii usually metamorphosed at the seventh molt which occurred about two weeks after hatching. Others, how- ever, required from two to four weeks and as many as 13 molts to complete larval development. The variation observed, therefore, was primarily one of tempo. Larvae of Palacmonctcs pugio The eggs carried by adult females were from 0.5 X 0.6 to 0.6 X 0.9 mm. in size. They usually hatched within a few days, but always within two weeks or not at all. The prezoeal molt occurs immediately before hatching ( Burkenroad, 1947), and the larva emerges from the egg as a zoea. First zoea (Figs. 1-11) : total length about 2.6 mm. Carapace, rostrum and abdomen without spines or teeth. Rostrum curved slightly downward at end. Abdomen of 6 somites, the last of which is not separate from the fan-shaped telson (Fig. 11). Telson with 14 spines. Eyes sessile, contained beneath carapace. Antennule (Fig. 3) simple; the single basal segment bears terminally a long seta and a short outer flagellum ; outer flagellum with three slender and one stout aesthetes and a short seta. Antenna (Fig. 4) biramous ; basis unsegmented; flagellum of one segment, shorter than scale with a long terminal seta ; scale of a long basal segment, which is convex on inner side, and four short terminal segments, with nine long setae on inner side and three short setae outside near tip. Mandible (Fig. 5) without palp; incisor process with three teeth at tip; molar process with fine-toothed cutting edge ; two movable teeth in angle between molar and incisor processes. First maxilla (Fig. 6) uniramous ; coxa with five inwardly directed spines ; basis with three spines and two teeth ; endopod simple, palp-like, with a terminal seta. Second maxilla plate-like, biramous ; protopod three-lobed, armed with three, two and four setae ; endopod unsegmented, bears on a lobe near the proximal end two and terminally one setae ; exopod a flattened gill bailer with three setae anteriorly, one laterally and one posteriorly. 148 A. C. BROAD LARVAL DEVELOPMENT OF PALAEMONETES 149 First maxilliped (Fig. 8) biramous; coxa reduced; basis with four medially directed setae; endopod two-segmented, the distal segment with four terminal and one median setae ; exopod longer than endopod with four apical and two sub-apical setae. Second maxilliped (Fig. 9) biramous; coxa reduced; basis with two setae; endopod three- segmented, with two strong spines at junction of ultimate and penultimate segments, ultimate segment with two smaller spines, a seta and a strong terminal claw ; exopod longer than endopod with a cylindrical proximal and a flattened paddle-like distal segment which bears four apical and three pairs of sub- apical setae. Third maxilliped (Fig. 10) biramous, larger than second maxilliped, but generally similar to it ; endopod with two setae on proximal segment ; exopod with four apical and three or four pairs of sub-apical setae. First and second pereiopods (Fig. 10) rudimentary. Other appendages lacking. Prominent groups of red and yellow-green chromatophores located dorsally at bases of eyes and at junction of abdominal somites 2 and 3. Paired groups of chromatophores ventrally on basal segment of antenna, on labrum, on thoracic sternites 1 and 8, on abdominal sternites 2 and 3 and on telson just anterior to anus. This larva corresponds very closely to the first zoea of P. vulgaris and to Faxon's (1879) description of the first stage larva of that species. It differs from the larva of P. vulgaris chiefly in the presence of a pair of chromatophores on abdominal sternite 2, which are lacking from the latter species. The basal segment of the antennal scale of P. vulgaris is less convex on its inner side and the scale is narrower than in P. f>ugio. Second soca (Figs. 12-18) : length about 2.8 mm. Differs from first zoea in the following : Carapace with supra-orbital and branchiostegal spines. Rostrum recurved at tip and with one dorsal rostral tooth located on the carapace just behind orbit. Pleurum of fifth abdominal somite terminates as a posteriorly directed tooth. Telson (Fig. 18) with 14 large and two minute spines. The outlines of the uropods often visible within telson. Eyes stalked with chromatophores on postero-ventral side of stalk. Antennule (Fig. 14) with peduncle segmented, segments marked by long setae on inner side, a long and a short seta on distal end of peduncle ; outer flagellum with two slender and two stout aesthetes. Antennal scale (Fig. 15) with three short segments at the distal end and 14 setae ; antennal flagellum terminates in a long and two short setae. First maxilla with four teeth and three spines on basis. Exopod of second maxilla with five anterior setae. Endopod of third maxilliped (Fig. 16) five- segmented. First pereiopod (Fig. 17) biramous; coxa reduced; basis with two setae; endopod five-segmented, ischium, carpus and dactylus with a seta each, two stout spines at junction of propodus and dactylus, dactylus terminates in a strong claw; exopod as in third maxilliped. PLATE i. Larval development of Palacmonetes pugio. Entire larvae X 19; appendages X 54. Figures 1-11 of first zoea. Fig. 1: ventral view. Fig. 2: dorsolateral view. Fig. 3: antennule. Fig. 4 : antenna. Fig. 5 : mandible. Fig. 6 : first maxilla. Fig. 7 : second maxilla. Fig. 8: first maxilliped. Fig. 9: second maxilliped. Fig. 10: third maxilliped and first and second pereiopods. Fig. 11: telson. Figures 12-18 of second zoea. Fig. 12: ventral view. Fig. 13: lateral view. Fig. 14: antennule. Fig. 15: antenna. Fig. 16: endopod of third maxil- liped. Fig. 17: first and second pereiopods. Fig. 18: telson. 150 A. C. BROAD LARVAL DEVELOPMENT OF PALAEMONETES 151 The second zoea corresponds to the second zoea of P. vulgaris and to Faxon's description of the second stage larva of that species. The second zoea of P. vulgaris differs from the corresponding larva of P. pugio in lacking chromatophores on abdominal sternite 2. All larvae which had molted once were in the form described. Third zoea (Figs. 19-25) : total length about 3.2 mm. Differs from the pre- vious larva in the following : Sixth abdominal somite separate from telson. Telson (Fig. 25) narrower than before, armed with 12 large and two small spines. Antennular peduncle (Fig. 21) with two long setae ventro-distally, two long setae on inner side, and, on a protuberance near the proximal end which will be the stylocerite, three short setae ; a rounded prominence bearing three or four short setae and located dorsally near the distal end of the peduncle is the antennular lobe ; outer flagellum with three stout aesthetes. Antennal scale (Fig. 22) with two short segments at distal end and 15 setae; antennal endopod separated into a short peduncle and an unsegmented flagellum which terminates in two short and two minute setae. Second pereiopod (Fig. 23) biramous; coxa reduced; basis with at least one seta ; endopod five-segmented, first and last segments with setae, two stout spines arise from junction of propodus and dactylus, dactylus terminates in a strong claw ; exopod shorter than endopod, similar in structure to other thoracic exopods. Third pereiopod (Fig. 24) biramous, rudimentary. Uropod (Fig. 25) biramous, unsegmented, with rudimentary inner ramus; outer ramus with 7 or 8 setae. The third zoea corresponds to the third zoea of P. vulgaris and to Faxon's third stage. It differs from the third zoea of P. vulgaris in the presence of chro- matophores on abdominal sternite 2. Variation in the form of larvae which had molted twice was noted in the total length and in the number of rudimentary pereiopods present. Larger larvae had, in addition to the appendages described above, rudiments of third and fourth pereiopods. All larvae which had molted twice were third zoeae. Fourth zoea (Figs. 26-31) : length about 3.5 mm. Differs from the third zoea in the following: Two dorsal rostral teeth on the carapace. Telson (Fig. 31) but little wider posteriorly than anteriorly, armed with eight stout and two small spines. Antennular peduncle with four long distal setae and a protuberance which is the rudiment of the inner flagellum. Antennal basis separated into two segments by an oblique fissure at the articulation of the peduncle ; scale (Fig. 27) not seg- mented, its disto-lateral tip (spine) projecting slightly, blade with 16 or 17 setae. Second pereiopod larger than before. Third and fourth pereiopods (Figs. 28 and 29) biramous, rudimentary. Fifth pereiopod (Fig. 30) uniramous, rudi- mentary. Uropod biramous; basis unsegmented; endopod snorter than exopod bears 8 setae ; exopod with 12 setae. PLATE n. Larval development of Palaemonetes pugio. Entire larvae X 19 ; appendages X 54. Figures 19-25 of third zoea. Fig. 19: ventral view. Fig. 20: lateral view. Fig. 21: antennule. Fig. 22: antenna. Fig. 23: second pereiopod. Fig. 24: third pereiopod. Fig. 25: uropods and telson. Figures 26-31 of fourth zoea. Fig. 26: ventral view. Fig. 27: tip of antennal scale, setae omitted. Fig. 28: third pereiopod. Fig. 29: fourth pereiopod. Fig. 30: fifth pereiopod. Fig. 31 : end of telson. Figures 32-35 of fifth zoea. Fig. 32 : ventral view. Fig. 33: tip of antennal scale, setae omitted. Fig. 34: third, fourth and fifth pereiopods. Fig. 35: telson. Figures 36-39 of sixth zoea. Fig. 36: ventral view. Fig. 37: third and fourth pereiopods. Fig. 38: fifth pereiopod. Fig. 39: telson. 152 A. C. BROAD LARVAL DEVELOPMENT OF PALAEMONETES 153 The fourth zoea of P. pugio corresponds to the fourth zoea of P. vulgaris but has no equivalent in Faxon's descriptions of the larvae of that species. The fourth zoea of P. pugio differs from that of P. vulgaris in the distribution of abdominal chromatophores and in the projecting tip of the spine of the antennal scale, which, in P. vulgaris larvae, does not extend beyond and free of the blade. Not all larvae which had molted three times correspond to the fourth zoea as described here. Among those larvae which molted the least number of times during development, this form was not represented by any intermolt. Fifth zoea (Figs. 32-35) : total length about 3.5 mm. The fifth zoea differs from the fourth in the following: Telson (Fig. 35) narrower posteriorly than anteriorly, armed with six stout and two slender spines. Antennular peduncle with 5 long setae distally; inner flagellum a separate segment tipped with a short seta. Antennal scale (Fig. 33) with 19 setae, tip projecting; flagellum divided into a short proximal and a longer distal segment. Mandible with three serrate movable teeth. Basis of first maxilla with five teeth and three setae. Middle lobe of basis of second maxilla with three setae; exopod with six to seven anterior setae. Third pereiopod (Fig. 34) biramous; coxa reduced; basis with at least one seta; endopod five-segmented, ischium, merus, carpus, and dactylus with setae, two stout spines at junction of propodus and dactylus, dactylus terminates in a claw ; exopod shorter than endopod with four apical and two pairs of sub-apical setae. Fifth pereiopod longer than fourth (Fig. 34), both rudiments. Uropodal endopod nearly as long as exopod, with 13 setae; exopod with a short seta on outside near proximal end and a short tooth in dorso-lateral corner, with 16 long setae. The fifth zoea of P. pugio differs from the fifth zoea of P. vulgaris as do fourth zoeae. There is no corresponding larval stage in Faxon's account of development of P. vulgaris, but a larva of this form was obtained by molt and considered ab- normal by Faxon. This form may be skipped in the development of either species. Sixth zoea (Figs. 36-39) : total length about 3.7 mm. Differs from the pre- vious larva in the following : Antennular peduncle with three plumose setae on inner side, angle of stylocerite is acute. Antennal scale with 20 plumose setae. Fifth pereiopod (Fig. 38) uniramous; coxa reduced; short setae on merus, propodus and dactylus, dactylus terminates in a long claw. Uropodal exopod with 16, endopod with 13 or 14 setae. The form described as the sixth zoea may be present in the development of either P. pugio or P. vulgaris. The species differ in the ways previously dis- cussed. This form is not represented among Faxon's larval stages, but was seen by him and considered abnormal. This form may be skipped in the development of either P. pugio or P. vulgaris. Seventh zoea (Figs. 40-45) : total length about 4.4 mm. The seventh zoea PLATE in. Larval development of Palacmonetes pugio. Entire larvae X 19; appendages X 54. Figures 40-45 of seventh zoea. Fig. 40 : ventral view. Fig. 41 : lateral view. Fig. 42 : antennal scale, setae omitted. Fig. 43 : third and fourth pereiopods. Fig. 44 : fifth pereiopod. Fig. 45 : telson. Figures 46-49 of eighth zoea. Fig. 46 : ventral view. Fig. 47 : first cheliped. Fig. 48: fourth pereiopod. Fig. 49: second pleopod. Figures 50-54 of ninth zoea. Fig. 50: ventral view. Fig. 51: first chela. Fig. 52: first pleopod. Fig. 53: second pleopod. Fig. 54: telson. 154 A. C. BROAD LARVAL DEVELOPMENT OF PALAEMONETES 155 differs from the sixth in the following : A minute anal spine present. Antennular peduncle with five setae on inner side ; outer antennular flagellum with three stout and one slender aesthetes. Antennal scale (Fig. 42) with 20 setae. Fifth perei- opod with a seta on each segment. First to fifth pleopods represented by small uniramous buds. The seventh zoea of P. pugio differs from the seventh zoea of P. vulgaris as the sixth zoeae differ and in regard to a small tooth on the ventral side of the anten- nular peduncle of the latter species. This tooth appears later in the development of P. pugio and is smaller than in P. vulgaris. Considerable variation in the devel- opment of the last three pereiopods was found in larvae first having pleopod buds. Among those larvae which, in their development, had skipped the forms described as fourth, fifth and sixth zoeae, pereiopods 3, 4 and 5 were rudiments. Among other larvae pereiopod 5 was still rudimentary at the time of the appearance of pleopod buds. The seventh zoea described here corresponds most closely to Faxon's fourth larval stage of P. vulgaris. Larvae of either species may have molted three, four, five or six times when the form described as the seventh zoea is achieved. Eighth zoea (Figs. 46-49) : total length about 4.9 mm. The eighth zoea differs from the previous larva in the following : The anal spine is stronger. The anten- nular peduncle bears, about mid-way of the proximal segment on the ventral side, a small tooth, 7 setae on inner side, 6 setae at distal end. Antennal basis with a tooth on ventral side at junction of scale; scale with 23 setae; flagellum of three or four segments. Propodi of first and second pereiopods (Fig. 47) swollen and protuberant at inner distal corner, forming, with dactylus, the beginning of a chela. Fourth pereiopod (Fig. 48) biramous ; coxa reduced, basis with a seta; endopod five-seg- mented, ischium, merus and dactylus with one seta each, carpus with two setae, junction of propodus and dactylus with three spines, dactylus tipped with a claw. Fifth pereiopod with a short spine arising from the middle of dactylus. First to fifth pleopods (Fig. 49) small, biramous, rudimentary. Uropodal exopod with 20, endopod with 18 setae. The eighth zoea of P. pugio corresponds to the eighth zoea of P. vulgaris from which it differs in the ways previously stated. This form corresponds to Faxon's fifth larval stage of P. vulgaris. There was considerable variation in the develop- ment of the pleopods, but all had non-setose, biramous pleopod rudiments. Ninth zoea (Figs. 50-54) : length about 5.1 mm. Differs from the previou? larva in the following : Rostrum setose in the angle of the anterior tooth. Outer antennular flagellum with four equal, sub-apical aesthetes and a slender aesthete arising from mid-way of the segment. Antennal flagellum longer than scale, five- segmented ; scale with 26 plumose setae. Entire outer edge of exopod of second maxilla setose. First maxilliped with a simple epipod, basal portion enlarged with 8 setae, proximal segment of exopod with two setae. PLATE iv. Larval development of Palaeinonetes pugio. Entire larvae and postlarva X 19; larval appendages X 54. Figures 55-58 of tenth (last) zoea. Fig. 55: ventral view. Fig. 56: lateral view. Fig. 57: second pleopod. Fig. 58: telson. Figures 59-63 of postlarva. Fig. 59: ventral view. Fig. 60: lateral view of rostrum and anterior portion of cephalothorax, X 19. Fig. 61 : lateral view of fourth and fifth abdominal pleurae, X 19. Fig. 62 : antennal scale, setae omitted, X 19. Fig. 63 : telson, X 54. 156 A. C. BROAD First and second pereiopods chelate (Fig. 51), the fixed finger tipped by the two spines formerly located at junction of propodus and dactylus. First to fifth pereiopods with arthrobranchs. First to fifth pleopods biramous. First pleopod (Fig. 52) rudimentary. Second (Fig. 53) to fifth pleopods with sparsely setose exopods. Uropodal exopod with 24, endopod with 17 setae. The ninth zoea of P. pugio differs from the ninth zoea of P. vulgaris as pre- viously described. The ninth zoea corresponds to Faxon's stage 6, first intermolt. Variation was noted in the extent of development of the inner ramus of the pleopods, the recurvature of the rostral tip, and the extent of development of the chelae. Tenth zoea (Figs. 55-58) : total length about 6.3 mm. The tenth zoea is the form representing full larval development. It differs from the previous larva in the following: Tip of rostrum curved slightly upward. Telson (Fig. 58) slender posteriorly, armed as before. Inner antennular flagellum with six aesthetes and two short apical setae. Antennal flagellum of 7 segments ; scale with 30 setae, its tip projecting or not. Mandible with four or five movable teeth in angle between incisor and molar processes. Basis of first maxilla with 6 teeth. Exopod of second maxilla fringed with 29 setae. Basis of first maxilliped with 9 setae and a bilobed epipod; proximal segment of exopod with 5 or 7 setae. Pereiopods essentially as before. First pleopod with rudimentary endopod. Second (Fig. 57) to fifth pleopods biramous with setose exopods and enclopods ; endopods with small appendices internae. Uropodal exopod with 29 and endopod with 26 plumose setae. The tenth zoea of P. pugio differs from the tenth zoea of P. vulgaris chiefly in the chromatophores of the ventral abdomen as previously discussed. This form corresponds to Faxon's sixth larval stage, intermolts 2 and 3. Some variation in the extent of development of the appendices internae of the pleopod endopods was noted. Postlarva (Figs. 59-63) : length about 6.2 mm. Rostrum shorter than antennal scale, with six dorsal teeth, the first of which is on the carapace directly over the posterior margin of the orbit, and two ventral teeth, the first of which is directly beneath the last dorsal tooth ; tip of rostrum free of teeth (Fig. 60). Carapace with antennal and branchiostegal spines. Posterior margins of abdominal pleurae rounded (Fig. 61). Anal spine present. Telson (Fig. 63) with a tooth extending back from the mid-point posteriorly, two pairs of terminal spines of which the inner pair are longer, a pair of setae mid-ventrally near the distal end, and two pairs of dorso-lateral spines about % and % of the way from the proximal end. Antennular peduncle of three segments ; stylocerite less than % the length of the basal segment of peduncle ; antero-lateral spine of basal segment exceeding anterior margin of segment; inner side of peduncle with 10 setae; basal segment containing a statocyst and a short ventral tooth. Inner antennular flagellum simple, five- segmented. Outer antennular flagellum four-segmented, bearing on the anti- penultimate segment two, and on the penultimate segment three aesthetes, and a tuft of setae on the ultimate segment. Length of antennal scale (Fig. 62) about four times its width, outer margin slightly concave; anterior end of spine projects free of blade and is slightly shorter. Antennal flagellum over half of total length. Mandible strong, incisor process stouter than in larval mandible ; teeth of molar process large, forming a triangular surface, with or without movable teeth in angle. LARVAL DEVELOPMENT OF PALAEMONETES 157 Basal portion of first maxilla bilobed, each lobe bearing on its inner surface numerous coarse setae ; endopod palp-like. Basal portion of second maxilla bilobed, each lobe bearing on its inner surface numerous coarse setae ; endopod unsegmented with neither lobes nor setae ; exopod setose around edge. Basal portion of first maxilliped large, bilobed, the lobes with coarse setae directed inwardly ; endopod reduced, bears two apical setae ; exopod with six setae on proximal segment and four long setae at tip of distal segment; epipod large, bilobed. Second maxilliped with five-segmented endopod, ultimate and penulti- mate segments wider than long, armed with coarse spines ; exopod with two setae ; epipod small, bilobed. Third maxilliped with four-segmented endopod, coarsely setose throughout ; endopod reduced ; epipod tiny, bilobed. First pereiopod chelate, somewhat stouter and shorter than second pereiopod; exopod a rudiment or lacking. Second pereiopod chelate, cutting edges of chela without serrations or teeth. Carpus shorter than palm ; exopod rudimentary if present. Third, fourth and fifth pereiopods not chelate, exopods rudimentary if present. Arthrobranchs at bases of pereiopods. Endopod of first pleopod rudimentary. Endopods of pleopods 2 to 5 with appendices internae. Uropodal exopod sparsely setose along outer edge with a tooth and a movable spine in the disto-lateral corner, numerous setae around the tip and on inner edge ; endopod with setae on inner edge and around tip. The distinctive distribution of chromatophores which characterized the larva is lost in the postlarva. The young prawn appears colorless to the unaided eye, but actually has numerous tiny chromatophores on the cephalothorax and abdomen. The postlarva of P. pugio is strikingly similar to the postlarva of P. vulgaris. The characters used to separate adults of these species are undeveloped in postlarvae. No attempt is made here to offer characters by which the two species may be dis- tinguished at this stage of development. The postlarva corresponds to Faxon's seventh stage. The sequence of larval intermolts The descriptions of zoea larvae given above were based on the structure of in- dividuals reared in the laboratory. Since the number of molts during development is not constant, it is obvious that certain of the described forms may be omitted in the life history of any individual. The relationship between the number of molts and the structure of intermolts observed in the laboratory is given in Table II. Column 4 of Table II shows that, for some larvae, the sequence of successive inter- molts was that given in the text descriptions. Columns 5 to 9 show progressive omission of more of the described forms. The data suggest a relationship between diet and the number and form of larval intermolts. DISCUSSION Larvae of Palaemonetes reared in the laboratory were structurally identical to those from nature described by Faxon (1879). This resemblance extended even to certain larvae considered abnormal by Faxon but shown by rearing to belong to series of intermolts which ultimately metamorphosed to produce normal post- larvae. The distinctive distribution of abdominal chromatophores, which was found to be diagnostic for the species treated in this paper, was not noted by Faxon 158 A. C. BROAD TABLE II The relationship between molting and form of Palaemonetes pugio and Palaemonetes vulgaris larvae fed various diets. Numbers in column 1 refer to text descriptions. Numbers in parentheses refer to described variations. The sequence of forms throtigh which larvae pass in development is given in columns 4 to 9. Zoea larva No. Approx. total length (mm.) Recognition character Non-living animal plus unicellular algae Intermolt number Non-living animal Artemia nauplii 1 2.6 Sessile eyes 1 1 1 1 1 1 2 3.0 Stalked eyes 2 2 2 2 2 2 3 3.2 Telson and uropods 3 3 3 (3) 3.3 4th and 5th pereiopod rudiments 3 3 3 4 3.5 2 dorsal rostral teeth 4 4 4 4 5 3.5 3rd pereiopod 5 5 6 3.7 5th pereiopod 6 6 5 7 4.4 Pleopod buds 7 7 6 5 4 4 8 4.9 Pleopod rudiments 8 7 6 (9) 5.0 Chelae 8 5 5 9 5.1 Pleopod exopod 9 9 8 7 6 6 10 6.1 Pleopod endopod 10 8 7 (10) 6.3 Appendices internae 11 10 9 9 8 7 PL 6.3 Postlarva 12 11 10 10 9 8 who found pigment spots to be variable in position. Minor morphological details, which might serve as indices of species, were not discussed or figured by Faxon. It seems possible that he may have dealt with larvae of more than a single species, although Holthuis (1952) feels that the adults and, presumably, the first stage larvae described by Faxon were P. vulgaris. The normality of larvae reared in the laboratory has been questioned by Gurney (1942) who believes that abnormal stages may be reared under artificial conditions. Gurney further states, however, that "extra stages," through which each individual need not pass in development, occur in nature. Numerous references to these extra stages are available (Faxon, 1879; Gurney and Lebour, 1941 ; Lebour, 1940). Fraser (1936) and Boden (1950, 1951) defined stages or norms of variation in euphausid Furcilia larvae on the basis of the frequency of occurrence of all the forms encountered. As a result of these analyses it is possible to state that some of the forms are normal and some abnormal in a purely statistical sense. No treat- ment of the frequency of occurrence of variation in larvae of a decapod is available. It is not presently possible to state with certainty that any individual decapod larva is either normal or abnormal except on the basis of the course of its development. Defining normal development, however, presents great difficulties. MacDonald (1927) and Fraser found numerical predominance of certain of the euphausid Furcilia stages over others. It was assumed that certain of the stages may be skipped in development. This assumption was confirmed by Fraser in the labora- tory. The normality of individual larvae and the course of larval development possibly depends upon extrinsic factors. Sandoz and Rogers (1944) found optimum temperature and salinity conditions for molting of Callinectes larvae and that the tempo of molting might be reduced by a sub-optimal diet. In view of the LARVAL DEVELOPMENT OF PALAEMONETES 159 lack of evidence to the contrary, the variation observed in the structure of larvae and the tempo of development in Palacinonetes is considered to be within the limits which may be considered normal for the species. The apparent discrepancy in the proposed developmental sequence, shown by the unfolding of pereiopods 3 and 5 before the appearance of pleopods in some larvae and after the pleopod buds have been formed in others, may be a function of rate rather than sequence of development. Pereiopod 3 appears after the second molt and is followed by pereiopods 4 and 5. These may appear all at once in the third intermolt among rapidly developing larvae, or their appearance may be in separate intermolts. All the fourth intermolt larvae have five pairs of pereiopods of which the last three are rudimentary. Among rapidly developing larvae the pleopod buds appear in the fourth intermolt, before the last three pereiopods have unfolded. If pleopods do not appear in fourth, fifth or sixth intermolt larvae, pereiopods 3 and 5 will have become functional before the pleopod buds are first seen. Pleopods always follow pereiopods in appearance. The length of time or number of inter- molts which intervene may permit some variation in the status of pereiopod un- folding at the time of the pleopod appearance. The present concept of the crustacean larval stage has contributed to confusion regarding development. Most authors refer to each larval intermolt as a stage. Numbers are assigned to these stages which presume a knowledge of the molting history of the individual. Thus, a larva is assumed, on the basis of structure alone, to have molted a certain number of times and, presumably, to be of a certain age. The basis for this implication is an assumed norm of development on which reason- able doubt may be cast. Development in arthropods is made to appear discontinuous by the inflexibility of the exoskeleton during the intermolt period. The morphology of the individual larva, which cannot change during the intermolt period, is determined by the extent of development completed at the time of the last molt. Fraser (1936) feels that, within limits, the time of molting may shift slightly backwards and forwards. This fluctuation, superimposed on a continuous process of development, was sug- gested as the cause of variation in euphausiid larvae. Heegaard (1953) has suggested variation in the rate of larval development in penaeids, but did not discuss molting. No variation in larvae is possible if either a causative or a casual relationship between molting and development exists so that each larval molt occurs at a precise time in development. The presence of larvae of the same molting history which vary widely in size and form argues in favor of the independence of the frequency of molting from the rate of larval development. Arbitrary stages may be defined in crustacean development but should not be thought of as inflexible, natural steps through which each individual passes. The choice between few or many stages is possible. If, as has frequently been the case, each form found is described as a stage, then many individuals may skip certain stages in development. If few stages are defined, certain individuals may pass through what might be called extra stages during the course of normal develop- ment. The inference that the variation in tempo of larval development is related to diet is inescapable. Trophic conditions may vary in nature during the breeding season of each species. At Beaufort, Palaemonetes larvae hatched in late April or May become part of a plankton community which is poor in total number of 160 A. C. BROAD organisms. A possible response to this sub-optimal condition might be prolonged larval life with a greater number of larval intermolts. Normal development of a decapod may itself vary according to the season of the year. SUMMARY 1. Palaemonetes pugio Holthuis and Palaemonetes vulgaris (Say) were reared in the laboratory from eggs through metamorphosis. 2. The larvae of these species are very nearly identical except for a pair of chromatophores found on abdominal sternite 2 of P. pugio but lacking from P. vulgaris. The sequence of development is the same for each species. 3. Individual larvae which were of the same age or which had molted the same number of times were not necessarily alike. Larvae of the same size, regardless of age or the number of molts completed, were alike. This discrepancy between age and development seems associated with the diet of the larvae. 4. The molting frequency of the larvae was independent of the rate of develop- ment. 5. Descriptions of a series of P. pugio larvae which illustrate the sequence of events in larval development are given. Some of these forms may be skipped. 6. The concept of the crustacean larval stage has assumed a constancy of de- velopment at variance with the facts observed in rearing experiments. The form of a larva alone may not be regarded as indicative of its age or previous molting history. LITERATURE CITED BODEN, B. P., 1950. The post-naupliar stages of the crustacean, Euphausia pacifica. Trans. Amer. Micr. Soc., 69 : 373-386. BODEN, B. P., 1951. The egg and larval stages of Nyctiphanes simplex, a euphausid crustacean from California. Proc. Zool. Soc. London, 121 : 515-527. BURKENROAD, M. D., 1947. Reproductive activity of decapod Crustacea. Amer. Nat., 81 : 392-398. BURKENROAD, M. D., 1949. Occurrence and life histories of commercial shrimp. Science, 110: 688-689. CHURCHILL, E. P., 1942. The zoeal stages of the blue crab, Callinectes sapidus Rathbun. Chesapeake Biol. Lab. Pub. No. 49 : 3-26. FAXON, W. A., 1879. On the development of Palaemonetes vulgaris. Bull. Mus. Comp. Zool. Harvard, 5 : 303-330. ERASER, F. C., 1936. On the development and distribution of the young stages of the krill (Euphausia superba). Discovery Repts., 14: 1-192. GURNEY, R., 1942. The larvae of decapod Crustacea. The Ray Society, London. GURNEY, R., AND MARIE V. LEBOUR, 1941. On the larvae of certain Crustacea Macrura, mainly from Bermuda. /. Linn. Soc., 41 : 89-181. HEEGAARD, P., 1953. Observations on spawning and larval history of the shrimp, Penaeus sctiferus (L.). Pub. Inst. Mar. Sci., 3: 75-105. HELDT, JEANNE, 1938. La reproduction chez les crustaces decapodes de la famille des peneides. Ann. Inst. Oceanogr., Monaco, 18: 31-306. HOLTHUIS, L. B., 1949. Note on the species of Palaemonetes (Crustacea Decapoda) found in the United States of America. Proc. Kon. Nederl. Akad., Wctensch. 52 : 87-95. HOLTHUIS, L. B., 1952. A general revision of the Palaemonidae (Crustacea Decapoda Natan- tia) of the Americas. II. The subfamily Palaemoninae. Allan Hancock Foundation Occ. Pap. No. 12: 231-249. HOPKINS, S. H., 1943. On the external morphology of the first and second zoeal stages of the blue crab, Callinectes sapidus Rathbun. Trans. Amer. Micr. Soc., 62 : 85-90. LARVAL DEVELOPMENT OF PALAEMONETES 161 HOPKINS, S. H., 1944. The external morphology of the third and fourth zoeal stages of the blue crab, Callincctes sapidus Rathbun. Biol. Bull., 87: 145-152. HUDINAGA, M., 1942. Reproduction, development and rearing of Penacus japonicus Bate. Jap. J. Zool, 10 : 305-392. LEBOUR, MARIE V., 1927. Studies of the Plymouth Brachyura. I. The rearing of crabs in captivity with a description of the larval stages of Inachus dorsettensis, Macropodia longirostris and Mala sqninado. J. Mar. Biol. Assoc. U. K., 14 : 795-821. LEBOUR, MARIE V., 1928. Studies of the Plymouth Brachyura. II. /. Mar. Biol. Assoc. U. K., 15: 109-118. LEBOUR, MARIE V., 1928. The larval stages of the Plymouth Brachyura. Proc. Zool. Soc. London, 1928 : 473-560. LEBOUR, MARIE V., 1930. The larval stages of Caridon. Proc. Zool. Soc. London, 1930 : 181-194. LEBOUR, MARIE V., 1932. The larval stages of the Plymouth Caridea. III. Proc. Zool. Soc. London, 1932: 131-137. LEBOUR, MARIE V., 1936 a. Notes on the Plymouth species of Spirontocaris. Proc. Zool. Soc. London, 1936 : 89-104. LEBOUR, MARIE V., 1936 b. Notes on the Plymouth Processa. Proc. Zool. Soc. London, 1936 : 609-617. LEBOUR, MARIE V., 1940. The larvae of the British species of Spirontocaris and their relation to Thor (Crustacea Decapoda). /. Mar. Biol. Assoc. U. K., 24: 505-514. LEBOUR, MARIE V., 1943. The larvae of the genus Porcellana and related forms. /. Mar. Biol. Assoc. U. K., 25 : 721-737. MACDONALD, R., 1927. Irregular development in the larval history of Meganyctiphanes norvegica. J. Mar. Biol. Assoc. U. K., 14 : 785-794. PEARSON, J. C., 1939. The early life histories of some American Penaeidae, chiefly the com- mercial shrimp, Penaeus setiferus (Linn.). Bull. U. S. Bur. Fish., 49: 1-73. SANDOZ, MILDRED, AND ROSALIE ROGERS, 1944. The effect of environmental factors on hatching, moulting and survival of the blue crab. Ecology, 25 : 216-228. THE RELATIONSHIP BETWEEN DIET AND LARVAL DEVELOPMENT OF PALAEMONETES *• 2 A. C. BROAD Duke University Marine Laboratory, Beaufort, N. C. Larval development of crustaceans consists, in its simplest form, of growth and the addition of somites and limbs. The primitive pattern has been obscured among higher crustaceans by the degree of development achieved by the embryo before hatching and by the magnitude of developmental change which may become evident after each larval molt. Gurney (1942) and most authors think of the mode of development for each species as fixed and regard the number and sequence of definitive larval stages as constant. Variation in the number and form of larval intermolts of Palaenwnetes pugio Holthuis and Palaemonetes vulgaris (Say) reared in the laboratory has been described by Broad (1957). The present paper is a consideration of the relation- ships between diet and survival, molting frequency, rate of larval development and the number and form of intermolts during development of these species. The author is indebted to Professor C. G. Bookhout, under whose guidance this work was done. He also wishes to express his thanks to Dr. T. R. Rice, who kindly furnished stocks of all species of unicellular marine algae used. METHODS Larvae dealt with were hatched in the laboratory from eggs carried by adult females and were reared through metamorphosis. Culture methods have already been discussed (Broad, 1957) and need not be repeated in detail. Each larva was fed the same diet throughout its life, but several different foods were offered to different individuals. The diets differed from one another gen- erally and specifically. There were five general diet categories : no food ; unicellular marine algae ; algae and non-living animal matter ; non-living animal tissue alone ; and living Artemia nauplii. Specific differences in diet were between the several combinations of the available foods in each general category. Larvae which were not fed nevertheless had available whatever food might be obtained from the raw sea water in which they were reared. The form of the mouthparts of these larvae makes it extremely unlikely that particles not visible to the unaided eye could be utilized as food. Some individuals were fed species or combinations of species of unicellular marine algae. These species were maintained in unialgal culture in the laboratory. 1 Supported in part by a contract with the Office of Naval Research, Nonr-1232(00), and a National Science Foundation grant, NSF-G-1214. 2 Part of a thesis submitted to the graduate faculty of Duke University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 162 DIET AND DEVELOPMENT OF PALAEMONETES 163 Clumps of cells which settle in older cultures were used as food. The algae avail- able were from T. R. Rice's stocks of Nltzschia dosteriuin and Nitzschia sp., (Bacillareae), Chlamydomonas sp. ; Thorocomonas sp. ; Nannochloris sp. (Chlo- rophyta) ; Porphyridhnn sp. (Rhodophyta) and Pyramimonas sp. (Chrysophyta). Other larvae were fed non-living animal tisues alone or in combination with one or more of the algal species. Animal matter used was obtained largely from the plankton. Zooplankton, collected by net, was killed by immersion in distilled water to prevent the fortuitous inclusion of living larvae which might later be confused with individuals being reared. The general zooplankton (and possibly a few con- taminating phytoplankton cells) was fed to some larvae. Others were fed on chaetognaths removed from the killed plankton. A few individuals were fed macerated gonad of the mud snail, Nassarius obsoletus. The larvae usually hatched at night. The day on which free-swimming in- dividuals were found was called the first day of larval life. Larvae and the bowls in which they were held were inspected daily. Only the presence of an exuvium was accepted as evidence of molting. Molts were considered to have occurred the day on wrhich the cast exoskeleton was found. Uneaten food was removed and fresh food added at the time of daily inspection. RESULTS Palacmonetes larvae were found to ingest almost any particulate matter with which they came in contact. There was no evidence of chemoreception or of selection of any type of food. Cannibalism was infrequently observed. In feeding, the zoea larvae grasp and hold objects with the maxillipeds while the maxillae and mandibles function as jaws. There was no indication of ability to obtain food by filtering. Larvae fed diets which differed in general composition showed different rates of survival and development and molted at different frequencies, but those fed diets which differed only in specific composition did not. All the larvae are grouped for treatment of data into five general categories according to the diet received. Diet and survival of larvae Figures 1 and 2 show the per cent of mortality observed at each molt among P. pugio and P. vulgaris larvae fed various diets. Most of the deaths occurred at the time of molting, but some individuals were lost during the intermolt phase. These are included among the larvae which died at the time of the next molt. The per cent of larval mortality at molt N is that fraction of the total number of larvae which completed molt N-l but did not survive molt N, and includes larvae lost during intermolt N. Loss of some intermolt larvae was due to removal of specimens for preservation or study, and the apparent mortality accordingly is biased upward when the total number of larvae involved is small or in the later molts. Larvae which were not fed did not survive nor did those which were fed only phytoplankton cells. Sixty P. pugio larvae were starved. Forty-two of these survived one molt and two individuals molted twice, but none lived longer than 10 days. Among 80 P. vulgaris larvae which were not fed, none molted and all died within 5 days. Feeding any of a variety of unicellular marine algae did not seem 164 A. C. BROAD 100- 80- 60- Z 40' 20- NO FOOD UNICELLULAR AL NON-LIVING ANIM ALGAE PLUS AN^ ARTCUIA NAUPLII I 2 3 MOLT NUMBER 100- 60 - O 5 20 UNICELLULAR ALGAE * ALGAE PLUS ANIMAL - ARTEMIA NAUPLII I 2 3 MOLT NUMBER FIGURE 1. Mortality at each of the first seven molts among Palaemonetes pugio larvae fed various diets. Per cent mortality at each molt is based on the number of larvae which survived the last molt. FIGURE 2. Mortality at each of the first seven molts among Palaemonetes vulgaris larvae fed various diets. Per cents computed as for P. pugio (Fig. 1). to improve survival or molting in either 280 P. pugio or 100 P. vulgaris larvae. The survival of algae-fed larvae shown in Figures 1 and 2 closely approximates that of starved larvae. Larvae that were fed foods of animal origin, either living or non-living, alone or in combination with algae, were able to survive through metamorphosis in the laboratory. The mortality of 608 P. pugio larvae that were fed diets of non-living animal matter, mostly zooplankton, combined with phytoplankton cells is shown by the dash-and-two-dots line in Figure 1. Sixteen of these individuals meta- morphosed. The dash-and-dot line in Figure 1 shows the mortality of 732 P. pugio larvae that were fed non-living animal matter alone. In general the two curves are alike. Both reflect, in peaks at the second molt, the ability of this species to survive a single molt without food. Only 6 individuals metamorphosed on a diet of freshly killed zooplankton or other non-living animal matter. By far the best survival was shown by larvae fed living Artemia nauplii. The mortality of 100 of these is shown by the dotted line in Figure 1. Sixty-five individuals survived metamorphosis. Another 40 P. pugio larvae, for which it was not possible to determine daily the state of development of each individual, were fed living Artemia nauplii. Mortality at each molt of these individuals is not shown, but 33 meta- morphosed. The mortality of 667 P. vulgaris larvae that received a diet of non-living animal matter combined with algal cells is shown by the dash-and-two-dots line in Figure 2. The initially high mortality shown by starved larvae is also evident for these larvae, and contrasts with the early independence of available food shown by P. pugio. Six postlarvae survived metamorphosis. The dotted line in Figure 2 shows the mortality observed among 390 P. vulgaris larvae fed living Artemia nauplii. One hundred twenty-two of these metamorphosed in the laboratory. Diet and the frequency of molting Molting of P. pugio larvae is shown in Figure 3. The differences in molting frequencies between larvae which survived and most of those which did not on DIET AND DEVELOPMENT OF PALAEMONETES 165 each diet are insignificant, although those individuals which did not maintain a regular molting schedule did not survive. In general, the range of days during which specific molts occurred among larvae which lived is more restricted for the earlier than for the later molts. This most likely results from some variation in the molting frequencies of larvae fed similarly. Since, except for the first two molts, there is little or no overlap in the means and standard deviations, or sometimes even the ranges, of corresponding molts by larvae fed different diets, the frequencies suggested by the diagram seem to be statistically separable. Average molting frequencies may be computed, although these become more or less meaningless since the interval between hatching and the first molt and that between the last larval molt and the molt of metamorphosis must be included. The interval between hatching and the first molt is usually somewhat longer than that between subsequent molts. The molt of metamorphosis, although not exactly comparable to other molts, is also included in the computations of average molting frequencies. Among larvae which survived metamorphosis on a diet of mixed plant and animal matter, the frequency of molting varied from one molt every 3.15 to 4.0 days with an average frequency of one molt every 3.70 days. Those larvae which survived on a diet of non-living animal matter alone molted once every 2.36 to 2.67 days with an average frequency of one molt every 2.51 days. Molting among larvae which survived metamorphosis on a diet of living Artemia DAY OF LARVAL LIFE 24 6 8 10 12 14 IS 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 j±L_49l 1 i,, |5 MOLTING OF 608 LARVAE FED UNICELLULAR ALGAE PLUS III ^T^ 16 r-t-i AfrA. i 1 1 ^A ^Fe ~^F~ 16 l-h 177 , 1 1 47 II V T6 "CP~ 16 III VI ' ^=^6 ' 1 ' 16 1 q? i — I — i 70 , 1 1 50 al u m 'l 65 '=P 6 •— t— ' 16 i R7 i 1 SO i 1 1 PA 2 II V VIII D 1 65 , 4-1 5 . ' 1 ' 16 III VI IX ~ 1— _l 165 u-l~l 6 , ' 1 16 db fl'i ' ' ?« i — — 1 1 17 0 IV VII X 5 M-1 65 •— t— ' 6 ' 1 ' 16 rfi fl? I 1 1 ?=, , 1 , 17 MOLTING OF V,^I°^ VIII ' XI T b5 •— t— ' b ' 1 ' |g 100 LARVAE FED H-i 82 i 1 1 14 ARTEMIA NAUPLII utj 65 •— t— ' 6 MOLTING OF 732 LARVAE FED ... H—i 69 V" -r-r-r- .. NON-LIVING ANIMAL TISSUE M-1 65 FIGURE 3. Frequency of the several molts among Palaemonetes pugio larvae fed three different diets. Number of each molt is given by Roman numbers. Upper line of each pair shows days during which molt occurred. Lower line gives days on which those larvae which completed metamorphosis molted. Means shown by vertical lines. Box on each side of mean is one standard deviation. Arabic numbers show sample size. 166 A. C. BROAD DAY OF LARVAL LIFE 2 4 6 8 10 12 !4 IS 18 20 22 24 26 28 30 32 34 i i i i i i i i i i i > i i i i i ^P 6 ,_ MOLTING OF 667 LARVAE FED II UNICELLULAR ALGAE PLUS i 1 1 n NON-I IVIN^ AN'MAI Jl^^lIF 1-4—1 |6 n IV ' ' | '6 cb 324 ' i T i II r-t-L 282 i 1 1 9 II cp_ |22 VI r_r-r 6 i-4—i Pfil i 1 1 R ot ||l .__ VII ._ i— I — i — 22 ' 6 CD •3O~7 i — 1 — i ££l i 1 1 R ^ IV is-) VIII r z "—1— ^^ |g| ' 1 ' o i— V , , 155 IX , _j o i — 1 122 i— 1 1 Q , 1 , 149 , 1 , 7 5 MOLTING OF VI X .,,. 1 1 ' \£d ' 1 ' 6 ^oo i AP\/AT rrn i 1 1 131 ARTEMIA NAUPLI1 ' 1 ' 122 FIGURE 4. Frequency of each molt among Palaemonetes vulgaris larvae fed two different diets. See legend of Figure 4 for explanation. nauplii was observed to occur with a frequency of once every 2.30 to 2.50 days with an average of one molt every 2.43 days. The molting of P. vulgaris larvae, shown in Figure 4, shows little statistical separateness between molting frequencies of larvae fed different diets. Because of the initially high mortality of these larvae the samples are even smaller than those for P. pugio. Fourth and fifth molts seem to be distinct between the two groups of larvae, but, possibly due to a slowing-down of the molting frequency as the time of metamorphosis approaches, the later molts are not. Among those larvae which survived on a diet of mixed animal and plant matter, the molting rate was one ecdysis every 2.4 to 3.1 days with an average of a molt every 2.62 days. The average molting frequency for larvae which metamorphosed on a diet of living Artemia nauplii was one molt every 2.67 days, but the molting of these varied from one molt every 2.1 to 3.6 days. Diet and the rate of larval development An approximation of the rate of larval development may be obtained from the number of days between hatching and metamorphosis. This, however, is not DIET AND DEVELOPMENT OF PALAEMONETES 167 completely satisfactory since metamorphosis need not always occur at what appears from morphological criteria to be the end of larval development. Thus, Faxon (1879) found "last stage" P. vulgaris larvae which molted in the laboratory gave rise to other "last stage" larvae morphologically indistinguishable from their pre- vious form. The larvae of both P. pugio and P. vulgaris reared in the present study sometimes passed through two or more identical intermolts at the end of development and before metamorphosis. At this time, depending upon other con- ditions not presently understood, the larva may metamorphose, as most of them do, or possibly may remain larval for some time. P. pugio larvae fed a diet of mixed animal and plant matter metamorphosed from 29 to 49 days after hatching. Most of the larvae metamorphosed at the eleventh or twelfth molt, but a few metamorphosed as early as the ninth or tenth molt, or as late as the thirteenth molt. Larvae of this species fed a diet of non- living animal matter alone metamorphosed from the nineteenth to the twenty- eighth day after hatching and at the eighth, ninth, tenth or eleventh molt. Those individuals fed a diet of living Artemia nauplii metamorphosed from 17 to 21 days after hatching, usually at the seventh, but sometimes at the eighth molt. P. vulgaris larvae fed a diet of mixed animal and plant matter metamorphosed from 24 to 34 days after hatching. The molt of metamorphosis was most often the tenth, but two individuals metamorphosed at the eleventh and thirteenth molts. Those larvae fed a diet of living Artemia nauplii metamorphosed from 14 to 30 days after hatching, usually at the seventh molt. Metamorphosis was also noted at the sixth, eighth, ninth and tenth molts. A second approximation of the rate of development might be obtained by com- paring the structure of larvae to age and molting history. In spite of the variation noted in duration and tempo, the sequence of development was the same for all larvae and for both species. Except for differences in color patterns which are specific, all newly-hatche.d P. pugio and P. vulgaris larvae were essentially alike. For the present purposes this first zoea larva may be characterized by the presence of all head appendages, three pairs of functional maxillipeds, two rudimentary pereiopods, sessile eyes, a fan-shaped telson and a lack of spines. After a single molt all larvae acquire stalked eyes, spines on the carapace and abdomen, and functional first pereiopods. Until after the second molt variation in form is almost non-existent, but, from the second molt to the end of larval development, there may be wide variation in the form of larvae of the same age or molting history. A table which summarizes the relationship between intermolt number and form of larvae fed variously has previously been presented, and the morphology of the several developmental steps or stages has been discussed in detail (Broad, 1957). For the present purpose it suffices to reiterate that the number of intermolts in develop- ment of either species dealt with may vary, but that the sequence of events in de- velopment does not. Approximate ages of intermolts discussed below may be obtained from Figures 3 and 4. Following the second molt, third intermolt, or third zoea larvae show variation in the number of pereiopod rudiments. Those individuals fed Artemia had rudi- ments of pereiopods 3, 4 and 5 while those fed other diets lacked pereiopods 4 and 5. Great variation was evident among fourth intermolt larvae. Those individuals fed Artemia bore rudiments of the pleopods but none of the others did. Among larvae fed non-living animal foods alone, pleopod buds first appeared in the fifth or 168 A. C. BROAD sixth intermolt and not until the seventh intermolt in larvae fed diets which com- bined algae with animal matter. The intervening intermolts, 4, 5 and 6, differed from one another in the development of pereiopods. Fifth intermolt larvae which had been fed Artemia had pleopod rudiments and chelae. The pleopod buds of fifth or sixth larval intermolts fed non-living animal tissue alone became rudiments after a single molt, but chelae did not appear until after two molts. Some of the eighth larvae fed algae and animal matter bore chelae, but others had none. The pleopods of all sixth intermolt larvae which had been fed Artemia bore setose exopods. These were first evident in seventh or eighth intermolts which had received non-living animal food and in ninth zoea larvae which had been fed algae plus non-living animal foods. The final larval form has been characterized by both setose endopods and ap- pendices intcrnae on pleopods. Most larvae fed Artemia nauplii achieved this final form in the seventh intermolt, although some individuals required two steps after the sixth zoea for appendices internae to appear. Metamorphosis occurred at the seventh or eighth molt. Appendices internae made their appearance in ninth intermolt larvae fed non-living animal matter, and metamorphosis occurred at the tenth molt. Among larvae fed algae and animal matter, the final larval form was reached in the tenth, eleventh, or even sometimes the twelfth intermolt. Meta- morphosis usually occurred at the following molt. DISCUSSION Since survival of larvae fed algal diets alone did not differ from that of zoeae which were not fed, it would seem that the algal species available had no value as food for either P. pugio or P. vulgaris larvae. In order to survive, the larvae must find some particulate food, probably animal in nature. A physiological distinction between P ' . pugio and P. vulgaris is possible in the ability of the former species to molt once and survive up to ten days without food while the latter neither molts nor lives longer than five days without food. Specu- lation regarding the survival value which might be associated with this relatively greater independence of trophic conditions, though interesting, is futile in view of the presently limited knowledge of the geographic and ecological distribution of the species of Palaemonetes. The ability of either species to adjust development to external conditions and the rather indefinite time of metamorphosis are both of positive survival value. It is possible that the varying rates of survival, molting and development noted among larvae reared in the laboratory may be due to differences in the total quantity rather than in the quality of food available. Two factors limited the amount of non-living animal tissue available to larvae fed this diet. In order to retard fouling of the water in the bowls, the total quantity of food added daily had to be kept within limits. Since food was always left, the quantity was at first thought to be sufficient. Non-motile food, however, sank to the bottom of the bowls. The larvae swam near the surface. It has already been stated that contact between zoea and food seemed to be the result of chance encounter rather than active search on the part of the larva. The IOWT probability of encounter between larvae swimming near the surface and food lying on the bottom might account for the uneaten food left each day. DIET AND DEVELOPMENT OF PALAEMONETES 169 If clumps of algal cells were added to the diet, a third limiting factor is also added. Larvae fed algae actually ingested the material offered. The red, green or brown color of the cells could be seen in the gut and feces of the larvae. Since the algal species used have been shown to be of no nutritive value, it seems possible that, where algae plus another food is offered, the ingestion of the nutritively inert material may restrict the intake of other foods which can be digested and utilized. Ne restrictions were placed on the total number of Artemia nauplii fed. The nauplii swam near the surface with the zoea larvae. The probability of encounter between larva and food was greatest when the diet consisted of living animals. It has been suggested that intraspecific variation in crustacean larvae may arise from extrinsic causes (Broad, 1957). Sandoz and Rogers (1944) found that poorly nourished Callinectcs zoeae molted only after a relatively long period and were smaller than other larvae which had received more food. Templeman (1936a, 1936b) found reducing the amount of food given Homanis larvae lengthened the intermolt period and sometimes resulted in the production of an "extra" larval intermolt. The present data suggest that the amount of food avail- able to Palaemonetes during its larval life may affect both the rate of development and the frequency of ecdysis. The independence of these variables is suggested by the differences in the magnitude of response to sub-optimal feeding. Although the longest regular interval between molts was only 1.9 times the least, the duration of larval life was extended 3.5 times over the most rapid successful development. Heegaard (1953) has suggested that different developmental rates among decapod larvae may be caused by "external as well as internal" factors. The amount of available food may be an external factor which affects both the rate of development and. independently, the frequency of molting. Variation in the form of larvae under these conditions might be restricted only by the morphological limitations of species. If norms of developmental stages exist among decapods, as Fraser (1936) has found among Euphausids, constancy of environment might be considered of prime importance in their establishment. Extra or abnormal larval stages found in nature or in the laboratory as wrell as stages skipped in development may reflect an adaptibility to environmental variation during development. SUMMARY 1. Larvae of Palaemonetes piigio Holthuis and Palaemonetes vulgaris (Say) reared in the laboratory showed differences in survival, frequency of molting and rate of development which may be associated with the amount of food available. 2. Larvae were unable to survive if fed diets of either single species or com- binations of species of several unicellular marine algae or if not fed. Starved P. piigio larvae were able to survive one molt without food but starved P. vulgaris larvae died without molting. 3. Larvae of both species lived through metamorphosis if fed a diet which included animal tissue. The best survival was obtained by feeding living Artemia nauplii. 4. The frequency of molting, the duration of larval life and the number of larval intermolts in the development of P. pngio and P. rulgaris vary according to the quantity of food available. The frequency of molting and the rate of develop- ment are suppressed by a reduction in intake of food. 170 A. C. BROAD 5. Variation in molting frequency independent of the rate of development makes possible variation in the form and number of larval intermolts. LITERATURE CITED BROAD, A. C., 1957. Larval development of Palaemonetcs pugio Holthuis. Biol. Bull., 112: 144-161. FAXON, W. A., 1879. On the development of Palaemonetes vulgaris. Bull. Mus. Comp. Zool., 5 : 303-330. FRASER, L. C., 1936. On the development and distribution of the young stages of the krill (Euphausia superba). Discovery Repts., 14: 1-192. GURNEY, R., 1942. The larvae of decapod Crustacea. The Ray Society, London. HEEGAARD, P., 1953. Observations on spawning and larval history of the shrimp, Penaeus setiferus (L.). Pub. Inst. Mar. Sci., 3: 75-105. SANDOZ, MILDRED, AND ROSALIE ROGERS, 1944. The effect of environmental factors on hatching, moulting and survival of the blue crab. Ecology, 25 : 216-228. TEMPLETON, W., 1936 a. Fourth stage larvae of Homarus americamis intermediate in form between normal third and fourth stages. /. Biol. Bd. Canada, 2 : 349-354. TEMPLETON, W., 1936 b. The influence of temperature, salinity, light and food conditions on survival and growth of the larvae of the lobster (Homarus americamis). J. Biol. Bd. Canada, 2 : 485-497. X-RAY EXPERIMENTS WITH MOLGULA MANHATTENSIS : ADULT SENSITIVITY AND INDUCED ZYGOTIC LETHALITY DANIEL S. GROSCHi AND ZOE H. SMITH 2 Marine Biological Laboratory, Woods Hole, Massachusetts Sessile organisms which pump large quantities of sea water through their bodies suggest themselves as ideal material in which to study biological effects of radio- active contaminants. Because of their relationship to the chordates, ascidians have particular attraction. Furthermore, many are functional hermaphrodites providing both sperm and eggs for simultaneous irradiation. Molgula manhattensis has the additional advantage of self-fertility so that crosses using gametes from the same individual can be made, as well as outcrosses. In the absence of radiobiological information on Molgula, the present x-ray experiments were performed rather than employing isotopes with their more difficult dosimetry. Isotope experiments may be performed in the future after a more complete knowledge of what may be expected from external radiations is at hand. MATERIALS AND METHODS Preliminary experiments were performed during the summer of 1955 with the assistance of Robert L. Sullivan. Specimens of Molgula were collected for us by personnel of the M. B. L. Supply Department. Irradiation was delivered by the M. B. L. generator in "A" position at 6000 r per minute (187 KV ; 28 ma ; filtration = 0.2 mm. Cu). To achieve massive doses without overheating, the animals were immersed in sea water within an inner chamber, and crushed ice was packed into the space between it and the outer wall of the plastic container. After irradiation, animals were kept in individual glass jars (50 X 80 mm.) in running sea water. Failure to contract in response to prodding with a blunt instrument was the criterion for death. An additional check on presumptive death was obtained by holding such animals until post-mortem flaccidity and degeneration became obvious. The results indicated that adults would be adequately resistant to whole-body irradiation which would allow the full range of dosages required for a genetic experiment on induced dominant lethality. In 1956 animals of adult size were obtained both through the M. B. L. Supply Department and by personal collection. The latter procedure seemed necessary when it was discovered that masses of Molgula held in the Eel Pond "live-cars" and in laboratory aquaria did not consistently exhibit gonads with mature gametes. The adequacy of the supply of eggs and sperm can be determined by examination with a low power microscope, usually after peeling off the test, always after removal of a portion of the mantle. 1 Academic affiliation : Genetics, N. C. State College, Raleigh, North Carolina. 2 Research assistant under the A.E.C. contract with the M. B. L., No. AT- (30-1) -1343. 171 172 DANIEL S. GROSCH AND ZOE H. SMITH Adults were irradiated in position "B" at 2500 r per minute, again using the Woods Hole machine, the Coolidge tubes this time not as close to the animals. Eggs were obtained by suction applied to a capillary pipette inserted into the lumen of an ovary. They were expelled into a stender dish of sea water. Immedi- ately after withdrawing eggs from both ovaries, sperm suspensions were prepared by macerating a portion of each testis in a separate dish of sea water (3-5 cc.). Eggs from each ovary were divided into three groups : ( 1 ) a control as a check against accidental fertilization during removal, (2) a sample which was fertilized with sperm from the adjacent testis, and (3) a sample which was cross-fertilized with sperm from another Molgula. Sixty-millimeter flat stender dishes, containing one inch of sea water, were used. They were placed on the sea table with their bases in running water. Fifteen hours later, tadpole development was scored using a stereoscopic dissecting-type (48 X ) microscope. Immediately after completion of these observations, the degree of cleavage for 100 objects was scored using a compound microscope (16 mm. objective, 10 X ocular). During July and the first half of August, pair-mating experiments were set up daily to obtain simultaneous data (a) for eggs from an irradiated animal fertilized by sperm from an untreated one, (b) for the reciprocal cross of treated sperm to untreated eggs, (c) from selfing the irradiated animal, and (d) selfing the non-irradiated member of the pair. The goal was to obtain data from at least five pairs of crosses for each of five chosen doses. T. H. Morgan's 1942 paper led us to expect a technical difference between the two ovaries in difficulty of removing unfertilized eggs. Therefore we kept separate controls and made separate crosses for each side of each animal. However, upon analysis of results, no significant differences between sides were demonstrable and the data for the two sides of each animal serve merely as replications. From August 13 through August 18 the daily plan of experiment was modified. On each day, five or more adult animals were simultaneously exposed to one of the five chosen doses. The gametes obtained from five treated animals were mixed to provide outcross data when both sperm and eggs were treated. At least five samples (averaging 800 objects/sample) were scored for each mass fertilization. On August 15 a mass outcross control was obtained. From August 20 through August 24, five animals were treated simultaneously at each of the five doses and selfed to obtain more information about the variability between material from different treated animals. From August 27 to September 1, miscellaneous experiments were set up: crosses of three different animals, each given a different dose, to a single untreated animal, and selfing crosses in which several doses were investigated each day. RESULTS Lethality of adults An exploratory experiment indicated that the critical dose of x-ray for the adult organism lay between 36,000 r and 60,000 r. This involved observations on 72 sea-squirts, 8 of which were controls, along with 8 samples of 8 animals given doses graded between 1000 r and 120,000 r. Two subsequent experiments of 40 each were set up with controls and samples irradiated at the following doses: 36,000 r, 42,000 r, 48,000 r, 54,000 r and X-RAY EXPERIMENTS WITH MOLGULA 173 60,000 r. In both the control and 36,000 r groups, individual animals were still alive more than a month after the date of irradiation. Also, although most of the animals given 42,000 r died during the first week, one lived almost a month. At higher doses no animals survived the fourth day, with the average time of death 2.4 days after treatment. This places the lethal dose between 42 and 48 kilo- roentgens. This is considerably less than the radiation required to kill adult insects (Sullivan and Grosch, 1953), brine shrimp (Grosch and Erdman, 1955) and vegetative microorganisms (Bacq and Alexander, 1955). On the other hand, such amounts of radiation are many times that required to kill mammals. Induced sygotic lethality A summary of the relative proportion of tadpoles obtained among the ova and zygotes studied is given in Figure 1. Consistently fewer swimming tadpoles emerged than developed to tadpole morphology. Furthermore, curves for data from irradiated eggs tend to lie below those representing sperm. This trend be- comes statistically significant at higher doses and it should be re-emphasized that the data come from paired matings. With self-fertilization, involving sperm and eggs from irradiated sea-squirts, the developmental yield is strikingly decreased (solid line, "Both Treated"). Along with the curve for selfing data, results from mass outcross experiments are shown by the broken line in Figure 1. With one exception, points obtained by calculating the mean are nearly identical whether selfing or outcrossing has been the procedure, provided both sperm and eggs are from irradiated animals. The standard errors omitted from the outcross curve in order not to further complicate Figure 1 are less than 2%. An additional group of data obtained from selfing five animals irradiated simultaneously at each dose also gives a curve similar to the solid line at the lower doses and identical with it at the three higher doses. Therefore it is not shown here. Since it has been corroborated by three separate sets of experiments, the curve above 5,000 r is a good representation of expectation when both sperm and eggs come from gonads irradiated in situ. Furthermore, this "Both-Treated" curve is predictable on the basis of one of the laws of probability : when two events are independent, the probability that both will occur simultaneously is the product of their separate probabilities. Thus, multiplying survival when sperm are x-rayed by survival when eggs are x-rayed we obtain the following : .33 X .075 = .0248 = 2.5% for 20,000 r .34 X. 185 =3.0629= 6.3% for 15,000 r .45 X .43 = .1935 = 19.4% for 10,000 r .545 X .51 = .2779 = 27.8% for 5,000 r These values calculated from Figure 1 data for the five higher doses are all within the range of one standard error from the "Both-Treated" curve shown in Figure 1. The theoretical value for 1000 r, 24.6% (.53 X .465), is lower than the "selfing" value obtained in pair experiments but falls within the range found in outcross experiments when both sperm and eggs are from treated animals. 174 DANIEL S. GROSCH AND ZOE H. SMITH SWIMMERS BOTH TREATED TOTAL TADPOLES X-RAY EXPERIMENTS WITH MOLGULA 175 TABLE I Development in the residuum of eggs and zygotes, tadpoles having been counted. The scoring was done on 100 objects -in each case. Values are given in per cent, representing failure to develop to tadpole morphology (means and standard errors) Both gametes treated X-ray dose Sperm Eggs in r Mass Self-cross Self-cross treated treated outcross 5 simultaneously pair experiments 0 99.5 ± 0.5 45.8 ± 11.9 1,000 84.3 ± 3.3 76.5 ± 0.5 54.2 ± 10.8 74.6 ± 6.3 63.8 ± 7.9 5,000 90.5 ± 2.0 72.2 ± 7.4 35.4 ± 12.9 55.4 ± 3.4 57.4 ± 9.8 10,000 89.9 ± 4.7 93.6 ± 2.9 59.2 ± 12.2 65.2 ± 5.6 64.8 ± 12.6 15,000 91.2 ± 1.2 79.8 ± 12.7 70.5 ± 8.4 65.9 ± 5.3 75.0 ± 6.8 20,000 86.1 ± 1.6 36.2 ± 10.2 76.0 ± 5.0 50.4 ± 11.7 No attempt was made to obtain a quantitative record of morphological aberra- tion. However, as an indication of developmental difficulty, thickened, bent and misshapen tails were typical for tadpoles in experiments above 10,000 r. Cleavage data In percentages, Table I presents a summary of results when objects other than tadpoles were scored. These are the averages from five or more experiments, in each of which 100 objects were carefully examined. Because of the time required and technical difficulty it is not feasible to examine all of the residuum. In order to understand Table I it must be realized that when few tadpoles develop from a group of eggs there are many undeveloped forms, and vice versa. Accordingly, since the amount of residuum varies, similar percentages with different doses may actually reflect differences. A conversion of the tabulated percentage values into numerical values, such as those plotted in Figure 2, helps to visualize the situation. The average total of eggs per stender dish, 800, is used as a common basis for presentation. The pertinent percentage of tadpoles (see Fig. 1) is subtracted. The applicable cleavage percentage (Table I) of the remainder gives the relative number of embryos plotted in Figure 2. All embryos considered iri Table I and Figure 2 appeared to be in the late gastrula or neurula stages, although at the two highest doses, structure was very disorganized. Presumably if embryos were able to begin development they could continue to such stages before facing an insurmountable developmental crisis. Extremely few embryos were found halted in an early cleavage stage. During the whole summer, when nearly 150,000 examinations were made, only 18 early cleav- age types were seen in selfing experiments, and 17 in outcrosses — exceptional in- dividuals making up only 0.02%. As might be expected, the number of gastrulae and neurulae increased as the number of tadpoles decreased at higher doses. Although this general trend is clear, unidentified sources of variability complicate the picture in selfing and pair- FIGURE 1. The relative proportions of tadpoles developing from gametes obtained from adult specimens of Molgula after irradiation. Results are contrasted when either or both types of gametes come from x-rayed parents. 176 DANIEL S. GROSCH AND ZOE H. SMITH 15 10 15 20 THOUSANDS OF ROENTGENS FIGURE 2. Post-cleavage zygotes which failed to develop to the tadpole stage. Results have been put on a common basis by calculations from the average total of eggs per sample, 800. Triangles indicate that the sperm were from treated animals ; circles, eggs. Squares represent selfed eggs and sperm from treated animals in pair experiments ; asterisks, data from 5 irradiated animals selfed on the same day. X's indicate outcross data, both parents x-rayed. Squares, circles and triangles represent results from pair experiments. mating experiments. When plotted as in Figure 2, zigzag lines are obtained. Furthermore, in the latter experiments, results when both gametes came from treated parents are not greatly different from those obtained when only one of the X-RAY EXPERIMENTS WITH MOLGULA 177 types of gametes has been exposed to radiation. Indeed, even after 5000 r, all three pair mating values were nearly identical. Therefore some general aspect of fertilization common to the experiments should be sought. The higher average values obtained after simultaneously selfing five animals given the identical dose of radiation are consistent with such a view. DISCUSSION Relative radiosensitivity of sperm and eggs In Molgula, relative radiosensitivity of eggs instead of sperm is an interesting parallel to Rugh's (1953) similar findings with the clam Spisula, although his crosses involved gametes which were irradiated after extraction from the parent. Such results are not expected, either on the basis of Henshaw's type of delayed cell division or that of chromosomal-gene effects. Sperm have been found more sensitive on either count. Mavor and De Forest (1924), who irradiated Arbacia gametes, found that samples scored two days later showed development ranging from gastrula stages to well-formed plutei. The retardation in development was greatest in those larvae developed from x-rayed sperm and increased with dose. Subsequently, Henshaw (1940) explained this phenomenon by a demonstration that radiation-delayed first cleavage is reflected in later development. Furthermore, the apparent difference in sensitivity was due to partial recovery occurring in the eggs prior to fertilization. Judging from insect experiments, dominant lethals are more readily induced in sperm. In fact, P. W. Whiting (1938) found that at dosages of about 10.000 r. practically every Habrobracon sperm contained at least one dominant lethal. Cer- tain of his experiments more closely resemble the conditions of the present experi- ment than any other investigations we have been able to find in the literature. In these experiments, female wasps mated before treatment contained both sperm and eggs when irradiated. Comparisons with data from females mated after irradia- tion, and with parthenogenetic data, indicated dominant lethals to be more readily induced in sperm than even recessive lethals in the eggs. In a definitive Habro- bracon paper, Heidenthal (1945) constructed the dominant lethal mutation curve for doses up to the asymptote and demonstrated that those secured for Drosophila (Sonnenblick, 1940; Demerec and Fano, 1944) were quite similar. An even steeper curve has been reported for Mellitobia (Kerschner, 1946). Muller and the Valencias (1949) have since presented Drosophila data which indicate that presumptive deficiencies are far less abundant if eggs are irradiated. Nucleic acid or its cycle is implicated in both the Arbacia cleavage delay experi- ments and the dominant lethal experiments with insects. Although usually dis- cussed separately, it seems possible that both types of damage may be reflected in the results obtained by scoring development at a specified time. However, it has been shown that neither phenomenon completely explains the present results. Per- haps a third and somewhat different aspect of cell division — other than chromosomal —is involved. The material may need to be considered from the standpoint of the general physiologist who studies the stimulus for initiating the process of cell division (Heilbrunn, 1955; Heilbrunn and Wilson, 1955; Rieser, 1955). Especially provocative is an earlier Arbacia paper (Heilbrunn and Young, 1935), which shows that irradiation in the presence of ovarian tissue produced a 178 DANIEL S. GROSCH AND ZOE H. SMITH considerably greater division delay than irradiation of eggs in sea water alone or in concentrated suspension. At least, sperm inactivation can be ruled out. This requires doses in excess of 100,000 r in marine forms as well as insects (Maxwell, 1938 ; Henshaw, 1940; Rugh, 1953). Self-sterility Another aspect of Molgula investigations which may turn out to be a problem in physiology rather than genetics is fertility-sterility. A range from perfect self- fertility to absolute self-sterility has attracted geneticists to ascidians from the early days of genetics research (Morgan, 1904). However, although T. H. Morgan himself devoted considerable attention to the problem, including experiments with Molgula (1942), neither the genetic basis nor the physiological mechanisms have been completely elucidated. Observations during the present experiments revise the Molgula picture. Self- incompatibility is not as extensive as previously believed, provided (1) that organisms with undeveloped or senile (degenerate?) gonads are not used, and (2) that no strong chemical cleaning solution or detergent is employed in cleaning glassware. In all our experiments in which these criteria were met, Molgula adults were self-fertile. The influence of a chemical agent was demonstrated dramatically one day when Alcanox had been used on the glassware. In spite of repeated water washings, as is the standard procedure in analytical chemistry, and over-night drying, no development occurred in selfing and only about 5% develop- ment in outcrosses. Ordinarily in outcrosses by far the great majority of eggs are fertilized and cleave. Although no details are available, at least one of Morgan's Molgula experiments bears a resemblance to this exceptional one of ours. He re- corded a case in which no eggs selfed and only two out of a large number of eggs cleaved when cross-fertilized. Perhaps hot water is the only safe cleaning agent, although the junior author feels that dilute HC1 rinses do much to offset the Alcanox type of hazard. SUMMARY 1. The lethal dose of x-rays for adult specimens of Molgula is placed in the neighborhood of 45,000 r (delivered at a rate of 6000 r/minute). 2. Radiation damage to gametes from irradiated adults can be measured in terms of tadpoles, unhatched or swimming. Eggs proved more sensitive than sperm. Curves when both gametes come from irradiated parents were similar no matter how obtained, pair matings or group matings, selfed or outcrossed. In the latter curves, 10,000 r is about the limit for swimmers, and 20,000 r for tadpole development (rate, 2500 r/minute). 3. The cleavage score for 100 objects residual to developed tadpoles did not provide a regular, clear-cut picture of radiation damage. It is suspected that un- investigated features of fertilization physiology cloud the issue. 4. Self-incompatibility in Molgula is not as extensive as previously believed. The condition of the gonads must be considered and chemical cleaning solutions should be avoided. 5. It is concluded that the physiology of spindle formation rather than that of nucleination or chromosomal continuity may be a most important aspect of results like the present. X-RAY EXPERIMENTS WITH MOLGULA 179 LITERATURE CITED BACQ, Z. M., AND P. ALEXANDER, 1955. Fundamentals of radiobiology. Academic Press, Inc., N. Y. DEMEREC, M., AND U. FANO, 1944. Frequency of dominant lethals induced by radiation in sperms of Drosophila melanogastcr. Genetics, 29 : 348-360. GROSCH, D. S., AND HOWARD E. ERDMAN, 1955. X-ray effects on adult Artemia. Biol. Bull., 108: 277-282. HEIDENTHAL, G., 1945. The occurrence of X-ray induced dominant lethal mutations in Habro- bracon. Genetics, 30: 197-205. HEILBRUNN, L. V., 1955. The dynamics of living protoplasm. Academic Press, Inc., N. Y. HEILBRUNN, L. V., AND W. L. WILSON, 1955. Changes in the protoplasm during maturation. Biol. Bull, 109 : 271-275. HEILBRUNN, L. V., AND R. A. YOUNG, 1935. Indirect effects of radiation on sea urchin eggs. Biol Bull., 69 : 274-276. HENSHAW, P. S., 1940. Further studies on the action of roentgen rays on the gametes of Arbacia punctulata. III. Fixation of irradiation effect by fertilization in the eggs. Amer. J. Roentgen. Rad. Therapy, 43 : 913-916. KERSCHER, JEAN, 1946. Dominant lethals induced by X-rays in sperm of the wasp Melitobia. Anat. Rec., 96 : 566. MAYOR, J. W., AND D. M. DE FOREST, 1924. The relative susceptibility to X-rays of the eggs and sperm of Arbacia. Proc. Soc. Exp. Biol. Med., 22 : 19-21. MAXWELL, JANE, 1938. Inactivation of sperm by X-radiation in Habrobracon. Biol. Bull., 74 : 253-255. MORGAN, T. H., 1904. Self-fertilization induced by artificial means. /. Exp. Zool., 1 : 135-178. MORGAN, T. H., 1942. Cross- and self-fertilization in the ascidian Molgula manhattensis. Biol Bull, 82 : 172-177. MULLER, H. J., R. M. VALENCIA AND J. I. VALENCIA, 1949. The production of mutations at individual loci in Drosophila by irradiation of oocytes and oogonia. Genetics, 35 : 126. RIESER, P., 1955. The effect of roentgen rays on the colloidal properties of the starfish egg. Biol Bull, 109: 108-112. RUGH, R., 1953. A study of the effects of x-irradiation at different levels on the germ cells of the clam, Spisula (formerly Mactra}. Biol Bull, 104: 197-209. SONNENBLICK, B. P., 1940. Cytology and development of the embryos of x-rayed adult Drosophila melanogaster. Proc. Nat. Acad. Scl, 26: 373-381. SULLIVAN, R. L., AND D. S. GROSCH, 1953. The radiation tolerance of an adult wasp. Nucle- onics, 11: No. 3: 21-23. WHITING, P. W., 1938. The induction of dominant and recessive lethals by radiation in Habro- bracon. Genetics, 23 : 562-572. ESTROGENS IN MARINE INVERTEBRATES DWAIX D. HAGERMAN.i FREDERICA M. WELLINGTON AND CLAUDE A. VILLEE Marine Biological Laboratory, Woods Hole, Mass., Department of Biological Chemistry, Harvard Medical School, and Research Laboratories, Boston Lying-in Hospital, Boston, Mass. Material with estrogenic activity demonstrable by bioassay in rodents has been found in marine invertebrate tissues. Steidle (1930), using a mouse bioassay, found traces of estrogens in a sea urchin, Echinus iniliaris, three molluscs, Aplysia, Octopus and Eledone, as well as in certain worms and arthropods. Similarly Schwerdtfeger (1932) found estrogens in a sea anemone, Actinia aquina, but none in the mollusc Chiton marginatus. Donahue (1940) reported small amounts of estrogenically active material to be present in extracts of the Bermuda urchin, Lyt echinus varicgatus, the reef urchin, Echinometria, a holothurian, Stichopns mobii, and a lobster, Palinurus argus. More recently, this same author (Donahue, 1948) made extracts of the shed eggs of another lobster, Homarus americanus, purified them by solvent partition, and made estrogen analyses by a fluorometric method. In this way he found 100 international units of estrogen per 100 gm. eggs. Gordon and Villee (1956) have described an enzymatic assay for estrogens, wrhich is as sensitive as the fluorometric methods now available. Their assay depends upon the fact that human placenta contains a DPN(diphosphopyridine nucleotide) -linked isocitric dehydrogenase which catalyzes the reaction. isocitrate + DPN ^ a-ketoglutarate + DPNH + CO2 and which is specifically activated by certain natural estrogens. In a limited range the degree of enzyme activation is a function of the amount of hormone present. Since they measured the appearance of reduced DPN spectrophotometrically, it was essential that the material being assayed give a clear solution, and for that reason they found it impossible to analyze blood and tissue extracts. The progress of the reaction can also be measured chemically, by analyzing the reaction mixture after incubation for total keto-acids produced, and in this way relatively impure tissue extracts can be assayed. Such assays clone on the ovaries of a number of .marine invertebrates are reported here. METHODS Invertebrate tissues were extracted for assay by the procedure used by Folch et al. (1954) for preparing total lipide extracts. The tissue was removed from the animal, blotted gently with filter paper, weighed accurately and homogenized in a Waring blendor containing 20 ml. 2 :1 chloroform :methanol per gram of tissue. The homogenate was filtered and the residue discarded. The filtrate was washed 1 Lalor Fellow, Marine Biological Laboratory, 1956. 180 ESTROGENS IN MARINE INVERTEBRATES 181 once with 0.2 volume of distilled water and once with 0.2 volume of 0.01 % aqueous calcium chloride which had previously been equilibrated with 2:1 chloroform :meth- anol. The extract was evaporated to dryness at room temperature under a gentle stream of clean air. The residue was taken up in ethanol for analysis and if, after thorough mixing, the solids did not go completely into solution, they were allowed to settle and the clear alcoholic extract was used for assay. A 20% w/v homogenate of term human placenta was made in ice-cold 0.25 M sucrose, and centrifuged for 10 minutes at 600 G to remove cell nuclei and debris. The supernatant was then centrifuged at 57,000 G for 60 minutes to remove cellular participate matter (Villee, 1955). The supernatant from the latter centrifugation contains the soluble matter of the cell, including the DPN-linked isocitric dehydro- genase. The enzyme-catalyzed reaction was carried out in 20-ml. beakers in a Dubnoff incubator at 37°, and was allowed to proceed for one hour. Each reaction vessel contained one ml. of the placental enzyme preparation ; one ml. buffer con- taining 30 micromoles K+, 10 micromoles Mg++, 20 micromoles phosphate, and 20 micromoles Cl~, adjusted to pH 7.3 ; 0.9 ml. water containing 0.75 micromole DPN and 3 micromoles cis-aconitate ; and 0.1 ml. ethanol in which the standard or unknown solution was dissolved. The reaction was started by adding the DPN. Crystalline estradiol- 17/? was used as a standard. Analyses of the reaction mixture for total keto-acid production were made by a modification of the method of Friedemann and Haugen (1943). The amount of keto-acid produced in vessels containing all components except estradiol was used as a blank correction for the standard, and that produced in vessels containing all components except DPN was used as a blank correction for the unknowns. Total nitrogen analyses of the enzyme preparation were made by a micro-Kjeldahl procedure. The keto-acid analysis results were calculated in micromoles keto-acid produced per milligram nitrogen per hour. Duplicate reaction vessels were incubated and analyzed for each of the standards and unknowns in each assay. RESULTS \ Separate standards were assayed with each set of unknowns, and the average results from 12 sets of standards are plotted in Figure 1. The least squares curve for these points is also shown. There is clearly a linear relationship between the logarithm of the amount of estradiol added and the amount of keto-acid produced over the range from 0.05 to 0.25 microgram estradiol per vessel. The index of precision, (A), for this curve is 0.3 (Bliss, 1944). Recoveries of estradiol added to tissue before extraction averaged 99%. The amount of estrogen in each unknowrn was calculated from a simultaneously determined standard curve for the same placental enzyme preparation. Each tissue extract was assayed with at least three different enzyme preparations from three different placentas. Of the tissues extracted, only the ovaries of Mactra (Spisula] solidissima contained significant amounts of estrogenic material, 1.1 ± 0.4 (mean ± standard error) milligrams estradiol equivalents per kilogram of fresh tissue. The other tissues, including the ovaries of Asterias forbesi, Arbacia punctulata, Strongylocentrotus drocbachiensis, Loligo pcalei, Busycon canalicula- tum, Carcinides inaenas, Homarus americanus, and Limn! us polyphemus, the whole tissue of Microciona prolijera and the liver of Homarus americanus all con- tained less than 50 micrograms estradiol equivalents per kilogram of fresh tissue. 182 HAGERMAN, WELLINGTON AND VILLEE 0.3 UJ o o O a: x 0.2 o o o o cr a. o o i o ui O.I 0.05 O.I 0.25 ESTRADIOL CONCENTR ATI ON, JLlGM./RE ACTION VESSEL FIGURE 1. Keto-acid production by placental isocitric dehydrogenase as a function of estradiol-17/3 concentration. DISCUSSION The results were calculated in terms of estradiol equivalents, since that com- pound was used as a standard. It has been shown (Villee and Gordon, 1956) that estrone, equilenin and equilin are as effective as estradiol in stimulating the enzyme, while a variety of other steroids and synthetic compounds do not affect the enzyme unless present in very large quantities. It is thus likely that the material present in Mactra ovary is one of these four compounds. The minimum amount of estrogen that can be detected in this assay is about 0.01 microgram of estradiol, and the tissue concentration that can be accurately determined depends, of course, on the amount of tissue extracted. With samples of tissue of about 10 grams, the analytical blanks are not unduly large and this mag- nitude of sample was used in these experiments. In all the tissues examined except Mactra ovary, the results of the assays indicated the presence of small amounts of active material, confirming the experience of earlier workers. The amounts are probably much smaller than the upper limit of 50 micrograms per kilogram which the present results make certain, but it would be necessary to extract larger quantities of tissue to make accurate estimates of the true amount present. The precision of which this assay method is capable depends on the same factors as any other bioassay, the index of precision of the standard curve and the number ESTROGENS IN MARINE INVERTEBRATES 183 of replicates which are made. The former is satisfactory and the effort expended in making many replicates by this method is less than with many other forms of bioassay. Even with the small number of assays that were done in the present work on the Mactra ovary extracts, one can be quite confident that this tissue contains a very large amount of estrogenic material, presumably estradiol or a closely related compound. One milligram per kilogram is ten times as much as the maximum previously reported (lobster eggs) for any species. Estradiol added in vitro has no effect on the metabolism of Mactra ovary (Hagerman, 1956) and apparently no physiologic effects of estrogens on molluscs have been described. The material may be present as an evolutionary freak, similar to the occurrence of uric acid as an excretory product in the Dalmatian coach hound, or may have some important physiologic function in this mollusc. Further speculation should await the isolation and complete chemical characterization of the material present in Mactra. SUMMARY 1 . An enzymatic method of assay for estrogens suitable for use with crude tissue extracts is described. 2. Of a variety of marine invertebrates examined, only the ovaries of the mollusc, Mactra (Spisula} solidissima, contained appreciable amounts of estro- genic material. LITERATURE CITED BLISS, C. I., 1944. Relative potency as applied to the assay of penicillin. Science, 100 : 577-578. DONAHUE, J. K., 1940. Occurrence of estrogens in the ovaries of certain marine invertebrates. Endocrinol, 27 : 149-152. DONAHUE, J. K., 1948. Fluorimetric and biological determination of estrogens in the eggs of the American lobster (Homarus americanus) . Proc. Soc. Exp. Biol. Med., 69: 179-181. FOLCH, J., M. LEES AND G. H. SLOANE-STANLEY, 1954. A simple method for preparation of total pure lipide extracts from brain. Fed. Proc., 13 : 209. FRIEDEMANN, T. E., AND G. E. HAUGEN, 1943. Pyruvic acid. II. The determination of keto- acids in blood and urine. /. Biol. Chem., 147: 415-442. GORDON, E. E., AND C. A. VILLEE, 1956. An in vitro assay for estradiol -17/3 and estrone. Endocrinol., 58: 150-157. HAGERMAN, D. D., 1956. Invertebrate metabolism in vitro not affected by estradiol. Biol. Bull, 111: 318-319. SCHWERDTFEGER, H., 1932. Bcitrage zum Vorkomen und zur Wirkung der weiblichen Sexual- hormone. Arch. f. Exp. Path, und Pharm., 163 : 487-492. STEIDLE, H., 1930. liber die Verbreitung des weiblichen Sexualhormons. Arch. f. Exp. Path. und Pharm., 157 : 89. VILLEE, C. A., 1955. An estradiol-induced stimulation of citrate utilization by placenta. /. Biol. Chem., 215: 171-182. VILLEE, C. A., AND E. E. GORDON, 1956. The stimulation by estrogens of a DPN-linked isocitric dehydrogenase from human placenta. Bull. Soc. Chim. Belg., 65: 186-201. THE EFFECTS OF X-IRRADIATION ON THE FERTILIZED EGGS OF THE ANNELID, CHAETOPTERUS * CATHERINE HENLEY AND DONALD P. COSTELLO Marine Biological Laboratory, Woods Hole, Mass., and Department of Zoology, University of North Carolina, Chapel Hill, N. C. The study of ionizing radiations and their effects on living systems has been of interest to biologists for many years. An important manifestation of such effects is evident in the mitosis of cells or tissues exposed to radiation, and excellent ex- perimental material for the analysis of cytological and developmental consequences of such exposure is available in the eggs of a number of marine invertebrate animals. One of the more favorable forms is the polychaete annelid, Chaetopterus pergatnent- aceus. The eggs of this form, although not transparent, are characterized by moderate amounts of yolk, so that it has been possible to make whole-mount prepa- rations of the irradiated and control eggs which show pertinent cytological detail clearly. Furthermore, the normal development of Chaetopterus has been studied by Mead (1898) and Lillie (1906), and there are available time-tables of develop- ment for given temperatures, so that irradiation can be begun at a known stage of mitosis, and subsequent deviations from the normal schedule of events studied in considerable detail. MATERIALS AND METHODS Ripe Chaetopterus males and females were collected by the M. B. L. Supply Department, and maintained in the laboratory in separate large fingerbowls supplied with running sea water. Eggs were obtained by clipping off two or three posterior parapodia from a single female ; these parapodia were transferred to pieces of cheesecloth wet with filtered sea water, through which the eggs passed into 100 cc. of filtered sea water contained in each of two (control and experimental) plastic boxes, Sy^" X 2%" X li/4" in size and provided with lids. The eggs were allowed to stand undisturbed for 15 minutes; during that period, the first maturation division proceeds to the metaphase (Lillie, 1902). Sperm were obtained by placing one parapodium from a male in 100 cc. of filtered sea water, 15 minutes before they were to be used for insemination. Immediately after insemination, the lids were placed on both boxes of egg sus- pension, and exactly thirty minutes after insemination, the experimental eggs were irradiated by the Laboratory x-ray technician, Mr. Alan P. Brockway. Samples from both control and experimental groups were examined at intervals with a binoc- ular dissecting microscope, to ascertain the progress of cleavage and later develop- ment. After irradiation, the contents of control and experimental boxes were transferred to two small fingerbowls, each containing an additional 100 cc. of 1 Work done under A. E. C. Contract AT- (40-1) -1085, and under a grant from the National Institutes of Health, RG-3907. 184 X-IRRADIATION OF CHAETOPTERUS EGG 185 filtered sea water; both fingerbowls were then kept on the sea water table, with running sea water flowing around them. The room air temperature varied from 21 to 25° C. Control and experimental cultures were examined for the final time 19 to 22 hours after insemination, when the controls were vigorously swimming trochophores. The x-ray generator used operates simultaneously two Coolidge tubes at 25 ma and 182 KVP, with an inherent filtration of 0.2 mm. copper. The plastic box (from which the lid was first removed) containing the experimental eggs was thus "cross-fired" between two x-ray beams. Since the eggs were distributed in an even layer on the bottom of the box, a fairly uniform field of irradiation was possible. The x-rays were delivered at two different rates. For total dosages below 3570 r, the machine was calibrated at 510 r per minute with the two tubes 48 cm. apart, the bottom of the Lucite box being approximately equidistant between the two tubes. For dosages above 3570 r, the x-rays were delivered at a rate of 2160 r per minute, with the tubes 16 cm. from one another. There was no appre- ciable rise in temperature during any of the irradiation treatments, and artificial cooling measures were therefore not used. The duration of treatment varied from one-half minute to eight minutes, and the dosages used were 255 r to 17,280 r Cytological preparations were made of samples from the control and experi- mental cultures, at times calculated to result in fixation of the eggs at the metaphase or anaphase of the first three cleavages. A modified squash whole-mount tech- nique was devised, which yielded excellent preparations in a minimum of time. The method involved fixation of the eggs in Kahle's fluid 2 (water, 30 parts; 95% alcohol. 16 parts; formalin, 8 parts; glacial acetic acid, 1 part) on cover-slips. A single drop of concentrated egg suspension was placed on one clean No. 1 square cover-slip, and a drop of fixative on a second cover-slip ; the second cover-slip (with the fixative) was inverted and dropped face-to-face onto the first cover-slip. No pressure was applied, and if drops of the correct size were used there was no distortion of the eggs. After about five minutes, the two cover-slips (still face- to-face) were transferred as a unit to a small staining jar containing fresh fixative, where they were kept for 15 to 30 minutes. During this period, the cover-slips were carefully separated from one another with watchmaker's forceps; approxi- mately one-half the eggs adhered to each of the two cover-slips, and very few were lost during the process of separation. After separation, the cover-slips were placed in porcelain racks, Chen Type A, provided with wire handles, and subsequent steps were carried out by transferring the racks to tall Stender dishes containing the necessary reagents. Hydration of the eggs through 70% alcohol, 50% alcohol and distilled water was followed by staining for four to six minutes in a solution of Harris' acid haematoxylin, diluted 1 :4 or 1 :5 with distilled water and filtered before each use. No counterstain was used. After staining, the eggs were "blued" in several changes of tap water alkalinized with 1% sodium bicarbonate solution, and dehydrated in three changes of triethyl phosphate,2 in carbol-xylol and in two changes of xylol; they were mounted in damar. The preparations were studied at magnifications of 1 50 X , 300 X and 660 X , using a Spencer compound binocular microscope with compensating oculars. Approximately 440 slides were prepared and studied. 3 We are indebted to Dr. Anna R. Whiting for suggesting the use of these reagents. 186 CATHERINE HENLEY AND DONALD P. COSTELLO RESULTS The results are summarized in Table I. It is apparent that relatively low doses of x-rays (255 to 1020 r) had no im- mediately obvious effects on fertilized Chaetopterus eggs. There was a very slight retardation (3-5 minutes) of the first three cleavages in the experimental eggs after 765 r, as compared with the control eggs, but no other visible gross or cytologi- cal effects. However, when the control and experimental cultures were examined 19 to 22 hours after insemination, the trochophores were very abnormal in cultures which had received 500 and 765 r, respectively. They were, for the most part, very disorganized masses, with marked ciliary defects ; they moved feebly, if at all. Almost all the larvae were dead in the groups which had received 1020 r, and dis- integration of the undifferentiated cytoplasm had occurred. In most of the experiments where the eggs were treated with 1275 r and higher dosages, there was at least a slight retardation (5-10 minutes) of the first three cleavages, and the majority of the embryos were dead 19 hours after insemination. The few surviving trochophores were very abnormal, with ciliary defects, cyto- plasmic blebs, etc. The occurrence of cleavage retardation reported here is in accordance with the results obtained by other workers. Thus, Packard (1918) observed delays in the division of Chaetopterus eggs irradiated with gamma rays from radium. Cook (1939) demonstrated a two- to five-hour delay in the first cleavage of Ascaris eggs x-irradiated at the one-cell stage. Henshaw (1940a, 1940b) and Henshaw and Cohen (1940) described marked cleavage retardation in Arbacia eggs, after x-irradiation of eggs, or sperm, or both gametes. Carlson (1938) described a cessation of mitosis in grasshopper neuroblasts treated with varying doses of x-rays (100-1000 r). The recovery time for return of mitosis (anaphases being used as the criterion) varied from three hours after 100 r to 22 hours after 1000 r. There must be a considerable difference in the radio-sensitivity of Chaetopterus eggs, as opposed to grasshopper neuroblasts, since complete inhibition or even pronounced delay of mitosis in our experiments required much higher dosages. X-ray dosages below 8640 r did not result in any obvious cytological damage to eggs fixed at the times of the first three cleavages ; at and above that dose level, however, there were often multipolar spindles, chromosome bridges at anaphase, and fragmentation, particularly in eggs fixed at the time of the third cleavage. These were very similar to the aberrations described by Costello, Henley and Kent (1952) in P32-treated fertilized Chaetopterus eggs. The chromosomes in the Chaetopterus egg are small, and the detection of minute cytological abnormalities is not always possible. Eggs treated with 15,120 r presented a striking cytological picture when they were fixed at the time of the second and third cleavages. At least some degree of karyokinesis had apparently taken place, but it was not accompanied by cytokinesis, so that there were often several interphase nuclei present in one undivided or in- completely divided mass of cytoplasm. 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COSTELLO cytoplasmic division, or with incomplete cytoplasmic division, is perhaps the most striking effect noted after treatment of these eggs with high dosages of x-rays. X-irradiation with 17,280 r resulted in marked effects (both gross and cyto- logical) on the first three cleavages. The division to two cells was very abnormal and noticeably retarded (5-7 minutes), and the two subsequent divisions were almost completely inhibited. There were some two- and three-cell stages present in the samples fixed at the times of second and third cleavage in the controls ; the stages fixed 75 minutes after insemination often had two interphase nuclei in each of two cells, again indicating a suppression of cytokinesis at an earlier stage. The three-cell stages (which are not normally found in the cleavage of Chaetopterus, although the polar lobe may simulate a third cell) usually had only one interphase nucleus in each of the three cells. It appeared that cytoplasmic division had taken place only in the smaller AB blastomere at the second cleavage, the larger CD blastomere remaining undivided. The possible significance of this observation will be discussed below. DISCUSSION Sensitivity of prophase chromosomes to irradiation The design of our experiments was such that treatment was begun at prophase of the first cleavage, a time which appears, according to the findings of many ob- servers utilizing a variety of materials, to be very susceptible to radiation. This fact, coupled with the circumstance that division in Chaetopterus eggs is predictable and synchronous (within certain limits imposed by temperature and other environ- mental factors), makes the material favorable for studies of radiation effects. One of the early reports on the susceptibility of prophase chromatin to irradia- tion came from Strangeways and Hopwood (1926), who irradiated chick tissue cultures with x-rays and found the most sensitive period to be at the time im- mediately before the onset of visible prophase. Sax (1938, 1943) reported the greatest frequency of x-ray-induced chromosome aberrations in Tradescantia microspores which were at the meiotic or mitotic prophase at the time of treatment. Luther (1938) x-irradiated frog eggs, and concluded that the maximum suscepti- bility was during prophase, the minimum susceptibility at metaphase. The findings of Marshak (1939) are somewhat at variance with the general observation that prophase is the stage of greatest sensitivity; x-irradiated onion seedlings, pre- treated with dilute solutions of ammonia, were examined cytologically and the results interpreted to indicate that the chromosomes were most sensitive at the resting stage. This was held to be the case regardless of whether or not ammonia pre-treatment was used. Henshaw and Cohen (1940) x-irradiated fertilized Arbacia eggs, and found that when cleavage retardation was the criterion, the time of greatest susceptibility was during the period after the male and female pronuclei had come together, and during early prophase. The late prophase stage, immediately before breakdown of the nuclear membrane, was found by Carlson (1941) to be the most sensitive period for grasshopper neuroblast cells, and Swanson (1942) made similar observations on Tradescantia pollen tube chromosomes. More recently, Bloom, Zirkle and Uretz (1955) utilized microbeams of protons and ultraviolet light for irradiation of individual chromosomes and parts of chromo- X-IRRADIATION OF CHAETOPTERUS EGG 189 somes, in cultures of Triturus heart tissue. They found that a given dose at the prophase stage produced "sticky" chromosomes and chromosome fragmentation; the same radiation to metaphase chromosomes resulted, again, in sticky chromo- somes but very few akinetic chromosomes or fragments were observed. Their findings are of great interest, not only because of the highly localized character of the radiation used, but also because they were able to follow the fate of a single irradiated chromosome in considerable and exact detail by the use of phase optics and time-lapse motion pictures. When Chaetopterus eggs are x-irradiated beginning 30 minutes after insemina- tion, both polar bodies have usually been given off (except in a few of the experi- ments performed early in the breeding season, when low temperatures slowed down all features of development of the egg). At 21° C, according to Heilbrunn and Wilson (1948), the male and female pronuclei have approached one another and are fusing, and by 40 minutes after insemination, the fusion nucleus is at the prophase of the first cleavage. This time-table of events proceeds at a considerably faster rate when the temperature is increased, and our data indicate that for most of the experiments reported here, the eggs were at the early prophase stage when irradiation was begun. Possible effects of irradiation on early development of the eggs In the ovary of the Chaetopterus female, a definite polarization of the egg is evident, the future animal pole being free in the lumen of the ovarian tubule, and the future vegetal pole being attached to the wall of the tubule (Lillie, 1906). Thus, the subsequent position of the polar bodies is "set" very early in develop- ment. The cleavage spindle is formed by the separation of the two sperm cen- trosomes (Mead, 1898), in a position in the egg which is determined by the position of the male pronucleus. This, in turn, is determined by the polarity of the egg (the sperm nearly always entering in the vegetal hemisphere), so that there is an orderly chain of events, each one of which is predicated on the original polarity of the egg. Lillie (1906) described the separation of large basophilic granules from the chromosomes of the first cleavage in Chaetopterus. The subsequent division of the egg segregates all these granules into the CD blastomere, none being left in the AB cell. X-irradiation with 17,280 r at prophase resulted, as noted above, in a failure of the CD blastomere to divide at the second or at the third cleavage, so that there appears to have been a drastic effect on the distribution of these granules, as well as on other cytoplasmic movements. Indeed, irradiation of eggs at the prophase of first cleavage could very well have other far-reaching effects, quite apart from those on the chromosomes. The in- fluence of x-irradiation on viscosity changes during mitosis of the Arbacia egg has been described by Wilson (1950). In unirradiated eggs, there is a marked increase in viscosity which reaches a value three or four times that of the unfertilized eggs at 15 minutes after insemination; this increased viscosity persists for a few minutes, and then drops to almost the level of viscosity of unfertilized eggs shortly before the first cleavage. Approximately the same magnitude of increase was observed in eggs irradiated and then inseminated, but it remained high for a period two to three times that of the control eggs, eventually decreasing shortly before 190 CATHERINE HENLEY AND DONALD P. COSTELLO cleavage. If viscosity changes occur in the fertilized Chaetopterus egg irradiated after insemination, they would be expected to have profound effects on the complex patterns of ooplasmic segregation characteristic of this form. Lillie (1906) demon- strated that there was an exact correlation between the distribution of ectoplasmic spherules (which, before the breakdown of the germinal vesicle, are distributed over the animal two-thirds of the egg, and which come to overflow the vegetal hemisphere after germinal vesicle breakdown) and the distribution of cilia in the trochophore larvae. The cilia apparently do not develop directly from the spher- ules, however. The fact that the later stages of development in our experimental cultures were almost invariably marked by abnormalities of cilia distribution (if, indeed, the embryos survived to the stage of cilia formation at all) indicates that the irradiation directly or indirectly affected some part of the cilia-producing mechanism. The phenomenon of polar lobe formation, which is characteristic of the eggs of certain annelids (including Chaetopterus) and molluscs, is a striking indication of the profound changes which occur in the cytoplasm of these eggs within the first few hours after fertilization. Although we observed no visible evidence of malformation of the polar lobe in the irradiated eggs, it is quite possible that there were inconspicuous disturbances in the apportionment of cytoplasmic materials to this structure. It is of interest in this connection to point out that even after 17,280 r, polar lobe formation immediately before the first cleavage was morpho- logically normal, although delayed. Formation of the second polar lobe after this dosage appears to have been completely suppressed. In any event, the deleterious effects of x-ray treatment on later development are, per se, an indication that irradiation interfered with early processes of differentia- tion. The possible role of cytoplasmic effects in radiation damage has been considered in the recent paper by Ord and Danielli (1956), in which they transferred x- irradiated Amoeba proteus nuclei to non-irradiated cytoplasm of the same form. The resulting organisms had a somewhat lower percentage of survival than did intact irradiated amoebae. When non-irradiated nuclei were transfered to irradi- ated cytoplasm, 18-48 hours after irradiation, the animals survived despite the fact that they had received very high doses of x-rays ; they were able to form new clones, although the time of first division was somewhat delayed. If the transfers of normal nuclei to irradiated cytoplasm were done sooner than 18 hours after treatment, however, the normal nuclei were apparently lethally damaged by the irradiated cytoplasm in most cases. Ord and Danielli point out that in this study, nuclear damage appears to be a direct result of x-ray damage to the nuclei, and an indirect result of contact with damaged cytoplasm. They suggest that perhaps the importance of nuclear damage from radiation, as opposed to cytoplasmic damage, may have been somewhat over-emphasized. Chromosome aberrations There have been many reports in the literature of chromosome aberrations which occur after irradiation from various sources. An early one was the paper by Packard (1918) ; he irradiated unfertilized Chaetopterus eggs with gamma rays from a radium source and observed multipolar spindles at the first cleavage, after X-IRRADIATION OF CHAETOPTERUS EGG 191 the treated eggs had been inseminated. Packard interpreted these multipolar spindles as being due to polyspermy, which he felt was facilitated in some way by the irradiation. Alberti and Politzer (1924a, 1924b) described and figured a variety of cytological abnormalities in the corneal epithelium of larval salamanders which had been x-irradiated ; among these anomalies were chromatin bridges at anaphase, telophase and interphase, akinetic fragments and chromosomes, accessory nuclei, multiple akinetic chromosomes, and multipolar spindles. Similar effects were described by Strangeways and Hopwood (1926) in x-irradiated chick tissue cultures, and by Sonnenblick (1940) in the embryos obtained after mating x-irradi- ated male and female Drosophila adults. Laznitzki (1943) also x-irradiated chick tissue cultures, and described a number of cytological abnormalities resulting from treatment; however, this author emphasizes the fact that no multipolar spindles were observed in irradiated material, which is in contrast to the findings of other investigators. Zirkle and Bloom (1953), utilizing proton bombardment of parts of amphibian heart cells in tissue culture, reported chromosome bridges, inhibition of cytokinesis, and unequal distribution of the daughter chromosomes. Chromo- some fragmentation after irradiation has been described by Carlson (1938) for grasshopper neuroblast cells, by Faberge (1940) for Tradescantia pollen grains, by Bishop (1942) for grasshopper spermatocytes, by Whiting and Murphy (1956) for Habrobracon oocytes, and by Lesley and Lesley (1956) in plants from treated tomato seeds. We have observed many of the aberrations described above in our material, especially after the higher doses of x-rays. Henshaw (1940d) studied the multipolar spindles which occurred in fertilized Arbacia eggs after x-irradiation ; it is interesting that in contrast to our findings (where multipolar spindles clearly attributable to irradiation were observed no sooner than the third cleavage), he described such effects at the first cleavage. This difference may be due to the fact that Henshaw used considerably higher doses (31,200 r) than were employed in the present study. In the same paper (1940d), he found no evidence of nuclear division without accompanying cytoplasmic division at the first cleavage in his treated eggs. We found karyokinesis without cytokinesis only at the second and third cleavages, which apparently were not studied by Henshaw. Recently, Bloom, Zirkle and Uretz (1955) irradiated parts of chromosomes of amphibian heart cells in tissue culture, using microbeams of protons or ultraviolet. Irradiation of the kinetochore region resulted in "drifting" of the chromosome until anaphase when it was incorporated as a lobe on the daughter nucleus, or be- came a small accessory nucleus. Chromosome "stickiness" and fragmentation were also reported. Chromosomes treated with beams of protons or ultraviolet outside the kinetochore region showed no such effects. Bombardment of extra-chromo- somal areas of the cells (cytoplasm and the ends of spindles) with relatively large numbers of protons produced no effects at the site of irradiation. Heterochromatic ultraviolet irradiation of the same regions, however, resulted in a disappearance of the spindle and derangement of the characteristic metaphase configuration ; a "false anaphase" followed, in which chromosomes, rather than chromatids, moved apart. It is of interest that many of the chromosome aberrations typical of radiation damage are produced also by a variety of other agents including, for example, low temperature (Callan, 1942; Book, 1945; Henley, 1950; Henley and Costello, 1949) ; 192 CATHERINE HENLEY AND DONALD P. COSTELLO high temperature (Briggs, 1947) ; colchicine (Callan, 1942) ; and ribonuclease (Kaufmann, MacDonald and Bernstein, 1955). Androgenesis after irradiation There has been considerable discussion in the literature as to the possibility of androgenesis occurring in fertilized irradiated eggs, as a consequence of irreparable damage to the egg chromatin, so that development proceeds with only the haploid, paternal complement of chromosomes. Packard (1918) reported that in Chaetop- terus eggs which had received heavy doses of gamma radiation before insemination, the female pronucleus remained in a polar position, often attached to the polar body material by a fine cytoplasmic strand in which chromatin threads can be dis- tinguished. Cleavage took place in such eggs in an orderly fashion, however, and the haploid number of chromosomes was present. Whiting (1948, 1955) found haploid androgenetic males in Habrobracon. They developed only from eggs x- rayed in first meiotic metaphase, thereby resembling Packard's results in Chae- topterus. Henshaw (1940d), on the other hand, reported that both pronuclei participated in the development of irradiated Arbacia eggs, even after heavy doses of x-rays. However, Arbacia eggs are fully mature, with the female pronucleus present, when in the fertilizable condition, whereas Chaetopterus eggs are at the metaphase of the first maturation division. Whiting (1955) studied Feulgen preparations of Chaetopterus eggs irradiated (during metaphase I) with 60,000 r and fertilized 3% minutes later with untreated sperm. The majority underwent continued cleavage, and of these 26% appeared to have sperm chromosomes, only, and were therefore androgenetic. Whenever chromatin abnormalities appeared in cleaving cells, there were more than nine chromosomes (the haploid number) present, and in all cells in which there were nine chromosomes, only, no aberrations were visible. Whiting therefore found no evidence of an injurious effect of irradiated cytoplasm upon untreated chromo- somes. In the experiments reported in the present paper, apposition of the pronuclei occurred normally in irradiated eggs, and the diploid number of chromo- somes appeared to be present. Since the eggs had been inseminated before irradia- tion, the occurrence of normal fertilization implies that no damage was suffered by the sperm, either directly or as a consequence of its passage through irradiated egg cytoplasm. The effects of irradiation on later development Lea (1955) points out that the death of a cell as a result of irradiation usually does not occur immediately but at, or following, the next division of the cell ; an immediate lethal effect requires much larger doses of radiation than a delayed lethal effect. The results obtained in the present study confirm this general state- ment, and are in accordance with the findings of other investigators. Cook (1939). for example, x-irradiated Ascaris eggs at the one-cell stage and obtained highly abnormal embryos, consisting of unorganized masses of cells. Sonnenblick (1940) reported the occurrence of undifferentiated non-viable masses of cells among the progency of adult fruit flies which had been treated with x-rays. Henshaw ( 1940c) found that x-irradiation of Arbacia eggs with 14,400-28,800 r resulted in the X-IRRADIATION OF CHAETOPTERUS EGG 193 appearance of a wide gradation of effects at the pluteus stage, ranging from shorten- ing of the skeletal arms to the formation of a disorganized mass of cytoplasm which subsequently disintegrated. Giese (1946) treated Chaetopterus sperm with 8000- 16,000 ergs/mm2 of ultraviolet; when such sperm were used to inseminate normal eggs, death ensued, at a stage which is not specified. Even after x-ray doses as low as 255 r in our experiments, there was some mortality in Chaetopterus trochophores examined approximately 22 hours after insemination. Above that dose level, the larvae were very abnormal or, more commonly, dead. This delayed action was especially striking after some of the lower doses of x-rays, where no particular gross or cytological effects (except for slight cleavage retardation) were discernible at earlier stages after treatment. SUMMARY 1. Fertilized eggs of Chaetopterus were x-irradiated, beginning 30 minutes after insemination; doses from 255 r to 17,280 r were used, and the duration of treatment was one-half minute to eight minutes. Observations were made of both living and fixed eggs, at various intervals after irradiation, and of living trochophore larvae 19-22 hours after irradiation. 2. The principal effects of relatively low doses (255-1020 r) were found to be a slight retardation of cleavage (3-5 minutes), and the production of abnormal trochophore larvae which were characterized by severe ciliary defects, cytoplasmic blebs, and very feeble movements (following doses of 255 to 765 r). Doses of 1020 r and above resulted in death of most of the larvae by the trochophore stage. The majority of the eggs irradiated with 1275 r and above showed at least a slight retardation of the first three cleavages. 3. Among the cytological abnormalities observed (especially after the higher doses) were multipolar spindles, chromosome fragmentation, and karyokinesis without cytokinesis. LITERATURE CITED ALBERTI, W., AND G. POLITZER, 1924a. Uber den Einfluss der Rontgenstrahlen auf die Zellteil- ung. Arch. Mikroskop. Anat. u. Entw., 100: 83-109. ALBERTI, W., AND G. POLITZER, 1924b. Uber den Einfluss der Rontgenstrahlen auf die Zellteil- ung. II. Mitteilung. Arch. Mikroskop. Anat. u. 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LUTHER, W., 1938. Radiosensitivity and cell division. Strahlcntherapic, 62 : 436. (Abstract in Radiology, 32: 249.) MARSHAK, A., 1939. The stage of mitosis at which chromosomes are rendered less sensitive to X-rays by ammonia. Proc. Soc. Exp. Biol. and Med., 39 : 194-198. MEAD, A. D., 1898. The origin and behavior of the centrosomes in the annelid egg. /. Morph., 14: 181-218. ORD, M. J., AND J. F. DANIELLI, 1956. The site of damage in amoebae exposed to X-rays. Quart. J. Micro. Sci., 97 : 29-37. PACKARD, C., 1918. The effect of radium radiations on the development of Chaetopterus. Biol. Bull, 35 : 50-70. SAX, K., 1938. Chromosome aberrations induced by X-rays. Genetics, 23 : 494-516. SAX, K., 1943. The effect of centrifuging upon the production of x-ray induced chromosomal aberrations. P. N. A. S., 29: 18-21. SONNENBLICK, B. P., 1940. Cytology and development of the embryos of x-rayed adult Drosophila melanogaster. P. N. A. S., 26: 373-381. STRANGEWAYS, T. S. P., AND F. L. HOPWOOD, 1926. The effects of X-rays upon mitotic division in tissue cultures in vitro. Proc. Roy. Soc. London, Ser. B, 100 : 283-293. X-IRRADIATION OF CHAETOPTERUS EGG 195 SWANSON, C. P., 1942. The effects of ultraviolet and x-ray treatment on the pollen tube chromosomes of Tradescantia. Genetics, 27 : 491-503. WHITING, A. R., 1948. Incidence and origin of androgenetic males in X-rayed Habrobracon eggs. Biol. Bull, 95 : 354-360. WHITING, A. R., 1955. Androgenesis as evidence for the nature of X-ray induced injury. Radiation Research, 2 : 71-78. WHITING, A. R., AND W. E. MURPHY, 1956. Differences in response of irradiated eggs and spermatozoa of Habrobracon to anoxia. /. Genetics, 54 : 297-303. WILSON, W. L., 1950. The effect of Roentgen rays on protoplasmic viscosity changes during mitosis. Protoplasma, 39: 305-317. ZIRKLE, R. E., AND W. BLOOM, 1953. Irradiation of parts of individual cells. Science 117: 487-493. THE TAXONOMY OF UNARMORED DINOPHYCEAE OF SHALLOW EMBAYMENTS ON CAPE COD, MASSACHUSETTS x EDWARD M. HULBURT Woods Hole Oceanographic Institution, Woods Hole, Mass. Several papers give brief accounts of unarmored Dinophyceae found along the eastern coast of the United States. Calkins (1902) described from Woods Hole three European species, with one as a new variety. Herdman (1924a) listed five European, sand-living species from Woods Hole. Lackey (1936) listed thirteen species, all European, in his account of Woods Hole protozoa. Martin (1929) described thirteen species, four of which were new, from Barnegat Bay. It would be expected that further study would show many more extensions of range from the east to the west side of the Atlantic. One wonders, though, whether new species would be few, as suggested by these figures, or on the contrary would be many, since studies hitherto have not been very detailed. Further, the Barnegat Bay list suggests that the shallow, estuarine type of habitat has as many species as coastal waters, since Martin's number is matched only by that of Lackey. The following study covers twenty-six species, of which twelve are completely ANTERIOR END _ _ APEX EPICONE. FLAOELLAR CHAMBER INTERCINGULAR REG-ION HVPOCONE POSTERIOR END NUCLEUS .ANTERIOR FLAGELLUM GIRDLE C+mOMATOPHORE SULCUS .LENS MELANOSOME _ ANTAPEX -POSTERIOR FLAOELLUM RIGHT LEFT VENTRAL VIEW FIGURE 1. Structure of a dinophycean. 1 Contribution No. 838 from the Woods Hole Oceanographic Institution. 196 TAXONOMY OF DINOPHYCEAE 197 new and nine show extensions of range from Europe. The collections which fur- nished the great majority of the material were from Great Pond, Falmouth Harbor, Salt Pond, and Uncatena Island Pond, all very shoal embayments on the southern shore of Cape Cod. The expectations, consequently, seem to be fulfilled. Water samples were concentrated by centrifuging and the Dinophyceae were studied alive in water mounts. At magnifications of X 440 and X 980 drawings were made of specimens which had ceased to swim but showed no deformity due to approaching disintegration. Outline, girdle, and sulcus were drawn with camera lucida ; the rest of the structures were drawn free-hand. For those unacquainted with the morphology of unarmored Dinophyceae and its special nomenclature, Figure 1 illustrates the typical structure of such a dino- phycean. The species studied fall into eight genera, which may be characterized as in the following key : KEY TO GEXERA Girdle and sulcus rudimentary Oxyrrhis Girdle and sulcus well-developed : Solitary : Without ocellus and nematocysts : Girdle not markedly displaced : a) Girdle in anterior third of body -Imphidinium b) Girdle in central portion of body Gymnodinium c) Girdle in posterior third of body Massartia Girdle markedly displaced Gyrodinmm With ocellus but without nematocysts Warnowia With ocellus and nematocysts Nematodinium Colonial Polykrikos OXYRRHIS Dujardin Oxyrrhis marina Dujardin Oxyrrhis marina Dujardin in Kofoid and Swezy, 1921, p. 117, text fig. R, 3. Oxyrrhis marina Dujardin in Lebour, 1925, p. 19, pi. 1, figs. 6a-6e. Woods Hole area: Uncatena Island, Salt Pond; March, August, October. New Jersey ; White Sea ; England ; brackish estuary near Nieuport, Belgium ; Marseilles harbor ; Genoa harbor. This species is distinguished by the posterior position of its flagella, by the broad excavation of the posterior sulcus, divided midway by a tentacle-like lobe, and by the partial encirclement of the girdle. Other characters are its elongate ellipsoidal form and absence of chromatophores. AMPHIDINIUM Claparede and Lachmann KEY TO SPECIES Chromatophores present : Chromatophore single 4. carteri Chromatophores many -1. wislouchi Chromatophores absent : Body stout, with rounded ends A. crassum Body very slender, with pointed ends A. sphenoides 198 EDWARD M. HULBURT PLATE 1 TAXONOMY OF DINOPHYCEAE 199 Amphidinium carter! nom. nov. (Plate 1, Figure 1) Amphidinium klebsi Carter, 1937, p. 58, pi. 8, figs. 12, 13, 14, 15. (non Kofoid & Swezy, 1921). Body dorsi-ventrally flattened, oval in outline in ventral view, elongate-elliptical in lateral view. Length 12-15 p, width 8-9 p. Epicone small, asymmetric, cres- cent-shaped in ventral view, somewhat flattened at apex ; in dorsal view beak-like, rising near right margin and projecting toward left margin. Hypocone truncate- elliptical in ventral or dorsal view, asymmetric, with its right margin convex and left straight or very slightly convex, the antapex broadly rounded. Left limb of girdle starting some distance above posterior end of epicone, running in an arching course anteriorly and laterally, then extending transversely around dorsal surface of epicone, finally leading posteriorly near right margin to round posterior end of epicone, failing, however, to meet the end of the left limb. Sulcus nearer right margin, extending from posterior end of epicone in curving course to antapex. Anterior flagellum inserted at the end of the left limb of girdle ; posterior flagellum inserted just below the anterior, separated from it by a "bridge" that separates girdle ends, extending 1.5 body lengths. Chromatophore single, covering whole inner surface, often perforate, golden brown. Nucleus at posterior end of hypocone, containing short chromatin cor- puscles. Pyrenoid present, in center of hypocone. Assimilate granules present or absent. Woods Hole area: Uncatena Island; October, July. Isle of Wight, England, in brackish pool. The species described here as Amphidinium carteri is identical with Carter's A. klebsi. It is considered an independent species since it is much smaller than the forms described as A. klebsi by Kofoid and Swezy (1921), Herdman (1924a), and Lebour (1925), and since it has only a single chromatophore. Amphidinium wislouchi n. sp. (Plate 1, Figure 2) Amphidinium sp. Wislouch, 1924, p. 121, pi. 3, fig. 11. Body dorsi-ventrally flattened, oval in outline in ventral view. Length 20-25 p., width 14—16.5 p. Epicone small, asymmetric, crescent-shaped in ventral view, PLATE 1 FIGURE 1. Amphidinium carteri nom. nov. FIGURE 2. Amphidinium wislouchi n. sp. FIGURE 3. Amphidinium crassum Lohmann FIGURES 4, 9, 13. Amphidinium sphenoides Wulff FIGURES 5, 6, 7, 8. Massartia rotundata (Lohmann) Schiller FIGURES 10, 14. Massartia asymmetrica (Massart) Schiller FIGURES 11, 12. Gyrodinium metum n. sp. FIGURES 15, 16. Gyrodinium estuariale n. sp. FIGURES 17, 18. Gyrodinium glaebum n. sp. 200 EDWARD M. HULBURT PLATE 2 TAXONOMY OF DINOPHYCEAE 201 somewhat flattened at apex ; in dorsal view beak-like, rising near right margin and projecting toward left margin. Hypocone truncate-elliptical in ventral or dorsal view, asymmetric, with its right margin convex and left almost straight, the antapex broadly rounded. Left limb of girdle starting some distance above posterior end of epicone, running in an arching course anteriorly and laterally, then extending transversely around dorsal surface of epicone, finally leading posteriorly near right margin to round posterior end of epicone, failing, however, to meet the end of the left limb. Sulcus nearer right margin, extending from posterior end of epicone in curving course to antapex. Anterior flagellum inserted at the end of the left limb of girdle; posterior flagellum inserted just below the anterior, separated from it by a "bridge" that separates girdle ends, extending 1.5 body lengths. Chromatophores many, elliptical, often arranged in a somewhat radiating manner, with pyrenoid as center. Woods Hole area : Uncatena Island, Great Pond ; October, March. Poland. This species is about the same as Wislouch's Amphidinium sp. and is quite similar to A. carteri except for having many chromatophores. Amphidinium crassum Lohmann (Plate 1, Figure 3) Amphidinium crassum Lohmann, 1908, p. 261, pi. 17, fig. 16. Amphidinium crassum Lohmann in Lebour, 1917, p. 188, fig. 2; 1925, p. 31, pi. 3, figs. 2a-2c. Body elongate elliptical, circular in cross-section. Length 23-30 p., width 11-17 p.. Epicone very small, 0.20-0.25 the body length, broadly conical with a pointed apex ; hypocone with its sides parallel in the anterior half, rounding into a broad antapex. Girdle not displaced, wide, shallow, its posterior margin wider than the anterior. Sulcus very narrow and shallow, straight, reaching from the apex to 0.66 of the length of hypocone. Flagellar chambers not seen. Anterior flagellum completely encircling body, posterior flagellum not seen. Chromatophores absent. Nucleus close to antapex, spherical, containing short chromatin corpuscles. Brown ingested bodies and assimilate bodies common. Woods Hole area : Great Pond, Falmouth Harbor ; April. May. Baltic Sea of? Kiel ; English Channel ; Plymouth Sound ; Adriatic Sea. Lohmann (1908) described two similar amphidiniums, A. crassum and A. longum, the second more slender and with a smaller, more pointed epicone than the first. Lebour (1917, 1925) described as A. crassum a type intermediate be- tween these two in proportion of length to width, but with the "fuller" epicone of Lohmann's A. crassum. Lebour's type is identical with that described here. PLATE 2 FIGURES 1, 2, 3, 4. Gymnodinium nelsoni Martin FIGURE 5. Gymnodinium lazulum n. sp. FIGURES 6, 7. Gyrodinium resplendens n. sp. FIGURES 8, 9. Gyrodinium aurcolum n. sp. 202 EDWARD M. HULBURT PLATE 3 TAXONOMY OF DINOPHYCEAE 203 Amphidinium sphenoides Wulff (Plate 1, Figures 4, 9, 13) Amphidinium sphenoides Wulff, 1916, p. 105, pi. 1, figs. 9a-9b. Body cylindrical, of slender proportions, tapered posteriorly to a sharp point, tapered anteriorly more abruptly to an equally sharp point. Length 35-42 p., width 12-13 ju.. Epicone 0.20-0.25 the total length, symmetrically diamond-shaped or triangular, respectively, in ventral or dorsal view, but asymmetrically triangular in side view, with the dorsal margin more sloping than the ventral ; outline contours concave. Hypocone similar but more elongate, triangular in dorsal and ventral view and more sloping along the dorsal than the ventral margin. Girdle deep, broad dorsally but narrowed ventrally, so that in side view the margins appear to converge. Sulcus narrow, running from near the apex to 0.66 the length of the hypocone. Anterior flagellar chamber a mere deepening of the left girdle end (posterior chamber not seen). Anterior flagellum encircling body; posterior flagellum somewhat shorter than the body. No chromatophores. Nucleus midway the length of hypocone. Assimilate spherules of various sizes present or absent. Woods Hole area : Great Pond ; January. Barents Sea. GYMNODINIUM Stein (emended by Kofoid and Swezy) KEY TO SPECIES Without striations : Chromatophores present G. nclsoni Chromatophores absent : Body globular G. laznlnm Body laterally flattened G. stellatuin With striations G. striatissimmn Gymnodinium nelsoni Martin (Plate 2, Figures 1, 2, 3, 4) Gymnodinium nelsoni Martin, 1929, p. 14, pi. 3, figs. 25-26. Body broadly fusoid, with truncate antapex, very much flattened dor si- ventrally. Length 50—70 /*, width 38-53 p. Epicone in ventral view sub-hemispherical to somewhat angled, its sides then straight or concave, and its apex broadly pointed. Hypocone trapezoidal, its sides convex, straight, or concave ; its apex wide, emargi- nate to broadly indented. In lateral view dorsal contour somewhat convex, ventral contour somewhat concave ; end-on view with a similar dorsal convexity and ventral concavity. Girdle narrow, deep, displaced one-two girdle widths. Sulcus not present on epicone, narrow and sigmoid in intercingular region, straight and PLATE 3 FIGURES 1, 2, 3. Gyrodinium dominans n. sp. FIGURE 4. Gyrodinium spirale (Bergh) Kofoid and Swezy FIGURES 5, 6. Gymnodinium striatissimum n. sp. FIGURES 7, 8, 9. Gyrodinium undulans n. sp. 204 EDWARD M. HULBURT PLATE 4 TAXONOMY OF DINOPHYCEAE 205 somewhat wider on hypocone, widening abruptly and showing a large excavation at antapex. Anterior and posterior flagellar chambers overlapping. Anterior flagellum not completely encircling cell ; posterior flagellum equal to the body length. Chromatophores many, rich brown, elliptical, radiating from center of cell. Nucleus central, wider than long, with numberless, elongate chromatin corpuscles. Cells usually free from assimilate and ingested bodies. Woods Hole area : Great Pond ; September. Barnegat Bay, United States east coast. G. nelsoni is distinguished from its close relative, G. splendens (Lebour, 1925), by having short, elliptical chromatophores instead of elongate slender ones. Gymnodinmm lazulum n. sp. (Plate 2, Figure 5) Body globular, circular in cross-section, epicone and hypocone subequal. Length 28-30 /x, width 30-32 p. Epicone conical bell-shaped, with marked con- cavity of outline near girdle; hypocone hemispherical bell-shaped, with concavity of contours less extensive but closer to girdle than in epicone. Girdle wide, deep, without displacement. Sulcus short, narrow, and faintly defined on epicone; on hypocone abruptly widening, more so on the right than the left, its margins fading out about halfway to antapex. Girdle ends dipping in markedly to the bottom of the sulcus, deeply excavating sulcus in girdle region. Anterior flagellum inserted at opening of a pore ; pore extending as small pustule from left girdle end posteriorly. Posterior flagellum inserted in trough-like depression of posterior sulcal floor. Anterior flagellum a delicate band completely encircling body; posterior flagellum short, 0.5 body length. Chromatophores absent. Nucleus not visible. Cytoplasm clear, quite trans- parent, smoke blue in color. Colored spherules, grading from copper-red through orange and brown to lemon-yellow, abundant, scattered throughout cytoplasm, often clustered near girdle. Angular, crystal-like, slate-gray bodies scattered at random through hypocone. Large, brown ingested bodies common. Woods Hole area: Great Pond; December, May. This species was the only one studied with a clear, blue, instead of a granular, gray, cytoplasm. The array of hues of the numberless small spherules, as well as the blue of the cytoplasm, are matched by similar structures in G. violescens (Kofoid and Swezy, 1921). Gymnodinium stellatum n. sp. (Plate 4, Figures 4, 5, 6) Body laterally flattened, elliptical in side view with sub-truncate anterior end. Length 25-47 /*, thickness 22-39 //,, width 17-25 /JL. Epicone in dorsi-ventral view taller than wide, with slightly sloping sides ; in lateral view as wide as tall, trape- PLATE 4 FIGURES 1, 2, 3. Gyrodinhim uncatenum n. sp. FIGURES 4, 5, 6. Gymnodinium stellatum n. sp. FIGURE 7. Polykrikos hartmanni Zimmermann FIGURE 8. Warnoivia parva (Lohmann) Lindemann FIGURES 9, 10. Nematodininm armatum (Dogiel) Lebour 206 EDWARD M. HULBURT zoidal, more sloping dorsally than ventrally. Hypocone similar to epicone in shape in dorsi-ventral view, with antapical notch dorsally ; in lateral view sub-hemispheri- cal, wider than tall. Girdle narrow, displaced 0.16-0.20 body length. Sulcus narrow, straight except for slight leftward divergence in girdle region, forming a deep hypoconal excavation with anterior limit marked laterally by a vague line running diagonally from posterior dorsal corner toward girdle region on ventral surface. Anterior flagellar chamber a long finger-like pocket extending posteriorly and somewhat dorsally; posterior flagellar chamber a narrow prolongation of hypoconal excavation, reaching almost to anterior chamber. Anterior flagellum encircling less than half the circumference, wide, band-shaped; posterior flagellum body length, sometimes double. Chromatophores absent. Nucleus within epicone, containing elongate chro- matin corpuscles. Peripheral cytoplasm with distinct radial structure. Assimilate occasionally abundant. Woods Hole area: Salt Pond; October, December, January. This species is very much like Gyrodinium uncatenum in this paper, differing principally in lacking chromatophores, in having a less displaced girdle, and a straighter sulcus. Among hitherto described species, it is similar only to Gym- nodinium bifurcatum (Kofoid and Swezy, 1921) in its lateral flattening and deep hypoconal excavation. Gymnodinium striatissimum n. sp. (Plate 3, Figures 5, 6) Body globular to elliptical, not flattened, its hypocone slightly larger than epicone. Length 29-43 p., width 23-31 p.. Epicone conical, pointed, or somewhat truncate. Hypocone similar but more truncate, varying from tapered type with moderately sloping sides and rounded antapex to type with slightly sloping sides and broad antapex. Surface with striations, fewer on epicone (15) than on hypocone (25). Girdle narrow, rather deep, slightly displaced, with distinctive posterior flexure to end of left limb. Sulcus shallow, narrow on epicone, some- what wider on hypocone, from near apex almost to antapex, distinguished by sharp leftward bend just anterior to girdle. Anterior flagellar chamber prolonged posteriorly. Anterior flagellum completely encircling body; posterior flagellum one body length, sometimes double. No chromatophores. Nucleus posterior, wholly within hypocone, with elongate chromatin corpuscles. Assimilate bodies of various sizes often abundant. Woods Hole area : Great Pond ; May. Gymnodinium striatissimum is similar in different number of striations on epicone and hypocone to the much larger G. multistriatum, G. rubruin, and G. translucens (Kofoid and Swezy, 1921). MASSARTIA Conrad KEY TO SPECIES Without striations : Chromatophores present M. rotundata Chromatophores absent M. asymmetnca With striations M. glauca TAXONOMY OF DINOPHYCEAE 207 Massartia rotitndata (Lohmann) Schiller (Plate 1, Figures 5, 6, 7, 8) Amphidinium rotundatuin Lohmann, 1908, p. 261, pi. 17, fig. 9. Amphidinium rotundatuin Lohmann in Wulff, 1916, p. 103, pi. 2, fig. 11. Amphidinium rotundatum Lohmann in Van Goor, 1925, p. 285, fig. 4. Gymnodinium minutum Lebour, 1925, p. 45, pi. 5, fig. 4. Massartia rotundata (Lohmann) Schiller in Conrad, 1939, p. 11, figs. 17-22. Body top-shaped, circular in cross-section. Length 8-17 /t, width 6-12 p. Epicone 2.5 times as long as hypocone, conical, with straight to gently convex sides, its apex pointed or somewhat rounded. Hypocone half as long as wide, broadly rounded, in lateral view asymmetric so that the dorsal portion is larger than the ventral one. Girdle very wide, very slightly displaced, its anterior margin over- hanging and of greater diameter than the posterior margin. Sulcus not discernible. Flagellar chambers absent. Anterior flagellum up to twice the girdle circumference in length ; posterior flagellum equal in length to the cell body. Chromatophores two, yellow-brown; one band-shaped, partially encircling pe- riphery of epicone; the other filling bottom of hypocone, extending on ventral surface to epicone. Nucleus not seen. Assimilate bodies present or absent. Pellicle occasionally present. Woods Hole area: Great Pond, Falmouth Harbor; January to April, August, September. Barents Sea ; White Sea ; Baltic off Kiel ; brackish estuaries near Nieuport (Belgium) and along the coast of Holland; Plymouth Sound; Adriatic Sea. Several different populations were studied. One had straight sides to the epicone and a pointed apex. Another had convex sides to the epicone and a pointed apex. A third was rather variable, rotund in appearance, often almost colorless, with apex rounded or pointed. In a similar way this distinctive species shows considerable variation as it receives treatment from various investigators. Lohmann describes it with relatively very small hypocone, whereas Wulff describes it with relatively very much larger one. In contrast to these, which have pointed apices, Conrad's has a rounded apex. Van Goor shows a very slender form. Lohmann's and Wulff's figures show a lobed epiconal chromatophore, whereas Lebour's shows a band-shaped chromatophore. Massartia asymmetrica (Massart) Schiller (Plate 1, Figures 10, 14) Gymnodinium asymtnetricum Massart, 1920, p. 132, figs. 22A— D. Massartia asymmetrica (Massart) Schiller in Carter, 1937, p. 59, pi. 8, figs. 17—18. Body globular, oval in outline, compressed dorsi-ventrally. Length 14-22 p., width 13-20 fj,. Epicone 0.66 body length, hemispherical, with small apical notch. Hypocone small, 0.33 body length, twice as wide as long, broadly rounded, often with slight oblique flattening in antapical region. Girdle very wide, shallow, displaced one girdle width, its anterior margin wider than posterior. Sulcus extending from girdle to antapex, widening abruptly during its course. Anterior 208 EDWARD M. HULBURT flagellum incompletely encircling body; posterior flagellum 1.0-1.5 body lengths. Chromatophores absent. Nucleus almost wholly within hypocone, wider than long, containing relatively few, short, unoriented chromatin corpuscles. Large, brown ingested body and smaller assimilate bodies frequent. Woods Hole area : Great Pond ; October, January, February. Isle of Wight, England ; estuary near Nieuport, Belgium. Massartia asymmetrica is similar to M. vorticella (Stein) Schiller and M. stigmaticum (Lindemann) Schiller, which, however, contain stigmas, and to M. glandula Herdman, which differs, however, in its larger size (20-35 /x long) and helmet-shaped instead of hemispherical epicone. Massart's and Carter's figures of M. asymmetrica show somewhat greater displacement of girdle than in the specimens described here. Massartia glauca (Lebour) Schiller Spirodinium glaucum Lebour, 1917, p. 196, fig. 13. Gyrodinium glaucum (Lebour) Kofoid and Swezy, 1921, p. 308, pi. 9, fig. 94, text fig. DD, 16. Gyrodinium glaucum (Lebour) Kofoid and Swezy in Lebour, 1925, p. 54, pi. 7, fig. 4, text fig. 15. Woods Hole area : Great Pond ; October, May. Plymouth Sound ; Adriatic Sea ; La Jolla, California. The specimens studied agreed closely in shape with those of Lebour and were not like Kofoid and Swezy's somewhat different form. They were, however, considerably smaller than Lebour's, 28-32 p, instead of 40-56 //, in length. This form is different from other species of Massartia in its striations ; it is distinguished by its slender form, the slight twist to the apex, and its absence of chromatophores. GYRODINIUM Kofoid and Swezy KEY TO SPECIES Without striations : Chromatophores present : Chromatophores 2 to 4 G. estuariale Chromatophores many : Body dorsi-ventrally flattened : Sulcus not deflected on epicone G. aureolum Sulcus deflected to right on epicone G. resplendens Body laterally flattened G. uncatenum Chromatophores absent : Sulcus sigmoid : Epicone rounded-conical G. me turn Epicone hemispherical G. glaebum Sulcus bisigmoid G. undulans With striations : Length 18-43 p G. dominant Length 66-96 /JL G. spirale TAXONOMY OF DINOPHYCEAE 209 Gyrodinimn estuariale n. sp. (Plate 1, Figures 15, 16) Body ellipsoid ; apex often somewhat pointed compared to broadly rounded antapex; often with slight asymmetry, the right side more convex than the left; slightly flattened dorsi-ventrally; with equal epicene and hypocone. Length 11-16 p., width 9-12 /x. Epicone broadly conical to sub-hemispherical, the right margin often a bit more sloping than the left ; hypocone hemispherical to somewhat trape- zoidal, with rounded to flattened, oblique, sometimes indented antapex. Girdle deep, moderately wide, displaced 0.25-0.33 body length, strongly posteriorly bend- ing in right limb. Sulcus slight on epicone, markedly deflected to right in inter- cingular area, widening, and proceeding straight to antapex on hypocone. Anterior flagellar chamber an elongate pocket diverging to right and running beneath posterior flagellar chamber which is a leftward underhollowing of sulcus between girdle ends. Anterior flagellum encircling body completely; posterior flagellum body length. Chromatophores yellow-brown, one or two in epicone, one or two in hypocone, distinctively inset from periphery. Nucleus not seen. Assimilate bodies usually sparse. Woods Hole area : Great Pond, Salt Pond, Uncatena Island ; July, August, October to January. Gyrodinium estuariale is very similar to Gymnodinium vitiligo and Gym- nodinium veneficum (Ballantine. 1956), differing in greater displacement of girdle ends, in wider, deeper girdle and sulcus, and in an oblique, instead of symmetrically rounded, antapex. In the intercingular region the sulcus is deflected to the right (passing from anterior to posterior end) in these species, contrary to most gyro- dinia. Gyrodinium estuariale is similar to Gymnodinium niarylandicum (Thomp- son, 1947) ; but the latter's sulcus follows the longitudinal axis or is deflected slightly to the left. Gyrodinium aureolum n. sp. (Plate 2, Figures 8, 9) Body essentially globular, its dorsi-ventral outline either somewhat ellipsoidal or somewhat fusiform, slightly dorsi-ventrally flattened, with subequal epicone and hypocone. Length 27-34 p., width 17-32 /*. Epicone hemispherical to broadly conical, sometimes slightly truncate. Hypocone similar, but usually distinctly truncate, with antapex faintly indented at times. Girdle wide, moderately deep, displaced 0.20 body length. Sulcus reaching from just behind apex all the way to antapex, with slight, left deflection in girdle region, rather narrow on epicone, wide on hypocone. Anterior flagellar chamber a posteriorly pointed, finger-shaped cavity ; posterior flagellar chamber an underhollowing of left sulcal margin opposite right girdle limb. Anterior flagellum completely encircling body; posterior flagellum very long, up to two body lengths. Numerous yellow-brown chromatophores present, elliptical in shape, usually arranged in a somewhat radiating manner. Nucleus spherical or wider than long, with elongate chromatin corpuscles. 210 EDWARD M. HULBURT Woods Hole area : Great Pond, Uncatena Island, Falmouth Harbor ; December to April. This species is rather similar to Gyrodinium aureum (Conrad, 1926). It is, however, different in its less elongate chromatophores, wider grooves, greater width for the same length, more conical outline of epicone and hypocone, and somewhat less displacement of girdle ends. Gyrodinium resplendens n. sp. (Plate 2, Figures 6, 7) Body broadly fusoid, with truncate apex and antapex, moderately flattened dorsi-ventrally. Length 36-62 p., width 32-48 //,. Epicone and hypocone similar, equal, trapezoidal in outline, their sides convex, straight, or concave; the apex somewhat rounded, the antapex with sulcal indentation. Girdle deep, moderately wide, displaced 0.20-0.25 body length. Sulcus extending onto epicone as very narrow superficial groove, diverging to right ; in intercingular region narrow, vertical or left deflected; on hypocone running straight to antapex, superficially narrow but broad beneath projecting lappet of left margin. Anterior and posterior flagellar chambers elongate pockets projecting toward but not reaching each other. Anterior flagellum completely encircling body ; posterior flagellum one body length. Chromatophores oval, rich brown, radiating, many-tiered. Nucleus somewhat anterior of center, wider than long, with numerous, elongate chromatin corpuscles. Ingested bodies occasional ; assimilate absent. Woods Hole area : Great Pond ; July, August. This species is close to Gyrodinium aureolum. It is also quite like Gym- nodiniwn nelsoni, differing in greater girdle displacement (so that it falls into the genus Gyrodinium}, less dorsi-ventral flattening with no ventral concavity, and truncate rather than hemispherical epicone. Gyrodinium uncatenum n. sp. (Plate 4, Figures 1, 2, 3) Body laterally flattened, elliptical to quadrangular in side view, elongate elliptical in ventral or dorsal view, with epicone and hypocone subequal. Length 40-54 p, width 28-33 p. Epicone in ventral and dorsal views helmet-shaped, taller than wide, broadly rounded at apex, its sides sloping gently, with slight concavities at girdle ; in lateral view, sub-hemispherical to trapezoidal, wider than tall. Hypocone very similar but often slightly more truncate at antapex and more distinctly trape- zoidal in side view. Girdle narrow, deep, displaced 0.33 body length, the right limb bending steeply posteriorly. Sulcus projecting slightly on epicone, curving to left in intercingular area, then sharply to right between right girdle end and antapex ; carried across antapex all the way to dorsal side ; deeply excavating hypocone, the anterior extent of excavation seen laterally as an oblique line running from dorsal end of sulcus toward ventral surface. Anterior flagellar chamber long, finger-like, projecting posteriorly and somewhat dorsally and rightward ; posterior flagellar chamber a long extension of sulcal excavation, reaching nearly to anterior chamber. Anterior flagellum wide and strap-shaped, completely en- TAXONOMY OF DINOPHYCEAE 211 circling body ; posterior flagellum occasionally double, twice the body length ; both flagella reaching all the way to the end of the chambers. Chromatophores elongate, yellow-brown, radiating from center, leaving marginal area clear (lateral view). Nucleus spherical, in the epicone, with slightly elongate chromatin corpuscles. Crytoplasm showing vague radiating structure in peripheral region. Countless, dark assimilate bodies often present. Woods Hole area : Great Pond, Uncatena Island ; July, August, October. This species is rendered distinctive by its lateral flattening, a rarity among gyrodinia. Very striking is the deep excavation in the hypocone, identical to that in Gymnodinium stellatum and G. bifurcation (Kofoid and Swezy, 1921). Gyrodinium metum n. sp. (Plate 1, Figures 11, 12) Body somewhat asymmetric, the right side more convex than the left, circular in cross-section, with hypocone slightly larger than epicone. Length 14.5-22 fi, width 11-16 p. Epicone rounded-conical in outline; hypocone truncate with straight to convex sides. Girdle deeply excavated, displaced about 0.20-0.25 body length, its right limb curving steeply posteriorly. Sulcus sigmoid, not extending onto epicone, narrow in intercingular region, and wide and deep on hypocone, flat- tening or indenting the antapex. Anterior flagellar chamber produced inward and posteriorly ; posterior flageller chamber a mere excavation in the sulcal floor. Ante- rior flagellum completely encircling the body; posterior flagellum 1.5 times body length. Chromatophores absent. Cytoplasm gray, foamy in texture. Nucleus not seen. Woods Hole area : Great Pond ; May, June, July, December, February. This species is distinguished by the Chinaman-hat shape of the epicone. A smaller-size variant was often seen. Its features are identical except respecting size— 9.5-12 p. X 9-7 /*. Gyrodinium glaebum n. sp. (Plate 1, Figures 17, 18) Body elliptical, very slightly compressed dorsi-ventrally, with equal epicone and hypocone. Length 17-25 /*, width 12-19 p. Epicone hemispherical but slightly asymmetric, with right contour more sloping or less fully curved than left. Hypocone similar, but broader, the asymmetry more marked, the left contour more sloping or less fully curved than right, often with oblique flattening in region of sulcus end. Girdle wide, rather deep, displaced two-three times its own width. Sulcus extending slightly onto epicone, narrow and leftward diverging in inter- cingular region, wide and deep in a straight course on hypocone. The two flagellar chambers overlapping each other, the anterior one a pronounced, posteriorly directed excavation, the posterior one an underhollowing of left girdle margin. Anterior flagellum only partially encircling body, posterior flagellum as long as body. Chromatophores absent. Nucleus somewhat anterior of girdle, with large and relatively few chromatin corpuscles. Large, brown ingested bodies often present, as well as small, refractive assimilate bodies. 212 EDWARD M. HULBURT Woods Hole area : Great Pond ; July, October. Gymnodinium variabile (Herdman, 1924a) is close in shape but lacks any marked girdle displacement. Gyrodinium undulans n. sp. (Plate 3, Figures 7, 8, 9) Body elliptical, somewhat dorsi-ventrally flattened with equal epicone and hypocone. Length 27-38 p., width 21-31 p. Epicone varying from sub-hemi- spherical to truncate-pyramidal, with rounded apex and straight but sloping sides. Hypocone asymmetric, truncate-pyramidal with rounded to flattened, oblique antapex, and sloping sides. Girdle wide, deep, displaced 0.20 body length, its right limb curving strongly posteriorly. Sulcus distinctive, forming a bi-sigmoid curve ; very narrow on epicone, swerving leftward and then rightward to meet left girdle end, curving again to left in intercingular area and widening, then bending to right on hypocone to form large overlapping lobe, finally returning leftward to antapex. Floor of sulcus on hypocone extending straight to antapex; and right margin of sulcus "bearing away" laterally to form a swelling. Anterior and posterior flagellar chambers projecting toward, but not reaching, each other. Anterior flagellum completely encircling body ; posterior flagellum one body length, occasionally double. Chromatophores absent. Nucleus large, principally within epicone, of elongate chromatin corpuscles. Assimilate bodies sometimes in form of large blocks. Woods Hole area : Great Pond ; February, January. Few Dinophyceae have either bi-sigmoid sulci or overlapping sulcal lobes. Gyrodinium dominans n. sp. (Plate 3, Figures 1, 2, 3) Body broadly fusiform, circular in cross-section, epicone and hypocone subequal. Length 18.5-43 /A, width 10—22 p. Epicone and hypocone conical to rotund-conical, their sides varying from convex to straight. Both epicone and hypocone occa- sionally with slight concavities near girdle. Surface with continuous striations, the number the same on epicone and hypocone, between 7 and 10 across ventral face. Girdle of moderate depth and width, displaced 0.25-0.33 body length. Sulcus sigmoid, deflected to left 0.25 transdiameter in intercingular region, reaching halfway up epicone, extending to posterior margin of hypocone on left side of antapex. Anterior flagellar chamber a posteriorly directed, finger-like projection from left end of girdle. Posterior flagellum inserted at posterior end of girdle (flagellar chamber not seen). Anterior flagellum not completely encircling body; posterior flagellum short, 0.50 body length. Chromatophores absent. Nucleus anterior, in epicone, containing elongate, oriented chromatin corpuscles. Woods Hole area: Great Pond, Falmouth Harbor, Salt Pond; April, July, August, October to December. Gyrodinium dominans is an ally to three very similar species of Gyrodinium: G. pingue (Schiitt, 1895, as Gymnodinium spirale var. pinguis; Wulff, 1916, as TAXONOMY OF DINOPHYCEAE 213 Spirodinium varians; Kofoid and Swezy, 1921; Lebour, 1925), G. obtnsiim (Schiitt, 1895, as Gymnodinium spirale var. obtitsa; Kofoid and Swezy, 1921 ; Lebour. 1925), and G. fissuin (Kofoid and Swezy, 1921). The chief distinction is the flexure of the sulcus in G. dominans, contrasting with the comparative straightness of the sulcus in the other three. Gyrodinhnn spirale (Bergh) Kofoid and Swezy (Plate 3, Figure 4) Gyrodinium spirale (Bergh) Kofoid and Swezy, 1921, p. 332, pi. 4, fig. 43, text fig. DD, 14. Gyrodinium spirale (Bergh) Kofoid and Swezy in Lebour, 1925, p. 56, pi. 8, fig. 1. Body slender fusiform ; circular in cross-section ; somewhat twisted on its longitudinal axis ; with epicone longer but narrower than hypocone. Length 66- 96 fjL, width 30-38 p. Epicone narrowly conical, convex or straight along left and dorsal contours. Hypocone with sides parallel anteriorly, convex posteriorly; its antapex pointed, excentric, to right of ventral, longitudinal axis ; with sulcal notch to left of antapex. Surface with continuous striations, 8—13 on epicone, 15-20 on hypocone. Girdle narrow, rather shallow, displaced more than 0.33 the body length, its right limb curving strongly posteriorly. Sulcus very narrow, from somewhat behind apex to left girdle end as a heavy line, a very narrow but distinct groove between girdle ends, widening and deepening from right girdle end to antapical notch. Anterior flagellum inserted at opening of a pustule, which is composed of a short, sac-shaped portion, extending posteriorly, and of a long, thread-like portion, extending somewhat anteriorly, then laterally, finally posteriorly along right contour to region of right girdle limb. Posterior flagellum likewise inserted at opening of a pustule having a long, thread-like extension anteriorly and leftward to region of left girdle limb. Anterior flagellum following girdle 0.33 or less of girdle length. Posterior flagellum short, about 0.25 of body length. Chromatophores absent. Nucleus elongate-ellipsoidal on left side (ventral view), in intercingular area, without apparent structure. Woods Hole area: Great Pond, Falmouth Harbor; December, April, May, August. Baltic Sea ; Norway ; Port Erin, Ireland ; Plymouth Sound ; Adriatic Sea ; La Jolla, California ; Indian Ocean ; coast of Australia. The organism defined here agrees closely with those described by Kofoid and Swezy and by Lebour. Distinctive characteristics are the slender form, twist of body, greater dorsal than ventral curvature, and a longer epicone than hypocone. WARNOWIA Lindemann Warnowia parva (Lohmann) Lindemann (Plate 4, Figure 8) Pouchetia parva Lohmann, 1908, p. 264, pi. 17, fig. 23. Body elliptical, circular in cross-section, slightly tapered posteriorly, epicone somewhat larger than hypocone. Length 22.5-30 p., width 15-18 p.. Epicone 214 EDWARD M. HULBURT hemispherical, hypocone similar but narrowed toward antapex and with sulcal indentation along left contour. Girdle shallow but very wide, displaced 0.50 the body length, with strongly descending right limb. Sulcus narrower than girdle, extending in sigmoid path from apex to antapex, bending from right margin toward center anterior to girdle, diagonal across ventral face between girdle ends, recurving from left margin toward center posterior to girdle. Anterior flagellar chamber an elongate pocket from left end of girdle limb, the posterior flagellar chamber not seen. Anterior flagellum not encircling body completely ; posterior flagellum short, 0.33 body length. Yellow bodies, resembling chromatophores in color and peripheral position but unlike in large size and irregular shape, sparse anteriorly but abundant posteriorly. Nucleus anterior, with elongate, oriented corpuscles. Melanosome near antapex, black, diffuse and spreading, or ellipsoidal and globular. Lens simple without evident laminations, projecting anteriorly on left side from melanosome. Nemato- cysts absent. Cell often within a pellice. Assimilate bodies frequent. Woods Hole area : Great Pond ; July. Baltic off Kiel. The organism studied here is probably a close match to Lohmann's Pouchetia parva, but Lohmann showed no girdle and sulcus. It is close to Nematodinium armatum, but the tapered hypocone, large irregular chromatophores, absence of nematocysts, and cyst differentiate it, as well as the characteristics mentioned under N. armatum. NEMATODINIUM Kofoid and Swezy Nematodinium armatum (Dogiel) Lebour (Plate 4, Figures 9, 10) Pouchetia armata Dogiel, 1906, p. 36, pi. 2, figs. 48-49. Nematodinium armatum (Dogiel) Lebour, 1925, p. 71, pi. 10, figs. 5a-5b Nematodinium armatum (Dogiel) Lebour in Martin, 1929, p. 19, pi. 2, figs. 5-7. Body elliptical, with equal epicone and hypocone. Length 33-53 p., width 20- 33 ja. Epicone evenly rounded, slightly asymmetric in ventral view, its right margin more sloping than the left ; hypocone also slightly asymmetric with more sloping left than right side, the antapex either somewhat pointed and off-center, or, usually, obliquely truncate. Girdle deep, moderately wide, displaced 0.33 the body length, with strongly descending right limb. Sulcus extending from near apex in a sigmoid path to antapex, widening on the oblique margin of antapex, a portion of it curving sharply to the right, delimiting a knob-like protuberance on ventral face of the hypocone. Anterior flagellar chamber an elongate pocket from left end of girdle limb (posterior chamber not seen). Posterior flagellum one body length (anterior flagellum not fully studied). Chromatophores yellow, circular or subcircular, few, and scattered. Nucleus large, anterior, of elongate chromatin corpuscles. Melanosome near antapex, black, diffuse and spreading, or ellipsoidal and globular. Lens single with concentric laminations, projecting anteriorly on left side from melanosome. Nematocysts present or absent, in region of right girdle limb. Woods Hole area: Great Pond; August. Barnegat Bay, New Jersey; Ply- mouth Sound; Naples. TAXONOMY OF DINOPHYCEAE 215 Some of the specimens were of full, ellipsoidal proportions, as in Dogiel's figure. Some were more slender, though not quite so slender as in Lebour's Figure 5a. Some had a slight tapering of hypocone and pointed antapex as in Martin's figures. They are all intermediate between the smaller Warnowia parva (Lohmann, 1908) without right ward curvature of sulcus on hypocone and with comparatively wider, shallower furrows, and the larger N ematodinium lebourae (Schiller, 1933; Kofoid and Swezy, 1921, as N. armatum), with rightward curva- ture of sulcus on hypocone and with comparatively greater transverse displacement of the sulcus. POLYKRIKOS Butschli KEY TO SPECIES Chromatophores present : Body cylindrical P. Jiartmanni Body laterally flattened P. lebourae Chromatophores absent P. schwartzi Polykrikos hartmanni Zimmermann (Plate 4, Figure 7) Polykrikos hartmanni Zimmermann, 1930, p. 436, figs. 8-9. Colony cylindrical in form with rounded ends, consisting of two zooids, de- limited by a slight constriction. Length 60—68 /*., width 42-47 p.. Epicone often smaller than hypocone in anterior zooid but equal to hypocone in posterior zooid. Girdles wide, rather shallow, displaced twice their width. Sulcus continuous from apex to antapex, roughly straight, narrowed at the constriction between zooids and in intercingular regions. Sulcus produced inward as anterior and posterior flagellar chambers, which extend posteriorly and anteriorly, respectively, their diverging ends overlapping. Anterior flagella incompletely encircling zooids ; posterior flagella about 0.66 as long as the colony. Chromatophores circular, small, numerous, yellow-brown in color. Nuclei always two, with elongate chromatin corpuscles. Several long nematocysts often, but not always, present in region just below anterior girdle. Innumerable black granules may fill peripheral cytoplasm of whole colony or may be restricted to posterior end. Woods Hole area : Great Pond ; August. Adriatic. Slight differences between our specimens and Zimmermann's are the smaller size of ours (Zimmermann's 80-120 //, X 55-75 ju.) and the yellow-brown instead of yellow-green Chromatophores. Polykrikos barnegatensis (Martin, 1929), also composed of two cells, is close to P. hartmanni but has a single nucleus and a more elliptical outline. Polykrikos schwartzi Butschli Polykrikos schwartzi Butschli in Kofoid and Swezy, 1921, p. 400, text fig. F, 4. Polykrikos schwartzi Butschli in Lebour, 1925, p. 67, pi. 10, figs. 2a-2b, text fig. 16c. Woods Hole area: Great Pond; August. Arctic near Iceland; off coast of Norway ; Skagerack ; Baltic off coast of Denmark ; Baltic off Kiel ; North Sea off 216 EDWARD M. HULBURT Helgoland ; Plymouth Sound ; Atlantic off Concarneau, France ; Mediterranean off French coast. This species is distinguished by its many cells (zooids), averaging about eight, with four nuclei ; by its cylindrical form ; and by the absence of chromatophores. Polykrikos lebourae E. C. Herdman Polykrikos lebourae E. C. Herdman, 1924b, p. 60, fig. 6. Polykrikos lebourae E. C. Herdman in Lebour, 1925, p. 68, pi. 10, fig. 3. Woods Hole area: Salt Pond, beach sand; November. Port Erin, Ireland, in sand. This species is quite distinctive in its lateral flattening. It has eight cells, two nuclei, and yellow-brown chromatophores. It is recorded from beach sand at Woods Hole by E. C. Herdman. DIAGNOSES OF NEW SPECIES Amphidinium carteri n. sp. Corpus dorsali-ventraliter compressum, a fronte visum ovale; epicono minuto, asymmetrico et rostroformi ; hypocono truncato-elliptico ; sulco prope marginem dextrum, leniter curvato ; chromatophoro uno, parietali, perforate, fulvo. Longi- tude 12-15 fj., latitudo 8-9 p.. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Amphidinium wislouchi n. sp. Corpus simile Amphidinio carteri sed paullo majus; chromatophoris multis, ellipticis, paululum radiatim ordinatis. Longitude 20-25 /*, latitudo 14—16.3 p.. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Gymnodinium lazulum n. sp. Corpus globosum ; epicono conico vel campaniformi ; hypocono hemispherico vel campaniformi ; sulco tenui ; corpore sine striis ; chromatophoris absentibus ; cytoplasmate pellucido, subcaeruleo, saepe cum multis corporibus multi-coloratis. Longitude 28-34 p., latitudo 30-32 p. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Gymnodinium stellatum n. sp. Corpus lateraliter compressum ; epicono quadrangulato et hypocono hemi- spherico; extremis cinguli 0.17 longitudinis corporis transpositis ; sulco a vicinitate apicis ad cavernam profundam hypoconi extendente ; corpore sine striis ; chroma- tophoris absentibus; structura cytoplasmatis exterioris perspicue radiata. Longi- tudo 25-47 /A, latitudo 22-39 p, crassitude 13-25 p. United States, in loco dicto Salt Pond, Barnstable County, Massachusetts. TAXONOMY OF DINOPHYCEAE 217 Gymnodinium striatissimum n. sp. Corpus globosum vel ellipticum; epicono et hypocono conico, aculeate, vel truncate ; cingulo angusto, flexuram posterioram extremi sinistri habente ; sulco a vicinitate apicis ad antapicem extendente ; corpore cum striis, in hypocono pluribus quam in epicono ornato ; chromatophoris absentibus. Longitudo 29-43 p., latitude 23-41 p,. United States, in loco dicto Great Pond, Barnstable County, Massa- chusetts. Gyrodinium estuariale n. sp. Corpus ellipticum, paululum asymmetricum, margine dextro convexiore quam margine sinistro ; epicono late conico vel sub-hemispherico ; hypocono hemispherico ; extremis cinguli 0.25-0.33 longitudinis corporis transpositis ; sulco ad latus dextrum inter extrema cinguli multum deflecto ; corpore sine striis ; chromatophoris fulvis, 2-4, paulum intra peripheriam positis. Longitudo 11-16 /A, latitude 9-12 ^. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Gyrodinium aureolum n. sp. Corpus globosum ; epicono et hypocono hemispherico vel conico ; extremis cinguli 0.33 longitudinis corporis transpositis ; sulco a vicinitate apicis ad antapicem extendente ; corpore sine striis ; chromatophoris multis, fulvis, plerumque omnibus plus minusve radiatim ordinatis. Longitudo 27-34 p, latitude 17-32 /A. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Gyrodinium resplendens n. sp. Corpus late fusiforme, apice et antapice truncate, dorsaliventraliter compressum ; extremis cinguli 0.20-0.25 longitudinis corporis transpositis ; sulco in epicono augusto, tenui ; ad latus dextrum curvato, in hypocono profundo et recto ; corpore sine striis ; chromatophoris multis, fulvis, radiatim ordinatis. Longitudo 36-62 /A, latitude 32-48 p. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Gyrodinium uncatenum n. sp. Corpus lateraliter compressum, a late visum ellipticum vel quadrangulum ; extremis cinguli 0.33 longitudinis corporis transpositis, parte dextra ad antapicem multum curvata; sulco in epicono tenui, ad sinistram prope extremitatem poste- riorem cinguli deflecto ; hypocono excavationem profundam antapicis praehente ; corpore sine striis ; chromatophoris luteo-fuscis, elongatis, radiatim ordinatis ; cytoplasmate circa peripheriam structuram radiatam habente. Longitudo 40-54 ^, latitude 28-33 /*. United States, in loco dicto Uncatena Island, Barnstable County, Massachusetts. Gyrodinium metum n. sp. Corpus paulum asymmetricum, latere dextro convexiore quam latere sinistro ; epicono conico, multo latiore quam hypocono; hypocono truncate, multo longiore quam epicono; cingulo profundo, extremis cinguli 0.20-0.25 longitudinis corporis 218 EDWARD M. HULBURT transpositis ; sulco S-curvato, in epicono absente, in hypocono lato ; corpora sine striae; chromatophoris absentibus. Longitude 14.5-2 ju,, latitude 11-16 /x. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Gyrodinium glaebum n. sp. Corpus ellipticum, paulum asymmetricum ; epicono hemispherico, hypocono late hemispherico; cingulo lato, extremis cinguli 2-3 latitudinibus cinguli trans- positis; sulco in epiconum vix extendente, in hypocono lato et profundo; corpore sine striis; chromatophoris absentibus. Longitude 17-25 //,, latitude 12-19 p. United States, in loco dicto Great Pond, Barnstable County, Massachusetts. Gyrodinium undulans n. sp. Corpus ellipticum ; epicono subhemispherico vel truncato-pyramidali ; hypocono truncato-pyramidali ; extremis cinguli 0.20 longitudinis corporis ; sulco in duo S- formata curvamina facto, margine sinistro in hypocono marginem dextrum super- posito ; corpore sine striis ; chromatophoris absentibus. Longitude 27-38 /*,, latitude 21-31 p.. United States, in loco dicto Great Pond, Barnstable County, Massachu- setts. Gyrodinium dominans n. sp. Corpus fusiforme ; epicono et hypocono conico ; extremis cinguli 0.25-0.33 longitudinis corporis transpositis; sulco S-curvato, in epiconum extendente, in hypocono ad marginem sinistrum prope antapicem extendente ; corpore cum striis, 7-10 a ventrale viso in epicono et hypocono; chromatophoris absentibus. Longi- tudo 18.5-43 p, latitude 10-22 p. United States, in loco dicto Great Pond, Barn- stable County, Massachusetts. The author wishes to express the greatest gratitude to Dr. William Randolph Taylor for his guidance in this study. He is also greatly obligated to Dr. Alfred C. Redfield and Dr. Trygve Braarud for their kindness in reading the manuscript. SUMMARY 1. Unarmored Dinophyceae were collected from very shallow embayments on the south shore of Cape Cod, Massachusetts. 2. Twenty-six species, distributed in eight genera, were studied. Twelve were considered as new species and nine showed extensions of range from Europe. LITERATURE CITED BALLANTYNE, D., 1956. Two new marine species of Gymnodinium isolated from the Plymouth area. /. Mar. Biol. Ass. U. K., 35 (3) : 467-474. CALKINS, G., 1902. Marine protozoa from Woods Hole. Bull. U. S. Fish Comm., 21 : 413-468. CARTER, N., 1937. New or interesting algae from brackish water. Archw. f. Protist., 90: 1-68. CONRAD, W., 1926. Recherches sur les Flagellates de nos eaux saumatres. le Partie, Dino- flagellates. Archiv. f. Protist., 55 : 63-100. CONRAD, W., 1939. Notes Protistologiques. X. Sur le schorre de Lilloo. Bull. Mus. Roy. Hist. Nat. Belg., 15 (41) : 1-18. TAXONOMY OF DINOPHYCEAE 219 DOGIEL, V., 1906. Beitrage zur Kenntnis Peridineen. Mitt. Zoo/. Stat. Ncapcl, 18: 1-45, pis. 1-2. HERDMAN, E. C, 1924a. Notes on dinoflagellates and other organisms causing discoloration of the sand at Port Erin. IV. Proc. Trans. Liverpool Biol. Soc., 38: 75-84. HERDMAN, E. C., 1924b. Notes on dinoflagellates and other organisms causing discoloration of the sand at Port Erin. III. Proc. Trans. Liverpool Biol. Soc.. 38: 58-63. KOFOID, C. A., AND O. SWEZY, 1921. The free-living unarmored Dinoflagellata. Mem. Univ. Calif., 5 : 1-562. LACKEY, J. B., 1936. Occurrence and distribution of the marine protozoan species in the Woods Hole area. Biol. Bull., 70 : 264-278. LEBOUR, M. V., 1917. The Peridiniales of Plymouth Sound from the region beyond the break- water. /. Mar. Biol. Assoc. U. K., U (2) : 183-200. LEBOUR, M. V., 1925. The dinoflagellates of Northern Seas. Pps. I-VIII, 1-250, Mayflower Press, Plymouth, England. LOHMANN, H., 1908. Untersuchungen zur Feststellung des vollstandigen Gehaltes des Meeres an Plankton. Wiss. Mecrcsimters., Abt. Kiel, N. F., 10: 129-370. MARTIN, G. W., 1929. Dinoflagellates from marine and brackish waters of New Jersey. Univ. lou-a Stud., Stud. Nat. Hist., 12 (9) : 1-32. MASSART, J., 1920. Recherches sur les organismes inferieurs. VIII. Sur la motilite des Flagellates. Bull. Acad. Roy. Bclgique, Cl. de 5V.. 5me, Ser. VI (4-5) : 116-141. SCHILLER, J., 1933. Dinoflagellatae. In L. Rabenhorst's "Kryptogamen-Flora von Deutschland, Osterreich und der Schweiz." 10 (3) (1) : 1-167. SCHUTT, F., 1895. Peridineen der Plankton-Expedition. Ergcbn. Plankton-Expedition der Humboldt-Stiftung, 4 (M,a,A) : 1-170. THOMPSON, R. H., 1947. Fresh-water dinoflagellates of Maryland. State of Maryland Board of Natural Resources, pub. no. 67 : 1-24. VAN GOOR, A. C. J., 1925. Einige bemerkenswerte Peridineen des hollandischen Brackwassers. Rec. Trav. Bot. Neerl., 22: 275-291. WISLOUCH, S., 1924. Beitrage zur Biologic und Entstehung von Heilschlamm der Salinem der Krim. Act. Soc. Bot. Polon., 2 (2) : 99-129. WULFF, A., 1916. Uber das Kleinplankton der Barentssee. Wiss. Mcrrcsimters., Kiel, N.F., Abt. Helgoland, 13 (1) : 97-117. ZIMMERMANN, W., 1930. Neue und wenig bekannte Kleinalgen von Neapel. I-V. Zeitschr. Botanik, 23 : 419-442. THE NATURE OF CERTAIN RED CELLS IN DROSOPHILA MELANOGASTER JACK COLVARD JONES 1 AND E. B. LEWIS 2 In a stock of the spineless (ss) mutant of Drosophila melanog aster, some of the flies were observed to have bright red cells under the cuticle. The presence of these pigmented cells has been found to depend upon a recessive mutant gene located at 26.0 ± in the second chromosome. The mutant has been named "red cells," symbol, re. This paper is a brief account of the location, histology, and cytology of the red-pigmented cells of the re mutant. * METHODS A stock homozygous for re and ss has been used for all studies unless otherwise specified. The .ys mutant serves merely as a marker to check on contamination of the stock and does not have any obvious effect on the expression of re. Under crowded culture conditions re may overlap wild type. To obtain maximum ex- pression of the re mutant, it is desirable to rear the larvae on an abundant supply of yeast. In the present work, additional dried yeast or paper towelling saturated with a thick fresh yeast suspension was added to the standard culture medium on the fourth day after introducing the parents. To study the effect of trypan blue, the re mutant was grown on standard culture media containing 1.5% trypan blue. Larvae of the third stage, pupae of various ages, and young adults of both sexes were studied. Intact larvae were immersed in 0.85% NaCl or in water, covered with a cover slip, and their various tissues examined in situ under high power (970 X). Pupae' and anaesthetized adults were pinned to a paraffin dish and dissected in Beadle-Ephrussi saline or in ethyl cellosolve. While a variety of fixatives were employed, cellosolve-fixed specimens were chiefly used. Fixed specimens were transferred to methyl benzoate and finally embedded in paraffin. Serial, cross, and sagittal sections were stained principally with picric acid since this stain did not interfere with the recognition of trypan blue uptake nor of red pigment dis- tribution. Several series were stained with methylene blue. One series was stained with hematoxylin and eosin, two series with Sudan III for fat, and two series according to the Bauer test for polysaccharides. OBSERVATIONS Third-instars and early pupae of the re strain, whether examined as whole mounts or as sectioned material, do not show any pigmented cells. In older pupae, *U. S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, National Institute of Allergy and Infectious Diseases, Laboratory of Tropical Diseases, Bethesda, Maryland. 2 Division of Biology, California Institute of Technology, Pasadena, California. 220 RED CELLS IN DROSOPHILA 221 • ' ""- FIGURE 1. Dorsal view of thorax of adult Drosophila re mutant showing alignment of red- pigmented cells. Unstained whole mount. X 87. FIGURE 2. Lateral view of re mutant showing clusters of pigmented cells. C ~ cardia, E = eve. Unstained whole mount. X 63. a pale yellow substance appears in the eye and in many of the cells destined to contain red pigment granules. Red granules first appear in scattered cells in the head before becoming visible in the facet cells of the eye (Fig. 3). Whether yellow material is itself converted directly into red pigment has not been determined. As pigment in the eye changes from pale brown to dark brown and then to red brown, the number of red granules in the cells of the head and thorax increases from one or two to many per cell. Before the granules actually form, or when only one or two are present, these thoracic cells are conspicuous by their yellow tinge. By the time the eye pigment is formed, pigment is present in the cells of the head, thorax, and abdomen. Living re flies of both sexes show red-pigmented cells under the cuticle in the thorax and head (Figs. 1 and 2). While red-pigmented cells may be widely dis- tributed throughout the body of young adults, they are most numerous and con- spicuous in the head and thorax. In the thorax, loose aggregations occur in two longitudinal rows along the dorsal midline of the mesonotum and scutellum (Fig. 1), and there are less conspicuous groups on either side of these. Small clusters of pigmented cells are sometimes seen in the legs (coxa and trochanter) and isolated pigmented cells occur within the abdomen (Fig. 5). The red-pigmented cells occur either singly or in definite groups of four or five or more, but they do not form syncytia (Fig. 4). When the cells are in definite locations, as in the supra-aortal masses and alongside the anterior part of the gut (Fig. 6), they are not bounded by connective tissue membrances and hence are extremely difficult to examine in fresh dissections. The pigment granules are round, ovoid, or irregular in shape (Fig. 4) and they vary in size from less than 0.5 to about 4 microns. The number of granules per cell ranges from a few to 50 or more. The cells bearing the granules are round or ovoid (Figs. 3-6), measure about 20 microns in diameter, and have a single 222 J. C. JONES AND E. B. LEWIS >, ,*' 5 *4 -"Si- Pi & FIGURE 3. Horizontal section through head of young pupa before eye pigment has formed, showing a red pigmented fat cell (RC). Hemocyte is shown at H. Picric acid. X 940. Ref. no. 2173. FIGURE 4. Typical red-pigmented cells in the thorax. Muscles are shown at M and an unpigmented fat cell at FC. Picric acid. X 1120. Ref. no. 2174. FIGURE 5. Fat cells in the abdomen of an adult fly, showing a single cell with red granules at R. Picric acid. X 1120. Ref. no. 2172-5. RED CELLS IN DROSOPHILA round nucleus. They are thus considerably larger than hemocytes which measure between 5 and 10 microns in diameter. The red cells, when examined in wet mounts, generally have large non-refringent droplets and other inclusions in their cytoplasm, in addition to pigment. Isolated pigment cells in saline do not send out lamellar extensions, and thus differ from certain kinds of hemocytes. When larvae are reared on trypan blue media enriched with added yeast, the resulting pupae and adults have blue dye in the gut lumen, hemocytes, scattered thoracic fat cells, garland cells, and thoracic and abdominal nephrocytes. In the nephrocytes the dye appears in irregular diffuse granular masses and/or in dense aggregates. Sometimes large dye droplets are seen free in the hemocoele. Only occasionally do cells having red pigment take up the dye. Abdominal fat cells do not take up trypan blue. Red-pigmented cells of the re strain have large fat droplets (Sudan III) and smaller deposits of polysaccharide (Bauer-positive material), and are in the same size range as typical fat cells in the head and thorax. Fat cells in the abdomen of adults are distinctly larger (25-35 microns) than those in the head or thorax (14-21 microns). COMBINATIONS OF re WITH OTHER MUTANT GENES The re mutant was combined with mutants which affect the eye color in order to determine whether the red granules in the fat cells are related to the eye pigments. When re is combined singly with mutants which remove the brown component of the eye pigment, namely, vermilion, cinnabar or scarlet, the homozygous double mutant combination has no pigment in the fat cells. When re is combined with the brown mutant, which removes the red component and leaves the brown component of the eye pigment, the homozygous double mutant has typical red fat cells. It should be added that re does not modify the eye color of wild-type nor of any of the above mutant types. The re mutant was combined with Microcephalus, a dominant mutant, locus 60.0 in the third chromosome which frequently results in a completely eyeless fly. Eyeless specimens thus obtained show the same degree of pigment development in the fat cells as do the cells of re flies with normal eyes. DISCUSSION The red-pigmented cells in Drosophila adults resemble typical peripheral fat body cells of the thorax and head in size, shape, general distribution, and possession of deposits of fat and polysaccharide.3 However, they do not usually stain vitally with trypan blue, while some other typical head and thoracic fat cells without red pigment often incorporate the dye. Those relatively few red-pigmented cells present in the abdomen are usually peripheral in location and of about the same 3 Dr. M. T. M. Rizki, who has examined our re mutant, independently concludes from his observation of the red-pigmented cells that they are fat cells (personal communication). FIGURE 6. Sagittal section through thorax of adult, showing clusters of cells lateral to the gut (G), some with trypan blue and others with red granules (RC). Picric acid. K 940. Ref. no. 2174-10. 224 J. C. JONES AND E. B. LEWIS size as the thoracic fat cells. Red fat cells are larger than typical hemocytes and do not send out lamellar extensions ("pseudopodia") in fresh dissections as many hemocytes do in vitro. None of the sessile or circulating hemocytes examined had red pigment. Indeed, so far as the authors are aware, there is no report of hemocytes with colored pigments among the insects. Red pigment in fat cells first appears in the pupal stage at about the same time as pigment forms in the eyes. Combinations of re with eye color mutants strongly suggest that the pigment in the fat cells is closely related to, or identical with, the browrn eye pigment. Microscopically, brown eye pigment is red in color. That the pigment has not diffused out of the eyes and been secondarily taken up by fat cells is shown by the presence of abundant red fat cells in eyeless re flies. SUMMARY A mutant of Drosophila mclanogastcr possesses pigmented, stationary cells in the body cavity of the pupal and adult stages. The pigment is present as numerous red granules in the cytoplasm. By a number of criteria, the pigmented cells are a type of fat cell. The evidence suggests that the pigment is related to, or identical with, the brown component of the eye pigment and that it develops in the fat cells autonomously. VASCULAR BUDDING, A NEW TYPE OF BUDDING IN BOTRYLLUS1.2 HIDEMITI OKA AND HIROSHI WATANABE Zoological Institute, Tokyo Kyoiku University, Tokyo, Japan In the early days of the investigation on compound ascidians, it was believed that botryllids propagate either by stolonial budding alone or by stolonial and palleal (peribranchial) budding. It was Seeliger (1907) who, in his monumental treatise on ascidians, denied once and for all the occurrence of stolonial budding in botryllids. According to him, the supposed stolonial budding in these ascidians is palleal budding which was misinterpreted. After Seeliger, all the workers on ascidians agreed that the only budding in botryllids is palleal (rf. Huus, 1937; Brien, 1948; Berrill, 1950; ct a/.), except perhaps E. C. Herdman, who maintained as late as 1924 that in Botryllus buds are occasionally formed from the blood vessels of the test. Since 1950 we often have had opportunities to observe that, in Botryllus primigenus at least, buds are formed from blood-cells gathered at the base of ampullae. It is proposed to name this type of budding "vascular" as distinct from "stolonial," for in Botryllus the blood vessels are generally called test vessels and not stolons, and, further, the buds are formed from blood-cells, and not from the mesenchymatous septum as in stolonial budding. In this paper the process of vascular budding will be described in some detail and a comparison will be made between this and the ordinary palleal budding. HISTORICAL 3 Following are the various views concerning budding in botryllids, arranged in chronological order. Savigny (1816), in his description of the marginal tubes (test vessels) in Botryllus, seems to have regarded them as an apparatus for the production of buds ; this view, which was more fully elaborated and established by Milne-Edwards (1842), was generally adopted until Metschnikoff (1869) demonstrated that in Botryllus gemmation takes place from the sides of the ascidiozooids, i.e., budding is palleal. This view of Metschnikoff was fully confirmed by later investigators such as Delia Valle (1881), Oka (1892), et al. Qanin (1870), however, maintained that the buds can be formed also "auf langen Stolonen und weit entfernt von dem Korper der Ascidien . . ." (p. 517). 1 Contributions from the Shimoda Marine Biological Station, No. 92. 2 The cost of this research has been partly defrayed from the Scientific Research Expendi- ture of the Department of Education of Japan. 3 Because the wartime literature is only poorly represented in Japan, we consulted Prof. N. J. Berrill of McGill University, Montreal. He assured us in a letter that since 1940 no paper had been published on the stolonial budding in Botryllus. We wish to express here our thanks to Prof. Berrill for his kind information. 225 226 HIDEMITI OKA AND HIROSHI WATANABE Giard (1872) also stated that in botryllids buds might be produced from blood vessels as well as from the body- walls of the ascidiozooids. In 1891, he still in- sisted that the inability of the test vessels to produce buds had not been sufficiently demonstrated. W. A. Herdman (1886) described for Sarcobotrylloides wyvillii (and Collela pedunculata) that buds were produced intravascularly from aggregations of blood- cells. He went so far as to make the stolonial budding one of the characteristics of the family Botryllidae. Bancroft (1903b) studied an aestivating colony of B otrylloides gascoi at Naples and found that many buds were formed in blood vessels apparently independently of zooids. He believed, however, that they were developed from zooids, not from blood vessels. He states (p. 151), "As no evidence in favor of an intravascular nor intra-ampullar origin of the isolated buds was detected. I feel convinced that they were developed from the zooids of the original colony before these had de- generated entirely. The buds must have severed their connections with the parent zooids, and must have been carried into the yellow lobe that was then being formed." According to the same author the buds described by Herdman (see above), too, might have been produced elsewhere and have migrated into the vessels. Seeliger (1907) denied once and for all the occurrence of the stolonial budding in botryllids. According to him, the budding once described as stolonial is in reality typical palleal budding. As to how misinterpretation has come into exist- ence, he says (p. 999) : "Ich glaube, dass dieser Irrtum darauf zuriickzufuhren ist, dass die haufig in Riickbildung begriffenen Zooide, die bereits an Grosse abgenom- men haben und mit den stoloahnlichen Mantelgefassen innig verwachsen sind, fiir neue Knospenanlagen gehalten wurden, die sich an und aus den erweiterten Gefassampullen entwickelt batten." E. C. Herdman (1924), however, could not reconcile herself to this view of Seeliger's and expressed herself in the following way (p. 4) : "It is quite possible, however, that occasionally certain swollen knobs on the blood vessels of the test may give rise to buds." She adds, however, that "it is certainly not usual in Botryllus and there is no definite proof that it has ever occurred." To sum up, it is today a well established fact that in botryllids buds are generally formed from the sides of ascidiozooids. Further, being organs for blood propulsion and respiration and perhaps also for excretion of test matrix, the ampullae are never transformed into new zooids. The problem is whether or not under certain circumstances buds are also formed from the walls of the blood vessels or the ampullae. MATERIALS AND METHODS The following observations were made principally on living colonies of Botryllus primigenus Oka, occurring in the vicinity of the Shimoda Marine Biological Station, Shimoda, Japan. As is well known, the zooids in Botryllus are generally grouped into systems. In Botryllus prhnigcnus, however, they are independent of one another, and each opens through its own atrial opening (cf. Oka, 1928). To facilitate observation, colonies were fixed on glass slides. These, except at the time of observation, were set out in the bay. The technique employed for fixing the colonies was the same as that described in the paper of Oka and Usui (1944). The colonies grew well on glass slides. VASCULAR BUDDING IN BOTRYLLUS 227 Various developmental stages of the bud were observed and sketched under a binocular (magnification: X 72) and an ordinary microscope (magnification: X 100). Since the buds are formed from blood-cells, vital staining of these was tried with methvlene blue and neutral red. j Observations on living materials were supplemented with examination of sections. In this case, strong Flemming's fluid wras used as the fixative and the sections were stained with Heidenhain's haematoxylin and eosin. Staining with thionin (1% aqueous solution) was also tried. All the observations were made at, or upon material obtained at, the Shimoda Marine Biological Station. It is our present duty to acknowledge our indebtedness to the Director and staff of that station for providing us facilities for carrying out this investigation. Thanks are also due to Miss Yoshiko Oshima who helped us in various ways. OBSERVATIONS Developmental cycle in the colony of Botryllus In Botryllus, buds (palleal buds) appear very precociously, so that the con- stituting members of a colony are not single individuals, but aggregates of in- dividuals belonging to three successive generations. They have been given the name "units" (Watanabe, 1953). Generally, a unit consists of more than three individuals. As a rule, two pairs of buds are formed by each individual, though not all of them develop. In the following text, as well as in Figure 1, each genera- tion is represented by only one individual. An individual has a definite life-span. Its life has been divided into 11 developmental stages by Berrill (1941a). A unit shows four different combinations of stages. On the first day, a bud of stage 1 is seen on the lateral wall of a bud of stage 6, which in its turn is connected with a zooid of stage 9 by means of the connecting B D FIGURE 1. Developmental cycle of the" unit in the colony of Botryllus (semi-diagrammatic). A, Phase A ; B, Phase B ; C, Phase C ; D, Phase D. HIDEMITI OKA AND HIROSHI WATANABE vessel, i.e., the colonial circulatory system. The combination of stages is 1-6-9. This phase is called phase A (Fig. 1, A). On the second day, the bud of stage 1 has developed into a bud of stage 3. The bud of stage 6, on the other hand, has grown into a bud of stage 8. This latter is attached to the parent zooid which is still in stage 9. The combination of stages is 3-8-9. The phase is called phase B (Fig. 1, B). Similarly, the phase on the third day is called phase C (combination of stages : 4—8-9) (Fig. 1, C), and that on the fourth day phase D (combination of stages: 5-9-11) (Fig. 1,D). On the fifth day, the bud of stage 5 has grown into stage 6 with a new bud of stage 1 formed upon it. It is attached to the zooid of stage 9. The zooid of stage 11 has completely disappeared. The unit thus returns to phase A and starts a new cycle. In each unit, the four phases are regularly repeated. Since all the units in a colony are exactly coordinated, we can also speak of the phases of the colony as a whole. A colony has four successive phases, which constitute a developmental cycle. Formation of the -vascular bud and its further development The formation of the bud is initiated by gathering of particular blood-cells (diameter ca. 3-4 ju.; see below) under the epidermis at the base of ampullae (Fig. 6). The number of cells is about 15-20. An intensive cell division follows, and, in one hour or so, a mass of cells (diameter ca. 20 /*) is formed (Figs. 2, 7). Since an ampulla is about 100-110 ^ in diameter, the mass occupies about one-fifth of its width. Active cell division continues, and, in two or three hours, a blastula-like struc- ture is formed (Figs. 3, 8), At the same time, the side-wall of the ampulla that lies over the blastogenic mass begins gradually to protrude, and, in four to five hourSj becomes distinctly visible as a bud (diameter 40-50 yu,) (Figs. 4, 9). At this stage, which will be designated stage 3, the anterior wall of the inner vesicle that faces the epidermis of the ampulla is two or three cells thick, while the remaining walls are very thin. Morphologically, the vascular bud of this stage exactly corresponds to the palleal bud of stage 3 (diameter ca. 48 /x) except that it has no ova. The cell layer constituting the outer vesicle or ectoderm of the bud is a direct continuation of the wall of the ampulla. The layer is at first closely applied to the inner vesicle, but later an ample space is formed between the two into which various kinds of blood-cells migrate, thus giving the impression that the inner vesicle is floating in the middle of the projecting part. Development of the inner vesicle is as follows. As the anterior wall continues to expand, two vertical folds appear. These gradually extend backwards until they divide the vesicle into a median and two lateral chambers, which become later the central pharyngeal chamber and a pair of lateral atrial chambers. Next, three evaginations are formed, representing the heart, the neural mass and the intestine, respectively. Later development is primarily an elaboration of these unit regions. Thus, the blastogenesis in vascular buds is an exact replica of that in palleal buds. VASCULAR BUDDING IN BOTRYLLUS 229 FIGURES 2-3. Development of the vascular buds. Figure 2 shows ampullae just before the appearance of vascular buds. X 120. 230 HIDEMITI OKA AND HIROSHI WATANABE FIGURES 4-5. Development of the vascular buds (continued). X 120. VASCULAR BUDDING IN BOTRYLLUS 231 6 7 FIGURES 6-7. Development of the vascular buds ; in sections. X 1200. 232 HIDEMITI OKA AND HIROSHI WATANABE 8 9 FIGURES 8-9. Development of the vascular buds ; in sections (continued) . Fig. 8, X 1200 ; Fig. 9, X 1000 VASCULAR BUDDING IN BOTRYLLUS 233 Nature of the blood-cells The blood-cells from which the inner vesicle originates are small, round cells. They show a strong affinity for thionin; further, they are more strongly stained by such vital stains as methylene blue (1/10,000 aq. sol.) or neutral red (1/10,000 aq. sol.) than other blood elements. In sections, it is seen that the cells have a hyaline cytoplasm; their nucleus is filled with a dense network of chromatin and lacks a nucleolus. The total picture suggests that they are cells of a primitive nature. They are the lymphocytes in the terminology of Sabbadin (1955). Time of appearance The appearance of the vascular buds is limited to a certain phase in the life cycle of the colony. They appear only during a short period (about 10 hours), extending from later phase B to early phase C. At that time, the colony is at the maximum of its activity and, accordingly, the growth of the ampullae is also very active. In other phases, no buds are formed even where they are expected to appear. In phase D we sometimes find small buds ; however, they are not new buds but older ones that have appeared in early phase C and are now on the way to dis- solution. Site of appearance In Botryllns colonies, numerous blood vessels traverse the test and terminate in contractile ampullae at the periphery of the colony; a flow of blood is maintained by them independently of heart action. The buds appear at the base of ampullae at a distance of about 0.6-0.7 mm. from the tip (Fig. 10). FIGURE 10. Vascular buds at the end of budding period. Note the various sizes. 234 HIDEMITI OKA AND HIROSHI WATANABE On each ampulla, only one bud is formed at a time. Not a single case has been recorded in which the buds are formed from the side-wall of the ordinary vessels. So far as is known, the appearance of the vascular buds is restricted to the most actively growing edges of the colony where the ampullae are especially numerous and active. Degeneration of the buds At the end of the budding period (early phase C) we often find tens and hundreds of newly formed buds, but not all of them continue to develop. Only those which surpass a certain size can develop and form perfect zooids, while the remaining ones undergo involution without even approximately reaching the stage wrhen the two vertical folds appear. For example, in a case out of 200 newly formed buds, only 70 developed into perfect ascidiozooids. Table I shows an example of different sizes of the buds in a colony at the end of the budding period. Of these 36 buds, those larger than 40 //, continued to develop (20 buds, or 56%), while those under 35 ^ soon began involution and disappeared completely (16 buds, or 44%). Relation between pallcal and vascular budding As is shown in Table II, the palleal bud appears in phase A. The vascular bud appears, as has already been pointed out, at the end of phase B, and attains stage 3 in phase C. It grows rapidly and becomes stage 5' (a stage a little behind 5) in phase D and stage 6' (a stage a little behind 6) in phase A of the following cycle. In phase B the vascular bud attains the stage 8, and from this moment on it develops exactly synchronously with the corresponding palleal bud. It also dies synchronously with the latter. In brief, the life of the zooid produced by palleal budding extends over 12 days, while that of the zooids produced by vascular budding lasts for ten days. The development, especially in its early stages, progresses a little more rapidly in the vascular than in the palleal bud. When the vascular bud has attained stage 6' we see buds of stage 1 appearing on its peribranchial walls. Thus the vascular bud, once formed, propagates by palleal budding. TABLE I Various sizes of vascular buds at the end of the budding period Diameter (in M) Number of buds ca. 70 3 ca. 50 10 ca. 40 7 ca. 35 4 ca. 30 8 ca. 20 4 Total 36 VASCULAR BUDDING IN BOTRYLLUS 235 TABLE II Relation between palleal and vascular budding, printed in bold-face type Vascular buds are Day Cycle Phase Palleal budding Vascular budding I A 1 II B 3 III n C 4 3 IV D 5 5' V A 1-6 1-6' VI B 3-8 3-8 VII n 1 C 4-8 4-8 3 VIII D 5-9 5-9 5' IX A 1-6-9 1-6-9 1-6' X B 3-8-9 3-8-9 3-8 XI n 2 C 4-8-9 4-8-9 4-8 3 XII D 5-9-11 5-9-11 5-9 5' Each vertical column represents the development of the same individual. In the growing edges of a colony, the test vessels grow out continuously. The distal end of the ampulla grows out and forms the new tip, while its basal part is continuously transformed into the ordinary vessel. This means that the vascular bud, though first formed at the base of the ampulla, is gradually removed from the latter. By the time the bud has attained stage 4-8, it is already at some distance from the ampulla. At the same time we see that a new vascular bud is being formed at the base of the ampulla (Fig. 11). Thus an ampulla forms only one bud at a time, yet it can produce many consecutively. As the vascular bud is formed a little later than the corresponding palleal one, it is at first smaller than the latter. At the end of development, however, no difference in size is perceptible between the two. The full-grown zooids formed by vascular budding are the same as those produced by palleal budding in almost every respect, even in the number of tentacles FIGURE 11. Two buds formed consecutively on the same ampulla. 236 HIDEMITI OKA AND HIROSHI WATANABE or rows of stigmata. The only difference is that in vascular buds no gonads are formed. In palleal buds, the gonads segregate as a mass from the lateral walls of the bud at an extremely precocious period. Such a segregation has never been observed in vascular buds. DISCUSSION Critical review of the earlier data With this fully established vascular budding at hand, a critical review of the earlier data concerning budding in botryllids will be attempted. Some of the earliest descriptions of the stolonial budding in Botryllns, such as those of Savigny or Milne-Edward, might have been founded on erroneous observa- tions, but what Herdman observed in Sarcobotrylloides wyi'illii and Bancroft in Botrylloides gascoi was in all probability vascular budding analogous to that described here. Some passages in Giard's paper (1872, p. 573) also make it probable that he really observed vascular budding in Botryllns. According to W. H. Herdman (1886; pp. 59, 90). the buds were formed from three sources : large cells which became ova, blood corpuscles, and the wall of blood vessels. In the case of our buds, large cells which were to become ova could not be detected in the mass of cells. The buds which were found in Botrylloides gascoi during aestivation, and which were entirely independent of parent zooids, are in our opinion certainly vascular buds, and not palleal buds developed from the zooids of the original colony and later carried into the yellow lobe, as was assumed bv Bancroft. Bancroft met J •' with the so-called yellow lobe only once, so he had no idea of its significance at the time when this was developing. He says (1903b, p. 152). "It is to be hoped that some future investigator will preserve and section the early stages of the develop- ment of the yellow lobe before the degeneration of the rest of the colony, and dis- cover why in this case the buds separate from the parent zooids at such an early stage." These buds in all probability were formed in situ from blood cells. There are some differences between our buds and those of Bancroft. a) The buds described by Bancroft were formed after all the zooids had died away. Our vascular buds, on the contrary, are formed in an active colony simultaneously with palleal buds. In passing, it may be noted that in our opinion degeneration of zooids in the case of Bancroft was not a phenomenon of aestivation, but was caused by some unfavorable conditions in the aquarium. b) The second difference is that in the case of Bancroft's material the usual coordination was entirely lost and the buds appeared so irregularly that it was practically impossible to tell to which generation they belonged, while our buds always appeared at the end of phase B of the colony. c) The third difference is that the buds of Bancroft were scattered all through the colony, while ours are located strictly at the bases of ampullae. The examples of Bancroft perhaps show that the epithelial wall of the vascular system can develop into the ectoderm of the new buds, wherever these are formed. Why is it that normally the formation of the buds is localized to the base of ampullae? It is because the flow of blood is most sluggish there, thus giving the blood-cells the best opportunity for settling. VASCULAR BUDDING IN BOTRYLLUS 237 Vascular budding in the system of budding of ascidians Whatever the type of budding may be, the initial stage of blastogenesis is a double-walled vesicle. The outer wall, called ectoblast, forms the epidermis, while the inner wall, called endoblast, forms the rest of the new zooid. The ectoblast invariably derives from the epidermis of the parent zooid, while the endoblast is variable in origin. According to this origin, the budding of ascidians is divided into three main groups : ectodermic, endodermic and mesoblastic. Vascular budding as described here, together with stolonial budding, belongs to the last group. Of all known cases of stolonial budding, that of Pcrophora comes nearest to the vascular budding of Botryllus. In Perophora the endoblast is formed from the mesen- chymatous septum of the stolon. Blood-cells in relation to budding Generally, blood-cells are considered to have nothing to do with bud formation. Berrill (1951), for example, in a survey on regeneration and budding in ascidians states: (p. 468): "Trophocytes, 'cell-packets,' and the various blood cells, other than those lymphotic cells which at times arise from septal mesenchyme or the epicardial epithelium, apparently play no part in any morphogenetic or histogenetic processes, except as victuallers." Neither epicardium nor mesenchymatous septum does exist in Botryllus. But, since the mesenchymatous septum in other forms, notably in Chn'clina, is known to gain or lose cells from or to the haemocoele as lymphochytes, the blood-cells of Botr\llns forming new buds may possibly be conceived as mesenchymatous septum dissolved into its cellular elements and cir- culating with the blood stream. According to the recent investigation of Sabbadin (1955), the blood-cells of Botryllus are of dual origin. One evolutionary series begins with a hemoblast. It has a vascular nucleus with a small amount of chromatin and cytoplasm rich in RNA-proteins. The other series begins with a lymphocyte. This is half the size of a hemoblast and is formed from the latter by division. Its nucleus is filled with a dense network of chromatin and lacks a nucleolus. Both series have their own leucocytes and vacuolated cells. As is clear from the foregoing descriptions, the blood-cells partaking in the formation of vascular buds are lymphocytes as defined by Sabbadin. It seems rather strange that buds are formed from lymphocytes, not from hemoblasts which seem to have a still greater evolutionary capacity. Size and inorpliogencsis The relation between size and morphogenesis in palleal buds has been studied by Berrill (1941b). The palleal bud appears first as a thickened disc of atrial epithelium, which rapidly transforms into a closed sphere surrounded by epidermis. The new organism is formed by a process of folding and local evaginations of this expanding sphere. Now there is seen a considerable variability in the size of the initial disc, and this determines, according to Berrill. the size and fate of the future zooid. Buds formed on too small a scale may fail to develop. Relatively small bud rudiments give rise to small zooids without gonads and with about six rows of stigmata, while 238 HIDEMITI OKA AND HIROSHI WATANABE large rudiments form a greater number of rows of stigmata and produce testes and ripening ova; between these two there are many intermediate forms. In general, discs and spheres produced by early generations are relatively small compared with those produced by later generations. According to Berrill this accounts for the fact that juvenile colonies are asexual, somewhat older colonies have well-developed testes but no ova, and only at the last do both testes and ova appear. The same explanation does not apply to the case of vascular buds. As has been demonstrated, the period during which the vascular buds appear is relatively short, extending from later phase B to early phase C. At the end of this period, we see buds of various sizes, but only those which surpass a certain size continue to develop, all the remainder degenerating. We have studied in sections the number of blood-cells taking part in the forma- tion of buds and found that variation is rather slight. The initial number of blood- cells, therefore, cannot be the cause of different sizes. The cause of the small size of some buds is to be sought in the belatedness of their appearance. Thus, small buds seem to have as full developmental capacity as larger ones. Their degenera- tion must, therefore, be understood as a manifestation of regulation of the colony as a whole. The absence of gonads in vascular buds is also not the sequence of small size, for the average size of vascular buds is of the same order as that of palleal buds, in which well-developed testes and ova appear. Regulating mechanism Of all colony-forming animals, Botrylhts is perhaps the one in which the zooids are most perfectly coordinated. We still know too little of the nature of the regulating mechanism, yet the fact that such a mechanism is working can be inferred from the following facts : a) In a colony, the zooids are exactly coordinated in budding and development. b) When two pieces of related colonies at different developmental phases are fused together, this difference is invariably equalized. This was first demonstrated by Bancroft (1903a) and is now being extensively studied by one of us (rf. Watanabe, 1953). c) The vascular buds are formed a little later than the corresponding palleal buds, but they are soon synchronized with the latter. d) Vascular buds formed too late are forced to degenerate, thus being elim- inated from the colony. Vascular budding in relation to the grou'th of the colony In Botryllus primigenus, palleal and vascular budding coexist in a colony. Maintenance and multiplication of zooids are brought about by palleal budding, while localized, rapid growth of the colony seeking a new substratum is effected exclusively by vascular budding. The colony can continue its existence by vascular budding alone. Such a state is realized when a piece of a colony containing no zooids but ampullae is isolated, or when all the zooids are experimentally removed, or again when, as in the case of Bancroft's material, all the zooids have died, owing to some unfavorable external VASCULAR BUDDING IN BOTRYLLUS 239 conditions. According to Bancroft (1899, p. 451) an isolated piece devoid of zooids never regenerated a colony, and none of the ampullae in such a piece showed the least tendency towards budding. On this point, therefore, we are quite at variance with him. Significance of our discovery for the general theory of budding in ascidians Our discovery is of significance in the following points : a) In Stolidobranchiata, to which the genus Botrylliis belongs, palleal budding has been considered the only way of asexual reproduction. We now know that Botrylliis propagates also by vascular budding. This indicates that budding of a rather primitive nature, as the vascular, has not completely disappeared even in such a highly specialized group as Stolidobranchiata. b) It is generally admitted that each species is given a single mode of asexual reproduction. Now, BotryUus propagates by two entirely different kinds of propa- gation, one ectodermic-peribranchial and one mesoblastic-vascular. c) The lymphocytes are capable themselves of organizing new individuals. Is vascular budding peculiar to Botrylliis primigenus? If we consider that of all the genera of compound ascidians it is BotryUus that has been most extensively studied till now, it is almost inconceivable that so typical a budding as the vascular has escaped the eyes of previous investigators. It is possible, although not very probable, that this type of budding is peculiar to our Japanese species. We have observed the same budding also in BotryUus communis, another Japanese species, which, however, in our opinion is homospecific with B. primigenus. SUMMARY 1. In Botrylliis primigenus, it has been found that, in addition to palleal budding, new buds are formed also from aggregations of blood-cells at the base of ampullae. 2. The blood-cells partaking in the formation of buds are lymphocytes as de- fined by Sabbadin. j 3. The formation of buds is possible only at a certain phase in the develop- mental cycle of the colony. 4. Even then, the buds are formed, not on all ampullae, but only on those lying in the most vigorously growing edges of the colony. 5. Our discovery is of significance in the following three points : a. The ability to form new buds from the vascular wall has not completely disappeared in Stolidobranchiata. b. A species can propagate by two entirely different kinds of budding. In our case, one is ectodermic-palleal and one mesoblastic-vascular. c. The lymphocytes are themselves capable of organizing new individuals. LITERATURE CITED BANCROFT, F. W., 1903a. Variation and fusion of colonies in compound ascidians. Proc. California Acad. Sci. (Scr. 3), 3: 137-186. BANCROFT, F. W., 1903b. Aestivation of Botr\lloldes gascoi Delia Valle. Mark Anniversary Volume, 147-166. 240 HIDEMITI OKA AND HIROSHI WATANABE BERRILL, N. J., 1941a. The development of the bud in Botryllits. Biol. Bull.. 80: 169-184. BERRILL, N. J., 1941b. Size and morphogenesis in the bud of Botryllits. Biol. Bull., 80: 185-193. BERRILL, N. J., 1950. The Tunicata with an account of the British species. London. Ray Society. BERRILL, N. J., 1951. Regeneration and budding in tunicates. Biol. Revicivs, 26: 456-475. BRIEX, P., 1948. Enbranchement des Tuniciers. /;; : Traite de Zoologie, edited by Pierre-P. Grasse. 11. Paris. Masson et Cie. DELLA VALLE, A., 1881. Nuove contribuzioni alia storia naturale delle ascidie composte del Golfo di Napoli. Atti Ace. Lined Mem. (Ser. 3), 10: 431-498. GANIN, M., 1870. Neue Tatsachen aus der Entwicklungsgeschichte der Ascidien. Zcitsehr. f. iviss. ZooL, 20: 512-518. GIARD, A., 1872. Recherches sur les ascidies composees ou synascidies. Arch. ZooL E.rp. ct Gen., 1 : 501-687. GIARD, A., 1891. Sur le bourgeonnement des larves d'Astellium spongiforme Gd. et sur la poecilogonie chez les Ascidies composees. C. R. Acad. Sci., 112: 301-304. HERDMAN, W. A., 1886. Report on the Tunicata collected during the voyage of H. M. S. "Challenger," during the years 1873-76. Vol. 2, Ascidiae compositae. Chall. Rep. ZooL, 14. Edinburgh. British Government. HERDMAN, E. CATHERINE, 1924. Botryllus. Liverpool Marine Biology Committee Memoirs, 26. London. Williams & Norgate. Huus, J., 1937. Tunicata: Ascidiacea. /;; : W. Kukenthal and T. Krumbach's Handbuch der Zoologie, 5. Berlin and Leipzig. Walter de Gruyter. METSCHNIKOFF, E., 1869. Uber die Larven und Knospen von Botryllus. Bull. Acad. St.- Petcrb., 13: 293-298. (Cited after W. A. Herdman. ) MILNE-EDWARDS, H., 1842. Observations sur les ascidies composees des cotes de la Manche. Mem. Acad. Sci. Paris, 18: 217-326. OKA, A., 1892. Uber die Knospung der Botrylliden. Zcitsehr. f. iviss. ZooL. 65: 521-547. OKA, A., 1928. Uber die merkwiirdige Botryllus-Art. B. primigcnus nor. sp. Proc. Imp. Aead. Japan, 4: 303-305. OKA, H., AND M. Usui, 1944. On the growth and propagation of colonies in Polycitor mutalnlis ( Ascidiae compositae). Sci. Rep. Tokyo Bunrika Daic/aku (Sec. B), 7: 23-53. SABBADIN, A., 1955. Studio sulle cellule del sangue di Botryllus selilosscri (Pallas). Arch. Ital. Anat. EmbrioL, 60: 33-67. DE SAVIGNY, M., 1816. Memoires sur les animaux sans vertebres. Paris. (Cited after W. A. Herdman. ) SEELIGER, O., 1893-1907. Tunicata (Manteltiere). In: Bronn's Klassen- und Ordnungen des Tierreichs, 3, Suppl. Leipzig. C. F. Winter'sche Verlagshandlung. WATANABE, H., 1953. Studies on the regulation in fused colonies in Botryllns primit/cnits. Sci. Rep. Tokyo Bunrika Daiuaku (Sec. B), 7: 183-198. OPTOKINETIC TESTING OF CYCLOPEAN AND SYNOPHTHALMIC FISH HATCHLINGS l K. T. ROGERS Department of Zoology, Obcrlin College, Obcrlin, Ohio, and The Marine Biological Laboratory, Woods Hole, Mass. Previous work on Fundulus heteroclitus embryos that were perfect cyclopeans, owing to treatment with ethyl alcohol, indicated that there is a strong tendency for the right side of the eye to function developmentally as a right eye would, in sending optic fibers to the left side of the brain, and for the left side of the eye to send fibers to the right side of the brain (Rogers, 1952). It seemed of interest to determine whether the right side of the cyclopean eye functions physiologically as a right eye would, and the left side, as a left eye. Optokinetic drum testing of optically abnormal amphibia (Sperry, 1944) suggested a means of making such tests. Even if positive optokinetic drum results were obtained with perfect cyclopean fish, it would be impossible to eliminate only one side of the retina sur- gically without damage to the other, but it might be possible to ablate one optic tectum to learn something further of the pathways involved. Nerve-stained sections could then be used to correlate the microscopic anatomy with the physio- logical results. Pearcy and Koppanyi (1924) moved the left eye of a large goldfish to an artificial orbit in the top of the cranium, just to the left of the midline, without severing the nerve. The effect on equilibrium was noted and vision tested during four weeks. They stated that they had produced a real "experimental cyclops." This eye, however, has few of the characteristics of the cyclopean eye. It is not formed from the medullary plate material on both sides of the midline, it is not in the typical ventral position, and it did not develop its nerve connections while it was in close relation to the diencephalic floor on both sides of the midline. Stockard (1909, p. 285) says in regard to cyclopean Fundulus heteroclitus treated with magnesium chloride solutions in the early stages : "Many embryos, showing the cyclopean defect in various degrees, hatched normally and were capable of swimming in a manner indistinguishable from ordinary two-eyed fish. These monsters gave many indications of ability to see. They went to the more brilliantly lighted side of the dish with the normal ones. They darted away in normal fashion when any object was placed in front of the eye, while similar objects put at equal distances from their tails caused no excitement." There appear to l)e no other reports in the literature of attempts to test cyclopean individuals for vision. MATERIALS AND METHODS In the 1955 season 16,987 Fundulus heteroclitus eggs that successfully passed the early cleavage stages were treated with magnesium chloride solutions. Of 1 This work was supported by Public Health Service grants, No. B-760 and No. B-760(C), and in 1955 by aid from the Committee on Productive Work, Oberlin College. 241 242 K. T. ROGERS 11,912 embryos surviving at least until the eyes were well formed, 0.3% were perfect cyclopeans with an eye of close to normal size, and only three such embryos hatched (Rogers, 1956). In the 1956 season 190 bowls containing somewhat larger numbers of eggs than in the 1955 work were treated in the same manner with magnesium chloride. An estimate was made that about 25,000 embryos survived until the eyes were well formed, 76 embryos (about 0.3%) were perfect cyclopeans with an eye close to normal size, and again only three such embryos hatched and swam. In 1955 the effect of temperatures between 11° and 16° C. was not tested. In 1956 water in bowls kept on the water table between June 6th and 12th was never below 15° or above 16° C. At this temperature seven bowls of 19/60 M MgCl2 solution, with a total of 1,020 eggs that were living when they reached the blastula stage, produced 7% optic abnormalities among the 560 embryos that survived until the eyes were formed. All 12 cyclopean embryos among them had reduced eyes and stunted bodies. Although the mortality rate rose to 45%, as compared with 20% in the 1955 series at 18° C., production of optic abnor- malities was increased by only 1%. In the material just described, three cyclopeans, three synophthalmics, and one anophthalmic in the 1955 series, and three cyclopeans and one synophthalmic in the 1956 series hatched and swam well enough to be tested in the optokinetic drum. In addition, from batches of Lucania parva eggs fertilized with Fundulus hctero- clitus sperm, one perfect cyclopean in 1955, and another in 1956, hatched and were tested. At first, a very large striped drum was tried, mounted on a horizontally- placed bicycle wheel revolving around a stationary central platform to hold the dishes of fish. Normal adults responded only moderately well, and hatchlings, not at all. Feeling that the hatchlings might not be affected by movement at such a distance, Mr. Michael Baron, helping with the technical work in 1955, lined an oatmeal carton only 10.5 cm. in diameter with vertical black and white stripes of one-half centimeter to one centimeter in width. When this drum was turned smoothly by hand around a small beaker containing normal hatchlings, the fish readily turned with the drum and quickly reversed directions when the drum was reversed. The fish were kept 6 to 10 days after hatching, and tested a number of times. When most of their yolk was used up, selected ones were drawn with camera lucida while under urethane anaesthesia, and all were fixed, sectioned, and stained with a modification of the Bodian protargol stain in the manner previously described (Rogers, 1952). RESULTS OF OPTOKINETIC TESTS Synophthalmic fish 89-1 (bowl number — individual fish number) and 89-2 (Fig. 4) responded directionally to drum rotation in the same manner as normal control hatchlings. Fish 89-1 did not start to turn with the drum as readily, and did not turn as rapidly as control fish, but otherwise exhibited normal optokinetic reflexes. Fish 89-2 responded nearly as well as controls. Synophthalmic fish 341-1, similar in appearance to the preceding two, did not respond to the drum, although it frequently swam at times when there were no apparent external stimuli. The unusual synophthalmic fish with right eye much reduced, 76-5 (Fig. 5), was slightly but permanently flexed toward its left. It did not orient well and often lay on its back on the bottom of the dish. Nevertheless, in any orientation, it OPTOKINETICS OF CYCLOPEAN FISH 243 could always be made to flex vigorously and repeatedly toward its own left with drum rotation in the opposite direction, whereas no movement could be elicited by drum rotation to its left. If nerve connections of such an eye are contralateral, as they normally are, the animal ought to flex or turn readily in the same direction as the' drum when the drum is rotated toward the blind side, and turn hesitantly or not at all when the drum is rotated toward the seeing side. The cyclopean "magnesium" fish 89-3, 105-1 (Fig. 1), 105-2, 340-2, 340-3, and 360-1, the cyclopean Lncania-Fitndnlns hybrids 263-1 (Figs. 2 and 3) and 478-1, and the anophthalmic "magnesium" fish 109-1 all failed to respond to any drum stimulation tried. Various tests were made on all these fish, but hatchling 105-1 was tested more extensively than the others, over a period of five days. Drums of various sizes with various widths of striping and turned at various speeds under various lighting conditions were used. The fish were sometimes positioned very close to the beaker side and the drum rotated so that its surface passed just outside the glass and very near the fish. Black-and-white, and red- and-white stripes, striped cones revolving under as well as around the beaker, and flat discs with alternate opaque cardboard and cut-away strips radiating from a central hub and rotated over a light bulb with the fish just above the disc, were also tried with uniformly negative results. Any movements on the part of the fish appeared to be fortuitous and unrelated to the drum rotation. When testing was not being carried on, the anophthalmic 109-1 appeared to swim around the dish at more frequent intervals than normal control fish. The failure of the perfect cyclopean fish to respond to the drum in 1955 led to the second season's work with the hope of obtaining positive results or of making the negative results more certain. The lack of response also made of prime im- portance the question as to whether the perfect cyclopean fish can see at all. Un- fortunately, all tests so far tried have failed to give rigorous proof. "Baiting" the fish from outside a glass container (Sperry, 1949) appears to be impossible with these hatchlings. Numerous attempts to observe the effect of a differentially lighted container (Stockard, 1909) failed to yield unequivocal results in the present work. The chromatophore response to visually perceived light in adult Fundulus (Butcher, 1938) was considered, but light does not have an obvious effect on the chromatophores of normal hatchlings. Potential-recording methods seem imprac- tical with these small fish. Fish 89-3 was placed in a glass container within the large drum mounted on the bicycle wheel, and the drum rotated for five-minute periods with three- to four-inch-wide black-and-white stripes visible, and then for similar periods with the stripes covered by a blank white paper. The number of times the fish swam, and the total length of time in seconds it was swimming, were recorded. A number of tests made it clear that these data were too variable to afford rigorous proof of ability to see. The only evidence obtained consisted of the apparent ability of perfect cyclopeans 89-3, 105-1, and 105-2 to localize the tip of a fine forceps when it was placed in front of them. These observations were not considered conclusive and therefore no attempt was made to repeat them on the perfect cyclopeans of the second season. Fish 105-1 responded 5 or 6 times in succession to a forceps placed close in front of it, by jerking quickly away. On the two succeeding tries, however, the fish slowly and deliberately came off the bottom and moved forward, accurately placing its tubular mouth against the forceps. 244 K. T. ROGERS FIGURE 1. View of right side of cyclopean Fundulus hctcroclitus hatchling 105-1. The anterior part of the body is flexed slightly to the right to show the eye. Drawn with camera lucida, urethane anaesthesia, X 20. FIGURE 2. View of right side of cyclopean Lucania pari: V * *sf.~£ljsi f 7V 6 8 OPTOKINETICS OF CYCLOPEAN FISH 247 retina projects posteriorly well into the third ventricle, instead of simply being continuous with the diencephalic wall. The pigment epithelium, however, is again a reflection of the diencephalic wall. In 340-2 the optic fibers clearly decussate as they leave the retinal tissue, with a large bundle from the right side of the eye going to the left tectum and a bundle half as large from the left side of the eye going to the right tectum. Fish 340-3 also has a large fiber bundle going to the left tectum and a small bundle to the right tectum, but fiber paths as they leave the eye cannot be followed clearly enough to see whether they decussate. All of the optic fibers of 360-1 go to the left tectum. Anophthalmic fish 109-1 has no eye tissue identifiable in section. DISCUSSION The two synophthalmic fish that responded directionally to the optokinetic drum in the same manner as control fish were found to have optic nerves that distribute equally to the two optic tecta after decussating at least in part, and possibly entirely. The synophthalmic fish of similar external appearance that failed to respond to the drum was found to have optic fibers distributing solely to the telencephalon on one side, and was probably blind. The failure of the perfect cyclopean fish to respond to the drum is harder to explain. Until the histological sections of the first season were studied, it was thought possible that in the four cases tested the optic nerve fibers from each side of the eye just happened to distribute about equally to both optic tecta. Horizontal optokinetic reflexes might then be cancelled out. This cannot be the explanation, however, because two of the total of eight perfect cyclopean fish tested have optic nerve fibers distributing to the optic tectum of one side only. No other explanation for the failure to respond is apparent. A right or left eye, even when displaced far toward the midline (fish 76-5), will send impulses that will initiate reflexes in response to the drum. Therefore, the two pupils or two separate retinal arcs of the typical synophthalmic condition are not requisite for the response. There is slight evidence that the cyclopean fish can see objects placed close in front of them. Comparison of their behavior with that of anophthalmics when objects are introduced near them, leads to the belief that the cyclopeans can see, but does not afTord rigorous proof. If they can see, the failure to respond to the drum remains perplexing. The individual with only one functional eye in the synophthalmic position and nerve distributing only homolaterally, responded directionally to a rotating drum in the same way as one-eyed anurans in which the regenerating optic nerve had been forced to grow to the homolateral optic centers (Sperry, 1945). Thus, Sperry's results in a regenerative situation are extended to a primary developmental situation. All figures are photomicrographs of Bodian preparations. FIGURE 6. Transverse section through synophthalmic eyes of fish 89-2. Note ventral fusion of sensory retinal arcs. X 115. FIGURE 7. Transverse section through perfect cyclopean eye of fish 105-1. Nasal pits are above, tubular mouth below. X 115. FIGURE 8. Transverse section of portion of eyes of fish 76-5. The large left eye sends a normal-sized nerve to the left side of the brain. X 800. 248 K. T. ROGERS SUMMARY 1. Perfect cyclopean, closely synophthalmic, and anophthalmic fish hatchlings were obtained by magnesium chloride treatment or by hybridization. 2. Synophthalmic fish, with distribution of optic nerve fibers generally similar to controls, responded essentially normally to a horizontally rotating optokinetic drum. 3. A synophthalmic fish, with optic fibers distributing to the telencephalon of one side, failed to respond to the drum. 4. A fish with only one functional eye in the synophthalmic position, and optic fibers distributing entirely homolaterally, responded in the opposite direction when the drum was rotated toward the blind side. 5. Although there was other, somewhat inconclusive, evidence that they could see, eight perfect cyclopean fish failed to respond to a horizontally rotating drum. LITERATURE CITED BUTCHER, E. O., 1938. The regions of the retina related to the different chromatophoric re- sponses in Fundnlns hctcroclitus. Bull. Mt. DCS. Is. Biol. Lai'., 1938: 18-19. PEARCY, J., AND T. KOPPANYI, 1924. The effects of dislocation of the eye upon the orientation of the goldfish (Carassins auratits). Science, 60: 502-503. ROGERS, K. T., 1952. Optic nerve pattern evidence for fusion of eye primordia in cyclopia in J:iindnlus hctcroclitus. J. E.i-p. Zool, 120: 287-310. ROGERS, K. T., 1956. Re-examination of the production of cyclopia in Funduliis hctcroclitus with magnesium chloride and ethyl alcohol. Biol. Bull., 110: 344-351. SPERRY, R. W., 1944. Optic nerve regeneration with return of vision in anurans. /. Ncuro- physiol., 1 : 57-70. SPERRY, R. W., 1945. Restoration of vision after crossing of optic nerves and after contralateral transplantation of eye. /. Ncnropliysiol., 8: 15-28. SPERRY, R. W., 1949. Reimplantation of eyes in fishes (Bathvc/ohiits soporator) \vith recovery of vision. Proc. Soc. E.rp. Biol. and Mcd., 71 : 80-8L STOCKARD, C. R., 1909. The development of artificially produced cyclopean fish — "the mag- nesium embryo." /. E.vp. Zool., 6 : 285-337. THE REVERSIBLE REPLACEMENT OF POTASSIUM BY RUBIDIUM IN ULVA LACTUCA 1 GEORGE T. SCOTT AND ROBERT DeVOE - Department of Zoology, Obcrlin College, Oberlin, Ohio and the Marine Biological Laboratory, Woods Hole, Massachusetts The substitution of rubidium ion for potassium ion in physiological processes has been studied for over 80 years. This subject is of special interest in view of the close chemical similarities of the two ions, rubidium being much more akin to potassium than is sodium. Permeability studies have revealed a high penetration rate of both rubidium and potassium ion (Brooks, 1932, 1939). Studies on the replacement of potassium by rubidium have compared, on the one hand, the amount of physical replacement of potassium by rubidium within the cell, and on the other hand the suitability of rubidium as a substitute for potas- sium physiologically. Complete physical replacement of potassium by rubidium has been shown for Chlorclla (Pirson, 1939), and yeast (Scott, unpublished data) ; partial physical replacement occurs in vertebrate muscle (Follis, 1943; Heppel and Schmidt, 1938; Mitchell, Wilson and Stanton, 1921) and in the duckweed Lcinna minor (Pirson and Kellner, 1952). Rubidium can completely substitute for potas- sium in the repolarization of nerve (Feng and Liu, 1951 ; Gallego and De No, 1947), the restoration of heart beat in the frog heart (Ringer, 1884; Zwaardemaker, 1919), the activation of spermatozoa (White. 1953). the transport into the eryth- rocyte (Love and Burch, 1953), the activation of zymase (Giordani, 1932), the respiration of mitochondria (Pressman and Lardy, 1952), the bacterial production of pyruvic acid from malic acid ( Lwoff and lonesco, 1947), the activation of pyruvic phosphoferase (Kachmar and Boyer. 1953), and for growth in the bacterium Streptococcus faccalis (MacLeod and Snell, 1948). Rubidium is less effective than potassium in supporting assimilation, chlorophyll formation and cell division in Chlorclla (Pirson, 1939), in supporting growth in Lactobacillus casei (MacLeod and Snell, 1948), or Nitzschia clostcriuui ( Stanberry, 1934). or for the secretion of adrenalin (Hermann, 1942). Rubidium is ineffective in supporting antibiotic activity in the subtilin-producing strain of Bacillus subtilis, although it promotes growth as well as does potassium in this bacterium (Feeney and Gari- baldi. 1948). The element is definitely toxic at high concentration in certain bacteria, being antagonized by potassium (Scharrer and Schropp, 1933), and is fatal to rats when it is substituted for up to 50-66 per cent of the potassium in the tissues (Follis, 1943; Heppel and Schmidt, 1938; Mitchell, Wilson and Stanton. 1921 ; Zipser and Freedberg, 1952). Previous work has been concerned with the extent of replacement of potassium 1 The research was aided by support from contract no. AT (11-1) -181, Division of Biology and Medicine, U. S. Atomic Energy Commission. 2 Present address : The Rockefeller Institute for Medical Research. 249 250 GEORGE T. SCOTT AND ROBERT DEVOE by rubidium or the physiological results thereof, rather than with the kinetics of replacement. The present work is a kinetic study of the reversible exchange of rubidium for potassium and of potassium for rubidium. MATERIALS AND METHODS The organism selected for this study was the green alga, Ulva lactiica. This marine organism, like most cells living in a high-sodium, low-potassium environ- ment, normally accumulates potassium and partially excludes sodium. Consisting of large membranous fronds two cell layers in thickness, thus presenting a large surface area for interchange with the environment, this alga is particularly well suited for investigation of this nature. Large fronds were collected from Perch Pond, near Falmouth, Mass., and con- ditioned for at least 24 hours in running sea water under incandescent illumination. Samples, cut from the same frond, were given a brief rinse in isotonic sucrose followed by a blot in absorbent toweling to remove most of the adherent sea water, then placed in large finger bowls with about 160 ml. per sample of an artificial sea water (Marine Biological Laboratory, Formulae and Methods IV, 1956), containing rubidium instead of potassium. The rubidium sea water was replaced with fresh rubidium sea water after 50 hours. Diffuse illumination was present during the experiment, which resulted in a slight drop in rubidium sea water pH. After 96 hours in rubidium sea water, the samples were placed in running sea \vater for 120 hours. Samples were removed in triplicate at various times, rinsed in isotonic sucrose for 30 seconds and blotted three times in absorbent tissue to remove extra-cellular sodium, potassium and rubidium ion. Wet and dry weights were taken and cell water calculated by difference. The samples were ground, extracted with 1 N HNO3 for two hours at 110° C., and the extracts diluted to volume in 50-ml. volumetric flasks. The extracts were analyzed for sodium, potas- sium and rubidium ions by the Beckman flame spectrophotometer. RESULTS The initial uptake of rubidium is both rapid and nearly complete within the first four hours, the time at which the first samples were taken. At 96 hours the rubidium ion concentration reaches a maximum of about 87 per cent of the control value of potassium ; longer immersion in rubidium sea \vater does not result in an increase above this maximal concentration value. The potassium loss is initially rapid and then continues to decrease in a manner parallel to that of the control, reaching a minimum of about 13 per cent of the control (Fig. 1). When the fronds are placed in running sea water, the kinetics of the replace- ment of rubidium by potassium are quite different from those involving the re- placement of potassium by rubidium. After an initial sharp drop in rubidium for 10 hours and a sharp rise in potassium for 10 hours, the rates of loss of rubidium and rise of potassium decrease. After 120 hours in running sea water the potas- sium content of the alga has practically reached that of the control, wrhereas the rubidium content has been reduced to only 30 per cent of its maximal value. Longer immersions were not practical, as the alga begins to show signs of aging. POTASSIUM AND RUBIDIUM EXCHANGE 251 100 120 HOURS 140 160 ISO 200 220 FIGURE 1. The replacement of potassium by rubidium within Ulva lactuca in rubidium sea water, and the replacement of rubidium by potassium on transfer of the alga to running sea water. The arrow indicates the time of transfer to running sea water. Concentrations are expressed on a cell water basis. Throughout the experiment the sodium concentration of the samples remained essentially constant at 26 ± 4 meq. per 100 gm. cell water. DISCUSSION The initial uptake of rubidium is too rapid to be explained on the basis of a new, separate uptake mechanism for rubidium alone, for such a mechanism would have to be much faster than that previously demonstrated for potassium in this alga (Scott and Hayward, 1954b). Rather, the rate of rubidium uptake is entirely consistent with the known rate of potassium turnover at 20° (Scott and Hayward, 1954a). Therefore, rubidium is being transported by the same mechanism as is potassium. The cessation of rubidium uptake is probably the result of the establish- ment of an equilibrium between the rubidium and the potassium concentrations, for the total alkali metal base (Na+ + K+ + Rb+) is constant within ± 5 meq. of that of the control (76-65 meq./lOO gm. cell water). Rubidium does not seem to affect the final level of potassium re-accumulation, but it would appear to have an effect on the kinetics of potassium re-accumulation. The re-accumulation of potassium is too slow to be accounted for solely on the basis of exchange of potassium ion for rubidium ion. Rather, it would seem that two factors might be operative : 1 ) the full maintenance of the potassium-accumula- tion mechanism is not supported by rubidium ion; 2) the age of the samples may be such as to render the postassium-accumulating mechanism sluggish. Since Ulva cycles from gametophyte to sporophyte generation on the order of every two weeks, it will be seen that the time course of the experiment (9 days) includes most of one phase of the cycle of the organism. (Whether a particular frond was gametophyte or sporophyte was not determined in this work, for the two generations are morpho- logically identical.) 252 GEORGE T. SCOTT AND ROBERT DEVOE In another experiment samples were left in rubidium sea water for as long as 144 hours before transfer to running sea water. The rate of potassium re-accumu- lation was identical to that found after 96 hours in the experiment reported above ; hence the increased time in rubidium sea water was not progressively detrimental to the potassium-accumulating mechanism. The presence of rubidium within this organism did not prevent the formation and discharge of a germinal ridge in some samples. These samples were not used in the analyses. SUMMARY 1. Rubidium ion replaces two-thirds of the potassium of Ulva lactuca within four hours after being placed in rubidium-containing sea water. 2. The rubidium concentration does not increase more than 5 meq. during the remainder of the experiment. 3. Potassium re-accumulation in running sea water is slower than the initial exchange of rubidium ion for potassium ion. 4. The disparity in the exchange kinetics is discussed. LITERATURE CITED BROOKS, S. C., 1932. The rate of penetration of rubidium into living cells of Valonia and its apparent ionic radii. /. Cell. Comp. Physiol., 2 : 223-231. BROOKS, S. C., 1939. Ion exchanges in accumulation and loss of certain ions by the living protoplasm of Nitella. J. Cell. Comp. Physiol., 14 : 383-401. FEENEY, R. E., AND J. A. GARIBALDI, 1948. Studies on the mineral nutrition of the subtilin- producing strain of Bacillus subtilis. Arch. Biochem., 17 : 447-458. FENG, T. P., AND Y. M. Liu, 1951. The membrane potential of potassium depleted nerve. Chinese J. Physiol., 18 : 61-70. FOLLIS, R. M., JR., 1943. Histological effects in rats resulting from adding rubidium or cesium to a diet deficient in potassium. Amer. J. Physiol., 138 : 246-250. GALLEGO, A., AND R. L. DE No, 1947. The effect of several univalent ions on frog nerve. /. Cell. Comp. Physiol, 29 : 189-206. GIORDAN:, M., 1932. Activation of zymase with rubidium chloride. Ann. Chim. Applicata, 22: 153-156. HEPPEL, L. A., AND C. L. A. SCHMIDT, 1938. Studies on the potassium metabolism of the rat during pregnancy, lactation, and growth. Univ. Calif. Pub. Physiol., 8 : 189-205. HERMANN, H., 1942. The effect of the cations of alkalies and alkaline earths on the secretion of adrenaline. Bull. A cad. Med., 126: 234-236. KACHMAR, J. F., AND P. D. BOYER, 1953. Kinetic analysis of enzyme reactions : the potassium activation and calcium inhibition of pyruvic phosphoferase. /. Biol. Chem., 200 : 669-682. LOVE, W. D., AND G. E. BURCH, 1953. A comparison of potassium42, rubidium86 and cesium134 as tracers of potassium in the study of cation metabolism of human erythrocytes in vitro. J. Lab. Clin. Med., 41 : 351-362. LWOFF, A., AND H. IONESCO, 1947. Replacement of potassium by rubidium and cesium in the bacterial production of pyruvic acid from malic acid. C. R. Acad. Scl., 225 : 77-79. MACLEOD, R. A., AND E. E. SNELL, 1948. The effect of related ions on the potassium require- ment of lactic acid bacteria. /. Biol. Chem., 176: 39-52. MARINE BIOLOGICAL LABORATORY, 1956. Formulae and Methods IV, p. 55. MITCHELL, P. H., J. W. WILSON AND R. E. STANTON, 1921. The selective absorption of potas- sium by animal cells. II. The cause of potassium selection as indicated by the ab- sorption of rubidium and cesium. /. Gen. Physiol., 4: 141-148. PIRSON, A., 1939. The effect of alkali ions upon the growth and metabolism of Chlorella. Planta, 29 : 231-261. POTASSIUM AND RUBIDIUM EXCHANGE 253 PIRSON, A., AND D. KELLNER, 1952. Physiological action of rubidium. Ber. deut. botan. Ges., 65 : 276-286. PRESSMAN, B. C, AND H. A. LARDY, 1952. Influence of potassium and other alkali cations on respiration of mitochondria. /. Biol. Chem., 197 : 547-556. RINGER, S., 1884. An investigation regarding the action of rubidium and cesium salts compared with the action of potassium salts on the ventricle of the frog's heart. /. Physiol., 4 : 370-379. SCHARRER, K., AND W. ScHROPP, 1933. Sand and water culture experiments with lithium and rubidium with special reference to the possibility of substituting these elements for potassium in plant nutrition. Ern'dhr. Pflanse, 29 : 413-425. SCOTT, G. T., AND H. R. HAYWARD, 1954a. The influence of temperature and illumination on the exchange of potassium ion in Ulva lactuca. Biochim. Biophys. Acta, 12 : 401. SCOTT, G. T., AND H. R. HAYWARD, 1954b. Evidence for the presence of separate mechanisms regulating potassium and sodium distribution in Ulva lactuca. J. Gen. Physiol., 37: 601. STANBERRY, F. A., 1934. The replacement of potassium by rubidium in Nitsschia closterium. J. Mar. Biol. Assoc., 19 : 931-940. WHITE, I. G., 1953. The alkali metal requirements of ram and bull spermatozoa. Australian J. Biol. Sci., 6 : 716-724. ZIPSER, A., AND A. S. FREEDBERG, 1952. The distribution of administered radioactive rubidium86 in normal and neoplastic tissue of mice and humans. Cancer Res., 12 : 867-870. ZWAARDEMAKER, H., 1919. On physiological radioactivity. /. Physiol, 53 : 273-289. THE MORPHOLOGY AND LIFE-HISTORY OF THE DIGENETIC TREMATODE, MICROPHALLUS SIMILIS (JAGERSKIOLD, 1900) BAER, 1943 x HORACE W. STUNKARD U. S. Fish and Wildlife Service and the American Museum of Natural History, Nczv York 24, N. Y. Although significant observations had been reported earlier, the first complete life-histories of microphallid trematodes were worked out by Cable and Hunninen (1940) and Rankin (1940b). MTntosh (1865) had described and figured larval worms from the tissues of the green crab, Carcinides niaenas (Linnaeus), taken at St. Andrews, Scotland, but the life-cycles of trematodes were quite unknown at the time and MTntosh described the structures as eggs, each of which contained a tiny worm that he surmised became a sexually mature distome in "such fishes as the Cotti, Gadi, and others," which feed on the crustaceans. He reported a speci- men of Cottus bulbalis, about a foot long, with (p. 204) "two entire specimens of Carcinus maenas, each upwards of two inches across the carapace, in its stomach, besides the partially digested debris of others." His descriptions and figures identify the worms as members of the genus Microphallus and with considerable certainty as M. similis (Jagerskiold, 1900). Although measurements were not given, his Figure 5 of an excysted specimen shows the suckers to be of approxi- mately equal size and the "small globule" (seminal vesicle) anterior to the acetab- ulum, together with the size of the gonads, evidence full development of the meta- cercaria. Levinsen (1881) described adult trematodes from the eider duck, Somateria molissima, taken at Egedesminde, Greenland, as Distoma pygmaeum and Brandes (1889) described similar worms from Tringa alpinus as Distoma clavijorme. Jagerskiold (1900) described specimens from Swedish gulls, Larus argentatus and L. fuscus, as Distoma pygmaeum var. simile. Other species have since been described from different hosts in other parts of the world. Ward (1894) described worms from American lake-fish as Distoma opacum and noted their similarity to D. pygmaeum. The parasites occurred in Amia calva, Ictalurus punctatus and Perca flavescens, and encysted larval stages were found in the crayfish, Cambarus propinquus. With the dismemberment of the old genus Distoma, Stossich (1899) erected the genus Levinsenia to include Distoma brachysomum Creplin, 1837, D. macrophallos von Linstow, 1875, D. pygmaeum Levinsen, 1881, and D. opacum Ward, 1894. Liihe (1899), but not Looss (1899), as has been claimed (cf. Looss, 1902: p. 704) designated L. brachysoma, and Jagerskiold (1900) proposed L. pygmaeum, as type of the genus Levinsenia. Noting differences between D. brachysomum and D. opacum. Ward (1901) named the latter species type of a 1 The experimental work was done at the Marine Biological Laboratory, Woods Hole, Massachusetts, during the period May 1 to October 31, 1956. 254 LIFE-CYCLE OF MICROPHALLUS 255 new genus, Microphallus. In this paper he reported that the name Levinsenia Stossich is a homonym of Levinsenia Mesnil, 1897, a polychaete annelid, and that Stiles and Hassall were to propose the name Levins eniella to replace it. Although the paper by Stiles and Hassall did not appear until 1902, the name Levinseniella Stiles and Hassall in Ward, 1901 was validated with L. brachysoina as type. Jagerskiold (1901), although aware of the announcement by Ward, proposed the name Spelotrema for the invalid name Levinsenia, with S. pygmaeum as type. Cable and Hunninen (1940), commenting on this action, wrote (p. 153), "If the law of priority should be applied to this case, Spelotrema Jagerskiold, 1901 should be suppressed as a synonym of Levinseniella Stiles and Hassall in Ward, 1901, since Jagerskiold stated subsequently (1904) 'Spelotrema (= Levinseniella)' and therefore certainly regarded them as synonymous. His later (1907) con- ception of two distinct genera is valid, however, and must be accepted although he should not have retained for them names which he had regarded previously as synonyms. To suppress Spelotrema as a synonym of Levinseniella, and propose a new generic name for the species at present allocated to the genus Spelotrema, would probably increase rather than diminish the present confusion. For this reason, the writers are inclined to let the matter stand." As noted, L. brachysoma and S\ pygmaeum are not congeneric and the characteristic features of the two genera were presented clearly by Rankin in two papers (1939, 1940a). The problem of nomenclature, stated by Cable and Hunninen, was resolved when Baer (1943) suppressed Spelotrema as a synonym of Microphallus and transferred all species from the former genus to the latter one. Baer described Microphallus gracilis from Neomys jodiens and formulated a key to thirteen members of the genus. Strandine (1943) and Rausch (1946a, 1946b, 1947) discussed morpho- logical features and host-specificity of species in the genus Microphallus. Rausch and Locker (1951) described Microphallus enhydrae n. sp., from the sea-otter, Enhydra lutris, and recognized fourteen species in the genus. Stunkard (1951, 1953) described metacercariae from the horseshoe crab, Limulus polyphemus, and their development to sexual maturity in white mice, golden hamsters, and the herring gull, Larus argentatus. Although the worms agreed closely with the description of M. clavijormis (Brandes, 1899), bionomic features seemed to pre- clude their allocation to that species and they were described as members of a new species, Microphallus limuli. Cable and Kuns (1951) erected a new genus, Carneophallus, and discussed the evolution and interrelationships of genera in the family Microphallidae. Lebour (1905) reported grape-like masses of "sporocysts" in the liver of Littorina rudis (= L. saxatilis}, filled with tail-less cercariae, doubled up in a curious manner. When extended, they measured 0.25 mm. in length and the figure shows them to be microphallid metacercariae. Miss Lebour regarded these larvae as identical with the encysted worms found by M'Intosh (1865) in the green crab, C. maenas. Brandes (1889) had suggested that the larva in the green crab is the encysted stage of Distoma clavijorme, described by him from Pelidna (Tringa) alpina and Aegialitis hiaticula. Nicoll (1906) described specimens from the ceca and intestine of Larus argentatus taken at St. Andrews, Scotland, which he identified as Levinsenia similis, the species that Jagerskiold (1900) had described as a variety of Dist. pygmaeum and (1907) raised to specific rank as type of Spelotrema. Nicoll noted that his specimens were somewhat larger than those of 256 HORACE W. STUNKARD Jagerskiold and that worms from the ceca were larger than those from the intestine. He declared that the metacercariae from C. maenas are as likely to prove larvae of S. similis as of 5\ clavijorme. The following year, Nicoll (1907) described the worms from L. argentatus at St. Andrews as a new species, Spelotrema excellens, distinct from S. similis. Other specimens from Pelidna alpina, Totanus calidris and Aegialitis hiaticula were described as a new species, Spelotrema feriatum. He gave a redescription of S. claviforme and in a footnote stated that the larvae from C. maenas are larger than the adults of 6". clavijorme and must belong to another species, perhaps S. excellens. The smaller metacercariae described by Lebour (1905) from the liver of Littorina rudis were suggested as the encysted stage of 6". clavijorme. Lebour (1908) designated the metacercaria from C. maenas as Cercaria carcini and recognized the difficulty of relating the larval forms of Spelotrema with any known adults. Nicoll and Small (1909) examined crabs at Millport on the Scottish west coast during August, 1908. They found three of four C. maenas and one of five Cancer pagurus infected with encysted metacercariae which they identified as a species of Spelotrema, most probably 5*. excellens Nicoll. At St. Andrews every green crab was infected, often with every tissue and organ riddled with cysts which occurred sometimes singly and sometimes in clusters in the liver, gonads, and along the blood vessels, nerves and intestine. They noted, as had Lebour (1908), that the cysts varied in size, shape and thickness of wall. They measured cysts of three size groups ; the small ones were oval with thin walls and the largest ones were spherical with thick walls composed of inner concentric and outer radial layers. Referring to the gradual increase in size of the cysts, the authors stated (p. 239), "From these figures there seems no reason to suppose that these groups are other than stages in the growth of the same cyst, and such being the case it is evident that the cercariae increase considerably in size during their sojourn in the crab." They measured a cyst from the original material of Prof. MTntosh which was 0.29 mm. in diameter. They suggested a possible error in the measurement given in his (1865) report, mentioned also by Guyenot et al. (1925). Lebour (1912) proposed that larval trematodes should be classified on bionomic grounds and arranged the British marine cercariae in two categories, dependent on whether they were produced in sporocysts or in rediae. Among those which develop in sporocysts, she gave a more complete account of Cercaria ubiquita, a larva which she previously (1907) had described from Paludestrina stagnalis at Fenham Flats and Loch Ryan on the west coast of Scotland. She reported that this species occurred also in Littorina sa.ratilis (syn. L. rudis), and at Millport the usual host was Littorina obtusata. The cercariae resembled C. cellulosa and C. pusilla of Looss (1896) and Lebour reported that they entered C. maenas and Cancer pagurus where they developed into "Spelotrema-like cercariae." The encysted larvae occupied (p. 432) "almost every tissue of the crab, liver, muscles, gonad and outside the blood vessels. Having settled down it grows considerably and the cyst with it, but the latter however is still very thin-walled. The stylet is lost when the cyst measures about 0.30 mm. across. The ventral sucker and alimentary canal appear and the body spines begin to form. The worm stops growing when the cyst is about 0.35 mm. across and then the cyst wall becomes very thick, 0.02 mm. thick, and the real resting stage begins. The cercaria is now of the ordinary Spelotrema form. The usual size of the thick-walled cyst is LIFE-CYCLE OF MICROPHALLUS 257 0.4—0.48 mm. across." After describing the excysted metacercaria, Lebour stated (p. 433), "From what has been said there can be little doubt that Cere aria ubiquita is the young form of 5". excellent the first host thus being Paludestrina stagnalis, Littorina obtusata and L. rudis. The intermediate host Carcinus maenas and Cancer pagurus and the final host probably the herring gull, Larus argentatus" In this paper Lebour also gave figures of Cercaria carcini Lebour, 1908 and Cercaria minor sp. inq., both from C. maenas. These species were encysted and therefore metacercariae ; furthermore, since the cyst walls were thin and the worms some- what smaller, it is probable, as suggested by Nicoll and Small (1909), that these larvae are identical with the ones identified by Lebour as Spelotrema excellens. Guyenot, Naville and Ponse (1925) described metacercariae which they found in a single, formalin-preserved specimen of C. maenas taken at Boulogne-sur-Mer, France. The cysts were oval, 0.40 by 0.35 mm., and the larvae were identified as Spelotrema carcini Lebour. These authors accepted Lebour's (1912) account, designating C. ubiquita as the larva of 5. excellens, but noted that when computed from the stated magnification of the figure, the metacercaria portrayed by MTntosh was only 0.13 by 0.16 mm. As noted earlier, M'Intosh gave no measurements and the presumed error in magnification may be explained by reduction in size of the drawing on publication. The French authors also described larger cysts, 0.90-1.2 mm. in diameter, in which the walls were weakened and the trematode larvae were filled with spores of a microsporidian, Nosema (Plistophora} spelo- tremae n. sp. Stunkard (1932) described cercariae from Littorina saxatilis and L. littorea at Roscoff, France as Cercaria ubiquitoides. Although the larvae were very similar to C. ubiquita, slight differences between these specimens and Miss Lebour's description prevented their allocation to that species. In C. ubiquita Lebour described two ducts on each side, "which run up the body springing from two masses of large cells which occupy the greater part of the body." In her figure, Lebour showed six gland cells on each side with the two median ducts crossing to open on opposite sides of the body. The cercariae at Roscoff were described as having four penetration glands on each side of the body, with ducts which lead forward, three associated in a common bundle that passes along the lateral face of the oral sucker while the other duct lies more mediad and passes over the sucker. The penetration glands did not stain with neutral red but the secretory granules were clearly visible. These granules, colorless in the cell bodies, stained a deep blood-red in the terminal portions of the ducts and frequently accumulated there to form enlargements. Stunkard also described metacercariae from C. maenas and Porcellana longicornis, which were referred provisionally to the genus Spelotrema, but no attempt was made to relate them to C. ubiquitoides. Rees (1936) described an ubiquitous cercaria from L. rudis, L. obtusata and L. littorea collected at Aberystwyth in February and March, 1935. He noted that the larvae were almost identical with those described by Stunkard (1932) from L. rudis and L. littorea at Roscoff. There were slight differences in measure- ment and other differences concerned the penetration glands, which in the speci- mens studied by Rees were, "more distinct, not lobed and the most anterior pair of cells is separated from the other three pairs." Rees declared (p. 621), "It is difficult to determine whether these differences are individual or specific because there are so few larval characters in cercariae of the Ubiquita group which can be 258 HORACE W. STUNKARD used for separating species. The problem is rendered more difficult by the close resemblance of the adult trematodes (species of the genus Spelotrema) ." In general, the measurements given by Rees agree with or overlap those given for C. ubiquitoides, except for the length of the stylet which measured 28 microns against "about 25 microns" in C. ubiquitoides. The size, shape and relative position of the gland cells are altered by degree of maturity of the larva, by contractions of the body musculature, and by the emission of secretion into their ducts. Since the ducts from the most anterior pair of penetration glands are separate from the ducts of the other cells, the cell bodies may be somewhat removed. The observation by Rees that only the two anterior pairs of cells and ducts take up neutral red stain, whereas the others do not, is a significant contribution to knowledge of the species. Obviously, I failed to note this feature in C. ubiquitoides, but the staining reaction differs with the constitution, concentration, age and condition of the neutral red solution. Accordingly, I am disposed to regard the larvae described by Rees and C. ubiquitoides as specifically identical. Since each penetration gland has its own duct, the account of Lebour is not precise and the lateral one of the two reported ducts on each side of the body is almost certainly a bundle of three ducts. Further- more, it appears that C. ubiquitoides can not be distinguished from C. ubiquita Lebour, 1907, and the name should be suppressed as a synonym. Lewis (1926) had reported Spelotrema simile as very common in gulls of the Aberystwyth area and the finding was confirmed by Rees (1936) who stated (p. 624), "The un- usually high percentage infestation of Littorina rudis with this cercaria, together with the high percentage of gulls parasitized by Spelotrema simile, suggests that this may be the larval form of this species." Cable and Hunninen (1938) described the successive stages in the life-cycle of a trematode which they identified as a new species, Spelotrema nicolli. The asexual generations were in the snail, Bittium alternatum, the metacercariae in the blue crab, Callinectes sapidus, and the sexual generation was developed in young herring gulls, Larus argentatus. These authors (1940) gave a more com- plete description of S. nicolli, the adult of which was compared with that of 6". pygmaeum, S. clavijorme, S. simile, S. excellens and 5". brevicaeca. In size and size of organs, 6". nicolli agrees closely with S. simile; the major difference is in the size of the male papilla, which is much smaller and similar to that of 6". pygmaeum. The metacercariae were found only in certain slender fibers which extend from the viscera to the bases of the legs. Cysts increase from 0.05 to 0.50 mm. in diameter ; the metacercariae become almost as large as the adults ; the excretory formula is 2 [(2 + 2) + (2 + 2)], which persists in the adult stage. The cercaria agrees closely with the description of C. ubiquitoides and of the species described by Rees ; the major difference is the shorter length of the stylet. Spelo- trema nicolli is identified by the size of the male papilla, the location of the meta- cercariae, and the taxonomic position of the first and second intermediate hosts. Timon-David (1949) reported metacercariae from the hepatopancreas of C. maenas in the Mediterranean at Marseille. The cysts were oval and on this feature and their size, they were identified as Spelotrema carcini Lebour. The present report covers part of a project on clam investigation conducted by the U. S. Fish and Wildlife Service. Since the green or shore-crab, C. maenas, is a serious predator of Mya arenaria on the New England coast, a survey of its parasites was undertaken in an attempt to determine whether or not some of them LIFE-CYCLE OF MICROPHALLUS 259 might serve as possible means of biological control. It has long been known that C. maenas in the Woods Hole area is infected by an undetermined, encysted meta- cercaria and experiments have been conducted to discover its identity, life-history, and biology. Metacercariae were fed to white mice and excysted specimens (Fig. 6) recovered after 24 hours. Other cysts were fed to recently hatched, uninfected birds, Sterna hirundo and Larus argentatus. Large numbers of worms were recovered, including all stages from juvenile to fully mature specimens. The structure of the metacercariae, especially of the smaller and recently excysted ones, suggested that they may be specifically identical with a minute, stylet-bearing cercaria (Figs. 1, la) reported by Stunkard (1950), which occurs in Littorina obtusata, L. saxatilis, and rarely in L. littorea. Small green crabs were exposed to these cercariae and became heavily infected ; enormous numbers of worms entered their tissues and developed to metacercariae, identical with those of natural in- fections. Small crabs exposed continuously with six to eight infected snails died in 10 to 20 days, and on dissection, each one yielded thousands of larvae. The parasites were present throughout the body of the crab, but the heaviest con- centration was in the digestive gland. When first encysted, the cyst is oval, the wall is very thin, and the stylet persists for a long time. Eggs of the parasite, recovered from the droppings of terns and gulls, and others teased from the uteri of gravid worms, were used to infect specimens of L. obtusata, collected near the laboratory from an area which is not frequented by birds and where examina- tion of 200 snails showed no infection. The eggs were embryonated under run- ning sea water for seven days and then spread on fronds of Fucus which were allowed to partially dry to ensure that the eggs would become attached to the slimy surface of the alga. These fronds were placed with 10 small specimens of L. obtusata in a gallon jar with a small opening and sharply curved upper shoulders, half filled with sea water and provided with a stream of fine bubbles from an air pump. The water was not changed for ten days and subsequently on alternate days the top water was siphoned off and replaced with fresh sea water. Since the eggs of microphallid trematodes do not hatch until they are eaten by a suitable snail, and since from the design of the experiment it is impossible to tell when eggs were eaten, the age of an infection is not known precisely. The snails were first exposed on July 31, and on October 1, five snails that were alive in the jar were crushed and examined. Three of them were infected ; two with large numbers of small daughter sporocysts but not yet producing cercariae, and the other contained three primary or mother sporocysts (Fig. 2) but no free daughters. The early stages of the infections afford clear evidence of their experimental nature and the complete life-cycle was thus consummated by laboratory infection of both inter- mediate and final hosts. The adult worms agree so completely with the descriptions of Microphallus similis (syn. Spelotrema simile) as given by Jagerskiold (1900) and Odhner (1905), that they are assigned to that species. The experimental demonstration of the life-cycle confirms the suggestion of Rees (1936), that Cercaria ubiquita Lebour is the cercarial stage of M. similis. Actually, the adults were described by Jagerskiold (1900), the metacercariae by MTntosh (1865), and the cercariae by Lebour (1907). Whether the specimens described by Nicoll (1907) as 6\ ex- cellens and S. feriatum are specifically distinct remains to be determined. Attempts to infect Limulus polyphemus were unsuccessful. Three small horse- 260 HORACE W. STUNKARD 1 LIFE-CYCLE OF MICROPHALLUS 261 shoe crabs with carapace widths of 17, 19, and 21 mm., collected near Orleans, Massachusetts, were exposed to cercariae for two weeks. On dissection, there were no recently entered, unencysted larvae although all three harbored mature cysts of Microphallus limit li in their livers. This finding supplements that of Stunkard (1953), who reported full-sized cysts in small L. polypheinus. In para- graph 3, line 4, of that report, 3 mm. should read 30 mm. DESCRIPTION OF STAGES IN THE LIFE-CYCLE Measurements in millimeters Adults (Fig. 7) Egg-bearing specimens from gulls and terns are about the same size. Fixed, stained and mounted they measured : length, 0.36—0.7 ; width, 0.22-0.36 ; acetabu- lum, 0.048-0.065; oral sucker, 0.05-0.065; pharynx, 0.02-0.03; male papilla, diameter 0.038-0.058; testes, 0.10 X 0.06 to 0.16 X 0.09; seminal vesicle, 0.06 X 0.04 to 0.09 X 0.064; ovary, 0.08 X 0.05 to 0.1 X 0.062; eggs colorless on the ovarian side, yellow on the antovarian side, 0.022-0.027 X 0.011-0.012, often collapsed and somewhat distorted in fixed and stained specimens. The body is oval to pyriform and either end may be wider; often there is a slight constriction between the more mobile forebody and the inert hindbody which is filled with reproductive organs and eggs. The dermomuscular wall is thin and weakly developed ; it consists of the usual circular, longitudinal and oblique layers, but the fibers are delicate and relatively few. The edges of the forebody tend to turn ventrad, forming an adhesive cup. The cuticula bears flattened spines which are conspicuous on the anterior half of the body but become smaller and sparser posteriorly. The suckers are approximately equal in size ; either may appear larger, depending on the degree of contraction by the sphincter and the size of the orifice. The esophagus is long and when the body is extended, it may be much longer than the ceca. The ceca are almost straight ; they diverge at an obtuse angle and ter- minate at the acetabular level. The anterior tips are constricted and lined with cuticula, continuous with that of the esophagus. The excretory system is un- changed from the metacercarial stage and has the formula 2[ (2 + 2) + (2+2) ]. The testes are dorsal, lateral, opposite; the vasa deferentia arise at the medial, anterior faces, pass mediad and anteriad where they unite to form the seminal FIGURE 1. Cercaria from L. obtusata, free-hand drawing of specimen stained with Nile blue sulphate; la, lateral aspect of stylet. FIGURE 2. Primary sporocyst from L. obtusata, experimental infection ; fixed and stained specimen, length 0.30 mm. FIGURE 3. Daughter or secondary sporocyst from L. obtusata; natural infection ; fixed and stained specimen, length, 0.375 mm. FIGURE 4. Cross-section of a mature worm to show the relative position and size of the acetabulum and of the male papilla. The metraterm opens into the genital atrium near the base of the papilla and in front of the left testis whose anterior portion appears in the section ; width of the worm, 0.28 mm. FIGURE 5. Metacercaria from C. macnas, natural infection ; cyst 0.46 mm. in diameter. FIGURE 6. Specimen, much flattened to study the excretory system, from intestine of a white mouse, 24 hours after the cyst was eaten ; length 0.55 mm. FIGURE 7. Adult specimen from intestine of Sterna hirundo, experimental infection ; one of the largest specimens, flattened under a cover glass, fixed and stained, length 0.69 mm. 262 HORACE W. STUNKARD vesicle, an oval sac which lies anterior and dorsal to the acetabulum. A coiled ejaculatory duct, enclosed in glandular cells, leads from the vesicle to the muscular male papilla which almost fills the genital atrium, situated at the left of the acetabulum. The ovary is oval to triangular, located on the right side of the body, between the seminal vesicle and the testis and cecum of the right side. The oviduct arises from the median posterior region, coils posteriad and ventrad where it expands to form a fertilization space from which Laurer's canal winds to the dorsal surface of the body, opening in the midline just behind the acetabulum. The oviduct then turns dorsad and anteriad, receives a short common vitelline duct and enlarges to form the ootype, lined with cilia and enclosed in the cells of Mehlis' gland. The vitellaria are large, lobed glands, situated below and behind the testes; ducts from the two sides pass mediad, anteriad and dorsad, uniting to form the common vitelline duct which discharges into the initial portion of the ootype. The uterus passes backward from the ootype almost to the posterior end of the body and then loops forward in coils on the ovarian side of the body as far as the end of the digestive cecum, then backward almost to the posterior end of the body where it crosses to the opposite side and forms a corresponding series of loops on the left side, with the terminal metratermal portion emptying into the left side of the genital atrium (Fig. 4). The extent of the uterus anteriorly is determined by the number of eggs ; in certain specimens the uterine coils may be below and behind the testes whereas in others the coils may underlie the ends of the digestive ceca. The egg and miracidium Egg-production begins almost immediately after the metacercaria is eaten by the final host. Figure 6 shows a worm which, fed as a metacercaria to a white mouse twenty-four hours earlier, already has eggs in the initial portion of the uterus. At first the egg-shell is thin, flexible and transparent ; but as the eggs traverse the uterus, the shells become thicker, harder, and bright yellow. This coloration of the shell obscures the larva in living eggs, but development can be followed by study of serial sections of gravid worms. The egg is operculate and the ovum is situated toward the opercular end of the egg. In eggs near the metraterm, the miracidium appears to be fully formed, but eggs used for infection experiments were kept for a week in running sea water to insure fully mature larvae. The miracidia emerge only after the eggs have been ingested by the snail host. Sporocyst generations (Figs. 2, 3) The amount of experimental material is limited, but three primary sporocysts were removed from a specimen of L. obtusata two months after exposure to eggs of M. siinilis. These sporocysts were sluggish, oval to cylindrical and 0.25 to 0.40 mm. long. One of them, fixed and stained, is shown in Figure 2. The presence in them of recognizable daughters identified them as sporocysts of the mother or primary generation. Daughter sporocysts obtained from two experi- mental infections were young, small, and very numerous. Daughter sporocysts of natural infection measured 0.10 to 0.60 mm. in length; they are oval, occur in LIFE-CYCLE OF MICROPHALLUS 263 large numbers, more than one hundred in a single snail. The wall often contains a yellowish pigment; it is thin and in older sporocysts change of shape results from movement of contained cercariae. Figure 3 was made from a daughter sporocyst, 0.375 mm. long. Cere aria (Figs. 1, la) Body length, 0.1-0.22 mm.; width, 0.02-0.05 mm.; the larva is very thin, delicate, colorless. The tail is 0.01-0.012 mm. wide at the base; contracted it is 0.05 mm. long, with fine cuticular annulations ; extended it may be 0.25 mm. long and very slender. There is no acetabulum; the larvae move by strokes of the tail but are unable to creep. They swim upward and sink when motionless. In swimming the body is contracted, bent ventrad, while the tail is extended and lashes violently. The body is covered with cuticular spines and the dorsal wall of the sucker bears a stylet, 0.023-0.026 mm. long and 0.009 mm. wide. The stylet (Fig. la) is asymmetrical in lateral aspect. The oral sucker measures 0.025-0.032 mm. ; other parts of the digestive system are not yet developed. The body contains numerous cystogenous glands and on either side, near the middle, there are four penetration glands. The two anterior cells on each side differ from the posterior ones ; the difference is demonstrated by the use of neutral red or Nile blue sulphate solutions, especially the latter, which stain the secretory granules of the anterior cells selectively while the contents of the posterior cells do not take the stain. Ducts from the anterior pair of cells pass forward beside those from the other cells for a short distance, but about halfway to the mouth they separate from the others and pass more mediad, crossing the dorsal side of the oral sucker, while the ducts from the other three penetration gland cells form a bundle that continues forward and passes at the side of the sucker. The ducts from the two anterior pairs of cells open to the surface ventrally, below the tip of the stylet, whereas the ducts from the posterior pairs open more anteriorly and at the sides of the tip of the stylet. The excretory system is shown in Figure 1 ; the vesicle is U- or V-shaped and the flame-cell formula is 2[ (1 + 1) + (1 +1) ]. The cercariae are carried by respiratory currents into the gill chambers of C. maenas where they enter the body at the bases of the gills and possibly at other non-cutic- ularized places, pass by way of the vascular system to all parts of the body, and localize principally in the connective tissue of the digestive gland. Mctacercaria (Fig. 5) The cysts increase in size and measure from 0.05 to 0.55 mm. in diameter; when the cercariae encyst they are bent ventrally ; the cyst is oval and the wall is very thin and flexible. The stylet is retained for some time and readily identifies the species. As the metacercaria grows, the digestive tract and actabulum are formed and the number of flame-cells is doubled. As the worm grows, the cyst becomes spherical and the wall increases in thickness. In a full grown cyst, the cavity may be 0.35-0.40 mm. in diameter and the wall consists of two layers, an inner hyaline one which may be resolved into strata and attains a thickness of 0.02 mm., and an outer radially striated layer which increases to a thickness of 0.06 mm. The substance of this layer, after digestion in a pancreatin solution, 264 HORACE W. STUNKARD appears to consist of parallel prisms. The worms become almost full grown as metacercariae ; the adults increase in size only by the further activity of the re- productive organs and the accumulation of eggs in the uterus. When younger and smaller metacercariae are eaten, the worms become gravid at a smaller size than when the metacercariae are older. The excretory vesicle of older meta- cercariae contains spherical concretions, the excretory wastes accumulated during the period of encystment. SUMMARY 1. The life-history of Microphallus similis has been worked out by experimental infection of both intermediate and final hosts. 2. Encysted metacercariae from Carcinides maenas developed to sexual maturity in Larus argentatus and Sterna hirundo. Eggs of the parasite developed in these hosts were used to infect Littorina obtusata. Two generations of sporocysts were recovered. 3. Littorina saxatilis and Littorina littorea also harbor the asexual generations at Woods Hole, Massachusetts. 4. The cercariae are minute, stylet-bearing monostomes and small green crabs, C. maenas, exposed to these cercariae became heavily infected; enormous numbers of larvae entered the tissues and developed into metacercariae identical with those of natural infections. Small crabs, each exposed continuously to the cercariae from six to eight infected snails, died in ten to twenty days and on dissection each yielded thousands of larvae. 5. The stages in the life-cycle of the parasite agree with descriptions by European investigators of corresponding stages : the metacercariae with meta- cercariae from C. maenas, described but not named by MTntosh (1865) ; the adults with M. similis from Swedish gulls, described and named by Jagerskiold (1900) ; and the cercariae with Cercaria ubiquita Lebour, 1907. The identity of these parasites as stages in the life-cycle of a single species is predicated. LITERATURE CITED BAER, JEAN G., 1943. Les trematodes parasites de la musaraigne d'eau Neomys fodiens (Schreb.). Bull. Soc. Neuchatel. Sci. Nat., 68: 34-84. BRANDES, G., 1889. Helminthologisches. Arch. Naturg., Jg., 54 : 247-251. CABLE, R. M., AND A. V. HUNNINEN, 1938. Observations on the life-history of Spelotrema nicolli n. sp. (Trematoda : Microphallidae) with the description of a new microphallid cercaria. /. Parasitol., 24 (Suppl.) : 29-30. CABLE, R. M., AND A. V. HUNNINEN, 1940. Studies on the life-history of Spelotrema nicolli (Trematoda :Microphallidae) with the description of a new microphallid cercaria. Biol.Bull., 78: 136-157. CABLE, R. M., AND M. L. KUNS, 1951. The trematode family Microphallidae with the descrip- tion of Carneophallus trilobatus gen. et sp. nov., from Mexico. /. Parasitol., 37 : 507-514. GUYENOT, E., A. NAVILLE AND K. PONSE, 1925. Deux microsporidies parasites de trematodes. Rev. Suisse Zoo/., 31 : 399-421. JAGERSKIOLD, L. A., 1900. Levinsenia (Distomum) pygmaea Levinsen, ein genitalnapftragendes Distomum. Centralbl. Bakt., I Abt., 27: 732-740. JAGERSKIOLD, L. A., 1901. Tocotrema expansum (Crepl.) (= Monostomum expansmn Crepl.) eine genitalnapftragende Distomide. Centralbl. Bakt., I Abt., 30 : 979-983. LIFE-CYCLE OF MICROPHALLUS 265 JAGERSKIOLD, L. A., 1907. Zur Kenntnis der Trematodengattung Levinseniella. Zool. Stud. Till. Tullberg, Uppsala, pp. 133-154. LEBOUR, MARIE V., 1905. Notes on Northumbrian trematodes. Report Northumberland Sea Fisheries Comm., pp. 100-105. LEBOUR, MARIE V., 1907. Larval trematodes of the Northumberland coast. Trans. Nat. Hist. Soc. Northumberland, Durham and Newcastle, N. S., 1 : 437-454. LEBOUR, MARIE V., 1908. Trematodes of the Northumberland coast, No. 2. Trans. Nat. Hist. Soc. Northumberland, Durham and Newcastle, N. S., 3 : 28-45. LEBOUR, MARIE V., 1912. A review of the British marine cercariae. Parasitology, 4 : 416-456. LEVINSEN, G. M. R., 1881. Bidrag til Kundskab om Gr0nlands Trematodfauna. Overs. K. Danske Vidensk. Selsk. Forh., pp. 52-84. LEWIS, E. A., 1926. Helminths of wild birds found in the Aberystwyth area. /. Helminth., 4 : 7-12. Looss, A., 1896. Recherches sur la faune parasitaire de 1'Egypte. Premiere partie. Mem. Inst. Egypt., 3 : 1-252. Looss, A., 1899. Weitere Beitrage zur Kenntnis der Trematoden-Fauna Aegyptens. Zool. Jahrb. Syst., 5 : 521-784. Looss, A., 1902. Ueber neue und bekannte Trematoden aus Seeschildkroten. Nebst Erorter- ungen zur Systematik und Nomenclatur. Zool. Jahrb. Syst., 16: 411-894. LUHE, M., 1899. Zur Kenntnis einiger Distomen. Zool. Anz., 22: 524-539. M'INTOSH, W. C., 1865. The trematode larva and Ascaris of the Carcinus maenas. Quart. J. Micros. Sci., N. S., 5 : 201-204. NICOLL, W., 1906. On some new and little known trematodes. Ann. Mag. Nat. Hist., 7 ser., 17: 513-527. NICOLL, W., 1907. Observations on the trematode parasites of British birds. Ann. Mag. Nat. Hist., 7 ser., 20: 245-271. NICOLL, W., AND W. SMALL, 1909. Notes on larval trematodes. Ann. Mag. Nat. Hist., 8 ser., 3 : 237-246. ODHNER, T., 1905. Die Trematoden des arktischen Gebietes. /w:R6mer and Schaudinn. Fauna Arctica, 4 : 291-372. RANKIN, J. S., JR., 1939. Studies on the trematode family Microphallidae Travassos, 1921. I. The genus Levinseniella Stiles and Hassall, 1901 and description of a new genus Cornucopula. Trans. Amer. Micros. Soc., 58 : 431-447. RANKIN, J. S., JR., 1940a. Studies on the trematode family Microphallidae Travassos, 1921. II. The genus Spelotrema Jagerskiold, 1901, and description of a new species, Spelotrema papillorobusta. Trans. Amer. Micros. Soc., 59 : 38-47. RANKIN, J. S., JR., 1940b. Studies on the trematode family Microphallidae Travassos, 1921. IV. The life-cycle and ecology of Gynaecotyla nassicola (Cable and Hunninen, 1938) Yamaguti, 1939. Biol. Bull., 79: 439-451. RAUSCH, R., 1946a. New host records for Microphallus ovatus Osborn, 1919. /. Parasitol., 32: 93-94. RAUSCH, R., 1946b. The raccoon, a new host for Microphallus sp. with additional notes on M. ovatus from turtles. /. Parasitol., 32 : 208-209. RAUSCH, R., 1947. Some observations on the host relationships of Microphallus opacus (Ward, 1894) (Trematoda:Microphallidae). Trans. Amer. Micros. Soc., 66: 59-63. RAUSCH, R., AND BETTY LOCKER, 1951. Studies on the helminth fauna of Alaska. II. On some helminths parasitic in the sea-otter, Enhydra lutris (L.). Proc. Helminth. Soc. Wash.,D.C.,18: 77-81. REES, W. J., 1936. Note on the ubiquitous cercaria from Littorina rudis, L. obtusata and L. littorea. J. Mar. Biol. Assoc., 20 : 621-624. STOSSICH, M., 1899. Los membramento dei Brachycoelium. Boll. Soc. Adriat. Sci. Nat. Trieste, 19 : 7-10. STRANDINE, E. J., 1943. Variation in Microphallus, a genus of trematodes, from fishes of Lake Lelanau, Michigan. Trans. Amer. Micros. Soc., 62 : 293-300. STUNKARD, H. W., 1932. Some larval trematodes from the coast in the region of Finistere. Parasitology, 24 : 321-343. STUNKARD, H. W., 1950. Further observations on Cercaria parvicaudata Stunkard and Shaw, 1931. Biol. Bull., 99: 136-142. 266 HORACE W. STUNKARD STUNKARD, H. W., 1951. Observations on the morphology and life-history of Microphallus limuli n. sp. (Trematoda :Microphallidae). Biol. Bull., 101: 307-318. STUNKARD, H. W., 1953. Natural hosts of Microphallus limuli Stunkard, 1951. /. ParasitoL, 39 : 225. STUNKARD, H. W., 1956. Studies on parasites of the green crab, Carcinides maenas. Biol. Bull, 111: 295. TIMON-DAVID, JEAN, 1949. Sur un trematode parasite des crabes en Mediterranee. Ann. ParasitoL, 24 : 25-28. WARD, H. B., 1894. On the parasites of the lake fish. I. Notes on the structure and life history of Distoma opacum n. sp. Proc. Amer. Micros. Soc., 15 : 173-182. WARD, H. B., 1901. Notes on parasites of the lake fish. III. On the structure of the cop- ulatory organs in Microphallus nov. gen. Trans. Amer. Micros. Soc., 22: 175-187. BIOLOGY MATERIALS The Supply Department of the Marine Biological Labora- tory has a complete stock of excellent plain preserved and injected materials, and would be pleased to quote prices on school needs. PRESERVED SPECIMENS for Zoology, Botany, Embryology, and Comparative Anatomy LIVING SPECIMENS for Zoology and Botany including Protozoan and Drosophila Cultures, and Animals for Experimental and Laboratory Use. MICROSCOPE SLIDES for Zoology, Botany, Embryology, Histology, Bacteriology, and Parasitology. CATALOGUES SENT ON REQUEST Supply Department MARINE BIOLOGICAL LABORATORY Woods Hole, Massachusetts CONTENTS Page ALLEN, BENNET M., AND MARION HUBBLE DEVICE Differences in susceptibility to whole-body gamma irradiation in the layers of the retina of Bufo 137 BROAD, A. C. Larval development of Palaemonetes pugio Holthuis 144 BROAD, A. C. The relationship between diet and larval development of Palaemonetes 162 GROSCH, DANIEL S., AND ZOE H. SMITH X-ray experiments with Molgula manhattensis : Adult sen- sitivity and induced zygotic lethality 171 HAGERMAN, DWAIN D., FREDERICA M. WELLINGTON AND CLAUDE A. VILLEE Estrogens in marine invertebrates 180 HENLEY, CATHERINE AND DONALD P. COSTELLO The effects of x-irradiation on the fertilized eggs of the annelid, Chaetopterus 184 HULBURT, EDWARD M. The taxonomy of unarmored Dinophyceae of shallow embay- ments on Cape Cod, Massachusetts 196 JONES, JACK COLVARD, AND E. B. LEWIS The nature of certain red cells in Drosophila melanogaster . . 220 OKA, HlDEMITI, AND HIROSHI WATANABE Vascular budding, a new type of budding in Botryllus 225 ROGERS, K. T. Optokinetic testing of cyclopean and synophthalmic fish hatchlings 241 SCOTT, GEORGE T., AND ROBERT DEVOE The reversible replacement of potassium by rubidium in Ulva lactuca 249 STUNKARD, HORACE W. The morphology and life-history of the digenetic trematode, Microphallus similis (Jagerskiold, 1900) Baer, 1943 254 Volume 112 Number 3 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, Vanderbilt University J. H. LOCHHEAD, University of Vermont JOHN B. BUCK, National Institutes of Health g. T. MOUL, Rutgers University T. H. BULLOCK, University of ^^J^- ARTHUR W. POLLISTER, Columbia University E. G. BUTLER, Princeton University ' MARY E' *A™s, Mns Hopkins University K. W. COOPER, University of Rochester A- R- WHITING, University of Pennsylvania M. E. KRAHL, University of Chicago CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor JUNE, 1957 Printed and Issued by LANCASTER PRESS, Inc. PRINCE 8C LEMON STS. LANCASTER, PA. INSTRUCTIONS TO AUTHORS The Biological Bulletin accepts papers on a variety of subjects of biological interest. In general, however, review papers (except those written at the specific invitation of the Editorial Board), short preliminary notes and papers which describe only a new technique or method without presenting substantial quantities of data resulting from the use of the new method cannot be accepted for publication. A paper will usually appear within three months of the date of its acceptance. 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Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa. under the Act of August 24, 1912. , , , , , PYREX brand ACCU-RED PIPETTES in our stock for imtnedi At savings up to 15% Tolerances within Federal Specification DD-V-581a Permanent red graduations Production of accurate, low cost Pipettes by a combination of uniform bore tubing and a modern calibration and marking method —envisioned and urged by us for a long time — has become a reality with the an- nouncement by Corning Glass Works of the new Pyrex brand ACCU-RED Pipettes. These new Pipettes have an accuracy well within the requirements of Federal Specification DD-V-581a. Calibrating and marking by one operation reduces the cost and results in savings up to 15%. They are made from accurate bore, uniform wall tubing of Pyrex brand glass 7740 and are therefore chemically stable, corrosion resistant, and unaffected by either hot air or steam pressure sterilization. Graduations and other markings are applied to the glass in a new permanent red which becomes part of the glass and remains sharply legible. Walls are extra heavy, and the sturdy tips have smooth, double bevels. A "sight line" behind each major graduation mark permits rapid and accurate meniscus readings by minimizing errors caused by parallax. 8161-D. Pipettes, Measuring (Mohr), Accu-red, Pyrex brand 10 25 1 /10 0.10 glass, graduated between points above tip. To deliver, ml. .. 1 1 2 5 Graduation interval, ml 1/10 1/100 1/10 1/10 1/10 Tolerance, ±ml. 0.02 0.02 0.02 0.04 0.06 Each 1.03 1.14 1.16 1.16 1.34 1.81 Per original "package of 18. 16.69 18.47 18.79 18.79 21.71 29.32 8169-L. Pipettes, Serological, Accu-red, Pyrex brand glass, graduated to extreme tip; with two bands at top indicating they are calibrated for blowing out the last drop. To deliver, ml 1 1 2 Graduation interval, ml 1/10 Tolerance, + ml. . . . Each Per original "package of 18 17.50 10 1/10 1 /1 00 1/10 i no i no 0.02 0.02 0.02 0.04 0.06 1.08 1.23 1.26 1.26 1.49 17.50 19.93 20.41 20.41 24.14 'May be assorted with items in Corning catalogue LP-36 and supplements thereto for maximum original package discounts. A.H.T.CO. ARTHUR H. THOMAS COMPANY More and more laboratories rely on Thomas I Laboratory Apparatus and Reagents I VINE ST. AT 3RD • PHILADELPHIA, PA. New AO-Baker INTERFERENCE MICROSCOPE If you need to examine or measure your material more effectively and precisely, we invite you to investigate this new advance in microscopy. American Optical • Instrument Division Buffalo 15, New York Dept. R-185 Please send me your catalog on the NEW AO-Baker Interference Microscope. NAME ADDRESS CITY ZONE STATE. The NEW AO-Baker Interference Microscope is the unique combina- tion of a double beam interferometer and polarizing microscope. It dra- matically provides for the precise examination of transparent speci- mens where detail is exhibited by variations in thickness or refractive index. With white light illumination, con- trast effects are greatly enhanced by brilliant and variable color contrasts. Details show up as if differentially stained. With monochromatic or filtered light, interference contrasts can be varied from bright to dark and rel- ative optical thicknesses are measur- able to an optimum accuracy of 1/300 wave length. Interference Contrast Microscopy like Phase Contrast Microscopy de- pends on the nature of the specimen detail to retard light — by virtue of refractive index and thickness — and does not depend on the property of the specimen to absorb light. In this connection the AO-Baker Interfer- ence Microscope is similar to the conventional Phase Contrast Micro- scope. The principle of the Phase Contrast Microscope depends upon light diffraction for its contrast effects — the AO-Baker Interference Micro- scope does not. By means of the unique built-in interferometer, mu- tually interfering beams are pro- duced, recombmed, and if the two beams suffer relative retardation, readily visible contrast results. The AO-Baker Interference Micro- scope has already won acclaim and recognition as an important aid to the solution of a great variety of bio- logical and industrial microscopical problems. Most scientific workers were initially of the opinion that the Interference Microscope would have its greatest utility for solving measurement problems. It now de- velops that equal or greater promise can be expected from its value as a method of variable phase and vari- able color contrast. American Optical Company INSTHUMINT DIVISION. 1UFFALO 15, NIW YORK THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, Vanderbilt University J. H. LOCHHEAD, University of Vermont JOHN B. BUCK, National Institutes of Health E. T. MOUL, Rutgers University T. H. BULLOCK, University of California, ARTHUR W. POLLISTER, Columbia University Los Angeles „ „ „ „ . TT . MARY E. RAWLES, Johns Hopkins University E. G. BUTLER, Princeton University K. W. COOPER, University of Rochester A. R. WHITING, University of Pennsylvania M. E. KRAHL, University of Chicago CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 112 FEBRUARY TO JUNE, 1957 Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. 11 THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press. Inc., Prince and Lemon Streets, Lancaster, Penn- sylvania. Subscriptions and similar matter should be addressed to The Biological Bulletin, Marine Biological Laboratory, Woods Hole, Massachusetts. Agent for Great Britain : Wheldon and Wesley, Limited, 2, 3 and 4 Arthur Street, New Oxford Street, London, W. C. 2. Single numbers $2.50. Subscription per volume (three issues), $6.00. Communications relative to manuscripts should be sent to the Managing Editor, Marine Biological Laboratory, Woods Hole, Massachusetts, between June 1 and September 1, and to Dr. Donald P. Costello. P.O. Box 429, Chapel Hill, North Carolina, during the remainder of the year. Entered as second-class matter May 17, 1930, at the post office at Lancaster, Pa., under the Act of August 24, 1912. LANCASTER PRESS, INC., LANCASTER, PA. CONTENTS No. 1. FEBRUARY, 1957 PAGE BONNER, JOHN TYLER, ALLAN A. HOFFMAN, WILFRED T. MORIOKA AND A. DUNCAN CHIQUOINE The distribution of polysaccharides and basophilic substances during the development of the mushroom Coprinus 1 FlNGERMAN, MlLTON Relation between position of burrows and tidal rhythm of Uca 7 FLICKINGER, REED A. Evidence from sea urchin-sand dollar hybrid embryos for a nuclear con- trol of alkaline phosphatase activity 21 GORDON, MALCOLM S. Observations on osmoregulation in the Arctic char (Salvelinus alpinus L.) 28 GRAY, I. E. A comparative study of the gill area of crabs 34 GROSS, WARREN J. An analysis of response to osmotic stress in selected decapod Crustacea 43 JENNINGS, J. B. Studies on feeding, digestion, and food storage in free-living flatworms (Platyhelminthes: Turbellaria) 63 JONES, RAYMOND F. Variation of nitrogen and carbohydrate constituents during the develop- ment of Himanthalia elongata (L.) S. F. Gray 81 MARONEY, SAMUEL P., JR., ALBERT A. BARBER AND KARL M. WILBUR Studies on shell formation. VI. The effects of dinitrophenol on mantle respiration and shell deposition 92 MEUWIS, A. L., AND M. J. HEUTS Temperature dependence of breathing rate in carp 97 PUNT, A., W. J. PARSER AND J. KUCHLEIN Oxygen uptake in insects with cyclic CO2 release 108 ROGICK, MARY DORA Studies on marine bryozoa. X. Hippadenella carsonae, N. sp 120 TRACER, WILLIAM Excystation of apostome ciliates in relation to molting of their crustacean hosts 132 No. 2. APRIL, 1957 ALLEN, BENNET M., AND MARION HUBBLE DEVICK Differences in susceptibility to whole-body gamma irradiation in the layers of the retina of Bufo 137 iv CONTENTS BROAD, A. C. Larval development of Palaemonetes pugio Holthuis 144 BROAD, A. C. The relationship between diet and larval development of Palaemonetes 162 GROSCH, DANIEL S., AND ZOE H. SMITH X-ray experiments with Molgula manhattensis: Adult sensitivity and induced zygotic lethality 171 HAGERMAN, DWAIN D., FREDERICA M. WELLINGTON AND CLAUDE A. VILLEE Estrogens in marine invertebrates 180 HENLEY, CATHERINE, AND DONALD P. COSTELLO The effects of x-irradiation on the fertilized eggs of the annelid, Chae- topterus 184 HULBURT, EDWARD M. The taxonomy of unarmored Dinophyceae of shallow embayments on Cape Cod, Massachusetts 196 JONES, JACK COLVARD, AND E. B. LEWIS The nature of certain red cells in Drosophila melanogaster 220 OKA, HlDEMITI, AND HlROSHI WATANABE Vascular budding, a new type of budding in Botryllus 225 ROGERS, K. T. Optokinetic testing of cyclopean and synophthalmic fish hatchlings. . 241 SCOTT, GEORGE T., AND ROBERT DEVOE The reversible replacement of potassium by rubidium in Ulva lactuca. 249 STUNKARD, HORACE W. The morphology and life-history of the digenetic trematode, Micro- phallus similis (jagerskiold, 1900) Baer, 1943 254 No. 3. JUNE, 1957 BENNETT, MIRIAM F., JOAN SHRINER AND ROBERT A. BROWN Persistent tidal cycles of spontaneous motor activity in the fiddler crab, Uca pugnax 267 BOLST, ALBERT L., AND ARTHUR H. WHITELEY Studies of the metabolism of phosphorus in the development of the sea urchin, Strongylocentrotus purpuratus 276 BROWN, F. A., JR. Response of a living organism, under "constant conditions" including pressure, to a barometric-pressure-correlated, cyclic, external variable. . 288 CHANDLER, ARTHUR C., JR. The immediate effects of low doses of x-radiation on the frequency of several mitotic stages in the Allium root tip 305 COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT Larval development of Balanus eburneus in the laboratory 313 DRISKO, RICHARD W., AND HARRY HOCHMAN Amino acid content of marine borers 325 HANKS, JAMES E. The rate of feeding of the common oyster drill, Urosalpinx cinerea Say, at controlled water temperatures 330 CONTENTS v GOULD, EDWIN Orientation in box turtles, Terrapene c. Carolina (Linnaeus) 336 HAND, CADET, AND MEREDITH L. JONES An example of reversal of polarity during asexual reproduction of a hydroid 349 IRISAWA, HIROSHI, AND AYA FUNAISHI IRISANVA The electrocardiogram of a stomatopod 358 JONES, RAYMOND F., AND L. R. BLINKS The amino acid constituents of the phycobilin chromoproteins of the red alga Porphyra 363 McWmNNiE, MARY A., AND MARY M. GLEASON Histological changes in regenerating pieces of Dugesia dorotocephala treated with colchicine 371 NAKAMURA, MITSURU Growth-promoting effects of hvdrolyzed nucleic acids, nucleotides, and nucleosides on Endamoeba histolytica 377 PAHL, GEORGE, AND C. S. BACHOFER Anaerobic recovery of Ascaris eggs from x-irradiation 383 SLATER, JOHN V. Radiocobalt accumulation in Tetrahymena 390 STADLER, JANICE, AND JOHN W. GOWEN Contributions to survival made by body cells of genetically differentiated strains of mice following x-irradiations 400 VALLOWE, HENRY H. Sexual differentiation in the teleost fish Xiphophorus hellerii, as modified by experimental treatment 422 Vol. 112, No. 3 June, 1957 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY PERSISTENT TIDAL CYCLES OF SPONTANEOUS MOTOR ACTIVITY IN THE FIDDLER CRAB, UCA PUGNAX 1 MIRIAM F. BENNETT, JOAN SHRINER AND ROBERT A. BROWN Szveet Briar College, Siveet Briar, Virginia, Northwestern University, Evanston, Illinois, and the Marine Biological Laboratory, Woods Hole, Massachusetts Early in the present century, it was reported that a number of littoral organisms showed cycles of behavior which persisted under so-called constant laboratory con- ditions with tidal frequencies and with phases adaptively related to tidal events of the areas from which the organisms were collected (Gamble and Keeble, 1903, 1904 ; Bohn, 1904, 1906). Later, Compel (1937) found that several species of animals display tidal rhythms of Oo-consumption which also persist under constant labora- tory conditions. These reports were not very successful in convincing the majority of biologists of the reality of persistent tidal rhythmicity. However, during the past few years, a number of studies have again pointed out that many organic processes, e.g., color change, spontaneous activity, and Oo-consumption, in a rather wide vari- ety of plants and animals do indeed vary with primary lunar or tidal frequency under constant conditions. Rao (1954) found that the filtering rate of species of Mytilns was greatest at the times of high tides in the areas of collection when the mussels were maintained in the laboratory. This rhythm of behavior was clearly apparent day by day. However, many of the lunar or tidal cycles that have been described are apparent only by sta- tistical analyses of 15 or 29 days of continuous data (Brown, Freeland and Ralph, 1955 ; Brown, Webb, Bennett and Sandeen, 1955 ; Brown, Shriner and Ralph, 1956). The results of the work to be described demonstrate that the fiddler crab, Uca pugna.v, does have an overt rhythm of primary lunar frequency, a rhythm of spon- taneous motor activity. MATERIALS AND METHODS In both 1955 and 1956, males of the species Uca pugnax were used in these studies. The crabs were collected from Chapoquoit beach or Sippiwisset beach on the Buzzards Bay side of Cape Cod. Tidal events on these two beaches occur roughly 10 minutes later than they do at New York City. In the laboratory the animals were kept in white-enamelled pans in a small amount of sea water until they 1 These studies were aided by contracts between the Office of Naval Research, Department of Navy, and Northwestern University, NONR 122803. 267 268 BENNETT, SHRINER AND BROWN I o I I.J I- D Z cc UJ I 2 HOURS 18 8/17 8/24 12 P M AM PM AM PM FIGURE 1. I. An illustration of part of the apparatus used to record the motor activity of an individual fiddler crab. For explanation of letters, see text. II. A, the average cycles of activity for a group of 20 crabs for the 15 consecutive days from August 15 through August 29, 1955. B, the average cycles of activity for a group of 20 crabs for the 15 consecutive days from August 12 through August 26, 1956. III. A and B, the mean, 29-day, tidal cycles of activity of fiddler crabs for July 6 through August 3, 1955 and August 2 through August 30, 1955, respec- tively. The arrows indicate the relative times of low tide at Chapoquoit Beach. C and D, the mean, 29-day, solar cycles of activity of fiddler crabs for July 6 through August 3, 1955 and August 2 through August 30, 1955, respectively. CYCLES OF ACTIVITY IN UCA 269 were placed in the recording apparatus. This was done usually within two days of their collection. Part of the recording apparatus employed is illustrated in Figure 1,1. A single crab was placed with a small amount of sea water in a plastic saucer (A), and covered with a circular piece of cardboard (B) which fitted the saucer tightly. Each of 10 saucers was supported on one side by metal bands (C) which were in turn fastened to a rigid horizontal bar (D). To the opposite side of each saucer was attached a nylon thread (E) which was fastened to the lever of a spring balance recording system (F) equipped with an ink- writing pen. The movements of the crabs in the finely balanced saucers were recorded on a kymograph which made one complete revolution every 24 hours. The spring balances and kymographs were of the type described by Brown (1954a). The experiments were carried on in an inner room in a brick building at the Marine Biological Laboratory in which the light intensity at the level of the record- ing apparatus was at all times essentially constant and less than two ft. c. The con- tainers with the inclosed crabs were shielded from movements and shadows in the laboratory. The air temperature in the room varied non-rhythmically from 21° to 24° C. through the summer months. During the summer of 1955, the activity of 10 crabs was recorded continuously from July 6 through August 13, and that of 20 crabs was recorded from August 14 through 30. Freshly collected crabs were placed in the recorders on July 5, July 14, and August 14. In 1956, 20 crabs, which were collected on June 16 were placed in the recorders on that day, and their activity was recorded through the afternoon of June 26. On August 11, another group was collected, and the activity of 20 of these was recorded from that day through August 28. The data recorded in 1955 were analyzed in the following manner: the number of minutes of each hour that each animal was active was determined from the kymo- graph recordings. From these figures was calculated the average number of min- utes per hour that the population (either 10 or 20 individuals) was active. The hourly data were converted into three-hour moving means as given in Table I. Mean, 29-day solar and lunar cycles of activity were analyzed by methods used ex- tensively in our laboratory (Brown, Bennett, Webb and Ralph, 1956; Brown, Free- land and Ralph, 1955) by which any possible solar or lunar cycles are synchronized day by day. The results for 1956, given in Table II, are in terms of activity units per hour. Activity units were derived as follows : for each hour, the number of animals of the group of 10 that was active, e.g., 8 out of 10, was recorded from the kymograph records. From these hourly values were calculated three-hour moving sums. The sums for the two groups of 10 each that were observed concurrently were added. For the periods August 14-30, 1955, June 17-26, 1956, and August 12-28, 1956, when the motor activity of 20 individuals was recorded, the average values per hour for the two groups of 10 crabs each were correlated on an hour-by-hour basis. However, in this report, all data are given as the average for the entire population that was observed at any particular time. RESULTS The hourly values of spontaneous motor activity for Uca pugna.v in 1955 are found in Table I, and those for the two periods of 1956 in Table II. By merely 270 BENNETT, SHRINER AND BROWN scanning these data, it is possible to observe that within many solar days there are two periods, each several hours in duration, of relatively high activity which are separated from one another by 12 to 13 hours, e.g., on July 7, 1955 (Table I), there was high activity during the first two hours of the day and again during hours 12 and 13. By noting the situation on two consecutive days, e.g., July 16 and 17, 1955 (Table I), it can be seen that the two periods of high activity occur later in the solar period on the second day than on the first. It is also evident from inspection of the data that there is a considerable range in the values, 0 minutes per hour active to 45 TABLE I The average number of minutes active /hour for groups of fiddler crabs for 1955 Hour 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 July 6 33 30 23 14 7 8 9 19 27 37 38 34 32 26 21 13 12 12 17 17 18 21 23 26 7 29 29 22 19 10 11 8 16 23 32 34 39 39 33 25 17 16 — — — 24 17 13 11 8 14 23 28 29 23 — — — — 15 16 18 14 18 18 17 11 8 8 8 10 8 10 12 9 17 20 16 17 12 10 6 4 3 3 6 10 17 20 14 9 6 6 5 4 8 9 8 7 10 10 14 15 13 13 11 8 6 6 10 10 13 11 14 13 13 9 6 5 8 10 11 6 5 11 6 9 12 14 13 10 9 7 7 6 6 4 4 7 11 10 8 5 6 5 6 6 8 5 12 4 4 6 8 10 12 14 12 10 6 4 2 1 1 4 6 7 8 6 6 2 4 4 5 13 3 3 3 7 10 10 7 5 — — — 13 10 7 5 1 6 11 13 8 4 3 3 4 14 3 2 2 3 7 9 10 7 — — 32 17 13 13 19 23 29 34 36 31 26 22 15 12 15 12 8 10 24 36 45 41 36 28 21 15 9 6 3 7 13 20 22 28 30 32 28 24 20 16 14 13 11 18 23 31 34 36 31 24 15 9 10 8 8 5 8 14 19 24 22 18 14 15 17 15 9 6 6 10 18 22 25 23 20 16 13 8 7 6 7 7 5 7 11 14 15 13 14 18 14 14 11 9 8 8 11 15 20 21 22 18 — . — — 6 6 5 3 6 8 11 13 18 19 17 15 8 6 8 8 8 7 13 15 19 14 12 5 2 1 1 4 4 5 3 5 9 11 20 15 16 16 10 7 6 7 9 12 19 16 14 11 13 10 5 2 2 2 2 1 1 3 6 21 7 5 5 6 12 12 12 8 5 3 5 7 — — — 9 5 3 2 2 4 4 5 4 22 5 8 12 12 14 12 12 10 12 10 6 5 7 8 11 12 12 7 5 3 4 3 3 2 23 4 6 9 15 17 19 12 9 7 8 9 6 4 4 4 4 5 5 3 1 3 6 9 7 24 5 4 8 6 5 3 10 18 19 18 15 15 10 6 6 8 9 10 12 11 5 1 1 2 25 5 6 6 5 3 4 3 11 23 31 26 17 8 8 7 7 7 7 7 9 9 12 9 8 26 5 5 4 3 4 8 11 20 19 21 13 8 3 1 — — — — 2 3 7 8 8 6 27 6 6 3 2 2 5 10 11 11 9 11 10 7 5 2 3 2 2 3 3 3 5 7 7 28 5 4 4 5 — — — — — 11 12 18 18 10 4 1 0 1 — — — — — - — 7O 94 71 1 f\ •3 9 1 e if If 30 8 4 5 5 7 6 6 3 4 11 16 Z*« 19 £, L 19 L " ' 19 16 11 6 J 4 & 3 4 5 6 X O 4 3 31 3 4 7 12 15 18 16 16 14 13 15 19 21 23 19 14 9 8 9 9 7 7 8 11 Aug. 1 11 9 5 6 9 13 12 13 12 12 12 14 17 19 22 22 19 14 17 18 14 10 5 3 2 3 5 6 7 6 6 6 10 11 8 3 5 12 19 22 20 20 20 18 16 11 10 6 7 3 6 7 8 10 11 — — — — — 13 9 6 10 11 19 24 30 30 25 23 20 13 11 4 12 12 12 11 14 15 14 12 7 10 12 12 9 7 10 8 10 11 11 11 9 12 10 11 5 11 12 12 9 6 5 6 7 6 5 4 4 3 4 6 8 9 10 11 11 10 9 10 11 6 10 9 5 5 4 6 7 7 8 8 8 4 3 4 5 4 3 3 5 6 6 7 9 9 7 8 7 6 7 5 8 7 8 5 6 10 14 14 9 8 10 11 11 9 9 11 8 7 5 8 9 8 7 5 7 10 8 8 7 8 9 8 7 5 3 5 7 8 7 6 9 11 11 10 9 9 7 4 5 8 12 10 7 8 7 7 3 3 6 6 8 11 16 15 9 4 6 6 13 10 15 19 18 17 15 13 11 8 6 3 2 3 7 10 13 15 17 17 17 15 11 5 10 15 11 16 14 12 15 19 21 20 18 18 16 10 5 3 3 5 8 16 16 11 4 1 2 1 2 12 7 11 11 9 11 16 18 19 19 17 12 7 7 6 9 9 11 14 16 16 15 13 11 7 13 5 10 10 9 10 6 9 8 9 9 8 8 9 9 9 7 5 7 6 9 10 12 12 9 1 O 91 93 90 9 ^ 9/=i 9fi 15 23 21 16 15 20 30 40 40 34 22 13 10 8 6 5 7 IV 16 20 Zl 33 £o 31 zu 36 zo 34 zo 29 zu 22 16 17 15 13 11 14 19 35 40 37 26 19 15 14 13 10 11 10 16 19 25 29 31 31 31 17 29 19 12 10 6 7 14 26 37 37 32 22 14 10 8 6 5 3 6 11 17 22 23 29 18 25 19 14 13 12 7 7 9 17 27 31 27 24 17 11 7 5 4 4 6 12 18 21 28 19 24 23 23 20 11 8 7 6 10 18 26 20 28 23 16 11 4 3 3 4 6 12 14 18 20 19 20 20 16 14 12 11 10 6 13 16 24 24 23 18 14 10 8 6 4 5 6 13 16 21 19 19 21 22 18 16 12 10 7 5 8 13 20 21 20 16 12 8 7 6 6 4 5 8 22 10 14 17 17 16 13 11 11 9 7 7 10 14 19 20 17 12 7 6 5 5 4 6 7 23 10 9 15 16 17 11 12 15 15 10 6 5 6 9 11 13 14 14 10 8 6 5 5 5 24 8 10 10 11 13 12 11 10 12 12 11 8 7 6 6 7 11 17 15 7 7 7 9 8 25 8 6 5 8 10 11 10 10 11 13 18 26 27 23 16 13 17 22 24 20 15 12 12 9 26 10 11 14 16 16 18 20 22 20 17 15 13 13 13 12 13 12 11 12 13 13 13 16 20 27 19 15 10 9 11 16 19 20 19 19 20 18 15 14 13 11 9 7 8 10 12 15 19 20 28 21 16 12 9 9 11 14 13 14 16 17 17 15 13 13 13 11 12 10 12 12 12 10 7 29 6 5 5 9 12 13 12 13 13 12 15 15 14 12 9 6 6 5 7 6 7 8 10 19 30 10 10 10 8 7 6 7 7 8 9 11 13 14 14 11 9 6 7 4 — CYCLES OF ACTIVITY IN UCA 271 TABLE II The activity units /hour for groups of fiddler carbs for 1956 Hour 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 June 17 20 28 36 41 37 30 27 23 22 18 18 17 18 24 30 37 35 33 30 29 26 24 25 28 18 26 24 29 36 41 37 31 26 23 22 19 17 17 18 17 24 30 37 35 30 25 24 28 29 19 28 26 25 31 38 44 42 — — . — — — 26 20 16 13 15 20 21 23 21 23 23 24 20 26 27 29 30 32 34 36 36 33 30 25 19 17 19 22 20 16 17 21 30 32 32 31 28 21 24 18 19 21 21 20 25 31 33 31 28 26 20 16 11 12 10 13 19 26 29 30 28 28 22 27 26 25 22 24 23 24 28 31 33 32 33 28 20 14 17 23 23 22 24 30 30 26 25 23 27 29 27 26 29 28 25 24 30 42 42 40 30 25 20 18 16 12 9 13 22 23 30 27 24 26 25 24 21 24 24 24 21 24 32 33 31 28 23 18 11 10 9 10 8 19 26 35 30 25 28 25 27 27 27 24 24 21 21 25 29 30 28 25 17 12 7 11 14 12 11 14 26 34 26 33 30 29 27 27 26 23 19 15 20 23 27 27 26 20 16 11 — — — — — — • — Aug. 12 57 56 53 51 43 35 28 27 25 28 37 50 55 56 54 50 48 47 52 50 48 44 49 54 13 58 56 53 48 44 — — . — — — 36 32 39 48 52 49 42 41 40 43 46 42 35 29 14 32 38 48 49 49 40 35 24 24 21 30 28 33 37 42 47 44 44 39 40 40 37 31 24 15 28 37 46 — — — 36 29 28 24 18 18 22 32 41 47 45 38 — — — — 38 32 16 24 20 24 32 37 40 35 27 16 13 13 11 9 9 15 29 42 44 43 39 43 36 26 20 17 17 26 24 29 29 — — — — 22 17 13 13 14 10 7 14 24 30 31 27 26 22 21 18 15 13 13 17 21 25 — — — — — 13 9 11 11 13 12 14 18 27 35 42 40 34 19 24 20 16 14 8 8 10 20 28 30 31 26 20 14 11 10 10 12 17 20 25 34 41 40 20 34 27 22 16 14 14 16 25 34 40 36 29 20 14 8 7 6 12 14 — — — 44 41 21 39 35 29 24 17 13 9 10 — — — — — — — — 14 9 9 7 7 11 23 35 22 38 34 26 23 19 16 10 10 15 23 28 32 31 29 23 16 11 8 10 10 11 16 23 30 23 36 35 34 27 24 20 21 17 17 17 19 — — — — 39 21 15 13 12 10 11 13 28 24 19 24 28 32 36 36 36 31 — — — 15 14 20 22 26 20 15 11 11 9 12 12 17 25 21 28 33 33 29 25 — — — — 13 12 — — — 32 30 22 18 13 13 11 11 11 26 16 20 23 — — — — — — . — — 22 22 19 23 29 34 33 28 23 21 17 17 19 27 o o 25 i A 25 1 Q 27 0 1 26 on 31 O ^ 32 •-) 7 38 -20 37 79 32 -2 ? 30 9Q 28 25 19 16 22 23 25 25 27 27 26 23 20 17 £Q lo lo Z\ ZU £J ~ / oZ JZ oo £y minutes per hour active in 1955, and 8 activity units per hour to 58 activity units per hour in 1956. Near the end of a particular recording period, both the mean daily activity and the amplitude of the cycles are lower than at the beginning of the same period. These results are shown graphically in Figure 1, II A and B, in which are plotted the cycles of motor activity for groups of 20 crabs for August 15 through 29, 1955 and August 12 through 26, 1956. In Figure 1, II A, one can follow the moving of the two peaks of activity across succeeding solar days from August 15 to 22. In this instance, the peak that occurred between hours 7 and 8 on August 15 occurs between hours 14 and 15 on the 22nd, and the evening peak of August 15 has moved to the early morning on August 22. For the cycles of August 23 through 29, it is more difficult to distinguish maxima or the movement of these maxima very precisely. There seems to be a warping and diminution of a peak as it moves into the afternoon and evening hours. The peak of the morning hours which can be identified on August 20, can be traced through August 29 ; however, it, too, shows warping and broadening. Generally, the same characteristics are to be seen in Figure 1, II B, especially for the cycles of August 12 through 16. After this time, the data are incomplete, and it is not advisable to complete the curves from only the available data. A period of high activity seen just after 0 hour on August 12 is identified as a rather sharp maximum at 12 on August 22, and similarly the afternoon high (hour 14) on August 12 is identified between hours 3 and 4 on August 25. Again, the warping and broadening of maxima, observed in 1955, are evident. 272 BENNETT, SHRINER AND BROWN In order to demonstrate the reality of the movement of peaks of activity across solar days at the average primary lunar or tidal rate of 51 minutes per day, the rate of movement for specific peaks was determined for 20 different intervals from the data of 1955 and 1956. The intervals used were in no case less than two days or more than 11 days. The average rate for these periods was 50.1 ± 3.58 minutes per day. Tidal phase relationships as well as frequency relationships are to be noted in these rhythms of activity. It was observed that in 1955, the peaks of activity oc- curred two to three hours before the times of low tide on the beaches from which the crabs were collected. In 1956, the maximum activity was, on the average, four to five hours before low tides. This phase relationship is shown in Figure 1, III in which are plotted the mean, 29-day tidal cycles of locomotor activity for July 6 through August 3, 1955 (A) and August 2 through 30, 1955 (B). These curves are plotted so that the times of low tides at Chapoquoit Beach, as indicated by arrows, lie directly under one an- other, although the tidal events actually occurred at different times on the two days with which the 29-day analyses were begun. The times of low tide were : 3 :22 and 15 :19 on July 6 and 1 :34 and 13 :37 on August 2. In these figures, the form of the tidal cycle, discussed previously, is again apparent. In A, a sharp peak of activity is seen at hour 1, or 2 hours and 22 minutes before low tide. The second maximum takes the form of a broad peak from hour 13 through 18, extending from 2 hours before until 3 hours after low tide. Activity fell rather steeply from 1 until 7 while the activity following the second maximum did not decrease so steeply nor to so great a degree. The cycle for August 1955 (Fig. 1, III B) was very much like that illustrated in Figure 1, III A, although the second period of high activity does not last so long in the former as in the latter. Again, the activity persisted at a higher level following the second maximum than it did following the first peak. In Figure 1, III C and D are plotted the mean, 29-day, solar cycles for the same periods of time as the lunar cycles. It is apparent that the amplitude of the solar cycles is less than that of the lunar cycles. From the lowest to the highest values there was a 2.4-fold increase in the tidal cycle for July 6 through August 3, and a 2.2-fold increase in the tidal cycle for August 2 through 30, whereas the increases for the solar cycles for the same periods were 1.6-fold and 1.3-fold, respectively. Although the amplitude of these solar cycles is low, the cycles indicate that activity between hours 6 and 12, other factors equal, is higher than at other times of the solar day. This tendency can be seen in Figure 1, II A by comparing the heights of the two peaks, i.e., the morning peak is higher than the afternoon peak from August 15 through 17. Coefficients of correlation for the activity of two groups of crabs, the activities of which were recorded independently during the same periods of time, were : + 0.768 ±0.028 for August 14 through 30, 1955; + 0.410 ± 0.058 for June 17 through 26, 1956; and + 0.687 ± 0.034 for August 12 through 28, 1956. DISCUSSION The cycle of spontaneous motor activity for the fiddler crab, Uca pugnax, de- scribed in this report, appears to be the first example of a clearly overt locomotor rhythm of primary lunar or tidal frequency. The occurrence of two peaks of ac- CYCLES OF ACTIVITY IN UCA 273 tivity, 12 to 13 hours apart, within one solar day, and the movement of these peaks across succeeding solar days at an average tidal rate establish the reality of a tidal cycle persisting under laboratory conditions. It must be pointed out that the day- to-day preciseness of this rhythm decreases somewhat after the crabs have been in the recording containers seven or eight days. The warping and broadening of peaks, as well as the diminution of activity, discussed previously, cannot be ex- plained satisfactorily at the present time. It is tempting to postulate that these phenomena are indications that an internal timing mechanism is not able to maintain a precise cycle longer than a week or so under constant conditions, and that after this time unknown external signals alone maintain only a less regular rhythm. Evi- dences for both endogenous and exogenous components of persistent rhythmicity in fiddler crabs have been reported recently (Brown, Webb and Bennett, 1955 ; Brown, Webb, Bennett and Sandeen, 1955). The difference between the phase relationships of actual tidal events and the peaks of activity observed in 1955 and 1956 brings up questions regarding the set- ting of phases of tidal cycles, and to what extent behavior cycles under constant con- ditions parallel those of the organisms in their natural environments. The lunar or tidal cycles described previously indicate that although many species have cycles of 12.4 and/or 24.8 hours, phase relationships are species specific. For example, the spontaneous activity of quahogs is low during the times of low tides when this spe- cies may not be covered by water (Bennett, 1954), while the pigment in the melano- phores of the fiddler crab, Uca pugnax, is most dispersed shortly before the times of low tide when these animals are typically active on the beaches (Brown, Finger- man, Sandeen and Webb, 1953). In these cases, the phases of the persistent tidal cycles seem to indicate some adaptiveness of the cycles to conditions which obtain under natural field conditions. On the other hand, Fingerman (1956) has reported that fiddler crabs, Uca pugilator and Uca speciosa, collected from regions of the Gulf coast where there is but one low tide per day, have persistent tidal cycles of color change characterized by twro peaks of pigment dispersion during each solar day, one shortly after low tide, and the second 12.4 hours later or shortly after high tide. The relationship observed between the fiddler crab activity and the times of low tide in 1955, i.e., maximum activity two to three hours before low tides, might sug- gest that the behavior of the crabs in the laboratory is much the same as that of the crabs on the beaches. Typically, these crabs begin to emerge from their burrows as the water recedes following a high tide, and great numbers of these crabs are usually running on the beaches until shortly before low tide. However, the ob- servations for 1956, i.e., that the peaks of activity occurred four to five hours be- fore low tide, suggest that the phase relationships of persistent tidal cycles change, and may not always reflect adaptive behavior of the species in its natural, changing, physical environment. It is possible that the difference in the rhythmic behavior of the crabs between the two years may reflect differences noted in field observations between these same two years. In 1955, as was usual, the crabs were collected, easily shortly before the times of low tides. In 1956, the animals were difficult to. collect since there were few running on the beaches before low tides. Many of the crabs used in 1956 had to be dug from their burrows. It is possible that stimuli other than those resulting from emergence and running of the crabs may set the phases of the observed tidal cycles. 274 BENNETT, SHRINER AND BROWN That the phases of a tidal cycle do shift in the absence of experimental modifi- cations is shown clearly in the report of Brown (1954b). In this work, it was found that the maxima of the tidal cycle of oysters collected from New Haven Har- bor and shipped to Evanston, Illinois correlated with the times of high tide in the native habitat during the first two weeks of maintenance under constant conditions. During the next month, the same group of oysters showed a rhythm with maxima at the times of lunar zenith and nadir. Two other reports contain information re- garding the experimental shifting of the phases of tidal cycles (Brown, Fingerman, Sandeen and Webb, 1953; Rao, 1954). However, since the problem of the setting of phases of persistent tidal cycles is one that has not been investigated to a great degree as yet, much more work must be done before any definite statements can be made. The overt tidal rhythm described here promises to be one by which not only the problem of phase setting but also experimental modifications of tidal cycles can be studied. The facts 1 ) that the cycle of locomotor activity repeats itself rather pre- cisely on a day-to-day basis for at least a week after the crabs are placed under con- stant conditions, and 2) that the cycles of two independent small groups correlate significantly to a high degree do point to its usefulness in such studies. SUMMARY AND CONCLUSIONS 1. The spontaneous locomotor activity of groups of fiddler crabs, Uca pugnax, was recorded during the summers of 1955 and 1956 under constant laboratory conditions. 2. This species shows an overt rhythm of activity of primary lunar or tidal fre- quency. Within solar days, there are two peaks of activity which are 12 to 13 hours apart. These maxima move across succeeding solar days at an average tidal rate. The cycles are precise for at least a week under constant conditions, but after this time, some warping and displacement of maxima occur. 3. A low amplitude solar rhythm of activity is apparent upon analysis of 29 days of continuous data. This rhythm is characterized by high activity betwreen hours 6 and 12 of the solar day. 4. There was observed a difference in phase relationships of the tidal rhythms be- tween 1955 and 1956. The state of the problem of the setting of phases of per- sistent tidal cycles is discussed. 5. Since this rhythm is precise for at least a week under constant conditions, and since the cycles of two groups of crabs recorded independently correlate to a high degree, this cycle appears to be an excellent one with which to study experimental modifications of persistent tidal rhythms. LITERATURE CITED BENNETT, M. F., 1954. The rhythmic activity of the quahog, Venus mercenaria, and its modifi- cation by light. Biol. Bull., 107 : 174-191. BOHN, G., 1904. Periodicite vitale des animaux soumis aux oscillations du niveau des hautes mers. C. R. Acad. Sci., Paris, 139 : 610-611. BOHN, G., 1906. La persistance du rythme des marees chez 1' 'Actinia equina. C. R. Soc. Biol., Paris, 61 : 661-663. BROWN, F. A., JR., 1954a. Simple, automatic, continuous-recording respirometer. Rev. Sci. Inst., 25 : 415-417. CYCLES OF ACTIVITY IN UCA 275 BROWN, F. A., JR., 1954b. Persistent activity rhythms in the oyster. Amcr. J. Physio!., 178: 510-514. BROWN, F. A., JR., M. F. BENNETT, H. M. WEBB AND C. L. RALPH, 1956. Persistent daily, monthly, and 27-day cycles of activity in the oyster and quahog. /. E.rp. Zool., 131 : 235-262. BROWN, F. A., JR., M. FINGERMAN, M. I. SANDEEN AND H. M. WEBB, 1953. Persistent diurnal and tidal rhythms of color change in the fiddler crab, Uca pugnax. J. Exp. Zool., 123 : 29-60. BROWN, F. A., JR., R. O. FREELAND AND C. L. RALPH, 1955. Persistent rhythms of O2-consump- tion in potatoes, carrots and the seaweed, Fucus. Plant Physiol., 30 : 280-292. BROWN, F. A., JR., J. SHRINER AND C. L. RALPH, 1956. Solar and lunar rhythmicity in the rat in 'constant conditions' and the mechanism of physiological time measurement. Amer. J. Physiol., 184 : 491-496. BROWN, F. A., JR., H. M. WEBB AND M. F. BENNETT, 1955. Proof for an endogenous component in persistent solar and lunar rhythmicity in organisms. Proc. Nat. Acad. Sci., 41 : 93-100. BROWN, F. A., JR., H. M. WEBB, M. F. BENNETT AND M. I. SANDEEN, 1955. Evidence for an exogenous contribution to persistent diurnal and lunar rhythmicity under so-called con- stant conditions. Biol. Bull., 109 : 238-254. FINGERMAN, M., 1956. Phase difference in the tidal rhythms of color change of two species of fiddler crab. Biol. Bull., 110: 274-290. GAMBLE, F. W., AND F. KEEBLE, 1903. The bionomics of Convoluta roscoffensis with special reference to its green cells. Proc. Roy. Soc., London, Ser. B, 72 : 93-98. GAMBLE, F. W., AND F. KEEBLE, 1904. The bionomics of Convoluta roscoffensis with special reference to its green cells. Quart. J. Micro. Sci., 47 : 363-431. COMPEL, M., 1937. Recherches sur la consommation d'oxygene de quelques animaux aquatiques littoraux. C. R. Acad. Sci., Paris, 205 : 816-818. RAO, K. P., 1954. Tidal rhythmicity of rate of water propulsion in Mytilus, and its modifiability by transplantation. Biol. Bull., 106 : 353-359. STUDIES OF THE METABOLISM OF PHOSPHORUS IN THE DEVELOPMENT OF THE SEA URCHIN, STRONGYLOCENTROTUS PURPURATUS 1 ALBERT L. BOLST 2 AND ARTHUR H. WHITELEY Department of Zoology and the Friday Harbor Laboratories, University of Washington, Seattle 5, Washington The eggs of sea urchins contain a group of acid-soluble phosphorylated com- pounds whose barium salts are soluble in alcohol. The magnitude of this fraction is unusually large in comparison with other animal tissues. The unfertilized eggs of various sea urchins and asteroids have been reported to have from 9.3% (Mende and Chambers, 1953) to 39.7% (Whiteley, 1949) of their acid-soluble phosphorus in the form of barium-soluble, alcohol-soluble compounds. In various vertebrate tissues these compounds comprise about 1 to 8.3% of the acid-soluble phosphorus (LePage, 1948; Sacks, 1949). Aside from quantitative measurements, very little is known about these compounds or the part they play in the metabolism of the echinoderm egg. Lindberg (1943) reported finding a compound in this fraction from the egg of the heart urchin, Brissopsis, which he subsequently (1946) iden- tified as 1, 2-propanediol phosphate, whose metabolism he studied. Horstadius and Gustafson (1947) reported some animalizing effect by both synthetic propanediol phosphate and a natural compound, and Borei (1948) has described the effect of this ester on egg respiration. However, Rudney (1952, 1954) has reported that the barium salt of propanediol phosphate is not soluble in alcohol, and the significance of the above observations, therefore, is not clear. Mende and Chambers (1953) have shown that some of the phosphorus of the barium-soluble, alcohol-soluble frac- tion of Asterias forbesii and Strongylocentrotus drobachicnsis is acid-labile. This paper supplies information relative to the extent to which the barium- soluble, alcohol-soluble fraction serves as a storage material for metabolism, and the rate of turnover of the compounds of this fraction in the eggs and embryos of the sea urchin, Strongylocentrotus purpuratus. The possibility has been explored that there are special periods in the developmental process when the metabolic activity of these compounds varies. The existence of propanediol phosphate as a component of this fraction has been examined, and preliminary chromatographic determination of the number of esters in the fraction has been made. As an outgrowth of the turnover studies, an unusual pattern of permeability of embryos to inorganic phos- phate during larval development has been found. 1 This investigation was supported in part by the Research Fund 171 of the University of Washington. A report of these studies was given at the meeting of the Western Society of Naturalists, December, 1952. 2 Currently on duty in the Navy. 276 METABOLISM OF PHOSPHORUS BY EGGS 277 MATERIALS AND METHODS Embryological. The sea urchins used in these experiments were Strongylo- centrotus purpuratus (Stimpson), collected from San Juan Island, Washington. Animals were induced to spawn by the injection of isotonic KC1 (Tyler, 1949). Batches of eggs less than 95 % fertilizable were not used. "Dry" sperm were di- luted to a 1 % suspension immediately before use. After insemination, excess sperm were removed from eggs by washing with fresh sea water. Filtered sea water was used throughout. In experiments on embryos older than the two-cell stage suspensions of devel- oping embryos were cultured in a four-liter Erlenmeyer flask which lay at an angle of about 30° in a water bath held to 11.0 ± 0.1° C. by a thermostat. Egg suspen- sions, ranging in concentration from 0.3 to 0.9% by volume, were gently agitated by rotating the flask at 28 rpm. For egg counts, 10-ml. aliquots were taken and, after appropriate dilution, ten individual counts were made and averaged. Aliquots of 300 ml. were taken from the stock suspensions at different stages of development for analysis of phosphates and for separate incubation with radioactive phosphate. Carrier-free radioactive phosphate was added to give a final radio- activity of 0.04 juc./ml., and incubation was continued in the same manner as de- scribed above, normally for 60 minutes. Duplicate 10-ml. aliquots were then taken for analysis of total phosphorus. The embryos were removed from the remainder of the suspension in a Incite centrifuge patterned after the Foerst plankton centri- fuge. After washing with fresh sea water the embryos were frozen and stored for later analysis. In experiments with eggs prior to the first cleavage the incubation times and radiophosphate concentrations varied and are given with the results. These sam- ples were analyzed immediately without freezing. Analytical. The extraction of the barium-soluble, alcohol-soluble material from the embryos followed the procedure of Sacks (1949) and that of Umbreit, Burris and Stauffer (1949). In some instances the other phosphate fractions identified below were also prepared. The sample was thawed and homogenized in a Potter- Elvehjem type of homogenizer in 2 ml. of 0.5 N HC1O4. This and the subsequent steps were carried out near 0° C. The homogenate was centrifuged and the residue (acid-insoluble fraction) re-homogenized twice with 1 ml. of 0.5 N HC1O4. The three supernatants were pooled, four volumes of re-distilled 95% ethyl alcohol added, and the mixture set aside for one hour. The extract was centrifuged, the residue (acid-soluble, alcohol-insoluble, probably polysaccharides) washed twice with acid- ethanol, the combined supernatants adjusted to pH 8.2, and 1 ml. of 25% barium acetate added. The precipitate (barium-insoluble plus barium-soluble, alcohol- insoluble fractions) that formed after one hour was centrifuged and washed with ethanol adjusted to pH 8.2. The supernatants were brought to 50.0 ml. This is the barium-soluble, alcohol-soluble fraction. Phosphorus was determined by the method of Berenblum and Chain (1938), and, in a few cases, by a modified Fiske and SubbaRow (1925) method with ferrous sulfate as reducing agent. The dried samples, blanks, and standards were digested in 70% HC1O4. The blue isobutanol extracts resulting from the phosphorus deter- minations were used for radioactivity assay. Aliquots were pipetted into planchets, dried, and counted with a Geiger-Miiller counter equipped with an end-window tube 278 ALBERT L. BOLST AND ARTHUR H. WHITELEY with window thickness of 3.26 mg./cm.2 Samples were counted to an error of less than \%. In experiments with pre-cleavage stages, where phosphate was analyzed by the Fiske and SubbaRow method, aliquots of eggs or extracts were dried directly on planchets. Paper chromatographic analysis for phosphate esters was carried out using a chromatographic system the details of which will be published elsewhere. The sol- vent system contained n-butanol, n-heptyl amine, and water. The descending method was used with washed Whatman No. 1 filter paper. The chromatograms were run at 1° C. for about 10 hours and developed by spraying with the Hanes- Isherwood molybdate reagent, heating at 80° C. for 5 minutes (Hanes and Isher- wood, 1949), and irradiating with ultraviolet light at wave-length 2537 A (Ban- durski and Axelrod, 1951). RESULTS If the phosphorus-containing compounds of the barium-soluble, alcohol-soluble fraction serve as a reserve of phosphorus, energy, or precursors of other substances during development, an indication of this function would probably be given by a de- crease in the concentration of the phosphorus in the fraction as development pro- gresses. Two experiments, each involving the eggs of a single sea urchin, were carried out to determine if this occurs. In each a large batch of fertilized eggs was cultured at 11° C. to the early pluteus stage. Aliquots were taken at intervals for determination of total phosphorus and barium-soluble, alcohol-soluble phosphorus. The results are given in columns three and four of Table I, and are shown in o X V) o or 0 o 1 >- a. (r CO CD * 12 10 01 to o o: o T T T • o- J_ _L 20 30 40 50 60 70 TIME AFTER FERTILIZATION - HOURS 80 90 FIGURE 1. Total (upper curve) and barium-soluble, alcohol-soluble phosphorus content (lower curve) of developing embryos of Strongylocentrotus purpuratus. Solid circles are data from Experiment 1 and open circles are from Experiment 2 of Table I. METABOLISM OF PHOSPHORUS BY EGGS 279 TABLE I Amount of phosphor iis and rates of incorporation of radioactive phosphorus in embryos and barium- soluble, alcohol-soluble compounds of embryos of the sea urchin, Strongylocentrotus purpuratus Embryos Phosphorus content /ig./egg Radioactive phosphorus content Specific activity Age Stage (cts./min. /egg/hr.) % BSAS- P32 (cts./min./ MgP/hr.) % BSAS- p32 BSAS-P Total P BSAS-P32 Total P82 BSAS-P32 Total P32 Experiment No. 1 0 hrs. UF* 3.0X10-4 8.5 X 10-" _ 4* 2-C1 2.9 7.2 6.5 X10-2 120X10-2 5.4 225 1710 13.2 17 UB 2.8 7.6 15.2 221 6.9 532 2910 18.3 41 EG 2.5 7.6 2.8** 36** 7.8 108** 472** 22.9 65 i LG 2.3 6.2 13.8 193 7.2 595 2240 26.6 89± EP 2.4 8.6 6.4 140 4.6 262 1550 16.9 Experiment No. 2 0 UF 3.9 6.7 _ _ 6 4-C1 2.9 7.4 3.9 56 7.0 134 732 18.3 21 UB 3.4 7.6 13.3 241 5.4 384 3090 12.4 34£ EG 2.9 8.4 24.8 392 6.4 863 4520 19.1 42| EG 2.2 8.5 19.0 332 5.7 868 3760 23.1 48 MG 2.6 8.8 17.3 248 7.0 657 3360 19.6 72 PR 2.2 8.2 7.2 141 5.1 324 1670 19.4 Experiment No. 3 43 EG 3.0 12.0 29.6 559 5.3 1120 5420 20.6 * Eggs from a single female used for each experiment. UF = unfertilized, 2-C1 = 2-cell stage, 4-C1 = 4-cell stage, UB = unhatched blastula, EG = early gastrula, MG = mid-gastrula, LG = late gastrula, PR = prism, EP = early pluteus. ** These data low, presumably by a factor of 10, due to a technical error. Experiment No. 3 was run at 43 hours to check this point. Figure 1, the curves of which are fitted by the method of least squares. The quantity of barium-soluble, alcohol-soluble phosphorus decreases at a uniform rate throughout the non-feeding stages of larval development. The rate of utilization in Experiment 1 is 7.4 X 10"7 microgram/egg/hour and in Experiment 2 is 20 X 10~7 microgram/egg/hour. The per cent of the fraction present at the beginning of development utilized in reaching the prism stage (72 hours) is 18.5% and 40.7% for Experiments 1 and 2, respectively (based on the fitted curves). It seems safe to conclude that the utilization of the material contributed appreciably to the general metabolism since it involves mobilization on the average of 13.6% of the total phos- phorus of the unfertilized egg. No significant variations in the rate of utilization correlated with visible aspects of morphogenesis are apparent in either experiment. 280 ALBERT L. BOLST AND ARTHUR H. WHITELEY TABLE II Distribution of radio phosphorus in barium-soluble, alcohol-soluble and other phosphorus-containing fractions in unfertilized and fertilized eggs. The per cent of radio phosphor us in each fraction is calculated with acid-insoluble plus acid-soluble equal to 100% Unfertilized eggs* Fertilized eggs** Fraction Specific Specific cts./min./ Per cent activity cts./min./ Per cent activity ml. p»2 cts./min./ ml. paz cts./min./ Mg P Mg P Total egg — — 188,500 100.3% 240 Acid-insoluble 24,050 29.2% 28.3 14,690 7.87 33.4 Acid-soluble 58,600 71.0 75.1 172,100 92.0 486 Barium-insoluble plus barium-soluble, alcohol- 60,100 73.0 95.5 160,500 85.9 596 insoluble Barium-soluble, alcohol- 1,180 1.40 4.37 1,350 0.72 11.6 soluble * Incubated 9 hours in P32 concentration of 0.04 juc/ml. ** Incubated one hour, before first cleavage, in P32 concentration of 0.02 me/ml. Among analyses done by both of the present authors, as well as those by others, the variation in the quantity of the barium-soluble, alcohol-soluble phosphorus is broad. Data of the present authors include values of 3.9, 3.0, 1.05 and 0.98 X 10~4 micrograms per egg, and many other analyses for which egg counts are not available indicate that the fraction from unfertilized eggs contains from 12.0% to 58% of the total phosphorus. Variations in multiplicate analyses rarely are of appreciable mag- nitude, usually amounting to only a few per cent ; it is believed the large differences from batch to batch represent true biological variations. Sacks (1949) reported variations of comparable magnitude in this fraction extracted from rat liver. Mende and Chambers (1953) found different batches of eggs of Asterias forbesii to contain 322, 153, and 138 /*g. P/ml., and Strongylocentrotus drobachiensis to con- tain 59 and 44 /xg. P/ml. During the period of development covered in these experiments there was an in- crease in the total egg phosphorus. In Experiment 1 this amounted to an increase of 9.1%, and in Experiment 2 of 23%, as calculated from the fitted curves. Although the barium-soluble, alcohol-soluble fraction shows an appreciable de- crease during development, the possibility exists that there is a simultaneous syn- thesis of some of the compounds in the fraction. The question of synthesis was ex- amined in unfertilized eggs and in embryos by adding radioactive inorganic phos- phate to cultures and determining the radioactivity of the fraction and of the whole eggs after appropriate time intervals. Unfertilized sea urchin eggs take up radioactive phosphate from sea water at an extremely low rate. To obtain sufficient activity in the phosphate fractions of such eggs, they were incubated for 9 hours at 12.0° C. in sea water containing 0.04 ^.c. radiophosphate/ml. The egg concentration was 0.4% by volume. Of a small sam- ple inseminated after this incubation, more than 95% fertilized and developed to the early pluteus. The data in Table II show that the incorporation into the barium- METABOLISM OF PHOSPHORUS BY EGGS 281 soluble, alcohol-soluble fraction prior to fertilization is very low ; even after pro- longed exposure to radiophosphate only \A% of the total activity of the egg was in this fraction. In a second experiment the figure was 0.78%. In confirmation of other investigators most of the activity enters the fraction containing inorganic phos- phate, nucleotide phosphate, and labile esters, though in the 9-hour experiment very much more is found in the acid-insoluble fraction than has been reported before (Chambers and White, 1954). The same low rate of incorporation of phosphate into this fraction persists di- rectly after fertilization, before the first cleavage. This is apparent from the second experiment of Table II, in which a 1% suspension of eggs inseminated 12 minutes earlier was incubated for 60 minutes with 0.02 /xc./ml. of radiophosphate. Although considerably more phosphate penetrated into these than into unfertilized eggs, the proportion in the barium-soluble, alcohol-soluble fraction is still only 0.7% of the whole. In comparable experiments, neither mono-iodoacetate nor 2,4-dinitrophenol, added with the radiophosphate, changed this pattern markedly, though both inhib- ited the uptake of phosphate by the egg. The subsequent embryonic period was examined from the two-celled embryo to the early pluteus stage at 90 hours in the experiments of Table I. In these the time of exposure to radiophosphate at each stage examined was 60 minutes. Columns 5 through 10 of Table I present the pertinent determinations of radioactivities. Of the radiophosphorus that enters the embryos during one hour, an average of 6.5% (4.6 to 7.8%, Column 7) is incorporated into the fraction. There is no consistent pattern of change in this value during the rest of the development. Considered in terms of specific activities, the fraction attains a level that is about 19% (12.4% to 26.6%, Column 10) of the specific activity of the total egg phosphorus, again with no consistent change during the rest of the development. This contrasts with 5% for the freshly fertilized eggs in the experiment of Table II. Although the com- ponents of this fraction are metabolically very inactive in the unfertilized egg and before the first cleavage, it is concluded that one or two hours after fertilization at least part of the barium-soluble, alcohol-soluble fraction becomes moderately stimu- lated metabolically relative to phosphorus compounds of the egg as a whole, and this increased level of metabolic activity is maintained with approximate constancy until the pluteus stage. The specific activities of the total egg phosphorus and of the barium-soluble, alcohol-soluble fraction are plotted against age of the embryos in Figure 2. It should be noted that these curves are not cumulative uptake curves, but rather are rate curves, each point representing the counts per minute per microgram of phos- phorus per 60 minutes exposure at the particular age indicated. The rate of incor- poration into the fraction is seen to increase to a maximum at about 40 hours, and then decline subsequent to this time. The peak of activity at 40 hours probably does not represent a special stimulation in metabolism within the fraction leading up to gastrulation, because, as was pointed out above, the activity of the fraction, when expressed as percentage of the whole embryo, is constant. Rather the peak reflects closely the uptake curve for the whole embryo, and is probably due to a change in permeability of the embryo to inorganic phosphate from the sea water. An unanticipated finding in these experiments is that the rate of uptake in both the total phosphorus and the barium-soluble, alcohol-soluble fraction varies markedly in the different phases of development. The rate of uptake which begins to increase 282 ALBERT L. BOLST AND ARTHUR H. WHITELEY 15 or 20 minutes after fertilization (Abelson, 1947; Brooks and Chambers, 1948; Whiteley, 1949) continues to increase markedly until the stage of early gastrulation, at 34 to 43 hours in the different experiments. The rate then begins to decrease 6000 10 20 30 40 50 60 70 AGE OF EMBRYOS AT EXPOSURE - HOURS 80 90 FIGURE 2. Rate of uptake of radiophosphate into the embryos of Strongylocentrotus pur- puratus (upper curve) and into the barium-soluble, alcohol-soluble phosphate fraction (lower curve) of these embryos. Solid circles are data from Experiment 1, open circles are from Experiment 2, and squares from Experiment 3 of Table I. The bracketed solid circles at 41 hours have been multiplied by 10 (see footnote to Table I). Specific activities are in counts/ min./Vg. P/hour. METABOLISM OF PHOSPHORUS BY EGGS 283 sharply, tending to level off in the prism and early pluteus stages. As nearly as could be determined, the inflection in the rate coincides with the onset of gastrula- tion. This pattern of uptake is shown when the data are considered either on the basis of counts per minute per egg or of specific activity. This change of permeability to orthophosphate is not, however, universal for all phosphate compounds. The permeability of embryos to propanediol phosphate, de- termined in an experiment similar to those with orthophosphate but using labeled ester instead, reaches a maximum rate earlier in development (mid-blastula) and thereafter does not decrease through the pluteus stage. Even in the absence of in- formation needed to calculate permeability constants for the ester and for ortho- phosphate, it appears that the ester penetrates much more slowly. Different mecha- nisms for the penetration of the two substances probably exist. The composition of the barium-soluble, alcohol-soluble fraction in this material is unknown. Lindberg (1943) had reported the identification of 1,2-propanediol phosphate in this fraction in sea urchin eggs, but Rudney (1952, 1954) presents cogent reasons for believing this ester separates into the barium-soluble, alcohol- insoluble fraction in rat liver. Sea urchin eggs possess very large amounts of barium-soluble, alcohol-soluble phosphates and of polysaccharides. Interference by the polysaccharides with the clean separation of esters might account for different distribution of propanediol phosphate reported by Lindberg and Rudney. To de- termine the solubility of propanediol phosphate under the exact conditions used in this investigation, fractionations were made of sea urchin egg homogenates contain- ing known amounts of synthetic 1,2-propanediol phosphate labeled with radioactive phosphorus. Two separate lots of propanediol phosphate labeled with P32 were synthesized by the method described by Lampson and Lardy (1949), using 0.188 and 0.150 millicurie P32 in the reaction tubes for the two syntheses. The lead salts obtained were converted to the free acid with dilute sulfuric acid in the first case and H2S in the other, and remaining traces of lead removed with Dowex-50 in the hydrogen form. The solution of lot 1 was neutralized to pH 7.0. This product contained no inorganic phosphate. The specific activity of the ester was 1439 counts per minute per microgram phosphorus under the standard counting conditions. The second lot, subjected to paper chromatographic analysis, showed a component with the same Rf as a commercial preparation (Nutritional Biochemicals Corp.) and a very faint unidentified second component with much less than 1% of the total activity. No inorganic phosphate was present. The specific activity was not determined. In each of two separate experiments,3 a mass of approximately a million un- fertilized eggs having an estimated barium-soluble, alcohol-soluble phosphorus con- tent of 300 micrograms was homogenized with cold 0.5 N HC1O4. Three hundred to 350 micrograms of phosphorus in the form of labeled propanediol phosphate were added to increase by a significant amount the content of supposed propanediol phos- phate already in the eggs. The fractionation was completed in the usual manner and the radioactivity in the various fractions measured with the results given in Table III. Essentially the entire amount of radioactivity wTas found in the barium- insoluble, plus barium-soluble, alcohol-insoluble fraction. It is concluded, in agree- 3 We are pleased to acknowledge the help of Miss Kathryn Eschenberg in one of these experiments. 284 ALBERT L. BOLST AND ARTHUR H. WHITELEY ment with Rudney, that the barium-soluble, alcohol-soluble fraction does not con- tain propanediol phosphate. This leaves us with no specific information as to the composition of this very large fraction of these eggs. A preliminary examination of the fraction has been made by paper chromatog- raphy. From a number of experiments in which, after isolation, it was subjected to various desalting pre-treatments, evidence for at least three components was de- rived. In all of these pre-treatments the samples were desalted with Dowex-50 in the hydrogen form and concentrated by evaporation. In some cases the phosphates precipitable by basic lead acetate were chromatographed, and in one the sample was treated with Dowex-2-OH. With different treatments, and depending on the TABLE III Recovery of phosphorus-labeled 1,2-propanediol phosphate when added to homogenates of unfertilized eggs in perchloric acid and subjected to barium and alcohol fractionation Experiment 1 Experiment 2 Fraction cts./min. % of total activity cts./min. % of total activity Acid-insoluble 31 0.3 38 0.4 Acid-soluble Alcohol-insoluble 104 1.0 60 0.7 Barium-insoluble and barium- soluble, alcohol-insoluble 10,203 96.4 8,825 97.8 Barium-soluble, alcohol-soluble 247 2.3 100 1.1 Totals ' 10,585* 100% 9,023 100% * 11,174 counts "were added to the homogenate. Recovery 94.6%. amount of sample, one or two clear spots were detectable. Compounds with Rf's of 0.12, 0.29, and 0.71 were found. With this system of chromatography, inorganic phosphate has an Rf of 0.54 and propanediol phosphate, either commercial or pre- pared by us, has an Rf of 0.73. The similarity between this Rf and that of the un- known at 0.71 is not taken as evidence of identity because other substances, for example glycerol phosphate, have the same Rf in this system. DISCUSSION The results of the present investigation indicate that there are at least three components in the barium-soluble, alcohol-soluble fraction. From its magnitude it is possible that one of these could serve as a storage compound of some kind, and the gradual, uniform utilization of the material during development would be in accord with this. The experiments with radioactive orthophosphate demonstrate an appreciable, though not great, synthesis in the fraction, probably in one or more components other than the storage ones. This synthesis is extremely low prior to fertilization and in the first hour there- after, but increases demonstrably beginning with the first cleavage. Cleavage ini- tiates an increase in activity in this fraction that is greater than the average increase METABOLISM OF PHOSPHORUS BY EGGS 285 for the phosphorus compounds of the egg ; before the first cleavage the specific ac- tivity in the fraction is 5% of that in the total egg phosphorus, while in subsequent stages it is about 19% of the total. The percentage of radioactive phosphorus that is incorporated into the fraction in embryos of different stages of development after cleavage starts is rather constant, despite large differences in total amount of radio- phosphorus that enters the embryo. This suggests that the limiting factor for the synthesis of the component is the availability of phosphate rather than the level of activity of the synthesizing enzymes. Chambers and White (1949) supply evidence that the eggs of this species have a very small inorganic phosphate pool, especially after fertilization, which would be in accord with this idea. The constancy indicates that there are no special periods during development after cleavage in which turn- over in this fraction is especially rapid relative to the turnover of the other phos- phorus compounds of the embryo. The evidence used by Lindberg (1946) for identification of propanediol phos- phate isolated from cow brain is extensive, but it is not clear in his 1943 or 1946 papers on what basis propanediol phosphate is considered to be a component of the barium-soluble, alcohol-soluble fraction, other than that the fraction has a high stabil- ity toward acid and alkaline hydrolysis. His identification of the ester in sea urchin eggs is also based on these features. LePage (1948) identified a phosphate com- pound in the barium-soluble, alcohol-soluble fraction of rat carcinoma as 1,2-pro- panediol phosphate on the basis of the phosphorus and lead content of its lead salt and its stability to hydrolysis in 1 N HC1. Against these observations, however, the experiments by Rudney (1952, 1954) and those reported here in which 96% to 98% recovery of labeled, synthetic ester was obtained in the barium-soluble, alcohol- insoluble fraction, demonstrate in a manner that seems unequivocal that this sub- stance is not a component of the barium-soluble, alcohol-soluble fraction. With the demonstration that this ester is not in the barium-soluble, alcohol-soluble fraction, there is, at present, no evidence that propanediol phosphate exists in sea urchin eggs. The existence of more than one compound in the fraction is further supported by the chromatographic studies which show a minimum of three components. In the related sea urchin, Strongylocentrotus drobachiensis , and in the star fish, As- terias forbesii, Mende and Chambers (1953) found that 23% and 60%, respectively, of the phosphorus of this fraction was hydrolyzed to orthophosphate in three hours at 100° C. in 1 N HC1. Further identification or characterization of these compo- nents has not been attempted. In several investigations of the permeability of sea urchin embryos to inorganic phosphate a greatly increased rate of penetration after fertilization has been re- ported, but subsequent changes in rate have not been followed beyond seven hours in 6\ purpuratus and four or five hours in Arbacia punctulata. In these early stages the uptake proceeds at a uniform rate. The experiments reported here show that during later cleavages and blastulation the rate of penetration continues to rise markedly, but that coincident with the onset of the first major form change, gas- trulation, the rate shows an abrupt and considerable drop which continues at least to the early pluteus. It will be of interest to determine if this new pattern reflects some profound change at gastrulation either of the metabolism within the cells of the embryo, or of a surface transport mechanism for phosphate correlated with the differentiation of the ectoderm. 286 ALBERT L. BOLST AND ARTHUR H. WHITELEY SUMMARY 1. The quantity of phosphorus in the barium-soluble, alcohol-soluble fraction of the acid-soluble phosphate compounds of the embryos of the sea urchin, Strongylo- centrotns purpuratiis (Stimpson), was measured until the formation of the early pluteus stage. In two experiments the phosphorus in the fraction decreased by 18.5% and 40.7% in reaching the late prism stage (72 hours). 2. During this period, the total phosphorus of the embryos increased an average of 14.4%. 3. The rate of penetration of radioactive phosphorus from sea water into the em- bryos during these experiments increased very greatly during cleavage and blastula- tion, reached a maximum at the onset of gastrulation, and decreased subsequently to a middle level at the prism stage. 4. The rate of uptake of radioactive phosphorus into the barium-soluble, alcohol- soluble fraction was extremely low in unfertilized eggs and in fertilized eggs before the first cleavage, amounting to 0.7% to 1.4% of the total uptake. 5. The rate of uptake of radioactive phosphorus into this fraction in cleaving eggs and embryos mirrored that of the total phosphorus, and the percentage of the labeled phosphorus in the fraction was relatively constant at all stages, averaging 6.5% of the total. 6. Chromatographic examination of the fraction has indicated the existence of at least three components. 7. It is concluded that there are several components in the fraction, at least one of which is a stored material that is steadily metabolized during development, and one or more others synthesized at a rate that is governed largely by the supply of available phosphate. 8. Synthetic radioactive propanediol phosphate added to a perchloric acid homogenate of eggs separated to the extent of 97% into the barium- and alcohol- insoluble, rather than the barium-soluble, alcohol-soluble fraction. It is concluded from this, in contrast to previous reports, that the latter fraction does not contain propanediol phosphate and that no evidence remains for the existence of this ester in sea urchin eggs. LITERATURE CITED ABELSON, P. H., 1947. Permeability of eggs of Arbacia punctulata to radioactive phosphorus. Biol. Bull, 93 : 203. BANDURSKI, R. S., AND B. AXELROD, 1951. The chromatographic identification of some bio- logically important phosphate esters. /. Biol. Chem., 193: 405-410. BERENBLUM, I., AND E. CHAIN, 1938. An improved method for the colorimetric determination of phosphate. Biochem. J ., 32 : 295-298. BOREI, H., 1948. Action of 1,2-propanediol phosphate on respiration in intact sea urchin eggs. Ark. Kami, Mineral, Geol, 26B : No. 17, 1-6. BROOKS, S. C., AND E. L. CHAMBERS, 1948. Penetration of radioactive phosphate into the eggs of Strongylocentrotus purpuratiis, S. franciscanus, and Urechis caupo. Biol. Bull., 95 : 262-263. CHAMBERS, E. L., AND W. E. WHITE, 1949. The accumulation of phosphate and evidence for synthesis of adenosinetriphosphate in the fertilized sea urchin egg. Biol. Bull., 97 : 225-226. CHAMBERS, E. L., AND W. E. WHITE, 1954. The accumulation of phosphate by fertilized sea urchin eggs. Biol. Bull., 106 : 297-307. METABOLISM OF PHOSPHORUS BY EGGS 287 FISKE, C. H., AND Y. SuBBARow, 1925. The colorimetric determination of phosphorus. /. Biol. Chem., 66 : 375-400. HANES, C. S., AND F. A. ISHERWOOD, 1949. Separation of the phosphoric esters on the filter paper chromatogram. Nature, 164: 1107-1112. HORSTADIUS, S., AND T. GusTAFSON, 1947. Change of determination in the sea urchin egg through the action of propanediol phosphate, phosphogluconic acid, and lactate. Zoo/. Bidrag Uppsala, 25 : 571-581. LAMPSON, G. P., AND H. A. LARDY, 1949. Phosphoric esters of biological importance. III. The synthesis of propanediol phosphate. /. Biol. Chan., 181 : 697-700. LEPAGE, G. A., 1948. Phosphorylated intermediates in tumor glycolysis. II. Isolation of phos- phate esters from tumors. Cancer Res., 8 : 197-200. LINDBERG, O., 1943. Studien iiber das Problem des Kohlenhydratabbaus und der Saiirebildung bei der Befruchtung des Seeigeleis. Ark. Kemi, Mineral., Geol., 16A: No. 15, 1-20. LINDBERG, O., 1946. On the occurrence of propanediol phosphate and its effect on the carbo- hydrate metabolism in animal tissues. Ark. Kemi, Mineral., Geol., 23A : No. 2, 1—45. MENDE, T. J., AND E. L. CHAMBERS, 1953. The occurrence of arginine phosphate in echinoderm eggs. Arch. Biochem. and Biophys., 45: 105-116. RUDNEY, H., 1952. Propanediol phosphate in acetone metabolism. Fed. Proc., 11: 279. RUDNEY, H., 1954. The synthesis of dl-propanediol-1 -phosphate and C14-labeled propanediol and their isolation from liver tissue. /. Biol. Chem., 210 : 353-360. SACKS, J., 1949. A fractionation procedure for the acid-soluble phosphorus compounds of liver. /. Biol. Chem., 181 : 655-666. TYLER, A., 1949. A simple, non-injurious method for inducing repeated spawning of sea urchins and sand dollars. Coll. Net, 19 : 19-20. UMBREIT, W. W., R. H. BURRIS AND J. F. STAUFFER, 1949. Manometric techniques and tissue metabolism. Second Ed., Burgess Publishing Co., Minneapolis. WHITELEY, A. H., 1949. The phosphorus compounds of sea urchin eggs and the uptake of radio- phosphate upon fertilization. Amer. Nat., 83 : 249-267. RESPONSE OF A LIVING ORGANISM, UNDER "CONSTANT CONDITIONS" INCLUDING PRESSURE, TO A BARO- METRIC-PRESSURE-CORRELATED, CYCLIC, EXTERNAL VARIABLE 1 F. A. BROWN, JR. Department of Biological Sciences, Northwestern University, Evanston, Illinois Many overt biological rhythms are known which exhibit cycles of 24 hours with such great precision, at least statistically, that the phases of the cycles do not be- come significantly altered with respect to external day-night even after weeks or months in conditions of constant darkness and temperature. Such rhythms have been reported in a wide variety of organisms and for many processes (see reviews by Welsh, 1938; Kleitman, 1949; Webb, 1950; Caspers, 1951). Less commonly known is the fact that persistent cycles of lunar-day frequency appear also wide- spread (e.g., Compel, 1937; Brown, Fingerman, Sandeen and Webb, 1953; Rao, 1954; and Ralph, 1956). One of the most striking properties of these rhythms ap- pears to be the temperature-independence of their frequency (Brown and Webb, 1948; Brown, Webb, Bennett and Sandeen, 1954; Pittendrigh, 1954). To account for these rhythms, either 1) there must be an astonishingly precise, temperature- independent clock-mechanism present within the organisms, or, 2) they are receiv- ing signals with the same average frequencies from external sources and which serve in some manner as pacemakers, or, 3) some combination of these two. Fluctuations in cycle-length from day to day under constant conditions in such regular overt cycles as running in the mouse (Johnson, 1939), and crab color change (Brown, Webb and Bennett, 1955), despite the great precision of the average lengths of the cycles, suggest strongly the operation of an external pacemaker. Proof for the ex- istence of at least a reasonably precise internal clock has been advanced (Brown, Webb and Bennett, 1955 ; Renner, 1955) through studying the results of rapid east- west geographic displacement of animals, but it is still not demonstrated that this is sufficiently precise to serve as the exclusive mechanism. Since there are known 24- hour physical cycles which appear to operate on universal time (e.g., atmospheric electrical potential), it may be argued that there is still lacking definitive proof that regular pacemakers are inoperative even during such rapid longitudinal displacement of organisms. In the course of seeking possible effective external rhythmic signals of the ap- propriate frequencies, average rhythms of O2-consumption of primary solar and pri- mary lunar frequencies have been found in all organisms so far examined for them. These have included organisms as widely diverse as seaweed, snails, crabs, Triturus, worms, carrots and potatoes (Brown, Bennett and Webb, 1954; Brown, Webb, Bennett and Sandeen, 1955; Brown, Freeland and Ralph, 1955). Studies of the 1 These studies were aided by a contract between the Office of Naval Research, Department of the Navy, and Northwestern University, NONR-122803. 288 CYCLIC FLUCTUATIONS IN METABOLISM 289 activities of quahogs, oysters and rats indicate that these, too, possess the same gen- eral types of average solar and lunar rhythms (Brown, 1954a; Brown, Shriner and Ralph, 1956; Brown, Bennett, Webb and Ralph, 1956). The fact that there are known to exist average rhythms of barometric pressure of solar- and lunar-day frequencies led to attempts to correlate barometric pressure changes with the observed biological cycles, and some definite correlations were found not only between the hourly rates of barometric pressure change and of con- current Oo-consumption, or activity, but also between mean daily levels of pressure and mean daily metabolic rates (Brown, Freeland and Ralph, 1955; Brown, Webb, Bennett and Sandeen, 1955). There were shown, furthermore, to be such distinct similarities between the general forms of the daily pressure changes and daily varia- tions in oyster and quahog activity (Brown, Bennett, Webb and Ralph, 1956) as to suggest strongly more than a fortuitous relationship. This resemblance was some- times a direct one and other times one of a mirror image. The experiments to be described herein were initially planned to resolve the problem of a possible direct role of barometric pressure variations. MATERIALS AND METHODS Potatoes, Solanum tuberosum, which were purchased at a local grocery store constituted the organism. With a cork-borer, cylinders 22 mm. in diameter and about l^o cm. high, each bearing an eye, were cut and the injured surfaces allowed to heal. In the experimental situation of constant temperature and constant very low illumination these developed sprouts. They were replaced during the experi- ments only after long periods when they grew too large for the respirometer vessels. The respirometers were of the type designed by Brown (1954b) which permitted continuous automatic recording of CX-consumption. The instrument was modified, however, in such a manner as to permit four respirometers to record as a unit, and the recorder was small enough to be sealed along with the respirometers in a baro- stat (Fig. 1). The barostat first successfully used was an 11 X 24-inch vertical autoclave sealed with an O-ring. Since it was not possible to view the interior once this was sealed, a simple barograph was included in the system to assure an absence of leakage. Later, five simplified barostats were constructed. These consisted of %o-mcn copper cylinders, 10% inches in diameter and 22 inches deep, with dished bottoms and a 1-inch-wide, %-inch-flat, ground-brass rim. These, covered by twelve-inch vacuum desiccator covers with an electrical inlet for the recorder motor and a glass stopcock passing through a rubber stopper, served as excellent barostats. A %-inch brass tripod ring, fitting snugly within the cylinder served as a platform to support the recorder, with its divers hanging into an enclosed, 10-inch-deep water bath. Each copper cylinder was supported in a 55-gallon steel drum full of water maintained at constant temperature. The temperature settings for the five baths all lay in the range 19.6 to 19.9° C. The apparatus was shielded from all light except that from a row of 7^o-watt, opalescent, incandescent lamps which provided a continuous illumination of about 1-2 ft. c. at the desiccator-cover surface, and obviously some far lower constant il- lumination at the surface of the organisms. One cylinder of potato was placed in each of the four respirometers of a re- cording unit and the whole lowered into a barostat, sealed, and the barostat as- 290 F. A. BROWN, JR. FIGURE 1. Four respirometers (R) hanging into a water bath (IB) from a spring scale (SC) ink recording system registering on a 24-hour drum (D) activated by an electric clock motor (M). A barograph (B) also registers on the same drum. The whole is supported by a stand (S) in a sealed copper-and-brass container which sets deeply in a constant-temperature bath (CTB). pirated to reduce the internal pressure to 28.00 in Hg. This was sufficiently below the lowest normal external pressure in Evanston, 111. (ca. 28.5) that the cover would always retain its seal. A barometer in each barostat permitted one to be continu- ously assured of the absence of leakage. Usually the apparatus was then left un- disturbed for three to five days. This was the length of time the recording needle required to spiral down the five-inch-length of the three-inch-diameter brass drum to the recording base-line, with the drum turned one revolution per day with an electric time-clock mechanism. The paper was a smooth-surface, bond, writing paper held in place with rubber cement. The pens were small L-shaped glass capil- laries with polished tips and contained a 50-50 glycerine-water mixture, colored with CYCLIC FLUCTUATIONS IN METABOLISM 291 neutral red. In more recent work, millimeter graph paper and commercially avail- able thermograph pens (counterbalanced) and ink have been found simpler to use. The springs used were wound from 0.014-inch, 8-18, stainless-steel wire, and were 5 cm. long and about % cm. in diameter. Over the recording range (10 gms./ cm.) in this mechanical recording system, there was only about a 10% variation from linearity from the first to the last day of recording. There was, undoubtedly, also, a very minor, gradual reduction in pressure in the barostat over a recording period as the O2-reservoirs of the respirometers were depleted, but this was not sig- nificant in view of the large size of the ratio : barostat gaseous volume/O2-reservoir volume. Respirometers were always loaded sometime between 8 A.M. and 8 P.M. In the analyses to follow, except when specifically stated otherwise, no data were used be- fore the first midnight after the sealing of a barostat, nor of the day they were opened. Hence, there were usually complete, undisturbed, calendar days of data. All times are central standard. From April 1 through April 26, 1955, only one barostat \vas in operation. From April 28 through June 8, five were in continuous operation. RESULTS It was apparent even after the first day of recording that the rate of O2-consump- tion in the potato is by no means uniform even under constant temperature, illumi- nation, oxygen, CO2, humidity and pressure. There were fluctuations usually with more than one conspicuous maximum a day. Eight sample days picked at random from the data, illustrating the variation in the two to five separate barostats in which recordings were complete on that particular day, are illustrated in Figure 2. These APRIL 30 MAY 7 MAY 10 MAY II MAY 14 _L MAY 16 MAY 18 MAY 19 _L J_ JL _L 6126 NOON 12 6 NOON 6126 NOON 12 NOON FIGURE 2. The forms of the daily fluctuations in oxygen consumption (three-hour sliding averages) measured on eight randomly selected days in all those (1 to 5) barostats from which records were complete for that day. The ordinate scales are comparable for all, but the pat- terns have been separated for clarity. 292 F. A. BROWN, JR. _ 1.0 ZEN. NAD. FIGURE 3. A. The mean rates of Oo-consumption for the 8 three-hour periods of the solar- day for all the data of the 154 barostat-d'ays. These are expressed as deviations from the daily means. B. Moving three-hour means of the daily variation for 100 randomly selected days in 1955 (solid line), compared with the comparable mean daily cycle obtained at the same time of year in 1954. CYCLIC FLUCTUATIONS IN METABOLISM 293 TABLE I The percentage increase from lowest to highest values of the daily cycles illustrated in Figure 2 Date Unit Apr. 30 May 7 May 10 May 11 May 14 May 16 May 18 May 19 1 133 38 71 33 73 49 2 37 73 26 35 84 31 3 67 35 77 52 4 100 46 38 34 89 54 5 67 23 10 80 45 72 32 Av. 82 50 35 40 47 54 78 45 are three-hour moving means. Table I includes the percentage increase from lowest to highest values of the days illustrated. One hundred and fifty-four complete baro- stat-days of data, including the 29 illustrated in Figure 2, were obtained, together with numerous fractional barostat-days. Inspection of the daily patterns revealed that there were clearly as many de- tailed patterns as there were days of data. But there appeared to be a suggestive generic similarity in the records obtained for any given day in entirely independent barostats with respect to amplitude of the fluctuations, gross trends, and approxi- mate times of the major maxima and minima. There seemed, apparently too com- monly to be attributed simply to chance, a tendency for one or more of the records to show on any given day an inversion of some of, or even the greater part of, the daily pattern relative to the other concurrent ones. In nearly every instance, how- ever, if one obtained the average daily pattern for a three- to five-day period of con- tinuous recording in one barostat, there was a distinct maximum about 6 or 7 A.M. and a minimum about 9 or 10 A.M. ; also, low values nearly always characterized the early morning hours. In Figure 3A is plotted the mean daily cycle for all barostat-days of data. This is plotted as the mean differences from the mean hourly rate for the whole day for each of the eight three-hour periods of the day. The mean daily fluctuation is seen to be about 8%. The mean hourly value for all these data was 7.44 with the range extending from about 5 to 14. For each value in the figure is indicated the standard error of the mean. The mean daily pattern is in large measure a mirror image of the one obtained without the use of a barostat during the 29-day period, May 12-June 9, 1954 (Brown, Freeland and Ralph, 1955) involving both inversion of the major 24-hour cycle and the secondary fluctuations superimposed on the larger cycle. Moving three-hour averages of 100 randomly picked days in 1955 and the 1954 cycle are seen in Figure 3B. In 1955 the highest rates for the day occurred in the afternoon ; in 1954 the highest rates were found in the morning. C. The mean lunar day cycle for the potato for the month of May, 1955 (solid line), com- pared with that obtained in May, 1954 (broken line). D. The mean daily cycles with sample standard deviations of the means, for the 99 positive barostat-days of O2-consumption, and the 55 negative ones. 294 F. A. BROWN, JR. 6 A.M. 6PM. FIGURE 4. A. The correlations between the rate of O2-consumption during the 4 to 7 A.M. period of a day and the mean rate and direction of barometric pressure change for the same day CYCLIC FLUCTUATIONS IN METABOLISM 295 There was also an apparent mean lunar-day cycle in the potatoes. It, too, showed a general major inversion relative to the comparable lunar-day cycle ob- tained in 1954. When only 29 consecutive days of data in May were used, in order exactly to randomize a daily cycle and hence to obtain the minimally distorted form of a lunar-day cycle, the results seen in Figure 3C were obtained. In the same fig- ure is the form of the lunar-day cycle obtained in 1954 without the use of a barostat. These are both three-hour moving means. Brown, Freeland and Ralph (1955) have shown that for the potato in respirom- eters subjected to normal fluctuations in pressure in 1954, there was present, even after all appropriate corrections for pressure changes had been made, a correlation coefficient of — 0.58 ± 0.035 between the hourly values of CX-consumption and the concurrent rates and direction of barometric pressure change over a month. That this demonstrated a direct response to some external factor fluctuating with baro- metric pressure changes was ascertained by obtaining the coefficient for the same monthly period using metabolism on the hours of day n and the pressure changes for day n + 1. Now, the value dropped to 0.217 ± 6.038, a value not significantly different from the auto-correlation of hourly pressure changes on two consecutive days. For reasons discussed in that paper it was considered unlikely that the re- sponse was a direct one to pressure on the part of the plant. This last presumption is supported amply by this work in which the pressure was kept constant during the recordings. Although the earlier work had shown a correlation between the hourly rates of Oo-consumption in the potato and the concurrent rate and direction of barometric- pressure change in organisms subjected to barometric-pressure fluctuation, using data available in the current experiments, no correlation with the hourly pressure change in the external environment was found. With 2374 hours of data, a value of r, 0.020 ± 0.021 was obtained. An attempt was made to test an hypothesis that there was not a correlation with the barometric pressure changes in these data obtained in 1955 because of abrupt 180° -shifts of the phases of at least one important component of daily rhythmicity in the plants. The 154 barostat-days of daily patterns of respiration were, by in- spection, divided into two categories on the basis of whether a maximum and mini- mum occurred about 6 and 9 A.M., respectively, or the cycle at these hours was ap- and the two preceding days for various two-hour periods of the day, for both the positive and negative groups of potatoes. B. The correlations between the mean rate and direction of barometric pressure change dur- ing the 2 to 4 A.M. period of a three-day period and the mean rate of Go-consumption for various three-hour periods on the last of the three days. C. The correlations between the mean rates of Oo-consumption during various three-hour periods of the day and the mean three-day changes in barometric pressure for the two preceding hours of the day. D. The correlations between the mean rate of Oo-consumption during the 4 to 7 P.M. period and the mean, three-day changes in barometric pressure during various two-hour times of day, for the negative group. E. The correlations between the mean rate of Oo-consumption during the period 5 to 8 P.M. and the mean three-day change in barometric pressure for various two-hour periods of the day for the positive group. The potatoes were maintained in barostats throughout the study. The values of Oo-con- sumption used are deviations from the daily mean in order to eliminate long-period trends in metabolic rate. 296 F. A. BROWN, JR. 30 >5 20 15 10 0.2 A. 0 0.2 0.4 + 0.2 A. 0 0.2 0.4 C. D. E. i i i i i i ! I I i I fO OJ — I | I Z Z Z CYCLIC FLUCTUATIONS IN METABOLISM 297 parently inverted, with a minimum about 6 and a maximum about 9 A.M. The latter were termed the negative group since in the form of the minor fluctuations this was more nearly the form of the 1954 patterns which showed the negative correlation with concurrent barometric pressure changes ; the former was termed the positive group. Barostat No. 1 yielded 14 negative days and 33 positive ones ; No. 2 gave 7 nega- tive and 23 positive ; No. 3 showed 17 negative and 10 positive ; No. 4 had 11 negative and 10 positive; and No. 5 provided 6 negative and 23 positive. Though both posi- tive and negative responses could be found in different barostats on a single calendar- day, there were two or three periods of three or four days, when there seemed to be a high percentage of negative cycles. In Figure 3D are seen the mean daily patterns for the 99 positive and 55 negative days expressed as mean hourly deviations from the daily means. It is very inter- esting to note that though these were based exclusively upon selection of an apparent inversion between about 4 and 11 A.M. between the two groups, the remainder of the daily patterns are in extraordinary agreement for the two groups even to most of the minor fluctuations. It was now assumed that, at least for the 4—11 A.M. period, the potatoes were divided into two separate, relatively homogeneous popu- lations with respect to any sign of correlation with any external pressure changes which might be present. On the other hand, it is very important to emphasize here that there was no a priori reason why a single value for either of these two groups for any arbitrarily selected single time of day should show any correlation whatso- ever with external atmospheric barometric pressure changes. First, an intensive search was made to learn whether the average rate of O2- consumption in the 55 days for potatoes of the negative group for the 4—7 A.M. period was correlated with external pressure changes in any manner. It was dis- covered that a correlation, — 0.46 ± 0.106, existed between the total respiration at this time on day n, expressed as deviation from the daily mean, and the algebraic sum of the rates of the pressure changes at 2-4 A.M. on days n, + (n — 1), + (w — 2). In Figure 4A it is readily seen that there is a rapid drop to no correla- tion as one moves away from 2-4 A.M. pressure changes in either direction. In Figure 4A it is also seen that not only did the 99 days of the positive group also show a significant correlation for essentially the same kind of relationship, but with op- posite sign, 0.326 ± 0.089. There was similarly a loss of correlation with pressure changes as one moved to other times of day. FIGURE 5. A. The correlations between the mean deviations in rate of O2-consumption dur- ing the 4 to 7 A.M. period and the mean change in barometric pressure during the 2 to 4 A.M. period during various three-day periods centering on days ranging from 31 days earlier to 5 days later than the day of correlated Go-consumption, for both positive and negative groups. B. The correlation between the rate of O2-consumption during the 4 to 7 A.M. period on day n with the changes in barometric pressure during the 2 to 4 A.M. period on single days ranging from day n to day n — 6. C. Correlations between O2-consumption and barometric pressure for the same hours as in B of the solar day, but now with the means of pressure including increasing numbers of earlier days up to three. D and E. The same as B and C except that the correlations of pressures at 2 to 4 P.M. with O2-consumption at S to 8 P.M. are used for the positive group and barometric pressure changes between 4 and 6 P.M. are correlated with O2-consumption for the 4 to 7 P.M. period for the nega- tive group. 298 F. A. BROWN, JR. Figure 5 demonstrates in two ways that the correlations that have been described are actually the result of a summation by the potato of three or more days of fluctua- tion in an external factor. First, for both the positive and the negative groups, the correlation not only increases to a maximum as one increases the number of days up to three, but there is a reduction beyond that point (Fig. 5C). Second, it is seen that the best single-day pressure change correlation occurs for both groups with pressure change on day n — 2, with lower or no correlation either earlier or later (Fig. 5B). In Figure 5A it is seen that there is a rapid reduction in correlation with summed three-day pressure changes for both positive and negative groups as one attempts the correlations with earlier days or later. The apparent drifts in correlations suggested for three-day periods earlier than n — 2, n — 3, and n — 4, and extending for nearly three weeks probably reflect only auto-correlations of pressures. However, only the correlation with (n) + (n — 1) + (n — 2) is sig- nificantly different from 0. Figure 4B demonstrates that the three-day sums of pressure changes at 2-4 A.M. (days n, n -- I, n — 2) are not significantly correlated with the rates of respiration (day n) for any time of the day other than the 4-7 A.M. period for either the posi- tive or negative groups. In Figure 4C the rates of CX-consumption at various pe- riods of day n are correlated with the sums of three-day (day n, n -- 1 and n — 2) pressure changes for the two-hour earlier period. There is a suggestion herein that there may be a second period in the day, in the afternoon, when there is a real cor- relation for the negative group, though a similar suggestion is lacking for the posi- tive one. When this possibility was explored in detail for the negative group (Fig. 4D), the highest correlation was found between the 4-7 P.M. deviation in O2- consumption and the 4-6 P.M. (day n, n -- 1, and n — 2) pressure change, -- 0.347 ± 0.12. There was also found an afternoon correlation for the positive group but not with the same temporal relationship (Fig. 4E). The highest correlation was found between the 2-4 P.M. pressure change and the 5-8 P.M. deviation in respira- tion, -- 0.273 ± 0.091. Just as the forms of the mean daily cycles for both groups of potatoes for this part of the days were quite similar, the correlations here also bore the same sign. It is seen from Figure 5E that the correlation in the afternoon is maximal with the summation of three days (n, n — 1, n — 2) of pressure changes, with a reduction with fewer or greater number of days. The correlations with single days (Fig. 5D) are relatively small, being about equal for days n, n — 1, and n — 2. Evidence supporting the earlier assumption of there being a tendency of the po- tatoes actually to be reversing from time to time the sign of their response to some external factor became apparent from a study of structure in correlation scatter- plots. One of these was the following. When one made no selection whatsoever of the 154 barostat-days of data and determined only the relationship between the extent of the fluctuation, positive or negative, from the daily mean for the 5-7 A.M. Oo-consumption on day n, and the algebraic sum of the barometric pressure changes from 2 to 6 A.M. for days n, n -- 1, and n — 2, a coefficient of 0.361 ± 0.070 was ob- tained. No correlation was obtained unless the sign of the response was ignored.2 - Further support of this hypothesis of inversions has been obtained since the preparation of this report. With 960 complete potato-days, including hourly data, obtained in barostats during October and November, 1956, the correlation between 3 X 3 X 3-day moving means of the 2 to 6 A.M. barometric pressure change on day n and the three-day moving mean of the 6 A.M. devia- CYCLIC FLUCTUATIONS IN METABOLISM 299 This highly significant result clearly justified the earlier division of the data into positive and negative cycles. Again, ignoring the sign of deviation of O2-consump- tion from daily mean, the correlation between the three-day (n, n -- 1, n — 2) 5-9 A.M. barometric pressure change and CX-consumption at 8-11 A.M. yielded a lower value of 0.223 ± 0.077. There was, further, with no selection of the 154 barostat days of data, found to be a correlation, 0.305 ± 0.073, between the algebraic sum of the 2-6 P.M. baro- metric changes of day n, n -- 1, and n — 2 and the deviation in CX-consumption of the potatoes from the daily mean at the 4—7 P.M. period, if one used simply average rate of pressure change, irrespective of sign. It was evident, however, from exami- nation of a scatterplot that the correlation for those 14 atypical days on which the pressure showed an overall rise at this time of day during the three-day period, was distinctly inferior to that for the 140 days of data of periods for which the pressure was (as typically) falling. The coefficient of correlation between the mean three- day rate of fall in afternoon barometric pressure and 4-7 P.M. deviation in rate of Oo-consumption was 0.355 ± 0.0734. TABLE II Correlation between rate of change of barometric pressure and O ^-consumption Bar. pressure Potato Correlation 11-3 A.M. 2-4 A.M. -0.087±0.08 2-6 A.M. 5-7 A.M. ±0.361 ±0.07 5-9 A.M. 8-11 A.M. ±0.223±0.077 8-12 A.M. 11-1 noon +0.024±0.08 11-3 P.M. 2-4 P.M. -0.056±0.08 2-6 P.M. 4-7 P.M. -0.305±0.073 5-9 P.M. 8-10 P.M. -0.084±0.08 8-12 P.M. 11-1 midnight -0.194±0.078 No other real correlations (except possibly for the 8-12 P.M. pressure change and midnight (11 p.M.-l A.M.) CX-consumption) could be found for the potatoes as a whole over the day, even when the structure of correlations was examined for pos- sible reversing responses. The foregoing results are included with some additional ones in Table II. The potatoes of barostat No. 3 for the 560 hours obtained during May and June showed an hourly correlation of their deviations from the daily means with the con- current deviations from the daily means of barometric pressure, of + 0.238 ± 0.040, a value highly significantly different from zero. That this was a correlation with the concurrent hourly values and not based simply upon similarities of the mean forms of two independent average cycles was readily apparent by finding no correla- tion for the same period with the pressures of day n + 3 (0.0923 ± 0.0451 ) , day n — 1 (0.100 ± 0.0425), and for half the period with day n + 1 (0.0118 ± 0.059), day tions from the daily means of 5 X 3 X 3-hour moving means of O=-consumption on day », yielded a coefficient of 0.58 ± 0.087. " This rather "high and unquestionably real correlation indicated that during this 60-day period, the potatoes must have been overwhelmingly of one sign in their cor- relation with the unknown external factor, namely positive, and, furthermore, this unknown effective force must have retained a high correlation with the morning barometric pressure change during this period. In no other lag or lead relationship, except for smaller and obviously explicable real correlations on days n + 1 and n — 1, did there appear from inspection to be cor- relations significantly different from zero. 300 F. A. BROWN, JR. n - 2 (0.0437 ± 0.059) and day n - 4 (0.0622 ± 0.057) of pressure change. The next highest correlation with hourly barometric pressure, and also highly signifi- cantly different from zero, was seen for barostat No. 5 for the same period. Baro- stats No. 1, 2 and 4 showed no comparable correlation. These results, together, clearly suggested that the failures to obtain correlations at some times with baro- metric pressure were not due to a lack of capacity of the potatoes to respond to a pressure-correlated external factor, but rather due to a failure to observe the re- sponse due to a mutual cancelling of opposite signs of response. Finally, when all the 2976 available hours of data obtained from May 1 through June 8 were correlated with the hourly differences from the daily means for baro- metric pressure, a value of -- 0.0697 ± 0.01825 (Fig. 6) was obtained. This value is obviously not zero though very small chiefly because it must be the residual after cancellation of the frequent changes of sign of response to the barometric-pressure- correlated variable. The calculated regressional relationship between the rate of O2-consumption, and the pressure, for all data, ignoring inversions, shows the rate of O2-consumption to increase from about 6.8 to about 8.2 arbitrary units, or about 0.8 0.6 OA . 0.2 O 0.0 O 0-2 9 3 0.4 x °0.6 OB 8 0 6 BAR. PRESS. 12 18 FIGURE 6. The relationship between the deviations from the daily mean of Oo-consumption of five groups of potatoes in barostats between May 1 and June 8, 1955, and the concurrent de- viations from the daily mean of barometric pressure (P < 0.002). CYCLIC FLUCTUATIONS IN METABOLISM 301 21%, as the pressure ranges from 0.15 inches Hg above to about 0.15 inch Hg below the daily mean, a moderately large range in the normal pressure changes for a single day. DISCUSSION It is quite clear from the preceding account that even when pressure, in addition to light, temperature, humidity and certain other factors are kept constant, there are still substantial fluctuations in CX-consumption in potatoes. Pressure changes are obviously not the factor responsible. This was strongly suggested in earlier studies (Brown, Freeland and Ralph, 1955) inasmuch as the day-by-day drift in CX-con- sumption at 4-7 P.M. appeared to parallel the day-by-day drift in mean barometric pressure, but apparently tending to lead the changes by one or two days. One might suspect that possibly pressure changes were in some manner respon- sible for the general inversion of the 1955 cycles relative to the 1954 ones or for the hour-by-hour correlations with pressure change obtained in 1954. That this was not so, has been ascertained through other experiments using the fiddler crab and Fucus in our laboratory during the summer of 1955. These results, to be pub- lished elsewhere, show the cycles of these species also to be essentially inverted rela- tive to comparable ones obtained in 1954 and similarly to show no longer the over- all hourly correlation with concurrent pressure changes. With the fiddler crab, however, parallel studies were made under conditions of fluctuating pressure ex- actly as was done in 1954 ; the cycles were, like those of the crabs in the barostats, similarly inverted relative to the 1954 ones, and similarly showed no significant over-all hourly correlation with pressure changes. There appears to be only one tenable hypothesis at this time concerning these inversions between the two years, namely, that some significant difference occurred between 1954 and 1955 with what- ever fluctuating external factors are responsible for determining the form of the mean biological cycles. All organisms studied in 1955 also appeared to exhibit in some degree, the phenomenon of phase inversions relative to other individuals of the same species being studied concurrently. Whatever the mechanism, it must include to some extent the organism as a biologically responding system. It is possible that various individuals possess different thresholds for some factor which is itself re- sponsible for the changes in sign of response to the external fluctuating factor. The existence of the inversions which have been described in this report may lead one to question whether these fluctuations in metabolism are strictly rhythmic. Fluctuations in barometric pressure show both solar and lunar tides, but these are, in temperate zones, in good measure obscured by relatively huge climatic fluctua- tions. The general form of the solar tidal fluctuations may be made evident, how- ever, through the averaging of two to five days of data. This, the potatoes seem also able to accomplish, judging from the results of this study. Hence they are ap- parently able to exhibit daily and lunar-day cycles of fluctuation, even though these are superimposed upon a much more randomly fluctuating background. But with the more or less erratic sign changes in 1955, the metabolism itself displays a true rhythm only for certain non-inverting components of the solar- and lunar-day cycles. The forms of the cycles are subject to the same fluctuations as are the mean daily pressure cycles. There is always the possibility, however, that the fluctuating fac- tors which produce these responses in organisms, possess some sharper frequency- 302 F. A. BROWN, JR. determining component than the pressure cycles, from which the organism can ob- tain an adequate pacemaking signal to regulate their own internal clocks. But just as the large-amplitude random fluctuations in atmospheric pressure make it very difficult to characterize the tides therein, so may externally induced large random fluctuations in the organism tend to obscure a more precise extant organismic meta- bolic cyclicity. One should ahvays bear in mind that the metabolic cycles which were the object of this study are not as regularly rhythmic as are numerous overt organismic cycles such as color-change or motor activity in many animals. These latter must be me- diated by an internal clock of the same precision as must be postulated to be func- tioning in these potatoes to integrate the effect of an external stimulus recurring at the same hours of the day over a few days. Such a clock is clearly needed to permit the organism to become an appropriate harmonic analyzer. Such a clock may also act as a buffer to filter out to some extent the large random elements of fluctuation in the external environment. The organism tends to retain for a time and repeat on a 24-hour cyclic basis an environmentally induced pattern of fluctuation. It is considered highly probable that the responses to the still unknown external factor which are proven to occur by these and earlier experiments in some manner regulate the frequencies of the endogenous clocks. SUMMARY 1. Fluctuations in CX-consumption in the potato under constant conditions, in- cluding pressure, were observed. The average solar-day cycle was determined and this was found to have in large measure a form which was the mirror image of that obtained during the same months of the preceding year. 2. The average lunar-day cycle was also determined and this, too, was in its principal features an inversion of that found for the same period of the preceding year. 3. A study of the patterns of daily variation for the 154 complete days of data revealed that all the patterns of fluctuation on any given day tended to exhibit a generic similarity to one another, tending to exhibit either parallel, or mirror image fluctuations, and of the same general amplitude. 4. All the daily patterns could be divided in two groups. One group (99 days) called the positive one (since its later discovered correlation with barometric pres- sure change was positive) possessed a maximum about 6 A.M. and a minimum about 9 A.M. ; the negative group (55 days) tended to show the mirror image of this form in the daily period 4 to 11 A.M. 5. The deviation in rate of Go-consumption from the daily mean for the positive group for the 4—7 A.M. period was found to show a positive correlation with the al- gebraic sum of the rates of change in barometric pressure during the three preced- ing 2-4 A.M. periods ; the negative group, on the other hand, showed a negative correlation for the comparable relationship. 6. A correlation between the deviation from the daily mean of O2-consumption at the 4—7 A.M. period and the algebraic sum of the pressure changes for the three preceding 2-4 A.M. periods was found for all 154 barostat-days if one ignored the sign of the deviation in rate of O2-consumption, proving true an earlier hypothesis CYCLIC FLUCTUATIONS IN METABOLISM 303 that the sign of the response of the organism to an external pressure-correlated fac- tor changed from time to time. 7. The deviation from the daily mean of (X-consumption for the 4—7 P.M. period showed a negative correlation with the algebraic sum of the rates of barometric pressure change for the preceding three 2-4 P.M. periods, and the 10-1, midnight de- viation, was correlated with the three-day pressure-change from 8-12 P.M. 8. Since it was demonstrated that the correlations were not with single days of an external factor, nor with any averaged three-day periods other than the three immediately preceding daily periods, it was evident that the potatoes were deriving an essential element of the form of their daily fluctuation from a response to an ex- ternal factor which, since pressure-correlated, clearly possessed average solar-day cycles. 9. The external factor appears to determine in the daily fluctuation of O2-con- sumption the amplitude of a morning oscillation (or its mirror image) with about a six-hour period, the height of the late-afternoon maximum, and probably also the extent of the midnight reduction in rate. 10. Possible relationships of the exogenous to endogenous cycles are discussed briefly with reference to the problem of biological clocks. LITERATURE CITED BROWN, F. A., JR., 1954a. Persistent activity rhythms in the oyster. Amer. J. PhysioL, 178: 510-514. BROWN, F. A., JR., 1954b. Simple, automatic continuous-recording respirometer. Rev. Sci. Instr., 25: 415-417. BROWN, F. A., JR., M. F. BENNETT AND H. M. WEBB, 1954. Persistent daily and tidal rhythms of O2-consumption in fiddler crabs. /. Cell. Comp. PhysioL, 44: 477-506. BROWN, F. A., JR., M. F. BENNETT, H. M. WEBB AND C. L. RALPH, 1956. Persistent daily, monthly, and 27-day cycles of activity in the oyster and quahog. /. E.rf>. Zool., 131 : 235-262. BROWN, F. A., JR., M. FINGERMAN, M. I. SANDEEN AND H. M. WEBB, 1953. Persistent diurnal and tidal rhythms of color change in the fiddler crab, Uca pugnax. J. E.rp. Zool., 123: 29-60. BROWN, F. A., JR., R. O. FREELAND AND C. L. RALPH, 1955. Persistent rhythms of O2-consump- tion in potatoes, carrots and the sea-weed, Fucus. Plant PhysioL. 30: 280-292. BROWN, F. A., JR., J. SHRINER AND C. L. RALPH, 1956. Solar and lunar rhythmicity in the rat in ''constant conditions" and the mechanism of physiological time measurement. Ainer. J. PhysioL, 184: 491-496. BROWN, F. A., JR., AND H. M. WEBB, 1948. Temperature relations of an endogenous daily rhythmicity in the fiddler crab, Uca. PhysioL Zool., 86: 371-381. BROWN, F. A., JR., H. M. WEBB AND M. F. BENNETT, 1955. Proof for an endogenous compo- nent in persistent solar and lunar rhythmicity in organisms. Proc. Nat. Acad. Sci., 41 : 93-100. BROWN, F. A., JR., H. M. WEBB, M. F. BENNETT AND M. I. SANDEEN, 1954. Temperature in- dependence of the frequency of the endogenous tidal rhythm of Uca. PhysioL Zool., 27 : 345-349. BROWN, F. A., JR., H. M. WEBB, M. F. BENNETT AND M. I. SANDEEN, 1955. Evidence for an exogenous contribution to persistent diurnal and lunar rhythmicity under so-called con- stant conditions. Biol. Bull., 109: 238-254. CASPERS, H., 1951. Rhythmische Erscheinungen in der Fortpflanzung von Clunio marinus (Dipt. Chiron.) und das Problem der lunaren Periodizitat bei Organismen. Arch. Hydrobiol. Suppl., 18: 415-594. COMPEL, M., 1937. Recherches sur la consommation d'oxygene de quelques animaux aquatique littoraux. C. R. Acad. Sci., France, 205 : 816-818. 304 F. A. BROWN, JR. JOHNSON, M. S., 1939. Effect of continuous light on periodic activity of white-footed mice (Peromyscus). /. Exp. Zool, 82: 315-328. KLEITMAN, N., 1949. Biological rhythms and cycles. Physiol. Rev., 29: 1-30. PITTENDRIGH, C. S., 1954. On temperature independence in the clock system controlling emer- gence time in Drosophila. Proc. Nat. Acad. Sci., 40 : 1018-1029. RALPH, C. L., 1956. Persistent rhythms of activity and O;, -consumption in the earthworm. Physiol. Zool., 30 : 41-55. RAO, K. P., 1954. Tidal rhythmicity of rate of water propulsion in Mytilus and its modifiability by transplantation. Biol. Bull., 106 : 353-359. RENNER, M., 1955. Ein Transoceanversuch zum Zeitsinn der Honigbiene. Naturwiss., 19 : 540-541. WEBB, H. M., 1950. Diurnal variations of response to light in the fiddler crab, Uca. Physiol. Zool., 23 : 316-337. WELSH, J. H., 1938. Diurnal rhythms. Quart. Rev. Biol., 13 : 123-139. THE IMMEDIATE EFFECTS OF LOW DOSES OF X-RADIATION ON THE FREQUENCY OF SEVERAL MITOTIC STAGES IN THE ALLIUM ROOT TIP ARTHUR C. CHANDLER, JR.*. 2 The Department of Zoology and Entomology, The University of Tennessee, Knoxville, Tennessee Many studies have been made of the effects of radiations on mitosis in plant cells, but the design of the experiments has been such that they could not be compared readily with effects on animal cells. In the present study an attempt has been made to deal with plant material in the same manner as has been frequently used for ani- mal tissues, namely, ( 1 ) to maintain the cells at a constant temperature before and after irradiation, (2) to make accurate dosage measurements at each treatment, (3) TABLE I The onion root tip cell mitotic cycle at 32° C. Stage Criteria by which the beginning of each stage Is dis- tinguished in the fixed, stained cell as observed with 4 mm. objective and 10 X ocular Relative frequency Interphase Prophase Early Late Prometaphase Metaphase Anaphase Telophase Densely granular appearance of nucleus caused by crowding of chromatin threads ; nucleoli visible Nucleus enlarged ; chromatin threads larger and less crowded; granules larger; nucleoli less distinct Chromosomes thicker and better separated ; nucleoli absent Nuclear membrane absent Chromosomes in equatorial plane Proximal ends of chromatids separated Newly-formed cell plate visible ; chromosomes less distinct .7546 (.7552) .1672 (.1692) .0184 (.0182) .0105 (.0112) .0133 (.0124) .0154 (.0102) .0211 (.0211) Figures in parentheses from Laughlin (1919). to subdivide certain of the longer mitotic stages, so that more detailed information on the mitotic effect could be obtained, (4) to define carefully the terminology used to designate the different stages (Table I), and (5) to make regular and frequent counts of both control and treated cells. 1 This study was submitted in partial fulfillment of the requirements for the degree of Master of Science in Zoology in the Department of Zoology and Entomology of the University of Ten- nessee. The author wishes to express gratitude to Dr. J. G. Carlson for his help in this study and his critical reading of the manuscript. 2 Present address : Box 2718, Duke University School of Medicine, Durham, North Carolina. 305 306 ARTHUR C. CHANDLER, JR. MATERIALS AND METHODS Ease in handling and in control of mold as well as bacterial infection of the roots dictated the use of seeds of Alliuin cepa in preference to bulbs. Preliminary experi- ments showed no significant differences in mitotic frequency between root tips of different seeds. Ultraviolet radiation was used to inhibit mold growth during germination. Preliminary tests indicated that the dose of ultraviolet used had no effect on the germination time of the seeds or the rapidity of root growth. Seeds were agitated continuously for a period of six minutes in a quartz flask at a distance of about four inches from a four-watt General Electric germicidal lamp. This was done in a closed, sterilized chamber, which was also used to make subsequent transfers. The seeds were then transferred, aseptically, to sterile petri dishes containing filter paper dampened with distilled water. The petri dishes and filter paper had been sterilized by dry heat and the water had been sterilized by ultraviolet radiation. A darkened incubator maintained at 26° C. was used to assure constant tem- perature during germination.3 After twenty-four hours the seeds were removed from the incubator long enough to receive a second ultraviolet treatment for one minute to further retard the growth of any mold that had succeeded in contaminating the dishes. No roots were in evidence at this time. After sixty hours, the roots had reached a length of 9 to 15 mm. At this time forty germinated seeds were transferred to each of two petri dishes. One of these sets was used as a control. The other was irradiated with either 128 r or 512 r of x-rays at the same dose rate, i.e., 86 r/min.4 The 128 r and 512 r doses were chosen because they were not large enough to cause death of the cells but sufficiently large to give easily measurable mitotic inhibition. Irradiation was done with a Coolidge tube (122 kv.p., 5 ma., with a 0.28-mm. aluminum filter). The dose rate was de- termined with a Victoreen dosimeter before each treatment. Immediately following x-irradiation, roots of two of the irradiated and two of the control seeds were fixed in a solution of three parts absolute alcohol, one part glacial acetic acid and one part chloroform for six hours. The remaining germi- nated seeds were immediately placed in an incubator maintained at 32° C. and kept there throughout a five-hour period. During this time control and treated root samples were removed and fixed at one-half-hour intervals. The material was stained with Feulgen's reagent and made into squash prepara- tions according to a method worked out by Dr. Mary Esther Gaulden of the Oak Ridge National Laboratory (personal communication). The numbers of the different mitotic stages occurring in ten fields at the center of each root tip squash preparation were recorded. A Howard disc was used to avoid any overlapping of these fields. The number of each of the seven mitotic stages recorded in all tips at a given half-hour interval was summated to give obser- 3 Preliminary experiments, in which the roots were allowed to grow in darkness at a con- stant temperature, showed that the diurnal mitotic rhythm, as described by Kellicott (1904), only occurs when Alliuin is allowed to grow in an environment where light is present. These experi- ments confirm that which was inferred by Gray and Scholes (1951). 4 This was the dose rate as determined with the dosimeter lying on a wooden table at the same level at which the petri dishes were placed during treatment. The scattering effect of the glass, as determined by measurements made with the dosimeter lying on a petri dish, added ap- proximately 1.3% to these doses and dose rates. LOW DOSAGE X-RAY AND ALLIUM MITOSIS 307 vational totals.5 All observations were made with a 4 mm. objective and 10 X oculars. A one-tailed chi-square test was run to determine when the experimental per- centages differed, at the 5 per cent level of significance, from the control percentages. Using one degree of freedom. P > 3.84 indicates this significance. The results of these tests will be found in Tables II and III. RESULTS Results of 128 r x-ray treatment The effects of 128 r of x-rays on the different mitotic stages are shown in Table II. Interphase was the only stage in which the frequencies of cells showed no sig- nificant differences from control frequencies within five hours following x-raying. TABLE II Ratios of experimental frequencies to control frequencies for 128 r Hours after x-raying Stage Interphase Early prophase Late prophase Prometa- phase Metaphase Anaphase Telophase 0.0 1.01 0.98 0.95 0.81 0.63 1.12 1.11 0.5 1.02 0.97 1.29 0.53 0.77 0.84 0.92 1.0 1.02 0.96 1.16 0.46 0.55 0.84 0.89 1.5 1.00 1.15 1.29 0.47 0.60 0.51 0.54 2.0 1.02 1.12 0.96 0.65 0.32 0.33 0.68 2.5 1.04 1.17 0.43 0.23 0.13 0.25 0.32 3.0 0.98 1.39 0.59 0.33 0.14 0.29 0.29 3.5 1.00 1.44 0.20 0.05 0.07 0.14 0.13 4.0 1.00 1.46 0.14 0.00 0.00 0.06 0.13 5.0 1.01 1.36 0.17 0.00 0.00 0.00 0.00 The italicized values indicate that the experimental percentage differs from the control per- centage at the five per cent level of significance. In all other stages, a period of "normal" mitotic activity ranging from a half- hour duration in prometaphase to between two and two and one-half hours in late prophase and telophase was followed by a significant change in frequency. The phrase, "normal mitotic activity," is used to denote the period in which no ob- servable changes in mitotic activity in relation to the control cells was evident. Early prophase cell frequencies increased significantly beginning one and one- half hours after x-raying, and remained significantly higher than the early prophase control cell counts throughout the five-hour sampling period. X-rayed late prophases showed no significant differences from the controls for twro hours, after which they gradually fell to a minimum at four hours. 5 These totals may be found in the thesis from which this paper is condensed, in the Univer- sity of Tennessee library. It is from these data that the ratios in Tables II and III were calcu- lated. Totals at each time interval range from 1296-2420 cells for the controls and 1767-2910 cells for the treated root tips in the 128 r experiment, and from 1340-1742 cells for the controls and 1431-2510 cells for the treated in the 512 r experiment. 308 ARTHUR C. CHANDLER, JR. Prometaphase, metaphase, anaphase, and telophase treated-to-control ratios de- crease more or less gradually, virtually reaching zero three to four hours after treatment. Results of 512 r x-ray treatment The effects of 512 r of x-rays on the different mitotic stages are shown in Table III. Following one and one-half hours of normal frequency, treated interphase cells increased in number and maintained a level significantly higher than the controls from two to four hours after treatment. TABLE III Ratios of experimental frequencies to control frequencies for 512 r Hours after x-raying Stage Interphase Early prophase Late prophase Prometa- phase Metaphase Anaphase Telophase 0.0 1.01 0.96 1.29 1.00 0.89 1.19 1.08 0.5 1.02 0.98 0.91 0.67 1.10 0.95 0.98 1.0 0.99 1.04 1.50 0.86 0.99 0.85 0.97 1.5 1.03 1.03 0.99 0.31 0.43 0.50 0.65 2.0 1.04 1.12 0.91 0.05 0.12 0.23 0.48 2.5 1.06 1.08 0.53 0.05 0.00 0.09 0.26 3.0 1.05 1.20 0.38 0.00 0.00 0.03 0.09 3.5 1.05 1.23 0.23 0.00 0.00 0.00 0.06 4.0 1.06 1.21 0.16 0.00 0.00 0.02 0.00 5.0 1.02 1.38 0.12 0.00 0.00 0.00 0.00 The italicized values indicate that the experimental percentage differs from the control per- centage at the five per cent level of significance. The early prophase frequency does not differ significantly from the control fre- quency for two and one-half hours. Subsequently it shows a significantly higher frequency, which is maintained through the five-hour sampling period. Late prophase, prometaphase, metaphase, anaphase, and telophase frequencies, on the other hand, gradually decrease after one to two hours at the normal level. Late prophases have virtually disappeared at the end of five hours ; prometaphases, metaphases, anaphases, and telophases at the end of two to three hours after irradiation. DISCUSSION It has been found in a variety of materials, both animal and plant, that if tissues are exposed to ionizing radiations, a decrease in the number of prometaphases, meta- phases, anaphases, and telophases follows, and if the dose of ionizing radiations is great enough to reduce the number of cells in these stages to zero, the order of dis- appearance of these stages will be the same as the order in which cells pass through them (Carlson, 1954). Ionizing radiations tend to cause a blockage in one of the mitotic stages. The cells that have passed this stage continue to progress through the mitotic cycle. Hence, it is obvious that the first stage after the blockage point LOW DOSAGE X-RAY AND ALLIUM MITOSIS 309 will be the first stage vacated by the cells in mitosis, and each subsequent stage would be vacated in the order in which they occur. The more or less gradual progression of cell frequencies toward zero in these stages, as determined in this paper, seems to be in complete agreement with Carlson's statement. Investigators working with Vicia root tips have reported different intervals of time between treatment with ionizing radiations and minimal mitotic activity. Deufel (1951) found that if the broad bean root tip was irradiated with 150 r of x-rays at a dose rate of 5 r /minute, and the tips were kept at 18° C. between treat- ment and fixation, the low point of mitotic activity was reached after 12 hours. Mottram (1936) states that the minimal mitotic activity of the root tip cells was reached nine hours following a 210 r dose of gamma-rays. According to the work of Juengling and Langendorff (1930), the minimal mitotic rates occur (1) fifteen hours following a 175 r dose of x-rays, (2) eighteen hours following treatment with 420 r of x-rays, and (3) thirty-three hours after irradiation with 550 r of x-rays, but there is no change in mitotic rate after treatment with doses of 40 or 80 r of x- radiation. All of Juengling and Langendorff's work was done with a dose rate of about 20 r/minute. Working with Allium root tips, Darlington and La Cour (1945) wrote that minimal mitotic activity occurs (1) about ten hours after treatment with 150 r of x-rays when the root tips are kept at a temperature of 24° C. after treatment, and (2) about twenty-four hours after x-irradiation with the same dosage if the root tips are kept at 16° C. after treatment. Marshak (1937), however, states that minimal mitotic activity occurs in Allium root tips three hours after treatment with either 70 or 220 r of x-rays at a dose rate of 20 r/minute. This is confirmed by Gray et al. (1940) using a small dosage of gamma-rays and maintaining a temperature of 25° C. following treatment. Gray, in a personal communication to Carlson (1954), however, states that "the true value could easily have been as late as six hours since the minimum tends to be rather flat." It has been concluded by Carlson (1942) that the low point of mitotic activity in the grasshopper neuroblast, at 26° C., occurs one hundred minutes after treat- ment with 31 r of x-radiation. Carlson, Snyder and Hollaender (1949) found that the low point of mitotic activity in the grasshopper neuroblasts is reached about sixty-six minutes after treatment with 32 r of gamma-rays when the material is maintained at 38° C. following treatment. It may be the effect of temperature on the time required for irradiated cells to complete mitosis that accounts for the ap- parent wide discrepancies in the results cited above. My studies show that minimal mitotic activity of Allium root tip cells occurs be- tween three and four hours after treatment with 128 r of x-rays, and between two and one-half and three hours after treatment with 512 r of x-radiation. The mate- rial was maintained at a temperature of 32° C. between treatment and fixation. Concurrence exists among investigators that, if the treatment dosage is suffi- ciently large, cells in interphase at the time of treatment may be prevented from entering early prophase. These findings are identical with those in the present study (see Tables II and III). Differences of opinion, however, exist concerning the immediate reaction of pro- phase cells to irradiation. Roller (1943), working with Tradescantia pollen grains, is among those who believe that prophase is not very sensitive to x-radiation since cells in prophase decrease in frequency following irradiation. These prophase cells, 310 ARTHUR C. CHANDLER, JR. according to him, complete mitosis with little or no delay as do the cells in prometa- phase through telophase. Roller, therefore, concludes that cells in interphase are the most sensitive to ionizing radiations. By dividing the grasshopper neuroblast prophase stage into five sub-stages, Carl- son (1940) concluded from statistical calculations that middle prophase is the stage most sensitive to mitotic inhibition. This same publication also indicated that cells treated while in middle and early prophase revert to earlier stages. This reversion of cells in middle and late prophase was later confirmed by direct observation of living cells (Carlson, 1942). St. Amand (1956) also confirmed this from studies of living cells. Deufel (1951) describes the slowing down of prophases in the Vicia root tip after irradiation. As is shown in Table II, A Ilium root tip cells, treated with 128 r of x-rays, ex- hibit no significant decrease in late prophase frequency until two and one-half hours after treatment. Obviously, they are not progressing into prometaphase because the frequency of cells in this stage has been decreasing, significantly, since the first one-half hour after treatment, and continue to do so until frequency reaches zero. Therefore, it must be concluded that late prophases have begun to revert, two and one-half hours subsequent to treatment, to early prophase. Early prophase has been increasing, significantly, since one and one-half hours after treatment. This was probably due ( 1 ) to an accumulation of cells passing into early prophase from interphase and being, at least partially, blocked from continuing to subsequent stages, and (2) to reversion of late prophases after two hours. Treatment of the root tip cells with 512 r of x-rays causes this same effect (see Table III) but to a greater degree. Late prophase cells could not be progressing to prometaphase after the third hour, at the latest, as prometaphase frequency is zero after that time. Therefore, as their count is dwindling, late prophase cells must be reverting to early prophase. Some early prophase cells are reverting to interphase as witnessed by a significant experimental increase in interphase cell counts at, and following, the second hour after treatment. The reversion of early prophase cells is indicated by the fact that late prophase is decreasing at the hours when early pro- phase is showing no significant change. Hence, reversion of early prophase cells would counterbalance the influx of cells into that stage from late prophase. Inter- phase is increasing significantly at this time. Of course, some of the interphase increase is due to the cells passing from telophase into interphase, but this influx ceases before interphase increase stops. If these hypotheses and conclusions are justified, they would indicate that late prophase is the most sensitive stage and early prophase is the second most sensitive stage to the effects of x-radiation. Since the criteria I used to designate late pro- phase seem to include the late and middle prophases of Carlson (1940, 1941, 1942) and St. Amand (1956), the stage sensitivities shown in this paper closely parallel the results reported by these two investigators. Reversion is not a limited phe- nomenon by any means. Beatty and Beatty (1954a and 1954b) have reported cases of it in Tradescantia, while Darlington and La Gour (1945) have noted evidence of reversion in Trillium. Reversion of early prophase to interphase may explain the long period in which no change in frequency is observed in early prophase. The period in which no significant difference between experimental and control frequencies occurred, demonstrated by cells in late prophase, both after 128 r and 512 r doses, could be explained, possibly, in this manner. The critical period must LOW DOSAGE X-RAY AND ALLIUM MITOSIS 311 be in late prophase if reversion occurs as has been previously stated. This period could not be too far into late prophase, however, as no piling-up of cells occurs in this stage. Some cells may pass through this block following treatment to give the "normal" mitotic period in late prophase. This passage will also contribute to the "normal" mitotic periods in subsequent stages for a short time following treatment. Piling-up in late prophase need not occur, even if the critical period is in late prophase, if all the cells very close to the critical period at the time of irradiation are so sensitive as to revert. SUMMARY 1. Root tip cells of germinated Allium seeds were given doses of 128 or 512 r of x-rays. The mitotic effects of this treatment were determined by making counts, in fixed preparations, of cells in several mitotic stages at regular intervals following x-raying. 2. After treatment with 128 r of x-rays virtual disappearance of cells in late prophase, prometaphase, metaphase, anaphase and telophase was observed between three and four hours. No observable change occurred in the interphase frequencies, but an increase was observed in early prophase one and one-half hours subsequent to treatment. 3. After receiving 512 r of x-racliation, late prophase, prometaphase, metaphase, anaphase and telophase cells fell to minimal frequency between one and one-half hours and three hours. The fall in frequencies after this dose was noticeably more rapid than following the 128 r dose. An increase was noted in interphase frequency two hours following treatment and in early prophase three hours following treat- ment. Again, late prophase frequency did not reach zero, but was at its lowest point five hours subsequent to treatment. 4. Minimal mitotic activity in the root tip cells of Allium ccpa is reached between three and four hours subsequent to treatment with 128 r of x-rays, and between two and one-half and three hours following treatment with 512 r of x-rays. Between treatment and fixation all root tips were maintained at 32° C. 5. Reversion of cells from late prophase to early prophase is indicated when the cells are treated with 128 or 512 r of x-radiation and from early prophase to inter- phase when the cells are treated with 512 r of x-rays. It is probably due to this latter reversion that the minimal mitotic rate was reached in less time by the cells treated with the larger dose of x-rays. 6. Late prophase, which includes the latter portion of middle prophase of certain other investigators, seems to be the most sensitive and early prophase the second most sensitive stage to the mitosis-inhibiting effect of x-radiation. LITERATURE CITED BEATTY, ALVIN V., AND JEANNE W. BEATTY, 1954a. The primary effect of X-radiation on Tradescantia microspores. /. Tenn. Acad. Sci., 29: 175. BEATTY, ALVIN V., AND JEANNE W. BEATTY, 1954b. Immediate effects of 200 r and 400 r of X-radiation on the microspores of Tradescantia paludosa. Amer. J . Botany, 41 : 242-250. CARLSON, J. GORDON, 1940. Immediate effects of 250 r of X-rays on the different stages of mito- sis in neuroblasts of the grasshopper, Chortophaga viridifasciata. J. Morph., 66: 11-23. CARLSON, J. GORDON, 1941. Effects of X-radiation on grasshopper chromosomes. Cold Spring Harbor Symp. Quant. Biol, 9: 104-111. 312 ARTHUR C. CHANDLER, JR. CARLSON, J. GORDON, 1942. Immediate effects of 31 r of X-rays on the different stages of mitosis in neuroblasts of Chortophaga. J. Morph., 71 : 449-462. CARLSON, J. GORDON, M. L. SNYDER AND A. HOLLAENDER, 1949. Relation of gamma-ray dosage rate to mitotic effect in the grasshopper neuroblast. /. Cell. Comp. Physiol., 33 : 365-372. CARLSON, J. GORDON, 1954. Immediate effects on division, morphology and viability of the cell. Radiation Biology (A. Hollaender, Ed.). McGraw-Hill Book Co., New York: 1: 763-824. DARLINGTON, C. D., AND L. F. LA COUR, 1945. Chromosome breakage and the nucleic acid cycle. /. Genetics, 46 : 180-267. DEUFEL, J., 1951. Untersuchungen ueber die Einfluss von Chemikalien und Roentgenstrahlen auf die Mitose von Vicia faba. Chromosoina, 4 : 239-272. GRAY, L. H., J. C. MOTTRAM, J. READ AND F. G. SPEAR, 1940. Some experiments upon the bio- logical effects of fast neutrons. Brit. J. Radiology, 13 : 371-388. GRAY, L. H., AND M. E. SCHOLES, 1951. The effect of ionizing radiations on the broad bean root. Brit. J. Radiology, 24: 28-92, 176-180, 228-236, 285-291, 348-352. JUENGLING, O., AND H. LANGENDORFF, 1930. Ueber die Wirkung verschiedenen hoher Roent- gendosen auf den Kernteinlungsablauf bei Vicia faba equina. Strahlentherapie, 38: 1-10. KELLICOTT, W. E., 1904. The daily periodicity of cell division and elongation in the root tip of A Ilium. Bull. Torrcy Bot. Club, 31 : 529-550. ROLLER, P. C., 1943. The effects of radiation in pollen grain development, differentiation, and germination. Proc. Roy. Soc. Edinburg, Ser. B, 61 : 398-429. LAUGHLIN, H. H., 1919. Duration of the several mitotic stages in the dividing root tip cells of the common onion. Carnegie Inst. of Washington. Publication 265, 48 pp. MARSHAK, A., 1937. The effect of X-rays on chromosomes in mitosis. Proc. Nat. Acad. Sci., 23 : 362-369. MOTTRAM, J. C., 1936. On the spacing of radiation according to variation in radiosensitivity. Brit. J. Radiology, 9 : 824-832. ST. AMAND, W., JR., 1956. Mitotic inhibition and chromosome breakage induced in grasshopper neuroblasts by X-irradiation at known mitotic stages. Radiation Research, 5 : 65-78. LARVAL DEVELOPMENT OF BALANUS EBURNEUS IN THE LABORATORY 1 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT Duke University Marine Laboratory, Beaufort, N. C.; and Department of Zoology, Duke University, Durham, N. C. Most of the descriptions of larval development of barnacles have been based upon material obtained from the plankton. By this method Willemoes-Suhm (1876) found that Lepas fascicularis passed through six free-swimming naupliar stages and one cyprid stage. Since his publication others have made similar studies on differ- ent species of acorn barnacles and reported that they may pass through from six to eight naupliar stages and one cyprid stage. Because of the similarity of naupliar and cyprid stages of different species in the plankton the sequence of stages in re- constructions has been questioned. Hence many investigators have attempted to verify reconstructed life cycles by rearing barnacles from the egg to the sessile stage in the laboratory. Attempts to rear nauplii from unhatched eggs or from naupliar stages in the plankton have met with limited success. Groom (1894a), for example, only main- tained Balanus perjoratus nauplii through the second stage and Treat (1937) was unable to rear Balanus balanoides beyond the third stage. Sandison (1954) was also unsuccessful in maintaining several South African barnacles (Balanus algicola, Balanus amphitrite denticulata, Balanus ma.rillaris, Balanus trigonus, Chthamalus dcntatus, Octomeris angulosa and Tetraclita serrata) beyond the third naupliar stage. Bassindale (1936) was able to rear Verruca stroemia to the cyprid stage by feeding the nauplii on Nitsschia sp. but the cyprids failed to settle. By using the same methods he could raise Balanus balanoides to the fifth naupliar stage only. Batham (1945) maintained the non-feeding nauplii of the goose-barnacle, Pollicipes spinosus, from the egg to a stage described as "post-cypris," but no attachment oc- curred. There have been two definite reports of barnacles being successfully reared from the egg to the settled pin-head : that of Herz (1933) for Balanus crenatus and Hudinaga and Kasahara (1941) for Balanus amphitrite hawaiiensis. Hudinaga and Kasahara also reared Tetraclita squamosa on Skelctonema costatum and Nitsschia closterium but they all died in the cyprid stage. Pyefinch (1948b, 1949a) refers to culturing and describes the larval stages of Balanus crenatus, giving the time of appearance after hatching for the individual stages in the laboratory. He does not discuss the duration of the stages or mention successful attachment and metamorphosis of the cyprid. All investigators to date have reared larvae in mass culture and have determined time and stage of molting by daily sampling. While this method may indicate the number of larval stages it does not give accurately the molting frequency or the variations in intermolt periods within the population nor does it take into account the per cent mortality. 1 These studies were aided by a contract between the Office of Naval Research, Department of the Navy, and Duke University NR 163-194. 313 314 J. D. COSTLOW, JR. AND C. G. BOOKHOUT The barnacle selected for this study was Balanus eburneus, for neither has it been reared in the laboratory nor have its larval stages been described, even though it has a range from Massachusetts to South America (Pilsbury, 1916). Its larval history is known only through the report of Grave (1933) who merely stated that it passes through naupliar, metanaupliar, and cyprid stages in 7 to 10 days at Woods Hole, Mass. Therefore, the present study was undertaken to determine the food which would support complete development, the number and description of the lar- val stages, the frequency of molting, duration and mortality of each intermolt and the length of time required for complete development. METHODS Intact adult Balanus eburneus were removed from pilings and cleaned of at- tached organisms. In the laboratory the basis of the barnacle was chipped away and the egg lamellae removed. The developing ova obtained and successfully reared had attained the distinct median eye spot and gray color which corresponds to the "H" stage of Groom (1894a). The lamellae were placed in finger bowls contain- ing filtered sea water with a salinity of 28.5 per thousand, Chlainydomonas sp., and 200,000 to 400,000 units of penicillin per liter. The bowls were then covered and maintained at 26° C., the average outside water temperature, in a constant-tempera- ture culture cabinet lighted by daylight fluorescent lamps. In order to obtain nauplii of known age only those which were observed to hatch were used. At the time of removal each nauplius was placed in a separate compartment of the rearing assem- bly. The assembly was made by drilling 100 holes in a piece of %" Incite with a second solid piece of lucite forming the bottom. Each well had a capacity of 1.2 cc. The assembly was then placed in a glass dish, covered, and maintained in the culture cabinet. The contents of the wells were checked two to three times daily with a binocular dissecting microscope. When an exuvium was found it was removed, placed in 70 per cent alcohol, and the time, number of the molt, and mortality re- corded for each nauplius. After the second molt freshly fertilized Arbacia punctu- lata eggs were introduced daily into the compartments in addition to the Chlamydo- monas sp. At this time the larval plutei, developed from the eggs of the previous day, were removed. The naupliar stages were determined from the number of molts under segre- gated conditions. These were drawn to scale on graph paper, with the aid of a Whipple disc, from the exuviae, fixed specimens, and, in a few cases, from the liv- ing nauplii. The setation formulae \vere obtained from the exuviae and dissected appendages of known stages. Measurements were made with an ocular microm- eter mounted in a compound microscope. In addition to the 121 nauplii raised in individual compartments, hundreds of newly hatched larvae were maintained in finger bowls and plastic compartmented boxes. From these sources specimens were fixed daily in 70 per cent alcohol and 60° -C. Benin's. After the stages had been de- termined from exuviae of segregated barnacles the fixed specimens were staged and studied to determine the consistency in appendage setation. RESULTS AND DISCUSSION The nauplii of Balanus eburneus, reared individually, pass through six stages and one cyprid stage, a conclusion which is consistent with Bassindale's (1936) rear- LARVAL STAGES OF BALANUS EBURNEUS 315 ,0.1 mm J FIGURE 1. Carapace, caudal and abdominal processes, and appendages of naupliar stages I, II, and III of Balanus eburneits reared in the laboratory. All swimming setae are cut short, a, lateral view of abdominal and caudal processes ; b, antennule ; c, antenna ; d, mandible. 316 J. D. COSTLOW, JR. AND C. G. BOOKHOUT ing of Verruca stroemia, and Chthamalus stellatus and the reconstructions of larval stages obtained from the plankton on Balanus perjoratus (Groom, 1894b), Balanus balanoides, Balanus crenatus, Verruca stroemia (Pyefinch, 1948a), Elminius modes- tus, Balanus improvisus, Balanus crenatus (Knight-Jones and Waugh, 1949), and Balanus algicola, Balanus trig onus, Octomeris arigulosa (Sandison, 1954). How- ever, our results, Bassindale's (1936), and those based on reconstructions from the plankton do not agree with those obtained from culture methods reported by Herz (1933) and Hudinaga and Kasahara (1941). Herz (1933) found that Balanus crenatus passed through eight naupliar stages and one cyprid stage and Hudinaga and Kasahara (1941) reported seven naupliar stages and one cyprid stage for Bala- nus amphitrite hawaiicnsis. The discrepancy between staged barnacle larvae ob- tained from the plankton and those taken from mass culture by Herz (1933) and Hudinaga and Kasahara (1941) may be due to the use of appendage setation and other morphological characteristics as the main criteria for staging rather than the use of the exact number of observed naupliar exuviae from the egg to the cyprid stage. As is shown below, setation and spine structure were considered in this study but the number of naupliar stages was based solely on the exact number of molts through which each individual passed. Nauplii. The most significant characters for each naupliar stage are given below. Stage I. (Fig. 1, I.) The small frontolateral horns appear slightly recessed and project caudally. The horns are frequently hidden on the ventral side by the antennules and antennae. The caudal process is short and blunt and the abdominal process terminates in two short spines (Fig. 1, la). All setae are devoid of setules. Stage II. (Fig. 1, II.) The frontolateral horns project from the carapace at approximately right angles. The bases of the horns appear slightly swollen. The abdominal process now bears one spine on each side of the base and is approxi- mately half the length of the caudal process (Fig. 1, Ila). All setae bear setules. Stage III. (Fig. 1, III.) The frontolateral horns are tapered gradually from the junction with the carapace. The abdominal process bears the same spines as in stage II but is now greater than half the length of the caudal process. While larger and slightly heavier than stage II the primary distinguishing features are differences in setation of the antennules and antennae. Stage IV. (Fig. 2, IV.) The posterior edge of the carapace is delimited from the caudal process for the first time and bears a pair of carapace spines. The ab- dominal process bears two spines on each side. One pair is located at the base and the second pair in line with the division between the caudal process and the ab- dominal process (Fig. 2, IVa). Stage V. (Fig. 2, V.) There are two pairs of spines near the terminal por- tion of the abdominal process, a large lateral pair and a smaller median pair. An- terior to these is a pair of spines between which is a small, mid-ventral spine. The base is quite swollen and shows partial segmentation. The maxillule first appear (Fig. 2, Va). Stage VI. (Fig. 2, VI.) The abdominal process is considerably enlarged over the fifth stage and six pairs of small spines mark the developing cirriform ap- pendages beneath the exoskeleton. The spines on each side of the mid-ventral LARVAL STAGES OF BALANUS EBURNEUS 317 FIGURE 2. Carapace, caudal and abdominal processes, and appendages of naupliar stages IV, V, and VI of Balanus eburncus reared in the laboratory. All swimming setae are cut short, a, lateral view of abdominal and caudal processes ; b, antennule ; c, antenna ; d, mandible. 318 J. D. COSTLOW, JR. AND C. G. BOOKHOUT spine, found in the fifth stage, are no longer present. The paired eyespots become quite distinct in the later sixth stage nauplii. Cyprid. No significant characteristics were observed which distinguish Balanus eburneus cyprids from those of Balanus amphitrite denticulata (unpublished data). Table I gives the setation formulae for each naupliar stage of Balanus eburneus. Since Bassindale's (1936) introduction of this system it has been applied to nauplii of many species of barnacles. Unfortunately, as asserted by Bassindale, the setation formulae alone do not give a definite indication of stage or species. Knight-Jones and Waugh (1949) point out the extreme differences between types of setae and believe it to be misleading. They note a remarkable similarity between the setation of earlier stages of several species. Stage I of Elminius modestus, Balanus improvi- sus and Balanus crenatus were found to be identical and stage II of Elminius modes- tus, Chthalamus stellatus, and Verruca stroemia had corresponding setation. San- TABLE I Setation formulae of the six naupliar stages of Balanus eburneus Stage Antennule Antenna Mandible I 04211 0.1.4.-0.3.2.2.2.G. 0.1.3.-0.3.2.2.2.G. II 04 2 1.1 0.2.4.-0.3.2.2.2.G. 0.1.4.-0.3.2.3.2.G. III 14211 025-03222G 0 1 4-0.3 2.3.2.G. IV 114211 035-05324G 0.1.4.-04.2.4.3.G. v 11142111 03.8-0 5 3 2 4 G 0.1.5.-0.4.4.4.3.G. VI 11142121 048-05324G 0.1.5.-0.4.4.4.3.G. dison (1954) gives the setation formulae for Balanus algicola, Octomeris angulosa, and Tetraclita serrata. The first stage of B. eburneus corresponds in setation to those forms listed by Sandison (1954) and also B. crenatus which in turn makes the first stage of B. eburneus identical to E. modestus and B. improvisus. The setation of the second to sixth stages of B. eburneus, to our knowledge, is different from that described for any other species. Norris, Jones, Lovegrove and Crisp (1951) found the setation formulae to be of very limited value in studies on Balanus perforatus, B. improvisus, and B. amphitrite denticulata and suggest setation to be a developmental feature rather than a specific one for the separation of barnacle larvae. In both B. improvisus and B. amphitrite denticulata Norris et al. (1951) found some variation in the setation of the mandibular exopodite of stage II. They observed that it may "sometimes" resemble the third stage by bearing five setae instead of four and sug- gest it as a possible explanation for the eight stages found by Herz (1933). Pye- finch (1949a), however, examined thousands of staged nauplii of Balanus crenatus and found setation to be uniform within each stage. Norris et al. (1951) propose that the rate of morphological development of eyes, limbs, and setae is more strongly influenced by temperature than the rate of growth and onset of ecdysis and conse- quently the latter do not always occur in step with the former. Hudinaga and Kasahara (1941) note that there is no change in setation of the three appendages between the sixth and seventh stages of B. amphitrite hawaiiensis. They discrimi- nate between the two stages primarily on the presence of completely developed paired eyes in the seventh stage as compared to the rudimentary nature in the sixth stage. The present study of B. eburneus, in addition to the accounts of other species LARVAL STAGES OF BALANUS EBURNEUS 319 by Pyefinch (1949a), Bassindale (1936), and Sandison (1954), shows that the change from rudimentary to completely developed paired eyes takes place in the sixth stage. The development of B. eburneus, under controlled temperature condi- tions, does not reveal any variation in setation within any one stage. Exuviae col- lected from nauplii undergoing a known molt showed consistency in the setation for that particular stage. Pyefinch (1949a) describes the third stage of B. crenatus as the first one with a delimited carapace bearing a pair of carapace spines. This feature is seen first in the fourth stage of B. eburneus and also in Balanus algicola, Balanus trig onus, Octo- tneris angulosa (Sandison, 1954) and Pollicipes spinosus (Batham, 1945). The consistency of the mandibular exopodite setation in all 6 naupliar stages of B. cre- natus appears to be an unusual feature when compared with the increase in setation described for other species. TABLE II Measurements of larval stages of Balanus eburneus reared under laboratory conditions Carapace Stage Total length (mm.) Width (mm.) Length (mm.) i 0.16-0.18 — 0.19-0.23 ii 0.21-0.23 — 0.32-0.34 in 0.23-0.27 — 0.35-0.38 IV 0.27-0.32 0.30-0.33 0.40-0.42 V 0.29-0.34 0.35-0.38 0.44-0.48 VI 0.36-0.39 0.42-0.50 0.54-0.60 Cyprid — — 0.46 Table II gives the range in size of the six naupliar stages and one cyprid stage of B. eburneus. Knight-Jones and Waugh (1949), using size distribution followed by setation formulae for staging planktonic material, found greater variation in size in the later stages. They also note that "total length" of nauplii is affected by flex- ing of the abdomen and thus this measurement may be erroneus. Greater variation in size of the later stages was also observed in this study of B. eburneus in the labo- ratory. With setation and observable internal development, such as paired eyes and cirriform appendages, the carapace width and length would be more reliable than total length for staging laboratory reared larvae and presumably planktonic material. The latter study, however, must be made before definite conclusions can be drawn. Figure 3 gives the variation in duration of intermolt period for the individual stages. Grave (1933) postulated 7 to 10 days as the time required for over-all de- velopment of B. eburneus at Woods Hole but did not include periods for each stage. Hudinaga and Kasahara (1941) estimated that the individual naupliar stages of B. amphitritc hawaiiensis lasted one day each and Pyefinch (1948b), using plankton samples taken at three-day intervals, postulated that each stage of Balanus balanoides was three days, or less, in duration. In the present study the duration of the first stage of B. eburneus ranged from 15 minutes to 4 hours. Many authors have re- ported that it is quite short in other species and Hudinaga and Kasahara (1941) 320 J. D. COSTLOW, JR. AND C. G. BOOKHOUT noted that it lasted for 15 minutes to 2 hours for B. amphitrite hawaiicnsis. The second stage of B. eburncus lasts approximately 24 hours and the third 36 hours but with greater variation (Fig. 3). Stage IV averages 2 days, stage V, 2.6 days, and stage VI, 2.5 days but with less uniformity than the earlier stages. n m STAGE FIGURE 3. Duration of intermolt of naupliar stages (I-VI) and cyprid stage (c) of 121 Balanus eburneus reared under segregated conditions. The vertical line indicates the range. The horizontal line indicates the mean. One hundred per cent of the first stage nauplii underwent ecdyses from 15 min- utes to 4 hours after hatching. This was true whether food was available or not. The second stage nauplii molt from 10 to 35 hours after hatching. During this stage the gut appeared green with Chlamydomonas sp. The third stage nauplii may molt from the second to fifth days (Fig. 4). As indicated by Sandison (1954) this is a period when many nauplii die if proper conditions are not present. Therefore, it was at this stage that we added to the diet of Chlamydomonas sp. fertilized Ar- bacia eggs to furnish food of animal origin. The latter were ingested and the gut took on the echinochrome color of the Arbacia eggs. Even so, there was a mortality of 6 per cent (Fig. 5). The fourth naupliar stage shows a greater degree of varia- tion in molting and ecdysis may occur from the third to eighth day with a majority molting on day 4 (Fig. 4). This is accompanied by an increase in mortality (Fig. LARVAL STAGES OF BALANUS EBURNEUS 321 IOO V 1 95 2 I 90 - 1 MOLTING PERIODS AND PERCENTAGE OF MOLTS IN NAUPLII OF BALANUS EBURNEUS 85 80 - 75 70 65 60 55 3 i '-, 50 i i O 2 \ \ * 45 i LL O r \ 0 4° — ' \ \ * \ Z i i • o 35 — . a Ld i a 30 - i \ i \ 25 _ i A A ^ « / \ i 1 \ \ / \ 20 _ I i * \ / \ i V \ 1 5 _ i \ \ ' i V \ I. ; \ 1 0 - ; \ \ 1 i \ \ 5 - \ \ v \ \ \ \ n ^ DAYS FIGURE 4. Molting frequency of naupliar stages of 121 Balanus eburncus reared under segregated conditions. Molts are indicated by numbers. 322 J. D. COSTLOW, JR. AND C. G. BOOKHOUT 5). Molting of the fifth naupliar stage occurs from day 5 to 9, with a majority on day 7. This stage showed greater mortality than any other stage, it being approxi- mately 33 per cent. The sixth and final naupliar stage molts into a cyprid on the seventh to twelfth day, with a mortality of 22 per cent. There is apparently considerable variation in the duration of the cyprid stage. Pyefinch (194Sb) reports that the cyprid of Balanus balanoidcs remains free- swimming in laboratory tanks for five days at 4-5° C. but estimated a shorter period in nature. Under laboratory conditions we found that those which settled and underwent metamorphosis to the pinhead did so within one to three days, whereas 30 I- OL §20 O I 5 5io u Ld °- 5 H 12 STAGE I2L FIGURE 5. Mortality in relation to naupliar stages (I-VI) and cyprid stage (c) of 121 Balanus cburneus reared under segregated conditions. those which persisted as cyprids for longer periods, up to 14 days, failed to settle and died. Mortality in the cyprid stage was approximately 16 per cent. In some cases the mortality was due to incomplete closing of the carapace following the sixth naupliar molt. These abnormal cyprids could live as swimming larvae for two to three days but they failed to settle. The over-all time of development for B. cburneus is quite short when compared with the times given for most other species. Bassindale (1936), although not giv- ing water temperatures, reported 13 days as the minimum time for completion of the sixth stage of Chthamalus stcllahts and a 22-day minimum for the sixth stage of Verruca stroemia. Unfortunately, comparison between species is not too reliable, for the effect of temperature on individual species has not been determined and can only be inferred from studies on other species at different temperatures. Hudinaga and Kasahara (1941) found that the minimum time for development of B. amphi- trite hawaiiensis, from the first naupliar stage to settling, was 7 days at 23-28° C. This falls within the range found by us for B. cburneus at 26° C. Grave (1933) postulated from planktonic material that B. eburnciis takes 7-10 days for complete development. This figure corresponds with ours even though the temperature at Woods Hole is normally 4 to 8° C. lower than at Beaufort. LARVAL STAGES OF BALANUS EBURNEUS 323 The best evidence to date that reduced temperatures increase the time of over-all development of barnacles is shown in the work of Pyefinch (1948b) and Batham (1945). Pyefinch (1948b) found that the over-all development of B. crcnatus took approximately 30 days at 4-5° C. whereas at 15° C., "or more," the time was re- duced to 16 days. Batham (1945) found that the goose-barnacle, Pollicipcs sf>ino- sus, takes 12 days to pass through all larval stages at 18° C. while in the "cold room" (temperature not given) it required 20 to 21 days. The times given by Batham (1945), however, did not include normal completion of the cyprid stage or settling. The relationships of the substratum and physical factors to settling are outside the scope of this paper. In the present study the only substratum offered was lucite and the physical factors of temperature and light were similar throughout the ex- periment, in that the temperature was maintained at 26° C. and the rearing assembly received constant illumination from below. There is always the question of normality when organisms are reared in the laboratory and the query of how survival compares with that in nature. In this experiment if a barnacle completed development, settled, and metamorphosed it was considered normal. Pyefinch (1949b) estimated the survival of barnacle larvae to be between 1 and 9 per cent, depending on the species with which he was working. Bousfield (1955) questions his estimate because of the methods used and was of the opinion that natural survival was approximately 10 per cent. Therefore, our observed survival of 16.3 per cent, under laboratory conditions, is higher than that estimated in nature. Whereas it is undoubtedly true that the chief sources of mor- j tality in nature are dispersal seaward and predation, improper food and bacteria are the chief causes in the laboratory. In preliminary experiments we found that B. ebitrneus, B. amphitrite denticulata, and Chthamalus frag His larvae could not be reared beyond the third stage on a diet of Chlamydomonas sp. alone. When devel- oping Arbacia eggs and penicillin were included, complete development occurred. The actual value of the penicillin is not known but it was observed that the tendency of nauplii to stick to surfaces of the rearing assembly was considerably reduced. SUMMARY AND CONCLUSIONS A technique has been devised for rearing segregated barnacle nauplii, under controlled laboratory conditions, which permits daily observations on the frequency of molting, the number of stages, and the specific characteristics of each stage. From a study of 121 segregated Balanus cburneiis, plus hundreds in mass culture, reared on Chlamydomonas sp. and Arbacia larvae at 26° C. the following conclu- sions may be drawn : 1. Ecdyses provide a definitive method for staging nauplii. The larval phase of B. eburneits consists of six naupliar stages and one cyprid stage. Secondary cri- teria, such as body size, spine structure, and appendage setation, are given for the larval stages. 2. The duration of the six naupliar stages is as follows : first stage, 15 minutes to 4 hours ; second stage, one to two days with an average of one day ; third stage, one to four days with an average of 1.5 days; fourth stage, one to four days with an average of two days ; fifth stage, one to five days with an average of 2.6 days ; and the sixth stage, two to four days with an average of 2.5 days. 324 J. D. COSTLOW, JR. AND C. G. BOOKHOUT 3. The cyprid stage ranges from one to fourteen days but successful attachment was observed only in those which settled one to three days following the final naupliar molt. 4. The over-all larval development in the laboratory ranges from 7 to 13 days. 5. The first ecdysis occurs from 15 minutes to 4 hours after hatching and is usually followed by the second molt during the first day. The third molt occurs from the second to the fifth days and the fourth molt takes place from the third to the eighth day. The fifth ecdysis occurs from the fifth to the ninth day with the sixth molt ranging from the seventh to the twelfth day. 6. Tables are given for the size of the nauplii and the setation of the appendages. 7. Successful metamorphosis and attachment was observed in 16.3 per cent of the 121 barnacle nauplii studied under segregated laboratory conditions. LITERATURE CITED BASSINDALE, R., 1936. The developmental stages of three English barnacles, Balanus bala- noides, Chtharnalus stellatus, and Verruca stroetnia. Proc. Zool. Soc. Lond., 1 : 57-74. BATHAM, E. J., 1945. Pollicipes spinosus Quoy and Gaimard. II. Embryonic and larval devel- opment. Trans. Roy. Soc. New Zealand, 75 : 405-418. BOUSFIELD, E. L., 1955. Ecological control of the occurrence of barnacles in the Miramichi es- tuary. National Museum of Canada, Bulletin 37. GROOM, T. T., 1894a. On the early development of the Cirripedia. Phil. Trans. Roy. Soc., 185 : 121-208. GROOM, T. T., 1894b. The life-history of the rock barnacle (Balanus). Part I. /. Marine Zool. and Microscopy, 1 : 81-86. GRAVE, B. H., 1933. Rate of growth, age at sexual maturity, and duration of life in sessile or- ganisms at Woods Hole, Mass. Biol. Bull., 65 : 375-386. HERZ, L. E., 1933. The morphology of the later stages of Balanus crenatus Bruguiere. Biol. Bull, 64 : 432-442. HUDINAGA, M., AND H. KASAHARA, 1941. Larval development of Balanus amphitrite hawaiien- sis. Zool. Mag. (Japan), 54: 108-118. KNIGHT-JONES, C. W., AND D. WAUGH, 1949. On the larval development of Elminius modestus Darwin. J. Mar. Biol. Assoc., 28: 413-428. NORRIS, E., L. W. G. JONES, T. LOVEGROVE AND D. J. CRISP, 1951. Variability in larval stages of Cirripedes. Nature, 167 : 444-445. PILSBURY, H. A., 1916. The sessile barnacles (Cirripedia) contained in the collections of the U. S. National Museum ; including a monograph of the American species. U. S. Na- tional Museum, Bulletin 93. PYEFINCH, K. A., 1948a. Methods of identification of the larvae of Balanus balanoides (L.), B. crenatus Brug., and Verruca stroemia O. F. Miiller. /. Mar. Biol. Assoc., 27 : 451^63. PYEFINCH, K. A., 1948b. Notes on the biology of Cirripedes. J. Mar. Biol. Assoc., 27 : 464-503. PYEFINCH, K. A., 1949a. The larval stages of Balanus crenatus. Proc. Zool. Soc. London, 118: 916-23. PYEFINCH, K. A., 1949b. Short-period fluctuations in the numbers of barnacle larvae, with notes on comparisons between pump and net plankton hauls. /. Afar. Biol. Assoc., 28 : 353-369. SANDISON, EYVOR E., 1954. The identification of the nauplii of some South African barnacles with notes on their life histories. Trans. Roy. Soc. S. Africa, 34: 69-101. TREAT, D. A., 1937. A comparative study of barnacle larvae. M.A. Thesis (unpublished), Department of Biology, Western Reserve University. VON WILLEMOES-SUHM, R., 1876. On the development of Lepas fascicularis and the Archizoea of Cirripedia. Phil. Trans. Roy. Soc., London, 166: 131-154. AMINO ACID CONTENT OF MARINE BORERS RICHARD W. DRISKO AND HARRY HOCHMAN Chemistry Division, U. S. Naval Civil Engineering Research and Evaluation Laboratory, Port Hueneme, California Although a great amount of study on the prevention of marine borer attack on wooden structures has been made in the last century, relatively little is known of the biochemical make-up of these organisms. The only published investigation of the amino acid content seems to be that of Lasker and Lane (1953) who reported eight amino acids present in Teredo bartschi Clapp. In view of the current idea (Fox, 1956) that primitive organisms have proteinaceous matter similar to that found in higher forms of life, it does not seem likely that several commonly occurring amino acids would be absent from the protein structure of Teredo. In the present investigation Teredo diegensis, Bankia setae ea (another member of the Teredinidae family) and two species of the crustacean borer Limnoria were investigated to determine their amino acid content. In all cases considerably more than the eight amino acids reported by Lasker and Lane were found to be present. In addition, southern yellow pine, redwood, and greenheart (Ocotea rodiaei) were hydrolyzed for identification of the amino acids present. The latter species of wood has been reported to possess a high protein content (Marchan, 1946) and to be highly resistant to marine borer attack (Baldwin, 1938). MATERIALS AND METHODS The marine borers investigated were Teredo diegensis, Bankia sctacea, and Limnoria tripunctata and quadripunctata. The first two were removed from both pine and redwood blocks, the Limnoria tripunctata from a creosoted log, and the quadripunctata from a redwood block in the same waters. Whole adult animals taken from the harbor and Teredo larvae produced in the laboratory were hydro- lyzed. The larvae were collected by screening the overflow sea water from a tank containing pine blocks infested with Teredo. In addition to analyzing whole ani- mals, Bankia was dissected into viscera, mantle, and gill, and Teredo into viscera, mantle, and reproductive organ containing larvae, and each section was analyzed individually. These dissected parts were extracted with 80 % alcohol prior to hy- drolysis in order to separate the free amino acids from those bound in the protein structure of the animals. The extracts were concentrated, the fatty substances ex- tracted with chloroform, and the residues spotted for chromatography without fur- ther treatment. Acid hydrolysis was in all cases effected by heating with 6 N HC1 in a boiling water bath for twenty hours. Excess of HC1 was removed by repeated evaporation to dryness under reduced pressure. Two-dimensional chromatograms using What- man No. 1 filter paper with phenol solvent (Block, 1950) in the first direction and lutidine-collidine (Dent et al., 1947) in the second were routinely run by the capil- 325 326 R. W. DRISKO AND HARRY HOCHMAN lary ascent method of Williams and Kirby (1948). The leucines were separated on a one-dimensional descending chromatogram using water saturated tert-a.my\ alcohol in an atmosphere of diethylamine. Confirmation of the identity of each spot was made by running suitable quantities of authentic amino acids simultaneously. The amino acids were revealed with the ninhydrin reagent of Levy and Chung (1953). Histidine and tyrosine were further identified by Block's (Block ct al., 1952) modi- fication of the Pauly reaction ; arginine, by one-dimensional chromatography using the solvent system of 77% alcohol and diethylamine (Block and Boiling, 1951) ; and the sulfur-containing amino acids, with the platinic iodide reagent (Toennies and Kolb, 1951). Dent's (1948) hydrogen peroxide treatment likewise proved to be useful in identifying the sulfur-containing amino acids. A spot corresponding to lanthionine was eluted by the method of Dent (1947) from an unsprayed two-dimensional chromatogram. It was then spotted on a one- dimensional descending paper and run for five days using w-butyl alcohol-acetic acid (Partridge, 1948) as the solvent. Glutamine, which was found to occur as a free amino acid in the Teredinidae, was further identified by elution (Dent, 1947) from an unsprayed two-dimensional chromatogram, by hydrolysis with acid, and by rechromatography. Glutamic acid was the only amino acid found on this latter chromatogram. The barium hydroxide hydrolysis method of Levy and Chung (1953) was used to detect tryptophan which is unstable to acid hydrolysis. The three woods investigated were hydrolyzed with 6 N HC1 in a boiling water bath for twenty hours. Before spotting, each hydrolyzate was partially purified by adsorption on a column of the hydrogen form of Dowex 50-X8 (200 to 400 mesh), washing with water, and elution with 4 N ammonia. A synthetic mixture of amino acids was similarly treated. RESULTS The amino acids found in the acid hydrolyzates of whole living organisms are summarized in Table I for each of the species studied. Taurine is also listed in this Table, as its spot was readily detected in these hydrolyzates. When the freshly- ground tissues of the Teredinidae were treated with 80% alcohol, taurine and /?- alanine were extracted into the alcohol. All other amino acids listed in Table I were found in the hydrolyzates of the 80% alcohol-insoluble residues. The alcoholic extracts of the dissected Teredinidae fragments all gave readily identifiable spots for a-alanine, /3-alanine, glutamic acid, glycine, and taurine. In addition, some of the fragments, notably the mantle, gave very faint tests for some of the other amino acids in Table I, but these were not further investigated. The Bankia gill, the Teredo reproductive organ, and the mantles from both organisms were found to contain glutamine. The acid hydrolyzates of both species of Teredinidae removed from redwood gave a spot corresponding to lanthionine, while those from pine did not. The spot gave a positive sulfur test with the platinic iodide reagent and proved to be chro- matographically indistinguishable from lanthionine in the solvent systems used. An unidentified spot was found in the acid hydrolyzates of both species of Limnoria. It is designated "A" in Table I. The spot persisted even when the tissues were hydrolyzed for an additional twenty-four hours. It was located on a AMINO ACIDS IN MARINE BORERS 327 TABLE I Amino acids in acid hydrolyzates of whole organisms Amino acid TP TR TL BP BR LT LQ a-Alanine* X X X X X X X /3-Alanine X X X X X o o Arginine* X X X X X X X Aspartic acid X X X X X X X Cystine-cysteine X X X X X X X Glutamic acid X X X X X X X Glycine X X X X X X X Histidine X X X X X X X Isoleucine X X X X X X X "Lanthionine" 0 X o o X o 0 Leucine* X X X X X X X Lysine X X X X X X X Methionine* X X X X X X X Phenylalanine* X X X X X X X Proline* X X X X X X X Serine X X X X X X X Taurine X X X X X X X Threonine X X X X X X X Tyrosine* X X X X X X X Valine* X X X X X X X Spot "A" o o o o o X X * Reported by Lasker and Lane to be present in Teredo bartschi Clapp. X Present in readily detectable amounts. O Not present in readily detectable amounts. TP — Teredo from pine; TR — Teredo from redwood; TL — Teredo larvae; BP — Bankia from pine; BR — Bankia from redwood; LT — Limnoria tripunctata; LQ — Limnoria quadripimdata. two-dimensional phenol and collidine paper between the spots for threonine and tyrosine. The Rf value in phenol was considerably lower when run in an atmos- phere of acetic acid (Dent, 1948) than in an atmosphere of ammonia. Tryptophan was not found in any of the alkaline hydrolyzates. This may have been because of its relative insensitivity to the ninhydrin reagent as compared to other commonly occurring amino acids. The amino acids found in the three species of wood are listed in Table II. The greenheart hydrolyzate gave a much stronger phenylalanine spot than did the hy- drolyzates of either pine or redwood. When a synthetic mixture of amino acids was treated with Dowex-50, all but lanthionine were eluted with ammonia. DISCUSSION These data indicate that there are at least eleven amino acids present in Teredo diegensis in addition to the eight reported present in Teredo bartschi Clapp by Lasker and Lane. It is considered unlikely that so many additional amino acids would occur in the one species and not the other. The fact that taurine and ^3-alanine are not present in any of the hydrolyzates of tissues that had been extracted with 80% alcohol indicates that these compounds are 328 R. W. DRISKO AND HARRY HOCHMAN not present in the protein of the borers investigated. Seventeen amino acids are common to each species. All the amino acids seem to be distributed throughout all the Teredinidae sections. In addition, spot "A" was detected in the hydrolyzates of both species of Limnoria, and a spot corresponding to lanthionine was found in the hydrolyzates of both species of Teredinidae which had been removed from red- wood. A corresponding spot was not found in the hydrolyzates of animals removed from pine. Cystathionine is reported (Dent, 1948) to have chromatographic prop- erties similar to those of lanthionine. Neither of these two amino acids has been found in nature (Dent, 1948). TABLE II Amino acids readily detected in acid hydrolyzates of woods studied a-Alanine Lysine Aspartic acid Phenylalanine Glutamic acid Proline Glycine Serine Hydroxyproline Threonine Isoleucine Valine Leucine The three species of wood studied contain thirteen amino acids in common. Hydroxyproline was found in all three woods but in none of the marine borers. A spot corresponding to lanthionine was not found in the chromatograms of the acid hydrolyzed redwood, but it was shown that lanthionine is not eluted with the other amino acids when a synthetic mixture is similarly treated. SUMMARY 1. Eighteen naturally-occurring amino acids and taurine have been identified chromatographically in the acid hydrolyzate of Teredo diegcnsis and Bankia sctacea, and seventeen naturally-occurring amino acids and taurine have been identified chromatographically in Limnoria tripunctata and qnadripunctata. A spot corre- sponding to lanthionine has been found in the above Teredinidae living in redwood blocks but not in those living in pine blocks. Glutamine has been found in the alcoholic extract of freshly-ground Teredinidae sections. 2. Three species of wood have been hydrolyzed with acid, and all were found to contain the same thirteen amino acids. LITERATURE CITED BALDWIN, C. E., 1938. Greenheart. Dock and Harbour Authority, 17: 112-115. BLOCK, R. J., 1950. Estimation of amino acids and amines on paper chromatograms. Anal. Chcm., 22 : 1327-1332. BLOCK, R. J., AND D. BOLLING, 1951. The amino acid composition of proteins and foods. Charles C. Thomas, Springfield. BLOCK, R. J., R. LESTRANGE AND G. ZWEIG, 1952. Paper chromatography. Academic Press Inc., New York. DENT, C. E., 1947. The aminoaciduria in Fanconi syndrome. A study making extensive use of techniques based on paper partition chromatography. Biochem. J ., 41 : 240-253. DENT, C. E., 1948. A study of the behavior of some sixty amino-acids and other ninhydrin- reacting substances on phenol-collidine filter paper chromatograms, with notes as to the occurrence of some of them in biological fluids. Biochem. J., 43 : 169-180. AMINO ACIDS IN MARINE BORERS 329 DENT, C. E., W. STEPKA AND F. C. STEWARD, 1947. Detection of the free amino acids of plant cells by partition chromatography. Nature, 160 : 682-683. Fox, S. W., 1956. Evolution of protein molecules and thermal synthesis of biochemical sub- stances. Amer. Sci., 44: 347-359. LASKER, R., AND C. E. LANE, 1953. The origin and distribution of nitrogen in Teredo bartschi Clapp. Biol. Bull, 105 : 316-319. LEVY, A. L., AND D. CHUNG, 1953. Two-dimensional chromatography of amino acids on buf- fered papers. Anal. Chan., 25 : 396-399. MARCHAN, F. J., 1946. The lignin, ash and protein content of some neotropical woods. Car- ribean Forester, 7 : 135-138. PARTRIDGE, S. M., 1948. Filter-paper partition chromatography of sugars. Biochem. J., 42 : 238-250. TOENNIES, G., AND J. J. KOLB, 1951. Techniques and reagents for paper chromatography. Anal. Chem., 23: 823-826. WILLIAMS, R. J., AND H. KIRBY, 1948. Paper chromatography using capillary ascent. Science, 107 : 481-483. THE RATE OF FEEDING OF THE COMMON OYSTER DRILL, UROSALPINX CINEREA (SAY), AT CONTROLLED WATER TEMPERATURES JAMES E. HANKS1 U. S. Fish and Wildlife Service, Milford, Conn. Information on the voracity of the oyster drill, Urosalpin.v cincrea (Say), within certain ranges of water temperature has been reported by a number of investigators, but evaluation of their destructiveness at specific temperatures maintained within close limits is virtually non-existent. In addition, the conclusions regarding the de- structiveness of drills within the different temperature ranges are drawn from ob- servations made at widely separated geographical areas (Carriker, 1955). Observations by Stauber (1950) and Loosanoff and Davis (1950-51) on cer- tain activities of drills from different areas of the North Atlantic Coast suggest the existence of distinct physiological races. If such races exist, a temperature-depend- ent activity, such as feeding, determined for drills from one area would not neces- sarily be applicable to drills of another habitat. Therefore, we have attempted to establish the feeding rate of a single geographical population of oyster drills at sev- eral constant temperatures. It was felt that such a study would not only provide more detailed information about the predation of drills, but also present a basis of comparison for investigators interested in the problem of physiological races. METHODS A bank of wooden frames, constructed to hold a number of enamel trays (18" X 20" X 3"), was arranged on a laboratory water table. Each tray was supplied with a separate, continuous flow of sea water. Temperatures were adjusted by mixing cold and heated sea water in glass cylinders, just above the frames, giving each bank of trays a separate supply of water at a constant temperature (Loosanoff, 1949). Water in the trays was maintained at approximately ± 1.0° C. of the temperature desired. Since the drills had a tendency to move up the sides of the tray and leave the water, covers were made to fit into each tray to keep the drills below the water line. They were constructed of Lucite plastic frames with a covering of %"-mesh Saran plastic screening. The salinity of the water deviated only slightly from 25%o throughout the ex- periments and is, therefore, considered to have exerted no influence on differential feeding at the various temperatures. To obtain a more comprehensive evaluation of feeding by drills at different tem- peratures, two species of bivalves, the common oyster, Crassostrea virginica Gmelin, and the mussel, Mytilus cdulis Linne, were used as foods in separate experiments. 1 Present address : Department of Zoology, University of New Hampshire, Durham, New Hampshire. 330 RATE OF FEEDING OF UROSALPINX 331 Each tray contained 20 adult, Long Island Sound drills measuring from 20.0 to 25.0 mm. in height, and either 30 to 40 oyster spat, ranging in size from 10.0 to 30.0 mm., or 40 mussels, ranging in size from 20.0 to 30.0 mm. Spat were growing on cultch (old oyster shells) in such numbers that four to five shells supplied the total needed. The oyster spat clusters or mussels were so distributed about the tray that no large concentration of food occurred at any one point. The drills were dispersed throughout the tray to decrease the opportunity for feeding by two or more drills on the same bivalve. At no time during the experiments did the drills con- sume all of the bivalves available in any tray, thereby indicating that feeding was maximal for each feeding period. The drills were brought to the experimental temperatures by keeping them for two to three hours at each five-degree level until the desired temperature was reached. After the experiment was begun the number of bored bivalves, as well as the number and condition of drills present, was ascertained at regular intervals. Following each such examination, oyster spat or mussels were added to replace those destroyed by the drills. Mortality of drills was low at all temperatures, except 30.0° C. All dead drills were replaced by drills from stocks kept at the same water temperature as the experimental groups to assure that their prior conditioning was the same. RESULTS The feeding rates are expressed as the number of bivalves destroyed per drill during one week of feeding. Since low temperatures could not be maintained dur- ing the spring and summer, observations on the groups at 5.0° C. were discontinued after 52 days, on the 10.0° C. groups, at 69 days, and on the 15.0° C. groups, at 90 days; while the groups at 20.0°, 25.0° and 30.0° C. were under observation for 102 days (Table I). At 5.0° C. the drills did not feed, nor did they show any tendency to attack the spat during the 52 days of the experiment. They usually remained in groups, each TABLE I Feeding rate of U. cinerea on oyster spat, size range 10-30 mm., at controlled water temperatures Temperature ° C 5. 0 10 .0 15 .0 20 .0 25 .0 3C .0 Tray 1 2 i 2 1 2 1 2 1 2 1 2 No. of drills 20 20 20 20 20 20 20 20 20 20 20 20 Feeding period (days) 52 52 69 69 90 90 102 102 102 102 102 102 No. of spat con- sumed 0 0 49 41 124 133 313 355 429 398 285 327 Spat consumed per drill per week 0 0 0.25 0.21 0.48 0.52 1.07 1.21 1.47 1.37 0.98 1.12 332 JAMES E. HANKS drill with the foot extended and attached firmly to the tray. Movement of a few inches by some drills, as shown by mucus paths, was noted on several occasions. Feeding at 10.0° C. appeared to be limited to occasional attacks by individual drills since the rate of feeding was only about one spat per drill every four to five weeks. The rate of feeding on oyster spat increased as the temperature increased from 10.0° to 25.0° C. The rate approximately doubled for each five-degree in- crease in water temperature from 10.0° to 20.0° C. However, the next five-degree rise, to 25.0° C., increased the rate of feeding only about 25 per cent over that at 20.0° C., and a distinct decrease in the rate of feeding occurred at 30.0° C. Thus, the optimum temperature for feeding of drills on oyster spat was at or near 25.0° C. The drills maintained at 30.0° C. were also slower in turning over and attaching to the tray than those kept at 25.0° C. On two occasions, when the temperature rose to about 34.0° C., 70 to 80 per cent of the drills were killed, although in neither TABLE II Feeding rate of U. cinerea on mussels, size range 20-30 mm., at controlled water temperatures Temperature ° C 1C o 15 0 2C 0 25 0 3C 0 Tray ... . i 2 1 2 1 2 1 2 1 2 No. of drills 20 20 20 20 20 20 20 20 20 20 Feeding period (days) 34 34 57 57 57 57 44 44 44 44 No. of mussels consumed 1 4 39 32 88 73 101 109 89 105 Mussels consumed per drill per week 0.01 0.04 0.24 0.20 0.54 0.45 0.80 0.87 0.71 0.82 instance were they exposed to this temperature for more than 16 hours. The sur- viving drills were not attached to the tray or to the oyster spat, as they usually were at 30.0° C. These observations suggest that while drills do feed at 30.0° C, this approaches the lethal temperature level. Comparable data for oyster drills feeding on mussels show that the optimum feeding temperature again lies near 25.0° C. (Table II). Apparently, only a few of all the drills kept at 10.0° C. fed during the 34 days of the experiment. The rate of feeding at 15.0° C. approximated one mussel per drill every five weeks. This rate was about doubled for each five-degree rise in temperature from 15.0° to 25.0° C. Although the rate of feeding at 30.0° C. was lower than that at 25.0° C., it was not as low as would be expected from the results when oyster spat were used as food. Probably, the rate of drill feeding at 30.0° C., in the mussel-fed experi- ment, was somewhat higher due to differences in handling. It was necessary to examine each tray and change the mussels daily to avoid a high mortality and re- sultant bacterial decomposition of mussels at 30.0° C. Thus, the drills may have been induced to attack more mussels because they were unable to feed without interruption. RATE OF FEEDING OF UROSALPINX 333 Statistical treatment of the data by analysis of variance indicates that differences in feeding rate between temperature levels are highly significant (F = 85.96 with df = 4,5 for mussels as food and F = 143.84 with df ==5,6 for oyster spat as food ; both values being significant beyond the 0.001 level). In order to determine which of the differences between temperatures were significant, a test for significance of gaps between means was applied (Bliss and Calhoun, 1954). Results of this test UJ tr UJ a. 84ft. 6941:22* I.TOmiles W:25*282ft,6OH FIGURE 2. Twelve cases in which two turtles with opposite homeward directions were re- leased from the same point at the same time. Sixteen different turtles were used. Symbols used: Release Points B, G, P. Time is EST. Turtle numbers at lower left. H is home direc- tion and distance to home. W is direction and total distance turtle went before being picked up. Second distance indicates the length of the last direction headed if there was a variation. If only one distance is shown the course was a straight line. 340 EDWIN GOULD SUNNY 50 Head ings 22 Turtles N OVERCAST 32 Head ings 17 Turtles PARTLY CLOUDY 40 Head ings 2 \ Turt les ORIENTATION IN TURTLES 341 mobile or bicycle. Between experiments they were kept in a small storage shed or in a pit dug in a woodlot. While in captivity they were well fed with fruits, vege- tables and cockroaches, and allowed to wallow in muddy water when they pleased. Observations were usually made during the morning and late afternoon because of the objectionable effect of heat. On very hot days the turtles would immediately head for the nearest shade or would burrow into a form. OBSERVATIONS Typical behavior after release In Figure 1 are shown two typical cases in which the headings of two turtles in clear weather were measured several times for about three-quarters of an hour after release. No. 14 started walking immediately after being set free, and No. 69 moved off after only two minutes' delay. No. 69 was returned to the same release point immediately after the heading shown in the figure, and it was observed to walk over a very similar course. No. 14 had been faced away from home, but it turned within 15 minutes and then continued in an essentially correct course until the observation was terminated. The heading chosen by a particular turtle was thus consistent from moment to moment, and was also very similar in most cases when the experiment was repeated on the same or on different days. On seven occasions a turtle was moved about 300 feet after it had indicated a definite heading, and in no case was there a significant change in the direction the turtle continued to head. To determine whether local factors unrelated to the home direction were guiding the turtles, a total of twelve experiments were performd in which two animals from quite different home ranges were released at the same time and at the same release point (often within ten feet of each other). The turtles were so selected that the homeward direction would be 90 to 180 degrees different for the members of each such pair. If some local factor were responsible for their initial headings, both should be more or less similarly affected ; if the direction of home was the important factor, they should separate. The results of these experiments are shown in Figure 2. While not all headings were perfectly correct, there is no doubt that each ani- mal selected the approximate direction of its own home independently of the direc- tion chosen by the other member of the pair. The importance of clear skies As soon as these observations of homeward headings of box turtles had been re- peated on several different days it became obvious that when the sun was clearly visible the headings were far better than when the sky was partly or wholly overcast. The results of the whole season's experiments under varying weather conditions are presented in Figure 3, which shows that the initial headings of these turtles are at least as accurately directed toward their homes as were those of pigeons and Manx shearwaters studied by Kramer and Matthews. The upper graph of Figure 3 con- FIGURE 3. Initial headings of box turtles (plotted as deviations to the right and left of the homeward direction) under sunny, overcast, and partly cloudy skies. Most of the seventeen tur- tles in the middle graph were released at two different release points with similar homeward di- rections under sunny skies. The small circle to the right of the upper graph indicates the distri- bution of the forty-one different homeward directions which were used in sunnv-weather releases. 342 EDWIN GOULD tains the observations of twenty-two turtles which in most cases were tested at two or more release points as described above. The actual homeward directions were widely distributed around the compass. As has already been made clear in Figure 2, these headings represent in most cases a definite choice of the homeward direction. It is of great interest that these accurate homeward headings largely disappeared under partly or completely overcast skies, for this at once indicates that the turtles were choosing the home direction by some form of celestial navigation similar to that studied in birds by Matthews, Kramer, and others. These releases were made at distances varying from 0.28 to 5.6 miles away from the place where the turtles b. c. d. 31-8/1653-1706 26-6/0615-0700 27-6/0630-1000 29.7/0525-0730 Turtle 9 No. 3 1 1 E 0° 180° 270° 0° 90° Hia ale — T 35m i G Id ' 1 1 1 1 b a. b. 29-6/0630-0730 10-7/1830-2000 "M Field S P I] i ill H? 1.64m .S3.95L a b m TEN DAYS • n . i i Homed 5-10/0700-1300 1 'HJ0.46m Plowed,Field , fl t : S w N a. b. c. 22-8/1 74 1-I8J5 22-8/1655-1740 28-8/1652-1755 Turtle 27 No. 69 r 2 1 1 V '0° 0° 90 0 180° 270° Mi. 70m p - 1 2 1 1 b a* b. c. d. 9^9/1120-1140 3- 10/12 15-1 228 18-9/1 1 30-1145 19-9/1 120-1152 : 4 i i HT0.55m !d G" * 3-10/^250-1315 ~T"~ i nio.35m Plowed Field y N E S W 24-9/1750-1 803 Turtle 9 No. 23 1 1 E 0° 180° 270° 0° 90° H TO.ISm [1 See Text 24-9/1807-1822 i r i See Text b 1 l : s W N E 9-lO/l208-\2\9 Turtle 9 No. 100 1 1 1 E 0° 180° 270° 0° 90° Hi b,70m ' LJL . s. 1 1 1 9-10/1223-1230 AirStrip i H! 0.30m J , • 9-10/1 235-1 245 ~r "RRYard H-0'.32|m . . r : s W N E 19-10/1145-1203 Turtle 2" No. 102 V ro° o° 90° 180° 270° i i f4o.43m I, ? 1 1 19-10/1220-1250 l-l'J- 0.61m' V N E S W 19-1 0/1 145-1204 Turtle ( No. Ul 1* 1 )° 90° 180° 270° 0° •HI**. ; 1 m 1 19-10/1220-1250 " Air Strip . J * M E S W N FIGURE 4. Type III orientation illustrated by graphic comparisons of turtles released from areas having different homeward directions. Release points: G, P, M field, Plowed field, RR yard and air strip. H is homeward direction. Distance from home in miles. Date and time in left column. 344 EDWIN GOULD had been picked up. In cases where turtles were released at short distances from home, the inability to orient in cloudy weather supported other indications that the turtles did not recognize terrain features from past experience. Alternating sunny- and cloudy-weather releases at irregular intervals resulted in the expected correct orientation in sunny weather and in poor orientation in cloudy weather. However, one turtle (No. 3) was released five times from the same place in sunny weather before it was able to head correctly in cloudy weather on the fifth release. The same result was achieved with another turtle (No. 30) under similar conditions. It may be noticed in Figure 3 that turtles released in cloudy weather headed in the direction opposite from home more frequently than would be expected by chance. Turtle 9 0° 180° 270° 0° 9O° 1-8/0645-0735 No. 30 1 dto.42m ' ,1 . 6 •' 1 I -8/1 500-1 540 I I ' .1 . HT 1.46m - 1 , P E : s w N E Turtle 9 0° 180° 270° 0° 90° a. 1-8/0745-0835 b. 8-9/0730-0755 No. 34 H.lo.3lm l_Jh_^J i b G -' Q • 20-9/1620- 1640 b. 1 -8/1500-1540 1 T | " °Illlb . H'il.SIm , P ]' 22-9/1628-1645 r ' IT Scout field H-l.05ri 1 E : s w N E >^^M-~MVM^H^KM^^Bi^B^»t^^_^M^K_lMHWMH,^^_nn_B_^K_aIK Turtle 27 0° 0° 90° 180° 270° 3 -8/1 725-1751 No. 49 1 HJI.40m : , i. G " i 8 -8/O730-0850 1 'HJS.Km ' , 1, r> i V / N E s w ORIENTATION IN TURTLES 345 22-9/0615-0638 Turtle C No. 84 1 f f 0 90° 180° 270° C )° 1 Hll.08m ,1 , , 6 " 22-9/1628-1645 Hl6.8Om Scout Field il i i 4 E S W N 18-9/1230-1247 Turtle 27 No.86 1 1 V '0° 0° 90° 180° 270° HTLOSm ' 1 . , a ' 22-9/1628-1645 1 H|6.80m ' " Scout Field i i II i V N E S W 22-9/0615-0638 Turtle l8 No.87 i 0° 270° 0° 90° 180° Wl-OSm • 11 . ,6 1 1 22-9/1628-1645 ' HJ 6.80m n Scout Field" i i > W N E S 15-10/1206-1244 Turtle c NoJOT 1 t )° 90° 180° 270° 0° Hlo.SOm1 " , J . a " 1 1 1 9-10/0835-0927 H-dWm "Air Strip , J 4 E S W N FIGURE 5. Type II orientation illustrated by graphic comparisons of turtles released from areas having different homeward directions. Release points : G, P, Scout field and air strip. H is homeward direction. Distance from home in miles. Date and time in left column. 346 EDWIN GOULD Others had a strong tendency to go in the direction they were faced when first re- leased, and in most cases this was opposite to the homeward direction. Facing the turtle in different directions in sunny weather had no effect, although very active turtles which move off immediately upon being released will often start in the direc- tion faced and then veer off to the homeward direction after a short distance. Turtles released under overcast skies showed other differences in behavior from those released in sunny weather. Eight turtles released in cloudy weather walked in circles ranging from seven to thirty feet in diameter. In a single case one turtle actually retraced its track three times in a circle seven feet across. One very obvi- ous difference was the hesitancy of the turtle immediately after release in cloudy weather as well as the shorter distance traveled and the general inactivity. The rate of movement was computed for sixteen turtles released twenty-four times in cloudy weather and twenty-eight times in sunny weather. The mean for cloudy weather was 3.2 feet per minute and the mean for sunny weather, 5.8 feet per minute ; stand- ard deviations were 1.6 and 2.5 ; variations (sd/x) were 50% and 43%, respectively. Evidence for Type II and Type III orientation Griffin (1952) distinguishes three types of orientation used by homing birds. Type I is a reliance on visual landmarks within familiar territory and the use of exploration or some form of undirected wandering wrhen released in unfamiliar ter- ritory. Type II is homing in which an animal goes in a certain direction even when it is carried into unfamiliar territory in a new and unaccustomed direction. Type III is the ability to choose the approximately correct direction of its home even when it is carried into unfamiliar territory in a new and unaccustomed direction. All the relevant data concerning individual turtles released from different home- ward directions have been illustrated in Figures 4 and 5. Nos. 3, 23, 69 and 100 in Figure 4 are the best examples of Type III orientation. All but two headings of No. 3 were well-directed toward home. At Release Point G on 29 July an incorrect heading of 0 degrees closely resembled the previous heading, 348 degrees, at Release Point P. This appears to be Type II orientation. In the second example, M field, 3.95 miles from home, had almost the same homeward direction as Release Point P. In the third example shown, No. 3 returned to its home after ten days or less. Taking into account the rugged terrain the turtle had to traverse, the difficulty was considerable. Deep ditches, logs and dense mats of honeysuckle presented numer- ous obstacles. Most of the headings of turtle No. 69 demonstrate good Type III orientation except for the 9 September record at Release Point G, which closely resembles pre- vious releases at P, apparently another example of Type II orientation. On 2 July, No. 23 was captured about three miles south of the pit where it was kept during the period of experimentation until the last week of August, when it escaped. On 24 September it was found only forty feet from the pit. On the as- sumption that it had made this place its new home, it was placed in a bag and taken to a large clearing 0.15 mile away. While walking to this release point I rotated and spun the turtle in various directions so as to remove any possibility of its re- membering the movements. It was in my pocket for the remainder of the short journey. Upon release, No. 23 headed accurately toward the spot where it had just been recaptured. Following a similar procedure, it was again released from a point having a homeward direction 72 degrees different from the previous release ORIENTATION IN TURTLES 347 point. Again the heading was toward the spot where it had just been recaptured. It started in the direction it had proceeded from the previous release point, but after a short distance it headed correctly. Neither the previous direction nor the home- ward direction corresponded to the direction the turtle was faced. No. 100 was released from three different release points within a period of thirty- seven minutes ; all headings are supportive evidence for Type III orientation. Figure 5 is a compilation of records of turtles released from different homeward directions which showed evidence of Type II orientation or of an ambiguous type which may have been Type II or Type III. The others in this figure are less obvi- ous but complete the collected data on this subject. Another instance of Type II orientation was demonstrated by ten releases of eight turtles from New Jersey and Massachusetts, 180 and 400 miles, respectively, from the home grounds. In six out of ten cases the headings closely resembled the same directions taken when last released from points approximately one mile from home. The differences between directions headed from New Jersey and Massachu- setts release points and those at close distances were 0, 3, 3, 10, 14, 17, 38, 43, 148, 155 degrees. DISCUSSION While the turtle is moving on its course the head is held high and is directed forward so that if it is walking in a direction opposite to the sun's position it is nec- essary for the turtle to turn its head in order to see the sun directly. This very thing was frequently observed. The turtles would stop, look about them for thirty seconds to a minute or more, and then continue on their way. These stops were often made every two to four minutes, varying considerably with the individual turtle. Many turtles showed ability to maintain a straight course. One, for ex- ample, traveled 200 feet and kept within three degrees of a straight line for twelve minutes. Seven turtles released before sunrise moved but little until the sun was visible. Turtles released just before sunset either stopped after the sun had set or changed to a wrong direction after having followed the correct course while the sun was visible. Twelve turtles were recovered after escape or release from a pit where they had been in captivity. Four turtles actually homed from distances of 405 to 563 yards. One returned to the pit from another release point after having been in captivity in the pit most of the summer. The remainder were picked up at points which were nearly in direct line with the homeward direction from their starting point. One of these had traveled 458 yards in less than six days in a presumed line which was only nine degrees from the homeward direction. The distance to home was 4.47 miles. In some preliminary experiments turtle headings were recorded under natural sunlight and then observed in the shadow of a person or tree when the sun was re- flected onto them from a mirror placed about 180 degrees from the sun's position. In all of twenty observations using ten turtles they changed their courses signifi- cantly when the sun's image fell upon them. In nearly all cases the turtles headed toward the mirror. Furthermore, in all cases the headings which were initially taken in the sun were resumed after the mirror was removed and the turtle was re- turned to natural conditions of direct sunlight. The exact meaning of these obser- vations remains obscure, but they do support the hypothesis that it is the sun which enables these turtles to exhibit Type II and Type III orientation. 348 EDWIN GOULD SUMMARY 1. An investigation was made to test the hypothesis that box turtles [Tcrrapcne c. Carolina (Linnaeus)] employ a means of sun orientation similar to that found in birds. Box turtles from different localities were taken in closed containers to un- familiar territory and released in large open fields 0.28 to 5.80 miles from their homes. (According to Stickel the home range is about 300 feet in diameter.) They were then observed over periods varying from ten minutes to two hours, and with the aid of a compass their headings during that period were plotted, and the distances traveled were paced off. After the observation they were again placed in closed containers and returned to a pit where they were kept until the next release. 2. Of forty-three turtles released and observed in this manner, twenty-two headed toward home ; seventeen of the latter were released under sunny and over- cast skies and in most cases this was done from at least two different release areas. Homeward headings were observed in sunny weather, but under overcast skies ori- entation broke down (Fig. 3). Twelve examples, two turtles each, of situations in which sixteen turtles with opposite homeward directions headed toward their respec- tive homes at the same time from the same place, demonstrated that the heading was not dependent on some local environmental factor at the release point (Fig. 2). 3. Of sixteen turtles released from several completely different homeward direc- tions, four showed definite ability to orient correctly (Fig. 4). 4. There were ten releases under sunny skies more than 150 miles from home. Nine turtles were used in these experiments. In seven of these headings the turtles went in a direction which seemed to correspond to the direction last chosen when close to home, regardless of the actual homeward direction. At short distances of a mile or less from home several turtles also headed in consistent direc- tions regardless of the homeward direction. 5. Ten turtles in twenty experiments were first observed for directional heading in natural sunlight, and then observed in the shade while the sun's image was re- flected upon them writh a mirror. The direction of heading was altered in all cases and usually resulted in the turtles' heading for the mirror. 6. While walking, turtles stopped frequently and turned their heads as though looking at the sun. 7. These findings seem to support the hypothesis that turtle orientation resembles the type of orientation found in birds ; however, more data are necessary to clarify numerous factors which may bring to light conflicting differences and close resem- blances. Work is continuing and new developments will be reported as they are observed. LITERATURE CITED BREDER, R. B., 1927. A new technique for studying the life habits of certain Testudinata. Zoologica, 9 : 231-243. CAGLE, F. R., 1939. A system of marking turtles for future identification. Copeia, No. 3 : 170-173. CARR, A. F., JR., 1952. Handbook of Turtles. Cornell University Press, Ithaca, New York. GRIFFIN, D. R., 1952. Bird navigation. Biol. Rev., 27 : 360-393. KRAMER, G., 1953. Wird die Sonnenhohe bei der Heimfindeorientierung verwertet? /. Orn., 94: 201-219. MATTHEWS, G. V. T., 1955. Bird navigation. Cambridge at the University Press. NICHOLS, J. T., 1939. Range and homing of individual box turtles. Copeia, No. 3: 125-127. STICKEL, LUCILLE F., 1950. Population and home range relationships of the box turtle, Terra- pene c. Carolina (Linnaeus). Ecol. Monog., 20: 351-378. AN EXAMPLE OF REVERSAL OF POLARITY DURING ASEXUAL REPRODUCTION OF A HYDROID CADET HAND AND MEREDITH L. JONES Department of Zoology, University of California, Berkeley, California A number of small and curious hydroids have been described over the years. These hydroids, in general, occur on soft bottoms as members of the fauna of the upper few millimeters and are of rather diverse asexual reproductive habits. This group, most of which are unrelated systematically, includes Boreohydra, Psammo- hydra, Corymorpha, Heteractis, Euphysa (the last three closely related), Halermita and Microhydra. In view of the apparently wide distribution of this type of hy- droid in soft bottom communities it is probably not surprising that still another form has been discovered in San Francisco Bay, California. On November 28, 1954, we recovered two live specimens of such a hydroid from a core sample 18 mm. in diameter taken at a depth of 30 feet off Pt. Richmond. The bottom at that location consists of grey mud overlain by about 5 mm. of loose debris, from which the hydroid to be described was taken. According to Sumner et al. (1914) the annual salinity in this area ranges from 18.0%o to 32.3%0 with a mean of 27.0%o. MORPHOLOGY AND REPRODUCTIVE BEHAVIOR The two specimens were a single polyp and a pair of polyps united at their base. Each had a small patch of debris adherent to its base. Figure 1 illustrates the pair. The polyps were quite extensible and were about 1-1.5 mm. long when maximally extended. The hydrocaulus was narrowest at the base and gradually enlarged to- ward the area of tentacular insertion, where the diameter was approximately twice that of the more proximal region. The tentacles were filiform and were capable of extending to about 1 mm. They were inserted in a single cycle at the base of the proboscis. The number of tentacles varied from as few as 4 to as many as 12, al- though, of the original specimens, the solitary individual had 6 and the pair had 9 and 10 at the time they were first observed. Due to the extreme contractility and extensibility of the hydroid, the relative length of the proboscis and the hydrocaulus was quite variable. In general, however, this relationship varied from 1:1 to 1:3 (i.e., with the hydrocaulus about three times the length of the proboscis, at times). The color of the hydroids was a light, flesh-pink, and the whole animal was translucent. The two original specimens were set aside in a stender dish to which was added a small amount of debris from the core sample. The specimens were observed at least once a week following this and were fed the nauplii of brine shrimp (Artemia) at each observation. Early in January it was noted that there were two pairs of polyps plus the single one in the culture dish. At the time it was assumed that the extra pair had been overlooked at the time of isolation. However, on January 24,, 349 350 CADET HAND AND MEREDITH L. JONES FIGURE 1. The original paired hydranths. The individual on the left has elongated preparatory to undergoing asexual division. FIGURE 2. A polyp pair in process of asexual division. REVERSAL OF POLARITY IN A HYDROID 351 _ F V D F •#% E V F A ¥ B 18 Jan. FIGURE 3. A schematic diagram showing results of observations on critical dates from January 18 to February 15, 1955. 1955, it was observed that a polyp of one of the pairs had become about twice the length of its partner and possessed two additional sets of tentacles, intercalated be- tween the base and the original whorl of tentacles, and further, at a point between the new tentacles and old hydranth, debris had collected and was adherent to the hydrocaulus (Fig. 2). Daily observations following this made it clear that we were witnessing an unusual mode of asexual reproduction which resulted in the produc- tion of pairs of polyps. By January 27, fission had occurred between the two new sets of tentacles, and the area of adherent debris had become the base of the new polyp pair. Pursuant to this discovery we began a series of daily observations ex- tending from January 24 to February 15. For convenience in record-keeping, letters were assigned to the polyps (these letters will be referred to in the text, where necessary), and the accompanying figure (Fig. 3) illustrates, in a diagrammatic fashion, the results of our observations from January 18 to February 15. At this time there were four pairs and a single polyp. The polyps "F" and "D" were the only polyps to divide during this period, and one of these probably gave rise to polyp pair "A-B." In each instance the elapsed time between the first appearance of new tentacles and actual divisions was three days. Following February 15 our observations became somewhat irregular, and a single observation on March 4 revealed that we now had seven pairs of polyps and that two of the pairs were preparing to divide once more (the single specimen had been sacrificed previously for nematocyst data). Immediately following this obser- vation, the dish containing the hydroids became badly fouled (presumably as a re- 352 CADET HAND AND MEREDITH L. JONES suit of over-feeding with Artemia larvae, and our subsequent failure to change the water), and on March 8, when this was discovered, the remaining hydroids were in what appeared to be very poor condition. Each polyp was tightly contracted to about one-third its normal height and was about 0.5 mm. in diameter. The ten- tacles were mere knobs and the animals had assumed a nearly hemispherical form. The water was changed, but this failed to save most of the specimens and by March 15 we were left with but a single pair of polyps. FIGURE 4. The monstrosity on March 25. On March 21 we observed that our remaining pair had once more produced two new polyps, but although these were well-formed, they still had not separated. On the next day (March 22), although the two new polyps had not yet parted, there were signs that still another pair of polyps was being formed, giving us six polyps in series, and this specimen was apparently becoming a monstrosity. We also noted that near the middle of the central hydrocaulus there was a single protuberance and a group of four structures which looked like still another pair of incipient polyps with irregularly placed tentacles. By the next day (March 23) our specimen had opened another mouth between a pair of orally-directed polyps and we could now count 12 sets of tentacles and 4 open mouths. By March 25 the animal had grown REVERSAL OF POLARITY IN A HYDROID 353 to about 4.5 mm. long (Fig. 4), was composed of 14 recognizable individuals and had 8 mouths which seemed to be functional (in that they were seen to open and close and brine shrimp larvae were ingested by several). On March 28 the specimen had finally succeeded in dividing into pieces, and we were able to count two single polyps, two separate double polyps, one triple polyp (three mouths and sets of tentacles with a single base), one quadruple (four mouths, three sets of tentacles and a base), and two groups of polyps which had three and four mouths each. These last two groups consisted of rather curious assemblages of mouths, misplaced tentacles, and bits of adherent debris (suggesting bases) at irregular points over the masses. This situation, that is, with eight variously assorted hydroids ranging from single individuals to groups of four, was maintained without further change until April 4. Following this date, the numbers were reduced by death, and by June 1, all the in- dividuals had died. As stated above, during the earlier part of our study, the original single polyp (polyp "G," Fig. 3) was sacrificed to study its nematocysts, and, unfortunately, none of the polyps or polyp pairs was preserved. Although we have taken more than 1200 core samples from the general area of the original collection, none have been found since. DESCRIPTION OF ASEXUAL REPRODUCTION The sequence of events leading to the production of new pairs of polyps in this hydroid has been closely observed. The earliest sign that the process was underway FIGURE 5. Development of tentacles prior to asexual division. A, the polyp pair with new tentacles barely recognizable (February 14) ; B, tentacle growth in 23 hours (February 15) ; C, tentacle growth in 40 hours (February 16). 354 CADET HAND AND MEREDITH L. JONES was the adherence of a small bit of debris to the hydrocaulus at about its mid-point, although this was commonly nearer the tentacles than the base (Fig. 5, A). This phenomenon was associated with a slight increase in the opacity of the tissues at this point, and the protrusion of a small knob of tissue. This knob within 24 hours of its appearance became completely obscured by adherent debris. In addition, during this first phase of development, two sets of minute protuberances were seen in the mid-region of the hydrocaulus, between the adherent debris and the original base (Fig. 5, A). Within 24 hours, the protuberances grew into recognizable ten- tacles, usually three or four per whorl (Fig. 5, B). With the appearance of the tentacles, there is a marked increase in the opacity of the tissue in this area. During the second 24-hour period, a translucent region developed in the area between the two new sets of tentacles, and subsequently, there was an indenting of the surface tissue of the hydrocaulus at this point. On the third day, there was a further continuation of the constriction of this area, leading to the actual separation of the two new polyps. This point of fission marked the distal end of the proboscis of each of the twTo new polyps, and each polyp possessed a functional mouth as soon as fission was completed ; in addition, the point of adherent debris had now become the base of the newly released pair. The result of this process of fission was two pairs of polyps, each with one member (the older) about one-third larger than the new polyp. DISCUSSION Many well-known studies have established the fact that hydroids possess gradi- ents, particularly those of regenerative ability, which are more marked or rapid in more distal pieces of stems. Steinberg (1954), in studies on Tiibularia, found that short pieces of stem (1-2 mm. long) regenerate bipolar or partial bipolar hydranths, while in longer pieces (2-12 mm.) a new hydranth is usually formed only at the original distal end of the stem, and, in still longer pieces (12-30 mm.), each end re- generates a hydranth. Steinberg also describes movements of cells toward the dis- tal ends of cut stems and states that these cell movements precede the process of regeneration. In the hydroid described above, since the whole animal is less than 2 mm. long, one wonders if there can be effective or actual gradients such as those obviously present in larger hydroids. However, numerous studies on Hydra, an equally small organism, have shown that well-developed gradients also exist in small hydroids. In the animal described here, the appearance of two new orally-directed hydroids interposed in series between an extant base and hydranth must mean that a re-organization, involving a reversal of the original polarity of the hydrocaulus, has occurred, and it would seem that information obtained from experimental studies already completed on other hydroids should help to explain this observed event. It should be pointed out that the reversal of polarity, which we assume to be present during the course of asexual reproduction, is a unique event. Hyman (1940, pp. 487-492) has reviewed much of the literature to that date relative to asexual reproduction and regulative processes in hydroids. She points out that in animals as small as hydras the polarity is wrell-developed and can only be altered by rather drastic treatments, i.e., electrical currents, burial of the apical end of a cut stem, or by treatment with cyanides, anesthetics, and other depressing chemicals. Further, it is well known that pieces of hydroid stems and hydras retain their origi- nal polarity whether free or grafted to other pieces. We assume, therefore, that our REVERSAL OF POLARITY IN A HYDROID 355 animal must also have a definite basal-oral polarity ; that in the course of asexual reproduction by this animal, the polarity is reversed, at least for a short part of the hydrocaulus ; and that this is a naturally-occurring and unusual event. After considering all the evidence available to us, we have come to the conclu- sion that we can explain our observations on this hydroid if we are allowed to make one hypothesis. This hypothesis is concerned with the first event we observed during the course of asexual reproduction (i.e., adherence of debris to the hydro- caulus) and is unique to the extent that it gives certain unusual features to the pre- sumptive base of the new polyp pair. We suggest that the appearance or formation of the new base, as the first step in asexual reproduction here, must represent an effective block in the existing gradient, and that this new base must compete (in a certain sense) with the old base for the hydrocaulus between them. Since it is well- established that distal portions of hydroid stems form new hydranths, we can now consider that the portion of the stem between the bases is distal to each base and that it proceeds to develop two new orally-directed polyps simultaneously. In other words, we believe that the two bases with the common piece of hydrocaulus between them, are, individually, behaving in the same fashion as would pieces of decapitate stem, i.e., that there will be regeneration of a new hydranth at the distal extremity. This, of course, must involve a reversal of the preexisting gradient in the piece of hydrocaulus closest to the newly formed base, and we might postulate that this is the result of some disorganizational or re-organizational power of the base. To the ex- tent that we observed a condensation or increasing opacity in the tissues at the points of formation of the two new polyps, our observations seem to fit the known facts, such as those presented by Steinberg (1954). If, therefore, our hypothesis is ac- ceptable, we can interpret asexual reproduction in this hydroid as a phenomenon involving regeneration at distal extremities. Cases of reversal of polarity do not seem to have been reported in nature for any other hydroids, unless we can interpret the study of Rees (1937) on Hetcrostepha- nus (now Heteractis aurata according to Kramp, 1949) as such. In H. aurata the polyp bud develops from the side of a hydranth, usually a tentacle base, in such a fashion that the free end of the bud is the future hydrocaulus. This same phe- nomenon has been observed by us in a similar hydroid (E\tf>h\sa sp.) from San Francisco Bay. In all other cases where transverse fission occurs, a new polyp de- velops at the distal end of the remaining hydrocaulus, and the new free individual develops a base at the proximal end of its own hydrocaulus. In one genus, Psam- mohydra, Schulz (1950) has found that the new hydranth develops tentacles before fission actually takes place. Recently Kinne (1956) has made observations on Cordylophom caspla, an athe- cate hydroid from fresh and brackish water. He noted that in approximately 20% of polyps adapted to a salinity of 24%c , there were various abnormalities developed. These consisted of: the formation of intercalary hydranths on a hydrocaulus ("uni- axial hydranth aggregates") ; fusion of polyps ("multiaxial hydranth aggregates") ; the formation of large coenenchymal bodies, which became detached from the colony and formed new hydranths; and "globular hydranth complexes," with no stolons and no stalks. The formation of the intercalary polyps is remarkably similar to the phenome- non observed in the hydroids from San Francisco Bay. However, there are points of difference, for in Cordylophora, the two new polyps are joined by the sides of the 356 CADET HAND AND MEREDITH L. JONES hypostome and the mouths are directed laterally, the original polyp is a member of a colony, and there is no interruption of the hydrocaulus between the old and new polyp head which might correspond to the new base of our local forms. Kinne mentions that when the hydranth groups are detached they frequently become estab- lished on the substrate and sprout new stalks, but no intimation is made that these persist as pairs, nor that further intercalary hydranths are formed. In an early section of this report we described a monstrosity which developed from a pair of polyps following their recovery from a period of depression. Hyman (1940) has reviewed the general effects of depression on hydroids, and notes that after recovery from depression, hydras often regenerate doubled parts or other anomalies, and that this is followed by fission processes. Kinne explains his vari- ous anomalous forms as the results of disturbances of growth and differentiation processes and a consideration of our observations leads us to evaluate our mon- strosities in the same light. However, since we noted some six apparently normal divisions, culminating in seven polyp pairs, and since the original pair was taken in the field and had not, so far as we know, been exposed to abnormal environmental conditions, we reserve judgment as to whether or not this process is abnormal, for this organism. The possibility does exist that the unknown hydroid, upon which we based our observa- tions, is actually a Cordylophora, for this genus has been collected at the Carquinez Straits, approximately 15 miles up the San Francisco Bay Estuary from Point Richmond. SYSTEMATICS We have little concrete information to guide us in the identification of the hy- droid we have described. Our study of the nematocysts revealed that it possesses a simple cnidom of desmonemes and eury teles, as follows : Small desmonemes (common) 4-5 X 3-4 /j. Large desmonemes (rare) 9-10 X 5-4 n Microbasic euryteles (common) 9-1 1 X 3-4 yu This cnidom suggests that the hydroid is a member of the gymnoblasts (antho- medusae) or limnomedusae (see Weill, 1934; Russell, 1938; Hand, 1954). The complete absence of perisarc also suggests that the hydroid may be a limnomedusan form. However, the perisarc apparently may be completely absent in some gymno- blasts and calyptoblasts. We also know that differences may occur between the cnidom of a hydroid and that of its medusa (Hand, 1954), but in this hydroid, we do not know what medusa, if any, is involved in the life history. Therefore, we can not place this hydroid in any order with certainty, although it seems clear that it can not be a calyptoblast. Because of the proximity of a source of Cordylophora, the possibility of the un- known form's being an abberant type of Cordylophora was explored. The main points of difference are that Cordylophora grows in extensive colonies, its tentacles are scattered, and it possesses a well-developed perisarc. None of these characters applies to the form described here. In addition, Hand and GwTilliam (1951) re- ported only a single size-class of desmonemes for Cordylophora, while we have found two size-groups. Thus, it seems doubtful that this is actually a growth vari- ant of Cordylophora. REVERSAL OF POLARITY IN A HYDROID 357 Since so little can be done with the hydroid from the standpoint of systematics, we will refrain from assigning it the status of a new species, pending its further collection and a more exact taxonomic determination. The authors wish to acknowledge the assistance of Mrs. Emily Reid, to whom we are indebted for the illustrations accompanying this report. SUMMARY 1. Asexual reproduction, involving reversal of the original oral-basal polarity, is described. 2. Asexual reproduction in this hydroid leads to the production of pairs of polyps sharing a common base. 3. The systematic position of the hydroid is not established, although the cnidom suggests possible affinities among the gymnoblasts. LITERATURE CITED HAND, C, 1954. Three Pacific species of "Lar" (including a new species), their hosts, medusae, and relationships. (Coelenterata, Hydrozoa). Pac. Sci., 8: 51-67. HAND, C., AND G. F. GWILLIAM, 1951. New distributional records for two athecate hydroids, Cordylophora lacustris and Candelabrum sp., from the west coast of North America, with revisions of their nomenclature. /. Wash. A cad. Sci., 41 : 206-209. HYMAN, L. H., 1940. The Invertebrates : Protozoa through Ctenophora. McGraw-Hill Book Co., New York. KINNE, O., 1956. Tiber den Einfluss des Salzgehaltes und der Temperatur auf Wachstum, Form und Vermehrung bei dem Hydroidpolypen Cordylophora caspia, (Pallas), Thecata. Clavidae. I. Mitteilung iiber den Einfluss des Salzgehaltes auf Wachstum und Entwick- lung mariner brackischer und limnischer Organismen. Zool. Jahrb., 66 : 565-638. KRAMP, P. L., 1949. Origin of the hydroid family Corymorphidae. Vidensk. Medd. fra. Dansk naturh. Foren., Ill: 183-215. REES, W. J., 1937. On a remarkable process of bud formation in a gymnoblastic hydroid (Heterostephanus sp.). /. Mar. Biol. Assoc., 21 : 747-752. RUSSELL, F. S., 1938. On the nematocysts of hydromedusae. /. Mar. Biol. Assoc., 23 : 145-165- SCHULZ, E., 1950. Psammohydra nanna, ein neues solitares Hydrozoon in der westlichen Belt- see. Kieler Meeres., 7 : 122-137. STEINBERG, M., 1954. Studies in the mechanism of physiological dominance in Tubidaria. J. Exp. Zool, 127 : 1-26. SUMNER, F. B., G. D. LOUDERBACK, W. L. SCHMITT AND E. C. JOHNSTON, 1914. A report upon the physical conditions in San Francisco Bay, based upon the operations of the United States Fisheries Steamer, "Albatross" during the years 1912 and 1913. Univ. Calif. Pub. Zool., 14 : 1-198. WEILL, R., 1934. Contribution a 1'etude des cnidaires et de leurs nematocystes. II. Valeur taxonomique du cnidome. Travaux de la Station Zoologique de Wimereux. Vol. XI. Paris. THE ELECTROCARDIOGRAM OF A STOMATOPOD HIROSHI IRISAWA AND AYA FUNAISHI IRISAWA Department of Physiology, School of Medicine, Hiroshima University, Hiroshima, Japan It is generally accepted that the crustacean heart beat is of neurogenic origin. This has been concluded from pharmacological (Krijgsman, 1952; Prosser, 1942), electrophysiological (Prosser, 1950; Hagiwara and Bullock, 1956) and anatomical evidence (Alexandrowicz, 1932, 1934). Both Limulus and the lobster have been extensively studied, but relatively few studies of other species of crustaceans have appeared in the literature. Alexandrowicz (1934) has described the innervation of the heart of stomato- pods, and showed that there are nerve cell bodies in the elongated ganglionic trunk, which he considers as an automatic apparatus which rules the heart beat. Since the ganglia of the stomatopod are of simpler structure than those of Limulus, a physio- logical study of the heart of the mantis shrimp may be of considerable assistance in explaining the origin of the heart beat of the arthropod neurogenic heart. This paper describes the electrocardiogram of the mantis shrimp and the detection and localization of the pacemakers of this heart. MATERIALS AND METHODS Marine mantis shrimps (Squilla oratorio, de Haan, Crustacea, Malacostraca, Stomatopoda) were used throughout the present study. They were fixed on a cork board, the shell opened dorsally and the preparations placed in a plastic dish filled with sea water. The dorsal muscles were dissected and removed to expose the long segmented tubiform heart which extends from the posterior to the thoracic part of the body cavity. Since the heart is closely attached to the digestive tract the ex- periments were carried out without isolation of the heart from the body. The preparations were decapitated at the level of the fifth segment, to avoid central nervous effects and suppress muscle activity. The indifferent electrode was placed remotely from the heart in the sea water, while the recording electrode was moved with the aid of a micromanipulator and microscope along the median line of the exposed heart. Low resistance micro- electrodes (tip diameter 10-15 /A), originally described by Tomita and Funaishi (1952), were used. The surface action potentials were amplified with a condenser- coupled amplifier, displayed on an oscilloscope and photographically recorded. When longer recordings were needed a smoked paper electrometer (Hatakeyama, 1954) was employed. RESULTS Structure of the heart. The heart consists of fourteen segments each of which has a single nerve cell, a pair of ostia and arteries. Except at the proximal and 358 THE ELECTROCARDIOGRAM OF A STOMATOPOD 359 distal ends of the heart tube, the single nerve cells of each segment lie individually in the median nerve trunk behind the ostial orifices. Alexandrowicz (1934) previ- ously pointed out that the size of the nerve cell varies with the segment. In this study, on the basis of thirteen preparations, it was found that the thirteenth segment always contained the largest cell (average value: length 80 p., width 63 /*,). There is a progressive decrease in the size of the nerve cells anterior and posterior to the cell of the thirteenth segment. The major components of the electrocardiograms. When the recording elec- trode was placed on the surface of the peripheral part of the heart, relatively slow triphasic action potentials were observed as shown in Figure 1 A. As can be seen FIGURE 1. Surface action potentials of the heart of the mantis shrimp. A. Record obtained at some distance from the nervous trunk. Small rhythmic waves and small spikes can be seen. B. Record from the central part of the heart tube, characterized by a train of spike potentials and the slow muscle potentials. C. When the contraction of the heart is small or absent, only a train of spike potentials is recorded. T. Time : 60 cycles per second. in this figure, the action potential usually reached its summit within 8 msec., and thereafter exhibited a slow exponential decay. However, when the recording elec- trode was placed on the mid-dorsal region of the heart, where the ganglionic trunks are located, a train of very rapid spike potentials was superimposed on the slow wave (Fig. IB). When the contraction of the heart tube was small or absent, only the spike train was recorded (Fig. 1 C). The spike components as observed in Figure 1, B and C had extremely brief durations not exceeding one millisecond. The height of these spikes remained relatively constant in the same preparation. However, attenuation of spikes was observed when the electrode was moved away from the median nervous trunks, as shown in Figure 1 A. The number of spikes in each heart beat varied widely from preparation to preparation, ranging from one 360 HIROSHI IRISAWA AND AYA FUNAISHI IRISAWA to about sixteen and averaging 7.2 spikes per heart beat in twenty-eight prepara- tions. The number of spikes seemed to decrease with the duration of each heart beat. The findings are compatible with the concept that the spike component arises from a stimulating discharge of the large single nerve cell of the heart segment and the slow potential from the heart muscle. Location of pacemaker. Transection is a valuable method for the detection of pacemakers in this type of heart because of its segmentation. When a part of the heart muscle was dissected, no remarkable change in action potentials or heart rate was observed ; however, when the nervous trunk was incised, definite changes oc- curred which varied according to the place of transection. It was not possible, how- ever, to transect the nerve trunk without injuring part of the heart muscle. TABLE I The influence of successive isolating transverse sections upon the rate of contraction of the segments so isolated Rates of various isolated segments after sectioning anterior to segment thirteen Preparation Rate of heart number before sectioning* Segment number 13 12 11 10 1 84 84 52 30 18 2 96 96 42 16 t 3 48 48 42 18 t 4 60 60 24 t t 5 60 60 30 t t 6 84 84 48 t t 7 54 54 30 14 t * Figures in the table are rates in contractions per minute. t Contractions stopped. Sections were made serially between successive segments starting with a cut between numbers 12 and 13. Following the cut, a recording of the rate was made and the process repeated with the next segment. Transection of the heart nerve trunk, starting with a cut anterior to segment thirteen, showed that the heart rate was reduced in sections anterior to the cut. The results of seven experiments in transection at successive levels are given in Table I. As can be seen from the table, the rate of beating of segment thirteen was unchanged by a section anterior to it but segments anterior to the cut beat at a slower rate. In terms of the rate of the thirteenth segment as 100 per cent, the first cut between seg- ments twelve and thirteen reduced the rate of segment twelve to an average of 56 per cent. The next cut between segments eleven and twelve stopped the beating in three out of seven cases, but in the four that continued to beat the rate was reduced to 29 per cent of the original. The next cut stopped beating in all but one preparation. These results clearly demonstrate that the thirteenth segment, which has the largest median nerve cell, has the fastest rate and governs the rate of the heart beat. THE ELECTROCARDIOGRAM OF A STOMATOPOD 361 DISCUSSION The electrocardiogram of the Stomatopoda resembles that of Limultis (Prosser, 1950) and Astacus (Hoffman, 1912). However, the electrocardiogram of the mantis shrimp differs in that it consists of two clearly distinguishable component potentials : one of muscle and one of nerve. Since the ganglionic trunk in Stomato- poda lies on the surface of the heart tube, pure nervous spikes are obtained. Prosser (1943) demonstrated bursts of impulses from the isolated Limulus heart ganglion. Welsh and Maynard (1951), and Maynard (1955) studied the cardiac ganglion of the lobster and showed that a burst of nerve impulses preceded and ac- companied the early part of the mechanical response. Matsui (1955) found that the number of spikes within one heart beat varied extensively from preparation to prepa- ration, ranging from several to eighty. Maynard (1955) distinguished two differ- ent spike potentials, namely, small and large components in his records. The type of spike-train which was obtained from a median ganglionic trunk of this stomato- pod seems to be simpler than any other pattern of impulses obtained through the surface electrode from other crustaceans, and only comparable with those records from a single nerve cell of lobster (Hagiwara and Bullock, 1955; Watanabe, per- sonal communication). Thus, the uniformity and the simplicity of this record seemed to be due to the simple structure of the ganglionic trunk : this was confirmed histologically (Irisawa and Irisawa, 1956). Since the heart ganglion frequency is unchanged after the removal of the influ- ence of the regulator nerve, the ordinary heart rate is probably regulated by the ac- tivity of the chain of ganglionic trunks. Possibly there is a dominating cell in thp ganglionic trunk that synchronizes heart activity. Maynard (1955) stated that both large and small cells are the pacemaker cells; Hagiwara and Bullock (1955) sug- gested that the posterior small cells may be the pacemaker cells. Our experiment demonstrated a remarkable gradient of the size of these heart ganglion cells and of the frequency of spontaneous firing by the respective segments when isolated from faster segments. Studies have also revealed variation in the sensitivity to a stimulus (Irisawa and Irisawa, 1956). In summation, the large cell of the thirteenth segment is the largest, most sensitive to a localized thermostimulus (Irisawa and Irisawa, unpublished data) and its segment has the highest automa- ticity. It is probable that this large cell of the thirteenth segment is the dominant cell and is the pacemaker in this heart. We wish to thank Prof. Y. Nisimaru for his encouragement, and also it is our privilege to acknowledge our sincere gratitude to Prof. C. A. G. Wiersma, Prof. T. H. Bullock and Dr. L. A. Woodbury for their help in reviewing the manuscript. We are also grateful to the grant of the Ministry of Education of Japan for a part of our apparatus which made the work possible. SUMMARY 1. The electrocardiogram of the tubular heart of the mantis shrimp, Squilla ora- toria de Haan, was studied. Structural findings are described which confirm Alex- androwicz's observation. 362 HIROSHI IRISAWA AND AYA FUNAISHI IRISAWA 2. The electrogram consists of rapid spike components and slow action potentials. The spikes originate from the median nervous system of this heart, and the slow potential from the muscle. 3. The results of transection experiments on the nerve trunk support the view that the nerve cell of the thirteenth segment has a dominant role in the pacemaker activity of the heart contraction. LITERATURE CITED ALEXANDROWICZ, J. S., 1932. The innervation of the heart of the Crustacea. I. Decapoda. Quart. J. Micr. Sci., 75: 181-249. ALEXANDROWICZ, J. S., 1934. The innervation of the heart of the Crustacea. II. Stomatopoda. Quart. J. Micr. Sci., 76: 511-548. HAGIWARA, S.. AND T. H. BULLOCK, 1955. Study of intracellular potentials in pacemaker and integrative neurons of the lobster cardiac ganglion. Biol. Bull., 109: 341. HATAKEYAMA, I., 1954. A simple paper electrometer. /. Physiol. Soc. Japan, 16: 124-126 (Japanese). HOFFMAN, P., 1912. Ueber den Herzschlag des Fluss Krebses mit besonderen Beriicksichtigung des systolischen Stillstandes. Zeitschr. f. Biol., 59: 297-313. IRISAWA, H., AND A. F. IRISAWA, 1956. Pacemakers of the invertebrate hearts. Medical Sci- ence, 7: 241-251 (Japanese). KRIJGSMAN, B. J., 1952. Contractile and pacemaker mechanisms of the heart of arthropods. Biol. Rev., 27 : 320-346. MATSUI, K., 1955. Spontaneous discharges of the isolated ganglionic trunk of the lobster heart (Pamtlirns japonicus). Sci. Rep. Tokyo Kyoiku Daigaku, B, 7: 256-268. MAYNARD, D. M., 1955. Activity in a crustacean ganglion. II. Pattern and interaction in burst formation. Biol. Bull., 109 : 420-436. PROSSER, C. L., 1942. An analysis of the action of acetylcholine on hearts, particularly in ar- thropods. Biol. Bull., 83 : 145-164. PROSSER, C. L., 1943. Single unit analysis of the heart ganglion discharge in Limulus polyphe- mus. J. Cell. Coinp. Physiol., 21 : 295-305. PROSSER, C. L., 1950. The electrocardiogram of Arenicola. Biol. Bull., 98: 254-257. TOMITA, T., AND A. FUNAISHI, 1952. Studies on intraretinal action potential with low resistance microelectrode. /. NeurophysioL, 15 : 75-84. WELSH, J. H., AND D. M. MAYNARD, 1951. Electrical activity of a simple ganglion. Fed. Proc., 10: 145. THE AMINO ACID CONSTITUENTS OF THE PHYCOBILIN CHROMOPROTEINS OF THE RED ALGA PORPHYRA1 RAYMOND F. JONES 2 AND L. R. BLINKS Hopkins Marine Station of Stanford University, Pacific Grove, Calif. The phycobilin chromoproteins, phycocyanin and phycoerythrin of red algae, because of their stability and water-solubility, have been studied physico-chemically by many investigators. They have been shown to have definite molecular weight, characteristic isoelectric points, mobility, diffusion and adsorption properties (Sved- berg and Lewis, 1928 ; Svedberg and Katsurai, 1929 ; Svedberg and Eriksson, 1932 ; Tiselius, 1930, 1937; Swingle and Tiselius, 1951). Their behavior as accessory pigments in photosynthesis is also well established (Haxo and Blinks, 1950; French and Young, 1952; Blinks, 1954a, 1954b; Yocum and Blinks, 1954). A compara- tive study of the chromatographically separated phycobilins of red and blue-green algae has recently revealed the occurrence of individual phycobilin pigments other than the classical varieties (Haxo, O'hEocha and Norris, 1955 ; Tiselius et al., 1956). The former authors also established the presence of allophycocyanin as a natural, (although minor) component of the chromoproteins of several of the red and blue- green algae. The water-soluble chromoproteins of Porphyra naiadum, after sepa- ration by column chromatography and electrophoresis, have been shown to contain an appreciable quantity of allophycocyanin as well as phycoerythrin and phycocyanin (Airth, 1955; Blinks and Airth, 1957). A highly ionized lavender fraction was also found to be present. This behaved as a homogeneous entity and moved as an anion in the electrophoretic cell even at a pH of 5.0 where phycocyanin and phyco- erythrin are nearly isoelectric. Quantitative analyses of crystallized chromoproteins by Kylin (1910), Kitasato (1925) and Fujiwara (1955) show very little difference in elementary composition. Wassink and Ragetli (1952) reported on the amino acid composition of phycocyanin isolated from a species of the blue-green alga Oscillatoria. These authors detected sixteen ninhydrin-reactive spots of which thirteen were identified. The present paper is concerned with the amino acid composition of several chro- matographically pure phycobilin chromoproteins isolated from Porphyra naiadum, Porphyra perforate, and Porphyra Nereocystis, all primitive red algae. MATERIAL AND METHOD The algae were freshly collected from the shores of the Monterey Peninsula, California. P. Nereocystis was found growing epiphytically upon the stripes of the large kelp Nereocystis Luetkeana. P. naiadum was collected from the leaves of the 1 Research supported under contract with the Office of Naval Research (Nonr 120-050). - Present address : Atomic Energy Authority of U. K., Windscale Works, Cumberland, England. 363 364 RAYMOND F. JONES AND L. R. BLINKS flowering plant, Phyllospadix growing at mean low tide. The species P. perforata was collected from the rocks high up in the intertidal zone. Each species occupied, therefore, a different ecological habitat. Extraction of pigments The algae were returned to the laboratory in sea water, then washed twice with distilled water before extraction. In the case of P. naiad urn the washed tissue was covered with distilled water and allowed to stand in the refrigerator for 24 hours at 5° C. (see Blinks and Airth, 1957). With P. Ncreocystis and P. perforata it was found necessary to macerate the algal tissue in a Waring Blendor for two minutes and to allow the macerated material to stand in the refrigerator for 48 hours at 5° C. to obtain maximum extraction. The material was then filtered through cheesecloth and the filtrate passed through Whatman No. 1 filter paper pulp to re- move chloroplasts and other fine organic debris. In each case the clear filtrate, ex- hibiting intense fluorescence, was precipitated with ammonium sulphate. Although the extracted chromoproteins of P. Nereocystis and P. perforata were precipitated with ammonium sulphate at 50% saturation, with P. naiadum precipitation was only complete at 90% saturation. After the purple-red precipitate was allowed to settle out overnight in the refrigerator it was removed by centrifugation, redissolved in distilled water and dialyzed against running tap water for 24 hours at 10° C., fol- lowed by 0.1 M acetate buffer at pH 5.0 for a further 24 hours. The non-dialyzable pigment solution was finally concentrated by pervaporation. Fractionation For the separation of the individual phycobilins the concentrated solution was subjected to column chromatography as employed by Airth (1955). Columns were prepared using one part tricalcium phosphate (dry weight) to five parts of washed Celite filter air. Gentle suction was used in forming the column which was washed with NaCl (1%) followed by 0.1 M acetate buffer at pH 5.0. The pigment extract was introduced onto the column and the individual phycobilins eluted with the ap- propriate buffer solutions, details of which are shown in Table I. Each fraction was further chromatographed on individual columns to ensure complete separation and elution. The phycobilins from P. naiadum were also separated using a Tiselius electro- phoresis apparatus. At pH 5.0 with acetate buffer, the individual chromoproteins were found to be homogeneous. Absorption spectra were determined for each pigment over the range 250-700 m/A using a Beckman Model DU spectrophotometer. The fractions, phycocyanin, phycoerythrin, allophycocyanin and the "highly ionized fraction" from P. naiadum were concentrated by pervaporation and a quan- titative analysis of the amino acids released on acid hydrolysis undertaken. Be- cause of the small amount of P. Nereocystis available at the time of the experiments, only phycocyanin and phycoerythrin were investigated from this alga. Acid hydrolysis The protein fractions were hydrolyzed using a mixture of equal volumes of con- centrated hydrochloric acid and glacial acetic acid containing 4 per cent of stannous AMINO ACIDS OF PHYCOBILINS 365 TABLE I Buffer solutions used in the chromato graphic separation of the various phycobilins Species Phycobilin Buffer Absorption data P. naiadum Phycocyanin 1 .!/ acetate pH 5 X max. 615 m/z Phycoerythrin 1 M acetate pH 5 X max. 545 m^i "Shoulder" 560-565 mM Highly ionized fraction 2 M acetate pH 5 X max. 565 and 615 m/x Allophycocyanin O.I M phosphate pH 7 X max. 650 m/x P. perforata Phycocyanin 1 M acetate pH 5 X max. 557 and 615 m/u Phycoerythrin 1 M acetate pH 5 X max. 495 and 565 m/z "Shoulder" 540-550 mM Allophycocyanin 0.1 M phosphate pH 7 X max. 650 m/u P. Nereocystis Phycocyanin 1 M acetate pH 5 X max. 557 and 615 m^t Phycoerythrin 1 M acetate pH 5 X max. 495 and 565 myu "Shoulder" 540-550 mM Allophycocyanin 0. 1 M phosphate pH 7 X max. 650 m/i chloride dihydrate (Fowden, 1954). Protein concentrations were adjusted to about 10 mg. per ml. and the hydrolysis performed in sealed tubes heated at 105° C. for 24 hours. After completion of hydrolysis the mixtures were evaporated to dryness in vacua to remove the volatile acids, and the residues redissolved in 5 ml. distilled water. The amino acids were absorbed on the cation exchange resin Zeo-Karb 225, eluted with 1 N NH4OH and finally dried in a vacuum desiccator over CaCL, and NaOH. Before chromatographing, the amino acids were taken up in 0.2 ml. iso- propanol (10%) to which was added a little HC1. Chromatographic procedure Whatman No. 1 chromatography paper was used throughout. Spots were ap- plied to the paper with calibrated micropipettes, the size of the spot being kept as small as possible (about 5 to 8 mm. diameter). For two-dimensional chromatography. n-butanol : acetic acid : water (4:1:5) was used for the first direction and phenol-water (80:20) containing 0.04% 8- hydroxyquinoline in an atmosphere of NH3 (1%) for the second direction. The butanol solvent was run for 28 hours at 20° C. and the solvent allowed to drip on the paper. The phenol solvent was run for 24 hours at a temperature of 20° C. The butanol was removed from the paper by drying the sheets in a current of warm air for two hours. The phenol was removed by the ether-wash technique of Fow- den (1951). The amino acids phenylalanine, leucine and isoleucine were resolved 366 RAYMOND F. JONES AND L. R. BLINKS 0-6- R perforate. S65 650 4OO 50O 6OO 7OO 400 0-6- > I- V) 0-4 -! a O O-2H R naiddum. 545 4OO 5OO 6OO 700 7OO FIGURE 1. Absorption spectra of isolated phycobilins from three species of Porphyra. phycoerythrin ; - phycocyanin ; allophycocyanin ; highly ionized fraction. by one-dimensional chromatography using continued development for four days in water-saturated tertiary amyl alcohol in an atmosphere of \% diethylamine. The solvent was removed from the paper by drying in a current of warm air for two hours. The amino acids were located by dipping the chromatograms in 0.2% isatin in acetone and heating at 100° C. for three minutes to reveal proline and then dipping them into 0.2% ninhydrin in acetone and heating at 100° C. for 15 minutes to detect the other amino acids (Jepson and Smith, 1953; Smith, 1953). AMINO ACIDS OF PHYCOBILINS 367 TABLE II Amino acid composition of phycobilin chromoproteins isolated from three species of Porphyra (Percentage by weight) Amino acid P. naiadum P. perforata P. Nereocystis PC PE A PC "I" PC PE APC PC PE Aspartic Glutamic 12.9 12.3 15.3 13.9 12.8 16.3 12.9 17.5 10.7 12.9 12.5 10.1 9.4 11.2 12.6 12.5 11.3 8.7 Serine 3.1 0.2 3.5 3.1 2.4 2.5 3.5 8.9 8.0 Glycine 5.4 5.7 7.2 5.2 6.8 9.1 9.2 11.9 13.5 Threonine 5.8 1.6 8.6 3.7 6.1 3.6 4.5 5.7 2.8 Alanine 12.3 18.3 10.9 9.5 12.6 20.3 12.5 12.3 11.7 Histidine 1.0 2.1 + 0.8 1.4 2.1 1.0 1.3 1.9 Lysine Arginine Proline 2.6 2.0 5.8 1.4 1.1 4.1 5.1 5.8 6.2 2.3 1.4 4.9 3.1 2.0 5.1 4.9 4.6 2.9 3.6 4.2 5.2 3.4 2.9 3.8 6.5 7.1 4.6 Valine + methionine 10.6 7.4 11.7 6.0 10.5 9.1 6.9 6.3 3.1 Phenylalanine Leucine 6.5 11.2 10.7 10.1 3.1 5.1 6.3 10.5 2.0 12.2 4.2 8.9 7.4 11.1 3.4 7.8 5.6 6.8 Isoleucine 9.1 6.3 3.9 8.8 6.8 4.4 7.8 3.1 5.2 Tyrosine Cystine — 2.0 + + 5.0 + 4.4 1.9 + + 4.1 3.7 + PC = phycocyanin; PE = phycoerythrin ; APC = allophycocyanin ; "I" = highly ionized fraction. + = present but too small to determine. - = not detected. Quantitative estimation The chromatograms, developed as described above, were utilized for quantitative estimation by a densitometric method. The maximum spot color density (i.e., aver- age blank reading minus the minimum reading for the given spot) multiplied by the spot area is a constant under the same conditions (Block, 1950). A densitometer suitable for the analysis of the chromatograms was constructed ; this consisted of a photoelectric cell, a constant voltage light source and a galvanometer (Weston Model 440 No. 10623). Rapidity of operation was improved by fixing the photo- electric cell on a movable arm which held it over a circular light source of diameter 0.5 cm. Before use the light source was adjusted to produce a suitable standard transmission. For the paper blanks percentage transmission readings of 90-100 were obtained while the amino acid spots varied between 5 and 80 per cent trans- mission for the concentrations employed. The area of the amino acid spot was de- termined by tracing the spot on uniform paper and weighing the cut-out spot with a torsion balance. Standard chromatograms of known amino acid composition were developed at the same time as the hydroly sates. For the standard amino acids linear relationships were obtained over the range of 1-25 jugm. amino acid with an error of ± 12%. All analyses were carried out in duplicate for each alga. RESULTS The three species of Porphyra investigated exhibit a graded increase in the ratio of phycocyanin to phycoerythrin. The deep water form, P. Nercoc\stis. has the 368 RAYMOND F. JONES AND L. R. BLINKS least phycocyanin. P. naiadum possesses comparatively large amounts of both phycocyanin and phycoerythrin. P '. perforata has proportionately the most phyco- cyanin and the least phycoerythrin. Allophycocyanin is present in all three species of Porphyra, but is most abundant in P. naiadum. The absorption spectra for the various isolated and chromatographically purified phycobilins are shown in Figure 1. The amino acid composition of these chromoproteins is given in Table II. The distribution of the amino acids is expressed as a percentage of the total weight of the amino acids in the protein. It is seen that, in the phycobilins isolated, the same m il FIGURE 2. The major amino acid components of four chromoprotein fractions of Porphyra naiadum. Acidic amino acids are shown in white, basic, cross-hatched, and neutral, in black. The amounts are indicated as percentage of the total. amino acids are present. Although tyrosine was not detected in the phycocyanin from P. naiadum it may well be due to the fact that this amino acid exists in concen- trations considerably lower than that of any other amino acid present. The quan- titative distribution of each amino acid varies for each protein analyzed. In all cases the dicarboxylic amino acids predominate and are comparable. Of the neutral amino acids, alanine and the two leucines are highest, although in the phycoerythrin isolated from P. Nereocystis glycine is present in relatively high concentration. It is interesting to note that alanine is present in greater amount than any other amino acid in the phycoerythrins isolated from P. naiadum and P. perforata. The basic amino acids of all the phycocyanins are low compared with the other phycobilin pro- teins. A histogram of the distribution in P. naiadum is shown in Figure 2. DISCUSSION The absorption characteristics of the isolated chromoproteins described in this work agree closely with those published by previous workers (Blinks, 1954a, 1954b ; Airth, 1955 ; Haxo, O'hEocha and Norris, 1955). Typical R-phycoerythrins of the AMIXO ACIDS OF PHYCOBILINS 369 higher red algae (Florideae) display absorption peaks at 495, 545, and 565 in//,. Of the lower red algae (Bangiales) here investigated, P. perforate and P. Nereocystis lack a pronounced second peak at 545 mp.. In P. naiad inn, the phycoerythrin is characterized by a single peak at 545 mp. and a small shoulder at 560 m/A. This phycobilin has been designated B-phycoerythrin (Blinks, 1954b; Airth and Blinks, 1956), and is closest to the C-phycoerythrin of the Cyanophyta which has a single peak at 550 mp.. The phycocyanins of P. perforate, and P. Nereocystis are similar in possessing the characteristic principal maximum at 615 mp, and a small one at 557 m/j,. P. naiadum, however, contains a phycocyanin which has but a single ab- sorption peak at 615 mp.. This is similar to the C-phycocyanin of blue-green algae. Allophycocyanin with an absorption maximum at 650 mp. is present in all three species of Porphyra, thereby substantiating the findings of Haxo, O'hEocha and Norris ( 1955) that it is a natural, although minor, component of the chromoproteins of marine algae. The highly ionized chromoprotein found only in P. naiadum has two absorption maxima, a major one at 545 m//, and a minor one at 615 m^, (prob- ably due to the presence of phycocyanin ) . The comparison of the above phycobilin proteins was undertaken to establish whether similar types of protein were present in the different species. The basis of comparison employed here, namely amino acid composition, although useful is sub- ject to certain limitations. The physico-chemical properties of proteins depend not only upon their amino acid composition, but upon the arrangement of the amino acid residues within the protein and the nature of the helical configuration of the mole- cule. It is therefore realized that an amino acid analysis alone cannot account for all the biological or physico-chemical characters of the proteins. The data presented show that the amino acid compositions of the various phy- cobilins differ significantly. Although P. perforate and P. Nereocvstis possess phycobilins of similar absorption spectra, the amino acid composition of the proteins varies. The amino acid analysis of the phycocyanin from P. naiadum differs widely from the analysis published by Wassink and Regetli (1952) for the C-phycocyanin of Oscillatoria which has similar absorption characteristics. Of particular note is the presence of arginine which was absent from Oscillatoria phycocyanin. P. naia- dum possesses phycobilin chromoproteins which differ from the other species of Porphyra, both in amino acid composition and absorption spectra. This is of par- ticular interest since Professor G. J. Hollenberg (University of Redlands) has noted several morphological peculiarities which will probably remove P. naiadum from its present genus. The amino acid analysis of the highly ionized fraction present in this species offers little explanation for the high mobility of the molecule when sub- ject to electrophoresis at pH 5.0. The degree of ionization is too great to be ac- counted for by the carboxyl groups of the amino acids. However, this fraction ex- hibited high absorption in the U. V. range of 265-280 mp. which suggests that the pigment may be attached to a nucleoprotein, in which case nucleic acid could be re- sponsible for the high mobility. SUMMARY 1. The phycobilin chromoproteins of three species of Porphyra have been sepa- rated by column chromatography and their individual absorption spectra recorded. These "chromatographically purified" chromoproteins were subjected to acid hy- drolysis and their constituent amino acids resolved by paper chromatography and determined quantitatively by a densitometric method. 370 RAYMOND F. JONES AND L. R. BLINKS 2. The quantitative amino acid composition of each chromoprotein differed. The dicarboxylic amino acids alanine, glycine and the two leucines were most abun- dant. Alanine was found to be present in high concentration in the phycoerythrin of P. naiadum and P. perforata. LITERATURE CITED AIRTH, R., 1955. The phycobilin pigments from Porphyra naiadum. Ph.D. Thesis, Stanford University, California. AIRTH, R. L., AND L. R. BLINKS, 1956. A new phycoerythrin from Porphyra naiadum. Biol. Bull, 111: 321-327. BLINKS, L. R., 1954a. The photosynthetic function of pigments other than chlorophyll. Ann. Rev. Plant Physiol., 5: 93-114. BLINKS, L. R., 1954b. The role of accessory pigments in photosynthesis. Symposium on Auto- trophic Micro-organisms. Cambridge at the University Press, Cambridge, England. BLINKS, L. R., AND R. L. AIRTH, 1957. Physico-chemical properties of phycobilins from Por- phyra naiadum. J. Gen. Physiol. (in press). BLOCK, R. J., 1950. Estimation of amino acids and amines on paper chromatograms. Anal. Chcm.. 22: 1327-1332. FOWDEN, L., 1951. The quantitative recovery and colorimetric estimation of amino acids sepa- rated by paper chromatography. Biochcm. J.. 48: 327-333. FOWDEN, L., 1954. A comparison of the compositions of some algal proteins. Ann. Bot. Loud., 18 : 257-266. FRENCH, C. S., AND V. K. YOUNG, 1952. The fluorescence spectra of red algae and the transfer of energy from phycoerythrin to phycocyanin and chlorophyll. /. Gen. Physiol., 35: 873-890. FUJIWARA, T., 1955. Chromoproteins in Japanese nori (Porphyra tencra) I. A new method for the crystallisation of phycoerythrin and phycocyanin. /. Biochem. (Japan), 42: 411-417. HAXO, F., AND L. R. BLINKS, 1950. Photosynthetic action spectra in marine algae. /. Gen. Physiol., 33 : 389-422. HAXO, F., C. O'nEocHA AND P. NORRIS, 1955. Comparative studies of chromatographically sep- arated phycoerythrins and phycocyanins. Arch. Biochcm. Biophys., 54: 162-173. JEPSON, J. B., AND I. SMITH, 1953. "Multiple Dipping" procedures in paper chromatography: a specific test for hydroxyproline. Nature, 172: 1100-1101. KITASATO, Z., 1925. Biochemische Studien iiber Phykoerythrin und Phykocyan. Acta Ph\to- chim. Tokyo II., 2 : 75-97. KYLIN, H., 1910. Uber Phykoerythrin und Phykocyan bei Ccramium nihnim. Hoppc-Seyl. Zeitschr., 69: 169-252. SMITH, L, 1953. Colour reactions on paper chromatograms by a dipping technique. Nature, 171 : 43-44. SVEDBERG, T., AND I. B. ERIKSSON, 1932. Molecular weights of phycocyan and phycoerythrin, III. /. Amer. Chcm. Soc., 54 : 3998-4010. SVEDBERG, T., AND T. KATSURI, 1929. The molecular weight of phycoerythrin from Porphyra tencra and of phycocyan from Aphanozomenon flosaquae. J. Amer. Chcm. Soc., 51 : 3573-3583. SVEDBERG, T., AND N. B. LEWIS, 1928. The molecular weights of phycoerythrin and phycyan. /. Amer. Chcm. Soc., 50 : 525-536. SWINGLE, S. M., AND A. TISELIUS, 1951. Tricalcium phosphate as an adsorbent in the chroma- tography of proteins. Biochem. J., 48: 171-174. TISELIUS, A., 1930. Moving boundary method of studying the electrophoresis of proteins. Nova Acta Reg. Soc. Sci. Upsal., 7: 1-107. TISELIUS, A., 1937. A new apparatus for electrophoresis analysis of colloidal mixtures. Trans. Faraday Soc., 33: 524-531. TISELIUS, A., S. HJERTEN AND O. LEVIN, 1956. Protein chromatography on calcium phosphate columns. Arch. Bioch. Biophys., 65: 132-155. WASSINK, E. C., AND H. W. J. RAGETLI, 1952. Paper chromatography of hydrolised Oscilla- toria phycocyanin. K. Nederl. Akad. Wetenschap. Proc. C., 4: 462-470. YOCUM, C. S., AND L. R. BLINKS, 1954. Photosynthetic efficiency of marine plants. /. Gen. Physiol., 38: 1-16. HISTOLOGICAL CHANGES IN REGENERATING PIECES OF DUGESIA DOROTOCEPHALA TREATED WITH COLCHICINE MARY A. McWHINNIE AND MARY M. GLEASON Department of Biological Sciences, DC Paul University, Chicago, Illinois It has been shown that colchicine inhibits or completely abolishes regenerative changes in pieces of Diigesia dorotocephahi (McWhinnie, 1955). Because of its selective stathmokinetic action it can be assumed that cellular studies, of colchicine- treated planarian pieces, should give evidence as to the source of blastema and re- generate cells. As early as 1902 Stevens reported numerous parenchymal cells in stages of mitosis, when untreated short transverse pieces of Planaria lugubris were regenerating. He suggested the embryonic nature of these cells which through multiplication and differentiation replaced the elements lost in section. With the use of x-rays Bardeen and Baetjer (1904) showed marked inhibition of regeneration in P. inaculata and P. lugubris. Histological study showed no change in the cells of muscle, nerve, endoderm or gonad and an absence of mitosis in parenchymal cells. Control pieces showed mitotic cells in the parenchyma. Wiegand (1930) also dem- onstrated this point with several planarian species. On an indirect basis Curtis and Schultze (1934) emphasized the role of parenchymal cells in regeneration by com- paring their number in species known to have high regenerative capacities (P. inacu- lata; P. agilis') with one limited in regenerative ability (P. fluviatilis) . Subsequent studies (Curtis, 1936) with x-rays showed a reduction of free parenchymal cells in proportion to the reduction in regeneration. Colchicine inhibition of development has been shown by Beams and Evans (1940). Fertilized eggs of Arbacia punctulata were unable to divide if exposed to colchicine during the pre-metaphase interval and also showed a considerable decrease in viscosity. Despite the evidence for the role of parenchymal cells in planarian regeneration, several workers have reported the absence of mitosis in normal planarian regenera- tion (Steinmann, 1926; Bandier, 1936; Clement, 1944). In an effort to demon- strate a specific source of cells which contribute to planarian regeneration, histologi- cal studies were made at selected intervals after colchicine treatment. MATERIALS AND METHODS A stock of Dugesia dorotocephala was collected and maintained in the manner previously described (McWhinnie, 1955). Animals were sectioned into two halves at the level of the mouth. After sectioning, the pieces were separated into two groups. In one group, anterior and posterior halves were placed into M/5000 col- chicine at the time of section. These were prepared for study at the end of exposure periods 3, 6 and 10 days. Pieces in the second group were placed into aerated tap water and were allowed to reconstitute for 24. 48 and 72 hours. At the end of each time interval these pieces were transferred to M/5000 colchicine where they were 371 372 MARY A. McWHINNIE AND MARY M. GLEASON PLATE I Explanation of Figures FIGURE 1. Mitotic figure in a free amoebocyte in a three-day regenerating planarian piece, exposed to J//5000 colchicine from the time of section. X 1425. COLCHICINE AND RECONSTITUTION 373 permitted to remain for 12 and 24 hours. All pieces to be prepared for cell study were narcotized in 0.05-0.1% chloretone, killed and fixed in Bouin's fluid and em- bedded using the standard paraffin technique. These were sectioned at 6 micra in such a manner that both frontal and sagittal sections were obtained. Delafield's hematoxylin was used without a counterstain. Study was with 1000 and 1500 di- ameters magnification. RESULTS The history of parenchymal cells in regenerating planarian pieces treated with colchicine demonstrates the paramount role of these cells in this developmental process. Planarian pieces treated with M/5000 colchicine for three days following section show many free amoebocytes in mitosis. These are uniformly distributed with extremely few in the area of the cut surface. Many of these cells appear elon- gate and in strands oriented to the cut surface, indicating migration. Mitotic fig- ures were normal (Fig. 1). However, after 6 days' exposure to colchicine from the time of section most of the mitotic amoebocytes had abnormal chromosomal configu- rations and considerable pycnosis (Fig. 2) . At 6 days these cells were still generally distributed throughout the parenchyma with never more than one to two in the re- gion of the cut surface. Also, the large free amoebocytes were oriented into longi- tudinal strands. At the end of 10 days' exposure to colchicine a larger number of cells were at metaphase or beyond. However, at this time there was extensive cellu- lar degeneration as evidenced by granulation of the nucleus, mitotic abberations and cytoplasmic vacuolation. Of the free amoebocytes undergoing degeneration many were oriented into migration strands. Despite the apparent increase in number of parenchymal cells in mitosis from three to ten days after section in treated regenerating planarian pieces, the untreated pieces do not show this same progressive increase in number of cells undergoing division in time. Three days after section, untreated planarian pieces show some free amoebocytes in mitosis (Fig. 3) but none were observed in the region of sec- tion. On a comparative basis the number of mitotic figures was less than in the three-day colchicinized pieces. A marked decrease in the proliferation of these cells is apparent by six days after section when pieces regenerate in aerated tap water. However, at this time orientation of migratory strands to the area of the cut is FIGURE 2. Mitotic figures in free amoebocytes in a six-day regenerating planarian piece, exposed to M/5000 colchicine from the time of section. X 1425. FIGURE 3. Mitotic figure in a free amoebocyte in an untreated three-day regenerating plana- rian piece. X 1425. FIGURE 4. A. Long section through the cut surface of a planarian piece treated with .17/5000 colchicine for 24 hours after 24 hours in water. < 150. B. Same as A. X 660. (A = amoebo- cyte.) FIGURE 5. Long section of an untreated regenerating planarian piece 48 hours after section. X660. FIGURE 6. Parenchyma of a planarian piece treated with colchicine for 24 hours after 48 hours in water. Note free amoebocytes in metaphase and evidence of migration. X 1425. FIGURE 7. Long section of an untreated regenerating planarian piece 72 hours after section. Note increased number of amoebocytes. X 660. FIGURE 8. Long section of a planarian piece treated with M/5000 colchicine for 24 hours after 72 hours in water. An increase in the number of cells can be seen in the region of the cut surface. An increase in gut degeneration is apparent. X 150. 374 MARY A. McWHINNIE AND MARY M. GLEASON notable. By ten days after section, when the regeneration process is conventionally considered complete, there is no evidence of mitotic activity. Regenerating pieces of planaria had no mitotic activity in epidermis, muscle or endoderm tissue, whether the pieces had been treated with colchicine or permitted to regenerate in water. When anterior and posterior halves of planarians were maintained in water for 24 hours and then transferred to Af/5000 colchicine for 12 and 24 hours, large num- bers of free parenchymal amoebocytes were in metaphase with considerably fewer in telophase. These cells had divided in situ as well as during migration to the area of the cut surface. Degenerative changes in the gut and parenchyma were not found after 12 hours and were minimal after 24 hours treatment. In this group some few mitotic cells showed degenerative changes as indicated by granulation of nuclear components and rupturing of the cytoplasm. Cells of the gut, fixed nuclei of the syncytium, muscle and epidermis were not in mitosis. Beneath the newly-formed epidermal covering there was a slightly larger number of free amoebocytes than in the rest of the parenchyma (Fig. 4 A and B) . At this same time interval untreated pieces had extremely few mitotic cells but a considerably greater number of amoebo- cytes at the cut surface (Fig. 5). A similar group of anterior and posterior halves was allowed to undergo regen- eration for 48 hours before treatment with colchicine for 12 and 24 hours. These pieces showed a still greater accumulation of free amoebocytes and more metaphase figures beneath the epidermal covering than in the previous group, i.e., colchicine treatment after 24 hours in water. Many amoebocytes in situ and in migration were at metaphase (Fig. 6). In untreated 72-hour regenerates there were few mitotic figures, but numerous amoebocytes were densely packed at the region of the blastema (Fig. 7). Pieces treated for 24 hours showed fewer mitotic figures than those treated for 12 hours. Treatment with colchicine for 12 and 24 hours, after a reconstitution period of 72 hours in water, resulted in pieces with a greater number of amoebocytes in the re- gion of the cut surface. At this time fewer mitotic cells were seen in all areas. Gut, as well as general parenchymal degeneration was more apparent than in the previous series (Fig. 8). On the other hand controls showed large numbers of amoebocytes in the blastema, little evidence of mitotic activity and no degenerative changes. DISCUSSION Through the use of colchicine on regenerating planarian pieces it can be con- cluded that the cellular elements involved in restoration to wholeness are primarily the free amoebocytes of the parenchyma. In all cases observed, after a minimum of 24 hours of reconstitution, epidermis, co-extensive with the epidermis of the rest of the piece, covered the wound surface. In no case was mitosis observed in this tissue. Similar wound epithelial covering without cell proliferation has been dem- onstrated in amphibian limb regeneration (Lash, 1955). The cut surface is readily distinguishable from the rest of the piece by the lack of sub-epidermal pigment as well as by the localized density of the parenchymal cells. With the use of colchicine it would appear that the time course of mitotic activity during regeneration explains the short interval in which there is no apparent change after section, the time of onset of greatest cellular proliferation and the known difference in susceptibility to toxic influences through the regeneration period. Some studies made in this work show COLCHICINE AND RECONSTITUTION 375 that mitotic cells increase in number from the third to the tenth day after section when pieces are placed into colchicine at the time of section. However, observation of untreated pieces at the same time intervals strongly indicates a greater number of parenchymal cells in division at the third day after isolation than in pieces 6 or 10 days after isolation. The progressively larger number of metaphase cells found at longer time intervals after isolation and introduction into colchicine would simply emphasize the sustained inhibition in the presence of the alkaloid and consequently the accumulation of inhibited cells. Evidence can be gained as to the time of greatest mitotic activity by permitting isolated pieces of planarians to initiate normal regeneration in water before placing them into colchicine. Halves of planarians placed into colchicine for 24 hours after initial regeneration in water for 24, 48, and 72 hours show a greater number of parenchymal cells in division at a final age of 72 hours (48 hours in water, 24 hours in colchicine). However, a substantial number of these cells were in division in pieces placed into colchicine for 24 hours after 72 hours' regeneration in water. While the number of mitotic figures found in this group was less than in the pre- ceding it is also true that parenchymal degeneration was considerably greater than in that group. Under these conditions there is extensive gut degeneration, granular degeneration in patches of parenchyma as well as in the area of the cut surface. Mitotic cells at this time showed a range from normal metaphase to cells with chro- mosomes widely dispersed and granular in appearance. These modifications were not so apparent in pieces placed into colchicine after 48 hours in water. The fixed 24-hour exposure to colchicine in these three groups with a greater susceptibility of pieces at the fourth day of reconstitution agrees with previous find- ings on the critical period in reconstitutional development. By studies of gross changes in planarian pieces and their susceptibility it was demonstrated that the fourth day in development is the most critical (McWhinnie, 1955). While the highest mitotic activity appears at the third day after isolation and both gross and microscopic evidence show a high susceptibility at the fourth day, it would appear that the increased population of parenchymal cells and their migration to the cut surface constitute the most active and therefore the most susceptible pe- riod in planarian regeneration. This is visibly expressed by the toxic effects of colchicine on the fourth day. It is suggested that the sequence of events in planarian regeneration includes an initial slow onset of division of parenchymal cells, rising to a peak at the third day after isolation. Associated with the rise in number of cells proliferating, oriented migrations of these cells to the cut surface follows. It would appear that the activ- ity of migration is greatest through the fourth to the sixth day afer section. Some oriented strands found in 48-hour pieces indicate the onset of migration at this time. By the sixth day of regeneration, mitotic activity and cell migrations are subsiding and the remainder of the reconstitution period represents the time of morphogenetic changes to complete species organization, both internal and external. It can be concluded also that the mechanism of colchicine inhibition of planarian regeneration is through its influence on parenchymal amoebocyte proliferation as well as reduced migration of those cells to the cut surface(s). It is entirely likely that colchicine-induced changes in viscosity (Beams and Evans, 1940) could ac- count, in part, for the marked difference in cell density in the blastema of normal and treated regenerating pieces. 376 MARY A. McWHINNIE AND MARY M. GLEASON SUMMARY 1. Histological studies show that the mechanism of colchicine inhibition of re- generation in pieces of Dugesia dorotocephala is the stathmokinetic action it exerts on free parenchymal amoebocytes. 2. Parenchymal amoebocytes are the only cells exhibiting mitotic activity during the period of regeneration. 3. Mitotic activity reaches a peak at the third day of development while oriented migrations of amoebocytes appear to set in at the second day with marked move- ment through the fourth to the sixth day after section. 4. The free amoebocytes of the parenchyma constitute the exclusive source of cells participating in the replacement of parts lost when planarian worms are sectioned. LITERATURE CITED BANDIER, J., 1936. Histologische Untersuchungen iiber die Regeneration von Landplanarien. Arch. f. Entw., 135 : 316-348. BARDEEN, C. R., AND F. H. BAETJER, 1904. The inhibitive action of roentgen rays on regenera- tion in planarians. /. E.vp. Zool., 1 : 191-195. BEAMS, H. W., AND T. C. EVANS, 1940. Some effects of colchicine upon the first cleavage in Arbacia punctulata. Biol. Bull, 79 : 188-198. CLEMENT, H., 1944. Les acides pentosenucleiques et la regeneration. Ann. Soc. Roy. Zool. de Bclg., 75 : 25-33. CURTIS, W. C., 1936. Effects of x-rays and radium upon regeneration. In: Biological effects of radiation. B. M. Duggar, edit. Chap. XII. McGraw-Hill Book Co., Inc., New York. CURTIS, W. C., AND L. M. SCHULZE, 1934. Studies upon regeneration, I. The contrasting powers of regeneration in Planaria and Procotyla. /. Morph., 55 : 477-513. LASH, J., 1955. Studies on wound closure in urodeles. /. Exp. Zool., 128: 13-28. MCWHINNIE, M. A., 1955. The effect of colchicine on reconstitutional development in Dugesia dorotocephala. Biol. Bull, 108 : 54-65. STEINMANN, F., 1926. Prospektive Analyse von Restitutionsvorgangen. I. Die Vorgange in den Zellen, Geweben und Organen wahrend der Restitution von Planarienfragmenten. Arch. f. Entiv., 108: 646-679. STEVENS, N. M., 1902. Notes on regeneration in Planaria lugubris. Arch. f. Entzv., 13: 396-409. WIEGAND, K., 1930. Regeneration bei Planarien und Clavelina unter dem Einfluss von Radium- strahlen. Zeitschr. f. ztriss. Zool, 136: 255-318. GROWTH-PROMOTING EFFECTS OF HYDROLYZED NUCLEIC ACIDS, NUCLEOTIDES, AND NUCLEOSIDES ON ENDAMOEBA HISTOLYTICA MITSURU NAKAMURA Department of Bacteriology, Montana State University, Missoula, Montana Endamoeba histolytica has not yet been maintained indefinitely in pure culture, although bacteria-free cultures have been carried for periods of up to one month in growth factor-fortified media (Nakamura and Baker, 1956). Jacobs (1947) and Shaffer and Frye (1948) have also grown the amebas in media containing no or relatively few multiplying bacteria. Therefore, it becomes apparent that although bacteria contribute tremendously to the growth and multiplication of the amebas, they are not absolutely essential and that perhaps amebic growth can occur in a semi-synthetic medium if supplied the necessary growth-promoting factors. It has been shown that purines, pyrimidines, citrovorum factor, and ribose-5-phosphate can substitute partially for bacterial association and permit bacteria-free cultures of E. histolytica to multiply for a limited period. However, ribonucleic acid (RNA) and desoxyribonucleic acid (DNA) tested singly or in combination failed to promote amebic growth ; on the other hand, dialysates of media containing RNA and DNA preconditioned by bacterial growth contained ameba-stimulatory substances which permitted seven sub-cultures of the amebas in the absence of associated bacteria (Nakamura and Baker, 1956). It was postulated that bacterial action on the nu- cleic acids produced catabolic intermediate (s) which were essential to the nutrition of the amebas. In order to determine more exactly the specific components in the nucleic acid digest which were ameba-stimulatory, nucleic acids were hydrolyzed by enzymatic, acid, and alkaline hydrolysis ; the dialysates of the hydrolysates were studied for their effects on E. histolytica under bacteria-free conditions. Further- more, nucleosides and nucleotides, obtained from commercial sources, were also as- sayed for their activity on the growth of the amebas. MATERIALS AND METHODS Organism and the assay medium Strains of E. histolytica employed in these experiments consisted of: (1) NRS, obtained from Dr. Quentin M. Geiman, Stanford University School of Medicine, San Francisco, California, (2) HUS-100, isolated from the stool of a carrier during an outbreak of amebiasis in Indiana in 1953, obtained from Dr. Chia-Tung Pan, De- partment of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts, and (3) UC, also obtained from Dr. Pan. Stock cultures of the amebas, containing a mixed bacterial flora, were maintained in a modified Boeck- Drbohlav (1925) medium. The assay methods were essentially identical with those described earlier (Nakamura, 1955; Nakamura and Baker, 1956; Nakamura and 377 378 MITSURU NAKAMURA Jonsson, 1956). Coagulated egg slants were overlaid with a liquid phase consisting of glucose (0.5%), sodium thioglycollate (0.3%), penicillin G (10,000 units/ ml. final concentration), streptomycin (5000 units/ ml. final concentration), horse serum-Ringer solution (1/5), rice powder (approximately 10 mg.), and a vaspar (vaseline and paraffin, 1/1 ) seal. The volume of the overlay fluid was four ml. The dialysates and the nucleosides and nucleotides assayed were added to the liquid phase of the medium. The inocula consisted of 2 drops of stock cultures adjusted to contain approxi- mately 15-20 amebas per low power field. The bacteria introduced with the ameba inocula were sterilized within 4—6 hours by the combinations of antibiotics used. The culture tubes were incubated for 3—4 days at 37° C. ; ameba counts were made by taking the sediment from each culture tube ( in duplicate ) , placing a few drops on a clean slide, covering with a cover slide (22 X 22 mm.) and counting the number of amebas per low power field. Ten fields were counted and an average count re- corded. At the same time the amebas were transferred to tubes containing identical nutritional components. Control tubes consisted of media lacking only the materials being assayed. Positive controls consisted of media fortified with the growth fac- tors ribose-5-phosphate and adenosinetriphosphate. Hydrolysis of nucleic acids The method of Kerr et al. (1949) was used for the acid hydrolysis of RNA. Fifty mg. of RNA were placed in a test tube with 5 ml. of 2 N sulfuric acid. The tube was placed in boiling water for 30 minutes. After hydrolysis the contents of the tube were diluted to 25 ml. with water. Acid hydrolysis of DNA was accom- plished by placing 50 mg. of DNA in 5 ml. N sulfuric acid and refluxing in boiling water for 2 hours. The hydrolysate was adjusted to pH 6.5 with alkali. The method of Volkin and Carter (1951) was used for the alkaline hydrolysis of RNA. Fifty mg. of RNA, dissolved in 3 ml. of 0.5 N NaOH, were kept at 37° C. for 17 hours. The digest was diluted with water to 0.02 N NaOH and fi- nally neutralized. DNA was hydrolyzed using a modified method of Marrian ct al. (1951). Enzymatic hydrolysis of RNA was accomplished by suspending 50 mg. of RNA in 5 ml. water and adjusting to pH 7.2 with dilute NaOH. Then 5 mg. of crystal- line ribonuclease, dissolved in 1 ml. of 0.1 M phosphate buffer at pH 7.2, was added to the nucleic acid solution under stirring. As the reaction progressed the solution was maintained at pH 7.2 with the addition of 0.05 N NaOH. The temperature of the digest was maintained between 25-27° C. Hydrolysis was complete in two hours. The method of Smith and Markham (1952) was used for the enzymatic digestion of DNA; the DNA was digested with desoxyribonuclease (20 ugm./ml.) in 0.005 M magnesium sulfate at pH 7.0 for 18 hours. The digests were dialyzed in water and the dialysates tested for their growth-promoting activity. RESULTS As is evident in Tables I, II, and III, enzyme-hydrolyzed nucleic acids (both RNA and DNA) yielded a product which was stimulatory to the growth of E. his- tolytica. In all of the experiments enzyme-hydrolyzed RNA consistently stimu- lated the amebas slightly more than the enzyme-hydrolyzed DNA preparation. Al- GROWTH FACTORS IN E. HISTOLYTICA 379 TABLE I Effect of hydrolyzed nucleic acids, nucleotides, and nucleosides on the growth of E. histolytica under bacteria-free conditions; strain NRS Aver, count Total no. of per low power Material assayed determinations field Basal (control) 15 1 Basal + enzyme-hydrolyzed RNA 4 79 Basal + enzyme-hydrolyzed DNA 4 55 Basal + alkali ne-hydrolyzed RNA 4 47 Basal + alkaline-hydrolyzed DNA 4 50 Basal + acid-hydrolyzed RNA 4 0 Basal + acid-hydrolyzed DNA 4 0 Basal + unhydrolyzed RNA 8 10 Basal -f- unhydrolyzed DNA 8 7 Basal + adenosine (0.1 mg./ml.) 4 41 Basal + guanosine (0.1 mg./ml.) 4 49 Basal + thymidine (0.1 mg./ml.) 4 59 Basal + adenylic acid (0.1 mg./ml.) 4 70 Basal + guanylic acid (0.1 mg./ml.) 4 66 Basal + thymidylic acid (0.1 mg./ml.) 4 83 Basal + uridylic acid (0.1 mg./ml.) 4 47 TABLE II Effect of hydrolyzed nucleic acids, nucleotides, and nucleosides on the growth of E. histolytica under bacteria-free conditions; strain HUS-100 Aver, count Total no. of per low power Material assayed determinations field Basal (control) 15 0.4 Basal + enzyme-hydrolyzed RNA 4 64 Basal + enzyme-hydrolyzed DNA 4 40 Basal + alkaline-hydrolyzed RNA 4 51 Basal + alkaline-hydrolyzed DNA 4 54 Basal + acid-hydrolyzed DNA 4 1 Basal + acid-hydrolyzed RNA 4 0 Basal + unhydrolyzed RNA 4 0 Basal + unhydrolyzed DNA 4 1 Basal + adenosine (0.1 mg./ml.) 4 39 Basal -f- guanosine (0.1 mg./ml.) 4 34 Basal + thymidine (0.1 mg./ml.) 4 43 Basal + adenylic acid (0.1 mg./ml.) 4 57 Basal + guanylic acid (0.1 mg./ml.) 4 68 Basal + thymidylic acid (0.1 mg./ml.) 4 49 Basal + uridylic acid (0.1 mg./ml.) 4 33 kaline hydrolysates of nucleic acids were also stimulatory to the amebas. However, acid-hydrolyzed nucleic acids were without ameba-stimulatory properties in studies on all three strains of E. histolytica. Unhydrolyzed nucleic acids were inactive, ex- cept for a slight effect on the NRS strain, as was to be expected according to the earlier data of Nakamura and Baker (1956). The nucleosides adenosine, guano- sine, and thymidine stimulated the HUS-100 and NRS strains but not the UC strain. The nucleotides adenylic acid, guanylic acid, thymidylic acid, and uridylic acid were active as growth factors for all three strains of amebas tested, although 380 MITSURU NAKAMURA TABLE III Effect of hydrolyzed nucleic acids, nncleotides, and nudeosides on the growth of E. histolytica under bacteria-free conditions; UC strain Aver, count Total no. of per low power Material assayed determinations field Basal (control) 18 2 Basal + enzyme-hydrolyzed RNA 4 83 Basal + enzyme-hydrolyzed DNA 4 69 Basal + alkaline-hydrolyzed RNA 4 40 Basal + alkaline-hydrolyzed DNA 4 40 Basal + acid-hydrolyzed RNA 4 3 Basal + acid-hydrolyzed DNA 4 2 Basal + unhydrolyzed RNA 4 1 Basal + unhydrolyzed DNA 4 0 Basal + adenosine (0.1 mg./ml.) 4 0 Basal + guanosine (0.1 mg./ml.) 4 Basal + thymidine (0.1 mg./ml.) 4 5 Basal + adenylic acid (0.1 mg./ml.) 4 61 Basal + guanylic acid (0.1 mg./ml.) 4 68 Basal + thymidylic acid (0.1 mg./ml.) 4 90 Basal + uridylic acid (0.1 mg./ml.) 4 56 the degree of activity as growth-promoting factors varied slightly from compound to compound. Attempts to maintain bacteria-free subcultures on media containing hydrolyzed nucleic acids, nucleotides, or nucleosides were generally unsuccessful. The longest culture maintained on enzyme-hydrolyzed RNA and enzyme-hydrolyzed DNA was 5 transfers for a total of approximately 15 days. Sterility tests indicated the absence of viable bacterial cells. In media containing alkaline-hydrolyzed nucleic acids, two to three subcultures were usually possible ; however, the total amebic populations were considerably lower than in the enzyme-treated nucleic acid media. Only one subculture with a meager ameba count was possible in the experiments containing nucleosides and nucleotides as growth factors. DISCUSSION The data in this report are in agreement with earlier reports that nucleic acids preconditioned by bacterial growth produce some catabolic metabolite (s) which are essential for amebic growth in the absence of living bacteria. In these experiments, enzymatic and alkaline digestion, rather than bacterial preconditioning, yielded ameba-growth-promoting factors. It is indeed difficult to explain the absence of similar stimulatory activity in the acid-hydrolyzed nucleic acid solutions. In studies with Trichomonas vaginalis, Sprince et al. (1953) reported that acid hydrolysis of RNA destroyed the growth-promoting effect of RNA whereas alkaline hydrolysis of RNA left intact this growth-promoting factor. They also reported that acid, alkaline, and enzymatic hydrolysis of DNA destroyed the growth-promoting effects of DNA. These results, however, are not quite analogous to the data in this paper since Sprince et al. (1953) were dealing with DNA and RNA which were estab- lished as growth-promoting factors for Trichomonas; in the case of Endamoeba his- tolytica, DNA and RNA in themselves do not stimulate amebic growth. GROWTH FACTORS IN E. HISTOLYTICA 381 It is highly probable that the ameba-growth-stimulatory action of nucleic acid hydrolysates was not due solely to the nucleosides and nucleotides formed during the digestion. Smith and Markham (1952) have found that enzyme digestion of DNA produces many dinucleoside monophosphates ; Markham and Smith (1952) reported that products of enzyme hydrolysis of RNA were largely cyclic pyrimidine nucleotides and only traces of adenylic and guanylic acids were found. On the other hand, alkaline digestion of RNA produces guanylic, adenylic, and uridylic acids (Magasanik and Chargaff, 1951 ). The growth factor effects of purified nucleosides and nucleotides indicate the im- portance of these substances in amebic nutrition ; these substances play a role in the synthesis of nucleic acids and pyridine nucleotides. Diphosphopyridine nucleotide has been shown to be necessary for amebic growth in the absence of bacteria (Naka- mura and Baker, 1956). Johnson (1953) similarly showed that cytidylic and guanylic acids were growth factors for Paraniecium multimicronudeatum. There is evidence that different strains of E. histolytica possess different growth factor requirements. The nucleosides, which were highly active for the NRS and HUS-100 strains, were without activity on the UC strain. It is possible that the UC strain can synthesize its own nucleoside but that it cannot phosphorylate the nucleoside into the nucleotide which it apparently requires. In the cases of the NRS and HUS-100 strains, it appears logical to assume that they can synthesize neither nucleosides nor nucleotides, yet when supplied these two growth factors exogenously, the amebas can synthesize their own nucleic acids. A strong point in favor of this assumption is the fact that pre-formed nucleic acids do not aid amebic growth appreciably whereas the nucleosides and nucleotides are highly stimulatory. SUMMARY 1. Enzyme and alkaline hydrolysates of ribonucleic and desoxyribonucleic acids contained growth-promoting factors for Endainocba histolvtica. Acid hydrolysates of nucleic acids, however, were without this stimulatory activity on the amebas. 2. Nucleosides. adenosine, guanosine, and thymidine, were stimulatory to the NRS and HUS-100 strains but not for the UC strain. Nucleotides, adenylic acid, guanylic acid, thymidylic acid, and uridylic acid, were highly stimulatory for the growth of all three strains of E. histolytica studied. LITERATURE CITED BOECK, W. C, AND T. DRBOHLAV, 1925. The cultivation of Endamocba histolvtica. Amcr J Hyg., 5 : 371-407. JACOBS, L., 1947. The elimination of viable bacteria from cultures of Endamoeba histolytica and the subsequent maintenance of such cultures. Amcr. J . Hyg., 46: 172-176. JOHNSON, W. H., 1953. Discussion of papers on ciliate nutrition. Ann. N. Y. Acad Set 56- 969-971. KERR, S. E., K. SERAIDARIAN AND M. WARGON, 1949. Studies on ribonucleic acid II Methods of analysis. /. Biol. Chem., 181 : 761-771. MAGASANIK, B., AND E. CHARGAFF, 1951. Studies on the structure of ribonucleic acids. Bio- chim. et Biophys. Acta, 7: 396-412. MARKHAM, R., AND J. D. SMITH, 1952. The structure of ribonucleic acids. I. Cyclic nucleo- tides produced by ribonuclease and by alkaline hydrolysis. Biochem. J., 52 : 552-557. MARRIAN, D. H., V. L. SPICER, M. E. BALIS AND G. B. BROWN, 1951. Purine incorporation into pentose nucleotides of the rat. /. Biol. Chem., 189: 533-541. 382 MITSURU NAKAMURA NAKAMURA, M., 1955. Growth factors for Endamoeba histolytica. Proc. Soc. Exp. Biol. Med., 89 : 680-682. NAKAMURA, M., AND E. E. BAKER, 1956. Nutritional requirements of Endamoeba histolytica. Amer. J. Hyg., 64 : 12-22. NAKAMURA, M., AND S. JONSSON, 1956. The effect of antimetabolites on the growth of Enda- moeba histolytica. I. Purine and pyrimidine analogs. Arch. Biochem. and Biophys., 66 : 183-189. SHAFFER, J. G., AND W. W. FRYE, 1948. Studies on the growth requirements of Endamoeba histolytica. I. Maintenance of a strain of E. histolytica through one hundred transplants in the absence of an actively multiplying bacterial flora. Amer. J. Hyg., 47: 214-221. SMITH, J. D., AND R. MARKHAM, 1952. The enzymatic breakdown of deoxyribosenucleic acids. Biochim. et Biophys. Ada, 8: 350-351. SPRINGE, H., R. GOLDBERG, G. KUCKER AND R. S. LOWY, 1953. The effect of ribonucleic acid and its nitrogenous constituents on the growth of Trichomonas vaginalis. Ann. N. Y. Acad. Sci., 56 : 1016-1027. VOLKIN, E., AND C. E. CARTER, 1951. The preparation and properties of mammalian ribonucleic acids. /. Amer. Chem. Soc., 72: 1516-1519. ANAEROBIC RECOVERY OF ASCARIS EGGS FROM X-IRRADIATION 1 GEORGE PAHL2 AND C. S. BACHOFER Department of Biology, University of Notre Dame, Notre Dame, Indiana Lea (1947) in his classic work on radiation biology has pointed out in his analysis of the work of Henshaw (1940) that the recovery of unfertilized, x-irradi- ated eggs of Arbacia is probably not due to diffusion out of the egg of inhibitory substances ; the correlation between recovery rate and oxygen uptake suggests that the effect of the radiation is to destroy some nuclear constituent, and recovery con- sists in the re-formation of this constituent as a result of the metabolic activity of the cell. The rate of oxygen uptake is presumably an indication of the general level of metabolic activity, and in Arbacia eggs appears to vary in different stages of the egg in much the same way as does whatever reaction is responsible for recovery. It does not follow necessarily that the rates of recovery in different organisms will be proportional to their respective rates of oxygen uptake. Some organisms have, in fact, been shown to consume oxygen at appreciable rates after irradiation al- though they do not show any recovery. The eggs of Ascaris lumbricoides suwn possess certain advantages for a test of the question whether oxygen is necessary for recovery from x-irradiation. Since they are facultative anaerobes they can be held for long periods of time in anaerobic conditions. Even at optimal temperatures for normal development, under anaero- bic conditions the eggs do not develop. If recovery should occur during the en- forced anaerobic metabolism, not only would the necessity of oxygen uptake for re- covery be disproved for Ascaris eggs, but some other possible mechanism of recovery would be suggested. The present paper complements a preliminary report (Pahl and Bachofer, 1954). MATERIALS AND METHODS A stock of eggs of Ascaris lumbricoides sun in in the one-cell stage was prepared according to methods already described by Bachofer and Pahl (1955). The source of x-rays was a beryllium- window tube operated at 100 kvp. and 8 ma., without added nitration. Each irradiated sample consisted of 105 eggs suspended in one ml. of distilled water and placed in an open, flat-bottom vial 2.7 cm. in diameter. The egg suspension was approximately 1.8 mm. deep with the eggs resting on the bottom during the exposure. The dose was calculated to be 12,000 r/min. at the center of the irradiated layer of eggs. This value was determined by exposing ferrous am- monium sulfate as a dosimeter to a 325-curie cobalt-60 source of gamma rays, as described by Weiss (1952). Ascaris eggs were then exposed to the gamma rays under the same conditions, and the biological response was correlated with dose. 1 This investigation was supported in part by Research Contract No. AT (11-1) -205 between the Atomic Energy Commission and the University of Notre Dame. 2 Present address : St. Mary's College, Winona, Minnesota. 383 384 GEORGE PAHL AND C. S. BACHOFER Aliquots of the same sample of eggs were then exposed to the x-ray beam, and from their response the output of the x-ray tube was determined. All subsequent ex- posures to x-rays were carried out under identical conditions at the same dose rate. Each irradiated sample was diluted 20-fold with Ascaris physiological saline so- lution (Baldwin and Moyle, 1947) immediately after irradiation. Aliquots of two ml. each were de-oxygenated by bubbling specially purified nitrogen through the sa- line solution containing the eggs. The eggs were then placed at the appropriate in- cubation temperatures. After certain designated incubation periods, the seals were broken and the supernatant de-oxygenated fluid was drawn off while the eggs re- mained settled on the bottom. Ascaris saline, in equilibrium with air, was then added and incubation completed at 30° C. The same procedure was followed for non-irradiated controls. TABLE I Effect of post-irradiation anaerobic treatment at 15° C., 20° C., and 30° C. over a period of two weeks on the survival of x-irradiated Ascaris eggs. Dose: 28,000 r Per cent survival Post-irradiation treatment Days under anaerobic conditions 0 i 7 14 Aerated 47.5 45.0 47.3 48.8 30° C. De-oxygenated 47.3 58.0 57.3 60.3 Aerated 49.8 46.5 43.0 35.0 20° C. De-oxygenated 45.0 48.3 50.0 38.8 Aerated 48.8 42.0 36.0 27.8 15° C. De-oxygenated 47.5 51.8 45.5 34.3 The two criteria used to determine the effects of irradiation and anaerobic treat- ment were the rate of first cleavage and the percentage of eggs which developed to the motile embryo stage. The term "survival" is used to designate the development of irradiated eggs into motile embryos. RESULTS Table I shows the results of irradiation of Ascaris eggs which were de-oxygen- ated immediately after irradiation and stored at temperatures of 15° C., 20° C., and 30° C. After periods of 1, 7, and 14 days at these temperatures, the de-oxygenated samples were aerated and incubated at 30° C. The results show that at any given temperature there was a higher survival for irradiated eggs given anaerobic treat- ment than for those incubated only aerobically. Keeping the eggs under anaerobic conditions for more than one day did not increase survival. Irradiated eggs kept at 30° C. throughout the entire post-irradiation period showed the highest survivals ANAEROBIC RECOVERY FROM X-IRRADIATION TABLE II Per cent survival of x-irradiated Ascaris eggs as affected by anaerobic treatment immediately after exposure 385 Per cent survival Dose in roentgens Days of post-irradiation anaerobic treatment 0 i i 2 7 0 97.5 96.8 97.0 97.3 96.6 28,000 47.3 56.7 58.0 57.1 57.3 40,000 26.9 36.3 36.4 36.6 36.4 48,000 12.5 20.8 21.1 21.7 22.0 60,000 2.5 6.7 6.4 6.8 6.6 for both anaerobic and aerobic treatment. This confirms a previous study of the authors (Bachofer and Pahl, 1955) on post-irradiation temperature treatment of Ascaris eggs. In view of this, all other experiments were performed at 30° C., which is approximately the optimal incubation temperature. Table II shows that anaerobiosis is able to bring about recovery over a wide dose range, even when untreated samples give survivals as low as 2.5%. When there was no anaerobiosis there was no recovery, recovery being represented by the dif- ference in survival between the first column (where there was no anaerobiosis) and the other columns (where there was anaerobiosis). The critical period of recovery is shown to occur during the first 24 hours of anaerobiosis, since survivals for 12 hours of treatment are not increased appreciably for longer periods of treatment. The effect of delaying the anaerobic treatment after irradiation was next inves- tigated, with both cleavage delay and survival as criteria of recovery. The results summarized in Table III for cleavage delay show that the eggs must be de-oxygen- ated before 15 hours have elapsed after irradiation in order to procure recovery. The low values in the column for no delay in anaerobic treatment indicate the great- est recovery. Conversely, in Table IV, the high values in the column for no delay in anaerobic treatment indicate the greatest recovery. The crucial period of ap- proximately 15 hours, therefore, affects both cleavage delay and survival. TABLE III 50% cleavage time in hours for x-irradiated Ascaris eggs as affected by 24-hour anaerobic treatment initiated at various intervals after exposure Delay before anaerobic treatment No anaerobic treatment Dose in roentgens 0 hrs. 15 hrs. 24 hrs. 50% cleavage time in hours 0 40.0 39.5 40.3 40.0 24,000 53.1 46.7 52.7 53.1 40,000 58.4 51.4 57.7 58.2 48,000 60.0 53.4 59.4 59.5 386 GEORGE PAHL AND C. S. BACHOFER The recovery phenomena summarized above have been verified over a dose range of 24 to 48 kr, both for delay of cleavage and for survival. Since anaerobic conditions during irradiation give high protection to Ascaris eggs, an experiment was designed to test whether post-irradiation anaerobic re- covery could be secured with eggs which had been protected by anaerobic conditions during irradiation. When a dose of 60,000 r was delivered to one sample of eggs TABLE IV Per cent survival of x-irradiated Ascaris eggs as affected by a 24-hour anaerobic treatment begun at various intervals after exposure Delay of anaerobic treatment No anaerobic treatment Dose in roentgens 0 hrs. 15 hrs. 24 hrs. 60 hrs. Per cent survival 0 97.0 97.0 97.3 97.5 97.0 24,000 63.5 73.9 64.8 62.3 62.0 40,000 26.9 36.4 27.2 27.6 25.8 48,000 12.5 21.1 11.8 12.5 12.0 that was in equilibrium with air and to another similar sample that was under an- aerobic conditions, the survival was increased in both cases by post-irradiation an- aerobiosis. These increases in survival were duplicated when treatment for iyz hours in 0.1 M KCN was substituted for post-irradiation anaerobiosis. Immedi- ately after the period of exposure to cyanide, the eggs were washed by centrifugation and incubated in Ascaris saline. The results in Table V clearly indicate that the same pattern of protection can be obtained with cyanide as with anaerobiosis. TABLE V Effect of 24-hour post-irradiation treatments immediately following the irradiation of Ascaris eggs under different conditions during irradiation. Dose: 60,000 r Treatment after irradiation Aerobic Anaerobic KCN Treatment during irradiation In equilibrium with air Anaerobic Per cent survival 2.5 6.4 6.5 74.3 82.1 82.2 In other studies (unpublished results) the authors have established that cyanide inhibits the oxygen consumption of Ascaris eggs. Cyanide, however, is a general inhibitor of respiratory cycles whether they include the cytochrome system or not. To demonstrate that Ascaris eggs do have a cytochrome system, they were sub- jected to the light-reversal inhibition test of carbon monoxide by use of a Warburg respirometer adapted to this purpose. The eggs showed the same rate of oxygen consumption in air and in a 5% oxygen-95% nitrogen mixture. When CO re- placed the nitrogen, however, there was an immediate and persistent drop in oxygen ANAEROBIC RECOVERY FROM X-IRRADIATION 387 consumption. Within a few minutes this leveled off at approximately 40% of normal consumption. In the presence of light this value rose to 75% of normal consumption. DISCUSSION Numerous studies on the effect of anaerobiosis and other factors during irradia- tion are not comparable to the present investigation, since the present study utilizes anaerobiosis after irradiation and is therefore concerned with recovery processes. Although a number of post-irradiation treatments have delayed the expression of injury or decreased its rate of development, most of them have had no effect on the final outcome. In work with mice, Bacq et al. (1950) found that NaCN given im- mediately after irradiation only delayed mortality, and they concluded that cyanide was ineffective when given after irradiation. Bachofer (1956) has shown that post- irradiation anaerobiosis of x-irradiated Ascaris eggs restores in part the normal rate of pronuclear fusion, which is slowed down considerably by x-irradiation ; the res- toration is a genuine recovery and is attributable to the period of anaerobiosis. The problem proposed by Lea (1947), as to whether the recovery of irradiated invertebrate eggs demands oxygen uptake or whether this uptake is a mere con- comitant action, has been solved for Ascaris eggs. The facultative anaerobic nature of Ascaris eggs makes possible a complete elimination of free oxygen during incuba- tion at optimum temperature. When x-irradiated eggs were subjected to post- irradiation anaerobiosis, their power of recovery surpassed that of x-irradiated aerated eggs as shown by decreased cleavage time and by higher survival (Tables I-V ) . It has been established, therefore, that oxygen is not necessary for recovery in this case. A possible mechanism to be considered is whether this recovery could be attrib- uted to the concomitant delay in cleavage brought about by anaerobic conditions. In studies concerned with cell cleavage, Schjeide and Allen (1951) found that tad- pole hematopoietic cells appear to be susceptible to x-rays in direct proportion to the amount of cell division allowed to proceed following the irradiation period. Re- covery of irradiated Arbacia eggs (Henshaw, 1940) was obtained only if they were kept unfertilized ; as the time between irradiation and fertilization was shortened, the recovery was likewise decreased. In unirradiated Arbacia eggs cleavage begins at optimal temperatures within an hour after fertilization. Cytological observations (Bachofer, 1956) show that all eggs of Ascaris lumbricoidcs removed from the ter- minal 25 mm. of the uteri, in which the sperm has entered the egg, are in the pro- nuclear stage. Upon incubation at optimal temperature, pronuclear fusion begins slowly and precedes first cleavage by approximately I1/-? hours. The eggs begin first cleavage only after 25 to 30 hours of incubation at optimal temperature, and achieve 50% cleavage after 40 hours. Since anaerobiosis had to be initiated before 15 hours had elapsed after irradiation in order to secure recovery, and anaerobic recovery was reduced if the treatment was delayed even a fewr hours following irradiation, it appears that the recovery process in question is not directly associated with the delay of first cleavage. Furthermore, if delaying the time of cleavage were the important factor in recovery, it would be expected that the survival of the irradiated eggs wrhich were de-oxygenated and placed at the various sub-optimal temperatures would have remained at the same peak as those placed at 30° C. The progressive decrease in survival of both the aerated and anaerobically incubated eggs kept for 388 GEORGE PAHL AND C. S. BACHOFER increasing lengths of time at temperatures lower than optimal (Table I) indicates that some factor other than cleavage delay is responsible. Further evidence that cleavage delay is not the contributing condition for re- covery is shown by the fact that the anaerobic incubation facilitates recovery only during the first 15 hours of this treatment (Table II). Likewise, eggs which have been allowed to incubate aerobically for 15 hours before being de-oxygenated give no evidence of recovery as indicated by cleavage delay (Table III) and survival (Table IV). During this 15 hours of aerobic incubation the eggs have not yet begun their first division. It appears that one must look to other conditions than delay of cleavage to explain the recovery. It should be borne in mind that cellular activity, including cell cleavage, is nec- essary in most cases to demonstrate the injury, since the injury is latent. Post- ponement of cellular activity after irradiation may not involve recovery ; once cellu- lar activity is allowed to proceed the damage may be manifested. If the damage is as great as that which would have been manifested by permitting cellular activity to proceed immediately after irradiation, then there was no genuine recovery. True recovery was reported by Cook (1939) for survival of irradiated eggs of Ascaris megalocephala held at low temperatures after irradiation, but the opposite was found to be true for Ascaris lumbricoidcs (Bachofer and Pahl, 1955). Both studies agreed, however, in that post-irradiation treatment did not affect the time required for first cleavage. Pertinent to the present case, therefore, is the fact that forestalling cell cleavage and cellular activity after irradiation does not in itself produce genuine recovery. The seat of the recovery from irradiation may involve various reactions of the respiratory cycle. The increased survival of irradiated eggs which have been sub- jected to cyanide or to anaerobiosis after irradiation suggests that the effects of ir- radiation operate to some extent through the cytochrome system, since both cyanide and anaerobiosis inhibit the cytochrome system. The mechanism of protection af- forded by respiratory inhibitors may be either the prevention of the products of ir- radiation from reacting with the cytochromes or the prevention of the radiation- affected cytochromes from participating in the chain of reactions that normally bring about the observed effects of irradiation. Insofar as the cytochromes may be in- volved, the second possibility appears more pertinent in the present study, since the anaerobic condition would be too late, in time, to prevent a highly activated radi- ation product from reacting with the cytochromes, but it could prevent the affected cytochromes from reacting further. There is, however, a function more important than holding the cytochromes in abeyance (Bachofer, 1956). It appears that checking aerobic metabolism permits anaerobic metabolism to restore essential molecules needed for normal development. The fact that recovery was greater for eggs held anaerobically at 30° C. than at sub-optimal temperatures indicates that anaerobic metabolism is associated with the recovery under consideration. SUMMARY 1. X-irradiation of Ascaris lumbricoides suum eggs produced delay of cell cleav- age and reduced the percentage of eggs that completed embryogenesis. The time required for cleavage of irradiated eggs was reduced by an anaerobic treatment after ANAEROBIC RECOVERY FROM X-IRRADIATION 389 irradiation. The percentage of eggs that completed embryogenesis was increased by the same post-irradiation anaerobiosis. After the anaerobic treatment, eggs must be incubated aerobically since there is no perceptible development under anaerobio- sis, although recovery takes place during this period. This recovery is greater at 30° C. than at sub-optimal temperatures. 2. Maximum recovery was obtained for eggs placed immediately after irradia- tion under anaerobiosis for periods of approximately 15 hours or more at 30° C. If the anaerobic treatment is delayed for 15 hours, the recovery is negligible. 3. Post-irradiation treatment with cyanide also fostered recovery from x-irradia- tion comparable to that secured with anaerobiosis. 4. Recovery was not due to delay of cleavage : the critical period for recovery took place long before cell division occurred even in air-saturated non-irradiated controls. 5. A cytochrome system in the eggs was demonstrated. The effects of cyanide treatment and anaerobiosis suggest that the mechanism of recovery may involve in- hibition of the cytochrome system, which is prevented from participation in the re- actions producing the expected deleterious effects of irradiation. There is, how- ever, a positive contribution attributable to anaerobic metabolism, since recovery is greatest at optimal temperatures under anaerobiosis. t LITERATURE CITED BACHOFER, C. S., 1956. Pronuclear fusion as affected by x-rays and by postirradiation anaero- biosis. Science, 123 : 139-140. BACHOFER, C. S., AND GEORGE PAHL, 1955. Influence of extended temperature treatments on recovery of x-irradiated Ascaris eggs. Radiation Res., 2 : 50-63. BACQ, Z. M., A. HERVE, J. LECOMTE AND P. FISCHER, 1950. Cyanide protection against x- irradiation. Science, 111 : 356-357. BALDWIN, E., AND V. MOYLE, 1947. An isolated nerve-muscle preparation from Ascaris lum- bricoides. J. Exp. Biol, 23 : 277-291. COOK, E. V., 1939. Influence of low temperature on recovery from roentgen rays. Radiology, 32 : 289-293. HENSHAW, P. S., 1940. Further studies on the action of roentgen rays on the gametes of Arbacia punctulata. V. The influence of low temperature on recovery from roentgen- ray effects in the eggs. Amer. J. Roent. and Rod. Ther., 43 : 921-922. LEA, D. E., 1947. Actions of radiations on living cells. The Macmillan Co., New York. PAHL, GEORGE, AND C. S. BACHOFER, 1954. Postirradiation anaerobiosis and recovery of Ascaris eggs. Radiation Res., 1: 555-556. SCHJEIDE, O. A., AND B. M. ALLEN, 1951. The relation of mitosis to the manifestation of x-ray damage in hematopoietic cells of tadpoles. /. Cell. Comp. Physiol., 38: 51-67. WEISS, J., 1952. Chemical dosimetry using ferrous and eerie sulfates. Nucleonics, 10: 28-31. RADIOCOBALT ACCUMULATION IN TETRAHYMENA JOHN V. SLATER Dept. of Biology, University of Buffalo, Buffalo 14, N. Y., and Biology Division, Oak Ridge National Laboratory x Previous studies have indicated that cobalt is essential for growth in Tetra- hymcna (Slater, 1952; Roth, 1956), but there is no information available about cation uptake in this animal. Although the major function of cobalt is believed to be that of serving as part of the vitamin B12 molecule (Marston, 1952), cobalt is also implicated as the element per se in important hydrolytic reactions (Johnson and Berger, 1942). The present experiments were designed to study the accumulation of cobalt in protozoans during growth. Elucidation of some of the factors regulating cobalt transport between the medium and the organism was also attempted. This study included the growth phase, the influence of deficient medium on exchange, the in- fluence of population density on uptake per animal, and the effect of ion concentra- tion on uptake. MATERIALS AND METHODS Strain E of Tetrahymena pyrifonnis was used in this investigation, and all the experiments were performed in synthetic medium (Slater, 1952). Calcium, uracil, and adenylic acid were omitted from the media in all instances and cytidylic and guanylic acids were reduced to 10 yugm./ml. levels. In some experiments, growth effects were eliminated by use of media deficient in essential growth factors. Cobalt-60 was used as a tracer and adjusted to 0.1 ^c./ml. (final concentration) except where indicated. Cultures were grown in 10 ml. of synthetic medium in 18-mm. Pyrex tubes, and growth was measured turbidimetrically with a Lumetron (Model 400) colorimeter equipped with a red (650 m/i) filter. Radiations were detected with a deep-well scintillation detector and a Nuclear Instrument and Chemi- cal Corporation Sealer (No. 162). Constriction chamber centrifuge tubes enabled clear separations of organisms from supernatant upon mild (100 G, one minute) centrifugation. The culture was washed with non-radioactive synthetic medium to remove excess fluid. The histidine in this medium is known to form a strong com- plex with cobalt (Burk ct al, 1946). Prior to the introduction of Co60, no cobalt was detected in the synthetic medium by ultraviolet emission spectroscopy, the porous cup technique being used. One 1 This investigation was performed in the Biology Division (Oak Ridge National Labora- tory, operated by Union Carbide Nuclear Company for the U. S. Atomic Energy Commission) while the author was a Research Participant in The Biology Division, from the University of Florida. My sincere appreciation is expressed to Drs. R. F. Kimball, William T. Burnett, Jr., and C. W. Sheppard for considerable aid and use of facilities. Dr. Norman G. Anderson was also very helpful in execution of a successful design for a constriction-chamber centrifuge tube. I am also very grateful to Dr. Cyrus Feldman for certain spectrographic analyses. 390 CO00 ACCUMULATION IN TETRAHYMENA 391 liter of synthetic medium was concentrated 500-fold by evaporation for this analysis, and it was estimated that less than 0.01 /Agm./ml. of cobalt ion was present. Radiation effects from the tracer used have, in many instances, been known to influence physiological processes. The extreme resistance of Tetrahymena to radi- ation (Elliott and Slater, 1951), however, makes it unlikely that any influence from the tracer's radiation was significant during these experiments. After the protozoans were separated from the supernatant by centrifugation, they were placed in two-mi, volumetric tubes and adjusted by micropipettes to 2.0-ml. volumes with distilled water. They were then transferred quantitatively to 120- 100- o o: 080- O m ID 0.60- UJ Q ^ 040- CL O 0.20- GROWTH K32 00 -2800 -2400 « M I -2000 | o c e 1600 -1200 800 .400 24 48 72 INCUBATION (hr) 96 FIGURE 1. Cobalt-60 uptake and release during growth in Tetrahymena. plastic tubes. Since the volume of the column of radioactive substance materially affected the number of counts registered by the scintillation detector, exactly 2.0-ml. volume adjustments were used throughout. RESULTS 1. Uptake during growth In the first series of experiments, Co60 at 0.01 /AC./ml. (final concentration) was introduced into each culture at the beginning of the experiment as a tracer for the movement of this element during growth. Spectrographic analysis revealed that the 392 JOHN V. SLATER added cobalt amounted to 3.7 /igm./ml. (final concentration). No growth effects were noticed from cobalt at this concentration in preliminary experiments. There was a steady uptake of cobalt during growth and an abrupt release of this ion shortly after the stationary phase was reached (Fig. 1). The temperature in the typical experiment reported was 27.5° ±0.5° C., and the initial inoculum from mid-log phase cultures amounted to 185,000 ± 5% animals per tube. The total up- take during 60 hours of growth amounted to 0.97 jugm. of Co per total mass of cells, or about 26% of the cobalt present. This amounted to 4.6 X 10"s% of the total co- balt present per hour per organism. Further calculations revealed that each Tetra- hyrncna at 60 hours possessed about 109 atoms of cobalt. The number of animals remained constant from the sixth to the twelfth hour after inoculation (Table I) al- though the optical density measurements increased steadily. Uptake of cobalt dur- ing this period was probably associated with the increase in volume of the individual TABLE I Cobalt-60 uptake during growth for the first 12 hours Time (hr. ) Optical density at 650 mjj Number of animals Uptake for total popula- tion (cts./min.) 0 1 2 185,000 77 208 4 252 6 0.04 479,000 381 8 0.07 459,000 489 10 0.11 457,000 743 12 0.14 466,000 806 organism. The volume of Tetrahymena reaches a maximum near the upper third of the log phase and falls off to about one-half this value upon reaching the stationary phase (Slater and Elliott, 1951). The release of Co60 during the stationary phase was studied for only two days to avoid the possibility of measuring cobalt release from disintegrating cells. Micro- scopic observation of the protozoans during this period failed to reveal any obvious morphological breakdown, although an imperceptible physiological breakdown is certainly not an impossibility. Fifty per cent of the accumulated cobalt was released into the medium in 36 hours with a rate amounting to 1.4 X 10-6%/hour/organism. 2. Effect of number of animals on uptake The influence of number of animals on uptake per animal was studied. Eight- hour periods of time were selected to minimize growth effects and any influence from the accumulation of metabolic wastes. The number of animals present had a definite effect on uptake (Fig. 2) per animal. Populations of the order of 104 ani- mals became nearly ten times as radioactive as populations of 2 X 10G organisms. At these high population densities it is not improbable that there was a great deal of competition for oxygen and also that harmful metabolic wastes resulted in inhibi- tory effects. Population densities of 2 X 105 cells, however, were far from being crowded under the experimental conditions and yet contained only one-third the ac- CO*' ACCUMULATION IN TETRAHYMENA 393 tivity of the lowest concentration. In the experiment shown, the temperature was maintained at 26.5° ± 0.5° C, and cobalt was introduced at the 0.01 ju.c./ml. level. 3. Cobalt release in deficient medium Release of cobalt was studied in media deficient in essential growth factors, salts, and glucose. Log-phase cultures were allowed to incubate initially in complete synthetic medium containing 0.01 /AC. /ml. of Co60 for 24 hours. The animals were 60 50- 25 50 75 PERCENTAGE OF 2,165,000 ANIMALS 100 FIGURE 2. Influence of number of animals on uptake per animal. then removed by centrifugation, washed once with deficient medium, and re- suspended in deficient medium. This medium was used to prevent growth effects. In the typical experiment illustrated (Fig. 3) the initial population amounted to 415,000 cells/tube. These had grown to about twice this number at the time of introduction to the "cold" medium. Two mechanisms are evident during cobalt release under these conditions. The first is very rapid and takes place within two hours. The rate of release during this time was 13% /hour/organism. The second mechanism is much slower and 394 JOHN V. SLATER amounts to 1.7% /hour/organism. Under these conditions, 50% of the cobalt was released in about 20 hours. 4. Cobalt concentration ability The ability of protoplasm to concentrate certain elements has been known for a long time. Tetrahywiena is no exception in this capacity. In the first series of ex- periments, uptake in relation to cobalt concentration was studied during 12 hours. (000 -, f> I e in I-LJ CO ui tr 100- o 2 800 LJ 1200- 400- 0.0010 0.0050 ,60, 0.0100 CONCN. OF Co*" (/AC /ml) FIGURE 4. Effect of cobalt-60 concentration on 12-hour uptake. TABLE II Influence of cobalt concentration on uptake in 12 hours Cone, of Co60 OiC./ml.) Co++ (Mgm./ml.) Cts./min. /total population Cts./min. /vol. occupied by population* Degree of cone.** cts./min. /total pop. cts./min./vol. occup. 0.0100 3.70 1104 221 4.9 0.0098 3.63 942 216 4.4 0.0096 3.55 1211 212 5.7 0.0094 3.48 1126 207 5.4 0.0092 3.40 1169 203 5.7 0.0090 3.33 1062 198 5.4 0.0080 2.96 910 176 5.2 0.0050 1.85 573 110 5.1 0.0010 0.37 117 22 5.3 0.0005 0.185 58 11 5.3 * Calculated from /ic./ml. times cts./min. //xc. times volume occupied by protozoans after 12 hours growth. One ml. containing 0.01 pc. of Co60 gives 10,500 cts./min. The volume occupied by these populations equaled 0.0212 ml. after 12 hours growth. ** Representing the concentration of cobalt by the entire population of animals over that contained in comparable volumes. 396 JOHN V. SLATER TABLE III Influence of cobalt concentration on maximum uptake Cone, of Co60 Oic./ml.) Co++ (Mgm./ml.) Max. uptake cts./min. /total population Cts./min. /vol. occupied by population* Degree of cone, cts./min. /total pop. cts./min. /vol. occup. 0.01 0.764 2064 265 7.8 0.005 0.508 1373 132 10.4 0.001 0.148 399 27 14.8 0.0005 0.061 165 13 12.7 0.0002 0.025 68 5 13.6 pH, 7.4; temperature, 25.7° ± 0.5° C. * Calculated from /zc./ml. times cts./min. /^c. times volume occupied by protozoans at maxi- mum uptake. One ml. containing 0.01 juc. of Co60 gives 10,500 cts./min. The volume occupied by these populations equaled 0.0253 ml. of maximum uptake (Slater, 1951). The influence of cobalt concentration on maximum uptake during growth was also studied. The greatest absolute amount of this ion was taken up by cultures in the presence of 0.764 ^igm./ml. of Co++ (Table III). The ability to concentrate this ion over that contained in the medium, though, reached maximum at 0.1-0.5 40 1 35- § 30 o. o 0. "5 0 25- | 20- H £ 15 u 10- 5- 0.0010 00050 O.CHOO CONCN. OFCo60(/tc/ml) FIGURE 5. Uptake of cobalt-60 with concentration at 36 hours. COm ACCUMULATION IN TETRAHYMENA 397 fj.gm./m\. of Co++. The maximum uptake was about one and three quarters times that with 0.764 /Agm./ml. of Co++. Correspondingly, the rate of uptake seemed to be linear with concentrations up to 0.508 /xgm./ml. of Co++ (Fig. 5) but was less at higher concentrations. DISCUSSION The uptake of cations by animal cells involves a complex spectrum of inter- related processes, any one of which may alter the total cellular absorptive capacity. Among these controlling factors are the rate of utilization and probably intracellular translocation. Probably the uptake of inorganic substances first involves a combi- nation with organic cell constituents (Sutcliff, 1954) ; specific metabolic pumps may also be involved. These cations then may become distributed to different cellular sites in response to varying chelation forces, which appear as the environmental mixture of cations changes. Thus during periods of major synthesis and growth, forces primarily concerned with cell divisions and external membrane changes might be most active, while during periods of relative inactivity, forces concerned with ex- change, turnover, and simple release might well become more active. Chelation forces may also be involved in mitochondrial physiology and are probably involved in ion transport in these participates. Studies of the relative binding abilities of various sites within the cell must certainly be done if the intricacies of cation trans- port and translocation are to be elucidated. In Tetrahymena, it was shown that the population density may greatly influence the uptake of cobalt per cell. At great densities, cellular chelate forces may compete for environmental cations. It is also considered likely that competition for dis- solved nutrients in general may inhibit the transport mechanism for any given cation. It was demonstrated earlier that cobalt was essential for growth in purified syn- thetic medium without glucose. The physiological role of this cation in protozoans where an extraneous source of the B12 molecule is not required is not clear. It has been suggested that cobalt ion in these experiments may act non-specifically to in- crease the availability of other cations by releasing them or displacing them from complexes (Ford and Hutner, 1955, pp. 101-136) at the cell surface (Hutner ct al.» 1950). Since Roth (1956) has shown that nickel does not have the same effect as cobalt on growth in Tetrahymena, and since the present experiments have demon- strated that cobalt is differentially bound to the animal, depending on the concentra- tion of the cation, any non-specific effect may be minimal. The uptake of cobalt during growth has been studied in Bacillus subtilis (Tanaka et al., 1952), Neurospora crassa (Ballentine and Stephens, 1951), and Saccharo- myces cerevisiae (Nickerson and Zerahn, 1949). No studies involving protozoan uptake of this ion seem to have been published. Sizable losses of cobalt were observed with advancing age of the population in all the above organisms. In both Tetrahymena and B. subtilis, the release of the ion began abruptly after the stationary phase began. A comparison of the concentrat- ing abilities of Saccharomyces and Tetrahymena showed that Saccharomyces had nearly 70 times the ability to concentrate cobalt possessed by the protozoan. Two washes of Tetrahymena did not appreciably remove the ion, and over 20 hours' washing of Saccharomyces also showed that cobalt was firmly held. Nickerson and Zerahn (1949) suggested that the presence of peripherally located metaphosphate might be significant in the accumulation of "metals" from dilute solution by yeasts. 398 JOHN V. SLATER In Neurospora (Ballentine and Stephens, 1951), at least 40% of the cobalt ac- cumulated was present in stable cobalto-proteins. Fractionation of these compounds revealed the presence of a soluble fraction comprising 57% of the stably bound cobalt and a nearly submicroscopic participate fraction. Similar cobalto-proteins were also found in Chlorclla milgaris and in the leaves of the musk melon and tomato. As with Neurospora, Saccharomyces, and B. subtilis, Tetrahymena concentrated cobalt against a concentration gradient. Scott and Ericson (1955) reported that cobalt was absorbed by the marine alga, Rhodymenia pahnata, and became bound within the plant as a complex quite differ- ent from B12. Analysis of this complex revealed the presence of several compo- nents. Thus cobalt may play a multiphysiological role in protoplasm. Ericson (1952) reported that sea weed possessed an unusual ability to absorb and concen- trate Co60 but the absorption of vitamin B12 was very limited and could not account for the concentration of the cation. In earlier work on the essentiality of cobalt for growth in Tetrahymena (Slater, 1952), it was shown that a definite response could be obtained in purified synthetic medium when as little as 0.5 /xgm./ml. of cobalt ion was present. Under the conditions of those experiments, this amount of cobalt was equivalent to about 5 X 1010 atoms of cobalt per animal. In the present experi- ments, nearly 10° atoms of cobalt were accumulated per animal, presumably for non- growth purposes since growth progressed in controls without added cobalt. Thus, in purified synthetic medium, when extraneous cations are removed to a large de- gree, nearly 50 times as much cobalt is used for growth as is accumulated when growth proceeds under the influence of other cations. In a study on cobalt localization in pooled white mouse cells, Rosenfeld and Tobias (1951) reported that most of the element was present in the cytoplasm and about 1% of it was firmly bound to cellular protein. Very little was discovered in the nuclei. Most of the cytoplasmic association was with the globulin in the bound fraction. The slowly released fraction of cobalt in Tetrahymena (Fig. 3) may be associated with a firmly bound fraction of this type, but this fraction appears to be about 75% of the total. The intracellular participate localization of cobalt remains to be elucidated. SUMMARY 1. A steady uptake of radioactive cobalt was observed during the growth of Tetrahymena in synthetic medium. When the initially-added amount of cobalt was 3.7 ngm./ml., nearly 26% of the total was accumulated during 60 hours of growth. This amounted to approximately one billion atoms of cobalt per animal. 2. Upon reaching the stationary phase, sizable amounts of cobalt were released from the population. Fifty per cent release was observed in 36 hours with a rate amounting to 1.4 X 10~6%/hour/organism. 3. The number of animals present had a definite effect on the accumulation of cobalt per animal. Populations of 104 animals became nearly ten times as radio- active as populations containing two million organisms. 4. In nutritionally-deficient media, the release of cobalt was biphasic. The first was very rapid and took place within two hours. The initial rate of release of cobalt amounted to 1 3 % /hour/organism. The second mechanism was much slower and amounted to 1.7% /hour/organism. Fifty per cent release was noticed in 20 hours under these conditions. CO00 ACCUMULATION IN TETRAHYMENA 399 5. As the concentration of cobalt was varied, the rates of uptake increased rapidly. Concentrations higher than 1.85 /xgm./ml. had little effect. The greatest absolute amount of cobalt was accumulated when 3.7 /xgm./ml. was initially present in the medium, but the ability to concentrate this ion over that contained in the en- vironment reached a peak at 0.37 /xgm./ml. of cobalt ion. LITERATURE CITED BALLENTINE, R., AND D. G. STEPHENS, 1951. The biosynthesis of cobalto-proteins by plants. /. Cell. Comp. Physio!., 37: 369-387. BURK, D., J. HEARON, L. CAROLINE AND A. L. SCHADE, 1946. Reversable complexes of cobalt, histidine and oxygen gas. /. Biol. Chan., 165 : 723-724. ELLIOTT, A. M., AND J. V. SLATER, 1951. Effect of radiations on survival in Tetrahymena. Proc. Amer. Soc. Protozool., 2 : 14. ERICSON, L. E., 1952. Uptake of radioactive cobalt and vitamin B,, by some marine algae. Chan. Indust., 34 : 829-830. FORD, J. E., AND S. H. HUTNER, 1955. Role of vitamin BI2 in the metabolism of microorganisms. In: Vitamins and Hormones, Vol. XIII. Academic Press, Inc., New York. HUTNER, S. H., L. PROVASOLI, A. SCHATZ AND C. P. HASKINS, 1950. Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc. Amcr. Phil. Soc., 94: 152-170. JOHNSON, M. J., AND J. BERGER, 1942. The enzymatic properties of peptidases. Adv. in En- zymol., 1 : 69-92. MARSTON, H. R., 1952. Cobalt, copper, and molybednum in the nutrition of animals and plants. Physiol. Rev., 32: 66-121. NICKERSON, W. J., AND K. ZERAHN, 1949. Accumulation of radioactive cobalt by dividing yeast cells. Biochim. Biophys. Acta, 3: 476-483. ROSENFELD, I., AND C. A. TOBIAS, 1951. Distributions of Co60, Cu04 and Zn"5 in the cytoplasm and nuclei of tissues. /. Biol. Cln-in., 191 : 339-349. ROTH, J. S., 1956. Studies on the function of intracellular ribonucleases. I. The action of cobalt and nickel on Tetrahymena pyriformis W. Exp. Ceil Res., 10: 146-154. SCOTT, R., AND L. E. ERICSON, 1955. Some aspects of cobalt metabolism by Rlwdymenia pal- inata with particular reference to vitamin B12 content. /. Exp. Bot., 6: 348-^361. SLATER, J. V., 1951. Growth in Tetrahymena in relation to carbohydrate metabolism. Ph.D. dissertation, Univ. of Michigan, Ann Arbor. SLATER, J. V., 1952. The influence of cobalt on the growth of the protozoan, Tetrahymena. Physiol. Zoo!., 25: 323-332. SLATER, J. V., AND A. M. ELLIOTT, 1951. Effect of culture age on the volume of Tetrahymena. Proc. Amer. Soc. Protosool., 2 : 14. SUTCLIFF, J. F., 1954. Cation absorption by non-growing plant cells. Syniip. Soc. Exp. Biol., 8 : 325-342. TANAKA, S., Y. SAWADA AND T. YAMAMOTO, 1952. Cobalt metabolism in Bacillus sitbtilis by using cobalt60. Bull. lust. Clicin. Research, Kyoto Univ., 30: 52-53. CONTRIBUTIONS TO SURVIVAL MADE BY BODY CELLS OF GENETICALLY DIFFERENTIATED STRAINS OF MICE FOLLOWING X-IRRADIATIONS x JANICE STABLER AND JOHN W. GOWEN Department of Genetics, loiva State College, Ames, loiva Our strains of inbred mice have inherited relatively stable differences in their sensitivities to absorbed radiant energy (Gowen, 1950; Gowen and Zelle, 1945; Gowen and Stadler, 1956; Grahn, 1954) from whole-body irradiation. These dif- ferences are related to fixed variations in the normal cell structures of the strains (Gowen, 1945; Gowen and Calhoun, 1943; Gowen, 1952; Weir, 1949). Progress in understanding the mechanisms of irradiation damage may be advanced if genetic differences in resistance are traced to the cells as a whole or to particular organ systems. Regional body irradiation affords a technique for localizing organs sig- nificant to radiation resistance. This technique along with surgical removal and/or shielding before organ exposure has been tried with some indications that given organs are significant to irradiation resistance. Examination of irradiation effects on genetic strains of mice having known ranges in resistance combined with partial body exposure without confounding by surgical interference offers a promising means of attacking this problem. MATERIALS AND METHODS The host constitutions in these experiments were differentiated into 5 distinct lines through 30 or more generations of brother-by-sister matings accompanied by selection for specific inherited types. The strains are homozygous albino but differ in coat color at the agouti locus. They are differentiated for resistance to Salmo- nella typhimurium, the typhoid-causing bacteria of mice. Under comparable con- ditions over a period of more than 20 years the mice of these strains maintain their relative resistances to 200,000 organisms to that observed in these experiments, S lOO/o, Z 45%, K 39%, Q 0% and Balb/Gw, hereafter called Ba, 0%. The strains differ genetically in body weight, growth rate, heart, kidney, liver, spleen and testis weights, serum globulins, leucocyte number, fixed phagocytic cells, macro- phages, and cells metabolizing fat and storing glycogen as well as other significant physiological characteristics. The environment of the animals throughout the ex- perimental period, as well as through many breeding generations, was that of a con- trolled laboratory where feed, water and management were uniform. All mice were 46 ± 3 days of age at the time of irradiation. Strains and sexes were randomly distributed across the different treatments. Thirty-seven weeks 1 Journal Paper No. J3128 of the Iowa Agricultural Experiment Station, Ames, Iowa. Project No. 1180 and 1187. This work has received assistance from Contract No. AT(11-1)107 from the Atomic Energy Commission. 400 CONTRIBUTIONS TO X-RAY RESISTANCE 401 were required to completely fill the experiment as designed. A General Electric Maxitron operated at 250 pkv, 30 ma with 0.25 mm. Cu + 1 mm. Al filtration at a distance of 47.5 cm. from anode to mid-mouse was used for the x-irradiation of all mice. The dose rate varied from 160 r/minute to 180 r/minute. Part of this range was accounted for by a change of x-ray tubes. Mice were held for x-irradia- tion in perforated plastic tubes with cork stoppers. These tubes were arranged in a wooden rack in two rows of eight tubes placed side by side with the closed ends of each row facing each other. The field of radiation covered by this rack of tubes permitted a range of only 12 r per minute over the entire area. The experiment performed and analyzed was designed as a factorial having four elements. Five inbred strains of mice having known differences in x-ray and ty- phoid sensitivities were utilized in comparable numbers. The numbers for the two sexes were balanced for each strain. There were four x-ray exposure doses : 0, 320, 480 and 640 roentgens. The levels of x-ray dosage were chosen to span the range from no effect to nearly complete lethality when the mice were exposed to whole- body irradiation. There were eight combinations of body coverage or exposure. The body of the mouse was divided into three regions — head, mid and rear. Shielding was with % inch-thick strips of lead laid across the tubes covering the region or regions of the mice for a given treatment. The eight combinations of regions exposed are shown below. Region Exposed None Head Mid Rear Head-Mid Head-Rear Mid-Rear Whole-Body The head region or anterior third of the body extended into the thorax. The middle third of the body, mid region, included that part of the body containing lower thorax, and abdominal cavity containing stomach and upper intestinal tract, liver, spleen, adrenals, ovaries and kidneys. The posterior third of the body, rear region, included the lower intestinal tract, bladder, and urinary system and testes of the males. These regions are illustrated in Plate 1. The eight groups of different regional exposures were treated with 320 r, 480 r and 640 r making a total of 24 different x-ray treatment groups. In addition the mice of one group wrere put in tubes and completely lead-covered for a time com- parable to that of the longest dose, 640 r, but were not exposed. This group acted as a control on handling as well as for un-irradiated, 0 r group. There were 25 treatment groups with 5 strains and 2 sexes making up 250 cells in the experiment. Each cell represented a different strain, sex, and treatment. A minimum of 25 mice were treated in each cell. Some cells contained a few extra animals. The completed experiment involved a total of 6904 mice. 402 JANICE STABLER AND JOHN W. GOWEN PLATE 1. Radiographs showing shielding marking off the three regions and their relation to the anatomy of the mouse. CONTRIBUTIONS TO X-RAY RESISTANCE 403 The immediate effects of x-ray were largely completed 12 days following irradia- tion. Although there were marked differences in survival times between the differ- ent strains, few deaths occurred after the twelfth day. A 15-day interval following irradiation was allowed to cover the direct effects of exposure. Deaths were re- corded daily and survivors of the fifteenth day were considered as surviving mice. The %-inch lead sheets used to delimit the body regions exposed to irradiation protected the shielded regions from the greater portion of irradiation. But this pro- tection was not complete. The ^-inch lead was sufficient to absorb all but 0.75 r per minute. The back scattering of the radiation into the shielded regions ac- counted for larger doses of radiant energy in the protected regions. Measurements made when the tubes were placed on a nylon mesh screen under the same conditions showed that the back scattering largely came from the wooden rack used to hold the plastic tubes containing the mice. Only 7.4 r or 1.2 per cent of the 640 r dose was absorbed in the lead-covered mid-region when head and rear regions were ex- posed, whereas 78 r or 12.2 per cent of the x-rays was absorbed when the wooden rack was used. The amount of radiation absorbed in the different shielded regions varied from 3 to 12.2 per cent of the dose given. The region receiving most scat- tered radiation was the mid-region when the head and rear regions were exposed. This could be expected as the scattering could enter from two directions. The amount of radiation absorbed in any shielded region should tend to magnify some- what the effects of the different treatments and reduce the differences between the treatments. The effects of scattered radiation were distributed through all treat- ment groups and tended to balance from one treatment to another. That this amount of radiation absorbed into the protected regions could lessen in some degree the recovery potentials of the mice treated in the region is recognized. The total roentgens scattered and absorbed should make this factor a minor contributor to the total variation. The results of localizing radiation to particular regions of the body have been determined by comparison of sexes, strains and dosages across all regional exposure treatments in the factorially designed experiment. Two separate criteria have been used to measure the effects of irradiation, the percentage survival of the treated mice and the length of survival in days within the observational period. Because of the unequal numbers of mice in the different treatment groups, all data have been analyzed throughout this paper by using disproportionate frequency analyses. Binomial analyses were used for the survival data and the customary methods for mean length cf survival within the observational periods. The data based on percentage survival did not allow for the full expression of the differences between strains, etc. Survival range had fixed limits at 0 and 100 per cent. These limits confined the quantitative estimates of the effects, i.e., strain or radiation, to values within this range. Consequently all conclusions drawn from the percentage survival data led to minimal estimates by the nature of these limitations of the scale. The data on length of survival were confined by one limiting measurement, the total length of the observation period. This fixed measurement again limited the full expression of the effects of irradiation on the mice. Any differences observed in both sets of data for each response were therefore minimal estimates of these con- sequences following the different treatments. 404 JANICE STABLER AND JOHN W. GOWEN EXPERIMENTAL RESULTS The main purpose of this experiment was to examine the effects of irradiation to particular organs (body regions) and to their summation of effects on the cells of the body as a whole. Two variables are intrinsic in the data. Known genetic dif- ferences may assist in these analyses by introducing reliable differences in the re- sistance levels of the mouse strains. Sex of the mice utilized in the tests could affect the results although previous experience has shown that this is a minor factor in the expression of the mouse typhoid syndrome. The effects of sex and strain will be analyzed separately. The sex effects are considered first. EFFECTS OF SEX Irradiation may cause acute effects leading to death. In the range of x-ray ex- posures of this experiment these radiation deaths were largely over by 12 days post- irradiation. Consequences of the x-ray treatments were measured by percentages TABLE I Effect of sex on radiation sensitivity. Per cent survival in 15 days Dose (r) Region exposed Mean per cent survived Variance analyses Within sex Between sex 1 d.f. Male Female d.f. M.S. M.S. F 0 None 100 100 289 — — — • 320 None 100 99 266 .004 .004 1.0 Head 100 100 270 — — — Mid 100 99 267 .004 .004 1.0 Rear 100 100 268 — • — — Head-mid 99 100 270 .004 .004 1.0 Head-rear 100 100 267 — — — Mid-rear 99 100 268 .007 .015 2.0 Whole-body 97 95 271 .035 .019 0.5 480 None 98 100 257 .011 .034 3.0 Head 100 100 258 . — • — — Mid 99 98 269 .018 .003 0.2 Rear 100 100 265 — . — — Head-mid 99 100 263 .004 .004 1.0 Head-rear 100 100 269 — — — Mid-rear 99 100 276 .007 .015 2.1 Whole-body 59 62 284 .241 .062 0.3 640 None 100 100 279 — Head 99 99 295 .010 .004 0.4 Mid 97 97 286 .027 .000 — Rear 99 100 289 .003 .004 1.0 Head-mid 92 95 284 .062 .092 1.5 Head-rear 99 99 286 .010 .003 0.3 Mid-rear 80 81 287 .159 .005 0.0 Whole-body 7 3 271 .045 .090 2.0 CONTRIBUTIONS TO X-RAY RESISTANCE 405 TABLE II Effect of sex on radiation sensitivity. Mean days survived in 15-day period Dose (r) Region exposed Mean days survived Variance analyses Within sex Between sex 1 d.f. Male Female d.f. M.S. M.S. F 0 None 15.0 15.0 289 — — — 320 None 15.0 15.0 266 0.06 0.06 1.0 Head 15.0 15.0 270 — — — Mid 15.0 15.0 267 0.00 0.00 1.0 Rear 15.0 15.0 268 — — — Head-mid 15.0 15.0 270 0.06 0.06 1.0 Head-rear 15.0 15.0 267 — — — Mid-rear 14.9 15.0 268 0.15 0.30 2.0 \Yhole-body 14.9 14.8 271 1.43 0.39 0.3 480 None 14.9 15.0 257 2.90 0.75 0.3 Head 15.0 15.0 258 — — — . Mid 14.9 14.8 269 1.20 1.02 0.9 Rear 15.0 15.0 265 — — — Head-mid 15.0 15.0 263 0.14 0.13 1.0 Head-rear 15.0 15.0 269 — — — Mid-rear 14.9 15.0 276 0.52 0.93 1.8 Whole-body 13.6 13.8 284 5.02 1.91 0.4 640 None 15.0 15.0 279 — Head 14.9 14.9 295 0.38 0.06 0.2 Mid 14.8 14.7 286 2.34 0.09 0.0 Rear 14.9 15.0 289 0.28 0.28 1.0 Head-mid 14.2 14.6 284 5.00 8.79 1.8 Head-rear 15.0 15.0 286 0.06 0.03 0.5 Mid-rear 13.2 13.3 287 14.18 0.42 0.0 Whole-body 9.4 8.9 271 9.52 12.70 1.3 of the animals which survived for 15 days following exposure and by the average lengths of survival of the irradiated groups. The data are subdivided for each sex into the x-ray dose and the region of the body in which the mouse received the ir- radiation. The strains were nearly balanced in numbers and were combined in these tests. Table I gives the data on the mice which survived the different x-ray treatments. The first column presents the x-ray dosage in air at the mid point of the mouse's body. Column 2 lists the body regions exposed to x-rays. Columns 3 and 4 give the mean survival as per cent of the mice surviving 15 days after the x-ray exposure according to the sex. The variance analyses give the degrees of freedom (d.f.) for the variances of the mice within sexes, the mean square (M.S.) between sexes with 1 d.f. and the F values for the mean squares. Throughout this paper * shows sig- nificance at the 0.05 level and ** at the 0.01 level. Table I shows that only animals which received whole-body irradiation had ap- preciable mortality. In the 15-day period no mice died in the untreated group; the 406 JANICE STABLER AND JOHN W. GOWEN 320 r whole-body exposed mice showed a few scattered deaths, 3 and 5 per cent ; the 480 r dose was more lethal, about 40 per cent of the mice died ; and the 640 r dose was almost completely lethal, only 3 and 7 per cent survived of the 273 mice treated. Consideration of sex effects on radiation survival was consequently almost entirely limited to the 480 r and 640 r whole-body irradiation groups. The differences in responses of the sexes to irradiation were small throughout the 25 comparisons. In no case was a significant difference observed. The 480 r and 640 r whole-body ex- posures were severe enough to test any real biological changes in the physiologies of the sexes suffered through irradiation exposure. No sex differences appeared. In view of these facts the data for sexes have been combined in further consideration of these experiments on survival to x-radiation. The severity of the x-ray effects may be measured by the number of days a mouse survives following x-ray exposure. Table II gives these data in the same form as Table I, with columns 3 and 4 showing the average number of days the males and females survived of the 15 days subsequent to x-irradiation. All treat- ment groups except the whole-body exposures at 320 r, 480 r and 640 r showed practically complete survival. The mean day survivals for whole-body exposures were 0 r— 15 days, 320 r— 14.9 days, 480 r— 13.7 days and 640 r— 9.1 days. The mean length of survival of the mice to whole-body irradiation was but slightly re- duced by 320 r, was lowered by 480 r and was severely reduced by 640 r. The tests for sex effects on length of survival were only critical for the 480 r and 640 r whole- body irradiation groups. The sexes showed comparable mean days of survival. In no case was there a significant difference between sexes. These results further support the conclusion that the sexes in mice are equally affected by x-irradiation. The conclusions on x-ray effects which are derived from these data will not be altered by combining the observations of the two sexes. These tests also furnish a measure of the degree of protection afforded the ani- mals by the coverage with lead plates. Animals of 0 r group were not exposed to x-rays but the bodies of the mice were completely shielded by %-inch lead plates. The unexposed and 640 r groups had 100 per cent survival. In the 320 r treatment group exposed and lead-shielded, one female out of 268 animals died ; the 480 r group had three males out of 259 mice die. Mortality at the three dose levels ap- peared fortuitous and unrelated to the irradiation. It was concluded that the pro- tection with the lead shields was adequate for all groups. GENETIC EFFECTS OF STRAINS The mice utilized in this experiment were known to exhibit differences in their abilities to withstand radiation. These differences have become isolated into dif- ferent strains. The data on the genetic effects on resistance to radiation as evi- denced by different strains within a treatment are presented in Table III. Five treatments displayed strain effects on x-ray sensitivity. These exposures were : whole-body at 320 r and 480 r and mid, head-mid and mid-rear regions at 640 r. Examination of the mean strain survivals indicates that these differences came largely from the high susceptibility of the Ba strain. All strains were affected by the whole-body exposures, and the severity increased with dose. At 640 r the effects were so severe as to have overreached strain differences as judged by the F test, even though the strains still retained their relative order of resistance. CONTRIBUTIONS TO X-RAY RESISTANCE TABLE III Genetic effects of strain differences on radiation sensitivity. Per cent survived 15 days post-irradiation 407 Dose (r) Region exposed Strains Per cent survival Variance analyses Within strains Between strains 4 d.f. s z K Q Ba d.f. M.S. M.S. F 0 None 100 100 100 100 100 286 — — — 320 None 100 98 100 100 100 263 .004 0.004 1.1 Head 100 100 100 100 100 267 — — — Mid 100 100 100 100 98 264 .004 0.004 1.0 Rear 100 100 100 100 100 265 — . — • — Head-mid 100 100 98 100 100 267 .004 0.004 1.0 Head-rear 100 100 100 100 100 264 — — — Mid-rear 100 100 100 100 97 265 .007 0.014 1.9 Whole-body 98 100 94 100 90 268 .034 0.107 3.2* 480 None 100 100 96 100 98 254 .011 0.015 1.3 Head 100 100 100 100 100 255 — — — Mid 98 100 98 98 97 266 .018 0.008 0.5 Rear 100 100 100 100 100 262 — — — Head-mid 100 98 100 100 100 260 .004 0.004 1.0 Head-rear 100 100 100 100 100 266 — — — Mid-rear 100 100 100 ' 100 97 273 .007 0.013 1.9 Whole-body 79 75 54 66 31 281 .213 2.116 9.9** 640 None 100 100 100 100 100 276 — — — Head 98 100 100 98 98 292 .010 0.005 0.5 Mid 100 100 95 100 92 283 .026 0.082 3.1* Rear 100 100 100 100 98 286 .003 0.003 1.0 Head-mid 98 98 96 100 75 281 .054 0.633 11.7** Head -rear 100 100 98 98 98 283 .010 0.006 0.5 Mid-rear 92 96 71 98 49 284 .124 2.624 21.1** Whole-body 11 4 4 6 0 268 .045 0.085 1.9 Strain differences were separated most clearly when the animals were exposed to the 640 r. These strain differences appeared in all treatments. The 480 r treated mice showed the strain effects only in the whole-body irradiated class. The 320 r treated groups were at the border line where a reduction in survival was only beginning to appear in the whole-body treated mice of the most susceptible strain. The \vhole-body 480 r irradiations ordered the strains for resistance ; the S was most resistant followed in order by the Z, Q, K and the most susceptible Ba. In the 640 r dose range S, Z and Q strains were not very different in their re- sponses. The Ba strain again showed the greatest radiation sensitivity with the K strain somewhat less sensitive than the Ba and noticeably less resistant than the other three strains. The mean days of survival for the 15-day interval following irradiation and the analyses of the strain differences are presented in Table IV. 408 JANICE STABLER AND JOHN W. GOWEN Table IV confirms the major features of Table III. Strain differences in re- sistance to irradiation were brought out in the 480 r whole-body treatment and with 640 r exposure when the mid, head-mid, mid-rear and whole body were treated. The significance tests identify a difference in radiation effects which made the two measures, per cent survival and length of survival, desirable. The 320 r whole-body exposure was sufficient to make the strain differences in per cent survival significant, whereas the mean length of survival did not show such differences. The mice that died, died late in the 15-day observation period. Following 640 r whole-body ex- posure, the mice showed only an insignificant difference in strain survivals even though all survival values were markedly reduced, whereas in length of survival the strain differences were accentuated to give a highly significant F value. The other three treatments, the mid, head-mid and mid-rear regions, suggest the mid region as the most sensitive to radiation damage. TABLE IV Genetic effects of strain differences on radiation sensitivity. Mean days survived in 15 days Dose (r) Region exposed Strains Mean days survived • Variance analyses Within strains Between strains S z K Q Ba d.f. M.S. M.S. F 0 None 15.0 15.0 15.0 15.0 15.0 286 — — — 320 None 15.0 14.9 15.0 15.0 15.0 263 0.06 0.07 1.1 Head 15.0 15.0 15.0 15.0 15.0 267 — — — Mid 15.0 15.0 15.0 15.0 15.0 264 0.00 0.00 1.0 Rear 15.0 15.0 15.0 15.0 15.0 265 — — — Head-mid 15.0 15.0 14.9 15.0 15.0 267 0.06 0.06 1.0 Head-rear 15.0 15.0 15.0 15.0 15.0 264 — — — Mid-rear 15.0 15.0 15.0 15.0 14.8 265 0.15 0.28 1.9 Whole-body 14.8 15.0 14.9 15.0 14.5 268 1.41 2.30 1.6 480 None 15.0 15.0 14.8 15.0 15.0 254 0.29 0.52 1.8 Head 15.0 15.0 15.0 15.0 15.0 255 — — . — Mid 14.9 15.0 14.8 15.0 14.7 266 1.20 0.91 0.8 Rear 15.0 15.0 15.0 15.0 15.0 262 — — — Head-mid 15.0 14.9 15.0 15.0 15.0 260 0.14 0.15 1.1 Head-rear 15.0 15.0 15.0 15.0 15.0 266 — . . — - — Mid-rear 15.0 15.0 15.0 15.0 14.7 273 0.52 0.84 1.6 Whole-body 14.4 14.2 13.4 14.2 12.2 281 4.44 45.52 10.3** 640 None 15.0 15.0 15.0 15.0 15.0 276 — — — Head 14.9 15.0 15.0 14.9 14.9 292 0.38 0.20 0.5 Mid 15.0 15.0 14.6 15.0 14.3 283 2.26 6.81 3.0* Rear 15.0 15.0 15.0 15.0 14.9 286 0.28 0.26 1.0 Head-mid 14.9 14.8 14.8 15.0 12.8 281 4.32 53.74 12.4** Head-rear 15.0 15.0 15.0 15.0 15.0 283 0.06 0.03 0.5 Mid-rear 14.4 14.7 12.4 14.8 10.2 284 11.01 235.54 21.4** Whole-body 10.6 10.4 8.8 10.4 5.4 268 5.72 265.42 46.4** CONTRIBUTIONS TO X-RAY RESISTANCE 409 TABLE V Effects of x-ray dosage to different body regions on survival 15 days after irradiation Region exposed Variance analyses Doses 3 d.f. Strains 4 d.f. Dose XStrain 12 d.f. Within dose and strain M.S. F M.S. F M.S. F d.f. M.S. Percentage survival for 15 days post-irradiation None Head Mid 1 No analysis — Practically all survived No analysis — Practically all survived .048 3.9** | .044 3.6** .017 1.4 1099 .012 Rear Head-mid No anal' .291 ysis — Prac 18.6** tically all .156 survived 10.0** .162 10.3** 1094 .016 Head-rear Mid-rear No anal' 2.66 /sis — Prac 75.3** ticallv all .873 survived 24.7** .593 16.8** 1108 .035 Whole-body 54.00 734.9** .869 11.8** .480 6.5** 1104 .074 Mean days survived in 15 day post irradiation period ' 1 None No analysis — Practically all survived Head No analysis — Practically all survived Mid 4.08 4.7** 3.27 3.7** 1.49 1.7 1099 .88 Rear No analysis — Practically all survived Head-mid 22.12 19.1** 13.56 11.7** 13.46 11.6** 1094 1.16 Head-rear No analysis — Practically all survived Mid-rear 220.15 73.7** 72.83 28.4** 54.61 18.3** 1108 2.99 Whole-body 2078.36 726.7** 131.31 45.9** 60.64 21.2** 1104 2.86 The mean days of survival of the five strains for the 25 different treatments showed somewhat less differentiation in the reactions of the strains to x-irradiation. The head-mid-rear exposure to 640 r showed the widest separation between Ba and K and these from the other three, S, Z, and Q strains. The order of the strains in resistance to radiation effects did not correspond to the order of these same strains in their natural resistance to mouse typhoid as noted earlier. REGIONAL EFFECTS OF IRRADIATIONS AND GENOTYPES Three elements were operating on the survival of the mice in this experiment, dosage of the x-rays, the region of the body exposed to x-rays and genotype as rep- resented by strain of mice under treatment. Table V measures these effects first in terms of the mice surviving for 15 days following irradiation and second, the lower half of the table, in terms of mean length of survival within the 15-day period. The data of Table V are presented for the body regions exposed, eight different categories in all. Of the eight groups four have not been analyzed as the deaths within these groups were few and scattered. When the mid region only was exposed to x-rays, differences in dosage and in strains were evident and of equal significance. The dosage X strain interaction 410 JANICE STABLER AND JOHN W. GOWEN was minor. Interaction between dosage X strain was a factor in survival when radiations were absorbed in the head-mid portion of the body. Both dosage and strain effects were large, with the dosage effects nearly double those of the strains. The interaction, however, was as large as the strain effects, indicating that the strains reacted differently to the different exposures. X-rays to the rear two-thirds of the body gave even more noticeable effects. The effects of the dosages were markedly greater than the strain differences and the strain effects showed more than random deaths. The whole-body irradiations, those involving the head, mid and rear regions, were more severe than those of other treatments. The x-ray effects were so pronounced at the 640 r level (Table III) that they overshadowed the strain differences. The strain effects were clearer in the 480 r and 320 r whole-body treatment groups. The data on length of survival within the 15-day interval following irradiation show essentially the same features as those for survival. Irradiation to the head- mid portion of the body was not as effective as that to the mid-rear portion, but both showed more change than when the x-rays were directed to the mid region alone. The mid region was sensitive but added irradiation to the rear or head regions increased the sensitivity. Irradiation to all regions leaves the body with no unexposed tissue. Under these conditions the survival values were materially reduced over those of all other types of treatment. QUANTITATIVE ANALYSES OF REGIONAL EFFECTS OF X-RAY EXPOSURE Quantitative estimates of the relative sensitivities of the different body regions were obtained by relating the survival values within the x-ray dosages for the different treatments. The basic theory was as follows. A factor common to mice of each strain was assumed to represent the natural resistance of the strain. This factor was considered as alone responsible for the survival values attained by the unexposed control groups. It was common to mice of all groups before treatment and was in a sense the potential resistance of the strain against which the x-ray or other treatments operated to reduce viability. The factor was designated a. Irradiation to the head region contributed a factor, h, to extend or reduce life. Its value depended upon the dosage of radiation to the head but within any one dosage its effect was regarded as a constant. In the same manner irradiation effects to the mid region were regarded as due to a factor, m, and those to the rear region were considered due to a factor, r. When two regions were irradiated the effects of radiation were assumed to be additive, i.e., h + m for total effects of irradiation to the head-mid regions. Any unexposed cells within the body would contribute possibilities of continuing normal functions. Even a small fraction of the cells having normal functions might be of vital impor- tance to the mouse. In consequence it was assumed that when the whole body was irradiated there were effects above and beyond those of h + in + r. These effects were represented by d. The full effects of whole-body irradiations were viewed as due to h + m + r + d. The d factor measured the importance of even a fraction of the body cells being normal in function. A system of eight equations was available for the analysis of these effects as expressed in the eight regional body treatments. CONTRIBUTIONS TO X-RAY RESISTANCE 411 a = value of control, unexposed mice a + h = value of control + head exposed a + m — value of control + mid exposed a + r — value of control + rear exposed a + h + m = value of control + head and mid exposed a -f- h + f — value of control + head and rear exposed a + m + r = value of control + mid and rear exposed a -\- h -}- m -\- r -{- d = value of control + whole body exposed The values for a, h, m, r and d were obtained from the system of five simul- taneous equations derived from the above by the method of least squares. These simultaneous equations are presented below. The P values in the equations were obtained as the sums of the observed values. Wherever a given factor entered into the basic equations it contributed to the corresponding P, i.e., Pl = the sum of the constants for all eight, P2 = the sum of four equations where h entered, etc. The general solution for each of the constants was as follows : a = 1/8 (5 Pi -- 3P2 -- 3P3 -- 3P4 + 4 P5) h = 1/8 (-3 Pi + 5 P2 + 1 Ps + 1 P4 -- 4 P5) m = 1/8 (-3Pi+ 1P2 + 5P3+ 1P4 -- 4P6) r = 1/8 (-3 Pi + 1P2+ 1P3 + 5P4 -- 4P5) d = 1/2 (1 Pi - - 1 P2 - - 1 P3 - - 1 P4 4- 4 P5) The constants for the effects of irradiation, as measured by percentage survival, for different intensities to the different regions are shown in Table VI. The con- stants fitted the observations well as shown by the variations accounted for by the five factors as against the residual variation left after the fits were made. As expected the severity of the effects of the x-rays to the different regions increased as dosage increased. The 320 r dose was not severe enough to separate the effects of exposure to particular body regions. The whole-body treatment even at this level of exposure showed the reduction in survival, d, to be greater than can be accounted for by the additive effects of h + in + r; d may be thought of as representing the recovery potential when any unexposed cells were present within the animal's body. In the 480 r dose range the separation of body regions by irradiation effects was clearer. The values of m confirmed the greater sensitivity of the mid region in lowering the survival rates of the mice following x-irradiation. The head and 412 JANICE STABLER AND JOHN W. GOWEN TABLE VI Effects of x-irradiation to particular body regions on percentage survival of 5 different strains of mice Constant Dose (r) Strains Average S z K Q Ba a 320 100 100 100.23 100 99.99 100.04 480 99.55 100.25 99.54 99.53 99.54 99.68 640 100.83 100.69 103.04 100.03 106.84 102.29 h 320 0 0 -0.68 0 0.88 0.04 480 0.45 -0.75 0.46 0.47 1.29 0.38 640 .01 -0.25 2.96 -0.92 -1.51 0.06 m 320 0 0 -0.68 0 -1.76 -0.49 480 -0.45 -0.75 -0.46 -0.47 -2.13 -0.85 640 -3.29 -2.07 -13.40 -0.04 -29.23 -9.61 r 320 0 0 0.23 0 -0.87 -0.13 480 0.45 0.25 0.46 0.47 -0.37 0.25 640 -2.47 -1.13 -9.91 -0.98 -14.63 -5.82 d 320 -1.75 0 -4.65 0 -8.41 -2.96 480 -21.05 -24.00 -45.90 -34.48 -67.30 -38.55 640 -84.37 -93.59 -78.91 -92.53 -61.46 -82.17 Variations in survival accounted for by the constants Dose d.f. Mean squares S z K Q Ba 320 r ace. 5f res. 3ft 15,935 0 16,000 0 15,711 1.7 16,000 0 15,404 1.0 480 r ace. 5 res. 3 15,175 0.5 15,045 0.5 14,512 0.6 14,783 0.6 13,923 1.5 640 r ace. 5 res. 3 13,574 9.8 13,784 1.5 12,547 73.4 13,783 1.1 11,004 196.0 f ace. = Variation accounted for by the different regions. ft res. = Residual or unaccounted for variation. rear regions appeared of almost equal resistance in the strains. With the two excep- tions of the h in the Z strain and r in the Ba strain, the h and r values for the other four strains showed a slightly stimulatory effect on viability. When the whole body was exposed to the 480 r x-rays the effects attributable to d were very severe. Since the effects as measured by the h, in and r values were not extreme and were quite consistent in the five strains, the order in magnitude of the d values for the five strains represented the levels of resistance of the strains to x-irradiation. The S mice were most resistant followed by the Z, Q, K and Ba in that order. The effects of irradiation were more marked in all regions when the exposure CONTRIBUTIONS TO X-RAY RESISTANCE 413 dose was 640 r. Except in the Q strain irradiation to the mid region, in, resulted in the severest reaction. The r values showed the rear region to be intermediate in resistance to irradiation. The head region as measured by h was least sensitive. The d values again portrayed the severity of exposure to x-rays in the absence of any unexposed cells or organs. The order of the strains in resistance to irradiation as observed from the d values with 480 r exposure and to a lesser degree with 320 r was not followed by the 640 r dose. As the h, m or r effects became greater the values of the d effects were decreased due to the limited range in which these con- stants operate. In the Ba strain survival from radiation had full expression from 0 per cent survival from whole-body exposure to 100 per cent survival for the un- irradiated controls. The sensitivity of the mid region accounted for 29 per cent of the mortality. The sensitivity of the rear region accounted for 1 5 per cent more and that of the head region only 1.5 per cent. The d effect contributed 61 per cent additional mortality and was restricted by the 0 limit for survival. The variation left unaccounted for after fitting the five constants was practically negligible when compared with that accounted for. The increased, but still minor, variation observed for the Ba strain at the 640 r exposure can best be attributed to the limitations of the scale for death and survival. A like analysis of the data on length of survival (Table VII) added a little information to that already gained (Table VI). The values for d were more con- sistent with the innate resistance levels of the strains than were those based on percentage survival because the scale of measurement was not so restricted. How- ever, at the lower x-ray doses the length of survival did not give more information than the percentage data because most mice lived the full 15 days. The mid region was again most susceptible to the x-rays with the rear next. Irradiations to the head showed little effect. DISCUSSION The analyses of these data showed that sex had only inconsequential acute effects on the response of mice to irradiation. This observation agrees with that of Abrams (1951) for whole-body irradiation. Kaplan and Brown (1952) also found like reactions of the sexes in an experiment involving 1700 mice when sexes were equally distributed across several x-ray doses and fractions of the doses. Sex differences may be greater when life span effects are considered. Comparisons of the mice without regard to strain show that the lethal effects of the whole-body x-rays increase with increase in kilovoltage, the 600 r at 250 pkv and 0.25 Cu + 1 Al filter being about as lethal as 960 r at 100 pkv Coolidge tube without filters (Gowen and Stadler, 1956). Differences between the responses of the five strains of mice to x-irradiation were evident. Strain differentiation depended upon the x-ray dose. The more susceptible strains, Ba and K, reacted to the lower dose, 320 r, which had no effect on the survival of the S, Z and O strains. As the exposure was increased to 480 r and 640 r the five strains were more clearly separated in their resistance levels. After exposure of the whole body to 480 r, 79 per cent of the S mice survived with a mean of 14.4 days. They were followed in order by Z, 75 per cent, 14.2 days; Q, 66 per cent, 14.2 days; K, 54 per cent, 13.4 days and Ba, 31 per cent with 12.2 days survival. The higher x-ray exposure of 640 r (250 kvp) delivered to the 414 JANICE STABLER AND JOHN W. GOWEN TABLE VII Effects of x-it 'radiation on body regions as measured by length of survival Constant Dose (D Strains Average S Z K Q Ba a 320 15.00 15.00 15.01 15.00 15.00 15.00 480 14.96 15.02 14.95 14.99 14.95 14.97 640 15.06 15.06 15.24 15.01 15.64 15.20 h 320 0 0 -0.03 0.0 0.02 0.00 480 0.03 -0.04 0.05 0.01 0.11 0.03 640 0.01 -0.03 0.34 -0.01 -0.06 0.05 m 320 0 0 -0.03 0 -0.06 -0.02 480 -0.03 -0.04 -0.05 -0.01 -0.18 -0.06 640 -0.24 -0.18 -1.15 -0.06 -2.74 -0.87 r 320 0 0 0.01 0 -0.05 -0.01 480 0.03 0.01 0.05 0.01 -0.02 0.02 640 -0.19 -0.08 -0.84 -0.08 -1.36 -0.49 d 320 -0.21 0 -0.11 0 -0.41 -0.15 480 -0.60 -0.73 -1.56 -0.83 -2.62 -1.27 640 -4.03 -4.32 -4.76 -4.50 -6.04 -5.53 Variations in survival accounted for by the constants Dose d.f. Mean squares S Z K Q Ba 320 r acc. 5f res. 3ft 360 0 360 0 359 0 360.0 0 356. .00 480 r acc. 5 356 355 350 355 341. res. 3 0 0 .01 0 .01 640 r acc. 5 333 334 312 335 277. res. 3 .06 .01 .61 .00 1.80 f acc. = Variation accounted for by the different regions, ft res. = Residual or unaccounted for variation. whole body was severe enough to appreciably narrow the range between the two extreme strains, S and Ba. The Z and Q strains interchanged positions in rank of resistance as measured by percentage survival, but were equal when the degree of severity was measured by length of survival. The strain differences were ex- pressed only in the treatment groups where the radiation was of sufficient intensity to reduce survival of the more sensitive strains (Tables III and IV). The strains responded differently to the graded x-ray doses as shown in Table V. The inter- actions between x-ray doses and strains in the exposure treatment groups were about equal to the effects of the strains alone. Each strain appeared to have a reaction curve of its own. CONTRIBUTIONS TO X-RAY RESISTANCE 415 This genetic differentiation of the strains in their response to radiation has been expanded by more recent observations on the LD50 values for 15 days under the same conditions of x-irradiation. The actual x-ray doses required to reduce sur- vival of each strain 50 per cent were determined by exposing mice of different strains to a range of x-ray doses. The LD50 values obtained experimentally for whole body irradiations were S, 537 r ; Q, 528 r ; Z, 522 r ; K, 481 r and Ba 438 r. From this information the Q and Z strains were more nearly alike in radiation sensitivity than was indicated in the 480 r whole-body treatment group of the experiment under discussion, but comparable to the levels determined by the 640 r exposure. The genetic differentiation of these five strains of mice in their resistance to x-irradiation confirms the observations of Gowen and Zelle (1945) on some of these same strains and by Henshaw (1944b) on other strains. Henshaw found that CoH mice were more sensitive than LAF, mice to whole-body irradiation as o J measured by survival and the effects on the blood picture. Kaplan and Paull (1952) showed differences between strains A and C57 black to radiation response. Differences between four strains were shown by Reinhard et al. (1954) with mini- mal lethal doses of x-irradiation. The four strains ranged in MLD values from 570 r for the Marsh strain to 492 r for C,H. Grahn (1954), in this laboratory, observed genetic differences between six strains of mice (including S, Z and Ba) in radiation response as related to body weight changes. With respect to dosage relationships these observations are in agreement with the findings of many other investigators. Heineke (1905) reported that increased x-ray exposure reduced the efficiency of the blood-forming organs in mice. Lawrence and Tennant (1937) concluded that length of life of Swiss mice following x-irradiation was directly related to the dose given. As dose was increased they noted the increased fre- quency of diarrhea in the mice. Like conclusions were reported by Osborne et al. (1952) and by Kaplan and Brown (1952). This latter work involved adequate samples of mice for each of nine x-ray doses, 283 r to 1131 r, given in single ex- posures and in fractions of these total doses. From the single exposures mortality increased from 4 per cent at 283 r to 83 per cent at the 566 r dose level. In this work the S, Z, K and Ba strains maintained the same order in both radiation response and in subsequent response to mouse typhoid. This experiment introduces a new strain, Q. The Q strain showed a difference in its reactions. It was quite resistant to x-rays but extremely susceptible to mouse typhoid. Q mice did not fit in the observed pattern of the other mice with respect to the two responses. The leucocyte level for the Q strain has not been determined but is of real interest. The level of the white blood count of this strain would be indica- tive of the causal relationship of leucocytes to disease resistance (Weir et al., 1953 ; Gowen and Calhoun, 1943) or to radiation sensitivity (Gowen, 1952). The K strain, not included in the six strains previously tested, followed in line with the other strains in both disease resistance and numbers of leucocytes (Thompson, 1952). Differences in regional sensitivities to x-rays became evident when the body regions and combinations thereof were irradiated. The order of increasing sensi- tivity was head, rear, head-rear, mid, head-mid, mid-rear and head-mid-rear or whole-body. The dosages were not adequate to appreciably affect survival when the head, rear or head-rear were x-rayed. Only those four groups in which the 416 JANICE STABLER AND JOHN W. GOWEN mid third was involved gave noticeable differences for the strains and dosages. Comparison of the mid-rear to the head-mid indicated a greater radiation sensitivity of the rear third as contrasted with the anterior third of the body. The reduction in survival and length of survival was between four and five times as severe fol- lowing exposure of the head-mid as it was following that of the mid alone. The mid- rear reaction was around 16 times as severe as that of the mid alone. The increased reduction was affected by the particular regions rather than by the pro- portion of the body exposed. Although no attempt was made in this investigation to associate specific organs to radiation sensitivity, specific organs or tissues were implicated by their inclusion within a given region as shown in Plate 1. The posterior third of the body implied the intestines, testes, bladder, etc., whereas the mid portion included the spleen, liver, etc. Our observations on the sensitivity of the mid-rear region in part con- firmed those of Warren and Whipple (1922). They found in dogs that the ab- domens, comparable to the rear plus a good portion of the mid region in these data, were more sensitive to x-irradiation than the head and thoracic region. They attributed this increase in mortality from x-rays to severe toxemia and septicemia enhanced by exposure of the intestines. Bond ct al. (1954) arrived at similar conclusions. Chrom (1935) separated the effects of exposure to the rear region from those to the rest of the abdomen. He concluded that the rear region, including the intestines, was not as sensitive as the upper part of the abdomen. These data also supported the conclusion of Osborne et al. (1952) that bacteremia and intes- tinal damage were not closely correlated. The observations of these investigators were supported by those which have been derived from our studies. The greater sensitivity of the mid region as shown in our data may be related to the response of the lymphoid tissues, spleen and nodes. Heineke's (1905) ob- servations, supported by those of Lawen (1909), showed the blood and blood- forming tissues to react strongly to x-radiation in rats, rabbits, mice and guinea pigs. Further emphasis on the hematopoietic tissues in radiation response as well as in recovery conies from the investigations of Lawrence and Tennant (1937), Ellinger (1945), Henshaw (1944a, 1944b, 1944c), Bloom and Jacobson (1948) and Barrow and Tullis (1952) to name but a few. Again the data of this paper may be interpreted as indicating the significance of the proper functioning of these organs as they affect survival. The important role of the spleen, a center of hematopoiesis, has been demon- strated by the increased survival obtained by shielding this organ from x-radiation (Jacobson, 1954; Wissler ct al, 1953; and Bond et al, 1950) and by the partial protection afforded irradiated animals by injections of splenic or bone marrow tissue homogenates (Jacobson, 1954; Cole and Ellis, 1953; Lorenz ct al, 1952; and Barnes and Loutit, 1955). Again the behavior of these organs under irradia- tion has parallel significance to the data on x-ray survival presented in this paper. The quantitative interpretation of these data is, however, somewhat different. The authors cited above have tended to consider each organ studied as all-important to irradiation survival. Our data show that while the different regions were sig- nificant, their effects on survival were less impressive than these other investigators suggested. The fitting of constants to the regional body effects offers a new approach to the evaluation of radiation effects to different regions of the body. This quantitative CONTRIBUTIONS TO X-RAY RESISTANCE 417 estimation of regional effects confirms our previous observations that exposure of the head region or anterior third of the body is less effective in reducing survival than either of the other regions. X-ray doses in the range used did not appear to influence this minor effect. Exposure of the rear region was shown to be detri- mental to survival at the higher exposure level of 640 r. This effect was consistent in the five strains although of minor importance to the Q strain. Irradiation to the mid region showed the greatest consistent reduction in survival for all strains. Increase in dosage increased the severity of the reaction in the strains. Greater mortality was observed in the more susceptible strains, Ba and K. Only the Q mice showed a different reaction ; exposures of the rear or head were nearly as detrimental as those to the mid region, although the effects of the three regions were small. The greatest reduction in survival resulted from whole-body irradiation. The amount of this reduction as measured by the constant, d, was above that due to the combined effects of head, mid, and rear exposures. The d values represent that percentage of the total mortality that resulted when all cells of the body were exposed and is in addition to the mortality resulting from the combined exposures to the three portions of the body. The magnitudes of these d values particularly after 640 r, but also after 480 r, indicate the importance of at least some unexposed cells in facilitating the recovery of the irradiated animal. The large differences between the combined effects of the three regions as compared to the body irradia- tion, d, suggest that any unexposed cells contribute materially to the protection of the mouse from irradiation. This was also indicated by the work of Gershon- Cohen et al. (1951), who showed that shielding areas comparable to 15 per cent of the total body area of mice resulted in reduced mortality from radiation. Almost equal protection was afforded the mice by shielding the liver, or lung or abdomen. They concluded that viability of the animal was increased by shielding any part of the hematopoietic system and was not confined to special organs. Jacobson et al. (1951) compared the hematopoietic recovery in mice irradiated with regions or organs shielded from exposure to 1025 r. Shielding of the spleen gave the greatest increase in survival as well as in hematopoietic recovery. Shielding of the liver lobe or portion of the intestines increased survival, but to a lesser degree. These results were associated with only somewhat less recovery of hematopoiesis. How- ever, shielding of the head gave nearly the same increase in survival as that of the intestine, but recovery in blood formation was only partial. The protection of the right hind limb, but not of the kidney, was also beneficial to survival. The results from the head and intestine shielding point to the influence of cells, tissues or systems, other than those involved in hematopoiesis, as contributing to the radiation response. Although injections of suspensions of splenic tissues con- tributed most, bone marrow and liver tissues as well as body tissues of embryos contributed to recovery of the irradiated host (Jacobson ct al., 1955). Our own observations do not rule out a major role for the hematopoietic system, but the increased severity of radiation to the whole body over that to the regions suggests that any cells, regardless of their apparent morphological specificity, could stimu- late recovery. The scattered radiation absorbed in the shielded regions would tend to increase the effects to the exposed regions. Consequently the presence of scattered radiation would tend to decrease the d value as obtained here (Table VI). In all cases the Ba mice show the largest effects of the regional exposures ; the 418 JANICE STABLER AND JOHN W. GOWEN d values have been minimized, however, by the limitation of 0 per cent survival. The K strain as previously shown is next in susceptibility to radiation followed by the Q, Z and S strains. The resistance of the S strain appeared related to the resistance of its cells to better withstand radiation even though exposures to the mid and rear regions were more detrimental to survival of the S mice than to the Z or O mice. The resistances of the Z and Q strains appeared to be determined by the interactions of all cells and regions. The increased susceptibility of the K and Ba strains was contributed to by the proportionately greater sensitivity of the mid and rear regions. The five strains, however, showed quite similar reactions but to different degrees in their responses to x-irradiation. These results were in contrast to those of Reinhard et al. (1954). They found marked differences in four strains of mice in radiation sensitivity of the head as compared to that of the remainder of the body. Kaplan and Paull (1952) also showed strain differences between A and C57 black mice in the results of spleen shielding. Protection of the spleen was more important to A mice than to C3H mice. This observation of the A strain was in accord with that of Lorenz et al. (1952). They observed differ- ences between four strains of mice in their responses to the protection afforded by bone marrow cell suspensions. Intravenous and intraperitoneal injections of the homogenate increased survival for two strains, L and LAFt. For strains A and C3H intraperitoneal injections were of little value in decreasing mortality. The data indicate the importance of maintaining at least some cells free of irradiation if the organism is to survive. The body cells retain a significant toti- potency which contributes to maintaining the organism as a whole even though the cells may have differentiated to extreme types anatomically or physiologically. SUMMARY AND CONCLUSIONS 1. The influence of x-irradiation absorbed in three body regions and in the combinations of these regions has been measured by three subsequent responses : survival to radiation, natural resistance to disease and ability to acquire resistance following contact with the disease agent, 5. typhimurium. The effects of irradia- tion are presented in this paper. Papers on natural and acquired resistance will follow. The experiment was designed as a factorial with five genetically differ- entiated strains of mice, S, Z, K, Q and Ba ; four levels of radiation : 0 r, 320 r, 480 r and 640 r ; eight treatment groups and two sexes. All mice were 46 ± 3 days of age when irradiated from a 250 pkv x-ray source operated at 30 ma with 0.25 mm. Cu + 1 mm. Al filter at a dose rate averaging 170 r/minute. For the initial treatment the strains and sexes were well balanced, at least 50 mice in each of the 25 different treatment groups. The bodies of the mice were marked off in three regions, head h, mid in, and rear r, each comprising one-third of the body length. These regions, their combinations and their controls with each irradiation account for the 25 treatment groups. Shielding was done with ^-inch lead. As most deaths occur between 7 and 12 days, an interval of 15 days was allowed for expression of any direct effects due to radiation. Deaths were recorded daily. 2. Percentage survival and length of survival were the two measurements used for determining the reactions in each response. 3. The sexes responded in like manner to x-irradiation. A penetration or wave- length effect was indicated in these data. The reactions of the mice to the whole CONTRIBUTIONS TO X-RAY RESISTANCE 419 body irradiation at 250 pkv, 0.25 Cu + 1 Al filter 600 r were similar to those for 100 pkv, Coolidge tube, no nitration, 960 r. 4. Within the x-ray dose range used the responses of the strains to x-irradiation were shown to be partially genetically determined. 5. The levels of radiation resistance were in the order from resistant to sensi- tive : S, Z, Q, K and Ba. After 480 r total-body exposure the survival percentages of the five strains were: S, 79; Z, 75 ; Q, 66; K, 54 and Ba, 31. This order does not coincide with the order known to be followed in natural resistance to mouse typhoid : S, Z, K, Q and Ba. 6. Shielding of one-third of the body protected the mice of the five strains from 320 r and 480 r x-radiation, and to much lesser degree, depending upon regional exposures, from 640 r. The dose of 640 r was not of sufficient intensity to allow full expression of strain differences for the different regional exposures. 7. Whole-body exposure to 320 r reduced the 15-day survival for the more sensitive strains Ba and K, 480 r decreased survival in the five strains ; 640 r was severe enough to largely overcome the genetic differences between the strains. 8. The mid region of the mouse was most sensitive of the three single regions, and more sensitive than the combined head and rear regions. The radiation effects were determined by the region rather than by the area of the body exposed. 9. The mid region in combination with the rear region showed greater sensi- tivity than the head-mid region. All strains were reduced in survival by exposure of the mid-rear to 640 r, whereas only the less resistant strains Ba and K showed the effects from the exposures of the less sensitive regions. 10. The decrease in survival showed the mid region as most sensitive for S, Z, K and Ba, followed by the rear portion with the anterior third of the body resistant. These four strains responded in the same manner but to different degrees. 11. The reactions of the Q strain separated it from the four other strains. Its level of radiation resistance with respect to the other strains was in contrast to its low level of natural resistance to mouse typhoid. Radiation resistance and natural resistance to this disease have been found highly correlated in seven of our strains of mice. The Q mice show comparable though slight sensitivity to x-radiation in the three body regions. Mortality in the Q strain was largely confined to whole- body exposures of 480 r and 640 r. This suggests that the Q mice have no par- ticular center of radiation sensitivity, but that mortality is the result of the inter- actions of the cells throughout the body. 12. The data on length of survival confirmed the results from percentage sur- vival and contributed additional information for those reactions that resulted in 0 or near 0 per cent survival. 13. The lead shielding, ^-inch in thickness, was adequate to protect the given regions from radiation. The three groups completely shielded when exposed to the three dosages of x-rays did not quite duplicate the 0 r group in their reactions. Mortality appeared unrelated to the x-ray dose, as 100 per cent survived 640 r, 98.4 per cent the 480 r and 99.6 per cent the 320 r. 14. The strains exhibited their own characteristic responses to different x-ray doses as was evidenced by the large values for dosage X strain interactions. These interactions were real, representing the expressions of genetic resistance and as such would contribute to the strain effects. 420 JANICE STABLER AND JOHN W. GOWEN 15. The effects of the relative sensitivities of the body regions were estimated quantitatively as well as qualitatively. The quantitative estimates compared favor- ably with the qualitative observations. 16. The additivity of the regional effects is supported by the little unaccounted- for variation remaining after fitting the constants derived on the assumption that h, m, r and d were additive in effect. 17. Mortality from whole-body irradiation was only partially accounted for by the combined mortalities resulting from the exposures to the different regions of the body. The effect of total-body exposure over and beyond that of the combined regional effects, d, was interpreted as a measure of the reaction when all cells of the body of the mouse had been exposed, or when all recovery potential had been affected. 18. The whole-body effect, d, was large and suggested that all cells may con- tribute to recovery regardless of the organ or system involved. As a consequence, protection of any cells of the body during exposure to radiant energy may stimu- late recovery. 19. In terms of host resistance the unexposed cells over-compensate. The extreme over-compensation initiated by cells in different unexposed regions when cells of other regions are inactivated points to the significance of all body cells in resistance whatever their degree of tissue or organ differentiation. 20. These results indicate that the body cells retained a totipotency to assist in maintaining the organism as a whole despite the differentiation which these cells may have undergone since their stem cells left the embryologically differentiating primitive tract. They further show the importance of maintaining at least a small portion of the body free from irradiation if irradiation exposure should occur through accident or calculated risk. LITERATURE CITED ABRAMS, H. L., 1951. Influence of age, body weight and sex on susceptibility of mice to lethal effects of X-radiation. Proc. Soc. Ex p. Biol. Mcd., 76: 729-732. BARNES, D. W. H., AND J. F. LOUTIT, 1955. Spleen protection; the cellular hypothesis. In: Radiobiology Symposium, Bacq, Z. M. and P. Alexander, eds. Liege, 1954. Pp. 134- 135. Academic Press Co., Inc., New York. BARROW, J., AND J. L. TULLIS, 1952. Sequence of cellular response to injury in mice exposed to 1100 r total body X-irradiation. Arch. Path., 53: 391-407. BLOOM, W., AND L. O. JACOBSON, 1948. Some hematologic effects of irradiation. Blood, 3: 586-592. BOND, V. P., M. N. SWIFT, A. C. ALLEN AND M. C. FISHLER, 1950. Sensitivity of abdomen of rat to X-irradiation. Amcr. J. Physiol., 161 : 323-330. BOND, V. P., M. S. SILVERMAN AND E. P. CRONKITE, 1954. Pathogenesis and pathology of post irradiation infection. Radiation Res., 1 : 389-400. CHROM, S. A., JR., 1935. Studies on the effect of roentgen rays upon intestinal epithelium and upon reticuloendothelial cells of the liver and spleen. Ada Radial. , 16: 641-660. COLE, L. J., AND M. E. ELLIS, 1953. Age, strain and species factors in post irradiation protec- tion by spleen homogenates. Amcr. J. Physiol., 175: 487-494. ELLINGER, F., 1945. Lethal dose studies with X-rays. Radiology, 44: 125-142. GERSHON-COHEN, J., M. B. HERMEL AND J. Q. GRIFFITH, JR., 1951. The value of small lead shields against injurious effect of total-body irradiation. Science, 114: 157-158. GOWEN, J. W., 1945. Genetic aspects of virulence in bacteria and viruses. Ann. Missouri Bot. Card,, 32: 187-211. GOWEN, J. W., 1950. Radiation effects on mice as related to survival. Genetics, 35: 112. CONTRIBUTIONS TO X-RAY RESISTANCE 421 GOWEN, J. W., 1952. Humoral and cellular elements in natural and acquired resistance to typhoid. /. Human Genetics, 4: 285-302. GOWEN, J. W., AND M. CALHOUN, 1943. Factors affecting genetic resistance of mice to mouse typhoid. /. Infect. Dis., 73: 40-56. GOWEN, J. W., AND J. STABLER, 1956. Life spans of different strains of mice as affected by acute irradiation by 100 pkv X-rays. /. Exp. Zool., 132: 133-155. GOWEN, J. W., AND M. R. ZELLE, 1945. Irradiation effects on genetic resistance of mice to mouse typhoid. /. Infect. Dis., 77: 85-91. GRAHN, D., 1954. Genetic variations in the response of mice to total body X-irradiation. I. Body weight response in six inbred strains. /. Exp. Zool., 125: 39-61. HEINEKE, H., 1905. Expermentelle Untersuchungen iiber die Einwirkung der Runtgenstrahlen auf innere Organe. Mitt, aus den Grensgebieten den Mcdisin und Chirurgie, 14: 21-94. HENSHAW, P. S., 1944a. Experimental roentgen injury. I. Effects on the tissues and blood of C3H mice produced with single small whole-body exposures. /. Nat. Cancer lust., 4: 477-484. HENSHAW, P. S., 1944b. Experimental roentgen injury. II. Changes produced with inter- mediate-range doses and a comparison of the relative susceptibility of different kinds of animals. /. Nat. Cancer Inst., 4: 485-501. HENSHAW, P. S., 1944c. Experimental roentgen injury. III. Tissue and cellular changes brought about with single massive doses of radiation. /. Nat. Cancer Inst., 4: 503-512. JACOBSON, L. O., 1954. Modification of radiation injury in experimental animals. Aincr. J. Roentgenol. and Rod. Thcr., 72: 543-555. JACOBSON, L. O., E. L. SIMMONS, E. K. MARKS, E. O. GASTON, M. J. ROBSON AND J. H. ELDREDGE, 1951. Further studies on recovery from irradiation injury. /. Lab. Clin. Med., 37 : 683-697. JACOBSON, L. O., E. K. MARKS AND E. O. GASTON, 1955. Observations on the effect of spleen shielding and the injection of cell suspensions on survival following irradiation. In: Radiobiology Symposium, Bacq, Z. M. and P. Alexander, eds. Liege, 1954. Pp. 122- 133. Academic Press Co., Inc., New York. KAPLAN, H. S., AND M. B. BROWN, 1952. Mortality of mice after total body irradiation as influenced by alterations in total dose fractionation and periodicity of treatment. /. Nat. Cancer Inst., 12: 765-775. KAPLAN, H. S., AND J. PAULL, 1952. Genetic modification of response to spleen shielding in irradiated mice. Proc. Soc. Exp. Biol. Med., 79: 670-672. LAWEN, A., 1909. Expermentelle Untersuchungen iiber des verhalten roentgentsierter Tiere gegen bakterielle Infektionen unter besonderer Beriicksichtigung der Bildung spezi- fischer Antikorper. Mitt, aus den Grensgebieten der Medisin und Chirurgie, 19: 141- 186. LAWRENCE, J. H., AND R. TENNANT, 1937. The comparative effects of neutrons and X-rays on the whole body. /. Exp. Med., 66: 667-687. LORENZ, E., C. CONGDON AND D. UpHOFF, 1952. Modification of acute irradiation injury in mice and guinea pigs by bone marrow injections. Radiology, 58: 863-877. OSBORNE, J. W., H. S. BRYAN, H. QUASTLER AND H. E. RHOADES, 1952. Studies on roentgen death in mice. IV. X-irradiation and bacteremia. Amer. J. Physiol., 170: 414-417. REINHARD, M. C., E. A. MIRAND, H. L. GOLTZ AND J. C. HOFFMAN, 1954. Mouse strain dif- ferences in response to radiation. Proc. Soc. Exp. Biol. Med., 85: 367-370. THOMPSON, S., 1952. Serum proteins, leukocytes and mortality of seven inbred mouse strains during cortisone administration and infection with Salmonella typhimurium. Unpub- lished Ph.D. Thesis, Iowa State College Library, Ames, Iowa. WARREN, S. L., AND G. H. WHIPPLE, 1922. Roentgen ray intoxication. I. Unit dose over thorax negative — over abdomen lethal. Epithelium of small intestine sensitive to X-rays. /. Exp. Med., 35: 187-202. WEIR, J. A., 1949. Blood pH as a factor in genetic resistance to mouse typhoid. /. Inject. Dis., 84: 252-274. WEIR, J. A., R. H. COOPER AND R. D. CLARK, 1953. The nature of genetic resistance to infec- tion in mice. Science, 117: 328-330. WISSLER, R. W., M. J. ROBSON, F. FITCH, W. NELSON AND L. O. JACOBSON, 1953. The effects of spleen shielding and subsequent splenectomy upon antibody formation in rats receiving total body X-radiation. J. Immunol., 70: 379-385. SEXUAL DIFFERENTIATION IN THE TELEOST FISH XIPHOPHORUS HELLERII, AS MODIFIED BY EXPERIMENTAL TREATMENT HENRY H. VALLOWE Department of Zoology, Ohio University, Athens, Ohio Attempts to alter the pattern of sexual differentiation in the Mexican swordtail fish, Xiphopoms hellerii, show a stability in sexual development usually not credited to this species. The swordtail has long been used as a classic example of lability of sexual differentiation and determination. The widespread reference to sex- reversal occurring in the species suggested a need for further investigation. This work was aided by the guidance and kind supervision given by the late Dr. Carl R. Moore of the University of Chicago. I am indebted to Dr. Myron Gordon for his generosity in supplying the fishes used in this study. It is a pleas- ure to acknowledge the cooperation of the Schering Corporation which supplied the hormone preparations used. ANIMALS AND THEIR TREATMENT The fishes used were descendants of ten pairs from the Genetics Laboratory of the New York Zoological Society. They were reared under conditions similar to those described in earlier papers (Vallowe, 1952, 1953). The original ten pairs produced 618 males and 490 females in a period of approximately three years. The presence of a modified anal fin was the criterion used to establish maleness. Fish not possessing the gonopodium were anesthetized in Chlorobutanol and ex- amined before a strong light. The characteristic amber color of the ova apparent in the ventral region of the posterior end of the body cavity was the criterion used to classify fish as females. The sex ratios which have been established for this species show wide varia- tions. Witchi (1939) considers X. hellerii to be a species without any hereditary sex determining mechanism although the presence of such mechanisms has been shown for other species in the genus. Geiser (1924) presents a comprehensive table showing the sex ratios reported by various authors. Harms (1926) gives a ratio of 24 males to 35 females. Bellamy (1922) reports 100 males to 66.7 females. Friess (1933) also reports a high proportion of males and considers temperature an important influence on the sex ratio. Breider (1935) made a study of the effects of light, nutrition, space and water and found that these had no certain influence on the sex ratio. In light of many conflicting reports, one cannot assume that a preponderance of one sex is due to sex-reversal unless all factors which may influence sexual differentiation are duly considered. As a basis for interpreting the experimental results reported, a large series of fish were killed at various stages of development, beginning two weeks before birth 422 SEXUAL DIFFERENTIATION IN XIPHOPHORUS 423 and continuing up to more than two years of age. The gonads of these fish were examined to determine the normal pattern of sexual development and differentia- tion. As early as three days after birth, the gonads could be recognized as poten- tial testes or ovaries by the relative size, number and arrangement of the primordial germ cells. In the young male the germ cells were slightly smaller than in the female. In addition, there were fewer germ cells in each gonad primordium and they were concentrated at the periphery of the gland. In the young female the slightly larger cells were in greater numbers and were well distributed. The em- bryonic development of this species closely parallels the description given by Goodrich ct al. (1934) and Dildine (1933, 1936) for L. reticulatus, that by Wolf (1931) for X. maculatus, and those by Geiser (1924) and Medlen (1950) for two species of Gambusia. Although sexual differentiation is conspicuous in these spe- cies shortly before birth, Essenberg (1923), Bailey (1933) and Regnier (1938) have shown it to be somewhat delayed in X. hcllerii. The differences which distinguish the early testes and ovaries are very subtle characteristics. The appearance of the early ovary and the indifferent gonad are so similar that Regnier (1938) considers that all the fish are born female and that the males later undergo a sex change. This introduces an unnecessary compli- cation which is easily resolved if one assumes a longer period of indifferent devel- opment. Once differentiation begins, it progresses in an orderly sequence of events. Chavin and Gordon (1951) describe the differentiation of the testes in X. maculatus by using a series of six stages. With only slight modifications, these stages were used in the present study to classify the testes of X. hcllerii. Gordon and Aronowitz (1951) make the observation that the histological structures of the testes of adult X. maculatus and X. hellerii are practically identical. The present study has shown that the development of the testis and the pattern of the differen- tiation of its structures are strikingly similar in the two species. Except for the time spent in the late stages of immaturity, the sequence of events is identical. The process of sexual differentiation in X. hellerii as described by Essenberg (1923) and Van Oordt (1925) traces the origin of the definitive sperms to the epithelial cells of the testis tubules. Wolf (1931), Goodrich ct al. (1934), and Chavin and Gordon (1951) were unable to find comparable stages of transforma- tion in the species which they studied. The testis in X. hellerii is the acinus-type characteristic of the vivaparous Cyprinodonts. The arrangement and development of the structures within the testis indicate that the acini form from pre-existing acini at the periphery of the testis. As the acini are formed, they are pushed along the tubules toward the central ducts by the growth of new acini at the periphery. The peritoneal covering of the testis, the stroma cells, and the epithelium of the ducts and tubules gave no evidence of transforming into germ cells. It is slightly more difficult to distinguish the newly differentiated ovary from the indifferent gonad because the arrangement of the cells remains about the same. However, the relative size and number of the cells characterize the gonad as an immature ovary. Early in the differentiation process the primordial germ cells are found scattered throughout the stroma of the ovary. When the fusion of the paired ovaries is completed, the germ cells come to lie within the wall of the cavity formed between them or in the stroma layer which supports this wall. In the mature female oogonia are found developing within the wall of the cavity, hence the name germinal epithelium. The larger and more advanced cells are pushed 424 HENRY H. VALLOWE outward toward the peritoneal covering. Essenberg (1923) feels that all the pri- mordial germ cells disintegrate and do not take part in the formation of the ger- minal epithelium. Although there is great difficulty in determining the origin of the cells within the germinal epithelium, no disintegrating primordial cells were encountered in the early stages of gonad fusion and cavity formation. The pri- mordial cells appeared to remain in normal active condition and were arranged in small groups surrounded by stroma cells. In the germinal epithelium it is possible to see all stages of transition in the shape of the nuclei from elongate oval, typical of the epithelial cells, to the spherical nuclei of oogonia. A comparable situation exists in L. reticulatus according to Goodrich ct al. (1934) and leads them to the conclusion that epithelial cells as \vell as primordial germ cells may give rise to the definitive ova. Wolf (1931) arrived at a similar conclusion. The origin of the sex cells appears to be different in the two sexes of these species. While the pri- mordial germ cells are the only source of definitive sperms, the peritoneal cells form- ing the wall of the ovarian cavity may contribute to the formation of definitive ova. The following series of experiments reports the effects of estrogenic and andro- genic hormones on gonad development. The results of the treatments described are interpreted in light of their effect on the normal sexual differentiation. EXPERIMENTS AND RESULTS Immature fish Young sexually immature fish (49 to 55 days old) were given nine weekly injections of 0.01 cc. of sesame oil containing 0.25 mg. of testosterone propionate into the body cavity. During this period of time there was a conspicuous thicken- ing and elongation of the anal fin rays, a growth and pigmentation of the sword- like extension of the caudal fin and an intensification of the lateral line and dorsal fin coloration. In short, the young fish appeared to be miniatures of the sexually mature adult males. The histological picture presented by the gonads of these fish was one of radical change in the immature ovaries and one of general stimulus in the young testes. The ovaries of the testosterone-injected immature females lacked any sign of ova. There were follicles present but these were filled with a loose collection of cells. This indicates that ova had been present previously, but that resorption had taken place. In other follicles primary spermatocytes, spermatids and spermato- phores were observed. The ovarian cavity was obscured and much stroma filled the gonad. The brood-mate control females showed ovaries in which fats and oils were being deposited in the maturing ova. The testes of immature males treated with this androgen showed an acceleration in spermatogenesis. The tubules and sperm ducts were filled with all stages of spermatogenesis including well formed spermatophores. The epithelium lining the sperm ducts was greatly hypertrophied but otherwise the testes appeared very similar to those of mature males. Brood-mate control males showed testes in less advanced stages of development. Another group of immature fish at comparable ages was given nine weekly injections of 0.01 cc. sesame oil containing 0.00083 mg. of estradiol benzoate. At the end of the treatment period these fish were deep-bodied, showed only faint SEXUAL DIFFERENTIATION IN XIPHOPHORUS 425 indications of coloration and, in general, appeared to be miniatures of the adult females. The histological picture presented by these fish was the reverse of the situation found in those injected with testosterone. The ovaries of the estradiol- treated females showed a general stimulus while the testes of the treated males showed radical changes. The testes of the estrogen-treated males showed a modification from the bipar- tite gonad to a fused structure in which the two lobes were no longer distinct. The peritoneal covering of the gonad had thickened, the germinal elements were no longer concentrated at the periphery of the gonad, the sperm ducts were obscured, and the blood vessels were enlarged. Some acini contained what appeared to be oogonia. Other acini contained disintegrating cells which resembled primary spermatocytes. The testes were larger than those of the brood-mate controls. In some of the gonads the sperm ducts appeared only as spaces with no organized epithelium. Two new dorso-lateral cavities had formed and were lined with a well organized epithelium. Oocytes were common in the testes showing this degree of modification. Ovaries of the estradiol-injected females showed ova in the stage of oil deposi- tion. There was a slight increase in the amount of stroma present but the blood vessels and follicular epithelium appeared normal. The germinal epithelium ap- peared very active and abundant oogenesis was observed. Mature fish The histological picture presented by the testes of sexually mature males after ten weekly injections of 0.02 cc. sesame oil containing 0.00166 mg. estradiol ben- zoate was one of general suppression and destruction. The most conspicuous effects obtained were those of an enlargement of the sperm ducts and the destruc- tion of the spermatogenic elements. In normal mature males the sperm ducts were paired, centrally located tubes which contained spermatophores suspended in a lightly staining, non-granular fluid. In estrogen-treated males the ducts were greatly enlarged and in some cases occupied most of the testes when viewed in cross-section. They seemed to be distended with fluid and contained spermato- phores in various stages of disintegration. Quantities of free spermatozoa were observed in the lumen of the ducts ; this condition is not found in normal mature males. However, free spermatozoa are found in the ducts of senile males which have passed the reproductive age and are no longer capable of fertilization. The effect on the germinal elements was most drastic in the intermediate, stages of spermatogenesis. While spermatozoa and spermatogonia were still abundant, primary and secondary spermatocytes and spermatids were usually reduced in number or entirely absent. The acini which, by their position, should have con- tained the intermediate stages of spermatogenesis, had hypertrophied walls in which the cell outlines were conspicuous. These acini were smaller than normal and were either filled with disintegrating germ cells or were completely empty. The reduction of the acini was accompanied by a slight increase in the amount of stroma tissue and the appearance of a fibrous tissue network. The blood vessels were numerous and enlarged. The testes appeared less bipartite than the normal con- dition, but none were found in which fusion was as complete as that found in the ovary. 426 HENRY H. VALLOWE The histological picture presented by the ovaries of females given ten weekly injections of 0.02 cc. sesame oil containing 0.5 mg. testosterone propionate was also one of general suppression and destruction. None of the ovaries showed any signs of spermatogenesis even when injections were continued for as long as eighteen weeks. A conspicuous activity was noted in the germinal epithelium but there was no indication that the primary germ cells being proliferated could be sper- matogonia. The cells were in groups of as many as ten, but their arrangement, size, and staining properties were strikingly similar to the oogonia found in normal ovarian development. The larger ova that were present in the gonad were in various stages of disintegration. Only a few of the ovaries examined showed the presence of mature ova ; in most cases, there remained only large, empty follicles which were in various stages of collapse. There seemed to be little effect of the hormone treatment upon the smaller ova ; they were still firm, were surrounded by a well organized follicle, and were in their normal arrangement in the ovary. There was very little increase in the amount of stroma in the ovary and only iso- lated areas in which a fibrous network was formed. On the other hand, the blood vessels had increased in size and were found throughout the ovarian tissue. In gross appearance, the ovaries of the androgen-treated females resembled those of immature normal females. In the empty follicles and ovarian cavity of many of the virgin ovaries the presence of a secretion was observed. This secretion appeared as a mass of crumpled membranes which is normally found only in the non-virgin ovary. The frequency of its appearance indicated that this is a typical response to androgenic injections and it may be indicative of an expulsion of mature ova from their follicles. However, no mature eggs were found in the ovarian cavities or ovi- ducts. If the eggs were completely evacuated from the body, they were never found in the aquaria. The ovaries of the gravid females did not differ from the ovaries of virgin females given the same treatment. In two cases where the females were killed three weeks after the initial injection of testosterone propionate, embryos in early stages of development still remained in the follicles. The characteristic membrane- like secretions were observed in the follicles of some of the gravid females. DISCUSSION The results obtained from the study of the normal sexual differentiation of X. hellerii, and from the attempts to alter the pattern of this differentiation by experimental treatment, point to a stability in sexual development and differentia- tion which has usually not been credited to this species. Much of the experi- mental and descriptive work dealing with Xiphophorin fishes has emphasized the ease with which the secondary sexual characteristics of these species can be made to respond to hormonal treatment. The effects of many hormone substances on the primary sex organs have also been described but the results are not always in agreement. However, in all cases of sex-reversal reported for adult X. hellerii the change has always been from female to male, whether such reversals were naturally occurring phenomena or whether they were induced by hormone treat- ments. During the course of this investigation no case of natural sex-reversal was observed in the laboratory population ; further, the sex ratio obtained for this SEXUAL DIFFERENTIATION IN XIPHOPHORUS 427 population indicates that unobserved sex-reversals could not have occurred in large numbers. The adult sex ratio and the sex distribution of the fishes used in the study of the normal development of the gonads did not indicate that a shift in the ratio occurred between the juvenile and mature populations. Essenberg (1923) postulated that a sex-reversal of 50 per cent of the immature females would explain the shift he observed in the sex ratio (a change from a ratio among immature fish of three females to one male to the adult condition of one female to three males). A possible explanation of naturally occurring sex-reversal in adult fish, in which virgin females become males, lies in improper initial classification. In this study only those fish which possessed amber ova that could be observed through the body wall when viewed against a strong light source were designated as females. This procedure reduced to a minimum the possibility of erroneous sex classification. The classification of immature males as females may account for a few, but cer- tainly not all, of the cases of hormone-induced sex-reversal reported by other investigators. Another factor which may play a significant role in the discrepancies of the results obtained from experiments with X. hellcrii lies with the variations in the fish used. The very important reviews by Gordon (1931, 1937) concerning the history of Xiphophorin species as aquarium fishes give substantial evidence to indicate that the X. hellcrii available from commercial hatcheries have possibly been produced by hybridization with X. maculatus. For the aquarist the hybrid fish has many traits that are desirable. The hybrid is usually more highly colored, more robust and larger than either parent species, and more prolific in the pro- duction of large broods of young. These factors would account for its selection and propagation by commercial hatcheries. Gordon and Rosen (1951) suggest that the hybrid possesses an unbalanced chromosomal arrangement that may have endocrinological significance in that this imbalance may initiate abnormal or non- functional gonads in the hybrid. Although hybrids may usually be identified by color or color patterns, some are practically identical with the wild-type swordtail. The few cases of natural sex-reversal that have come to my attention have always been in hybrid fishes or in fish with unknown origins. A strong possibility exists that the sex-reversals reported by Essenberg (1926) occurred in hybrid stocks. (He gives the origin of the stock as the Crescent Fish Farm, a commercial fish hatchery.) Further application of this possibility suggests that the work of other investigators may also be based on commercially available hybrid strains rather than on pure species stocks. The results of the experimental treatments given to the pure species used in this study were found to be in close accord with the results obtained by many inves- tigators using the closely related species which are not known for their tendencies to produce sex-inversions. The response to the techniques employed indicated that these fish were in no way aberrant but were in close agreement with the other members of the viviparous Cyprinodont group. Although the immature fish developed the germ cells of the opposite sex under hormone treatment, the sexually mature fish did not when subjected to the same hormone preparations. Since the gonads are homologous structures, it is possible that they retain a few common properties until differentiation becomes so advanced that these must be sacrificed. Evidence from the treatment of maturing males 428 HENRY H. VALLOWE with estradiol benzoate shows that the ability of the testis to produce ova is re- tained from a short time after gonopodium elongation. However, this response is lost after the later stage of male development, i.e., gonopodium differentiation, is initiated. In the mature male the effects of estrogenic treatment were a destruc- tion and suppression of existing elements. Unlike the results obtained in immature and maturing males, there was no stimulus to develop female characteristics. Immature females produced spermatozoa within their ovaries under the influ- ence of testosterone propionate. This response, however, was not found in the ovaries of mature females given twice the amount of the same hormone preparation. SUMMARY 1. The normal sexual development and differentiation of Xiphophorus hellerii have been observed in the light of the adult sex ratio and the differentiation of the gonads from birth to maturity. The sexual differentiation has been shown to follow a definite pattern in both sexes. The gonads of both sexes are homologous and indifferent at birth, but within a few days testes and ovaries can be distin- guished by the relative size, number and arrangement of the primordial germ cells. No atypical gonads were discovered in the course of this study which would indi- cate the possibility of sex-reversal. 2. Injections of testosterone propionate induced spermatogenesis in the ovaries, of immature females but had no comparable effect in mature specimens. 3. Injections of estradiol benzoate induced oogenesis in the testes of immature males but had no comparable effect in mature specimens. 4. Improper initial sex classification and hybrid origin of the fish used are suggested as possible explanations for some of the discrepancies between the results obtained in this study and those reported in earlier investigations. 5. The results of the study of the normal sexual development and the attempts to alter the differentiation pattern by experimental means indicate a stable sexual development and differentiation for this species. LITERATURE CITED BAILEY, RALPH J., 1933. The ovarian cycle in the viviparous teleost, Xiphophorus hcllcri. Biol. Bull., 64: 206-225. BELLAMY, A. W., 1922. Breeding experiments with the viviparous teleosts Xiphophorus hcllcri and Platypoccilus maculatus (Giinth.)- Anat. Rec., 23: suppl. 98-99. BREIDER, HANS, 1935. t)ber Aussenfaktoren, die das Geschlechtsverhaltnis bei X. helleri Heckel kontrollieren sollen. Zcitschr. U'iss. Zool., 146: 383-416. CHAVIN, WALTER, AND MYRON GORDON, 1951. Sex determination in Platypoccilus maculatus. I. Differentiation of the gonads in members of all-male broods. Zoologica, 36: 135-146. DILDINE, GLENN C, 1933. Germ cell origin and gonad differentiation in the viviparous top minnow, Lcbistcs rcticulatus. Anat. Rcc., 57: suppl. 88. DILDINE, GLENN C., 1936. Studies in teleostean reproduction. I. Embryonic hermaphroditism in Lcbistcs rcticulatus. J. Morph., 60: 261-277. ESSENBERG, JACOB M., 1923. Sex-differentiation in the viviparous teleost, Xiphophorus hcllcri Heckel. Biol. Bull, 45: 46-96. ESSENBERG, JACOB M., 1926. Complete sex-reversal in the viviparous teleost, Xiphophorus hcllcri. Biol. Bull, 51: 98-111. FRIESS, ELSE, 1933. Untersuchungen iiber die Geschlechtsumkehr bei Xiphophorus helleri Heckel. Arch. f. Entw., 129: 255-355. GEISER, S. W., 1924. The sex ratio in Gambusia holbrooki. Biol. Bull, 47: 175-212. SEXUAL DIFFERENTIATION IN XIPHOPHORUS 429 GOODRICH, H. B., J. E. DEE, C. M. FLYNN AND ROWENA N. MERCER, 1934. Germ cells and sex differentiation in Lebistcs reticulatus. Biol. Bull., 67: 83-96. GORDON, MYRON, 1931. Hereditary bases of melanosis in hybrid fishes. Amer. J. Cancer, 15: 1495-1523. GORDON, MYRON, 1937. Heritable color variations in the Mexican swordtail fish. /. Hcrcd., 28: 222-230. GORDON, MYRON, AND OLGA ARONOWITZ, 1951. Sex determination in Platypoccilus maculatus. II. History of a male platyfish that sired all-female broods. Zoologica, 36: 147-153. GORDON, MYRON, AND DONN E. ROSEN, 1951. Genetics of species differences in the morphol- ogy of the male genitalia of xiphophorin fishes. Bull. Amer. Mus. Nat. Hist., 95: 409- 464. HARMS, J. W., 1926. Beobachtungen ueber Geschlechtsumwandlungen reif der Tiere und deren Fx Generation. Zool. Anz., 67: 67-79. MEDLEN, AMMON B., 1950. Sperm formation in Gambusia affinis. Texas Jour. Sci., 2: 395-399. REGNIER, M. T., 1938. Contribution a 1'etude de la sexualite des cyprinodont vivipares (Xipho- phorus hellcri, Lebistes reticulatus). Bull. Biol., 72: 385-493. VALLOWE, HENRY H., 1952. Boiled egg yolk an essential ingredient. Aquarium, 21: 350. VALLOWE, HENRY H., 1953. Some physiological aspects of reproduction in Xiphophorus macu- latus. Biol. Bull., 104: 240-249. VAN OORDT, G. J., 1925. The relation between the development of the secondary sex characters and the structure of the testis in the teleost Xiphophorus hclleri. J. Exp. Biol., 3: 43-59. WITCHI, EMIL, 1939. Modification of the development of sex in lower vertebrates and in mam- mals. In: Sex and internal secretions, ed. by Edgar Allen. Baltimore. Williams and Wilkens. WOLF, L. E., 1931. The history of the germ cells in the viviparous teleost Platypoccilus macu- latus. J. Morph. and Physiol, 52: 115-153. INDEX ACCUMULATION of radiocobalt in Tetra- hymena, 390. Action potential of stomatopod heart, 358. Activity, tidal cycles of, in Uca, 267. Adult sensitivity to x-rays, of Molgula, 171. Alanine, content of, in Porphyra, 363. Alga, marine, replacement of potassium by rubidium in, 249. Alga, red, amino acids of, 363. Algae, variations in chemical constituents of, during development, 81. Algae as food for Palaemonetes, 162. Alkaline phosphatase activity of echinoderm hybrid embryos, 21. ALLEN, B. M., AND M. H. DEVICK. Differ- ences in susceptibility to whole-body gamma irradiation in the layers of the retina of Bufo, 137. Allium, effects of x-irradiation on mitosis in root tips of, 305. Amino acid constituents of Porphyra, 363. Amino acid content of marine borers, 325. Amoebocytes, role of, in Dugesia regenera- tion, 371. Amphibian, gamma irradiation of, 137. Amphidinium, taxonomy of, 196. Anaerobic recovery of Ascaris eggs from x- irradiation, 383. Analysis of response to osmotic stress by Crustacea, 43. Anatomy of ectoproct bryozoan, 120. Androgens, role of, in sexual differentiation of Xiphophorus, 422. Annelid, x-irradiation of fertilized eggs of, 184. Anophthalmia in Fundulus hatchlings, 241. Antarctic Bryozoa, 120. Apostome ciliates, excystation of, 132. Arbacia embryos as food for Balanus, 313. Arctic fish, osmoregulation in, 28. Artemia nauplii as food for Palaemonetes larvae, 162. Ascaris eggs, x-irradiation of, 383. Ascidian, vascular budding in, 225. Asexual reproduction of Botryllus, 225. Asexual reproduction of hydroid, 349. Assay of estrogens in marine invertebrates, 180. , C. S. Sec G. PAHL, 383. Balanus, larval development of, 313. Bankia, amino acid content of, 325. BARBER, A. A. See S. P. MARONEY, JR., 92. Barnacle, larval development of, 313. Barometric pressure, effects of, on oxygen consumption of potato, 288. Basophilic substances, distribution of during development of mushroom, 1. BENNETT, M. F., J. SHRINER AND R. A. BROWN. Persistent tidal cycles of spon- taneous motor activity in the fiddler crab, Uca, 267. "Biological clocks," 288. Birgus, osmotic regulation in, 43. Bivalves as food for Urosalpinx, 330. Blastula, sea urchin, uptake of phosphate in, 276. BLINKS, L. R. See R. F. JONES, 363. Blood-cells as site of budding in Botryllus, 225. Blood freezing point of Arctic fish, 28. Body cells, role of, in survival of x-irradiated mice, 400. BOLST, A. L., AND A. H. WHITELEY. Studies on the metabolism of phosphorus in the development of the sea urchin, Strongylo- centrotus, 276. BONNER, J. T., A. A. HOFFMAN, W. T. MORIOKA AND A. D. CHIQUOINE. The distribution of polysaccharides and baso- philic substances during the development of the mushroom, Coprinus, 1. BOOKHOUT, C. G. Sec J. D. COSTLOW, JR., 313. Borers, marine, amino acid content of, 325. Botryllus, vascular budding in, 225. Box turtles, orientation in, 336. Brachyuran crabs, gill area of, 34. Breathing rate in carp, 97. BROAD, A. C. Larval development of Palae- monetes, 144. BROAD, A. C. The relationship between diet and larval development of Palaemonetes, 162. BROWN, F. A., JR. Response of a living organism under "constant conditions" in- cluding pressure, to a barometric-pres- sure-correlated, cyclic, external variable, 288. 430 INDEX 431 BROWN, R. A. Sec M. F. BENNETT, 267. Brown algae, variations of chemical con- stituents of, 81. Bryozoa, marine, 120. Budding of hydroid, 349. Budding, vascular, in Botryllus, 225. Bufo, gamma irradiation of, 137. Burrows, position of, in Uca, 7. (CALCIUM, uptake of by oyster mantle, 92. Callianassa, osmotic regulation in, 43. Cancer, osmotic regulation in, 43. Cape Cod Dinophyceae, 196. Carabus, oxygen uptake in, 108. Carbohydrate constituents, variations in, dur- ing development of alga, 81. Carbon dioxide production by insects, 108. Carcinides, trematode parasite of, 254. Carp, dependence of breathing rate of, on temperature, 97. Cecropia silkworm, oxygen uptake of, 108. Cell division in onion root tips, effects of x- irradiation on, 305. Cercariae of Microphallus, 254. Chaetopterus, x-irradiation of fertilized esss of, 184. CHANDLER, A. C, JR. The immediate effects of low doses of x-radiation on the fre- quency of several mitotic stages in the Allium root tip, 305. Char, Arctic, osmoregulation in, 28. CHIQUOINE, A. D. Sec J. T. BONNER, 1. Chlamydomonas as food for Balanus, 313. Chlamydomonas as food for Palaemonetes larvae, 162. Chloride concentrations in Arctic fish, 28. Chromatographic identification of amino acids in borers, 325. Chromatography of Porphyra amino acids, 363. Chromatography of sea urchin egg extracts 276. Chromatophore rhythms in Uca, 7. Chromatophores of Palaemonetes, 144. Chromoproteins of Porphyra, 363. Chromosome configurations in colchicine- treated Dugesia tissues, 371. Chromosomes, effects of x-irradiation on, in Chaetopterus eggs, 184. Ciliates, excystation of, 132. Cirripede, development of, 313. Cleavage, uptake of phosphate during, in sea urchin, 276 Cleavage of x-irradiated Ascaris eggs, 383. Cleavage of x-irradiated Chaetopterus eggs, 184. Cleavage of x-irradiated Molgula eggs, 171. Cobalt-60, accumulation of, in Tetrahymena 390. Cobalt-60 irradiation of toad, 137. Coelenterate, reversal of polarity in, 349. Colchicine treatment of Dugesia, 371. Cold, role of, in feeding of Urosalpinx, 330. Comparative study of gill area of crabs, 34. Compound ascidian, vascular budding in, 225. "Constant conditions," variation of oxygen consumption of potato during, 288. Convoluta, feeding in, 63. Coprinus, distribution of substances during development of, 1. COSTELLO, D. P. Sec C. HENLEY, 184. COSTLOW, J. D., JR., AND C. G. BoOKHOUT. Larval development of Balanus in the laboratory, 313. Crab, fiddler, tidal cycles of activity in, 267. Crab, relation between burrow position and tidal rhythm of, 7. Crab, trematode parasite of, 254. Crabs, apostome ciliate parasites of, 132. Crabs, gill area of, 34. Crabs, response to osmotic stress by, 43. Crassostrea as food for Urosalpinx, 330. Crustacea, gill area of, 34. Crustacea, response to osmotic stress by, 43. Crustacean, amino acid content of, 325. Crustacean, apostome ciliate parasites of, 132. Crustacean, Cirripede, development of, 313. Crustacean, electrocardiogram of, 358. Crustacean, larval development of, 144, 162. Crustacean, relation between burrow position and tidal rhythm of, 7. Crustacean, tidal cycles of activity in, 267. Crustacean, trematode parasite of, 254. Culture of Balanus, 313. Culture of Endamoeba, 377. Cyanide, protective effect of, against x-ir- radiation damage to Ascaris eggs, 383. Cycles of Uca, 7. Cycles, tidal, in Uca, 267. Cyclic carbon dioxide release in insects, 108. Cyclic changes in oxygen consumption of potato, 288. Cyclopean fish, optokinetic testing of, 241. Cycloporus, feeding in, 63. Cyprinus, breathing rate in, 97. Cytochrome system, role of, in recovery of Ascaris eggs from x-irradiation, 383. Cytology of x-irradiated Chaetopterus eggs 184. Cytology of x-irradiated onion root tips, 305. as growth-promoting factor for Enda- moeba, 377. DNP, effects of, on oyster mantle respiration. 92. 432 INDEX Decapod Crustacea, response of, to osmotic stress, 43. Delay of cleavage in x-irradiated Ascaris eggs, 383. Delay of cleavage in x-irradiated Chaetop- terus eggs, 184. Dendraster-Strongylocentrotus hybrid em- bryos, 21. Dependence of carp breathing rate on tem- perature, 97. Deposition of oyster shell, 92. Developing sea urchin egg, metabolism of phosphorus in, 276. Development of Balanus, 313. Development of Botryllus, 225. Development of Chaetopterus, effects of x-rays on, 184. Development of Himanthalia, 81. Development of Molgula eggs, effects of x- irradiation on, 171. Development of mushroom, 1. Development of Palaemonetes, 144, 162. DEVICK, M. H. Sec B. M. ALLEN, 137. DEVOE, R. See G. T. SCOTT, 249. Diagnostic features of unarmored Dinophyceae, 196. Diet as factor in larval development of Palae- monetes, 162. Differential susceptibility to gamma irradia- tion, 137. Differentiation, sexual, in Xiphophorus, 422. Digenetic trematode, morphology and life- history of, 254. Digestion in flatworms, 63. Dinitrophenol, effects of, on oyster mantle respiration, 92. Dinophyceae, unarmored, taxonomy of, 196. Dipteran, red cells in, 220. Distribution of unarmored Dinophyceae, 196. Drill, oyster, rate of feeding of, 330. DRISKO, R. W., AND H. HOCHMAN. Amino acid content of marine borers, 325. Drosophila, red cells in, 220. Dugesia, regeneration of, after colchicine treatment, 371. gCHINODERM egg, metabolism of phos- phorus in, 276. Echinoderm embryos, hybrid, alkaline phos- phatase activity of, 21. Ecology of Bryozoa, 120. Ecology of crabs, 34. Ecology of Uca, 7. Ectoproct bryozoan, new species of, 120. Effects of dinitrophenol on oyster mantle res- piration, 92. Effects of x-rays on Allium mitosis, 305. Effects of x-rays on fertilized Chaetopterus eggs, 184. Effects of x-rays on Molgula development, 171. Eggs, Ascaris, x-irradiation of, 383. Eggs, Chaetopterus, x-irradiation of, 184. Eggs, Molgula, x-irradiation of, 171. Eggs, sea urchin, phosphorus metabolism in, 276. Electrocardiogram of stomatopod, 358. Embayments, Dinophyceae of, 196. Embryo, sea urchin, phosphorus metabolism in, 276. Embryogenesis of Ascaris, effects of x-rays on, 383. Embryology of echinoderm hybrids, 21. Emerita, osmotic regulation in, 43. Endamoeba histolytica, growth-promoting fac- tors for, 377. Environment, effect of, on oxygen consumption of potato, 288. Enzyme studies on marine invertebrates, 180. Estradiol, effect of, on sexual differentiation of Xiphophorus, 422. Estradiol equivalents in marine invertebrates, 180. Estrogens, role of, in sexual differentiation of Xiphophorus, 422. Estrogens in marine invertebrates, 180. Exchange of rubidium and potassium in Ulva, 249. Excystation of apostome ciliates, 132. pAT cells in Drosophila, 220. Feeding of flatworms, 63. Feeding of oyster drill, 330. Fertilization, effect of, on uptake of phos- phorus by sea urchin egg, 276. Fiddler crab, apostome ciliate parasites of, 132. Fiddler crab, relation between burrow position and tidal rhythm of, 7. Fiddler crab, tidal cycles of activity in, 267. FINGERMAN, M. Relation between position of burrows and tidal rhythm of Uca, 7. Fish, Arctic, osmoregulation in, 28. Fish, breathing rate of, 97. Fish, sexual differentiation in, 422. Fish hatchlings, optokinetic testing of, 241. Flatworms, free-living, feeding of, 63. FLICKINGER, R. A. Evidence from sea urchin- sand dollar hybrid embryos for a nuclear control of alkaline phosphatase activity, 21. Food of Balanus, 313. Food of Palaemonetes, 162. Formation of shell in oysters, 92. Free-living flatworms, digestion in, 63. INDEX 433 Frequency of mitotic stages in Allium after x-irradiation, 305. Fruit fly, red cells in, 363. Fundulus hatchlings, optokinetic testing of, 241. Fungi, development of, 1. QAMETES, Molgula, x-irradiation of, 171. Gamma irradiation of toad, 137. Gas exchange by insects, 108. Genetically differentiated strains of mice, sur- vival of, following x-irradiation, 400. Genetics of red cells in Drosophila, 220. Geographical distribution of unarmored Dino- phyceae, 196. Gill area of crabs, 34. GLEASON, M. M. See M. A. McWniNNiE, 371. Glycine content of Porphyra, 363. Glycogen distribution during mushroom de- velopment, 1. Gonad, Spisula, estrogenic material in, 180. Gonad differentiation in Xiphophorus, 422. GORDON, M. S. Observations on osmoregula- tion in the Arctic char, Salvelinus, 28. GOULD, E. Orientation in box turtles, Ter- rapene, 336. GOWEN, J. W. See J. STADLER, 400. GRAY, I. E. A comparative study of the gill area of crabs, 34. Green crab, trematode parasite of, 254. GROSCH, D. S., and Z. H. SMITH. X-ray experiments with Molgula manhattensis : adult sensitivity and induced zygotic lethality, 171. GROSS, W. J. An analysis of response to osmotic stress in selected decapod Crus- tacea, 43. Growth of Palaemonetes larvae, 144, 162. Growth of radiocobalt-treated Tetrahymena, 390. Growth-promoting factors for Endamoeba, 377. Gymnodinium, taxonomy of, 196. Gymnodinoides, excystation of, 132. Gyrodinium, taxonomy of, 196. T-JABITAT, correlation of, with gill area of crabs, 34. Habitat of alga with respect to amino acid content, 363. Hadrocarabus, oxygen uptake in, 108. HAGERMAN, D. D., F. M. WELLINGTON AND C. A. VILLEE. Estrogens in marine in- vertebrates, 180. HAND, C., AND M. L. JONES. An example of reversal of polarity during asexual re- production of a hydroid, 349. HANKS, J. E. The rate of feeding of the common oyster drill, Urosalpinx, at con- trolled water temperatures, 330. Hatchlings, fish, optokinetic testing of, 241. Heart-beat of Squilla, 358. Hematopoietic system of mice, role of, in x-irradiation damage, 400. Hemigrapsus, osmotic regulation in, 43. Hemolymph, insect, dissociation curve of, 108. HENLEY, C., AND D. P. COSTELLO. The ef- fects of x-irradiation on the fertilized eggs of Chaetopterus, 184. Hermit crab, apostome ciliate parasites of, 132. HEUTS, M. J. See A. I. Meuwis, 97. Himanthalia, development of, 81. Hippadenella, new species of, 120. Histochemistry of echinoderm hybrid em- bryos, 21. Histochemistry of mushroom development, 1. Histology of colchicine-treated Dugesia pieces, 371. Histology of cyclopean and synophthalmic fish, 241. HOCHMAN, H. See R. W. DRISKO, 325. HOFFMAN, A. A. Sec J. T. BONNER, 1. Homing ability of box turtle, 336. Hormones, role of, in sexual differentiation of Xiphophorus, 422. Hormones in marine invertebrates, 180. Host-parasite relationship between crustaceans and apostome ciliates, 132. HULBURT, E. M. The taxonomy of unarmored Dinophyceae of shallow embayments on Cape Cod, 196. Hybrids, fish, optokinetic testing of, 241. Hybrid embryos of echinoderms, 21. Hydroid, reversal of polarity in, 349. Hyrolyzed nucleic acids, growth-promoting effects of, on Endamoeba, 377. IMMEDIATE effects of low x-ray doses on Allium mitosis, 305. Induced zygotic lethality in Molgula, follow- ing x-irradiation, 171. Inhibition of mitosis in x-irradiated Chaetop- terus eggs, 184. Inhibition of regeneration in Dugesia after colchicine treatment, 371. Innervation of stomatopod heart, 358. Insects, oxygen uptake in, 108. Invertebrates, marine, estrogens in, 180. IRISAWA, H., AND A. F. IRISAWA. The elec- trocardiogram of a stomatopod, 358. Irradiation of Ascaris eggs, 383. Irradiation of Chaetopterus eggs, 184. Irradiation of mice, 400. Irradiation of Molgula, 171. 434 INDEX Irradiation of onion root tips, 305. Irradiation of Tetrahymena, 390. Irradiation of toad, 137. JAPANESE species of Botryllus, vascular budding in, 225. JENNINGS, J. B. Studies on feeding, diges- tion, and food storage in free-living flat- worms, 63. JONES, J. C., AND E. B. LEWIS. The nature of certain red cells in Drosophila, 220. JONES, M. L. See C. HAND, 349. JONES, R. F. Variation of nitrogen and car- bohydrate constituents during the de- velopment of Himanthalia, 81. JONES, R. F., AND L. R. BLINKS. The amino acid constituents of the phycobilin chromo- proteins of the red alga Porphyra, 363. 17" ETO-ACID production of marine inverte- " brates, 180. Kinetics of replacement of potassium by ru- bidium in Ulva, 249. KUCHLEIN, J. Sec A. PUNT, 108. LABORATORY culture of Balanus, 313. Larval development of Balanus, 313. Larval development of Palaemonetes, 144, 162. Leptoplana, feeding in, 63. Lethal temperature for carp, 97. Lethality of x-irradiation of Molgula, 171. Leucine, content of, in Porphyra, 363. LEWIS, E. B. Sec J. C. JONES, 220. Life-history of Himanthalia, 81. Life-history of trematode, 254. Limnoria, amino acid content of, 325. Littorina as intermediate host for trematode parasite of crab, 254. Low doses of x-irradiation, effects of, on Al- lium mitosis, 305. Low temperature, role of, in feeding of Uro- salpinx, 330. Lucania-Fundulus hybrids, optokinetic testing of, 241. Lunar-day cycles in oxygen consumption of potato, 288. Lunar rhythms in Uca, 267. Lymphocytes of Botryllus as sources of budding, 225. \t ACTRA, estrogenic material in ovary of, 1V1 180. "Magnesium embryos" of Fundulus, 241. Mantle respiration of oyster, 92. Marine alga, amino acids of, 363. Marine alga, reversible replacement of potas- sium by rubidium in, 249. Marine borers, amino acid content of, 325. Marine Bryozoa, 120. Marine invertebrates, estrogens in, 180. Marine shrimp, development of, 144, 162. MARONEY, S. P., JR., A. A. BARBER AND K. M. WILBUR. Studies on shell formation. VI., 92. Massachusetts Dinophyceae, 196. Massartia, taxonomy of, 196. McWniNNiE, M. A., AND M. M. GLEASON. Histological changes in regenerating pieces of Dugesia treated with colchicine, 371. Mesostoma, feeding of, 63. Metabolic activity of crabs, correlation of, with gill areas, 34. Metabolism of phosphorus in sea urchin eggs, 276. Metabolism of potato, 288. Metabolism of x-irradiated Ascaris eggs, 383. Metacercariae of Microphallus, 254. Metamorphosis of Palaemonetes, 144, 162. MEUWIS, A. L, AND M. J. HEUTS. Tempera- ture dependence of breathing rate in carp, 97. Mice, survival of, following x-irradiation, 400. Microphallus, life-history and morphology of, 254. Mitosis in Allium, effects of x-irradiation on, 305. Mitotic activity in regenerating Dugesia, 371. Mitotic effects of x-irradiation of Chaetop- terus eggs, 184. Modification of sexual differentiation in Xi- phophorus, 422. Molgula, x-irradiation of, 171. Mollusc, amino acid content of, 325. Mollusc, estrogenic material in ovary of, 180. Mollusc, feeding of, 330. Mollusc, shell formation in, 92. Mollusc as intermediate host for trematode parasite of crab, 254. Molting of Balanus, 313. Molting of crustacean hosts in relation to excystation of apostome ciliates, 132. Molting stages of Palaemonetes, 144. MORIOKA, W. T. See J. T. BONNER, 1. Morphology of ectoproct bryozoan, 120. Morphology of Palaemonetes larvae, 144. Morphology of trematode, 254. Morphology of unarmored Dinophyceae, 196. Mortality of x-irradiated mice, 400. Motor activity, tidal cycles of, in Uca, 267. Mushroom, development of, 1. Mutant (red cells) of Drosophila, 220. Mytilus as food for Urosalpinx, 330. INDEX 435 , M. Growth-promoting ef- fects of hydrolyzed nucleic acids, nu- cleotides and nucleosides on Endamoeba, 377. Nannochloris as food for Palaemonetes larvae, 162. Nature of certain red cells in Drosophila, 220. Naupliar stages of Balanus, 313. Nematode eggs, x-irradiation of, 383. Nematodinium, taxonomy of, 196. Nerve distribution, optic, in cyclopic and synophthalmic fish, 241. Neurophysiology of Squilla, 358. New species of Dinophyceae, 196. New type of budding in Botryllus, 225. Nitrogen constituents of Himanthalia, 81. Nitzschia as food for Palaemonetes larvae, 162. Nuclear control of alkaline phosphatase ac- tivity, 21. Nucleic acids, growth-promoting effects of, on Endamoeba, 377. Nucleotides and nucleosides, growth-promot- ing effects of, on Endamoeba, 377. Nutrition, role of, in accumulation of radio- cobalt in Tetrahymena, 390. , H., AND H. WATANABE. Vascular budding, a new type of budding in Botryl- lus, 225. Onion root tip, effects of x-irradiation on mitosis in, 305. Optokinetic testing of cyclopean fish, 241. Orientation in box turtles, 336. Orthophosphate, permeability of sea urchin egg to, 276. Osmoregulation in Arctic fish, 28. Osmotic stress, response to, by Crustacea, 43. Ova, Ascaris, x-irradiation of, 383. Ova, Chaetopterus, x-irradiation of, 184. Ova, sea urchin, phosphorus metabolism in, 276. Ovary, Spisula, estrogenic material in, 180. Oxygen, role of, in recovery of Ascaris eggs from x-irradiation, 383. Oxygen consumption of potato, 288. Oxygen uptake of insects, 108. Oxygen uptake of oyster mantle, 92. Oxyrrhis, taxonomy of, 196. Oyster drill, rate of feeding of, 330. Oyster mantle, respiration of, 92. p -32 uptake by sea urchin eggs, 276. Pachygrapsus, response to osmotic stress by, 43. Pagurus, apostome ciliate parasites of, 132. PAHL, G., AND C. S. BACHOFER. Anaerobic recovery of Ascaris eggs from x-irradia- tion, 383. Palaemonetes, larval development of, 144, 162. Parasite of crabs, 132. Parasitic protozoan, growth-promoting fac- tors for, 377. Parenchyma, role of, in regeneration of Dugesia, 371. PARSER, W. ]. Sec A. PUNT, 108. Periodicity in Uca, 7. Periplaneta, oxygen uptake in, 108. Permeability of sea urchin egg to phosphate, 276. Permeability studies on Ulva, 249. Persistent tidal cycles of motor activity in Uca, 267. Phosphatase activity of echinoderm hybrid embryos, 21. Phosphorus, metabolism of, by sea urchin egg, 276. Phycobilin chromoproteins of Porphyra, 363. Physiology of Arctic fish, 28. Pigment, amino acid content of, in Porphyra, 363. Pigment, red, in Drosophila cells, 220. Pigment dispersion rhythms in Uca, 7. Planarian, regeneration of, after colchicine treatment, 371. Plankton as food for Palaemonetes, 162. Platyhelminth, feeding in, 63. Platyhelminth, morphology and life-history of, 254. Platyhelminth, regeneration of, after col- chicine treatment, 371. Polarity, reversal of, in hydroid, 349. Polycelis, feeding in, 63. Polykrikos, taxonomy of, 196. Polysaccharides, distribution of, during de- velopment of mushroom, 1. Population density, role of, in radiocobalt accumulation in Tetrahymena, 390. Porphyra, amino acids of, 363. Porphyridium as food for Palaemonetes, 162. Position of burrows of Uca, 7. Post-irradiation treatment of Ascaris eggs, 383. Potassium, replacement of, by rubidium, in Ulva, 249. Potato, oxygen consumption of, 288. Potential, action, of stomatopod heart, 358. Pressure, barometric, effects of, on oxygen consumption of potato, 288. Propanediol phosphate in sea urchin eggs, 276. Prophase, sensitivity of to x-irradiation, 184, 305. Protection of mice against radiation damage, 400. 436 INDEX Protective effects of anaerobiosis and cyanide against x-irradiation damage to Ascaris eggs, 383. Protein metabolism of marine borers, 325. Protein synthesis in Himanthalia, 81. Protochordate, vascular budding in, 225. Protozoan parasites of Crustacea, 132. Protozoan, parasitic, growth-promoting fac- tors for, 377. Protozoan, radiocobalt accumulation in, 390. Pugettia, osmotic regulation in, 43. PUNT, A., W. J. PARSER AND J. KUCHLEIN. Oxygen uptake in insects with cyclic carbon dioxide release, 108. Pupae, insect, oxygen uptake of, 108. T? NA as growth-promoting factor for Enda- moeba, 377. Radiation effects on Chaetopterus eggs, 184. Radiation effects on Molgula gametes, 171. Radiation resistance of mice, 400. Radiocalcium uptake by oyster, 92. Radiocobalt accumulation in Tetrahymena, 390. Radiophosphorus, uptake of, by sea urchin eggs, 276. Rate of breathing of carp, 97. Rate of feeding of oyster drill, 330. Rate of larval development of Palaemonetes, 144. Recessive mutant in Drosophila, 220. Reconstitution of colchicine-treated Dugesia, 371. Recovery of Ascaris eggs from x-irradiation, 383. Red alga, amino acids of, 363. Red cells in Drosophila, 220. Regeneration of colchicine-treated Dugesia, 371. Relationship between burrow position and tidal rhythm of Uca, 7. Relationship between diet and development of Palaemonetes, 162. Replacement of potassium by rubidium in Ulva, 249. Reproduction, asexual, in Botryllus, 225. Reproduction, asexual, in hydroid, 349. Reptile, orientation in, 336. Respiration of carp, 97. Respiration of insects, 108. Respiration of irradiated Ascaris eggs, 383. Respiration of oyster mantle, 92. Respiration of potato, 288. Respiratory rates of crabs, 43. Response of potato to barometric pressure, 288. Response to osmotic stress by Crustacea, 43. Retinal layers of toad, susceptibility of, to gamma irradiation, 137. Reversal of polarity in hydroid, 349. Reversible replacement of potassium by ru- bidium in Ulva, 249. Rhythms of activity in Uca, 267. Rhythms of oxygen-consumption by potato, 288. Rhythmicity of Uca, 7. Roentgen irradiation of Ascaris eggs, 383. Roentgen irradiation of Chaetopterus eggs, 184. Roentgen irradiation of mice, 400. Roentgen irradiation of Molgula gametes, 171. Roentgen irradiation of onion root tips, 305. ROGERS, K. T. Optokinetic testing of cyclo- pean and synophthalmic fish hatchlings, 241. ROGICK, M. D. Studies on marine Bryozoa. X., 120. Root tip, Allium, effects of x-irradiation on, 305. Round-worm eggs, x-irradiation of, 383. Rubidium, replacement of potassium by, in Ulva, 249. QALT exchange in Crustacea, 43. Salvelinus, osmoregulation in, 28. Sand dollar-sea urchin hybrid embryos, 21. SCOTT, G. T., AND R. DEVOE. The reversible replacement of potassium by rubidium in Ulva, 249. Sea urchin egg, metabolism of phosphorus in, 276. Sea urchin-sand dollar hybrid embryos, 21. Sea weed, nitrogen and carbohydrate con- stituents of, 81. Semilunar rhythms in Uca, 7. Sensitivity of adult Molgula to x-rays, 171. Sexual differentiation in Xiphophorus, 422. Sexual dimorphism in gill area of crabs, 34. Shell formation in oysters, 92. Shrimp, larval development of, 144, 162. Shrimp, mantis, electrocardiogram of, 358. SHRINER, J. See M. F. BENNETT, 267. SLATER, J. V. Radiocobalt accumulation in Tetrahymena, 390. SMITH, Z. H. See D. S. GROSCH, 171. Solanum, oxygen consumption of, 288. Solar-day cycles in oxygen consumption of potato, 288. Sperm, Molgula, x-irradiation of, 171. Sphinx, oxygen uptake of, 108. Spiracle movements in insects, 108. Spisula, estrogenic material in ovary of, 180. Spontaneous motor activity, cycles of, in Uca, 267. INDEX 437 Speculation in mushroom, 1. Squilla, electrocardiogram of, 358. STABLER, J., AND J. W. GOWEN. Contribu- tions to survival made by body cells of genetically differentiated strains of mice following x-irradiations, 400. Stenostomum, feeding in, 63. Stomatopod, electrocardiogram of, 358. Storage of food in flatworms, 63. Strain differences in survival of x-irradiated mice, 400. Stress, osmotic, response to, by Crustacea, 43. Strongylocentrotus-Dendraster hybrid em- bryos, 21. Strongylocentrotus, metabolism of phosphorus in eggs of, 276. Studies in metabolism of phosphorus in sea urchin eggs, 276. Studies on marine Bryozoa, 120. STUNKARD, H. W. The morphology and life- history of the digenetic trematode, Micro- phallus, 254. Sun, role of, in orientation of box turtles, 336. Survival of x-irradiated mice, 400. Susceptibility to gamma irradiation of toad retina, 137. Swordtail fish, sexual differentiation in, 422. Synophthalmic fish, optokinetic testing of, 241. 'pADPOLES, Molgula, development of, from x-irradiated gametes, 171. Taxonomy of Bryozoa, 120. Taxonomy of trematodes, 254. Taxonomy of unarmored Dinophyceae, 196. Teleost, sexual differentiation in, 422. Teleost hatchlings, optokinetic testing of, 241. Temperature, role of, in irradiation damage to Ascaris eggs, 383. Temperature, role of, in x-irradiation damage to onion root tips, 305. Temperature, water, role of, in feeding of Urosalpinx, 330. Temperature dependence of breathing rate in carp, 97. Teredo, amino acid content of, 325. Terrapene, orientation in, 336. Testing, optokinetic, of cyclopean fish, 241. Testosterone, effect of, on sexual differentia- tion of Xiphophorus, 422. Tetrahymena, radiocobalt accumulation in, 390. Thorocomonas as food for Palaemonetes larvae, 162. Tidal cycles in Uca, 267. Tidal rhythm and burrow position of Uca, 7. Toad, gamma irradiation of, 137. TRACER, W. Excystation of apostome ciliates in relation to molting of their crustacean hosts, 132. Trematode, morphology and life-history of, 254. Triclad turbellarian, feeding in, 63. Tunicate, vascular budding in, 225. Turbellaria, feeding in, 63. Turtles, orientation in, 336. , apostome ciliate parasite of, 132. Uca, osmotic regulation in, 43. Uca, relation between burrow position and tidal rhythm of, 7. Uca, tidal cycles of activity in, 267. Ulva, reversible replacement of potassium by rubidium in, 249. Upogebia, osmotic regulation in, 43. Uptake of oxygen in insects, 108. Uptake of radiocobalt by Tetrahymena, 390. Urosalpinx, rate of feeding of, 330. yALLOWE, H. H. Sexual differentiation in the teleost fish Xiphophorus, as modi- fied by experimental treatment, 422. Variation of nitrogen and carbohydrate con- stituents during development of Himan- thalia, 81. Vascular budding in Botryllus, 225. VILLEE, C. A. See D. D. HAGERMAN, 180. \yARNOWIA, taxonomy of, 196. WATANABE, H. Sec H. OKA, 225. Water temperature, role of, in feeding of Uro- salpinx, 330. Wave-length, role of, in survival of x-ir- radiated mice, 400. WELLINGTON, F. M. See D. D. HAGERMAN, 180. WHITELEY, A. H. Sec A. L. BOLST, 276. Whole-body gamma irradiation of toad, 137. Whole-body x-irradiation of mice, 400. Whole-body x-irradiation of Molgula, 171. WILBUR, K. M. See S. P. MARONEY, JR., 92. Worm, planarian, regeneration of, 371. Worms, feeding of, 63. X-IRRADIATION of Aiiium, 305. X-irradiation of Ascaris eggs, 383. X-irradiation of Chaetopterus eggs, 184. X-irradiation of mice, 400. X-irradiation of Molgula, 171. Xiphophorus, sexual differentiation in, 422. £OOPLANKTON as food for Palae- monetes, 162. Zygotic lethality in Molgula after x-irradia- tion, 171. BIOLOGY MATERIALS The Supply Department of the Marine Biological Labora- tory has a complete stock of excellent plain preserved and injected materials, and would be pleased to quote prices on school needs. PRESERVED SPECIMENS for Zoology, Botany, Embryology, and Comparative Anatomy LIVING SPECIMENS for Zoology and Botany including Protozoan and Drosophila Cultures, and Animals for Experimental and Laboratory Use. MICROSCOPE SLIDES for Zoology, Botany, Embryology, Histology, Bacteriology, and Parasitology. CATALOGUES SENT ON REQUEST Supply Department MARINE BIOLOGICAL LABORATORY Woods Hole, Massachusetts CONTENTS Page BENNETT, MIRIAM F., JOAN SHRINER AND ROBERT A. BROWN Persistent tidal cycles of spontaneous motor activity in the fiddler crab, Uca pugnax 267 BOLST, ALBERT L., AND ARTHUR H. WHITELEY Studies of the metabolism of phosphorus in the development of the sea urchin, Strongylocentrotus purpuratus 276 BROWN, F. A., JR. Response of a living organism, under "constant conditions" including pressure, to a barometric-pressure-correlated, cyclic, external variable 288 CHANDLER, ARTHUR C., JR. The immediate effects of low doses of x-radiation on the frequency of several mitotic stages in the Allium root tip 305 COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT Larval development of Balanus eburneus in the laboratory 313 DRISKO, RICHARD W., AND HARRY HOCHMAN Amino acid content of marine borers 325 HANKS, JAMES E. The rate of feeding of the common oyster drill, Urosalpinx cinerea Say, at controlled water temperatures 330 GOULD, EDWIN Orientation in box turtles, Terrapene c. Carolina (Linnaeus) 336 HAND, CADET, AND MEREDITH L. JONES An example of reversal of polarity during asexual reproduction of a hydroid 349 IRISAWA, HlROSHI, AND AYA FUNAISHI IRISAWA The electrocardiogram of a stomatopod 358 JONES, RAYMOND F., AND L. R. BLINKS The amino acid constituents of the phycobilin chromoproteins of the red alga Porphyra 363 MCWHINNIE, MARY A., AND MARY M. GLEASON Histological changes in regenerating pieces of Dugesia dorotocephala treated with colchicine 371 NAKAMURA, MITSURU Growth-promoting effects of hydrolyzed nucleic acids, nucleotides, and nucleosides on Endamoeba histolytica 377 PAHL, GEORGE, AND C. S. BACHOFER Anaerobic recovery of Ascaris eggs from x-irradiation 383 SLATER, JOHN V. Radiocobalt accumulation in Tetrahymena 390 STADLER, JANICE, AND JOHN W. GOWEN Contributions to survival made by body cells of genetically differen- tiated strains of mice following x-irradiations 400 VALLOWE, HENRY H. Sexual differentiation in the teleost fish Xiphophorus hellerii, as modi- fied by experimental treatment 422 . MBL WHO! LIBRAK UH 1AZH 0