THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, University of Texas ARTHUR W. POLLISTER, Columbia University FRANK A. BROWN, JR., Northwestern University C. L. PROSSER, University of Illinois JOHN B. BUCK, National Institutes of Health MARY E. RAWLES, Carnegie Institution of T. H. BULLOCK, University of California, Washington Los Angeles WM. RANDOLPH TAYLOR, University of Michigan E. G. BUTLER, Princeton University A. R. WHITING, University of Pennsylvania J. H. LOCHHEAD, University of Vermont CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor VOLUME 114 FEBRUARY TO JUNE, 1958 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, 1958 PAGE GRANT, WILLIAM C., JR., AND JOAN A. GRANT Water drive studies on hypophysectomized efts of Diemyctylus viri- descens GRINNELL, ALAN D., AND DONALD R. GRIFFIN The sensitivity of echolocation in bats 10 HARVEY, WILLIAM R., AND CARROLL M. WILLIAMS Physiology of insect diapause. XI. Cyanide-sensitivity of the heart- v beat of the Cecropia silkworm, with special reference to the anaerobic capacity of the heart 23 HARVEY, WILLIAM R., AND CARROLL M. WILLIAMS Physiology of insect diapause. XII. The mechanism of carbon mon- oxide-sensitivity and -insensitivity during the pupal diapause of the Cecropia silkworm 36 HYMAN, LIBBIE H. Notes on the biology of the five-lunuled sand dollar 54 LOQSAJiOFF, V. L. Some aspects of behavior of oysters at different temperatures 57 MCDONALD, BARBARA BROWN Quantitative aspects of deoxyribose nucleic acid (DNA) metabolism in an amicronucleate strain of Tetrahymena 71 MARUYAMA, K. Contractile protein from crayfish tail muscle 95 HYMAN, LIBBIE H. The occurrence of chitin in the lophophorate phyla 106 No. 2. APRIL, 1958 ALLEN, ROBERT D., AND EDWARD C. ROWE The dependence of pigment granule migration on the cortical reaction in the eggs of Arbacia punctulata 113 BUCK, JOHN Cyclic CO2 release in insects. IV. A theory of mechanism 118 CHACE, FENNER A., JR. A new stomatopod crustacean of the genus Lysiosquilla from Cape Cod, Massachusetts 141 CHRISTENSEN, AAGE MILLER, AND JOHN J. MCDERMOTT ^~ Life-history and biology of the oyster crab, Pinnotheres ostreum Say. . (l46y CLARK, A. M., AND M. J. PAPA Some effects of oxygen upon the white pupae of Habrobracon 180 OSG^A iv CONTENTS GANAROS, ANTHONY E. On development of early stages of Urosalpinx cinerea (Say) at constant temperatures and their tolerance to low temperatures 188 HSIAO, SIDNEY C., AND HOWARD BOROUGHS The uptake of radioactive calcium by sea urchin eggs. I. Entrance of Ca45 into unfertilized egg cytoplasm 196 JOHNSON, T. W., JR. A fungus parasite in ova of the barnacle Chthamalus fragilis denticulata . . 205 LYNCH, WILLIAM F. The effect of x-rays, irradiated sea water, and oxidizing agents on the rate of attachment of Bugula larvae 215 MALAMED, SASHA Gastrular blockage in frogs' eggs produced by oxygen poisoning 226 MAZIA, DANIEL The production of twin embryos in Dendraster by means of mercapto- ethanol (monothioethylene glycol) 247 TWEEDELL, KENYON S. Inhibitors of regeneration in Tubularia 255 No. 3. JUNE, 1958 BRYAN, JOHN H. D., AND JOHN W. Go WEN The effects of 2560 r of x-rays on spermatogenesis in the mouse 271 COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT Larval development of Balanus amphitrite var. denticulata Broch reared in the laboratory 284 DAVIS, H. C. Survival and growth of clam and oyster larvae at different salinities. . . 296 EKBERG, DONALD R. Respiration in tissues of goldfish adapted to high and low temperatures 308 FlNGERMAN, MlLTON, AND MlLDRED E. LOWE Stability of the chromatophorotropins of the dwarf crayfish, Cambarel- lus shufeldti, and their effects on another crayfish 317 GROSS, WARREN J. Potassium and sodium regulation in an intertidal crab 334 MCFARLAND, WILLIAM N., AND FREDERICK W. MUNZ A re-examination of the osmotic properties of the Pacific hagfish, Poli- stotrema stouti 348 MOULTON, JAMES M. The acoustical behavior of some fishes in the Bimini area 357 PROVASOLI, L. Effect of plant hormones on Ulva 375 RUGH, ROBERTS The so-called "recovery" phenomenon and "protection" against x- irradiation at the cellular level 385 YOUNG, RICHARD S. Development of pigment in the larva of the sea urchin, Lytechinus variegatus. . 394 Vol. 114, No. 1 February, 1958 ^ i i THE X^1C/ BIOLOGICAL BULLETIN LJ^.?Ah rt*/ PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY WATER DRIVE STUDIES ON HYPOPHYSECTOMIZED E OF DIEMYCTYLUS VIRIDESCENS. PART I. THE ROLE OF THE LACTOGENIC HORMONE WILLIAM C. GRANT, JR. AND JOAN A. GRANT Department of Biology, Williams College, H'illutinstoza'n, Massachusetts It is well known that following metamorphosis the eastern, spotted newt D'ie- myctylits viridesccns passes into a terrestrial or red eft stage which lasts from three to four years before the animals migrate to water where they become sexually mature. In certain parts of Long Island and in the Woods Hole area, however, Noble (1926, 1929) found that the eft stage failed to develop. There has been considerable interest in the role played by the endocrine glands in the events as- sociated with the migration of efts from land to water. Adams (1932) was able to induce adult skin texture and pigmentation in efts injected with an anterior lobe preparation (phyone), while Dawson (1936) showed that pituitary preparations administered to efts brought about the maturation of the lateral line system. The studies of Reinke and Chadwick (1939) demonstrated that efts receiving implants of whole adult pituitaries or anterior lobes voluntarily migrated to water from 2 to 6 days following treatment. The test animals acquired a smooth, moist skin and showed a tendency toward the olive pigmentation of the adult. In certain cases keeling of the tail was evident after an extended period. The thyroids showred some stimulation as the result of the implants but the gonads remained unaffected. Molting usually occurred on the second to fourth day following implantation. Gonadectomized efts molted and entered water from 4 to 8 days after implantation of adult pituitaries according to Reinke and Chadwick (1940). Thyroidectomized individuals and animals which had been both thyroidectomized and gonadectomized were forced to water following similar treatment, although in these cases molting was abnormal with pieces of cornified epithelium sloughing off in patches after the animals had assumed the aquatic habitat. The failure of thyroidectomized efts to undergo a normal molt is understandable in the light of investigation by Adams and her co-workers. Adams ct al. (1932) and Adams and Grierson (1932) have shown that a pituitary-thyroid relationship is necessary for proper molting. Changes in cutaneous circulation, rather than the stimulation of secretion of the cutaneous glands, may be of major importance to the molting process. According to Chadwick (1948), however, the thyroid exerts a direct effect on molting by the stimulation of the inter-papillary skin glands. An increased incidence in molting, noted by Chadwick and Jackson (1948) following 1 Copyright © 1958, by the Marine Biological Laboratory 2 WILLIAM C. GRANT, JR. AND JOAN A. GRANT the injection of efts with prolactin, may have been due to the stimulation of cell division in the epidermis. Chadwick (1940a) induced water drive in efts ranging from 60 to 95 mm. in length with injections of Antuitrin G (Parke Davis). This preparation was less effective than implants of adult newt pituitaries, as it failed to induce water drive in smaller animals or those which had been thyroidectomized. Prolactin was iden- tified with the active water drive principle of the anterior lobe by Chadwick (1940b). Injections of 14 to 20 mg. of prolactin (Eli Lilly unidentified lot) caused water drive in all normal, thyroidectomized and gonadectomized efts within a period of 10 days. Chadwick (1940c, 1941) obtained the migration of efts to water following intramuscular and intraperitoneal implants of hypophyses of a variety of vertebrates such as the water snake (Natrix), the domestic fowl and several genera of urodele and anuran amphibians. That the water drive reaction may be more complex is indicated by Dr. Richard W. Payne (unpublished data) who obtained positive results after the injection of a wide number of pituitary prep- arations, several of which showed negative prolactin activity by the pigeon crop assay. Tuchmann-Duplessis (1948, 1949) has shown that the administration of 60-120 Riddle-Bates units of prolactin to the land stage of Tritnnts cristatits and T. alpestris resulted in the migration of the animals to water and the assumption of pigmentation and morphological characters associated with the aquatic, repro- ductive phase. The cloaca became enlarged while the gonads and prostate became active. Prolactin administered to castrated males produced only water drive and color changes. The results reported above indicate that prolactin is probably the active prin- ciple concerned with the initiation of water drive and that the thyroid, while not necessary to this reaction, facilitates the process by conditioning normal molting. Nevertheless, the situation remains confused, considering that a number of hormone preparations other than prolactin have produced water drive activity, and it is not clear which of the various phenomena accompanying migration (pigment changes, etc.) are stimulated directly by the water drive factor and which result from en- dogenous release of other endocrine substances through stimulation of the pituitary. The present investigation is part of an extended study seeking to clarify the complex situation involved in the transformation of the terrestrial eft to the aquatic adult. The use of hypophysectomized test animals has been necessary in order to rule out the endogenous release of prolactin itself by a specific testing agent or non- specific "shock" effect and to eliminate synergic reaction within the pituitary. Grant and Grant (1956) have previously indicated that prolactin causes water migration and skin changes in hypophysectomized efts but none of the other changes toward the adult condition. "Water drive" is used below to indicate the actual migration of efts to water and "water drive syndrome" to designate migration plus associated morphological changes, etc. The term "drive" is used loosely and is not necessarily meant to imply the operation of precise directive factors. The authors wish to express their indebtedness to Dr. Grace E. Pickford, Bingham Oceanographic Laboratory, Yale University, for suggesting the project and for furnishing many of the preparations used. We are further indebted to WATER DRIVE STUDIES ON DIEMYCTYLUS 3 Dr. C. H. Li of the Hormone Research Laboratory, University of California, for the donation of a quantity of his highly purified prolactin used in the tests. This investigation was sponsored, in part, by a grant from the Mearl Corporation of New York City, through the kindness of Mr. Harry E. Mattin and Dr. Leon M. Greenstein, while a generous Grant-in- Aid from the Signa Xi-RESA Research Fund helped finance investigations through the past year. Our thanks are extended to Williams College and to Dr. S. A. Matthews, Chairman of the Department of Biology, for laboratory space and numerous facilities used during this investigation. MATERIALS AND METHODS Efts were collected near Lyme Center, New Hampshire ; Quechee, New York ; Honesdale, Pennsylvania, and Williamstown, Massachusetts. Animals from dif- ferent localities were segregated and kept in plastic boxes, 15-20 animals per box, on a thick bed of moss (visually Polytrichum). Room temperature ranged from 21°-24° C. Illumination was that of the room except for a few hours each day when the containers were subjected to direct illumination from an overhead lamp. This procedure aided the growth of the moss, the presence of which seems to be extremely helpful in keeping animals healthy. Attempts made to keep efts on a floor of wet, cellulose sponge proved quite unsuccessful. Animals were fed En- chytraeus worms and blowfly larvae. Some test animals were kept without feed- ing for extended periods in a refrigerator at approximately 5° C. Hypophysec- tomized efts are particularly susceptible to infection, but 1 c/f, solutions of potassium bichromate or malachite green diluted 1:15,000 have proved efficient prophylactic agents. The total length of experimental animals ranged from 40 to 74 mm. with weight varying from 0.5 to 1.4 gm. The probable age of such animals was from 1 to 2.5 years and all were well removed from the naturally occurring aquatic phase of their cycle. Snout-vent measurements, which have proved an accurate standard in herpetological work, were also recorded. Animals were anesthetized in a 0.5 (/c solution of chloretone and hypophysec- tomized with the aid of a suction pipette attached to an aspiration unit. At the termination of the experiments hypophysectomy was verified histologically on most specimens. Two weeks after hypophysectomy subcutaneous injections were made with a 27-gauge, Huber point needle. In general, injections were made every other day at various concentrations of solution, but a constant volume of 0.1 cc. at each injection was maintained. Water drive responses were studied in con- tainers possessing equal areas of land and water. The time taken for the assump- tion of the aquatic habitat, the number of molts and the duration of the aquatic phase of life were recorded for all individuals as far as possible. The various preparations used in the injections were made up in a standard amphibian Ringer's, with controls receiving the same volume of saline as experi- mental animals. The following pituitary preparations were tested: FSH (swine, Armour Lot No. K45208R), LH (sheep, Armour Lot 227-80), TSH (Armour Lot 317-51), Antuitrin S (Parke Davis Lot P459D), ACTH (hog. Armour Lot K 52204), posterior pituitary preparation (hog, Lot 503), GH (beef growth hor- mone, Wilhelmi Lot B168), prolactin (Sheep, Schering Lot 4g Hyex 4, Armour sheep Lot 759-CCC and the highly purified sheep preparation of Li), MSH WILLIAM C. GRANT, JR. AND JOAN A. GRANT TABLE I Results of water drive studies following the treatment of land phase Diemyctylus viridescens with various pituitary preparations Treatment Hypophy- sectomy Wt., gm. Length, mm. Total/Standard Total dose, mg. Results Molting FSH Armour* — 0.55 55/26 8 9 days to water + — 0.80 55/29 8 10 days to water + + 1.20 67/36 8 partial response abnormal + 1.40 69/36 8 8 days to water abnormal + 0.85 67/34 0.8 negative response — + 0.72 63/32 0.8 negative response — + 0.47 56/29 0.8 negative response — LH Armour — . 0.81 56/33 8 negative response — — 0.47 48/23 8 negative response — + 1.10 73/36 8 negative response — + 1.12 68/37 8 negative response — GH Wilhelmi + 1.10 73/38 0.8 negative response + + 0.53 60/29 0.8 negative response — + 0.53 59/32 0.8 negative response — + 0.72 63/33 0.8 negative response — + 0.74 63/32 0.8 negative response — + 0.53 51/31 0.8 negative response — A CTH A IDI our + 0.62 57/26 0.8 negative response — + 0.71 64/32 0.8 negative response + + 0.65 59/32 0.8 negative response — + 0.67 62/34 0.8 negative response — + 0.68 64/33 0.8 negative response — A corticotropin (Li) + 0.84 65/35 0.8 negative response — Posterior pituitary Armour + 1.22 72/37 0.8 negative response — + 0.68 58/32 0.8 negative response — + 0.50 52/31 0.8 negative response — + 0.62 54/31 0.8 negative response — TSH A rmour + 0.61 58/32 0.8 negative response — + 0.74 61/34 0.8 negative response + + 0.84 65/35 0.8 negative response + + 0.57 54/33 0.8 negative response + + 0.62 60/33 0.8 negative response + Antuitrin S l';irke & Davis — 0.65 52/30 6 dead 7th day — — 0.72 53/35 4 dead 5th day — MS1I A rmour + 0.88 66/35 2 negative response — + 0.47 51/28 2 negative response — + 0.55 55/30 2 negative response — Intermedia (Li) + 0.60 58/39 0.8 negative response — ' All animals receiving 8 mg. of FSH showed a tendency toward the olive pigmentation of the adult. WATER DRIVE STUDIES ON DIEMYCTYLUS (melanophore stimulating hormone, Armour Lot R 527225). According to Steel- man et al. (1953), the assay for the FSH preparation shows it to be contaminated with 0.5 I.U. of prolactin per mg., a fact which is of importance in interpreting the results given below. RESULTS (a) Various mammalian pituitary preparations The detailed results of this series of injections are given in Table I. LH, TSH, ACTH, MSH, posterior pituitary, GH and Antuitrin S failed to induce water drive in any of the animals tested. One animal given 0.8 mg. of ACTH-free Intermedin (Li) also gave a negative response. Most efts treated with TSH underwent a normal molt following injections, while one eft of the GH series and one of the ACTH series showed this reaction. All other preparations failed to produce normal molts in hypophysectomized individuals with the result that the FIGURE 1. A normal eft (A) is shown beside a hypophysectomized animal (B), which having failed to molt is covered with a thick layer of cornified epithelium. WILLIAM C. GRANT, JR. AND JOAN A. GRANT TABLE II Results of water drive studies following the treatment of land phase Diemyctylus viridescens with prolactin Treatment Wt., gm. Length, mm. Total/Standard Total dose. tng. Effective dose I.U.* Results Molting Xon-hypophysecto- mized Schering prolactin 30 I.U./mg. 0.54 50/28 8 240 7 days to water + 0.64 51/29 8 240 7 days to water + keeling of tail and olive pigmentation Hvpophysectomized Armour prolactin 25-30 I.U./mg. 1.12 74/39 8 216 8 days to water abnormal 0.41 56/29 8 162 6 days to water — 0.61 63/33 0.8 23 10 days to water abnormal 0.43 55/28 0.8 23 8 days to water — 0.76 57/31 0.8 23 7 days to water abnormal 0.80 60/29 0.08 — partial response abnormal Hvpophysectomized Prolactin (C.H.Ln 35 I.U./mg. 0.66 57/32 0.4 14 10 days to water — 1.14 69/36 0.4 10.5 5 days to water — 0.63 59/32 0.4 10.5 5 days to water abnormal 0.75 65/34 0.4 7 4 days to water abnormal 0.59 58/32 0.4 10.5 5 days to water — 0.48 55/29 0.4 — Dead on 3rd day 0.73 60/32 0.4 — Dead on 4th day 0.80 62/34 0.4 14 7 days to water — 0.71 57/31 0.4 14 8 days to water abnormal 0.35 40/26 0.4 14 7 days to water abnormal 0.60 57/30 0.4 14 7 days to water — 0.61 54/29 0.4 14 8 days to water abnormal 0.97 69/36 0.4 10.5 6 days to water abnormal Hypophysectomized Prolactin (C.H.Li} 35 I.U./mg. 0.87 58/34 0.04 1.4 8 days to water — 0.92 64/35 0.04 1.4 10 days to water — 0.63 57/29 0.04 1.4 8 days to water abnormal 0.82 65/34 0.04 1.4 8 days to water — 0.44 50/27 0.04 1.4 8 days to water abnormal 0.78 70/35 0.04 1.05 6 days to water — * Effective dose is estimated as the amount of prolactin efts had received at the time of their assumption of an aquatic habitat. efts rapidly became covered with a thick, black layer of cornified epithelium until even the eyes were obscured (Fig. 1). Both normal and hypophysectomized efts receiving a total of 8 mg. of FSH showed water drive activity from 8 to 10 days following the initial injections. One WATER DRIVE STUDIES ON DIEMYCTYLUS / animal failed to give a complete reaction and was in and out of water for several weeks before returning permanently to land. In these animals molting was ab- normal with the skin sloughing off in irregular patches after the efts had entered water. There was a trend in the pigmentation of all individuals toward the adult olive, though this was more marked in the non-operated efts. It is interesting that tests for water drive in animals receiving 0.8 mg. of FSH were completely negative, and that molting failed to occur. (/> ) Tests with prolactin Two unhypophysectomized animals receiving 8 mg. (240 I.U.) of Schering prolactin migrated to water on the seventh day following the initial injections and within a fewr weeks had acquired many features associated with the water drive syndrome (i.e., smooth, moist skin, olive pigmentation and keeling of the tail). Other prolactin preparations were injected into hypophysectomized efts in doses varying from 8.0 to 0.04 mg. as shown in Table II. In all cases where death did not occur before the injections were completed, the tests were positive. The ani- mals assumed the aquatic habitat from 4 to 10 days following the first injection. It should be noted that several animals migrated before all injections had been com- pleted and it is therefore desirable to give results in terms of the effective dose (i.e., the dose animals had received at the time of the water-drive response) rather than total dose. The range in effective dose was from 216 to 1.05 I.U. The water drive reaction is very positive as animals giving the reaction remain completely submerged, take food under water and will immediately return to water if placed on land. One eft receiving 0.08 mg. (2.3 I.U.) failed to give the complete reaction but migrated alternately between land and water over the time observed. Records on the duration of water drive are far from complete as most test animals died before leaving water. However, in a number of cases animals actually did return to land after periods varying from two to five weeks. It is of particular significance to note that whereas all hypophysectomized animals showed positive water drive in response to treatment with prolactin, they failed to assume the olive pigmentation and tail keel associated with the water drive syn- drome. When molting occurred it was abnormal, but beneath the patches of thick- ened corneum the smooth, moist skin retained the orange pigmentation of the eft and showed no tendency toward the adult olive. No keeling of the tail was appar- ent in any individual even after extended periods in water. CONCLUSIONS The primary concern of the present investigation was to determine as precisely as possible the endocrine factor responsible for water drive in the red eft. From the results reported above we are in agreement with Chadwick (1940c) that pro- lactin is the active principle. All animals treated with this substance migrated to water and assumed a smooth skin texture similar to that of the adult. As tests were conducted on hypophysectomized efts the possibility of hypophyseal synergy or en- dogenous release must be ruled out. All other pituitary preparations administered gave a negative reaction with the exception of the 8-mg. dose of FSH. This is understandable, as the assay for the gonadotropin shows it to be contaminated with 8 WILLIAM C. GRANT, JR. AND JOAN A. GRANT lactogenic hormone. Both the Armour prolactin and the homogeneous preparation of Li produced positive results in animals receiving as little as 2.3 to 1.4 I.U. per effective dose. The 4 I.U. of prolactin contained in the FSH were therefore quite sufficient to initiate water drive. No minimum dosage level for the water drive re- action has yet been established but the failure of 0.8 mg. FSH to elicit positive re- sults may indicate it to be about 0.4 I.U. Though there was some variability in the time animals responded to treatment with prolactin, there is at present no indication of a dose-response relationship and it is suggested that the reaction may follow the all-or-none principle. Reinke and Chadwick (1940) have shown that the thyroid and gonads are not directly involved in the water drive response and initial histological survey of our prolactin tests shows no thyrotropic or gonadotropic activity. The lactogenic hor- mone is not effective in promoting molt in hypophysectomized animals as it was in normal efts studied by Chadwick and Jackson (1948). However, as molting did occur in efts receiving injections of TSH it suggests that the increased molting re- ported by Chadwick and Jackson (1948) in intact animals was due to the endoge- nous release of TSH resulting from treatment. Though our investigations are in general agreement with those of Chadwick, we cannot support his assumption that prolactin effects the entire water drive syndrome. Work on hypophysectomized animals indicates that the problem is considerably more complex and can tentatively be divided into four major steps. 1. Migration to water and change of skin texture : induced by prolactin. 2. Normal molting which facilitates but does not directly affect water drive : release of thyroid hormone mediated through the pituitary (TSH). 3. Appearance of olive pigmentation : unknown principle involved, possibly MSH. 4. Morphological characteristics associated with water drive such as keeling of the tail and development of the lateral line system : unknown principle or principles involved. It is tempting to suggest that prolactin may initiate the entire water drive syn- drome by triggering the endogenous release of other endocrines which induce many changes associated with the aquatic phase. The identification of these substances, the parts of the cycle they effect and possible interrelationships involved will be taken up in future papers. In conclusion it appears safe to say that the lactogenic hormone produces water drive and skin change, and that the red eft test for the presence of prolactin (1 I.U. or above) is a positive and reliable one. SUMMARY 1. Other investigators have shown that the land (eft) stage of Diemyctylus viridesccns can be induced to enter water and assume adult pigmentation and morphological characteristics following treatment with various pituitary prepara- tions. Hypophysectomized efts were used in the present experiment in order to assure positive identification of the active, water drive principle. 2. Operated animals treated with LH, growth hormone, ACTH, posterior pi- tuitary preparation, TSH, Antuitrin S and melanophore-stimulating hormone gave a negative response. Eight-milligram injections of FSH produced water drive in WATER DRIVE STUDIES ON DIEMYCTYLUS several animals, but this was most probably clue to the contamination of the sub- stance with prolactin. 3. Most hypophysectomized efts, with the exception of those receiving TSH, either failed to molt or underwent an abnormal molt after the animals had been induced to enter water. 4. Operated animals receiving injections of prolactin (240 to 1.05 I.U.) migrated to water from 4 to 10 days following treatment. However, they failed to acquire adult pigmentation and associated characteristics. 5. The lactogenic hormone has been identified as the principle which initiates the migration of efts to water and the water drive test for prolactin is considered to be reliable. LITERATURE CITED ADAMS, A. E., 1932. Observations on the effect of anterior pituitary extract (phyone) on the 'red phase' of Triturus viridcsccns. Anat. Rec., 52: 46. (Abst.) ADAMS, A. E., AND M. C. GRIERSON, 1932. Cornification and molting in Triturus. Proc. Soc. Exp. Biol. Med., 30 : 341-344. ADAMS, A. E., A. KUDER AND L. RICHARDS, 1932. The endocrine glands and molting in Tri- turus viridcsccns. J. Exp. Zool., 63 : 1-55. CHADWICK, C. S., 1940a. Induction of water drive in Triturus viridescens with anterior pitui- tary extract. Proc. Soc, Exp. Biol. Med., 43: 509-511. CHADWICK, C. S., 1940b. The water drive in Triturus viridcsccns as an effect of the growth promoting hormone of the anterior hypophysis. /. Tenn. Acad. Sci., 15: 412. CHADWICK, C. S., 1940c. Identity of prolactin with water drive factor in Triturus viridcsccns. Proc. Soc. Exp. Biol. Med., 45 : 335-337. CHADWICK, C. S., 1941. Further observations on the water drive in Triturus viridcsccns. II. Induction of the water drive with the lactogenic hormone. /. Exp. Zool., 86: 175-187. CHADWICK, C. S., 1948. Evidence of a thyroid-skin gland relationship in the induction of molt- ing in the red eft of Triturus viridcsccns. Anat. Rcc.. 101 : 678. (Abst.) CHADWICK, C. S., AND H. R. JACKSON, 1948. Acceleration of skin growth and molting in the red eft of Triturus viridcsccns by means of prolactin injections. Anat. Rcc.. 101 : 718. (Abst.) DAWSON, A. B., 1936. Changes in the lateral line organs during the life of the newt Triturus viridescens. A consideration of the endocrine factors involved in the maintenance of differentiation. /. Exp. Zool., 74 : 221-237. GRANT, W. C., AND J. A. GRANT, 1956. The induction of water drive in the land stage of Triturus viridcsccns following hypophysectomy. Anat. Rcc., 125 : 604. (Abst.) NOBLE, G. K., 1926. The Long Island newt ; a contribution to the life history of Triturus viri- desccns. Amer. Mus. Nov., No. 228. NOBLE, G. K., 1929. Further observations on the life-history of the newt Triturus viridescens. Amcr. Mus. Nov., No. 348. REINKE, E. E., AND C. S. CHADWICK, 1939. Inducing land stage of Triturus viridcsccns to as- sume water habitat by pituitary implants. Proc. Soc. Exp. Biol. Med., 40 : 691-693. REINKE, E. E., AND C. S. CHADWICK, 1940. The origin of water drive in Triturus viridescens. I. Induction of the water drive in thyroidectomized and gonadectomized land phases by pituitary implantations. /. Exp. Zool.. 83 : 224-233. STEELMAN, S. L., W. A. LAMONT, W. A. DITTMAN AND E. J. HAWRYLEWICZ, 1953. Fractiona- tion of the swine follicle stimulating hormone. Proc. Soc. Exp. Biol. Med., 82 : 645-647. TucHMANN-DuPLESSis, H., 1948. Developpement des caracteres sexuels du Triton traite par des hormones hypophysaires gonadtropes et lactogenes. C. R. Soc. Biol., 142: 629-630. TUCHMANN-DUPLESSIS, H., 1949. Action de 1'hormone gonadtrope et lactogene sur le com- portment et les caracteres sexuels secondaires du triton normal et castre. Arch. Anat. Micr. Morph. Exp., 38: 302-317. THE SENSITIVITY OF ECHOLOCATION IN BATS ALAN D. GRINNELL AND DONALD R. GRIFFIN Biological Laboratories, Harvard University, Cambridge 38, Mass. The full significance of acoustic orientation in bats can only be understood when we know what kinds of objects are detected and at what distances. Is it true, as is often assumed, that echolocation is limited to very close ranges of a foot or two? To what extent can bats discriminate between different objects? Are they merely aware that something is or is not directly ahead, or does echolocation inform them about the distance, size, numbers, direction and speed of motion of whatever is re- turning the echoes ? Insectivorous bats seem to use echolocation in the pursuit and capture of flying insects; do they distinguish between various kinds of insects? Some continue to hunt insects in the rain ; how can they tell the beetles from the raindrops ? It would also be helpful to know how the acuity of echolocation varies among the several groups of bats which employ quite different intensities and pat- terns of sound for echolocation (Mohres, 1953; Griffin and Novick, 1955; Griffin, 1958). These and related questions call for a better understanding of the sensitivity and effective range of echolocation, and this paper describes some new measurements of the distances at which bats first react to the presence of small wires. Although the smaller species of bats often fly very close to large objects such as the walls of a room before showing any sign of awareness that something is ahead, they do change the pattern of their orientation sounds at somewhat greater distances. For example, a My otis htcifugus commonly increases its pulse repetition rate from perhaps 5 to 10 per second before take-off to 15 or 20 per second during ordinary flight and to 50 or more per second when landing or dodging small obstacles. This increase is closely correlated with success in avoiding objects such as wires. The rate rises every time a normal or blindfolded bat approaches the wires, but deafened br.ts show no such increase as they fly up to wires which they cannot detect (Galambos and Griffin, 1942). We have utilized this characteristic increase in pulse repetition rate to determine the distance at which bats first react to obstacles of various sizes by thus changing the pattern of their emitted sounds, and the results demonstrate a greater range and sensitivity of echolocation than had previously been recognized. We are happy to acknowledge our gratitude to the Office of Naval Research for the support of these studies through a contract with Harvard University. Repro- duction in whole or in part is permitted for any purpose of the United States Government. METHODS Bats were allowed to fly in the rectangular room shown in Figure 1. This room is 10 meters long, 3.7 meters wide, and 2.4 meters high; it was free from furniture or obstructions other than the wires, three observers, and a microphone and camera 10 SENSITIVITY OF ECHOLOCATION 11 mounted on tripods. Seven meters from the end of the room, marked A, was a row of vertical wires 30 cm. apart, and 5.5 meters from the same end, in an indentation in one wall, was an Auricon model CM-72 16 mm. sound motion picture camera with a 9.5 mm. lens. In all cases the flying bats stood out against the white back- ground formed by the opposite wall, which was marked only by conspicuously num- bered vertical stripes placed at 60-cm. intervals to provide a frame of reference. In each flight used in the present measurements, the bat was released close to point A and flew approximately straight towards the opposite wall, B, passing the wires 7 meters away from its starting point. Usually it turned in the remaining 3 meters or else landed on the end wall. The observer who released the bat at point A noted its approximate distance from the wall opposite the camera as it flew towards and past the wires. A second observer turned the camera to follow the bat during its flight, which in a typical VERTICAL STRIPES / \ WIRES 4- MICRO- PHONE B SOUND CAMERA AMPLIFIERS AND FILTERS FIGURE 1. Diagram of room used for measurements of the distance of vocal reaction to the wires. case approximated the dashed line of Figure 1. The third observer kept the micro- phone pointed towards the flying bat. The motion pictures showed the flight path of the bat as it appeared from the position of the camera silhouetted against the op- posite wall, but a parallax correction (based on the first observer's notes of the bat's distance from the white wall) was necessary except when the animal was directly opposite the camera. The camera was so placed that any errors introduced by parallax were minimized in the region where the pulse repetition rate was beginning to change. The high frequency sounds emitted by the bat as it flew the length of the room were picked up by a 640AA Western Electric condenser microphone placed 2.1 meters beyond the wires. The amplifiers and associated apparatus were similar to those used in previous studies of bat sounds (Griffin, 1950, 1953). The frequency band from 30 to 80 kc was selected by Spencer-Kennedy Laboratories model 301 and 302 variable electronic filters, 54 db/octave slope at 30 kc high pass and 18 db/octave slope at 80 kc low pass. The amplified signal was then passed through 12 ALAN D. GRINNELL AND DONALD R. GRIFFIN a detecting circuit (the pulse detector used by Griffin and Novick, 1955), and the resulting clicks were recorded directly on the sound track of the same film contain- ing the photographs. The developed film was studied with a time-motion study projector, the single frames being projected one at a time while the corresponding portion of the sound track was moved past a celluloid time scale calibrated in milli- seconds. It was thus possible to measure directly for every pulse the position of the bat and the elapsed time since the last pulse. 50 3 2 Distance from wires (meters) FIGURE 2. Variation of the pulse-to-pulse interval during one flight of a Myotis lucifugus through a row of 3 -mm. wires along approximately the flight path shown in Figure 1. The arrow indicates the distance of the first vocal reaction. The bats used in these experiments were Myotis lucifugus which had been in captivity for no more than one day, and all were in excellent condition. Six sizes of wire were used: 3 mm., 1.07 mm., 0.65 mm., 0.46 mm., 0.28 mm. and 0.18 mm. in diameter. The 3-mm. wire \vas rubber-covered, but all the others were bare iron or copper. Out of about 650 flights photographed, 146 were selected for analysis because the bats did react to the wires as demonstrated by straight flights through the wires, usually with a clear effort to dodge them. For this reason there was a larger proportion of misses than would otherwise have been the case. Flights with appreciable turns and flights near the walls, ceiling, or floor were excluded since a close approach to any object is likely to involve a change in pulse repetition rate. \\ e also excluded flights in which the record of the pulses on the sound track was SENSITIVITY OF ECHOLOCATION 13 of low amplitude or was complicated by noise, so that there was a danger that some of the pulses might be overlooked in studying the record. Other flights were ex- cluded because the pulse repetition rate varied widely during the 3 to 4 meters of straight flight from point A to the vicinity of the wires or did not return to approxi- mately the same level after the bat passed through the wires. Nor were any flights used unless we were confident that the photographs established the bat's position with an accuracy of ± 10-15 cm. over at least the major part of its flight through the wires. The time required for sound to travel from bat to microphone was only 10 1) Q- to TJ c O u 0> at 32IQI Distance from wires (meters) FIGURE 3. Intervals between pulses emitted by a bat flying through a row of 0.65-mm. wires. about 0.03 second at the very most, and it decreased gradually as the animal flew towards the wires. Hence the acoustic delay had no appreciable effect on the meas- urement of the interval between pulses. RESULTS More than 500 flights through the wires showed the characteristic increase in pulse repetition rate with only two or three exceptions, all of which occurred with the 0.18-mm. wire. For the 146 flights selected for analysis the bat's position was determined at the time each pulse was emitted, and the pattern of sound emission in typical flights is shown graphically in Figures 2-6, where each point represents a single pulse. Since the repetition rate varies rapidly it is more appropriate to consider the data in terms of the time interval between pulses. Therefore the or- 14 ALAN D. GRINNELL AND DONALD R. GRIFFIN dinate of these graphs shows the time elapsed since the previous pulse, together with the corresponding repetition rate. Figures 2-6 show typical examples of these curves with five of the six sizes of wire studied, including cases when the pulse-to- pulse interval was both relatively constant (Fig. 6) or rather variable (Fig. 3) be- fore the approach to the wires, cases in which the actual values of the interval were high (Fig. 2) or low (Fig. 3), and cases in which the drop was slight (Fig. 5) as well as others in which it was very marked (Fig. 4). In the present experiments the wires were hung from small screw hooks in the ceiling, but the vocal reactions occurred when the bats were flying more than a meter below the ceiling, and thus were most unlikely to be reacting to the hooks. Further- 100 Q) — O. o> .2 2 80 cr. 60 40 20 wires 0.46 mm wire 10 15 20 25 30 40 50 100 9 a (A 0) — d! 5432101 Distance from wires (meters) FIGURE 4. Intervals between pulses emitted by a bat flying through a row of 0.46-mm. wires. more, there were many similar hooks elsewhere on the ceiling which caused no change in the emitted sounds, and control tests with no wires hanging from the hooks showed no significant variations in the pulse-to-pulse interval. Each of four- teen bats for which clear records of the first flights are available yielded a typical curve like those shown in Figures 2-6 the first time it flew through the experimental room, showing that the change in pulse repetition pattern was not merely the result of familiarity with the position of the wires. The actual values of the pulse-to-pulse interval varied considerably. With a few individuals it was as high as 150-170 msec during the straight flight towards the wires (Fig. 2), with others it was approximately constant at 40 to 60 msec (Fig. 3). Just at the wires the interval sometimes fell to about 10 msec, but in SENSITIVITY OF ECHOLOCATION -100 432101 Distance from wires (meters) FIGURE 5. Intervals between pulses emitted by a bat flying through a row of 0.28-mm. wires. «ioo 32101 Distance from wires (meters) FIGURE 6. Intervals between pulses emitted by a bat flying through a row of 0.18-mm. wires. 16 ALAN D. GRINNELL AND DONALD R. GRIFFIN other cases, especially with the smaller sizes, it fell only to 40 or 50 msec. Two methods are available for estimating the distance from the wires at which a first vocal reaction occurs with sufficient regularity to be significant. The first is to judge for each curve the approximate point at which the interval first fell signifi- cantly below the previous level and the level to which it returned after the wires had been passed. Such estimates could be made with some confidence within ±15- 20 cm., and examples are shown by small arrows on Figures 2-6. The average, minimum and maximum values of such estimates for each of the six sizes of wire are listed in Table I. It was encouraging to obtain nearly the same average dis- tances of first reaction in completely independent series of photographs with the same wires taken several months apart and using different bats. TABLE I Distance from wires of various sizes at which Myotis lucifugus first reacted by decreasing the interval between pulses. The wires used were vertical and spaced 30 cm. apart. The individual estimates of the distance of detection were made from curves similar to Figures 2-6; and average, minimum, maximum values of such estimates are shown below. Owing to the uncertainty of such estimates their average is lower than the distance of first reaction to the wires obtained from Figure 7 Diameter of wire (mm.) 3.0 1.07 0.65 0.46 0.28 0.18 No. of bats 10 17 6 6 3 3 No. of flights 29 42 21 17 13 19 Per cent misses 93% 97% 76% 82% 77% 74% Average est. distance of detection (cm.) 186 144 133 118 92 66 Range (66-294) (49-197) (66-245) (69-180) (30-138) (30-117) Distance of detection obtained from Fig. 7 (cm.) 215 185 150 120 105 90 A second, and probably better, method is to average the values of the pulse-to- pulse interval measured at various distances from the wires. The results of this type of averaging are shown in Figure 7, together with arrows to indicate the dis- tances at which these average curves first showed a definite drop below the level characteristic of flight before and after approach to the wires. As shown in Table I these estimates of the average distance of first reaction are somewhat greater than those obtained by the first method, presumably because variation due to other fac- tors than proximity to the wires was cancelled out to some extent in the averaging process. Therefore the values obtained from Figure 7 provide the best available measure of the distance at which alert and successful bats of this species first react to wires of these six sizes. Consideration of Figure 7 is somewhat complicated, however, by the fact that not all of the individual curves covered the same range of distances from the wires. Hence the ends of the average curves are based on a smaller number of flights than is listed in Table I. Careful examination of the in- dividual curves did not disclose any significant effects on the average curve of this change in number of flights, and in those more important portions of the average curves that are shown in Figure 7 by solid dots, 90% or more of the number of flights listed in Table I were included in the averages. Table I shows that with all six sizes of wire there was a large proportion of SENSITIVITY OF ECHOLOCATION 17 misses, the remaining flights being "touches" or "hits" as defined by Griffin and Galambos (1941). Study of the few individual curves for touches or hits in the present series showed no appreciable difference from those ending in a miss. This is not inconsistent with the observation of Galambos and Griffin (1942) that there was less likely to be a change in rate in flights ending in a hit, because the present series was initially selected to include only straight flights by bats that were regis- tering a high degree of success at dodging the wires. DISCUSSION These measurements are an extension of earlier experiments in which the pulse repetition rate was shown to increase as bats approached wires that were about 1.2 millimeters in diameter. It is therefore important to point out certain differences in the methods used and in some aspects of the results obtained. The apparatus available for the earlier studies was not capable of reliably registering bat pulses at a sufficient distance to provide information such as that presented in Figures 2-7, even if the bat's distance from the wires had been recorded. Furthermore the room was smaller (4.5 meters long instead of 10 meters in the present experiments), and the flights studied were not limited to straight approaches by bats at their optimal level of skill at echolocation. This is probably why a higher proportion of the pres- ent series were misses, and why almost every one of the present trials showed a clear decrease in pulse-to-pulse interval as the bat approached the wires. More sensitive apparatus might well have revealed a larger proportion of cases with a slight but detectable change in repetition rate, had it been available in 1941. But this does not alter the fact, demonstrated at that time, that successful bats are much more likely to show a marked change in repetition rate on approaching small obstacles than are those which collide with the wires. In the original experiments it was our impression from visual observation that the bats ordinarily reverted to a distinctly lower pulse repetition rate just before passing through the wires. It is therefore of interest to examine the more extensive and accurate data obtained in the present experiments with respect to the positions at which the pulse-to-pulse interval rose again to approximately the value measured before the bat approached within two meters of the wires. It is clear from the indi- vidual flights illustrated in Figures 2-6, as well as from the average curve for each size of wire, that the interval did not completely return to its earlier level until some distance past the wires. In several individual curves, however, the pulse-to-pulse interval appears to have risen shortly before the wires were passed, as in Figure 3. But many other individual curves, such as Figures 2 and 4, show that the pulse- to-pulse interval did not rise appreciably until the wires had been passed. It should be recalled in this connection that the position of the bat was determined only within ± 10-15 cm., and in a majority of individual curves the increase in interval oc- curred within this distance of the wires. In a substantial minority of cases the first definite increase appeared to be delayed until the bat was more than 15 cm. past the wires (8 out of 29 flights with 3-mm. wire, 17 out of 42 with 1.07-mm., 8 out of 21 with 0.65-mm., 4 out of 13 with 0.46-mm., but only one out of 13 with the 0.28-mm. and 3 out of 19 flights with the 0.18-mm. wire). In only one case, with the 0.28-mm. wire, the curve began to rise more than 15 cm. before the plane of the wires. 18 100 o. c o •o C o o 0> V) ALAN D. GRINNELL AND DONALD R. GRIFFIN 654321 wires I FIGURE 7. 54321 0 I Distance from wires (meters) Average pulse-to-pulse intervals of bats flying past wires ranging from 0.18 to 3 mm, in diameter. The arrows indicate the distance of first vocal reaction. SENSITIVITY OF ECHOLOCATION 19 The average curves of Figure 7 show a slight increase in the interval just as the bats flew past the wires. But since the possible error of the determinations of the bat's position was ± 10-15 cm. this small difference is only barely significant. In short, these measurements demonstrate only that the rise in the interval between pulses occurred on the average within 15 cm. of the wires, and was apparently more likely to begin shortly before passage through the plane of the wires than shortly after. Yet the 1.07-mm. wire was approximately the same size as the wires used in the earlier tests, and the spacing of the wires was the same. It is not clear whether the difference between our strong impression from observing the first ex- periments and the results of these more accurate measurements resulted from the selection of more alert and skillful bats for the present series, from the larger size of the room, or from some other factor. In many flights the pulse-to-pulse interval just before the bat reached the wires alternated somewhat regularly between two quite different values, as in Figure 2. In other words the pulses tended to come in pairs, each pair separated from the TABLE II Number of flights showing a marked alternation in the interval between pulses, similar to that illustrated in Figure 2 Definite alternation No alternation Doubtful Diameter of wire (mm.) Number Per cent Number Per cent Number Per cent 3.0 25 86% 2 7% 2 7% 1.07 23 55% 11 26% 8 19% 0.65 12 57% 4 19% 5 24% 0.46 5 26% 10 53% 2 11% 0.28 1 8% 10 77% 2 15% 0.18 2 10.5% 15 79% 2 10.5% next by an interval somewhat greater than that between the members of each pair. A similar tendency for pulses to occur in groups of two, three or occasionally four, was apparent in the first graphic records of bats' orientation pulses (Galambos and Griffin, 1942). Perhaps these groups correspond to the respiratory cycle. In the present series of curves the presence of this feature is clearly correlated with the size of the wires themselves, as shown in Table II. Whatever significance this alternation may have, it was more likely to occur with the larger sizes of wire. Perhaps there was simply not time during the last quarter of a second or so of flight up to the 0.18- or 0.28-mm. wire for so compli- cated a vocal reaction. Indeed, if the decrease in pulse-to-pulse interval did not occur until the bat was closer than one meter to the wires, the period of increased repetition rate often contained only five or six pulses. Whatever additional in- formation the bat obtained from the extra pulses over and above those that would have been produced at the previous rate, its vocal reaction was a brief and limited one. The pattern of sound emission has been discussed above in terms of time, but it is also of interest to consider it in terms of space. The same photographs show how fast the bats were moving towards and past the wires, and the average of 54 20 ALAN D. GRINNELL AND DONALD R. GRIFFIN velocity measurements was 3.9 meters per second, with the extremes of the series 2.4 and 6.3 meters/second. The speed did not vary significantly with the different sizes of wire, nor at different portions of the individual flights, except in a few cases when a late turn to dodge a wire caused momentary slowing and fluttering. Since the interval between pulses averaged about 70 milliseconds before the rep- etition rate had increased in proximity to the wires, a flight velocity of four meters per second means that one pulse was emitted about every 28 cm. As the bats flew within 0.5 meter of the wires the interval often fell to 20-30 msec, and the lower figure corresponds to one pulse every 8 cm. of flight at 4 meters/sec., or approximately once every time the bat moved through a distance equal to its own length. When the flight slowed in front of the wires, even shorter distances sep- arated the positions at which successive pulses were emitted. The actual detection of an echo from the wires must of course have preceded the first vocal reaction of the bat, and hence the distance of detection was somewhat greater than the distance of reaction discussed above. To consider the 3-mm. wire, for example, the average distance of reaction was 215 cm. But the first pulse to occur after a shortened interval was not registered until it had travelled to the microphone located 210 cm. beyond the wires, or 425 cm. from the bat. The acoustic delay for this distance is about 13 msec. A further correction should be made for the bat's reaction time, which might have been as little as 15 msec (the approximate time required for the contraction of the intra-aural muscles at the onset of a loud sound), or perhaps it was as long as 200 msec (the order of magnitude of minimum human reaction times). A conservative estimate of 25 msec for the sum of acoustic delay and reaction time indicates that when a bat detected the echoes of the 3-mm. wires it was at least 10 cm. farther from the wires than our data demonstrate directly. A similar estimate for the 0.18-mm. wires places the bat about 100 cm. when their echoes first became audible. If this does not appear to be a very impressive range of detection it is well to bear in mind that it is about 5500 times the diameter of the wires themselves. The success of bats in avoiding wires naturally varies with the diameter of the wire ; but even when wires are spaced 30 cm. apart the percentage of misses of an alert Myotis lucijugus does not fall sharply until the diameter of the wire is reduced below about 0.3 mm. In a long series of experiments with this species performed by Curtis (1952) in a smaller flight room, the average percentage of misses of wires spaced the same distance apart varied as follows with the wire di- ameter: 4.8 mm., 85%, 1.21 mm., 85%, 0.68 mm., 77%, 0.35 mm., 72%, 0.26 mm., 52%, 0.12 mm., 39%, and 0.07 mm., 36%. The chance score at this spacing is about 35%. These bats had been less highly selected for skillful flight and ob- stacle avoidance than those in the present series. For example, the three bats tested with 0.18-mm. wire registered 32 misses out of 46 flights photographed. The only selection involved in this series was the decision that the bat was flying well and tending to head straight towards the wires so that it was worthwhile to take pictures of it. In view of the small size of the wires, relative to the wave-lengths of the emitted sounds, it is surprising that the distance of detection did not vary more with wire size. While the ratio of wire diameters was about 17:1, the distances of reaction varied only by less than 2.5:1. If Raleigh scattering was the chief source of the echoes, the ratio of echo intensities at a fixed distance should have been (17)* :1 SENSITIVITY OF ECHOLOCATION 21 (Morse, 1948). To be sure, the echo intensity also varies inversely as the cube of the distance (since the echo radiates from wires as a series of cylindrical waves), and atmospheric attenuation reduces the echo somewhat further. Even if we as- sume the echo intensity to vary inversely as the fourth power of the distance, we still face a puzzling discrepancy of (17)4/(2.5)4 or more than two thousand. One way to escape from this dilemma is to postulate that much higher frequencies or shorter wave-lengths are used to detect these small wires, perhaps harmonics of the fundamental frequencies in the bat's orientation pulse. This might bring the wire diameters up to the order of one wave-length so that Raleigh scattering would not predominate, and the echo intensity would vary more slowly with wire diameter. But all available evidence indicates that the maximum intensity of the emitted pulse, and the maximum sensitivity of hearing, both occur at about 50-60 kc, or a wave-length of 6 to 7 mm., where all but the two larger sizes should be within the range of Raleigh scattering. At higher frequencies the echo intensity and the sensitivity of hearing would probably both fall off fairly rapidly. Another and perhaps better explanation would be that the bats could actually detect the wires at greater distances than our data indicate, but that they do not trouble themselves to increase the pulse repetition rate until they come within a meter or two. The relatively small increase in distance of vocal reaction between the 1.07- and 3-mm. wire could be explained about equally well by assuming that the echo strength increased less rapidly as the wire diameter approached one wave- length, or by postulating that the 3-mm. wire was detected at a greater distance but did not elicit a vocal reaction until about two meters. We cannot resolve this question without new and more refined experimental evidence. It is interesting to note in this connection a suggestion of a double break in the curves for the 1.07- and 3-mm. wires. It is possible that a slight reduction in the pulse-to-pulse interval occurs somewhat earlier than the onset of the pronounced drop which is apparent at all six wire sizes. About one-third of the individual curves for the 3-mm. wire have a distinct double break, as shown, for example, in Figure 2. Since these insectivorous bats apparently detect small wires at 1.0 to 2.25 meters it is natural to inquire whether larger objects can be located at correspond- ingly greater distances. One factor which limits a simple extrapolation to larger sizes and longer ranges of detection is the attenuation of high frequency sound in air. (For values of the coefficient of atmospheric absorption in the bats' frequency range see Beranek, 1949, pages 64—72.) Furthermore the intensity of the echo falls off as approximately the third or fourth power of the distance, depending upon the geometry of the object reflecting or scattering the sound. Nevertheless it must be possible for these bats to detect objects several centimeters in size at considerably greater distances, and really large objects such as trees or buildings are presumably detectable at distances of many meters. No methods have yet been devised, however, to determine objectively the maximum distances at which such objects are first detected by bats, and this fact presents a real challenge to future students of echolocation and bat behavior. SUMMARY 1. The distance at which bats (Myotis lucifugns) react to the presence of a row of small wires has been measured by a photographic determination of the 22 ALAN D. GRINNELL AND DONALD R. GRIFFIN distance at which the pulse repetition rate first increases as the bats fly towards the wires. Distinct changes in this rate were measured in almost every flight towards wires spaced 30 cm. apart and ranging in diameter from 0.18 to 3 mm. 2. The interval between successive pulses averaged 60 to 80 msec as the bats flew along the room towards the row of wires, and dropped to 20-40 msec just before the barrier. The intervals decreased less with the smaller sizes of wire. 3. All but the largest of these wires are well below one wave-length of the emitted sounds of these bats (50-60 kc, or 6-7 mm., at the peak intensity and 120 kc, or about 3 mm., at the very beginning of some pulses). 4. Clear evidence that the wires had been detected was furnished at the point where the interval between pulses first dropped significantly below the level that prevailed before and after the approach to the row of wires. This average distance of first vocal reaction to the row of wires was 215 cm. for 3-mm. wire, 185 cm. for 1.07-mm., 150 cm. for 0.65-mm., 120 for 0.54-mm., 105 cm. for 0.28-mm., and 90 cm. for 0.18-mm. A conservative correction for reaction time and the acoustic delay between the bat and the microphone indicates that the distance of first de- tection must have been at least 10 cm. greater than these distances of reaction. 5. Since small wires can be detected at distances of as much as 5500 times the wire diameter, and well before the bat gives evidence by its flight pattern that it is aware of them, it appears likely that larger objects are detected at considerably greater distances. LITERATURE CITED BERANEK, L. L., 1949. Acoustic measurements. John Wiley and Sons, New York. CURTIS, W. E., 1952. Quantitative studies of echolocation and vision in bats and owls. Thesis deposited in Library of Cornell University, Ithaca, N. Y. GALAMBOS, R., AND D. R. GRIFFIN, 1942. Obstacle avoidance by flying bats ; the cries of bats. /. Exp. Zool., 89 : 475-490. GRIFFIN, D. R., 1950. Measurements of the ultrasonic cries of bats. /. Acoust. Soc. Amer., 22: 247-255. GRIFFIN, D. R., 1953. Bat sounds under natural conditions, with evidence for the echolocation of insect prey. /. Exp. Zool, 123 : 435-466. GRIFFIN, D. R., 1958. Listening in the dark. Yale University Press, New Haven, Conn. GRIFFIN, D. R., AND R. GALAMBOS, 1941. The sensory basis of obstacle avoidance by flying bats. /. Exp. Zool, 86 : 481-506. GRIFFIN, D. R., AND A. NOVICK. 1955. Acoustic orientation of neotropical bats. /. Exp. Zool, 130: 251-300. MOHRES, F. P., 1953. Uber die Ultraschallorientierung der Hufeisennasen (Chiroptera-Rhino- lophidae). Zeitschr. f. vergl. Physio!., 34: 547-588. MORSE, P. M., 1948. Vibration and sound. McGraw Hill Book Co., New York. Sec. Edit. PHYSIOLOGY OF INSECT DIAPAUSE. XI. CYANIDE-SENSI- TIVITY OF THE HEARTBEAT OF THE CECROPIA SILK- WORM, WITH SPECIAL REFERENCE TO THE ANAEROBIC CAPACITY OF THE HEART 1 WILLIAM R. HARVEY 2 AND CARROLL M. WILLIAMS The Biological Laboratories, Harvard University, Cambridge 38, Massachusetts Among the metabolic changes which accompany the onset of insect diapause is a pronounced decrease in sensitivity to cyanide and carbon monoxide. This fact was first discovered by Bodine and Boell (1934a, 1934b) in diapausing eggs of the grasshopper Melanoplus, and has subsequently been studied in further detail in Melanoplus (Robbie et al., 1938; Robbie, 1941) and in the Cecropia silkworm. The situation in the case of Cecropia may be summarized as follows. Cyanide and carbon monoxide are lethal agents for the caterpillar of the Ce- cropia silkworm — a fact which mirrors the presence in the larval insect of an intact and functional cytochrome system. However, immediately after pupation the cytochrome system undergoes partial breakdown in all tissues except the inter- segmental muscles of the abdomen. Simultaneously, the over-all metabolism of the diapausing pupa becomes substantially insensitive to cyanide and high pressures of car- bon monoxide. This state of affairs persists throughout the prolonged period of pupal diapause. Finally, months later, the termination of diapause and initiation of adult development are accompanied by re-synthesis of cytochromes and the appear- ance of a fresh increment of metabolism which is sensitive to carbon monoxide. If one blocks this increment with cyanide or carbon monoxide, the insect's development is brought to a standstill. On the basis of these findings one may infer that the metabolism during the growing, non-diapausing stages in the life history is mediated by the usual cyanide- and carbon monoxide-sensitive cytochrome oxidase. In this sense there is nothing remarkable about the insect's metabolism before and after the pupal diapause. But what is remarkable is the character of the metabolism of the diapausing insect itself. The clear-cut resistance to cyanide and carbon monoxide suggests that the metabolism of the diapausing insect proceeds via some simpler and more primordial system of electron transfer making use of a terminal oxidase other than cytochrome oxidase. Under this point of view, the metabolism of the diapausing pupa is conceived to differ, not only quantitatively, but also qualitatively, from that before and after diapause. This prospect has been examined experimentally by Schneider- man and Williams (1954a, 1954b) and incorporated into a comprehensive theory of the biochemistry of diapause. Crucial to this interpretation is the breakdown of the cytochrome system at 1 This investigation was supported, in part, by a grant from the National Cancer Institute of the U. S. Public Health Service. 2 Predoctoral Fellow of the Public Health Service and the Lalor Foundation. 23 24 WILLIAM R. HARVEY AND CARROLL M. WILLIAMS the outset of pupal diapause — a matter which has recently been re-examined by Shappirio and Williams (1957a, 1957b) in individual tissues of the Cecropia silk- worm. Spectrophotometric studies at room temperature and spectroscopic studies at the temperature of liquid nitrogen confirm that, in all tissues except the inter- segmental muscles of the abdomen, rapid changes in the cytochrome system take place at the outset of pupal diapause. Within 24 hours after the pupal molt, cyto- chromes b and c decrease at least 30-fold and become indetectable ; cytochrome b5 and cytochrome oxidase (a + a3) likewise decrease at this same time, but then stabilize at low but finite levels. The net effect is that throughout the pupal diapause the tissues contain cytochrome oxidase in large excess over cytochrome c. Consequently, if the cyanide- and carbon monoxide-sensitive system fails to par- ticipate in the metabolism of the diapausing tissues, then the block in electron transfer must be localized at the level of cytochrome c rather than at the level of cytochrome oxidase itself. Whether cytochrome c actually disappears at the outset of diapause is a matter which lies beyond the resolution of the most sensititive methods of assay available at the present time. This is a question of decisive importance because a low concentration of c in the presence of a tremendous excess of oxidase might camou- flage the participation of the cytochrome oxidase system in the metabolism of diapause. Thus, by means of carbon monoxide or cyanide one could poison, say, 95 per cent of cytochrome oxidase activity and the residual 5 per cent of active oxidase might still be able to saturate cytochrome c and sustain the low and "carbon monoxide-insensitive" metabolism of the diapausing insect. Because of the limitation inherent in methods for the assay of cytochrome c, the problem appeared to be intractable to further biochemical analysis at the pres- ent time. Therefore, we have directed attention back to the insect itself. We have selected for intensive study the physiology of a particular tissue, the insect heart. Through an investigation of this tissue we have been able to bypass many of the above-mentioned difficulties and to comprehend what appears to be the mechanism of cyanide and carbon monoxide-sensitivity and -insensitivity in the Cecropia silkworm. In the present paper attention is directed to the effects of cyanide on the heartbeat of the insect during metamorphosis. In the following paper (Harvey and Williams, 1958) the effects of carbon monoxide will be considered. MATERIALS AND METHODS 1. Experimental animals The experimental animals, Platysamia cecropia L., were reared and managed according to methods described previously (Williams, 1946, 1956). Experiments were performed on the insect at the following stages : mature larvae shortly before the initiation of spinning; unchilled pupae which had been stored at 25° C. ; chilled pupae which had been stored at 6° C. ; chilled pupae which had been stored for 4 to 6 months at 6° C. and then returned to 25° C. for one week; post-diapausing individuals at successive stages in adult development at 25° C. ; and adult moths which had developed and emerged at 25° C. Certain experiments were per- formed in parallel on the related Polyphemus silkworm (Telea polyphemus Cram.). CYANIDE AND HEARTBEAT 25 2. Methods A. Exposure of isolated hearts to increasing concentrations of cyanide The dorsal half of the abdomen was excised with scissors and pinned by its lateral margins to a wax layer in the bottom of a circular dish of Lucite (poly- methyl methacrylate). Each dish was provided with a Lucite cover and with inlet and outlet tubes arranged in such a manner that the preparation was auto- matically bathed in 20 ml. of gently flowing insect Ringer's solution (Ephrussi and Beadle, 1936). The latter was slightly modified by the substitution of 0.001 M potassium phosphate buffer (pH 7.0) for a corresponding proportion of the potassium chloride. To the solution prior to use were added a few milligrams of a 1 : 1 mixture of crystalline phenylthiourea and streptomycin sulfate — the former to block tyrosinase activity and the latter to oppose bacterial growth. The gut and gonads were removed from the preparation, thereby exposing the heart and alary muscles. The paired masses of fat body were pressed aside so that the heart could be viewed in situ through a dissecting microscope. The physiological solution was aerated continuously by a gentle stream of oxygen introduced into the fluid by a 20-gauge hypodermic needle passing through the lateral wall of the dish. A 26- gauge needle passing through the plastic cover permitted the addition of a solution of hydrogen cyanide; a reservoir of the latter was stored in a one-liter Pyrex wash bottle which was connected by Tygon tubing to the hypodermic needle. The preparation was first equilibrated with insect Ringer until the heartbeat was stabilized. This ordinarily required one to two hours. The flow of Ringer was stopped and the cyanide solution was then dripped into the perfusion fluid at a rate of approximately ten drops per minute. The concentration of cyanide in this stock solution was 10 to 100 times the inhibitory level, as ascertained in pre- liminary experiments. The dropwise addition of cyanide was continued until the heartbeat was strongly inhibited. The oxygen flow was shut off and a two-mi, sample of the perfusion fluid wras then immediately withdrawn into a hypodermic syringe and analyzed for cyanide by the phenolphthalin technique described by Robbie (1944). B. Exposure of isolated hearts in a flowing system An elongate plastic tube, 1.9 cm. in outside diameter, was cut longitudinally to form two semi-cylindrical troughs. The depression was then filled with melted wax. A series of hearts was isolated and pinned to the wax bottom of the plastic trough ; the latter was then slipped into a glass tube (60 cm. long and I. D. 2 cm.). The glass tube was equipped with ground glass joints at its two ends. One end was connected by the ground joint to a stoppered reservoir containing the solution to be tested. The latter was forced from the reservoir by a slight positive pressure of overlying oxygen or nitrogen. The solution, after flowing slowly over the abdomens, made exit from the ground joint at the distal end of the glass tube and was passed in rubber tubing into a five-gallon bottle containing strong alkali. As the occasion required, samples of solution were withdrawn from the rubber tube with a hypodermic syringe and analyzed for cyanide or for oxygen. 26 WILLIAM R. HARVEY AND CARROLL M. WILLIAMS C. Appraisal of heartbeat In Method A the constant agitation of the oxygen bubbles caused considerable irregularity in the frequency of beat. Therefore, in appraising the heartbeat in experiments utilizing Method A, primary attention was centered on the amplitude of the beat rather than its frequency. This method of study was soon abandoned in favor of Method B. Here the frequency of heartbeat was found to be far more constant and predictable. Under constant conditions the variability of heart rate was small compared to that brought about by the experimental treatment. Rou- tinely the frequency of heartbeat was counted for each individual over a period of from one to five minutes and averaged as beats per minute. The strength (amplitude) of the beat was also scored as normal (3), subnormal (2), barely detectable (1), and absent (0). In order to obtain an over-all index of heart function, the recorded frequencies were divided by 1 when the heartbeat was normal, by 2 when the beat was subnormal, and by 3 when the beat was barely detectable. We shall hereafter refer to this value as the "heartbeat index." D. Reagents Cyanide was obtained as potassium cyanide (Mallinckrodt) assaying not less than 96.0%. Fresh solutions were prepared daily in oxygenated Ringer, neutral- ized with 1 N hydrochloric acid to pH 7.0, and stored in stoppered containers in the cold. At this pH, 98% of the cyanide is present as hydrocyanic acid. The experimental gases were obtained in pressure cylinders and assayed as follows: "pre-purified nitrogen" (Airco), 99.998%; oxygen (Airco), 99.5%. RESULTS 1. Acute poisoning of the isolated heart Isolated hearts of Cecropia, at successive stages in metamorphosis, were exposed during a period of one-half hour to increasing concentrations of cyanide by Method A. The concentration required to inhibit the heartbeat during this half hour was ascertained for each of a series of animals at each of seven stages in metamorphosis. When judged in this manner, the cyanide-sensitivity of the heartbeat is found to undergo large and systematic changes during the course of metamorphosis. As recorded in Table I and Figure 1, the heartbeat of the mature larva is blocked within 0.5 hour by cyanide concentrations somewhat less than 10~3 M. However, immediately after the pupal molt, a remarkable resistance to cyanide becomes evi- dent. Thus, within one day after the molt, the inhibitory cyanide concentration in- creases to 5 X 10~3 M. This trend continues until, some two to three weeks later, the inhibitory concentration is not far short of 10"1 M. The net effect is that the transition of the larva into a diapausing pupa is accompanied by a 100-fold decrease in sensitivity to acute poisoning by cyanide. This condition then persists during the months of pupal diapause. After prolonged exposure to 6° C., the pupal diapause is terminated ; only one or two days at 25° C. are then required for the visible initiation of adult develop- ment (Williams, 1956). Though pupae of this type show no detectable develop- ment when examined immediately after their return to 25° C., it is of interest that CYANIDE AND HEARTBEAT 27 resistance to cyanide has already begun to decline (Table I and Fig. 1). By the first or second day of adult development the heart is approximately as sensitive to cyanide as the larval heart. During the three-week period of adult development at 25° C., one records an ever-increasing sensitivity to acute poisoning by cyanide. Finally, the heart of the freshly emerged adult moth is blocked by cyanide at con- centrations as low as 10~5 M — a sensitivity 8,000 times that recorded for the dia- pausing pupa. TABLE I Acute toxicity of cyanide for Cecropia and Polyphemus hearts: cyanide concentrations which block* the heartbeat during 0.5-hour exposure P. cecropia Stage No. of preparations Final concentration of cyanide (X10-* M) Reversibility of effects Fifth instar larva 7 7.5 ± 0.46** + One-day-old pupa Pupa after 2-3 weeks at 25° C. Pupa after 8 months at 6° C. 6 6 12 50.0 ± 4.80 770.0 ± 170.00 77.0 ± 6.90 0 0 0 First or second day of adult develop- ment at 25 °C. Fifteenth or sixteenth day of adult development at 25° C. 5 6 5.1 ± 1.20 3.0 ± 1.70 + + Adult moth 21 0.1 ± 0.04 + T. polyphemus Stage No. of preparations Final concentration of cyanide (XlO-i M) Reversibility of effects Pupa after 5 months at 25° C. Pupa after 7 months at 6° C. 6 11 350.0 ± 44.00 31.0 ± 8.30 0 0 Eleventh or twelfth day of adult development at 25° C. 4 12.0 ± 3.10 0 Adult moth 8 0.5 ± 0.21 + * No beat or only trace of beat during one minute of observation. ** Standard error. As summarized in Table I, the observations were repeated on pupae and adults of the Polyphemus silkworm (Tclea polyphemus}. Here again, the cyanide resist- ance of the pupal heart is evident. For both these species the response of the pupal hearts to acute poisoning by cyanide is remarkable, not only in terms of the high concentrations required to in- hibit the heartbeat, but also in terms of the irreversibility of this inhibition (Table I). Whereas the heartbeat of larvae, developing adults, and adults is promptly re- 28 WILLIAM R. HARVEY AND CARROLL M. WILLIAMS 10" u u. O O I- < O z O u < O 10 ,-3 10 r4 10*5 5TH INSTAR LARVA FRESH PUPA PUPA AFTER 2-3 WEEKS AT 25 °C. PUPA AFTER 8 MONTHS AT 6«C. 1-2 DAYS OF ADULT DEVEL- OPMENT AT 2.5° C. 6™ TO I6TH DAY OF ADULT DEVELOPMENT AT 25 °C. ADULT MOTH FIGURE 1. Cyanide concentrations required to block the beat of the isolated heart of the Cecropia silkworm within 0.5 hour. The resistance of the heart to acute cyanide-poisoning is seen to undergo major changes during the larval-pupal-adult transformation. gained when returned to cyanide-free Ringer, the pupal hearts are evidently killed by the high concentrations required to inhibit them. The experiment therefore directs attention to the paradoxical behavior of the pupal heart in relation to poisoning by cyanide. As illustrated in Figure 1, the pupal heart continues to beat normally for at least a half hour in cyanide concen- trations far exceeding 10"3 M; that is, under conditions where one would anticipate CYANIDE AND HEARTBEAT 29 the inhibition of the vast majority of cytochrome oxidase activity. How can one account for this resistance of the pupal heart to cyanide? One possibility is that the pupal heart contains a terminal oxidase other than cytochrome oxidase, and that this unknown oxidase is insensitive to cyanide. How- ever, it was necessary to consider an even simpler explanation ; namely, that the pupal heartbeat can be sustained by strictly anaerobic processes. 2. Pupal heartbeat under anaerobic conditions The hearts of four diapausing pupae were isolated and pinned in the bottom of the glass tube described under Method B above. The tube was first perfused with a gently flowing stream of oxygenated Ringer, and the heartbeat of each individual ascertained. The 400-ml. tube was then perfused rapidly with oxygen-free insect Ringer ; the perfusion was then continued at the lower rate of approximately 500 ml. per hour. Special attention was given to the total removal of oxygen from the physiological solution prior to its use. For this purpose, pre-purified nitrogen was bubbled through the Ringer for at least two hours ; moreover, after traversing the solution the nitrogen was bubbled through a solution of reduced methylene blue (Fildes. 1931). The absence of color change gave assurance that all oxygen had been removed from the Ringer. The latter was then stored under a slight positive pressure of pre-purified nitrogen, and displaced by this pressure through the tube containing the hearts. The hearts of four diapausing pupae were studied — first in air, then for 5^ hours in oxygen-free Ringer, and, finally, for 43 hours in oxygenated Ringer. The various measurements are summarized in Table II, along with the average heartbeat indices. One is immediately impressed by the striking resistance of these diapausing hearts to strictly anaerobic conditions. After 0.5 hour of anaerobiosis, none of the hearts showed any detectable depression. After one hour, only one of the four was depressed. Between the first and second hours the over-all index valvte decreased TABLE II Effects of strictly anaerobic conditions on isolated hearts of brainless diapausing Cecropia pupae Animal No. Rate (beats/min.) and amplitude* of heartbeat Air Hours in Ringer equilibrated with pre-purified nitrogen Subsequent hours in oxygenated Ringer Yz l 2 3 4H SK Y< 1H 25 43 1 2 3 4 12(3) 13(3) 17(3) 12(3) 17(3) 13(3) 19(3) 20(3) KD 12(3) 19(3) 19(3) 0(0) 12(2) 13(2) 14(3) 0(0) 13(3) 0(0) 12(2) 0(0) 5(3) 14(2) 8(1) 0(0) 4(2) 0(0) 0(0) 8(3) 14(3) 20(3) 26(3) 22(3) 17(3) 4(3) 19(3) 17(3) 13(3) 6(3) 13(3) 10(3) 9(3) 7(3) 8(3) Average heartbeat index 13.5 17.2 12.5 6.8 4.8 3.8 0.5 17.0 15.5 12.2 8.5 See Methods. 30 WILLIAM R. HARVEY AND CARROLL M. WILLIAMS markedly ; however, one of the hearts showed a normal beat after two hours of anaerobiosis and another, after three hours. Three of the four hearts were still beating after 4*/> hours of anaerobiosis, and one, after S1/^ hours. It will be observed in Table II that the effects of 5l/2 hours of anaerobiosis were promptly reversed when the hearts were returned to oxygenated Ringer. Indeed, the average index values actually increased for about an hour over the level at the BRAINLESS v CHILLED * -4 -2 0 2 4 6 8 10 12 14 16 16 20 22 PUPAE PUPAE DAYS OF ADULT DEVELOPMENT AT 25 °C FIGURE 2. The "anaerobic reserve" of the isolated Cecropia heart is plotted as a function of pupal-adult development. The discontinuities in the curve correspond to days or weeks of storage under the conditions noted on the X-axis. The anaerobic reserve declines almost to zero during the course of adult development. Each datum is the average derived from the hearts of four to eleven individuals. outset, and then stabilized at or near the initial, normal level. The absence of any permanent damage attributable to anaerobiosis is also confirmed by the continuation of heartbeat for l1/^ further days until the experiment was abandoned. 3. Heartbeat of chilled pupae, developing adults, and adults under anaerobic con- ditions Is the high degree of facultative anaerobism peculiar to the heart of the dia- pausing pupa? In order to answer this question the experiment, just considered, was repeated on the hearts of : previously chilled pupae ; chilled pupae that had been returned to 25° C. and were just prior to the initiation of adult development; de- veloping adults ; and adults. The results are recorded in Figure 2 in terms of the period of anaerobiosis required for the reversible inhibition of 50 per cent of the average heartbeat index. The method of arriving at this value is illustrated in Figure 3. The average heartbeat indices of the diapausing pupae are here plotted CYANIDE AND HEARTBEAT 31 as a function of the duration of exposure to oxygen-free Ringer. A smooth curve is drawn by inspection through the series of points and the time for 50 per cent inhibition is ascertained from the curve. In the results summarized in Figure 2, it is clear that a considerable capacity for anaerobism persists within the pupa during the months of chilling at 6° C. When pupae of this type are placed at room temperature, the capacity for anaerobism actually appears to increase slightly. By the fourth day of adult development the "anaerobic capacity" has returned to the level observed in diapausing pupae. This trend continues and by the eleventh day of adult development the time for 50 per cent inhibition under anaerobic conditions has dropped to 1.5 hours. On or about the eleventh day of adult development the anaerobic capacity decreases precipitously to the low level characteristic of the adult moth. The adult moth, emerging after 21 days of development at 25° C., is maximally sensitive to oxygen lack in that the heart is able to beat less than 10 minutes in the total absence of oxygen. In the experiments just considered, the anaerobic condition was established by the use of oxygen-free Ringer's solution. The observations on pupal and adult hearts were repeated in a series of experiments in which the anaerobic condition was established by the ventilation of the tube with a flowing stream of pre-purified nitro- gen gas (300 ml. per hour). Precisely the same results were observed. In a further series of experiments making use of pre-purified nitrogen, the find- ings were confirmed in studies of the pupal and adult hearts of Telca polyphemus. Consequently, for both these species, it is clear that the pupal heart, unlike the adult heart, possesses a substantial "anaerobic reserve" which can sustain the beat- ing of the heart for as long as S1/^ hours in the total absence of oxygen. Aside from the intrinsic interest of this new finding, the anaerobic capacity of the pupal heart is obviously critical in the design of experiments testing the aerobic metabolism of the pupal heart. 4. Sensitivity of the pit pal heart to prolonged exposure to cyanide In Section 1 the pupal heart was found to be extremely resistant to cyanide. However, it will be recalled that this result was based on experiments of short dura- tion in which the heart was exposed to increasing concentrations of cyanide during a period of 0.5 hour. We now see that the pupal heart can beat for up to 51/o hours in the total absence of oxygen. The earlier experiments were therefore inadequate as a test of the cyanide sensitivity of the pupal heart. For this reason the effects of cyanide on the pupal heartbeat were re-examined in experiments of prolonged duration. Isolated pupal hearts were placed in the flow tube (Method B) and subjected to a flowing stream of oxygenated insect Ringer containing a precise concentration of cyanide. The reservoir of Ringer was prepared in a stoppered five-gallon bottle and stored under oxygen. In order to cause the Ringer to flow through the ex- perimental tube, the reservoir was slightly compressed by the addition of a stream of oxygen ; the latter was bubbled through an aqueous solution of 10"1 or 10"2 M potassium cyanide before entering the reservoir. In this manner it was possible to prevent any significant change in the cyanide concentration in the Ringer during prolonged experiments. This fact was confirmed by cyanide assays performed on fluid that had traversed the chamber. 32 WILLIAM R. HARVEY AND CARROLL M. WILLIAMS Cyanide at two specific final concentrations was studied in detail ; namely, 10~2 M and 10~3 M. It will be recalled that both these concentrations were without detect- able effects on the pupal heartbeat in experiments of short duration. The effects of 10 2 M cyanide are summarized in Figure 4. In terms of the average index values, the heartbeat remained normal for li/o hours. Two of the six hearts stopped beating after 2 hours. After 31/2 hours, all hearts showed con- siderable depression, and three of the six had stopped. The average time required for 50 per cent inhibition was 2.25 hours. The tube was then flushed with cyanide- free Ringer. Three hearts showed a slight recovery at this time. This was a temporary effect, however, for all six hearts were in standstill after a total of two hours in cyanide-free Ringer. In like manner the effects of perfusion with oxygenated Ringer containing 10~3 M cyanide were studied. A considerable depression was first evident after 2^4 hours, but all four hearts were still beating after 4 hours. At the end of S1/^ hours, two of the hearts stopped beating and the other two showed only a trace of beat. At this time the system was flushed with cyanide-free Ringer. All four hearts showed a delayed recovery and three of the four were beating normally after a total of 16 hours in cyanide-free Ringer. DISCUSSION The heartbeat of the larva and the adult Cecropia silkworm is blocked in a re- versible manner by brief exposure to cyanide in concentrations less than thousandth molar. Therefore, on the basis of this classical test, it seems safe to conclude that the hearts of the larva and the adult moth make use of cytochrome oxidase as "terminal oxidase." When the same criterion is applied to the pupal heart, the latter is found to beat normally when immersed in cyanide at concentrations not far short of tenth molar. Here, then, is a tissue which appears to be totally insensitive to cyanide over the range of concentrations at which cyanide is an inhibitor of cytochrome oxidase. Consequently, the pupal heart has appeared to be a clear instance of a cyanide- insensitive tissue whose function is not dependent on metabolism mediated by cyto- chrome oxidase. Prior to the present investigation, we have routinely thought that this was so (Williams, 1951 ; Harvey and Williams, 1953). On the basis of the experimental results described above, it is now clear that the pupal heart is by no means insensitive to cyanide. The crux of the matter is that the true cyanide-sensitivity of the heart is camouflaged most effectively by an anaerobic capacity peculiar to the pupal heart. Whereas the adult heart is able to beat for less than ten minutes in the total absence of oxygen, the pupal heart, by contrast, beats normally for one or more hours under the same conditions. The experimental conditions leave little room for doubt that the pupal heartbeat, during this prolonged period of facultative anaerobism, is sustained by strictly anaerobic metabolism. Under this circumstance the heart can scarcely require the function of cytochrome oxidase or, for that matter, any other enzyme concerned with the utilization of atmospheric oxygen. Therefore, for a corresponding period the pupal heart is found to be totally insensitive to physiological concentrations of cyanide. The true sensitivity of the pupal heart to cyanide is unmasked only when one CYANIDE AND HEARTBEAT 33 continues the experiment sufficiently long to use up the anaerohic reserve. This fact is evident in a comparison of Figures 3 and 4. The inhibition of the pupal heart by cyanide (Fig. 4) shows precisely the same time-course as that observed under anaerobic conditions in the absence of cyanide (Fig. 3). As the heart ex- hausts its anaerobic reserve, it becomes progressively more dependent on aerobic metabolism and progressively more sensitive to cyanide. These observations strongly argue that the aerobic metabolism of the diapausing heart requires the presence and function of cytochrome oxidase. 14 12 id- UJ ffl o IT UJ I I I -1.5 -1.0 -05 Of NITROGEN 40.5 41.0 1.5 420 425 430 | 43.5 OXYGEN 44 0 HOURS FIGURE 3. Technique for determining the time required for the 50 per cent inhibition of the heartbeat index during exposure of isolated hearts to oxygen-free Ringer. Each datum is the average from the hearts of eight brainless diapausing pupae. On the basis of our present data we are unable to state the lower limit of cyanide concentration which inhibits the pupal heart in experiments of this type. However, for reasons which will be considered in detail in the following paper, we doubt that the pupal heart can ever be inhibited by the very low cyanide concentrations (10~5 M) which suffice to block the heartbeat of the adult moth. While clarifying the problem of the cyanide-insensitivity of the pupal heart, the present study directs attention to a fresh problem — the changes occurring in the insect's capacity for anaerobic metabolism during the course of metamorphosis. As illustrated in Figure 2. these changes are large and systematic. Of particular interest is the rapid loss of "anaerobic reserve" which supervenes approximately midway in adult development. We suspect that this change is not peculiar to the heart. Thus, according to Schneiderman and Williams (1954b), mature larvae and adult moths of the Ce- 34 WILLIAM R. HARVEY AND CARROLL M. WILLIAMS cropia silkworm are killed in less than one day when exposed to "tank nitrogen" containing less than 0.5% oxygen. Diapausing pupae, by contrast, survive more prolonged exposures, the L.D. 50 per cent being three days. On the basis of present inadequate information we are unable to comprehend the full meaning of this shift in the capacity for anaerobic metabolism. The quantita- CYANIDE HOURS FIGURE 4. Effects of 0.01 M cyanide on the beat of hearts isolated from diapausing Cecropia pupae. Each datum is the average derived from the hearts of six brainless dia- pausing pupae. tive changes suggested in Figure 2 are almost precisely the reverse of those occur- ring in the over-all aerobic metabolism and in the concentration of such typically aerobic enzymes as the cytochromes. The problem obviously merits further study. SUMMARY 1. During the course of metamorphosis the heart of the Cecropia silkworm ap- pears to undergo pronounced shifts in its sensitivity to cyanide. 2. In the mature larva the heartbeat is promptly blocked by 10~3 M cyanide; in the adult moth it is even more sensitive and is brought to a standstill by 10~5 M cyanide. 3. In the intervening pupal stage the heart is insensitive to acute poisoning by physiological concentrations of cyanide. 4. This insensitivity is observed only in experiments of short duration. When the exposure to cyanide is continued for many hours, the pupal heartbeat is blocked by 10-- or 10-3 M cyanide. CYANIDE AND HEARTBEAT 35 5. The paradoxical response of the pupal heart can he accounted for in terms of a pronounced capacity for anaerobic metabolism which is peculiar to this particular stage. The pupal heart can beat for as long as 5V2 hours in the complete absence of oxygen. During this same period the heart is insensitive to cyanide. 6. While discounting any true insensitivity of the pupal heart to cyanide, the experimental results direct attention to major and previously unsuspected changes in the anaerobic capacity of the Cecropia silkworm during the course of metamorphosis. LITERATURE CITED BODIXE, J. H., AND E. J. BOELL, 1934a. Carbon monoxide and respiration: action of carbon monoxide on the respiration of normal and blocked embryonic cells ( Orthoptera). /. Cell. Comp. Physiol., 4 : 475-482. BODIXE, J. H., AND E. J. BOELL, 1934b. Respiratory mechanisms of normally developing and blocked embryonic cells (Orthoptera). /. Cell. Comp. Physiol., 5: 97-113. EPHRUSSI, B., AXD G. W. BEADLE, 1936. A technique of transplantation for Drosophihi. Aincr. Nat., 52 : 218-225. FILDES, P., 1931. Anaerobic cultivation. Chapter VI in System of Bacteriology, 9 ( Gt. Brit.) Medical Research Council : 92-99. HARVEY, W. R., AND C. M. WILLIAMS, 1953. Changes in the cyanide sensitivity of the heart- beat of the Cecropia silkworm during the course of metamorphosis. Anat. Rcc., 117: 544. HARVEY, W. R., AND C. M. WILLIAMS, 1958. Physiology of insect diapause. XII. The mech- anism of carbon monoxide-sensitivity and -insensitivity during the pupal diapause of the Cecropia silkworm. Biol. Bull.. 114: 36-53. ROBBIE, W. A., 1941. The action of cyanide on eggs and embryos of the grasshopper, Mchino- plus differential is. J. Cell. Coin p. Physiol.. 17 : 369-384. ROBBIE, W. A., 1944. An improved phenolphthalin technique for the microdetermination of cyanide. Arch. Biochein., 5 : 49-58. ROBBIE, W. A., E. J. BOELL AXD J. H. BODIXE, 1938. A study of the mechanism of cyanide inhibition: I. Effect of concentration on the egg of Mclanoplns differentinlis. Phvsiol. Zoo!.. 11: 54-62. SCHXEIDERMAX, H. A., AXD C. M. WILLIAMS, 1954a. The physiology of insect diapause. VIII. Qualitative changes in the metabolism of the Cecropia silkworm during diapause and development. Biol. Bull.. 106: 210-229. SCHXEIDERMAN, H. A., AXD C. M. WILLIAMS, 1954b. The physiology of insect diapause. IX. The cytochrome oxidase system in relation to the diapause and development of the Cecropia silkworm. Biol. Bull.. 106: 238-252. SHAPPIRIO, D. G., AXD C. M. WILLIAMS, 1957a. The cytochrome system of the Cecropia silk- worm. I. Spectroscopic studies of individual tissues. Proc. Ro\. Soc. London. Scr.B. 147: 218-232. SHAPPIKIO, D. G., AND C. M. WILLIAMS, 1957b. The cytochrome system of the Cecropia silk- worm. II. Spectrophotometric studies of oxidative enzyme systems in the wing epithelium. Proc. Roy. Soc. London, Scr. B, 147: 233-246. WILLIAMS, C. M., 1946. Physiology of insect diapause : the role of the brain in the production and termination of pupal dormancy in the giant silkworm, Platvsauiia cecrnpin. Biol. Bull.. 90: 234-243. WILLIAMS, C. M., 1951. Biochemical mechanisms in insect growth and metamorphosis. l:cIE\ j S1I \ _*"»*_ ' V " 444 ?CHRO XIDAS m **co- ?y H;0 LARGE .« DEDUCED CYTOCMHOMf 01 IDAS t COMPLEXED «"TH CO OX (CO ABSENT) SOX I LOW CO PRESSURE) 99 X (HIGH CO PRESSURE) STEADY STATE CONDITION Hj 0 KM1BITION OF VERY LARGE REDUCED CYTOCMROME OXIDASE COMPLEXED WITH CO STEADY STATE CONDITION MtBITION OF RC9PRATKM \ /CYTOCMHOME\ /CYTOCMROME]/ II C 1 OXIDASE / / V irm ; V / '' ' NONE OX I/2 O; IRESPIHATION (CO ABSENT) + + DEPRESSED BECAUSE Of tOW OXYGEN TENSK>N) (T£l , ^^, M'° \ / CYTOCMROME \ (CYTOCHROME \| ^ 1 OXIDASE 11 SOX / \ - / \ /v 1 LOW CO PRESSURE) + + I/2 O2 MODERATE I NEARLY 5OH.I «• CO' 1 1 •*<« HJO \ / 1* * 1 \ / \ /CYTOCMROME \ F". OCMROM£\ [ XIDASE 1 « / * VERY v - ( 99X 1 HIGH CO TTV / ^ I/2 Oj LARGE (NEARLY PRESSURE) /// 99%) *4CO '//, ///, 0 X (CO ABSENT) REDUCED CYTOCMROME OXJDASE COMPLEXED WITH CO 50 X I LOW CO PRESSURE) 99X ( HIGH CO PRESSURE) STEADY STATE CONDITION H20 INHIBITION RESPKATON NEARLY 50X NEARLY 99X FIGURE 4. CO-inhibition of the respiration of diapausing Cecropia pupae. Diagram of the steady-state condition of the electron transport system when the oxygen tension is not limiting respiration (5% atm. oxygen or above). CARBON MONOXIDE AND HEARTBEAT 49 cytochrome c a correspondingly low concentration of reduced oxidase exists in the normal pupal heart at ordinary oxygen tensions (Fig. 4A). Under this circum- stance carbon monoxide finds a limited target within the diapausing heart. The addition of sufficient carbon monoxide to complex, say, 50 per cent of the reduced oxidase transiently slows the rate of oxidation of reduced cytochrome oxidase. This causes additional oxidase to accumulate in the reduced form until the amount of reduced oxidase is twice that present in the absence of carbon monoxide (Fig. 4B). Although 50 per cent continues to be complexed by carbon monoxide, in the new steady-state the amount of uncomplexed reduced oxidase becomes the same as it had been in the total absence of carbon monoxide. Consequently, the respiration is uninhibited. At very high pressures of carbon monoxide sufficient to complex, say, 99 per cent of reduced oxidase, the reserve of oxidase becomes limiting and the system can no longer undergo the necessary degree of internal compensation (Fig. 4C). Therefore, the rates of electron transfer, oxygen consumption, and water formation are slowed down. B. CO -inhibition at low oxygen pressures As we have just seen, the concentration of reduced cytochrome oxidase and the sensitivity to carbon monoxide can be enhanced experimentally by lowering the oxygen tension. Let us consider the hypothetical case where the oxygen pressure is lowered until 50 per cent of the oxidase is in the reduced form (Fig. 5A). The rate of oxygen consumption and water formation remains the same as at higher oxygen pressures. If one now adds enough carbon monoxide to complex 50 per cent of the re- duced oxidase, a new steady-state results in which nearly all the oxidase shifts to the reduced condition (Fig. 5B). Whether the respiration will be inhibited will be determined by whether sufficient oxidase is present to supply the necessary degree of compensation. In the case considered in Figure 5B, this condition is fulfilled and the rate of water formation is diagrammed as uninhibited. However, as shown in Figure 5C, the compensatory mechanism breaks down if the pressure of carbon monoxide is further increased. A strong inhibition of respiration and of water formation is then observed. C. CO -inhibition at very low oxygen pressures Attention is finally directed to the set of circumstances diagrammed in Figure 6. The oxygen pressure at the outset is reduced to a very low level (0.18% atm.) so that the respiration is already inhibited and most of the cytochrome oxidase is present in the reduced form (Fig. 6A). The reserves of oxidized FIGURE 5. CO-inhibition of the respiration of diapausing Cecropia pupae. Diagram of the electron transport system when the oxygen tension is low but not limiting respiration (1% atm. oxygen). FIGURE 6. CO-inhibition of the respiration of diapausing Cecropia pupae. Diagram of the electron transport system when the oxygen tension is limiting respiration (0.18% atm. oxygen). FIGURE 7. CO-inhibition of the respiration of developing adults of the Cecropia silkworm. Diagram of the steady-state condition of the electron transport system when the oxygen tension is not limiting the respiration. The resynthesis of cytochrome c enhances the metabolism and the sensitivity to carbon monoxide. 50 WILLIAM R. HARVEY AND CARROLL M. WILLIAMS oxidase have already been exhausted and the system is immediately sensitive to low pressures of carbon monoxide (Figs. 6B and 6C). 5. Carbon monoxide and the pupal heart Under experimental conditions the pupal heart is found to respond to carbon monoxide in accordance with theory. In Figure 8 the experimental results of 100 - X UJ O UJ CD UJ UJ 0.8S Number of clones 30- 35 7 4 40- 65 3 7 1 70- 95 3 5 9 100-135 4 3 7 140-165 1 4 17 170-195 1 — 8 200-225 — 1 2 >230 4 1 2 Time to €2 div. (B) (after Ci fixed) (min.) >235 205-230 — . — - — - 175-200 2 4 — - 135-170 6 3 1 105-140 4 7 2 85-110 3 8 6 55- 80 — 3 13 25- 50 — — - 18 <20 1 6 c Age of Ci Cz interphase 0.0-.09 .1-.19 5 2 — • .2-.29 5 6 — .3-39 1 4 2 .4-.49 4 3 1 .5-.S9 3 4 5 .6-69 — 3 10 .7-.79 — 3 10 .8-89 — — 12 .9-99 — — • 6 very unequal division of DNA had occurred between the sister cells, thus masking the start of synthesis. In three other cases (Nos. 12, 15, 48), Q was fixed so early in interphase (65 minutes or less) that the low ratios probably have little signifi- cance. The last three clones (Nos. 82, 84, 85) are unexplained exceptions; pos- sibly the apparent lack of synthesis in Q is related to the unusually long generation times. The data obtained in the course of this study suggest that cells duplicate each unit of DNA during their growth cycles. The presence of 25 clones in the "partial synthesis" group (Q/C, 0.60 to 0.84) indicates that the synthesis is not instan- 90 BARBARA BROWN McDONALD taneous. In the "completed synthesis" group there are 43 clones with C±/C2 ratios of 0.85 to 1.17. (To be sure, some of the low ratios might represent Q cells still in the process of synthesis.) Of these 43 clones, 21 have ratios of 0.99 or less (in- dicating that Q contained the lower amount of DNA), 4 have ratios of 1.00, and 18 have ratios above 1.00 (indicating that Q contained the higher amount of DNA) ; selection of the cell with the lower or higher amount of DNA for Q was thus perfectly random. In 16 of these clones (37%) the sisters contained nearly equal amounts of DNA (Q/C,, ratios of 0.95-1.04) ; in 12 (28%) the ratios were less equal (0.90-0.94, and 1.05-1.10) ; and in 15 (35%) the ratios were even less equal (0.85-0.89, and 1.11-1.17). These figures take on special significance when compared with the 70 C2a-C2b cells falling in the same ratio groups (Table III, lines 3, 4, 5)— 29 (41%) are in the first group, 20 (29%) in the second, and 21 (30%) in the third. Such close correspondence between these two sets of cells— sisters just at the end of the division process, and sisters of the ensuing life cycle — offers good evidence that, following division, the DNA content of a cell is precisely duplicated. This investigation of DNA synthesis in Tetrahymena indicates, further, that a considerable time elapses from the completion of synthesis to the end of the inter- phase period. It is intriguing to consider the implications of this time period. Cytological examination shows no obvious, complicated mitotic apparatus being formed, although the chromatin does undergo a fairly regular condensation. Cel- lular reorganization, however, occurs before the mother cell divides into two daugh- ters. The most striking change which takes place before the start of division is the formation of a new mouth (Furgason, 1940), allotted to the posterior daughter of the dividing pair. By studying growing cells under a phase microscope, it is hoped to determine the amount of time necessary for this process. DISCUSSION The synthesis of DNA by Tetrahymena pyriformis H clearly occurs during the interphase period. Other workers, in some cases using very different methods of analysis, have come to similar conclusions for cells with mitotically dividing nuclei — the micronuclei of a ciliate (Chilodonella uncinatus — Seshachar, 1950), vertebrate cells (Swift, 1950), chick fibroblasts in tissue culture (Walker and Yates, 1952), sea urchin embryos (McMaster, 1955), Vicia faba root tips (Howard and Pelc, 1951 ; Deeley et al., 1957), onion root tips (Patau and Swift, 1953). After the end of a division (the beginning of the new interphase) a period of time elapses before DNA synthesis begins. This pause was particularly obvious in some of the individual clones with long generation times, when several hours might elapse before synthesis could be detected. Synthesis appears to be completed more than an hour before the cells begin to divide, a considerable period when compared to their average generation time of around four hours. The exact duplication of DNA during interphase is suggested strongly (1) in mass cultures, by the amounts of DNA in the macronuclei of dividing daughter cells during log phase, as compared with the amounts in older, non-dividing cells, and (2) in individual clones, by the similar DNA ratios in pairs of dividing daughters, as compared with those in sister pairs after synthesis of DNA had occurred. DNA IN TETRAHYMENA 91 In general, the amounts of DNA allotted to sister nuclei are remarkably close. Frequently, however, division is quite unequal ; furthermore, very often a piece of chromatin is left behind in the cytoplasm. Such irregularities might be expected to result in genie imbalance, and death ; instead, they seem to result in the wide range of DNA values found among non-clonal cells. Very occasional deaths have been noted among isolated cells (2 among a set of 101 cells, for example), which might be attributable either to cellular abnormality, or to some external factor. Among cells fixed during growth, occasional diffuse-looking nuclei have been found which were unsuitable for photometric analysis — possibly they represented dying cells, or possibly they resulted from faulty fixation. That unequal division of the macro- nucleus does not usually have an adverse effect is shown by the fact that the DNA ratios between apparently healthy sister cells, after synthesis of new DNA, are as variable as those between dividing daughters. It seems reasonable, however, that some method for a fairly orderly distribution of chromatin must be present in this micro-organism, with no micronucleus but only an amitotically dividing macro- nucleus, which has successfully propagated itself for almost 30 years in the labora- tory. At division, the appearance of the Tctrahymena macronucleus does suggest some sort of organization. Unfortunately, the basic structure of ciliate macronuclei is difficult to interpret. In Paramecium aurelia, however, Sonneborn (1947) obtained genetic evidence (the regeneration of parts of degenerating macronuclei) for genome segregation, from which he concluded that the macronucleus must contain about 40 diploid "sub- nuclei," distributed at amitotic division in intact units. (It will be recalled that Moses, 1950, estimated the macronucleus of P. caudatum to contain about 40 times as much nucleoprotein as the micronucleus.) Kimball (1953) could find no cyto- logical evidence for subunits in the macronucleus of P. aurelia, however, but only for a high degree of polyploidy. Grell, too, has been unable to find cytological evidence for subnuclei in the macro- nuclei of suctoreans (for example, Grell, 1953b). The budding by which these organisms reproduce vegetatively, however, suggests that genome segregation must occur. Particularly in the free-living stage of Tachyblaston ephelotensis, Grell (1950) noted the similarity in size and structure of the parts budded off the parent macronucleus, with its linear arrangement of chromatin (he reported that there appeared to be 8 chromatin elements in each bud). In another type of protozoan— a radiolarian, Aulacantha scolymantha — Grell (1953c) has found clear cytological evidence for a method by which genome segregation could occur. In the highly polyploid nucleus of this organism, the chromosomes appear to be linearly arranged in complete, individual sets, forming numerous chains of "Sammelchromosomen." Random separation of these intact genomes necessarily would result in perfectly balanced daughter nuclei. It is possible that the occurrence of "Sammel" chromo- somes might also explain the efficiency of amitosis in ciliates, although the division figure of the Aulacantha nucleus, which also has no spindle, appears to be quite different from those of ciliate macronuclei. Purely in the realm of speculation, it has occurred to the present author that, rather than "Sammel" chromosomes, the Feulgen-positive granular strands which appear to extend the length of the dividing macronucleus in Tetrahymena might each represent a row of identical chromosomes, held together by forces of attraction. If this were the case, the daughter nuclei would be assured of a fairly well balanced 92 BARBARA BROWN McDONALD assortment of chromosomes, no matter how unequal the division (assuming that all the strands separated in approximately the same region). The chromatin frag- ments left behind at division would probably cause no serious imbalance, and might, indeed, be a way of correcting a nucleus somewhat unbalanced by the previous di- vision. If a similar chromosome arrangement also occurred in the degenerating macronuclear skein of Paramecium, the parts breaking off would very likely contain complements of chromosomes, as has been suggested by the regeneration experi- ments. A condition such as this could also explain the efficiency of the budding of suctoreans. Another characteristic of T. pyrifonnis H which has been demonstrated in the present experiments is the variability in generation times among different clones, as compared with the usual close similarity between sister cells. The length of generation time seems to have no relation to the amount of DNA. Once synthesis of DNA has been completed, the photometric measurements of mass culture cells indicate that under some conditions division does not necessarily follow immediately. In general, cells from the one-week- and two-week-old culture contained approximately twice as much DNA as individual dividing daughters from log phase. Prescott's recent studies (1957) of generation time and lag phase in strain HS indicate that cells from stationary phase may divide soon after inocula- tion into fresh medium, and may then undergo a lag phase before logarithmic growth begins. Such preliminary division seems a reasonable consequence if the inocu- lated cells contained a doubled amount of DNA. By alternating temperature changes, Scherbaum and Zeuthen (1954) caused lo- garithmically growing cells (strain GL) essentially to stop dividing until the final return to optimal temperature. The following synchronous (85%) division oc- curred 90 ± 10 minutes later, a time period remarkably similar to that found in the present experiments (about 80 minutes) to occur between the end of synthesis and the end of the interphase period. They report that the following two somewhat less synchronous divisions of the treated cells occurred about 1.7 hours (100 min- utes) apart. Correlated with these interesting data is the fact that they found (Zeuthen and Scherbaum, 1954) by Hoff-Jp'rgensen microbiological assay that cells at the end of treatment contained about four times as much DNA as normally grow- ing cells. By Schmitt-Thannhauser analysis, Ducoff (1956) has confirmed the unusual amount of DNA synthesis by temperature-treated cells. In view of the degree of DNA synthesis and the time elapsing before the first synchronous division, the question arises, as in the present experiments, about the length of time required for cellular changes (such as the formation of a new mouth) which must take place before division can begin. The author wishes to express her appreciation to Professor A. W. Pollister, in whose laboratories this work was carried out, for his stimulating advice and guid- ance, and to Professor F. J. Ryan for the provision of additional laboratory facilities. SUMMARY 1. In a mass culture of TctraJiyniena [>yriformis H which has stopped growing, the macronuclei contain approximately twice as much DNA as do newly divided macronuclei in a logarithmically growing culture. DNA IN TETRAHYMENA 93 2. Non-clonal cells show considerable variability as to DNA content and gen- eration time. 3. Cells in small clones show close similarity as to DNA content and generation time. 4. Duplication of DNA occurs during an intermediate part of interphase, start- ing some time after the end of the previous cell division, and reaching completion a considerable period of time before the next division begins. LITERATURE CITED ALFERT, M., AND I. I. GESCHWIND, 1953. A selective staining method for the basic proteins of cell nuclei. Proc. Nat. Acad. Sci., 39: 991-999. ALFERT, M., AND N. O. GOLDSTEIN, 1955. Cytochemical properties of nucleoproteins in Tetra- hymena pyriformis; a difference in protein composition between macro and micronuclei. /. Exp. Zool, 130: 403-421. CHEN, T. T., 1940. Conjugation in Paramecium bursaria between animals with very different chromosome numbers and between animals with and without micronuclei. Proc. Nat. Acad. Sci., 26 : 243-246. CHEN, T. T., 1944. Staining nuclei and chromosomes in protozoa. Stain Tech., 19 : 83-90. CORLISS, J. O., 1953. Comparative studies on holotrichous ciliates in the Colpidium-Glaucoma- Leucophrys-Tetrahymcna group. II. Morphology, life cycles and systematic status of strains in pure culture. Parasitology, 43 : 49-87. DEELEY, E. M., H. G. DAVIES AND J. CHAYEN, 1957. The DNA content of cells in the root of Vina faba. Exp. Cell Res., 12 : 582-591. Di STEFANO, H. S., 1948. A cytochemical study of the Feulgen nucleal reaction. Chromosoma, 3: 282-301. DUCOFF, H. S., 1956. Radiation-induced fission block in synchronous cultures of Tetrahymena pyriformis W. Exp. Cell Res., 11 : 218-220. ELLIOTT, A. M., AND R. E. HAYES, 1953. Mating types in Tetrahymena. Biol. Bull., 105: 269-284. FURGASOX, W., 1940. Significant cytostomal pattern of "Glaucoma-Colpidium group" and a proposed new genus and species, Tetrahymena geleii. Arch. f. Protist., 94 : 224-266. GRELL, K. G., 1950. Der Generationswechsel des parasitischen Suktors Tachyblaston ephelo- tcnsis Martin. Zeitschr. f. Parasit., 14: 499-534. GRELL, K. G., 1953a. Die Konjugation von Ephelota gemmipara R. Hertwig. Arch. f. Protist., 98: 287-326. GRELL, K. 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In: Recent Developments in Cell Physiology, J. A. Kitching, ed. ; Proceedings of the 7th Sym- posium of the Colston Research Society, London, 141-157. CONTRACTILE PROTEIN FROM CRAYFISH TAIL MUSCLE K. MARUYAMA Biological Institute, College of General Education, University of Tokyo, Komaba, Meguro, Tokyo, Japan Physicochemical studies on vertebrate striated muscle clearly indicate that the interaction of actomyosin, the muscle contractile protein, with adenosine triphos- phate (ATP) may be the fundamental phenomenon on the molecular level in mus- cular contraction (cf. Szent-Gyorgyi, 1951; Weber and Portzehl, 1954). More and more evidence to support this thesis has been obtained in the field of compara- tive biochemistry in the animal kingdom (Maruyama and Tonomura, 1957). In invertebrates, the highly developed muscles of arthropods and molluscs have been investigated biochemically in detail. In arthropods, however, there are very few reports on crustacean contractile protein, whereas the characteristics of insect actomyosin have been well established (Gilmour and Calaby, 1953 ; Maruyama, 1954, 1957b, 1957c). Edsall and Mehl (1940) investigated flow birefringence properties of lobster myosin in relation to the protein denaturation. Humphrey (1948) prepared myosin from the crab, Maia, and briefly described its adenosine- triphosphatase (ATPase) properties. Shrinkage of the glycerine-treated muscle fibers of Limulus with ATP was observed by Sarkar (1950). On the physico- chemical properties of crustacean tropomyosin, detailed works have been recently published (Tsao, Tan and Peng, 1956; Laki, 1957; Kominz, Saad and Laki, 1957). The present article is concerned with the results of a comparative biochemical study on the ATP-myosin B system and several associated enzymes in crayfish tail muscle. MATERIALS AND METHODS Materials. The crayfish, Cambarus clarkii, was used as material. Tail muscles were carefully isolated from exoskeleton and well washed with cold de-ionized water. In a few cases, jaw muscles were also dissected out. Preparation of contractile protein. The so-called myosin B or natural acto- myosin was extracted and purified as established in rabbit skeletal muscle (Szent- Gyorgyi, 1945). Muscles were homogenized in ten times the volume of cold 0.05 M KC1 in a Waring Blendor and the water-extractable portion was removed by centrifugating at 3000 G for 5 minutes at 0° C. The residue was washed twice more and finally suspended in five times the volume of the Weber-Edsall solution (0.6 M KC1, 0.04 M NaHCO3, 0.01 M Na2CO3). After being placed for 24 hours at 0° C., the suspension turned so viscous that it was diluted with an equal volume of 0.6 M KC1. The suspension was centrifuged for 10 minutes to remove the in- soluble matter. The viscous supernatant was neutralized to pH 6.5-6.8 with dilute acetic acid and diluted in ten times the volume of cold de-ionized water. The flocculant precipitate was collected by centrifugation, dissolved in 0.6 M KC1 and diluted in water. This dilution-precipitation procedure was repeated three times 95 96 K. MARUYAMA and finally the substances insoluble in 0.6 M KC1 were removed by centrifugation at 8000 G for 30 minutes at 0° C. Methods of assaying physicocliemical properties. The measurements of ultra- violet absorption spectra were carried out with a Shimazu spectrophotometer. Purine and pentose contents were estimated according to Schneider's procedure (Schneider, 1946). Solubility in KC1 was tested at pH 7.0 at 0° C., as described in an earlier paper (Maruyama, 1957a). Salting-out analysis was carried out ac- cording to Snellman and Tenow (1954), as modified by Tonomura and his associ- ates (1956). Methods of observing pliysical changes with ATP. Super-precipitation was observed in a test tube, containing 0.03 M tris-(hydroxymethyl)-aminomethane (Tris) buffer, 1 mM ATP and 1.5-2.5 mg. protein at pH 7.0 and 25° C. Viscosity was measured with viscosimeters of the usual Ostwald type at pH 6.4 at 5-10° C. Turbidity was determined in a Hitachi turbidimeter at pH 6.4 and 15° C. For investigation of viscosity and turbidity, the concentration of myosin B was suitably adjusted by dilution with 0.6 M KC1. A small amount of concentrated ATP was added to test the ATP effect. Enzyme tests. The ATPase activity was determined by measuring the increase in inorganic phosphorus (P) after a specified time, usually 5 minutes at 30° C., in a system containing the protein dissolved in 0.6 M KC1, 1 mM ATP, 0.033 M Tris buffer (pH 7.0), 0.6 M KC1, and 10 mM CaQ2 or some other ions, as specified. Total volume of the reaction mixture was 1.5 ml. In order to investigate the effect of pH of incubation, 0.05 M histidine was substituted for Tris. The reaction was started by the addition of substrate and stopped by the addition of 0.5 ml. of 20% trichloroacetic acid (TCA). Appropriate blanks were always run simultaneously with the experimentals. The water-extractable apyrase activity was determined on the 0.05 M KC1 ex- tract of muscle suspensions. On proving the occurrence of adenylate kinase in crayfish muscle, the 0.05 M KC1 extract was treated with heat and acid according to Colowick and Kalckar (1943) and incubated in the ATPase-assaying system with and without crayfish myosin B. Adenylate deaminase activity was tested as described before (Maruyama, 1957a). ATP was purchased as crystalline disodium salt from Sigma and AMP from Schwarz Labs. Protein was estimated by multiplying nitrogen values, obtained by a micro- Kjeldahl procedure, by the factor 6. Inorganic phosphorus was measured on a 1.0-ml. aliquot of the TCA super- natant by a micro-modification of the method of Lohmann and Jendrassik (1926), as described by Moriwaki (1956). RESULTS Some Physicocliemical Properties Absorption spectra. Ultraviolet absorption spectra of crayfish myosin B dis- solved in 0.6 M KC1 showed the characteristics of protein nature : a maximal ab- sorption was found around 275 m^ and a minimal one was at 255 m/i. Extinction coefficient (e) on basis of gram N per /, and 1.0 cm. light path was 9.0 at 275 CRAYFISH MYOSIN B 97 which was somewhat higher than that of rabbit myosin B (Tarver and Morales, 1951). The ratio of absorption coefficient at 275 mp. to that at 255 m/t is 1.3. Purine and pentose contents. The results described above suggest that some minute amounts of purine-containing substances such as nucleotides or nucleic acids were present as contaminants in the preparations. The acid-soluble nucleotide frac- tion contained 1.5 X 10~5 moles purine per gram myosin B and the nucleic acid fraction contained 1.0 X 10~5 moles. The determinations of pentose showed ap- proximately equimolar amounts and any detectable amounts of desoxyribose were not present. These values are a little higher than those for rabbit myosin B (Buchthal et al, 1951). Solubility in KCl. Crayfish myosin B was completely soluble in concentrations of KCl higher than 0.4 M. The solubility curve is quite in accord with that of rabbit myosin B (Szent-Gyorgyi, 1945). Myosin B dissolved in 0.6 M KCl was slightly yellowish white in color. TABLE I Effect of varied concentrations of Ca, Mg and EDTA on the super -precipitation of crayfish myosin B Concn. M Ca Mg EDTA 0 ++ ++ + + IO-6 ++ ++ + + io-B ++ + + + + 10-" ++ ± ± io-3 + io-2 ± Observed within three minutes after the addition of 1 mM ATP at pH 7.0 and 20° C., in the presence of 0.10 M KCl. + + + , + + : intense ; + : moderate ; ± : weak ; — : negative. Salting-out analysis. Under experimental conditions similar to those of Snell- man and Tenow (1954), nearly all the proteins were precipitated between 30—38% of saturated ammonium sulfate in the present preparation. Physical Changes with ATP Super-precipitation. Under the optimal conditions, e.g., at 20° C. and pH 7.0 in the presence of 0.10 M KCl, a typical super-precipitation was found to take place within one minute after the addition of 1 mM ATP, and to reach the end within five minutes. The super-precipitation was evidently recognized in the presence of KCl in concentrations between 0.06 and 0.16 M. The optimal KCl concentrations were 0.10-0.12 M, which were quite in accordance with those of rabbit myosin B (Szent-Gyorgyi, 1945). The effects of Ca, Mg and ethylenediaminetetraacetic acid (EDTA), were tested (Table I). These agents in a high concentration (10 mM) inhibited the super-precipitation. Calcium ions affected little, having no ac- celerating effect. Magnesium ions greatly speeded up the precipitation in 10~5 M, but retarded in over 10~* M. On the other hand, EDTA inhibited in concentra- tions higher than IO"4 M. It should be noted that the inhibitory effects of high concentrations of Mg or Ca and of EDTA were qualitatively different : after several hours, the precipitation took place even in the presence of a high concentration of Mg or Ca, but in the presence of EDTA no precipitation was observed to occur. 98 K. MARUYAMA Change of viscosity. The relative viscosity of crayfish myosin B dissolved in 0.6 M KC1 was highly anomalous and ATP caused a marked drop in the viscosity (Fig. 1). According to Weber and Portzehl (1952), the extent of the viscosity change of actomyosin with ATP (ATP sensitivity) can be expressed as follows: ATP sensitivity = Zn x 100 ATP Here Z?/ = In^rel/C; r/rel = relative viscosity in the absence of ATP; ZJ/ATP = that in the presence of a sufficient amount of ATP to cause the maximal viscosity change ; and C = concentration of protein (grams/liter). The viscosity data derived from Figure 1 are as follows : Zr, = 0.40, Zr,ATP = 0.20 and the ATP sensitivity = 100%. The viscosity of myosin B solution drops rapidly with the addition of ATP and gradually rises again when ATP is split by the ATPase action of myosin B. The recovery process followed an S-shaped curve and the recovered viscosity reached about 50 c/c of the drop. Calcium ions, which activate the ATPase action, strongly accelerated the recovery process, while magnesium, an inhibitor of the ATPase ac- tion, retarded it. These tendencies are in good accord with those in rabbit or insect myosin B (cf. Mommaerts, 1948; Maruyama, 1957b). On the other hand, EDTA, in 10 mM, completely inhibited the viscosity change with ATP. Change of turbidity. On addition of ATP, the apparent turbidity of myosin B solution decreases because of the decrease of intensity of scattered light (cf. Tono- mura, 1956). Figure 2 shows the change of turbidity in crayfish myosin B, which FIGURE 1. Drop of viscosity of crayfish myosin B with ATP; pH 6.4; 6° C. ; 0.6 M KC1. O, control: •, 1 mM ATP added. C, protein concentration, g./7. CRAYFISH MYOSIN B 99 10 5 OQ o: D 0 1 2 TIME IN MIN. FIGURE 2. Change of turbidity of crayfish myosin B with ATP; pH 6.4; 15° C. ; 1 mM ATP ; 0.6 M KC1. \ i t i i ] i 1 I T ©„ o^^ o „ o,^ O ^ o ^ o ^_^ ^_^ o ^^ o ^ ^ ^H »H 1-1 .-« T-t ^7 *-« "^ »H bo M T^ ^ '-^ '"r bo X-a x-a x-a x-a x-a x-a X K W x-a x-a K J° m DO w CO (B CO w E^ •sg P CJ CJ •-' to S"d ^£ %> C o U OJ •^ M « -v^ Ed ^% E~d ^K e* ^ S £ B ^ — p"> c o ^^ fiu -sg E x_x 5.1 5.1 5.1 2.1 2.1 2.1 1953 Agapema 3.6 3.6 3.6 1.76 1.76 L76 2.0 0.5 0.25 0.6 0.2 0.1 2.5 10 20 3.5 10 20 3.2 2.6 2.45 1.23 1.06 1.02 1.6 3.1 3.35 1.2 2.1 2.2 16.3 13.3 12.8 1954 Agapema 1.2 1.56 1.66 0.63 0.86 0.92 6.3 5.4 5.2 99 88 86 96 87 85 593 604 606 596 605 607 1.0 1.64 L73 0.5 0.69 0.73 0.96 1.68 1.76 0.5 0.69 0.74 0.08 0.2 0.23 0.27 0.51 0.59 Hyalophora 45 86 93 68 89 94 14 11.8 2.46 5.7 7.66 9.34 5.2 78 92 600 3.9 4.1 0.03 79 14 11.8 1.4 10 7.2 10.4 5.8 73 88 604 4.5 4.6 0.038 88 14 11.8 0.7 20 6.9 11.1 6.2 70 85 607 4.9 5.0 0.043 94 126 JOHN BUCK tion of spiracular constriction and in-flow of air, without limiting the rate of O2 up- take. Similar serial computations indicate the theoretical feasibility of widely dif- ferent degrees of relative CO2 retention, both in systems with several parameters the same as in Agapema, and in systems involving known respiratory data from Hyalophora (Table III). V. THEORY IN RELATION TO INDUCED CHANGES IN INTERBURST CO2 RELEASE RATE In response to certain environmental and endogenous alterations the rate of CO, release during the interburst period undergoes definite and reproducible changes. These must be accounted for by any theory purporting to explain cyclic CO2 re- lease. Of these, all that involve a reduction in demand for O2 (Table IV, re- sponses No. 2, 4), or increase in availability of O2 (No. 5), decrease the rate of TABLE IV Effects of certain environmental and endogenous changes on rate of CO* release during the interburst period Response No. Change IBRCO2 1 Increasing temperature Increase1-2'3 2 Decreasing temperature Decrease1 '2'3'5 3 Increasing metabolic rate (injury; dev.) I ncrease3 4 Decreasing metabolic rate Decrease2'3 5 Increased ambient pO2 above 21% Decrease3'4 6 Decreased ambient pO2 (below 21%, but above about 10%) Increase3'4 1 Punt, 1944. 2 Punt, 1950. 3 Schneiderman and Williams, 1955. 4 Buck and Keister, 1955. 5 Buck and Keister, 1958. CO2 release; and all tending to decrease the availability of O2 (No. 6), or increase the call for O2 (Nos. 1, 3), have the opposite effect. Now we know (Buck and Keister, 1955) that environmental O2 concentrations between \% and 100% neither increase nor decrease rate of O2 consumption (CO2 production), so the observed changes in CO2 release rate must be due to changes in rate of CO2 retention. The retention changes in turn must involve changes in air in-flow and CO2 out-diffusion due to alterations in either spiracular valve area or effective CO2 gradient, or both. Although no direct measurements of spiracular valve areas in pupae with different metabolic rates or in different O2 tensions or temperatures have been reported, the fact that increase in ambient O, concentration above the normal atmospheric level reduces the rate of interburst CO2 release (Table IV, resp. No. 5) without increasing the rate of O2 uptake, suggests that the maintained, steady-state valve area of the interburst period is regulated so as to keep the rate of entry of O2 at the minimum that will fully supply respiration (see also Buck and Keister, 1955, 1958; Schneiderman, 1956). A teleological support THEORY OF CYCLIC CO, RETENTION 127 for this simplifying assumption is the fact that such regulation would also minimize water loss, a particularly acute problem in diapausing pupae since they are denied water intake for many months, and hence would be in line \vith a main and well established function of spiracles (e.g., Hazelhoff, 1926). The assumption is also compatible with flow-diffusion, and in fact appears to be the only way of reconciling the role of O2 uptake in inducing air flow with the fact that the rate of O2 uptake is not O2-limited except at very low ambient concentrations. Even for responses not involving changed metabolic rate the expected changes in CO2 are not easy to predict. For example, insofar as purely diffusive transfer is concerned, a decrease in ambient pO2, since it induces spiracular dilation, would be expected to increase the interburst rate of CO2 escape (see Fick equation). How- ever, if the pupa regulates valve area so as to keep the rate of O2 entry constant, ambient pO2 will be inversely proportional to area and the effects of changing pO2 on interburst CO2 out-diffusion rate will be similarly non-linear (Buck and Keister, 1955, p. 160). On the same basis changing ambient pO2 will have an inversely proportional effect on RF (i.e. on CO2 flow-retention), but with the additional com- plication of a different proportionality constant (RD /—' A, whereas RF — 'A X r2 ; see Poiseuille equation). Hence the over-all effect of changing ambient pO2 in a flo\v-diffusion system is not immediately obvious, either qualitatively or quanti- tatively. The interrelated changes in valve area, air-flow rate, interburst CO2 release rate and areal retention rate can be visualized by constructing a family of curves relating valve area and air-flow rate at different pressures (computed from the Poiseuille equation). Figure 1 shows such a plot, to which have been added (a) the straight line relating diffusion rate of CO2 (RdCO2) to valve area for the valve length and trans-spiracular CO2 gradient of Agapema, and (b) the fitted values of air-flow rate and valve area for the respiratory parameters measured in the 1953 and 1954 samples of Agapema and for several hypothetical situations involving greater degrees of CO2 retention (Table III). Figure 2 is a similar plot, with different isobars and R 5 10 CD o U. 5 O I 4 h- O I J BOTTOM TtMP. -C 10T OYSTtRS- PltRCSS PT OYSTERS- B S.C.B. CRABS- PIERCES PT. CRABS- B.S.C B M 1955 ~M ' 7 1956 I 45 t- LJ t/1 40 Ul 2} - X o 25 0 AIR 15 20 25 30 OXYGEN(POUNDS PRESSURE) FIGURE 2. Oxygen consumption for white pupae that were exposed to 100 per cent oxygen (15 to 30 pounds for one minute). EFFECTS OF OXYGEN ON HABROBRACON WHITE PUPAE 183 White pupae treated with thirty pounds of oxygen are arrested at this stage of development. They remain as white pupae for about a week and after this time may become somewhat pigmented. They appear to be alive for at least two weeks as indicated by the lack of discoloration or the absence of drying-out of the pupae. Further, such pupae showed the same magnitude of oxygen consumption after two days as after one hour. White pupae exposed to 15 pounds of oxygen be- come pigmented before they are arrested in development while white pupae treated with 30 pounds of oxygen are arrested immediately as white pupae. Within these TABLE I Respiratory quotients of white pupae exposed to oxygen Treatment Expressed as ul/25 pupae/hr. Gas Pressure (pounds) No. of experiments O2 consumed CO2 liberated R. Q. Air 15 6 40 27 .67 Oxygen Oxygen Oxygen 15 20 25 6 6 6 29 15 10 18 10 8 .61 .70 .77 extremes the amount of delay in pigmentation is influenced by the pressure of oxygen that is applied. Groups of pupae (25 pupae/group) were treated with oxygen and then measured for oxygen consumption with a Warburg respirometer. The amount of oxygen utilized by the pupae was found to decrease as the oxygen pressure was increased (Fig. 2). Comparison of the per cent of eclosion (Fig. 1) and the oxygen consumption (Fig. 2) shows that the degree of decrease is not of the same magnitude in each. The eclosion ratio drops off faster than the rate of oxygen uptake. After exposure TABLE II Oxygen uptake for white pupae exposed to oxygen (measured 1 and 24 hours after treatment) Treatment Gas Air Oo O, 02 Pressure (pounds) 15 15 20 25 ul O2/25 pupae/hr. 1 hour 24 hours 38 32 28 26 11 12 8 8 to an oxygen pressure of 20 pounds the eclosion falls to about 5 per cent of the air controls while for the oxygen uptake it falls to about 75 per cent of the air controls. Thus, the decrease in the eclosion percentage is most marked in the oxygen dosage range from air to 20 pounds of oxygen, while the greatest decrease in oxygen con- sumption does not occur until after treatment within the dosage range of 20 to 30 pounds of oxygen. The data indicate that there is no difference in oxygen uptake between pupae treated with 18 pounds or with 20 pounds of oxygen (Fig. 2). The respiratory quotient (R. O.) was determined after exposure to various pres- 184 A. M. CLARK AND M. J. PAPA TABLE III Eclosion ratios of -white pupae exposed to oxygen at 10° C. and 26° C. Eclosion ratio Gas Air Oo Oo O, 02 Treatment Pressure (pounds) 15 15 18 20 25 26° C. 74-82 12-58 7-41 5-101 0-36 10° C. 41-47 48-57 47-55 79-93 34-50 sures of oxygen (Table I). In these experiments groups of pupae were measured for oxygen consumption for three hours, after which the KOH was removed from the center well and the amount of carbon dioxide liberated was determined. With increasing pressures of oxygen there is a decrease both in the oxygen consumption and in the liberation of carbon dioxide. There is no change in the R. Q. with in- creased oxygen pressure (Table I). It seemed of interest to inquire whether there was any recovery of the ability to consume oxygen following treatment with oxygen. In order to test this, pupae whose oxygen consumption had been measured within one hour after treatment with oxygen pressures from 15 to 25 pounds were kept in an incubator for 24 TABLE IV Oxygen uptake of -white pupae exposed to oxygen at 10° C. and 26° C. Treatment Gas Air 02 Pressure (pounds) 15 25 ul Oi/25 pupae/hr. 26° C. 10° C. 44 46 21 47 hours and then re-measured for oxygen consumption. These data appear in Table II and show that there is no change in oxygen uptake after 24 hours. Experiments not reported here have shown that the oxygen consumption does not increase after two days. Thus, the decrease in oxygen consumption following oxygen treatment is irreversible. Groups of white pupae were placed into a refrigerator at 10° C. for % hour or kept at room temperature (26° C.) for the same length of time. They were then exposed immediately to oxygen of known pressure and placed into the incubator (30° C.) to observe for developmental effects. Pupae treated when cold wrere much more resistant to oxygen than were the pupae that were treated when warm (Table III). For example, of 50 pupae that were treated with 25 pounds of oxy- TABLE V Eclosion ratios of white pupae treated with oxygen before and after exposure to 10° C. and 26° C. Eclosion ratio Oxygen pressure (pounds) Treatment 15 20 10° C., then O2, then 10° C. 26° C., then O2, then 10° C. 10° C., then O2, then 26° C. 26° C., then O2) then 26° C. 91-106, 34-90 84-96 35-76 62-72 1-58 34-44 3-42 25 26-43 0-30 33-41 0-35 EFFECTS OF OXYGEN ON HABROBRACON WHITE PUPAE 185 gen after exposure to 10° C., 35 developed to the adult stage and emerged from their cocoons while of 36 pupae treated in the same manner after exposure to 26° C., none developed to the adult stage and eclosed. Other groups of pupae were treated in the same manner and measured for oxygen uptake. Pupae exposed to 25 pounds of oxygen after cold exposure consumed as much oxygen as the controls while pupae treated after exposure to warm temperature showed a marked decrease in oxygen consumption (Table IV). These data on oxygen consumption are in agreement with the data on eclosion (Table III). Since temperature has an effect on the sensitivity of pupae to oxygen, the pos- sibility that this oxygen-sensitivity could be modified by exposure to different tem- peratures after oxygen treatment was considered. Groups of white pupae were placed either at 10° C. or 26° C. for YO hour, then exposed to oxygen of known pres- sure and then placed at 10° C. or at 26° C. for one hour (Table V). Eclosion ra- tios were obtained from the pupae so treated and showed that the post-treatment with temperature had no effect upon recovery. Thus, the temperature at the time of treatment with oxygen modified the oxygen-sensitivity. Whether longer periods of post-treatment with temperatures of 10° C. would be effective has not been tried. The metabolic state of the organism at the time of treatment seems, therefore, to de- termine the extent of its sensitivity. DISCUSSION Habrobracon white pupae when exposed to oxygen show an immediate and marked decrease in oxygen consumption and, subsequently, an arrestment of develop- ment and of pigmentation. The magnitude of these effects can be correlated to a certain degree with the dosage of oxygen that is applied to these organisms. It seems clear that the arrestment of pigmentation is due to the lack of sufficient oxygen in the tissues to allow for the enzymatic oxidation of tyrosine to melanin. This seems to be indicated by the following observations. The steep drop in the pig- ment-forming ability occurs after those dosages of oxygen where a marked decrease in oxygen consumption occurs (between 20-30 pounds pressure, Figure 2). At doses of less than 20 pounds of oxygen, there is relatively little decrease in pig- mentation and in oxygen consumption. The arrestment of pigmentation in Habro- bracon white pupae can be brought about also by exposure of the pupae to lowered concentrations of oxygen. There is no recovery in the rate of oxygen consumption for pupae whose oxygen consumption has been lowered by exposure to oxygen. It is generally realized that an arrestment of pigmentation may be caused by exposure of insects to environments with less oxygen tension. The fact that arrestment of pigmentation may be caused by increased oxygen pressures was reported by Linden in 1906 (Sussman, 1949). The events that are responsible for the arrestment of development may be dif- ferent from those responsible for the arrestment of pigmentation since pupae that are exposed to 15 pounds of oxygen show an arrested development but exhibit no delay in the acquiring of pigment. It seems difficult to relate this arrested devel- opment to a decrease in available oxygen since the incidence of pupae that develop to the adult stage after exposure to 20 pounds of oxygen is low (5 per cent of controls) while their rate of oxygen consumption is relatively high (75 per cent of controls). It is possible that the arrested development may be due to the inac- 186 A. M. CLARK AND M. J. PAPA tivation of a substance that has some control over development or to an increased concentration of some toxic materials. Various authors (see Bean, 1945) have suggested that the primary effect of exposure to oxygen gas is the inactivation of oxidative enzymes with a resultant generalized tissue anoxia. The fact that there is an immediate decrease in oxygen consumption for Habrobracon pupae indicates that this hypothesis may be valid. Studies on the oxygen uptake and enzyme activity of tissue homogenates, at present in progress, are needed to show this. To date, however, no decrease in oxygen consumption or in succinic dehydrogenase activity of homogenates from oxygen- treated pupae has been observed. Extensive experiments bearing on this hypothe- sis of tissue anoxia have been carried out by Stadie, Riggs and Haugaard (1944) with negative results. They found no immediate reduction in oxygen uptake in tissues from rats that had been killed by 7 atmospheres of oxygen. They assume, therefore, that generalized tissue anoxia is not the cause of acute oxygen poisoning. Despite this, it is not possible to eliminate the possibility that localized tissue anoxia may occur. All stages of development in Habrobracon are not equally sensitive to the in- jurious effects of oxygen (Clark and Herr, 1954). The larval and prepupal stages are not affected by 30 pounds of oxygen, while almost all of the pupae are injured. The reason for this difference in stage-sensitivity is not known at present. It seems clear, however, that it is not due simply to a difference in the rate of metabolism. Based upon oxygen consumption studies one can show that the oxygen-resistant larvae are more active than are the oxygen-sensitive pupae. In the present paper, however, experiments have been given that show that pupae that have been made less active by exposure to a temperature of 10° C. are more resistant to the toxic effects of oxygen than are pupae that were kept at 26° C. immediately before treatment. It seems that some qualitative difference in the metabolism of larvae and pupae exists that can be related to this difference in sensitivity. Our primary aim. then, is to determine the nature of these differences during development. The marked and immediate decrease in oxygen consumption for Habrobracon pupae and the absence of a compensating recovery is surprising. In the wasp Mormoniella vitripennis, exposure of black pupae to 5 atmospheres of oxygen for from 4 to 6 hours prevented 50 per cent from emerging but their oxygen uptake was unimpaired (Goldsmith and Schneiderman, 1956). We have treated pupae of other insect species with 2 atmospheres of oxygen under conditions comparable to those that we used for Habrobracon but no obvious effects on oxygen uptake or on development have been observed. The species tested were Drosophila inelano- gaster, Muse a domestica, Ephestia kuhniella and Polities sp. It is hard to imagine that other species of insects do not exist that exhibit strong oxygen-sensitivity and, therefore, our search for other insects in this category continues. The authors wish to express their appreciation to Dr. James B. Krause and to Dr. Richard Darsie for helpful suggestions concerning the manuscript. SUMMARY 1. Habrobracon were exposed as white pupae to oxygen and studied for effects upon development, oxygen consumption and pigmentation. EFFECTS OF OXYGEN ON HABROBRACON WHITE PUPAE 187 2. A marked decrease in the incidence of pupae that complete development occurs after exposure to oxygen within the range from air to 20 pounds. The greatest decrease in the rate of oxygen uptake and pigmentation occurs after ex- posure within the range from 20 to 30 pounds. 3. The decrease in oxygen uptake following treatment is immediate. No sub- sequent recovery of oxygen uptake was observed 24 hours after treatment. 4. There is no modification of the respiratory quotient following treatment with oxygen. With increasing pressures of oxygen both the oxygen consumption and carbon dioxide liberation decrease at the same rate. 5. The sensitivity of white pupae to oxygen is modified by temperature. Pupae treated when cold are more resistant than pupae treated when warm. Thus, low- ering the metabolic state of the pupae increases their resistance to oxygen. 6. The inability of the oxygen-treated pupae to acquire pigmentation has been explained on the basis of insufficient oxygen to allow for the oxidation of tyrosine to melanin. The effect of the oxygen treatment upon oxygen consumption and on development is unexplained and at present obscure. LITERATURE CITED BEAN, J. W., 1945. Effects of oxygen at increased pressure. Physiol. Rci'.. 25: 1-147. CLARK, A. M., AND E. B. HERR, JR., 1954. The sensitivity of developing Habrobracon to oxy- gen. Biol. Bull., 107: 329-334. CLARK, A. M., AND C. J. MITCHELL, 1951. Radiosensitivity of haploid and diploid Habrobracon during pupal development. /. Exp. Zoo/., 17: 489-498. GLASS, B., AND H. L. PLAINE, 1952. The role of oxygen concentration in determining the ef- fectiveness of X-rays on the action of a specific gene in Drosophila melanogaster. Proc. Nat. Acad. Sci., 38 : 697-705. GOLDSMITH, M. H., AND H. A. SCHNEIDERMAN, 1956. Oxygen poisoning in an insect. Anat. Rec., 125 : 560. STADIE, W. C., B. C. RIGGS AND N. HAUGAARD, 1944. Oxygen poisoning. Amer. J. Med. Sci., 207: 84-114. SUSSMAN, A. S., 1949. The functions of tyrosinase in insects. Quart. Rev. Biol.. 24: 328-341. WILLIAMS, C. M., AND H. K. BEECHER, 1944. Sensitivity of Drosophila to poisoning by oxygen. Amcr. J. Phvsiol.. HO: 566-573. ON DEVELOPMENT OF EARLY STAGES OF UROSALPINX CINEREA (SAY) AT CONSTANT TEMPERATURES AND THEIR TOLERANCE TO LOW TEMPERATURES ANTHONY E. GANAROS U. S. Fish and Wildlife Service, Milford, Conn. One of the most destructive predators of young oysters in Long Island Sound is the oyster drill, Urosalpin.v cinerea (Say). However, our knowledge of its early development and the tolerance of its egg cases to winter temperatures, when de- posited late in the season, remains incomplete. Carriker's (1955) comprehensive review of the literature on oyster drills clearly shows that most workers, while mentioning the time needed for ova to develop into young conchs, neglect to give the temperature ranges at which development occurs. Among the few who offer information on this subject, Haskin (1935) states that within the temperature range of 23.3° to 29.1° C., 18 to 25 days are needed for the first protoconch to hatch. Federighi (1931) reports that within a range of 18.0° to 32.0° C., it takes approximately 40 days to complete the development. Stauber's field data (Car- riker, 1955) indicate that within the temperature range of 15.0° to 25.0° C., from 45 to 78 days are required for drill eggs to develop. Cole (1942) reports 27 to 32 days at 22.6° C., and 44 to 55 days at 18.3° C. As can be seen from the above references, the information is insufficient to form precise conclusions. We, therefore, devised experiments to determine more accurately the rate of early development of drills at several constant temperatures, which may be encountered within the temperature range of Long Island Sound or adjacent waters. It was also considered of theoretical interest and practical importance to learn the fate of the eggs deposited so late in the fall that they cannot complete develop- ment. Such egg cases, collected during the winter from subtidal and intertidal zones, frequently contain live ova and veligers. Yet, no systematic observations on whether these eggs and embryos can survive the winter and be released in the spring have ever been made. If these embryos could develop, they would have an early start, thus adding to the destructive potential of the next year-class of drills. METHODS Egg cases were obtained from drills maintained in the laboratory at 20.0° C. Preliminary experiments were made with egg cases scraped from the shells of the oysters used to feed the drills and from the glass sides of the aquarium. They were examined under a dissecting microscope and only those that had non-segmented ova, a soft pliable outer membrane, and a translucent bluish-white appearance were selected. It was estimated that such egg cases had been deposited within three days. Later, clusters of young oysters were placed in the aquarium overnight, and the egg cases deposited on them were vised in the experiments. Thus, the age of these egg cases was known to be not more than 16 hours. 188 DEVELOPMENT OF UROSALPINX 189 To determine the rate of development of ova at different constant temperatures, 20 egg cases were placed in perforated, transparent, plastic containers weighted with lead, which were then put in trays. Each tray was supplied with a constant flow of sea water at a salinity of about 25%o, and the temperature of the water was maintained at 7.5°. 10.0°, 15.0°, 20.0°, 25.0° and 30.0° C, controlled to within ± 1.0° C. (Loosanoff, 1949). In addition to conducting experiments at the above constant temperatures, egg cases were also kept at chilling winter temperatures just above freezing, and others were exposed to sub-freezing temperatures in and out of sea water. For observa- tions on the effects of the chilling temperature, 26 egg cases, dredged from New Haven Harbor on December 5, 1955, were suspended in outdoor tidal tanks and systematically examined until March 22, 1956. Egg cases were also taken in winter from an aquarium kept at 20.0° C. and gradually conditioned to the Harbor water temperature which, at that time, was approximately 1.3° C. On January 7, 1956, they were placed in the Harbor and kept there until July 20, 1956. To expose the egg cases to sub-freezing temperatures, they were placed in the freezing compartment of a refrigerator. In the first experiment the temperature of the compartment was - - 12.0° C. (± 1.0° C.). and in the second, - 16.0° C. (± 1.0° C.). In each experiment the egg cases, with ova and veligers, were pre- conditioned in sea water to 3.0° C. Altogether nine plastic boxes, each contain- ing 20 egg cases, were used. Six containers were without water and three con- tained 100 ml. of sea water each. One container without water was removed at the end of the first half hour and the others at half-hour intervals thereafter, up to three hours. The first group of egg cases kept in sea water was removed after one hour of exposure and the others at hourly intervals. In later, similar experiments, the egg cases were left at both temperatures for two, four and six hours. DEVELOPMENT AT Six CONSTANT TEMPERATURES We determined the number of days required at different temperatures for de- velopment of ova (Fig. 1) to the following embryological stages: early larvae (Fig. 2), shelled veliger (Fig. 3), and protoconchs (Fig. 4). The time needed to reach the early veliger stage decreased with increases in temperature from 10.0° to 30.0° C. (Table I). The groups kept at 30.0° and 25.0° C. attained the shelled veliger stage between the fourth and seventh days. However, it required eight additional days for those kept at 20.0° C. to reach the same stage, and even longer for those at 15.0° and 10.0° C. In spite of the longer time required for ova at 20.0° C. to develop to the shelled veliger stage, they had reached the protoconch stage by the 22nd day, the same as the groups kept at 25.0° and 30.0° C. Nevertheless, the egg cases kept at 30.0° and 25.0° C. started releasing young conchs on the 22nd day, and continued for 16 days for both temperature groups. None of those kept at 20.0° C. were released until the 30th day, and those kept at 15.0° C. were not released until the 56th day. Thus, while there was only eight days' difference in the time required to reach the protoconch stage between the egg cases kept at 20.0° C. and those at 15.0° C., there was 26 days' difference in the time of release of the first young conchs. Moreover, at 20.0° C. the period between the first conch released and the last was only 13 days, while for the 15.0° C. group 22 days were needed. 190 ANTHONY E. GANAROS FIGURE 1. Egg case of I', cincrca with five ova. X 10. At 10.0° C. the ova required 66 days to reach the early veliger stage, and 84 days before the shelled veligers appeared. At 7.5° C. no development occurred during the 54 days. At the end of those periods both the 10.0° C. and 7.5° C. groups were placed in water of 20.0° C. to determine whether they would develop under the new temperature condition, regardless of their previous treatment. Only 65 per cent of the egg cases kept originally at 10.0° C. produced conchs, while no development occurred in the former group at 7.5° C. Since the egg cases at 7.5° C. were apparently adversely affected before being placed at 20.0° C., it may be inferred that temperatures at 7.5° C. and lower not only arrested development, but killed the eggs after a prolonged period of exposure. Our results indicated that the optimum temperature for drill development was TABLE I Number of days for ova to develop to various stages at constant temperatures and the percentage of egg cases producing young conchs Temperatures 7.5° C. 10.0° C. 15.0° C. 20.0° C. 25.0° C. 30.0° C. Early veliger 54* 66 10 7 4 2 Shelled veliger — 84** 25 15 7 7 Protoconch — — 30 22 22 22 Release of young conchs — — 56-78 30-43 22-38 22-38 Percentage of egg cases producing young conchs o% 65% 90% 100% 95% 90% * No development. ** Placed in water of 20.0° C. DEVELOPMENT OF UROSALPINX 191 FIGURE 2. Egg case, with outer membrane removed, containing early motile larvae just before a true velum is developed. Larvae at this stage have a gut and can feed on particulate matter by means of currents created by the cilia. X 10. FIGURE 3. Egg case, with outer membrane removed, containing veliger larvae, with foot and velum present, after torsion has occurred. The shell has begun to form along the outer edge of the mantle. X 10. 192 ANTHONY E. GANAROS about 20.0° C. (Table I). However, since the rate of development was faster at 25.0° C. and the difference in the percentage survival of egg cases producing young conchs between 20.0° C. and 25.0° C. was small, it was considered possible that the optimum was above 20.0° C. Therefore, a second experiment was conducted at 20.0° C. and 25.0° C., using 20 egg cases in each temperature group, and the percentage survival of ova determined. At 20.0° C., 139 out of 176 ova, or 78.9 per cent, developed to the protoconch stage, as compared to 96 out of 168 ova, or 56.8 per cent development, at 25.0° C. Thus, although the rate of development was faster at 25.0° C.. the optimum temperature, from the standpoint of successful development to the young conch stage, appeared to be about 20.0° C. Moreover, at 20.0° C. the period for protoconch release was shorter than at any other temperature. FIGURE 4. Egg case, with outer membrane removed, containing three early protoconch larvae which show partial spiral development and pigmentation. Note also the two undeveloped ova in the same egg case. X 10. Since the egg cases in the first experiment were deposited within a period of three days, their age alone could not account for the difference of 13 to 22 days between the release of the first and last young conch. The conclusion that slight differences in the age of the cases are not responsible for pronounced differences in the time needed for release of young conchs of the same groups is further substan- tiated by our second experiment at 15.0° C. in which egg cases collected within a 16-hour period started releasing young conchs at 62 days and continued to do so for 19 days. TOLERANCE TO Low TEMPERATURES To study the tolerance of egg cases to winter temperatures when exposed at low tide and to simulate tide pool conditions, cases containing ova and veligers were placed in water and exposed to air at the same sub-freezing air temperatures. DEVELOPMENT OF UROSALPINX 193 Because oyster drills in the intertidal zone stay close to the low water level and deposit their egg cases there, the latter are seldom exposed to air for more than an hour or two. Nevertheless, even under these conditions all the eggs would die if exposed to air temperatures around - 15.0° C. However, if the time of exposure at this temperature is reduced to a half hour, approximately 40 per cent may survive. At temperatures around - 12.0° C., five per cent could survive after two hours of exposure. The survival was greater when the egg cases were protected by water (Table II). At exposure periods of two hours at air temperatures around - 12.0° and - 15.0° C., about 95 per cent of the eggs survived. The chance of survival of eggs in frozen tide pools would be much greater than in air, not only because of the TABLE II Survival of ova and veligers in egg cases of U. cinerea (a) submerged in sea water, and (b) when exposed in air, subjected to two sub-freezing temperatures. Survival is expressed in percentage of egg cases in which shelled veligers developed after returned to 20.0° C. sea water Air temperature -12.0° C. (±1.0° C.) Air temperature -16.0° C. (±1.0° C.) Length of exposure Temperature at end of exposure (°C.) Survival (%) Length of exposure Temperature at end of exposure (°C.) Survival (%) (a) submerged in sea water 1 Hr. 2 Hrs. 3 Hrs. 4 Hrs. 6 Hrs. 1.8 100 1 Hr. 2.8 95 2 Hrs. 3.0 95 3 Hrs. 3.5 95 4 Hrs. 7.5 85 6 Hrs. 5.0 90 5.0 95 8.1 25 12.0 25 16.0 0 (b) exposed in air i Hr. -11.0 75 JHr. -15.0 40 1 Hr. -11.0 20 1 Hr. -15.0 0 H Hrs. -11.2 5 H Hrs. -15.0 0 2 Hrs. -11.5 5 2 Hrs. -15.3 0 2i Hrs. -11.8 0 2J Hrs. -16.2 0 3 Hrs. -12.4 0 3 Hrs. -16.0 0 warmer temperatures of the water, but also because desiccation of the egg cases is inhibited. Usually when drill cases were exposed to the sub-freezing air tempera- tures they were desiccated to such an extent that their walls collapsed. In gen- eral, experiments showed that the percentage survival in both air and water decreased with increases in time of exposure and with decreases in temperature (Table II). The resistance of eggs and embryos to low temperature was tested under more natural conditions when, on December 5, 1955, 26 egg cases, 11 of which contained ova to segmented stages and 15 contained veligers and shelled veligers, were dredged from New Haven Harbor. The bottom temperature was 7.1° C. and the veligers were observed revolving within their capsule. These cases were placed 194 ANTHONY E. CAN ARCS in tidal tanks in water of 7.5° C. on December 6, 1955, and the veligers were still motile on February 16, 1956, 63 days later. The average water temperature dur- ing this period was 2.3° C. with a range from •- 0.1° to 7.5° C. By March 22, however, the ova and segmented stages within the cases were disintegrated and the veligers were dead. To supplement these observations 120 egg cases collected from the laboratory aquaria were conditioned gradually to the outdoor water temperature of 1.3° C. and placed in the Harbor on January 7, 1956. These egg cases were kept until July 20, 1956, but showed no evidence of development. DISCUSSION The incubation times found in our experiments are in general agreement with the data of other authors. Thus, Raskin's (1935) report of 18 to 25 days as the time for the first young conchs to hatch at 23.3° to 29.1° C. agrees with our 22-day period at 25.0° C. Cole's (1942) findings of 27 to 32 days at 22.6° C. and 44 to 50 days at 18.3° C. for drills are again in agreement with our observations. If we interpolate our data, we obtain 30 to 38 days at 22.5° C. and 43 to 56 days at 17.5° C. Compared to our results, Cole (1942) found that the surprisingly short period of only five days was required for all the young conchs to be released at 22.6° C. and only six days at 18.3° C. Cole does not give the number of egg cases used in this experiment and the short period he records may be the result of having very few egg cases, since we found considerable variation between egg cases at any one temperature. Pope (Carriker, 1955) found that the period for protoconch release from an egg case or group of egg cases, which we assume were deposited at the same time and kept under identical conditions, extended from four to 38 days. He attributes this to an uneven development of the embryos, but we found the embryonic devel- opment within a single egg case to be generally uniform, except for malformed embryos and undeveloped ova. By the time of release, some of these are partly eaten by the normally developing drills or remain undeveloped to such an extent that they never emerge (Fig. 4). Considerable variation in the period of incu- bation, however, does occur between the egg cases deposited within a 16-hour period. We observed that the greatest variation in hatching time between egg cases occurs after the protoconch stage, which may not be caused entirely by an uneven development of the embryos. Some protoconchs remain in their cases longer than others, which may be due to a variation in some mechanism releasing the egg case operculum. Variations in hatching may also be caused by the physical obstruc- tion of the operculum opening which a protoconch may find too small. On one occasion wre saw a young drill, emerging from a case, become entrapped in the operculum opening and block the passage for the remaining protoconchs for eight days. Our experiments showed that although egg cases can withstand sub-freezing water temperatures for short periods, they cannot survive prolonged periods of chilling. At first it appears to be more drastic to subject the egg cases to sub- freezing temperatures rather than to temperatures above freezing but if, for ex- DEVELOPMENT OF UROSALPINX 195 ample, the permeability of the egg cases is affected, the osmotic malfunction pro- gresses at a faster rate at chilling temperatures than at sub-freezing temperatures (Luyet and Gehenio, 1940). Chilling temperatures, as these authors point out, become lethal to protoplasm only after long periods. This may explain the sur- vival of the egg cases which we kept in the tidal tanks for a period of 63 days and also the presence of egg cases observed by other workers during the winter months (Carriker, 1955). The author wishes to thank Dr. V. L. Loosanoff for his suggestions and construc- tive criticism throughout the experimental work and both Dr. V. L. Loosanoff and Mr. H. C. Davis for the critical reading of this paper. I also wish to thank Mr. C. A. Nomejko for preparing the photomicrographs and tables. SUMMARY 1. The rate of ova development increases directly with the increase in tempera- ture from 15.0° to 25.0° C. No increase in the rate of ova development \vas ob- served above 25.0° C. 2. Optimum temperature for ova development of U. cinerea of Long Island Sound appears to be 20.0° C., or between 20.0° and 25.0° C. 3. Egg cases of U. cinerea kept in sea water can withstand sub-freezing tem- peratures for longer periods than egg cases exposed to sub-freezing air temperatures. 4. In sub-freezing temperatures, the percentage mortality increases with the period of exposure and with a decrease in temperature. 5. Egg cases kept at 10.0° C. for as long as 84 days showed partial development and were capable of producing normal protoconchs when returned to 20.0° C. ; whereas, egg cases kept at 7.5° C. for 54 days were not viable. 6. Our experiments and observations suggest that egg cases remaining through the winter in Long Island Sound will not contain viable ova in the spring. LITERATURE CITED CARRIKER, M. R., 1955. Critical review of biology and control of oyster drills, Urosalpinx and Eupleura. Sp. Sci. Kept., Fisli. 148. COLE, H. A., 1942. The American whelk tingle, Urosalpinx cinerea (Say), on British oyster beds. /. Mar. Biol. Assoc., 25 : 477-508. FEDERIGHI, H., 1931. Studies on the oyster drill (Urosalpinx cinerea, Say). Bull. U. S. Bur. Fish., 47: 85-115. HASKIN, H. H., 1935. Investigations on the boring and reproduction activities of oyster drills, Urosalpinx cinerea (Say) and Eupleura sp. Unpub. Rept. U. S. Bur. Fish. LOOSANOFF, V. L., 1949. Method for supplying a laboratory with warm sea water in winter. Science, 110: 192-193. LUYET, B. J., AND P. H. GEHENIO, 1940. Life and death at low temperatures. Biodynamica, Normandy, Missouri. THE UPTAKE OF RADIOACTIVE CALCIUM BY SEA URCHIN EGGS. I. ENTRANCE OF CA45 INTO UNFERTILIZED EGG CYTOPLASM 1 SIDNEY C. HSIAO AND HOWARD BOROUGHS Department of Zoology, University of Hawaii and Hawaii Marine Laboratory, Honolulu. T. H. In studying the physiology of calcium accumulation of sea urchin eggs and its ultimate utilization in the formation of the calcareous skeleton, the use of radio- active calcium as a tracer offers many advantages. But a search of the literature on the uptake of radiocalcium by sea urchin eggs revealed only one report which was a study using an indirect method (Rudenberg, 1953). In his study of the role of the jelly coat in the uptake of calcium by the eggs of Arbacia punctulata Rudenberg observed the radioactivity present in his samples by a Geiger tube placed above the incubation medium. He concluded that Ca45 was accumulated only by eggs with jelly coats and for up to six hours after fertilization. About six hours after fertilization a loss of calcium from the eggs was noted. But eggs with- out jelly coats showed no uptake and no loss of calcium. In the present study, experiments have been designed to give more direct answers to the following series of questions: (1) Will sea urchin eggs, with and without jelly coats, take up radio- calcium from the medium ? (2) If either or both types of eggs take up radiocalcium from the medium, are the Ca*5 ions simply adsorbed on the surface or can they be demonstrated in the egg cytoplasm? (3) If there is an uptake of radiocalcium, is it due to a net accumulation of calcium or simply to an exchange of Ca45 with stable Ca40? MATERIALS AND METHODS Mature, unfertilized eggs of Tripneustes gratilla (Linnaeus), a large, common species of sea urchin in Hawaiian waters, were used. Freshly collected females were induced to shed their eggs by the KC1 injection method (Tyler, 1949). One female could provide 30-50 ml. of eggs, more than enough for a whole series of experiments. The eggs were strained through bolting cloth or several layers of cheesecloth to remove extraneous matter, and washed with filtered sea water with the help of gentle centrifugation before use in the following experiments. (1) Comparison of Ca45 uptake by intact eggs and by eggs without jelly coats. To compare the time course of the uptake of radiocalcium by these two types of eggs, a freshly washed batch of eggs from one female was divided into two approximately equal aliquots. One aliquot was treated with HC1 to remove the jelly coats by the method of Harvey (1941). A stock Ca45 solution with an activity of 5 ^c/ml. was made by dilution of Oak Ridge Ca45CL solution having a specific activity of 73.81 mc/g. with an appropriate quantity of filtered normal sea water. One-half ml. of a 1:10 suspension of eggs with jelly coats removed was put into a 50-ml. 1 Contribution no. 100 from Hawaii Alarine Laboratory, University of Hawaii. 196 CA-45 UPTAKE BY SEA URCHIN EGGS 197 beaker already containing 4 ml. of filtered sea water. One-half ml. of the stock Ca45 solution was then added, giving a final volume of 5 ml. and a Ca45 concentra- tion of 0.5 juc/ml. Twelve beakers were similarly prepared. The content of the first to the twelfth beaker was incubated at room temperature (24-27° C.) with occasional stirring according to the following schedule : Beaker No. 12345678 9 10 11 12 Incubation time in minutes 0 1 2 4 8 16 32 64 128 256 512 1024 At the end of the specified time of incubation, the content of each beaker was centrifuged to separate the eggs from the incubation medium. As it took two minutes to effect this separation, the actual time of contact between the eggs and Ca45 varied from 2 to 1026 minutes. A 0.5-ml. aliquot of the supernatant medium after each time interval was trans- ferred to an aluminum planchette, dried and counted with a thin window (1.4 mg. cm2) Geiger tube and a commercial sealer. All samples were prepared in triplicate. Dose planchettes were made of the incubation medium without eggs by diluting it ten-fold and triplicate of 0.5-ml. volume used for counting. A con- venient aliquot of the incubated eggs from each time interval was transferred from the centrifuge tube, spread on a weighed planchette, dried to constant weight and the activity of the eggs counted in the same way as the medium. After correction for background and for self-absorption the counts were expressed in counts per minute per mg. dry weight. A similar series was run using eggs with jelly coats intact. These two experiments were repeated, but the incubated eggs were washed in normal filtered sea water before being counted for Ca45 activity. As a control, eggs from the same female were incubated in normal filtered sea water and prepared and counted in a similar manner. (2) Removal of Ca45 jrom eggs. In order to ascertain whether the activity of eggs incubated in radiocalcium-containing sea water could be removed from the egg surface, radioactive eggs, with and without jelly coats, were washed with (a) sea water, (b) a 0.02 per cent V/V solution in sea water of a commercial surface active wetting agent, "Sterox SK" (supplied by Monsanto Chemical Co.), and (c) a 0.05 per cent W/V solution in sea water of a commercial detergent "Tide," manufactured by Procter and Gamble Co. Samples of eggs without jelly coats and of intact eggs were washed separately by re-suspending the radioactive eggs in 10 ml. of the washing fluid in a centrifuge tube, and stirring them gently for 5 minutes. At the end of 5 minutes a 0.5-ml. aliquot of the well-mixed egg sus- pension was transferred to a second tube, centrifuged to remove the washing liquid from the eggs which were then spread on a planchette in order to measure egg radioactivity. The 9.5-ml. egg suspension in the original tube was centrifuged at a RCF of 2330 G to pack down the eggs. The sedimented eggs were completely drained by inverting the tube whose inside wall was wiped dry with absorbent tissue. The washing procedure was repeated five times with each sample of eggs. (3) Autoradiograpliy of eggs. Intact eggs and eggs without jelly coats were made radioactive by incubating them in sea water containing Ca4r'. They were fixed separately with neutral formalin, dehydrated by direct transfer into dioxane (diethylene dioxide) which was changed once. Infiltration was made with two changes of paraffin. Sections of eggs 5 or 10 microns thick were mounted on slides 198 SIDNEY C. HSIAO AND HOWARD BOROUGHS from some of which the paraffin was removed by xylene, and the remaining ma- terial coated with a thin veneer of gelatin and autoradiographs made on Kodak medical (No Screen) x-ray films. Other sections were treated by Townsley's method (personal communication). In this method, which is similar to that worked out by Belanger and Leblond (1946), the paraffin sections wrere floated on slides previously coated with gelatin (Kodak photographic inactive gelatin, 0.5% and chrome alum, 0.05% solution) and allowed to attach by drying. After dis- solving the paraffin in xylene the sections were run down through grades of alcohol to double glass-distilled water. The slides were next coated with Ilford nuclear research emulsion, type G.5, which was warmed to 38° C.. thinned with a little distilled water and applied in a very thin layer with an artist's brush. The coated slides were dried under an electric fan and placed in a slide box made light proof by being wrapped in aluminum foil and black paper. Exposure of the emulsion to the /^-radiation from egg sections was completed in a refrigerator and later de- veloped photographically. Examination of the autoradiographs was made with both light and phase contrast microscopes. (4) Total calcium determination. The total calcium content of ordinary and radiocalcium-containing eggs was estimated after the eggs were dried to constant weight. Aliquots of the dried specimens were wet-ashed by the method of Norris and Lawrence (1953). The egg calcium was precipitated with ammonium oxalate according to Holth's (1949) recommendation to exclude magnesium. The pre- cipitated calcium oxalate was quantitatively estimated by flame spectrophotometry with a Beckman model DU spectrophotometer according to the method published in "Application Data for Beckman Instruments" by the manufacturer. A series of eggs having various radioactivities, from zero to 686 cpm per mg. dry weight, was used for analysis. RESULTS When exposed to Ca45 in the medium eggs with and without jelly coats showed high rates of radiocalcium uptake during the first few minutes. After this initial phase the quantity of Ca45 taken up by the eggs increased with the increased time of incubation. Figure 1 represents the results of a typical experiment. Three things are shown by this figure: (1) unfertilized eggs, with and without jelly coats, took up radiocalcium; (2) unfertilized eggs without jelly coats took up more radio- calcium than intact eggs when both were treated similarly, as shown by the heights of curves A and C in contrast to those of curves B and D of this figure. This was true throughout the whole time course at all the different time intervals of incuba- tion used. (3) When the eggs were washed with filtered sea water after incuba- tion and before being placed on the planchettes for counting, both types of eggs retained Ca45, but the activity due to Ca45 retained by eggs without jelly coats was much higher than that of intact eggs, as shown by curves C and D. Eggs from the same female but incubated in normal sea \vater as control showed no radioac- tivity at all. Table I shows the results of washing the treated eggs in order to see if the Ca45 taken up by the two types of unfertilized eggs can be removed, assuming that the radiocalcium ions are on the surface of the jelly or in the jelly coats in the case of intact eggs, and on the surface of the vitelline membrane in the case of eggs CA-45 UPTAKE BY SEA URCHIN EGGS 199 400 .? 300 o> 5 >. 200 6> 100 e 200 100 50 40 30 20 10 ~t 1 1 r T 1 1 T -V 1 1 T A. Eggs without jelly B. Eggs with jelly coats C. Eggs without jelly (washed) D. Eggs with jelly coats (washed ) 2345678 Time in hundreds of minutes 10 FIGURE 1. Radiocalcium uptake by sea urchin eggs with and without jelly coats for different periods of incubation. without jelly. It will be seen from this table that eggs without jelly coats had approximately 1.5 times as much activity as intact eggs. Intact eggs washed once with detergent for five minutes lost about 80 per cent of the original radioactivity. On the other hand, eggs without jelly similarly treated lost only about half of the 200 SIDNEY C. HSIAO AND HOWARD BOROUGHS original activity. After the fourth wash the washing fluid showed practically no activity, indicating that a surface active agent could not remove any more Ca45 from these eggs. But it will be noticed from Figure 2 that in washing with the wetting agent "Sterox SK," eggs without jelly coats consistently retained more radioactivity after each washing than did eggs with jell}- coats. At the end of the fifth washing eggs without jelly coats retained 11 times as much activity as did similarly treated intact eggs, while the washing fluid showed no activity in either case. In washing with a solution of "Tide" (see Table I) about six times as much activity was retained by jelly-free eggs as by eggs with jelly coats. It was noticed during these washing experiments that the first washing of intact eggs removed all the jelly coats. That the activity remaining in the detergent washed eggs was due to Ca45 in the cytoplasm and not removable by surface active agents is shown by autoradio- TABLE I The loss of radiocalcium from eggs washed with detergents showing that eggs without jelly coats retained more Ca45 than did intact eggs Material Washing fluid Washing No. Egg activity. Material Washing fluid Washing No. Egg activity, cpm cpm Eggs with "Wetting 0 499 Eggs with- "Wetting 0 736 jelly coats agent" 1st 103 out jelly agent" 1st 321 intact 6 drops, 2nd 34 6 drops, 2nd 137 sea water 3rd 34 sea water 3rd 83 10 ml. 4th 33 10 ml. 4th 80 5th 5 5th 56 Eggs with "Tide" 0 499 Eggs with- "Tide" 0 736 jelly coats l%w/v 1st 121 out jelly l%w/v 1st 306 intact solution 2nd 31 solution 2nd 199 0.5 ml., 3rd 173 (?) 0.5 ml., 3rd 81 sea water 4th 46 sea water 4th 82 9.5 ml. 5th 9 9.5 ml. 5th 52 graphs of sectioned eggs. In Figure 3 are shown photomicrographs of some of the autoradiographs. Figure 3A is a photomicrograph of an autoradiograph made by three whole unfertilized eggs on an Ilford emulsion. The dark regions around these eggs show the presence of Ca45 in the jelly coats. Figure 3B shows an auto- radiograph made on x-ray film by an intact egg incubated in Ca4r'-containing sea water. In this figure the outline of the jelly coat and the egg can be seen. Figure 3C is a lower power photomicrograph made with a light microscope. In the center of this photomicrograph is a section of an untreated egg included among treated eggs whose sections show the presence of Ca4rj inside the cytoplasm. Figures 3D- 3F are photomicrographs made with a phase contrast microscope. In Figure 3D the egg membrane and cytoplasm are seen to contain radiocalcium. Figure 3E is a higher power photomicrograph of the autoradiograph of another group of eggs, while Figure 3F is a detailed view of a single section of a radioactive egg. The results of chemical estimation of total calcium content of eggs having taken CA-45 UPTAKE BY SEA URCHIN EGGS 201 up various quantities of radiocalcium are shown in Figure 4. This figure shows that of the 15 groups of radioactive eggs analyzed, no group showed significantly different content of total calcium from the average value of all the groups, although the radioactivity of the highest group was about 340 times that of the lowest. It also shows that increase in Ca45 taken into the eggs was not followed by increase cr> cn 0) 800 700 c o E 600 500 400 Eggs without jelly coats E CL O > o 300- 200- ntact eggs Washing FIGURE 2. Decrease in radiocalcium in sea urchin eggs after washing with detergent solution (Sterox SK). in total calcium. Total calcium fluctuated independently of the Ca45 content of the eggs about a mean value of 2.7 jug per mg. dry weight. The calcium content of ordinary, i.e., unincubated, eggs was not lower than that of radioactive eggs. This indicates that there is no calcium accumulation, but an exchange of ordinary Ca40 for radioactive Ca45 leaving the total calcium content more or less unaltered. 202 SIDNEY C. HSIAO AND HOWARD BOROUGHS A R ' * ». * D FIGURE 3. Photomicrographs showing Ca45 inside egg jelly and in sections of egg cyto- plasm. 3A, autoradiograph of three intact eggs showing Ca45 among the jelly coats, magni- fication 100 X. 3B, autoradiograph on Kodak x-ray film of one intact egg, magnification 150 X. 3C, autoradiograph of 10 /u thick sections of eggs, magnification 37 X. 3D, phase contrast photomicrograph of similar egg sections, magnification 70 X. 3E similar to 3D, magnification 140 X. 3F, detailed picture of a single section of egg, magnification 140 X. CA-45 UPTAKE BY SEA URCHIN EGGS 203 c? cu 4 £ \ cr 3 2.7 2 2 o o o "o O - O mean value 0 0 © S groups O 100 200 Ca4S content n 300 400 500 600 cpm/mg. dry wt. of eggs FIGURE 4. Total calcium content of ordinary and radioactive eggs showing no increase in total calcium with increase of Ca45 uptake. DISCUSSION The consistently greater amount of Ca45 taken up by eggs without jelly coats than that by intact eggs indicates that not only is jelly coat unnecessary for the uptake of radiocalcium, but it acts as a hindrance to the entrance of these ions into the egg cytoplasm. As the total diameter of intact eggs, inclusive of jelly coat, measures 126-130 microns, and that of the vitellus about 82 microns, an ion on the surface of the jelly coat would theoretically have to travel 63-65 , microns to reach the geometrical center of the egg, while in the case of eggs without jelly, an ion on the surface of the vitelline membrane would have to travel only a distance of 41 microns to reach the same point. It is hence not surprising that for an equal period of incubation in sea water containing radiocalcium, eggs without jelly could take up more Ca45 than intact eggs. In other words, in eggs without jelly coats the Ca40 of the egg cytoplasm could be exchanged directly with the Ca45 in the incubation medium or on the vitelline surface. In intact eggs, the first place of Ca45 entry would be the jelly where the entering Ca45 could either exchange with the Ca40 of the jelly coats or continue to move into the vitellus. When eggs with jelly coats were washed with detergent, the first washing re- moved most, if not all, of the jelly. If these eggs were first made radioactive, approximately 80 per cent of the radioactivity came off with the jelly. This ob- servation suggests a possible explanation of the loss of calcium by fertilized Arbacia eggs after 6 hours incubation reported by Rudenberg (1953). As the intact radio- active eggs were shaken for 6 or more hours, a good deal of the jelly coats would 204 SIDNEY C. HSIAO AND HOWARD BOROUGHS be detached which, when brought to the surface by stirring, would account for the reported increase in radioactivity in the surface layer of the incubation medium. Although the autoradiographs of sections of the radioactive eggs do not give the exact location of the Ca45 ions in the cytoplasm, because the denaturation of the egg protein by formaldehyde fixation and subsequent histological procedures applied to the eggs and their sections may have altered their positions, the fact that these histological treatments and photographic developments used in connection with Ilford emulsion film coatings did not remove them indicates that the Ca45 must be firmly attached to the organic compounds of the eggs. An approximate idea of the site of the uptaken Ca45 is provided by the autoradiographs. Further investigations are in progress to determine which fraction or fractions of the egg substance com- bined with the radiocalcium. SUMMARY AND CONCLUSIONS 1. The entrance of radiocalcium into unfertilized eggs of Tripncustes gratilla (Linnaeus) has been investigated in this study. By direct measurements of the radioactivity of intact eggs and of eggs without jelly coats it was found that al- though both types of eggs took up Ca45, eggs without jelly coats took up much more than intact eggs. 2. By washing with detergent both types of eggs made radioactive by incuba- tion in radiocalcium-containing sea water, it was found that jelly-free eggs retained 1 1 times as much Ca45 as intact eggs when washed with a commercial wetting agent "Sterox SK," and 6 times as much when washed with a dilute solution of "Tide." 3. It is concluded that jelly is not essential to the uptake of radiocalcium by sea urchin eggs and the presence of jelly coats reduces the amount of penetration into the egg cytoplasm. 4. Autoradiographs of whole eggs showed the presence of Ca45 in the jelly coats if intact eggs were incubated in radiocalcium-containing sea water. Auto- radiographs of sections of both types of eggs showed that Ca45 was inside the egg cytoplasm. 5. No significant difference in total calcium was found by chemical analysis of groups of eggs incubated for various periods of time in Ca45-containing sea water, i.e., made to take up varying amounts of radiocalcium. None of the groups showed significantly different contents of total calcium from that of ordinary unfertilized eggs. It is concluded that Ca*5 enters the egg cytoplasm by exchange with Ca40 of the eggs. LITERATURE CITED BELANGER, L. F., AND C. P. LEBLOND, 1946. A method for locating radioactive elements in tissues with a photographic emulsion. Endocrinology, 39: 8-13. HARVEY, E. B., 1941. Vital staining of centrifuged Arbacia punctulata egg. Biol. Bull., 81 : 114-118. HOLTH, T., 1949. Separation of calcium from magnesium by oxalate method. A critical study. Anal Chem., 21: 1221-1226. NORRIS, W. P., AND B. J. LAWRENCE, 1953. Determination of calcium in biological material. Anal. Chem., 25 : 956-960. RUDENBERG, F. H., 1953. The role of the jelly coat in the uptake of calcium by eggs of Arbacia punctulata before and after fertilization. Exp. Cell Res., 4: 116-126. TYLER, A., 1949. A simple non-injurious method for inducing repeated spawning of sea urchins and sand dollars. Coll. Net, 19: 19-20. A FUNGUS PARASITE IN OVA OF THE BARNACLE CHTHAMALUS FRAGILIS DENTICULATA T. W. JOHNSON, JR. Dct>art]>icnt of Botany, Duke University, Durham, Nortli (. aroliiui Species of fungi in marine animals are apparently not numerous, although some are very destructive parasites. Outstanding among the latter are Ichthyosporidium hojeri (Plehn and Mulsow, 1911 ; Sproston, 1944) in herring, salmon, and flounder, and Dennocystidium mar-inn tn (Mackin ct al., 1950; Ray and Chandler, 1955) in oysters. Among other reports of marine zoophagous fungi worthy of mention are : Cycloptericola marina in Cyclopterus hiinpns (Apstein, 1910) ; Leptolegnia marina in the pea crab, Pinnotheres pisum (Atkins, 1929, 1954a) ; a saprolegniaceous and a pythiaceous fungus in the calcareous shells of molluscs (Bornet and Flahault, 1889) ; Spongiophaga sp. in sponges ( Galtsoff, 1940) ; three species of Nephro- myces in the ductless kidneys of various ascidians (Giard, 1888) ; a pink yeast (Torula) in oysters (Hunter, 1920) ; boring fungi in various shell-forming animals (Kolliker, 1860) ; two species of Thalassomyces in the decapod Pasipliaea (Nieza- bitowski, 1913) ; a marine Laboulbenia on Aepus robini (Picard, 1908) ; an Asco- mycete, Didymella conchae (Bonar, 1936) in mollusc shells (a marine lichen ac- cording to Santesson, 1939) ; Sirolpidhim zoophthorum in lamellibranch larvae (Vishniac, 1955), and unnamed marine eccrinids in Panopeus hcrbstii and Eincrita talpoida (\\rolf and Wolf, 1947). A very few species of fungi occur in the ova of marine invertebrates: Lagenidium callincctcs (Couch. 1942) in the blue crab, Callinectes sapidns; Plectospira dubia and P\thinin thalassiiuti (Atkins, 1954b, 1955) in the pea crab, and an unnamed fungus suggestive of Sirolpidium zooph- thoruni and Plectospira dubia in the oyster drill, Urosalpinx cincrca (Ganaros, 1957). Two fungi have been described from barnacles. The Ascomycete Phar- cidia marina (Santesson, 1939, places this organism in the lichen genus Artho- pyrenia) occurs on the shells of Balanus balanoides (Bommer, 1891) ; a second Ascomycete, Didymella balani (also renamed as a lichen) develops on the test of Chthdmalus stcllatus (Hariot, 1887). A new species of Phycomycete, Lagenidium chthamalophilnm, parasitic in the ova of Chthamalus fragilis var. dcnticnlata, is described in this paper. Lagenidium chthamalophilum sp. nov. Hyphae crassae, contortae vel irregulares, ramosae ; intra- et extramatricales, vacuolis et guttulis multis, pallide flavae usque ad hyalinas; plerumque 10-18 //, in diam. Sporangium ex septatione hyphae forniatum, tubulo singulo apice dilato in vesicam sphaericam. Sporae reniformes, a latere biflagellatae, in vesica for- mantur et vesica deliquescente emittuntur. Oogonia rara ; lateralia vel terminalia vel intercalaria sed semper in hyphis intramatricalibus ; globosa vel subglobosa vel elongato-irregularia ; 19-47 ^ diam. Oosporae singulae, vel raro binae; sphaericae 205 206 T. W. JOHNSON, JR. FIGURES 1-9. FUNGUS PARASITE OF BARNACLE OVA 207 (21-25 /A diam.) usque ad ellipsoidales (18-27 X 16-23 //.), guttulas centricas vel eccentricas includunt. Germinatio oosporarum adhuc incognita. Antheridia adhuc non observata. Hyphae stout, contorted or irregular, branched ; filling the ova and emergent from them ; generally reticulately vacuolate, with numerous minute cytoplasmic oil bodies, infrequently with very diffuse cytoplasm containing oil bodies ; pallid golden- yellow to hyaline ; occasionally constricted at points of penetration through the egg membrane ; variable in diameter, generally 10-18 ^ ; occasionally producing globose, lateral swellings up to 39 /*, in diameter. Sporangia formed by segmentation of intramatrical hyphae, very rarely produced on extramatrical hyphae; variable in length, diameter coincident with that of hypha ; each producing one stout emergent discharge tube expanded apically into a spherical vesicle. Planonts reniform, lat- erally biflagellate, 8.5-10.2 X 6.8-8.5 /A; cleaved from sporangial protoplasm within the vesicle ; aplerotic ; discharged upon rapid deliquescence of vesicle. Oogonia rare ; lateral, terminal, or intercalary on intramatrical hyphae ; globose, subglobose, or slightly elongate-irregular; 19-47 p, in diameter. Oospores 1, rarely 2 ; spherical, blunt-conic, or nearly ellipsoid ; containing a single centric or eccentric mass of small oil globules; 18-27 X 16-23 /A, spherical ones 21-25 /JL in diameter; germination unknown. Antheridia not observed. Parasitic in ova of Chthauialus jragilis dcnticulata, Beaufort Inlet, North Caro- lina, June 17, 1957 (TYPE), leg. J. D. Costlow. Chthamalus jragilis dcnticulata, common in the Beaufort, North Carolina region, is a small, pallid-brown to grayish-white, sessile barnacle attaching to pilings, rocks, sea walls, to other barnacles, and to the stems and leaves of Spartina alt crni flora. The animal occurs only on the uppermost portions of pilings, for example, near the high water line. These barnacles, among the last to be covered by water at incom- ing tide, are submerged for the short slack high water period, and are the first to be exposed on ebb tide. CJithamalus jragilis dcnticulata occurs with, in fact often attaches to, a second equally abundant barnacle, Balanus amphitrite. The lamellae (egg cases) of C. jragilis denticulata are usually paired and lie free within the mantle. The larval planktonic stage, the nauplius, develops and is liberated within the parent barnacle. Uninfected eggs (in mass) change color as the em- bryo develops : bright orange-yellow, pallid yellow, pale cream. Infected lamellae, however, are often pallid gray or grayish-green. Lagenidium chthamalophilum may develop in the ova of Chthamalus jragilis denticulata. at any time between late gastrulation and emergence of the nauplii. Released nauplii are apparently not infected, nor are there any somatic tissues of the parent animal invaded by the fungus. Ova showing three or more appendage buds are generally most often infected (Fig. 6). Early stage embryos (one or more appendage buds) in entire egg masses may be destroyed, leaving only a cluster of egg membranes filled with fungus mycelium (Figs. 1,2). On the other hand, if lamellae with more mature or differentiated embryos within the egg mass become infected, some embryos escape invasion by the fungus and develop into FIGURES 1-9. Lagenidium chthamalophilum. 1, 2, hyphae within ova membrane; 3, early infection stage showing branching of hypha ; 4, emergent hyphae with a sporangial and oogonial initial ; 5, coiled extramatrical hypha ; 6, infection by two spores of an embryo with three ap- pendage buds ; 7, vacuolate extramatrical hypha ; 8, guttulate extramatrical hypha ; 9, intra- and extramatrical hyphae showing constrictions and two sporangial initials. 208 T. W. JOHNSON, JR. FUNGUS PARASITE OF BARNACLE OVA 209 the planktonic stage. Infection visible in one or two peripheral ova in a lamella spreads rapidly through the entire cluster so that within two days (continual sub- mersion in raw sea water) all embryos are invaded. Inoculation is brought about by laterally biflagellate planonts. After a 10-15 minute period of active swimming (in 50 ml. of raw, aerated sea water, at 25° C.) the spores settle on an ovum (Fig. ISa) without rounding up. Within three min- utes after attaching to the egg membrane, the spore protoplasm penetrates the membrane (Fig. ISb), enlarges rapidly into a foot-like hyphal rudiment (Fig. 18c), and grows along the embryo. Within 25 minutes after inoculation, infection has been established and the young hypha is developed (Fig. 6). Whether hyphae actually penetrate the embryo is not known ; the dense, opaque embryonic host cells prevent direct observation, and a suitable technique for fixing and sectioning in- fected eggs has not been developed. The vegetative hyphae of Lagenidiuin chthamalophilum are very characteristic. Early in the incubation period, the hyphae become highly vacuolate, and generally maintain a reticulate vacuolation throughout development. Hyphae are charac- teristically "foamy" in appearance, suggestive of those of Monoblepharis. Emer- gent hyphae, similarly, are usually extremely vacuolate (Figs. 4, 7), but occasionally have very diffuse, strand-like cytoplasm (Fig. 8). In either case, the many minute refractive oil bodies in the cytoplasm give it a readily discernible golden-yellow cast. Emergent hyphae (other than the sporangial discharge tubes and vesicles) are not often observed in the lamellae immediately after dissection from the animal. Such hyphae form in abundance, and sporangial discharge occurs frequently, how- ever, when infected lamellae are placed in sterile sea water and incubated for 12-18 hours at room temperature. Hyphae emerging from an ovum penetrate the mem- brane either with or without constriction (Figs. 2, 10). The extramatrical hyphae are generally stout and somewhat contorted, freely branched, and of a diameter coincident with that of the intramatrical hyphae. Occasionally, the emergent hyphae are very slender and coiled (Fig. 5), as in Pythium thalassium (Atkins, 1955). Neither the intra- nor extramatrical hyphae are septate except where reproductive cells are delimited. Sporangia are formed by segmentation of the intramatrical hyphae almost ex- clusively. During formation of the delimiting septa, that portion of the hypha destined to become a sporangium accumulates protoplasm, and often has a few large vacuoles (Fig. 12). These vacuoles disappear prior to movement of the protoplasm into the apical vesicle. A stout discharge or exit tube (Fig. 11) de- velops from the cylindrical sporangium, penetrates the egg membrane (without constriction) and elongates. The apex of the exit tube enlarges to form, at first, a subglobose or slightly irregular swelling (Figs. 12, 20) containing very diffuse, vacuolated cytoplasm (Fig. 12). The bulbous discharge tube apex subsequently becomes perfectly spherical ; it is completely formed before the sporangial content flows into the tube. The vesicle wall is cellulose as is the basal sporangium and vegetative hypha wall. FIGURES 10-27. Lagenidium chthamalophilum. 10, infection in a pre-emergence embryo with eye spot; 11-17, stages in formation of vesicle and spores, and spore discharge (see text) ; 18, germination and penetration of spore protoplast ; 19, planonts ; 20, sporangial discharge tube and immature vesicle; 21, immature oogonium ; 22-27, mature oogonia ; Fig. 19, scale a, others, scale b. 210 T. W. JOHNSON, JR. Sporogenesis begins with movement of the sporangial protoplasm through the discharge tube and into the vesicle. Occasionally, the protoplasmic stream sepa- rates partially, leaving one or more fusiform masses connected to the main comple- ment by a slender strand (Fig. 13). The sporangial content and the cytoplasm of the tube aggregate into a slightly irregular mass centrally located in the vesicle (Fig. 14). Spores are cleaved out in the protoplasmic mass, appearing first as polygonal units, then as definite spherical or reniform cells (Figs. 15, 16). In no instances observed did the spores fill the vesicle. Spore discharge is initiated with a slow "shimmering" motion of the vesicular spore mass. The movement then becomes undulating and increases in rapidity until the spores are moving rapidly over and around one another in the center of the vesicle. For one or two minutes the spores are moving extremely rapidly. If such spores are killed with osmic acid fumes and stained with acid fuchsin or gen- tian violet, short, stubby flagella are visible on the peripheral spores. The vesicle deliquesces (Fig. 16) within 30 seconds, leaving the rapidly moving spores hanging together momentarily. One or two peripheral spores dart away, and subsequently, within a few seconds the spore mass breaks up to liberate rapidly but evenly swim- ming spores. The entire process, from migration of the undifferentiated proto- plasm to spore discharge, is completed within 20 minutes, in raw, aerated sea water at 25-27° C. Sexual reproductive cells are rarely produced in vivo. Short lateral branches with enlarged apices mature into oogonia containing a single oospore (Fig. 27), but oogonia may also develop as intercalary (Figs. 22, 26) or terminal (Figs. 23. 25) hyphal segments. Intercalary or terminal oogonia often contain two oospores. Whether antheridia are produced by L. chthamalophilum is not known; hypogynous antheridial cells, certainly, are not in evidence. In a few instances, short hyphae were observed near oogonia and in contact with them (Figs. 21, 27), but no antheridial cells were evident. These hyphal branches may be nonfunctional an- theridial branches ; if so, they are of monoclinous and androgynous origin (Johnson, 1955, pp. 14, 15). Extramatrical hyphae do not produce sex cells. Attempts to culture Lagenidium chthamalophilum were successful. Spores ger- minated well on aged sea water agar, and on sea w7ater agar fortified with 0.1% glucose and 0.05% yeast extract. Subsequent growth was very sparse, though extensive, and neither sexual nor asexual cells developed in culture. Very slender, sparingly branched, contorted, vacuolate hyphae are produced on the agar media. The parasite can, in my opinion, be assigned equally well to Pythium (Mid- dleton, 1943) or Lagenidium (see Sparrow, 1943) as these genera are presently imclerstood and circumscribed. In both genera, planont maturation occurs, gen- erally, in an evanescent vesicle produced at the apex of a sporangium or sporangial discharge tube. In neither genus, however, is the vesicle pre-formed. This fact alone, were it to be considered significant at the generic level, would exclude the barnacle parasite from both genera. On the other hand, the evidence is stronger in favor of assigning the fungus to Lagenidium than to Pythium. The lagenidia- ceous features of the parasite are : the "foamy," granular cytoplasmic content of the stout, branched hyphae ; sporangial delimitation by hyphal segmentation, and oospore formation by contraction of hyphal segment content. The nonseptate na- ture of the hyphae, of course, suggests Pythium rather than Lagenidium, although members of the latter genus having pythiaceous mycelium are known (Sparrow, FUNGUS PARASITE OF BARNACLE OVA 211 1943). The fungus in barnacle ova, while suggestive vegetatively of the Aphrag- mium type of Pythium, has a simplified sexual apparatus, that is, no well defined antheridium, and no periplasm in the oogonium. In the final analysis, assignment of the fungus to Lagenidium turns, I believe, on simplicity of the sexual structures. Two marine species of Lagenidium are known. Lagenidium sp. (Johnson, 1957) produces sporangia formed by hyphal segmentation just as does L. chthama- lophilum, but the hyphae of the former are not stout and vacuolate, and the vesicle is not pre-formed. These two features also separate L. chthamalophilum from L. callinectes (Couch, 1942), parasitic in ova of Callinectes sapidus. Furthermore, L. callinectes has a persistent vesicle, the barnacle parasite does not. Lagenidium chthamalophilum differs in several significant respects from all other known species in the genus. The irregular, contorted, stout hyphae (with lateral lobulations) of L. entophytum (Pringsheim) Zopf suggests L. chthamalophihnn, but other features separate the two immediately. Lagenidium closterii deWildeman produces an ex- tramatrical sporangial discharge tube, as in L. clitliamalophilum ; the hyphae of the former are more delicate and the discharge tube is bulbous at the base. The pythiaceous hyphae of L. marchalianum deWildeman are very slender and markedly constricted; these differ significantly from the stout, vacuolate hyphae of L. chthamalophilum. The only other myceloid member of Lagenidium suggestive of the barnacle parasite is L. gigantcum (Couch, 1935). Couch's species, however, has segmented mycelium, and lacks a pre-formed vesicle. Vegetatively, Lagenidiinn chthamalophilum resembles Plectospira dubia (Atkins, 1954b), particularly in the stout, irregularly branched and swollen hyphae. In other characteristics, notably those of sporogenesis and discharge, these two fungi are obviously dissimilar. Pythium thalassium, parasitic in Pinnotlicrcs pisum ova (Atkins, 1955), produces very stout hyphae resembling those of L. clitharnalo- philum, but the Pythium has two major characteristics distinguishing it from the barnacle parasite : the sporangia of P. thalassium are filamentous and proliferate internally, and the hyphae are not highly vacuolate. The geographical distribution of Lagenidium chthamalophilum is not known, inasmuch as only host barnacles in the immediate vicinity of the Duke Marine Lab- oratory have been examined. Forty-four collections (totalling 1284 individuals) of Chthamalns jragilis dcnticulata were made within a five-mile radius of the Labo- ratory, including two series of collections on the outer banks of the Inlet region. The number of egg-bearing parent barnacles, and the number of infected lamellae in any one collection varied considerably. A sample series of ten collections, show- ing infection incidence, is given in Table I (each animal with paired lamellae). Occasionally, barnacles were collected in which only one egg mass was found. Twenty-one per cent of such masses were infected. It should be noted that some infected lamellae may have been overlooked, especially if they had been inoculated shortly before the animals were collected. For example, in eight dissections out of twenty-nine, visually uninfected ova masses showed infection after three days in storage in sterile, filtered sea water. Thirty-four per cent of all examined C. jragilis dcnticulata lamellae (1016 individual cases) were infected. Percentages of infection are based on hosts collected from pilings and mooring stakes. The same species of barnacle occurring on Spartina alterniflora showed some infection, but only infrequently. Only 3 of 86 barnacles (with one or two lamellae) from Spartina were invaded by the Lagenidiinn. 212 T. W. JOHNSON, JR. A replicated experiment was performed to determine whether Lagenidium chthanialophilum is actively parasitic in ova of Chthamalns, or is an invader of moribund eggs. Egg masses were dissected from the barnacles with sterilized needles, examined immediately with a dissecting microscope, and separated into two lots, infected and uninfected. As each lamella was removed, it was placed in a drop of sterile sea water to eliminate inadvertent inoculation in handling infected and uninfected eggs. The obviously parasitized egg masses were easily detected with the dissecting microscope. Infected lamellae were placed in small Petri plates, covered with sterile sea water, and incubated overnight. This short period of in- cubation induced sporulation. The uninfected lamellae were kept in pairs, as they were dissected from the parent animal, and incubated for 12-24 hours in drops of sterile sea water on slides in damp chambers. The masses were then examined; if no infection was visible, one lamella of the pair was placed in the dish with the sporulating fungus, the other lamella (from the same animal) in a separate Petri plate of sterile sea water, and utilized as the control. No specific stages of embryo TABLE I Incidence and percentage infection of Chthamalns fragi] is denticulata lamellae by Lagenidinm chthanialophilnni No. animals in sample No. animals with ova No. of lamellae No. infected lamellae Percentage infection 41 14 28 8 28.5 34 31 62 42 67.7 15 15 30 28 93.3 13 12 24 16 66.6 43 37 74 54 72.9 22 3 6 6 100.0 11 6 12 2 16.6 41 40 80 62 77.5 65 49 98 84 85.6 23 8 16 2 12.5 development were selected for the tests. The plates were incubated at room tem- perature, and examined at 1, 3, and 5 days. Some plates containing a parasitized and nonparasitized egg mass were discarded when the control lamella ( one of the uninfected pair) showed signs of the fungus. No infection was visible in the "uninfected" egg cases by the end of 24 hours. At 72 hours, however, all naturally uninfected lamellae had been invaded by Lage- nidium chthaiiialopJiiliun. Two controls showed infection, and three lamellae in the dishes with the fungus had matured into nauplii ; these plates were discarded. In sea water, in the laboratory, visible infection develops between 24 and 72 hours ; inoculation, presumably, may occur during the first 24-hour period. Under nat- ural conditions, inoculation must occur (since the "inoculum" is a motile spore) during the short periods (twice in approximately 24 hours) that the opercula of the barnacle are open while the animal is submerged at high tides. This period of time varies roughly — during the neap tide periods, at least — between 45 and 90 minutes, diurnally, for those individuals highest on the substratum. It is true that an exposed, closed animal retains sufficient water within the mantle to enable the FUNGUS PARASITE OF BARNACLE OVA 213 planonts of the fungus to swim about and presumably cause infection. This seems a less likely time for infection to occur, however, since tests show that heavily fouled, oxygen-depleted sea water has a retarding effect on spore discharge and movement. The close natural association of Chthamalus fragilis denticulata and Balanus amphitrite prompted a replicated series of artificial inoculations using infected lamellae from C. fragilis denticulata and uninfected masses from B. amphitrite. Lamellae were dissected and incubated in the manner described previously. Forty- three attempts at inducing infection in B. amphitrite ova were made. No infection in B. amphitrite eggs was evident at the end of 21 days, although the infected ova of C. fragilis denticulata were completely destroyed at the end of the three-week incubation period. While some lamellae of B. amphitrite were actually inoculated with planonts of the Lagenidium (visual inspection of individual ova), these spores did not germinate, or, if they germinated, did not penetrate the egg membrane. Many B. amphitrite egg masses were examined from animals collected in the same localities as C. fragilis denticulata, but none was infected. Dr. J. D. Costlow has never observed infection in the lamellae of B. amphitrite, although he has used ova from this species in extensive studies on larval development. These observations, in view of the proximity of B. amphitrite and C. fragilis denticulata in natural habitats, suggest the hypothesis that the ova of the former are, if not immune, certainly highly resistant to L. clithanialophilum. The importance of Lagenidium clithamalophilum in reducing Chthamalus fragilis denticulata populations cannot be judged from this preliminary investigation. Cer- tain further studies on the fungus and its host, however, may be of significance in elucidating the effect of the fungus. Significant among these studies are : distri- bution and severity of infection ; conditions favorable to establishment and spread of infection ; the period, in the reproductive cycle of the host, at which the animal is most susceptible; any fluctuations (and causes thereof) in percentage of infec- tion, and a search for other suscepts. The support of the National Science Foundation, through Grant G-2324, is gratefully acknowledged. I am indebted to Dr. J. D. Costlow, Duke University Marine Laboratory, for technical guidance in the zoological aspects, and to several of my colleagues for opinions and criticisms of the mycological portions of the investigation. Mr. Thomas M. Simkins, Jr., Duke University Library, very kindly prepared the Latin diagnosis. SUMMARY 1. Lagenidium cJitJiamalopJiiliim is described as a parasite of the ova of Chthama- lus fragilis denticulata. The pathogen is characterized by the formation of a vesicle before sporangial protoplasm migration, and by highly vacuolate, stout vegetative hyphae. In these features, L. chthamalophilum differs from the usual interpretation of members of the genus. The fungus is compared with other Phyco- mycetes known to parasitize crustacean ova. 2. Artificial inoculation experiments show L. clithamalopliilum to be specific for C. fragilis denticulata. The associated barnacle, Balanus amphitrite, is resistant to the fungus. 214 T. W. JOHNSON, JR. LITERATURE CITED APSTEIN, C., 1910. Cyclopterus lumpus, der Seehase. Seine Fischerei und sein Wageninhalt. Mitteil. Deutsch. Seefischerei-Vereins, 26: 450-465. ATKINS, D., 1929. On a fungus allied to the Saprolegniaceae found in the pea-crab Pinnotheres. J. Mar. Biol Assoc., 16: 203-219. ATKINS, D., 1954a. Further notes on a marine member of the Saprolegniaceae, Leptolegnia marina n. sp., infecting certain invertebrates. /. Mar. Biol. Assoc., 33 : 613-625. ATKINS, D., 1954b. A marine fungus Plectospira dubia n. sp. (Saprolegniaceae), infecting crustacean eggs and small Crustacea. /. Mar. Biol. Assoc., 33 : 721-732. ATKINS, D., 1955. Pythium thalassium sp. nov. infecting the egg-mass of the pea-crab, Pinno- theres pisum. Trans. Brit. Myc. Soc., 38 : 31-46. BOMMER, C., 1891. Un champignon pyrenomycete se developpant sur le test des Balanes. Bull, dcs Seances Soc. Belg. Micr., 17: 151-154. BONAR, L., 1936. An unusual Ascomycete in the shells of marine animals. Univ. California Publ. Bot., 19 : 187-194. BORNET, E., AND C. FLAHAULT, 1889. Sur quelques plantes vivant dans le test calcaire des Mollusques. Bull Soc. Bot. France, 36: CXLVII-CLXXVII. COUCH, J. N., 1935. A new saprophytic species of Lagenidium, with notes on other forms. Mycologia, 27: 376-387. COUCH, J. N., 1942. A new fungus on crab eggs. /. Elisha Mitchell Sci. Soc., 58: 158-162. GALTSOFF, P. S., 1940. Wasting disease causing mortality of sponges in the West Indies and Gulf of Mexico. Proc. 8th Amer. Sci. Cong., 3: 411-421. GANAROS, A. E., 1957. Marine fungus infecting eggs and embryos of Urosalpinx cinerea. Science, 125: 1194. GIARD, A., 1888. Sur les Nephromyces, genre nouveau de champignons parasites du rein des Molgulidees. C. R. Acad. ~Sci., Paris, 106: 1180-1182. HARIOT, P., 1887. Note sur le genre Mastodia. J. dc Bot., 1 : 231-234. HUNTER, A. C., 1920. A pink yeast causing spoilage in oysters. U. S. D. A. Bull. No. 819, pp. 1-24. JOHNSON, T. W., JR., 1955. The Genus Achlya: Morphology and Taxonomy. 180 pp. Univ. Mich Press : Ann Arbor. JOHNSON, T. W., JR., 1957. Marine fungi. III. Phycomycetes. Mycologia, 49: 392-400. KOLLIKER, A., 1860. On the frequent occurrence of vegetable parasites in the hard tissues of the lower animals. Quart. J. Micr. Sci., 8: 171-187. MACKIN, J. G., H. M. OWEN AND A. COLLIER, 1950. Preliminary note on the occurrence of a new protistan parasite, Dermocystidium marinum n. sp., in Crassostrea virginica (Gmelin). Science, 111: 328-329. MIDDLETON, J. T., 1943. The taxonomy, host range and geographic distribution of the genus Pythium. Mem. Torrey Bot. Club, 20: 1-171. NIEZABITOWSKI, E. L., 1913. Pasorzyty roslinne morskich rakow glfbinowych z rodzaju Pasiphaca. (Die pflanzlichen Parasiten der Tiefsee-Decapoden-Gattung Pasiphaea). Kosmos, 38: 1563-1572. PICARD, F., 1908. Sur une Laboulbeniacee marine (Laboulbenia marina n. sp.) parasite d'Aepus Robini Laboulbene. C. R. Seances et Mem. Soc. Biol., Paris, 65 : 484-486. PLEHN, M., AND M. MULSOW, 1911. Der Erreger der "Taumelkrankheit" der Salmoniden. Centrabl. Bakt. Parasitcnk., 59: 63-68. RAY, S. M., AND A. C. CHANDLER, 1955. Dermocystidium marinum, a parasite of oysters. Exp. Parasitol, 4: 172-200. SANTESSON, R., 1939. Amphibious Pyrenolichens I. Arkiv for Bot., 29A : 1-67. SPARROW, F. K., JR., 1943. Aquatic Phycomycetes exclusive of the Saprolegniaceae and Pythium. 785 pp. Univ. Michigan Press: Ann Arbor. SPROSTON, NORA G., 1944. Ichthyosporidhtm hoferi (Plehn & Mulsow, 1911), an internal fungoid parasite of the mackerel. /. Mar. Biol. Assoc., 26 : 72-98. VISHNIAC, HELEN S., 1955. The morphology and nutrition of a new species of Sirolpidium. Mycologia, 47 : 633-645. WOLF, F. A., AND F. T. WOLF, 1947. The Fungi. Vol. II, 538 pp. Wiley : New York. THE EFFECT OF X-RAYS, IRRADIATED SEA WATER, AND OXIDIZING AGENTS ON THE RATE OF ATTACHMENT OF BUGULA LARVAE * WILLIAM F. LYNCH St. Ambrose College, Davenport, loiva, and the Marine Biological Laboratory, Woods Hole, Mass. Few observations concerning the effects of ionizing radiations have been made on bryozoan tissues. Oka (1954) x-rayed various regions of the fresh-water bryozoan, Lophopodella carteri, and noted that the more active embryonic tissues had greater radiosensitivity than other parts. But no observations known to the writer have been reported concerning the effects of x-radiation on the attachment and metamorphosis of bryozoan larvae. It is well known that some of the biological effects of ionizing radiations have been attributed to the production of H2O2 or organic peroxides when aqueous solu- tions are x-rayed (Evans, 1947; Barren et al., 1949; Kimball and Gaither, 1952). Barren and his co-workers found that both irradiated sea water and hydrogen peroxide inhibited oxygen uptake in sea urchin sperm ; but the latter had two op- posite effects, increasing respiration at great dilution and inhibiting oxygen uptake in higher concentration. Furthermore, Blum (1941, p. 96) has emphasized the probability that the effects of certain photodynamic dyes may be mediated through the production of H,O2. Since some of the basic dyes had been found to be potent inductors of attachment and metamorphosis of Bugnla larvae when sea water solu- tions of these dyes were exposed to light (Lynch, 1955a), it was of interest to de- termine whether x-rays and hydrogen peroxide would have any effect on setting. If ionizing radiations were found to affect the rate of attachment of the larvae, the problem of determining whether the action of these rays was a direct or an indirect one would naturally arise. After x-raying the larvae proved to have a positive effect on the rate of setting, it was decided to employ sea water seeded immediately after being x-rayed with non-irradiated larvae. The results of these experiments led to an investigation of the possible effects of adding H2O2 to sea water and finally to observations on the action of two other oxidizing agents, sodium 2,6-dichloro- benzenoneindophenol and 2,3,5-triphenyltetrazolium chloride, on the attachment and metamorphosis of Bugula larvae. MATERIALS AND METHODS Each experiment on the effects of radiation involved three dishes : the first con- tained larvae x-rayed in filtered sea water; the second contained sea water that was seeded immediately after being x-rayed with non-irradiated larvae, and the 1 This investigation was aided by a grant from the National Science Foundation, NSF 1728, and by a scholarship given by the Marine Biological Laboratory, Woods Hole, Massa- chusetts. The writer is grateful to Dr. Albert Kind for supplying some of the oxidizing agents used for these observations. 215 216 WILLIAM F. LYNCH third had control larvae in their natural medium. For these experiments plastic containers 7 cm. in diameter and 1.6 cm. high were employed. Each dish con- tained 30 ml. of sea water and was covered with a plastic top ; all three containers were kept close together on a table and were wrapped with paper towels until the time of counting. Light was excluded to delay the decomposition of any photo- labile by-products, especially peroxides, that might be formed by irradiation. The x-ray data are as follows: the machine operates on 182 kv. pk. and 25 ma. with an equivalent filtration of 0.2 mm. of copper. During the summer of 1956, when larvae of B. turrita were employed, the output of the tubes (position A) was 4724 r per minute and the organisms were irradiated for three minutes and twenty seconds to give a total of 15,733 r. During the previous summer the x-ray dosage for larvae of B. flabellata was 18,333 r and the organisms were also irradiated for three minutes and twenty seconds at 5500 r per minute. The tubes were water- cooled and an electric fan was directed upon them. Since the temperature was found to rise only a fraction of a degree, the irradiated material was not cooled by an ice chamber. Sea water solutions of the three oxidizing agents were made in the following concentrations found to be most effective : 1 : 14,000 parts by volume of 30% H,O2 (7 X 10-4 M), 1 X 10-5 M 2,3,5-triphenyltetrazolium chloride (TTC) and 0.01 mg~ of sodium 2,6-dichlorobenzenoneindophenol (SDBI) per liter (3.4 X 10~8 M). The pH was that of natural sea water. Stender dishes 6 cm. in diameter contain- ing 30 ml. of solution were seeded with larvae and covered with glass lids. The controls were placed in the same amount of sea water in similar containers and kept as near as possible to the experimental dishes. In the experiments with H2O2 both experimental and control dishes were shielded from light by wrapping them in paper towels. The others were exposed to diffuse daylight coming from a win- dow about three feet from the region where the Stender dishes were placed. RESULTS I. X-rays and irradiated sea water Table I shows that x-raying larvae of B. flabellata induced more rapid setting than that which occurred in the controls, the t ratio for the difference of the two groups being 5.12 (P=.005). For these experiments the number of attached organisms was counted thirty minutes after irradiation with 18,333 r. The three experiments in which larvae were placed in sea water immediately after it had been irradiated suggest that the accelerated rate of attachment is an indirect effect presumably caused by the action of ionizing radiations on sea water. Table II gives more convincing evidence of an indirect effect of ionizing radia- tions. For these experiments larvae of B. turrita were irradiated with 15,733 r and the number of attached organisms was counted at the end of eight hours. The time of irradiation was the same for both groups of larvae, but it was found that the output of the x-ray tubes had dropped during the course of a year when the machine was calibrated towards the end of the period of experimentation. Al- though the rate of attachment of the larvae of both B. flabellata and B. turrita was accelerated either by x-raying the larvae or by seeding them in irradiated sea water, the time at which the effects could be detected differed considerably. Larvae of INDUCED SETTING OF BUGULA LARVAE 217 B. turrita showed no notable acceleration of the rate of attachment by thirty min- utes after either being x-rayed or being placed in irradiated sea water, but by eight hours there were always more attached organisms in the experimental dishes than in the controls. The t ratio for counts made at this time indicates a significant difference in the mean number of attached organisms in the experimental and con- trol dishes, being 6.64 (P = .001) for irradiated larvae and 4.09 (P = .005) for organisms seeded in irradiated sea water. It does not appear that the difference in the rate of setting of B. flabellata and B. turrita can be ascribed to the lower x-ray dosage of the latter, for 20,000 r did not produce effects appreciably different from those which followed irradiation with 15,733 r. After studying the effects of vari- ous agents on both types of larvae, one gains the general impression that it is both TABLE I The effects of x-rays (18,333 r) and of irradiated sea water on the rate of attachment of larvae of B. flabellata. The larvae were irradiated thirty minutes after the adult colonies had been exposed to light and the irradiated sea water was seeded with larvae at the same time. The number of attached organisms in the three groups (x-rayed larvae, larvae in irradiated sea water and those in natural sea water} was counted thirty minutes later X-rayed larvae Larvae in irradiated sea water Control larvae No. of exp. No. of larvae No. attached Per cent attached No. of larvae No. attached Per cent attached No. of larvae No. attached Per cent attached 1 104 87 84 87 85 98 100 50 50 2 214 207 97 66 63 95 63 62 98 3 46 44 96 191 190 99 145 48 33 4 150 143 95 — — — 349 183 52 5 248 242 98 . — • — — 247 158 64 6 340 330 97 — — — 257 130 50 7 170 158 93 — — - — . 167 55 33 8 151 141 93 — — — - 94 20 21 Average per cent 94 ± 4 50 ±24 The t ratio for the significance of the difference of the means of the x-rayed and control larvae = 5.12; P = <.005. The / ratio was computed from percentages carried out to one decimal point (not the rounded percentages shown in columns 4 and 10). more difficult to induce metamorphosis and easier to inhibit fixation in larvae of B. turrita than in those of B. flabellata. These differences may be correlated with the longer natatory period of B. turrita in natural sea water. It is difficult to determine, however, whether these differences are actually specific or whether they can be attributed to altered environmental conditions that prevailed during the two summers when each type was used almost exclusively. Although either x-raying larvae of B. turrita or seeding them in irradiated sea water induced more rapid attachment of the organisms, subsequent development was seriously impeded. Frequently larvae that had been x-rayed failed to develop after attachment. In other cases undifferentiated growth occurred at a much retarded rate. Instead of forming normal zoids, the larvae merely developed elongated masses of clear, gelatinous, stolon-like material without any internal organization. 218 WILLIAM F. LYNCH Usually these growths formed at opposite sides of the body of the larva. One growth evidently corresponded to the stolon for attachment of the organism and the other developed in the region where the body of a normal zoid usually forms. Both growths were abnormally long and slender. The material corresponding to the stolon of a normal zoid rarely differentiated into the four knob-like projections, symmetrically placed and each branching dichotomously, which are characteristic of stolons of B. turrita. (Stolons of B. flabellata have three rather than four parts.) The material which grew out from the region where the zooecium normally forms did not differentiate into a gut and tentacles. TABLE II The effect of x-rays (15,733 r) and of irradiated sea water on the rate of attachment of larvae of B. turrita. The larvae were irradiated thirty minutes after the adult colonies had been exposed to light and the irradiated sea water was seeded with larvae at the same time. The number of attached organisms in the three groups (x-rayed larvae, larvae in irradiated sea water and those in natural sea water) was counted eight hours later Temp. = 24-26° C. X-rayed larvae Larvae in irradiated sea water Control larvae No. of exp. No. of larvae No. attached Per cent attached No. of larvae No. attached Per cent attached No. of larvae No. attached Per cent attached 1 102 100 99 75 65 87 36 12 33 2 60 56 93 194 192 99 48 38 79 3 26 23 88 140 130 93 31 17 55 4 41 35 85 102 101 99 46 28 61 5 15 14 93 56 48 86 26 16 62 6 45 36 80 131 76 58 43 15 35 7 32 28 88 180 154 85 28 20 71 8 57 43 75 96 65 88 47 19 40 9 49 43 88 78 69 88 50 20 40 10 24 21 88 20 10 50 22 15 68 11 45 40 89 23 14 61 55 22 40 12 50 45 90 24 19 79 41 29 71 Average per cent 88 ± 6 81 ± 16 54 ± 16 The / ratio for the significance of the difference of the means of the x-rayed and control larvae = 6.64; P = <.001. The t ratio for larvae in irradiated sea water vs. the controls = 4.09; P = .005. The t ratios were computed by using the rounded percentages in columns 4, 7, and 10. Larvae that attached in irradiated sea water developed similar undifferentiated growths. These zoids, however, elongated more than those formed from larvae that had been x-rayed. In fact, growth sometimes exceeded that of the controls, but the transparent gelatinous material, often peculiarly twisted, lacked internal organization. Larvae that attached to the surface film sometimes developed normal stolons. Those that attached to the bottom frequently formed long slender stolons, about three times normal length, with a spherical mass in the region where they were attached to the substrate ; and some were attached by two stolons. A few developed a bud in the zooecial wall for a second zoid. But even when the original irradiated sea water had been replaced several times by fresh sea water, the growth INDUCED SETTING OF BUGULA LARVAE 219 corresponding to the zooecium failed to differentiate a gut and tentacles. Only a single larva formed a well-developed zoid with everted tentacles ; and this differen- tiation occurred only after six days, whereas internal organization can readily be detected in a normal zoid by the end of forty-eight hours. Thus, a notable feature of larvae that were either x-rayed or placed in irradiated sea water was growth without differentiation ; and the development of x-rayed larvae was more drastically impeded than that of organisms seeded in irradiated sea water. These observations seem to be reasonably consistent. Nevertheless, it would be premature to ascribe the failure of the zoids to differentiate in a normal manner solely to ionizing radiations. X-raying the larvae not only markedly reduces their motility but also causes them to settle on the bottom of the container, and larvae which attach geopositively usually do not develop as well as those which affix themselves to the surface film. Factors affecting larval differentiation are at present largely unknown. And judgments concerning degrees of growth are more subjective than those based on numerical data. In an almost unexplored field of larval differentiation, experimental designs that appear to have only one variable may be deceptive unless similar replications can be obtained during two different summers and with more than one species. Since the chief purpose of the experi- ments was to determine the effects of x-rays and irradiated sea water on the attachment of the larvae, it would be inadvisable to draw definitive conclusions concerning the influence of these agents on development until further observations have been made. A few experiments were performed to determine what role the surface of the plastic containers might play in attachment of the larvae. Both x-rayed larvae and organisms irradiated in sea water were emptied from the plastic containers into Stender dishes and the latter were also used for the controls. Although the larvae attached somewhat less readily in the Stender dishes, there was more rapid fixation of both x-rayed larvae and those in irradiated sea water than in the con- trols. It is not unlikely that some of the great variability in the time of attach- ment of the controls, always a puzzling situation that occurs yearly in almost every experiment, can be attributed to differences in roughness of the various Stender dishes used. The temperature of the water in which the adult colonies are kept also seems to cause variability in the time of setting (cf. Lynch, 1955b). II. Oxidizing agents One of the problems encountered in determining the possible effects of oxidiz- ing agents on the rate of fixation of the larvae was that of getting solutions dilute enough to prevent cytolysis. Both strong and weak solutions greatly reduced the motility of the larvae. But in solutions that were too strong the larvae merely became immobilized on the bottoms of the containers without attaching themselves ; and these organisms eventually cytolyzed. With solutions that were weaker the time of counting was an important factor. If counts were made too soon, no ob- servable differences in control and experimental larvae could be detected. If counts were made too late, when the controls had metamorphosed in large num- bers, again no differences could be observed. After a wide variety of concentra- tions had been tested, the right dilution for inducing attachment was eventually found. 220 WILLIAM F. LYNCH Hydrogen peroxide. Table III shows that by twenty-one hours the number of attached larvae (Bugula turrita) in sea water containing 1 : 14,000 parts of 30% H2O2 was significantly greater than that of the controls (P = .001). Since there were no notable differences in the number of attached organisms in the control and experimental dishes by twelve hours, H2O2 had a delayed action in inducing fixa- tion, somewhat similar to that observed after one-minute exposures of bryozoan larvae to urea (Lynch, 1957). The cause of this delayed action is unknown. Zoids formed from larvae exposed to sea water containing H2O2 resembled those in irradiated sea water. Elongation sometimes exceeded that of the controls, but the zoids were usually misshapen. The larvae generally attached in greater numbers to the surface film than to the bottoms of the containers and the majority TABLE III The effects of 30% H20>2 (1:14,000 parts by volume = 0.0072 volume per cent) in sea water on the rate of attachment of larvae of B. turrita. The experimental and control solutions were seeded with larvae thirty minutes after the adult colonies had been exposed to light (except no. 1, which was seeded at fifty minutes). The number of attached organisms was counted twenty-one hours later. The pH was that of sea water Experimental larvae Control larvae No. of exp. No. of larvae No. attached Per cent attached No. of larvae No. attached Per cent attached 1 28 28 100 23 12 52 2 62 54 87 32 15 47 3 35 34 97 72 38 53 4 16 16 100 13 6 46 5 31 31 100 17 11 65 6 20 20 100 20 18 90 7 37 36 97 43 20 46 8 35 35 100 44 22 50 Average per cent 98 ± 2 56 ± 15 The t ratio for the significance of the difference of the means of experimental and control larvae (computed by using fractional percentages) = 7.07; P = <.001 of these formed normal tetrapod stolons with dichotomous branches at their ends, but the zooecial region failed to differentiate internal organs. Geopositive larvae produced much gelatinous, stolon-like material without differentiation. None of the organisms observed during a period of a week developed tentacles, although these normally form by about forty-eight hours. Larvae of B. flab el la ta, observed during the previous summer, were less adversely affected than those of B. turrita. Although H2O, considerably reduced the motility of the larvae, it apparently was not excessively injurious to the cilia ; otherwise the organisms would have dropped to the bottoms of the containers and remained there. The effects of TTC. Table IV shows that sea water solutions of TTC in con- centrations of 1 X 10"5 M (pH = 7.9-8.0) also induced more rapid fixation of the experimental larvae (B. turrita) than that which occurred in the controls. In solu- tions of 5 X 10~5 M the larvae attached more readily than in the weaker medium, INDUCED SETTING OF BUGULA LARVAE 221 TABLE IV The effect of 0.00001 M 2,3,5 -triphenyltetrazolium chloride in sea water (pH = 7.9-8.0) on the rate of attachment of larvae of B. turrita. Control and experimental dishes were seeded with larvae thirty minutes after the adult colonies had been exposed to light and the number of attached organisms in each group was counted at the end of eight hours Experimental larvae Control larvae No. of exp. No. of larvae No. attached Per cent attached No. of larvae No. attached Per cent attached 1 61 49 80 33 17 52 2 36 24 67 35 11 31 3 41 19 46 27 8 30 4 55 35 64 23 8 35 5 50 42 84 37 17 46 6 30 26 87 56 38 68 7 44 41 93 58 34 59 Average per cent 74 ± 15 46 ± 16 The t ratio for the significance of the difference of the means of the control and experimental larvae (computed by using fractional percentages) = 3.47; P = .015 but subsequent development was considerably retarded. If the Stender dishes were flooded with fresh sea water after the larvae had attached, growth took place at a markedly reduced rate after exposure to either concentration of TTC. Some of these organisms formed tentacles. Larvae left in the weaker of the two TTC TABLE V The effect of sodium 2, 6-dichlorobenzenoneindophenol in concentrations of 0.01 mg. /liter of sea water (3.4 X 10~& M) on the rate of attachment of larvae of B. turrita. The experimental and control Stender dishes were seeded with larvae thirty minutes after the adult colonies had been exposed to light and the number of attached organisms was counted seven hours later. Temp. = 24-26° C. The pH of the experimental solution was that of sea water Experimental larvae Control larvae No. of exp. No. of larvae No. attached Per cent attached No. of larvae No. attached Per cent attached 1 56 50 89 42 24 57 2 81 59 73 53 16 30 3 64 31 48 35 11 31 4 49 42 86 115 28 24 5 82 46 56 97 55 57 6 128 92 72 96 47 49 7 82 53 65 197 132 67 8 205 127 61 61 36 59 9 58 44 76 64 34 53 10 18 17 94 44 27 61 Average per cent 72 ±15 49 ± 17 The / ratio for the significance of the difference of the means of the experimental and control larvae (obtained by using rounded percentages in columns 4 and 7) = 3.08; P = .015. WILLIAM F. LYNCH solutions elongated slightly by twenty-four hours and these zoids resembled those in sea water containing H2O2 insofar as there was an abnormal amount of gelatinous material without differentiation; organisms left in the stronger solution did not develop. Larvae that attached in solutions of 5 X 10~5 M TTC became slightly pink, indicating a reduction of the solution ; those in the weaker solution became faintly pink. Although TTC solutions exposed to air turned somewhat pink by eight hours, either with or without organisms in them, the larvae were always more deeply colored than the solutions. The effects of SDIB. This oxidizing agent, while inducing fixation of the larvae at a rate significantly higher than that of the controls (P = .015), as shown in Table V, was less injurious to the larvae than any of the other agents discussed in this paper. Larvae that were left in solutions of 0.01 mg./liter of sea water (pH — 7.9) generally developed normally but at a rate somewhat slower than the controls. A preponderance of settings occurred at the surface film, and these developed better than those on the bottom. Larvae that attached to the bottoms of the Stender dishes formed zoids without any differentiation by forty-eight hours. But development was variable in these solutions, sometimes equalling that of the controls and sometimes being inferior. ///. Reducing agents A very limited number of preliminary experiments was carried out with two reducing agents, sodium bisulfite and sodium thiosulfate, to determine whether their action on attachment would be opposite to that of oxidants. But neither reducing agent prevented attachment. Sea water solutions of sodium thiosulfate as strong as 10 mg./liter and concentrations of sodium bisulfite of 0.0001 M, 0.001 M and 0.005 M did not prevent attachment and metamorphosis. Nor did these solutions appear to have any inhibitory effects on the larvae. Although it seems unlikely that further experimentation with these solutions will show that they inhibit at- tachment, a greater variety of concentrations should be tested before a definitive conclusion can be reached concerning their action. DISCUSSION Since the action of x-rays in inducing fixation of Bugula larvae can be simu- lated by irradiated sea water, the effect on attachment appears to be an indirect one. Other instances of a similar indirect effect of ionizing radiations in living material have been reported in the literature. Barren and his co-workers, for instance, believed that the inhibiting effect of x-rayed sea water on the respiration of sea urchin sperm was attributable to stable organic peroxides formed during irradiation of the medium (Barren et al., 1949). Similarly, Wichterman and Figge (1954) found that when paramecia were x-rayed the lethality of the radiations was correlated with the extent of exposure to air of the culture medium. These investigators concluded that the lethal factor was probably H2O2 or some other oxidation product formed in the moist air surrounding the culture fluid ; their paper contains a brief review of the literature. The apparently indirect action of x-rays in inducing fixation of bryozoan larvae may offer a possible explanation of the seemingly strange observation of Bertholf and Mast (1944) that extracts of INDUCED SETTING OF BUGULA LARVAE muscle tissue from rabbits killed with x-rays had an accelerating effect on ascidian metamorphosis. The action of hydrogen peroxide and the other oxidizing agents discussed in this paper poses an interesting question concerning the effects of four other agents in inducing metamorphosis: copper, basic dyes, iodine and quinone (Lynch, 1949, 1952, 1956, 1957). These substances are also inhibitors of the dehydrogenases, and the action of some of them can be reversed by cysteine (references for copper and iodine are given by Needham, 1950, p. 425 ; for quinone see White et al., 1954 ; and for basic dyes cf. Quastel and Wheatley, 1931). The difference in effect on attachment produced by cysteine on the one hand (Lynch, 1957) and by iodine, copper and the basic dyes on the other, might be an indication that the latter inac- tivate an enzyme system, such as succinic dehydrogenase, and that this inactivation abruptly ends larval life allowing the adult action system to gain control. Such a hypothesis has been proposed by Glaser and Anslow (1949) as a possible explana- tion of the action of copper in inducing metamorphosis in ascidian tadpoles; these investigators have suggested that copper may operate alternately as an electron donor or acceptor in the prosthetic group of some oxidizing enzyme, which they visualize as being a porphyrin-like ring compound. The data on bryozoan meta- morphosis do not preclude the possibility that oxidizing enzymes may be involved, but it seems unlikely that succinic dehydrogenase plays an important role in the fixation of Bugiila larvae, for this enzyme is inhibited by urethane (White ct al., 1954). If the effect were primarily on this enzyme system, one would expect urethane to act like quinone, copper, iodine and the basic dyes ; but urethane in- hibits fixation, whereas the other four agents induce attachment (Lynch, 1957). The effect of these agents on succinic dehydrogenase in other organisms, how- ever, may give a clue concerning their action on the colloidal state of protoplasm. It has long been suspected that oxidizing and reducing agents play an important part in blood clotting (Matthews, 1936). And it has been suggested that the conversion of fibrinogen into fibrin involves an oxidation process. Chargaff and Bendich (1943) believe that this oxidation involves aminoacyl groups of proteins; Baumberger (1941), on the other hand, considers that the oxidation of sulphydryl groups is of prime importance in the clotting mechanism. And the significance of certain reducing agents wThich inhibit the conversion of prothrombin into thrombin has been emphasized by Carter and Warner (1954). Recently Mazia and Dan (1952) have shown that the spindle fibers of cells undergoing mitosis can be iso- lated by creating artificial disulfide bonds when these cells are treated with HL>O, (or iodine) before the rest of the cell content is solubilized by a detergent. These investigators believe that H.,O2 removes hydrogen from the sulphydryl groups of proteins and converts these substances into less soluble material by joining them together by — S — S — bonds. Thus there appears to be a polymerization of smaller molecules through these disulfide bridges. Calcutt (1951), likewise, thinks that certain photodynamic dyes affect the exposed — SH groups of the protein molecule. The diversity of factors capable of inducing attachment of bryzoan larvae seems to indicate that these agents have a direct effect on the fluid of attachment. In many of the sessile organisms the cementing substance appears to be a mucoprotein (cf. Pyefinch and Downing, 1949), and Knight-Jones (1953) has found evidence that barnacles attach by means of a substance which he considered to be a quinone- 224 WILLIAM F. LYNCH tanned protein, a compound similar to the material in the hardened cuticle of an insect. The action of x-rays in inducing fixation of Bngula larvae would not be out of harmony with the working hypothesis that attachment, when artificially in- duced, is brought about by agents which cause coagulation. Such a coagulating effect of x-rays has been reported for such varied types of protoplasm as sea urchin eggs (Rieser, 1955) and paramecia (\Yichterman and Figge, 1954). On the other hand, irradiation of fibrinogen prolongs the clotting time (Rieser, 1956). The possibilities just discussed may form a link which would connect the effect of photodynamic dyes with that of x-rays, since both agents may release H2O2 or organic peroxides (Blum. 1941, p. 96; Barren ct al., 1949). It would be reason- able to suspect that the action of both iodine and quinone would be similar to that of H2O2. The excellent development of zoids formed from larvae whose metamorphosis had been induced by treatment with sea water containing neutral red in parts of 1:100.000 (Lynch, 1952) and the poor development of zoids in solutions of H2O2 may be attributed, perhaps, either to unrecognized extrinsic factors affecting the latter or to the higher concentration of H2O2 (1 : 14,000 parts). Experiments with concentrations of neutral red that were ten times stronger than those used for the observations previously reported showed that larvae in these media also failed to develop after attachment. SUMMARY 1. X-raying larvae of either B. flabcllata (18,333 r) or B. turrita (15.733 r) within thirty minutes after the organisms began to emerge from the parental colonies induced more rapid setting than that which occurred in the controls (P = .005 and .001, respectively). Irradiated sea water had a similar, but slightly less pronounced, effect (P == .005). In these experiments the subsequent devel- opment of larvae of B. turrita into zoids was drastically impeded. Slow growth, usually without differentiation, was observed. 2. Sea \vater solutions of H2O2 (7 X 10"4 Af), of 2,3,5-triphenyltetrazolium chloride (1 X 10~5 M) and of sodium 2,6-dichlorobenzenoneindophenol (3.4 X 10~8 M) at a pH of 7.8-8.0 also induced more rapid setting of the experimental larvae (P -- .001, .015 and .015, respectively). The subsequent development of larvae exposed to H2O2 and to 2,3,5-triphenyltetrazolium chloride resembled that of organisms that were either x-rayed or placed in irradiated sea water. Sodium 2,6-dichlorobenzenoneindophenol was less injurious to the larvae than the other agents used. An explanation of the possible role of these agents in inducing an accelerated rate of setting is presented. LITERATURE CITED BARRON, E. S. G., VERONICA FLOOD AND BETTY GASVODA, 1949. Studies on the mechanism of action of ionizing radiations. V. The effect of hydrogen peroxide and of x-ray ir- radiated sea water on the respiration of sea urchin sperm and eggs. B'wl. Bull., 97 : 51-56. BAUMBERGER, J. P., 1941. 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The effects of certain organic compounds and of antimitotic agents on the rate of metamorphosis of Bugula and Amaroecium larvae. /. Exp. Zool., in press. MATTHEWS, A. P., 1936. Principles of Biochemistry. Wood, Baltimore. MAZIA, DANIEL, AND KATSUMA DAN, 1952. The isolation and biochemical characterization of the mitotic apparatus of dividing cells. Proc. Nat. Acad. Sci., 38 : 826-838. NEEDHAM, JOSEPH, 1950. Biochemistry and Morphogenesis. Cambridge at the University Press. OKA, SHUZITU, 1954. Radiosensitivity in Lophopodella cartcri, a fresh water bryozoan. Sci. Rep. Tokyo Bunrika Daigaku, 111 : 211-217. PYEFINCH, K. A., AND S. F. DOWNING, 1949. Notes on the general biology of Tubularia larynx Ellis and Solander. /. Mar. Biol. Assoc., 28: 21-43. QUASTEL, J. H., AND H. M. WHEATLEY, 1931. The action of dyestuffs on enzymes. Biochcm. J., 85 : 629-639. RIESER, PETER, 1955. The effect of roentgen rays on the colloidal properties of the starfish egg. Biol. Bull., 109: 108-112. RIESER, PETER, 1956. Radiation-induced alteration of fibrinogen clotting rate and clot lability. Proc. Soc. Exp. Biol. Med,, 91 : 654-657. WHITE, ABRAHAM, PHILIP HANDLER, E. L. SMITH AND DEWrrr STETTEN, 1954. Principles of Biochemistry. McGraw-Hill Book Co., Inc., New York. WICHTERMAN, RALPH, AND F. H. J. FiGGE, 1954. Lethality and the biological effects of x-rays in Paramecium; radiation resistance and its variability. Biol. Bull., 106: 253-263. GASTRULAR BLOCKAGE IN FROGS' EGGS PRODUCED BY OXYGEN POISONING1 SASHA MALAMED -• s Department of Zoology, Columbia Unii'ersitv. New York 27, N. Y. Seldom do embryos stop developing without dying. Occasionally, however, maintenance becomes temporarily independent of gross morphological change as in the diapause of insects. Although non-reversible, this condition may be experi- mentally produced in amphibians by a few methods which result in highly uniform populations of arrested embryos which stay alive, that is, do not cytolyze for a rela- tively long time. Well-known among these techniques are CN~ or azide treat- ment (Spiegelman and Moog, 1945), and^certain hybridizations (Moore, 1941; Brachet, 1944). In the late forties, however, Nelsen (1947, 1948, 1949, 1950) obtained a gastrular block in frogs' eggs by using 3 atmospheres of oxygen added to air at standard pressure. This method offers certain advantages toward a causal analysis of development. First, the agent's effect is not immediate as in the case of azide, nor is it nec- essary to treat the embryos continuously as with CN~. After 24 hours of treat- ment with oxygen pressure, the embryos are in the early cleavage stages. Until late blastulation they cannot be distinguished from the controls. Development stops just before dorsal lip formation and cytolysis does not set in for at least 30 hours. Embryos are thus obtainable with chemical aberrations which have not yet ap- peared at the morphological level. Second, while each of the other methods except that employing azide has an all-or-none effect, the influence of oxygen pressure may be varied by controlling the dosage. By reducing the latter, "incompletely blocked" embryos are produced. These develop into larvae of normal appearance except for the scar of an abnormal gastrulation in the form of a persistent yolk plug. Third, azide will affect any pre-gastrular stage. Although CN~~ and hybridi- zation have effects which are largely specific for gastrulation, oxygen pressure's specificity is even sharper. Clayton (1950) has shown in embryos pre-treated with the appropriate dose of oxygen that the movements of the notochord anlage are not prevented, though some of the other gastrular movements cease. Thus, in- completely blocked embryos do form neural tubes. With either CN~~ or hybridiza- tion, however, gastrulation is completely blocked and all the movements stop. Before exploiting these advantages afforded by the oxygen pressure effect, cer- tain preliminary problems needed clarification. Accordingly, the present report is 1 Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, of Columbia University. 2 Public Health Service (Predoctorate) Research Fellow of the National Cancer Institute. 3 Present address : Department of Physiology, Western Reserve University School of Medi- cine, Cleveland 6, Ohio. 226 OXYGEN POISONING OF FROGS' EGGS 227 an investigation of 1) whether it is oxygen or pressure or both that affects the embryos, 2) dosage requirements, and 3) whether there is any effect on develop- mental rate prior to the gastrular block as in the case of CN~. Although the latter does not stop pre-gastrular development, the fact that it slows these stages shows that it attacks a system on which development is dependent before as well as during gastrulation and thus detracts from its value as an analytical tool for the study of chemical events peculiar to the process of gastrulation. MATERIALS AND METHODS Eggs of Rana pipicns from Vermont were obtained by the pituitary injection method of Rugh (1948, pp. 102-106) and fertilized by his method with slight modifications. After injection the females were kept at 12° to 14° C. until stripped ; insemination was at room temperature. Fertilization and development of the em- bryos was in 10% frog Ringer's solution. A few experiments were performed shortly after the natural breeding season of R. pipiens. For this purpose gravid females were stored at 5° C. from January until used as above. These "summer'! frogs were kept in a 0.03% tap water solution of sodium sulfadiazine until injection. Stages of development were designated according to Shumway (1940) and with reference to Rugh (1948, pp. 63-65). The designated stage was the latest one which at least one-half the embryos had reached. APPARATUS An apparatus was constructed consisting of 6 glass pressure chambers which were shaken and kept at constant temperature. The chambers were continuous or discontinuous with each other in various combinations. Thus 6 levels of either of 2 variables could be studied at the same time, that is, on samples of a single clutch of eggs. These dosage variables were pressure and hours of treatment. In addition it was possible to have 3 chambers stationary with simultaneous shaking of the others. This permitted the effect of shaking to be determined on one clutch of embryos at 3 levels of either of the 2 dosage variables. Since the apparatus in- cluded only one water bath, comparative temperature studies on single clutches were precluded. Provision was made for the inclusion of 4 control bottles, shaken and unshaken, and containing eggs under no increased oxygen pressure. The pressure manifold with its 3 gauges was mounted above a rectangular Warburg apparatus (Fig. 1). The latter provided the temperature regulation and shaking mechanism needed. Rubber pressure tubing connected the manifold to the pressure chamber assemblies submerged in the water bath. On each bank of the Warburg apparatus were mounted a few manometer supports connected by means of an aluminum rod fastened to their horizontal arms. Shaking of the pressure chambers was effected by clamping the assemblies to this rod. The pressure chamber assemblies (Fig. 2) were slightly modified units of the Parr hydrogenation apparatus. In each assembly, a 500-ml. Pyrex glass bottle (surrounded by a perforated steel shield) served as the pressure chamber for the embryos. The pressure manifold (Fig. 1) was assembled of ^-inch galvanized iron pipe and brass fittings. The nozzles of the manifold and of the pressure chamber inlet 228 SASHA MALAMED FIGURE 1. Constant-temperature pressure apparatus with provision for shaking. tube permitted a flexible connection of rubber pressure tubing and this, in turn, allowed for shaking of the pressure chamber assemblies while the manifold was stationary. The gauges supplied by the Parr Instrument Co. were of the Bourdon type and read from 0 to 100 p.s.i. (pounds per square inch) in units of 1 which were large enough for estimation of the needle position to the nearest 0.2 p.s.i. ^g^f^ioK^f-t- .-—a •y . FIGURE 2. Pressure chamber assembly. OXYGEN POISONING OF FROGS' EGGS 229 They had a specified accuracy of at least ± 0.5 p.s.i. When in a closed system, with gas supplied from a single source, the gauge readings up to 50 p.s.i. agreed to 0.2 p.s.i. The 10 valves with stainless steel needles were from Hoke, Inc. A commercial pressure cylinder supplied the gas which entered the manifold through either terminal valve ; by shutting the other one, a closed system was effected. To decompress the first pressure chamber in a time series the appropriate terminal valve was opened. For the others in the series, non-terminal valves were used. The specified purity of the gases obtained from the Ohio Chemical and Surgical Equipment Co. and from the Matheson Co. was 99.5%. The principal impurity of the oxygen was nitrogen and vice versa. For each experimental run, the set- tings of the needle valves were determined by the variables to be studied. The system permitted positive pressures up to 50 p.s.i. The total leakage dur- ing the course of any run never exceeded 5% of the gauge readings registered at the start. Actually, more than 2% leakage seldom occurred. Temperature control was maintained as in ordinary procedures with the War- burg apparatus, with one exception. Since all the runs were below room tempera- ture, cooling coils were added to the floor of the water bath. Through these coils flowed water which was cooled in a separate water bath by a portable refrigerator. This "cooling bath" was temperature-controlled at about 5° C. below the temperature desired in the Warburg bath. The heating unit of the latter operated intermittently against this continuous cooling. The Warburg bath was run at 18.0°, 12.0°, or 8.0° C. with a variability of ± 0.05° C. in each case. Shaking was at a rate consistent with normal development of the embryos and rapid gas diffusion between the liquid phase containing the eggs and the gas phase above it. The stationary surface area wTas 4.9 square inches and 50 ml. of 10% Ringer's solution were used. The depth in the pressure bottle of the 10% Ringer's solution plus the embryos was 1 inch. The Warburg was altered to permit shaking on each bank at 30 ± i c.p.m. in a horizontal plane with amplitude of H inches. In a few runs with a preliminary apparatus, the shaking rate was 36 ± 1 c.p.m. PROCEDURE Each experimental run may be generally divided into 3 phases : 1 ) fertilization and compression, 2) decompression and selection of embryos, and 3) tabulation of abnormalities. Table I summarizes these steps. 1) At room temperature and 30-45 minutes after insemination, the clutch of eggs is rinsed with 10% Ringer's solution and then cut up into groups of 20-40 eggs. Only those clutches of eggs in which at least 80% rotate are used. The animals are distributed about 300 to a pressure bottle, each of the latter containing 50 ml. of 10% Ringer's solution. Including one control, 7 bottles are usually loaded. The metal-shielded bottles are fitted to the rubber stoppers attached to the manifold of the apparatus, then the clamps to the bottles. The former are tight- ened and, with the addition of the control bottle (s), are attached to the aluminum rods, thus submerging them all in the water bath, the latter at 18.0°, 12.0°, or 8.0° C. Now shaking is begun and pressure is built up gradually and simultane- ously in all the bottles over a period of 20 minutes. The midpoint of this pe- riod is noted as the start of pressure treatment. The bottles are not flushed ; the oxygen is added to the air in them ; hence the total pressure in each bottle 230 SASHA MALAMED equals the sum of the gauge reading for oxygen plus 1 atmosphere of air. After application of pressure the remaining eggs are observed to make sure that the first cleavage has not yet occurred. The bottles are shaken continuously until the time for decompression except in certain experiments where the effect of shaking is studied. 2) Decompression is gradual over one hour, and the midpoint of this period of time is taken as the end of pressure treatment. The order and times of de- compression of the individual chambers in any one run varies, of course, with the purpose of the latter. While pressure is being released, work is progressively begun on the contents of each bottle already decompressed and immediately un- TABLE I Summary of procedure Phases Steps Temperature changes of eggs Relative time approximately^ Stage of development of normal eggs 1. Bath on Fertilization Rotation Room temp. 0 min 45 min Loading Shaker on Bath temp. 45 min -100 min. Compression 100 min -120 min. Before 3 2. Decompression Unloading Fresh Ringer's Selection (Fresh Ringer's) Room temp. 18° C. First Last Cleavage 8,9 Bottle 11-12 hrs. 13 hrs. 16-17 hrs. *(A) *(B) 19 hrs. 3. Tabulation Room temp. 71-74 hrs. 14 * Staging of controls and last treated sample to be decompressed for developmental rate studies. A For relative times of controls follow sub-column for last bottle, and disregard times for compression and decompression. loaded. The contents of the bottles containing embryos plus medium still at a temperature near that of the water bath are emptied into a finger bowl containing about 50 ml. of fresh 10% Ringer's solution at room temperature. From this finger bowl, when they are at the mid- or late blastula stage, 100 (or, in a few experiments, 50) apparently normal eggs are selected. If, after decompression, less than 80% of the embryos appear normal in either the control or treated samples, no embryos are selected and the run is discontinued. In the selection process, the blastulae are transferred to a second finger bowl containing fresh 10% Ringer's solution. After staging of the embryos, all the finger bowls are placed at 18.0° C. This results in a set of finger bowls, each of which corresponds to one of the pressure bottles. The 100 eggs in each appear normal and are in a uniform stage OXYGEN POISONING OF FROGS' EGGS 231 of development earlier than gastrulation. These embryos are left to develop at 18.0° C. until neural fold formation of the controls. Because of the selection process, pre-gastrular abnormalities are eliminated from the populations to be counted at the end of each run. Actually, oxygen treat- ment and shaking, either singly or in combination, are found by comparison with control groups of eggs to have little if any effect on development prior to gastrula- tion. The great majority of unhealthy eggs selected out are unfertilized. The selection process is completed before gastrulation. Selection (as well as each of the other steps) is the same for the controls as for the treated eggs. The former are removed from the Warburg bath with the last of the series to be decompressed. Thus each member of these pairs of oxygen- treated and untreated embryos will have had the same history of temperature en- vironments at the time of neurulation when abnormalities are counted. Collation of "stagings" on these sets from different runs constitutes the developmental rate studies. 3) When the control eggs are neurulas, cytolysis has not yet occurred in the abnormal embryos. At this time all the ringer bowls are transferred to room tem- perature and the numbers of normal and neurulating embryos in each finger bowl are counted. If less than 95% of the controls are normal, the run is disregarded. In the abnormal class, evidence of developmental arrest or abnormality before stage 9 or 9 + is never found, and since only rarely does an abnormal neurula occur which does not show a gastrular aberration, that is, unincorporated yolk, the results are expressed as numbers or per cents of normal gastrulae. RESULTS 1. Identification of the Effective Agent When Rana pipiens embryos are treated with oxygen during early cleavage they stop developing normally at the late blastula stage, well after decompression. The columns for oxygen in Table II show typical results with various dosage conditions. Since effective oxygen treatment involves the use of pressure, the question arose as to the role that is played by this factor. Experiments were run using nitrogen instead of oxygen. They were otherwise identical in procedure and equal or greater in dosage than the oxygen runs which invariably produce embryos which fail to gastrulate normally. In none of the nitrogen experiments did more than 3% of the embryos block at gastrulation. Table II, which includes results with oxygen for comparison, shows no mechanical effect of pressure on development. The ineffectiveness of pressure "alone" was confirmed by another type of ex- periment in which one bottle of a pair was shaken and the other was not. Except for this difference, the embryos in the two bottles had the same oxygen treatment. Several pairs of bottles were used, each pair for a different duration of treatment. Table III shows that even though pressure (and here the gas was oxygen) was the same in the stationary bottles as in the shaken ones, only in the former was gastru- lation normal. It might appear from consideration of Table III that shaking is the effective factor that we seek. But control embryos in air at atmospheric pressure are rou- tinely shaken and show better than 95% normal development, for otherwise an 232 SASHA MALAMED TABLE II Per cent normal gastrulae with comparable treatments of oxygen and nitrogen. Air at 1 atmosphere. Treatment at 8.0° C. ; 100 eggs per sample except at 45 p.s.i. of oxygen •with 23 hours where 50 eggs used. Nitrogen samples from same cross. Oxygen- samples from different crosses. All samples shaken at 30 c.p.m. P.S.I, added to air 45 30 Gas O2 N2 O2 N2 O2 N2 N2 Hours of treatment 14 14 23 24 23 24 38 Per cent normal gastrulae 25 97 0 99 5 99 99 experiment is disregarded. Gastrulation is normal even when shaking is combined with nitrogen pressure (see Table II). Since shaking and pressure treatment singly or together are ineffective, and yet the two together with oxygen produce gastrular blockage, it becomes apparent that oxygen at the given pressures is the effective agent. The reason why the given pressures are required may be explained by Dalton's Law which states that in a two-phase system the solubility of a given gas in the liquid phase is directly pro- portional to its partial pressure in the gas phase above the liquid. Raising the oxygen pressure to the given hyperatmospheric levels, then, is one way to increase the solubility of oxygen in the medium so that at equilibrium the oxygen concen- tration is at a toxic level. Shaking the system speeds saturation of the medium after oxygen pressure is built up over it. If, after oxygen pressure is applied, shaking serves merely as an aid in bringing the toxic agent, oxygen, through the 10% Ringer's solution to the embryos, then embryos which are not shaken should also be poisoned when treated for an additional period to allow for the slow reaching of equilibrium between the gas and liquid phases. To illustrate this point two series were run, one with shaking and the other without it. In each series several durations of oxygen treatment were used, a dif- ferent sample of 50 embryos for each dosage. The two curves of Figure 3 show that to produce a given number of abnormal gastrulae it took roughly 5 hours more under oxygen pressure without shaking than it did with shaking. Two control series were also run at ambient air pressure, one series with shaking, and the other without shaking. In either series, at least 48 of the 50 embryos in each sample developed normally. Before concluding that gastrular blockage is caused by excess oxygen in the liquid environment of the embryos, two miscellaneous possibilities must be elimi- nated. These are 1 ) that the embryos suffer from shock resulting from short com- pression and decompression periods and 2) that they are overcrowded (see Barth, 1946). As for the first consideration, reference to Table III, Figure 3, and the data in the next section (Dosage) reveals the many short doses of oxygen treat- ment, including those with shaking, which did not produce abnormal gastrulae. In TABLE III Per cent normal gastrulae with oxygen treatment with and without shaking. Treatment at 18.0° C. with 45 p.s.i. of oxygen added to air at 1 atmosphere; 100 eggs per sample. All samples from same cross. Shaking at 30 c.p.m. Hours of oxygen treatment 10 13 16 Shaking + + + Per cent normal gastrulae 92 0 93 0 95 0 OXYGEN POISONING OF FROGS' EGGS 233 all these cases, just as with the longer, harmful treatments, compression and decom- pression were gradual over 20 and 60 minutes, respectively. In addition, the em- bryos were treated in the same manner with nitrogen (cf. Table II) and develop- ment was normal. The possibility of overcrowding is precluded by these same data for, again, normal gastrulation resulted from conditions which were just as crowded as those which produced gastrular blockage. Furthermore, even the non- shaken, non-pressure-treated embryos showed at least 95% normal gastrulae as, of course, the shaken controls did. Actually, as far as oxygen concentration in the medium was concerned, the oxygen-treated embryos were "undercrowded." Thus it appears that neither duration of pressure change nor population density affected the embryos deleteriously. It appears, then, that gastrular blockage is effected by a pressure-produced high oxygen concentration in the liquid environment of the embryos. Indeed, as 50 _ 40 d E o 30 o _0 E 20 10 o - x Shaken o Not shaken 8 12 16 20 Hours of Op Treatment * FIGURE 3. Effect of duration of oxygen treatment on gastrulation with and without shak- ing. Treatment at room temperature with 45 p.s.i. of oxygen added to air at 1 atmosphere ; 50 eggs per sample. A different cross (no common parentage) used for each time dosage. Shaken and non-shaken samples at a given time dosage are from same cross. Shaking at 36 c.p.m. will be shown below, the percentage of normal gastrulae varies inversely with the partial pressure of oxygen at a given duration of treatment (see Figure 8). If pres- sure does have some effect other than via Dalton's Law, certainly it is not through an increase in the force per unit area in the mechanical sense. 2. Dosage Regardless of whether duration of oxygen treatment or pressure was varied, in each series or run, pressure was applied to all the samples simultaneously and before the first cleavage. Each dosage datum obtained represented a different sample of embryos since by the time the per cent normal gastrulae of a sample was determined, the normal embryos were too advanced to be blocked at the late 234 SASHA MALAMED 100 0 50 O O 0 18.0 °C O Means from all Crosses ...» Single Cross 0 O O 5 10 Hours of Oo Treatment 15 FIGURE 4. Effect of duration of oxygen treatment on gastrulation. The points represent the means from Table IV. Treatment at 18.0° C. with 45 p.s.i. of oxygen added to air at 1 atmosphere. Shaking at 30 c.p.m. Single cross from Figure 5. blastula stage; thus a second dose on the same sample was precluded, and cor- respondingly, a second datum. With few exceptions, the size of each sample was 100 embryos. Increasing a given sample to 200 made a difference of no more than 5% normal gastrulae. Oc- casional recounts of the same sample agreed within 2%. All the data in this sec- tion are from shaken samples. 100 Q) _d 10 O O O 6 50 0 0 10 15 Hours of Treatment FIGURE 5. Effect of duration of oxygen treatment at various temperatures on gastrula- tion. Pressure is 45 p.s.i. of oxygen added to air at 1 atmosphere; 100 eggs per sample. A different cross (no common parentage) used for each temperature. Shaking at 30 c.p.m. OXYGEN POISONING OF FROGS' EGGS 235 Curves of per cent normal gastrulae versus degrees of dosage of either kind in which all the embryos had the same parents were the most valuable, for genetic variability was thus avoided. Where, alternatively, the arithmetic averages of sets of repeat experiments were used, information was provided concerning any R. pipiens embryo of any parents, but the contours of the curve were softened and sharp breaks were obscured (see Figure 4). TABLE IV Per cent normal gastrulae with increasing durations of oxygen treatment and different parental backgrounds. Treatment at 18.0° C. with 45 p.s.i. of oxygen added to air at 1 atmosphere. Shaking at 30 c.p.m. Exptl. Hours of oxygen treatment no 6 7 8 81 9 10 11 12 13 16 i 9 1 cf 1 0 0 0 2 9 2 cf 2 83 43 3 9 3 cf 3 92 83 86 71 0 0 9 4 cf 4 95 71 4 9 5 cf 4 90 81 9 6 cf 4 0 0 9 7 cf 5 0 0 0 9 8 cf 5 0 5 9 9 cf 5 78 9 10 cf 5 10 9 7 cf 6 0 0 0 9 8 cf 6 0 0 9 11 cf 7 98 79 7 9 11 cf 8 96 97 9 1 1 cf 9 99 91 Means 92 83 86 0 74 11 60 0 0 0 No. of samples 1 2 1 3 8 8 7 2 1 1 34 samples: 15 crosses: 100 embryos/sample 11 9 9 (13 clutches; 97, 9 8 stripped twice) 9 cf cf 7 9 cf (No common parentage) a. Duration of treatment (time dosage and per cent abnormality} With increasing time dosage the per cent normal gastrulae dropped sharply. In Figure 4, a mean curve is plotted showing all the data at 18.0° C., with 45 p.s.i. of oxygen added to air at 1 atmosphere; 3400 embryos and 15 crosses are repre- sented (see also Table IV). A curve of samples drawn from one of the 15 crosses ($3 c?3 in Table IV, 18.0° C. curve in Figure 5) is also presented. In either case, as soon as the embryos had been treated long enough to affect a few, it took 236 SASHA MALAMED a relatively short additional dose ("effective time dosage") to affect them all. For the curve of the means this was 6 hours. The steepness of the slopes in Figure 4 is confirmed by Figure 5. The latter shows curves from three sets of samples, each set from a different cross with no common parents. Each set was run at a different temperature but with the same pressure dosage. It is seen that from 100% to 0% normal gastrulae took 5 hours at 8.0° C., 3 hours at 12.0° C., and 4 hours at 18.0° C. (except that in the 18.0° C. set the highest figure is only 92%). As a matter of fact these figures are maximal. If they are in error due to the points being taken at intervals of no less than an hour, correction would only shorten the effective time dosage. Additional evidence of the shortness of the effective time dosage is provided by Figure 6 which shows the data from all crosses run at 8.0° and at 18.0° C. At each temperature the pressure dosage was the same. Each cluster of points is concen- trated along the time axis. 00 d O ~d £ 50 0 o o 3 8 0* O O 0 I8.0°C. • 8.0° C. g 6 9 Hours of O, 12 15 Treatment 18 FIGURE 6. Effect of temperature on oxygen poisoning. Pressure is 45 p.s.i. of oxygen added to air at 1 atmosphere; 100 eggs per sample. Data for 18.0° C. from 15 crosses; for 8.0° C. from 5 crosses. Shaking at 30 c.p.m. The other interesting aspect of these curves (Figs. 4, 5, 6) is the "lag dosage" before the oxygen is effective (see also Table IV). Its length of about 8 hours stands in contrast to the shortness of the effective time dosage. (Since all these data are from shaken samples, this lag is independent of that due to slow diffusion which is shown in the curve of Figure 3 for non-shaken eggs.) b. The effects of temperature on time dosage Oxygen solubility varies inversely with temperature. The increment in con- centration of this gas is especially large from 18.0° to 8.0° C. in a saline solution (Umbreit, Burris and Stauffer, 1949, p. 5). Thus lowering the temperature has the same effect as increasing the pressure (cf. Dalton's Law) and this, it will be OXYGEN POISONING OF FROGS' EGGS 237 seen in a later section, decreases the duration of treatment necessary for gastrular blockage (see Figure 8). Also lowering the time dosage is a second effect of a drop in temperature. Sensitivity to oxygen decreases with developmental age (Nelsen, 1949). Since lowered temperature also slows development, any given duration of treatment is more effective at a lower temperature than at a higher one, for that part of the time at 18.0° C. spent on later stages is expended at 8.0° C. on the earlier, more sensitive stages. Consequently, less hours of treatment are required to produce gastrular blockage at 8.0° than at 18.0° C. In two ways, then, temperature decrease enhances the effectiveness of oxygen treatment and tends to shift to the left a curve of per cent normal gastrulae versus hours of treatment (see Figure 7). On the other hand, unless they are very atypical, the actual chemical reactions resulting from oxygen treatment are slowed by a temperature decrease (Getman and Daniels, 1943, p. 363). This third effect tends to cancel out the other two. Thus any separation along the time axis of curves at different temperatures is a net effect. 100 d O 50 d 0 Staqe Sensitivity O Concen- tration ( B.V. Chemical Rate B.V.) Hours of Treatment FIGURE 7. Postulated compensatory effects of decreased temperature on oxygen poisoning. B. V. stands for "Biological Variability." See text for explanation. As far as lag dosage is concerned, neither Figure 5 nor Figure 6 shows any net effect of temperature. The former shows the results of oxygen treatment at three temperatures over a 10.0° C. range. The slight separation of the three curves is well within the variability inherent in the biological material (see Table IV) and therefore cannot be considered significant. Moreover, in Figure 6 error due to this variability is reduced through the use of samples from many crosses and here the cluster of points for 18.0° C. and those for 8.0° C. show the same lag dosage. In Figures 5 and 6, although the lag dosage remains unaffected, the effective time dosage is increased by a temperature drop. In the former figure the slope of the 8.0° C. curve is less than that of the curves of 12.0° and 18.0° C. Although error is introduced due to the large time intervals (1 hour) between points, this error, as well as that due to biological variability, is reduced in Figure 6. Here, confirming the data of Figure 5, the cluster for 8.0° C. is more spread out along the abscissa than is the one for 18.0° C. In addition, with a fast drop in percentage 238 SASHA MALAMED as compared to a slow drop, there is smaller probability at a random time dosage of a point falling midway between 100% and 0% normal gastrulae. The points for 18.0° C. actually do aggregate at the ends of the percentage range while those for 8.0° C. are more evenly spread along the ordinate. Thus in the range from 8.0° to 18.0° C. the several effects of temperature are fully compensatory for lag dosage, while for the effective time dosage the chemical rate effect is greater than the combination of the opposite two (see Figure 7) and a net positive temperature coefficient for oxygen poisoning is demonstrated. c. The effect of time dosage on type of abnormality Even though the embryos from different crosses varied greatly in their oxygen sensitivity (see Table IV and Figure 3), a rather striking uniformity of response to treatment was demonstrated among siblings. This was especially well shown when, after tabulating the per cent of normal gastrulae (class 1), the abnormal embryos in each sample of a time dosage series were broken down according to TABLE V Per cent of gastrulae of each class in samples exposed to increasing durations of oxygen treatment at 12.0° C. Pressure is 45 p.s.i. of oxygen added to air at 1 atmosphere; 100 eggs per sample. All samples from same cross and shaken at 30 c.p.m. Hours of oxygen treatment Classes of gastrulae 7f 81 9! 101 ill 12| 1. Normal 99 60 7 0 0 0 2. Incompletely blocked 1 40 93 63 42 1 3. Completely blocked 0 0 0 37 58 99 type of gastrular blockage as follows : class 2 : incompletely blocked (abnormal gastrulation) and class 3: completely blocked (stage 9 or 9 +, no dorsal lip). As usual, each sample was drawn from the progeny of the same cross and represented a different time dosage ; except for the latter, the conditions of treatment were kept constant. As is shown in Table V, with increasing time dosage each of the three classes is progressively filled, leaving always at least one null class. After class 2 reaches nearly 100% it decreases as class 3 increases. At 9f hours there are two null classes (1 and 3) for practically all the embryos have been treated long enough to prevent normal gastrulation, but none have yet been affected so badly as to prevent it completely. The change from 8| to 9f hours of treatment is entirely from class 1 to class 2. Those embryos already in class 2 at 8f hours do not enter class 3 with the additional hour of treatment. They "wait" for the embryos still normal with 8f hours of treatment to "catch up" and become incompletely blocked at 9f hours, that is, until class 2 is full before becoming completely blocked at 10f hours. OXYGEN POISONING OF FROGS' EGGS 239 These data reveal two discrete thresholds of time dosage, an earlier one for incom- plete blockage of gastrulation, and a later one for complete blockage. In the data of Table V, these occur, respectively, between 7\ and 8f, and between 9% and 10!| hours of oxygen treatment. Occasionally in runs at 8.0° C. with 45 p.s.i. of oxygen added to 1 atmosphere of air the embryos are distributed among all three classes at one time dosage. Otherwise, however, the pattern of progression from 100% normal to 100% in- completely blocked to 100% completely blocked gastrulae recurs in time dosage series run at 18.0° or 12.0° C. with 45 p.s.i. of oxygen added to air at ambient pres- sure or at 8.0° C. with 30 or 15 p.s.i. added to air. In addition, the same kind of results are obtained when the abnormal embryos are further subdivided into four classes. d. Pressure dosage In these studies. 45, 30, and 15 p.s.i. of oxygen were used; each was added to air at 1 atmosphere. Since the partial pressure of oxygen in the latter is about IUU x >< w d 0 «_ ' S in d (D A O 0 "5 50 E ^ Solid : ? D o Z 3.2 atm. Op Open : * 2.2 atm. 02 0 • ^ i i i i i i i 6 9 12 15 18 21 24 Hours of Op Treatment FIGURE 8. Gastrular blockage as a function of partial pressure of oxygen for various durations of treatment. Treatment at 8.0° C. ; 100 eggs per sample. Samples from different crosses (no common parentage) represented by symbols of different shape. Shaking at 30 c.p.m. 0.2 atmosphere, the addition of pure oxygen in the several cases resulted in partial pressures of approximately 3.2. 2.2, and 1.2 atmospheres. Various time dosages were used with each pressure dosage except that of 1.2 atmospheres of oxygen. Each sample of embryos went through one period of treatment. The pressure was not changed during this period. Either one or several pressures of oxygen were used in a single run. All the samples in a run were from the same cross and a different cross (no common parentage) was used for each run. 240 SASHA MALAMED Earlier, a partial pressure of 3.2 atmospheres of oxygen was found to be effec- tive at 18.0° C. and at 12.0° C. These studies, however, were to include lower pressures and correspondingly weaker oxygen tensions in the 10% Ringer's solu- tion. (Dalton's Law holds for oxygen to about 99% of the theoretical values in the pressure range of this work (Moore, 1950, p. 121).) In compensation, there- fore, a temperature of 8.0° C. was used to ensure effectiveness of the treatment. The temperature reduction was expected ( see Figure 7 ) to act in these ways : 1 ) to increase the oxygen concentration in the 10% Ringer's solution, and 2) because of decreased developmental rate, a) to concentrate the treatment on the earlier, more sensitive stages, and b) to increase the number of hours of treatment possible before gastrulation. These effects were considered of more importance than the antagonistic one of decreased chemical rate. Figure 8 presents all the data with 3.2 and 2.2 atmospheres of oxygen plotted as per cent normal gastrulae against duration of treatment. The points fall into two separate clusters corresponding to the oxygen dosages used. An effect was also obtained with 1.2 atmospheres of oxygen. After 88 hours of treatment the embryos were in the mid-blastula stage and appeared normal. After selection at stage 9, however, none gastrulated normally. These data show that for a given time dosage, the percentage of embryos poisoned by oxygen varies directly with the partial pressure of that gas. This, of course, is consistent with the evidence presented in a previous section that the role of pressure in effective oxygen treatment lies in its increasing the oxygen con- centration in the egg medium. c. Pressure-tiine-dosage relationships Increased duration of treatment compensates for reduced partial pressure of oxygen. The most extreme demonstration was the experiment in which a dosage of 1.2 atmospheres resulted in gastrular blockage with 88 hours of treatment. Thus, for a given effectiveness of oxygen poisoning in terms of per cent normal gastrulae, pressure dosage varies inversely with time dosage. This may be seen by extending a horizontal line through the 2 clusters of Figure 8 and comparing their time and pressure dosages at that level. 3. Rate of Development In almost every experimental run, the stages of the embryos were determined at one or two developmental ages before gastrulation. Each run provided one or more sets of one untreated, or control, and one oxygen-treated sample, each set representing a different parental cross. The members of a given set were of equal sample sizes. At the times of comparative staging, the two samples in each set had the same history of temperature environments. The results of 35 stagings on 22 crosses are collated in Table VI. In those cases in which retardation did occur, it was of the oxygen-treated eggs. With a few exceptions (cross no. 9, 10, 21), retardation did not occur in those 11 sets in which the treated samples went on to show some percentage of normal gastrulae. Even in the exceptional cases, the retardation appeared only in the later (B) staging. In 11 crosses providing 17 stagings, the oxygen treatment resulted in 0% normal gastrulae. In 7 of these 11 crosses, the treated sample OXYGEN POISONING OF FROGS' EGGS 241 TABLE VI Comparison of developmental stages of oxygen-treated and untreated embryos, the samples of a given cross having the same history of temperature environments at the time of staging. All samples in a given run fertilized at the same time. One treated and one untreated sample per cross. Staging shortly after decompression denoted by letter A; after selection by letter B (cf. Table I). About 300 eggs per sample at A staging; 100 eggs per sample at B staging except for cross no. 18 where 50 eggs were used and cross no. 21 where 192 treated and 200 untreated eggs were used. All samples shaken at 30 c.p.m. Run no. Cross no. Oxygen treatment Per cent nor- mal gastrulae of treated eggs Developmental stages of eggs Temperature Atm. Hours Treated Untreated 1 IB 18.0° C. 3.2 16 0 9- 9 2 2B tt 1 1 9 43 8 + 8 + 3 3B tt u 11 0 9- 9- 4 4B 5B 6B u i ( 1 1 i 1 tt tt tt tt H 71 81 0 9- 9- 9- 9- 9- 9- 5 7A 7B < i (I tt tt 10 4 1 0 1 1 8 + 9 8 + 9 + 8A 8B it It tt tt 10 I ( 0 ( t 8 + 9 8 + 9 + 9A 9B n n ft (4 10 1 t 78 1 1 8 + 9 8 + 9 + 10A 10B 1 ( tt f i I ( 10 tt 10 < i 8 + 9 8 + 9 + 6 11A 11B 1 ( ( i tt tt 12 u 0 tt 8 9 8 + 9 + 7 12A 12B 1 ( tt tt I C 11 t f 79 tt 8 + 9 + 8 + 9 + 13A 13B 1 1 ft ( t tf 11 tt 97 1 1 8 + 9 + 8 + 9 + 14A 14B ( ( ft tt tt 11 tt 91 tt 8 + 9 + 8 + 9 + 8 15B 12.0° C. ( f 14 0 8 8 9 16B tt tt 12| 0 8 + 9- 10 17B 8.0° C. tt 12* 94 8 + 8 + 11 18A 18B ft ft f f ft 23* tt 0 1 1 5 7 + 6 8 242 SASHA MALAMED TABLE VI (Continued) Run no. Cross no. Oxygen treatment Per cent nor- mal gastmlde of treated eggs Developmental stages of eggs Temperature Atm. Hours Treated Untreated 12 19A 19B 8.0° C. 1 1 3.2 ( ( 131 ( i 0 i ( 4 9- 4 9- 13 20A 1 1 2.2 23 5 7 7 20B 1 4 1 1 (I ( i 8 8 14 21 A ( ( 1 1 19 47 6 6 21B ( 1 i ( ( ( ( ( 9- 9 15 22A 1 ( 1.2 88 0 9- 9 22B I 1 1 1 1 1 i * 9- 9 + was retarded. Of the 17 stagings, in 10 cases the controls were ahead of the treated animals. Where retardation did appear (in 13 of the 35 stagings), it was slight. Except for one staging (no. ISA), the delayed samples were less than one Shumway (1940) stage behind the controls. In 10 of the 22 crosses development of the oxygen-treated samples was delayed as compared to that of the controls. However, only in cross no. 18 was retardation shown before blastulation. In all cases where the beginning of the lag could be well localized (cross no. 7, 8, 9, 10), it first appeared in the blastula stage. The substaging (9 — , 9, 9 +) technique was neither very accurate nor precise from run to run. On the other hand, comparing the stages of the two samples at any given time was cjuite reliable. In other words, in Table VI, comparison of stages in horizontal rows is more dependable than in the vertical ones. Therefore, it is harder to accurately compare sets of samples from many crosses in order to tell when retardation first appears, than it is to tell in how many of all the crosses retardation does occur, and how slight it is. A few experiments without shaking were performed. Here again, in those cases when it occurred, retardation of the oxygen-treated eggs was slight and during blastulation. In the runs using nitro- gen (see Table II) instead of oxygen, the gas-treated samples showed no lag in development. Comparative staging was not as accurately performed at neurulation when the percentages of normal gastrulae were determined as at the pre-gastrular stages recorded in Table VI. Nevertheless, at that time no significant differences in de- velopmental stage between the control, and treated but unharmed eggs were noticed. It is seen, therefore, that in many but not all cases, oxygen-treated embryos are retarded as compared to untreated controls. Generally, however, the decrease in developmental rate is slight and begins during the late blastula stage. DISCUSSION Amphibian embryos subjected at fertilization or during cleavage to any one of a variety of treatments will not gastrulate. This process seems to be a critical OXYGEN POISONING OF FROGS' EGGS 243 one in early development and if not all, certainly larger percentages of experi- mentally treated eggs die or first appear abnormal at this stage than at any other. Conditions bringing this about include certain hybridizations (Moore, 1941) ; Brachet, 1944), CN~ treatment (Spiegelman and Moog, 1945), parthogenesis (Parmenter, 1933), and uterine over-ripening (Briggs, 1941). The developmental block produced by oxygen pressure in R. pipicns embryos is another which is manifested at or near gastrulation. The normal development demonstrated in the experiments using nitrogen pres- sure, some of those using oxygen pressure without shaking, and those of Nelsen (1948) using air pressure, shows that the inhibition of gastrulation through the use of oxygen pressure is not a mechanical effect ; oxygen poisoning is not the result of the exertion of a high force per unit area. This is not surprising, for living systems are relatively unaffected by non-localized pressures applied and re- leased gradually (Heilbrunn, 1952, pp. 503-509). With few exceptions, the lowest such pressures having a biological effect are about 100 times those used in the present studies. At a given temperature the oxygen tension of the embryos' culture medium is directly proportional to the partial pressure of the gas (Dalton's Law) when equilibrium is established, and the results of these experiments can be explained by assuming that the gastrular abnormalities studied are, in turn, functions of oxygen tension. This assumption is confirmed by the experiments using oxygen with and without shaking. Equilibrium between gas and liquid phases is more rapidly established with shaking and the oxygen tension quickly reaches its satura- tion level in the 10% Ringer's solution. Thus for threshold durations of oxygen treatment, gastrular blockage occurs only in the shaken embryos. As would be expected, non-shaken embryos will be affected only if they are treated with oxygen for longer periods of time. Furthermore, with higher oxygen pressure, the per- centage of normal gastrulae falls (see Figure 8). Additional confirmation is pro- vided by experiments of Nelsen (1949) which have been repeated by the present writer. These employed a vertically suspended string-like mass of eggs exposed to oxygen pressure without shaking. Those at the top of the string near the surface of the medium, and therefore in contact with a saturated solution of oxygen, stopped developing at gastrulation. The lower the position of the embryos and, corre- spondingly, the lower the oxygen tension, the more normal were their fates at gastrulation. The dosage studies reveal a threshold whose significance is not clear. For, it takes a relatively long duration of treatment (about 8 hours) to produce any ab- normal gastrulae ; yet after this dosage is completed, treatment of only about 4 more hours results in no normal gastrulae. Frequently the dosage necessary to produce 100% completely blocked gastrulae is greatly exceeded. Yet developmental arrest never occurs earlier than at the late blastula stage. This suggests that until the very end of the pre-gastrular period, as opposed to the post-gastrular stages, normal development of the embryo is not dependent upon systems which are sensitive to high oxygen tensions. The developmental rate studies confirm this view. Almost without exception, oxygen-treated embryos develop at the same rate as do untreated ones — until late blastulation. At that time, some, but not all, embryos are slightly retarded. This is understandable if some system sensitive to high oxygen tension, although nee- 244 SASHA MALAMED essary for normal development after gastrulation, may be dispensed with before this process if, indeed, it operates during the pre-gastrular stages at all. It should be pointed out that results of this sort are not obtained with all agents causing developmental arrest at gastrulation. Although certain hybrids, as well as oxygen-poisoned embryos, do stop developing abruptly (Moore, 1941), CN~- treated animals are invariably retarded by several stages beginning in early cleavage (Spiegelman and Moog, 1945). Thus in the case of CN=^ it cannot be said that what is being affected at the chemical level is correlated in the normal embryo specifically with the events beginning at gastrulation. The interpretation for the CN'~" experiments may be that before gastrulation the poison inhibits a cytochrome oxidase-limiting system which controls develop- mental rate to an extent such that the latter is merely decreased. At gastrula- tion, however, the level of inhibition relative to the heightened energy demands (see Barth and Barth, 1954) is such that development ceases entirely. This is a concept of a quantitative change at gastrulation. With oxygen poisoning the interpretation is that a qualitative change occurs at or just prior to gastrulation such that a chemical system comes into play whose operation is necessary for development to proceed, but which is not needed for even unretarded pre-gastrular development. What is inhibited during early cleavage is either this system sensitive to high oxygen tension or the conditions necessary for the system's establishment. These studies were designed as the preliminary steps toward analyses at the cellular and chemical levels. As to the former, the possibility must be entertained that in oxygen-poisoned embryos, gastrular blockage is mediated through chromo- somal aberrations. For, increased oxygen tensions enhance x-irradiation effects (Giles and Riley, 1950), and Conger and Fairchild (1952) showed that the chromo- some breakage produced by oxygen in Tradescantia microspores was identical to that caused by x-rays. Thus it has been suggested (Gerschman, Gilbert, Nye, Dwyer and Fenn, 1954) that high oxygen tensions act similarly to x-rays. As for the chemical considerations, Brachet, who has long emphasized the role of — SH in development (1950, pp. 170-184), has suggested that the — SH enzymes are inactivated in the oxygen-poisoned embryos (1949). This seems quite prob- able for Haugaard (1946), using adult mammalian tissue slices and homogenates, demonstrated a close correlation between susceptibility to inactivation by high oxy- gen pressure and the presence of essential — SH groups in some 20 oxidative and non-oxidative enzymes. Dickens, also in 1946, presented similar results. Non- protein — SH groups are also affected by oxygen, the rate of oxidation being pro- portional to the oxygen pressure (Barron, 1955). It is generally believed that the inactivation operates through an irreversible oxidation of — SH to — S — S — . With this body of work as a guide, a metabolic analysis has been started (Malamed, 1954). It was found that oxygen-treated embryos had the same oxy- gen uptake rate as controls, from shortly after decompression until the late blastula stage. After this stage the controls continued to rise in respiratory rate. At this point, however, corresponding to the time when all the treated embryos were com- pletely blocked, their rate of oxygen consumption levelled off. It then stayed con- stant until cytolysis set in, about the time the controls developed tailbuds. These results, the same as obtained with a frog hybrid by Barth (1946), indicate that what the oxygen-sensitive system is needed for is the (aerobic) production of OXYGEN POISONING OF FROGS' EGGS 245 energy, which is in turn presumably necessary for the cell movements or, more properly, the mechanical work which constitutes gastrulation. I wish to express my appreciation for the encouragement and guidance, at both the theoretical and technical levels, of Prof. L. G. Earth. To Prof. O. E. Nelsen I am indebted for the first stimulation of an interest in embryos and oxygen poisoning. Special thanks are due to Prof. J. R. Gregg, Drs. R. McMaster, J. Reiner, and A. Kostellow for their criticisms and suggestions. Mr. A. Pfeiffer was most helpful in the design and construction of the apparatus. SUMMARY 1. The effect of oxygen poisoning on gastrulation in Rana pipicns eggs has been studied using an apparatus consisting of 6 pressure systems continuous with each other or not, in various combinations. The apparatus permitted the embryos to be kept at constant temperature. Shaking and non-shaking samples could be run simultaneously. Oxygen treatment started before the first cleavage and ended during the early cleavage stages. 2. In the mechanical sense, pressure has no effect on gastrulation, for gastrula- tion is normal in experiments using nitrogen and in others using oxygen without shaking. 3. The role of pressure is via an increase in the oxygen tension of the eggs' medium, according to Dalton's Law. That gastrular blockage is a function of oxygen tension is shown by comparing results with and without shaking for various durations of treatment and by the higher percentage of abnormal gastrulae with higher partial pressure of oxygen. 4. With shaking and 45 p.s.i. of oxygen added to air at 1 atmosphere, durations of treatment of less than 8 hours are without effect on gastrulation. At this threshold, additional treatment of about 4 hours results in no normal gastrulae. 5. Temperature has little if any (net) effect on oxygen poisoning. This is explained on the basis of several temperature effects which are largely compensatory. 6. With 2.2 atmospheres partial pressure of oxygen a longer duration of treat- ment is required to affect gastrulation than with 3.2 atmospheres. An effect has been obtained using 1.2 atmospheres. 7. Comparison with controls shows that after oxygen treatment the embryos are not always retarded before gastrulation. When there is a developmental delay, it is slight and does not begin before the late blastula stage. 8. These results are interpreted as follows : at gastrulation a qualitative change occurs such that a new chemical system on which development is dependent comes into play. During early cleavage high oxygen concentrations inhibit either this system or conditions necessary for its establishment. LITERATURE CITED BARRON, E. S. G., 1955. Oxidation of some oxidation-reduction systems by oxygen at high pressures. Arch. Biochem. Biophys., 59: 502-510. EARTH, L. G., 1946. Studies on the metabolism of development. /. Exp. ZooL, 103 : 463-486. EARTH, L. G., AND L. J. EARTH, 1954. The Energetics of Development. Columbia University Press, New York. 246 SASHA MALAMED BRACKET, J., 1944. Acides nucleiques et morphogenese, la polyspermie et 1'hybridation chez les anoures. Ann. Soc. Roy. Zool. Belg., 75 : 49-74. BRACKET, J., 1949. L'hypothese des plasmagenes dans le developpement et la differentiation Colloq. Intern. Centre Nat. Recherche Sci. (Paris), VIII: 145-162. BRACKET, J., 1950. Chemical Embryology (translated from the French by L. G. Barth). Interscience Publishing Co., New York. BRIGGS, R. W., 1941. The development of abnormal growths in Rana pipiens following delayed fertilization. Anat. Record, 81 : 121-136. CLAYTON, J. W., 1950. Oxygen pressure effects on gastrular movements and tail organization in Rana pipiens. Master's Thesis, University of Pennsylvania. CONGER, A. D., AND L. M. FAIRCHILD, 1952. Breakage of chromosomes by oxygen. Proc. Nat. Acad. Sci., 38: 289-299. DICKENS, F., 1946. The toxic effects of oxygen on brain metabolism and on tissue enzymes. 2. Tissue enzymes. Biochcm. J., 40: 171-187. GERSCHMAN, R., D. L. GILBERT, S. W. NYE, P. DWYER AND W. O. FENN, 1954. Oxygen poison- ing and x-irradiation : a mechanism in common. Science, 119: 623-626. GETMAN, F. H., AND F. DANIELS, 1943. Outlines of Physical Chemistry. Seventh edition. John Wiley and Sons, Inc., New York. GILES, N. H., JR., AND H. P. RILEY, 1950. Studies on the mechanism of the oxygen effect on the radiosensitivity of Tradescantia chromosomes. Proc. Nat. Acad. Sci., 36: 337-344. HAUGAARD, N., 1946. Oxygen poisoning. XL The relation between inactivation of enzymes by oxygen and essential sulfhydryl groups. /. Biol. Chcin., 164 : 265-270. HEILBRUNN, L. V., 1952. An Outline of General Physiology. Third edition. W. B. Saunders Co., Philadelphia. MALAMED, S., 1954. Influence of oxygen poisoning on development of frog embryos. Federa- tion Proc., 13: 93-94. MOORE, J. A., 1941. Developmental rate of hybrid frogs. /. Exp. Zool., 86: 405-422. MOORE, W. J., 1950. Physical Chemistry. Prentice-Hall, Inc., New York. NELSEN, O. E., 1947. Oxygen and air pressure effects upon the early development of the frog's egg. Science, 106: 295-296. NELSEN, O. E., 1948. Changes in the form of the blastopore in blocked gastrulae of Rana pipiens. Anat. Record, 101 : 710. NELSEN, O. E., 1949. The cumulative effect of oxygen-pressure in the blocking of gastrulation in the embryo of Rana pipiens. Anat. Record, 105 : 599. NELSEN, O. E., 1950. Temperature and the oxygen pressure inhibition of the early development of Rana pipiens. Anat. Record,' 108: 583. PARMENTER, C. L., 1933. Haploid, diploid, triploid, and tetraploid chromosome numbers, and their origin in parthenogenetically developed larvae and frogs of Rana pipiens and R. palustris. J. Exp. Zool., 66: 409-454. RUGH, R., 1948. Experimental Embryology. Revised edition. Burgess Publishing Co., Minneapolis. SHUMWAY, W., 1940. Stages in the normal development of Rana pipiens. I. External form. Anat. Record, 78: 139-148. SPIEGELMAN, S., AND F. MOOG, 1945. A comparison of the effects of cyanide and azide on the development of frogs' eggs. Biol. Bull, 89: 122-130. UMBREIT, W. W., R. H. BURRIS AND J. F. STAUFFER, 1949. Manometric Techniques and Tis- sue Metabolism. Second edition. Burgess Publishing Co., Minneapolis. THE PRODUCTION OF TWIN EMBRYOS IN DENDR ASTER BY MEANS OF MERCAPTOETHANOL (MONOTHIOETHYLENE GLYCOL)2 DANIEL MAZIA * Scripps Institution of Oceanography, University of California, La Jolla, California, and Department of Zoolo//y, University of California, Berkeley, California The problem of individuation in the earliest embryonic development of certain animal groups resolves itself into questions concerning the interaction of blastomeres. Some transaction between the blastomeres determines that the first division will produce an individual composed of two cells rather than two individual embryos. Physical contiguity is a factor by definition, for, in those cases where the blasto- meres are capable of producing complete embryos, such "twinning" can always be achieved by complete separation of the blastomeres. But complete physical sepa- ration is not necessary for functional isolation of the blastomeres ; from studies of echinoid eggs we have a variety of experimental conditions under which twin em- bryos are produced from sister blastomeres in contact with each other (summarized by Schleip, 1929; Harvey, 1940). The experimental problem is to define the means — not necessarily a single one — whereby adjacent cells can mutually influence or restrict each other's behavior. The question is of interest in research on cell division as well as on developmental problems, and probably has much broader implications relative to the behavior of multicellular systems. In the case of echinoid eggs, it has received a good deal of attention, particularly in studies on cell division, and some of the ideas regarding the mechanisms are reviewed in a paper by Dan and Ono (1952). Our chemical insights into the mechanisms of blastomere interaction are rather rudimentary, centering on the study of "extracellular coats" or "intercellular ce- ments" which have, for good reasons, been characterized as calcium proteinates. The present work is part of a series of studies in which mercaptoethanol (mono- thioethylene glycol) was employed as an agent which was expected to interfere with the association of protein molecules through thiol groups. The considerations underlying the study and the selection of this agent are discussed in another paper (Mazia, 1958). It was found, with the eggs of Dcndrastcr e.rccntrictts. that treat- ment with mercaptoethanol at the proper time would produce twins in very high yields even though the blastomeres remained in contact within the fertilization 2 Contributions from the Scripps Institution of Oceanography, New Series, No. 995. 1 Permanent address : Department of Zoology, University of California, Berkeley. The major part of this work was done during the tenure of a visiting professorship at the Scripps Institution of Oceanography, University of California, La Jolla, California. The author thanks the director and staff of that institution for this opportunity and for their valued cooperation. The work was completed during the tenure of a research professorship in the Adolph C. and Mary Sprague Miller Institute for Basic Research in Science at Berkeley. Material support from the American Cancer Society and the Office of Naval Research is gratefully acknowledged. 247 248 DANIEL MAZIA FIGURE 1. Twin blastulae. Dcndrastcr eggs had been placed in 0.1 M mercaptoethanol in sea water at 41 minutes after insemination, and exposed for 28 minutes. Photographed alive at 7 hours 15 minutes after insemination. Twins are hatching in embryo at top of photograph. TWINNING IN DENDRASTER 249 membrane. The points of interest in the following discussion are not only the interpretation of the effect as one implicating thiol groups in blastomere interaction, but also the fact that processes determining the twinning or non-twinning may be restricted to a short period during the cleavage of the eggs. METHODS The details of the methods used will be found in a previous paper (Mazia, 1958). The eggs of Dcndrastcr c.rccntricits, obtained in Mission Bay, San Diego, Cali- fornia, were used. At various times after fertilization, nine volumes of egg sus- pension were mixed with one volume of 1 M 2-mercaptoethanol (Eastman) in sea water. A common synonym for mercaptoethanol (HSCHXHoOH) is monothio- ethylene glycol. After various times of exposure, the eggs were washed in sea water and their development was followed. When the fertilization membrane was to be removed, this was done by treatment with a solution of Worthington "Crude Protease" in sea water (0.1 mg. per ml.). In the case of the Dcndrastcr egg, the protease may be introduced a few minutes after fertilization, and the dissolution of the fertilization membrane may be observed visually. The fact that the membrane of Dcndrastcr eggs is susceptible to protease for some time after fertilization was called to my attention by Dr. William E. Berg. RESULTS The over-all study of which this is a part concerned the blockage of mitosis by mercaptoethanol. The essential finding was that 0.1 M solutions would block division completely if applied at any time up to the time of metaphase, which takes place at about 35—40 minutes after fertilization, at 24° C. If the mercaptoethanol is applied at any time after this critical point, the cells divide without delay, and if left in the mercaptoethanol are blocked reversibly in the two-cell stage. In the course of observations on the reversibility of the block it was noted that a large proportion of those eggs which had cleaved while in the mercaptoethanol gave rise to twrin blastulae when returned to sea water. Such a population containing the twin blastulae is shown in Figure 1. These blastulae gastrulate and develop into normal plutei (Fig. 2). In order to obtain twinning, the mercaptoethanol must be applied during the period of furrowing. This is shown in Table I, where the yield of twins from eggs placed in mercaptoethanol at various times from metaphase to the completion of furrowing is given. At 35 minutes after fertilization, half of the eggs are blocked FIGURE 1A. Another group of twin blastulae, fixed in 1 per cent formaldehyde in sea water. In this experiment, evidence of incomplete twinning is seen in some individuals. FIGURE 2. Plutei produced by twinned embryos. The small plutei are the products of twinning. The large pluteus, from an egg which failed to produce twins, serves as a control. FIGURE 3. Second cleavage of Dcndrastcr egg in Ca-free sea water, showing irregular positions of blastomeres. Eggs had been placed in Ca-free sea water at 30 minutes after fertilization and exposed for 60 minutes, during which time the first and second cleavages occurred. FIGURE 4. Blastulae from eggs that had been exposed to Ca-free sea water from thirtieth to ninetieth minute after fertilization (<:/. Fig. 3). This experiment paralleled that shown in Figure 1, used the same lot of fertilized eggs and was photographed at the same time as Figure 1. Rotating blastulae gave blurred photographs. 250 DANIEL MAZIA and half have passed into the insensitive stage following metaphase. The latter are blocked in the two-cell stage. Upon return to sea water after 30 minutes in mercaptoethanol, those that had divided in the mercaptoethanol gave rise to twin embryos. Those that were blocked before the first division gave single embryos. By the thirty-eighth minute after fertilization, all of the cells had passed the critical stage, divided in mercaptoethanol and gave rise to a large proportion of twin embryos. Around 45 minutes after fertilization, when most of the cells were well advanced in cleavage at the time the mercaptoethanol was introduced, the yield of twin embryos began to decrease rapidly. If mercaptoethanol was introduced 10 minutes later, the number of twins produced was small. The critical time for twinning thus comes immediately after the critical time for mitotic blockage, as determined in the previous study (Mazia, 1958). The maximum yield of twins is obtained when the mercaptoethanol is introduced just at the time of the mitotic elongation of the cleavage furrows. The duration of the TABLE I Production of twin embryos from Dendraster eggs placed into O.I M mercaptoethanol at various times after fertilization Time after fertilization when mercaptoethanol was introduced* (minutes) Per cent cleavage in mercaptoethanol Per cent twin blastulae** 35 50 50 38 90 85 41 95 + 90 44 95 + 45 47 95 + 30 50 95 + 8 53 95 + 8 * Duration of mercaptoethanol treatment: 30 minutes. ** These percentages are relative to the total number of blastulae, and do not take into con- sideration individuals which degenerated before reaching the blastula stage. In the particular experiment from which Table I is taken, about 15 per cent of the eggs treated at 38, 41 and 44 minutes degenerated. s exposure to mercaptoethanol seems to have relatively little significance. What is important is that it acts during the brief effective period: the 5-10 minutes during which mitosis is completed and furrowing is going on. Another kind of variation of "sensitivity" to mercaptoethanol with time should be mentioned, though it has not yet received adequate study. This is evidenced by the failure of some of the eggs to form normal blastulae, single or double. This was not recorded in Table I, where the fraction of twin blastulae is given relative to the total number of blastulae. To illustrate, in the experiment given in Table I, a yield of 90 per cent twins from eggs exposed at 41 minutes after fertilization was given. In the whole population, 15 per cent of the eggs failed to form normal blastulae, so that the yield of twins could also be given as 78 per cent — still a very high figure. The best-studied methods of obtaining multiple embryos from echinoderm eggs involve modification of the ionic content of the environment, whether by removing TWINNING IN DENDRASTER 251 Ca or other ions or by varying the concentration of the sea water in the direction of hypotonicity or hypertonicity. In the present study, the effects of Ca-free sea water were compared with those of mercaptoethanol. A small volume of fertilized eggs (less than 0.5 ml.) was washed by centrifuging and re-suspending in 15 ml. of Ca-free sea water four times, beginning at 30 minutes after fertilization. The jX-"* • 7 , FIGURE 5. Dcndraster eggs with fertilization membranes cleaving in 0.1 M mercapto- ethanol. Blastomeres are not so firmly apposed as in control (Fig. 6), hut appear to be con- nected by strands of clear material (arrows). FIGURE 6. Control for Figure 5. Eggs have just completed cleavage in normal sea water. FIGURE 7. Dendr aster eggs without fertilization membranes just after cleavage in 0.1 M mercaptoethanol. FIGURE 8. Quadruplets produced when mercaptoethanol treatment is applied at both the first and second divisions. 252 DANIEL MAZIA fertilization membranes were not removed. The eggs were permitted to go to the four-cell stage in Ca-free sea water before being returned to normal sea water. It was clear by this time that the Ca-deficiency was having its expected effect on blastomere adhesion. The first cleavage blastomeres were not flatly apposed, as was the case in the control, and the planes of the second cleavages did not coincide (Fig. 3). Nevertheless, long treatment with Ca-free sea water did not cause twinning (Fig. 4). Apparently, the embryo can organize itself to form a single blastula, following the treatment with Ca-free sea water, as long as the blastomeres are held together within the fertilization membrane. This corresponds with Har- vey's (1940) experience with hypertonic sea water. It should be mentioned that the results in Figure 1 and Figure 4 were obtained with the same lot of eggs. The mercaptoethanol does visibly affect the adhesion of the blastomeres. Figure 5 shows eggs that have cleaved in mercaptoethanol, the fertilization membrane being- present. While they are compressed together, we do see rather more separation than in the controls (Fig. 6), and also see strands of glassy-appearing material between the blastomere surfaces in the furrow. If the fertilization membranes have been removed by protease, the cleavage in mercaptoethanol gives fully spherical blastomeres (Fig. 7), connected by tenuous strands, an appearance almost identical with that of membrane-free eggs that have cleaved in Ca-free sea water. It would be predicted that if the mercaptoethanol was applied again at the time of the second cleavage, quadruplets would be produced. This was the case, as shown in Figure 8. The yield of quadruplets was never as high as that of twins. This would be expected from the fact that the eggs were not as synchronous in their second cleavage. In a desynchronized population a good man}' of the embryos will either be in a stage earlier than metaphase, at which they will merely be blocked, or at a stage later than the sensitive part of cleavage (Table I ), in which case the mercaptoethanol will not be effective. Finally, it should be mentioned that the effect of mercaptoethanol could not be duplicated with ethanol or with ethylene glycol, the analogs lacking the SH group. The latter may be considered the active center, and other SH compounds might have similar effects. The writer has found none that is comparably nontoxic and therefore usable at such high concentrations. DISCUSSION Two questions demand discussion: (1) the chemical interpretation of the effect of mercaptoethanol in inducing twinning, and (2) the relation of the results to the earlier observations on twinning and on blastomere adhesion. The most reason- able interpretation of the chemistry of the observed effect is that the mercapto- ethanol is affecting some interaction between the blastomeres that involves the formation of S — S bonds. This reagent is commonly used for reducing S — S bonds in proteins (Olcott, 1942). It has been seen that its analogs lacking the SH group are ineffective in inducing twinning. The results would be in accord with the hypothesis that the blastomere interaction depends on the formation of a gel serving as a cement between the blastomeres, and would fit equally well with a hypothesis calling for the establishment of fibrous connections between the blasto- meres. The formation of protein gels by the establishment of intermolecular S — S bonds has been described by Huggins ct al. (1951). The role of S — S bonds in TWINNING IN DENDRASTER 253 the formation of protein fibers is well known from studies on the keratins. Mercap- toethanol would be expected to block such intermolecular bonding by preventing the oxidation of the SH or by competing with protein SH. The fact that the mercaptoethanol is effective only during a short period and is ineffective later would lead to the conclusion that we are dealing with the forma- tion of the inter-blastomere links during the cleavage process itself. The mercapto- ethanol is effective in preventing the formation of the links but not in splitting them once they are formed. This might mean that it acts by competition with protein SH in the formation of S — S bonds, not by reduction of S — S. It might also mean that the protein S — S becomes inaccessible to the reagent or that sufficient secon- dary bonds are formed, following the establishment of the intermolecular S — S links, to hold the structure together in the face of the reduction of the latter. In any event, the results imply that during cleavage, connections are formed between the blastomeres. The alternative explanation is that pre-existing factors tending to hold the blastomeres together (e.g. the hyaline layer as envisaged by Dan and Ono. 1952) undergo a change that renders them susceptible to mercaptothanol during the brief period of cleavage. These results do not stand in contradiction to any of the previous observations regarding the induction of twinning by other means and especially by variations in the ionic environment. These have been interpreted, with some difference of opinion as to the details, as reflecting the significance of extracellular layers having the character of calcium proteinates (Moore, 1949; Hagstrom and Hagstrom, 1954). The physical properties of such layers are known to be affected by the ionic composition of the medium ; obviously they will also depend on the protein- to-protein links that make their existence as stable masses possible. The contrast between the effects of Ca-free sea water and of mercaptoethanol in the present experiments is explicable on the assumption that the Ca-free sea water softens the layers but does not dissolve them quickly, while the mercaptoethanol actually solu- bilizes newly-appearing or pre-existing protein that would normally function in holding the blastomeres together. Thus the effect of Ca-free sea water might be reversible where the effect of mercaptoethanol was not. The most striking feature of the mercaptoethanol effect is its complete irreversi- bility with respect to the division during which the reagent was applied, and its complete lack of effect on subsequent divisions. If it is applied at first cleavage, the blastomeres are "isolated" but subsequent divisions are normal, and the end result is fully normal twins in a large proportion of the population. If it is applied again at the second division, there is a fair yield of normal quadruplets. The re- sults are not perfect : some incomplete twinning and occasional quadruplets are observed when the treatment takes place at the first division and some triplets are obtained when the treatment is given at the first and second divisions. On the whole, however, the results may be described as the effective and irreversible iso- lation of blastomeres by chemical means. SUMMARY 1. When Dcndrastcr eggs are permitted to cleave in 0.1 M mercaptoethanol in sea water and then restored to normal sea water, a large proportion of the embryos develops as twins, producing normal twin plutei. 254 DANIEL MAZIA 2. The effectiveness of the mercaptoethanol treatment is restricted to the short period during which the first cleavage furrows are forming. 3. If the treatment is repeated at the time of the second cleavage, quadruplets are produced. 4. Twins are not produced when the eggs cleave in Ca-free sea water. 5. The results are discussed in terms of the significance of the thiol groups of proteins for the interactions of blastomeres. LITERATURE CITED DAN, K., AND T. Oxo, 1952. Cyto-embryological studies of sea urchins. I. The means of fixation of the mutual positions among the blastomeres of sea urchin larvae. Biol. Bull., 102: 58-73. HAGSTROM, B., AND BRITT HAGSTROM, 1954. The action of trypsin and chymotrypsin on the sea urchin egg. E.rf. Cell Res., 6 : 532-534. HARVEY, E. B., 1940. A new method of producing twins, triplets, and quadruplets in Arbacia punctulata, and their development. Biol. Bull., 78: 202-216. HARVEY, E. B., 1956. The American Arbacia and Other Sea Urchins. Princeton University Press, Princeton, New Jersey. MUGGINS, C, D. F. TAPLEY AND E. V. JENSEN, 1951. Sulphydryl-disulphide relationships in the induction of gels in proteins by urea. Xnturc, 167: 592-593. MAZIA, D., 1958. SH compounds in mitosis. I. The action of mercaptoethanol on the eggs of the sand dollar Doidrastcr c.rcciitricus. E.vp. Cell Res. (in press). MOORE, A. R., 1949. On the precursors of the fertilization and the hyaline membranes in the eggs of the sea urchin Stronglycentrotus purpuratits. Biodynamica, 6: 107-212. OLCOTT, H. S., 1942. Monothioglycol. 'Science, 96: 454. SCHLEIP, W., 1929. Die Determination der Primitiventwicklung. Akademische Verlagsgesell- schaft, Leipzig. INHIBITORS OF REGENERATION IN TUBULARIA1 KENYON S. TWEEDELL Department of Zoology, University of Maine, Orono, Me., and Marine Biological Laboratory, IVoods Hole, Mass. When stems of Tubularia are removed from the colony and isolated from their hydranths, regeneration will occur in the isolated stems preferentially at the distal ends as regulated by an inherent polarity gradient (Child, 1941). Several factors, both intrinsic and extrinsic to the isolated stem, may govern and in many cases prevent regeneration. Of the parameters known to have an inhibitory effect, low- ering the temperature will decrease the rate of regeneration (Moore, 1939; Moog, 1941 ; Berrill, 1948) but it increases the size of the reconstituting hydranths (Moog, 1941). Similarly, Torrey (1912), Miller (1937, 1939), Earth (1937, 1938, 1940), and Rose and Rose (1941) all found that a lowering of the oxygen tension will inhibit regeneration. However, certain respiratory poisons such as cyanide or urethane (Moog and Spiegelman, 1942) will inhibit regeneration without any parallel effect on respiration. Miller (1939) and Goldin (1942a) indicated that increased hydrogen ion concentration of the sea water would reverse the normal polarity of the stems or inhibit regeneration. Later, Goldin (1942b) found that at oxygen tensions favorable to regeneration, an increase of the hydrogen ion con- centration by the addition of CO2, would cause complete inhibition. It was shown by Rose and Rose (1941) that oxygen alone will not assure regeneration unless there is sufficient cut surface of the stem open to the sea water to allow release of an inhibitor, believed to be produced by tissues of the adult organism. Similar results were reported by Goldin (1942a) using explanted coenosarc fragments and Miller (1942) who varied metabolic exchange by covering portions of the perisarc. This inhibitor, presumably a metabolic substance, was collected by Rose (1940) from colony water and later (Rose and Rose, 1941) produced from an aerated, saturated mixture of cut stems and hydranths in sea water. When this water was applied to freshly amputated stems, regeneration was blocked. The active factor was rather unstable, being heat-labile but non-volatile. Hydranths alone were found to be active but there was evidence that stems also produced a substance which made them inhibitory upon one another. Later, Steinberg (1954) showed by ligaturing stems at intervals after amputation that inhibitor production within the regenerating stem begins around 30 hours post-amputation, when the distal hydranth has become well determined. Recently, Tardent (1955) has been able to produce inhibition with "hydranth equivalent" extracts made from tissue breis of mature hydranths of Tubularia larynx. This substance does not lose its activity after sterilization or refrigerated storage. While the tissue extracts and the inhibitor water have the same effect, namely general inhibition of regeneration, certain evidence suggested that the two factors 1 This investigation was supported in part by the Coe Research Fund, University of Maine. 255 256 KENYON S. TWEEDELL were not identical. The present experiments were designed to localize the source and further identify the active factor in inhibitor water, and secondly to compare it with the inhibitory factor produced from tissue extracts of the adult organism. MATERIALS AND METHODS Throughout the experiments, Tubularia crocea collected in the Woods Hole area was used. Since its appearance can be greatly altered according to the time of year it is collected and the prevailing seasonal conditions, often considerable difficulty is attached to its identification. As originally described by Agassiz (1862) and later by Nutting (1899) and Fraser (1944), Tubularia (Parypha} crocea Agassiz grows from a dense stolon mass and is separated into long pale, almost white stems from 8 to 10 cm. high. The stems are unbranched or slightly branched, annulated sparsely at intervals and the pedicel is distinctly swollen just below the base of the hydranth. The hydranths are red, with 20 to 24 proximal and the same number of distal tentacles. The gonophores (when mature) hang in long racemes between the proximal tentacles. They consist of 10 to 12 slender branches, each of which by successive branching may bear up to 8 or more medusae. The medusae are sessile, with no apparent radiating canals. At the oral end of the female medusae there are 6 to 10 crested tentacles or apical processes which are laterally compressed. The male medusae do not possess these crested structures. The coelenteron may have one or more longitudinal endodermal partitions which form two to four incomplete channels. Additional observations have shown that the amount of branching found in this species ranges from thickly branched specimens (often caused by settling actinulae) to almost totally unbranched individuals. The mature hydranth may measure 8 to 15 mm. from tip to tip of the proximal tentacles. Secondly, as observed by Cohen (1952) and Rose (1957) the pigmentation of the hydranth can vary from red through various intergrades of orange and yellow. In the past three summers we have noted hydranths viewed with incident light ranging from rose to orange red earlier in the summer along with various intergrades of orange, yellow or white later in the summer. The latter material tends to have long pale stems and is very sparsely branched. The color in the proboscides of the medusae usually conforms to the color of the hydranth. Another late summer variety conforming to the above description has deep red-wine colored hydranths which often exhibit medusae with a brown or golden proboscis or "core" in contrast to the hydranth color. One important difference of the late summer varieties is their resistance to higher temperature. When the sea water temperature reaches about 21° C., the former variety dies out and the warm water forms survive. Collection of inhibitor zvater Inhibitor water was obtained from stems amputated from the stolon mass with the hydranths left intact. Each stem was cut separately and transferred to fresh sea water accompanied by a minimum of debris, small organisms, etc. The hy- dranths were washed in filtered sea water and then transferred to an aspirator flask containing twice-filtered sea water. The number of mature hydranths varied from two to four per nil. of collecting fluid. TUBULARIA REGENERATION INHIBITORS 257 Air bubbles which kept the stems turning over continually were generated through the flask in one of two ways. Initially, the top of the aspirator bottle was attached to a faucet vacuum aspirator and the base of the flask fitted with a clamp- regulated tube for the air intake. Later, an ordinary aquarium aerator was at- tached directly to the base spout of the bottle. In both cases the bottle was inclined with the spout down and submerged in a pan of running sea water. In this manner, during operation of the pump the hydranths and stems were continually rotated and aerated. Inhibitor water was harvested after 18 to 24 hours. The water collected was then filtered twice through No. 1 and No. 50 Whatman filter paper in a Buchner funnel, before being submitted to any other treatment. This will be referred to as plain filtered inhibitor. This solution appears slightly opaque and has a distinctive pungent odor. Microscopic examination shows that breakdown products of cellular cytolysis, bacteria and ciliate protozoans are present. Preparation of tissue extracts Large numbers of mature hydranths (250 to 550) were collected, washed in sterile sea water, drained and homogenized in a teflon-glass tissue homogenizer. The resultant brei was then centrifuged for 15 minutes at 1560 G. Several layers were produced. Floating at the top was a tough, dark red foam layer of intact cells, fibers and pigment, immediately followed by a short cap of fatty material. The major portion consisted of an opaque brown supernatant solution and at the bottom there was a dark red pigment layer covered by a white layer. The super- natant solution was re-centrifuged at 15,000 G for 15 minutes. The first and second sediment layers were re-suspended in filtered sea water and again centri- fuged at 15,000 G for 5 minutes. The combined supernatants were re-centrifuged at 21,000 G for 15 minutes. The final supernatant was then made up to 100 ml. in filtered, bacteria-free sea water. In the final solution, each ml. of extract was equal to a known number of hydranths depending on the original number. Throughout the following experiments, the criterion for regeneration was the degree of differentiation, i.e., the number of fully differentiated hydranths per input of freshly amputated stems in standing sea water. Stages of regeneration referred to are adopted from those described in detail by Davidson and Berrill (1948), Rose and Rose (1941) and Steinberg (1954). They are referred to as the inactive stage, pigmented band (primordia of the tentacles), proximal ridge (proximal tentacle striations), proximal-distal ridge (proximal-distal tentacle striation), pinched (constriction of the hydranth) and emerged (fully developed regenerate) stages. RESULTS Before attempting to analyze the active substance in inhibitor water it was deemed necessary to determine from which tissues the inhibitor originated and which were the best sources. This necessitated finding if there was any mutual inhibitory effect of the cut stems upon each other. Both Rose and Rose (1941) and Earth (1938) pointed out that crowding freshly amputated stems would retard their regeneration. In this experiment, a series of Stender dishes containing 18 ml. of standing sea water were filled with increasing numbers of freshly amputated stems. The 258 KENYON S. TWEEDELL results of 6 experiments can be seen in Figure 1. All of the stems regenerated in the dishes up to 16 stems per volume and at 24 stems per dish, at least 90 % of the cut stems went on to form fully differentiated hydranths at the same time as the controls, about 48 hours post-amputation. Beyond this, the number of regen- erates slowly dropped off. Above an input of 24 stems, the stems which were able to regenerate, did so at a considerable time after the controls. The last two points are readings taken 70 hours after amputation. While it was remarkable that so many stems would regenerate under such crowded conditions, the rate of regeneration had clearly lagged. These results suggested that if an inhibitor was being produced, it was occur- ring in sub-threshold quantities or it must come from more differentiated tissues of the regenerating hydranth. This seemed to validate an earlier report by Stein- 3 • I a. u UJ > 8 16 24 32 40 NUMBER OF STEMS PER DISH FIGURE 1. The effect of crowding upon regenerating stems in standing sea water. berg (1954) that only later stages of differentiation produce substances which inhibit earlier stages of regenerating stems. Living tissue explants It had also been reported by Rose and Rose (1941) that the presence of living adult hydranths almost completely inhibited the regeneration of stems. The effect of mature hydranths alone was therefore tested on a relatively simple level of biological assay. Fixed numbers of freshly cut stems were added to standard volumes of standing sea water. To these dishes, freshly amputated hydranths were added in increasing numbers. The purpose was to find the minimum number of hydranths which would show an inhibitory effect upon the regenerating stems. This group of experiments was conducted at temperatures between 18° and 22° C. In the first series of experiments, amputated hydranths only were placed in 18 ml. of standing sea water in Stender dishes. The number of amputated stems added was either 1, 2, 4 or 8 stems per dish as depicted by solid lines in Figure 2. Each point represents the average of six experiments. There was no inhibitory effect up to the addition of 4 hydranths per dish but as the number of freshly amputated hydranths was increased, the number of regenerates began to decrease. TUBULARIA REGENERATION INHIBITORS 259 Between the addition of 16 to 32 hydranths, the maximum number of regenerates was around 3 regardless of stem input. Complete inhibition of all stems occurred with the addition of 40 or more hydranths. This is in agreement with Tardent (1955) who found that tissue extracts of adult hydranths in approximately the same volume of sea water produced almost total inhibition with the addition of 40 hydranth equivalents. When the volume of the culture medium was increased ten-fold, as represented by the dotted curve B in Figure 2, the first indication of inhibition was seen with the addition of 16 hydranths per bowl with 8 stems. Three out of 8 stems were still able to regenerate along with 64 hydranth explants. In a second series of experiments the effects of explanted cut stems with intact hydranths upon freshly amputated stems were examined. Total prevention of re- 64 NUMBER OF HYDRANTHS PER DISH FIGURE 2. The effect of living hydranth explants upon increased numbers of regenerating stems in standing sea water. Each point represents the average of six experiments. generation became evident when the ratio of tissue explants to regenerating stems became 4:1. In all cases the stems which were inhibited almost always stopped their develop- ment at the stage when proximal and distal ridges were first becoming apparent. The stems which did regenerate in the presence of an increased amount of hydranth material, did so at a considerably later time than the controls. It was obvious that the rate of regeneration was retarded. Inhibition occurred only when freshly cut stems or those in the very early stages of regeneration were tested. If the previously amputated stems had reached the stage of proximal ridge or later before being added to the culture medium, they were unaffected by the addition of hydranths. The sensitive period to the explants appears early in the regenerative phase. These results suggested that inhibition might be caused by either an increase 260 KENYON S. TWEEDELL in tissue mass which could result in an accumulation of metabolites, a reduction in the oxygen tension or an accumulation of CO2. It has already been pointed out that a reduction in available oxygen and an increase in CO2 will prevent regeneration. Subsequently a duplicate series of dishes were set up in which the culture medium was aerated. Each dish contained 180 ml. of standing sea water and 8 stems. The dishes were aerated with an aquarium aerator and air stones. A com- parison of aerated (curve A) and the non-aerated series (curve B) can be seen in Figure 2. Whereas inhibition of regenerates becomes evident in the non-aerated dishes, the effect of additional hydranths, up to the limit studied, was abrogated by aeration in all cases. The principal effect of aeration is to drive off CO, from the water (Emmens, 1953) and the above experiments strongly indicate that it is the factor acting here. Since inhibitor water is usually collected in the presence of vigorous aeration, it did not seem likely that the results above were due to the same factor that is collected in inhibitor water. This conclusion was supported when identical groups of hydranths without stems were placed in standing sea water. After 24 hours, the culture solutions were harvested minus the hydranths and freshly amputated stems were added to the solution. None of the harvested solutions had any ap- parent inhibitor action upon the regenerating stems. It can be concluded that retardation and prevention of regeneration from both crowded stems and the addi- tion of extra living hydranths to amputated stems are not due to the same factor which is found in inhibitor water. Effects of inhibitor zt'ater From a practical standpoint, the most effective source of active inhibitor water was from cut stems with the hydranths intact, obtained by the method already described. Repeated harvests of sea water obtained from cut stems only, without the hydranths, were collected under identical conditions. These solutions had no inhibitory effect when applied to freshly amputated stems. The following sets of experiments were therefore run concurrently from re- peated harvests of inhibitor water. The results from each modification of the inhib- itor water are treated separately and the tabulations represent single experiments of ten amputated stems each. Effect of plain filtered inhibitor. In each experiment, 50 ml. of newly collected inhibitor were applied to a series of freshly cut stems, 10 stems per finger bowl. The inhibitor solution used in some experiments completely inactivated most of the stems or arrested development prior to complete regeneration in the remaining stems. Other solutions caused only inactivation of some stems and retarded regen- eration in other stems. Partial or total inhibition was in general correlated with the length of collecting time and the number of equivalent hydranths per ml. of collecting fluid. Occasionally a weak inhibitor solution was produced if the num- ber of hydranths/ml. were two or less. Strong inhibitor solutions were always obtained when three or more hydranths/ml. were vised. The results in Table I-A show that when most control stems had completely differentiated or emerged, the majority of the inhibitor-treated stems remained inactive. After 50 to 70 hours post-amputation, many of the stems which had begun a retarded development reached the pinched or emerged stage. Only 9% TUBULARIA REGENERATION INHIBITORS 261 of the treated stems actually emerged. It can be seen further that once the treated stems were inactivated, only 7% of them recovered to begin regeneration. It was found that if these inactivated stems are removed and placed in running sea water, they will recover and go on to regenerate. TABLE I Comparative effects of treated inhibitor water upon regeneration of cut stems in standing sea water Type of inhibitor preparation Hours after am- putation Number in regenerative stages Emerge Pinch Prox.-dist. ridge Prox. ridge Pigmen. band Pre- primordia In- active A. Plain filtrate 28-46 50-70 5 18 2 26 1 11 5 27 22 28 6 132 118 Control 30-52 52-69 56 79 41 18 3 3 B. Dialysis of plain filtrate 30-46 65-70 2 5 2 3 4 52 52 Control 45 15 3 2 Dialysis control 50 12 6 2 C. Norite "A" adsorption of plain filtrate 40-70 28 9 2 Filtered inhibitor 40-70 7 8 5 29 Control 40-56 30 D. Bacterial filtrate of inhibitor 32-46 56-70 5 13 6 7 6 3 70 65 Control 37-48 69 13 7 E. Dialysis of bacteria- free inhibitor 30-46 56-70 10 4 11 3 1 2 5 81 63 F. Dialysis of bacteria- free inhibitor in sea water plus chloro- mycetin 32-46 56-70 1 14 9 18 7 3 5 2 30 23 3 Control: Stems in plain sea water 45-56 52 8 4 6 Since Goldin (1942a, 1942b) and Miller (1942) had demonstrated that an increase in hydrogen ion concentration results in a decrease of regeneration, por- tions of the most potent filtered inhibitor were tested for a decrease in the pH. It was postulated that inhibition might be caused by an accumulation of CO2 in the inhibitor water or, as both Goldin (1942a) and Miller (1942) suggested, by a 262 KENYON S. TWEEDELL lowering of the pH due to the accumulation of acid metabolites. Several checks of the pH of plain sea water and freshly collected inhibitor water showed that there was a maximum drop of 0.57 in pH from that of normal sea water, pH 7.96. Part of the inhibition encountered might be the result of this increase in hydrogen ion tension either from CO2 accumulation or acid-producing metabolites. The present method of collecting inhibitor water by means of vigorous aeration would tend to drive off any excess CO2 produced in the medium which would minimize it as a source of inhibition. Goldin's figures show that a drop in initial pH of 1.12, by the addition of HC1, only reduced the number of regenerates to 7 out of 10 as com- pared to 9 out of 10 in the controls. He did not obtain complete inhibition with CO. 2 until he had decreased the initial pH of his solutions from 1.3 to 1.8 pH units depending on the oxygen concentration. It is therefore difficult to assess the effect of increased acidity presumably brought about by acid metabolites in the present experiments. Dialysis of filtered inhibitor. Routine preliminary tests for proteins made upon the filtered inhibitor were in general negative with the exception of a weak ninhydrin positive result. However, the latter result could be caused by various other con- taminates in the inhibitor water. The inhibitory action of plain filtered inhibitor water was therefore tested on newly cut stems after dialysis. Cellulose dialyzer tubing, 1" flat, was thoroughly washed with running sea water, both inside and out. (This procedure was found absolutely necessary since dialyzer tubing contains a water-soluble plasticizer that is quite toxic to Tubular ia. In fact, it was found that the rinsings of dialysis tubing would completely inhibit regeneration.) Each tube, containing 25 or 50 ml. of plain filtered inhibitor, was placed in 150 ml. of standing sea water in a finger bowl. In some cases the solution outside of the dialysis bag was a bacterial filtrate of plain sea water. Ten amputated stems were added to each bowl on the outside of the dialyzer tubing. Control dishes of amputated stems in plain bacteria-free sea water and a second control of sea water dialyzed against standing sea water containing stems were included. The results showed that the inhibitor filtrate does dialyze and the effect of in- hibition is still strongly evident after dialysis as indicated in Table I-B. When these results are compared to the inhibition produced after the direct application of the filtrate, it is seen that there was little decrease in its effectiveness even after dilution against 3 to 6 times its volume. Treatment with Norite. Concurrent with the previous experiments, part of the filtered inhibitor was treated with Norite "A." The mixture was then filtered through Whatman No. 1 filter paper producing a clear filtrate. This solution was then applied to freshly amputated stems. The results are shown in Table I-C. It is clear that adsorption by Norite almost completely removed the effect of the in- hibitor. While this treatment did eliminate the factor or factors which cause inhibi- tion, their identity was far from established. Likewise, microscopical examination of the clear filtrate also indicated that cells in suspension and microorganisms were removed. It was quite possible that the presence of cells or microorganisms was linked to activity of the inhibitor and experiments were devised to eliminate them. Earlier, the plain filtered inhibitor was subjected to high speed centrifugation at 21,000 G for one hour and the supernatant decanted off. Most of the bacteria would be TUBULARIA REGENERATION INHIBITORS 263 eliminated by this procedure. Application of this solution of cut stems showed no alteration in inhibitor activity. Since Rose (1940) had shown that heating the inhibitor to 90 or 100° C. completely inactivated it, bacterial filtration was utilized. Action of inhibitor after bacterial filtration. A clear bacteria- and cell-free solution was obtained after passing the inhibitor solution through a Handler bac- terial filter. In each experiment, ten newly amputated stems were introduced into 50 ml. of this solution. While bacteria and other microorganisms are introduced along with the stems, their growth was never observed in the straight filtrate and the solution remained clear. After 48 hours most of the controls had regenerated but the majority of the stems in the bacteria-free inhibitor were inactive or retarded in their development. See Table I-D. It was observed that many of the stems which began cell move- ment formed curious bulbular outgrowths or blebs at the distal and sometimes proximal ends of the stem. These abortive attempts to regenerate are evidence that tissue migration does occur but even the early signs of hydranth differentiation are lacking. Even so, a considerable number of retarded stems reached the fully emerged regenerative stage. It was becoming apparent that the active factor in the inhibitor was not dependent upon a continuous interaction with microorganisms. This conclusion was further supported by the next experiment. Dialysis of bacteria-free inhibitor. In experiments which were run concur- rently with those using bacteria-free inhibitor, 50 ml. of bacteria-free preparations of inhibitor were sealed in well washed dialyzer tubing. The tubing was rinsed in sterile sea water and then placed in 150 ml. of standing bacteria-free sea water in finger bowls. The inhibitive action of the bacteria-free inhibitor was still effective after dialysis as shown in Table I-E. When almost all controls had regenerated (from 45 to 56 hours), none of the treated stems had emerged. Here, again, a certain number of the retarded stems were able to recover and regenerated tardily. It can be seen by comparison with the action of filtered inhibitor alone that little activity was lost from the dilution of the inhibitor after dialysis. Since micro- organisms were presumably blocked from the bacteria-free inhibitor water con- tained in the dialysis tubing, there does not appear to be a direct interaction between them and the inhibitor factor. As a precaution against possible growth of microorganisms introduced on the amputated stems, part of the bacteria-free sea water was prepared with 0.002% of chloromycetin. The results shown indicated that the antibiotic offers some pro- tection against the inhibitor. Whereas only 23% of the stems ever regenerated when treated with the bacteria-free filtrate, 53% of the stems treated with chloro- mycetin eventually regenerated. The greatest recovery was seen when there was partial inhibition caused by a less active inhibitor. The possible explanation for this unusual result will be discussed later. Inhibition with adult tissue extracts Extracts prepared from tissue breis of the entire adult hydranth were made according to the procedure stated earlier and adopted from the technique of Tardent (1955). The hydranth extracts were found highly resistant to heat of steriliza- tion or boiling. Such treatment caused a denaturation of proteins while the re- maining filtrate was still active. This filtrate could be refrigerated for several 264 KENYON S. TWEEDELL days at 7° C. with no diminution of activity. All subsequent extracts were there- fore subjected to heat sterilization and centrifuged to throw down the precipitate. Further high speed centrifugation of the extract at 21,000 G for one hour had no effect on the inhibitor activity. Tardent's experiments were based on the addition of hydranth equivalents per 15 ml. of culture medium in which he measured the regeneration rate (length/time) of the cut stem. He produced complete inhibition after adding 20 to 40 hydranth equivalents. In experiments designed to duplicate Tardent's, we found that even the addition of one ml. of extract (equivalent to 5 hydranths) would completely TABLE II Effects of increasing amounts of hydranth tissue extracts on regenerating stems in standing sea water Amount of Stages reached Hours added sterile after extract. amputation 1 ml. = 5 hydranth equiv. Emerge Pinch Proximal- distal Proximal Pigment band Inactive 5 ml. 2 4 14 (bulbous) +50 7 10 8 12 (bulbous) 20 (bulbous) control 20 1 ml. 20 2 20 3 20 (stunted) 4 14 6 + 70 5 2 6 12 7 2 4 12 10 20 control 20 1 ml. 20 2 20 3 20 (stunted) 4 20 " +96 5 20 7 8 2 10 10 20 control 20 stop all regeneration. This amount is much lower than that which Tardent re- ported necessary for complete inhibition. In a second series of experiments shown in Table II, each bowl contained 10 stems in sea water made up to 100 ml. with increasing concentrations of extract. Each ml. of extract was equivalent to 5 hydranths. It was found that up to the addition of 3 ml. of extract, all stems could regenerate after 72 hours although they were stunted in size. The effective concentration which blocked part of the stems fell between 20 and 25 hydranth equivalents. As the concentration was increased from 5 to 7 ml. (£ to ^ hydranth equivalent per ml. of culture solution) none of the treated stems had emerged after 50 hours when all the control stems had re- TUBULARIA REGENERATION INHIBITORS 265 generated. In spite of this, examination of the experimental stems after 96 hours showed that some of the retarded stems were capable of regeneration. Those which did regenerate did so at an extremely slow rate and always resulted in stunted individuals. In particular, the proximal and distal tentacles were smaller. Com- plete inhibition, with no individuals regenerating, occurred between a hydranth equivalent concentration of 35 to 50 per 100 ml. of culture fluid (a concentration of ^ to ^ hydranth per ml. of the culture medium) . Most of the retarded stems, particularly those in hydranth equivalent concen- trations of 25 or higher, manifested large bulbous protrusions, often accompanied by concentrations of pigment at the tip but without other signs of differentiation. These were not unlike those produced by the action of inhibitor water. Dialysis of tissue extracts. Preparations of full strength hydranth extract (25 ml.) were placed in dialysis tubing and dialyzed against 125 ml. of standing TABLE III Action of hydranth tissue extracts on regenerating stems after dialysis in standing sea water Tissue extract preparation Hours after amputation Number regenerated Control (stems only) Dialysis of extract after 24 hour dialysis in running sea water + 50 + 50 10/10 Dialysis of plain extract + 36 0/10 + 50 0/10 Dialysis of boiled, precipitated, and + 36 0/20 centrifuged extract + 50 0/20 + 62 1/20 + 68 1/20 + 100 1/20 7/10 Control — dialysis of plain sea water + 37 + 62 14/20 20/20 sea water along with 10 freshly amputated stems. Identical preparations of steri- lized, precipitated and centrifuged tissue extracts were also tested. From Table III it is evident that both preparations of the hydranth extract can dialyze and inhibit regeneration of the cut stems. Dialysis tubing filled with plain sea water had no effect on the stems. Likewise, if the extract was first dialyzed against running sea water for 24 hours, the subsequent application of the dialysate to regenerating stems showed a total loss of inhibitory activity. Preliminary identification of the inhibitor. Bacterial filtrates of inhibitor water were prepared and these submitted to general biochemical tests. Routine tests of the filtered inhibitor for protein were negative and it has been shown that the active factor in the inhibitor water is dialyzable. The filtrate consistently gave a positive test with Schiff's reagent, usually regarded as specific for aldehydes. Numerous investigators have questioned this specificity and have suggested that the reagent 266 KENYON S. TWEEDELL will react with ketones and other substances with unsaturated hydrogen bonds but the evidence for this is quite conflicting (Hale, 1957). Other tests of the inhibitor solution for ketones were found to be negative. Since the active factor in inhibitor water could be adsorbed on activated char- coal, preliminary investigations were made with a variety of adsorbants in a chromatographic column. A Pyrex column was employed, 24" long X ^" internal diameter, and fitted to a suction flask attached to a faucet vacuum. Occasionally, filtration was aided by a positive pressure head supplied from an aerator. The inhibitor solutions were first adsorbed on Norite "A" and amberlite resins CG-45 and CG-50 and then eluted with either 2% ammonia in 50% ethanol or 1 % acetic acid in sea water. The resultant elutants gave positive tests with Schiff's reagent. Application of the neutralized elutants to freshly amputated stems re- sulted in inhibition but these results were not conclusive due to the nature of the solvents. As it was desired to apply these elutants to cut stems for assay, water-soluble adsorbents, magnesium oxide and aluminum oxide were used. When these col- umns were eluted with sea water, the elutants gave negative tests with Schiff's reagent and still retained some of the inhibitor activity. At this point it is not certain if the test substances positive to Schiff's reagent are identical with the inhibitor fraction. Further chromatographic analyses are anticipated. DISCUSSION Throughout the present experiments three principal observations were asso- ciated with the inhibition of amputated stems. When the inhibitory effect fell short of causing complete inhibition, a reduction in the rate of regeneration was always noted. This was measured by the length of time necessary for the regenerate to reach the fully differentiated stage of hydranth formation. Such an effect has been reported with almost every inhibitory parameter of regeneration investigated. Very often reduced rate was accompanied by a reduction in the size of the re- generating hydranths. Generally, size reduction may be correlated with a reduced rate but, as Moog (1941) has observed, a lowering of the temperature allows an increase in the size of the regenerate in Tubularia. Any regeneration rate based on length per time could therefore be subject to this and other errors. For this reason the criterion of stages in differentiation was used. Another characteristic of inhibition was the evident prevention of differentiation even though the stems displayed activity usually associated with it. In many of the non-regenerating stems knob-like blebs of tissue were formed beyond the perisarc at the distal end. Quite often both ends of the stem were so affected. These projections were probably caused by a migration of the coenosarc since the coenosarc became visibly thinner in the center of the stem but they were never accompanied by visible differentiation. A shifting movement of the entire coenosarc toward the distal end in normal regeneration, as reflected in a gradient of optical density, has been thoroughly described by Steinberg (1954, 1955). It is likely that the cellular movement seen here is of the same nature but the prevention of regeneration in the present experiments seems to be a suppression of differentiation rather than a restriction of cell movement. Inhibition of differentiation in an active regenerate can be produced in other TUBULARIA REGENERATION INHIBITORS 267 ways. Specific inhibition of differentiated parts has been strikingly demonstrated with living tissue grafts by Rose (1955, 1957). He found if grafts of developing hydranth primordia were properly orientated in a distal position to a regenerating hydranth, the grafts suppressed the differentiation of the specific like parts in the host. The influence qt the graft was so strong that it could cause the regression of specific like parts already formed. A group of experiments have been performed recently by C. Fulton (personal communication) in which he collected inhibitor water in low concentrations of streptomycin or penicillin. In most cases the growth of microorganisms was re- stricted and the water collected was not effective against regenerating stems. These results suggested that the inhibitor production was linked to microorganisms which are known to multiply during the collection of inhibitor water. In the present experiments precautions were taken against inclusion of microorganisms in the active portion of the inhibitor water but this does not rule out the significance of Fulton's observations, that the activity of inhibitor water might be due to by- products of bacteria. However, there are several reasons why the inhibitor effect may not be a direct toxic agent of bacteria or other microorganisms. First, the experiments of Rose (1940), Tardent (1955) and our own show that stems alone, devoid of hydranths, do not produce inhibitor water when collected under identical conditions. Since microorganisms do grow in stem water, this water should also be inhibitory if the bacteria are producing a toxic factor. Secondly, from the present experiments it was seen that inhibition is not always a total all-or-none effect. In a weak solution of inhibitor all stems may eventually regenerate long after the controls. Occa- sionally 1 out of 10 treated stems will regenerate along with total regeneration in the controls. If the inhibitor were a toxic factor produced by bacteria, it would completely stop all stems from regenerating. Other observations of Rose and Rose (1941), Steinberg (1954) and our own indicated that the inhibitor acts only during the early stages of regeneration. It is not likely that a toxic substance produced by bacteria would be so specific as to affect only one portion of the regenerative phase. Lastly, the introduction of antibiotics to the collecting water, in addition to having a bacteriostatic action, might also greatly reduce the effectiveness of the inhibitor. As seen in the present experiments, the addition of an antibiotic, chloromycetin, somewhat suppressed the activity of the inhibitor. Antibiotics are sometimes used to remove biologically active substances from solution. Kutsky (1953, Kutsky et al., 1956), working on the isolation of nucleoproteins from chick embryo extracts, used streptomycin to cause a specific precipitation of the nucleo- protein fraction from the supernatant fluid. The possibility that the same kind of action is occurring when inhibitor water is collected in the presence of antibiotics should not be overlooked. SUMMARY 1. A study has been made of the effects of living tissue explants, inhibitor water solutions and tissue extracts as inhibitors of regeneration in Tubularia crocea. 2. Stems alone have little inhibitory effect upon one another. 3. Living hydranth explants can cause complete inhibition of cut stems. The effective sensitive period or the period of developmental arrest extends from the 268 KENYON S. TWEEDELL time of amputation to just before the proximal ridge stage. Inhibition in these cases can be cancelled by aeration and is not due to metabolic inhibitors. 4. Complete inhibition of cut stems can be produced with harvests of culture solutions taken from cut stems with intact hydranths. No inhibition was obtained with solutions taken from stems only. There is little loss in potency after nitration, centrifugation or sterilization. The active factor withstands bacterial nitration and is dialyzable. The fresh nitrate gives a positive reaction with the Schiff reagent. It is heat-labile, susceptible to cold storage and can be adsorbed on Norite "A." Part of its activity can be removed with antibiotics and it is possible to completely adsorb the inhibitor on inorganic salts and ion exchange resins. 5. The supernatants obtained from breis of hydranth tissue were also an effec- tive inhibitor. A threshold concentration of 20 to 25 hydranth equivalents/ 100 ml. initiated inhibition and complete inhibition resulted from the addition of ^ to -J hydranth equivalent/ml, of culture medium. Fresh or boiled tissue extract will dialyze and cause complete inhibition and prior dialysis of the extract against run- ning sea water does alter its potency as an inhibitor. The extract is highly resistant to sterilization, centrifugation and storage. LITERATURE CITED AGASSIZ, L., 1862. Contributions to the natural history of the United States of America, IV, pp. 249-265, 342. BARTH, L. G., 1937. Oxygen as a controlling factor in the regeneration of Tubularia. Biol. Bull, 73: 381. BARTH, L. G., 1938. Oxygen as controlling factor in the regeneration of Tubularia. Physiol. Zool., 11: 179-186. BARTH, L. G., 1940. The role of oxygen in regeneration in Tubularia. Biol. Bull., 79 : 360. BERRILL, N. J., 1948. Temperature and size in the reorganization of Tubularia. J. E.vp. Zool., 107: 455. CHILD, C. M., 1941. Patterns and Problems of Development. Univ. of Chicago Press, Chicago. COHEN, A. L, 1952. Studies on the pigmentation changes during reconstitution in Tubularia. Biol. Bull, 102: 91-99. DAVIDSON, M. E., AND N. J. BERRILL, 1948. Regeneration of primordia and developing hydranths of Tubularia. J. E.vp. Zool., 107: 465-477. EMMENS, C. W., 1953. Keeping and Breeding Aquarium Fishes. Academic Press, New York. ERASER, C. MCLEAN, 1944. Hydroids of the Atlantic Coast of North America. Univ. of Toronto Press, Toronto. Pp. 94-102. GOLDIN, A., 1942a. Factors influencing regeneration and polarity determination in Tubularia crocea. Biol. Bull, 82: 243-254. GOLDIN, A., 1942b. A quantitative study of the interrelationship of oxygen and hydrogen ion concentration in influencing Tubularia regeneration. Biol. Bull., 82 : 340-346. HALE, A. J., 1957. The histochemistry of polysaccharides. In: International Review of Cytology, VI: pp. 193-263. KUTSKY, R. J., 1953. Stimulating effect of nucleoprotein fraction of chick embryo extract on homologous heart fibroblasts. Proc. Soc. E.vp. Biol. Med., 83 : 390-395. KUTSKY, R. J., R. TRAUTMAN, M. LIEBERMAN AND R. M. CAILLEAU, 1956. Nucleoprotein fractions from various embryonic tissues ; a comparison of physiochemical character- istics and biological activity in tissue culture. Exp. Cell Res., 10 : 48-54. MILLER, J. A., 1937. Some effects of oxygen on polarity in Tubularia crocea. Biol. Bull., 73 : 369. MILLER, J. A., 1939. Experiments on polarity determination in Tubularia regenerates. Anat. Rec., 75: (4), Suppl. 38-39. MILLER, J. A., 1942. Some effects of covering the perisarc upon Tubularia regeneration. Biol. Bull, 83: 416-427. TUBULARIA REGENERATION INHIBITORS 269 MOOG, F., 1941. The influence of temperature on reconstitution in Tubularia. Biol. Bull., 81 : 300-301. MOOG, F., AND S. SPEIGELMAN, 1942. Effects of some respiratory inhibitors on respiration and reconstitution in Tubularia. Proc. Soc. Exp. Biol. Med. N. Y ., 49 : 392. MOORE, J. A., 1939. The role of temperature in hydranth formation in Tubularia. Biol. Bull., 76: 104-107. NUTTING, C. C., 1899. The hydroids of the Woods Hole region. Bull. U. S. Fish Comm., 19: 325-386. ROSE, S. M., 1940. A regeneration-inhibiting substance released by Tubularia tissue. Biol. Bull., 79 : 359-360. ROSE, S. M., AND F. C. ROSE, 1941. The role of a cut surface in Tubularia regeneration. Physiol. Zobl, 14: 328-343. ROSE, S. M., 1955. Specific inhibition during differentiation. Ann. N. Y. Acad. Sci., 60: 1136-1159. ROSE, S. M., 1957. Polarized inhibitory effects during regeneration in Tubularia. J. Morph., 100: 187-205. STEINBERG, M. S., 1954. Studies on the mechanism of physiological dominance in Tubularia. J. Exp. Zool, 127: 1-26. STEINBERG, M. S., 1955. Cell movement, rate of regeneration, and the axial gradient in Tubularia. Biol. Bull, 108: 219-234. TARDENT, P., 1955. Zum Nachweis eines regenerationshemmenden Stoffes im Hydranth von Tubularia. Rev. Suisse de Zool., 62 : 289-294. TARDENT, P., 1956. Pfropf-Experimente zur Untersuchung des regenerationshemmenden Stoffes von Tubularia. Rev. Suisse de Zool., 63 : 229-236. TORREY, H. B., 1912. Oxygen and polarity in Tubularia. Univ. of Calif. Publ. Zool., 9 : 249. Vol. 114, No. 3 June, 1958 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY THE EFFECTS OF 2560 r OF X-RAYS ON SPERMATOGENESIS IN THE MOUSE1 JOHN H. D. BRYAN AND JOHN W. GOWEN Department of Genetics, Iowa State College, Ames, Iowa The effects of irradiation on the mammalian testis have been the subject of numerous investigations (for example Regaud and Blanc, 1906; Hertwig, 1938; Eschenbrenner and Miller, 1950; Oakberg, 1955; and Bryan and Gowen, 1956). The more recent papers of this series have been concerned with quantitative aspects of the problem. The foregoing studies have established the fact that the spermato- gonia are the most radiation-sensitive constituents of the seminiferous tubules. After exposure to radiation, spermatogonial proliferation is progressively reduced and the frequency of spermatogonia declines to a very low level. Following this irradiation-induced decrease in spermatogonia, the other cell types (spermatocytes, spermatids and sperm) disappear in the order of their development. Knowledge of the nature of the spermatogonial response is therefore of considerable importance to an approach toward an understanding of the action of irradiation on cells and tissues. Evidence has accrued which suggests that spermatogonial necrosis may be an important factor (Regaud and Lacassagne, 1927; Hertwig, 1938; and Oak- berg, 1955). In this regard conclusions based on tracer studies must also be considered. The studies of Holmes (1947), Howard and Pelc (1953), Forssberg and Klein (1954), Smellie et al. (1955) and others, clearly show that irradiation effectively inhibits DNA synthesis together with mitotic activity. Furthermore the data of Howard and Pelc (1953) indicate that the mitotic inhibition (or delay) is brought about by the failure of cells in interphase to enter the synthetic phase, rather than by interruption of synthetic processes already going on. These studies would suggest that in the case of the testis, the cessation of spermatogonial activity (through inhibition of DNA synthesis) should be a major factor in bringing about the irradiation-induced depletion of spermatogonia. The data of Bryan and Gowen (1956), derived from quantitative histological and from cytophotometric studies, are in accord with these ideas — as are the earlier conclusions of Eschen- brenner and Miller (1950) and Shaver (1953). There are, then, two rather dif- ferent responses to irradiation which have been advanced as explanations for the observed behavior of irradiated mammalian seminiferous tubules. The important 1 Journal Paper No. J-3333 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa. Project No. 1187. This work has received assistance from Contract No. AT (11-1) 107 from the Atomic Energy Commission. 271- 272 JOHN H. D. BRYAN AND JOHN W. GOWEN point is : what are the relative levels of importance which may be ascribed to either process ? It appeared likely that a comparison of results following exposure to different dose levels of x-rays would shed more light on the nature of any rela- tionship between these proposed mechanisms. In our previous paper results ob- tained following exposure to 320 r of x-rays were reported. This present paper reports data obtained following exposure to a high dose of x-rays (2560 r). These data are, where feasible, presented together with corresponding data from our 320 r experiment. As will be seen, the available evidence suggests that both mechanisms play a role in the observed radiation response of spermatogonia, the level of importance ascribable to either one depending upon the dosage levels of radiation employed. O oc \- z O O 200- 150- • — • SPERMATOGONIA-INTERPHASE O — O SPERMATOGONIA-MITOTIC A— A SPERMATOCYTE -CHROMATIN Ld O o: LU Q. 10 35 10 DAYS AFTER X-RAYS (2560r) 28 FIGURE 1. Strain Ba. Incidence of spermatogonia and spermatocytes at different times following 2560 r of x-rays. MATERIALS AND METHODS The animals chosen were 58-day-old males of strains BALB/Gw (hereinafter referred to as Ba) and S. These inbred strains of mice differ in their sensitivity to mouse typhoid. The experimental animals were irradiated in plastic tubes and were exposed to a dose of 2560 r (250 pkv, 30 ma; filtration 0.25 mm. Cu, 1 mm. Al; anode-target distance 47.5 cm., dose rate 430 r/min.). The irradia- tion was delivered to the pelvic region only, the rest of the body being shielded with lead. These conditions of irradiation are, except for the x-ray dose, identical with those of our previous studies (Bryan and Go wen, 1956). Control and irradiated animals were killed at 1, 8 and 24 hours, 3, 5, 10, 16 EFFECTS OF X-RAYS ON MOUSE TESTIS 273 and 28 days following exposure. The 28-day material is missing from the S series due to death of animals prior to this sampling time. From each animal the testes were rapidly removed and weighed. One testis was then fixed in Carnoy's acetic-alcohol (1:3) and the other used for dry weight determinations. I900-] 18001 o cc h- z o o UJ o a: I700- I600 300- 200^ IOO- SPERMATID NUCLEI O — O SPERMHEADS A— -A SERTOLI NUCLEI 135 10 16 DAYS AFTER X-RAYS (2560r) FIGURE 2. Strain Ba. Incidence of spermatids, sperm and Sertoli nuclei at different times following 2560 r of x-rays. The histological material was processed, and slides were stained, as described earlier (Bryan and Gowen, 1956). As in our previous work, the procedure of Chalkley (1943) was used to obtain estimates of the relative areas of the tubules occupied by each stage of spermatogenesis. This procedure was also used to provide data with respect to spermatogonial and non-spermatogonial necrosis during the first 24 hours following exposure to x-rays. RESULTS The data obtained are summarized in Tables I-III. Data pertaining to both strains are presented together for ease of comparison. In Table I all values are 274 JOHN H. D. BRYAN AND JOHN W. GOWEN expressed in terms of per cent of control values. These data are also expressed in graphical form in Figures 1-4. The data of Table I indicate that the relative area occupied by spermatogonia in interphase undergoes little change during the first hour after irradiation. There- after there is a pronounced and progressive decline which reaches a low point by 5 days following exposure. The data further suggest that there may be an abortive attempt at regeneration during the period of 5-10 days after x-rays, fol- lowed by a further decline (absence of spermatogonia at 16 days). With respect to the mitotically active spermatogonia, a marked contrast in response is evident. 200- o QL \- Z o o 150- • — • SPERMATOGONIA-INTERPHASE O — O SPERMATOGONIA- MITOTIC A— A SPERMATOCYTE CHROMATIN \ \ \ \ 135 10 16 DAYS AFTER X-RAYS (2560r) FIGURE 3. Strain S. Incidence of spermatogonia and spermatocytes at different times following 2560 r of x-rays. Thus by one hour after irradiation the area occupied by this class has declined to 65% of the control in the case of strain S and 30% of the control in strain Ba. In both strains at one day after exposure, the area has further declined to less than 10% of the control value. Thereafter no spermatogonial mitotic activity was recorded for strain S during the remainder of the experiment. In the case of strain Ba, the response is essentially the same except that a low level of spermato- gonial activity (4% of control) was encountered in the 10-day material. Two other cell classes disappear from the seminiferous epithelium following exposure to 2560 r of x-rays. These are the spermatocytes and spermatids. The respective areas occupied by these cells have declined to zero levels by 16 days after exposure. EFFECTS OF X-RAYS ON MOUSE TESTIS 275 In view of the recorded differences in behavior of the sperm fraction of the two strains (see Table I, and Figures 2 and 4), it is unfortunate that 28-day material from strain S was not available. The response over the period of 1-10 days is rather similar. Over the period 10-28 days, strain Ba data indicate a progressive decline to a very low level at 28 days whereas strain S, in contrast, shows a marked increase during the 10-16-day period. IOOCH 500- O a: o o 400H 300- UJ o (T UJ CL 200- SPERMATID NUCLEI* O — O SPERM HEADS / A— A SERTOLI NUCLEI / 35 10 16 DAYS AFTER X-RAYS (2560r) FIGURE 4. Strain S. Incidence of spermatids, sperm and Sertoli nuclei at different times following 2560 r of x-rays. The data pertaining to the Sertoli cell fraction indicate that the trend is similar in both strains but differs in magnitude. Thus in the case of strain Ba the rela- tive area occupied by this class undergoes a steady increase over the period 0-5 days at which time the level reached is about 2.5 times the control. This value then falls to about 2 times the control value by 10 days, and then increases again reaching at 16 days a level about 17 times the control value. In the case of strain S 276 JOHN H. D. BRYAN AND JOHN W. GOWEN TABLE I Frequency of cell types at various times after 2560 r of x-rays* Stage and strain 1 hour 8 hours 1 day 3 days 5 days 10 days 16 days 28 days Spermatogonia Ba in interphase S 103.5 90.1 72.5 73.5 51.6 29.9 13.4 5.5 7.7 1.1 13.3 8.9 0.0 0.0 0.0 Spermatogonia Ba in mitosis S 30.5 65.4 9.7 41.9 1.7 6.9 5.1 0.0 0.0 0.0 4.3 0.0 0.0 0.0 0.0 Spermatocyte Ba chromatin S 105.6 100.9 111.4 100.0 95.1 115.1 128.5 114.8 99.2 79.0 49.7 104.0 0.0 0.0 0.0 Spermatid Ba nuclei S 113.5 106.2 105.6 110.2 110.7 97.8 72.4 91.6 68.1 69.7 138.1 97.7 0.0 0.0 0.0 Sperm Ba heads S 59.9 89.8 88.9 91.2 106.3 107.3 120.8 113.5 222.2 227.5 178.2 139.7 78.4 521.6 4.4 Sertoli Ba nuclei S 97.9 121.6 107.6 143.6 181.6 172.1 223.2 293.4 250.1 510.5 212.5 227.2 1,702 1,036 1,860 2 Values expressed as per cent of controls. the corresponding values are at 5 days 5 times, and at 10 days 2.2 times the control levels. The value attained by 16 days is about 11.4 times the control level. The cause of this striking difference between the strains, at 5 days, is not clear. These changes in relative areas of the Sertoli fraction are, within limits, reflections of changes in area of the spermatogenic cells. In Table II are presented the data with respect to spermatogonial and non- spermatogonial necrosis. Data from our previous experiment (320 r) are included TABLE II Spermatogonial and non-spermatogonial necrosis at various times after 320 r or 2560 r of x-rays Strain and time after x-rays 320 r 2560 r spermatogonial necrosis non-spermatogonial necrosis spermatogonial necrosis non-spermatogonial necrosis J?a Control 3.8 4.0 2.1 2.5 3.8 4.0 2.1 2.5 ^a 1 hour O 3.4 5.8 3.0 2.4 6.0 6.5 3.7 1.2 ^a 8 hours O 9.7 8.8 2.2 1.7 15.3 14.9 3.0 2.0 ^a 24 hours 7.4 4.6 2.3 2.2 0.0 0.0 4.5 3.2 EFFECTS OF X-RAYS ON MOUSE TESTIS 277 in this table for purposes of comparison. All data in this table are expressed as percentages. Changes in area occupied by the spermatogonial fraction (normal interphasic + mitotic + necrotic spermatogonia) following exposure to x-rays are presented in summary form in Table III. TABLE III A comparison of expected and observed frequencies of spermatogonia at various times following exposure to x-rays of different dose levels Time after x-rays Strain Ba S % total spermatogonia Difference from control Difference as % of control % total spermatogonia Difference from control Difference as % of control Control 12.35 15.10 320 r 1 hour 8 hours 24 hours 14.47 +2.12 17.17 13.55 -1.55 10.26 9.89 -2.46 19.92 12.29 -2.81 18.61 10.39 -1.96 15.87 6.78 -8.32 55.10 2560 r 1 hour 8 hours 24 hours 9.11 -3.24 26.23 13.21 -1.79 11.85 10.27 -2.08 16.84 11.61 -3.49 23.11 3.32 -9.03 73.12 3.01 -12.09 80.07 DISCUSSION In the case of the testis it is clear that any treatment which interferes with, or prevents, spermatogonial mitotic activity will bring about partial or complete maturation depletion of the seminiferous tubules. Temporary depletion will follow if, for a short period of time, the level of spermatogonia is reduced much below normal thereby preventing the quantitative replacement of the spermatocyte frac- tion. Such a reduction may be brought about either by inhibition of chromosomal reduplication (and therefore of mitosis), a relatively high level of spermatogonial necrosis or by some combination of these. Depletion of the permanent type may be brought about in the same manner but with the added proviso that spermato- gonial regeneration must also be prevented. There is ample evidence on hand that exposure to x-rays brings about the onset of maturation depletion (see introduction for references). It then follows that the effects of a dose of x-rays large enough to produce a permanent absence of spermatogonia must differ quantitatively and/or qualitatively from the effects of a dose causing only temporary changes. The present work is concerned with 278 JOHN H. D. BRYAN AND JOHN W. GOWEN the response of the mouse testis to a dose of x-rays large enough to induce per- manent depletion of the seminiferous tubules. In order to facilitate comparison with the present results, a brief resume of previous results utilizing a dose of x-rays of 320 r (Bryan and Go wen, 1956) is included here. With respect to interphasic spermatogonia, our 320 r data indicate a marked decline to less than 10 % of the control value by three days following exposure. This low level remains in effect until 10 days, at which time regenera- tion commences. Spermatogonial mitotic activity follows a different course. There is an initial decline reaching a low point 8 hours after exposure, then a marked rise during the 8-24-hour period. This is followed by a further and more exten- sive decline during 1-3 days post-irradiation. Thereafter the pattern of response is essentially the same as for the non-dividing cells. The 2560 r data show marked deviations from this pattern. The area occupied by interphasic spermatogonia undergoes a rapid decline reaching a very low level by 5 days. There is a slight rise during the 5-10-day period, but the level then declines to zero by 16 days. The mitotic spermatogonia follow a similar pattern but the rate of decline during the early post-irradiation period (1-24 hours) is much more rapid. There is, then, no rise in mitotic activity during the 8-24-hour period ; nor does repopulation of the seminiferous tubules take place. These facts lend themselves to the inter- pretation, that following exposure to 2560 r of x-rays, spermatogonial mitosis must be delayed or inhibited for a longer period of time than in the case of the 320 r experiment. Furthermore the surviving spermatogonia must be unable to sustain a regenerative phase after the initial inhibitory effects of the irradiation have worn off. As stated above, spermatogonial necrosis may contribute to some extent to the depletion process. It is also to be expected that large doses of x-rays would be likely to produce a greater frequency of cell death than small doses. Hence it is probable that spermatogonial cell death may play a more prominent role in the initiation of maturation depletion following exposure to large doses of x-rays. With these points in mind, the pertinent sections were analyzed (by the Chalkley method) for spermatogonial and non-spermatogonial necrosis. These data are listed in Table II together with corresponding data obtained from the 320 r experi- ment. Exposure to either dose of x-rays increases the frequency of necrosis above control levels by one hour after irradiation. Similarly, a peak is reached at the 8-hour period followed by a return to lower levels at 24 hours. The pattern of response is therefore about the same for either dose of x-rays ; however, there is a difference in the magnitude of this response. At 8 hours after irradiation with 2560 r, the frequency of spermatogonial necrosis is almost double that found fol- lowing exposure to 320 r. Then at 24 hours following exposure no cells were encountered which could be classified as necrotic spermatogonia. These observa- tions may be interpreted in two ways. On the one hand it may mean that a large fraction of cells are heavily damaged and undergo degenerative changes even though they are far removed, in time, from mitotic activity. The remaining cells, then, constitute a less severely damaged fraction which may undergo degeneration with the onset of mitosis. This interpretation is in accord with the views of Lasnitski (1943) concerning the effect of large doses of x-rays (2500-10,000 r). The pos- sibility also exists that the observed absence of necrotic spermatogonia is correlated EFFECTS OF X-RAYS ON MOUSE TESTIS 279 with the very low mitotic rate 24 hours following exposure. This view receives some support from the reports of Glucksmann and Spear (1939) and others, to the effect that irradiation-induced cellular degeneration occurs at about the same time as the onset of mitotic activity. Then it follows that under conditions where mitotic levels are low (as in the present case) the chances of encountering necrotic cells are likewise much reduced. It is obvious from the present data that spermato- gonial nuclei survive for different periods of time (some are present at 5-10 days following exposure). This must mean that some cells have suffered less damage than others, yet this fraction also is destined to be eliminated from the tubules by 16 days following x-ray exposure. From this we may conclude that severely damaged cells may undergo degeneration prior to the onset of mitosis (in agree- ment with Lasnitski), while less severely damaged cells do not degenerate until they attempt to enter mitosis. With respect to the foregoing discussion, the relations between the levels of necrosis induced by different doses of x-rays are of significance. As Table II shows, an 8-fold increase in dose approximately doubles the frequency of necrotic cells at the 8-hour period. Although the conditions of the present experiments are quite different from those of Lasnitski (1943), who used tissue cultures of chick fibroblasts, nevertheless the results are fairly similar. This author's results indicate that a four-fold increase in dose increased the frequency of necrosis to about 1.4 times that of the low dose, whereas a 1.1 -times increase in necrosis resulted when the dose wras doubled. If a curve is fitted to these data it is found that an 8-fold increase in dose should result in the approximate doubling of the necrotic level (as observed in the present work). This offers further support for the idea that, as the x-ray dose is increased, cells further removed from the sensitive period are likely to suffer lethal injury. It may be argued that fixed and stained preparations do not allow a very accurate estimation of the frequency of necrosis. Unless the intervals between the fixation times are less than the time necessary for cells to undergo lysis and be eliminated, such estimates are likely to be minimum values. However this is open to verification. Thus from control data the total area of the tubules occupied by spermatogonia (normal + necrotic) can be determined. A comparison of this value with similar determinations on irradiated material will reveal the goodness of fit existing between the expected and observed frequencies. The important point is whether or not any irradiation-induced decrease in area occupied by normal spermatogonia can be accounted for by an increase in the necrotic value. An analysis of this kind is summarized in Table III. It can be seen that, with the exception of the Ba 320 r, one-hour material, each time period shows a deficiency of spermatogonia. Following exposure to 320 r, these deficiencies range from about 10% to 20% of control values during the first 8 hours. With respect to the 2560 r experiment, the corresponding values range from about 12% to 26% of controls. It is clear that these changes in area are, during the first 8 hours, quite similar despite the difference in dose levels employed. Since the data in Table III take into account spermatogonial necrosis, the observed deficiencies cannot be accounted for on the basis of the increased frequency of necrosis as reported in Table II. On a priori grounds it would be logical to impute the observed difference to 280 JOHN H. D. BRYAN AND JOHN W. GOWEN additional and "unobserved" spermatogonial necrosis. This is not an entirely satisfactory explanation. The stage at which cells are most sensitive to irradiation corresponds to that portion of the mitotic cycle during which chromosomal redupli- cation is taking place. Irradiation does not inhibit DNA synthesis in cells which are already in the period of synthesis, but delays these cells in entering division (see Howard and Pelc, 1953). At the time of irradiation, then, there is a fraction of spermatogonia which has passed the critical stage. These cells are most prob- ably those observed in mitosis during the first few hours following exposure. In confirmation of this are the results of Bullough and Van Oordt (1950) and others, which indicate that, in the mouse, the duration of mitosis (prophase-telophase) is of the order of three hours. Now a certain proportion of these dividing spermato- gonia transform into spermatocytes. These products of division are, therefore, lost from the spermatogonial fraction. Thus it follows that shortly after irradia- tion, the spermatogonial fraction will be decreased both by loss of these cells and by the reduction in frequency of replacement divisions. Unfortunately it is not possible to calculate the decrease in the spermatogonial fraction to be expected on these grounds. However it is evident that the deficiencies reported in Table III cannot entirely be ascribed to "unobserved" necrosis. The estimates of necrosis as determined in the present work are, therefore, reliable indices of irradiation- induced cellular degeneration. The effects described above are cumulative, and therefore any deficiency should become progressively more marked with time following exposure to large doses of x-rays. Reference to Table III shows that this is the case. Exposure to 2560 r results in reduction of spermatogonial area to 20-27% of control values by 24 hours. In addition, it was expected that the 2560 r data would show trends similar to those following exposure to 320 r, but of greater magnitude. The data of Tables II and III are in agreement with this, but the relation is somewhat obscured by varia- tion in the level of spermatogonia scored at 24 hours following exposure to 320 r. Since relatively few animals were used in these experiments, sampling errors un- doubtedly contribute to this observed variation between strains. In the case of the 320 r experiment, the surviving fraction of spermatogonia eventually repopulate the tubules. This suggests that recovery has occurred prior to the onset of mitotic activity 10 days following exposure. It is also possible to interpret these findings to mean that the surviving spermatogonia constitute a relatively more resistant fraction. Such an interpretation would be in accord with the conclusions of Eschenbrenner et al. (1948). In marked contrast are the results of the 2560 r experiment. Here, also, a small fraction of spermatogonia are present at 5 days following exposure. At 10 days, both strains show a slight increase in spermatogonia over the 5-day levels, but by 16 days, spermatogonia have been completely eliminated. These observations may be explained by the assumption of a further period of necrosis during the 10-16-day interval. This implies, in con- trast to the 320 r case, that the spermatogonia present at 5 days subsequently attempt to undergo mitosis at which time latent damage expresses itself. The spermatogonia present at 5 days following exposure to either dose of x-rays have the morphological characteristics of the so-called type A or "dusty" spermatogonia. Type A cells are regarded as "stem-line" germ cells by Clermont and Leblond (1953). These authors point out that a small fraction of Type A EFFECTS OF X-RAYS ON MOUSE TESTIS 281 spermatogonia after one division cycle become "dormant." Such "dormant" cells do not divide again until later in the spermatogenic cycle. In other words these spermatogonia remain "dormant" for about 6 days following the initiation of this inactive phase. This means that, in these cells, recovery from the effects of radia- tion exposure should be possible before mitosis recommences. With respect to our 320 r material, this apparently is the case. On the other hand, any recovery following exposure to 2560 r must only be partial since repopulation does not occur — despite the suggestion of an increase in frequency of spermatogonia by 10 days post-irradiation. On these grounds the spermatogonia present at 5 days following exposure to 2560 r (some or all of which may in fact be "dormant" type A cells) must be capable of limited division. Otherwise, the frequency of spermatogonia would not undergo the changes observed during the 5-16 day period. Further evidence which points up the more widespread damage produced by exposure to this high dose of x-rays is provided by a consideration of non-spermato- gonial necrosis. Reference to Table II shows that at 24 hours, the necrotic level has risen to 1.5-2.0 times that of the control. This increase can be largely ac- counted for on the basis of the very high frequency of degenerating metaphase I spermatocytes. It was observed that practically all metaphase I plates present in sections of these tubules were necrotic. In the 320 r experiment, the frequency of non-spermatogonial necrosis remained close to control levels. Very few necrotic metaphase I spermatocytes were encountered in this material. The present data, when considered together with the results of our 320 r experi- ment, allow several conclusions to be drawn with respect to the manner in which the histological effects of radiation exposure are brought about. Certain of these conclusions take on added significance when considered in the light of other ex- perimental approaches. Cells are prevented from entering division following ir- radiation. This may come about either through inhibition of DNA synthesis (chromosomal reduplication) or by death of the cells. Both mechanisms play a role in the irradiation-induced depletion of spermatogonia. The death of cells plays a more important role in this process following high doses of irradiation — such as 2560 r — than following exposure to low doses (320 r in the present case). Irradiation-induced mitotic inhibition would appear to be the major factor following exposure to relatively low doses of x-rays. As is readily apparent, this effect of x-rays on cells is of a very basic nature. It must perforce be taken into considera- tion if the nature of the effects of irradiation on biological systems is to be evaluated in a proper manner. It is clearly evident that different levels of injury are produced by the irradiation treatments used here. Thus following 320 r, the surviving spermatogonial fraction is capable of mitotic activity to the extent necessary for the initiation of tubule repopulation. After exposure to 2560 r, on the other hand, the survivors are incapable of such a sustained effort. SUMMARY 1. Changes in the cellular composition of the seminiferous tubules induced by exposure to 2560 r of x-rays have been analyzed by a quantitative histological pro- cedure. These data have been compared with the results obtained following ex- 282 JOHN H. D. BRYAN AND JOHN W. GOWEN posure to a much lower dose (320 r) in an attempt to gain further insight with respect to the manner in which the observed changes are brought about. 2. Exposure to 320 r results in a temporary maturation depletion of the semi- niferous epithelium. This is brought about mainly by the inhibition of spermato- gonial mitosis with irradiation-induced spermatogonial necrosis playing only a minor role. In contrast, exposure to 2560 r produces a permanent depletion due to the fact that surviving spermatogonia are incapable of sustained regenerative efforts. 3. The frequency of necrotic spermatogonia, following 2560 r, was found to be double the peak value attained in the 320 r material, or four times that of the corresponding controls. 4. Taken together, the data for the 320 r and 2560 r experiments suggest that spermatogonial depletion is brought about in two ways : ( 1 ) by suppression of mitosis due to inhibition of DNA synthesis, and (2) the killing of cells. Irradiation- induced necrosis plays a much more important role following exposure to high doses of x-rays. Even so the frequency of necrosis in either experiment did not reach very high levels, being about 9% after the low dose and about 15% in the case of the high x-ray dose. 5. Further evidence was obtained in support of the view that relatively heavily damaged cells may undergo degenerative changes prior to the onset of division, while less heavily damaged cells manifest degenerative changes only at about the time of entry into mitosis. LITERATURE CITED BRYAN, J. H. D., AND J. W. GOWEN, 1956. A histological and cytophotometric study of the effects of x-rays on the mouse testis. Biol. Bull., 110: 229-242. BULLOUGH, W. S., AND G. J. VAN OoRDT, 1950. The mitogenic actions of testosterone pro- pionate and oestrone on the epidermis of the adult male mouse. Ada Endocrinol., 4 : 291-305. CHALKLEY, H. W., 1943. Method for quantitative morphologic analysis of tissues. /. Nat. Cancer Inst., 4 : 47-53. CLERMONT, Y., AND C. P. LEBLOND, 1953. Renewal of spermatogonia in the rat. Amer. J. Anat., 93: 475-501. ESCHENBRENNER, A. B., E. MILLER AND E. LORENZ, 1948. Quantitative histologic analysis of the effect of chronic whole-body irradiation with gamma rays on the spermatogenic elements and the interstitial tissues of the testes of mice. /. Nat. Cancer Inst., 9 : 133-148. ESCHENBRENNER, A. B., AND E. MILLER, 1950. Effect of roentgen rays on the testis : Quanti- tative histological analysis following whole-body exposure of mice. Arch. Pathol., 50: 736-749. FORSSBERG, A., AND G. KLEIN, 1954. Studies on the effects of x-rays on the biochemistry and cellular composition of ascites tumors. II. Changes in the pattern of glycine-2-C" incorporation during the first two hours after irradiation in vivo. Exp. Cell Res., 7 : 480-497. GLUCKSMANN, A., AND F. G. SPEAR, 1939. The effect of gamma radiation on cells in vivo. Part II. 1. Single exposures of the fasting tadpole at room temperature. 2. Single exposures of the normal animal at low temperature. Brit. J. Radio!., 12 : 486-498. HERTWIG, P., 1938. Die Regeneration des Samenepithels der Maus nach Rontgenbestrahlung, unter besonderer Berucksichtigung der Spermatogonien. Arch. exp. Zellforsch., 22 : 68-73. HOLMES, B. E., 1947. The inhibition of ribo and thymo-nucleic acid synthesis in tumour tis- sue by irradiation with x-rays. Brit. J. Radio!., 20 : 450-453. EFFECTS OF X-RAYS ON MOUSE TESTIS 283 HOWARD, A., AND S. R. PELC, 1953. Synthesis of desoxyribonucleic acid in normal and irradiated cells and its relation to chromosome breakage. Heredity, 6 (Suppl.) : 261-274. LASNITSKI, I., 1943. The response of cells in vitro to variations in x-ray dosage. Brit. J. Radiol., 19: 250-256. OAKBERG, E. F., 1955. Sensitivity and time of degeneration of spermatogenic cells irradiated in various stages of maturation in the mouse. Radiation Res., 2 : 369-391. REGAUD, C. L., AND J. BLANC, 1906. Action des rayons X sur les diverses generations de la lignee spermatique. Extreme sensibilite des spermatogonies a ces rayons. C. R. Soc. Biol, 61 : 163-165. REGAUD, C. L., AND A. LACASSAGNE, 1927. Effects histophysiologiques des Rayons de Roentgen et de Becquerel-Curie sur les tissus adultes normaux des animaux superieurs. Arch. de I'lnstitiit du Radium, I, 1 et seq. SHAVER, S. L., 1953. X-irradiation injury and repair in the germinal epithelium of male rats. I. Injury and repair in adult rats. Amer. J. Anat., 92 : 391-432. SMELLIE, R. M. S., G. F. HUMPHREY, E. R. M. KAY AND J. N. DAVIDSON, 1955. The incorpora- tion of radioactive phosphorus into the nucleic acids of different rabbit tissues. Biochem. J., 60: 177-193. LARVAL DEVELOPMENT OF BALANUS AMPHITRITE VAR. DENTICULATA BROCH REARED IN THE LABORATORY i JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT Duke University Marine Laboratory, Beaufort, N. C., and Department of Zoology, Duke University, Durham, N. C. While there have been numerous studies on Cirripedia larvae based on recon- structed life-histories, to date there have been only three reports on nauplii reared from the egg through all larval stages to the sessile form, that of Herz (1933) for Balanus crenatus, Hudinaga and Kasahara (1941) for Balanus amphitrite hawaii- ensis, and Costlow and Bookhout (1957) for Balanus eburneus. This method has the advantage over reconstructed life-histories in that one can be sure of the identity of the adult, the source of eggs and future larvae. Once the life-histories of all species of barnacles in a given area have been described, ecological studies may be made with a greater degree of assurance. Ecological investigations based on sam- pling, such as that of Bousfield (1955), may provide the number of stages, the approximate duration and mortality of the individual stages, and the distribution and fluctuations in large populations. Laboratory studies on individually reared larvae, however, can give more detailed information on all phases of the life-history other than distribution and population fluctuations. Both types of research are required before a complete picture can be obtained. Laboratory studies are neces- sarily prerequisite to physiological and genetic investigations. Balanus amphitrite denticulata Broch is one of the most widely distributed acorn barnacles. It has been reported from the tidal waters of Britain which are artificially heated by industrial effluents (Crisp and Molesworth, 1951), the estuaries of South Africa (Sandison, 1954), and the East and West coasts of North America (Dr. Dora Henry, personal communications). In spite of its world-wide distribu- tion only the first two larval stages have been described (Sandison, 1954). Bishop (1950) believes that Balanus amphitrite denticulata merits a more thorough study and questions its position as a variety of B. amphitrite. Thus it should be of interest to compare the larval development of this variety with the descriptions of larvae of Balinus amphitrite albicostatus (Ishida and Yasugi, 1937) and Balanus amphitrite hawaiiensis '(Hudinaga and Kasahara, 1941). Balanus amphitrite denticulata is the most abundant fouling organism in the inter-tidal region at Beaufort, North Carolina and breeds during the same summer months as Balanus eburneus. During the past two years we have followed the larval development of B. amphitrite denticulata in the laboratory to determine the number of stages, the frequency of molting, and the duration of the intermolt periods. Our secondary objectives were to compare the appendage setation and body form with the corresponding naupliar stages of the other two varieties of Balanus amphitrite which have been described and with the larvae of barnacles belonging to different species and genera. 1 These studies were aided by a contract between the Office of Naval Research, Department of the Navy, and Duke University NR 104-194. 284 LARVAE OF B. AMPHITRITE DENTICULATA 285 The technique of rearing individual larvae of Balanus ainphitrite denticulata was identical to that described for Balanus eburneus (Costlow and Bookhout, 1957). Mass cultures of the larvae were maintained on a diet of CJilainydomonas sp. and fertilized Arbacia eggs at a constant temperature of 26° C. RESULTS AND DISCUSSION The nauplii of Balanus amphitrite denticulata reared individually in the labora- tory pass through 6 stages and one cyprid stage. Nauplii. The most significant morphological characteristics of each naupliar stage are given below. Stage I. (Fig. 1, I.) The curved frontolateral horns project caudo-laterally, are relatively long, and situated close to the carapace. The carapace is rounded and the abdominal process terminates in two short spines (Fig. 1, la). All setae are devoid of setules (Fig. 1, Ib, c, d). Stage II. (Fig. 1, II.) The frontolateral horns project laterally. Anterior to the small lateral spines the carapace has relatively straight lateral borders. Pos- terior to the spines the carapace tapers into the caudal process which bears a small dorsal and ventral tooth at its midpoint (Fig. 1, I la). The abdominal process bears one spine on each side of the base and the maxillules are prominent (Fig. 1, Ila). The majority of the setae now bear setules (Fig. 1, lib, c, d). Stage III. (Fig. 1, III.) The frontolateral horns are tapered from a slightly swollen basal portion adjoining the carapace. The lateral edges of the carapace are rounded and the lateral spines of stage II are missing. The abdominal process bears the same spines as in stage II (Fig. 1, Ila). Stage IV. (Fig. 2, IV.) A pair of short carapace spines marks the posterior edge where the carapace is delimited from the caudal process. Two pairs of spines are located on the abdominal process. The posterior pair is located at the base of the abdominal process and the anterior pair is in line with the division between the caudal and abdominal processes. A pair of small teeth appears just anterior to the bifurcated end of the abdominal process. Six rows of minute bristles are found on the ventral surface of the abdominal process (Fig. 2, IVa). Stage V. (Fig. 2, V.) The anterior and posterior pairs of spines of the abdominal process remain as in stage IV. Lateral to the posterior abdominal spine, however, a smaller spine is added on each side. The sub-terminal teeth and bristles remain as in the previous stage (Fig. 2, Va). Stage VI. (Fig. 2, VI.) Six pairs of small spines on the ventral surface of the abdominal process replace the bristles of the previous stage. The developing cirriform appendages are visible beneath the exoskeleton, but are not shown in Figure 2, Via. The anterior pairs of spines, found in stage V, are not present but the small paired lateral spines and large paired median spines remain. Paired eyespots develop in the later sixth stage nauplii. Cyprid. The rounded anterior end of the cyprid is the widest portion of the carapace, curving gradually to the posterior end. The degree of pigmentation varies considerably but some brown pigment was always observed. When the bivalve carapace is closed both the anterior and posterior ends are smooth. The number of naupliar stages observed for B. ainphitrite denticulata is con- sistent with our findings for Balanus eburneus (Costlow and Bookhout, 1957) and 286 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT O.I mm a a FIGURE 1. Carapace, caudal and abdominal processes, and appendages of naupliar stages I, II, and III of Balanus amphitrite dcnticulata reared in the laboratory. All swimming setae are cut short, a, lateral view of abdominal and caudal processes ; b, antennule ; c, antenna ; d, mandible. LARVAE OF B. AMPHITRITE DENTICULATA 287 a FIGURE 2. Carapace, caudal and abdominal processes, and appendages of naupliar stages IV, V, and VI of Balamis amphitrite dcnticulata reared in the laboratory. All swimming setae are cut short, a, lateral view of abdominal and caudal processes ; b, antennule ; c, antenna ; d, mandible. 288 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT for numerous species whose larval stages have been reconstructed from planktonic material. The seventh naupliar stage described for Balanus amphitrite albicostatus by Ishida and Yasugi (1937) and for Balanus amphitrite hawaiiensis by Hudinaga and Kasahara (1941) differed from the sixth stage only in the presence of com- pletely developed paired eyes. In B. amphitrite denticulata, as in B. cburneusf the development of paired eyes occurs within the sixth naupliar stage. TABLE I Comparison of appendage setation for nauplii of B. amphitrite denticulata (B.a.d.), B. amphitrite albicostatus (B.a.a.} and B. amphitrite hawaiiensis (B.a.h.) Stage Antennule Antenna Mandible Bad 04211 023-0 3222 G. 0 1 3 -03.2 2.2 G I B a a 0421- 0 1 4 -0 3 2 2 2 G. 0 1.3.-0.3.2.3.2.G. Bah 04211 023-032 2.3.G 0.1 3.-03 2.3.3.G. Bad 04211 025 -03.2 2.3.G. 0.1.4.-0.3.2.3.2.G. II B a a 04211 0 1 6 -03. 2. 2. 2. G. 0.1.3.-0.3.2.3.2.G. Bah 04211 025 -0.3. 2. 2.3. G. 0.1.4.-0.3.2.3.3.G. Bad 14211 02 5-0.3.2.2.4.G. 0.1.4.-0.3.3.3.2.G. III Baa 14211. 0 1 6-0.3.2.2.3.G. 0.1.3.-0.4.2.3.2.G. Bah 14211 025-03224G 0 1 4 -0 3 3 3 3 G Bad 1142 1.1. 03.6.-0.5.3.2.4.G. 0.1. 4.-0.4. 3.4.3. G. IV Baa 114211 027-0432 3 G 0 1 4.-0 423 3.G Bah 1.1.4.2.1.1. 0.3.6.-0.5.3.2.4.G. 0.1.4.-0.4.3.3.3.G. Bad 11142111 038-05324G 0 1.5 -04.4 4.3. G. V Baa 11142111 029-043 2.3.G. 0.1.5.-0.4.2.3.3.G. Bah 11142111 038-0 5.3 2.4 G. 0.1.5.-0.4.4.4.3.G. Bad 11142121. 048-0 5.3.2.4.G. 0.1.5.-0.4.4.4.3.G. VI B a a. 1.1 1 4 2 1 2 1. 03 9.-0.5.3.2.4.G. 0.1.5.-0.4.2.4.4.G. B a h. 1 1 1.4.2.1.2.1. 0.3 9.-0.5.3.2.4.G. 0.1.5.-0.4.4.4.3.G. B.a.d. VII Baa none 11142121 none 039-0532 4.G. none 0 1.5-0.4.2.4.4.G. Bah 11142121. 039-0532 4.G 0.1. 5 -0.4.4.4. 3. G. Since the introduction of setation formulae for appendages of barnacle nauplii by Bassindale (1936) all investigators have maintained that setation alone was not a reliable criterion for separating larval stages of different species. Some in- vestigators believe it is a useful criterion when used with other morphological characters, such as length of whole body, length and width of carapace, and body spine structure. Others believe it is merely a developmental feature which is of no value in separating larvae. Nevertheless, most authors have continued to give the setation formulae for the naupliar stages described. Knight- Jones and Waugh (1949) summarized the setation formulae for the first two naupliar stages of seven species of barnacles. They conclude that small differences and similarities in setation during the early stages are of no systematic significance and may be- LARVAE OF B. AMPHITRITE DENTICULATA 289 used only as secondary criteria for identification of stages of a given species. In order to evaluate whether setation formulae are significant or not, even as secon- dary characters, we believe it would be helpful to first determine if the formulae of closely related varieties of B. amphitrite are identical or different. If they prove to be different, and useful in separating varieties, they may well be used to separate species as well as genera. Table I gives the appendage setation for each stage of B. amphitrite denticulata and compares this variety with B. aniphitrite hawaiiensis and B. amphitrite albico- status. The antennule setation is identical for each corresponding stage of all three varieties but can be used to determine whether one is examining stage III, IV, V or VI. Using the setation of the antenna and mandible, however, all stages of the three varieties other than the fifth of B. aniphitrite hawaiiensis and B. amphi- trite denticulata can be separated from one another with the setation of the mandible being more consistently different than that of the antenna. Table II gives the other species of Balanus, as well as species of other genera, which have the same setation of individual appendages as B. amphitrite denticulata. Setation formulae from the available literature indicate that when all three pairs of appendages are considered, the first stage of B. amphitrite denticulata can be separated from all other species described. For example, B. amphitrite albico- status has identical antennule and antenna setations but the mandible is different (Table II). Stage II of B. amphitrite denticulata is identical in setation of all appendages to Balanus improvisus, Chthamalus dentatus, and Octomeris angulosa as shown in Table II by the appearance of these three species in all three columns. While the third stage may be separated from all other species by setation, the fourth stage of B. amphitrite denticulata is identical to the fourth stage of Balanus trig onus. The fifth naupliar stage has setation identical to B. amphitrite hawaii- ensis, B. eburneus, and B. improvisus, while the sixth stage nauplius is identical only to B. improvisus (Jones and Crisp, 1954). Thus, in areas where two or more of these species of barnacles are associated and there is an overlapping of the breeding season, setation could not be relied upon as the only criterion for identi- fication. It is significant, however, that after the second naupliar stage, identical setation of all appendages is confined to the genus Balanus. In B. eburneus (Costlow and Bookhout, 1957), as in Balanus crenatus (Pye- finch, 1949) the setation of each individual stage was found to be consistent. This was true also of B. amphitrite denticulata and the variability described by Norris et al. (1951) for second stage Balanus improvisus and B. amphitrite denticulata was not observed. Sandison (1954) gives the setation formulae for the appendages of the first two stages of B. amphitrite denticulata. Our findings differ from Sandison's only in the antennary exopodite of the second stage. Sandison (1954) gives 0.2.5— O.3.2.2.2.G. whereas we found 0.2.5— O.3.2.2.3.G. Another character which has proved to be useful when combined with other criteria is carapace width and length and total length, although the latter is less reliable. In cases where setation is identical, marked differences in carapace dimen- sions may be useful in identification. When dimensions are similar or overlap, however, as they do for B. amphitrite denticulata and B. eburneus (Table III), this character cannot be used. Both of these species have been reared on Chlamy- domonas sp. and Arbacia eggs at 26° C. in a continuously lighted culture cabinet. 290 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT TABLE II Setation of B. amphitrite denticulata nauplii and other species which have identical setation in one or more appendages Stage Antennule Antenna Mandible I 0.4.2.1.1. 0.2.3.-03 2 2 2.G. 0.1.3.-0.3.2.2 2.G. B. a. denticulata B. a. denticulata B. a. denticulata B. a. albicostatus B. a. albicostatus B. algicola B. a. hawaiiensis B. crenatus B. algicola B. eburneus B. balanoides B. improvisus B. crenalus B. perforatus B. eburneus B. trigonus B. improvisus C. dentatus B. perforatus* C. stellatus B. trigonus E. modestus C. dentatus M. mitella E. modestus 0. angulosa M. mitella T. serrata 0. angulosa T. squamosa T. serrata V. stroemia T. squamosa V. stroemia II 0.4.2.1.1. 0 2 5 -0 3 2.2 3 G 014-03232G B. a. denticulata B. a. denticulata B. a. denticulata B. a. albicostatus B. a. hawaiiensis B. algicola B. a. hawaiiensis B. improvisus B. improvisus B. algicola B. perforatus C. dentatus B. balanoides B. trigonus 0. angulosa B. crenatus C. dentatus T. serrata B. eburneus O. angulosa B. improvisus B. perforatus B. trigonus C. dentatus C. stellatus E. modestus M. mitella O. angulosa T. serrata T. squamosa V. stroemia III 1.4.2.1.1. 0.2.5.-0.3.2.2.4.G. 0.1.4.-0.3.3.3.2.G. B. a. denticulata B. a. denticulata B. a. denticulata B. a. hawaiiensis B. a. hawaiiensis B. a. albicostatus B. improvisus B. algicola B. perforatus B. eburneus B. trigonus B. improvisus 0. angulosa B. perforatus B. trigonus C. stellatus E. modestus M. mitella 0. angulosa T. squamosa V. stroemia LARVAE OF B. AMPHITRITE DENTICULATA 291 TABLE II — Continued Stage Antennule Antenna Mandible IV 114211. 036-05324G 0 1 4.-0.4.3.4.3.G. B. a. denticulata B. a. denticulata B. a. denticulata B. a. albicostatus B. a. hawaiiensis B. improvisus B. a. hawaiiensis B. trigonus B. trigonus B. algicola B. balanoides B. eburneus B. improvisus B. perforatus B. trigonus C. stellatus E. modestus M. mitella O. angulosa T. squamosa v 11142111 0 3.8.-0.5.3.2.4.G. 0.1.5.-0.4.4.4.3.G. B. a. denticulata B. a. denticulata B. a. denticulata B. a. albicostatus B. a. hawaiiensis B. a. hawaiiensis B. a. hawaiiensis B. eburneus B. eburneus B. algicola B. improvisus B. improvisus B. eburneus E. modestus B, perforatus B. improvisus E. modestus B. perforatus B. trigonus O. angulosa VI 11142121 04.8.-0.5.3.2.4.G. 0.1.5.-0.4.4.4.3.G. B. a. denticulata B. a. denticulata B. a. denticulata B. a. albicostatus B. eburneus B. a. hawaiiensis B. a. hawaiiensis B. improvisus B. improvisus B. algicola E. modestus B. perforatus B. crenatus B. trigonus B. eburneus E. modestus B. improvisus B. trigonus B. perforatus 0. angulosa * Norris and Crisp, 1954. All other sources indicated in text. B — Balanus 0 — Octomeris C — Chthamalus T — Tetraclita E — Elminius V — Verruca M — Mitella We have no personal observations on how these larvae compare in size with those from the natural environment. The first two stages of B. amphitrite denticulata reared by us at Beaufort are slightly larger than those described by Sandison (1954) from South Africa which in turn were larger than those found by Crisp (Sandison, 1954) in Great Britain. Body form and spine structure should also be considered with setation and body dimensions. The general body form of all nauplii of the three varieties of 292 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT Balanus ainphitrite is similar but there are differences in spine structure. The lateral spines of the second stage of B. aniphitrite denticulata (Fig. 1, II), observed also by Sandison (1954), are not found in B. aniphitrite albicostatus or B. amphi- trite hawaiiensis. Ishida and Yasugi (1937) show six pairs of small spines on the ventral surface of the abdominal process in the third stage of B. aniphitrite albico- status. These do not appear in B. ainphitrite denticulata or in B. eburneus (Cost- low and Bookhout, 1957) until the sixth naupliar stage. Earlier workers introduced into general usage the term "metanauplius," ap- parently referring to the sixth stage barnacle nauplius. Inasmuch as additional functional appendages are not added during the sixth naupliar stage, it cannot correctly be identified as a metanauplius. It is true that the six pairs of cirriform appendages appear during the sixth stage but they are underneath the exoskeleton and do not function until the cyprid stage. In the larval Cirripedia described to date no true metanaupliar stage has been described. TABLE III Measurements of larval stages of B. aniphitrite denticulata (B.a.d.) and B. eburneus (B.e.) reared under laboratory conditions at 26° C. Carapace Total length (mm.) Stage Width (mm.) Length (mm.) Bn A R /» B.a.d. B.e. B.a.d. B.e. I 0.14-0.20 0.16-0.18 0.16-0.24 0.19-0.23 II 0.19-0.24 0.21-0.23 — — 0.25-0.30 0.32-0.34 III 0.22-0.27 0.23-0.27 — — 0.30-0.34 0.35-0.38 IV 0.25-0.32 0.27-0.32 0.25-0.32 0.30-0.33 0.35-0.39 0.40-0.42 V 0.29-0.36 0.29-0.34 0.30-0.35 0.35-0.38 0.41-0.47 0.44-0.48 VI 0.35-0.43 0.36-0.39 0.35-0.40 0.42-0.50 0.47-0.53 0.54-0.60 C — — — — 0.49 0.46 The duration of individual stages of B. aniphitrite denticulata was similar to that found for B. eburneus at 26° C. The first stage was short, as in many other species, and lasted from ten minutes to six hours. The duration of the second stage was one day and that of the third stage, one to three days with an average of 1.5 days. Stage IV lasted from one to two days with an average of one day. Stage V averages 1.5 days and stage VI, 2.5 days but with greater variation than in the earlier stages. The molting frequency was similar to that observed for B. eburneus (Costlow and Bookhout, 1957). The size range of B. aniphitrite dcnticulata cyprids, 0.45 mm.-0.53 mm., over- laps the range of B. eburneus cyprids. Doochin (1951) reported an average length of 0.53 mm. for cyprids of B. ainphitrite niz>cus and notes that because of the varia- tion in length it could not safely be used to distinguish cyprids of B. aniphitrite niveus from those of B. iniprovisus. Barnes (1953) found "at least" two distinct modes in size frequency curves for B. balanoidcs and B. crenatus cyprids and interprets them as two populations of distinct sizes, developing in different environ- ments. Pyefinch (1948), noting the discrepancy in Balanus balanoidcs cyprid LARVAE OF B. AMPHITRITE DENTICULATA 293 sizes as reported by Runnstrom (1925) and Bassindale (1936), suggests that the cyprid attains a greater length in more northern waters. Pyefinch (1948) uses pigmentation and position of eyes to separate the cyprids of B. balanoides and B. crcnatus. Unfortunately, these characters are similar in B. amphitrite denticulate, and B. eburneus cyprids. In both species the anterior end is broader and the dorsal surface curves back to the posterior end. Both posterior and anterior ends are smooth when the bivalve carapace is closed, as in B. amphitrite niveus, and the notched appearance of B. improvisus cyprids (Doochin, 1951) has not been observed. No definite external characteristic has been found in this study which may be relied upon to differentiate cyprids of B. amphitrite denticulata and B. eburneus. The cyprid stage of B. amphitrite denticulata could be maintained in the labo- ratory from one to eight days but successful attachment was observed only in those which settled one to three days following the final naupliar molt. Hudinaga and Kasahara (1941) noted that the settling time of B. amphitrite hazvaiiensis varied considerably, ranging from a few hours to five days after the final naupliar ecdysis. The over-all time of development at 26° C. of B. amphitrite denticulata from hatching to the settled stage, ranged from seven to ten days, the same time interval observed for B. eburneus (Costlow and Bookhout, 1957), when reared at the same temperature. Hudinaga and Kasahara (1941) found that B. amphitrite hawaiiensis required seven days at 23-28° C. to attain the cyprid stage and usually one additional day to settle and metamorphose. Ishida and Yasugi (1937) ob- served that B. a in phi trite albicostatus attained the cyprid stage in approximately two weeks at 20-25° C. None of their cyprids settled and metamorphosed. Hudinaga and Kasahara (1941) note that B. amphitrite albicostatus required only one week for complete development to the young barnacle when fed enough Skele- tonema costatum. Yasugi (1937) reported that the goose barnacle, Mitella mitella L., required nine days for development to the cyprid at 26—28° C. but at 23-26° C. the period was extended to twelve days. Mortality in B. amphitrite denticulata was higher than that of B. eburneus. In B. eburneus the greatest mortality occurred in the fifth and sixth stages. In B. amphitrite denticulata there was no mortality in the fifth stage, 22.2 per cent in the sixth stage, and 49.2 per cent in the cyprid. While Hudinaga and Kasahara (1941) do not give the approximate number of either B. amphitrite hawaiiensis or B. amphitrite albicostatus that successfully settled, they do point out that if the food was not adequate, the time for development to the cyprid stage was increased and that survival was reduced. Survival of B. amphitrite denticulata under laboratory condition was 12.7 per cent. SUMMARY AND CONCLUSIONS From a study of 126 isolated Balanus amphitrite denticulata nauplii reared in the laboratory, plus hundreds in mass culture, the following conclusions may be drawn : 1. The free-swimming larvae of Balanus amphitrite denticulata consist of six naupliar stages and one cyprid stage. 2. Carapace size, appendage setation, and spine structure are given for each naupliar stage. 294 JOHN D. COSTLOW, JR. AND C. G. BOOKHOUT 3. Most naupliar stages of Balanus amphitrite denticulata may be separated from the larvae of B. amphitrite albicostatus and B. amphitrite hawaiiensis on the basis of setation formulae ; the fifth stage of Balanus amphitrite denticulata and Balanus amphitrite hawaiinensis have identical setation of all appendages. 4. A comparison of appendage setation with larvae of other species and genera of barnacles described to date indicates that setation formulae are not definitive in distinguishing all stages of all species. Setation is, however, a valuable criterion for identification of nauplii and may be used to separate certain stages and species. 5. At 26° C., the duration of the six naupliar stages of Balanus amphitrite denticulata is as follows : first stage, ten minutes to six hours ; second stage, one day; third stage, one to three days with an average of 1.5 days; fourth stage, one to two days with an average of one day; fifth stage, one to three days with an average of 1.5 days ; and the sixth stage, one to five days with an average of 2.5 days. 6. The duration of the cyprid stage ranges from one to eight days. Successful settling and metamorphosis were observed only in those which settled one to three days after the final naupliar molt. 7. The time required for complete larval development in the laboratory at 26° C. was seven to ten days after hatching. Successful settling and metamorphosis were observed in 12.7 per cent of the 126 nauplii studied under segregated laboratory conditions. 8. Mortality, under laboratory conditions, was highest during the sixth naupliar stage (22.2 per cent) and the cyprid stage (49.2 per cent). LITERATURE CITED BARNES, H., 1953. Size variation in the cyprids of some common barnacles. /. Mar. Biol. ' Assoc., 32 : 297-304. BASSINDALE, R., 1936. The developmental stages of three English barnacles, Balanus balanoides, Chthamalns stellatus and Verruca stroemia. Proc. Zool. Sue. London, 106: 57-74. BISHOP, M. W. H., 1950. Distribution of Balanus amphitrite Darwin var. denticulata Broch. Nature, 165: 409. BOUSFIELD, E. L., 1955. Ecological control of the occurrence of barnacles in the Miramichi estuary. National Museum of Canada, Bulletin 37. COSTLOW, JOHN D., JR., AND C. G. BOOKHOUT, 1957. Larval development of Balanus eburneus in the laboratory. Biol. Bull., 112: 313-324. CRISP, D. J., AND A. H. N. MOLESWORTH, 1951. Habitat of Balanus amphitrite var. denticulata in Britain. Nature, 167: 489. DOOCHIN, H. D., 1951. The morphology of Balanus improvisus Darwin and Balanus amphitrite niveus Darwin during initial attachment and metamorphosis. Bull. Mar. Sci. Gulf and Caribbean, 1 : 15-39. 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 haivaii- ensis. Zool. Mag. (Japan), 54: 108-118. ISHIDA, S., AND R. YASUGI, 1937. Free-swimming stages of B. amphitrite albicostatus. Botany and Zool, Tokyo, V: 1659-1666. JONES, L. W. G., AND D. J. CRISP, 1954. The larval stages of the barnacle Balanus improvisus Darwin. Proc. Zool. Soc. London, 123 : 765-780. KNIGHT-JONES, C. W., AND D. WAUGH, 1949. On the larval development of Elminius modestus Darwin. /. Mar. Biol. Assoc., 28: 413^28. NORRIS, E., L. W. G. JONES, T. LOVEGROVE AND D. J. CRISP, 1951. Variability in larval stages of Cirripedes. Nature, 167 : 444-445. NORRIS, E., AND D. J. CRISP, 1954. The distribution and planktonic stages of the Cirripede Balanus perforatus Bruguiere. Proc. Zool. Soc. London, 123 : 393-409. LARVAE OF B. AMPHITRITE DENTICULATA 295 PYEFINCH, K. A., 1948. Methods of identification of the larvae of Balanus balanoides (L.), B. crenatus Brug., and Verruca stroemia O. F. Muller. /. Mar. Biol. Assoc. 27 : 451^63. PYEFINCH, K. A., 1949. The larval stages of Balanus crenatus. Proc. Zool. Soc. London, 118: 916-23. RUNNSTROM, S., 1925. Zur Biologic und Entwicklung von Balanus balanoides (Linne). Bergen Mus. Aarbok., Naturv. Raekke, Nr. 5. 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. YASUGI, R., 1937. On the free-swimming larvae of Mitella mitella L. Botany and Zool, Tokyo, V: 792-796. SURVIVAL AND GROWTH OF CLAM AND OYSTER LARVAE AT DIFFERENT SALINITIES H. C. DAVIS U. S. Fish and Wildlife Service, Milford, Conn. Adult clams (Venus mere enaria} and oysters (Crassostrea virginica) are found, both in areas where the salinity is almost oceanic, and in areas where it is low. There is little published information on the effects of salinity on the reproductive processes of the hard clam. Turner and George (1955) reported an experiment in which early larvae of V . mercenaries were introduced into the bottom of a glass tube in which layers of sea water of diminishing salinity were placed one above the other. The larvae swam upward, through the sharp gradients that separated the layers, with no loss in velocity until they had passed the boundary between the sea water at 20.0 parts per thousand and that at 15.0 p.p.t. In the latter their velocity decreased and they no longer moved upward. Instead, they swam in a circular pattern just above the interface. Turner, in a personal communication, reported rearing clam larvae to metamorphosis at salinities of 31.0, 28.0, 24.0 and 20.0 p.p.t. He reports, however, that he had a constant mortality in 20.0 p.p.t. until, by the tenth day, he had only about 20 per cent as many living larvae at this salinity as were still living at the higher salinities. Since Korringa (1941) has reviewed the literature relating to the effects of salinity on several species of oysters, only those works dealing with American oysters will be mentioned here. Ryder (1885), Nelson (1921), Hopkins (1931), Loosanoff (1932) and other investigators have attempted to evaluate, from field data, some of the effects of salinity on various phases of oyster physiology. In addition, Loosanoff (1948, 1952) found experimentally that adult Long Island Sound oysters developed functional spermatozoa and fertilizable eggs at a salinity of 7.5 p.p.t. but that these eggs did not develop normally. In lower salinities gonad development was arrested. He found, however, in one experiment that Long Island Sound oysters, which were already ripe, spawned at salinities as low as 5.0 p.p.t. Butler (1949), in a study of oysters from upper Chesapeake Bay, con- cluded that gametogenesis was inhibited in 90 per cent of the surviving population until salinity levels rose to about 6.0 p.p.t. Amemiya (1926) and Clark (1935) studied the salinity range for the develop- ment of fertilized eggs, of the American oyster, into shelled larvae. Both concluded that 14.5 or 15.0 p.p.t. was the lower limit for normal development and that 39.0 p.p.t was the upper limit. Amemiya, however, believed the optimum salinity for development was from 25.0 to 29.0 p.p.t., whereas Clark thought the optimum was at 23.0 p.p.t. Nelson (1921), in New Jersey waters, observed active free-swimming larvae in salinities ranging from 5.17 to 28.80 p.p.t. From this and his observation that the adult oyster closed and refused to feed at salinities below 10.42 p.p.t., 296 BIVALVE LARVAE AND LOW SALINITIES 297 Nelson concluded that oyster larvae (p. 38) "may become accustomed to much lower densities than the adult animal will stand, and still remain active." Prytherch (1934) studied the salinity limits for the attachment and meta- morphosis of oyster larvae and found that they could attach in salinities from 5.6 to 32.2 p.p.t., but that beyond these limits (p. 71) "no setting occurred though many of the larvae crawled for periods of over four hours." Moreover, although (p. 71) "setting was accomplished with considerable regularity in salinities ranging from 9.0 to 29.0 p.p.t.," beyond these limits only a small percentage of the larvae was able to complete the process. He believed that the salinity range from 16.0 to 18.0 p.p.t. was optimum for setting. In the present study we have re-investigated the effect of salinity on develop- ment of fertilized eggs of the American oyster, C. virginica, into shelled larvae to obtain quantitative data for estimating the relative percentage of eggs developing normally in different salinities. Similar studies were also made on eggs of the hard clam, V . mercenaria. In addition, we determined the effects of several lowered salinities on the survival and growth of free-swimming larvae of both clams and oysters after they had reached the straight-hinge stage. DEVELOPMENT OF FERTILIZED EGGS AT DIFFERENT SALINITIES Methods Our laboratory tap water cannot be used, without treatment, to lower the salinity because it contains enough metallic ions, chiefly copper, to be toxic to de- veloping eggs. In Experiment No. 1 we did use tap water to lower the salinity but added a chelator to bind up the excess metal ions. In Experiment No. 2 we used distilled water to dilute our usual sea water. This water was from a Stokes still that discharged into a tin-lined storage tank. In all subsequent experiments a Barnstead BD-2 demineralizer was our source of salt-free water, and this water was stored either in Pyrex carboys or polyethylene tanks. High salinity water was obtained by evaporation of our sea water in poly- ethylene containers until the salinity was 44.52 p.p.t. This water was then stored in a Pyrex carboy until used in these experiments. The salinities tested in Experi- ment No. 3 were obtained by making appropriate dilutions of this high salinity water with our usual sea water (27.0 p.p.t.) . In Experiment No. 4 all the salinities were obtained by diluting the high salinity water with demineralized water. The salinities tested in Experiment No. 5 were obtained by diluting our usual sea water with demineralized water. The animals used in these experiments were spawned in the usual manner (Loosanoff and Davis, 1950; Davis, 1953). Fertilized eggs of both oysters and clams were thus obtained free of the body tissues and excessive sperm that may have affected the results of earlier workers, who used stripped eggs and sperm. For each experiment eggs from several females were pooled and an equal number of eggs was taken from this mixed lot to start cultures at each of the different salinities. All containers were then covered, to prevent loss of water by evapora- tion, and placed in the constant temperature bath at 23.0° C. Forty-eight hours later the contents of each culture vessel were screened and the number of normal straight-hinge larvae determined. Experimental errors, including transfer of the 298 H. C. DAVIS eggs to the experimental culture vessels, recovery of the larvae and sampling, can probably account for differences of not more than ±10 per cent in any individual experiment. Salinity tolerance of developing eggs of V . mercenaria Eggs of the hard clam of Long Island Sound can develop into normal straight- hinge larvae only within the relatively narrow salinity range of 20.0 to 32.5 p.p.t. TABLE I Comparison of the percentage of eggs, of oysters and clams, that develops to normal straight-hinge larvae in sea water of different salinities. Highest number developing to straight-hinge larvae from each spawning taken as 100 per cent. Experiments No. 3, No. 4 and No. 5 from same spawning Salinity (in p.p.t.) Eggs of Long Island Sound oysters Eggs of Peconic Bay oysters Eggs of Long Island Sound clams Exp. No. 1 Exp. No. 2 Exp. No. 3 Exp. No. 4 Exp. No. 5 Exp. No. 3 Exp. No. 4 Exp. No. 5 44.5 0 40.0 0 35.0 56 29 0 1.0 32.5 99 35 52 34 30.0 92 78 54 84 27.5 75 100 Control* 89 87 88 88 92 92 92 100 (26.0- 27.0) 25.0 85 91 22.5 100 100 94 100 80 84 20.0 90 82 84 76 21 16 17.5 79 91 58 63 0 0 15.0 64 73 48 0 12.5 <0.1 <0.1 18 0 10.0 0 0 0 0 7.5 0 0 0 0 5.0 0 0 2.5 0 0 * Control — Milford Laboratory sea water. (Table I). At a salinity of 35.0 p.p.t. only one per cent or less of the eggs de- veloped into shelled larvae, and at 17.5 p.p.t. none of the eggs developed into normal shelled larvae. Even at 20.0 p.p.t. only 16 to 21 per cent of the eggs developed into straight-hinge larvae and at 32.5 p.p.t. only 34 to 52 per cent reached this stage. Thus, the salinity range for practical work, extending from 22.5 to 30.0 p.p.t., is narrower than the biological or absolute range (20.0 to 32.5 p.p.t.). In our experiments, the optimum salinity for the development of clam eggs was about 26.5 to 27.5 p.p.t. ' BIVALVE LARVAE AND LOW SALINITIES 299 Salinity tolerance of developing eggs of C. virginica Some eggs of Long Island Sound oysters, conditioned at 26.0 to 27.0 p.p.t., developed into normal straight-hinge larvae in salinities as low as 12.5 p.p.t. (Ex- periments No. 1 and No. 2, Table I, and Experiment No. 6, Table II). The highest percentage of normal straight-hinge larvae was obtained at a salinity of 22.5 p.p.t. The percentage of normal larvae obtained in salinities below 22.5 p.p.t. decreased progressively, in most experiments, with each successive decrease in salinity down to 15.0 p.p.t. Below 15.0 p.p.t. the percentage of normal larvae decreased abruptly and in a salinity of 12.5 p.p.t. less than 0.1 per cent of the eggs developed normally. TABLE II Comparison of the relative percentage of normal straight-hinge larvae obtained at salinities (a) from eggs of oysters from different areas at the same salinity and (b) from eggs of oysters from the same area at different salinities Oysters conditioned and spawned at Hodges Bar oysters that developed 26.0-27.0 p.p.t. gonads at about 8.74 p.p.t.* Oysters spawned in Long Island Peconic Hodges Hodges Sound Bay Bar Bar Exp. No. 6 Exp. No. 7 Exp. No. 6 Exp. No. 7 7.5 p.p.t. 10.0 p.p.t. 15.0 p.p.t. Exp. No. 8 Exp. No. 8 Exp. No. 8 Control 26.0-27.0 p.p.t. 100 100 82 91 0 0 0 25.0 p.p.t. 99 86 80 94 0 0 0 22.5 p.p.t. 100 87 90 100 0 0 26 20.0 p.p.t. 100 86 100 91 7 26 72 17.5 p.p.t. 92 76 87 81 50 76 92 15.0 p.p.t. 37 58 60 45 48 100 100 12.5 p.p.t. <0.1 13 1.4 11 83 92 89 10.0 p.p.t. 0 0 0 0 100 98 78 7.5 p.p.t. 0 0 0 0 99 96 72 5.0 p.p.t. 0 0 0 0 0 0 0 2.5 p.p.t. 0 0 0 0 0 0 0 * These oysters were kept, for four days prior to spawning, at the salinities at which they were induced to spawn. * Eggs of oysters from Peconic Bay, where the salinity may be as high as 31.0 p.p.t., showed about the same percentage developing in each of the lower salinities as did eggs of Long Island Sound oysters, except that a slightly higher percentage of these eggs developed at a salinity of 12.5 p.p.t. (Experiments No. 4 and No. 5, Table I, and Experiment No. 7, Table II). In salinities above 22.5 p.p.t. the percentage of normal larvae again decreased progressively with each successive increase in salinity up to 35.0 p.p.t. Because of the comparatively high percentage of eggs that developed normally in 35.0 p.p.t., we suspect that at least a few eggs would have developed in 37.5 p.p.t. (not tested), although in 40.0 p.p.t. none developed normally. The salinity tolerance of eggs of Maryland oysters from Hodges Bar, a low salinity area, differed only slightly from that of eggs of Long Island Sound oysters when both groups of parent oysters were conditioned at 26.0-27.0 p.p.t. (Experi- 300 H. C. DAVIS merits No. 6 and No. 7, Table II). However, when Maryland oysters from the same area developed gonads in their native habitat, where the salinity was only 8.74 p.p.t. at the time they were collected, and were spawned in salinities of 7.5, 10.0 and 15.0 p.p.t., the eggs developed into normal straight-hinge larvae at 10.0 p.p.t., and larvae, normal in shape and only slightly smaller in size, developed at 7.5 p.p.t. (Experiment No. 8, Table II). Even at 5.0 p.p.t. many of the eggs developed into very early shelled stages before they died. The upper salinity limit for development of normal larvae from eggs of oysters which developed gonads and were spawned at low salinities was also appreciably lower than for eggs of oysters from the same area that developed gonads and were spawned at 26.0— 27.0 p.p.t. None of the eggs produced at low salinities developed into normal larvae at salinities above 22.5 p.p.t. EFFECT OF LOWERED SALINITIES ON GROWTH OF LARVAE Methods To determine the effect of lowered salinities on growth of larvae we started with straight-hinge clam and oyster larvae 48 hours old. These larvae were obtained by spawning clams and oysters in sea water at our normal salinity (26.0-27.0 p.p.t.). Several 18-liter cultures of the eggs of each species were then set up in sea water at that salinity and permitted to develop for 48 hours. At the end of this period the larvae from all the cultures were collected on stainless steel screens to give a single combined culture of clam larvae and one of oyster larvae. The number of larvae per ml. in each combined culture was determined and appropriate volumes used to start duplicate cultures of clam larvae and duplicate cultures of oyster larvae at each of the salinities tested. In both experiments the water in which the larvae were kept was changed every second day. Since the food used was grown in sea water of our normal salinity, it was added before the salinity was adjusted after a change of water. In the first ex- periment additional food was given to the cultures on the days between changes, as was our usual practice. The salinity could not be adjusted after this additional food was given, however, and it was found that this increased the salinity of the cultures appreciably. The increase in salinity, while only about 0.5 p.p.t. in cul- tures in which the nominal salinity was 22.5 p.p.t., was as much as 1.5 p.p.t. in cul- tures nominally at 10.0 p.p.t. and lower. In the second experiment the salinities were maintained at the nominal level. About iy2 times the usual amount of food was given on the days when the water was changed and the salinity adjusted, but no additional food was given until the next change of water. Effect on clam larvae The results of the two experiments on clam larvae were in general agreement in that growth of larvae was comparatively good at salinities of 20.0 p.p.t. and higher (Fig. 1). In both experiments larvae were reared to metamorphosis at these salini- ties. In the first experiment there was no appreciable difference in growth of the larvae at 20.0 p.p.t., 22.5 p.p.t. and at our normal salinity (26.0-27.0 p.p.t.). How- ever, in the second experiment, in which the salinities were controlled more care- BIVALVE LARVAE AND LOW SALINITIES 301 fully, the rate of growth of larvae decreased progressively at each successively lower salinity, as shown by the average size at each measuiing period. At a salinity of 17.5 p.p.t., although growth of the clam larvae was significantly slower than at normal salinity, some larvae did reach metamorphosis. These larvae were sluggish and, apparently, more susceptible to disease than were larvae kept at higher salini- ties. Thus, even though some larvae reached metamorphosis, such a high mortality occurred during and immediately after setting that we were unable to follow their growth further. FIRST EXPERIMENT 27.0 PPT. (CONTROL) 22.5 PPT. .. .:....: •..' '- -"- '.--..• ""'".. ' ' '--'-'i 1 " " ".". — — — — : — .•. — ; 7-] 20.0 PPT. •,.. ;., ,,","•;• : ,"":; ,•; ', ;.' "•„•,•,", : ;-r.t 17.5 PPT. GROWTH INCREMENT F^2ND TO I2TH DAY 15.0 PPT. '•Ki - - - •• ~ Z ; ! • : . S3 12.5 PPT. ALL DEAD BY I2TH DAY SECOND EXPERIMENT 27.0 PPT. (CONTROL) 22.5 PPT. 20.0 PPT 175 PPT. GROWTH INCREMENTS C3$332ND TO 4TH DAY 15.0 PPT. ESD4TH TO 6TH DAY |=]6TH TO 8TH DAY ES]8TH TO IOTH DAY r~niOTH TO I2TH DAY 12.5 PPT. 95% MORTALITY BY 8TH DAY A i t i r i 0 110 120 130 140 ISO 160 170 180 190 200 210 220 MEAN LENGTH IN MICRONS FIGURE 1. Growth of clam larvae at different salinities. Samples were taken and meas- urements made only at the beginning and termination of the first experiment. In the second experiment, salinities were more carefully controlled and samples, from each of the duplicate cultures at each salinity, were taken every second day. One hundred larvae from each sample were measured. Clam larvae kept at a salinity of 15.0 p.p.t. grew even more slowly than those kept at 17.5 p.p.t. They were sluggish and susceptible to attack by protozoa, fungus and bacteria. In each experiment some larvae lived more than 12 days, but all died before reaching setting size. At a salinity of 12.5 p.p.t., a few clam larvae survived for 10 or 12 days, but did not grow. In the second experiment, when samples were taken every two days, a slight but progressive decrease in size of larvae kept at this salinity was noted. In both experiments, it was observed that at salinities of 12.5 p.p.t. and lower, the shells of dead clam larvae were completely disintegrated in approximately 48 hours. The progressive decrease in size of larvae at 12.5 p.p.t., therefore, suggests that the shells, even of living larvae, were being slowly dissolved. 302 H. C. DAVIS 27.O PPT. (CONTROL) GROWTH INCREMENTS E222ND T06TH DAY 22.5 PPT. EIS36TH TO 10 TH DAY BZZZiJBZBJiZ^^ 1 MOTH TO 14 TH DAY 20.0 PPT. 17.5 PPT. 15.0 PPT. &%^VW^^MW^w&VM^W£t-*-';':*;*:"- •:•:•:•: •:-:•:•: •:-:-:-:-:-:»:-:-:*:-:-:i 1 1 12.5 PPT. 1 10.0 PPT. 1 7.5 PPT. B(M:::;:::::::::tz=| A. , 1 _x_, ._ i i , 80 90 100 no 120 130 140 ISO 160 MEAN LENGTH IN MICRONS FIGURE 2. Growth of oyster larvae at different salinities. Samples, from each of the duplicate cultures at each salinity, were taken on the sixth, tenth and fourteenth days. One hundred larvae from each sample were measured. (CONTROL) 22.5 PPT. 200 PPT 175 PPT. 15.0 PPT 12.5 PPT IO3 P ' PT i90-95% MORTALITY BY I4TH DAY 7.5 PPT. MORTALITY BY I2TH DAY 5.0 PPT. NO GROWTH -100% MORTALITY BY 12 TH DAY 2.5 PPT. 100% MORTALITY BY 6TH DAY GROWTH INCREMENTS E222ND TO 6TH DAY TO IOTH DAY IOTH TO I4TH DAY 70 80 90 100 110 120 130 MEAN LENGTH IN MICRONS 140 ISO 160 FIGURE 3. Growth of oyster larvae at different salinities. Salinities were more carefully controlled than in the previous experiment. Samples from each of the duplicate cultures at each salinity, were taken on the sixth, tenth and fourteenth days. One hundred larvae from each sample were measured. BIVALVE LARVAE AND LOW SALINITIES 303 At salinities of 10.0 p.p.t. or lower, clam larvae showed no growth and all were dead within six days. The lower borderline salinity for clam larvae appears to be about 17.5 p.p.t. Clam larvae and set would probably survive and grow slowly at this salinity if all other conditions were nearly ideal, but would probably die if some other environ- mental factor were unfavorable. It would seem exceedingly doubtful that condi- tions in nature would ever be so favorable that clam larvae could survive, reach setting stage, and continue to grow at salinities of 15.0 p.p.t. or lower. Effect on oyster larvae Oyster larvae grew at a comparatively normal rate in all salinities down to and including 12.5 p.p.t. However, a salinity of 17.5 p.p.t. was optimum, by a slight margin, for growth of larvae at least through the tenth day (Figs. 2 and 3). In both experiments larvae kept in a salinity of 15.0 p.p.t. had, by the fourteenth day, attained an average length equal to or slightly greater than those kept at 17.5 p.p.t. In the first experiment the larvae kept at 12.5 p.p.t. were, by the fourteenth day, slightly larger than those at any other salinity, and even the larvae kept at 10.0 p.p.t. were almost as large as the controls (Fig. 2). In the first experiment, however, due to the addition of food on the days when the water was not changed, the salinities of these cultures actually ranged from 12.5 to about 14.0 p.p.t. and from 10.0 to 11.5 p.p.t., respectively. In the second and other experiments, in which the salinity wras more carefully controlled, growth of larvae at 12.5 p.p.t. was appreciably slower than at 15.0 p.p.t., while larvae kept at 10.0 p.p.t. grew con- siderably slower than the controls, and in some experiments mortality was high. In the first experiment, in which the salinity of the cultures nominally at 7.5 p.p.t. actually ranged from 7.5 to 9.0 p.p.t., some oyster larvae lived through the 14 days of the experiment, although growth was slow and mortality high (Fig. 2). In the second experiment, and others in which the salinity was held at 7.5 p.p.t., the larvae appeared to feed and seemed quite normal for the first few days, even though growth was very slow (Fig. 3). By the eighth to tenth day at this salinity, how- ever, oyster larvae appeared moribund and by the twelfth day mortality was almost complete. At a salinity of 5.0 p.p.t. oyster larvae appeared moribund within 48 hours. After four days almost all were completely dead, and in only a few could ciliary motion be detected. Thus far, we have the results of only one experiment using larvae of oysters from low salinity areas in Maryland. While the results of a single experiment are not always reliable, this experiment indicated that when these oysters are con- ditioned at a salinity of 26.0-27.0 p.p.t. their larvae do not tolerate any lower salinities than do larvae from Long Island Sound oysters conditioned at the same salinity. As a matter of fact, in this experiment the optimum growth of the Maryland larvae was at 22.5 p.p.t., and the rate of growth decreased progressively with each successive decrease in salinity below this optimum. Effect on older oyster larvae The results of the above experiments, in which we started with straight-hinge larvae, could be interpreted as indicating that larger larvae (10 to 14 days old) 304 H. C. DAVIS can grow as fast as or faster, at a salinity of 15.0 p.p.t. (or even 12.5 p.p.t. in the first experiment), than at 17.5 p.p.t. We believed, however, that this was because the larvae at 17.5 p.p.t., being larger at 10 days, were more severely handicapped by insufficient food. Davis and Guillard (in press) have shown that it requires approximately four times the quantity of food given these cultures to maintain maximal growth of larvae after they reach an average length of 140 p, yet such a quantity of food would have handicapped the smaller or slower growing larvae at the other experimental salinities. To test the above hypothesis in another experiment we determined the rate of growth of older larvae at salinities of 26.0-27.0 p.p.t. (control), 17.5, 15.0, 12.5, 10.0 and 7.5 p.p.t. A culture of oyster larvae, that had been reared to a mean length of 165 p. at normal salinity, was divided into six smaller cultures and one TABLE III Comparison of growth of older oyster larvae at different salinities. Measurements are in microns Control 26.0-27.0 p.p.t. 17.5 p.p.t. 15.0 p.p.t. 12.5 p.p.t. 10.0 p.p.t. 7.5 p.p.t. Initial mean length 165.55 165.55 165.55 165.55 165.55 165.55 Length after 8 days at different 206.30 214.00 203.05 205.75 186.80 175.49 salinities Increase 40.75 48.45 37.50 40.20 21.25 9.94 of these was kept at each of the above salinities. Eight days later it was found that the larvae kept at 17.5 p.p.t. had grown the fastest, while there was very little difference in size between the controls and those kept at 15.0 or 12.5 p.p.t. (Table III). The larvae kept at 10.0 p.p.t., however, had increased in size only about one-half as much as the controls, and those kept at 7.5 p.p.t. had increased only about one-fourth as much as the controls. These cultures were continued for several more days to get an indication of the setting rate of larvae in the different salinities. Those kept in 17.5 p.p.t. gave significantly more spat than any of the other cultures, but setting was also good at 15.0 and 12.5 p.p.t. A few spat were obtained at 10.0 p.p.t. but the larvae kept at 7.5 p.p.t. all died before metamorphosis. DISCUSSION Our experiments on the development of fertilized clam eggs indicate that even with a salinity as high as 22.5 p.p.t., the reproductive potential of clams may be reduced as much as 15 to 20 per cent. If the salinity is reduced to 20.0 p.p.t., the reproductive potential of clams is reduced 80 to 85 per cent and if the salinity over the spawning beds should be as low as 17.5 p.p.t., there appears to be no possibility of obtaining normal larvae. BIVALVE LARVAE AND LOW SALINITIES 305 Once clam larvae have attained the straight-hinge stage, we find, as did Turner (personal communication), that the larvae grow quite well at 20.0 p.p.t. but con- trary to Turner's results we find no significant mortality at this salinity. Turner and George's (1955) observation that the larvae swam upward, the normal reac- tion, until they came into the sea water at 15.0 p.p.t. also appears significant. We find that at both 17.5 and 15.0 p.p.t. the larvae appear sluggish, grow slowly and suffer high mortality either prior to reaching setting stage (15.0 p.p.t.) or during metamorphosis (17.5 p.p.t.). The minimum salinity at which a good percentage of clam eggs develops into straight-hinge larvae is 22.5 p.p.t. Once the larvae have attained this stage, however, they survive and grow well at a salinity as low as 20.0 p.p.t. Thus, as Loosanoff, Miller and Smith (1951) showed for the temperature requirements of eggs and larvae of V . mercenaries, the embryonic stages cannot tolerate as wide a range of salinities as can the larval stages. Similarly, Chanley (in press) found that small juvenile clams (1.8 to 3.6 mm. in length) survived for a month or more at 15.0 p.p.t. but died in salinities of 12.5 p.p.t. or lower, while larger juveniles (5.0 to 21.5 mm. in length) survived at 12.5 p.p.t. Much additional research is needed to find the minimum salinities at which adult clams develop gonads, to find whether the salinity at which the parents develop gonads influences the salinity tolerance of the eggs and larvae, and to find whether races tolerant of low salinities exist or can be developed. By comparison with the status of research on clams, the relation of salinity to the reproductive processes of oysters appears to be fairly well documented. Thus, the findings of Loosanoff (1948, 1952) and Butler (1949) appear to agree quite well that 6.0 to 7.5 p.p.t. is the minimum salinity for the development of gametes by the American oyster. Additional research is required, however, to determine more clearly the value of gametes produced at low salinities. In general, our results on the development of fertilized eggs of oysters condi- tioned at a salinity of 27.0 p.p.t. are in close agreement with those of Amemiya (1926) and Clark (1935). However, possibly because of improvements in tech- nique, use of larger cultures and repeated trials, or due to differences in the salinities at which the oysters developed gonads, we have been able to demonstrate that a few eggs of such oysters will develop normally at 12.5 p.p.t. or about 2.5 p.p.t. lower than found by earlier workers. The very close agreement of the results of Amemiya (1926), Clark (1935), and ours with oysters conditioned at 26.0-27.0 p.p.t., researches so widely separated in time, space and populations sampled, suggested that the salinity tolerance of eggs of the American oyster was quite similar throughout the range of the mollusk. Preliminary tests with Maryland oysters from Hodges Bar, a low salinity area, that had been artificially conditioned at 27.0 p.p.t., appeared to confirm this sug- gestion. Additional research, however, showed that this was not true when these oysters developed gonads in their native habitat where the salinity was only 8.74 p.p.t. at the time the oysters were collected. When these oysters were spawned at salinities of 7.5, 10.0 or 15.0 p.p.t., comparatively normal larvae were obtained in a salinity as low as 7.5 p.p.t. It may be that eggs of oysters developing gonads at even lower salinities can develop at salinities below 7.5 p.p.t. Additional research is needed to determine the value of larvae developing at 306 H. C. DAVIS such low salinities. Will such larvae survive, grow and set? Will they grow and set at lower salinities than larvae developing at higher salinities? Our observation that, once they have reached the straight-hinge stage, oyster larvae may live for some time at salinities as low as 5.0 p.p.t. suggests that the larvae Nelson (1921) reported swimming at a salinity of 5.17 p.p.t., and those observed by many other workers at low salinities, may simply be survivors of larval populations accidentally carried into these low salinities. Our observations, even of older larvae, at 10.0 p.p.t., indicate that growth at this salinity is extremely slow although a very few of the larvae kept at this salinity did set. Perhaps larvae from oysters that develop gonads at low salinities survive better and grow faster at salinities of 10.0 p.p.t. and lower than do larvae from oysters conditioned at 26.0-27.0 p.p.t. Otherwise, our observations on growth, coupled with Prytherch's (1934) observations on the setting of larvae at different salinities, would seem to suggest that oyster sets, occurring in areas where the salinity is only 10.0 p.p.t. or lower, are dependent upon larvae that grow almost to setting size at higher salinities and are carried to low salinity setting areas as practically fully mature larvae. Moreover, Chanley's results (in press) indicate that recently set spat, like the larvae, grow best at salinities near 17.5 p.p.t. and that growth is significantly retarded by salinities of 10.0 p.p.t. or lower. Unlike the larvae some of his spat grew slowly at a salinity of 5.0 p.p.t. although only about 40 per cent survived at this salinity. The author wishes to express his deep appreciation of the valuable counsel and assistance given by Dr. V. L. Loosanoff, Director of Milford Laboratory. Many thanks are also due to Mr. J. B. Clancy for the Peconic Bay oysters, to our col- leagues at the Annapolis laboratory for the Maryland oysters, to Mr. C. A. Nomejko for preparing the figures and slides, to Miss Norma Pritchard and Miss Beverly Boyne for many of the larval measurements, and to Miss Rita Riccio for her careful editing of the manuscript. SUMMARY 1. The optimum salinity for the development of straight-hinge larvae from eggs of clams from Long Island Sound is about 27.5 p.p.t. 2. The salinity range for development of eggs of these clams is from 20.0 p.p.t., at which salinity only 16 per cent to 21 per cent of the eggs develop, to 35.0 p.p.t., at which salinity only 1 per cent or less of the eggs develops normally. 3. The optimum salinity for growth of clam larvae after they reach the straight- hinge stage is 27.5 p.p.t. or higher, while 15.0 p.p.t. is the lowest salinity at which appreciable growth occurs, and 17.5 p.p.t. is the lowest at which we were successful in rearing clam larvae to metamorphosis. 4. Both the optimum salinity and the salinity range for the development of straight-hinge larvae from eggs of the American oyster appear to be governed by the salinity at which the parent oysters develop gonads. 5. The optimum salinity for the development of eggs of oysters from Long Island Sound, Peconic Bay, and Hodges Bar, Maryland was about 22.5 p.p.t. when these oysters developed gonads at a salinity of 26.0-27.0 p.p.t. 6. When Hodges Bar oysters developed gonads at a salinity of approximately 8.74 p.p.t. the optimum salinity for the development of their eggs was between BIVALVE LARVAE AND LOW SALINITIES 307 10.0 and 15.0 p.p.t. and appeared to be dependent upon the salinity at which the parent oysters were kept immediately prior to spawning. 7 . The salinity range for development of normal straight-hinge larvae from eggs of these low salinity oysters was from 7.5 to 22.5 p.p.t., whereas the range for eggs from oysters conditioned at 26.0-27.0 p.p.t. was from 12.5 to above 35.0 p.p.t. 8. The optimum salinity for growth of larvae of Long Island Sound oysters, conditioned and spawned at 26.0-27.0 p.p.t., was 17.5 p.p.t. 9. The optimum salinity for growth of larvae of Hodges Bar oysters, conditioned and spawned at 26.0-27.0 p.p.t., appeared to be about 22.5 p.p.t. 10. It is still undetermined whether the optimum salinity for growth of larvae is influenced by the salinity at which the parent oysters develop gonads. LITERATURE CITED AMEMIYA, I., 1926. Notes on experiments on the early developmental stages of the Portuguese, American and English native oysters, with special reference to the effect of varying salinity. /. Mar. Biol. Assoc., 14: 61-175. BUTLER, P. A., 1949. Gametogenesis in the oyster under conditions of depressed salinity. Biol. Bull., 96: 263-269. CHANLEY, P. E., 1957. Survival of some juvenile bivalves in water of low salinity. Proc. Nat. Shellfish. Assoc., Vol. 48 (in press). CLARK, A. E., 1935. Effects of temperature and salinity on early development of the oyster. Prog. Kept., Atl. Biol. St., St. Andrews N. B., No. 16: 10. DAVIS, H. C., 1953. On food and feeding of larvae of the American oyster, C. virginica. Biol. Bull., 104: 334-350. DAVIS, H. C., AND R. R. GUILLARD. Relative value of ten genera of micro-organisms as foods for clam and oyster larvae. U. S. Dcpt. of Interior, Fish and Wildlife Service (in press). HOPKINS, A. E., 1931. Factors influencing the spawning and setting of oysters in Galveston Bay, Texas. Bull. Bur. Fisheries, 47: 57-83. KORRINGA, P., 1941. Experiments and observations on swarming, pelagic life and setting of the European flat oyster, Ostrea edulis L. Arch. Neer. Zool., 5: 1-249. LOOSANOFF, V. L., 1932. Observations on propagation of oysters in James and Corrotoman Rivers and the Seaside of Virginia. Va. Comm. of Fish., Neivport News, Va., 1-46. LOOSANOFF, V. L., 1948. Gonad development and spawning of oysters (O. virginica) in low salinities. Anat. Rec., 101 : 55. LOOSANOFF, V. L., 1952. Behavior of oysters in water of low salinities. Conv. Address, Nat. Shellfish. Assoc., 135-151. LOOSANOFF, V. L., AND H. C. DAVIS, 1950. Conditioning V . mercenaria for spawning in winter and breeding its larvae in the laboratory. Biol. Bull., 98: 60-65. LOOSANOFF, V. L., W. S. MILLER AND P. B. SMITH, 1951. Growth and setting of larvae of Venus mercenaria in relation to temperature. /. Alar. Res., 10: 59-81. NELSON, T. C., 1921. Aids to successful oyster culture. I. Procuring the seed. N. J. Agric. Exp. Sta., Bull. 351, 1-59. PRYTHERCH, H. F., 1934. The role of copper in the setting, metamorphosis and distribution of the American oyster, Ostrea virginica. Ecol. Monogr., 4: 49-107. RYDER, JOHN A., 1885. New system of oyster culture. Science, 6: 465-467. TURNER, H. J., AND C. J. GEORGE, 1955. Some aspects of the behavior of the quahaug, Venus mercenaria, during the early stages. Eighth Rept. on Invests, of Shellfish, of Mass., Dept. of Nat. Res., 5-14. RESPIRATION IN TISSUES OF GOLDFISH ADAPTED TO HIGH AND LOW TEMPERATURES DONALD R. EKBERG Department of Physiology, University of Illinois, Urbana, Illinois Poikilothermic animals adapted to low temperatures are very different physio- logically from warm-adapted animals. These differences have been detected in terms of lethal temperatures, metabolic variations, and other gross changes. In recent years an interest has arisen in the cellular mechanisms of these changes. Peiss and Field (1950) measured the oxygen consumption of minced brains and sliced livers from cold- (0°-1° C.) adapted cod (Boreogadus saida) and from warm- (25° C.) adapted golden orfe (Idus melanotus}. Differences in Q02 of both liver and brain were in the same direction as for intact animals. Liver and brain excised from the cold-adapted animals consumed oxygen at a higher rate than did the same tissues excised from the warm-adapted animals when measured at the same temperature. The Q10 of the orfe tissues was greater than that of the cod tissues, particularly in the 0° C.-100 C. temperature range. Similar results were reported for goldfish (Carasshis auratus) brain breis by Freeman (1950). However, for muscle tissue, the oxygen consumption was greater in muscle from warm-adapted than from cold-adapted fish. These findings on muscle were corroborated by the studies of Suhrman (1955). In an attempt to localize the cellular mechanisms of temperature adaptation, Precht and his co-workers have recently shown alterations in enzyme activity in tissues from animals adapted to various temperatures (Precht, Christophersen and Hensel, 1955). Christophersen and Precht (1952) observed that the catalase activity of the liver of the carp (Carrasius vulgaris Nils) adapted to 1° C. was greater than that of liver from fish adapted to 26° C. However, the dehydrogenase activity (rate of decolorization of methylene blue) was found to be greater in the liver of the warm-adapted animals. On the other hand, Precht (1951) found that the liver catalase activity of the eel (Anguilla vulgaris L.) adapted to 26° C. was greater than that of liver catalase from animals adapted to 11° C. ; the dehydro- genase activity of liver or muscle brei decreased as the adaptation temperature increased; the catalase activity of eel muscle appeared to be independent of the adaptation temperature. In potato beetles (Leptinotarsa decemlineata Say) adapted to 3° C. and 24° C. Precht (1953) found that blood catalase and tissue succinic dehydrogenase were greater in their activity in the cold-adapted than in the warm-adapted insects. The high temperature of adaptation wras only 17.5° C. ; Precht also observed that higher temperatures (24° C.) greatly increased locomotor activity in these insects with a concomitant increase in dehydrogenase activity. MATERIALS AND METHODS Adaptation of animals. The experimental animals (Carassius auratus} were obtained from Grassyfork, Inc.. Martinsville, Indiana. These goldfish varied in 308 RESPIRATION IN TISSUES OF GOLDFISH 309 weight from 20 to 80 grams. Upon arrival at the laboratory they were put into a series of tanks which were continually being renewed with aerated dechlorinated water. Pulverized Purina Dog Chow was placed into these tanks for one day. After this period the fish were transferred to continuously aerated water in the adaptation tanks, which consisted of seven-gallon aquaria immersed in water at 10° and 30° C. The fish were kept in these adaptation tanks for two weeks without feeding prior to experimentation. The water in the tanks was completely changed every week. Preparation of tissues for manometric studies. Following the adaptation period a fish was removed from the adaptation tank, blotted with a paper towel, and weighed. The animals were then killed by severing the spinal cord immediately posterior to the head with a pair of scissors. The brain was removed first and blotted to remove blood and meninges; then the body cavity was opened by two connecting incisions — one median ventral and one ventral transverse. Thus, the liver could be removed piece-wise by scraping it away from the intestine, spleen, and gall bladder. Then the gill filaments were cut away from the gill arches, washed in frog Ringer's, and placed in the medium described below. Gills are quite thin and thus may be used in a Warburg vessel without homogenization or mincing. Ringer's Solution NaCl 6.5 g./l. KC1 0.15 g./l. CaCl2 0.12 g./l. 70 parts NaHCO3 0.20 g./l. Na2HPO4 0.01 g./l. 0.3 M glucose 5 parts 0.16 M Na-pyruvate 4 parts 0.1 M Na-succinate 7 parts 0.16 M Na-L glutamate 4 parts Phosphate buffer pH 7.5 13 parts 0.021 M Na2HPO4 1 part 0.160MKH2PO4 The above mixture was kept frozen in the refrigerator prior to use. New mixtures were made up weekly. The medium is similar to that prepared by Krebs (1950) for mammalian tissues. Each vessel contained 2.5 ml. of the above medium and gill filaments plus 0.2 ml. of 10 per cent KOH and fluted Whatman No. 1 filter paper in the center well. Brain and liver homogenates did not survive well at temperatures above 20° C. Thus, if the oxygen consumption were measured at temperatures of 10°, 18°, and 26° C. successively with the same tissues, it was observed that the oxygen con- sumption at 26° C. was decidedly lower than if the Q02 had been measured initially at this temperature. The homogenate technique was discarded, therefore, in favor of the following: The tisues were removed from the animal and placed on filter paper on a Petri dish over ice. The tissues were then minced with a razor blade and placed into the previously mentioned medium. It was found that the oxygen consumption of a given sample of tissue could be measured at two tem- 310 DONALD R. EKBERG peratures (14° and 22° C., 10° and 30° C.) without appreciable loss of activity at the higher temperature. Cyanide inhibition of respiration. The method used in these studies was es- sentially that of Robbie (1948). The center well contained 0.4 ml. of 0.42 M Ca(CN)2 in 10 per cent Ca(OH)2. This mixture equilibrates with the vessel fluid to give a final cyanide concentration of 10~3 M. However, the equilibration time is about one hour at 26° C. and to alleviate this delay 0.1 ml. of 2.8 X 10~2 M KCN was placed into the vessel fluid of 2.7 ml., thus giving a final concentration of 10~3 M cyanide. Samples of the vessel fluid were removed at the end of an experiment and analyzed for cyanide by the phenolphthalin method of Robbie (1948). It was found that the equilibrium concentration of cyanide was not appreciably altered by the above procedure. The control vessels contained 0.4 ml. of 10 per cent Ca(OH),. Iodoacetate inhibition of respiration. Since iodoacetate produces its effect on living systems by combining with SH groups of various enzymes, it is to be expected that its action will be non-specific. However, Adler and co-workers (1938) have shown that triose phosphate dehydrogenase (TDH) is more sensitive to iodoacetate than a number of other sulfhydryl enzymes. Recently Kelly and co-workers (1955) have used iodoacetate in small concentration (5.4 X 10~4 M) to block glycolysis in adrenal tissue Thus, it was hoped that a similar study utilizing goldfish gills would yield information concerning the activity of the hexose monophosphate shunt in this tissue. The following medium was used : Control 0.3 M glucose Phosphate buffer Ringer's solution 5 parts 30 parts 70 parts Experimental 0.3 M glucose Phosphate buffer 15 mg. iodoacetic acid in 100 ml. Ringer's solution 5 parts 30 parts 70 parts Thus, the final concentration of iodoacetate in the vessel was 5.4 X 10 4 M. It was found that iodoacetate produced only a small inhibitory effect on the oxygen consumption of whole gills. Before considering that this lack of marked inhibition is due to a high level of activity of the hexose monophosphate shunt it was necessary to know whether the iodoacetate was entering the cells. At pH 7.5 the iodoacetic acid is highly ionized, and if one assumes that only the un-ionized form enters the cells, then a method for increasing the amount of the un-ionized form would be to lower the pH. However, a drop of 2-3 pH units would decidedly inhibit oxygen consumption ; thus the homogenate technique was employed to facilitate diffusion of the iodoacetic acid into the cells. The results of the rate-temperature studies for goldfish brain, liver, and gills are given in tabular form in Tables I and II and in graphic form in Figure 1. The data for the cyanide and iodoacetate inhibition studies are given in Table III. Two tests were used for the level of significance : Student's Test as modified by Fisher and reported by Patau (1943) and the Mann- Whitney U Test as reported by Siegel (1956). The latter test is much simpler to run than the t test, and since it is a nonparametric test as opposed to the parametric t test, one need not concern RESPIRATION IN TISSUES OF GOLDFISH 311 TABLE I Oxygen consumption of brain and liver from goldjish adapted to 10° C. and from goldfish adapted to 30° C. Temperature of measurement in ° C. Qoj (jjl./mg. dry weight/hour) P value Adaptation temperatures 10° C. Number of animals 30° C. Number of animals / M-W Brain 10 0.813 5 0.700 5 0.37 0.11 14 0.992 5 0.887 5 0.90 0.42 22 1.53 5 1.50 4 0.93 0.45 30 2.27 4 2.18 4 0.92 0.17 Liver 10 0.621 4 0.454 4 0.76 0.10 14 0.907 6 0.649 5 0.04 0.09 22 1.39 3 0.849 3 0.08 0.05 26 1.36 8 1.19 6 — 0.286 30 1.44 4 1.41 4 — 0.56 himself with the stringent assumptions of the t test. The probabilities for assuming random distribution are given for both tests in Tables I-III. The five per cent level is taken as the minimum level of significance. Brain and liver oxygen consumption. The differences in oxygen consumption for brain and for liver from goldfish adapted to 10° C. and 30° C. as shown in Table I and Figure 1 are small and not statistically significant. Gill oxygen consumption. The oxygen consumption of gills from goldfish adapted to 10° C. was in all cases greater than that of gills from fish adapted to 30° C. as shown in Table II and Figure Ic. However, in the sequence of meas- TABLE II Oxygen consumption of gills from goldfish adapted to 10° C. and from goldfish adapted to 30° C. Qoj (pl./mg. dry weight/hour) Tempera- P value ture of Month of Adaptation temperature measure- ment in study °C. 10° C. Number of animals 30° C. Number of animals t M-W 10 August 0.405 3 0.255 3 0.07 0.002 14 July 0.706 3 0.448 3 0.14 0.10 22 July 1.37 4 0.858 4 0.001 0.014 30 August 1.60 3 1.00 3 0.013 0.05 10 February 0.257 8 0.106 6 0.002 0.002 18 February 0.557 8 0.262 6 0.0006 0.001 26 February 1.074 8 0.522 4 0.0002 0.002 312 DONALD R. EKBERG urements from July to February all of the curves (Fig. Ic) are shifted down on the rate axis. This indicates a seasonal variation upon which temperature acclima- tion is superimposed. Homogenized gills (controls for the IAA experiments) had a lower oxygen consumption, 0.553 /xl/mg./hr. for the 10° C. gills and 0.193 jul/mg./hr. for the 30° C. gills than did whole gills (Table II), but the Q02 3000 t 2000 10 «-• O ^ - * 1000 CM o o 500 - 30°C -30° 26° 22°C. I I I 14° 10°- I I I I I 330 340 350 I/T X 105 3000 E 2000 \ ro »_• O .c CM O O 1000 500 LIVER IO°C 30° C -30° 26° 22°C I I I I 330 340 350 I/T X 105 2000 » E ro O 1000 800 600 400 CM O O 200 100 C AUGUST GILLS FEBRUARY 30° C. 26°C. 22°C. I I \ FEBRUARY V-7 I8°C. I4°\ IO°C. I I ^i 330 n^ 340 I/T X I05 350 FIGURE 1. Arrhenius plot of the oxygen consumption of brain (a), liver (b), and gills (c) from goldfish adapted to 10° C. (solid lines) and from goldfish adapted to 30° C. (broken lines). See Tables I and II for statistical data. RESPIRATION IN TISSUES OF GOLDFISH 313 for gill homogenates from cold-adapted fish was significantly higher than homoge- nates from warm-adapted fish. Cyanide inhibition of oxygen consumption. The results of the effects of 10~3 M cyanide on the oxygen consumption of goldfish liver and gills are summarized in Table III. A small non-significant difference is noted for liver, while the gills from fish adapted to 30° C. were more resistant to cyanide poisoning than gills from fish adapted to 10° C. lodoacetate inhibition of oxygen consumption. The data for this group of experiments are summarized in Table II. lodoacetate had little or no effect on the oxygen consumption of intact gills, yet IAA strongly inhibited the gill homoge- nates. This suggests that permeability is the limiting factor. This is in contrast to the cyanide studies in which the effect of cyanide was fairly rapid (inhibition was maximum after a 10-minute equilibration period). Since no definite state- TABLE III The effect of 10~3 M cyanide (Nov.-Dec.~Jan.) and 5.4 X 10~* M iodoacetic acid (Feb.-March) on the oxygen consumption of tissues from goldfish adapted to 10° C. and from goldfish adapted to 30° C. when measured at 26° C. Tissues Per cent inhibition of oxygen consumption P value Adaptation temperature 10° C. Number of animals 30° C. Number of animals / M-W Cyanide Liver 85.9 7 81.2 7 0.17 0.13 Gills (whole) 79.1 7 57.5 7 0.024 0.009 lodoacetate Gills (homoge- nized) 52.6 7 77.4 7 — 0.027 ments can be made concerning the number of cells broken in the homogenate or of the amount of iodoacetate that actually entered the cells, it is impossible to estimate accurately the amount of metabolic activity accounted for by the hexose mono- phosphate shunt. However, these results do indicate that gill homogenates from 10° C.-adapted fish are more resistant to iodoacetate poisoning than are gills from fish adapted to 30° C. ; this is opposite to the effect of cyanide. DISCUSSION In view of the negligible temperature adaptation differences in oxygen con- sumption observed in goldfish liver and brain, it appears that further studies of oxygen consumption in these tissues would be of doubtful value. On the other hand goldfish gills appear to be an ideal tissue for this type of study in that gills from fish adapted to 10° C. do show a significantly higher rate of oxygen consump- tion than do gills from fish adapted to 30° C. when measured at temperatures between 10° C. and 30° C. 314 DONALD R. EKBERG Figure 2 indicates that there is a definite decrease in oxygen consumption by goldfish gills during the period from July to February regardless of the adaptation temperature. Seasonal variations in oxygen consumption of intact goldfish have not been reported. However, Hoar (1955, 1956) has demonstrated a greater resistance to high temperature in summer than in winter for goldfish adapted to the same temperature. A comparison of the rate-temperature curves reveals no apparent difference between slopes of oxygen consumption by gills from animals adapted to 10° C. or 30°. C. for any given month of measurement. Yet, this does not exclude the pos- sibility that an enzyme or a group of enzymes may change in such a way that the net effect is unnoticeable. Precht et al. (1955), as previously mentioned, have shown that enzymes such as dehydrogenases and catalase change their activity with a change in adaptation temperature. However, he has worked with homogenates and thus may not con- clude that a certain enzyme has increased or decreased in concentration, but only that its activity has increased or decreased. One must consider the following pos- sibilities as affecting the activity of a certain enzyme : ( 1 ) change in permeability of the cell membrane to substrate molecules ; (2) release within the cell of some activating agent or of some inhibitor; (3) inactivation of a chemical inhibitor or activating agent initially present within the cell ; (4) increase or decrease of the actual amount of enzyme present ; (5) change in intrinsic properties of the enzyme itself. Present data do not allow a distinction to be made among these possibilities. However, Kaplan (1954, 1955), Fraser and Kaplan (1955), and Kaplan and Paik (1956) have attempted to describe the Euler effect (an increase in enzyme activity when the enzyme is extracted from the cell) on yeast catalase by progressive elimination of (1) to (4) above, thus leaving (5) as the operating mechanism. Cyanide inhibition of gill oxygen consumption. The increased resistance of gills from 30° C.-adapted goldfish to 10~3 M cyanide, as measured by oxygen con- sumption, may be interpreted in a number of ways. (1) There is an increase in the amount of one or more of the cytochromes in the gills from 30° C.-adapted fish. (2) The cytochrome system of the gills from 30° C.-adapted fish is changed in such a manner that it is less sensitive to cyanide. (3) An oxidative pathway which is less sensitive to cyanide than is the cyto- chrome system is increased in activity in the gills from 30° C.-adapted fish. In view of the fact that the oxygen consumption of the gills from 30° C.-adapted fish is lower than that of gills from 10° C.-adapted fish, it does not appear that ( 1 ) offers an adequate explanation of the observed differences in cyanide sensitivity. The second possibility (2) must be considered as a possible explanation and merits further study. It seems more probable, however, that in the gills of fish adapted to high temperatures the oxidative pathway is shifted awray from the cytochrome system (3). If the activity of the oxidases and aerobic dehydrogenases were increased, this in turn could increase the activity of the peroxidases and catalase. lodoacetate inhibition of gill oxygen consumption. If one may assume that iodoacetic acid is primarily blocking triose phosphate dehydrogenase, then it would RESPIRATION IN TISSUES OF GOLDFISH 315 appear that the hexose monophosphate shunt is operating at a higher capacity in the gills of 10° C.-adapted fish. Such an increase in shunt activity suggests (Clock and McClean, 1954) a correlation between protein synthesis and the shunt. In cold-adapted fish gills there may exist a higher rate of protein synthesis than in warm-adapted fish gills. This in turn would increase the need of cold-adapted fish gills for oxygen. These inhibitor studies suggest that the cytochrome system has become less sensitive to cyanide poisoning or that an alternate pathway less sensitive to cyanide than the cytochrome system has increased in activity in gills from fish adapted to 30° C., and that the hexose monophosphate shunt may be more active in gills from 10° C.-adapted fish. The writer is deeply indebted to Professor C. Ladd Prosser for his helpful suggestions during the experimentation period and during the preparation of the manuscript. SUMMARY AND CONCLUSIONS 1 . No statistical differences were observed in the oxygen consumption by either brain or liver homogenates from goldfish adapted to 10° C. and from fish adapted to 30° C., when measurements were at temperatures between 10° and 30° C. 2. The oxygen consumption of gills from goldfish adapted to 10° C. was found to be significantly higher at all temperatures of measurement, than the oxygen consumption of gills from goldfish adapted to 30° C. 3. No difference was noted in the slopes of the rate-temperature curves of the gills from fish adapted to 10° and from fish adapted to 30° C. as measured by oxygen consumption. 4. A seasonal variation in the oxygen consumption of goldfish gills was noted, in that the oxygen consumption of the gills decreased from July to February. This was true regardless of the temperature of adaptation. However, significant dif- ferences were observed between 10° C. and 30° C. gills throughout the experimental period. 5. The oxygen consumption of gills from fish adapted to 30° C. was inhibited to a lesser extent by 10'3 M cyanide than was the oxygen consumption of gills from fish adapted to 10° C. 6. The oxygen consumption of gills from goldfish adapted to 10° C. was in- hibited to a lesser extent by 5.4 X 10"* M iodoacetate than was the oxygen con- sumption of gills from fish adapted to 30° C. 7. It is suggested that temperature acclimation may be associated with changes in relative activities of CN~ and IAA sensitive enzymatic pathways. LITERATURE CITED ADLER, E., H. VON EULER AND G. GUNTHER, 1938. Dehydrasen und Jodessigsaure. Skand. Arch. Physiol, 80: 1-15. CHRISTOPHERSEN, J., AND H. PRECHT, 1952. Untersuchungen zum Problem der Hitzeresistenz I. Versuche an Karauschen (Carassiits vnlgaris Nils). Biol. Zcntralbl., 7: 313-326. FRAZER, M. J., AND J. C. KAPLAN, 1955. The alteration of intracellular enzymes. III. The effect of temperature on the kinetics of altered and unaltered yeast catalase. /. Gen. Physiol., 38: 515-547. 316 DONALD R. EKBERG FREEMAN, J. A., 1950. Oxygen consumption, brain metabolism and respiratory movements of goldfish during temperature acclimatizations; with special reference to lowered tem- peratures. Biol. Bull, 99: 416-424. CLOCK, G. E., AND P. McCLEAN, 1954. Levels of enzymes of the direct oxidative pathway in mammalian tissues and tumours. Biochem. J., 56: 171-175. HOAR, WILLIAM S., 1955. Seasonal variations in the resistance of goldfish to temperature. Trans. Royal Soc. Canad., 49 : 25-34. HOAR, WILLIAM S., 1956. Photoperiodism and thermal resistance of goldfish. Nature, 178: 364-365. KAPLAN, J. G., 1954. The alteration of intracellular enzymes. II : The relation between the surface and the biological activities of altering agents. /. Gen. Physiol., 38: 197-211. KAPLAN, J. G., 1955. The alteration of intracellular enzymes. I. Yeast catalase and the Euler effect. Exp. Cell Res., 8 : 305-328. KAPLAN, J. G., AND Wooo-Ki PAIK, 1956. The alteration of intracellular enzymes. IV. Kinetics of catalase alteration induced by chemical agents. Canad. J. Biochem. Physiol., 34 : 25-38. KELLY, T. L., E. D. NIELSON, R. B. JOHNSON AND C. S. VESTLING, 1955. Glucose-6-phosphate dehydrogenase of adrenal tissue. /. Biol. Chem., 212 : 545-554. KREBS, H. A., 1950. Body size and tissue respiration. Biochem. Biophys. Acta, 4: 249-269. PATAU, K., 1943. Zur statischen Buerteilung von Messungereihen (Eine neue t-Tafel). Biol. Zcntralbl., 63: 152-168. PEISS, C. N., AND J. FIELD, 1950. The respiratory metabolism of excised tissues of warm- and cold-adapted fishes. Biol. Bull., 99: 213-224. PRECHT, H., 1951. Der Einfluss der Temperatur auf die Atmung und auf einige Fermente beim Aal (Anguilla vulgaris L.). Biol. Zentralbl., 70: 71-85. PRECHT, H., 1953. Uber ruhestadien erwachsener Insekten. I. Versuche an Kartoffelkafern (Leptinotarsa decemlineata Say). Zeitschr. vergl. Physiol., 35: 326-343. PRECHT, H., J. CHRISTOPHERSEN AND H. HENSEL, 1955. Temperatur und Leben. Springer, Berlin. ROBBIE, W. A., 1948. Use of cyanide in tissue respiration studies. Meth. Med. Res., 1 : 307-316. SIEGEL, S., 1956. Nonparametric Statistics for the Behavioral Sciences. McGraw-Hill Book Co., New York. SUHRMAN, R., 1955. Weitere Versuche iiber die Temperatur Adaptation der Karauschen (Carrassius vulgaris Nils). Biol Zentralbl, 74: 432-448. STABILITY OF THE CHROMATOPHOROTROPINS OF THE DWARF CRAYFISH, CAMBARELLUS SHUFELDTI, AND THEIR EFFECTS ON ANOTHER CRAYFISH * MILTON FINGERMAN AND MILDRED E. LOWE Department of Zoology, Newcomb College, Tulane University, Netv Orleans 18, Louisiana The chromatophore systems of three crayfishes have been investigated to a con- siderable degree. Of these most is known about color changes in the dwarf crayfish, Cambarellus shufeldti. Brown and Meglitsch (1940) found that the sinus gland in the eyestalk of the crayfish Orconectes immunis contained a chromatophorotropin that concentrated red pigment. McVay (1942) demonstrated that the central nervous system of this crayfish produced a material that concentrated red pigment. The chromatophore system of the dwarf crayfish has been the subject of previous investigations by Fingerman (1957a, 1957b) and Fingerman and Lowe (1957a, 1957b). The red pigment dispersed maximally when dwarf crayfish were put on a black background and concentrated maximally when the crayfish were put on a white background. In the supraesophageal ganglia, circumesophageal con- nectives, and tissues of the eyestalks were chromatophorotropins that dispersed as well as concentrated red pigment (Fingerman, 1957a). Behavior of red pigment on isolated portions of the carapace of Cambarellus that had been on black and on white backgrounds was also investigated (Fingerman, 1957b). Red pigment had an inherent tendency to concentrate nearly maximally when the chromatophores were no longer controlled by hormones. Reciprocal blood transfusions between specimens of Cambarellus that had been on black and on white backgrounds for two hours revealed that the blood always contained pigment- dispersing and -concentrating hormones. The degree of pigment dispersion at any time appeared to be due to the relative quantity of each antagonist in the blood. Fingerman and Lowe (1957a) determined the effects of maintenance of speci- mens of Cambarellus on black and on white backgrounds for periods up to three weeks. The rates of red pigment dispersion and concentration in intact crayfish decreased progressively. In addition, red pigment gradually lost its inherent ability to concentrate after isolation. The titers of chromatophorotropins in the circumesophageal connectives of dwarf crayfish that had been on a black or a white background for two weeks changed sig- nificantly relative to the titers in crayfish that had been on the same shade of back- ground for two hours (Fingerman and Lowe, 1957a). They found that the hor- mone not needed for proper adaptation to one of the backgrounds, e.g., red pigment- dispersing hormone of crayfish on a white background, was stored in the circum- esophageal connectives. The quantities of chromatophorotropins in the blood also 1 This investigation was supported by Grant No. B-838 from the National Institutes of Health. 317 318 MILTON FINGERMAN AND MILDRED E. LOWE changed. The hormone that was stored in the circumesophageal connectives of crayfish kept on a black or a white background for two weeks decreased in the blood and the hormone needed in the blood for background adaptation increased. These data provided evidence that the secretory products of the central nervous system were physiologically involved in the color change process. Fingerman and Lowe (1957b) suggested that the chromatophorotropins in the eyestalks of dwarf crayfish were different from those in the circumesophageal con- nectives. Evidence was based on differences in rates of disappearance of the hormones in extracts of the eyestalks and circumesophageal connectives maintained at room temperature. Chromatophorotropins that dispersed and concentrated red pigment were found in the eyestalks, supraesophageal ganglia, and circumesophageal connectives of the crayfish Orconectes clypeatus by Fingerman (1958). When specimens of Orco- nectes clypeatus were kept on a black background their red pigment dispersed maxi- mally but when the crayfish were transferred to a white pan this pigment concen- trated to an intermediate state only. Maximal red pigment concentration did not occur in specimens kept under constant illumination on a white background for 32 days. Hormones that dispersed red pigment could be demonstrated only by direct application of extracts to chromatophores on isolated portions of carapace. Direct injection of extracts did not produce red pigment dispersion. The current concept of the origin of chromatophorotropins is that they are all neurosecretory products. Those found in the sinus gland originate in the medulla terminalis X organ as granules which migrate through axons to the sinus gland where they are stored (Bliss, Durand and Welsh, 1954). Fingerman and Aoto (unpublished observations) have found cytological evidence of neurosecretion in the supraesophageal ganglia, circumesophageal connectives, and optic ganglia of dwarf crayfish. The present investigation was undertaken to shed light on several questions that have arisen as a result of the studies summarized above. For example, (1) since direct injection of tissue extracts of Orconectes into specimens of Orconectes did not cause pigment dispersion, we wished to learn whether tissue extracts from Cambarellus could disperse red pigment in specimens of Orconectes and vice versa. (2) The possibility that the chromatophorotropins found in the eyestalk are different from those found in the supraesophageal ganglia and circumesophageal connectives was also investigated further. MATERIALS AND METHODS Adult specimens of the dwarf crayfish, Cambarellus shujcldti, and immature specimens of the crayfish Orconectes clypeatus were collected at Hickory, Louisiana, for use in the experiments described below. The specimens of both species used in the experiments were about 20 mm. long. The crayfishes were kept in aquaria that contained tap water approximately one inch deep. Air-conditioning kept the temperature of the laboratory between 25 and 27° C. The dark red chromatophores in the portion of the carapace dorsal to the heart were staged according to the sys- tem of Hogben and Slome (1931) by the senior author. Stage 1 represented maxi- mal concentration of pigment, stage 5 maximal dispersion, and stages 2, 3, and 4 the intermediate conditions. RED CHROMATOPHORES OF CRAYFISHES 319 Tissue extracts were routinely prepared as follows. The organs to be assayed were dissected out and placed in Van Harreveld's solution (Van Harreveld, 1936). When the desired number of each organ was available the organs were transferred with a minimum of saline to a glass mortar, triturated, and suspended in a suffi- cient volume of Van Harreveld's solution to yield the desired concentration. Crayfish that received injections of extracts had one eyestalk removed at least 12 hours prior to the experiment. Brown, Webb and Sandeen (1952) and Finger- man (1957a) found that the responses of one-eyed individuals to chromatophoro- tropins were greater than the responses of intact specimens, presumably because the presence of both eyestalks made the organisms more capable of antagonizing in- jected hormones. Brown and Meglitsch (1940) and McVay (1942) were forced to use large specimens of Orconectes immitnis whose exoskeleton was opaque. These investi- gators, therefore, applied extracts directly to the chromatophores on portions of the carapace removed from the body. The specimens used herein were small enough so that their carapaces were sufficiently transparent to allow accurate, direct ob- servation of the underlying chromatophores. For convenience and to save space the hormone that concentrated red pigment will be referred to as RPCH (red pigment-concentrating hormone) and that which dispersed red pigment as RPDH (red pigment-dispersing hormone). Use of the same letters for hormones in the eyestalks and in the supraesophageal ganglia plus the circumesophageal connectives does not imply that the chromatophorotropins have the same molecular structure. EXPERIMENTS AND RESULTS Analysis of variation to be expected when the same extract is injected into two groups of dwarf crayfish The aim of this experiment was to determine how much of the variance between two sets of data in the results presented below would be due to differential response of the crayfish used for assay and how much to differences in titers of chromato- phorotropins in the extracts. To solve this problem an extract of the supraesopha- geal ganglia, with the circumesophageal connectives attached, of Cambarellus was prepared with a final concentration of one-third of a complement per 0.02 ml. Van Harreveld's solution. Ten one-eyed dwarf crayfish were then placed into each of two white enameled pans containing tap water ; five were then placed into a third white pan. In like manner 25 were distributed among three black enameled pans. The crayfish in the white pans had been on a white background for at least one hour and were inspected prior to the experiment to be certain that their red pigment was maximally concentrated (stage 1). Crayfish in black pans had maxi- mally dispersed red pigment (stage 5). The crayfish in the two pans with five crayfish were each injected with 0.02 ml. Van Harreveld's solution as a control. The crayfish in the remaining pans were then injected with the extracts. Each crayfish in one white and one black pan was injected from the same syringe with 0.02 ml. of extract. The average stage of the red chromatophores of the crayfish in each pan was determined 15, 30, 60, 90 and 120 minutes from the time of injection. 320 MILTON FINGERMAN AND MILDRED E. LOWE The results are presented in Figure 1. As evident from inspection of the figure, RPCH and RPDH were present. The maximal effect of the former preceded that of the latter, the typical situation with extracts of supraesophageal ganglia and circumesophageal connectives (Fingerman, 1957a). The results of the two groups that received extracts were extremely similar, if not identical, since the difference between two consecutive determinations of the average chromatophore stage of the same group of crayfish may be 0.2 of a unit, but never more. The sum of the differences between the total of the average chromatophore indices for corresponding LJ O UJ Cd O £3 O cd i O 0 I 2 HOURS FIGURE 1. Responses of red chromatophores of one-eyed Cambarellus on black and on white backgrounds to the same extract of supraesophageal ganglia, with the circumesophageal connectives attached. Dots and circles, two groups that received the same extract; half-filled circles, control. groups of crayfish was 0.6 unit for dispersing activity and 0.1 unit for concentrating activity. Results appreciably different from these values may, therefore, be con- sidered as due to a significant difference in the quantities of chromatophorotropins in the extracts themselves and not to the crayfish used for the assay. Responses of dzuarf crayfish to chromatophorotropins of Cambarellus and Orconectes The aim of this experiment was to compare the effects of extracts of eyestalks and supraesophageal ganglia, with the circumesophageal connectives attached, of dwarf crayfish and Orconectes upon the dark red chromatophores of dwarf crayfish. RED CHROMATOPHORES OF CRAYFISHES 321 Since direct injection of freshly prepared extracts of eyestalks and supraesophageal ganglia plus the circumesophageal connectives of Orconectes into specimens of Orconectes never produced red pigment dispersion, we wished to learn whether these extracts could disperse red pigment in specimens of Cambarellus. Both eyestalks and the supraesophageal ganglia, with the circumesophageal con- nectives attached, of dwarf crayfish and Orconectes were removed and extracted as described above, so that the final concentration was one-third of a complement per 0.02 ml. Five one-eyed dwarf crayfish with maximally concentrated red pigment were placed into each of five white pans that contained aerated tap water. In like manner five one-eyed crayfish with maximally dispersed red pigment were placed into each of five black pans. The crayfish were injected and the chromatophores staged in the manner described in the first experiment. F 4 UJ cr O I U -B 0 I 20 I 2 HOURS FIGURE 2. Responses of the red chromatophores of one-eyed Cambarellus on a white background (A) and on a black background (B) to extracts of eyestalks and supraesophageal ganglia, with the circumesophageal connectives attached, of Cambarellus shufeldti and Orco- nectes clypeatus. Half -filled circles are the controls. Open symbols are eyestalks, closed sym- bols nervous tissue. Circles, tissue of Cambarellus; triangles, tissue of Orconectes. The results are presented in Figure 2 where each point represents the average of 10 crayfish. As evident from the figure, each extract contained RPCH and RPDH. The eyestalks of dwarf crayfish and Orconectes contained more RPDH and less RPCH than the supraesophageal ganglia plus the circumesophageal connectives. Significantly, the eyestalks and supraesophageal ganglia plus the circumesophageal connectives of Orconectes contained sufficient RPDH to produce an appreciable response in dwarf crayfish but no red pigment dispersion occurred when extracts of these tissues were injected into specimens of Orconectes (Fingerman, 1958). Responses of Orconectes to chromatophorotropins of dwarf crayfish and Orconectes Since direct injection of extracts of eyestalks and supraesophageal ganglia plus the circumesophageal connectives of Orconectes into dwarf crayfish did produce red 322 MILTON FINGERMAN AND MILDRED E. LOWE pigment dispersion, we wished to determine the results of the reciprocal experiment. Red pigment of the specimens of Orconectes used in the assay was in an intermediate state of dispersion since red pigment in this species does not concentrate maximally when specimens are placed on a white background (Fingerman, 1958). The results are presented in Figure 3 where each point represents the average of 10 crayfish. Extracts of tissues from Orconectes did not produce red pigment dispersion; RPCH alone was evident. As had been found previously (Fingerman, 1958), supraesophageal ganglia and circumesophageal connectives were more potent sources of RPCH than eyestalks. UJ UJ or 82 cr o I A -B 0 I 20 I 2 HOURS FIGURE 3. Responses of the red chromatophores of one-eyed Orconectes on a white back- ground (A) and on a black background (B) to extracts of eyestalks and supraesophageal ganglia, with the circumesophageal connectives attached, of Orconectes clypeatus and Cambarcl- lus shufeldti. Half-filled circles are the control. Open symbols are eyestalk, closed symbols nervous tissue. Circles, tissue of Orconectes; triangles, tissue of Cambarellus. The responses of specimens of Orconectes on black and on white backgrounds to extracts of supraesophageal ganglia, with the circumesophageal connectives at- tached, of dwarf crayfish and Orconectes were qualitatively similar. Concentration but no dispersion of red pigment in Orconectes was evident in spite of the fact that RPCH and RPDH were present in these extracts (Fig. 2). Eyestalk extracts of Cambarellus, however, caused a transitory concentration of red pigment in Orconectes that was followed by dispersion. Concentration must have been caused by the small amount of RPCH in the eyestalks of dwarf crayfish and dispersion by the large amount of RPDH shown in Figure 2. Extracts of eye- stalks of both species produced equal red pigment dispersion in dwarf crayfish but the eyestalk extract of Orconectes contained much more RPCH (Fig. 2). This difference was probably a contributing factor to the lack of red pigment dispersion in Orconectes with extracts of tissues from Orconectes, because the large amount of RPCH would hinder the expression of RPDH whereas in the eyestalk of Cambarel- lus the titer of RPDH so overbalanced the titer of RPCH that dispersion could not occur. The failure of extracts of Orconectes to disperse red pigment in Orconectes RED CHROMATOPHORES OF CRAYFISHES 323 suggests that the titers of chromatophorotropins in the blood at the time of injection are important or else when the extracts are injected materials are secreted to an- tagonize the added hormones. Influence of time and temperature upon chromatophorotropins of dwarf crayfish This experiment was designed to determine the effects of boiling and mainte- nance at room temperature upon the titers of RPCH and RPDH in the supraesopha- geal ganglia with the circumesophageal connectives attached of dwarf crayfish. Twenty organs were removed and extracted in 1.2 ml. Van Harreveld's solution. The extract was then divided into two equal portions. One fraction was placed in 5 - 2 0 HOURS FIGURE 4. Responses of the red chromatophores of one-eyed Cambarellus on white (A) and on black (B) backgrounds to boiled (dots) and unboiled (circles) portions of the same extract of supraesophageal ganglia, with the circumesophageal connectives attached, of Camba- rellus injected immediately after preparation. Half -filled circles, control. boiling water for 30 seconds and cooled to room temperature. The boiled and un- boiled extracts were then assayed on specimens in black and in white pans. The extracts were kept in the syringes on a table top at room temperature for 120 min- utes and then assayed again. The results are presented in Figures 4 (zero time injection) and 5 (120 minute injection) . Boiling appeared to activate an inactive form of RPDH but had little, if any, effect on RPCH. Even more striking differences were apparent between the responses of the crayfish to boiled and unboiled extracts kept for 120 minutes at room temperature. The responses of the crayfish injected with the unboiled aged extract were the same as those observed by Fingerman and Lowe (1957b). The amount of RPCH had decreased considerably and the amount of RPDH had in- creased. In contrast, RPDH in the boiled preparation disappeared at a faster rate than RPCH. The latter showed no decreased potency. If anything, a slight increase was evident, probably due to the degeneration of its antagonist. 324 MILTON FINGERMAN AND MILDRED E. LOWE These results can be explained by assuming that RPDH exists in the neuro- secretory cells in a functional form and a non-functional one which may be activated either by boiling or being kept at room temperature for two hours. Activation probably represents release of bound hormone from the interior of neurosecretory granules as has been shown with catechol amines by Hillarp and Nilson (1954) and Blaschko, Hagen and Welch (1955). Decrease in the quantity of the active form because of its instability occurs simultaneously with release of additional hor- mone from the neurosecretory granules in unboiled extracts. Inactivation of RPDH would occur in boiled extracts but no further release from the granules because boiling caused the immediate release of all bound RPDH from the neurosecretory granules. To illustrate, at the start of the experiment boiled extract caused more red pigment dispersion than unboiled (Fig. 4A). During the two hours at room O a: S3 Q. O I- < 22 O cr 0 I 20 I 2 HOURS FIGURE 5. Responses of the red chromatophores of dwarf crayfish on white (A) and on black (B) backgrounds to the same extracts of Figure 4 but injected after 120 minutes at room temperature. See Figure 4 for key to symbols. temperature RPDH was becoming inactivated. Since in the boiled extract no in- active hormone would be available, degeneration alone occurred so that the net effect was decreased potency (Fig. 5 A). In the case of the unboiled extract, dur- ing the two hours at room temperature the active form was being released from the neurosecretory granules at a faster rate than the functional hormone was becoming inactivated so that the net effect was an increased titer of RPDH. The same logic apparently does not apply to RPCH. Boiling seemed to stabilize the molecule so that it was not inactivated as was RPCH of unboiled extracts. Stability of chroniatophorotropins in dried tissue The object of this experiment was to determine the effects of drying for two hours on RPCH and RPDH to rule out the possibility that the results of the previ- ous experiment were due to the use of triturated tissues and not to the nature of RED CHROMATOPHORES OF CRAYFISHES 325 the neurosecretory granules or the hormones. Supraesophageal ganglia, with the circumesophageal connectives attached, of dwarf crayfish were dissected out and cut in half longitudinally. One batch was triturated immediately and re-suspended in sufficient Van Harreveld's solution to yield a final concentration of one-third of a complement per 0.02 ml. The second set was dispersed in the bottom of a glass mortar and allowed to dry at room temperature for 120 minutes, then triturated and suspended in sufficient Van Harreveld's solution for a final concentration of one- third of a complement per 0.02 ml. The freshly prepared extract was assayed and two hours later the extract of the dried tissues was assayed. The results are presented in Figure 6 where each point represents the average of 10 crayfish. The results of drying the tissue for two hours were qualitatively the same as those observed with extracts left at room temperature for two hours. The 2 0 HOURS FIGURE 6. Responses of the red chromatophores of one-eyed dwarf crayfish on white (A) and on black (B) backgrounds to extracts of supraesophageal ganglia, with the circumesopha- geal connectives attached of Cambarellus. Circles, extracted at time of removal ; dots, dried at room temperature for 120 minutes ; half-filled circles, control. extracts of dried tissues contained more RPDH and less RPCH than the extracts of fresh tissue. The same result was obtained when the extracts were left at room temperature for two hours. Therefore, the changes depicted in Figures 4 and 5 were not due solely to the aqueous environment of the extract but were mainly due to the instability of the hormones and probably of the neurosecretory granules in which the hormones are present when first formed. Fractionation of chromatophorotropins in the sinus glands and supraesophageal ganglia plus the circumesophageal connectives of dwarf crayfish The object of this experiment was to determine the relative solubilities of RPDH and RPCH from the sinus gland and the supraesophageal ganglia plus the circum- esophageal connectives in absolute ethyl alcohol. In this way further information concerning the similarity of the corresponding molecules from the two sources would be obtained since Fingerman and Lowe (19S7b) had obtained preliminary 326 MILTON FINGERMAN AND MILDRED E. LOWE evidence that RPDH and RPCH in the eyestalks were different from the materials in the circumesophageal connectives of dwarf crayfish with the same functions. The method employed was described in detail by Fingerman (1956). Six su- praesophageal ganglia, with the circumesophageal connectives attached, were placed on a glass slide. The excess moisture was removed and the tissue was then smeared with a glass pestle. In like manner, six sinus glands were removed with a mini- mum of other tissues from eyestalks and smeared on a glass slide. The hormones in the smeared tissues were then extracted by dropping one ml. 100% ethyl alcohol from a syringe onto the tilted slide and collecting the alcohol in a watch glass. The alcohol was allowed to evaporate and the residue was re-suspended in 0.24 ml. Van Harreveld's solution. u O U a: 02 a: u • *> B I 2 0 HOURS FIGURE 7. Responses of the red chromatophores of dwarf crayfish on black and on white backgrounds to extracts of supraesophageal ganglia, with the circumesophageal connectives attached (A), and sinus glands (B). Dots, alcohol-soluble fraction; circles, alcohol-insoluble fraction ; half -filled circles, control. After the few remaining drops of alcohol had evaporated from the glass slide the tissues were again extracted by placing 0.24 ml. Van Harreveld's solution on the tissues for two to three minutes. The fluid was then taken up in a syringe. The four fractions were then assayed. The results are presented in Figure 7 where each point represents the average of 10 crayfish. As evident from the left half of the figure (nervous tissue), the alcohol-soluble fraction contained more RPCH and less RPDH than the alcohol-in- soluble fraction. Consideration of the results obtained with the sinus glands re- vealed the reverse situation prevailed. The conclusion may, therefore, be drawn that the chromatophorotropins of the supraesophageal ganglia plus the circumesoph- ageal connectives are different from those of the sinus gland. Enzymatic inactivation of chromatophorotropins of Cambarelhis This set of experiments was designed to shed further light on what happens to chromatophorotropins after they have been secreted into the blood. Carstam RED CHROMATOPHORES OF CRAYFISHES 327 (1951) showed that the hypodermis of the prawn Leander adspersus contains an enzyme capable of inactivating chromatophorotropins from the sinus gland. Finger- man and Lowe (19S7b) showed that the presence of hypodermis of dwarf cray- fish likewise hastened the inactivation of chromatophorotropins in nervous tissue. We decided, therefore, to investigate further the nature of this inactivation. For the first experiment of the series supraesophageal ganglia, with the circum- esophageal connectives attached, were removed from dwarf crayfish and triturated with a sufficient volume of Van Harreveld's solution to yield 0.90 ml. extract with a concentration of one-third complement per 0.02 ml. Two-hundredths ml. of this extract was then injected into each of five one-eyed crayfish on a white background and into a like number on a black background. The entire carapace had, in the o co4 u a: o i Q. -a i U 0 I 20 ! 2 HOURS FIGURE 8. Responses of the red chromatophores of Cambarellus on white (A) and on black (B) backgrounds to unboiled extracts of supraesophageal ganglia, with the circumesopha- geal connectives attached, of dwarf crayfish. Circles, extract administered immediately after boiling ; dots, after two hours exposure to boiled carapace ; circles filled on left side, after two hours exposure to unboiled carapace ; circles filled on right side, control. meantime, been removed from six dwarf crayfish. Three of the carapaces were boiled to destroy the proteins in the hypodermis. The six carapaces were then placed on individual glass depression slides. Into each depression containing a cara- pace was placed 0.1 ml. of the extract. The slides were then covered to minimize evaporation. After the extract had been exposed to the carapaces for two hours, the fractions that had been exposed to boiled and unboiled carapace were collected separately and assayed. The results are presented in Figure 8 where each point represents the average of 10 crayfish. The unboiled extract exposed to the boiled and unboiled pieces of carapace be- haved just as the unboiled extract shown in Figures 4 and 5. After two hours of exposure to both boiled and unboiled carapace the unboiled extract showed an in- crease in red pigment-dispersing potency and a decrease in RPCH. The same results were obtained when the unboiled extract was simply left at room temperature 328 MILTON FINGERMAN AND MILDRED E. LOWE for two hours. The extract that had been exposed to unboiled carapace, however, contained more RPDH and less RPCH than the extract that had been in contact with boiled carapace. Presumably, this difference was due to preferential inactiva- tion of RPCH by an enzyme in the hypodermis of the unboiled carapace. This en- zyme must have been destroyed in the boiled carapaces. Apparently, the enzyme destroyed RPCH in preference to RPDH and for this reason the latter was more abundant after two hours of exposure. Spontaneous release of active RPDH from the neurosecretory granules also must have occurred during the two hours of ex- posure to the carapace. Since boiled extract kept for two hours had less RPDH than was present at the time of heating (Figs. 4 and 5), the logical sequel to the experiment just described wherein inactivation of the hormones was not great, was to expose boiled extracts to 2 0 HOURS FIGURE 9. Responses of the red chromatophores of Cambarellus on white (A) and on black (B) backgrounds to boiled extracts of supraesophageal ganglia, with the circumesopha- geal connectives attached, of dwarf crayfish. Circles, extract administered immediately after boiling ; dots, after two hours exposure to boiled carapace ; circles filled on left side, after two hours exposure to unboiled carapace ; circles filled on right side, control. boiled and unboiled carapaces for two hours. Furthermore, as mentioned above, the titer of RPCH in boiled extracts showed no tendency to decrease for at least two hours at room temperature so that any change in the titer of this hormone would have been enzymatically induced. Extracts for this experiment were prepared and handled in the same manner as those for the first experiment of this series with the one exception that the ex- tract was immersed in boiling water for 30 seconds, cooled, and then placed on the depression slides. The results of the second experiment are shown in Figure 9. Each point represents the average of 10 crayfish. The extract exposed to boiled carapace for two hours behaved exactly like the boiled extract of Figure 5 ; the titer of RPDH decreased markedly whereas no tend- ency for RPCH to diminish was apparent. However, the results with the boiled extract exposed to unboiled carapace were quite different from those obtained with RED CHROMATOPHORES OF CRAYFISHES 329 extracts exposed to unboiled carapace. The quantity of RPCH decreased mark- edly, presumably due to enzymatic inactivation. The quantity of RPDH showed little or no decrease, probably because so much of its antagonist was destroyed that a lower titer of RPDH was able to exert a sizeable effect. Here also a preferential inactivation of RPCH occurred. Ld o LJ o: ° x Q_ § ^ o a: x u r O 0 I HOURS FIGURE 10. Responses of the red chromatophores of dwarf crayfish on black and on white backgrounds to epinephrine. Responses of the red chromatophores of dwarf crayfish to epinephrine An epinephrine solution (adrenalin chloride) manufactured by Parke, Davis and Co. was diluted with Van Harreveld's solution so that the final concentration was 2 X 10~5 gram epinephrine per 0.02 ml. Ten one-eyed dwarf crayfish with maxi- mally concentrated red pigment and a like number with maximally dispersed red pigment were each injected with 0.02 ml. of the solution of epinephrine. The aver- age stage of the red chromatophores of the crayfish in each pan was then deter- mined 15, 30, 60, 90, and 120 minutes after they had been injected. The experi- ment was repeated once. The results are presented in Figure 10 where each point represents the average of 20 crayfish. As is evident from inspection of the figure, epinephrine produced considerable pigment dispersion. No tendency for the red pigment to concentrate was apparent. 330 MILTON FINGERMAN AND MILDRED E. LOWE Filterability and adsorbability of the chromatophorotropic factors present in the supraesophageal ganglia and circumesophageal connectives of dwarf crayfish An extract of the supraesophageal ganglia, with the circumesophageal connec- tives attached, of dwarf crayfish was prepared so that the final concentration was one-third of a complement per 0.02 ml. of fluid. The extract was then divided into three portions. One part was untreated. The second aliquot was filtered once through "Aloe Standard American" filter paper, catalogue no. 42700. The third was shaken through crushed charcoal and then centrifuged to remove the small par- ticles of charcoal. The fractions were then assayed. The experiment was done twice. UJ P 4 (/) HI or o -, I2 0 I 20 I 2 HOURS FIGURE 11. Responses of the red chromatophores of dwarf crayfish on white (A) and on black (B) backgrounds to extracts of supraesophageal ganglia, with the circumesophageal con- nectives attached. Circles filled on the left side, pure extract ; circles, extract filtered through paper ; dots, extract shaken with powdered charcoal ; circles filled on right side, control. The results are presented in Figure 1 1 where each point represents the average of 10 individuals. No striking differences were found among the titers of RPDH in the extracts. A slight adsorption of RPDH on filter paper may have occurred. In contrast, a striking decrease of RPCH in the extract shaken with charcoal was apparent whereas this substance was not adsorbed on filter paper. A slight aug- mentation of the titer of RPCH in the filtered extract was apparent although the reason for this is not clear since an appreciable quantity of its antagonist (RPDH) was still in the extract. DISCUSSION The experiments described above were based upon some problems that investi- gators of color changes in crustaceans have been considering for several years. One such is why an extract that concentrates pigment in the animal from which it RED CHROMATOPHORES OF CRAYFISHES 331 was obtained disperses pigment when injected into another species. This problem is strikingly exemplified by the results obtained when extracts of Orconectes and dwarf crayfish were injected into the donor species as well as interspecifically. Extracts of tissues of specimens of Orconectes never produced red pigment dis- persion in specimens of Orconectes yet they produced striking concentration and dispersion of the pigment of dwarf crayfish. On the other hand, extracts of the eyestalk of dwarf crayfish were capable of producing dispersion of red pigment in specimens of both Orconectes and dwarf crayfish. Specimens of Orconectes may have an excellent feed-back mechanism associated with the chromatophore system so that any displacement of the red pigment in cray- fish on a white background to a more dispersed state is rapidly met with release of RPCH to maintain a steady-state of the red pigment. Extracts of the eyestalks of dwarf crayfish alone were able to overcome this mechanism because of the large amount of RPDH present in the extract relative to RPCH. The other extracts used had more RPCH present relative to the quantity of RPDH so that the Orco- nectes could easily antagonize the RPDH in these extracts by secretion of some RPCH. In support of this hypothesis are the results of Fingerman (1958) who showed that the blood of Orconectes always contained RPCH and RPDH and that the state of the pigment at any given time appeared to be due to the relative quantity of each hormone in the blood. He was able to produce slight dispersion of red pigment in specimens of Orconectes with blood from specimens on a white back- ground and considerable dispersion with blood from Orconectes on a black back- ground. The conclusive evidence presented herein that the RPCH and RPDH found in the sinus gland are different from the materials found in the supraesophageal ganglia plus the circumesophageal connectives that concentrate and disperse red pigment adds another complication to the already complicated study of control of color changes in crustaceans. The problem immediately arises why an organism should produce two hormones to accomplish the same function. Since the maximal red pigment-dispersing effect obtained when extracts of eyestalks or sinus glands are used occurs sooner than that of the supraesophageal ganglia plus the circumesopha- geal connectives (Figs. 2 and 7), RPDH of the sinus gland may be used to move the pigment rapidly to the desired state of dispersion and once at this state the fac- tor of the supraesophageal ganglia plus the circumesophageal connectives may take charge. Such a situation may explain the results of Fingerman and Lowe (1957b) who assayed the amount of RPDH in the blood of dwarf crayfish after a background change and found that the amount in the blood 30 minutes after transfer of crayfish from a white to a black background was more than needed to maintain the pigment in the maximally dispersed condition. Perhaps the excess titer was due to RPDH from the sinus gland and once the pigment had become maximally dispersed the pigment was kept in this state by a lower titer of RPDH from the supraesophageal ganglia and the circumesophageal connectives. Fingerman (1956) demonstrated that in the blue crab, Callincctcs sapidus, the factor in the sinus gland that dispersed red pigment was different from the hormone in the circumesophageal connectives that accomplished the same effect. Perhaps this difference in hormones in the sinus gland and central nervous organs is a general thing among crustaceans. 332 MILTON FINGERMAN AND MILDRED E. LOWE Carstam (1951) showed that an enzyme that inactivated the red pigment concen- trating hormone of the sinus gland of the prawn Leander adspersus was present in the hypodermis of the following crustaceans, Leander adspersus, Cancer pagurus, Homarus vulgaris, and Idothea neglecta. Presumably inactivation of RPCH of Cambarellus (Fig. 9) was due to the presence of this enzyme in the hypodermis of the unboiled carapace. Results obtained by different investigators with mammalian hormones injected into crustaceans do not form a consistent pattern. Ostlund and Fange (1956) found that epinephrine dispersed red pigment in Leander adspersus. The same results were obtained with dwarf crayfish (Fig. 10). In contrast, Nagano (1950) found that adrenalin produced pigment concentration in the shrimp • Par 'at y hyraena barracuda Comment 1 specimen 1 specimen 1 specimen Several individuals 1 specimen 1 specimen A 4" specimen Large school and individual speci- mens 2 specimens 1 specimen Several specimens Feeding sounds recorded from a group. Male known to call in the breeding season (Tavolga, 1956). Several specimens Several specimens Feeding sounds recorded 1 specimen 1 specimen Feeding sounds recorded 1 specimen 1 specimen 1 specimen 1 specimen 1 specimen 1 specimen 1 specimen 1 specimen 1 specimen salt hydrophone and a modified Heathkit amplifier Model A-7C, or an Ekotape microphone Model 205. Recordings were made at 7V-2 and 33/4 in. /sec. on the Ekotape tape recorder and selected recordings have been analyzed on a vibration frequency analyzer, the Kay Vibralyzer. Sound-generating equipment used during the investigation consisted of a Hewlett-Packard audio oscillator Model LAJ or the Ekotape tape recorder, a Craftsman C550 amplifier, and a QBG transducer. All observations at sea were made in calm weather from the 30-foot motor launch RESEARCH of the Lerner Marine Laboratory, between 0800 and 1400 hours from 9 July to 12 August. Suitcase amplifier settings varied from 0 + 14 -- 20 to 0 + 10 — 5. During work at sea, the hydrophone was suspended just above the bottom, or so that it cleared the bottom in the shoalest water in the listening area at drift stations. Forty species of fishes, distributed among 29 families, were studied at Bimini ACOUSTICAL BEHAVIOR OF BIMINI FISHES 359 (Tables I and II). The stimuli used in eliciting sound in the laboratory were those of handling, capture in a net or feeding, or a combination of these. All sounds reported were recorded from isolated, submerged specimens, except the pectoral fin drumming and tooth striclulation of individual triggerfishes (Balistidae) which were not heard while the fish were being handled under water. All other sounds described were produced in air as well as under water, except the snap of the demoiselle, Pomacentrus leucosticits (Mueller and Troschel), which was recorded only from submerged specimens. The species which produced no detectable sounds other than those of chewing TABLE II Characteristics of fish sounds recorded at Bimini Family Species Vibration frequency analysis Balistidae Carangidac Chaetodontidae Batistes vetula in air at micro- phone Melichthys picens in air at mi- crophone B. vetula in air at microphone M. piceus in air at microphone Feeding of a mixed group of the two spp. at AX-58-C hydro- phone Caranx hippos hand-held in aquarium at hydrophone Angelifhthyes ciliaris in cement tank at AX-58-C hydro- phone Pomacanthus arcuatus W. of Turtle Rocks at AX-58-C hydrophone Sound: Toothplate stridulation Duration: .12 sec. /stridulation Frequency span: 0 to above 8 kc. Predominant intensities: .7-1.8 kc., 2.1— 3.8 kc. Sound: Toothplate stridulation Duration: .06 to .1 sec. /stridulation Freq. span : 0 to above 8 kc. Pred. int.: 1.2 to 2.3 kc. Sound : Pectoral fin-drumming Duration: .03 sec. /pulse Freq. span: 0 to 5.8 kc. Pred. int.: 1 to 1.7 kc. Sound : Pectoral fin-drumming Duration: .02 to .04 sec. /pulse Freq. span : 0 to above 8 kc. Pred. int.: .7 to 2.2 kc. Freq. span: 0 to above 8 kc. Pred. int. ; .6 to 2.9 kc. Sound: Stridulation, pharyngeal teeth Duration: .06 sec. /stridulation Freq. span: 0 to above 8 kc. Pred. int.: .3 to 1.2 kc., 1.7 to 3.3 kc. Sound: Grunt, single or repeated Duration: .06 to .1 sec. /grunt Freq. span: 0 to 1.1 kc. Pred. int. : Below .5 kc. Sound: Grunt or moan-like sound Duration: .04 to .2 sec. Freq. span: 0 to 1.5 kc. Pred. int. : Below .5 kc. I 360 JAMES M. MOULTON TABLE II — Continued Family Species Vibration frequency analysis Diodontidae Haemulidae Holocentridae Pomacentridae Serranidae Tetradontidae Diodon hystrix hand-held in aquarium at hydrophone Haemulon sciurus hand-held in aquarium at hydrophone Holocentrus ascensionis in a- quarium (Fig. 10) and at Turtle Rocks (Fig. 12); AX- 58-C hydrophone Pomacentrus leucostictiis in a- quarium; male pursuing others of species at hydro- phone Epinephalus adsencionis in ce- ment tank at AX-58-C hy- drophone Epinephalus striatus in live car at AX-58-C hydrophone Spheroides spengleri hand-held in aquarium at hydrophone Sound: Toothplate stridulation Duration: .09 sec. /stridulation Freq. span: 0 to 8 kc. Pred. int.: 2.5 to 5.5 kc., with narrow inten- sity bands at 3, 4.3 and 5 kc. Sound : Stridulation, pharyngeal teeth Duration: .02 to .1 sec. /stridulation Freq. span: 0 to above 8 kc. Pred. int.: 1.5 to 4 kc. Sound : Thump-like single, or volleyed Duration: .04 to .1 sec. /thump Freq. span : 0 to above 4 kc. Pred. int.: Below 1 kc. Sound: Single or repeated snaps Duration: .02 sec. /snap Freq. span: 0 to 1.5 kc. Pred. int. : 0 to .8 kc. Sound: Vibrant grunt, single or repeated Duration: .04 to .1 sec. /grunt Freq. span: 0 to .9 kc. Pred. int. : 0 to .3 kc. Sound: Vibrant grunt, single or repeated Duration: .1 to .2 sec. /grunt Freq. span: 0 to 2 kc. Pred. int. : 0 to .4 kc. Sound : Toothplate stridulation Duration: .07 sec. /stridulation Freq. span : 0 to above 8 kc. Pred. int. : 1.3 to 3 kc. are shown in Table I. Twelve species producing other-than-chewing sounds are listed in Table II, together with data derived from vibration frequency analysis. Of this latter group, only three species were identified as sources of sounds recorded during listening at sea — squirrelfish, Nassau grouper and black angelfish, Poma- canthus arcuatus (Linnaeus), all of which use the air bladder in sound production. No individual free fishes were identified at sea as sources of stridulatory noises. Vibration frequency analysis of these latter sounds from recordings made at sea is rendered somewhat difficult by a broad band of sound with predominant intensities between 2 and 6 kc., which is characteristic of warmer seas, and which has gen- erally been ascribed to snapping shrimp. It is not unlikely, however, that sounds created by the teeth of reef fishes (Table II — feeding of balistids) may contribute to this sound band, which on vibration frequency analysis tends to obscure upper frequency ranges of various sounds clearly discernible below the 2-kc level. ACOUSTICAL BEHAVIOR OF BIMINI FISHES 361 SECONDS SECONDS FIGURE 1. The pectoral fin-drum of Balistes vetula in air. FIGURE 2. The pectoral fin-drum of Mclichthys piccus in air. SOUND PRODUCTION AND BEHAVIOR OF BIMINI FISHES In the following account, each family of which sound-producing representatives were studied at Bimini, is dealt with from the points of view of the sounds pro- duced and the correlated behavior. Initial references are to earlier descriptions dealing with sound production in the families concerned. Balistidae (Bridge, 1910, pp. 357, 361 ; Fish, 1948, pp. 15-19, 1954, pp. 62-65; Schultz and Stern, 1948, p. 132). The queen triggerfish, Balistes vctitla L., and the black triggerfish, Melichthys piccus (Poey), each possesses above the base of the pectoral fin a thin membrane lying lateral to the air bladder and covered by scales larger and more plate-like than those elsewhere on the body (Figs. 3, 4), a characteristic of these genera of the Balistidae (Evermann and Marsh, 1900). Males and females removed from the water and handled, frequently elevated and rapidly fluttered the pectoral fins against this region, as described by Schultz and Stern (1948), resulting in the production of a throbbing sound (Table II; Figs. 1, 2). Bridge (1910, p. 357) attributes a throbbing sound primarily to move- ments of the pectoral girdle. .;'#|3^:%, FIGURE 3. Outline of M. piccus showing (a) position of drumming membrane posterior to the gill opening. X 3. FIGURE 4. Detail of the drumming membrane of M. piccus. X 2.5. 362 JAMES M. MOULTON Differentiation of the "drumming membrane" is not apparent externally in the ocean triggerfish (Canthidcnnis sabaco Poey), nor does this species during handling move the pectoral fins to the drumming position. Similarly, the toothplate stridula- tion readily demonstrated by the queen and black triggerfishes (Table II; Figs. 5, 6 ) during handling out of water was not performed by the several ocean trigger- fish studied. While tooth stridulation and pectoral fin drumming were not heard from iso- lated triggerfishes handled underwater, recordings made during the feeding about the hydrophone of a captive population of queen and black triggerfishes showed predominant intensities of accompanying sounds between .6 and 2.9 kc. (Table II), which essentially spans the frequency ranges of predominant intensities obtained during tooth stridulation by these species in air. It was not possible to ascribe any specific sounds recorded at sea to triggerfishes, although their feeding activity probably contributed to background sounds recorded. They are common in the area studied. e- 7- 6- 5- 4- 3' 3 ' > i t . SECONDS SECONDS FIGURE 5. Tooth stridulation of B. I'ctula in air. FIGURE 6. Tooth stridulation of M. piceus in air. Carangidac (Bridge, 1910, p. 363; Fish, 1948, pp. 25-30). The pharyngeal tooth stridulation of Caranx hippos (Linnaeus) was recorded in a laboratory aquarium during handling of a 3.5-inch individual (Table II; Fig. 7). The sound recorded was not identified in any recording made at sea. Similar sound produc- tion in a related species, Caranx crysos (Mitchill), has been described by Fish (1954), but the thump she describes as occurring during shock was not heard during handling of the small specimen, nor was any detectable sound recorded from adult carangids swimming around and past the hydrophone at sea. A local fisherman related the striclulatory sound to "rattling of the ear bones" and asserted that a hooked specimen making this noise attracts other individuals of the species. Chaetodontidae (Bridge, 1910, p. 361). The vibrant deep grunts of the queen, Angelichthyes ciliaris (Linnaeus), and black, PomacantJins arcnatns (L.), angel- fishes (Table II) are not easily distinguished from those of the serranids with which they may occur. Vibration frequency analysis shows (Fig. 8) a tendency for highest frequencies of the angelfish grunts to be located in the middle of the call, whereas serranid grunts tend to be initiated with high frequency spikes. Each ACOUSTICAL BEHAVIOR OF BIMINI FISHES 363 may vary in the direction of the other, however, and since serranids and angelfishes tend to occur in similar areas, the sounds of the two may be confused. Field and laboratory observations indicated that serranids are far more prolific in call produc- tion under ordinary circumstances than are the angelfishes. Under laboratory conditions, both queen and black angelfishes produce the grunt during feeding on bits of conch, and when startled to quickened swimming by an observer. The deeply recessed air bladder, as seen in the black angelfish, bears no intrinsic muscles, and sound production is due to the contraction of axial musculature adjacent to the air bladder. Each quick motion of a black angelfish nibbling at the hydrophone at sea resulted in a brief grunt, although more leisurely swimming of both species in laboratory tanks was not accompanied by sound pro- duction. Handling of black angelfish under water brings forth brief grunts of low intensity, coinciding with body muscle contractions. Uniquely among species studied, the black angelfish, usually in pairs, readily approached and butted against the hydrophone at sea. SECONDS 8 SECONDS FIGURE 7. Pharyiigeal tooth-stridulation of Caranx hippos in aquarium. FIGURE 8. A short and two longer calls of Pomacanthus arena tits west of Turtle Rocks, the latter during approach of another P. arcuatus. Snapping shrimp in background. The deeply recessed position of the air bladder brings it into intimate associa- tion with surrounding peritoneum, and to the latter attach many of the axial muscle fibers heavily surrounding the slender ribs. These attached fibers appear to main- tain a tension on the wall of the air bladder ; cutting of the fibers creates a resonance within the bladder, and results in its partial collapse. The maximum duration of angelfish grunts obtained during this investigation occurred west of Turtle Rocks on 10 July (Fig. 8), when an adult black angelfish examining the hydrophone and butting gently at its rubber case suddenly gave vent to prolonged, rather moan-like sounds, each of .2-sec. duration, and swam toward an approaching fish of the same species. The two fish faced each other for a few moments, after which both came quietly to the hydrophone and finally swam off together. The interpretation of a recognition signal in the prolonged grunts wras rather difficult to avoid, for prior to production of the longer grunts, the first fish produced shorter, sharper sounds during its examination of the hydrophone. On another occasion, 12 July in the same area, sounds similar to the shorter grunts 364 JAMES M. MOULTON 7- 6- 5- > t |»tr M'tt s s .2 .3 .4 SECONDS SECONDS FIGURE 9. Toothplate stridulation of Diodon hystrix in aquarium. FIGURE 10. Toothplate stridulation of Sphcroidcs spcngleri in aquarium. were recorded as a pair of black angelfishes butted several times against a pair of cowfish, Lactophrys tricornis (L.), approximately 6 feet from the hydrophone. No sounds were recorded from the single species of butterflyfish studied, Chaetodon striatus L., nor were sounds recorded from an immature specimen (4-inch, total length) of the French angelfish, Poinacanthits parn (Bloch). Diodontidac, Tetradontidae (Fish, 1948, 1954; Burkenroad, 1931 ). The porcu- pinefish, Diodon hystri.v L., and the puffer, Sphcroidcs spcngleri (Bloch), were so similar in acoustical behavior as to merit a single discussion (Table II). Both produce sound during and after inflation by stridulation of the toothplates, the sound being of a klaxon-like variety (Figs. 9, 10). Only feeding sounds were recorded from undisturbed individuals. Frequencies of greatest intensities are similar in chewing and stridulation noises, although these levels vary between the two species (Table II) and may be expected to vary with size. Only a single individual of each was recorded at Bimini. Observations on these species and on the common puffer, Sphcroidcs inaculatus (Bloch and Schneider) of the Woods Hole area suggest that the stridulatory sound is more readily elicited from smaller individuals than from full-grown animals. .4 SECONDS FIGURE 11. Pharyngeal tooth stridulation of Hacmulon sciitnts in aquarium. FIGURE 12. Single thump-like sounds of Holocentrus ascensionis in cement tank. ACOUSTICAL BEHAVIOR OF BIMINI FISHES 365 Haemnlidac (Burkenroad, 1930, 1931; Fish, 1948, pp. 63-66; Schultz and Stern, 1948, p. 131). Although the fishes called grunts are notoriously noisy species of warmer marine waters, due to the rasping stridulation of the pharyngeal teeth, observations at Bimini indicated that not all species of the Haemulidae are equally important sound producers (Tables I and II). The haemulid rasp was not identified in any recordings made at sea; it was heard only from hand-held specimens of the blue-striped (Table II; Fig. 11) and yellow grunts, Hacnudon sciurus (Shaw) and H. flavolineatum (Desmarest), respectively; no recordings V&'JffHi'HffV'T &1H • > 1* - » i w 4 -«* v»r,> •", * *• 4 .1 '(Iff 4 '4. ..^ .tA" SECONDS FIGURE 13. Grunts of Epincphalus striatus in cement tank. FIGURE 14. Two E. striatus grunts (left) and a volley of thump-like sounds of H. asccn- sionis west of Turtle Rocks. Snapping shrimp in background. FIGURE 15. Snaps of male Pomacentrus leucostictus in aquarium. of the latter were obtained. The observations of Burkenroad (1930) which related the sounds of Hacnmlon to movements of the pharyngeal teeth were confirmed, with the exception that the association between the dorsal pharyngeal teeth and the anterior end of the air bladder seems more important in resonating the rasping sound than does the varying relation between the air bladder and the lower pharyn- geal teeth. The opercula are somewhat extended during sound production. Rub- bing together of dissected pharyngeal toothplates produces a much fainter sound than that produced by the living fish. Holocentridae (Fish, 1948). Sound production by the squirrelfish, Holo- 366 JAMES M. MOULTON centrns ascensionis, said to have derived its name from its chattering call (D. de Sylva, personal communication), is chaiacteristic of the western edge of the Great Bahama Bank (Table II; Fig. 12, 14). It was recorded only along, and mainly to the west of, the island and rock chain extending from North Bimini south to South Cat Cay (Tables III and IV; Fig. 16). In rocky areas of the bottom along a relatively narrow area probably extending not far below the limits of visibility from the surface, squirrelfish are characteristically found in the daytime each hover- ing near a depression approximately large enough to receive a single fish. Gen- erally living in higher parts of ledges than the grouper, an occasional squirrelfish inhabits a rocky fissure with E. striatus. Collection of squirrelfish in traps up to TABLE III Grouper and squirrelfish sound production along the western edge of the Great Bahama Bank in the Bimini area Recording station Duration of recording Calls/minute Grouper Squirrelfish Both 1) At 15 fa. depth W of Moselle Shoal, drifting N 13 min. .08 0 .08 2) At 25 fa. depth SW of (1), drifting N 18 min. A 0 .4 3) W of gap between Round and Turtle Rocks 6 min. 1.0 5.2 6.2 4) W of Turtle Rocks 5 min. .2 7.2 7.4 5) W of Turtle Rocks 1 1.5 min. .8 6.3 7.1 6) W of Turtle Rocks 45 min. 1.9 9.5 11.4 7) W of Turtle Rocks, drifting SE 14 min. .6 3.4 4.0 8) W of Triangle Rocks, drifting NE 10 min. 1.3 3.3 4.6 9) West of Triangle Rocks 13 min. 1.7 3.2 4.9 10) W of gap between Piquet and Triangle Rocks 15 min. 2.5 8.6 11.1 11) W of Piquet Rocks 12 min. 2.4 4.1 6.5 Average number of calls/minute (11 stations): Grouper 1.2 Squirrelfish 4.6 Both 5.8 a mile east of Turtle Rocks and occasional sightings in deeper reaches east of the Bimini-Cat Cay chain indicated that the species may move beyond its more com- mon daytime habitat along the outer face of the Bank. Holocentrids are mainly nocturnal in their habits (Barbour, 1905, p. 119; Randall, 1955, pp. 33, 38) and at night their local distribution may be considerably more dispersed than during the daytime, although the species considered here is said to return to the same hole each day (Ray and Ciampi, 1956, p. 207). Squirrelfish sounds are produced by contractions of body wall musculature against the rather firm-walled air bladder which is closely associated with the rib cage. The first three ribs are expanded, flattened and thinned dorsally, and are intimately associated with the air bladder wall ; more posterior ribs are easily ACOUSTICAL BEHAVIOR OF BIMINI FISHES 367 separated from the bladder. Physiological stimulation indicated that the muscula- ture chiefly responsible for sound production was that attaching to the dorsal por- tions of the first three ribs, which appear to serve as drumheads. The structure of the air bladder, and its close relationship with the auditory region of the skull, have been described by Nelson (1955). The thump-like sounds of squirrelfish may be repeated singly at irregular inter- vals or produced in rapid volleys ; they are more sharply peaked on vibration fre- quency analysis than are the sounds of angelfish and grouper. When produced by TABLE IV Grouper and squirrelfish sound production over the Great Bahama Bank in the Bimini Area Recording station Duration of recording Calls /minute Grouper Squirrelfish Both 1) On Moselle Shoal 23 min. .04 .06 .1 2) Over wreck EXE of North Rock 27 min. .4 0 .4 3) At North Rock, E side 27.5 min. .04 .2 .2 4) At Crossing Rocks, W side 37.5 min. .1 1.0 1.1 5) In Bimini Harbor entrance, drift to N 6.5 min. 0 .6 .6 6) 50' off S. Bimini, W side 13 min. .7 .2 .9 7) At Round Rock, W side 8 min. 1.0 0 1.0 8) At Round Rock, E side 9 min. .3 0 .3 9) On line between Turtle and Round Rocks, drift N\V 10 min. .9 1.9 2.8 10) Same station 30 min. 1.4 1.8 3.2 11) North end of Turtle Rocks, E side 4 min. 0 0 0 12) Same station 3 min. 0 0 0 13) On line between Triangle and Turtle Rocks, drift NE 12 min. .4 2.2 2.6 14) Concrete shipwreck, E of Turtle Rocks 9 min. 0 0 0 15) Triangle Rocks, E side 11 min. 0 .6 .6 16) Dollar Harbor 5.5 min. 0 0 0 17) Wedge Rocks, NE side 8 min. .8 1.4 2.2 Average number of calls/minute (17 stations): Grouper .3 Squirrelfish .5 Both .8 startled or handled squirrelfish, the sounds are volleyed in bursts of 4 to 20. Singly-produced sounds are of longer duration than those produced in volleys ; these singly-produced sounds are not dissimilar to the ear from the sounds of Nassau grouper, although they cover a greater frequency span than do those of the grouper when both are recorded at the hydrophone. Undisturbed squirrelfish confined in laboratory aquaria and outdoor tanks re- mained silent during listening periods of up to three hours ; confined specimens produced sounds only when handled, fed or startled. Quickened swimming during feeding was characterized by sound production of a considerably lower intensity 368 JAMES M. MOULTON than that produced during handling of the fish. Sudden startling of confined squirrelfish generally resulted in volleys of sound production. At sea, squirrelfish were sometimes heard when no individuals were sighted, but the acoustical behavior of the species was characteristic when specimens were approached by the suspended hydrophone at drift stations. This behavior included Moselle Shoal North Rock Sunken Ship * Crossing Rocks NORTH BIMlNt N SOUTH BIMINI North Cat Coy South Cat Cay Dollar Harbor *. Wedge Rocks KEY 10 Fathoms 100 Fathoms 5 Miles FIGURE 16. The Bimini area from Moselle Shoal to South Cat Cay. ACOUSTICAL BEHAVIOR OF BIMINI FISHES 369 erection of the spiny fins, adjustment of position so that an eye was directed toward the approaching hydrophone, movement toward a rocky depression near which the fish hovered, and production of the call in volleys of 3 to 20 individual pulses. The continuing approach of the hydrophone resulted in the fish's moving into its rocky shelter. Pomacentridae (Fish, 1948, pp. 59-63). The sharp but rather faint knocks or snaps produced probably only by males of the demoiselle, Pomacentrus leuco- stictus (Mueller and Troschel), were recorded when a given individual suddenly dashed from cover to pursue other individuals approaching its place of conceal- ment (Table II; Fig. 15). These were heard on several occasions. The behavior was similar to that of a sciaenid, Corvina nigra (Bloch) described by Dijkgraaf from Naples (1947). Serranidae (Fish, 1954, pp. 36-44). The air bladder of the Nassau grouper, Epinephalus striatits, is thin-walled and lacks intrinsic muscles, yet this species equals the squirrelfish in its importance as a source of marine sound in the Bimini area (Table II; Figs. 13, 14). Contractions of body wall musculature appear to be responsible for the sound production, while the unusually heavy peritoneum stretched over the air bladder and adjacent organs apparently acts as the sounding- board. Appropriate stimulation of opened fishes brought about some sound pro- duction, even after deflation of the air bladder. The strong contractions of body wall musculature accompanying production of the call are visible externally, and are easily elicited by startling or handling the fish in laboratory tanks. Sounds similar to those produced on alarm, but of a somewhat lower intensity, are produced during quickened swimming accompanying feeding. Like the squirrelfish, the Nassau grouper inhabits the underwater ledges west of the Bimini-Cat Cay chain of rocks and island (Fig. 16). Its distribution is broader from east to west than that of the squirrelfish, since it was sighted where rocky fissures occurred in an otherwise sandy bottom in the Bimini lagoon, and it was seen and recorded in a shipwreck approximately 1 1 miles northeast of North Bimini Island on the Great Bahama Bank (Station 2, Table IV; Fig. 16). Un- identified sounds thought to originate from serranids were recorded at depths exceeding 15 fathoms west of North Bimini Island (Stations 1 and 2, Table III), from which depths serranids have been obtained at Bimini (Scholander ct al., 1951 ; Scholander and van Dam, 1954). The Nassau grouper tends to be more secretive in its habits than the squirrel- fish, ordinarily lying deeper in rocky fissures or well beneath overhanging ledges on the outer faces of wrhich squirrelfish are more frequently seen. In acoustical behavior, the two are similar. As the suspended hydrophone approaches a grouper, the fins are erected and the fish retreats to cover, accompanying the retreat with the characteristic vibrant grunts. Like the call of the squirrelfish, that of the grouper was sometimes heard when no individual was sighted. The call is a characteristic marine sound of the area immediately west of the Bimini-Cat Cay chain. The sound of the rock hind, Epinephalus adsencionis (Osbeck) (Table II), while of markedly lower intensity than that of the Nassau grouper, is produced under similar circumstances in laboratory tanks and is of similar characteristics. It was never specifically recognized during recording at sea. No sounds whatever were recorded from a captive Promicrops itaiara (Lichtenstein) weighing in the 370 JAMES M. MOULTON neighborhood of 300 pounds, although the fish on one occasion very nearly swallowed the hydrophone. DISTRIBUTION OF GROUPER AND SQUIRRELFISH SOUNDS IN THE BIMINI AREA The distribution of squirrelfish and Nassau grouper in the Bimini area has been summarized above. Determination of this distribution was based on both visual and aural observations. The sounds of these species were selected to explore the possibility of determining the distribution of sonic species by aural means alone. The total area of observation extended approximately 20 miles along the north- west edge of the Great Bahama Bank (Fig. 16), from Moselle Shoal on the north to Wedge Rocks on the south, and in an east-west direction from the location of a sunken freighter on the Great Bahama Bank (25° 4?/ N, 79° 7' W) 10.7 miles on a heading of 75 degrees from the northern tip of the Bimini Islands, to a station over an approximate depth of 150 feet southwest of Moselle Shoal. Recordings were made at 28 stations (Tables III and IV), all but two (Stations 1 and 2, Table III) being approximately on or within the 6-fathom line to permit observa- tion of fishes during recording. Table III includes those stations, fixed and drift, at which recordings began on or were wholly confined to the edge of the Great Bahama Bank, to the west of the Bimini-Cat Cay chain; Table IV includes those stations located further in upon the Bank, in most cases immediately to the east of the Bimini-Cat Cay chain. The stations are in each case listed in order from north to south, and are referable to Chart No. 1854 of the United States Hydro- graphic Office, and to U. S. Coast and Geodetic Survey Chart No. 1112. While not each call ascribed to either grouper or squirrelfish in Tables III and IV has been analyzed by vibration frequency analysis, a broad sample of recordings made at sea has been thus treated, and the results have indicated that the inter- pretations of recordings upon which Tables III and IV are based are correct. In erecting Tables III and IV, a rapid volley of either grouper or squirrelfish sounds has been characterized as a single call. Closely consecutive calls of the same species but of different individuals are usually interpretable as such on the basis of intensity differences, due to the origins' being at different distances from the hydrophone. It will be noted in Table III that, although squirrelfish calls were lacking in the deepest stations recorded (1 and 2), along the sloping edge of the Bank they predominated over grouper calls by a factor of nearly 4. Moving of the listening station from the slope side of the Bimini-Cat Cay chain immediately to the Bank side (Table IV) resulted in an abrupt drop in incidence of calling by both species, but particularly by the squirrelfish. On the Bank itself, where squirrelfish were much less frequently sighted, their calls predominated over grouper calls by a factor of less than 2. The calling of these two species together was over 7 times as frequent along the edge of the Bank as to the east of its edge (Tables III and IV) . The difference in incidence of calling between slope and Bank sides of the Bimini- Cat Cay chain is exemplified especially by comparison of Table III, Stations 4, 5 and 6, with Table IV, Stations 9, 10, 11 and 12. Short moves of the listening station resulted in marked differences in the incidence of underwater biological sounds. ACOUSTICAL BEHAVIOR OF BIMINI FISHES 371 Although not indicated by the data presented in Tables III and IV, observations at drift stations (Table III, Stations 7 and 8; Table IV, Stations 9 and 13) indi- cated that as the hydrophone moved over alternately sandy and rocky bottom, the incidence of calling rose markedly as the boat moved over underwater ledges and fell over the sandy stretches ; the incidence of calling thus provided an indication as to the type of bottom beneath the boat, and correlated with sightings of grouper and squirrelfish. OTHER UNDERWATER SOUNDS OF THE BIMINI AREA Sounds most frequently heard during the underwater listening in the Bimini area, other than those described, are ( 1 ) the snapping and crackling characteristic of tropical seas, generally attributed to snapping shrimp, but actually indistinguish- able by methods commonly employed from the sounds of some stomatopods (John- son, et al., 1947; Moulton, 1957) ; (2) a rattling sound like that produced by the spiny lobster, Pannlinis argus (Moulton, 1957) ; and (3) another unidentified rattling sound with its predominant frequencies lying between .5 and 1.3 kc., each pulse being of .02 second duration, and repeated at intervals of approximately .12 second in volleys of varying length. In addition to these, a distinctive series of sounds was recorded twice the same day (12 July 1956 at Stations 6 and 9, Table IV). At Station 6, the sound was a buzz-like whine singly produced; at Station 9, the same sound was preceded by a number of sharp metallic raps and was followed by a number of brief chirps of somewhat lower frequency than the raps. The metallic raps were so similar to the sound of pounding on a steel hull that two observers, prone at the glass panels, concluded a vessel to be bearing down on the listening post. Since the listening boat was quiet except for water noise along the hull, and since no other boats were within view to the horizon, it is assumed that the sounds were of biological origin, but the source is unknown. They do not correspond with known cetacean sounds (Mr. William Schevill, personal communication). DISCUSSION The behavior of the Nassau grouper and squirrelfish in the Bimini area fur- nishes a marked exception to the generalization (Fish, 1954, p. 7) that fishes in the field are silenced by strange contacts. Both of these species were obviously stimulated to active sound production by approach of the boat and suspended hydro- phone at drift stations over shallow waters. The spiny fin erection and movement toward concealment of these species upon approach of the hydrophone were strongly suggestive that the sounds described are related to self-protection, a probability further suggested by production of the same sounds during handling of these fishes. Although some fishes may use echo-location (Griffin, 1955), there is no evidence at present of its being involved in the cases under discussion. Cir- cumstances surrounding production of grouper and squirrelfish calls, as observed at Bimini, were similar to those that surround production of sea robin grunts (Moulton, 1956b). While the grunts are readily produced during handling by most specimens of the sea robins common at Woods Hole, Prionotits cvolans (L.) and P. caroUnus (L.), the grunts are also produced by sea robins contained to- gether in live cars and living on the bottom. Sea robin grunts are not, however, 372 JAMES M. MOULTON so easily stimulated by startling as are the sounds of Nassau grouper and squirrel- fish which readily produced sounds recorded at sea after periods of confinement of two weeks in laboratory aquaria. The squirrelfish is the most significant producer of underwater sound among fishes in the Bimini area, and if the 20-mile extent of the Bank area studied may be considered typical of the whole, of the edges of the Great Bahama Bank gen- erally. In view of this significance, Fish's ( 1948, p. 44) estimate of the sonic importance of the Holocentridae as "probably none" must be rejected. Since holo- centrids are of wide distribution in tropical and sub-tropical waters, it seems prob- able that their sonic importance extends to other waters than those of the Bimini area. The acoustical behavior of the angelfishes (Chaetodontidae) has not been hitherto adequately described, but there can be little doubt that the behavior of the single specimen recorded at Turtle Rocks on 10 July furnished evidence of a call accompanying recognition behavior in this species. The black angelfish which is most common of the angelfishes in the Bimini area, has a tendency to examine underwater objects (hydrophone, swimming cowfishes), such examination being accompanied by sounds of briefer duration than those proposed as a part of recog- nition behavior. The black angelfish is usually observed in pairs during July and August at Bimini. It seems likely that chaetodontids may contribute to under- water sound in other tropical and sub-tropical areas, although they are not dis- cussed among sound-producing fishes of the Pacific by Fish (1948). Further evidence of the tendency of captive fishes to become silent unless dis- turbed was provided by all species studied during the summer of 1956. The only species to produce sound spontaneously in laboratory aquaria during listening periods of up to three hours, other than those produced during feeding and being startled, was the small pomacentrid, Pouiaccntnts Icucostictiis, probably a male, as it pursued other individuals encroaching on its hiding place. As is the case along the northeastern coast of the United States where the most significant fish producers of underwater sound which have been identified (sciaenids, triglids, and batrachoidids) are fishes using, rather than skeletal stridu- latory mechanisms, muscles in close association with the air bladder (Tower. 1908; Fish, 1954; Moulton, 1956b), a holocentrid. a serranid and a chaetodontid are the most frequently calling fishes of the area immediately about the Bimini-Cat Cay chain. Of the former, however, both sea robins and toadfish produce sounds with muscles intrinsic to the air bladder, while all three of the most important calling fishes of the Bimini area use muscles extrinsic to the air bladder in producing their sounds. The structural specializations and behavioral patterns of the sound producing species studied at Bimini have provided further striking evidence of the significance of sound in the biology of the species concerned, and the consistent incorporation of sound production into behavioral patterns observed in the clear water about Bimini (Nassau grouper, squirrelfish, angelfish) strengthens a conclusion that sound is of significance to the species concerned. Yet, as has repeatedly been affirmed, clear evidence of effectiveness of the sounds concerned in modifying the movements of fishes in nature is still lacking, although Tavolga (1956) has observed the females of a goby to demonstrate increased activity during sound production ACOUSTICAL BEHAVIOR OF BIMINI FISHES 373 by the male in breeding. The sounds of all calling species studied at Bimini are produced by both sexes, except for the snaps of Por.iaccntrus Icucostictus. Experiments of playing into the water artificial sounds and recordings of natural sounds such as those that have elsewhere modified fish behavior (Moulton, 1956a, 1956b; Tavolga, 1956) had no notable effect in the Bimini area. During all listening at sea in the Bimini area, all during daylight, sound production was prolific or rare, depending on the distribution of the species concerned and not on alternating periods of quiet and of sound production which seem to characterize production of the staccato call of sea robins at Woods Hole (Moulton, 1956b). Therefore, these experiments are not reported in detail. The Nassau grouper and squirrelfish were never observed in areas where their calls were not heard at Bimini. The most characteristic component of background noise in the Bimini area is the "crackle" so characteristic of warmer seas, and which has generally been ascribed to snapping shrimp (Johnson ct al., 1947). By the analysis methods used, this noise in the Bimini area presents components cumulatively spanning the fre- quency range examined (up to 8 kc.). The invertebrates largely responsible are a common stomatopod (Gonodactylus oerstedi} and several kinds of snapping shrimps, including Alpheus armatns and Synalpheus spp. (Johnson ct al., 1947; Pearse, 1950; Moulton, 1957). The sound spectra obtained during this study from recordings of feeding and of stridulation sounds of various fishes indicate that in regions where producers of such sounds are numerous, they may contribute extensively to the spectrum of underwater sounds generally attributed to inverte- brate sound producers. I am much indebted to Dr. R. H. Backus for constructive criticism of the manuscript of this paper. I am also indebted to Mr. Donald de Sylva for assistance in identification of several of the fishes studied, and to Mr. E. R. Powell for assist- ance with the illustrations. Use of the generous facilities of the Lerner Marine Laboratory of the American Museum of Natural History is gratefully acknowledged. SUMMARY 1. On the basis of observations and recordings at sea and in the laboratory, the acoustical behavior of 13 species of Bahamian fishes is described, and their sounds are defined. Twenty-six species producing no calls in the course of this study are specified. 2. The most important sound-producers among fishes of the Bimini area are the squirrelfish, Holoccntrus ascensionis, and the Nassau grouper, Epinephalus stnatus. Their characteristic sounds may be anticipated when these species en- counter a strange object at sea, and probably generally during the daytime along the edges of the Great Bahama Bank. 3. A single observation has indicated that calling is a component of recognition behavior in the black angelfish, Pomacanthus arcuatus. The families Chaeto- dontidae and Holocentridae should be added to lists of fish families containing calling members. 4. The usefulness of underwater listening in studying the distribution of some calling fishes has been demonstrated in the cases of the squirrelfish and Nassau grouper. 374 JAMES M. MOULTON LITERATURE CITED BARBOUR, T., 1905. Notes on Bermudian fishes. Bull. Mus. Coinp. Zool., 46: 109-134. BRIDGE, T. W., 1910. Fishes. Cambridge Natural History, 7 : 139-537. Macmillan and Co., Ltd., London. BURKENROAD, M. D., 1930. Sound production in the Huciintlidae. Copeia, 1930, No. 1 : 17-18. BURKENROAD, M. D., 1931. Notes on the sound producing marine fishes of Louisiana. Copeia, 1931, No. 1 : 20-28. DIJKGRAAF, S., 1947. Ein Tone erzeugender Fisch im Neapler Aquarium. Expericntia, 3: 493. DOBRIN, M. B., 1947. Measurements of underwater noise produced by marine life. Science, 128 (105 N.S.) : 19-23. EVERMANN, B. W., AND M. C. MARSH, 1900. The fishes of Porto Rico. Bull. U. S. Fish Comm. for 1900: 1-350. FISH, M. P., 1948. Sonic fishes of the Pacific. ONR Contr. N6 ori-195, T.O. 1, Tech. Report No. 2. FISH, M. P., 1954. The character and significance of sound production among fishes of the western North Atlantic. Bull. Bingliain Oceanographic Collection, 14 ; Art. 3 : 1-109. FISH, M. P., A. S. KELSEY, JR. AND W. H. MOWBRAY, 1952. Studies on the production of underwater sound by North Atlantic coastal fishes. /. Mar. Res., 11 : 180-193. GRIFFIN, D. R., 1955. Hearing and acoustic orientation in marine animals. Papers Mar. Biol. and Oceanogr., Deep-Sea Research, suppl. to Vol. 3, pp. 406-417. JOHNSON, M. W., F. A. EVEREST AND R. W. YOUNG, 1947. The role of snapping shrimp (Crangon and Synalpheus ) in the production of underwater noise in the sea. Biol. Bull, 93: 122-138. MOULTON, J. M., 1956a. The movements of menhaden and butterfish in a sound field. Anat. Rec., 125: 592. MOULTON, J. M., 1956b. Influencing the calling of sea robins (Prionotus spp.) with sound. Biol. Bull., Ill: 393-398. MOULTON, J. M., 1957. Sound production in the spiny lobster Panulirus argus (Latreille). Biol Bull, 113: 286-295. MOULTON, J. M., AND R. H. BACKUS, 1955. Annotated references concerning the effects of man-made sounds on the movements of fishes. Fisheries Circ. No. 17, Dep't of Sea and Shore Fisheries, Augusta, Maine. NELSON, E. M., 1955. The morphology of the swim bladder and auditory bulla in the Holo- centridac. Fieldiana: Zoology, 37: 121-130. PEARSE, A. S., 1950. Notes on the inhabitants of certain sponges at Bimini. Ecology, 31 : 149-151. RANDALL, J. E., 1955. Fishes of the Gilbert Islands. Atoll Research Bull., No. 47, Pacific Science Board, Natl. Acad. of Sci., National Research Council, Washington. RAY, C., AND E. CIAMPI, 1956. The Underwater Guide to Marine Life. A. S. Barnes and Co., New York. SCHOLANDER, P. F., C. L. C'LAFF, C. T. TENG AND V. WALTERS, 1951. Nitrogen tension in the swimbladder of marine fishes in relation to the depth. Biol. Bull, 101 : 178-193. SCHOLANDER, P. F., AND L. VAN DAM, 1954. Secretion of gases against high pressures in the swimbladder of deep sea fishes. I. Oxygen dissociation in blood. Biol. Bull., 107 : 247-259. SCHULTZ, L. P., AND E. M. STERN, 1948. The Ways of Fishes. D. Van Nostrand Co., Inc., New York. TAVOLGA, W. N., 1956. Visual, chemical and sound stimuli as cues in the sex discriminatory behavior of the gobiid fish Bathygobius separator. Zoologica, 41, Part 2: 49-64. TOWER, R. W., 1908. The production of sound in the drum-fishes, the sea-robin and the toadfish. Annals N. V. Acad. Sci., 18: 149-180. EFFECT OF PLANT HORMONES ON ULVA1 L. PROVASOLI Haskins Laboratories, 305 East 43rd Street, Nciv York 17, N. V. Foyn (1934a) in his early attempts to grow Ulva lactiica found that Ulva, like Cladophora suhriana (Foyn, 1934b), grows poorly in sea water enriched with nitrates and phosphates (Schreiber, 1927) and that the addition of soil extract to Schreiber's medium is necessary to obtain normal growth and the entire life-cycle. This medium ("Erdschreiber") later became the standard medium for growing marine flagellates in bacterized cultures (Gross, 1937; Parke, 1949). Kylin (1941) employed Ulva lactiica to analyze the biological activity of dif- ferent samples of sea water : he found that sea water at 70 meters depth is inade- quate to support normal growth and that addition of nitrates, phosphates and trace metals made it suitable for the germination of the zoospores of Ulva and elicited as rapid growth of the germlings to the stage of 15-20 cells as did the "Erdschreiber." Levring (1946), employing the same technique and test organism, formulated a synthetic sea water which, similarly enriched, allowed normal development of the germlings of Ulva, thus confirming, with a chemically defined medium, Kylin's conclusion. Levring's medium was the starting point for the formulation of sev- eral synthetic marine media which do not precipitate and are suitable for the cultivation of a number of marine and brackish algal flagellates in bacteria-free culture (Provasoli, McLaughlin and Droop, 1957). Since Foyn, Kylin and Levring worked with bacterized cultures, I wondered if Ulva, when bacteria-free, would grow in mineral media or if it would require organic factors. Many other algae which, like Ulva, were previously cultured in Erdschreiber + bacteria, require, besides nitrates and phosphates, growth factors and trace metals when cultured in synthetic media without bacteria (Provasoli and Pintner, 1953; Sweeney, 1954; Lewin. 1954; Droop, 1955a, 1955b, 1957; Provasoli, 1957). In exploratory attempts to grow Ulva in bacteria-free culture, I failed to obtain a typical foliaceous thallus but in trying to obtain it, I found that Ulva germlings respond to plant hormones. MATERIAL AND METHODS Bacteria-free cultures of Ulva were obtained by placing pieces of thallus on the surface of agar media containing various concentrations of an antibiotic mixture (1 ml. of the concentrated antibiotic solution contains: K penicillin G 12,000 units; chloramphenicol 50 ^g. ; polymyxin B 50 /xg. ; neomycin 60 /xg.). I recognized from the beginning the necessity of employing thalli free from epiphytic organisms : the pieces of thallus were selected and inspected under the dissecting microscope and, as an additional precaution, were cleaned by brushing 1 Aided in part by Contract NR 163-202 with the Office of Naval Research. 375 376 L. PROVASOLI the two surfaces with a thick, soft water-color brush. Even so, the epiphytes could not always be eliminated and several bacteria-free cultures of Ulva were infected with small diatoms (mainly Nitsschia). The concentrated antibiotic mixture was sterilized by nitration through a glass filter, and 0.1-, 0.2-, 0.3-ml. aliquots were dispensed on the bottom of sterile Petri dishes; 20 ml. of sterile \A% agar media, kept at 45° C., were added and thoroughly mixed with the antibiotic by twirling. The pieces of Ulva thallus (5-mm. squares) were left on this agar for 7 days, removed aseptically with a spatula, placed in depression slides containing sterile media, cut in several narrow strips with an iridectomy scalpel, and transferred to liquid media. The pieces treated with the lower concentrations of antibiotics (0.1-0.2 ml. in 20 ml. of agar media) were infected with either bacteria, a pink yeast, or diatoms. All the pieces treated with 0.3 ml. of antibiotic mixture (final concentration of antibiotics per ml. of agar medium : K penicillin 200 units, and 1 ^.g. each of chloramphenicol, neo- mycin, and polymyxin) were bacteria-free but about % were infected by diatoms. Several liquid media were tried : the most successful were ASW III and ASW 8 (Table I) ; both are enriched sea water media similar to, but richer than, Erd- schreiber. ASW III (richer in organics) was employed in the early experiments; later I employed ASW 8 which allows better growrth. The cultures are carried in screw-cap tubes (125 X 20 mm.) with 10 ml. of medium ; to avoid chemical contamination we employ plastic caps without liners. At first, Ulva was grown in continuous light (200 foot-candles; fluorescent tubes) at 18-20° C., later in alternate light (16 hours) and darkness (8 hours). The sample of gibberellins was kindly supplied by Dr. Nickell, of Chas. Pfizer & Co., and the one of kinetin bought from the California Foundation for Biochemical Research. RESULTS The purified strips of thallus, when transferred to liquid media, began to pro- duce thin filamentous germlings from their surface and looked like pincushions. That many zoospores were also set free was evident from the many small germlings that covered the walls of the test tubes. The germlings arising from the thallus were round, thin, solid, and never became more than 2^4 mm. long; after two months they bleached, leaving a few intensely green spots which dotted their surface at random. The germlings on the walls of the tubes in certain media behaved similarly and produced a number of rhizoids, many of them colorless ; in other media, the germlings developed only rhizoids and looked like stellate colonies or like an elongated root system in minia- ture. Pieces of bleached stubby germlings, or the stellate rhizoidal colonies, when transferred to new media, produce new germlings from the islands of intensely green cells which are scattered among the bleached tissues ; these green cells remain dormant and viable for a year (longest time tried) in the old medium. The germlings of the second generation underwent a similar cycle, they grew a few millimeters and later bleached partially ; no zoospores were produced by these germlings. Serial transfers are carried out by removing the old germlings asepti- cally from the culture tubes, cutting them in pieces and inoculating the pieces in different media. EFFECT OF PLANT HORMONES ON ULVA 377 Foyn obtained normal development of the thallus, production of zoospores and gametes of Ulva, in bacterized cultures grown in Erdschreiber. ASW III, an Erdschreiber enriched with vitamins, liver extract, and carbon sources, allowed only the formation of germlings ; the typical thallus was never obtained in bacteria- free cultures in this medium. The beginning of a .thallus (the formation of two short, thick "rabbit ears" or a fan-like curly, thick, small thallus) was obtained in TABLE I Media for Ulva Foyn's Erdschreiber ASW III ASW 8 Sea water 100 ml. 100 ml. 80 ml. H2O 20 ml. NaNO3 10 mg. 30 mg. KNO3 20 mg. Na2HPO4 • 12 H2O 2 mg. K2HPO4 2 mg. Na2 glycerophosphate 3 mg. Mn (as Cl) 0.04 mg. Fe (as Cl) 0.01 mg. 0.05 mg. P II metals* 3 ml. Vitamin mix No. 8** 0.1 ml. Vitamin mix S. 3*** 0.5 ml. Bi2 0.01 Mg. Liver 1 : 20f 1. mg. Soil extract 5 ml. 4 ml. Na H glutamate 50 mg. Glycine 50 mg. Tris (hydroxymethyl) amino-methaneft 100 mg. 100 mg. pH 8.0 7.5 8.0 * One ml. of P II metal contains: ethylenediamine tetraacetic acid, 1 mg. ; Fe (as Cl) 0.01 mg. ; B (as H3BO3) 0.2 mg. ; Mn (as Cl) 0.04 mg. ; Zn (as Cl) 0.005 mg. ; Co (as Cl) 0.001 mg. ** One ml. of Vitamin mix No. 8 contains: thiamine HC1, 0.2 mg. ; nicotinic acid, 0.1 mg. ; putrescine 2 HC1, 0.04 mg. ; Ca pantothenate, 0.1 mg. ; riboflavin, 5.0 jug- ; pyridoxine 2 HC1, 0.04 mg. ; pyridoxamine 2 HC1, 0.02 mg. ; para-aminobenzoic acid, 0.01 mg. ; biotin, 0.5 jug- ; choline H2 citrate, 0.5 mg. ; inositol, 1.0 mg. ; thymine, 0.8 mg. ; orotic acid, 0.26 mg. ; Bi2, 0.05 /ug. ; folinic acid, 0.2 jug. ; folic acid, 2.5 pg. *** One ml. of Vitamin mix S. 3 contains: thiamine HC1, 0.05 mg. ; nicotinic acid, 0.01 mg. ; Ca pantothenate, 0.01 mg. ; p.-aminobenzoic acid, 1.0 yug- ; biotin, 0.1 ^g- ; inositol, 0.5 mg. ; folic acid 0.2 ^g- ; thymine, 0.3 mg. f Nutritional Biochemical Corporation, Cleveland, Ohio, U. S. A. ft "Sigma 7-9 biochemical buffer," Sigma Chemical Co., St. Louis, Missouri, U. S. A. two of the tubes which were infected with a pink yeast or a diatom. However, re-infection of the axenic cultures of Ulva germlings with clonal cultures of the yeast or diatom failed to produce a thallus. Disappointed by the failure to obtain normal thalli, I re-examined Foyn's cultural methods and noted that he had found (1955) that only the northern European variety of Ulva can be grown in continuous light, that the southern variety (which he proposed to call Ulva thureti) bleached if grown in continuous light and that a normal thallus was formed only with a daily period of 6 or more hours of darkness. From then on, all cultures had an 8-hour 378 L. PROVASOLI 1 8 14 18 10 15 11 12 16 19 20 FIGURES 1-21. 13 17 21 EFFECT OF PLANT HORMONES ON ULVA 379 dark period and 16 hours light but no thallus formed and the germlings bleached as before. The repeated failure to obtain normal morphogenesis of the thallus in bacteria- free culture, in media similar to Erdschreiber, suggested that the failure was due to a lack of morphogenetic regulators ; some of them, like indolacetic acid and gib- berellin, can be produced by microorganisms. I tried various concentrations and combinations of adenine, indolacetic acid (IAA) and kinetin which influence growth and differentiation in higher plants. In the first experiments we found that adenine by itself favored the production of more germlings from the dormant green cells and induced longer filaments ; the best concentration was 3 mg.'/r (6 mg.% is inhibitory). In the presence of 1 ju.g.% kinetin, indolacetic acid induced a large number of germlings whose length tended to increase proportionally with the concentration of IAA: 5 fj.g.% was the best. When 3 mg.% adenine was superadded, the most effective concentration of IAA was lO/xg.% ; 30/xg.% inhibited the length and number of germlings. In the presence of 5/xg. IAA, increasing concentrations of kinetin also favor the elongation of germlings; the highest concentration tried (10 /tg.%) induced the longest germlings obtained in these early experiments (Fig. 5). When adenine was superadded, the number of germlings induced by the combined action of IAA and kinetin is apparently not affected, but adenine completely inhibits the sharp elongation produced by 10 /xg.% kinetin (Fig. 8). Perhaps in Uh'a these morpho- genetic determinants interact and have a limited specific action paralleling their activities on the tissues of higher plants. At this point, we tried to substitute sea water media with synthetic mineral EXPLANATION OF PLATE I FIGURES 1-8. Medium ASW III : forty-five days' growth. The new growth is repre- sented by the lateral filaments budding from the old pieces. FIGURE 1 : ASW III alone. FIGURE 2: + kinetin 2.5 fj.g.%. FIGURES 3-5. Kinetin curve: basal medium = ASW III + indolacetic acid 5 Mg-% ; FIGURE 3: + kinetin 1 /xg.% ; FIGURE 4: + kinetin 5 Mg-% ; FIGURE 5: + kinetin 10 Mg-%- FIGURES 6-8. Kinetin curve : basal medium = ASW III + indolacetic acid 5 Mg-% + adenine 3 mg.%; FIGURE 6: + kinetin 1 Mg-% ; FIGURE 7: + kinetin 5 Mg-% ', FIGURE 8: + kinetin 10 Mg.%. FIGURE 9. ASW 8 medium + adenine 3 mg.% + kinetin 20 Mg-%- Same as Figure 15 but after 120 days' growth. Note islands of green, resistant cells interspersed in the bleached tissue of the blade. FIGURES 10-20. ASW 8 medium: sixty days' growth. FIGURES 10-13. Indolacetic acid curve: basal medium = ASW 8 + kinetin 10 Mg-% ; FIGURE 10: No addition; FIGURE 11: + in- dolacetic acid 5 /J.g.% ', FIGURE 12: + indolacetic acid 10 /J.g.%; FIGURE 13: + indolactic acid 20 Mg-%.. FIGURES 14-15. ASW 8 medium + adenine 3 mg.%; FIGURE 14: No addition; FIGURE 15: + kinetin 20 Mg-%- FIGURES 16-20. Gibberellins curve: basal medium = ASW 8 + indolacetic acid 5 Mg.% + kinetin 10 Mg.%. FIGURE 16: No addition; FIGURE 17: + gibberellins 1 Mg-% ; FIGURE 18: + gibberellins 10 Mg-% ; FIGURE 19: + gibberellins 40 Mg-% : FIGURE 20: + gibberellins 100 Mg-% : note that all the filaments are bleached and that only the rhizoids of the disc of attachment are still green. FIGURE 21. Same as Figure 18, but after 120 days' growth; note the many knobby islands of resistant green cells interspersed on the bleached filaments. Enlargement of all figures 2 X natural size. 380 L. PROVASOLI media (Provasoli et el., 1957) or with other types of enriched sea water to which we added the most effective hormone combination (i.e., IAA 5 pg.% and kinetin 10 p-g.%). ASW 8 was far better than both ASW III and the synthetic media, and was used from then on. In ASW 8, formation of germlings and germ ling elongation was again favored by the combination of kinetin and IAA. With kinetin constant at 10 p.g.%, only rhizoids were formed when IAA was absent; 10 fj.g.% IAA elicited longer germ- lings than 5 //.g.% IAA after 30 days growth, but at this concentration the tips of the germlings became brown in 60 days and the germlings were completely brown in 90 days (Fig. 12). The combination of kinetin 10 p.g.% and IAA 5 ng.% produced healthy green germlings which kept on growing and the tips began to flatten, as happens normally in nature, at an earlier stage (Fig. 11); at higher concentrations (20 /ig.%) IAA inhibited and only rhizoids were produced (Fig. 13). Gibberellin, superimposed on the favorable concentrations of kinetin and IAA, induced a dramatic elongation. As with IAA, concentrations of gibberellin ap- proaching the lethal induce a more rapid elongation : thus gibberellin at 100 pg.% produced the longest and thinnest filaments at 30 days growth, but growth stopped at this time and the filaments were totally bleached at 60 days (Fig. 20) ; only the rhizoids of the attachment disc remained green. Gibberellin at 10 /xg.% elicited maximum elongation; concentrations between 10 and 40 /xg.% neither inhibited nor reduced the number of green islands of cells left when the germlings bleached at the end of growth (Fig. 21). Gibberellin at 1 /j.g.% seemed to produce a definite response as compared with the control, but this may be due to a difference in inoculum (compare Fig. 17 with Fig. 11). So far, the response of Ulva to morphogenetic substances had been to induce few or many, and shorter or longer, atypical solid filaments — a far cry from what happens in nature ; still, a beginning of blade formation could be detected in the flattening at the tips of the germlings grown in kinetin 10 pg.% + IAA 5 p.g.%. An elongated flat blade, probably composed of two layers of cells and similar to the one normally occurring in nature, was obtained by the addition of 20 /u.g.% kinetin to 3 mg.'/c adenine (Fig. 15). Adenine alone and lower concentrations of kinetin (2.5, 5, 10 p.g.% ) + adenine were completely ineffective; only rhizoids and lumpy growth around the inoculum appeared ; the atypical elongated germlings were com- pletely lacking (Fig. 14). DISCUSSION Though the studies of Ulva in bacteria-free culture are just beginning, two unexpected findings emerge : 1 ) a sea weed under our experimental conditions re- quires exogenous hormones for normal morphogenesis; 2) the thalli. typical and atypical alike, reach only an extremely small size as compared with the natural one, then bleach, but only partially, leaving islands of green cells which, when transferred to new medium, can originate new germlings. Thuret (1878) described only two morphological types of cells in Ulva: the oblong cells constituting the major portion of the thallus and the rhizoids which make up the disc of attachment. The rhizoids are formed by "tubular cells" originating in the basal part of the foliaceous thallus : these cells elongate, push their tips downward between the two cell layers of thallus, reach the substratum to which EFFECT OF PLANT HORMONES ON ULVA 381 the thallus is attached, and form a mat of filaments which anchors the thallus solidly. Delf (1912) found that the tubular cells differ clearly from the other cells of Ulva in being multinucleate (they have 3-5 nuclei in the upper portion, few in the tubular portion and 2-5 nuclei in the rhizoidal portion). These observations were made on discs of Ulva growing on thalli of Polysiphonia; the material had been fixed in the early spring (i.e., before the appearance of foliaceous thalli) yet the tubular cells were undoubtedly alive when fixed. Schiller (1907) believes that new germlings can originate from the rhizoids and Cotton (1910) and Delf (1912) postulate that the foliaceous part of the thallus of Ulva is annual while the disc of attachment is perennial. Similarly, our experiments show that there are two types of cells : one which bleaches and dies easily, and a very resistant one. However, the resistant cells are located in two regions: 1) the disc of attachment, and 2) the erect elongated por- tion of the germling in which islands of cells remain green when the whole germling bleaches. Though both of these permanently green cells produce new germlings, they seem to have a different resistance to unfavorable conditions. At inhibitory concentrations of IAA (10 p.g.% ; Fig. 12) and gibberellin (100 p.g.% ; Fig. 20) no green islands appeared, all the cells of the erect part of the germling died, but the rhizoids remained green; at higher concentrations of IAA (20 pg.% ; Fig. 13) the green islands of the inoculum did not produce new germlings but only a mat of rhizoids. It is most probable that the cells constituting the mat of the disc of attachment in our cultures are rhizoids, nonetheless we intend to test this hypothesis cytologically and see whether they are polynucleate. One would be tempted to consider that the cells of the green islands are also polynucleate because they are able to produce new germlings directly and without passing through the zoospore stage. However, they could also be morphologically identical with the oval cells which normally produce zoospores, but have a different potency. These cells do not appear only in the atypical germlings obtained in the laboratory; the original pieces of Ulva, from which our cultures derive, were small squares cut from the upper median part of the foliaceous thallus which is supposedly composed only of cells producing zoospores or gametes, yet not only zoospores were produced but a number of germlings originated directly from the piece of thallus which took the appearance, as noted, of a pincushion. Islands of permanently green cells in our cultures not only appeared in the atypical filamenteous germlings (Fig. 21), but also in the bleaching flat blade obtained with adenine + kinetin (Fig. 9). We can conclude then that another type of cell (different at least in its physiological potencies) exists among the oblong cells of the growing germlings and of the foliaceous thallus. These observations invite new studies on the morphology and potencies of the cells of Ulva, and on the localization and distribution in the various parts of the thallus of the various morphological and physiological types of cells. The resistant rhizoids of the attachment disc may prove the commonest and most valuable way of surviving winter and other hardships. The presence of other resistant cells in the foliaceous part of the thallus may be equally important eco- logically in providing a more efficient way of spreading the species : pieces of thallus, fragmented by waves and transported by currents, can easily colonize distant sites far beyond the reach of the short-lived swimming zoospore stage. Skoog and Miller (1957), in a penetrating review, conclude that regulation of 382 L. PROVASOLI growth may depend more upon the quantitative interactions than upon the quali- tative action of the single plant hormones. This contrasts with the previous ideas that there are specific organ-forming substances and that "determination" is an irreversible loss in the regenerative abilities of cells and tissues. We have not yet explored separately the action of each morphogenetic sub- stance in its active range, nor the effects of kinetin at higher concentrations nor all the various combinations of morphogenetic agents. It seems, at this stage, that production of more germlings and, especially, the elongation into atypical germlings result from the combined action of indolacetic acid and kinetin ; adenine and indolacetic acid appear antagonistic. The narrow effective range of IAA is puzzling. Judging from the elongation of the atypical filaments, kinetin and gibberellin are not toxic over a wide range, while indolacetic acid is effective only in a narrow range (1 ju.g.% IAA is barely active, 5 are optimal, and 10 /xg.% induce rapid growth followed by rapid death). The formation of a flat thallus, so far, has been obtained by combining adenine with kinetin, but in this combination kinetin is inactive up to 20 /*g.%, while in combina- tion with indolacetic acid it elicits elongation of atypical germlings at 10 jug.% (Fig. 5). So far, only adenine + kinetin have given normal growth while growth of atypical germlings results from the combined action of kinetin and IAA. Distinguishing between specific actions, interactions, and mixed actions of plant hormones is a complicated task in higher plants : isolated specialized tissues — an artificial situation — may be misleading for morphogenetic conclusions ; mixed tis- sues represent different potencies, while organs are too highly specialized and reflect the interdependency of many distinct tissues. If other algae respond to plant hormones as one may expect, they may become excellent experimental material. The Chlorophyceae, because of their closeness to the primitive land plants, may be the best choice : they abound in species repre- senting practically all the early steps of increasing structural complexity — the simple filament ; different types of heterotrichous filaments ; complex branched filamentous thalli in which the prostrate and the erect system may be unequally developed; thalli with specialized oogamy; and foliaceous thalli. With algae, one can work with whole organisms, and not with parts of highly evolved organisms artificially avulsed from the whole, simply by selecting species in order of increasing morphological complexity. The activity of plant hormones on Ulva raises the question of precisely where in the algal line of evolution toward the land plants, plant hormones were first employed as morphogenetic regulators. Earlier studies on the action of indolacetic on unicellular algae seem uncon- clusive or negative. Preliminary experiments, done in collaboration with J. J. Pintner and K. Gold, show that several fresh water and marine unicellular algae and even the colonial Volvox globator do not respond to indolacetic acid, kinetin and gibberellin : growth rate, final growth and morphology are unchanged within the concentration range effective for Ulva. It is not surprising that flagellates which are considered morphologically the primitive form from which the vegetal and animal tendencies of the algae have evolved, do not respond to morphogenetic hormones. Hormones are concerned with the balanced growth of a cellular or- ganism— how can one expect to find visible changes in an organism which has no cellular parts ? The lack of effect on Volvox supports the generally held idea that EFFECT OF PLANT HORMONES ON ULVA 383 this line is an evolutionary cul-de-sac and that Volvox is a colony of individuals. However, since the cell is the site of action of the hormones, "unicellular" algae may be the material of choice for studying the mode of action of plant hormones at the cellular level, but then, we need powerful specific antagonists to plant hormones. It has been fortunate that plant hormones under our experimental conditions are indispensable for normal morphogenesis of Ulva. Quite likely this will hold for other media but, if it were not so, some nutritional factors upset the normal morphological development; the study of their role in morphogenesis could then allow a deeper insight into the action of plant hormones. ASW 8 allows better growth and the action of plant hormones is more evident in ASW 8 than in ASW III. The main difference between the two media is the presence in ASW III of an aqueous liver extract and soil extract, both of which introduce purines. Some purines, as adenine and kinetin, are important morphogenetic agents for Ulva, but it is conceivable that other purines may interfere with the normal processes of growth. Definitive results can be obtained only by substituting for sea water a chemically defined medium to eliminate the unknown organic constituents of sea water. The Ulva data suggest that these plant hormones may be as ecologically im- portant for other sea weeds as vitamins are for phytoplanktonts. The auxins and gibberellins are microbial products (see Brian's review, 1957) and the unknown natural purines, which act like kinetin, may also be significantly contributed in natural waters by microbial action. It is possible therefore that the coastal zone, because of the land drainage which favors microbial growth, may never be so poor in plant hormones as to limit sea weed growth severely, but fluctuation in their level may control speed of growth and size of crop. This may be of economic importance to nations, like Japan and Ireland, which farm and use sea weeds extensively. To resolve these issues, not only are extensive pure culture studies needed but also convenient sensitive methods for assaying plant hormones in sea water. SUMMARY 1. Bacteria-free Ulva lactuca, in sea water media enriched with vitamins, grows as atypical, short, filamentous germlings which do not develop into a foliaceous thallus. These filaments reach a few millimeters, then bleach, leaving a few islands of intensely green cells which, upon transfer to new media, produce new germlings. 2. The initiation of new germlings from these green islands and the length of the atypical filaments are increased by the combination of kinetin + indolacetic acid ; adenine and indolacetic acid seem antagonistic. Conspicuous elongation of the filaments is promoted by the addition of gibberellins to the kinetin-indolacetic acid combination. 3. A normal flat blade was obtained so far only with adenine + kinetin. 4. The responses depend both on the interaction and concentrations of these morphogenetic agents : indolacetic acid is effective only in a very narrow range of concentrations and a blade is produced only with relatively high concentrations of kinetin. 5. The rhizoids of Ulva and the cells of the green islands can produce directly new germlings and are far more resistant to unfavorable conditions than the other 384 L. PROVASOLI cells of the thallus which can originate zoospores or gametes. The morphogenetic and ecological significance of the resistant cells is discussed. 6. These responses of a relatively simply organized sea weed to plant hormones link even more tightly the green algae to the higher land plants. 7. The variety of evolutionary steps toward increased morphological complexity in the algae suggests that whole organisms, because of their relative morphological simplicity, may be valuable experimental material for studying the mode of action of plant hormones. LITERATURE CITED BRIAN, P. W., 1957. The effects of some microbial metabolic products on plant growth. Symp. Soc. Ex p. Bio/., 11: 166-182. COTTON, A. D., 1910. On the growth of Ulva latissima in water polluted by sewage. Kew Bull. DELF, F. M., 1912. The attaching discs of the Ulvaceae. Ann. Bot., 26: 403^08. DROOP, M. R., 195Sa. Cobalamin requirement in Chrysophyceae. Nature, 174: 520. DROOP, M. R., 1955b. A pelagic marine diatom requiring cobalamin. /. Mar. Biol. Assoc., 34 : 229-231. DROOP, M. R., 1957. Auxotrophy and organic compounds in the nutrition of marine phyto- plankton. /. Gen. Microbiol, 16: 286-293. FOYN, B., 1934a. Lebenszyklus and Sexualitat der Chlorophycee Ulva lactuca L. Arch. /. Protistenk., 83: 154-177. FOYN, B., 1934b. Lebenszyklus, Cytologie und Sexualitat der Chlorophycee Cladophora suhriana. Arch. f. Protistenk., 83 : 1-56. FOYN, B., 1955. Specific differences between northern and southern European populations of the green alga Ulva lactuca L. Pubbl. Stas. *ZooL, Napoli, 27: 261-270. GROSS, F., 1937. Notes on the culture of some marine plankton organisms. /. Mar. Biol. Assoc., 21 : 753-768. KYLIN, H., 1941. Biologische Analyse des Meerwasser. Fysiogr. Sallsk. Fb'rhandl., 11 : No. 21. LEVRING, T., 1946. Some culture experiments with Ulva and artificial sea water. Fysiogr. Sallsk. Forhandl, 16, No. 7:1-12. LEWIN, R. A., 1954. A marine Stichococcus sp. which requires vitamin BJ-. /. Gen. Microbiol., 10: 93-96. PARKE, M., 1949. Studies on marine flagellates. /. Mar. Biol. Assoc., 38: 255-286. PROVASOLI, L., 1957. Alcune considerazioni sui caratteri morfologici e fisiologici delle Alghe. Boll. Zool. Agrar. e Bachich., 22 : 143-188. PROVASOLI, L., J. J. A. MCLAUGHLIN AND M. R. DROOP, 1957. The development of artificial media for marine algae. Arch. f. Mikrobiol., 25 : 392-A28. PROVASOLI, L., AND I. J. PINTNER, 1953. Ecological implications of in vitro nutritional re- quirements of algal flagellates. Ann. N. Y. Acad. Sci., 56: 839-851. SCHILLER, J., 1907. Beitrage zur Kenntnis der Entwicklung der Gattung Ulva. Sitzber. d. k. Akad. d. Wiss. in Wien., 116: 1691-1706. SCHREIBER, E., 1927. Die Reinkultur von marinen Phytoplankton und deren Bedeutung fur die Erforschung der Produktionsfahigkeit der Meerwassers. Wissench. Meeres unters. Helgoland N. F., 10: 1-34. SKOOG, F., AND C. O. MILLER, 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol., 11 : 118-131. SWEENEY, B. M., 1954. Gymnodinium splendens, a marine dinoflagellate requiring E^. Amer. J. Bot., 41: 821-824. THURET, G., 1878. fitudes phycologiques. Analyses d'algues marines. Masson et Cie., Paris. THE SO-CALLED "RECOVERY" PHENOMENON AND "PROTECTION" AGAINST X-IRRADIATION AT THE CELLULAR LEVEL ROBERTS RUGHi Marine Biological Laboratory, Woods Hole, Mass., and Radiological Research Laboratory, Columbia University, New York 32, N. Y. There has been some evidence that if the sea urchin egg is allowed to remain unfertilized for several hours following x-irradiation, there will result a higher percentage of cleavage than when eggs are fertilized immediately after exposure. This has been designated as evidence of "recovery" of the egg by Henshaw ( 1932- 1941). It presumes that such damage as is inflicted upon the egg can be rectified if the egg is given time. Among higher forms many chemical agents have been examined to determine whether they might either "protect" the organism against x-irradiation lethality, or enhance the "recovery" from radiation insult. Among the first of the effective agents was cysteine (Patt, 1955) but more recently another — SH compound, namely cysteinamine,2 has proven to be at least as effective. No drug has given complete protection but many have increased the dose necessary to kill a given per- centage of the exposed animals, or to increase the probability of survival. The mechanism of either the so-called "recovery" or of the chemical "protection" against x-irradiation has not been revealed. Since, among higher forms, the hematopoietic and reticuloendothelial systems are the more radiosensitive, and since the "protected" animals show quick regeneration of these systems, it has been pre- sumed by some investigators that the so-called protective drugs somehow substi- tuted for or masked the enzymes critically important in respiration. Since the sea urchin (Arbacia) egg showed the "recovery" phenomenon but did not have any hematopoietic system, it seemed appropriate to study the possible "protective" value of cysteinamine followed by x-irradiation and prior to fertilization of the Arbacia egg, and its possible effect on the "recovery" phenomenon. MATERIALS AND METHOD The eggs of Arbacia are readily available during the early summer and are easily fertilized to give nearly 100% cleavage and normal development. In every instance eggs from the same female were used for controls and for x-irradiation with and without benefit of the drug, so that there was no variation in the biological material. Fertilization or subsequent fixation of all the eggs of any single experi- ment was accomplished in 60 seconds, and in the same order in the various con- 1 This work was done under Contract AT-30-1-GEN-70 for the Atomic Energy Commission. 2 Cysteinamine is also known as cysteamine or beta-mercaptoethylamine or, commercially, as Becaptan hydrochloride. It was made available through Dr. W. Christiansen, representing the Labaz Laboratory, in Belgium. 385 386 ROBERTS RUGH tainers. The fixation was at stated intervals after insemination for the purpose of determining the cleavage percentage, and since the order of fixation was the same as the order of fertilization, and in the same elapsed time, the interval for all eggs between fertilization and fixation was the same. It is obvious that when one deals with the cleavage percentage at a specified time after fertilization of an egg so temperature-sensitive as is the egg of Arbacia, that each set of data from the eggs of a single female on any particular day must be considered separately from other data similarly collected on other days for the simple reason of temperature variability, even though the range of temperature variation was very low. By maintaining controls for each set of data separately, effects of temperature fluctuation were obviated. This was considered to be more accurate than using a temperature control bath and averaging the data. The eggs from a single adult Arbacia were removed by forceps and placed over cheesecloth in a beaker of filtered sea water. Within 15-20 minutes all of the mature eggs were dehisced from the ovaries and settled through the cloth into the beaker. When most of the eggs had settled on the bottom, the supernatant sea water was decanted off, and the eggs were transferred to a 500-cc. graduate which was filled with fresh, filtered sea water so that the eggs were thoroughly washed. This was repeated twice more. Finally, the eggs were diluted to 800 cc. of sea water and divided into two lots, one of which was considered the control lot. To the other was added 1 cc. of cysteamine from a freshly opened vial. In this way the concentration of eggs was the same in the control and experimental batches. The cysteamine was added within one-half hour of the beginning of irradiation so as to allow complete penetration into the egg. One cc. of cysteamine in 400 cc. of suspended eggs made a 0.0025 gram per cent solution which was non-toxic and in which eggs could be fertilized and would develop to plutei. With the above con- ditions, the variables were reduced to two, namely the level of exposure with or without the chemical agent. Eggs in uniform concentration were placed in covered plastic fly boxes measur- ing 67 mm. in diameter and 20 mm. in depth. The controls were placed in one box and those in the chemical agent in the other, and the two boxes were super- imposed during x-irradiation. When half the desired dose had been delivered, the position of the two boxes was reversed so that the slight difference in geometry of the two, with respect to the radiation, would be balanced out, and the exposure would be equated. The x-irradiation facilities used were those provided by the Marine Biological Laboratory at Woods Hole, Mass., and consisted of two alternate-parallel x-ray tubes run at 182 KVP and 25 MA and having an equivalent filtration of 0.2 mm. of copper.3 The output of the combined tubes at position "A" was 5,184 r/min. and at position "B" was 2,160 r/min. in air. In position "A" the distance between the targets was 18 cm. and at position "B" it was 29 cm. The maximum interval of exposure was 57.52 minutes but during this time there were brief interruptions during which eggs were removed. At all times a fan blew cool air over the dishes to dissipate any heat from the x-ray tubes. Should there have been any heat effect it would be the same for experimentals and controls. The exposures ranged from 25,000 r to 125,000 r. 3 The author expresses here his appreciation of the efficient irradiation service rendered by Mr. Alan Brockway. IRREVOCABLE X-IRRADIATION EFFECTS 387 Fertilization of all eggs from a single experiment was accomplished within a period of less than one minute. At designated intervals following fertilization, samples of eggs were removed from the dishes by pipette and fixed in 10% formalin in sea water in Syracuse dishes to which had been added a small amount of acetic acid. Fixation was immediate. The order of fertilization was followed when the samples of eggs were fixed so that the time variable during the fertilization procedure was cancelled out by the same time interval and order of fixation. The percentage of cleavage was determined subsequently by counting groups of 200 eggs in each Syracuse dish, and the percentage was recorded to the nearest 5%. It was found that under the temperature conditions of the laboratory, fixation at six hours post-fertilization would include all the eggs which would ever enter cleavage, taking into consideration the matter of delay in cleavage which could be attributed to x-irradiation alone. TABLE I "Recovery" in cleavage rate of Arbacia eggs following x-irradiation, due to delay in the time of fertilization* Time: Pert, to 24 hrs fixation is irs. 2J irs. Time: Irrad. to fertilization (hrs.). . . 5 3 2 i 5 3 2 i 5 Controls 90 99 95 95 95 99 99 99 100** 25,000 r 75 85 60 10 85 99 90 90 100** 50,000 r 5 10 1 2 90 80 80 80 50 75,000 r 0 0 0 0 40 30 45 30 10 100,000 r 2 1 0 0 80 80 35 10 5 * Values in percentage cleaved eggs. ** See footnote No. 4. Note: Maximum cleavage was achieved in eggs allowed to stand in fresh sea water for three hours after irradiation and before fertilization. After 100,000 r some 80% of the eggs did cleave in the time interval of two and one-third hours. The fact that at 75,000 r there was lower per- centage of cleavage may indicate that some egg nuclei were functioning while at 100,000 r there may have been complete parthenogenesis. EXPERIMENTAL DATA The experimental data consist of percentage cleavage and development at stated intervals following delayed fertilization and also following irradiation in cysteinamine, as well as a combination of the two variables. "Recovery": At average laboratory temperatures 50 per cent of the eggs of Arbacia will achieve the first cleavage within one hour after fertilization. If one examines fertilized eggs at one and one-third hours, the maximum percentage of cleavage will be observed. When eggs are x-irradiated and then fertilized by normal sperm, a delay in cleavage is observed, rather directly related to the level of irradiation. In the experiments reported here, eggs were x-irradiated from one 4 The fact that there was a higher percentage of blastula than of cleavage is explained on the basis of delay in cleavage by the specific times of fixation for determining cleavage per- centage. If time is not limited, the blastula percentages can be regarded as evidence of delayed but ultimate development. This apparent disparity occurred even slightly for the controls. 388 ROBERTS RUGH to five hours before insemination and the percentage cleavage was determined at one and one-third, two and one-third, three and twenty-four hours thereafter. The data are found in Table I below. The term "recovery" has been used to describe the above phenomenon, since at any given time there is a higher percentage of cleavage in those eggs which had the greatest delay between x-irradiation and fertilization. The fact that even after 100,000 r the eggs do eventually cleave is of interest, indicating that the changes brought about by x-irradiation may be cytoplasmic, indirectly affecting the mech- anisms of mitosis. In other words, the cytoplasm may show some evidence of "recovery." The mechanism for mitosis (e.g., the chromosomes and spindle) TABLE II Cysteinamine "protection" of the Arbacia egg against x-irradiation. Effect on the time of the first cleavage Percentage cleavage (400 eggs) at 2.5 hrs. post-insemination Date Controls 51,840 r 69,120 r 86,400 r Sea wat. Chem. S.W. Chem. S.W. Chem. S.W. Chem. 6/20 91 82 90 4 94 1 63 6/21 88 82 0 8 0 12 0 2 6/23 90 82 39 87 4 85 0 54 6/25 83 80 33 69 24 54 4 44 6/26 71 83 20 81 4 84 6/27 33 83 6 84 4 61 6/27 9 78 3 73 1 32 Averages : 88 82 31 70 9 69 2 48 Note: Any single experiment is complete in itself, but the data may vary with those from another day for reasons of temperature fluctuations alone. Thus, data on any horizontal line should be compared and it will be seen that in every instance there is a higher percentage of cleavage at 2.5 hours in eggs x-irradiated in the chemical than in the sea water alone. This is positive proof of qualified "protection" at the cellular level. The averages, below the table, have only relative significance but confirm the above. could be contributed by the unirradiated sperm. Since none of the eggs x-irradiated above 50,000 r developed into plutei, it must be assumed that the damage to the egg nucleus is beyond "recovery," preventing normal development. Since there is no "recovery" of the developmental potentialities it may be pos- sible to explain the phenomenon in terms- of temporary interference with insemina- tion and fusion of the protonuclei, or with the mechanics of mitosis, the degree of interference being related to the level of x-irradiation. It is not likely that this delay is due to any effects of the x-irradiated egg, exudates, or sea water on the spermatozoa, for their time span of activity in dilute suspensions is very short at best. It is more probable that the egg membrane, the cytoplasm, or even the elements involved in the kinetics of pronuclear fusion and mitosis may be tem- porarily altered so as to delay the progression of the sperm nucleus toward the egg nucleus. Whatever this effect of x-irradiation, it is reversible in time and even IRREVOCABLE X-IRRADIATION EFFECTS 389 after 100,000 r some 80% of the eggs will achieve the first cleavage at least. To call this "recovery" is misleading, simply because the word generally implies much more than is demonstrated here. No egg exposed to more than 50,000 r x-rays ever "recovers" in the sense that it can develop past the critical stage of gastrulation. There is restoration of the conditions necessary for early mitosis only. Chemical protection: Likewise the word "protection" must be qualified for in no permanent sense does the chemical "protect" animals (or eggs) against the effects of ionizing radiations, although it may lessen the effects. Nevertheless, if one x-irradiates Arbacia eggs in a tolerable solution of cysteinamine, then transfers TABLE III The effect of time between irradiation and fertilization as well as immersion of eggs in cysteinamine during exposure, on the percentage of cleavage (200 eggs to nearest 5%) Eggs fixed 2 hours after insemination Time : Irradia- tion to 6 hrs. 5 hrs. 2 hrs. 1 hr. fertilization Control Exp. Control Exp. Control Exp. Control Exp. Control 100 100 100 100 100 100 100 100 50,000 r 60 90 40 90 25 100 1 90 75,000 r 5 95 2 95 0 90 0 30 100,000 r 10 80 0 90 0 2 0 0 125,000 r 0 30 0 30 0 0 0 0 Note : Experimental eggs were those immersed and irradiated in the chemical while the con- trols were x-irradiated in filtered sea water. In every instance where data are available, eggs x-irradiated while in the chemical show much better percentage of cleavage than did the controls. That both "protection" by the chemical and time for "recovery" were working is indicated by the fact that at 75,000 r we have a range of cleavage 30% to 95%, depending upon the time interval between irradiation and fertilization. Cleavage percentage values include the 2- to 8-cell stages. At 24 hours those which had been chemically "protected" during x-irradiation were normal-appearing blastulae while the control eggs were disintegrated or abnormal blastulae. Even after 125,000 r 10 per cent of the experi- mentals were normal-appearing blastulae while the controls were 100 per cent abnormal. them to filtered sea water and inseminates them, there is evidence that a higher percentage of eggs will cleave than in the "unprotected" controls, no matter what level of irradiation. Table II below illustrates this point. When one combines the two variables of time between x-irradiation and fertiliza- tion as well as the presence of the so-called "protective" agent during x-irradiation, data of Table III are obtained. When one delays examining the eggs until about three hours after insemination, further facts become clear. An exposure of 50,000 r does not prevent cleavage in the slightest, although there is a delay. Above this level of exposure there is a drop in ultimate cleavage percentage so that at 125,000 r the maximum level of cleavage is 50% at three hours after insemination in those eggs which had an interval of five hours between irradiation and insemination. A further delay in insemination (to six hours) was deleterious so that no eggs developed. 390 ROBERTS RUGH In every instance where eggs were x-irradiated in cysteinamine, there was higher percentage of cleavage than in the "unprotected" controls. In fact, the level of cleavage was the same as that of the controls or 100% even after 125,000 r, providing there was a delay in fertilization. Thus, we see two forces acting in favor of the egg, with additive effects up to the point of five hours delay in fertilisation. Those eggs which were inseminated within one hour of exposure to 125,000 r, while suspended in cysteinamine, showed 90% cleavage at three hours. This is certainly evidence of better "recovery" by means of the "protective" action of the TABLE IV The effect of time between x-irradiation and fertilization, as well as presence of cysteinamine during exposure, on the percentage of cleavage of Arbacia eggs Eggs fixed 2 hours after insemination Time: Irradia- tion to 6 hrs. 5 hrs. 2 hrs. 1 hr. fertilization Control Exp. Control Exp. Control Exp. Control Exp. Control 100 100 100 100 100 100 100 100 50,000 r 100 100 100 100 100 100 100 100 75,000 r 80 100 80 100 90 100 80 100 100,000 r 30 100 30 100 20 90 30 90 125,000 r 0 80 50 100 40 80 10 90 Note: There is evidence from these data that cysteinamine did not alter the tendency of eggs to "recover" if time was allowed between irradiation and insemination but that the two factors were additive. At 24 hours all controls above 50,000 r were dead while those x-irradiated to this level in cysteinamine showed 100% ciliated motile blastulae (even after 125,000 r). After 48 hours a few from 50,000 r and 75,000 r plus cysteinamine developed into crude plutei. This would never happen with "unprotected" control eggs. drug than by time lapse between x-irradiation and fertilization. True, this was increased slightly to 100% by adding the time factor, but these figures are too close to be significantly different. Immersion of eggs in cysteinamine following x-irradiation was invariably deleterious, the eggs going to pieces during the early attempts to cleave. DISCUSSION The words "recovery" and "protection" when used in radiobiology must be qualified or clearly defined. "Recovery" has been used to imply a return to the pre-irradiated state. It is defined as a "restoration to the normal state." It is very doubtful that this ever occurs following x-irradiation-induced morphological change at the cellular level. In a complex and multi-cellular mammal, for instance, there may be "recovery" in the sense that hair returns after epilation, lymphocytes appear after lymphopenia, and even the sterile testis may again become functional. Nevertheless, in every one of these examples it is more likely that there has been regeneration from un- IRREVOCABLE X-IRRADIATION EFFECTS 391 damaged cells and that the originally damaged cells have been removed. The organism as a whole does have remarkable powers of restoration, but there is no evidence that the once damaged cells can themselves be "restored to the normal state." There is no doubt (as Henshaw first pointed out) that a delay in fertilization after x-irradiation of the Arbacia egg will allow a greater percentage to undergo the early cleavages. This was confirmed up to a delay of three hours. However, ultimate cleavage was not improved and development was never achieved beyond the gastrula stage in eggs exposed to 50,000 r or more. Thus, the so-called "re- covery" relates to counteracting somehow the delaying effects of x-irradiation on the mechanism of cleavage in the egg. It does not mean that the radiation-insulted egg can develop as does an unirradiated egg, a true recovery situation. The word "protection" is also used variously (Bacq and Alexander, 1955). With mice, rats, guinea pigs, etc., it generally refers to the ability of a larger per- centage of animals to survive a given dose of radiation, or to tolerate a larger dose than usual. The data are generally based upon a thirty-day survival. Certainly such animals exhibit most of the expected sequelae of x-irradiation such as shorten- ing of life, higher incidence of cataracts, sterility, etc. It has been erroneously pre- sumed that animals surviving thirty days after exposure are "normal" and the word "protection" is used. Hollaender and Doudney (1955) found that when E. coll were x-irradiated in cysteinamine, they could tolerate twelve times as much of an exposure. That is, a dose of 60,000 r with the chemical had the same effect as 5000 r without its presence. Patt (1955) has reviewed the thesis that it is the sulfhydryl group ( — SH ) that is protective, by virtue of its ability to either protect or substitute for an enzyme, or to reactivate the enzyme after x-irradiation. The difficulty lies in the fact that there are many compounds containing the -— SH group which have no protective value. Hollaender and Stapleton (1953) and Gray (1956) suggested that chemicals such as cysteine, entering the cell, require oxygen in order to be me- tabolized and thereby render the intracellular substance anoxic. But anoxia, while favoring survival of x-irradiated cells, probably does not alone explain the mecha- nism of cysteinamine protection. It might be suggested here that the delay in initiation of cleavage is due to cytoplasmic effects of ionizing radiations on the egg, and those cytoplasmic effects are protected by such a chemical agent as cysteinamine. Certainly the results herein reported indicate that cleavage time is better "protected" by this chemical than by the time-lapse between x-irradiation and insemination. It is probable that the so-called "recovery" of the egg in time, with respect to initial cleavage and without benefit of chemical agent, is achieved largely by effects in the cytoplasm. One would tend to agree with Blum et al. (1951) that in addition to the cytoplasmic damage there is more serious damage, concerned with embryonic development, affecting the nucleoproteins. However, this is not manifest until the critical phase of gastrulation. It is of interest that the only eggs exposed to x-irradiation above 50,000 r which reached the recognizable pluteus stage were those which had been "protected" by cysteinamine and none of those benefited by a delay in insemination. This again indicates that the two mechanisms may act in a different manner but are not in conflict. In fact, they may be additive at lower levels of exposure. 392 ROBERTS RUGH Finally, it has been shown (see Bacq and Alexander, 1955) that glutathione plays a metabolic role in the process of cell division by effecting a fermentation within the cell which stimulates cell division. Specifically, cell division is not pos- sible without the prior denaturation of protein by means of the — SH radical and the consequent reduction of the store of oxidized glutathione. Glutathione changes the oxidation-reduction level which in turn alters the fermentative metabolism that leads to cell division. The presence of thiol poisons in the cell, which could inhibit cell division, can be reversed by some of the — SH compounds, such as cysteine (Hammett, 1929) and presumably also by cysteinamine. That the — SH radical is vitally concerned in cell division has been well demonstrated. There is no doubt that there is an increase in the soluble thiol compounds prior to cell division and also a high concentration of -— SH radicals before, during and after mitosis. It is therefore conceivable that cysteinamine, like glutathione (Stern, 1956), might play a critical metabolic role in affecting mitosis. If the x-irradiated cell is given some time to repair the interference with the mitotic mechanism, or if the cell is provided with an excess of the — SH radical in the form of cysteinamine, the expected effect of cleavage delay caused by x-irradiation is not experienced. But, making available the — SH radical certainly does not allow true and full "recovery" nor is it fundamental "protection" of the cell since we would be ignoring herein all effects of x-irradiation on the chromosomes and genes which have to do with normal developmental processes. Therefore so far as development is con- cerned, there is neither "recovery" nor "protection." SUMMARY AND CONCLUSIONS 1. The often used terms "recovery" and "protection" in radiobiology are spe- cifically defined. As circumscribed, recovery at the cellular level does not occur following x-irradiation damage. Protection simply refers to better survival and in no way implies saving the exposed cell from the sequelae of x-irradiation insult. 2. The prior finding (of Henshaw) that a delay in insemination of Arbacia eggs following x-irradiation will allow for better initial cleavage percentage has been confirmed. However, it has been shown that there was no actual increase in ulti- mate cleavage percentage but rather, the x-irradiation factors which caused a delay in cleavage were neutralized. X-irradiated eggs never "recovered" because they could not develop through gastrulation to plutei. 3. Cysteinamine, if available to the Arbacia egg during x-irradiation, will coun- teract the delaying effect of x-irradiation on cleavage following insemination and will, in addition, allow further development. Some embryos will achieve the pluteus stage. This suggests that in addition to the apparent "recovery" by delay in insemination, the — SH cysteinamine must in some way reduce or prevent ir- radiation damage to the nucleus in some eggs, allowing them to become ciliated blastulae and even stunted plutei. They do not develop further than abnormal plutei following x-irradiations above 50,000 r, even with the benefit of the cysteina- mine, so that protection of the egg to allow normal development did not occur. 4. Cysteinamine imposed on the Arbacia egg after x-irradiation was so dele- terious that the early cleavage stages disintegrated rapidly even in concentrations which were tolerated well by unirradiated embryos. This may be due to the anoxia caused by cysteinamine. IRREVOCABLE X-IRRADIATION EFFECTS 393 5. It is concluded that "recovery" from x-irradiation damage defined as "resto- ration of the normal state" does not occur even with a delay in insemination. There may be some evidence of chemical nuclear "protection" to the extent that some eggs can develop to the early pluteus stage, but no further. This is hardly "protection" in the common usage of the word. It is concluded, therefore, that nuclear damage by x-irradiation is irrevocable and irreparable and that neither "recovery" nor "protection," properly defined, occurs at the cellular level following x-irradiation insult. LITERATURE CITED BACQ, Z. M., AND P. ALEXANDER, 1955. Fundamentals of Radiobiology. Academic Press, N. Y. BLUM, H. F., J. C. ROBINSON AND G. M. Loos, 1951. The loci of action of ultraviolet and x-radiation and photorecovery in the egg and sperm of the sea urchin Arbacia punctu- lata. J. Gen. Physiol., 35 : 323. GRAY, L. H., 1956. A method of oxygen assay applied to a study of the removal of dissolved oxygen by cysteine and cysteamine. In: Progress in Radiobiology, ed. Mitchell, Holmes, Smith, Oliver and Boyd, London. HAMMETT, F. S.,1929. The chemical stimulus essential for growth by increase in cell number. Protoplasma, 7: 297-322. HENSHAW, P. S., 1932. Studies of the effect of roentgen rays on the time of the first cleavage on some marine invertebrate eggs. I. Recovery from roentgen ray effects in Arbacia eggs. Amer. J. Roentgen. Rod. Therapy, 27: 890-898. HENSHAW, P. S., 1933. The effect of roentgen rays on the time of the first cleavage in marine invertebrate eggs. II. Differential recovery and its influence when different methods of exposure are used. Radiology, 21 : 533-541. HENSHAW, P. S., AND D. S. FRANCIS, 1936. The effect of x-rays on cleavage of Arbacia eggs : Evidence of nuclear control of division rate. Biol. Bull., 70 : 28-35. HENSHAW, P. S., 1940. Further studies on the action of roentgen rays on the gametes of Arbacia punctulata (6 parts). Amer. J. Roentgen. Rod. Therapy, 43: 899-933. HENSHAW, P. S., 1941. The induction of multipolar cell division with x-rays and its possible significance. Radiology, 36 : 717-724. HOLLAENDER, A., AND C. O. DOUDNEY, 1954. Studies on the mechanism of radiation protection and recovery in the cysteamine and /3-Mercaptoethanol. Symp. Radiobiol. Liege, pp. 112-121, Butterworths, London. HOLLAENDER, A., AND G. E. STAPLETON, 1953. Fundamental aspects of radiation protection from a microbiological point of view. Physiol. Rev., 33 : 77. PATT, H. M., 1955. Remarks concerning sulfhydryl-protection against mammalian radiation injury. Symp. Radiobiol. Liege, pp. 105-109, Butterworths, London. STERN, H., 1956. Sulfhydryl groups and cell division. Science, 124: 1292-1293. DEVELOPMENT OF PIGMENT IN THE LARVA OF THE SEA URCHIN, LYTECHINUS VARIEGATUS *• 2 RICHARD S. YOUNG « Department of Zoology, Florida State University, Tallahassee, Florida Very little is known concerning the origin of invertebrate pigment cells. Most invertebrate eggs contain varying amounts of carotenoid pigments, which are suffi- cient to mask the appearance of any new pigments. The unsegmented egg of the sea urchin Lyt echinus variegatus, however, contains very small amounts of caro- tenoid or other pigment material and formation of a new pigment (echinochrome) in the early gastrula stage is readily observed. The striking appearance of pig- ment in early development suggests that the eggs of this animal might be unusually favorable material for a study of the cellular origin and chemo-differentiation of a defined substance — echinochrome. Echinochrome is a substituted naphtho- quinone, red-purple in color, found in the test, spines, epidermis and various internal organs of sea urchins. The chemical structure and physical and chemical proper- ties of certain echinochromes have been established by various investigators (Ball, 1936 ; Lederer and Glaser, 1938 ; Glaser and Lederer, 1939 ; Kuhn and Wallenfels, 1939, 1940; Wallenfels and Gauhe, 1943; Goodwin and Srisukh, 1950). A num- ber of physiological functions have been ascribed to echinochrome. However, ques- tions concerning the embryological and biochemical derivation, metabolism, and possible physiological functions of polyhydroxynaphthoquinones in echinoids are mainly unanswered. The present study is an attempt to determine the embryonic origin of the echino- chrome-forming cells, and to throw some light on the intracellular mechanisms affecting echinochrome synthesis. MATERIALS AND METHODS The eggs and sperm of the sea urchin Lyt echinus variegatus were used through- out this work. Arbacia punctulata was used in some instances for comparison. The animals were collected in the Gulf of Mexico at the mouth of Alligator Harbor, Florida, using the facilities of the Florida State University Marine Laboratory. The animals were maintained in running sea water or jugs of continually aerated sea water at about 15 degrees centigrade. The eggs and sperm were obtained from the animals by the KC1 injection method. Eggs were fertilized in filtered sea water with approximately 0.5% sperm suspensions. 1 Supported in part by a grant from the National Science Foundation to Dr. Charles B. Metz. 2 Part of a thesis submitted to the graduate faculty of Florida State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 3 Food and Drug Administration, Bureau of Biological and Physical Sciences, Pharmacology Division, Washington, D. C. 394 DEVELOPMENT OF PIGMENT IN LYTECHINUS 395 Development was observed with the dissecting microscope and the phase con- trast microscope. The ultra violet absorption spectrum of the echinochrome was determined by means of the Beckman DU Spectrophotometer. All results are based on experiments involving at least 200 eggs. Each experi- ment was repeated at least twice. Any deviation from these numbers will be discussed in the text. The various techniques used in the individual experiments will be described with the results of the experiments for the sake of continuity and clarity. RESULTS Normal development The fertilized egg of Lytechinns is relatively pigment-free. There is a very faint yellowish cast to the egg, probably due to carotenoids as in most echinoderm eggs, but the nucleus and cellular inclusions are clearly visible under the dissecting microscope. The echinochrome-containing cells (echinophores) are first noticeable under the microscope during early gastrulation, when the veg2 cell layer is invaginating to form the gut. They appear to be in the ectodermal layer and at first only in the region of the invagination. As gastrulation proceeds, the echinophores become dispersed throughout the gastrula, apparently in the ectodermal layer. In the pluteus, these cells are evenly dispersed in the outer body wall, with usually some concentration in the arm tips. A series of experiments was designed to determine more precisely the origin of these cells in the course of development of the embryo and perhaps what intra- or inter-cellular mechanisms were involved. Lithium experiments Exogastrulae were produced using the technique of Herbst (1892) (treatment of fertilized eggs in a 0.1 M LiCl solution in sea water for five hours). This pre- sented an opportunity to determine the effect of LiCl on pigmentation and to determine if the normal association of the germ layers is necessary for pigmenta- tion. These exogastrulae were striking in that echinophores always appeared in the ectodermal portion of the larvae and never in the endoderm. The pigment appeared first at the site of evagination and by the time the evaginated gut was completely formed these cells were dispersed throughout the ectodermal wall of the larvae. The evaginated gut wall never contained pigment cells, although occa- sionally pigment could be seen inside the lumen where apparently it had migrated. This particular phenomenon will be discussed in more detail later, in connection with the amoeboid movements of these cells. From this experiment it may be said that pigment is associated with the ectoderm (at least in exogastrulae) and that a normal association of the germ layers is not necessary for pigment formation. LiCl in the concentrations (0.1 M-0.5 M) used had no apparent effect on pigmentation. 396 RICHARD S. YOUNG Thiocyanate experiments Since the pigment cells were found only in the ectodermal portions of the LiCl-induced exogastrulae, it would be interesting to know if the ectoderm alone is able to give rise to these echinophores. "Dauer" blastulae (permanent blastulae with no gut or skeleton) were formed using NaSCN (0.125 M) and the technique of Lindahl (1936). Those embryos which developed into "Dauer" blastulae were never pigmented. They remained as colorless hollow balls of ciliated ectodermal cells, whereas if there was any sign of invagination and appearance of endodermal derivatives, pigment was always formed in the ectodermal regions. In these cases, where very little endoderm was present, pigment cells were few and widely scattered. However, as in previous experiments, the pigment first appeared around the invaginating region, and later gradually became dispersed throughout the larval wall. Apparently the ectoderm alone is not capable of giving rise to pigment, at least not in those "Dauer" blastulae formed by treatment with NaSCN. Isolation experiments It is possible to study the effects of ectodermization on pigmentation, without the influence of a chemical agent such as NaSCN. In these experiments, the tech- nique of Horstadius (1928) was used. The most satisfactory method for removing the fertilization membranes was found to be the shaking method of Driesch (1891). Since Lytechinus eggs have no pigment band and removal of the fertilization membrane also removes the polar bodies at the animal pole, there is no sure way of determining which is the animal and which is the vegetal pole at the 8-cell stage. However, at the 16-cell stage, the micromeres have appeared and identification of the vegetal pole is quite easy. For this reason, most of the operations were done at the 16-cell stage. Some, however, were done at the 8-cell stage and will be discussed later. About 10 eggs at the 16-cell stage were placed in a drop of sea water in the shallow depression in the lid of a small stender dish, by means of a capillary pipette. The drop of sea water was kept as small as possible so that the surface tension served to hold the embryos in place while the cuts were being made. Under the dissecting microscope the desired blastomeres were separated with glass dissecting needles. The separated cells were picked up with the capillary pipette, transferred to sea water in Syracuse dishes and allowed to develop. By means of this technique, 30 animal and 30 vegetal halves were isolated at the 16-cell stage. Invariably, the animal halves formed typical "Dauer" blastulae, while the vegetal halves gastrulated and sometimes formed miniature plutei. These were usually defective in that they often had only one arm or a poorly formed gut and skeleton. The "Dauer" blastulae from isolated animal halves were never pig- mented and remained unpigmented until they died. The vegetal half -embryos were always pigmented in a typical fashion. Similar results were obtained in 40 cases in which halves of the 8-cell stage were isolated. However, since there was no way of identifying the animal and vegetal halves, the assumption was made that some of the cuts would isolate halves containing two animal and two vegetal cells, depending on the plane of the cut. This assumption appeared to be well founded, since some halves formed "Dauer" DEVELOPMENT OF PIGMENT IN LYTECHINUS 397 blastulae, while most developed into pigmented plutei. Only the animal halves form "Dauer" blastulae whereas the vegetal halves and those halves containing two animal and two vegetal cells gastrulate and develop pigment. These experiments confirmed the results obtained in the NaSCN experiments, that is, that the ectoderm alone does not produce pigment. Pigment is formed when the embryo gastrulates (regardless of what fraction of the original egg is present) and only if the embryo gastrulates. Vital staining experiments In order to determine exactly what cells are responsible for the production of these echinophores, the technique of vital staining was employed. The technique of Horstadius (1935) was used. The dye used was Griiblers "Neutral Rot," since this was the only dye found to penetrate the larvae satisfactorily. The eggs were stained at the 16-cell stage. A fine glass capillary which was filled with agar containing a one per cent solution of neutral red was put into a drop of sea water in a stender dish lid. The egg to be stained was moved into position and held in place for about one minute, at which time enough dye was absorbed to render it readily identifiable. At the 16-cell stage the eight animal cells were stained in this way in a series of 12 eggs. These stained cells never became pigmented after gastrulation. The four micromeres in 12 eggs were stained in the same way and here, too, the stained cells never produced echino- phores. When the four macromeres (of 12 eggs) were stained, the pigment cells in the pluteus showed traces of the dye. The macromeres do not divide until the end of the 32-cell stage, so that staining of the vegx and veg2 cells derived from the macromeres is impossible until the 64-cell stage. At the time of the 64-cell stage, the individual blastomeres are extremely small and difficult to stain indi- vidually. However, an attempt was made to stain the cells comprising vegx in one series of eight eggs, and veg2 in another series of eight eggs. It was found that the veg2 cells and the micromere material always invaginated at gastrulation and gave rise to no pigmented cells. Although it was almost impossible to stain only veg1 cells since some of the stain invariably got into veg2 cells, it was none the less possible to see that it was from this material that the echinophores ulti- mately arose, since the concentration of the dye in the vegt cells was much greater. The material from vegl did not invaginate. but at the time of gastrulation, it re- mained near the site of invagination and ultimately gave rise to the ventral ectoderm of the pluteus. Therefore, it would seem that the echinophores originate from the vegx cells but only under conditions permitting axial differentiation. The question then arises as to how these pigment cells become dispersed throughout the ectoderm of the pluteus, if their origin is localized in material which is ultimately destined to become only the ventral ectoderm of the pluteus. A series of observations gave an answer to this question. Phase microscope observations Boveri (1901) noticed amoeboid cells, of a brick red color, appearing in the late gastrula stage of the sea urchin Paracentrotus lividus. He considered the 398 RICHARD S. YOUNG pigment the same as that in the egg. Monroy et al. (1951) also noted these amoeboid cells and suggested the possibility that the pigment in them might be echinochrome, although they were not able to obtain enough material to prove its presence. Neither of these workers reported a detailed study of the movements of the pigment cells, or their location. These cells were studied in Lyt echinus variegatus under the phase contrast microscope. In the early gastrulae when the echinophores first appeared, it was found that the cells were large, irregularly shaped structures in the region of the invagination. This would be the material derived from vegi cells. First, small pale orange pigment granules appeared. These were not easily visible except under high magnification. After a few hours, when the invagination process was almost complete, the pigment in these cells became darker red in color and much more concentrated, while the cells themselves increased greatly in number. At this time, it could be seen that the pigment cells were amoeboid in nature, apparently able to move freely within the ectodermal layer in any dimension. However, they were never seen to enter the endodermal layer underneath. By the pluteus stage, they had invaded the ectoderm of the larva and appeared to be most concentrated in the more actively growing arm tips of the pluteus. By the time the pluteus was fully formed, the echinophores had become much less motile and migration had virtually ceased. In observations of eight exogastrulae, the same pattern was seen, except that the endoderm was no longer immediately under the ectoderm. The echinophores were seen occasionally to work themselves completely free of the ectoderm and come to lie in the cavity of the blastocoel and eventually even in the everted gut lumen. This explains the occasional appearance of pigment in the gut cavity of exogastrulae. The next series of experiments was designed to determine what physical and chemical factors in the developing egg were responsible for the formation of the pigment and to obtain some information as to the relative roles played by the nucleus and cytoplasm in this synthesis. Since the pigment is elaborated long before the organism begins to take in food material from the outside, the pigment must be synthesized from pre-existing materials present in either the egg or sperm, or both. Hybridisation experiments A series of experiments was designed to determine the effect of Arbacia X Ly- t echinus hybridization on subsequent pigmentation. Tennents' (1912) method (ageing of gametes for two hours followed by a five-minute treatment with alkaline sea water before fertilization) was used in obtaining these hybrids. The larvae from Arbacia male X Lytechinits female were all maternal in appearance. Pig- mentation, size and general body structure of gastrulae (very few reached the pluteus stage) were typical of Lyt echinus. The reverse cross, Lytechinus sperm X Arbacia egg, was unsuccessful in that no gastrulae were obtained and nothing could be seen concerning any newly formed pigment cells at gastrulation. These experiments were repeated three times with practically identical results. It can be said only that Arbacia sperm cannot affect normal pigmentation in the DEVELOPMENT OF PIGMENT IN LYTECHINUS 399 Lyt echinus egg, in Arbacia sperm X Lyt echinus egg hybrids. The question then arises as to the normal role, if any, played by the sperm in pigmentation. One way of answering this question is to study the development of artificially activated eggs. Parthenogenesis experiments The (butyric acid-hypertonic sea water) method of Tennent (1912) was found to be the most satisfactory for producing parthenogenetic larvae. Again, it was noted that if gastrulation occurred, pigment was formed and apparently in the same fashion as described for normal fertilized eggs. The parthenogenetic plutei were normally pigmented and quite like the fertilized controls. These experiments were also repeated three times with the same results. It would seem that the presence of the sperm cell is not essential for pigment formation. Identification of the pigment The assumption has been made so far that the pigment in question is the substi- tuted naphthoquinone, echinochrome. The proof of this lies in isolation, physical and chemical properties and spectrophotometric analysis. Echinochrome may be extracted by treatment with slightly acidified, organic solvents such as 80 per cent acetone or ether containing one per cent of HC1. The pigment may be then trans- ferred by dilution into diethyl ether and chromatogrammed to remove impurities. The free compound shows very slight solubility in water or petroleum ether but is readily restored by shaking in air or by any of a number of mild oxidizing agents (Ball, 1936). Clearly defined absorption bands are exhibited by solutions of echinochrome in various solvents. According to Kuhn and Wallenfels (1939), the absorption maxima of an echinochrome solution in carbon disulphide were 535, 499 and 464 m/A, in chloroform 532, 497 and 462 m/t, in benzene 532, 494 and 461 m/t and in concentrated sulfuric acid, 502 and 469 m^. The following evidence shows that the pigment appearing at gastrulation in the sea urchin, Lyt echinus variegatus, has the properties of echinochrome. ( 1 ) The pigment is orange-red in color. (2) The pigment may be extracted from gastrulae and plutei by the above described procedure. (3) The pigment, when extracted and dried, is nearly insoluble in water and petroleum ether. (4) The pigment turns red in acid solution and violet in the alkaline range. (5) The pigment is soluble in diethyl ether, acetone, ethanol and carbon disulfide. (6) The pigment is very slightly soluble in chloroform. (7) The addition of 5 mg. of sodium hydrosulfite to 10 ml. of a brick red solution of the pigment quickly bleaches the solution. (8) The addition of small amounts of an oxidizing compound (hydrogen perox- ide) or shaking in air quickly restores the color to the solution. (9) The absorption spectrum of a carbon disulfide-pigment solution showed peaks on the Beckman DU Spectrophotometer at 530, 491, and 460 m/x. 400 RICHARD S. YOUNG Carotenoids, chromolipids, melanins and flavins may also be reddish in color (Sumner and Doudoroff, 1943). However, melanins and flavins (Mayer and Cook, 1943) are insoluble in almost all organic solvents. Flavins are water-soluble. The carotenoids may be bleached (Fox, 1936) but only by oxidizing agents rather than reducing agents. The absorption spectrum is typical of echinochrome and not carotenoids, since the carotenoid absorption peaks are around 510 and 485 m^t (Fox and Scheer, 1941). The change in color with changes in pH is also typical of quinone pigments. It was not possible to isolate and crystallize a sufficient amount of the pigment from plutei to permit further analysis of its physical and chemical properties but on the basis of the above evidence, the pigment appears to be echinochrome. Chewier-differentiation study The effect of a large number of inhibitors was studied in an attempt to specifi- cally inhibit pigmentation and perhaps learn somethong of the metabolic pathway involved in its synthesis. It was found, however, that only those inhibitors which stopped development at gastrulation or ectodermized the eggs, such as 2-4-dinitro- phenol, pyocyanine and iodosobenzoic acid, had an effect on pigmentation. DISCUSSION Boveri (1901) first noticed in the late gastrula stage of the sea urchin Para- centrotus lividus the appearance of amoeboid cells, rather heavily loaded with large pigment granules which differed from those of the unsegmented egg both with respect to their larger size and to their color. He noted that the number of these cells increased rapidly with the age of the embryo. Boveri, however, considered the pigment of the same nature as that of the egg. Monroy et al. (1951) noted that starting from the stage when the new pigment appeared, the embryos still retained a red-violet color after having been exhaustively extracted for carotenoids and that the remaining color was due to pigment still present in the amoeboid cells. These workers showed that the pigment could not be extracted with chloroform, acetone, methanol or pyridine but on slight acidification with dilute HC1, it could be taken up quantitatively in ether. It was thought the pigment was probably echinochrome but they were not able to obtain sufficient amounts to prove it spec- trophotometrically. They also noted that the eggs of one female developed normally up to the beginning of gastrulation when exogastrulation occurred. In this case, they were unable to detect echinochrome. This observation seems unlikely in view of the exogastrulation experiments of the present study and was probably due to masking by other pigment. Gustafson and Lenique (1951), using Psammechinus miliaris, mentioned pig- ment formation in the gastrula stage. They did not identify the pigment. How- ever, they did mention that the red pigment cells became especially concentrated in the arm tips and the apical region, where the ectoderm is characterized by high mitochondrial activity. This observation is confirmed in the present study as previously mentioned, where echinophores are concentrated in the arm tips and apical region. It was also noted that in advanced starving plutei, the amount of echinochrome is appreciably reduced, suggesting the use of this protein-echino- chrome complex as a food source under extreme conditions. DEVELOPMENT OF PIGMENT IN LYTECHINUS 401 Using the Lytechinus egg in which the pigment is not sufficient to mask pigment elaboration subsequent to fertilization, it is possible to trace the differentiation of chromatophores with considerable accuracy. It was found that a pigment was synthesized in the embryo in the gastrula stage. This pigment was echinochrome. The particular cells in which the pigment appeared were shown by vital staining to originate from vegi and to be amoeboid in nature. With the use of isolation techniques and chemical treatment it was shown that pigment formation occurred only in association with gastrulation. Evidently, pigment cell differentiation is related to gastrulation in some way. Since the pigment cells differentiate in exo- gastrulae as well as in normal embryos, the differentiation does not appear to be an induction effect, at least to the extent that it requires the juxtaposition of endo- derm with the other germ layers during gastrulation. It appears more likely that pigment cell differentiation, including the formation of pigment itself, is under the same control system as that governing the differentiation of other parts of the embryo (e.g., skeleton, muscle, gut, coelom, etc.). From the work of Runnstrom (1933) and especially Horstadius (1939) this over-all differentiation appears to depend upon the quantitative interaction of some sort of double gradient system. This system may function to produce pigment cells from vegt in normal develop- ment, but when the system is modified experimentally, for example by surgical or chemical treatments that result in "Dauer" blastulae, then pigment cells as well as other types of tissue fail to differentiate. The production of echinochrome by the differentiating chromatophores presents an interesting problem in the chemo-differentiation of a defined substance. Un- fortunately, no information is available concerning the pathways of echinochrome synthesis. However, echinochrome production may be correlated with protein synthesis, at least to the extent that new enzymes required for echinochrome syn- thesis may be elaborated by the embryo. Furthermore it is known that echino- chrome occurs in the form of a protein complex (Kuhn and Wallenfels, 1940; Shapiro, 1946), and extensive protein synthesis begins at the same time that echinochrome first appears in the embryo, namely, at the time of gastrulation (Caspersson, 1947 ; Brachet, 1941; Zeuthen, 1951; Hultin, 1950; Perlmann, 1954). It is of interest to note the similarities between melanophore development in the vertebrates and echinophore development in the sea urchin. DuShane (1935) proved the neural crest origin of pigment cells in the amphibian. The formation of the neural crest and subsequent pigmentation are dependent on gastrulation in the amphibian and both amphibian and Lytechinus pigment cells are ectodermal in origin. In the amphibian, however, pigment cell formation is more complex, in that gastrulation induces the formation and differentiation of the neural crest, which in turn differentiates still further, giving rise to a number of structures, among which are the pigment cells. These cells, too, are amoeboid and migrate to their definitive position (Twitty and Niu, 1954) where they apparently lose their amoeboid capabilities and come to rest. SUMMARY 1. A pigment having the properties of echinochrome is synthesized in the embryo of the sea urchin Lytechinus variegatus. Differentiation of the echino- phores and synthesis of the echinochrome begins at the gastrula stage. 402 RICHARD S. YOUNG 2. Echinophores differentiate from the vegx cell layer of the embryo, become amoeboid and migrate into other ectodermal regions. 3. Echinophore differentiation appears to depend upon a normal relation of the "double gradient" system of the embryo. Since echinophores were produced in exogastrulae, normal juxtaposition of the germ layers is not essential. 4. The sperm nucleus was found to have no essential role in the pigmentation process. Pigment formed according to the maternal pattern in hybrid and partheno- genetic embryos. 5. Of a variety of chemical substances tested, including several respiratory and other inhibitors, only those agents which inhibited gastrulation of the embryo caused failure of pigment formation. 6. Echinochrome synthesis is apparently related to protein synthesis in the embryo. LITERATURE CITED BALL, E. B., 1936. Echinochrome, its isolation and composition. /. Biol. Chem., 114: Proc. 30, vi. BOVERI, T., 1901. Die Polaritat von Oocyte, Ei tmd Larve des Strongylocentrotus lividus. Zool. Jahrb., 14: 630-653. BRACHET, J., 1941. La detection histochimique et le microdosage des acides pentosenucleiques. Ensymol, 10: 87-98. CASPERSSON, T., 1947. Relations between nucleic acid and protein synthesis. Symp. Soc. Exp. Biol, 1 : 66-73. DRIESCH, H., 1891. Entwickelungsmechanische Studien I. Zeitschr. f. zviss. Zool., 53: 600-637. DuSnANE, G. P., 1935. An experimental study of the origin of pigment cells in Amphibia. /. Exp. Zool., 72 : 1-31. Fox, D. L., 1936. Structural and chemical aspects of animal coloration. Amer. Nat., 70: 477-493. Fox, D. L., AND B. T. SCHEER, 1941. Comparative studies of the pigments of some Pacific coast echinoderms. Biol. Bull, 80: 441^155. GLASER, R., AND E. LEDERER, 1939. Echinochrome et spinochrome ; derives methyles ; distribu- tion; pigments associes. C. R. Acad. Sci., Paris, 208: 1939-1942. GOODWIN, T. W., AND S. SRISUKH, 1950. A study of the pigments of the sea urchins, Echinus esculentus and Paracentrotus lividus. Biochem. J ., 47 : 69-76. GUSTAFSON, T., AND P. LENIQUE, 1951. Studies on mitochondria in the developing sea urchin egg. Exp. Cell Res., 3 : 251-274. HERBST, C., 1892. Experimentelle Untersuchungen iiber den Einfluss der veranderten chemischen Zusammensetzung des umgebenden Mediums auf die Entwicklung der Tiere. I. Zeitschr. f. wiss. Zool, 55: 446-518. HORSTADIUS, S., 1928. Ueber die Determination des Keimes bei Echinodermen. Acta Zool, Stockh., 9 : 1-33. HORSTADIUS, S., 1935. Ueber die Determination im Verlaufe der Eiachse bei Seeigeln. Pubbl. Stas. Zool NapoH, 14: 251^78. HORSTADIUS, S., 1939. The mechanics of sea urchin development, studied by operative methods. Biol Rev., 14: 132-179. HULTIN, T., 1950. The protein metabolism of sea urchin eggs during early development studied by means of N-labeled ammonia. Exp. Cell Res., 1 : 599-602. KUHN, R., AND K. WALLENFELS, 1939. liber die chemische Natur des Stoffes im Eier des Seeigels (Arbacia f^ustulosa) absondern, urn die Spermatozoen anzulocken. Berh. deutsch. chem. Gcs., 72: 1407-1413. KUHN, R., AND K. WALLENFELS, 1940. Echinochromes as prosthetic groups of high molecular symplexes in eggs of Arbacia pustulosa. Chem. Abstr., 37: 369-371. LEDERER, E., AND R. GLASER, 1938. Sur 1'echinochrome et le spinochrome. C. R. Acad. Sci. Paris, 207 : 454-456. DEVELOPMENT OF PIGMENT IN LYTECHINUS 403 LINDAHL, P. E., 1936. Zur Kenntnis der physiologischen Grundlagen der Determination im Seeigelkeim. Ada Zool, Stockh., 17: 179-188. MAYER, F., AND A. H. COOK, 1943. The Chemistry of Natural Coloring Matters. Rheinhold, New York. 273 p. MONROY, A., A. M. ODDO AND M. DENICOLA, 1951. The carotenoid pigments during early development of the egg of the sea urchin Paracentrotus lividus. Exp. Cell Res., 2 : 700-702. PERLMANN, P., 1954. Study on the effect of antisera on unfertilized sea urchin eggs. Exp. Cell Res., 6 : 485-490. RUNNSTROM, J., 1933. Kunze Mitteilung zur Physiologic der Determination des Seeigelkeimes. Arch f. Entw., 129 : 442-459. SHAPIRO, H., 1946. The extracellular release of echinochrome. /. Gen. Physiol., 29 : 267-275. SUMNER, F. B., AND P. DouDOROFF, 1943. An improved method of assaying melanin in fishes. Biol. Bull., 84: 187-194. TENNENT, D. H., 1912. Studies in cytology. II. /. Exp. Zool., 12: 79-93. T WITTY, V. C., AND M. C. Niu, 1954. The motivation of cell migration, studied by isolation of embryonic pigment cells singly and in small groups in vitro. /. Exp. Zool., 125 : 541-574. WALLENFELS, K., AND A. GAUHE, 1943. Synthesis of echinochrome. Chem. Abstr., 37 : 5963. ZEUTHEN, E., 1951. Segmentation, nuclear growth, and cytoplasmic storage in eggs of echino- derms and amphibia. Pubbl. Stas. Zool. Napoli, 23 : 47-69. INDEX y\TP in crayfish tail muscle, 95. Acclimation to temperature in goldfish tissues, 308. Acorn barnacle, larval development of, 284. Acoustical behavior of Bimini fishes, 357. Acoustical orientation in bats, 10. Activity of oysters at different temperatures, 57. Actomyosin from crayfish tail muscle, 95. Adaptation of goldfish tissues to high and low temperatures, 308. Adenine, effect of on development of Ulva, 375. Adenosine triphosphate in crayfish tail muscle, 95. Adenylate kinase activity of crayfish tail muscle, 95. Adult characteristics, assumption of by sala- mander efts, 1. Agapema, cyclic carbon dioxide release in, 118. Alga, marine, effect of plant hormones on, 375. ALLEN, R. D., AND E. C. ROWE. The depend- ence of pigment granule migration on the cortical reaction in the eggs of Arbacia, 113. Amicronucleate strain of Tetrahymena, DNA metabolism in, 71. Amphibian, water drive in, 1. Anaerobic capacity of Cecropia pupal heart, 23. Anatomy of Balanus larvae, 284. Anatomy of stomatopods, 141. Apyrase activity of crayfish tail muscle, 95. Arbacia, development of pigment in larvae of, 394. Arbacia eggs, cortical reaction in, 113. Arbacia eggs, x-irradiation of, 385. Arbacia plutei as food for Balanus larvae, 284. Attachment of Bugula larvae, 215. Auditory phenomena in bats, 10. Auditory phenomena in fishes, 357. Autoradiography of sea urchin eggs, 196. BACTERIA, role of in inhibition of regen- eration in Tubularia, 255. Bacteria-free cultures of Ulva, 375. Bahamian fishes, acoustical behavior of, 357. Balanus, larval development of, 284. Barnacle, larval development of, 284. Barnacle ova, fungus parasite in, 205. Bats, echolocation in, 10. Behavior, acoustical, of Bimini fishes, 357. Behavior of oysters at different temperatures, 57. Beta-mercaptoethylamine, "protective" effects of against x-irradiation damage to Ar- bacia eggs, 385. Bimini fishes, acoustical behavior of, 357. Bioassay of prolactin, 1. Biology of five-lunuled sand dollar, 54. Biology of oyster crab, 146. Bivalve, effect of temperature on behavior of, 57. Blastomere interaction in Dendraster, 247. Blockage to development in frogs' eggs, 226. Blood analyses of hag-fish, 348. Blood sodium and potassium in Pachygrapsus, 334. BOOKHOUT, C. G. See J. D. COSTLOW, JR., 284. BOROUGHS, H. Sec S. C. HSIAO, 196. Brachiopoda, chitin in, 106. Brain respiration of goldfish, 308. Bryozoa, chitin in, 106. Bryozoan larvae, rate of attachment of, 215. BUCK, J. Cyclic carbon dioxide release in insects. IV., 118. Bugula larvae, rate of attachment of, 215. Burrowing activities of Mellita, 54. BRYAN, J. H. D., AND J. W. GOWEN. The effects of 2560 r of x-rays on spermato- genesis in the mouse, 271. (CALCIUM, radioactive, uptake of by sea urchin eggs, 196. Calcium, role of in Dendraster twinning, 247. Cambarellus, chromatophorotropins of, 317. Cambarus, contractile protein from muscle of, 95. Cape Cod, new stomatopod from, 141. Carbon dioxide release by oxygen-treated Ha- brobracon white pupae, 180. Carbon dioxide release in insects, 118. Carbon monoxide sensitivity of silkworm pu- pae, 36. Cecropia, cyclic carbon dioxide release by, 118. Cecropia, diapause in, 23, 36. Cell division in mouse testis, effects of x-rays on, 271. 404 INDEX 405 Cell division in x-irradiated Arbacia eggs, 385. CHACE, F. A., JR. A new stomatopod crusta- cean of the genus Lysiosquilla from Cape Cod, Mass., 141. Chitin in lophophorate phyla, 106. Chlamydomonas as food for Balanus larvae, 284. Chloride concentrations in hagfish blood, 348. CHRISTENSEN, A. M., AND J. J. MCDERMOTT. Life-history and biology of the oyster crab, Pinnotheres, 146. Chromatophorotropins of crayfishes, 317. Chthamalus ova, fungus parasite in, 205. Cirripede, larval development of, 284. Clam larvae, survival and growth of at dif- ferent salinities, 296. CLARK, A. M., AND M. J. PAPA. Some effects of oxygen upon the white pupae of Habro- bracon, 180. Cleavage of mercaptoethanol-treated Den- draster eggs, 247. Cleavage of x-irradiated Arbacia eggs, 385. Coelenterate, inhibition of regeneration in, 255. Cold, effect of on behavior of oysters, 57. Cold, effect of on development of Urosalpinx, 188. Cold, effect of on eclosion rate of Habrobra- con white pupae, 180. Cold, effect of on goldfish tissue respiration, 308. Cold, role of in oxygen-poisoning of frogs' eggs, 226. Color changes in crayfishes, 317. Contractile protein from crayfish tail muscle, 95. Copulation in Pinnotheres, 146. Cortical reaction in Arbacia eggs, 113. COSTLOW, J. D., JR., AND C. G. BOOKHOUT. Larval development of B. amphitrite var. denticulata reared in the laboratory, 284. Crab, ion regulation in, 334. Crab, oyster, life-history of, 146. Crassostrea, effect of temperature on, 57. Crassostrea, pea crab from, 146. Crassostrea larvae, survival and growth of at different salinities, 296. Crayfish tail muscle, contractile protein from, 95. Crowding, role of in Tubularia regeneration, 255. Crustacean, ion regulation in, 334. Crustacean, life-history of, 146. Crustacean, stomatopod, from Cape Cod, 141. Crustacean ehromatophorotropins, 317. Crustacean eggs, fungus parasite of, 205. Crustacean muscle, contractile protein from, 95. Crustacean tissues, enzyme activity of, 95. Cyanide-sensitivity of Cecropia pupal heart- beat, 23. Cyclic carbon dioxide release in insects, 118. Cyprid larvae of Balanus, 284. Cysteinamine, "protective" effects of against x-irradiation damage to Arbacia eggs, 385. Cytochemistry of Tetrahymena, 71. Cytochrome system in Cecropia pupa, 23, 36. Cytoplasm, egg, uptake of radiocalcium by, 196. Cytoplasmic granules in Arbacia egg, 113. metabolism in Tetrahymena, 71. DNA synthesis in mouse testis, effects of x- rays on, 271. Darkness, role of in development of Ulva, 375. "Dauerblastulae" of Lytechinus, 394. DAVIS, H. C. Survival and growth of clam and oyster larvae at different salinities, 296. Desiccation, effects of on Pachygrapsus, 334. Dendraster, twinning in, 247. Desoxyribonucleic acid metabolism of Tetra- hymena, 71. Detergent, effect of on radiocalcium uptake of sea urchin eggs, 196. Development of Balanus, 284. Development of bivalve larvae in relation to salinity, 296. Development of Dendraster twins, 247. Development of Habrobracon pupae, 180. Development of oxygen-poisoned frogs' eggs, 226. Development of parasitized barnacle eggs, 205. Development of pigment in Lytechinus, 394. Development of Pinnotheres, 146. Development of sperm in mouse testis, effects of x-rays on, 271. Development of Ulva, effect of plant hormones on, 375. Development of Urosalpinx, 188. Development of x-irradiated Arbacia eggs, 385. Diapause, insect, physiology of, 23, 36. Dichlorobenzenonindophenol, effect of on at- tachment of Bugula larvae, 215. Diemyctylus efts, water drive studies on, 1. Differentiation in regenerating Tubularia, 255. Differentiation of x-irradiated Bugula larvae, 215. Diffusion of gases in insects, 118. Distribution of fishes in Bimini area, 357. Division, cell, in Dendraster, blockage of by mercaptoethanol, 247. Division of Tetrahymena, 71. Drill, oyster, development of, 188. Dwarf crayfish, ehromatophorotropins of, 317. 406 INDEX JfCHINOCHROME migration in Arbacia eggs, 113. Echinoderm, pigment development in larva of, 394. Echinoderm eggs, cortical reaction in, 113. Echinoderm eggs, twinning in, 247. Echinoderm eggs, uptake of radiocalcium by, 196. Echinoid, biology of, 54. Echolocation in bats, 10. Eclosion of Habrobracon white pupae, 180. Ecological factors in relation to behavior of oysters, 57. Ecology of Chthamalus in relation to fungus parasite, 205. Ecology of Pinnotheres, 146. Ecology of Urosalpinx, 188. Ectoprocta, chitin in, 106. Effect of plant hormones on Ulva, 375. Effect of x-irradiation on Bugula attachment, 215. Effects of oxygen on Habrobracon white pu- pae, 180. Effects of x-rays on mouse testis, 271. Efts, water drive studies on, 1. Egg-deposition of Pinnotheres, 146. Eggs, Arbacia, cortical reaction in, 113. Eggs, Arbacia, x-irradiation of, 385. Eggs, barnacle, fungus parasite in, 205. Eggs, frog, oxygen poisoning of, 226. Eggs, sea urchin, uptake of radiocalcium by, 196. Eggs, Urosalpinx, development of, 188. EKBERG, D. R. Respiration in tissues of gold- fish adapted to high and low tempera- tures, 308. Electrolytes in hagfish blood, 348. Embryo, sea urchin, development of pigment in, 394. Embryology of mercaptoethanol-treated Den- draster eggs, 247. Embryology of parasitized barnacle eggs, 205. Embryology of Urosalpinx, 188. Embryology of x-irradiated Arbacia eggs, 385. Embryos, frog, oxygen poisoning of, 226. Embryos, twin, in Dendraster, 247. Endocrine studies on salamander efts, 1. Enzyme kinetics in crayfish muscle extracts, 95. Epinephalus, acoustical behavior of, 357. Epinephrine, effect of on red chromatophoro- tropins of crayfish, 317. Erdschreiber medium for culture of Ulva, 375. Evolution of telson in stomatopods, 141. Evolutionary significance of hagfish osmotic properties, 348. Exogastrulation in Lytechinus eggs, 394. PEEDING of MeiHta, 54. Feeding of oysters at different temperatures, 57. Fertilization of Arbacia eggs before x-irradia- tion, 385. Fertilization reaction in Arbacia eggs, 113. FINGERMAN, M., AND M. E. LOWE. Stability of the chromatophorotropins of the dwarf crayfish, Cambarellus, and their effects on another crayfish, 317. Fishes, Bimini, acoustical behavior of, 357. Five-lunuled sand dollar, biology of, 54. Flow of water through oysters at different temperatures, 57. Flowing gases, effects of on silkworm heart- beat, 23, 36. Food in relation to survival of bivalve larvae at different salinities, 296. Freezing-point measurements of hagfish blood, 348. Frogs' eggs, oxygen poisoning of, 226. Fungus parasite in barnacle ova, 205. ("JAMETES, methods for obtaining, from clams and oysters, 296. GANAROS, A. E. On development of early stages of Urosalpinx at constant tempera- tures and their tolerance to low tempera- tures, 188. Gas exchange in insects, 118. Gases, effects of on silkworm pupal heartbeat, 23, 36. Gastrular blockage in frogs' eggs, 226. Gibberellin, effect of on development of Ulva, 375. Gill oxygen consumption of goldfish, 308. Goldfish tissues, respiration in, 308. Gonad, mouse, effects of x-rays on, 271. Gonadotropin, role of in water drive of sala- mander eft, 1. GOWEN, J. W. See J. H. D. BRYAN, 271. GRANT, W. C., JR., AND J. A. GRANT. Water drive studies on hypophysectomized efts of Diemyctylus. I., 1. Granule migration in Arbacia egg cortex, 113. GRIFFIN, D. R. See A. D. GRINNELL, 10. GRINNELL, A. D., AND D. R. GRIFFIN. The sensitivity of echolocation in bats, 10. GROSS, W. J. Potassium and sodium regula- tion in an intertidal crab, 334. Grouper, acoustical behavior of, 357. Growth cycle of Tetrahymena, 71. Growth of bivalve larvae at different salini- ties, 296. Growth of Pinnotheres, 146. Growth of regenerating Tubularia, 255. INDEX 407 Growth of Ulva germlings, 375. Growth of x-irradiated Bugula larvae, 215. ABROBRACON, effects of oxygen on white pupae of, 180. Hagfish, osmotic properties of, 348. HARVEY, W. R., AND C. M. WILLIAMS. Phys- iology of insect diapause. XL, XII., 23, 36. Hatching of Pinnotheres, 146. Hawaiian sea urchin, uptake of radiocalcium by eggs of, 196. Heartbeat of Cecropia pupa, 23, 36. Heat, effect of on development of Urosalpinx, 188. Heat, effect of on eclosion of Habrobracon white pupae, 180. Heat, effect of on goldfish tissue respiration, 308. High temperature, effect of on behavior of oysters, 57. High temperature, effect of on goldfish tissue respiration, 308. Histochemistry of chitin in lophophorate phyla, 106. Holocentrus, acoustical behavior of, 357. Hormone, lactogenic, role of in water drive of efts, 1. Hormones, chromatophorotropic, in crayfish, 317. Hormones, plant, effect of on development of Ulva, 375. Host size in relation to growth and develop- ment of Pinnotheres, 146. HSIAO, S. C., AND H. BOROUGHS. The uptake of radiocalcium by sea urchin eggs. I., 196. Hyalophora, cyclic carbon dioxide release in, 118. Hybridization of Lytechinus and Arbacia, 394. Hydrogen peroxide, role of in attachment of Bugula larvae, 215. HYMAN, L. H. Notes on the biology of the five-lunuled sand dollar, 54. HYMAN, L. H. The occurrence of chitin in the lophophorate phyla, 106. Hypophysectomized efts, water drive studies on, 1. JAA, effect of on development of Ulva, 375. IAA, effect of on goldfish tissue respiration, 308. Indolacetic acid, effect of on development of Ulva, 375. lodoacetic acid, effect of on goldfish tissue respiration, 308. Infection of barnacle eggs with fungus para- site, 205. Infection of oysters with Pinnotheres, 146. Inhibitors of regeneration in Tubularia, 255. Insect diapause, physiology of, 23, 36. Insects, cyclic carbon dioxide release in, 118. Insemination reaction in Arbacia eggs, 113. Instars of Pinnotheres, 146. Intertidal crab, sodium and potassium regula- tion in, 334. Ion regulation in crab, 334. Ionizing radiations, effect of on attachment of Bugula larvae, 215. Ionizing radiations, effect of on sea urchin eggs, 385. Irradiation, roentgen, of Arbacia egg, 385. Irradiation, roentgen, of Bugula larvae, 215. Isolation, "chemical," of Dendraster blasto- meres, 247. Isolation of animal and vegetal halves of Lytechinus embryos, 394. Isotope, radioactive, uptake of by sea urchin eggs, 196. JELLY coat of sea urchin egg, role of in radiocalcium uptake, 196. JOHNSON, T. W., JR. A fungus parasite in ova of the barnacle Chthamalus, 205. T^EY to stomatopods, 141. Kinetics of enzyme action in crayfish tail muscle extracts, 95. Kinetin, effect of on development of Ulva, 375. LABORATORY culture of Balanus, 284. Lactogenic hormone, role of in water drive of efts, 1. Lagenidium as parasite of barnacle eggs, 205. Larva of Lytechinus, pigment development in, 394. Larvae, bivalve, survival and growth of at different salinities, 296. Larvae, Bugula, rate of attachment of, 215. Larval development of Balanus, 284. Larval stages of Pinnotheres, 146. Lethal effects of carbon monoxide on silk- worm pupae, 36. Lethal effects of cyanide on silkworm pupae, 23. Life-history of Balanus, 284. Life-history of oyster crab, 146. Light, role of in development of Ulva, 375. Light-reversible inhibition by carbon monox- ide in silkworm pupae, 36. Lithium-treatment of Lytechinus eggs, 394. Liver respiration of goldfish, 308. 408 INDEX LOOSANOFF, V. L. Some aspects of behavior of oysters at different temperatures, 57. Lophophorate phyla, chitin in, 106. Low temperature, effect of on behavior of oysters, 57. Low temperature, effect of on development of Urosalpinx, 188. Low temperature, effect of on eclosion rate of Habrobracon white pupae, 180. Low temperature, effect of on goldfish tissue respiration, 308. Low temperature, role of in oxygen poisoning of frogs' eggs, 226. LOWE, M. E. See M. FINGERMAN, 317. Lunules of Mellita, 54. LYNCH, W. F. The effect of x-rays, irradi- ated sea water, and oxidizing agents on the rate of attachment of Bugula larvae, 215. Lysiosquilla, new species of, 141. Lytechinus, development of pigment in larvae of, 394. MACRONUCLEAR division in Tetrahy- mena, 71. MALAMED, S. Gastrular blockage in frogs' eggs produced by oxygen poisoning, 226. Mammalian testis, effects of x-rays on, 271. Marine alga, effect of plant hormones on, 375. Marine bryozoa, attachment of, 215. Marine eggs, fungus parasite in, 205. Marine fishes, sound production by, 357. Marine hagfishes, osmotic properties of, 348. MARUYAMA, K. Contractile protein from crayfish tail muscle, 95. Maturation of mouse sperm, effects of x-rays on, 271. MAZIA, D. The production of twin embryos in Dendraster by means of mercapto- ethanol, 247. McDERMOTT, J. J. See A. M. CHRISTENSEN, 146. MCDONALD, B. B. Quantitative aspects of DNA metabolism in an amicronucleate strain of Tetrahymena, 71. McFARLAND, W. N., AND F. W. MUNZ. A re-examination of the osmotic properties of the Pacific hagfish, Polistotrema, 348. Mechanism of cyclic carbon dioxide release in insects, 118. Meiosis, effects of x-rays on, in mouse testis, 271. Mellita, biology of, 54. Mercaptoethanol, use of in producing Den- draster twins, 247. Metabolism of Cecropia diapause, 23, 36. Metabolism of DNA in Tetrahymena, 71. Metabolism of goldfish tissues, 308. Metabolism of Habrobracon white pupae, 180. Metabolism of insects, 118. Metamorphosis of Bugula larvae, effects of various agents on, 215. Metamorphosis of silkworm pupae, 23, 36. Migration of pigment granules in Arbacia eggs, 113. Migration of salamander efts to water, 1. Mitosis, effects of x-rays on, in mouse testis, 271. Mitosis in Dendraster eggs, blockage of by mercaptoethanol, 247. Mitosis in x-irradiated Arbacia eggs, 385. Mollusc, behavior of at different temperatures, 57. Mollusc larvae, survival and growth of at different salinities, 296. Molting of hypophysectomized salamander efts, 1. Molting stages of Pinnotheres, 146. Monothioethylene glycol, use of in producing Dendraster twins, 247. Morphogenesis of Ulva, effect of plant hor- mones on, 375. Morphogenesis of x-irradiated Arbacia eggs, 385. Morphology of Balanus larvae, 284. Morphology of barnacle egg fungus parasite, 205. Morphology of Pinnotheres developmental stages, 146. Morphology of stomatopods, 141. MOULTON, J. M. The acoustical behavior of some fishes in the Bimini area, 357. Mouse, effects of x-rays on spermatogenesis in, 271. Movement of oyster shells at different tem- peratures, 57. MUNZ, F. W. See W. N. MCFARLAND, 348. Muscle, crayfish, contractile protein from, 95. Myosin in crayfish tail muscle, 95. Myotis, sound emission by, 10. Myxinoid, osmotic properties of, 348. JS^AUPLII of Balanus, 284. Neurosecretory system of dwarf crayfish, 317. New stomatopod from Cape Cod, 141. Nitrogen treatment of frog eggs, 226. Nucleic acid metabolism of Tetrahymena, 71. OCCURRENCE of chitin in lophophorate phyla, 106. Orconectes, effect of Cambarellus chromato- phorotropins on, 317. Orientation in bats, 10. Origin of pigment in Lytechinus, 394. INDEX 409 Osmotic properties of hagfish, 348. Osmotic regulation in Pachygrapsus, 334. Ova, Arbacia, cortical reaction in, 113. Ova, Arbacia, x-irradiation of, 385. Ova, barnacle, fungus parasite in, 205. Ova, Dendraster, treatment of with mercapto- ethanol, 247. Ova, frog, oxygen poisoning of, 226. Ova, sea urchin, uptake of radiocalcium by, 196. Ova, Urosalpinx, development of, 188. Oxidizing agents, effect of on rate of attach- ment of Bugula larvae, 215. Oxygen, effect of on silkworm pupal heart, 23, 36. Oxygen, effects of on Habrobracon white pupae, 180. Oxygen poisoning of frogs' eggs, 226. Oxygen uptake of goldfish tissues, 308. Oxygen uptake in insects, 118. Oyster crab, life-history of, 146. Oyster drill, development of, 188. Oyster larvae, survival and growth of at dif- ferent salinities, 296. Oysters, behavior of at different tempera- tures, 57. pACHYGRAPSUS, ion regulation in, 334. Pacific hagfish, osmotic properties of, 348. PAPA, M. J. See A. M. CLARK, 180. Parasite, fungus, in barnacle ova, 205. Parasitic wasp pupae, effects of oxygen on, 180. Parthenogenesis in Lytechinus, 394. Partial fertilization of Arbacia eggs, 113. Pea crab, life-history of, 146. Peroxide, role of in attachment of Bugula larvae, 215. Phoronida, chitin in, 106. Phosphorus in crayfish tail muscle, 95. Photo-reversal of CO-inhibition in silkworm pupae, 36. Phycomycete parasite of barnacle eggs, 205. Phylogenetic significance of chitin, 106. Physiology of insect diapause, 23, 36. Pigment-concentrating and -dispersing hor- mones in crayfishes, 317. Pigment-development in Lytechinus, 394. Pigment granule migration in Arbacia eggs, 113. Pigmentation, adult, assumption of by sala- mander efts, 1. Pigmentation of developing Ulva, effect of plant hormones on, 375. Pigmentation of Habrobracon pupae, effects of oxygen on, 180. Pinnotheres, life-history of, 146. Pituitary hormones, role of in eft water drive, 1. Plant hormones, effect of on development of Ulva, 375. Platysamia, physiology of insect diapause in, 23, 36. Podia of Mellita, 54. Polistotrema, osmotic properties of, 348. Polyphemus, physiology of diapause in, 23, 36. Population differences in survival of bivalve larvae at different salinities, 296. Potassium regulation in crab, 334. Pressure, effects of on development of frog eggs, 226. Pressure, effects of on Habrobracon white pupae, 180. Production of Dendraster twins, 247. Prolactin, role of in water drive of efts, 1. Protease, use of in removing Dendraster fer- tilization membrane, 247. "Protection" against x-irradiation effects in Arbacia eggs, 385. Protein, contractile, from crayfish tail muscle, 95. Protozoan, DNA metabolism in, 71. PROVASOLI, L. Effect of plant hormones on Ulva, 375. Pumping activity of oysters at different tem- peratures, 57. Pupae of Habrobracon, effects of oxygen on, 180. Pupal diapause, physiology of, 23, 36. Q UANTITATIVE aspects of DNA metab- olism in Tetrahymena, 71. D ADIATION effects on Arbacia egg, 385. Radiation-sensitive constituents of mouse tes- tis, 271. Radiocalcium, uptake of by sea urchin eggs, 196. Radiosensitivity of Arbacia eggs, 385. Rana eggs, oxygen poisoning of, 226. Rate of attachment of Bugula larvae, 215. Rate of development of oxygen-poisoned frog eggs, 226. Recognition behavior in black angelfish, 357. Recording of fish acoustical behavior, 357. "Recovery" phenomenon in x-irradiated Ar- bacia eggs, 385. Red efts, water drive of, 1. Reducing agents, effects of on Bugula larvae, 215. Re-examination of hagfish osmotic properties, 348. Regeneration inhibitors in Tubularia, 255. 410 INDEX Regulation of sodium and potassium in crab, 334. Release of carbon dioxide in insects, 118. Reproductive potential of bivalves in relation to salinity, 296. Respiration of Habrobracon white pupae, 180. Respiration in insects, 118. Respiration in tissues of goldfish, 308. Roentgen irradiation of Arbacia eggs, 385. Roentgen irradiation of Bugula larvae, 215. Roentgen irradiation of mouse testis, 271. ROWE, E. C. See R. D. ALLEN, 113. RUGH, R. The so-called "recovery" phe- nomenon and "protection" against x-irra- diation at the cellular level, 385. SALAMANDER eft, water drive in, 1. Salinity in relation to survival and growth of bivalve larvae, 296. "Salt pools" in Pachygrapsus, 334. Sand dollar, five-lunuled, biology of, 54. Sea urchin eggs, cortical reaction in, 113. Sea urchin eggs, twinning in, 247. Sea urchin eggs, uptake of radiocalcium by, 196. Sea urchin eggs, x-irradiation of, 385. Sea urchin larvae, pigment development in, 394. Sea water, irradiated, effect of on attachment of Bugula larvae, 215. Sea weed, effect of plant hormones on, 375. Seasonal variations in goldfish oxygen con- sumption, 308. Seminiferous tubules of mouse, effects of x- rays on, 271. Sensitivity of echolocation in bats, 10. Sensitivity of Habrobracon pupae to oxygen, 180. Serum chloride and sodium concentrations of hagfish blood, 348. Setation formulae of larval barnacles, 284. Setting of x-irradiated Bugula larvae, 215. Sexual reproduction in fungus parasite of bar- nacle egg, 205. Shaking, role of in oxygen poisoning of frog eggs, 226. Shell-opening of oysters at different tempera- tures, 57. Shore crab, ion regulation in, 334. Silkworm, diapause in, 23, 36. Slime, role of in osmotic properties of hagfish blood, 348. Sodium concentrations in hagfish blood, 348. Sodium regulation in crab, 334. Sonic experiments with Bimini fishes, 357. Sound emission by bats, 10. Spat, oyster, infection of with Pinnotheres, 146. Spermatogenesis in mouse, effects of x-rays on, 271. Spiracles, role of in cyclic release of carbon dioxide by insects, 118. Spontaneous production of sound by Bimini fishes, 357. Squirrelfish, acoustical behavior of, 357. Stability of crayfish chromatophorotropins, 317. Stomatopod from Cape Cod, 141. Stress, osmotic, in crab, 334. Sulfhydryl bond, possible role of in oxygen poisoning of frog embryos, 226. Sulfhydryl bond, possible role of in x-irradia- tion of Bugula larvae, 215. Sulfhydryl group, importance of in x-irradi- ated Arbacia eggs, 385. Sulfhydryl groups, importance of in Den- draster twinning, 247. Survival of bivalve larvae at different salini- ties, 296. Synthesis of DNA in Tetrahymena, 71. , effect of on attachment of Bugula larvae, 215. Taxonomy of stomatopods, 141. Telea, physiology of diapause of, 23, 36. Teleost, fresh-water, respiration of tissues of, 308. Teleosts, sound production by, 357. Temperature, effect of in cyanide-sensitivity of silkworm pupae, 23. Temperature, effect of on behavior of oysters, 57. Temperature, effect of on goldfish tissue respi- ration, 308. Temperature, effect of on pigment concentra- tion in crayfish, 317. Temperature, relation of to development of Urosalpinx, 188. Temperature, relation of to eclosion of Habro- bracon white pupae, 180. Temperature, role of in oxygen-poisoning of frogs' eggs, 226. Terrestrial stage of Diemyctylus, water drive in, 1. Tetrahymena, DNA metabolism in, 71. Thiol groups, importance of in Dendraster twinning, 247. Tissue culture of Ulva, 375. Tissue extracts of Tubularia as inhibiting agents, 255. Tissues of goldfish, respiration in, 308. Toothplate stridulation in marine fishes, 357. Triphenyltetrazolium chloride, effect of on attachment of Bugula larvae, 215. Tripneustes, uptake of radiocalcium by eggs of, 196. INDEX 411 Triturus, water drive of, 1. Tropical fishes, acoustical behavior of, 357. Tube feet of Mellita, 54. Tubularia, inhibition of regeneration in, 255. TWEEDELL, K. Inhibitors of regeneration in Tubularia, 255. Twinning in Dendraster, 247. ULTRAVIOLET absorption spectra of crayfish tail muscle extracts, 95. Ulva, effect of plant hormones on, 375. Underwater sounds in Bimini area, 357. Unfertilized sea urchin eggs, uptake of radio- calcium by, 196. Uptake of radiocalcium by unfertilized sea urchin eggs, 196. \7"ENUS larvae, survival and growth of, at different salinities, 296. Vital staining of Lytechinus embryos, 394. \yARM-ADAPTED goldfish tissues, respi- ration of, 308. Wasp pupae, effects of oxygen on, 180. Water drive studies on hypophysectomized efts, 1. Water flow through oysters at different tem- peratures, 57. WILLIAMS, C. M. See W. R. HARVEY, 23, 36. of Arbacia eggs, 385. X-irradiation of Bugula larvae, 215. X-rays, effects of on mouse testis, 271. VOUNG, R. S. Development of pigment in the larva of the sea urchin, Lytechinus, 394. r7 OID metamorphosis after x-irradiation, 215. Volume 114 Number 1 THE BIOLOGICAL BULLETIN PUBLISHED BY THE MARINE BIOLOGICAL LABORATORY Editorial Board HAROLD C. BOLD, University of Texas FRANK A. BROWN, JR., Northwestern University JOHN B. BUCK, National Institutes of Health T. H. BULLOCK, University of California, Los Angeles E. G. BUTLER, Princeton University J. H. LOCHHEAD, University of Vermont ARTHUR W. POLLISTER, Columbia University C. L. PROSSER, University of Illinois MARY E. RAWLES, Carnegie Institution of Washington WM. RANDOLPH TAYLOR, University of Michigan A. R. WHITING, University of Pennsylvania CARROLL M. WILLIAMS, Harvard University DONALD P. COSTELLO, University of North Carolina Managing Editor FEBRUARY, 1958 Marine Biological Laborati l ' 1958 WOODS HOLE, MASS. Printed and Issued by LANCASTER PRESS, Inc. PRINCE & LEMON STS. LANCASTER, PA. 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They should be carefully proof-read before being submitted and all typographical errors corrected legibly in black ink. Pages should be numbered. A left-hand margin of at least 1$ inches should be allowed. 2. Tables, Foot-Notes, Figure Legends, etc. Tables should be typed on separate sheets and placed in correct sequence in the text. Because of the high cost of setting such material in type, authors are earnestly requested to limit tabular material as much as possible. Similarly, foot- notes to tables should be avoided wherever possible. If they are essential, they should be indi- cated by asterisks, daggers, etc., rather than by numbers. Foot-notes in the body of the text should also be avoided unless they are absolutely necessary, and the material incorporated into the text. Text foot-notes should be numbered consecutively and typed double-spaced on a sepa- rate sheet. Explanations of figures should be typed double-spaced and placed on separate sheets at the end of the paper. 3. A condensed title or running head of no more than 35 letters and spaces should be included. 4. Literature Cited. The list of papers cited should conform exactly to the style set in a recent issue of The Biological Bulletin; this list should be headed LITERATURE CITED, and typed double-spaced on separate pages. 5. Figures. The dimensions of the printed page, 5 by 7| inches, should be kept in mind in preparing figures for publication. Illustrations should be large enough so that all details will be clear after appropriate reduction. Explanatory matter should be included in legends as far as possible, not lettered on the illustrations. Figures should be prepared for reproduction as line cuts or halftones; other methods will be used only at the author's expense. 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THE BIOLOGICAL BULLETIN THE BIOLOGICAL BULLETIN is issued six times a year at the Lancaster Press, Inc., Prince and Lemon Streets, Lancaster, Pennsylvania. 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 num- bers, $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. 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 GRANT, WILLIAM C., JR., AND JOAN A. GRANT Water drive studies on hypophysectomized efts of Die- myctylus viridescens 1 GRINNELL, ALAN D., AND DONALD R. GRIFFIN The sensitivity of echolocation in bats 10 HARVEY, WILLIAM R., AND CARROLL M. WILLIAMS Physiology of insect diapause. XI. Cyanide-sensitivity of the heartbeat of the Cecropia silkworm, with special reference to the anaerobic capacity of the heart 23 HARVEY, WILLIAM R., AND CARROLL M. WILLIAMS Physiology of insect diapause. XII. The mechanism of car- bon monoxide-sensitivity and -insensitivity during the pupal diapause of the Cecropia silkworm 36 HYMAN, LIBBIE H. Notes on the biology of the five-lunuled sand dollar 54 LOOSANOFF, V. L. Some aspects of behavior of oysters at different temperatures 57 MCDONALD, BARBARA BROWN Quantitative aspects of deoxyribose nucleic acid (DNA) metabolism in an amicronucleate strain of Tetrahymena. . . 71 MARUYAMA, K. Contractile protein from crayfish tail muscle 95 HYMAN, LIBBIE H. The occurrence of chitin in the lophophorate phyla 106 M.?J; WHO.l LIBRARY WH 1AZJ S