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 CO 2 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 Ca 45 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^1 C / 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 show r ed 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 few r 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 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 6472.) 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 b 5 and cytochrome oxidase (a + a 3 ) 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 w r as 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 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 Eleventh or twelfth day of adult development at 25 C. 4 12.0 3.10 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 6C. 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 S 1 /^ hours. It will be observed in Table II that the effects of 5 l /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 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 l 1 /^ 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 S 1 /^ 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 5 1/ 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 S 1 /^ 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 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 5V 2 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 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) (Tl , ^^, 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 '//, ///, X (CO ABSENT) REDUCED CYTOCMROME OXJDASE COMPLEXED WITH CO 50 X I LOW CO PRESSURE) 99X ( HIGH CO PRESSURE) STEADY STATE CONDITION H 2 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/C 2 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 C 2a -C 2 b 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. 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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 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 NaHCO 3 , 0.01 M Na 2 CO 3 ). After being placed for 24 hours at 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 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 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 CaQ 2 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 ++ ++ + + 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 3038% 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 = Z n 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 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 O 2 up- take. Similar serial computations indicate the theoretical feasibility of widely dif- ferent degrees of relative CO 2 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 CO 2 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 CO 2 re- lease. Of these, all that involve a reduction in demand for O 2 (Table IV, re- sponses No. 2, 4), or increase in availability of O 2 (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 IBRCO 2 1 Increasing temperature Increase 1 - 2 ' 3 2 Decreasing temperature Decrease 1 ' 2 ' 3 ' 5 3 Increasing metabolic rate (injury; dev.) I ncrease 3 4 Decreasing metabolic rate Decrease 2 ' 3 5 Increased ambient pO 2 above 21% Decrease 3 ' 4 6 Decreased ambient pO 2 (below 21%, but above about 10%) Increase 3 ' 4 1 Punt, 1944. 2 Punt, 1950. 3 Schneiderman and Williams, 1955. 4 Buck and Keister, 1955. 5 Buck and Keister, 1958. CO 2 release; and all tending to decrease the availability of O 2 (No. 6), or increase the call for O 2 (Nos. 1, 3), have the opposite effect. Now we know (Buck and Keister, 1955) that environmental O 2 concentrations between \% and 100% neither increase nor decrease rate of O 2 consumption (CO 2 production), so the observed changes in CO 2 release rate must be due to changes in rate of CO 2 retention. The retention changes in turn must involve changes in air in-flow and CO 2 out-diffusion due to alterations in either spiracular valve area or effective CO 2 gradient, or both. Although no direct measurements of spiracular valve areas in pupae with different metabolic rates or in different O 2 tensions or temperatures have been reported, the fact that increase in ambient O, concentration above the normal atmospheric level reduces the rate of interburst CO 2 release (Table IV, resp. No. 5) without increasing the rate of O 2 uptake, suggests that the maintained, steady-state valve area of the interburst period is regulated so as to keep the rate of entry of O 2 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 O 2 uptake in inducing air flow with the fact that the rate of O 2 uptake is not O 2 -limited except at very low ambient concentrations. Even for responses not involving changed metabolic rate the expected changes in CO 2 are not easy to predict. For example, insofar as purely diffusive transfer is concerned, a decrease in ambient pO 2 , since it induces spiracular dilation, would be expected to increase the interburst rate of CO 2 escape (see Fick equation). How- ever, if the pupa regulates valve area so as to keep the rate of O 2 entry constant, ambient pO 2 will be inversely proportional to area and the effects of changing pO 2 on interburst CO 2 out-diffusion rate will be similarly non-linear (Buck and Keister, 1955, p. 160). On the same basis changing ambient pO 2 will have an inversely proportional effect on R F (i.e. on CO 2 flow-retention), but with the additional com- plication of a different proportionality constant (R D /' A, whereas RF 'A X r 2 ; see Poiseuille equation). Hence the over-all effect of changing ambient pO 2 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 CO 2 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 CO 2 (RdCO 2 ) to valve area for the valve length and trans-spiracular CO 2 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 CO 2 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 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 TAB