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Temperature resistance and thyroid activity in goldfish maintained under controlled photoperiods Robertson, Georgina Beth 1958

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TEMPERATURE RESISTANCE AND THYROID ACTIVITY IN GOLDFISH MAINTAINED UNDER CONTROLLED PHOTOPERIODS by G-eorgina Beth Robertson B.A., University of B r i t i s h Columbia, 1956 A thesis submitted i n p a r t i a l fulfilment of the requirements for the Degree of Master of Arts i n the Department of Zoology We accept this thesis as conforming to the standard required from candidates for the degree of Master of Arts Members of the Department of Zoology The University of B r i t i s h Columbia A p r i l , 1958 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allov/ed. without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date F\pn\ 3 4 , V \ S S - This m a t e r i a l i s not to be reproduced without the w r i t t e n permission of the L i b r a r i a n of the U n i v e r s i t y of B r i t i s h Columbia. TEMPERATURE RESISTANCE AND THYROID ACTIVITY IN GOLDFISH MAINTAINED UNDER CONTROLLED PHOTOPERIODS ABSTRACT Temperature resistance was measured i n goldfish which had been maintained under controlled photoperiods during different seasons over a twelve month period. A consistently higher heat resistance was found i n f i s h which had been kept under the longer photoperiod. The cold resistance of short-day f i s h was greater than that of long-day f i s h i n the f a l l and winter, but i n the spring and summer the relationship seemed to be reversed. Thyroid uptake of radioiodine was determined i n f i s h which had been subjected to the same photoperiod treatment. Short-day 131 f i s h showed a consistently higher uptake of I , although the differences were not s t a t i s t i c a l l y significant. Photoperiodically adapted f i s h treated with thyroxine or TSH showed increased resistance to cold* Thiouracil caused the reverse effect, but thiourea caused increased resistance i n the long-day group. Tissue cholesterol analyses were carried out on f i s h treated with thyroid materials. Gonad weight/body weight relationships were compared i n f i s h maintained under the two photoperiods to determine possible correlations between the state of sexual maturity and thermal resistance. i i i Table of Contents Page Acknowledgements , Introduction 1 Materials and Methods General 3 Photoperiod Control 3 Thermal Resistance 5 Cold Resistance Test 6 Heat Test 6 Thyroid Activity 7 Cholesterol 8 Results Temperature Resistance Heat Resistance 9 Cold Resistance 9 Variability in Samples as Possible Causes of Irregularities in Effect of Photoperiod 9 Ratio of Narcotized to "truly dead" Fish 12 Thyroid Activity 15 Effects of Thyroxine, Thyroid Inhibitors, and TSH Temperature Resistance Heat Resistance 17 Cold Resistance 20 Thyroid Activity 20 Cholesterol 21 Moisture Changes 21 Gonad Weight-Body Weight Relationships 24 Discussion Changes in Temperature Resistance 25 Thyroid Activity 28 Effects of Thyroid Treatment 30 Sexual Development 33 Summary 35 Appendix Literature Cited i v L i s t of Tables Page Table I Mean weight i n grams of goldfish used i n 10 temperature resistance tests. I I Mean weight i n grams of hormone treated 11 f i s h used i n temperature resistance tests. I l l Ratio of males to females i n groups of 13 f i s h used i n temperature tolerance tests. IV Ratio of narcotized f i s h to "truly dead" 14 f i s h i n temperature tolerance tests. V Percentage uptake of radioiodine by goldfish 16 thyroid. VI Maximum radioiodine uptake within f i r s t 24 18 hours expressed as percent of injected dose. VII Heat resistance measured as minutes difference 19 from the control group required to k i l l different percentages of samples of 20 goldfish after 77 days controlled photoperiod. VIII Effect of thyroid materials on tissue 22 cholesterol expressed as fo difference from control value. LX Effect of thyroid materials on percent moisture 23 content of goldfish. List of Figures Effect of photoperiod on heat resistance of goldfish. Effect of photoperiod on cold resistance of goldfish. Percent uptake of radioiodine by 8- and 16-hour goldfish. Effect of thyroxine, TSH, thiourea, or thiouracil on cold resistance of goldfish. Effect of thyroxine or thiourea on percent uptake of radioiodine by goldfish. Gonad weight-body weight relationships i n female goldfish maintained under 8-hour or 16-hour photoperiods. Gonad weight-body weight relationships in male goldfish maintained under 8-hour or 16-hour photoperiods. v i List of Appendices Page Appendix I In i t i a l temperatures and temperature ranges 37 of lethal temperature baths. II Time required for 25%, 50%, and 75% mortality in cold tests. 38 III Time required for 25%, 50%, and 75% mortality in heat tests. 39 IV Time required for 25%, 50%, and 75% mortality of thyroid treated fish in cold tests. 40 V Time required for 25%, 50%, and 75% mortality of thyroid treated fish in heat tests. 41 VI Mean weights and standard deviations of samples used for cholesterol analysis. 42 VII Mean weights of total body and of gonads with mean ratios of these two weights for photoperiod goldfish. 43 ACKNOWLEDGEMENTS I wish to extend my sincere thanks to Dr. W. S. Hoar for suggestion of the problem and supervision of the work. His continued interest and valuable assistance i n a l l phases of the study and i n preparation of the manuscript are greatly appreciated. For c r i t i c i s m of the manuscript I am indebted to Dr. P. A. Dehnel and Dr. W. B. Malcolm. I have greatly appreciated the assistance of my fellow workers i n this laboratory, particularly Dr. B. Baggerman, P. Canagaratnam and C. P. Hickman. I also wish to thank Miss R. Miletich who iyped the manuscript. Financial assistance from the National Research Council made this project possible and i s gratefully acknowledged. INTRODUCTION Seasonal changes i n physiological processes have been shown i n many animals. The metabolic rate, heart rate, or numbers of red or white blood cells have been shown to vary seasonally in various animals (Bullock, 1955). Seasonal variations i n temperature tolerance have also been noted. Brett (1946) observed seasonal variations in the lethal temperature of fi s h , and related these to acclimation of the fish to changing environmental temperatures. Johannes (1957) found seasonal variations in the heat tolerance of scallops acclimated to the same temperature and suggested that these might be related to the spawning cycle. In this laboratory goldfish maintained at a constant temperature showed seasonal changes in thermal resistance (Hoar, 1955). It was suggested that photoperiodically controlled changes i n endocrine physiology were responsible for these changes in temperature resistance. In experiments designed to test this hypothesis, goldfish of identical thermal and dietary history were subjected to long-day (16 hour) or short day (8 hour) light periods. In the f a l l and winter the 8-hour day fish were consistently more resistant to cold and less resistant to heat than the 16-hour day fish. In the spring and summer the differences between the two groups were less marked, and were generally in the opposite direction (Hollands, 1956). The present study was undertaken in an attempt to obtain a complete annual picture of the effects of controlled photoperiod on temperature resistance. Since the photoperiod effect demonstrated by Hollands (1956) 2 suggested an endocrine mechanism controlling the temperature resistance, an attempt was made to measure the endocrine a c t i v i t y i n the thyroid gland and the gonads, and to relate this to changes i n the temperature resistance. Thyroid a c t i v i t y of f i s h subjected to different photoperiods was determined by measuring the uptake of radioiodine. In addition, determinations were made of the temperature resistance of goldfish which had been kept under the two photoperiods and then treated with thyroid materials. I t has been suggested that temperature death results from a disorganization of the protein-lipid-calcium complex which forms the plasma membrane (Heilbrunn, 1952). Temperature acclimation may change the permeability properties of c e l l membranes by altering the metabolism 0 of the l i p i d s (Hoar and Cottle, 1952a). Irvine et a l (1957) showed that dietary supplements of cholesterol or phosopholipid increase both the heat and cold resistance of goldfish. Pish subjected to 8-hour photoperiods generally show higher tissue cholesterol and tissue phosopholipid than those subjected to 16 hours l i g h t (Hollands, 1956). To explore further the possible mechanism of changing thermal resistance and to relate the endocrine picture to the tissue chemistry, the tissues of thyroid treated f i s h were analyzed for cholesterol. These experiments were designed to determine whether the changes found i n the temperature resistance of thyroid treated f i s h were related to changes i n the f a t metabolism. 3 MATERIALS AND METHODS A. General Goldfish (Carassius auratus) were obtained from the Goldfish Supply Company. S t o u f f v i l l e , Ontario. One l o t of 800 f i s h arrived i n October, 1956 and was used for the experiments from November, 1956 to July, 1957. Another group of 300 f i s h , obtained i n A p r i l , 1957, was used for the experiments carried out i n the l a t t e r part of July, 1957, while a th i r d group of 300 f i s h , obtained i n July, 1957, was used for the experiments carried out i n the f a l l of 1957. Details of sample size, dates of testing, etc. are given i n Appendices I to V. The stock f i s h were kept i n large concrete indoor tanks at 10-15° C and fed regularly on a mixture of Pablura and Clark's Fish Pood. The water was changed at least once a week. B. Photoperiod Control Photoperiod acclimation was carried out i n a darkened room provided with two wooden cupboards, each containing three aquaria. Complete l i g h t control within the cupboards was ensured by a sliding door i n front of each aquarium. Two different photoperiod treatments were used. In one cupboard the f i s h received 8 hours l i g h t and 16 hours darkness per day, while i n the other the f i s h received 16 hours li g h t and 8 hours darkness. A r t i f i c i a l l i g h t was provided by fluorescent lamps, one above each aquarium. The l i g h t intensity was 58-66 foot candles at the water surface. The ligh t s were controlled by an automatic time clock. 4 Each aquarium was provided with a heater and a "thermostat which maintained the temperature at 20^ 2° C. The temperature was checked twice daily. The f i s h were fed a mixture of Pablum and Clark's Fish Food ("Grower Crumbles" - J . R. Clark and Co., Salt Lake City, Utah) allowing 0.4 grams of food per f i s h per day. The f i s h were fed three times a day. The aquaria were cleaned at least once a week. An attempt was made to obtain an annual picture of the effect of photoperiod on temperature resistance. Groups of 65 f i s h were placed under each photoperiod at various intervals throughout the year. After 35 to 75 days (generally 40-50 days) of controlled l i g h t , temperature resistance was measured using 20 or 21 f i s h from each photoperiod group for each test. Another 20 f i s h from each of the original 65 were used for determinations of thyroid a c t i v i t y . Experiments were carried out to determine the effects of thyroxine, thiourea, and thiouracil on the temperature resistance of photoperiodically acclimated f i s h . In the f i r s t experiment only thyroxine and thiourea were used, but i n a second experiment thiouracil was also used because i t was f e l t that thiourea might have other metabolic effects on the animal besides inhibiting thyroid a c t i v i t y . A th i r d experiment was designed to test the effect of TSH on temperature resistance. Thyroxine, thiourea, or thiouracil treatments were carried out on f i s h which had been under controlled l i g h t conditions for 77 days. Thyroxine (B.D.H. synthetic thyroxine sodium) was used i n a concentration of 1 part i n 2 mi l l i o n , while thiourea (Fisher S c i e n t i f i c Co.) and thiouracil (Nutritional Biochemical Corporation) were used i n concentrations of 0.05%. The thyroxine and thiourea were dissolved i n a small amount 5 of water which was then added to the water in the aquaria. Thiouracil is only slightly soluble in water and was apparently not completely dissolved. The photoperiod treatment was continued until the temperature resistance tests were carried out. The cold resistance tests were carried out after 8 or 9 days of thyroxine, thiourea, or thiouracil treatment, while the heat resistance tests were carried out after 12 or 13 days. Fifteen 8-hour and 13 16-hour fish which had been kept under controlled photoperiods for 90 days were injected with Parke Davis Thyrotropic Preparation. Each fish was injected with 0.5 ml. of a solution containing 0.3 U.S.P. units (as used by Chavin, 1956a). The same number of controls were injected with 0.5 ml. of isotonic saline. The injections were repeated two days later, and four days after the f i r s t injection the cold resistance of the fish was tested. C, Thermal Resistance During the day prior to the thermal resistance tests, 40 or 42 fish were randomly chosen from each of the two photoperiod groups. The fish were marked with f i n clips. In about half of the experiments a caudal clip was used for the 8-hour group and a pelvic c l i p for the 16-hour group. In the other tests the clips were reversed. When more than two groups of fish were to be used, other f i n clips, and combinations of clips had to be used. After f i n clipping, fish were moved to holding baths at 19^ 3° C, half of the 8-hour, and half of the 16-hour fish being placed in one bath, and the remainder i n a second one. The fish remained in these holding baths overnight. The marking and moving of the fish was done the day before the test, in order to avoid exciting the fish 6 just prior to the thermal resistance tests. 1. Cold Resistance Test Twenty or 21 f i s h from each photoperiod group were placed at one time i n a thermostatically controlled refrigeration bath. The i n i t i a l temperature of the bath was about 2.0° C (once as high as 5.8° C). The temperature of the bath was lowered at the rate of about 1° per hour, and was usually maintained at about 1° C, although i t occassionally went as low as 0.2° C for short periods. I t i s emphasized that the f i s h being compared for thermal resistance were exposed together, and consequently were subjected to identical temperature stress. The f i s h were checked hourly during the f i r s t part of the test, and at less frequent intervals after the f i r s t 10 hours. The following c r i t e r i o n was used to determine which animals were to be removed. The f i s h which did not respond to prodding with a cold glass rod while s t i l l i n the cold bath were placed on a cold glass plate and tapped three times on the caudal peduncle. Fish which showed no response were removed and those which responded were returned to the cold bath. The time and temperature were recorded with each removal. The f i s h which had been removed were immediately placed i n a bucket of aerated water at room temperature. Those which recovered after at least 30 minutes were recorded as "survivors" and those which did not recover as "truly dead". The length, weight, and sex of each f i s h was then recorded, and the animals preserved i n 10% formal in for gonad studies. 2. Heat Test The remaining f i s h from each group were placed i n a heated bath with an i n i t i a l temperature of 34.7° to 36.4° C. The temperature of 7 the bath was maintained at 36^2° C. The test was watched constantly and the f i s h removed as soon as they ceased to respond to tapping. The animals which were removed were transferred to aerated water at room temperature and after 15 minutes or more were checked for "recovery". Size and sex were recorded. D. Thyroid Ac t i v i t y The thyroid a c t i v i t y was measured by the uptake of tracer doses of radioiodine. Twenty f i s h from each photoperiod group were injected 131 intraperitoneally with approximately two micro curies of I . Standards 131 were prepared by adding the same amount of I to 100 ml. volumetric 131 flasks and diluting to 100 ml. The a c t i v i t y of the dose of I varied s l i g h t l y from one experiment to another but was between 2 and 4 microcuries. At each of fiv e time intervals (6, 12, 24, 48 and 96 hours after injection) three to five f i s h were k i l l e d and the thyroid glands removed. The length, weight, and sex of each f i s h was recorded. The thyroids were then wet ashed by boiling with 2N NaOH and the samples were diluted to a constant volume. A 1 ml. aliquot of the diluted digest was dried i n a nickel planehet and the radioactivity measured using a Philips Electronic Counter. The results were expressed as per cent uptake of the administered dose. The effect of thyroxine and thiourea on thyroid a c t i v i t y was determined by treating the f i s h with thyroxine (1 part i n 2 million) 131 or thiourea (0.05%) for ten days prior to the injection of I . 8 E. Cholesterol Cholesterol analysis was carried out on f i s h which had been treated with thyroxine ( l part i n 2 million) or thiourea (0.05$) or thiouracil (0.05$). Twenty f i s h were removed from each treatment group and from a control group at intervals of 4, 8, and 16 days after the beginning of the treatment. The length, weight, and sex of each f i s h was recorded and they were divided into male and female samples. After maceration i n a meat grinder, the f i s h were pulped i n a Waring Blender. Duplicate moisture determinations were made on samples of the pulped tissue, using a Cenco Moisture Balance. The tissue mash was mixed with anhydrous sodium sulfate and the l i p i d s extracted with anhydrous peroxide-free ether, using the Waring Blender. Each sample was extracted three times. Duplicate cholesterol determinations were carried out using the method of Sperry and Webb (1950) with the modifications suggested by Hollands (1956). Cholesterol values were expressed as per cent of the tota l l i p i d i n the ether extract. Total l i p i d s were determined i n t r i p l i c a t e by evaporating 4 ml aliquots of the ether extract to dryness i n a vacuum oven and then weighing to constant weight. 9 RESULTS A. Temperature Resistance 1. Heat Resistance Seasonal changes i n the effect of the length of the photoperiod on heat resistance are shown i n Figure 1. At the 50$ mortality l e v e l , the 16-hour f i s h showed a consistently greater resistance to high temperature. However, the magnitude of the difference i n the resistance between the two groups appears to vary seasonally, being greater i n f a l l and winter than i n summer, with the exception of the group tested i n February. 2. Cold Resistance Figure 2 shows the seasonal variation i n the effects of controlled photoperiod on the cold resistance. On seven occasions the 8-hour f i s h were more resistant to cold and on fi v e occasions the 16-hour groups were more resistant. In general, however, the 8-hour f i s h tested i n winter were considerably more resistant to cold than the 16-hour groups, but i n the summer the situation was reversed. Two exceptions to this were noted; the groups tested i n February, and i n early October, 1957. 3. V a r i a b i l i t y i n Samples as Possible Causes of Irregularities  i n Effect of Photoperiod Since size and sex as well as season have been shown to influence the temperature resistance of the goldfish (Hoar, 1955) these factors were considered as possible causes of these i r r e g u l a r i t i e s i n the thermal resistance tests. I t has been shown (Irvine, 1954; Hoar, 1955; Hollands, 1956) that larger f i s h are more resistant to temperature than are smaller ones. Tables I and I I show the mean weight for each group of f i s h used i n the temperature Figure 1. EFFECT OF PHOTOPERIOD ON HEAT RESISTANCE OF GOLDFISH. Heat resistance measured as difference i n survival time between 8-hour and 16-hour f i s h , values above line indicating longer survival by 16-hour f i s h . Three bars i n each series are 25%, 50% (black) and 75% mortality levels. X - level of mortality not achieved by more resistant group. Figure 2. EFFECT OF PHOTOPERIOD ON COLD RESISTANCE OF GOLDFISH. Cold resistance measured as difference i n survival time between 8-hour and 16-hour f i s h , values above the line indicating longer survival by 8-hour f i s h . Three bars i n each series are 25$, 50$ (black) and 75$ mortality levels. X - level of mortality not achieved by more resistant group. TABLE I Mean, weight i n grams of goldfish used i n temperature resistance tests. Cold Test Heat Test 8 Hour 16 Hour 8 Hour 16 Hour Date Mean Weight Standard Deviation Mean Weight Standard Deviation Mean Weight Standard Deviation Mean Weight Standard Deviatic Nov. 29 17.7 3.69 17.2 4.09 14.6 4.96 15.4 3.83 Dec. 13 17.3 4.35 17.9 4.23 15.5 3.36 15.6 2.18 Feb. 18 15.5 4.00 16.7 4.78 16.3 4.77 15.9 4.06 March 25 17.4 5.54 19.3 5.27 16.8 2.02 18.1 4.25 Ap r i l 4 16.5 4.85 17.7 3.83 May 16 16.6 3.90 17.5 3.58 May 23 15.6 4.27 16.4 3.54 18.7 3.36 17.4 4.65 July 5 16.7 3.99 16.0 4.11 15.8* 2.78 18.4* 4.64 Julyl9-23 16.0 3.60 15.2 3.36 15.2 2.86 16.5 3.36 Oct. 4 7.6 2.60 8.1 2.66 7.6 2.57 8.9 2.59 Oct. 18-22 10.3 2.85 10.0 3.87 9.1 2.69 9.5 3.02 Nov. 1 13.6 4.73 10.7 2.46 Entire sample not weighed. TABLE I I Mean weight i n grams of hormone treated f i s h used i n temperature resistance tests. Cold Test Heat Test 8 Hour 16 Hour 8 Hour 16 Hour Date and Mean Standard Mean Standard Mean Standard Mean Standard Treatment Weight Deviation Weight Deviation Weight Deviation Weight Deviation July Thyroxine 16.2 3.95 17.9 3.87 14.1 2.08 15.6 3.48 Thiourea 15.2 4.25 15.3 3.38 14.9 4.24 14.9 4.28 Control 16.0 3.60 15.2 3.36 15.2 2.86 16.5 3.36 October Thyroxine 9.6 2.45 9.4 2.28 8.0 3.08 9.4 3.19 Thiourea 9.2 2.38 9.1 1.57 9.0 2.97 8.3 2.04 Thiouracil 9.5 2.70 10.7 2.76 Control 10.3 2.85 10.0 3.87 9.1 2.69 9.5 3.02 November TSH 12.7 3.49 14.15 4.22 Control 13.6 4.73 10.7 2.46 resistance tests. There seems to be no evident correlation between the ir r e g u l a r i t i e s i n the temperature resistance and the weight differences between the 8-hour and 16-hour groups. In general, the sizes of the 8-hour and 16-hour f i s h were of the same order. The f i s h i n the l a s t shipment (tested i n October and November, 1957) were much smaller than those used i n the previous tests. Irvine (1954) f i r s t showed that male f i s h were more resistant to cold than female f i s h maintained and tested under the same conditions. In the heat tests the females showed a greater tendency to survive than the males (based on the sex rat i o of the survivors) but the females tended to be more quickly narcotized (Hollands, 1956). The ratio of males to females i n the 8-hour and 16-hour groups i s shown i n Table I I I . There i s considerable variation i n the ra t i o s , but no correlation can be seen between differences i n the ratios and the i r r e g u l a r i t i e s noted i n the heat and cold tests. 4. Ratio of Narcotized to "Truly dead" Pish There i s also a p o s s i b i l i t y that the tendency of the f i s h to become narcotized i s greater at certain seasons than at others and that the "removal" data may have different meanings at different seasons. The rati o of narcotized ( i . e . those which recovered when placed i n water at room temperature after removal from the heat or cold test) to "truly dead" f i s h i s shown i n Table IV. In most of the experiments the majority of the f i s h were narcotized. In the experiments where the 16-hour f i s h were found to be more resistant to cold than the 8-hour groups (February, May, early July, and early October) the 8-hour groups had a much higher rati o of narcotized to "truly dead" f i s h than was found i n the 16-hour groups. This indicates that the 8-hour f i s h had a greater tendency to TABLE I I I Ratio of males to females i n groups'of f i s h used i n temperature tolerance tests. Cold Test Heat Test 8 Hour Ratio 16 Hour Ratio 8 Hour Ratio 16 Hour Ratio Males Females M/F Males Females M/F Males Females M/F Males Females M/F Nov. 29 9 12 .75 10 11 .91 8 13 .62 14 7 2.00 Dec. 13 4 16 .25 10 13 .77 5 15 .33 14 7 2.00 Feb. 18 12 8 1.50 9 10 .90 11 9 1.22 13 7 1.86 Mar. 25 10 10 1.00 7 13 .54 7 13 .54 9 11 .82 A p r i l 4 6 14 .43 9 11 .82 May 16 9 9 1.00 12 8 1.50 May 23 9 11 .82 7 13 .54 10 10 1.00 9 11 .82 July 5 7* 12 .58 8 12 .67 9* 7 1.28 3* 6 .50 July 19-23 13 7 1.86 10 10 1.00 14 5 2.80 8 12 .67 Oct. 4 10 10 1.00 9 11 .82 10 10 1.00 11 9 1.22 Oct. 18-22 14 8 1.75 7 13 .54 11 10 1.10 7* 12 .58 Nov. 1 9 5 1.80 5 7 .71 * Not a l l determined. TABLE IV Ratio of narcotized f i s h to "truly dead" f i s h Cold Test 8 Hour 16 Hour Narco-tized Dead Ratio N/D Narco-tized Dead Ratio N/D Nov. 29 7 12 .58 11 10 1.10 Dec. 13 14 6 2.33 12 11 1.09 Feb. 18 16 3 5.33 9 6 1.50 Mar. 25 5 8 .62 9 10 .90 Apr i l 4 11 4 2.75 13 5 2.60 May 16 17 1 17.0 14 4 3.50 May 23 14 4 3.50 13 5 2.60 July 5 17 3 5.67 15 5 3.00 July 19-23 6 11 .54 9 11 .82 Oct. 4 15 5 3.00 11 7 1.57 Oct. 18-22 3 6 .50 7 8 .88 Nov. 1 5 8 .62 7 4 1.75 * Not a l l determined. temperature tolerance tests. Heat Test 8 Hour 16 Hour Narco- Ratio Narco- Ratio tized Dead N/D tized Dead N/D 15 3 5.00 8 10 .80 12 8 1.50 17 4 4.25 16 4 4.00 15 5 3.00 14 6 2.33 9 9 1.00 3 16 .19 7 13 .54 10 7 1.43 7* 4 1.75 10 9 1.11 8 12 .67 11 6 1.83 5 14 .36 16 5 3.20 14 2 4.67 15 enter cold narcosis during these months. One factor which would influence the number of narcotized fish i s the time interval at which the fish were checked. This i s particularly important in the case of the cold tests where the animals were checked every hour for the f i r s t part of the test, and then at considerably longer intervals. In this case animals which became narcotized near the beginning of the interval between checks might be dead by the time the next check was made. B. Thyroid Activity An attempt was made to determine the effect of the length of the photoperiod on the thyroid activity, and seasonal variations in the effect of the photoperiod. Typical uptake curves are shown i n Figure 5. The uptake of reached a maximum within the f i r s t 24 hours, then f e l l slightly, and i n most cases rose again at the end of the test period (48 or 96 hours). Other workers in this laboratory have found the same -type of uptake curve in f l a t f i s h and salmon (Hickman and Baggerman, unpublished data). It i s possible that this may indicate a different mechanism in fishes than that occurring in mammals where the uptake curve reaches a maximum after 24 hours and then fa l l s gradually. 131 Table V shows the mean I uptake for the males and the females at 6, 12, 24, and 48 hours after the injection of radioiodine. The values presented are based on a l l the experiments carried out (8 tests). The same results are shown in graphical form in Figure 3. As indicated by the magnitude of the standard deviation, there was extreme variability 131 i n the uptake of I . When the mean values were compared by a t-test the Figure 3. PERCENT UPTAKE OF RADIOIODINE BY 8 AND 16 HOUR GOLDFISH. Values are mean figures for eight determinations made at about 6 week intervals. 12 2 4 3 6 H O U R S TABLE V Percentage uptake of radioiodine by goldfish thyroid. Sex Hours after Short-day f i s h Long-day f i s h injection 131 131 Number of Mean I Standard Number of Mean I Standard f i s h uptake Deviation f i s h uptake Deviation 6 8 2.63 0.77 • 8 2.14 1.30 12 8 2.88 0.97 10 2.50 1.14 Male 24 17 2.02 1.23 12 1.77 0.80 48 23 2.39 1.36 20 2.11 0.98 Female 6 12 24 48 16 16 15 16 3.19 3.00 3.48 2.70 0.93 1.34 2.20 2.06 16 14 19 20 3.30 2.69 2.57 2.56 1.52 2.33 1.52 2.88 a* 17 differences between the uptake i n the 8-hour and 16-hour groups were not statistically significant. However, the small average differences are consistently in one direction and i n comparing the uptake curves for the two groups used in each experiment, the 8-hour fi s h regularly showed a higher uptake. The maximum iodine uptake occurring within the f i r s t 24 hours after 131 injection of I i s shown in Table VI. In this case values for the males and females were combined. No significant seasonal changes are evident. There are several possible explanations for the extreme variability found in the thyroid activity of the goldfish. In particular, Chavin (1956a,b) described normal thyroid f o l l i c l e s occurring in the head kidney of the goldfish, and found that i n 47% of the fish which he tested the 131 uptake of I was greater in the head kidney than in the throat thyroid. Only the throat thyroid was considered in this series of experiments. C. Effects of Thyroxine, Thyroid Inhibitors, and TSH 1. Temperature Resistance a. Heat Resistance The effects of thyroxine and thiourea on the heat resistance of photoperiodically adapted fish are shown in Table VII. In the summer experiment thyroxine had l i t t l e effect on the heat resistance of the 8-hour day fish, while in the f a l l i t caused an increased resistance. The heat resistance of the 16-hour day fish was decreased by treatment with thyroxine in both the summer and f a l l tests. Treatment with thiourea caused decreased resistance to heat in both the 8-hour and TABLE VI Date Maximum radioiodine uptake within f i r s t 24 hours expressed as percent of injected dose. Short-day f i s h Long-day f i s h Dec. 1 Dec. 14 Feb. 18 Mar. 26 Apr. 9 May 29 June 21 Oct. 8 Hours after injection of j.131 6 12 12 6 12 6 24 12 Mean I uptake 3.25 3.05 3.15 3.23 3.84 2.99 4.42 3.85 131 Hours after injection of j.131 6 6 24 6 24 12 6 6 Mean I uptake 3.41 2.44 2.49 2.79 3.09 3.08 2.44 5.51 131 00 TABLE VII Heat resistance measured as minutes difference from the control group required to k i l l different percentages of samples of 20 goldfish after 77 days controlled photoperiod. Thyroxine Thiourea 8 Hour 16 Hour 8 Hour 16 Hour 25$ -14 +2 -22 -11 July 19 50$ 0 -13 -28 -23 75$ 0 -19 -28 -29 Oct. 18 25$ 50$ 75$ +22.9 +20.6 +11.0 -22.5 -20.0 -17.5 -1,4 -14.1 -8.5 -17.0 -9.0 -15.5 VO 20 16-hour groups i n the summer and i n the f a l l * b. Cold Resistance Experiments were also carried out to determine the effects of thyroxine, thiourea, thiouracil and thyrotropic hormone on the cold resistance of goldfish. The results are shown i n Figure 4. In the summer test, thyroxine decreased the cold resistance of the 8-hour day group at the 25% and 50% mortality levels, and increased the cold resistance at the 75% mortality l e v e l . In the f a l l experiment thyroxine caused a considerable increase i n the cold resistance of the 8-hour group, less than 25% of the animals being k i l l e d after 72 hours i n the cold test. In both the summer and f a l l tests treatment with thyroxine resulted i n increased cold resistance i n the 16-hour day groups. Injection of TSH resulted i n increased cold resistance i n the 16-hour group. The f i s h of the 8-hour groups died more rapidly than the controls up to the 50% mortality l e v e l , but at the 75% mortality level showed more resistance than the controls. Thiourea caused decreased cold resistance i n the 8-hour day f i s h i n both summer and f a l l . On the other hand, the 16-hour day f i s h showed consistently increased resistance when treated with thiourea. Treatment with thiouracil resulted i n decreased resistance i n both the 8-hour and 16-hour groups. 2. Thyroid Act i v i t y 131 The uptake of I was determined i n f i s h which had previously been treated with thyroxine or thiourea. In line with the work of others (Chavin 1956a), both substances were found to cause a decrease i n thyroid a c t i v i t y , a s l i g h t l y greater effect being seen i n the case of the thyroxine treated f i s h (Figure 5). Figure 4 . EFFECT OF THYROXINE, TSH, THIOUREA OR THIOURACIL ON COLD RESISTANCE OF GOLDFISH. Cold resistance measured as difference i n survival time between treated f i s h and controls maintained under the same photoperiod. Values above line indicate increased resistance by-treated f i s h . Three bars i n each series are 25$, 50$ (black) and 75$ mortality levels. X - level of mortality not achieved by more resistant group. Figure 5. EFFECT OF THYROXINE OR THIOUREA ON PERCENT UPTAKE OF RADIOIODINE BY GOLDFISH. Control Thiourea Thyroxine H O U R S 21 3. Cholesterol Cholesterol analysis was carried out on f i s h which had been treated with thyroxine, thiourea, or thiou r a c i l . The results are shown i n Table VIII; the mean weights of the f i s h are shown i n Appendix VI. In the f i r s t experiment (June) treatment with thyroxine resulted i n a decrease i n tissue cholesterol i n the female f i s h . The male f i s h also showed a decrease i n cholesterol after 4 days treatment, but an increase after 8 and 16 days of treatment. In experiments B and C (August and November) "the cholesterol was increased after thyroxine treatment i n both the female and male f i s h . The effect of thiourea was extremely variable. In experiment A the cholesterol level i n the females increased after 4 and 8 days treatment, but was lower than i n the controls after 16 days treatment. The males showed a lower cholesterol level than the controls after 4 days treatment, but a higher value than the controls after 8 and 16 days treatment. In the second experiment the females showed a decrease i n tissue cholesterol after thiourea treatment while the males showed an increase i n cholesterol after 4 and 8 days treatment and a decrease after 16 days. The thiouracil treated females (tested i n November) showed an increase i n tissue cholesterol, while the males showed an increase after 7 days treatment and a decrease after 14 days treatment. 4. Moisture Changes Moisture determinations were made on samples of the macerated tissue from the groups used for cholesterol analysis. No significant changes i n the moisture content of the hormone treated f i s h were noted (Table IX). TABLE T i l l Effect of thyroid materials on tissue cholesterol expressed as °/o difference from control value. Control expressed as percent cholesterol i n total l i p i d . Experiment November Days of treatment Females fo change from control Males fo change from control Control Thyroxine Thiourea Thiouracil Control Thyroxine Thiourea Thiouracil A 4 4.56 -21.05 +8.55 5.56 -26.44 -29.67 June 8 3.39 -35.40 +24.48 2.68 +16.42 +88.06 16 3.75 -38.18 -14.67 3.09 +26.21 +47.90 B 4 13.11 -49.88 13.73 +19.74 +13.69 August 8 14.87 -11.84 12.33 +23.68 +23.11 16 7.76 -11.98 9.79 +6.13 -9.91 C 7 8.35 +36.53 9.86 +20.18 +21.91 14 8.45 +81.42 14.73 +2.85 -50.10 S3 TABLE IX Effect of thyroid materials on percent moisture content of goldfish. Females Males Days of Experiment treatment Control Thyroxine Thiourea Thiouracil Control Thyroxine Thiourea Thiours A 4 75.1 . 76.2 77.1 75.8 77.7 75.2 June 8 74.0 78.6 78.0 76.3 74.1 76.6 16 74.0 . 77.9 75.8 76.3 76.4 76.0 B 4 79.6. 80.4 , 79.0 80.5 79.6 80.5 August 8 79.9 80.2 80.4 79.6 80.2 79.7 16 77.9 78.1 78.5 78.5 78.3 78.4 C 7 78.6 78.2 79.2 79.7 78.8 78.5 November 14 78.7 78.3 78.6 77.5 78.6 78.3 to 24 D. Gonad Weight - Body Weight Relationships Fish which were used for the temperature resistance tests were preserved i n formalin, and a study was made of the relationships of the gonad weights to the body weights. The mean ratio of the gonad weight (mgm.) to body weight (gm.) i s shown i n Figures 6 and 7 for groups tested at various intervals from February to November. Mean body weights, gonad weights, ratios of gonad weight/body weight, and sample sizes are shown i n Appendices VII and VIII. In the spring (February, March, and April) the longer photoperiod (16 hours of light) resulted i n accelerated gonadal development i n the females i n the 16-hour groups as compared with those i n the 8-hour groups. In A p r i l the ratio i s more than twice as great f o r the 16-hour f i s h , indicating a marked stimulation of ovary growth i n "this group. In the f i s h tested from May u n t i l November the ratio of gonad weight to body weight was s l i g h t l y higher i n the 8-hour group although i n view of the mixed size (age) of samples and the v a r i a b i l i t y i n the ratio i t i s doubtful whether significance can be attached to these differences. Much less variation was noted i n the gonad weight/body weight ratios of the male f i s h . This could be largely due to the fact that the male gonad does not show the large relative increase i n weight at maturity. Figure 6. GONAD WEIGHT-BODY WEIGHT RELATIONSHIPS IN FEMALE GOLDFISH MAINTAINED UNDER 8-HOUR OR 16-HOUR PHOTOPERIODS. Solid bars - 8-hour f i s h Open bars - 16-hour f i s h Figure 7. GONAD WEIGHT-BODY WEIGHT RELATIONSHIPS IN MALE GOLDFISH MAINTAINED UNDER 8-HOUR OR 16-HOUR PHOTOPERIODS. Solid bars - 8-hour f i s h Open baxs - 16-hour f i s h F E B . M A R . A P R . M A Y M A Y J U L Y O C T . N O V . 25 DISCUSSION Changes i n Temperature Resistance I t can be seen from Figures 1 and 2 that manipulation of the photoperiod modifies both the heat and cold resistance of goldfish. In general, the data confirm earlier findings i n this laboratory showing increased heat resistance with the longer photoperiod and increased cold resistance with the shorter photoperiod, at least when the experiments are performed during the f a l l and winter (Hoar, 1956; Hollands, 1956). Seasonal changes i n the temperature resistance have been shown previously (Brett, 1946) but these had been attributed to acclimation to changing environmental temperatures. I t i s possible that the photoperiod-induced changes i n temperature resistance are important i n preparing f i s h for sudden temperature changes i n the spring and f a l l . This would enable the animals to survive sudden temperature fluctuations which may occur too rapidly for acclimation to take place. The effect of the length of the photoperiod on temperature resistance appears to vary seasonally. Fish subjected to 16 hours of l i g h t per day were more resistant to heat than those subjected to 8 hours, but the magnitude of the difference i n the heat resistance was greater i n winter than i n summer, except i n the group tested i n February. The cold resistance showed an even greater seasonal variation. In fact data presented i n Figure 2 leave some doubt as to the r e a l i t y of the postulated effect. In the winter months, the 8-hour f i s h were much more resistant to cold than the 16-hour groups, but i n the summer (May and the early part of July) the 16-hour f i s h were more resistant. Moreover, 26 there were two marked exceptions - the groups tested i n February and early October, 1957. These data can best be considered together with those obtained during the previous year (Hoar, 1956; Hollands, 1956). In this case again fish tested in November and December showed a consistent relationship between photoperiod and cold resistance while no relationship, or even a reversal, was found in fish tested during the spring. Thus, i t i s indicated that the effect i s easily produced i n f a l l and winter, but other factors appear to alter this effect in the spring. Irvine (1954) showed that male fish were more resistant to cold than female fish maintained and tested under the same conditions, while the female fish were more resistant to heat. The larger fish were shown to be more resistant to temperature than "the smaller ones. The mean weights and the male to female ratios of the groups of fish used in these experiments were considered as possible sources of some of the variability. The mean weights of the 16-hour and 8-hour groups were very similar (Tables I and II) and there i s no apparent correlation between the seasonal variations in the temperature resistance and weight differences between the two groups. Although the sex ratios varied considerably, differences between the 8-hour and 16-hour groups could not be correlated with variation i n the temperature resistance (Table III). Differences in the mean weights or the sex ratios of the 8-hour and 16-hour groups were also considered as possible explanations for the inconsistencies found in the temperature resistance tests. In the groups tested for heat resistance in February, 1957 the 16-hour fish were found to be more resistant than the 8-hour fish, but the magnitude of the difference was much smaller than in the other winter months. This could 27 not be explained on the basis of differences i n the weights or sex ratios of the two groups. Two exceptions were found to the apparent seasonal variation i n cold resistance. In the group tested i n February, the 16-hour f i s h were more resistant to cold than the 8-hour f i s h . The mean weight of the 16-hour group was greater than that of the 8-hour group, but the 8-hour f i s h had a higher ratio of males to females. Thus the 16-hour group had a slight advantage with respect to weight, but the 8-hour group had the advantage as regards the relative numbers of males and females. I t may be significant that i n this test the 8-hour group showed a much higher ratio of narcotized to "truly dead" f i s h than did the 16-hour group. The 8-hour f i s h were being removed while s t i l l narcotized and this would reduce their apparent cold resistance. I t may also be of significance that there were r e l a t i v e l y more narcotized f i s h of both groups during the summer and that the rati o (Table IV) i s always greater i n the 8-hour f i s h . This may, at least partly, account for the apparent decreased resistance of this group during the summer. I t i s impossible to attach much significance to these data because of the d i f f i c u l t y of separating the narcotized from the dead f i s h unless the test i s watched continuously for periods as long as two days. A possible explanation for the irregularity found i n the cold test i n early October, 1957, l i e s i n the fact that the f i s h used i n this test were from a new shipment of animals and were much smaller and evidently i n poorer condition than those used i n a l l the previous tests. Since the seasonal variation i n the temperature resistance could not be s a t i s f a c t o r i l y explained on the basis of biased samples, i t appears l i k e l y that i t i s related to seasonal changes i n the physiology of the goldfish. These w i l l now be examined. 28 Thyroid A c t i v i t y Various authors have noted seasonal changes occurring i n the thyroid a c t i v i t y of cold-blooded vertebrates. Carter (1933) described the winter condition of the frog as one i n which the amount of thyroid hormone i n the circulation was decreased. Hoar (1939) described seasonal changes i n the histology of the thyroid i n Atlantic salmon. The thyroid a c t i v i t y was increased i n the spring and summer, and decreased i n the f a l l and winter. The effect of increased and decreased l i g h t on the thyroid gland of the salamander was studied by Stein and Carpenter (1943). They concluded that probably both l i g h t and temperature were involved i n the control of the natural seasonal cycle of thyroid a c t i v i t y . The suggestion was made that the effect of light may be an indirect influence by way of the p i t u i t a r y , and may involve both the thyroid gland and the gonads. 131 Because of the extreme v a r i a b i l i t y i n the uptake of I i n the goldfish, i t was not possible to show s t a t i s t i c a l l y significant differences between the thyroid a c t i v i t y of the 8-hour and the 16-hour f i s h . However, i n comparing the results from each experiment (20 f i s h from each photoperiod treatment) i t was found that the 8-hour f i s h nearly always showed a 131 s l i g h t l y higher uptake of I . N o seasonal variation i n the effect of the length of the photoperiod on thyroid a c t i v i t y was observed. There are several possible explanations for the extreme v a r i a b i l i t y 131 i n the I uptake. Two d i f f i c u l t i e s were encountered i n the technique. The thyroid gland i n f i s h i s very diffuse, with scattered thyroid f o l l i c l e s located around the ventral aorta i n the lower jaw. I t i s therefore impossible to remove the entire gland without taking variable amounts of the surrounding tissue. Because the digested samples contained much non-thyroid tissue, 29 131 there may have been considerable self-absorption of I when the counts were being made. The amount of sif-absorption varies with the amount of tissue present, and w i l l probably be greater i n the larger f i s h . Chavin (1956-a and b) has described functional thyroid f o l l i c l e s occurring i n the lymphoids! pronephric remnants, the head kidneys. He found that the relative a c t i v i t y of the throat thyroid and the head kidney thyroid was variable. The head kidney thyroid was actually more active than the throat thyroid i n 47% of the f i s h . The throat thyroid was more active i n 23%, and the two showed approximately equal a c t i v i t y i n 20" of the f i s h . In 10% of the f i s h studied, the head kidneys, accumulated no radioiodine. F o l l i c u l a r counts revealed no correlation between the 131 number of f o l l i c l e s and the I uptake. Since only "the throat thyroid was considered i n our experiments, t h i s w i l l certainly account for some of the v a r i a b i l i t y found. Other workers have also found low thyroid a c t i v i t y i n the goldfish. Most freshwater fishes, which experience a r e l a t i v e l y low iodine concentration i n the normal environment, accumulate up to 30% of an injected dose of I 1 3 1 , compared to 2-3% i n marine fishes. The goldfish, however, appears to be an exception. Berg and Gorbman (1954) found that the maximum uptake 131 of I i n the goldfish was 3%, and this occurred 12-24 hours after the 131 injection of the tracer dose. Chavin (1956a) reported that the I uptake of the throat thyroid reached a mean maximum value of approximately 3% 131 within 1 hour after injection of Nal . In the head kidney a maximum of 5% was reached after 24 hours. Fortune (1956) likewise considers the goldfish thyroid to be r e l a t i v e l y inactive. In normal goldfish thyroids, the radioiodine i s found mostly i n 30 the form of monoiodotyrosine and diiodotyrosine, with l i t t l e , i f any, thyroxine formed even after one week (Berg and Gorbman, 1954). Goldfish then are poor thyroxine producers, and are very sluggish in their response to iodine levels in the water. Since TSH increased "the proportion of radiothyroxine, these writers suggested that the low efficiency of the thyroid gland may be at least partially due to lack of pituitary thyrotropin. Pickford (1957) states that exposure to low temperatures has no striking effect on the thyroid metabolism of f i s h . Fish are able to maintain a relatively constant level of thyroid function irrespective of the external temperature, and this must be mediated through a regulation of thyrotropic secretion. It i s suggested (Pickford, 1957) that in order to maintain a constant level of thyroid function, the release of thyrotropin must be diminished at higher temperatures, since even small amounts would have a much greater effect than at lower temperatures. The regulation of thyroid function can be correlated with the ability of the cold-blooded vertebrates to maintain a relatively constant metabolic level over a wide range of environmental temperatures. From the results of various authors i t seems probable that illumination, mediated through the pineal organ, exerts an inhibitory action on the release of thyrotropin i n fish. This inhibition can be removed by pinealectomy or by keeping the fish in total darkness (Pickford, 1957). Effects of thyroid treatment It has been shown previously (Hoar, 1954) that in the summer, fish treated with thyroxine showed an increased resistance to cold. Thiourea was shown to exert an even greater protective effect. However, fish tested 31 in winter failed to show increased resistance when treated with these drugs. Pickford (1957) cites further work on the effect of -thyroid activity on temperature resistance. For example, i t has been shown (LaRoche and Leblond, 1954) that although radiation thyroidectomy impaired the ability of salmondid parr to withstand rising temperature, the opposite effect occurred with antithyroid drugs. Suhrmann (1955) found that thiourea promoted the resistance of cold-adapted goldfish to high temperatures. In our experiments thyroxine was found to cause a decrease in the heat resistance of 16-hour fish tested in both the summer and f a l l . This was accompanied by an increase in the cold resistance. This i s in agreement with the previous work reported by Hoar (1954). Thus, treatment with •thyroxine appears to have made the "summer" fish (i.e. those subjected to the 16-hour photoperiod) more like the "winter" fish . This seems to be in agreement with the evidence that the 8-hour fish were characterized by a higher level of thyroid activity. However, i t must be taken into consideration that the photoperiod fish tested in summer were tested during the season in which the 8-hour fish showed a lower resistance to cold than did the 16-hour fish. In the 8-hour fish tested in the summer, thyroxine had l i t t l e effect on the heat resistance. There was a decrease in the cold resistance at the 25% and 50% mortality levels, and an increase at the 75% mortality level. The 8-hour fish showed an increased resistance to both heat and cold when tested in the f a l l . Treatment with thiourea resulted in decreased heat resistance and increased cold resistance i n the 16-hour fish, the same effect as was observed after treatment with thyroxine. The 8-hour fish showed decreased 32 resistance to both heat and cold after treatment with thiourea. Treatment with thiouracil resulted in decreased cold resistance in both the 8-hour and 16-hour groups tested in the f a l l . TSH caused increased resistance to cold in the 16-hour group, and in the 8-hour group above 25$ mortality level. Thus i t appears that thyroxine and TSH can cause increased resistance to cold in both the 16-hour and 8-hour groups although, as might be expected, the effect is more marked in the 16-hour group. Thiouracil reversed this effect, but thiourea exerted a protective effect in the 16-hour groups. The protective effect of thiourea has been noted previously with "natural" summer fish (Hoar, 1956). It i s possible that thiourea in this case i s not acting as a thyroid inhibitor but producing some quite different physiological or pharmacological effect. Thyroid activity is known to influence fat metabolism in mammals and the same is probably true in fish. Hollands (1956) showed that short-day goldfish generally showed a higher tissue cholesterol than the long-day fish, although no correlation was found between the thermal resistance and the biochemical constituents of the tissues. Irvine (1957) showed that dietary supplements of cholesterol increased the heat and cold resistance of goldfish. In the present study i t was found that the short-day fish generally had a higher thyroid activity than the long-day fi s h , and that thyroxine increased the cold resistance. It therefore seems possible that increased thyroxine in the winter fish may increase cold resistance by causing changes in the fat metabolism. However, on the basis of the evidence available at present i t is not possible to come to any definite conclusions as to the relationships between the length of the photoperiod, 33 thyroid a c t i v i t y , and l i p i d metabolism. In the cholesterol studies reported here, thyroxine-treated f i s h usually had higher tissue cholesterol after 16 days of treatment (Table VII) but the results were so variable that no great significance can be attached to them. Sexual Development The effect of the length of the photoperiod on the breeding cycle i n many animals i s well established (Bullough, 1951; Marshall, 1956). The effect of l i g h t may be an indirect influence by way of the pituitary and involving both the thyroid gland and the gonads (Stein and Carpenter, 1943). Since i t was f e l t that the temperature resistance of photoperiodically acclimated f i s h might be related to changes i n the endocrine a c t i v i t y of the gonads as well as of the thyroid gland, the gonad weight-body weight relationships of 8-hour and 16-hour f i s h were compared. Although the ratio of gonad weight to body weight i n the females was higher i n the 16-hour f i s h than i n the 8-hour f i s h i n February, March and A p r i l , these differences could not be correlated with changes i n the heat and cold resistance. In later experiments (May to November) the 8-hour f i s h showed a s l i g h t l y greater degree of gonadal development. The f i s h used i n the July, October, and November experiments were from different shipments than the f i s h used i n the previous experiments and were much smaller than the previous groups and appeared to be immature. The effect of the day length up to the time at which the photoperiod treatment began must also be considered, as the endocrine mechanisms controlling gonadal development may already have been set into motion at the time that the f i s h were placed under the controlled light conditions. Furthermore, f i s h which were placed under the long photoperiod i n the winter were subjected to a greater change i n day length than those placed under the shorter day length, and the reverse was true in the summer. On the whole, data presented in Figure suggest that the longer photoperiod produced a marked gonad stimulation in the four month period prior to spawning, but that the fish were refractory to such stimulation at other seasons. This type of seasonal variation in response to controlled photoperiod has been observed by several investigators (eg. Harrington, 1957). SUMMARY Photoperiod control was found to modify the heat and cold resistance of the goldfish. This effect appeared to vary seasonally. Fish exposed to 16 hours of l i g h t per day were more resistant to heat than those receiving 8 hours of l i g h t , but the difference between the two groups was more pronounced i n the winter than i n the summer. During the winter the short-day f i s h were more resistant to cold than the long-day f i s h , but the relationship appears to be reversed i n the summer. Biased samples were considered as a possible reason for the v a r i a b i l i t y . I t i s more l i k e l y that the seasonal variations i n the temperature resistance are related to changes i n the physiology and a variable tendency to enter narcosis. Thyroid uptake of radioiodine was measured i n f i s h which had been exposed to short day length or long day length. The results suggested a higher uptake of radioiodine by the short-day f i s h but no definite correlation with temperature resistance was possible. Treatment of the photoperiodically adapted f i s h with thyroxine, TSH, thiourea, or thiouracil prior to testing thermal resistance gave extremely variable results. However, i t appears that thyroxine and TSH increase cold resistance, particularly i n the less resistant 16-hour f i s h , and that thiourea causes the reverse effect. Thiourea exerted a protective action on the 16-hour f i s h . Fish which had been treated with thyroid materials were analyzed for tissue cholesterol to determine whether the increased cold resistance i n the thyroxine treated f i s h could be related to f a t metabolism. The data 36 were inconsistent and no conclusions could be drawn. In comparing the gonad weight/body weight relationships of the short-day and the long-day fish, no definite correlation could be found between the state of sexual maturity and the thermal resistance. In the late winter and early spring the longer photoperiod stimulated gonad development. APPENDIX I I n i t i a l temperatures and temperature ranges of lethal temperature baths. Pish used from Nov. 29, 1956 to July 5, 1957 obtained i n October, 1956; those used on July 19-23 obtained i n A p r i l , 1957 and those used from Oct. 4, 1957 to Nov. 1, 1957 obtained i n July, 1957. Date Days under Heat Test Cold Test controlled photoperiod I n i t i a l Temperature Degrees Centigrade Range I n i t i a l Temperature Degrees Centigrade Range Nov. 29 40 34.7 34.7 - 38.0 3.8 0.9 - 3.8 Dec. 13 41 35.8 35.8 - 37.8 2.5 0.4 - 2.5 Feb. 18 42 35.8 35.6 - 36.9 3.3 0.8 - 3.3 Mar. 25 35 36.4 36.0 - 37.0 5.8 0.3 - 5.8 Ap r i l 4 42 3.5 0.8 - 3.5 May 16 75 2.1 0.3 - 2.1 May 23 42 36.4 36.4 - 36.6 2.0 0.3 - 2.0 July 5 49 36.0 35.5 - 37.5 3.2 0.8 - 3.2 July 19-23 77 35.0 35.0 - 37.6 3-5 0.5 - 3.5 Oct. 4 65 35.6 35.6 - 37.2 3.1 0.2 - 3.1 Oct. 18-22 77 - 35.5 35.4 - 36.0 3.2 0.3 - 3.2 Nov. 1 90 3.3 0.3 - 3.3 APPENDIX II Time (Hours) required for 25%, 50%, and 75% mortality i n Cold Tests. 8 Hour 16 Hour Date 25% 50% 75% 25% 50% 75% Nov. 29, 1956 17.5 20.8 23.5 15.0 18.5 21.5 Dec. 13, 1956 22.5 28.3 * 6.1 8.6 22.0 Feb. 18, 1957 6.3 10.2 14.3 8.1 14.3 22.4 Mar. 25, 1957 28.5 49.0 * 27.0 29.0 39.5 Apr. 4 , 1957 18.6 24.0 30.8 9.5 18.7 25.5 May 16, 1957 .7 3.7 7.1 2.2 8.3 24.0 May 23, 1957 4.4 12.2 25.3 6.2 18.2 32.0 July 5, 1957 7.8 10.4 13.8 7.8 13.0 * July 19, 1957 41.0 61.0 69.0 17.0 24.8 35.0 Oct. 4 , 1957 4.6 6.8 11.8 6.4 10.0 15,4 Oct. 18, 1957 54.0 82.0 * 23.0 48.0 * Nov. 1, 1957 15.0 47.5 62.0 3.2' 8.6 22.5 00 \ APPENDIX I I I Time (Minutes) required for 25%, 50%, and 75% mortality i n Heat Tests. Date 8 Hour 16 Hour 25% 50% 75% 25% 50% 75% Nov. 29, 1956 88 123 160 92 143 170 Dec. 13, 1956 17 29 35 35 49 72 Feb. 18, 1957 50 61 70 46 63 74 Mar. 25, 1957 21.5 30 44 44 66 90 May 23, 1957 15.5 25 33 26 33.5 46 July 5, 1957 50 68 92 54 80 * July 22, 1957 52 61 73 49 71 88 Oct. 4, 1957 56 81 126 53 91 148 Oct. 22, 1957 4.5 27.5 42 33 56 73 * Level of mortality not reached. so APPENDIX IV Time (Hours) required for 25$, 50$, and 75$ mortality i n Cold Tests. Date Treatment 8 Hour 16 Hour 25$ 50$ 75$ 25$ 50$ 75$ July 19 Control 4.10 61.0 69.0 17.0 24.8 35.0 Thyroxine 39.5 59.0 * 34.0 43.5 51.5 Thiourea 26.5 39.5 53.0 28.5 50.0 62.0 Oct. 18 Control 54.0 82.0 * 23.0 48.0 Thyroxine # * * 45.0 82.0 * Thiourea 39.5 70.0 28.0 60.0 * Thiouracil 6.4 12.6 17.5 9.6 20.5 * Nov. 1 Control 15.0 47.5 62.0 3.2 8.6 22.5 T.S.H. 6.6 61.0 73.0 27.0 43.0 52.0 * Level of mortality not reached. APPENDIX V Time (Minutes) required for 25$, 50$, and 75$ mortality i n Heat Tests. Date Treatment July 22 Control Thyroxine Thiourea Oct. 22 Control Thyroxine Thiourea 8 Hour 25$ 50 $ 75$ 52 61 73 38 62 73 30 33 45 4.5 27.5 42 35.5 48 53 3 13.5 33.5 16 Hour 25$ 50$ 75$ 49 71 88 51 58 69 38 48 59 33 56 73 10.5 36 55 16 47 57 APPENDIX VI Mean weights and standard deviations of samples Experiment Treatment Days of Males treatment Number Weight of f i s h A Control 4 7 , . 12.8 June 8 6" 10.8 16 7 10.4 Thyroxine 4 8 10.9 8 10 10.3 16 6 13.3 Thiourea 4 5 12.5 8 8 13.9 16 6 13.3 B Control 4 14 7.1 August 8 11 7.1 16 10 7.2 Thyroxine 4 9 6.8 8 11 7.1 16 9 7.3 Thiourea 4 14 7.3 8 11 6.9 16 10 6.6 C Control 7 8 11.7 November 14 5 10.2 Thyroxine 7 8 11.7 14 10 13.8 Thiouracil 7 9 10.9 14 7 11.3 ed for cholesterol analysis. Females Standard Number Weight Standard Deviation of f i s h Deviation 2.38 8 12.4 3.92 1.49 9 14.6 3.78 3.62 8 16.0 4.35 3.39 7 13.7 3.52 2.19 5 13.3 4.10 3.67 10 9.5 3.07 2.86 10 11.1 3.85 3.52 7 11.6 2*95 3.67 9 13.2 3.26 1.30 6 10.4 3.97 1.29 9 6.9 1.69 2,10 9 .7.7 2.32 1.64 11 9.1 2.79 2.11 9 7.0 1.68 1.35 10 9.2 2.28 1.94 6 8.4 1.39 2.06 9 7.9 1.44 1.24 9 7.3 2.08 3.32 12 13.9 4.76 2.11 16 13.2 2.59 2.90 12 13.1 4.82 3.32 15 12.0 4.44 4.59 11 16.2 6.16 4.84 12 11.4 4.68 APPENDIX VII Mean weights of total body and of gonads with mean ratios of these two weights for photoperiod goldfish k i l l e d on dates indicated. Material preserved i n 10% formaldehyde. Males Date Number Body Gonad Ratio Number Body Gonad Ratio of f i s h Weight Weight Gonad Weight/ of f i s h Weight Weight Gonad Weig gm. mgm. Body Weight gm. mgm. Body Weigl: Feb. 18 23 16.9 287.6 19.65 23 16.6 242.0 14.42 Mar. 25 16 17.8 279.0 15.50 14 19.4 303.1 15.32 Apr. 4 7 14.4 209.2 14.58 9 17.7 270.4 15.73 May 16 9 18.8 355.6 19.50 12 17.2 423.7 24.85 May 23 19 17.3 290.5 15.86 18 17.6 321.4 18.19 July 19-23 12 15.0 132.2 8.70 12 16.1 116.6 7.29 Oct. 4 10 8.5 64.5 7.04 10 9.9 45.7 4.56 Nov. 1 19 12.6 141.0 11.31 8 11.1 74.9 7.05 Females Feb. 18 17 17.6 521.6 29.48 17 19.2 964,2 48.42 Mar. 25 23 18.6 566.7 30.86 23 19.6 851.5 44.54 Apr. 4 13 19.5 576.7 31.43 11 19.9 1183.6 64.08 May 16 10 16.0 413.1 24.54 6 19.8 442.2 23.97 May 23 22 18.3 1011.1 55.89 21 17.1 968.1 52.66 July 19-23 12 14.2 320.5 20.68 12 16.5 309,6 19.16 Oct. 4 11 9.8 220.1 22.69 12 11.3 208.3 18.32 Nov. 1 13 14.6 430.3 27.23 12 14.6 267.6 18.76 44 LITERATURE CITED BERG, 0. and A. GORBMAN. 1954. Normal and altered thyroidal function in domesticated goldfish, Carassius auratus. Proc. Soc. Exp. Biol. Med. 86: 156-159. BRETT, J. R. 1946. Rate of gain of heat tolerance in goldfish (Carassius auratus). Univ. Toronto Stud., Biol. Ser., no. 53 (Pub. Ont. Fish. Res. Lab., no. 64), 9-28. BULLOCK, T. H. 1955. Compensation for temperature in the metabolism and activity of poikilotherms. Biol. Rev. 30: 311-342. BULLOUGH, W. S. 1951. Vertebrate Sexual Cycles. Methuen and Co., Ltd., London. CARTER, G. S. 1933. On the control of the level of activity in the animal body. I. The endocrine control of seasonal variation in activity in the frog. J. Exp. Biol. 10: 256-273. CHAVIN, W. 1956a. Thyroid distribution and function i n the goldfish, Carassius auratus L. J. Exp. Zool. 133: 259-280. CHAVIN, W. 1956b. Thyroid distribution and function in goldfish, Carassius auratus L. as determined by the uptake of tracer doses of radioiodine. Anat. Rec. 124 (2): 272. FORTUNE, P. Y. 1956. An inactive thyroid gland in Carassius auratus. Nature 178: 98. HARRINGTON, R. W., Jr. 1957. Sexual photoperiodicity of the Cyprinid fish, Notropis bifrenatus (Cope), in relation to the phases of i t s annual reproductive cycle. J. Exp. Zool. 135: 529-556. HEILBRUNN, L. Y. 1952. An outline of general physiology. Third edition. W. B. Saunders Co., Philadelphia. HOAR, W. S. 1939. The thyroid gland of the Atlantic salmon. J. Morph. 65: 257-292. 45 HOAR, W. S. 1954. Fish endocrinology and behaviour. N.R.C. MS. report. Mimeo. HOAR, W. S. 1955. Seasonal variations i n the resistance of goldfish to temperature. Trans. Roy. Soc, Canada, V, 49: 25-34. HOAR, W. S. 1956. Fish endocrinology and behaviour. NiR.C. MS. report. Mimeo. HOAR, W. S. and M. K. COTTLE. 1952a. Some effects of temperature acclimatization on the chemical constitution of goldfish tissues. Can. J . Zool. 30: 49-54. HOAR, W. S. and M. K. COTTLE. 1952b. Dietary fat and temperature tolerance of goldfish. Can. J. Zool. 30: 41-48. HOLLANDS, M. 1956. The effect of photoperiod on the goldfish (Carassius auratus). M.A. Thesis, University of B.C. Unpublished. IRVINE, D. G. 1954. The cold resistance of goldfish (Carassius auratus) fed certain l i p i d diets. M.A. Thesis, University of B.C. Unpublished. IRVINE, D. G., K. NEWMAN and W. S. HOAR. 1957. Effects of dietary phospholipid and cholesterol on the temperature resistance of goldfish. Jan. J . Zool. 35: 691-709. JOHANNES, R. E. 1957. High temperature as a factor i n scallop mass mortality. F.R.B.C. MS. Report Series B i o l . , no. 638. Sept. 30, 1957. LaROCHE, G. and C. P. LEBLOND. 1954. Destruction of thyroid gland of Atlantic salmon (Salmo salar L.) by means of radioiodine. Proc. Soc. Exp. B i o l . Med., 87: 273-276. MARSHALL, F. H. A. 1956. Physiology of Reproduction. Vol. I. Part 1. 3rd. ed. Edited by A.S. Parkes. Longman Green and Co., Ltd., London. PICKFORD, G. E. 1957. The physiology of the pitui t a r y gland of fishes. New York Zoological Society, New York. 46 SPERRY, W. M. and M. WEBB. 1950. A revision of the Schoenheimer-Sperry method for cholesterol determination. J . B i o l . Chem. 187: 97-106. STEIN, K. F. and E. CARPENTER. 1943. The effect of increased and decreased l i g h t on the thyroid gland of Triturus viridescens. J . Morph. 72: 491-515. * SUHRMANN, R. 1955. Weitere Tersuche fiber die Temperaturadaption der Karauschen (Carassius vulgaris N i l s ) . B i o l . Zbl., 74: 432-448. * Original paper not consulted. 

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