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Meristic variation in the medaka (oryzias latipes) produced by temperature and by chemicals affecting… Ali, Mohammed Youssouf 1962

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MERISTIC VARIATION IN THE MEDAKA (ORYZIAS LATIPES) PRODUCED BT TEMPERATURE AND BY CHEMICALS AFFECTING METABOLISM by MOHAMMED IOUSSOUF ALI B.Sc. (Hons.) University of Calcutta, 1945 M.Sc., University of British Columbia, 1959 A Thesis Submitted in Partial Fulfilment of The Requirements for the Degree of Doctor of Philosophy in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1962 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 o f the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t 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 b]r the Head of my Department o r by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t 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 g a i n s h a l l not be allowed without my w r i t t e n permission. Department of Zoology,  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date August 25, 1962. GRADUATE STUDIES F i e l d of Study: Zoology "Fisheries B i o l o g y and Management Problems i n Icthyology Marine Zoogeography Experimental Zoology Q u a n t i t a t i v e Methods i n Zoology Limnology Other Studies: F i s h e r i e s Economics F i s h e r i e s Law F i s h e r i e s H y d r a u l i c s F i s h e r i e s Anthropology I n t r o d u c t i o n to Synoptic Oceanography Marine Benthonic, Organisms and t h e i r Environment Problems i n Philosophy P. A. L a r k i n C. C. Lindsey J . C. Briggs W. S. Hoar P. A. L a r k i n T. G. Northcote A. D. Scott G.,F..Curtis E. S. P r e t i o u s H, B. Hawthorn G. L. P i c k a r d R. F. Scagel B. Savery The U n i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of MOHAMMED YOUSSOUF ALI B.Sc. (Hons)., U n i v e r s i t y of C a l c u t t a M.Sc., U n i v e r s i t y of B r i t i s h Columbia FRIDAY, SEPTEMBER 7, 1962, at 2:30 P.M. IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING COMMITTEE IN CHARGE Chairman: F. H„ SOWARD D. CHITTY I. McT. COWAN Rs W. DUNNING C. V. FINNEGAN P. A. LARKIN C. C. LINDSEY N. TOMLINSON N. J . WILIMOVSKY Ex t e r n a l Examiner: F e r r i s NEAVE F i s h e r i e s Research Board of Canada PARALLELS BETWEEN MERISTIC VARIATION IN THE MEDAKA (ORYZIAS LATIPES) PRODUCED BY TEMPERATURE AND BY CHEMICALS AFFECTING METABOLISM ABSTRACT M e r i s t i c characters i n o f f s p r i n g from 27 p a i r s of medaka were i n v e s t i g a t e d w i t h respect to some f a c t o r s known to a l t e r metabolism. Temperature, l i g h t , thyroxine,''thiourea, d i n i t r o p h e n o l , urethan and s a l i n i t y were test e d . Egg s i z e , egg d e n s i t y , nature of r e a r i n g c o n t a i n e r s , q u a l i t y of succ-e s s i v e day's egg batches from the same parent, mechanical shock of developing eggs and p r i c k i n g the chorion of eggs were a l s o s t u d i e d as p o s s i b l e f a c t o r s producing m e r i s t i c v a r i a t i o n s . Mean v e r t e b r a l counts showed a V-shaped r e l a t i o n to temperature i n 9 out of 15 r e p l i c a t i o n s , inverse r e l a t i o n i n 2, and no c o n s i s t e n t r e l a t i o n i n 4. P e c t o r a l f i n ray counts were i n v e r s e l y r e l a t e d to temperature. Degree and d i r e c t i o n of change of other f i n rays w i t h temperature v a r i e d between genotypes. V e r t e b r a l counts were not a f f e c t e d by v a r i a t i o n s i n l i g h t i n t e n s i t i e s or d u r a t i o n ; f i n ray counts were a l t e r e d but t h e i r r e a c t i o n lacked u n i f o r m i t y . Mean t o t a l v e r t e b r a l counts of 8 out of 11 r e p l i c a t i o n s were a l t e r e d i n thyroxine s o l u t i o n , but magnitude and d i r e c t i o n of change d i f f e r e d between genotypes. When eggs were hatched i n thyroxine s o l u t i o n , p e c t o r a l ray counts were lowered. Exposure of larvae to thy r o x i n e produced s i g n i f i c a n t decrease i n p e c t o r a l , a n a l , d o r s a l , and caudal f i n ray counts. Rearing eggs to hatching i n t h i o u r e a produced s i g n i f i c a n t increase i n mean t o t a l v e r t e b r a l counts i n only 2 of 11 r e p l i c a t i o n s . P e c t o r a l and anal f i n ray counts increased, but t o t a l caudal rays decreased, i n samples from t r e a t e d eggs as w e l l as from larvae t r e a t e d i n t h i o u r e a a f t e r hatching. Rearing of eggs to hatching i n d i n i t r o p h e n o l , urethan, or sea water r e s u l t e d i n an increase i n mean v e r t e b r a l counts. P e c t o r a l rays increased i n lower concentrations of d i n i t r o p h e n o l , or i n urethan, but were unaffected i n sea water. Anal, d o r s a l , and t o t a l caudal rays were not a l t e r e d i n sea water, but v a r i a b l e e f f e c t s r e s u l t e d from d i n i t r o p h e n o l or urethan. No c o r r e l a t i o n was found between m e r i s t i c counts and egg s i z e . V e r t e b r a l and p e c t o r a l ray counts seemed to f o l l o w those of the f a t h e r ; p a t e r n a l i n f l u e n c e was very pronounced i n i n h e r i t a n c e of p e c t o r a l rays. V e r t e b r a l , p e c t o r a l and d o r s a l ray counts were not a f f e c t e d by other extraneous f a c t o r s t e s t e d . E f f e c t of egg density of anal and t o t a l caudal rays was v a r i a b l e . F i n a l f i x a t i o n of t o t a l vertebrae occurred at the embryonic stage when eye pigmentation commenced and p e c t o r a l buds had appeared. Other characters remained s e n s i t i v e to environmental i n f l u e n c e even a f t e r hatching The r e l a t i o n of metabolism to m e r i s t i c c h a r a c t e r s , and evident p a r a l l e l s between e f f e c t s . o f the s e v e r a l f a c t o r s used are discussed. ABSTRACT M e r i s t i c characters i n o f f s p r i n g from 27 p a i r s of medaka were i n v e s t i g a t e d w i t h respect to some f a c t o r s known to a l t e r metabolism. Temperature, l i g h t , t h yroxine, t h i o u r e a , d i n i t r o p h e n o l , urethan and s a l i n i t y *?ere t e s t e d . Egg s i z e , egg d e n s i t y , nature of r e a r i n g con-t a i n e r s , q u a l i t y of successive days egg batches from the same parent, mechanical shock of developing eggs and p r i c k i n g the chorion of eggs were a l s o studied as p o s s i b l e f a c t o r s producing m e r i s t i c v a r i a t i o n s . Mean v e r t e b r a l counts showed a V-shaped r e l a t i o n to temperature i n 9 out of 15 r e p l i c a t i o n s , inverse r e l a t i o n i n 2, and no co n s i s t e n t r e l a t i o n i n 4. Pe c t o r a l f i n ray counts were i n v e r s e l y r e l a t e d to tem-perature. Degree and d i r e c t i o n of change of other f i n rays w i t h tem-perature v a r i e d between genotypes. V e r t e b r a l counts were not a f f e c t e d by v a r i a t i o n s i n l i g h t i n t e n -s i t i e s or d u r a t i o n ; f i n ray counts were a l t e r e d but t h e i r r e a c t i o n lacked u n i f o r m i t y . Mean t o t a l v e r t e b r a l counts of 8 out of 11 r e p l i c a t i o n s were a l t e r e d i n thyroxine s o l u t i o n , but magnitude and d i r e c t i o n of change d i f f e r e d between genotypes. When eggs were hatched i n thyroxine s o l u t i o n , p e c t o r a l ray counts were lowered. Exposure of larvae to thyroxine pro-duced s i g n i f i c a n t decrease i n p e c t o r a l , a n a l , and caudal f i n ray counts. Rearing eggs to hatching i n thiourea produced s i g n i f i c a n t increase i n mean t o t a l v e r t e b r a l counts i n only 2 of 11 r e p l i c a t i o n s . P e c t o r a l and anal f i n ray counts increased, but t o t a l caudal rays decreased, i n samples from tr e a t e d eggs as w e l l as from larvae t r e a t e d i n thiourea a f t e r hatching. ( i i ) Rearing of eggs to hatching i n d i n i t r o p h e n o l , urethan, or sea water r e s u l t e d i n an increase i n mean v e r t e b r a l counts. P e c t o r a l rays increased i n lower concentrations of d i n i t r o p h e n o l , or i n urethan, but were unaffected i n sea water. Anal , d o r s a l , and t o t a l caudal rays were not a l t e r e d i n sea water, but v a r i a b l e e f f e c t s . r e s u l t e d from d i n i t r o p h e n o l or urethan. No c o r r e l a t i o n was found between m e r i s t i c counts and egg s i z e . V e r t e b r a l and p e c t o r a l ray counts seemed to f o l l o w those of the f a t h e r ; p a t e r n a l i n f l u e n c e was very pronounced i n i n h e r i t a n c e of p e c t o r a l rays. V e r t e b r a l , p e c t o r a l and d o r s a l ray counts were not a f f e c t e d by other extraneous f a c t o r s t e s t e d . E f f e c t of egg d e n s i t y on anal and t o t a l caudal rays was v a r i a b l e . F i n a l f i x a t i o n of t o t a l vertebrae occurred at the embryonic stage when eye pigmentation commenced and p e c t o r a l buds had appeared. Other chara c t e r s remained s e n s i t i v e to environmental i n f l u e n c e even a f t e r hatching. The r e l a t i o n of metabolism to m e r i s t i c c h a r a c t e r s , and evident p a r a l l e l s between e f f e c t s of the several f a c t o r s used are discussed. (xil) ACKNOWLEDGEMENTS This work was done under the supervision of Dr. Casimir C. Lindsey, Department of Zoology, University of British Columbia. His guidance and assistance during a l l phases of the experimental work and writing of the thesis are gratefully acknowledged. The author also wishes to express his gratitude to Dr. William S. Hoar, Dr. Cyril V. Finnegan, Dr. Dennis Chitty and Dr. Peter A. Larkin who offered valuable counsel and stimulation throughout the study and during the preparation of the manuscript. Thanks are also due to Dr. John J. Magnuson and Mr. J. Ruffelle for assisting in culturing tnedaka and to Mr. T. Pletcher for assisting in photomicrography. Grateful thanks are also extended to the External Aid Office, Government of Canada, and Government of East Pakistan for allowing the author to extend his stay and undertake the project. The project was financed by the National Research Council of Canada through grants-in-aid to Dr. Casimir C. Lindsey. ( i i i ) TABLE OF CONTENTS page ACKNOWLEDGEMENTS INTRODUCTION 1 MATERIALS AND METHODS 3 Outline of experiments . . . . . . . . . . 3 Experimental fi s h . . . . . . 5 Laboratory installations 5 Containers for rearing eggs and young . . . 7 Breeding and hatching . . 9 Care of eggs 10 Foods and feeding 11 Clearing and counting of meristic series . 11 Calculation of hatching time IS EFFECT OF MALACHITE GREEN (EXPERIMENT I) . . . . 17 Introduction 17 Description of experiment 17 Results 17 EFFECT OF EGG REARING CONTAINERS AND VARIABILITY OF AERATION (EXPERIMENT II) 25 Introduction 25 Description of experiment 25 Results 25 EFFECT OF SUCCESSIVE DAYS EGGS (EXPERIMENT III) 29 Introduction 29 Description of experiment 29 Results 29 (iv) Page EFFECT OF MECHANICAL SHOCK (EXPERIMENT IV) . . . . 31 Introduction 31 Description of experiment 31 Results 31 EFFECT OF PRICKING THE CHORION (EXPERIMENT V) . . 39 Introduction . . . . . . . . 39 Description of experiment 39 Results 39 EFFECT OF EGG DENSITY (EXPERIMENT VI) 46 Introduction 46 Description of experiment 46 Results 46 EFFECT OF EGG SIZE (EXPERIMENT VII) 53 Introduction . . . . . 53 Description of experiment 53 Results 55 Conclusion 60 STAGE OF FIXATION OF MERISTIC SERIES (EXPERIMENT VIII) 72 Introduction 72 Description of experiment 72 Results 72 Conclusion 76 EFFECT OF SUSTAINED TEMPERATURE (EXPERIMENT IX) . 83 Introduction • 83 Description of experiment 83 Results 84 Conclusions . . 96 (V) Page EFFECT OF INCREASED LIGHT (EXPERIMENT X) 103 Introduction 103 Results 103 Conclusion . . . . . . . 106 EFFECT OF THYROXINE AND THIOUREA (EXPERIMENT XI) . . . . 113 Introduction 113 Description of experiment 113 Results 118 Conclusion 141 EFFECT OF 2,4-DINITROPHENOL (EXPERIMENT XII) 146 Introduction 146 Description of experiment 146 Results 149 Conclusion 149 EFFECT OF URETHAN (EXPERIMENT XIII) 150 Introduction 150 Description of experiment 150 Results 151 Conclusion 151 EFFECT OF SALINITY (EXPERIMENT XIV) 152 Introduction 152 Description of experiment 152 Results 152 HATCHING TIME AND MERISTIC VARIATION 153 SIZE HIERARCHY AND MERISTIC VARIATION 153 SUMMARY OF RESULTS 155 DISCUSSION 159 LITERATURE CITED 180 APPENDICES (vi) LIST OF FIGURES Figure Page 1 Arrangement of controlled environment unit. 4 2 Instrument board and arsangement of small cloth baskets. 4 3 Arrangement of bottles in a tank for experiments XI - XIV. 8 4 Vertebral column showing the f i r s t vertebra in a cleared juvenile medaka. 8 5 Vertebral column and caudal f i n rays in a cleared juvenile medaka. 12 6 Pectoral f i n rays in a cleared juvenile medaka. 12 7 Anal f i n rays in a cleared juvenile medaka. 13 8 Dorsal f i n rays in a cleared juvenile medaka. 13 9 Effect of egg density on mean total vertebrae and pectoral and anal f i n rays of genotype Y (Experiment VI) 44 10 Effect of egg density on mean dorsal and total caudal f i n rays of genotype Y (Experiment VI) 45 11 Mean vertebral counts of f i s h transferred from 20Q to 30°C and 30° to 20°C (Experiment VIII) 69 12 Effect of transfer of developing embryo from 20° to 30°Con mean pectoral and anal f i n rays of genotype U (Experiment VIII). 70 13 Effect of transfer of developing embryo from 20° to 30°C on mean dorsal and total caudal f i n rays of genotype U (Experiment VIII) 71 14 Effect of sustained temperature on mean total vertebrae (Experiment IX) 85 15 Effect of sustained temperature on mean pectoral f i n rays (Experiment IX) 87 16 Effect of sustained temperature on mean anal f i n rays (Experiment IX) 89 17 Effect of sustained temperature on mean dorsal f i n rays (Experiment IX) 92 ( v i i ) Figure Page 18 Effect of sustained temperature on mean total caudal f i n rays (Experiment IX) 94 19 Effect of increased light on mean total vertebrae and pectoral and anal f i n rays (Experiment X) 104 20 Effect of increased light on mean dorsal and total caudal f i n rays (Experiment X) 105 21a Effect of thyroxine and thiourea to hatching on mean total vertebrae of genotypes reared in 24°C (Experiment XI) 119 b Effect of thyroxine and thiourea on mean total vertebrae of genotypes reared in 26° and 30°C (Experiment XI) 120 22a Effect of thyroxine and thiourea to hatching on mean pectoral f i n rays of genotypes reared in 24°C (Experiment XI) 124 b Effect of thyroxine and thiourea on mean pectoral f i n rays of genotypes reared in 26° and 30°C (Experiment XI) 125 23a Effect of thyroxine and thiourea to hatching on mean anal f i n rays of genotypes reared in 24°C (Experiment XI) 130 b Effect of thyroxine and thiourea on mean anal f i n rays of genotypes reared in 26° and 30°C (Experiment XI) - 131 24 Effect of thyroxine and thiourea on mean dorsal f i n rays (Experiment XI) 135 25a Effect of thyroxine and thiourea to hatching on mean total caudal f i n rays of genotypes reared in 24°C (Experiment (XI) 137 b Effect of thyroxine and thiourea on mean total caudal f i n rays of genotypes reared in 26° and 30°C (Experiment XI) 138 26 Effect of urethan and dinitrophenol on mean total vertebrae and pectoral and anal f i n rays of genotype Y (Experiments XII and XIII) 147 '2& Effect of urethan and dinitrophenol on mean dorsal and total caudal f i n rays of genotype Y (Experiments XII and XIII) 148 ( v i i i ) LIST OF TABLES Table Page I Egg numbers and mortality in experiment I 19 II Frequency distribution of total vertebrae in experiment I 20 III Frequency distribution of pectoral rays in experiment I 21 IV Frequency distribution of anal rays in experiment I 22 V Frequency distribution of dorsal rays in experiment I 23 VI Frequency distribution of total caudal rays in experiment I 24 VII Egg number and mortality in experiment II 19 VIII Frequency distribution of total vertebrae in experiment II 20 IX Frequency distribution of pectoral rays in experiment II 21 X Frequency distribution of anal rays in experiment II 22 XI Frequency distribution of dorsal rays in experiment II 23 XII Frequency distribution of total caudal rays in experiment II 24 XIII Egg numbers and mortality in experiment III 27 XIV Frequency distribution of total vertebrae in experiment III 27 XV Frequency distribution of pectoral rays in experiment III 27 XVI Frequency distribution of anal rays in experiment III 28 XVII Frequency distribution of dorsal rays in experiment III 28 XVIII Frequency distribution of total caudal rays in experiment III 28 XIX Egg numbers and mortality in experiment IV 33 XX Frequency distribution of total vertebrae in experiment IV 34 (ix) Table Page XXI Frequency distribution of pectoral rays in experiment IV 35 XXII Frequency distribution of anal rays in experiment IV 36 XXIII Frequency distribution of dorsal rays in experiment IV 37 XXIV Frequency distribution of total caudal rays in experiment IV 38 XXV Egg aamber and mortality in experiment V 33 XXVI Frequency distribution of total vertebrae in experiment V 34 XXVII Frequency distribution of pectoral rays in experiment V 35 XXVIII Frequency distribution of anal rays in experiment V 36 XXIX Frequency distribution of dorsal rays in experiment V 37 XXX Frequency distribution of total caudal rays in experiment V 38 XXXI Egg numbers and. mortality in experiment VI 41 XXXII Frequency distribution of total vertebrae in experiment VI 41 XXXIII Frequency distribution of pectoral rays in experiment VI 41 XXXIV Frequency distribution of anal rays in experiment VI 42 XXXV Frequency distribution of dorsal rays in experiment VI 42 XXXVI Frequency distribution of total caudal rays in experiment VI 43 XXXVII Egg numbers and mortality in experiment VII 48 XXXVIII Frequency distribution of total vertebrae in experiment VII 48 <x) Table Page XXXIX Frequency distribution of pectoral rays in experiment VII 50 XL Frequency distribution of anal rays in experiment VII 50 XLI Frequency distribution of dorsal rays in experiment VII 51 XLII Frequency distribution of total caudal rays in experiment VII 52 XLIII Egg numbers and mortality in experiment VIII 61 XLIV Frequency distribution of total vertebrae in experiment VIII (a) Transfer of eggs from 20° to 30°C 63 (b) Transfer of eggs from 30° to 20°C 65 XLV Frequency distribution of pectoral rays in experiment VIII (a) Transfer of eggs from 20° to 30°C 66 XLVI Frequency distribution of anal rays in experiment VIII (a) Transfer of eggs from 20° to 30°C 66 XLVII Frequency distribution of dorsal rays in experiment VIII (a) Transfer of eggs from 20° to 30°C 67 XLVIII Frequency distribution of total caudal rays in experiment VIII (a) Transfer of eggs from 20° to 30°C 68 XLIX Egg numbers and mortality in experiment IX 77 L Egg numbers and mortality in experiment X 97 LI Frequency distribution of total vertebrae in experiment X 98 LII Frequency distribution of pectoral rays in experiment X 99 LIII Frequency distribution of anal rays in experiment X 100 LIV Frequency distribution of dorsal rays in experiment X 101 LV Frequency distribution of total caudal rays in experiment X 102 LVI Egg numbers and mortality in experiment XI 108 (a) Fertilized eggs reared in the solutions up to hatching 108 (b) Eggs f e r t i l i z e d and reared in the solution up to hatching 111 (c) Chorion pricked eggs reared in the solution up to hatching 111 (d) Larvae reared in the solutions up to hatching 112 (xi) Table Page LVII Egg numbers and mortality in experiment XII 142 LVTII Frequency distribution of total vertebrae in experiment XII 143 LIX Frequency distribution of pectoral rays in experiment XII 144 LX Frequency distribution of anal rays in experiment XII 144 LXI Frequency distribution of dorsal rays in experiment XII 145 LXII Frequency distribution of total caudal rays in experiment XII 145 LXIII Egg numbers and mortality in experiment XIII 142 LXIV Frequency distribution of total vertebrae in experiment XIII 143 LXV Frequency distribution of pectoral rays in experiment XIII 144 LXVT Egg numbers and mortality in experiment XIV 142 LXVII Frequency distribution of total vertebrae in experiment XIV 143 INTRODUCTION I t i s w e l l known that the m e r i s t i c characters of c o l d blooded v e r t e b r a t e s , p a r t i c u l a r l y f i s h , vary w i t h i n a s i n g l e s p e cies. E a r l i e r workers (Heincke, 1898; Schnackenbeck, 1931) a t t r i b u t e d such v a r i a t i o n s to genetic d i f f e r e n c e s among the populations. But subsequent f i n d i n g s have demonstrated that the c h a r a c t e r s , though t h e i r l i m i t s of v a r i a b i l i t y are g e n e t i c a l l y c o n t r o l l e d , may be modified by environmental i n f l u e n c e s o perating at the time of t h e i r f i x a t i o n . Inf;uences of d i f f e r e n t environmental f a c t o r s - e.g. temperature (Schmidt 1917, 1919, 1921; Mottley 1934; G a b r i e l 1944; Dannevig 1950; Lindsey 1954, 1962, B l a x t e r , 1957, Seymour 1959; Itazawa 1959), temperature and/or s a l i n i t y (Schmidt, 1920). temperature, oxygen and carbon d i o x i d e (Taning, 1944, 1946 and 1950), s a l i n i t y and temperature (Heuts 1947 .and 1949), and l i g h t (McHugh 1954; Lindsey 1958; Canagaratnam 1959 and Lyubitskaya 1956 and 1961) have been st u d i e d . Although the i n f l u e n c e of the environmental f a c t o r s i n determining the f i n a l expression of d i f f e r e n t m e r i s t i c s e r i e s i s e s t a b l i s h e d , i t i s not c l e a r hox* these f a c t o r s operate. Marckmann (1954) suggested that temperature a l t e r s m e r i s t i c counts by a l t e r i n g the metabolism of the developing embryo. According to t h i s hypothesis, the lowest v e r t e b r a l counts obtained i n the intermediate temperature i n sea t r o u t , Salmo t r u t t a was the r e s u l t of a most economic metabolism of the i n d i v i d u a l s at t h i s temperature. Canagaratnam (1959) suggested that there i s probably a r e l a t i o n s h i p between the a c t i v i t y of the p i t u i t a r y - t h y r o i d complex and the f i n a l f i x a t i o n of many m e r i s t i c s e r i e s . The present work was undertaken to study the r e l a t i o n s h i p of t h y r o i d a c t i v i t y , metabolism.-and the f i x a t i o n of d i f f e r e n t m e r i s t i c s e r i e s i n the medaka, Oryzias l a t i p e s (Temminck and S c h l e g e l ) , a Japanese cyprinodont, by r e a r i n g eggs and larvae i n thyroxine and thiourea s o l u t i o n s . In a d d i t i o n experiments were conducted to observe the e f f e c t s of d i n i t r o p h e n o l , urethan, temperature, l i g h t and some' e"xtraHeous sources of v a r i a t i o n . Several d i f f e r e n t m e r i s t i c s e r i e s x^ere examined which y i e l d e d i n f o r m a t i o n on the genotypic and phenotypic c o n t r o l of the i n d i v i d u a l s e r i e s . P o s s i b l e r o l e of metabolism and t h y r o i d a c t i v i t y i n r e l a t i o n to f i x a t i o n of m e r i s t i c characters have been considered i n the d i s c u s s i o n . S i z e h i e r a r c h i e s and s e l e c t i v e m o r t a l i t y have a l s o been considered i n r e l a t i o n to m e r i s t i c v a r i a t i o n . -3-HATERIALS AND METHODS Ou t l i n e of experiments. The number and purpose of each experiment are l i s t e d below. Experiment 1: e f f e c t of malachite green, treatment on developing eggs. Experiment 11: e f f e c t of the nature of egg r e a r i n g c o n t a i n e r s ( b o t t l e s , baskets) and v a r i a t i o n i n a e r a t i o n . Experiment 111: e f f e c t of the q u a l i t y of eggs obtained on successive days from the same parent. Experiment IV: e f f e c t of mechanical shock given to developing eggs. Experiment V: e f f e c t of p r i c k i n g the chorion of f e r t i l i z e d eggs. Experiment VI: e f f e c t of the d e n s i t y of egg and young i n the r e a r i n g c o n t a i n e r s . Experiment V l l : . ' e f f e c t of yolk diameter (egg s i z e ) . Experiment V l l l : determination of the s e n s i t i v e period f o r d i f -f e r e n t m e r i s t i c characters during development by t r a n s f e r of eggs from high to low and low to high temperatures. Experiment IX: e f f e c t of sustained temperature. Experiment X: e f f e c t of increased l i g h t ( i n t e n s i t y and d u r a t i o n ) . Experiment XI: e f f e c t of thyroxine and th i o u r e a . Experiment X l l : e f f e c t of 2, 4-Dinitrophenol. Experiment X l l l : e f f e c t of urethan. Experiment XIV: e f f e c t of s a l i n i t y . Arrangements of c o n t r o l l e d environment u n i t s . A-Aminco relay;' 3-Light hood; C-Tank; D-Water i n l e t ; E-Outlet pipe. Instrument board and arrangement of small c l o t h baskets. A - A i r valve; E-Heater t e r m i n a l s ; F-Water i n l e t ; G-Indicator l i g h t ; H-Thermoregulator; I - S n a l l c l o t h basket. A l l experiments except I I , I I I , IV, VI and V I I I were repeated more than once w i t h eggs from d i f f e r e n t genotypes i n order to ob t a i n a b e t t e r p i c t u r e of the complex r e l a t i o n s h u p between environment and genotype. Experimental f i s h A domestic stock of medaka, Oryzias l a t i p e s (Temminck and S c h l e g e l ) , a small Japanese cyprinodont was used f o r a l l the experiments. Briggs and Egami (1959) o u t l i n e some of the f e a t u r e s which make i t u s e f u l as an experimental animal. Medaka are q u i t e hardy and can withstand a wi range of temperature. The temperature range of the species, according to Rugh (1962), i s from 7° to 39°C w i t h the optimum between 20° and 25°C. Time of breeding and egg production can be regulated by c o n t r o l l i n g the i H u m i l i a t i o n . . Number of eggs produced at a s i n g l e spawning vary w i d e l y (1-30 eggs according to Rugh, 1962). Depending upon food, temperature and space, the time required to a t t a i n m a t u r i t y v a r i e s from 4 to 6 months. Upon m a t u r i t y , the female breeds r e a d i l y on successive days f o r 3 or 4 months. Laboratory i n s t a l l a t i o n s C o n t r o l l e d environment apparatus was set up i n the B i o l o g i c a l Sciences B u i l d i n g , U n i v e r s i t y of B r i t i s h Columbia i n the w i n t e r of 1959 and the experiments were conducted from March, 1960 through May, 1962. Each u n i t of the c o n t r o l l e d apparatus c o n s i s t e d of a tank (54x54x38cm) made of 3/4" plywood and l i n e d w i t h non-toxic neopreme p a i n t ( f i g u r e 1). Each tank held approximately 70 l i t e r s of water. -6-Freshwater from the U n i v e r s i t y mains was de c h l o r i n a t e d and f i l t e r e d before being taken i n t o the l a b o r a t o r y pipes. Water was guided i n t o every tank w i t h 'tygon'tubing and g l a s s T-tubes from a s i n g l e f a u c e t . The m a j o r i t y of the experiments were conducted i n d e c h l o r i n a t e d and f i l t e r e d water but a few \?ere done i n s t r a i g h t tap water owing to the removal of the d e c h l o r i n a t i n g u n i t . T h i s d i f f e r e n c e i n water, however, d i d not a f f e c t e i t h e r s u r v i v a l and growth of f i s h or the r e s u l t s . L i t t l e or no c h l o r i n e can be detected i n the raw water. Water flow i n t o each tank was maintained at 3 l i t e r s per hour, which replaced the e n t i r e volume i n a tank every 20 hours. Compressed a i r was provided i n each tank by two a i r d i f f u s e r stones ( f i g u r e 2 ) , which provided both a e r a t i o n and mixing. Both water and a i r flow i n t o each tank were checked twice d a i l y . Each tank was provided w i t h a 500 watt s t a i n l e s s immersion heater (Aminco) w i t h 80cm e f f e c t i v e heating surface and a "Quickset" bimetal thermo r e g u l a t o r (Aminci product). Heating was c o n t r o l l e d by a s u p e r s e n s i t i v e r e l a y (Aminco). Both the thermoregulator and the heating element were suspended i n t o water from a plywood instrument board, which a l s o contained an i n d i c a t o r l i g h t to s i g n a l the working of the heater. Temperature i n every tankrwas recorded twice d a i l y w i t h a thermomeitefc c a l i b r a t e d to 0.1°c. from three p o i n t s at depths where eggs remained suspended. The temperature was maintained w i t h i n +0.1°c. i n every tank throughout the experiments. In a few cases where the temperature of a tank had departed widely from the set temperature, the experimental l o t s were discarded. Each tank was provided w i t h a l i g h t hood (54x35x22.5cm) made from plywood and painted white i n s i d e . L i g h t hoods f i t t e d the top of each -7-tank except the area occupied by the instrument board. The edges of the hood were l i n e d w i t h 1 cm t h i c k foam rubber s t r i p s to insure-l i g h t p r o o f i n g . The i n s i d e centre of the hood was provided w i t h an e l e c t r i c lamp holder i n t o which was f i t t e d a 7.5 watt, f r o s t e d g l a s s f i l a m e n t bulb (G.E.). This bulb, l y i n g a t a distance of approximately 17 cm. from the water s u r f a c e , provided 9 f t . - c . of l i g h t on the water surface i n the centre of the tank. For a l l experiments, except the increased l i g h t i n t e n s i t y and d u r a t i o n , the above-mentioned l i g h t i n t e n s i t y was maintained by a time switch f o r a day-length of 16 hours. Containers f o r r e a r i n g eggs and young For experiments I=X eggs were reared i n c l o t h baskets (10x10x15cm) made from nylon " h o r s e h a i r " c r i n o l i n e (10 meshes/cm) w i t h nylon c h i f f o n l i n e r s (30 meshes/cm). Eight baskets were suspended i n water i n a tank i n two rows ( f i g u r e 2) w i t h 10 cm of the height (out of 15 cm) under-water. Baskets i n the f i r s t row (nearer to instrument panel) were always used f o r r e a r i n g the eggs u n t i l hatching. Eggs r e s t e d on the bottom of the baskets. A f t e r hatching, the young were reared i n the baskets i n the second row (near the o u t l e t ) u n t i l they were ready f o r p r e s e r v a t i o n . In some r e p l i c a t i o n s of experiment, young were reared i n s l i g h t l y l a r g e r baskets (12x12x15.5cm). Except f o r s i z e , these baskets were s i m i l a r to the smaller ones. Approximately 12 cm out of 15.5 cm of the height of the l a r g e r basket remained under water when suspended i n tanks. In a s i n g l e experiment, the c o n t r o l and the treatments were reared i n the same s i z e of baskets to avoid any e f f e c t s of "space f a c t o r " on development (Brown 1946; Comfort 1956). Figure 3. Arrangement of b o t t l e s i n a tank f o r experiments XI - XIV. J - A i r j e t ; L - B o t t l e . Figure 4. Vertebral column showing the f i r s t vertebra (I) i n a cleared j u v e n i l e tnedaka. For experiments XI-XIV, eggs were reared i n 710 ml wide-mouthed b o t t l e s . Each b o t t l e c o n t a i n i n g 300 ml of l i q u i d ( s o l u t i o n or water) was f l o a t e d i n a tank and provided w i t h an a i r j e t ( f i g u r e 3 ) . A f t e r hatching the young were reared i n baskets except i n experiment XI(a) where the young were reared i n the appropriate s o l u t i o n s i n . b o t t l e s . A l t o g e t h e r 8 b o t t l e s were kept i n one tank. Breeding and hatching One of the fourteen tanks was used f o r h o l d i n g the brook stock at 24°C. Eggs from 27 p a i r s of parents were used f o r the experiment. Experiment 1 ( e f f e c t of malachite green) was repeated twice w i t h eggs of parent W and Y, w h i l e f o r experiment I I eggs of parent Y and A were u t i l i s e d . Experiment I I I was performed w i t h eggs of parent U. Only a s i n g l e r e p l i c a t i o n of experiment IV was conducted w i t h eggs of parent T but experiment V was repeated twice x^ith eggs of parent R and V. In experiment VI,-eggs of parent Y were used. Parent F and J and r e c i p r o c a l crosses thereof g i v i n g small and l a r g e eggs r e s p e c t i v e l y were used f o r the f i r s t r e p l i c a t i o n of experiment V I I . . For the second, eggs of parent N and S and r e c i p r o c a l crosses thereof were used. E f f e c t s of sustained temperature (experiment IX) were test e d on eggs of parents A, B, C, D, E, G, H, I , K, Q, R, V, W, X and Y. Eggs of parent W, Y and A were reared i n d i f f e r e n t i n t e n s i t i e s and durations of l i g h t i n experiment X. In thyroxine and thiourea treatments, eggs of parents G, H, 0, F, Q, S, V, Y, a and b were u t i l i z e d . Eggs of parent Y were used f o r experiment X I I ( d i n i t r o p h e n o l e f f e c t ) and X I I I (urethan e f f e c t ) . E f f e c t of s a l i n i t y (experiment XIV) was tested w i t h eggs of parent a. -10-Breecling and hatching (con'd) Breeding occurred every morning soon a f t e r the l i g h t went on. The eggs adhered to the female's abdomen. Some of the parents l a i d eggs three to four hours a f t e r the l i g h t s were on. The number of eggs l a i d , and t h e i r frequency, were not uniform. As a r e s u l t , i n some experiments egg numbers were not uniform (see ta b l e s on the record of the number and m o r t a l i t y of eggs). As the number of eggs given by a female was sm a l l , eggs obtained on several successive days were used f o r a s i n g l e treatment i n any experiment. Every morning eggs were removed from the female's abdomen w i t h i n 15 to 30 minutes of spawning. Each egg was then separated, counted and t r a n s f e r r e d to the desired treatment basket or b o t t l e . Eggs of d i f f e r e n t parent were reared i n separate c o n t a i n e r s , and u s u a l l y allowed to hatch n a t u r a l l y . In. a few instances,hatching was induced by g i v i n g the eggs a temperature shock (Hagnuson 1961) or by-p u t t i n g them i n a beaker away from aerated water (Kinne and Kinne 1962) ( D e t a i l s i n the tables on eggs numbers and m o r t a l i t y ) . Care of eggs To prevent fungus a t t a c k , eggs i n baskets were t r e a t e d twice d a i l y w i t h one eye-dropped f u l l of malachite green s o l u t i o n (1:200,000). As a r e s u l t of the c i r c u l a t i o n and mixing of water by a e r a t i o n , the s o l u t i o n was spread unifor m l y i n s i d e the basket and was replaced by freshwater i n f i f t e e n to twenty minutes. Baskets were checked every day f o r dead eggs which were discarded when discovered. M a l a c h i t e green was not used on eggs reared i n b o t t l e s . The dead or fungus attacked eggs were simply removed w i t h a p i p e t t e . -11-Foods and feeding In a l l experiments, l i v i n g Paramecium sp were fed to the newly hatched medaka four times each day f o r at l e a s t 7 days. Commencing from the t h i r d day a f t e r hatching, the young were a l s o supplied w i t h l i v e b r i n e shrimp n a u p l i i (Artemia s a l i n a ) twice every day i n conjunc-t i o n w i t h Paramecium. At the end of the seventh day of hatching the young were fed w i t h bri n e shrimp n a u p l i i only and t h i s was continued u n t i l the f i s h were preserved. Brine shrimp n a u p l i i was always supplied i n excess and the l e f t o v e r , food i n the baskets from one day was removed on the morning of the next day. The young were reared f o r s i x to ei g h t weeks a f t e r hatching and then preserved i n 4% n e u t r a l f o r m a l i n . Rearing time was a l i t t l e longer i n lower than i n the higher temperature so that samples of approximately the same length frequency were obtained from a l l treatments. C l e a r i n g and counting of m e r i s t i c s e r i e s . Two to three weeks a f t e r p r e s e r v a t i o n , f i s h were dyed w i t h A l i z a r i n and cl e a r e d under an u l t r a v i o l e t lamp i n KOH s o l u t i o n s . In c l e a r i n g and s t a i n i n g , the procedure o u t l i n e d by H o l l i s t e r (1934) was followed w i t h minor m o d i f i c a t i o n s as to the concentration of KOH s o l u t i o n s and d e v i a t i o n of exposure to u l t r a v i o l e t ray. A f t e r c l e a r i n g , specimens were f i r s t measured and then counted i n g l y c e r i n e under a b i n o c u l a r microscope f i t t e d w i t h c r o s s h a i r and v e r n i e r stage. The f o l l o w i n g m e r i s t i c s e r i e s wer counted: (a) Vertebrae; Figure 5. Ve r t e b r a l column and caudal f i n rays i n a cleared j u v e n i l e medaka. L - Last v e r t e b r a ; U - U r o s t y l e ; S - Secondary caudal f i n rays. Figure 6. P e c t o r a l f i n :ays i n a cleared j u v e n i l e medaka. I - F i r s t ray ; L - Last ray. -13-Figure 8. Dorsal f i n rays i n a cleared j u v e n i l e medaka showing the f i r s t and l a s t ray counted. I -F i r s t ray; L - Last ray. -14-(b) P e c t o r a l rays; (c) Anal r a y s ; (d) Dorsal rays and (e) Caudal r a y s . (a) Vertebrae ... A l l vertebrae from the b a s i - o c c i p i t a l to the u r o s t y l e were counted. Since the centrum of the f i r s t v e rtebra was not c l e a r l y v i s i b l e , the f i r s t neural process behind the o c c i p i t a l r e g i o n was considered to represent the f i r s t vertebra ( f i g u r e 4 ) . Thereafter each element having a c l e a r neural process and r i b s or haemal process was counted as a s i n g l e v e r t e b r a . The u r o s t y l e was counted as one v e r t e b r a . The v e r t e b r a l count was subdivided i n t o abdominal and caudal elements; the f i r s t centrum showing a haemal process was treated as the f i r s t caudal vertebrae. In some cases where two centra were fused or c l o s e l y apposed, these were counted as two only i f there were two d i s t i n c t neural and haemal processes-. (b) P e c t o r a l f i n rays ... The p e c t o r a l rays stained w e l l and were c l e a r l y v i s i b l e . When s t a i n i n g was poor, the rays were not counted. Rays from both f i n s were counted i n the m a j o r i t y of the experimental l o t s hut i n a few cases only p e c t o r a l rays of the l e f t f i n were counted. The rays a t the periphery on e i t h e r side of a p e c t o r a l f i n were very small but they were included i n the count ( f i g u r e 6). (c) Anal and d o r s a l f i n rays ... These were d i s t i n c t and a l l were counted. The l a s t two were counted as two independent rays instead of one as counted by others (Taning, 1944; Seymour, 1956). ( f i g u r e s 7 and 8 ) . (d) Caudal f i n rays ... Here two types of rays - i . e . primary and secondary rays were d i s t i n g u i s h e d ; rays attached to the lower and upper halves of the hypural were treated as the primary rays. These rays tended to branch at t h e i r t i p . Unbranched and smaller rays on the outer side of both the upper and lower primary rays were considered as secondary rays ( f i g u r e 5). -15-The data were analysed mainly by comparing the means by • t' test using the method outlined by Dixon and Massey (1957). Two means were considered different only when the calculated value of 't' was greater than the tabled value of *t* at P=.01. Calculation of hatching time A wide variation in the time of hatching of eggs from the same spawning was observed in medaka. This was similar to the results found for sea-trout (Marckmann, 1954) and other fishes. Although most eggs hatch within a short period of time, some required much longer time. In order to avoid the bias introduced by the late hatching eggs in the calculation of hatching time, the time required for 50% hatching was used in comparisons of the time required for different treatments. This calculation was further complicated by the fact that the eggs for the samples were accumulated over three or four days. The mean time to 50% hatching was therefore calculated as shown in the following example: Time to 50% hatching of the eggs of genotype X in 30°C Date Time Total Eggs E g g s L a i d 21 January 62 0945 7 22 " " 0945 29 23 " " 09 50 39_ 75 -16-Total hours for a l l eggs 7 x 0 - 0 29 x 24 » 696 39 x 72.08 o 2811 3507 Average time •» 3507 -r 75 • 47.0 hours Total hatched » 72 Time required for 36 eggs to hatch - February 1, ,962 .... 1700 hours Total time from 0945 hours January 21, 1962 to 1700 hours February 1, 1962 =271 hours Time to 50% hatching » 271.00-47.0 = 224 hours -17-EFFECT OF MALACHITE GREEN (EXPERIMENT I) In t r o d u c t i o n In a l l experiments where eggs were reared i n c l o t h baskets, eggs x^ere treated xcith malachite green s o l u t i o n (1:200,000) tx^ice d a i l y to c o n t r o l fungus. This experiment \<ias performed to t e s t the e f f e c t of malachite green treatment on the m e r i s t i c s e r i e s . D e s c r i p t i o n of experiment Eggs of parent W and Y were reared i n small c l o t h baskets u n t i l hatching. In a l l except one l o t ( c o n t r o l of genotype W) eggs hatched n a t u r a l l y . The c o n t r o l l o t of genotype W had not done so long a f t e r the hatching xras due. These eggs-x^ere, t h e r e f o r e , induced to hatch by simply removing them i n t o a beaker from the basket. The beaker was f l o a t e d i n the same temperature ( i . e . , 30°C) bath but a e r a t i o n was dis c o n t i n u e d . Eggs hatched r a p i d l y and the larvae x^ere then reared i n lar g e c l o t h baskets i n both r e p l i c a t i o n s . R e s u lts As may be expected, s u r v i v a l of eggs up to hatching x^as lox?er i n both genotypes reared without malachite green treatment (Table I ) . In genotype W, s u r v i v a l of the young u n t i l p r e s e r v a t i o n x^as l e s s than 50% i n the l o t x^ithout malachite green treatment. D i f f e r e n c e s between the mean v e r t e b r a l (Table I I ) , p e c t o r a l ray (Table I I I ) , anal ray (Table IV) d o r s a l ray (Table V) and t o t a l caudal rays (Table VI) of the samples from treated and untreated eggs were not s i g n i f i c a n t (P>.05) i n e i t h e r genotypes. 18-Thus i t i s concluded that malachite green as used i n the present s e r i e s of experiments does not a l t e r the m e r i s t i c counts of the medaka. -19-Table I . Egg numbers and m o r t a l i t y i n experiment I: E f f e c t of malachite green. Treatment No. of No. As % of Time to No. sur- As % of Remarks f e r t d . hat- f e r t d . 50% hat- v i v e d to f e r t d . eggs ched egg ching (hrs.) preser- eggs v a t i o n (a) Parent: W Control 100 79 79 460 42 42 Hatching induced by removing eggs i n t o beaker wi th -out a e r a t i o n Eggs t r e a t e d w i t h m. 100 96 96 226 92 92 green (b) Parent: Y Control 100 85 85 270 80 80 Eggs t r e a -ted w i t h 100 98 98 226 95 95 m. green Table V I I : Egg number and m o r t a l i t y i n experiment I I . E f f e c t of egg r e a r i n g / c o n t a i n e r s and v a r i a b i l i t y of a e r a t i o n . (c) Parent: a Nature of c o n t a i n e r In b o t t l e w i t h aer- 50 43 86 328 32 64 In b o t t l e up a t i o n to hatching In b o t t l e without 50 38 76 156 35 70 In b o t t l e up a e r a t i o n to hatching In c l o t h basket 50 42 84 387 39 78 w i t h aer-a t i o n -20-Table I I . Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment I : E f f e c t of malachite green. Treatment Temp (°C) Total vertebrae 29 30 31 32 Number Mean Remarks (a) Parent: W Control 30° 1 21 20 42 30.45 Eggs t r e a -ted w i t h m. green 30° 33 17 50 30.34 (b) Parent: Y Cont r o l 30° 13 65 2 80 30.86 Eggs t r e a -ted w i t h m. green 30° 13 79 3 95 30.89 Table V I I I . Frequency E f f e c t of d i s t r i b u t i o n of t o t a l vertebrae i n experiment I I : egg r e a r i n g c o n t a i n e r s and v a r i a b i l i t y of a e r a t i o n . (a) Parent: a In b o t t l e w i t h aer-a t i o n 30° 21 12 33 30.36 In b o t t l e wi thout a e r a t i o n 30° 18 14 1 33 30.51 In c l o t h basket w i t h aer-a t i o n 30° 27 12 39 30.31 (b) Parent: Y In b o t t l e w i t h aer-a t i o n 26° 12 54 4 70 30.89 In c l o t h basket 26° 3 71 3 87 31.00 w i t h aer-a t i o n 21-Table I I I . Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment I: E f f e c t of malachite green. Treatment Temp (°C) Pe c t o r a l rays Number . Mean Remarks 10 11 12 13 14 (a) Parent: W Contr o l 30° 19 59 6 84 10.84 Eggs t r e a -ted w i t h 30° 22 77 1 100 10.79 m. green (b) Parent: Y Control 30° 7 128 25 160 12.11 Eggs t r e a -ted w i t h 30° 3 140 46 1 190 12.24 m. green Table IX. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment I I : E f f e c t of egg r e a r i n g c o n t a i n e r s and v a r i a b i l i t y of a e r a t i o n . (a) Parent: a In b o t t l e w i t h aer- 30° 2 45 19 66 11.26 a t i o n In b o t t l e wi thout 30° 2 47 17 66 11.23 a e r a t i o n In c l o t h basket wi th 30° 3 57 18 78 11.19 a e r a t i o n (b) Parent: Y In b o t t l e w i t h aer- 26° 63 77 140 12.55 a t i o n In basket w i t h aer- 26° 63 106 3 172 12.65 a t i o n -22-Table IV. Frequency d i s t r i b u t i o n of anal rays i n experiment I: E f f e c t of malachite green. Treatment Temp (°C) 16 17 Anal rays 18 19 20 21 Number Mean Remarks (a) Parent: W Co n t r o l 30° 1 3 21 17 42 18.29 Eggs t r e a -ted w i t h 30° 7 29 14 50 18.14 m. green (b) Parent: Y Control 30° 4 37 37 2 18.46 Eggs t r e a -ted w i t h 30° 39 52 4 95 18.63 m. green Table X. Frequency d i s t r i b u t i o n of anal rays i n experiment I I . E f f e c t of egg r e a r i n g containers and v a r i a b i l i t y of a e r a t i o n . (a) Parent: a In b o t t l e w i t h aer- 30° 4 16 11 2 33 17.36 Not d i f f e r e n t fromU«et a t i o n sample (P>.05) In b o t t l e without 30° 3 13 14 3 33 17.51 a e r a t i o n In basket w i t h aer- 30° 4 14 15 6 39 17.59 a t i o n (b) Parent: Y In b o t t l e w i t h aer- 26° 2 18 37 12 1 70 18.89 a t i o n In basket w i t h aer- 26° 2 28 37 12 1 87 18.79 a t i o n -23-Table V. Frequency d i s t r i b u t i o n of do r s a l rays i n experiment I : E f f e c t of malachite green. Treatment Temp C ° c ) Dorsal rays Number Mean Remarks 5 6 7 8 (a) Parent: w Control 30° 34 8 42 6.19 Eggs t r e a t e d w i t h m. green 30° 33 16 1 50 6.36 (b) Parent: Y Contr o l 30° 69 10 1 80 6.15 Eggs tre a t e d w i t h m. green 30° 76 18 1 95 6.21 Table X I . Frequency d i s t r i b u t i o n of do r s a l rays i n experiment I I : E f f e c t of egg r e a r i n g containers and v a r i a b i l i t y of a e r a t i o n . (a) Parent: a In b o t t l e w i t h aer- 30° 1 32 33 5.97 a t i o n In b o t t l e without a e r a t i o n 30° 32 1 33 6.03 In c l o t h basket 30° 38 1 39 6.03 w i t h aer-a t i o n (b) Parent: Y In b o t t l e w i t h aer- 26° 50 20 70 6.29 a t i o n In c l o t h basket 26° 1 64 22 87 6.24 w i t h a e r a t i o n -24-Table VI. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment I : E f f e c t of malachite green. Treatment Temp (°C) 19 20 21 Total caudal 22 23 24 rays 25 26 27 28 Number Mean Remark s (a) Parent: VJ Co n t r o l 30° 2 10 13 13 3 1 42 22.19 Eggs tre a t e d 30° 9 10 23 6 1 1 50 22.64 Not d i f f e r e n t w i t h m. green from con-t r o l (P>.05) (b) Parent: Y Co n t r o l 30° 7 18 35 15 4 1 80 22.96 Eggs t r e a -ted w i t h 30° 9 17 42 19 6 93 22.96 m. green Table X I I . Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment I I . E f f e c t of egg r e a r i n g containers and v a r i a b i l i t y of a e r a t i o n . (a) Parent a In b o t t l e w i t h aer- 30° 3 15 10 4 1 33 23.54 a t i o n In b o t t l e without 30° 1 2 10 12 6 2 33 23.81 a e r a t i o n In c l o t h basket 30 C w i t h a e r a t i o n (b) Parent: Y In b o t t l e w i t h aer- 26 c a t i o n 17 2 23 23 16 39 23.51 Tend to be lower than basket 70 21.94 sample (P<.05; >.02) In c l o t h basket w i t h 26° 5 5 32 29 13 3 a e r a t i o n 87 21.56 -25-EFFECT OF EGG REARING CONTAINERS AND VARIABILITY OF AERATION (EXPERIMENT I I ) . I n t r o d u c t i o n This experiment was conducted to t e s t the p o s s i b l e e f f e c t s of d i f f e r e n c e s between containers used f o r r e a r i n g eggs i n the present s e r i e s of experiments on m e r i s t i c characters. D e s c r i p t i o n of Experiment. Eggs of genotype a " were reared to hatching i n three sets of c o n d i t i o n s ; i n c l o t h basket i n the tank x^here water was being replaced every 20 hours; i n 300 ml of water i n a b o t t l e w i t h continuous a e r a t i o n ; and i n a b o t t l e i n 300 ml of stagnant water without any a i r supply. Upon hatching, larvae from a l l c ontainers were t r a n s f e r r e d to small c l o t h baskets and reared t h e r e i n u n t i l p r e s e r v a t i o n . A l l three l o t s were reared i n the same tank i n 30°C temperature bath throughout the experiment. Data obtained from genotype Y reared i n basket and i n b o t t l e w i t h a e r a t i o n i n connection w i t h experiments IX and XI are a l s o included here f o r comparison. R e s u l t s S u r v i v a l of eggs up to hatching i n a l l three l o t s of genotype a was h i g h . S u r v i v a l i n the l o t s i n aerated b o t t l e and c l o t h basket was almost i d e n t i c a l but i n the l o t i n stagnant and non-aerated water, t h i s was somewhat lower (Table V I I ) . V a r i a t i o n i n the method of r e a r i n g eggs d i d not a f f e c t the mean v e r t e b r a l counts i n any of the genotypes (Table V I I I ) . D i f f e r e n c e s between the p e c t o r a l (Table I X ) , anal (Table X), and d o r s a l rays (Table X) was a l s o not s i g n i f i c a n t (P> .05). In genotype a -26-mean t o t a l caudal ray counts of the three samples (Table X I I ) d i d not d i f f e r from each other s i g n i f i c a n t l y but the mean count of the l o t of genotype Y i n b o t t l e tended to be higher than the mean of the l o t reared i n basket a l l through (P = .02 - .05). Typeof containers used t h e r e f o r e , introduced no b i a s i n m e r i s t i c s e r i e s w i t h the p o s s i b l e exception/caudal ray count. -27-Table X I I I . Egg numbers and m o r t a l i t y i n experiment I I I : E f f e c t of q u a l i t y of eggs on successive days from same parent. Date Parent No. of No. As % of Time No. sur- As % of Remarl eggs f e r t d . hat- f e r t d . to 50% vived to f e r t d . obtained eggs ched eggs hatch- preser- eggs ing (hrs.) v a t i o n March 23, 1961 U 47 45 96 265 36 77 A p r i l 8, 1961 U 55 54 98 262 37 67 Table XIV. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment I I I . E f f e c t of q u a l i t y of eggs on successive days from same parent. Date eggs obtained Temp. <°C) Total 30 vertebrae 31 32 Number Mean Remarks March 23, 1961 24° 2 27 1 30 30.97 A p r i l 18, 1961 o 24 3 30 3 36 31.00 Table XV. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment I I I . E f f e c t of q u a l i t y of eggs on successive days from same parent. Date Temp P e c t o r a l rays Number Mean Remarks eggs obtained (°C) 11 12 13 March 23, 1961 24° 14 42 4 60 11.83 A p r i l 8, 1961 24° 13 49 10 72 11.96 -28-Table XVI. Frequency d i s t r i b u t i o n of anal rays i n experiment I I I . E f f e c t of q u a l i t y of eggs on successive days from same parent. Date eggs obtained Temp Anal rays Number Mean Remarks (°C) 19 20 21 Parent U March 23, 1961 24° 5 16 9 30 20.13 A p r i l 8, 1961 24° 11 12 3 36 19.78 Tends to be lower. (P<.05; >.02) Table XVII. Frequency d i s t r i b u t i o n of dorsal rays i n experiment I I I . Date eggs obtained Temp Dorsal rays Number Mean Remarks ( C) 6 7 March 23, 1961 24° 29 1 30 6.03 A p r i l 8, 1961 24° 34 2 36 6.06 Table X V I I I . Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment I I I . Date eggs obtained Temp To t a l caudal rays Number Mean Remarks (°C) 21 22 23 24 25 26 March 23, 1961 2 11 14 3 30 23.60 A p r i l 8, 1961 1 11 12 9 2 1 36 23.08 Tends to be lower. (P<.05; >.02) -29-EFFECT OF SUCCESSIVE DAYS EGGS (EXPERIMENT I I I ) I n t r o d u c t i o n The number of eggs l a i d by a female on successive days was always s m a l l . This n e c e s s i t a t e d the use of several days eggs f o r any one treatment and f o r d i f f e r e n t treatments. I t was therefore necessary to test the e f f e c t of successive l o t s of eggs 6f d i f f e r e n t days on the m e r i s t i c s e r i e s considered. D e s c r i p t i o n of Experiment. Two batches of eggs from genotype U, obtained on two d i f f e r e n t dates w i t h an i n t e r v a l of 15 days i n between, were reai-ed i n 24°C under i d e n t i c a l c o n d i t i o n s ( w i t h the exception that the number of f e r t i l i z e d eggs put i n was d i f f e r e n t ) . R e s u lts S u r v i v a l to hatching was more than 957=, i n both l o t s . There xtfas a l s o no d i f f e r e n c e between the two l o t s i n terms of s u r v i v a l at the time these were preserved (Table X I I I ) . Mean v e r t e b r a l (Table XIV), p e c t o r a l (Table XV) and d o r s a l ray (Table XVII.) counts of the two l o t s showed no s i g n i f i c a n t d i f f e r e n c e . Mean anal ray count of the sample from eggs of l a t e r date tended to be lower than that of the other (P<^.05; Table XVI. T o t a l caudal ray count of the l o t from l a t e r date eggs a l s o tended to be lower (P<.05) than the other. Despite the tendency of d i f f e r e n c e d i s p l a y e d by anal and caudal counts, i t i s concluded that successive days eggs has no e f f e c t -30-on most m e r i s t i c c h a r a c t e r s . Anal caudal counts remain s e n s i t i v e even a f t e r hatching (as w i l l be shown l a t e r ) and v a r i a t i o n may be due to some unknown f a c t o r s not dependent on the time elapsed between the two egg batches t e s t e d . -31-EFFECT OF MECHANICAL SHOCK (EXPERIMENT IV) In t r o d u c t i o n Because d i f f e r e n t egg l o t s were probably subjected to s l i g h t l y d i f f e r e n t handling, t h i s experiment was conducted to te s t the e f f e c t of mechanical shocks during care of eggs on the formation of m e r i s t i c characters. D e s c r i p t i o n of experiment E f f e c t s of disturbance was tested by shaking the developing eggs v i g o r o u s l y . Eggs were picked up from the r e a r i n g basket and placed i n an S-ounce b o t t l e p a r t l y f i l l e d w i t h water (approximately 3 ounces) taken from the temperature bath. The b o t t l e was then shaken v i g o r o u s l y w i t h i t s l i d t i g h t l y screwed. A f t e r four minutes of continuous shaking, eggs were put back i n the appropriate baskets. Three l o t s of eggs of parent T were used f o r t h j s experiment. The f i r s t batch was used as c o n t r o l and allowed to hatch without any disturbance. The second l o t of eggs were subjected 'to shaking immediately a f t e r f e r t i l i z a t i o n and t h e r e a f t e r shaking was repeated d a i l y f o r four minutes u n t i l hatching. Eggs of the t h i r d l o t were allowed to develop undisturbed f o r the f i r s t four days f o l l o w i n g f e r t i l i z a t i o n . A f t e r t h i s p e r i o d , these eggs were a l s o subjected to four minutes of continuous shaking d a i l y u n t i l hatching. A l l three l o t s were reared i n the same temperature bath. Results Shaking eggs from f e r t i l i z a t i o n to hatching d i d not a f f e c t s u r v i v a l to hatching. Compared to the c o n t r o l s u r v i v a l of the l o t -32-subjected to shaking (from the date of f e r t i l i z a t i o n ) was s l i g h t l y higher (Table XIX). In the t h i r d l o t shaken a f t e r 4 days of undisturbed development, s u r v i v a l at the corresponding stage was lower. Mean t o t a l v e r t e b r a l count (Table XX), p e c t o r a l ray (Table XXI), d o r s a l ray (Table XXIII) and t o t a l caudal ray counts (Table XXIV) of the three l o t s d i d not d i f f e r from each other s i g n i f i c a n t l y . Mean anal ray count of the sample from eggs shaken d a i l y from f e r t i l i z a t i o n was not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l mean but the mean of the sample from eggs shaken a f t e r four days undisturbed development tended to be higher (P<. 05) than the mean of the c o n t r o l (Table X X I I ) . The above r e s u l t s f o r a l l characters except anal rays show that shaking of eggs does not a l t e r the m e r i s t i c counts. Anal rays are f i n a l l y f i x e d a f t e r hatching and as such, observed v a r i a t i o n may not be a t t r i b u t a b l e to shaking of eggs. -33-Table XIX. Egg numbers and m o r t a l i t y i n experiment IV. E f f e c t of mechanical shock. Treatment No. of No. As % of Time to No. sur- As % of f e r t d . hat- f e r t d . 50% hat- vived to f e r t d . eggs ched eggs ching h r s . p r e s e r - eggs v a t i o n Parent T Con t r o l 40 30 75 755 23 57 Shaken 4 minutes 55 42 76 287 40 73 d a i l y from f e r t i l i z a t i o n F i r s t 4 days undisturbed; t h e r e a f t e r 39 26 67 287 21 54 shaken 4 min-utes d a i l y Table XXV. Egg numbers and m o r t a l i t y i n experiment V: E f f e c t of p r i c k i n g the chorion. (a) Parent R Con t r o l 100 84 84 477 74 74 Chorion p r i c k e d 57 17 30 275 12 21 (b) Parent V Control 75 71 95 317 70 93 Chorion p r i c k e d 75 29 39 321 29 39 -34-Table XX. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment IV: E f f e c t of mechanical shock. Treatment Temp ( ° c ) Total vertebrae 29 30 31 32 Number Mean Remark s Parent T Control 24° 17 5 22 30.23 Shaken 4 minutes d a i l y from f e r t i l i z -a t i o n 24° 27 13 40 30.32 Not d i f f e r e n t from c o n t r o l (P>.05) F i r s t 4 days un-dis t u r b e d ; shaken 4 minutes d a i l y 24° 1 15 5 21 30.19 Do (P?.05) Table XXVI. Frequency E f f e c t of d i s t r i b u t i o n p r i c k i n g the of t o t a l c h orion. vertebrae i n experiment V: (a) Parent R Control 26° 19 54 1 74 30.76 Chorion p r i c k e d 26° 3 7 2 12 30.92 (b) Parent V Control 26° 2 63 5 70 31.04 Chorion p r i c k e d 26° 25 4 29 31.14 Not d i f f e r e n t from c o n t r o l (P>.05) -35-Table XXI. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment IV: E f f e c t of mechanical shock. Treatment Temp C ° c ) P e c t o r a l 10 11 rays 12 Number Mean Remarks Parent T Contr o l 24° 9 11 20 11.55 Shaken 4 minutes d a i l y from f e r t i l i z -a t i o n 24° 27 13 40 11.33 Not d i f f e r e n t from c o n t r o l (P>.05) F i r s t 4 days undi sturbed; t h e r e a f t e r shaken 4 min-utes d a i l y . 24° 14 7 21 11.33 Do (P7.05) Table XXVII. Frequency d i s t r i b u t i o n of pe c t o r a l of p r i c k i n g the chorion. . rays i n experiment V: E f f e c t (a) Parent R Contr o l 26° 3 88 57 148 11.37 Chorion p r i c k e d 26° 15 9 24 11.37 (b) Parent V Contr o l 26° 8 113 19 140 11.08 Chorion p r i c k e d 26° 2 42 14 58 11.21 Not d i f f e r e n t from control(P>.05) -36-Table XXII. Frequency d i s t r i b u t i o n of anal rays i n experiment mechanical shocks. IV: E f f e c t of Treatment Temp Anal rays (°C) 17 18 19 20 21 Number Mean 22 Remarks Parent T Control 24° 1 11 7 3 22 18.53 shaken 4 min-utes d a i l y from f e r t i l -i z a t i o n 24° 14 20 6 40 18.80 Not d i f f e r e n t from c o n t r o l (P>.05) F i r s t 4 days undisturbed; thereaf t e r shaken 4 min-utes d a i l y . ' 24° 5 10 5 1 21 19.09 Tends to be higher than c o n t r o l (P<.05;>.02) Table XXVIII. Frequency d i s t r i b u t i o n of p r i c k i n g the chorion. anal rays i n experiment V: E f f e c t of (a) Parent R Contr o l 26° 4 14 48 7 1 74 19.82 Chorion p r i eked 26° 3 7 2 12 13.92 Lower than con t r o l (P<.01) (b) Parent V Contr o l 26° S 34 26 2 70 19.31 Chorion 26° 4 13 5 4 3 29 18.62 Do (P<.01) p r i eked -37-Table X X I I I . Frequency d i s t r i b u t i o n of do r s a l rays of mechanical shock. i n experiment IV: E f f e c t Treatment Temp (°C) Dorsal rays 6 7 8 Number Mean Remarks Parent T Con t r o l 24° 13 9 22 6.42 Shaken 4 min-utes d a i l y from f e r t i l i z a t i o n . 24° 26 14 40 6.35 F i r s t 4 days undisturbed; t h e r e a f t e r shaken 4 min-utes d a i l y 24° 12 9 21 6.43 Table XXIX. Frequency d i s t r i b u t i o n p r i c k i n g the chorion. of d o r s a l rays i n experiment V: E f f e c t of (a) Parent R Co n t r o l 26° 30 43 1 74 6.60 Chorion p r i c k e d 26° 11 1 12 6.08 Lower than c o n t r o l . (P<.01) (b) Parent V Control 26° 53 13 *70 6.13 "5 f i s h w i t h 5 do r s a l rays. Chorion p r i c k e d 26° 16 7 **29 6.03 **6 f i s h w i t h 5 do r s a l rays. -38-Table XXIV. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment IV. E f f e c t of mechanical shock. Treatment Temp (°C) 20 Total caudal rays 21 22 23 24 25 Number Mean Remarks Parent T Contr o l 24° 4 4 9 4 1 22 22.73 Shaken 4 min' utes d a i l y from f e r t i l -i z a t i o n " 24° 2 5 k7 13 3 40 23.25 Not d i f f e r e n t from c o n t r o l (P>.05) F i r s t 4 days undisturbed; t h e r e a f t e r shaken 4 min utes d a i l y 24° 1 5 9 3 3 21 23.09 Not d i f f e r e n t from c o n t r o l . (P>.05) Table XXX. Frequency E f f e c t of d i s t r i b u t i o n of t o t a l p r i c k i n g the chorion. caudal rays i n experiment V. (a) Parent R Contr o l 26° 1 22 15 28 7 1 74 22.28 Chorion p r i c k e d 26° 1 3 5 2 1 12 21.92 Not d i f f e r e n t from c o n t r o l (P>.05) (b) Parent V Contr o l 26° 5 8 41 13 2 69 23.50 Chorion p r i c k e d 26° 5 10 12 2 29 23.38 -39-EFFECT OF PRICKING THE CHORION (EXPERIMENT V) In t r o d u c t i o n In one experiment w i t h thyroxine and th i o u r e a , chorion p r i c k e d eggs were used. I t was ther e f o r e considered necessary to t e s t i f p r i c k i n g of chorion as such a l t e r s any of the m e r i s t i c characters. D e s c r i p t i o n of experiment This experiment was r e p l i c a t e d twice w i t h eggs of genotypes R and V. The chorion of each egg was pr i c k e d under a bi n o c u l a r microscope w i t h a very sharp d i s s e c t i n g needle s h o r t l y a f t e r f e r t i l i z a t i o n . Both c o n t r o l and the chorion p r i c k e d eggs of genotype R were reared i n small baskets i n 26°C i n the same tank from f e r t i l i z a t i o n to p r e s e r v a t i o n . In the case of genotype V, control and chorion p r i c k e d egg l o t s were reared i n 300 ml of aerated water i n b o t t l e i n 26°C bath. A f t e r hatching the young were reared i n small c l o t h baskets u n t i l they were f i n a l l y preserved. Results In both r e p l i c a t i o n s , m o r t a l i t y before hatching was greater i n the chorion p r i c k e d egg l o t s (Table XXV). Mean v e r t e b r a l (Table XXVI), p e c t o r a l ray (Table XXVII) and t o t a l caudal ray (Table XXX) counts of the samples from chorion pricked eggs were not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l means i n e i t h e r of the r e p l i c a t i o n s . Mean anal ray counts of the sample from chorion p r i c k e d eggs i n both r e p l i c a t e s were lower (P^.01) than the r e s p e c t i v e c o n t r o l mean (Table XXVIII). Mean d o r s a l ray count of the sample from p r i c k e d eggs of genotype R was lower than the c o n t r o l mean (P<.01) but no d i f f e r e n c e was found i n the other r e p l i c a t i o n (Table XXIX). -40-From the above, i t appears that most m e r i s t i c counts are not a f f e c t e d by p r i c k i n g of the chorion- As i n other experiments, the anal ray counts produced ambiguous r e s u l t s . Because of t h e i r l a t e f i x a t i o n (demonstrated l a t e r ) , they may have been influenced by other v a r i a b l e s than that under i n v e s t i g a t i o n . -41-Table XXXI. Egg numbers and m o r t a l i t y i n experiment VI. E f f e c t of egg d e n s i t y . Parent No. of No. hat- As % of Time to No. sur- As % of Remarks f e r t d . ched f e r t d . 50% hat- vived to f e r t d . eggs eggs ching(hrs). preser- eggs v a t i o n Y 25 25 100 297 25 100 Y 50 50 100 273 50 100 Y 100 96 96 291 90 90 Y 200 193 97 283 178 89 Table XXXII. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment VI. No. of eggs (°C) Temp To t a l vertebrae Number Mean Remarks 30 31 32 25 24° 1 22 2 25 31.04 50 24° 3 42 5 50 31.04 100 24° 5 79 6 90 31.01 200 24° 12 155 11 178 30.99 Table XXXIII. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment VI. No. of eggs Temp (°C) Pe c t o r a l 11 12 rays Number Mean Remarks 13 14 25 o 24 10 38 2 50 12.84 50 24° 1 20 76 2 99 12.80 100 24° 41 136 3 180 12.79 200 24° 3 120 215 14 352 12.68 Not d i f f e r e n t from 25 egg sample. (P>.05) -42-Table XXXIV., Frequency d i s t r i b u t i o n of egg d e n s i t y . of anal rays i n experiment VI. E f f e c t No. of Temp eggs (°C) Anal rays 17 18 19 20 21 Number Mean Remarks Parent Y o 25 24 1 13 8 3 25 19.52 50 24° 1 4 30 15 50 19.18 Not d i f f e r e n t from 25 eggs sample (P>.05) 100 24° 21 50 18 1 90 18.99 (1) Lower than 25 eggs sample (P<.01) (2) Not d i f f e r e n t from 50 & 200 eggs sample (P>.05) 200 24° 2 33 101 39 1 176 19.02 (1) Lower than 25 egg sample (P<.01) (2) Not d i f f e r e n t from 50 eggs sample (P>.05) Table XXXV.. Frequency d i s t r i b u t i o n of dorsal rays i n experiment VI. No. of eggs (°C) Temp Dorsal 6 7 rays 8 Number Mean Remarks Parent Y 25 24° 15 10 25 6.40 50 24° 33 16 1 50 6.36 100 24° 68 22 90 6.24 Not d i f f e r e n t from any other sample (P>.05) 200 24° 122 54 176 6.31 -43-Table XXXVI. Frequency distribution of total caudal rays in experiment VI. No. of eggs Temp (°C) Total 17 18 19 20 caudal 21 22 rays 23 24 25 Number Mean Parent 25 Y 24° 1 3 6 10 5 25 22.60 50 24° 17 12 14 5 2 50 22.26 1 100 24° 2 5 35 33 11 3 1 90 21.67 2 200 24° 1 1 3 7 55 64 33 9 1 174 21.81 3 (1) Not different from 25 egg sample (P>.05); higher than 100 egg sample (P<.01). (2) Lower than 25 eggs and 50 eggs samples (P<.01); not different from 200 egg samples. (3) Lower than 25 eggs sample $P<.01); tends to be lower than 50 eggs sample (P<.02; > .01). -44-Figure 9. Effect of egg density on mean total vertebrae and pectoral and anal f i n rays of genotype Y (Experiment VI) -45-Figure 10. Effect of egg density on mean dorsal and total caudal f i n rays of genotype Y (Experiment VI) -46-EFFECT OF EGG DENSITY (EXPERIMENT VI) I n t r o d u c t i o n The purpose of experiment VI was to explore the extent and d i r e c t i o n of the i n f l u e n c e of the d i f f e r e n t d e n s i t y of eggs and young upon d i f f e r e n t m e r i s t i c s e r i e s , since egg numbers were not i d e n t i c a l i n other experiments designed to t e s t other v a r i a b l e s . D e s c r i p t i o n of experiment Eggs of genotype Y were Used. Eggs x^ere reared i n l o t s of 200, 100, 50 and 25. For the f i r s t three l o t s , eggs obtained -in consecutive spawning days were used. The l o t i n the l a s t sample was from a s i n g l e day's spawning. A l l the l o t s were reared i n small c l o t h baskets i n 24°C bath i n the same tank, both before and a f t e r hatching. Results S u r v i v a l up to hatching i n d i f f e r e n t l o t s ranged from 96 to 100 percent (Table XXXI). From 25 and 50 eggs, 25 and 50 young survived up to p r e s e r v a t i o n . S u r v i v a l up to hatching i n the l o t s of 100 and 200 eggs was s l i g h t l y lower (96%). Mean t o t a l v e r t e b r a l (Table XXXII: Figure 9 ) , p e c t o r a l ray (Table XXXIII: Figure 9) and d o r s a l ray (Table XXXV: Figure 10) were not a f f e c t e d by egg d e n s i t y . A four or e i g h t f o l d increase i n density appeared to i n f l u e n c e the expression of f i n a l anal ray counts (Table XXXIV: Figure 9 ) . Mean count of the l o t from 25 eggs was s i g n i f i c a n t l y higher than the means from l o t s of -47-100 and 200 eggs. S t a t i s t i c a l t e s t s f a i l e d to r e v e a l any d i f f e r e n c e between the means of the samples from 25 and 50 eggs but the l a t t e r showed a trend towards decrease. Mean counts of samples from 50, 100 and 200 egg l o t s were compared w i t h each other, but no s i g n i f i c a n t d i f f e r e n c e was observed. Results w i t h respect to t o t a l caudal rays are presented i n Table XXXVI and Figure 10. Mean t o t a l caudal rays of the sample from 25 egg-lot was not d i f f e r e n t from that of the 50 egg l o t sample but was s i g n i f i c a n t l y higher than the mean counts of samples from 100 and 200 egg l o t s (P<.01). There was s i g n i f i c a n t d i f f e r e n c e between the mean caudal rays of the samples from 50 and 100 eggs-lot where the mean of the l a t t e r was lower (P<.01). The mean counts of the samples from 50-eggs l o t and 200-eggs l o t revealed a strong tendency of being d i f f e r e n t (P<.02). The means of 100 and 200-eggs l o t samples showed no d i f f e r e n c e (P>.05). Density of eggs or of the young does not therefore i n f l u e n c e the number of vertebrae and p e c t o r a l or d o r s a l f i n rays. E f f e c t s of d e n s i t y alone on the number of anal and t o t a l caudal rays are not c o n c l u s i v e . The e f f e c t becomes apparent perhaps only when the d e n s i t y has reached a c e r t a i n c r i t i c a l l e v e l , not reached i n most' other experiments. -48-Table XXXVII. Egg numbers and m o r t a l i t y i n experiment V I I . E f f e c t of egg s i z e . Parent Ho. of f e r t d . eggs No. hat ched :- As % of f e r t d . eggs Time to 50% hat-ching No. sur-vived to preser-v a t i o n As % of Mean egg f e r t d . diameter eggs (mm) R e p l i c a t i o n I F 98 48 49 263 38 39 1.03 F? J<? 116 58 50 280 54 47 1.06 164 116 71 273 96 59 1.13 J 105 63 60 302 36 34 1.14 R e p l i c a t i o n I I N 100 72 72 360 40 40 1.09 N<? Sc5 100 78 72 360 40 40 1.09 sg Nc? 100 67 67 524 47 47 1.16 s 100 53 53 316 41 41 1.14 Table XXXVIII. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment V I I . Parent Egg s i z e Temp (°C) Total vertebrae Number 29 30 31 32 Mean Remarks R e p l i c a t i o n I F small 24° 4 30 4 38 31.00 Lower than J(P<.01) F£ J<? small 24° 1 23 12 36 30.31 (1) Lower than F (P<.01) (2) Not d i f f e r e n t from J(P>.01) J $ F<? lar g e 24° 1 19 15 1 36 30.44 (1) Lower than F (P<.01) (2) Higher than J(P<.01) J l a r g e 24° 3 28 5 36 30.05 Lower than F(P<.01) -49-Table XXXVIII continued. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experimetn V I I . Parent Egg s i z e Temp To t a l vertebrae Number Mean Remarks (°C) 29 30 31 32 R e p l i c a t i o n I I N small 24° 18 22 40 30.53 Lower than S(P<.01). N?Se? small 24° 6 47 1 54 30.91 (1) Higher than N(P<.01) (2) Lower than S(P<.01) (3) Lower than S?N^ (P<.01) S?N<? large 24° 1 27 14 42 31.31 Tends to be lower than S (P4.02;>.01) S large 24° 17 23 40 31.56 Table XXXIX. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n Table XL. Frequency d i s t r i b u t i o n of anal rays experiment V I I : E f f e c t of egg s i z e . i n experiment V I I . Parent Egg s i z e Temp (°C) P e c t o r a l rays 10 11 12 13 Number Mean Anal rays 16 17 18 19 20 21 22 Number Mean R e p l i c a t i o n I F small 24° 3 31 4 38 11.03 1 20 14 3 38 17.50 F9 J S small 24° 19 17 36 11.47 1 9 24 3 36 17.83 1 J ? Fc? large 24° 29 7 36 11.19 2 10 20 6 36 18.89 J l a r g e 24° 23 12 1 36 11.39 3 6 23 6 1 36 19.06 2 R e p l i c a t i o n I I N small 24° 17 57 5 79 11.85 4 8 16 10 6 40 19.35 3 N9 So* small 24° 70 34 104 5 11.33 2 17 24 11 54 4 19.81 S? Nc? large 24° 7 64 11 82 12.05' 6 26 9 1 42 20.12 5 S large 24° 29 39 2 70 11.61 6 2 8 13 15 1 39 20.13 Note: 1. Higher than F(P<.01); not d i f f e r e n t from J . 2. Not d i f f e r e n t from J(P>.05); tend to be lower than F?J<?(P<.02;>.01) 3. Higher than F(P<.01) 4. Higher than S(P<.01) and N?So*(P<r.01) 5. Lower than S?Nc?and S(P<.01) 6. Lower than S?N<?(P<.01) Note: 1. Lower than J(P<.01); tend to be higher than F(P<.05;>*02); lower than J$Fo"(P<.01) 2. Higher than F(P<.01) 3. Lower than S(P<.01) 4. Tends to be higher than N(P<.02;>.01); not d i f f e r e n t from S?Nc?(P>.05); tend to be lower than S(P<.05;>.02) 5. Not d i f f e r e n t from S(p>.05) -51-Table X LI. Frequency d i s t r i b u t i o n of do r s a l rays i n experiment V I I : E f f e c t of egg s i z e . Parent Egg s i z e Temp (°C) Dorsal 5 6 rays 7 Number Mean R e p l i c a t i o n I F small 24° 5 33 38 5.86 F$ J <? small 24° 34 2 36 6.06 1 J ? Fc? large 24° 28 3 36 2 6.22 J l a r g e 24° 1 34 1 36 6.00 R e p l i c a t i o n I I N small 24° t 20 20 40 3 6.50 Ns? Sc? small 24° 50 4 54 4 6.35 S9 Nc? large 24° 22 20 42 6.47 5 S large 24° 5 27 7 39 6.05 Note: 1. Not d i f f e r e n t from F 2. Not d i f f e r e n t from J 3. Higher than S (P<.01) 4. Not d i f f e r e n t from N (P>.05); higher than S (P<.01) 5. Higher than S (P<.01); Not d i f f e r e n t from N and NS S3 (P>.05) -52-Table XLII. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment VII: E f f e c t of egg s i z e . Parent Egg s i z e Temp (°C) Total caudal 17 18 19 20 21 22 rays 23 24 25 26 Number Mean R e p l i c a t i o n I F smal 1 24° 1 8 13 12 4 38 22.24 F? J o" small 24° 4 15 14 3 36 23.44 J 9- F<? l a r g e 24° 4 11 13 7 1 36 22.72 J l a r g e 24° 2 8 13 10 3 36 23.11 R e p l i c a t i o n II N small 24° 1 12 10 15 1 1 40 23.15 small 24° 1 1 2 9 14 19 6 2 54 23.31 la r g e 24° 2 2 8 18 6 5 41 21.96 s large 24° 2 1 4 1 16 10 5 39 21.82 Note: 1. Higher than F(P<.01) 2. Not d i f f e r e n t from J(P>.05) 3. Higher than S(P<.01) 4. Not d i f f e r e n t from N(P>.05) 5. Not d i f f e r e n t from S(P>.05); lower than N and N?S<? (P<.01) -53-EFFECT OF EGG SIZE (EXPERIMENT VII) I n t r o d u c t i o n This experiment was designed to study the p o s s i b l e e f f e c t s of yolk s i z e , expressed as egg s i z e , on d i f f e r e n t m e r i s t i c s e r i e s , o Taning (1952) found no r e l a t i o n s h i p between yolk s i z e and the v e r t e b r a l number, but Garside and Fry (1959) hypothesized that yolk s i z e of the egg i n f l u e n c e s the number of myomeres. D e s c r i p t i o n of experiment As yolk diameter was the f a c t o r under c o n s i d e r a t i o n , each i n d i v i d u a l egg used was measured under a b i n o c u l a r microscope equipped w i t h an o c u l a r micrometer. The o c u l a r micrometer was c a l i b r a t e d to a piece of c i r c u l a r stage micrometer placed i n s i d e a Syracuse watch g l a s s under water. Quantity of water i n the watchglass was j u s t enough f o r immersing one egg. In making the c a l i b r a t i o n , an egg under water i n the watch g l a s s was brought i n t o sharpest focus by a d j u s t i n g the height of the eye p i e c e s . Then.the stage micrometer was placed i n s i d e ivater i n g l a s s and i t was brought i n t o sharpest focus (same as that f o r the egg) by a l t e r i n g the height of the watchglass and l e a v i n g the eye piece height undisturbed. The l e v e l of the watchglass w i t h the stage micrometer i n it'was a l t e r e d by p u t t i n g g l a s s s l i d e s and cover glasses under i t . The same c a l i b r a t i o n was used f o r measuring the yolk diameter of a l l eggs used i n both the r e p l i c a t i o n s . The yolk diameter of medaka eggs i n s i d e the chorion i s c l e a r l y v i s i b l e . The diameter of the yolk was measured twice from -54-two p o s i t i o n s before the completion of f i r s t cleavage and the mean of the two was recorded as the diameter of that egg. While measuring the yolk diameter, the animal pole of the egg was avoided i n a l l cases. This observation was repeated twice w i t h d i f f e r e n t sets of parents. In each r e p l i c a t i o n two sets of parents were s e l e c t e d on the b a s i s of the y o l k diameter of the females. In the f i r s t r e p l i c a t i o n , parents F and J were used. The mean yolk diameter of eggs from the female of parent F was s i g n i f i c a n t l y smaller (P<.01) than that of the eggs from the female of parent J . A f t e r o b t a i n i n g eggs from these sets of parents, a r e c i p r o c a l cross of the two was made; female of F \?as crossed w i t h the male of J and v i c e versa. Mean diameter of eggs obtained from the two sets of parents a f t e r c r o s s i n g was s t i l l s i g n i f i c a n t l y d i f f e r e n t (P<.01), female of F and J g i v i n g small and large eggs r e s p e c t i v e l y . Small and la r g e egg l o t s from the o r i g i n a l parents and t h e i r crosses were reared i n the same tank i n 24°C temperature bath. For r e a r i n g the f e r t i l i z e d eggs and the young,small c l o t h baskets were used. Parents N and S g i v i n g small and large eggs r e s p e c t i v e l y were used f o r the second r e p l i c a t i o n of the experiment. Here a l s o the female:, g i v i n g smaller egg (N) was crossed w i t h the male of the parent g i v i n g l a r g e egg and v i c e versa. Crossing was done a f t e r o b t a i n i n g samples of eggs from the o r i g i n a l parent. Mean yolk diameter of the eggs was s i g n i f i c a n t l y d i f f e r e n t (P<.01) i n the o r i g i n a l parent and i n the crosses. A l l four l o t s i n the second r e p l i c a t i o n were reared i n 24°C i n small c l o t h baskets in;the same tank. -55-R e s u l t s S u r v i v a l : Percent s u r v i v a l up to hatching was lower than i n some of the other experiments and ranged from 49 to 78 (Table XXXVII). A f t e r h a t c hing, m o r t a l i t y of the young too was comparatively higher and s u r v i v a l up to the time of p r e s e r v a t i o n ranged from 34 to 58% i n the f i r s t r e p l i c a t e and 39 t i 48% i n the second one. T o t a l vertebrae: Mean v e r t e b r a l counts of the d i f f e r e n t samples are presented i n Table XXXVIII. Mean vertebrae of the sample from smaller egg (genotype F) was s i g n i f i c a n t l y higher than that from l a r g e egg (genotype J ) . Upon c r o s s i n g , t h i s d i f f e r e n c e i n the mean v e r t e b r a l counts between the sample from small and large eggs was removed (F>.05) Crossing of the female of parent J (l a r g e egg) w i t h the male of parent (small egg) r e s u l t e d i n a s i g n i f i c a n t r i s e i n the mean v e r t e b r a l count. On the other hand, c r o s s i n g the female of parent F (small egg) w i t h the male of J ( l a r g e egg parent) reduced the v e r t e b r a l count (P<.01). In both cases i n f l u e n c e of the f a t h e r i n the f i n a l determination of the vertebrae of the o f f s p r i n g was apparent and fo l l o w e d a p a t t e r n of blending i n h e r i t a n c e . In the second r e p l i c a t i o n , mean vertebrae of the sample from eggs w i t h large yolk diameter (parent S) was higher (P<.01) than that from eggs w i t h smaller yolk diameter (parent N). As i n the f i r s t r e p l i c a t i o n , r e c i p r o c a l c r o s s i n g of the females r e s u l t e d i n a decrease i n the higher mean count (from large egg: parent S? and No* ) and an increase i n the mean of the sample from smaller egg (parent: N? So*). This increase was s t a t i s t i c a l l y s i g n i f i c a n t (P<.01) but the decrease brought about i n the mean of the sample from l a r g e egg ( l a r g e egg of -56-p a r e n t S v s t h e l a r g e egg f rom S 9 and No* ) o n l y t ended t o be s i g n i f i c a n t (P = . 0 1 - . 0 2 ) . A l t h o u g h the c r o s s i n g o f t h e g e n o t y p e s i n the s e c o n d r e p l i c a t i o n r e s u l t e d i n i n t e r m e d i a t e mean c o u n t s , the d i f f e r e n c e be tween t h e s e two means ( s m a l l egg f rom N $ S$ and l a r g e egg f rom S ? M<? ) was s t i l l s i g n i f i c a n t ( P < . 0 1 ) . T h i s was d i f f e r e n t f rom t h e r e s u l t o f c r o s s i n g i n the r e p l i c a t i o n I where t h e i n i t i a l d i f f e r e n c e be tween t h e mean c o u n t s d i s a p p e a r e d c o m p l e t e l y a f t e r r e c i p r o c a l c r o s s i n g o f t h e p a r e n t s . P e c t o r a l r a y s : P . e s u l t s a r e p r e s e n t e d i n ' T a b l e .XXXIX. Mean , p e c t o r a l r a y s o f the sample f r o m eggs w i t h l a r g e y o l k d i a m e t e r (parent J ) i n the f i r s t r e p l i c a t e was h i g h e r (P< .01) t h a n the mean o f t h e sample f r o m eggs x ^ i t h s m a l l e r y o l k d i a m e t e r ( p a r e n t F ) . Upon c r o s s i n g the f e m a l e o f p a r e n t F ( g i v i n g s m a l l eggs) x-?ith t h e m a l e o f p a r e n t J ( g i v i n g l a r g e e g g s ) , mean c o u n t s o f t h e sample f r o m s m a l l eggs i n c r e a s e d s i g n i f i c a n t l y ( P < . 0 1 ) . S i m i l a r l y i n t h e o t h e r c r o s s ( l a r g e egg y i e l d i n g f e m a l e o f p a r e n t J w i t h the m a l e o f the s m a l l egg y i e l d i n g p a r e n t F) the mean p e c t o r a l c o u n t shox-?ed a s t r o n g t e n d e n c y o f d e c r e a s e (P = . 0 1 - . 0 2 ) a s compared to the mean o f t h e sample f r o m o r i g i n a l p a r e n t J . I n b o t h c a s e s the m a l e a p p e a r e d t o d e c i d e t h e f i n a l e x p r e s s i o n o f the p e c t o r a l r a y s . The o r i g i n a l d i f f e r e n c e between t h e means o f the sample f r o m eggs x c i t h s m a l l and l a r g e y o l k ( p a r e n t F v s J ) t e n d e d to become s l i g h t l y r e d u c e d a f t e r t h e r e c i p r o c a l c r o s s (F<J? Jo " vs J $ Fc? : P = . 0 1 - . 0 2 ) . I n t h e s econd r e p l i c a t i o n w i t h p a r e n t s N (eggs w i t h s m a l l e r y o l k d i a m e t e r ) and S (eggs w i t h l a r g e y o l k d i a m e t e r ) , mean p e c t o r a l -57-ray counts of the former was s i g n i f i c a n t l y higher than the l a t t e r (P<.01). Upon c r o s s i n g the female of N x^ith the male of S, t h i s higher p e c t o r a l ray was reduced s i g n i f i c a n t l y (P<.01) although the eggs s t i l l remained s m a l l . The i n f l u e n c e of the male i n t h i s case x>?as very strong and the r e s u l t a n t mean p e c t o r a l ray of the cross x«?as even loxrer (P<.01) than the mean of the parent S ( l a r g e yolk diameter). The other cross ( i . e . c r o s s i n g the female of parent S g i v i n g eggs x«th lar g e yolk diameter x-?ith the male of the parent g i v i n g small egg i.e.N) increased the mean count (F<1.01) of large egg sample (P<..01; parent S vs parent S$ No"). Here a l s o , the mean count was higher (P<.01) than the mean p e c t o r a l ray of the o f f s p r i n g of parent (parent N) x^hose f a t h e r c o n t r i b u t e d to t h i s i n c r e a s e . In both crosses i n t h i s r e p l i c a t i o n , the in f l u e n q e of male's genetic c o n s t i t u t i o n was strong and pronounced i n i n h e r i t a n c e of p e c t o r a l r a y s . Anal r a y s : Mean anal ray counts of d i f f e r e n t samples are presented i n Table XL. Mean anal ray count of the sample from eggs v?ith small yolk diameter was loxver than the mean of the sample "from large yolked egg (P<.01) i n the f i r s t r e p l i c a t i o n (parents F and J ) . Crossing the female g i v i n g smaller egg xvith the male of the parent g i v i n g l a r g e r egg ( F ? v 7 i t h J<0 d i d not change the mean ray count from the eggs w i t h smaller yolk s i g n i f i c a n t l y although a strong trend f o r increase (P = .02-.05) was observed as compared w i t h the o r i g i n a l sample from small eggs. Crossing the female g i v i n g large egg w i t h the male from the small egg parent ( J $ w i t h fS ) d i d not produce any d i f f e r e n c e i n the mean anal ray counts of the samples from eggs with l a r g e yolk diameter. -58-Th e mean anal ray counts of the samples from eggs w i t h l a r g e yolk diameter and from eggs w i t h small yolk diameter were s t i l l d i f f e r e n t (P<.01) a f t e r c r o s s i n g of the males. Re s u l t s obtained i n the second r e p l i c a t i o n were almost i d e n t i c a l w i t h those of the f i r s t . Here a l s o , mean count of the sample from smaller egg ( a f t e r crossing) showed a tendency to become higher than the mean of the sample from smaller egg from the o r i g i n a l parent (N and N9-So": P = .01-.02). The mean count of the sample from smaller eggs a f t e r c r o s s i n g became higher to the extent that i t s d i f f e r e n c e w i t h the mean of the sample from the large egg ( o r i g i n a l ) and l a r g e egg (crossing) was r e s p e c t i v e l y reduced and o b l i t e r a t e d . In the other cross ( l a r g e egg female w i t h small egg male), the anal count was not a l t e r e d s i g n i f i c a n t l y . R e s ults i n the case of the samples from eggs w i t h l a r g e r yolk diameter i n both the r e p l i c a t i o n s suggest that anal rays are dependent on the mother. Insofar as the samples'from eggs w i t h smaller yolk are concerned, c r o s s i n g the female w i t h the male from the la r g e egg parent tended to increase the mean counts i n both the r e p l i c a t e s but the increase was not s i g n i f i c a n t , (not a t P<.01). Mean anal ray counts of the samples from smaller eggs i n both r e p l i c a t e s were lower than the means of samples from large egg and t h i s d i f f e r e n c e remained the same even a f t e r c r o s s i n g i n the f i r s t r e p l i c a t e w h i l e i n the second r e p l i c a t e the d i f f e r e n c e disappeared. Dorsal rays: In the f i r s t r e p l i c a t i o n there was no d i f f e r e n c e i n the mean do r s a l ray of the samples from eggs x<dth small and l a r g e yolk diameters. The c r o s s i n g of the parents a l s o d i d not a l t e r the counts i n any s i g n i f i c a n t manner (Table X L I ) . -59-In the second r e p l i c a t i o n w i t h parents N and S g i v i n g eggs w i t h small and large yolk diameter r e s p e c t i v e l y , the mean count of the o f f s p r i n g of N was s i g n i f i c a n t l y , higher. No s i g n i f i c a n t a l t e r a t i o n i n d o r s a l ray counts occurred i n the sample from eggs w i t h small yolk diameter when the female of N was crossed w i t h the male of S. The mean dorsal ray count showed an increase (P<.01) i n .the case of the samples, from eggs w i t h large yolk diameter a f t e r c r o s s i n g as compared to the mean of the o f f s p r i n g from o r i g i n a l l a rge egg sample. Results from the second r e p l i c a t i o n i n d i c a t e some i n f l u e n c e of the f a t h e r ' s genetic c o n s t i t u t i o n on do r s a l rays. Total caudal r a y s : Data on mean t o t a l caudal rays are presented i n Table X L I I . Compared to the sample from eggs w i t h l a r g e yolk diameter (parent J ) , the mean of the sample from eggs w i t h small yolk diameter (parent F) i n the f i r s t r e p l i c a t i o n was s i g n i f i c a n t l y lower (F<.01). But a f t e r c r o s s i n g , (F£ Ja* ) the mean count from the sample from eggs w i t h small yolk diameter increased a p p r e c i a b l y (by 1.20 r a y s ) . The increase i n ' t h i s mean was even s l i g h t l y higher than the mean of the sample from l a r g e egg (parent J ) . Mean caudal rays of the sample from eggs w i t h large yolk diameter showed a s i g n i f i c a n t decrease (P<.01) as a r e s u l t of c r o s s i n g the female with.>'the male from the other genotypel R e s u l t s from t h i s r e p l i c a t i o n i n d i c a t e a marked paternal i n f l u e n c e on the determination of the t o t a l caudal rays of the o f f s p r i n g . But the r e s u l t s of the second r e p l i c a t i o n showed a d i f f e r e n t trend i n that the mean counts of the sample from eggs w i t h smaller yolk diameters were higher (P<.01). R e c i p r o c a l c r o s s i n g d i d not produce any app r e c i a b l e a l t e r a t i o n i n the mean counts of samples from eggs w i t h l a r g e and small yolk diameters. -60-A comparison of the r e s u l t s of both r e p l i c a t i o n s showed no r e l a t i o n s h i p between yolk s i z e and caudal rays. These r e s u l t s a l s o f a i l e d to i n d i c a t e any consistency i n the r e l a t i o n s h i p between the caudal ray and the i n f l u e n c e of the genetic make-up of the parents. Conclusion There i s no d i r e c t casual r e l a t i o n between yolk s i z e of the egg and d i f f e r e n t m e r i s t i c characters of medaka. -61-Table X L I I I . Egg numbers and m o r t a l i t y i n experiment V I I I : (a) Transfer from 20° C to 30° C: Parent U Period No. of No. of No. *As % of Time to No. sur- -As % of of dev.' eggs i n eggs to hat- f e r t d . 50% hat- vived to f e r t d . i n 20°C 20°C 30°C ched eggs ching i n preser- P O O S (hrs) 30°C (hrs) v a t i o n 24 43 36 36 95 161 32 84 48 49 44 41 93 144 41 93 72 41 35 32 89 143 24 67 96 46 41 39 95 123 34 33 120 40 35 33 94 128 33 94 144 34 28 28 97 118 23 79 168 39 25 24 71 96 22 65 192 39 34 34 100 9S 31 91 216 48 41 41 95 95 36 84 240 40 34 33 94 83 30 86 264 51 46 46 100 60 39 35 238 32 27 27 100 46 22 81 "'•'Assuming 5 eggs preserved at time of t r a n s f e r as a l i v e . -62-Table X L I I I . Egg numbers and m o r t a l i t y i n experiment V I I I : (cont'd) (b) Transfer from 30°C to 20°C. Period No. of No. of No. "As % of Time to No. sur- *As % of of dev. eggs i n eggs to hat- f e r t d . 50% hat- vived to f e r t d . i n 30°C 30°C 20°C ched eggs ching preser- eggs (hrs) v a t i o n 24 62 53 40 70 441 18 32 48 49 38 , 25 57 388 15 34 72 46 41 37 90 419 23 56 96 33 27 25 76 263 8 24 "Assuming 5 eggs preserved at time of t r a n s f e r as a l i v e . (c) Control samples. Eemp No. of No. hat- As % of Time to No. sur- As % of (°C) eggs ched f e r t d . 50% hat- vived to f e r t d . eggs ching (hrs) preser- egg 5 v a t i o n 20° 40 40 100 483 36 90 30° 35 34 97 168 30 86 -63-Table XLIV . Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment V I I I : (a) Transfer of eggs from 20° to 30°C: Parent U. Hours (and day degrees) i n 20°C before t r a n s f e r Develop- Total vertebrae mental* 30 31 32 33 stage at t r a n s f e r Number Mean D i f f . wi th 30°C c o n t r o l D i f f . w i t h 20°C c o n t r o l 7 22 1 30 30.80 30°C c o n t r o l yes P<.01 24 (20) B l a s t u l a 11 complete. 21 32 30.65 None P>.C5 Yes P<.01 48 (40) Embryonic 28 s h i e l d 13 41 30.32 Yes P<.01 Yes P<C.01 72 (60) Optic cup 7 forming;3 somi tes 16 1 24 30.75 None P>.05 Yes P<.01 96 (SO) Somites 5 20 Audi tory v e s i c l e formed' t a i l bud 28 1 34 30.88 None P>.05 Yes P<.01 120 (100) C i r c u l a t i o n s t a r t e d ; eyes s l i g h t -l y rounded 25 8 33 31.24 Yes P<.01 None P>.05 144 (120) Elongated embryo; pec-t o r a l buds seen; eye pigmentation commenced 9 14 23 31.61 Yes P<.01 Strong trend P<.05; >.01 168 (140) Po s t. par t f r e e and mov-10 12 22 31.54 Yes P<.01 None P>.05 in g . R e c t o r a l s t r i a n g u l a r . -64-Table XLIV. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment V I I I : (Cont'd). .Transfer of eggs from 20°"to 30°C: Parent U. Hours (and day degrees) i n 20°C before t r a n s f e r Develop-mental stage at t r a n s f e r Total vertebrae 30 31 32 33 Number Mean D i f f . D i f f . w i t h w i t h 30°C 20°C c o n t r o l c o n t r o l 192 (160) Eyes d a r k l y 16 pigmented;pec-t o r a l s d i s t i n c t 13 2 31 31.55 Yes P<.01 None P>.05 216 (180) not recorded 16 20 36 31.35 Yes P<.01 l-'one F>.05 2^0 (200) not recor-ded 15 15 30 31 .50 Yes F<.01 None P>.05 264 (220) 25 14 39 31.36 it t i 288 (240) 1 11 10 22 E n t i r e 1 22 13 36 31.33 * 20°C Control -65-Table XLIV. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment V I I I . (b) Transfer of eggs from 30°C to 20°C: Parent U. Hours (and day degrees) i n 30°C before t r a n s f e r Developmental Total stage at 30 St r a n s f e r vertebrae 31 32 Number Mean D i f f . w i t h 30°C Control D i f f wi th 20°C con-t r o l 0 1 22 13 36 31.33 Yes P<.01 20°C con-t r o l 24 (30) Optic cup f o r -ming; 5-6 somites 8 10 18 31.55 Yes PO01 None P>.05 48 (60) C i r c u l a t i o n s t a r t e d ; a u d i -tory v e s i c l e d i s t i n c t 1 12 2 15 31.07 None P>.05 Tends P<.05 >.01 72 (90) Embryo elongated; p e c t o r a l s t r i -a ngular; p o s t e r i o r p a r t f r e e and mov-ing 5 18 23 30.78 None P>.05 Yes P<.01 96 (120) P e c t o r a l s moving; melanophore along 1 7 8 30.87 None P>.05 Yes P<.01 d o r s a l l i n e E n t i r e 1 22 7 30 30.80 30°C con. Yes P<.01 Table XLV. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n Table XLVI. Frequency d i s t r i b u t i o n of anal rays i n experiment V I I I . experiment V I I I . (a) Transfer of eggs from 20°C to 30 C, (a) Transfer of eggs from 20°C to 30°C: Parent U Parent U. Period of Pe c t o r a l rays Number Mean D i f f . D i f f . Anal : rays Number Mean D i f f . D i f f . dev. i n 10 11 12 13 wi th wi th 18 19 20 21 22 w i t h wi th 20°C before 30°C 20°C 30 C 20°C t r a n s f e r con- con- con- con-(hrs) t r o l t r o l t r o l t r o l 0 23 7 30 11.23 30°C Yes P<.01 1 9 17 3 30 19.73 30°C None P>.05 24 4 47 7 53 11.05 None None P>.05 II 3 12 15 2 32 19.50 P>.05 it 48 1 72 9 82 11.01 tt II 17 16 8 41 19.78 it it i 72 6 33 9 48 11.06 tt it 1 7 13 3 24 19.74 it ON It 1 96 4 37 5 46 11.02 ti •t 8 16 9 1 34 19.09 Yes P<.01 Yes P<.ol 120 2 27 15 44 11.29 t i tt 4 14 9 6 33 19.21 Tends P<.02 ti None None 144 1 14 6 21 11.24 »i it 6 8 9 23 20.13 P>.05 P>.05 Tends 168 10 10 20 11.50 P=.05 it 1 4 12 4 1 22 20.00 it it Yes -192 30 32 62 11.51 P<.01 t i 1 10 12 8 31 19.87 it it 216 15 52 5 72 11.86 i i it 15 17 4 36 19.69 tt tt 240 7 65 6 78 11.99 n tt 3 13 11 1 2 30 19.53 t i it 19.41 t i Tends 264 7 65 6 78 11.99 II tt 5 16 15 3 39 P<C.05;>.02 288 13 28 3 44 11.77 it it 2 11 8 1 22 19.36 II tt E n t i r e 2 50 20 72 12.25 II 20°C 2 9 18 5 2 36 19.89 it 2 0°C c o n t r o l -67-Table XLVII. Frequency d i s t r i b u t i o n of d o r s a l rays i n experiment V I I I : (a) Transfer of eggs from 20°C to 30°C: Parent U. Period o f dev. i n 20°C before t r a n s f e r (hrs) Dorsal rays 6 7 Number Mean D i f f . w i t h 30°C c o n t r o l D i f f . w i t h 20°C c o n t r o l 0 25 5 30 6.17 30°C c o n t r o l None P>.05 24 25 7 32 6.22 None P>.05 t i 48 33 8 41 6.19 i i i i 72 23 1 24 6.04 i i II 96 31 3 34 6.09 II II 120 30 3 33 6.09 II 1! 144 19 4 23 6.17 II »l 168 20 2 22 6.09 II II 192 23 8 31 6.23 216 23 13 36 6.08 n It 240 18 12 30 6. AO Tends P=.05 Yes P<.01 264 25 14 39 6.36 None P*.05 None P,>.05 288 16 6 22 6.27 it if E n t i r e 31 4 36* 6.08 it 20°C c o n t r o l * 1 f i s h w i t h 5 rays -68-Table X L V I I I . Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment V I I I : (a) Transfer of eggs from 20°C to 30°C: Parent U. Period of dev. i n 30°C before t r a n s f e r (hrs.) 21 Total ( 22 23 caudal 24 ( rays 25 26 Number Mean D i f f . w i t h 30°C c o n t r o l D i f f wi 20°C c o n t r o l 0 1 7 13 8 29 22.96 30°C c o n t r o l None P>.05 24 3 3 20 6 32 22.91 None P>.05 tt 48 3 5 22 10 1 41 23.00 II it 72 2 12 10 24 23.33 tt ii 96 4 15 14 1 34 23.35 120 3 12 16 2 33 23.51 Yes P<.01 Tends P=.05 144 2 4 8 7 2 23 23.13 None P>.05 None P>.05 168 1 9 3 8 1 22 22.95 it ti 192 2 6 11 11 1 31 23.10 ii ti 216 1 8 15 11 1 36 23.08 n ii 240 9 11 8 2 30 23.10 II it 264 1 10 15 12 1 39 23.05 ir it 288 5 10 7 22 23.09 ii II E n t i r e 1 7 20 6 2 36 23.08 it 20°C c o n t r o l -69-P E R I O D O F D E V E L O P M E N T IN 2 0 ° C B E F O R E T R A N S F E R H O U R S 0 4 8 9 6 144 192 2 4 0 288 t oc -//—n —T~ 0 < o i -~ 31.5 LU 31.0 < 3 0 . 5 CD or w 31.5 > 31.0 30 .5 4 0 8 0 120 160 D A Y D E G R E E S 2 0 0 2 4 0 D A Y D E G R E E S 6 0 120 -U U--u- •U U 0 4 8 9 6 H O U RS P E R I O D O F D E V E L O P M E N T IN 3 0 ° C B E F O R E T R A N S F E R Figure 11. Mean vertebral counts of f i s h transferred from 20° to 30°C (top) and from 30° to 20°C (bottom). Shaded area indicates period between embryonic shield and v i t e l l i n e circulation. (Experiment VIII). -70-i 1 1 1 1 1 1 1 1 1 1 1 H t—r £ 12.0 < oc < or o u UJ Q . Ui S 2 0 . 0 or < 19.5 2 19.0 U" .u- -U U •u. •u 40 D A Y D E G R E E S 8 0 120 160 2 0 0 _i_ 48 9 6 2 4 0 U l 1 r 2 4 0 o c 2 8 8 144 f 9 2 H O U R S P E R I O D O F D E V E L O P M E N T IN 2 0 ° C B E F O R E T R A N S F E R Figure 12. Effect of transfer of developing embryo from 20° to 30°C on mean pectoral and anal f i n rays of genotype U. (Experiment VIII). - 7 1 ->-< < to or O a <r UJ 6.5 U" 6.0 H 23.5 O < 23.0 < a < U < 2 2.5 UJ 5 0 • T 1 1 1 r U u .U-U' •u u T 1 1 1 r 4 0 D A Y D E G R E E S 8 0 120 160 U T 1 1 1 r -V/-r u — u — u ^ y ^ u - Z A u 2 0 0 2 4 0 «c i i 48 96 2 4 0 2 8 8 144 192 H O U R S P E R I O D O F D E V E L O P M E N T IN 2 0 ° C . B E F O R E T R A N S F E R Figure 1 3 . Effect of transfer of developing embryo from 2 0 ° to 3 0 ° C on mean dorsal and total caudal f i n rays of genotype U (Experiment V I I I ) -72-STAGE OF FIXATION OF MERISTIC SERIES (EXPERIMENT VI I I ) I n t r o d u c t i o n T h i s experiment was performed to determine the time of v e r t e b r a l f i x a t i o n i n medaka. Batches of eggs at d i f f e r e n t stages of development were t r a n s f e r r e d from low to high and from h i g h to low temperature. A comparison of the mean v e r t e b r a l count w i t h the mean count of c o n t r o l s at high and low temperature was expected to r e v e a l i f the number of vertebrae had become f i n a l l y determined at c e r t a i n stage of development. D e s c r i p t i o n of experiment Eggs from parent U were used f o r a l l the t r a n s f e r s made from 20°C to 30°C and v i c e versa. For each t r a n s f e r and c o n t r o l ( i n 30° and 20°C) eggs obtained on a s i n g l e day's spawning were used. As che number of eggs obtained each day v a r i e d , the number i n i t i a l l y used f o r each t r a n s f e r was not the same. The c o n t r o l l o t i n 30°C was reared e n t i r e l y i n that temperature w h i l e the corresponding l o t i n 20°C were hatched and reared i n 20°C (15.5 to 22.5 days a f t e r hatching) up to the stage when the a n a l , caudal and d o r s a l f i n s were d i s t i n c t l y v i s i b l e to the naked eye. Thereafter, the young were reared i n 24°C u n t i l such time that they could be preserved. Transfers i n the two d i r e c t i o n s were made at the end of periods of development as shown i n Tablex X L I I I and XLIV. At the time of each t r a n s f e r , f i v e developing eggs were taken from the basket and preserved. Before p r e s e r v a t i o n , the stage of development of these f i v e eggs were observed under a b i n o c u l a r microscope and recorded (Column 2: Table XLIV. The remaining eggs x\?ere t r a n s f e r r e d to the d e s i r e d -73-temperature by removing the baskets d i r e c t l y from the low to high or high to low temperatures without any c o n d i t i o n i n g . A l l the samples t r a n s f e r r e d to high temperature were reared i n that temperature u n t i l they could be preserved. The l o t s t r a n s f e r r e d to lower temperature (20°C) were reared i n the low temperature u n t i l hatching. Thereafter, these l o t s were reared i n 24°C f o r f u r t h e r growth and as such, only v e r t e b r a l counts (which were found to be f i x e d before hatching) were considered. Eggs and young i n a l l cases were reared i n sma11 c l o t h baskets. In c a l c u l a t i n g percentage s u r v i v a l , the 5 eggs that were preserved at the time of t r a n s f e r were deducted from the i n i t i a l number of eggs used f o r r e a r i n g and i n the c a l c u l a t i o n s shown i n Table X L I I I the remaining t r a n s f e r r e d eggs were treated as the i n i t i a l number of eggs. Results In a l l the l o t s t r a n s f e r r e d from low to high temperature, s u r v i v a l u n t i l hatching and up to the time of p r e s e r v a t i o n ranged from 70 to 100% and 66 to 94% r e s p e c t i v e l y (Table X L I I I ) . Transfers i n the opposite d i r e c t i o n a f f e c t e d the s u r v i v a l r a t e and were not as s a t i s f a c t o r y . T o t a l Vertebrae: a) Transfer from 20 to 30°C. Mean v e r t e b r a l counts of samples of d i f f e r e n t days t r a n s f e r s from low to high temperature are presented i n Figure I I and Table XLIVa. The s e n s i t i v e p e r i o d f o r the vertebrae appeared to extend from the embryonic s h i e l d stage to the stage xjhen eye pigmentation commenced and p e c t o r a l buds had appeared. -74-These stages of development corresponded to 48 hours (40 day degrees) and 144 hours (120 day degrees) of complete development i n 20°C. Furthermore, these stages correspond roughly to Fundulus stages 14 to 22 (Oppenheimer 1937). The mean v e r t e b r a l count of the l o t t r a n s f e r r e d at the end of 48 hours (beginning of s e n s i t i v e period) was s i g n i f i c a n t l y lower than the mean of the c o n t r o l a t 30°C. The mean count of the l o t t r a n s f e r r e d at the end of 144 hours (120 day degrees), on the other hand, showed a strong tendency to become higher than the c o n t r o l i n 20°C (P<.05; >.01). Mean counts of a l l t r a n s f e r s made a f t e r 168, 192, 216, 240, 264 and 288 hours of development d i d not d i f f e r from the mean of the c o n t r o l i n 20°C (P>.05 i n a l l cases). b) Transfer from 30° to 20°C: The number of vertebrae seemed almost f i x e d a f t e r 48 hours in c u b a t i o n i n 30°C (P>.05 as compared to 30°C; P<.05 as compared to the 20°C mean count) (Table XLIVb and Figure I I ) . The stage of development reached by the eggs at the end of 48 hours i n 30° corresponded more or l e s s to the stage obtained i n 20° i n 120 hours (100 day degrees). The mean count of the sample t r a n s f e r r e d at the end of 72 hours (90 day degrees) showed that the vertebrae has become f i n a l l y f i x e d by t h i s time i n 30°C. The developmental stage at t h i s time c l o s e l y corresponded to the stage obtained i n 168 hours development i n 20°C. -7 5-From the data the s e n s i t i v e period f o r the f i x a t i o n of the number of vertebrae appears to extend between the stage when the embryonic s h i e l d i s formed and the time when p e c t o r a l buds appear and eye pigmentation s t a r t s , a f t e r which changes i n the temperature f a i l to a l t e r the number. Other m e r i s t i c s e r i e s : Although t h i s experiment was designed to determine time of f i x a t i o n of vertenrae, other m e r i s t i c s e r i e s were counted only f o r the l o t s t r a n s f e r r e d from low to high temperature. F e c t o r a l rays: Mean p e c t o r a l rays of the samples t r a n s f e r r e d at the end of 24, 48, 72, 96 and 120 hours were lower (P<.01) than the mean of 20°C c o n t r o l but were not s i g n i f i c a n t l y d i f f e r e n t from 30°C c o n t r o l (Table XLV: Figure 12). Mean count of the l o t t r a n s f e r r e d at the end of 168 hours (7 days) development tended to be higher (P=.05) than the mean of the l o t i n 30°C ( c o n t r o l ) ^ Mean counts of a l l the l o t s t r a n s f e r r e d t h e r e a f t e r were higher than that of 30°C c o n t r o l but lower than the mean of the c o n t r o l i n 20°C. Anal rays: Mean anal ray counts of the sample t r a n s f e r r e d at the end of 96 hours was lower (P<.01) than the mean of the high and low temperature c o n t r o l (Table XLVI: Figure 12). Mean of the sample t r a n s f e r r e d a f t e r 120 hours was lower than the mean of the c o n t r o l i n 20°C and showed a marked tendency of being lower (P=.01-.02) than the mean of the c o n t r o l i n 30°C. Compared to the c o n t r o l i n 20°C, the mean counts of the l o t s t r a n s f e r r e d at the end of 264 and 238 hours were somewhat lower (P=.02-.05) but a comparison w i t h the c o n t r o l i n the high temperature showed no d i f f e r e n c e . Mean counts of the remaining samples e x h i b i t e d no d i f f e r e n c e w i t h the mean of the c o n t r o l s at 20° or 30°C. -76-Dorsal rays: Mean do r s a l count of the sample t r a n s f e r r e d a t the end of 240 hours was higher than the mean of the low temperature c o n t r o l (P<.01) and a l s o showed a tendency to be higher than the mean of the c o n t r o l i n 30°C (Table XLVII: Figure 13). Mean counts of the other t r a n s f e r l o t s showed no d i f f e r e n c e w i t h e i t h e r of the c o n t r o l mean counts. Total caudal rays: The mean count of the l o t t r a n s f e r r e d at the end of 120 hours was higher (P<.01) than the mean of the c o n t r o l i n 30°C and furthermore d i s p l a y e d a tendency of being higher (P=.05) than the mean count of the c o n t r o l i n 20°C (Table X L V I I I : Figure 13). T o t a l mean caudal ray counts of the remaining t r a n s f e r l o t s d i d not rev e a l any s i g n i f i c a n t d i f f e r e n c e w i t h the mean of e i t h e r the c o n t r o l i n 20° or 30°C. Conclusion Transfer of developing embryo revealed that the s e n s i t i v e period f o r the v e r t e b r a l number i n medaka extends between the embryonic s h i e l d stage and the stage when p e c t o r a l buds had appeared. High temperature shock i n the beginning of the s e n s i t i v e period lowers the number of vertebrae but an increase i n the number r e s u l t s when s i m i l a r shock i s a p p l i e d towards the end of the s e n s i t i v e p e r i o d . S e n s i t i v e p e r i o d f o r the f i x a t i o n of p e c t o r a l and other f i n r a y s , although commencing e a r l y , extends to periods a f t e r hatching. -77-Table XLIX. Egg numbers and m o r t a l i t y i n experiment IX: E f f e c t of temperature. Temp No. of No. hat- As % of Time to No. sur- As % of Remarks <°C) f e r t d . ched f e r t d . 50% hat- vi v e d to f e r t d . eggs eggs ching (hrs.) preser-v a t i o n eggs (1) Parent A 20° 49 28 57 16 33 414 h r s . i n 20°C;hatched and reared i n 24°C 22 (a) 65 26 40 661 10 15 22° (b) 3S 40 45 26 29 Hatched by temp, shock a f t e r 512 hrs 24° 133 40 30 366 33 30 26° 33 6 18 28° 73 42 58 339 28 38 30° 95 ' 27 28 276 6 6 32°(a) 95 27 28 188 11 12 32°(b) 80 19 24 200 10 13 Reared i n 26° a f t e r hatchin; (2) Parent B o 22 31 19 61 15 48 28° 32 26 81 26 81 32° 35 24 69 274 16 46 -78-Table XLIX continued. Egg numbers and m o r t a l i t y i n experiment IX: E f f e c t of temperature. Temp (°C) No. of f e r t d . eggs No. hat-ched As % of f e r t d . eggs Time to 507, hat-ching (hrs) No. sur-vived to preser-v a t i o n As % of f e r t d . eggs Remarks (3) Parent C 20°(a) 156 94 60 39 25 64 hatched i n 20 ; 30 hat-ched i n 26 ; 13 reared en-t i r e l y i n 20°( 20°(b) 40 21 52 9 23 22° 33 20 61 24°(a) 68 56 82 50 74 24°(b) 37 28 76 311 26 70 26°(a) 82 66 80 60 73 26°(b) 30 30 100 26 87 30° 76 35 46 35 46 32° 35 28 80 28 80 (4) Parent D 22° 20 14 70 724 11 55 24 (a) 22 12 54 311 11 50 24°(b) 17 13 76 288 10 59 24°(c) 13 6 46 26° 20 20 100 20 100 32° 16 9 56 -79-Table XLIX continued. Egg numbers and m o r t a l i t y i n experiment IX: E f f e c t of temperature. Temp ( C) No. of f e r t d . eggs No. hat-ched As % of f e r t d . eggs Time to 50% hat-ching (hrs) No. sur-vived to preser-v a t i o n As % of f e r t d . eggs Remarks (5) Parent E 552 hours i n 20° 71 56 79 36 51 20°C. Hat-ched and reare< i n 24°C. 24° 29 17 59 299 16 55 28° 23 22 96 198 21 91 32° 27 20 74 312 10 37 (6) Parent G 20° 23 5 22 386 4 17 Reared i n 24°C a f t e r hatching 22° 20 12 60 601 8 40 24° 66 55 83 498 46 70 26° 90 67 74 277 34 51 28° 40 17 43 266 17 43 32° 94 67 71 213 33 35 (7) Parent H 20° 125 104 83 459 65 52 Reared i n 24°C a f t e r hatching 22° 119 100 84 360 52 44 24° 125 144 91 80 64 26° 75 69 92 357 36 48 28° 77 58 75 227 40 52 30° 125 110 88 221 71 57 32° 150 137 91 185 126 84 -80-Table XLIX continued. Egg numbers and m o r t a l i t y i n experiment IX: E f f e c t of temperature. Temp (°C) No. of f e r t d . No. hat-ched As % of f e r t d . Time to 50% hat-No. sur-vived to As % of f e r t d . Remarks eggs eggs ching (hrs.) preser-v a t i o n eggs (8) Parent I 0 20 62 20 32 465 14 23 22° 103 55 53 433 33 32 24° 100 82 82 268 61 61 26° 104 41 39 263 28 27 28° 114 72 63 265 54 47 30° 101 34 83 176 75 74 32° 100 75 75 177 60 60 34° 150 24 16 204 7 5 Reared i n 32°C a f t e r hatching (9) Parent K 504 hrs. i n 20°C 20° 107 42 39 34 32 Hatched and reared i n 24°C. 22° 104 60 58 432 28 27 24° 164 116 71 273 96 59 26° 44 27 61 243 27 61 28° 41 28 6S 292 22 54 30° 83 46 55 236 46 55 32°(a) 77 48 62 164 20 26 32°(b) 103 47 46 168 28 27 (10) Parent Q 24° 50 42 84 317 30 60 28° 93 58 62 228 27 29 32° 108 35 32 174 26 24 -81-Table XLIX continued. Egg numbers and m o r t a l i t y i n experiment IX: E f f e c t of temperature. Temp <°C) No. of f e r t d . eggs No. hat-ched As 7. of f e r t d . eggs Time to 507. hatching (hrs.) No. sur-vived to preser-v a t i o n As 7. of f e r t d . eggs Remarks (11) Parent R 20° 100 31 31 1059 14 14 Reared i n 24° a f t e r hatching 22° 100 61 61 730 36 36 o 26 100 84 84 477 74 74 28° 100 60 60 490 41 41 30° 100 48 48 560 29 29 32° 100 69 69 457 19 19 34° 100 50 50 290 18 18 Reared i n 32°C a f t e r hatching. (12) Parent U • 20° 40 40 100 483 36 90 24°(a) 47 45 96 265 36 77 24° (b) 55 54 98 262 46 84 28° 53 53 100- 202 51 96 30° 35 34 97 168 31 89 32° 88 81 92 162 71 81 -82-Table XLIX continued. Egg numbers and m o r t a l i t y i n experiment IX. E f f e c t of temperature. Temp (°C) No. of f e r t d . eggs No. hat-ched As % of f e r t d . eggs Time to 50% hatching (hrs.) No. sur-v i ved to preser-v a t i o n As % of f e r t d . eggs Remarks (13) Parent W 22° 100 99 99 439 88 88 26° 100 '100 100 365 93 93 30° 100 96 96 226 92 92 32° 100 90 90 383 60 60 34° 100 20 20 331 7 7 Reared i n 32°C a f t e r hatching. (14) Parent X 22° 75 70 93 478 62 83 26° 75 59 79 316 51 68 30° 75 72 96 200 70 93 32° 75 71 95 221 - 66 88 34° 75 61 81 198 41 55 (15) Parent Y 521 h r s . i n 20° 20° 100 95 95 554 90 90 hatched and reared i n 26 C. 24° 100 96 96 289 90 90 26° 100 93 93 346 87 87 30° 100 98 98 226 95 95 32° 100 93 93 226 64 64 34°(a) 100 a. 31 b. 43 74 279 343 7 9 a. 16 b. From 50 eggs hatched & rea-red i n 34 C. From 50 eggs hatched & rea-red i n 30°C. 34°(b) 100 67 67 325 4 4 34°(c) 100 43 43 255 9 9 Reared i n 32°C a f t e r hatching. -83-EFFECT OF SUSTAINED TEMPERATURE (EXPERIMENT IX) I n t r o d u c t i o n The purpose of t h i s experiment was to determine the e f f e c t of sustained r e a r i n g temperature on the d i f f e r e n t m e r i s t i c c h a r a c t e r s . Information on the e f f e c t of temperature was a l s o necessary to determine whether temperature induced changes are comparable to changes induced by t h y r o x i n e , thiourea or d i n i t r o p h e n o l . This experiment was repeated w i t h several genotypes to discover d i f f e r e n c e s i n t h e i r response. D e s c r i p t i o n of experiment Temperatures of 20°, 22°, 24°, 26°, 28°, 30°, 32° and 34°C were used. A l t o g e t h e r , f i f t e e n r e p l i c a t i o n s of the experiment were made. Eggs of each r e p l i c a t e could not be tr e a t e d i n a l l the above-noted temperatures. A l s o , i n some of the r e p l i c a t e s , the number of eggs put i n each temperature was not the same. These departures from u n i f o r m i t y were imposed by the i r r e g u l a r i t y i n the number of eggs obtained d a i l y and l o s s of breeding a c t i v i t y of d i f f e r e n t sets of parents. D e t a i l s of the genotypes used, number of eggs treated i n d i f f e r e n t temperatures e t c . are o u t l i n e d i n Table XLIX. Eggs obtained on several successive days were placed i n one p a r t i c u l a r temperature treatment. Spawning always occurred at 24°C and the eggs were put i n t o d i f f e r e n t temperatures d i r e c t l y without any c o n d i t i o n i n g i n the temperatures concerned. As w i l l appear from Table XLIX t h i s technique of egg t r a n s f e r d i d not a f f e c t the m o r t a l i t y of eggs a p p r e c i a b l y . -84-Eggs were allowed to hatch n a t u r a l l y i n the baskets i n a l l temperatures except i n some instances i n 20°C bath where developed eggs d i d not hatch out even a f t e r a prolonged in c u b a t i o n i n that temperature. Hatching i n those l o t s was induced by t r a n s f e r r i n g the eggs to a higher temperature, i . e . 24°C The temperature shock induced hatching of the eggs w i t h i n about an hour of t r a n s f e r . A f t e r hatching such l o t s were reared i n the higher temperature u n t i l presei-vation. P a r t i c u l a r s of such t r a n s f e r s are recorded i n Table XLI In 34°C, the highest temperature used, m o r t a l i t y a f t e r hatching was very high f o r most genotypes. In some cases, as shown i n the t a b l e , the young were t r a n s f e r r e d to lower temperatures f o r f u r t h e r growth and f a t t e n i n g . For l o t s so t r a n s f e r r e d i n the course of t h e i r growth and d i f f e r e n t i a t i o n , only vertebrae x<rere taken i n t o account f o r the present a n a l y s i s as the number of vertebrae was found to become f i x e d very e a r l y i n development (Experiment V I I I ) . Young i n a l l r e p l i c a t e s x-?ere reared i n the small baskets (10 x 10 x 15 cm) except samples of r e p l i c a t e s number X I I I to XV, which were reared i n the l a r g e r basket (12 x 12 x 15.5 cm) a f t e r hatching. Results S u r v i v a l of eggs to hatching and s u r v i v a l of young to the stage when these Xvere preserved mere recorded f o r every genotype (Table XLIX). S u r v i v a l x-?as greatest i n temperatures from 24° to 30°C i n almost a l l genotypes. In extreme high or low temperatures s u r v i v a l was loxv-er; here a l s o , the genotypes responded d i f f e r e n t l y . -85-Figure 14. Effect of sustained temperature on mean total vertebrae. Letters indicate genotypes (Experiment IX) -86-In genotypes U, W, X and Y, percentage s u r v i v a l up to hatching i n a l l temperatures ranged between 67 and 100 (except i n 34°C f o r W). The percentage of f r y s u r v i v i n g i n these genotypes was a l s o higher and ranged between 55 and 95 (except f o r genotypes W and Y i n 34°C). In s p i t e of t h i s , s i g n i f i c a n t v a r i a t i o n was found i n the m e r i s t i c characters of these genotypes. T o t a l vertebrae: Main v e r t e b r a l counts of each genotype are. p l o t t e d a g a i n s t the appropriate temperatures (Figure 14) and are a l s o shown i n appendix I. In some cases where there was more than one r e p l i c a t i o n of one genotype i n the same temperature, samples from a l l r e p l i c a t i o n s are lumped together i f the means of i n d i v i d u a l samples were not d i f f e r e n t . Of the f i f t e e n genotypes, mean counts of nine were high i n the lowest and highest temperature w i t h the lowest mean i n an intermediate temperature. The mean count i n the lowest temperature was s i g n i f i c a n t l y higher (P<.01) than the lowest mean count i n the intermediate temperature i n a l l the nine genotypes but t h i s i s not true of the d i f f e r e n c e between the mean counts i n the h i g e s t temperature and intermediate temperature. In genotypes I and Y, d i f f e r e n c e between the means i n the highest and intermediate temperature (lowest mean) was s i g n i f i c a n t (P<.01) and i n genotype VI, the d i f f e r e n c e was i n d i c a t i v e of being s i g n i f i c a n t (P<.05). D i f f e r e n c e between the means i n highest and intermediate temperature i n the remaining s i x genotypes were not s t a t i s t i c a l l y s i g n i f i c a n t . The intermediate temperature that produced the lowest mean vertebrae was d i f f e r e n t f o r d i f f e r e n t genotypes and v a r i e d -87--88-between 26° and 32°C except i n genotype 2 where the lowest count was obtained i n 24°C; but f o r t h i s genotype no data are a v a i l a b l e a t 26°C. Mean counts of three out of f i f t e e n genotypes (G, H and Q) were not a f f e c t e d by temperature treatments. Mean counts of two out of f i f t e e n genotypes (U and VI) showed a progressive decrease w i t h increase i n the temperature. The mean counts i n the highest temperature (32°C f o r U and 34°C f o r >J) were the lowest and both the means were s i g n i f i c a n t l y lower than those i n the lowest temperature (P<,01). In genotype A the mean counts showed a V-shaped r e l a t i o n s h i p to temperature i n a l l treatment except i n the two highest temperatures where the mean tended to decrease again. D i f f e r e n c e between the mean at the highest temperature and high mean counts i n che previous two temperatures was not s i g n i f i c a n t . A l a r g e v a r i a t i o n i n the mean v e r t e b r a l count between the genotypes i n any s i n g l e temperature was al s o found. V e r t e b r a l count of a l l the r e p l i c a t i o n s were al s o analysed f o r abdominal and caudal vertebrae s e p a r a t e l y . Except f o r genotype H and U, the abdominal vertebrae d i d not a l t e r s i g n i f i c a n t l y i n the temperature treatments; v a r i a t i o n s were due almost e n t i r e l y to a l t e r a t i o n s i n the caudal vertebrae (data not i n c l u d e d ) . P e c t o r a l r a y s : Mean p e c t o r a l ray counts of i n d i v i d u a l s reared i n d i f f e r e n t sustained temperatures are shown i n Figure 15 and appendix I I . Mean counts of samples from 20° or 34°C where the eggs x^ere hatched or reared i n higher or lower temperature r e s p e c t i v e l y are omitted from -89-Figure 16. Effect of sustained temperature on mean anal f i n rays. Letters indicate genotypes (Experiment IX) -90-c o n s i d e r a t i o n s . U n l i k e the v e r t e b r a l s e r i e s , p e c t o r a l rays were p r o g r e s s i v e l y reduced by the increase i n temperature. A l l genotypes except E and G responded to the treatment i n a s i m i l a r manner, i . e . a prog r e s s i v e decrease w i t h increase i n temperature. In E and G, no d i f f e r e n c e was caused by the treatments. I t may be r e c a l l e d that the v e r t e b r a l counts of genotype G and I-I were a l s o not a l t e r e d by the temperature treatments. S i g n i f i c a n t v a r i a t i o n i n the p e c t o r a l ray counts between genotypes were n o t i c e a b l e ; most genotypes having a higher v e r t e b r a l count a l s o gave a higher p e c t o r a l ray count. In a l l but the two r e p l i c a t e s mentioned above, the d i f f e r e n c e between the mean counts i n the lowest and. highest temperature was s i g n i f i c a n t (P<.01). Anal rays: Mean anal ray counts i n d i f f e r e n t temperatures are shown i n Figure 16 and appendix I I I . Mean counts of samples reared i n two d i f f e r e n t temperatures during pre- and post- hatching period of development and growth,(i.e. 20°C samples r a i s e d i n higher temperature a f t e r hatching) have not been taken i n t o account. The genotypic i n f l u e n c e w i t h respect to temperature e f f e c t was most pronounced i n t h i s m e r i s t i c character. Temperature - e f f e c t on anal rays can be summarized i n t o the f o l l o w i n g c a t e g o r i e s : a) Mean anal ray count increased w i t h increase i n temperature. The increase was progressive i n genotypes D and I . Anal rays increased w i t h increase i n temperature up to a poi n t and beyond that, the increase was not p r o p o r t i o n a l to the temperature r i s e . This type of response -91-was obtained i n genotypes B and K. In A, C, G and K, although there was an increase, the p a t t e r n was not c o n s i s t e n t . D i f f e r e n c e s between the low mean count ( i n lower temperature) and highest mean count i n the high temperature was s t a t i s t i c a l l y s i g n i f i c a n t . b) Mean anal counts decreased p r o g r e s s i v e l y w i t h increase i n temperature. This e f f e c t was pronounced i n genotype Y. Mean count i n 24°C (lowest temperature) was higher (P<.01) than the mean i n 34°C (highest temperature). c) Anal ray counts of genotypes E, Q and E showed an in v e r t e d V-shaped r e l a t i o n s h i p to temperature. The mean count of genotype E and 0 i n the intermediate temperature was higher than the mean count i n the highest temperature (P<.01) and tended to be higher than that i n the lowest temperature (P<.05; > .02). d) In genotypes U, W and X, anal counts were not appr e c i a b l y a l t e r e d by temperature d i f f e r e n c e s . In a d d i t i o n to the v a r i a t i o n i n response between the genotypes, v a r i a b l e r e s u l t s i n anal count were obtained from samples of the same genotype reared i n two separate l o t s at the same temperature. Of the two samples of genotype C i n 26°, the sample w i t h the higher number of f i s h i n basket gave a higher mean anal ray count (P<.01). Mean counts of two samples of the same genotype i n 24°C e x h i b i t e d no d i f f e r e n c e , though the popu l a t i o n s i z e of one x^as double the other. S i m i l a r l y , no d i f f e r e n c e x^as found i n the means of three l o t s of genotype D i n 24°C :(P<«05). The mean counts of txro samples of genotype K i n 32° tended to be d i f f e r e n t (P = .02-.05), mean count of the sample from -92 5.5 22 2 6 T E M P E R A T U R E 3 0 ° C . 3 4 Figure 17. Effect of sustained temperature on mean dorsal f i n rays. Letters indicate genotypes (Experiment IX) -93-clenser p o p u l a t i o n being lower i n t h i s case. This was i d e n t i c a l to the r e s u l t obtained i n experiment VI but opposite to that found i n genotype C i n 26°C (see above). Mean counts of the two samples of genotype U i n 24°C a l s o showed a tendency to be d i f f e r e n t (P = .02-.05) and here the sample w i t h denser population had a lower mean count. S i g n i f i c a n t v a r i a t i o n was a l s o found between genotypes i n respect of mean ray counts i n any s i n g l e temperature. R e l a t i o n s h i p between anal rays and other m e r i s t i c s e r i e s was not very c l e a r . Some genotypes w i t h high v e r t e b r a l and/or p e c t o r a l rays a l s o had high anal rays. In othe r s , the r e l a t i o n s h i p was in v e r s e . Dorsay r a y s : In ten out of f i f t e e n r e p l i c a t e s , the mean counts formed an i n v e r t e d V when p l o t t e d against temperatures (Figure 17; Appendix IV). In others s i m i l a r p l o t t i n g of the counts revealed no p a t t e r n . In genotypes C and R, the count i n the intermediate temperature was s i g n i f i c a n t l y higher than both i n the lowest and highest temperature. Most of remaining r e p l i c a t i o n s showed the same p a t t e r n but d i f f e r e n c e s i n d i f f e r e m t temperatures were not s t a t i s t i c a l l y s i g n i f i c a n t . The v a r i a t i o n between genotypes i n t h e i r mean do r s a l count was a l s o not s i g n i f i c a n t except f o r genotypes C and R i n which the mean counts were higher than i n the r e s t . I nsofar as the r e l a t i o n s h i p of the do r s a l rays to other m e r i s t i c s e r i e s i s concerned, no d e f i n i t e trend was di s p l a y e d by the genotypes. Total caudal rays: As i n the anal ra y s , a l a r g e genotypic v a r i a b i l i t y was observed f o r t o t a l caudal rays (Appendix V and Figure 18). -94 P l o t t i n g of the mean t o t a l caudal rays against temperatures r e s u l t e d i n an i n v e r t e d V-shaped curve i n genotypes A, B, K, W, X and Y. Compared to the means i n the intermediate temperature, mean counts i n the lowest temperature were lower (P<.01) f o r these genotypes but the same was not true f o r the means i n the highest temperature. In genotypes E, Q and R, t o t a l caudal rays tended to increase w i t h r i s e i n temperature. D i f f e r e n c e between the means i n lowest and highest temperature was s i g n i f i c a n t i n E and R (P<.01) wh i l e i n Q, the means showed a tendency to d i f f e r (F = .02-.05). In genotype D, the mean t o t a l caudal rays i n the highest and lowest temperature were higher than i n the intermediate temperatures showing a V-shaped r e l a t i o n s h i p to temperature. D i f f e r e n c e s between the mean counts, however, were not s i g n i f i c a n t . In the remaining genotypes, i . e . C, G, K, I and U, no d e f i n i t e trend of temperature e f f e c t x^as observed. In a d d i t i o n to the genotypic v a r i a t i o n s described above, v a r i a b l e r e s u l t s i n t o t a l caudal ray counts were obtained by r e a r i n g eggs of the same genotype i n separate l o t s at the same temperature. Mean counts of the two samples of genotype K i n 32° tended to be d i f f e r e n t (P<.05»£02), where mean t o t a l caudal ray of the sample w i t h denser population tended to be higher. This trend was i n contrast to the r e s u l t s obtained i n experiment VI. Of the two samples of genotype U i n 2A°Cj mean count of the sample w i t h denser population tended to be higher (P<".05; > .02). -96-Conclusion Mean v e r t e b r a l counts i n extreme low and high sustained temperature become higher than i n the intermediate temperature. The intermediate temperature producing the lowest count v a r i e s w i t h genotypes. The V-shaped r e l a t i o n s h i p of v e r t e b r a l counts to temperature can be obtained i n most genotypes i f extreme temperatures nearer the upper and lower l e t h a l l i m i t s are used. Ve r t e b r a l counts of some genotypes, however, are not temperature l a b i l e . P e c t o r a l f i n ray counts i n medaka decreases c o n s i s t e n t l y w i t h increase i n temperature. Dorsal ray count shows i n general an in v e r t e d V-shaped r e l a t i o n to temperature, although mean counts i n d i f f e r e n t temperatures are not s t r i k i n g l y d i f f e r e n t . E f f e c t s of sustained temperature on anal and t o t a l caudal ray counts do not reveal any c l e a r p a t t e r n . 97-Table L. Egg numbers and m o r t a l i t y i n experiment X: e f f e c t of increased l i g h t ( i n t e n s i t y and d u r a t i o n . Treatment No. of f e r t d . eggs No. hat-ched As % of f e r t d . eggs Time to 507=. hatching (hrs.) No. sur-v i v e d to preser-v a t i o n As 7= of f e r t d . eggs (a) Parent: W 9 f t . - c . f o r 16 h r s . 100 96 96 226 92 92 9 f t . - c . f o r 24 h r s . 100 91 91 301 67 67 170 f t . - c . f o r 16 h r s . 100 82 82 370 53 53 (b) Parent: Y 9 f t . - c . f o r 16 h r s . 100 98 98 226 95 95 9 f t . - c . f o r 24 h r s . 100 100 100 278 97 97 170 f t . - c . f o r 16 h r s . 100 99 99 223 97 97 (c) Parent: a 9 f t . - c . f o r 16 h r s . 50 42 84 387 39 78 9 f t . - c . f o r 24 h r s . 50 44 88 395 34 68 170 f t . - c . f o r 16 h r s . 50 49 98 208 44 88 -98-Table L I . Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment X: E f f e c t of increased l i g h t ( i n t e n s i t y and d u r a t i o n ) . Treatment Temp (°C) T o t a l 30 vertebrae 31 32 Number Mean Remarks (a) Parent: W 9 f t . - c . f o r 16 h r s . 30° 33 17 50 30.34 9 f t . - c . f o r 24 h r s . 30° 23 27 50 30.54 Not d i f f e r e n t from c o n t r o l (P>.05) 170 f t . - c . f o r 16 h r s . 30° 35 15 50 30.30 (b) Parent: Y 9 f t . - c . f o r 16 h r s . 30° 13 79 3 95 30.89 9 f t . - c . f o r 24 h r s . 30° 13 36 1 50 30.76 170 f t . - c . f o r 16 h r s . 30° 11 38 1 50 30.80 (c) Parent: a 9 f t . - c . f o r 16 h r s . 30° 27 12 39 30.31 9 f t . - c . 30° 20 14 34 30.41 f o r 24 h r s . 170 f t . - c . 30° 32 11 1 44 30.29 f o r 16 h r s . -99-Table LIT. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment X: E f f e c t of increased l i g h t ( i n t e n s i t y and duration) Treatment Temp ( ° c ) 10 P e c t o r a l rays 11 12 13 14 Number Mean (a) Parent: W 9 f t . - c . f o r 16 h r s . 30° 22 77 1 100 10.79 9 f t . - c . f o r 24 h r s . 30° 13 82 5 100 10.92 1 170 f t . - c . f o r 16 h r s . 30° 19 78 3 100 10.84 (b) Parent: Y 9 f t . - c . f o r 16 h r s . 30° 3 140 46 1 190 12.24 9 f t . - c . f o r 24 h r s . 30° 10 72 18 100 2 12.08 170 f t . - c . f o r 16 h r s . 30° 1 20 77 2 100 11.80 3 (c) Parent: a 9 f t . - c . f o r 16 h r s . 30° 3 57 18 78 11.19 9 f t . - c . f o r 24 h r s . 30° 2 49 16 67 11.21 170 f t . - c . f o r 16 h r s . 30° 12 69 7 88 10.94 4 Note: 1. Not d i f f e r e n t from c o n t r o l (PX05) ue.,i« hrs. 9-ft.-c. 2. Lower than 16 h r s . 9 ft.-c.(P<.01). Higher than 16 h r s . 170 f t . - c . (P<.01). 3. Lower than 16 h r s . 9 f t . - c . (P<.01). 4. Lower than 16 h r s . 9 f t . - c . (P<.01). -100-Table L I I I . Frequency d i s t r i b u t i o n of anal rays i n experiment X: E f f e c t of increased l i g h t ( i n t e n s i t y and d u r a t i o n ) . Treatment Temp (°C) 16 Anal rays 17 18 19 20 Number Mean (a) Parent: W 9 f t . - c . f o r 16 h r s . 30° 7 29 14 50 18.14 9 f t . - c . f o r 24 h r s . 30° 1 12 27 9 1 50 17.94 170 f t . - c . f o r 16 h r s . 30° 2 11 26 10 1 50 17.94 (b) Parent: Y 9 f t . - c . f o r 16 h r s . 30° 39 52 4 95 18.63 9 f t . - c . f o r 24 h r s . 30° 2 13 25 5 50 18.66 170 f t . - c . f o r 16 h r s . 30° 9 30 11 50 19.04 1 (c) Parent: a 9 f t . - c . f o r 16 h r s . o 30 4 14 15 6 39 17.59 9 f t . - c . f o r 24 h r s . 30° 3 18 13 34 17.29 170 f t . - c . f o r 16 h r s . 0 30 1 15 21 7 44 17.77 Note: 1. Higher than 16 h r s . 9 f t . - c . (P<.01). -101-Table LIV. Frequency d i s t r i b u t i o n of d o r s a l rays i n experiment X: E f f e c t of - increased l i g h t ( i n t e n s i t y and d u r a t i o n ) . Treatment Temp <°C) Dorsal rays Number 5 6 7 S Mean (a) Parent: w 9 f t . - c . 30° 33 16 1 50 6.36 f o r 16 h r s . 9 f t . - c . 30° 43 6 1 50 6.16 1 f o r 24 h r s . 170 f t . - c . 30° 38 12 50 6.24 f o r 16 h r s . (b) Parent: y 9 f t . - c . 0 30 76 18 1 95 6.21 f o r 16 h r s . 9 f t . - c . 30° 36 13 1 50 6.30 f o r 24 h r s . 170 f t . - c . 30° 29 21 50 6.42 2 f o r 16 h r s . (c) Parent: a 9 f t . - c . o 30 38 1 39 6.03 f o r 16 h r s . 9 f t . - c . 0 30 1 32 1 34 6.00 f o r 24 h r s . 170 f t . - c . o 30 41 3 44 6.07 f o r 16 h r s . Note: 1. Tends to be lower than 16 h r s . 9 f t . - c . (P<.05). 2. Higher than 16 h r s . 9 f t . - c . (P<T.01). -102-Table LV. Frequency E f f e c t of d i s t r i b u t i o n of increased l i g h t t o t a l caudal rays i n experiment X: ( i n t e n s i t y and d u r a t i o n ) . Treatment Tgtnp ( C) To t a l 20 21 22 23 caudal rays Number ' 24 25 26 27 28 Mean (a) Parent: w 9 f t . - c . f o r 16 h r s . 30° 9 10 23 6 1 1 50 22.64 9 f t . - c . f o r 24 h r s . o 30 4 13 24 8 1 50 22.78 170 f t . - c . f o r 16 h r s . 30° 2 5 6 31 5 1 50 22.72 (b) Parent: Y 9 f t . - c . f o r 16 h r s . o 30 9 17 42 19 6 93 22.96 9 f t . - c . f o r 24 h r s . 30° 4 2 28 10 6 50 23.24 170 f t . - c . f o r 16 h r s . 30° 2 7 17 16 8 50 1 23.44 (c) Parent: a 9 f t . - c , f o r 16 h r s . o 30 7 17 8 4 2 1 39 23.51 9 f t . - c . f o r 24 h r s . 30° 1 1 15 14 3 34 23.53 170 f t . - c . f o r 16 h r s . 30° 1 7 20 13 3 44 24.23 2 Note: 1. Higher than 16 h r s . 9 f t . - c . (P<.01). 2. Higher than 16 h r s . 9 f t . - c . (P<.01). -103-EFFECT OF INCREASED LIGHT (EXPERIMENT X) Introduction This experiment was designed to determine i f altered light intensity or duration w i l l change any one or a l l of the meristic series. The experiment was repeated with genotypes W, Y, and a. Treatments in 9 ft.»c. of light for 16 hours a day were used as the controls. In the second treatment, duration of light of the same intensity as the control was increased to 24 hours a day. In the third treatment,dntensity of light was increased to 170 f t . - c . by replacing the 7.5 watt lamp with a 60 watt lamp. The increased intensity was maintained for 16 hours daily during the entire course of the experiment. Eggs in a l l the replications were reared in small baskets (10x10x15 cm.) up to hatching. Except for genotype a, the young were transferred upon hatching to the larger rearing baskets (12x12x15.5cm.) and reared therein until preservation. Young of the genotype a were reared in* the small basket. Total number of f e r t i l i z e d eggs used, percentage of hatching and other relevant information are presented In Table L. A l l the lots were reared in 30°C temperature bath a l l throughout the experiment. Results Survival in a l l the treatments was satisfactory for a l l genotypes. Survival up to hatching ranged from 82 to 100 percent but the result was variable with respect to survival up to preservation. Three genotypes showed a different proportion of survival under identical conditions (Table L»). Mean vertebral counts were not 104-Figure 19. Effect of increased light on mean total vertebrae and pectoral and anal f i n rays. Letters indicate genotypes (Experiment X) -105-< 6.51 2 2 . 5 1 1 ' 1— 144 216 2 7 2 0 L I G H T ( F O O T C A N D L E H O U R S ) Figure 20. Effect of increased light on mean dorsal and total caudal f i n rays. Letters indicate genotypes (Experiment X) -106-affeeted by either increased duration or increased intensity of light (Table LI and Figure 19). Effects of the treatments on pectoral ray counts were variable on the genotypes used (Table LII and Figure 19). The mean ray count was no\t.altered by increased light duration or intensity in genotype W, while in genotype Y, under increased intensity and duration, mean counts were reduced (P. .02). HThe effect of increased intensity was most pronounced in this case and greatest reduction of pectoral ray was obtained. Genotype a was affected only by increased light intensity where the mean count decreased (P£< .01). Mean anal ray counts also varied with genotypes (Table LIII and Figure 19). No effect was apparent in genotype W. The control (in 9 f t . - c . for 16 hours) mean of genotype a was not different from the means of either of the treatment lots. In case of genotype Y, an. increase (P<.01) in the mean count occurred under increased light intensity. Increased light duration tended to decrease (P<.05) the dorsal rays of genotype W (Table LIV and Figure 20,)., In others no difference was produced. The effect of increased light intensity was apparent only in genotype Y where the dorsal ray count increased (P<.01). Effects of treatments on the total caudal rays were variable (Table LIV and Figure 20). Mean counts of genotype W were not affected by increased duration, or.increased intensity. Increased intensity increased the caudal ray counts in genotype Y and a (P<.01) but increased light duration had no effect on either of these genotypes. Conclusion Increased light conditions do not affect vertebral counts -107-in medaka raised in 30°C temperature bath, but effects on pectoral, anal and other f i n rays are variable. -108-Table LVI. Egg numbers and mortality in experiment XI: Effect of thyroxine and thiourea. (a) Fertilized eggs reared in the solutions up to hatching. Treatment No. of No. hat- As % of Time to 50% No. sur- As % of fertd. ched fertd. hatching vived to fertd. eggs eggs (hrs.) preser- eggs. vation (1) Parent: G .0025% thiourea 75 33 44 276 21 28 .005% " 75 43 57 244 34 45 .01% " 42 31 74 314 27 64 .02% " 18 16 89 274 16 89 .04% " 27 17 63 288 12 44 .05% " 26 18 69 290 17 65 (2) Parent: M Fresh water control 44 32 75 283 28 64 0.1 PPM thyroxine 100 68 68 289 46 46 0.2 " " 99 37 37 395 16 16 0.4 " " 103 48 46 323 29 28 (3) Parent: 0 Fresh water control 26 17 65 317 9 35 Tapwater control 97 66 68 382 46 47 0.2 PPM thyroxine 71 39 55 333 8 11 0.4 " " 70 29 41 288 20 29 0.8 " " 56 17 30 ' 360 15 27 1.6 " " 60 12 20 352 8 13 .01% thiourea 73 31 42 346 19 26 .02% " 61 26 43 278 21 34 .04% 52 29 56 307 24 46 -109-Table LVI. Egg numbers and m o r t a l i t y i n experiment XI: E f f e c t of thyroxine and t h i o u r e a . (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment No. of No. hat- As % of Time to 50% No. sur- As % of f e r t d . ched f e r t d . hatching v i v e d to f e r t d . eggs eggs (hrs.) preser- eggs v a t i o n (4) Parent: P Fresh water c o n t r o l 40 26 65 288 23 58 0.2'. PPM thyroxine 94 33 35 348 23 24 0.4 " " 51 29 57 323 20 39 0.8 " " 50 23 46 320 15 30 .01% thio u r e a 46 33 72 312 30 65 .02% " 50 22 44 269 17 34 .04% " 50 27 54 302 15 30 (5) Parent: Q Fresh water c o n t r o l 50 33 66 269 26 52 0.8 PPM thyroxine 61 33 54 260 21 34 .02% t h i o u r e a 54 31 57 251 22 41 (6) Parent: S Fresh water c o n t r o l 52 51 98 278 33 63 0.2 PPM thyroxine 119 92 77 272 48 40 0.4 PPM " 71 68 96 276 62 87 0.8 " »» 66 57 86 312 51 77 1.6 " " 50 26 52 406 25 50 .01% thiourea 74 69 93 283 64 86 .02% " 64 60 94 264 57 89 .04% " 66 57 96 279 55 83 -110-Table LVI continued. Egg number and m o r t a l i t y i n experiment X I : E f f e c t of thyroxine and ^thiourea. (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment No. of No. hat- As % of Time to 507= No. sur- As % of f e r t d . ched f e r t d . hatching v i v e d to f e r t d . eggs eggs (hrs.) preser- eggs v a t i o n (7) Parent: V Fresh water c o n t r o l 75 71 95 317 70 93 0.4 PPM thyroxine 75 55 73 489 52 69 0.8 " " 75 71 95 409 66 88 3.2 " " 75 54 72 497 50 67 1 .057« th i o u r e a 75 67 89 371 63 84 (8) Parent: Y Fresh water c o n t r o l 100 80 80 223 70 70 0.8 PPM thyroxine 100 91 91 291 88 88 .027o th i o u r e a 100 89 89 252 42 42 Note: 1. 43 hatched i n water a f t e r 489 hours i n the s o l u t i o n . 11 hatched i n water by temperature shock a f t e r 379 hours i n the s o l u t i o n . - I l l -Table LVI continued. Egg number and m o r t a l i t y i n experiment X I : E f f e c t of thy r o x i n e and t h i o u r e a . (b) Eggs f e r t i l i z e d and reared i n the s o l u t i o n s up to h hatching. Treatment No. of No. hat-f e r t d . ched. eggs As % of f e r t d . eggs Time to 50% hatching (hrs.) No. sur-vived to preser-v a t i o n As % i f e r t d eggs (1) Parent: Y Fresh water c o n t r o l 100 80 80 223 70 70 0.8 PPM thyroxine 100 97 97 350 90 90 0.2% t h i o u r e a 100 100 100 249 95 95 (2) Parent: a Fresh water c o n t r o l 50 43 86 328 32 64 0.8 PPM thyroxine 50 33 66 343 33 66 .04% thiou r e a 50 43 186 362 32 64 (3) Parent: b Fresh water c o n t r o l . 50 49 93 270 46 92 0.8 PPM thyroxine 50 27 54 439 27 54 .04% t h i o u r e a 50 19 38 328 15 30 Parent: V <c) Chorion p r i c k e d eggs hatching. reared i n the s o l u t i o n s up to Fresh water c o n t r o l 75 29 39 321 29 39 3.2 PPM thyroxine 75 7 397 6 & (2> 22 16 12 29 39 22 29 .02% t h i o u r e a 75 29 39 356 21 28 Note: Z l . Hatched i n s o l u t i o n . ( l . Hatched i n water a f t e r 47 5 hours i n s o l u t i o n . -112-Table LVI continued. Egg numbers and m o r t a l i t y i n experiment X I : E f f e c t of thyroxine and th i o u r e a . (d) Larvae reared i n the s o l u t i o n s a f t e r hatching. A. Number of eggs used. No. of f e r t d . eggs No. hatched As 7, of f e r t d . eggs Time to 50 7» hatching Remarks Parent: V 150 123 82 328 Reared & hatched i n tapwater ( i n b o t t l e ) B. Number of lar v a e reared. Treatment No. of larv a e i n s o l -u t i o n Mean r e a r -i n g time i n s o l u t i o n (hrs.) No. sur-vived to preser-v a t i o n As 7. of la r v a e Parent: V Fresh water 41 663 37 90 3.2 PPM thy-r o x i n e 41 227 29 71 .057. th i o u r e a 41 650 37 90 -113-EFFECT OF THYROXINE AND THIOUREA (EXPERIMENT XI) Introduction The object of this experiment was to determine the effect of thyroxine and thiourea solutions upon different meristic series in medaka. Marckmann (1954 and 1958) implied that meristic variation i s related to the rate of metabolism. Canagaratnam (1959) speculated that some of the variations are under the control of thyroid activity. Dales and Hoar (1954) suggested that the thyroid hormone of cold blooded vertebrates i s more directly concerned with growth and differentiation than i t i s with general metabolism. But according to Hoar (1951) the thyroid i s evidently involved in the regulation of the metabolism of f i s h whether or not i t i s involved in! the control of oxidative metabolism. On the basis of these suggestions, i t was expected that rearing of eggs and young in thyroxine and thiourea solutions would alter some or a l l of the meristic series, i f these were under the control of the activity of thyroid or metabolism. Description of experiments This experiment was performed in four different manners with eggs from several genotypes as detailed below. Thyroxine and thiourea used in the experiments were the products manufactured by the British Drug Housed and the Eastman Organic Chemicals respectively. Solutions of thyroxine and thiourea were made in tapwater in a l l cases. The following concentrations of thyroxine and thiourea solutions were used although a l l the concentrations were not used for a l l the replications (Table LVI). -114-Thyroxine: 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 parts per million Thiourea: .0025%, .005%, .01%, .02%, .04% and .05% a) Rearing of f e r t i l i z e d eggs in the solutions up to hatching. This was replicated with eggs of genotypes G, M, 0, P, Q, S, V and Y. In a l l cases, eggs were reared in 300 ml of the appropriate solution or tapwater (controls) in a 710 ml bottle. The bottles were floated in the desired temperature bath and were provided with a i r jets. The control and the treatment lots of a single genotype were kept in the same waterbath. Solutions and water in the bottles were replaced every sixth day with fresh solutions or water until the eggs hatched out. Particulars of the concentrations of solutions used are given in Table LVT. Temperature baths used for the different replications were not the same. Replications involving genotypes G, M, 0, P, Q and S were made in 24°C waterbath while those of genotypes V and Y were reared in 26°C bath. Except for the egg lot of genotype V in 3.2 thyroxine solutions, eggs were allowed to hatch naturally in the bottles. In case of V in 3.2 parts per million thyroxine, the eggs did not show any sign of hatching even after 490 hours in the solution (over 20 days in 26°C); 53 out of the total of 75 eggs were, therefore, washed thoroughly in water and hatched naturally in water in the bottle in about 8 hours. Remaining 21 eggs of this treatment were also transferred to water earlier (at the end of 378 hours) but the hatching was induced by temperature shock in 30^C. -115-Upon hatching, the larvae from the treatments and the control of any one genotype were reared until preservation in small cloth baskets in the same tank. Larvae of genotype Y were reared up to preservation in three large cloth baskets in the same waterbath. The amount of time the larvae spent in the bottles after hatching varied from 0 - 8 hours, depending on' the time of the day the eggs hatched. Eight hours exposure to the solution occurred only in such cases where the egg had hatched out shortly after the light went off at night. During the 8-hour dark period, hoods were not l i f t e d from the tank. But this comparatively long period of exposure was rare. In most cases, eggs hatched out during the period of daylight (16 hours). In the sample from eggs of genotype V in 3.2 ppm thyroxine solutions, larvae hatched naturally in water were reared separately from the ones hatched by temperature shock. The lot of f i s h obtained by temperature induced hatching was not used for analysis of any of the meristic series considered. b) Eggs f e r t i l i z e d and reared in the solutions up to hatching. Although the chorion of medaka eggs i s known to be freely permeable to crystalloids and small molecules (Yamamoto 1936) i t was considered useful to f e r t i l i z e the eggs in the solutions and find out i f any variation in the result could be obtained. Three replications of this experiment were made with eggs of genotypes Y, a and b. In the three replications, parents were placed in a bottle containing the appropriate solution where the eggs were lai d and f e r t i l i z e d . Eggs were then removed from the female and transferred to the rearing bottle containing the proper solution. The entire procedure of transferring -116-th e eggs was done in the appropriate solution. In a l l cases, eggs were allowed to hatch naturally in the solutions. On hatching, the young were placed in baskets and reared in water for further growth and fattening. Small baskets were used for rearing the young of genotypes a and b, while the offspring of genotype Y were reared in large baskets. Control and treatment lots of the same genotype were reared in the same tank. Samples of genotype Y and b were raised in 26°C. Egg lots of genotype a were reared in 30°C. Solutions and water in the control lots were replaced with fresh solution and water every sixth day until hatching, Particulars of the number of eggs and mortality are presented in Table LVI(*|). c) Chorion pricked hatching eggs reared in the solutions up  to hatching. Eggs of genotype V were used for this experiment. Chorion of a l l the eggs used for control and treatments were pricked with a sharp dissecting needle soon after f e r t i l i z a t i o n under a binocular : microscope. Immediately after pricking the chorion, the eggs were transferred into 710 ml bottles containing 360 ml of appropriate solution. Eggs were allowed to hatch normally inside the solution, but in 3.2 ppm thyroxine treatment, only 7 eggs hatched naturally. The remaining eggs were allowed to remain in the solution for 475 hours (over a mean period of 19 days) and then washed thoroughly in water and hatched in water in a bottle. Eggs and young of this experiment were reared in 26°C. Upon hatching, the larvae were transferred to small baskets and reared therein until young were large enough to be preserved. -117-d) Larvae reared in the solutions after hatching. 150 eggs of genotype V were reared and hatched in 710 ml bottleo containing 300 ml of freshwater. A total of 123 eggs hatched (Table LVId.) and the larvae were equally distributed into three 710 ml bottles containing 275 ml of freshwater, 275 ml of / 3.2 ppm thyroxine and 275 ml of .05% thiourea. The liquid in each bottle was brought to 300 ml by adding 25 ml of water containing Paramecium culture in order to feed the larvae. Water containing Paramecium was added to each bottle once daily for seven days. Feeding with brine shrimp nauplii was also started after two days of hatching of larvae in each case. Brine shrimp was given twice daily. The solution containing Paramecium and l e f t over brine shrimp was replaced with fresh solutions every night approximately two hours before the lights went off. Thus the young were exposed to pure solutions of thyroxine and thiourea for at least 10 hours daily for the entire period they were reared in the solutions. Young reared in thyroxine became emaciated and unhealthy. Mortality of young in this solution was higher. Consequently, the young f i s h of this bottle were transferred to water in small cloth baskets after they were exposed to thyroxine for a mean period of 227 hours. The larvae in water and in thiourea lots did not show any sign of distress or i l l health and they were reared in the bottles for 663 and 650 hours respectively. Thereafter, these two samples were transferred to baskets in tank and were reared there until time of preservation. -118-Results As shown in Table LVI the i n i t i a l egg numbers reared in different treatments and the control in a single replication were not uniform in several replications of experiment XIa. In order to find out i f i n i t i a l egg density has in any way influenced the number of vertebrae and pectoral rays, the data for all.'the replications (1-VI) with variable number of eggs were tested for an association. Chi-square test for vertebral count and pectoral ray count showed no relationship between the i n i t i a l egg density and either of these two characters. In replications numbers VI, VII, and VIII in experiment XIa, survival ranged from 52 to 92 percent. Except for two instances (genotype S in 0.2 ppm thyroxine and genotype Y in .027. thiourea) survival up to preservation in these replications ranged from 50 to 93%. In other replications of this experiment, rate of survival was not as satisfactory. In a l l the replications except no.l (where a control.1 lot i s lacking) the control lots showed a tendency to survive better up to hatching than the treatment lots. This pattern was lost when survival rates up to preservation were compared. In the second set of replications, where the eggs were f e r t i l i z e d in the solution and reared therein until hatching,percent survival was greater than 507. in different treatment in a l l the three replicates except for genotype b in .04% thiourea (Table LVIb.). Compared to the egg lot of genotype V in experiment XIa, survival of chorion pricked eggs of genotype V in experiment XIc was appreciably lower in the control as well as in the treated lots. There was no difference in survival between the control and treated lots. -119-31.5 -»-o UJ < CE CD UJ »-oc UJ > UJ 31.0 3 0 . 5 -3 0 . 0 Figure 21a. q • • o o - tn _i C J sr. 9 o o o o o cc GO 6 THIOUREA (%) g THYROXINE (ppm) Effect of thyroxine and thiourea to hatching on mean total vertebral of genotypes reared in 24°C. Letters indicate genotypes (Experiment XI) -120-a-b-Y-Y-V-•a -b •Y •Y -V •V V V Eggs fer t i l ized and reared in solutions up to hatching. Eggs reared in solutions up to hatching. Chorion pricked eggs reared in solutions up to hatching. La rvae reared in solutions. 31.0 u e 10. CVJ < o UJ < 01 as UJ H rr > < UJ S 3 0 . 5 3 0 . 0 m Q CM THIOUREA (%) Figure 21b. O cr i -z o IP CD 6 -II- CVJ THYROXINE (ppm) Effect of thyroxine and thiourea on mean total vertebrae of genotypes reared in 26° and 30OC. Letters indicate genotypes (Experiment XI) -121-Lower survival in these lots was apparently caused by the shock involved in pricking the chorion of eggs. Compared to the control, survival up to preservation was lower in both thyroxine and thiourea treated lots. Rearing of the larvae and young in .05% thiourea solution did not affect the survival rate. But in 3.2 ppm thyroxine solution, survival rate was affected and only 71% survived as against 90% in thiourea and in water (control). Total vertebrae a) Fertilized eggs reared in the solutions up to hatching. Response of the genotypes to thyroxine treatment was variable (Appendix VI and Figure 21a and b). The effect was apparent in lots treated in high concentrations of the solution. Mean vertebral counts of the samples of genotype 0 in 1.6 and 0.8 ppm solution were lower (P4.01) than the control. Mean count of the sample of genotype P in 0.8 ppm solution was not altered, but the mean of the lot of this genotype in 0.4 ppm (next lower concentration) was lower (P<.01) than the control. In concentrations of 0.1 and 0.2 ppm solutions, vertebral counts of both 0 and P remained unaffected. Mean count of the sample of genotype V in 3.2 ppm solution was significantly lower (P<C.01) than the control, even though the eggs were washed and hatched in water after being in the solution for a period of 489 hours. Mean count of the sample of V in 0.8 ppm solution was not altered but the same of the sample in 0.4 ppm solution showed a strong tendency of being lower (P - .02-.05) than the control. -122-Alteration in the vertebral count in thyroxine treated samples was not always in the same direction. In genotype S, mean vertebral counts of samples reared in a l l the different concentrations of thyroxine solution were higher (P<.01) than the mean of the control l o t . Although mean count in each concentration was higher than the control, there was no difference between the mean counts of the samples in different concentrations of the solution. In two other ge'-notypes, vertebrat counts of thyroxine treated samples tended to increase. In genotype Y, the mean of the sample in 0.8 ppm solution tended to be higher (P « .02-.05) than the control. Mean count of the lot of genotype M in 0.1 ppm solution also tended to increase (P=.05). Contrary to thyroxine, vertebral counts in thiourea treated lots were not affected except in genotype S in 017. solution, where the mean was higher (P<.01) than the control. Mean of the sample of genotype Y in 027. solution showed at.tendency to be higher (P=.05) than the control. In others, no variation in the means between control and thiourea treated lot and between lots treated in different concentrations of thiourea was observed. b) Eggs f e r t i l i z e d and reared in the solutions up to hatching. Results are presented in appendix VI and figure 21b. Although there was a decrease in the mean vertebrae in the thyroxine (0.8 ppm) treated samples of genotypes a and b, the decrease was not significant (P>.05), Mean count of the lot of genotype Y in 0.8 ppm solution, on the other hand, tended to be higher (P = .02-.05) than the control. This was similar to the result obtained for this genotype in experiment XIa. where eggs were reared in thyroxine after f e r t i l i z a t i o n . -123-Mean vertebral counts of genotypes Y and b in .04% thiourea solution in 26°C temperature-bath were not affected (P>.05) (Figure 21b). But the mean of the sample of genotype a in .04% and reared in 30°C bath was significantly higher (P<.01) than the control (Figure 21b). c) Chorion pricked f e r t i l i z e d eggs reared in the solutions  to hatching. Mean vertebral count of the sample reared in 3.2 ppm thyroxine solution showed a strong tendency of being lower (P=.02) than the control (Figure 21b and Appendix Vic). Comparison of this result with that obtained by simply rearing the f e r t i l i z e d eggs in 3.2 ppm solution indicated that pricking the chorion of egg did not alter the effect of thyroxine solution. Mean count of the present sample, though slightly higher, was not significantly different from the mean of sample from eggs with intact chorion. Mean count of the control sample from chorion pricked eggs was similarly slightly higher (but not significant) than the mean of the control lot from intact eggs. In contrast to the other results in thiourea solutions already described, the mean vertebral count of the sample in this case in .02% solution was lower (P-.02) than the control mean (Figure 21b and Appendix Vic). Difference between the means of this sample from chorion pricked egg and the sample from eggs reared in thiourea with chorion intact was not significant (P>.05) d) Larvae reared in the solutions after hatching Mean vertebral counts of the samples reared in thyroxine and thiourea solutions after hatching did not differ from the mean of the control lot (Appendix VId and Figure 21b). This result was expected -124-12.0 -CO > < or < or o t-o UJ 0. UJ S 11.0 -8.0 10.0 -Figure 22a. THIOUREA (%) | THYROXINE (ppm) u Effect of thyroxine and thiourea to hatching on mean pectoral f i n rays of genotypes reared in 24°C. Letters indicate genotypes (Experiment XI) -125-5.0 Figure 22b. .o o CM 9, i T H I O U R E A (%) i_ CO d CM ro i • 2 T H Y R O X I N E Ippm) O o Effect of thyroxine and thiourea on mean pectoral f i n rays of genotypes reared in 26° and 30°C (only a). Legends are same as in Figure 21b. (Experiment XI) -126-in the perspective of the findings in experiment VIII. Pectoral rays. a) Fertilized eggs reared in the solutions to hatching. Results are presented in appendix Vila and figure 22a and b. In 1.6 and 3.2 ppm thyroxine solutions, decrease in the mean pectoral rays was very pronounced in a l l genotypes. In lower concentrations, the effect was variable and the reduction in the mean counts was not as pronounced. Fertilized eggs of only one genotype - (i.e. genotype V) were reared in 3.2 ppm thyroxine solution. Although the eggs were removed from the solution after 489 hours of rearing and then washed and hatched in water, the pectoral ray count of the sample was reduced drastically. The mean pectoral ray of this sample was lower than the control by 3.04 rays. The next lower concentration of the solution was 1.6 ppm and eggs of genotype 0 and S were reared therein. In both cases, the effect on the pectoral rays was very pronounced. Mean pectoral counts in both were reduced by about 3 rays. A solution of the strength of 0.8 ppm was used for genotypes 0, P, Q, S, V and Y. Effects of this concentration upon the pectoral rays were variable. The decrease in the mean pectoral ray count in a l l genotypes reared in this solution was significant (P<.01) except, in the case of genotype Q where pectoral rays remained unaffected (P>.05). Eggs of genotypes M, 0, P, S, V and Y were also reared in 0.4 ppm thyroxine solution. Effect of the concentration was variable. -127-In three genotypes - i.e. 0, P and S, pectoral rays were decreased and in P the decrease was even greater than that obtained in the lot reared in 0.8 ppm solution. In the remaining three genotypes, pectoral counts were not affected in this concentration except in M where a tendency of decrease was noticeable (P • .02-05). Thyroxine solutions in concentrations lower than 0.4 ppm had no effect on the fixation of pectoral ray in genotypes M, P and S. In genotype 0, a significant decrease in the mean count resulted in the sample reared in 0.2 ppm solution. Treatment in thiourea solutions of different concentrations as shown in the table produced no effect on the mean pectoral ray count of most genotypes. Of the eight genotypes used, mean pectoral ray count of genotype V in .057. thiourea solution was significantly higher than /the control (P<.01). Mean counts of the sample of genotype Y in .027. thiourea solution tended to be higher than the control (P = .02-.05). In genotype G, the mean total pectoral ray in .057, solution tended to be higher (P => .02-.05) than that in .00257. solution. b) Eggs f e r t i l i z e d and reared in the solutions up to hatching. Results are summarized in appendix Vllb and figure 22b. In a l l replicates, eggs were reared only in 0.8 ppm thyroxine solution. Mean pectoral ray counts in genotypes a and b were significantly lower than the respective controls ,(P<.01). In genotype Y*»there was no difference between the mean rays of the control and thyroxine treated sample, although the mean count of the sample of genotype in identical concentration of thyroxine solution in experiment XIa was lower than the:control. -128-The mean pectoral ray count of the sample of genotype Y in thiourea solution (.02%)was higher than the control (P<.01). In the other replication of this genotype in identical solution in experiment XIa mean pectoral ray of the sample in thiourea solution showed merely a strong tendency to become higher than control. In both cases, the direction of response was identical. Of the remaining replications, the mean pectoral ray count of the thiourea treated sample of genotype b was higher than the control (P - .01-.02). The mean count of thiourea treated sample of genotype a in 30°C bath showed no difference (P>.05) with the mean count of the control. c) Chorion pricked f e r t i l i z e d eggs reared in the solutions to hatching. Results are presented in appendix Vllc.and figure 22b. The mean pectoral ray count in 3.2 ppm thyroxine solution was significantly lower than the control in spite of the fact that the eggs were washed and hatched in water after 475 hours in thyroxine. This was similar to the result obtained in experiment XIa. But compared to the mean pectoral ray in 3.2 ppm in experiment XIa this mean count was higher, although the eggs were from the same genotype and the concentrations of thyroxine solution were identical. Eggs of the present sample were, however, exposed to thyroxine solution for a lesser period of time (475 as against 489 hours in XIa). The mean pectoral ray count of the sample reared in .02% thiourea solution was significantly higher (P<.01) than the control. Compared with the mean of the sample of this genotype in thiourea -129-solution (.05%) in experiment XIa, mean pectoral ray count of the present sample showed a strong tendency of being higher (P • .02-.05) in spite of the fact that the solution of thiourea here was weaker (.02%). From the overall comparison of mean pectoral counts of the control, thyroxine and thiourea treated lots, from eggs with and without the chorion pricked, i t i s observed that pricking the chorion of eggs did not alter the effect of thyroxine or thiourea upon the pectoral rays. d) Larvae reared in the solutions after hatching. Results are presented in appendix Vlld and figure 22b. Mean pectoral count of the lot reared in 3.2 ppm thyroxine solution was lower than the mean of the control by 5.46 rays. This mean count was also lower than the mean counts of the samples from eggs of this genotype reared in the same solution up to hatching without and with their chorion pricked. The mean pectoral ray of the sample reared in .05% thiourea solution was higher (P<.01) than the control. Compared to the mean count of the sample from eggs reared in an identical solution up to hatching (experiment XIa) the mean count of the lot reared in the solution after hatching was higher (P<.01).But there was no difference between this mean and the mean count of the sample from eggs reared with their chorion pricked (experiment Xlc). Anal rays. a) Fertilized eggs reared in the solution up to hatching. Results are summarized in appendix VIII and figures 23a and b. -130-21.5 20.5h CO > < tr. —' 19.5h z UJ S I 8.5 r-18.0 m o o CM o CO d 10 THIOUREA — mm CM <t O O N - • • O O ° ° ° •oi _ _ J . C _ _ . (%) > S THYROXINE (ppm) Figure 23a. Effect of thyroxine and thiourea to hatching on mean anal f i n rays of genotypes reared in 24°C. Letters indicate genotypes (Experiment XI) -131 Figure 23b. Effect of thyroxine and thiourea on mean anal f i n rays of genotypes reared in 26° and 30° C (only a). Legends are same as in Figure 21b (Experiment XI). -132-As the larvae were to be removed from the solutions soon after hatching and reared in renewable tap water, no effect of the solutions on anal counts was anticipated. But the results obtained showed that in at least some genotypes, effects were produced on anal rays. Anal ray counts of genotype M in different concentrations of thyroxine did not show any variation. In case of genotype 0 the mean count of the sample in 0.8 ppm tended to be lower (P =.05) than the control but the mean in the next higher concentration (in 1.6 ppm) showed no difference (P/>.05); size of this latter sample was however very small (8 fi s h ) . In 0,4 ppm thyroxine solution, the anal ray count showed a strong tendency of increase (P = .01-.02) whereas in the lowest concentration of thyroxine, i.e. in 0.2 ppm, the count remained unaffected. Mean anal counts of the samples of genotype P in 0.8 and 0.4 ppm thyroxine were lower than the control in each case (P<.01). The mean of the sample from eggs reared in 0.2 ppm was not affected. In genotype Q the mean anal ray count of the sample from thyroxine (0.8 ppm) treated eggs tended to be lower than control (P<.05). Mean anal ray of genotype S remained unaffected in a l l concentrations of the thyroxine solution. Contrary to the above, the mean anal ray count of each sample of genotype V obtained by rearing eggs in 3.2 ppm, 0.8 ppm and 0.4 ppm thyroxine solution was lower than the control (P<.01). Compared to the mean count of the control lot, mean of the samples of genotype Y in 0.8 ppm and 0.4 ppm thyroxine solution was not different. -133-Rearing and hatching the eggs in thiourea solutions produced no effect on the anal ray counts in the majority of the genotypes cited above. In genotype 0 the mean count in the sample in .04% thiourea solution showed a strong tendency to increase (P = .02-.05). Mean counts of the samples of genotype S in .04% and .02% thiourea solutions were higher (P<.01) than the mean of the control. b) Eggs f e r t i l i z e d and reared in the solutions up to  hatching. Results are shown inappendix VIII and figure 23b. The mean anal ray count of genotype Y in 0.8 ppm tended to be higher (P •> .02-.05) than the control. Sample of genotype b in 0.8 ppm thyroxine had a mean anal ray count lower than the control (P<.01). In the third replication of this experiment in 30°C bath, the mean anal ray of the sample of genotype a in 0.8 ppm thyroxine solution was the same as that of the control. The mean count of the thiourea treated sample of genotype a strongly tended to be higher than the control (P = .01-.02). In the other two replicates there was no difference between the mean anal ray counts of the control and respective samples from eggs f e r t i l i z e d and hatched in thiourea solutions. c) Chorion pricked f e r t i l i z e d eggs reared in the solutions to hatching. Results are summarized in appendix VIIIc and figure 23b. The mean anal ray count of the sample in 3.2 ppm thyroxine solution was not different from the control (P>.05). This was in contrast to the result obtained by rearing the f e r t i l i z e d eggs of the genotype in -134-thyroxine solution of identical concentration where the mean anal ray count was significantly lower than the corresponding control (experiment Xla). In both, eggs were reared in thyroxine solution for a period of time (489 hours in Xla and 475 hours in this case) but hatched in water. The mean anal ray of the sample from eggs reared and hatched in .027. thiourea showed no difference (P>.05) from the mean of the control. This mean again i s also not different from the mean of the sample of this genotype reared in .057. thiourea solution in experiment Xla. d) Larvae reared in the solutions after hatching. Results are presented in appendix VIII and figure 23b. The effect of thyroxine solution on the anal ray of the young was pronounced. Compared to the mean anal rays of the control, the mean of the sample reared in 3.2 ppm thyroxine was lower by 2.98 rays. This was also lower than the mean count of the sample in identical thyroxine solution in experiments XL a and c. Comparison of the mean anal ray counts of the three respective control lots showed no difference (P .05) between them. Mean anal ray of the lot reared in .057. thiourea solution strangely tended to be higher than the control (P =• .01-.02). This was not seen in the replicates of this genotype V in experiments where eggs were reared and hatched in thiourea solution (experiments XI a and c). Dorsal rays a) Fertilized eggs reared in the solutions up to hatching. Results are summarized in appendix IX and figure 24. In -135-Figure 24. Effect of thyroxine and thiourea to hatching on mean dorsal f i n rays of genotypes reared in 24°C (upper) and in 26° and 30°C (lower). For lower figure, legends are same as in Figure 21b. (Experiment XI) -136-none of the samples treated in thyroxine solution, the mean dorsal ray count differed from the mean of the control lots. But the mean count of the samples in thiourea solution was altered in some of the genotypes and this alteration was in the direction of increase in every such case. The mean count of the sample of genotype G in .047. thiourea was higher (P<.01) than the mean of the lot in .00257. thiourea solution. But the mean count of the sample in the highest concentration of thiourea solution (.057.) did not di f f e r from that in .00257. solution. Samples of genotype 0 reared in .047. and .017. thiourea solutions had a mean dorsal ray count higher (P<.01 in each case) than the control but the mean of the lot in .02% solution did not differ from the control. Although the mean of the sample of genotype P in the highest (.04%) concentration of thiourea did not diffe r from the control, the mean of the sample reared in .02% solution was higher (P<.01) than the control. A significant increase in the mean dorsal ray count of genotype Y was also obtained in .02% thiourea solution, b) Eggs f e r t i l i z e d and reared up to hatching in the solutions. Results are presented in appendix IXb and figure 24. As in experiment Xla, treatment in thyroxine showed no effect on the dorsal ray counts of any of the replicates of this experiment. Rearing in thiourea solutions also had no effect on the dorsal ray counts of a l l the genotypes including Y, the mean count of the sample of which in identical solution of thiourea in experiment Xla showed an increase. In the present case, however, there was a weak suggestion of increase in the mean count in thiourea solution but this was not significant. -137-2 4 . 5 CO < rr o < z < UJ 2 3 . 2 2 . 5 -T H I O U R E A (%) T H Y R O X I N E Figure 25a. Effect of thyroxine and thiourea to hatching on mean total caudal f i n rays of genotypes reared in 24°C. Letters indicate genotypes (Experiment XI) -138-17.0 in o o CM O THIOUREA 1%) Figure 25b. o rr. »-z o u d CD d -VA-CM THYROXINE (ppm) Effect of thyroxine and thiourea on mean total caudal f i n rays of genotypes reared in 26° and 30°C (only a), Legends as in Figure 21b (Experiment XI) -139-c) Chorion pricked f e r t i l i z e d eggs in the solution, to  hatching. Rearing of the chorion pricked eggs in thyroxine and thiourea solution caused no difference in the mean dorsal ray counts of the samples (Appendix IXc and Figure 24). The results were also not different from those where eggs were reared with intact chorion (experiment Xla). d) Larvae reared in the solutions after hatching. Mean count of the lot in 3.2 ppm thyroxine solution was significantly lower (P<.01) than the control (Appendix IXd and Figure 24). Though the mean of the lot in thiourea solution was higher than the control, the difference was not significant (P>.05). Total caudal rays a) Fertilized eggs reared in the solutions up to hatching. Results are presented in appendix Xa and figure 25 a and b. In four of the replicates, the mean caudal ray counts of samples reared in thyroxine solutions differed from the controls or showed some tendency of becoming different. But this difference was not in the same direction in different genotypes. Mean counts of genotype M in 0.4 ppm solution tended to be lower (P<.05). Mean total caudal ray of genotype P was lower in only 0.4 ppm solution but the same in the higher 0.8 ppm) and lower (0.2 ppm) concentration merely showed a tendency of being lower (P<.05). Mean counts of the samples of genotype V in a l l concentrations- of the solution were lower (P<.01) than the control. Mean of the sample of genotype Y was higher (P<.01) than control in 0.4 ppm solution but the same in the higher concen-tration (0.8 ppm) was not significantly different from control. -140-In a l l but two genotypes, mean total caudal rays of lots reared In thiourea solution did not diffe r from the controls. Of the genotypes showing difference, mean count of the sample of genotype P in .01% solution was lower (P<.01) than control. Means of the samples of this genotype reared in .02% and .04% solutions showed a tendency fc6 decrease (P<.05). In genotype V, mean count of the sample in thiourea solution showed a tendency of being lower than the mean of the control (P<.05). b) Eggs f e r t i l i z e d and reared up to hatching in the solution. In a l l three replicates with genotypes Y, a and b, the rearing of the eggs in thyroxine and thiourea solutions did not alter the mean total caudal rays in any significant manner (Appendix Xb and Figure 25b). Results obtained here for genotype Y did not dif f e r from those obtained in experiment XIa in identical concen-trations of the solutions. c) Chorion pricked f e r t i l i z e d eggs reared in the solution  up to hatching. Although the mean total caudal ray of the sample in 3.2 ppm thyroxine was somewhat lower than the control, the difference was not significant (P>.05: Appendix Xc and Figure 25b). This result was different from that obtained in experiment XIa with the same genotype, where the thyroxine treated sample had a lower mean than the control. Mean count of the sample hatched in .02% thiourea solution was slightly higher than the control but the increase was not st a t i s t i c a l l y significant (P>.05). This result also differed from the result in XIa where the mean in .05% solution was lower than the control. -141-d) Larvae reared in the solutions after hatching. Mean count of the sample in thyroxine solution was lower (P<.01) than the control by 4.77 rays (Appendix Xd and Figure 25b. This decrease was also larger than that found in the sample of the genotype from eggs hatched in similar solution in experiment Xla. The mean count of the lot raised in .05% thiourea solution was lower (P<.01) than the control. This result i s also similar to that obtained with this genotype in the replication in connection with experiment Xla, but the decrease in the mean in the present case was greater. Conclusions Both thyroxine and thiourea alter meristic characters of medaka. In high concentrations, thyroxine generally decreases the meristic characters whereas thiourea tends to increase them. This i s , however, not always true. The effect of thyroxine on vertebral number was a duplication of high (or low) temperature whereas on pectoral ray counts, the effect appeared to be parallel to that obtained by increase in temperarures. The effect of thiourea on the pectoral ray appeared to be a duplication of the effect produced by low temperatures. In respect of anal, dorsal and total caudal ray counts, the effects of thyroxine and thiourea are not clear and consistent, particularly in replications where eggs were reared only to hatching in the solutions. -142-Table LV I I . Egg number and m o r t a l i t y i n experiment XH: E f f e c t of 2, 4-Dinitrophenol. Treatment No. of No. hat- As % of Time to 507= No. sur- As % of f e r t d . ched f e r t d . hatching vived to f e r t d . eggs eggs (hrs.) preser- eggs v a t i o n Parent: Y Fresh water c o n t r o l 100 80 80 223 70 70 1: 1,000,000 d i n i t r o p h e n o l 100 24 24 229 18 18 1: 800,000 d i n i t r o p h e n a l 100 70 70 231 51 51 Table L X I I I . Egg number and m o r t a l i t y E f f e c t of urethan. i n experiment X I I I : Parent: Y Fresh water c o n t r o l 100 80 80 223 70 70 0.57, Urethan 100 48 48 255 17 17 Table UXVI. Egg number and m o r t a l i t y i E f f e c t of s a l i n i t y . n experiment XIV: Parent: a Fresh water 50 43 86 431 39 78 Sea water 50 43 86 460 35 70 -143-Table LVIII. Frequency distribution of total vertebrae in experiment XII: Effect of 2,4-Dinitrophenol. Treatment Temp (°C) Total 30 vertebrae 31 32 Number Mean Remarks Parent: Y Fresh water control 26° 12 54 4 70 30.89 1: 1,000,000 dinitrophenol 26° 4 14 18 30.78 1: 800,000 dinitrophenol 26° 2 39 10 51 Higher than fresh-water (P<.01). 31.16 Table LXIV. Frequency distribution of total Effect of urethan. vertebrae in experiment XIII: Parent: Y Freshwater control 26° 12 54 4 70 30.89 0.57. Urethan o 26 2 10 5 17 Tends to be higher 31.18 than fresh water (P<.05;>.02). Table LXVII. Frequency distribution of total vertebrae in experiment XIV: Effect of salinity. Parent: a Fresh water control 26° 17 22 39 30.56 Sea Water 26° 5 29 1 35 30.89 Higher than fresh' water (P<.01). -144-Table LIX. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment X I I : E f f e c t of 2,4 - d i n i t r o p h e n o l . Treatment Temp P e c t o r a l rays (°C) 11 12 13 14 15 Number Mean Parent: Y Fresh water c o n t r o l 26o 63 77 140 12.55 1: 1,000,000 d i n i t r o p h e n o l 26° 8 26 2 36 12.83 1 1: 800,000 di n i n t r o p h e n o l 26° 38 63 1 1102 12.64 Table LXV. Frequency d i s t r i b u t i o n of p e c t o r a l E f f e c t of urethan. rays i n experiment X I I I : Parent: Y Freshwater c o n t r o l 26° 63 77 140 12.55 0.5% Urethan 26° 7 22 4 1 34 12.97 2 Table LX. Frequency E f f e c t of d i s t r i b u t i o n of anal rays 2.4-Dini trophenol. i n experiment X I I . Treatment Temp Anal rays (°C) 17 18 19 20 21 Number Mean Parent: Y -Freshwater c o n t r o l 26° 2 18 37 12 1 70 18.89 1: 1,000,000 d i n i t r o p h e n o l 26° 6 8 4 18 18.89 1: 800,000 d i n i t r o p h e n o l 26° 4 24 20 2 1 51 18.45 3 Note: 1. Higher than f r e s h water (p<.01). 2. Higher than f r e s h water (P<.01). 3. Lower than f r e s h w a t e r (P<.01). -145-Table LXI. Frequency d i s t r i b u t i o n of do r s a l rays i n experiment X I I : E f f e c t of 2,4-Dinitrophenal. Treatment Temp (°C) Dorsal 5 6 rays 7 Number Mean Parent: Y Fresh water c o n t r o l 26° 50 20 70 6.29 1: 1,000,000 d i n i t r o p h e n o l 26° 13 5 18 6.28 1: 800,000 d i n i t r o p h e n o l 26° 37 14 51 6.27 Table L X I I . Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment X I I : E f f e c t of 2,4-Dinitrophenol. Treatment Temp (°C) T 19 20 To t a l caudal 21 22 23 rays 24 25 26 Number Mean Parent: Y Fresh water c o n t r o l 26° 1 2 23 23 16 5 70 21.94 1: 1,000,000 d i n i t r o p h e n o l 26° 3 9 5 1 18 22.22 1: 800,000 d i n i t r o p h e n o l 26° 6 24 13 7 1 51 22.47 1 Note: 1. Higher than f r e s h water (P<.01). -146-EFFECT OF 2, 4 - DINITROPHENOL (EXPERIMENT XII) Introduction This experiment was performed to study the effect of dinitrophenol on the fixation of different meristic series and to ascertain i f the effect of the chemical parallels the effect of temperature in this respect. Dinitrophenol increases oxygen consumption by uncoupling oxidative phosphorylation and probably makes less energy available for chemical work. It was anticipated that the resultant alteration in the metabolic pattern of the eggs would produce changes in the meristic series fixed early in development. Description of experiment Eggs of genotype Y were reared in two concentrations (1: 1,000,000 and 1: 800,000) of dinitrophenol. The chemical used i s the product manufactured by Eastman Kodak Company, Rochester, New York, and solutions were made in tap water?,;. Fertilized eggs were put into 300 ml of the solution of required concentration and reared therein until hatching. After hatching, the young were transferred to small baskets in tap water for further growth. A control lot was reared in an identical manner in a bottle containing 300 ml of water up to hatching and reared thereafter in a basket. A l l the lots were reared in 26°C temperature both before and after hatching. -147-Figure 26. Effect of urethan and dinitrophenol on mean total vertebrae and pectoral and anal f i n rays of genotype Y (Experiments XII and XIII) -148-co 6.5 | 1 1 1 1 1 r cc U R E T H A N D I N I T R O P H E N O L Figure 27. Effect of urethan and dinitrophenol on mean dorsal and total cauday f i n rays of genotype Y (Experiments XII and XIII) -149-Results 707. of the eggs survived to hatching in the higher concentration of the solution while in the lower concentration a large mortality occurred as a result of heavy fungus attack (Table LVII). Mean vertebral count of the lot hatched in the stronger solution was higher (P<.01) than the control (Table LVTII and Figure 26). Mean pectoral ray count of the sample in the weaker solution was higher than the control (P<.01) but the stronger solution produced no significant difference (Table LIX and Figure 26). In lower concentration, the mean anal ray count was not altered but in the higher, mean count decreased significantly (P<.01. Table LX and Figure 26). While mean dorsal ray counts remained unaffected in dinitrophenol solutions (Table LXI and Figure 27), total caudal ray count of the sample from eggs hatched in stronger solution was higher than the control mean (Table LXII and figure 27). Conclusion Dinitrophenol alters the meristic counts by upsetting the availa b i l i t y of energy in the developing embryo. The effect of the chemical on vertebrae appears to be parallel to that obtained in extreme low (or high) temperature whereas the same on pectoral rays resembles the effect of low temperature. In view of the inconsistent results obtained for anal and total caudal rays in temperature, effects on these characters cannot be compared. -150-EFFECT OF URETHAN (EXPERIMENT XIII) Introduction Urethan (Ethyl carbamate) i s known as a mitotic poison and i t s a b i l i t y to arrest or reduce mitotic activity was demon-strated in case of sea urchin eggs ( L i l l i e 1941; Cornman 1950) and in c i l i a t e s (Burt 1945). Urethan exerted a retarding effect on growth and differentiation of the embryonic structures.in Zebra fi s h , Brachydenio rerio (Battle and Hisaoka 1952). The object of the present experiment was to find out how urethan influences the meristic characters, particularly the vertebrae, when eggs are reared in the solution. Description of experiment Eggs of genotype Y were used for this experiment. 100 eggs were placed in 300 ml of 0.5% urethan solution in a 710 ml bottle and reared in 26°C temperature bath. The solution was made in tap water with urethan manufactured by Fisher Scientific Company. The solution in the bottle was replaced every six days unt-il the eggs hatched out. Eggs were placed inside the solution immediately after f e r t i l i z a t i o n and reared continuously therein until hatching. Immediately upon hatching, the larvae were transferred to small cloth basket in tap water and reared there until preservation. The lot used as control for thyroxine,thiourea and dinitrophenol was also used as a control for this experiment. Aeration was maintained in the bottles containing the eggs. -151-Results 48% survived up to hatching but a great mortality occurred after hatching. The number that survived up to preservation was less than 50% of the number hatched (Table LXIII). Mean vertebral count of the urethan treated sample displayed a strong tendency to become higher.(P » .02-.05; Table LXIV and Figure 26). "An increase in the mean pectoral ray count also was obtained by the treatment in urethan (P<.01: Table LXV and Figure 26) but anal, dorsal and total caudal ray counts remained unaffected (Figures 26 and 27). Conclusion Urethan alters the meristic characters of medaka and the effect resembles those obtained in low temperature. -152-EFFECT OF SALINITY (EXPERIMENT XIV) Introduction In an aquatic organism which must regulate both water and salt, there w i l l probably be some effect of salinity on metabolism. This experiment was conducted to test the effect of salinity and resultant altered metabolism on the meristic characters in medaka. Description of experiment Eggs of genotype a were used for this experiment. Sea water (25.88%.salinity) available in the Biological Sciences Building of the University was used for the treatment. 50 eggs were placed in 300 ml of sea water in a 170 ml bottle and floated in a 26°C bath. For comparison, another lot of 50 eggs were reared in 300 ml of fresh water (tap water) in a similar bottle alongside the treatment bottle. Water in both bottles was aerated. On hatching, larvae from both bottles were transferred to small cloth basket in fresh (tap) water and reared therein until preservation. Results Survival of eggs up to hatching was 86% in both lots but mortality of fry was slightly greater in the lot hatched in sea water (Table LXVI). Mean vertebral count of the sample from eggs reared in sea water was higher (P<.01) than that in f r e s h L ^ t e r (Table LXVII). Pectoral, anal, dorsal and total caudal ray counts were not altered significantly by sea water. Salinity induced alteration of metabolism therefore, affects the meristic counts in medaka. -153-HATCHING TIME AND MERISTIC VARIATION The data of a l l experiments were analysed with respect to hatching time. Tines to 507. hatching are recorded in Tables I, VII, XIII, XIX, XXV, XXXI, XXXVII, XLIII, XLIX, L, LVI, LVII, LXIII and LXV1. In some of these experiments marked differences occurred in hatching times of lots from different parents or treatments, while in others hatching times were similar. Analysis for association between hatching time and meristic characters were made for different genotypes in the same treatment, and for different egg lots of the same genotype reared separ-ately but under identical conditions. In a l l the experiments, no correlation was found between time to hatching and number of meristic parts formed. SIZE HIERARCHY AND MERISTIC VARIATION As expected,a considerable variation in the relative sizes of individuals occurred within a single lot. This variation was found in lots where young f i s h were obtained from eggs accumulated on successive days as well as in lots where these were from eggs of a single day*s spawning. To test for correlation between relative size and meristic differences of the individuals within the same lot, data of the following lots were analysed: (a) Individuals from 25 egg lot, 100 egg lot and 200 egg lot in experiment VI. (b) Lots from eggs obtained on March 23 and April 8, 1961 in experiment III and a l l lots of genotype J, F and crosses thereof in experiment VII. Individuals in each lot were divided into small and large f i s h on the basis of their standard length. Individual count of each -154-character was then recorded against the length and then a chi-square test (Dixon and Massey 1957) was made for each character separately. If the calculated chi-square value was higher than the tabled value with appropriate degrees of freedom, an association between the character and length was concluded. These tests revealed a significant positive association between size and the total caudal ray of individuals (within the same lot) in the lot from 200 eggs (egg density experiment) and in the lot from eggs of April 8, 1961 (genotype U). In other lots, there was no correlation between relative size and caudal rays. A significant positive association between anal ray and-length was found in the lot obtained from 200 eggs (genotype Y; egg density experiment) and in. the lot obtained by the cross of J? and Fcf (Egg size experiment replicate #1). No correlation between length and vertebrae, pectoral rays and dorsal rays was found in any of the lots analysed. SUMMARY OF RESULTS Effect of treatments on the mean counts of different meristic series. Treatments and number Total Pectoral Anal f i n Dorsal Total caudal of replications vertebrae f i n rays f i n f i n rays (in figure) rays rays  Malachite green treat-ments of eggs up to hatching: 2 None None None None None Nature of egg rearing None None None None Sample from bottle containers and varia- reared eggs tends to tions in aeration up have lover mean to hatching: 2 for f i r s t part and 1 for second part Successive days eggs None None Mean of earlier None Mean of earlier lot from same parent: 1 lot tends to be tends to be higher hi gher Mechanical shock to None eggs (shaking) up to hatching: 1 None Mean of lot None None shaken after 4 days undisturbed development tends to be higher Pricking chorion of None None Mean lower in Mean lower in None fe r t i l i z e d eggs: 2 both pricked pricked chorion chorion lots. lot in one. Summary of results (cont'd) Treatments and number Total of replications vertebrae (in figure)  Pectoral f i n rays Anal f i n rays Dorsal f i n rays Total caudal f i n rays Egg density effect: 1 None None Erratic. Fourfold increase in density decreased mean in one lot: in others no effect None Increased density reduced mean count but effect not progressive Egg size effect: 2 Independent of Independent egg size. of egg size. In one, large egg lot gave higher rays, in the other Independent Independent of egg size, of egg size. Temperature transfer of developing eggs (a) Low to high tem-perature In early part Not fixed of sensitive before period temper- hat-ching ature shock reduced verte-brae. Towards end of sensi-tive period,ve-rtebrae increased. Not fixed before hatching. Not fixed before hatching. Not fixed before hatching. (b) High to low temperature Vertebrae fixed by 100-120 day degrees. ON Sustained temperature. 15. V-shaped curve in Inversely 9; no effect in 3, related to decreased with temperature increase in tern- in 13. In 2 perature in 2. effect irregular Erratic in the last. Altered, but no consistent pattern. Alteration not statist-i c a l l y signif-icant. But majority gave an inverted V curve against temperature. Altered but no consistent pattern. Summary of results (Cont'd) Treatments and number Total Pectoral Anal f i n Dorsal Total caudal of replications Vertebrae f i n rays rays f i n rays f i n rays .. (in figure) Increased light (a) Duration:3 None Lowered in 1 None None None (b) Intensity: 3 None Lowered in 2 Increased Increased Increased in 2 in 1 in 1 A. Thyroxine (a) Fertilized eggs hatched in solutions. 7 Decreased in 3; increased in 3; no effect in 1 Decreased Reduced in 3; None no effect in the rest. Reduced in 3; increased in 1; and no effect on the rest. (b) Fertilized and hatched in solution, 3 (c) chorion pricked eggs hatched in solution. 1. No effect on 2. Tended to increase in 1. Tended strongly to decrease. Decreased in 2. No effect in 1. Decreased Reduced in 3; None no effect in the rest None None None None U l i (d) Larvae reared in solution. 1. B. Thiourea (a) Fertilized eggs hatched in solution 7 (b) Fertilized and hatched in solution. 3. No effect Increased in 1; no effect on the rest. Increased in 1; no effect on the rest. Decreased Increased in 3; no effect in the rest. Increased in 2; no effect in the rest. Decreased Increased in 2; no effect in the rest. Tended to increase in 1; no effect in the rest. Decreased Increased in 4; ho effect in the rest None Decreased Tended to reduce in 2; no effect in the rest. None (c) Chorion pricked None eggs hatched in solution. 1. (d) Larvae reared None in solution. 1 Increased Increased None Tended to increase None None None Reduced Summary of results (cont'g) Treatments and number Total Pectoral Anal f i n Dorsal Total caudal of replications Vertebrae f i n rays rays f i n rays f i n rays (in figure)  2,4-Dinitrophenol; 1 Increased in higher concentrations. Increased in lower concentrations Decreased in higher . concentrations. No effect Increased in.* high concentrations. Urethan; 1 Tended to increase. Increased. No effect. No effect. No effect. Seawater; 1 Increased. No effect. No effect. No effect. No effect. -159-DISCUSSION Metabolism and meristic characters: A large number of diversified chemical processes are basic to the ac t i v i t i e s of a l l living organisms. These processes,which may be collectively called metabolism, are controlled by a number of widely different interlocked enzyme systems. Some metabolic processes are very general in nature and supply the energy whereby the organism merely continues to exist,while others are more specific, providing energy with which the organism can undergo growth or differentiation, or carrying on some special activity such as muscular movement. The former has been termed as the 'standard' or resting metabolism while the latter has been expressed as 'active metabolism' (Fry 1957). The difference between the active and standard rates of metabolism has been termed as 'scope of activity' (Fry 1947). Metabolism, whether 'active* or 'resting', w i l l become altered as a result of the alteration of the activity of the complex interlocking enzyme system that together constitute metabolism. Alteration of the processes would determine the amount of energy available for growth or differentiation. In other words, growth and differentiation in a developing embryo are the resultant effects of the activity of the various enzyme systems. The rate of activity of a single enzymatic reaction or a complex of enzyme reactions i s subject to alteration by various environmental conditions of which temperature i s the most important. Temperature acts as a general stimulant for a l l processes that constitute metabolism. Over a wide range of temperature, the rate of activity f i r s t increases with increasing temperature up to a certain optimum, beyond which the rate drops abruptly in spite of further increase -160-in temperature. In extreme high temperature, a depression in metabolism may be expected and therefore a reverse effect on the process of differantiation or growth. Unlike temperature, effect of certain other factors on metabolism is not generalized in nature. Photoperiod, for example, has been suggested to influence metabolism by increasing the activity of thyroid. Role of thyroid activity in the regulation of metabolism i s not clear. Thyroxine is known to stimulate metabolism but i t probably does so by affecting specific points on any chemical cycle or by acting upon some specific aspects of metabolism. Thiourea depresses metabolism probably by interfering with the production of thyroid hormone. Dinitrophenol alters the energetics of a living c e l l by uncoupling phosphorylation and reducing the availability of ATP (Gorbman and Bern,1962) Urethan, like many anaesthetics, is known to depress rate of respiration at relatively high concentration (Giese 1961) but in low concentration, i t acts as a stimulant and increases oxygen consumption (Heilbrunn 1955). Salinity has been found to alter the metabolic activity of certain aquatic organism probably as a result of the altered needs of energy for osmotic or ionic regulation (Hickman, 1959). Vertebrae: In the present investigations with sustained temperature, mean vertebral counts in 9 out of 15 replications decreased gradually with increase in temperature and then showed an abrupt rise with further increase in temperature. This pattern of response of vertebral count suggests that increase in temperature up to a certain level increases the metabolism by accelerating the activity of the enzyme systems responsible -161-for the reduction of the number of vertebrae. An optimum acceleration of these biochemical act i v i t i e s occurred in the intermediate temperatures where lowest mean count was obtained but further increase in temperature perhaps inactivated or depressed the reaction systems whereby an increase in the mean count resulted. Similarity of response of vertebral count in extreme high and low temperature is probably due to the fact that in both the extreme conditions metabolism was uneconomic and greater part of available energy had to be spent in maintaining the basic activity of l i f e . It i s not, however, clear why lowest number of vertebrae should result in the intermediate temperature producing economic metabolism. Similar results are reported by Marckmann (1954). He found that the sea trout larvae having lox<rest mean vertebral count in the inter-mediate temperature also had maximum body weight while rearing the larvae at both higher and lower temperature produced higher number of vertebrae and lower body weights. According to him, larvae with maximum body weight in the intermediate temperature had the lowest or most economic metabolism per day degree, possibly conditioned by the most harmonic interaction between single processes taking place during the development of the larvae. But in both higher and lower temperature, the metabolism was greater and uneconomic and this was reflected in higher vertebral number and lower body weight. This hypothesis was corroborated by Marckmann (1958) by actual measurement of oxygen consumption of sea trout larvae in different temperatures. The V-shaped relationship of vertebral counts to temperature as obtained in medaka can also be -162-explained in the light of the above hypothesis. Although the V-shaped relationship of vertebral number to temperature was obtained in a large * number of similar experiments o with different species of fi s h (Taning, 1944 in sea trout; Seymour, 1956 in Chinook salmon, Oncorhynchus tshawytscha; Lindsey,1952 in paradise fish , Macropodus operculcris; Molander and Molander - Swedmark, :L957? in plaice, Pleuronectes platessa'^Biazawa, 1959 in Channa argus and Lindsey, 1962, in three spine stickleback, Gasterosteus aculeatus), this i s not the invariable rule. In some investigations inverse relationship between the vertebral number and temperature was observed (Gabriel,1944 in k i l l i f i s h , Fundulus heteroclitas; Blaxter (1957) and Hempel and Blaxter (1961) in herring, Clupea harengus). In the present investigation 2 out of 15 replications made with different genotypes showed a similar inverse relationship to temperature. This was probably due to the fact that the highest temperature used for these genotypes (32°C for U and 34° for W) were not intense enough to bring about inactivation of the enzymatic processes involved. Rearing eggs in s t i l l higher temperature would probably have resulted in a V-shaped curve for these genotypes as well. This seemed to be the situation when the temperature effect on genotype Y is considered in which case, an inverse relationship would have been the conclusion i f eggs were reared only in temperatures up to 32°C. In the remaining 4 out of 15 genotypes subjected to sustained temperature treatment, mean vertebral counts failed to reflect a consistent pattern of the effect of temperature. The activity systems regulating the vertebral numbers were probably not la b i l e to the temperatures used. -163-Light, like temperature, also influences chemical reaction systems in a living organism. The processes by which various physiological systems are modified may be, according to Eisler (1961), attributed to thermal and photochemical properties of the experimental light source. In the present study, mean vertebral counts of the samples reared under longer and brighter light conditions was not significantly altered. A l l the lots for this experiment were reared in a high temperature(30°c)bath. Between light and temperature, the latter perhaps acted as the dominant controlling factor and altered the vertebral numbers to the maximum of the genetic limit. Canagaratnam (1959) obtained similar results with the vertebral counts of sockeye salmon. Light effect was negated or neutralized in high temperature (12°C) although the same altered the vertebral count significantly at a lower temperature (8°C). This suggests that under appropriate conditions, light may modify the activity of the reaction systems responsible for vertebral numbers probably through the pituit&ry-thyroid axis.' This is further evident from the results obtained in other species of f i s h reared under experimental light conditions (Dannevig, 1932; McHugh,1954,* Lindsey, 1958 and Eisler, 1961). The mechanism through which light controls metabolism is not well understood. Canagaratnam (1959). however, indicated that photoperiod or length of period of illumination may alter metabolism by altering the activity of the thyroid. If this hypothesis were correct, i t would be possible to alter the meristic characters by altering the thyroid activity by other known agents. Results of the present investigations with thyroxine and thiourea support this hypothesis. -164-In 8 out of 12 replications of the experiment where eggs were reared in thyroxine solution, mean vertebral counts of the samples were significantly altered as shown by st a t i s t i c a l analysis. The direction of change was however, not the same in a l l of them (in 4 the count decreased and in the remaining 4, this increased). The alterations in the vertebral counts in thyroxine treated eggs probably resulted from changed metabolic pattern induced by thyroxine through i t s influence upon the thyroid tissue. Canagaratnam (1959) found the existence of thyroid f o l l i c l e s in sockeye embryos of the stage (with optic vesicle, otic capsule and pectoral buds) when the number of vertebrae was being finally fixed. The sensitive period for vertebrae in medaka approximately corresponded to similar stages in sockeye, and i t can be assumed that development of endocrine organs in these embryos also reached the same level. Variation in the direction of change of the vertebral count i s , however, d i f f i c u l t to explain. As opposed to thyroxine,, .'. thiourea treatment was effective, only in two replications (genotype S in .017. solution in 24°C and genotype a in .047. solution in 30°C) where the mean counts were significantly higher. Thiourea is known to depress metabolism in f i s h . From this point of view, effect of thiourea i s perhaps similar to that of low temperature which increased.the" vertebral count by altering the metabolism to an uneconomic level. As for the instances where no alteration of vertebral count occurred, i t may be suggested that thiourea in the low concentrations used in the present investigation was probably not strong enough to induce alterations in the activity complex in these genotypes or the target organ in those perhaps had not reached a definitive stage of development to respond to thiourea. -165-Although the thyroid i s evidently involved in the regulation of the metabolism of fis h (Hoar 1951), results of earlier investigations vary as to the specific aspects of metabolism that are under i t s control (Pickford and Atz, 1957). In so far as the effect of thyroid hormone on the respiratory metabolism of f i s h is concerned, conflicting results were obtained in different as well as in the same species. Etkin, Roof and Mofskin (1940) and Hasler and Meyer (1942) found no change in the rate of oxygen consumption in gold fish, Carassius aucatus treated in thyroxine or thyroid preparations but Muller (1953J cited in Pickford and Atz, 1957) observed a significant increase in oxygen uptake in the same species after injecting the individuals with thyroxine. Similar conflicting results on the effects of anti-thyroid drugs ( thiourea, thiouracil) on the respiratory metabolism in f i s h have also been reported (no effect in gold fi s h , , Chavin and Rossmoore, 1956; reduction in oxygen consumption in thiouracil treated specimens of Campostoma anomalum. Osborn. 1951). Though the role of thyroid on respiratory metabolism i s not clear, there are evidences that other metabolic functions (nitrogen and protein metabolism, carbohydrate metabolism, fat metabolism etc) are under the control of the thyroid (Pickford and Atz, 1957). One or more of these metabolic functions may be instrumental in "the final expression"of the number of vertebrae and other meristic characters. Trifonova, Vernidube and Phillipov (1939) suggested that periods of growth and differentiation in f i s h -166-embryos were characterized by differences in carbohydrate metab-olism. In the developing eggs of medaka, evidence for the early use of fat was found (Yamamoto, 1951; Nakano, 1953) and according to Smith (1957) fat metabolism i s of major importance to the development of fi s h embryo. Alterations in the vertebral counts obtained in thyroxine and thiourea treated samples may therefore be attributable to influence of the drugs upon different aspects of the metabolism of developing medaka embryos. x Increase in the vertebral count of the sample in 2,4-Dinitrophenol was perhaps due to lack of energy for growth or differentiation. Dinitrophenol acts by uncoupling respiratory oxidation from phosphorylation and thus reduces the supply of ATP (Gorbman and Bern, 1962). The net result of this process would be uneconomic metabolism for the embryo as a whole, and growth . •.1 or differentiation would be slowed down or inhibited. Ishida (1951) also found that the development of medaka eggs was inhibited in dinitrophenol although the oxygen consumption was greatly increased. From the point of metabolism, this effect of dinitrophenol is comparable to that obtained in low (or high) temperature and thiourea. The present result is not, however, in agreement with those of Waterman (1939) who, by rearing eggs of medaka in dinitrophenol solutions (1: 40,000 to 1: 200,000), obtained a reduction in the number of myotomes as well as deformities in the body axis. In the present case, no deformities in the body axis of any of the individuals from the treated sample was found. The differences in the two results may be attributed to the weaker solutions (1: 800,00 and 1: 1,000,000) used in the present case. In higher concentration, -167-dinitrophenol probably exerts a toxic effect in addition to un-coupling phosphorylation and this may account for deformities and reduction in myotomes. In urethan solution (0.5%) there was also an increase in the number of vertebrae. Urethan in very low concentrations acts as a stimulating agent (Heilbrunn, 1955) but in relatively high concentrations (0.1 to 0.5 molar) decreases respiration (Giese, 1961). Bodine and Fitzgerald (1948) found that in low concentration, ethyl urethan causes an increase in the oxygen consumption of grasshopper embryos whereas higher concentrations tend to decrease respiration. The solution used in the present investigation was very low (0.055 molar) and the effect produced on vertebrae was perhaps the outcome of metabolic imbalance induced by increased oxygen consumption. The increased oxygen consumption indicates that more energy was perhaps required by the embryos for maintaining basic act i v i t i e s leaving no surplus for growth or differentiation. Thus the action of urethan upon the metabolism of embryos ,.ig'} comparable to those caused by dinitrophenol, low or extreme high temperature, and thiourea, insofar as the different-iation of vertebrae is concerned. Results of the present investigation differed from the findings of Battle and Hisaoka (1952) with eggs of zebra fish, Brachydenio rerio. By treating eggs in concentrations from 0.25% to 1.00% urethan, they obtained embryos with lesser number of myotomes and shorter body axis. They also observed deformities in the embryos reared in the solution but in the present case, none of the surviving individuals showed any deformity in their body axis. -168-Vertebral count was also increased by rearing eggs in sea water (25.88%) in the present investigation. In saline medium, osmoregulatory activity of the embryo demands more energy (Hickman, 1959) and this resulted in an uneconomic metabolism which caused an increase in the mean vertebral count. Thus salinity effect on the formation of vertebrae by altering the metabolic pattern may be said to be parallel to those of low or extreme high temperature, thiourea, dinitrophenol (in low concentration) and urethan. Increase in mean vertebral count in higher salinity was also obtained in threespine sticklebacks(Lindsey, 1962). Pectoral rays: As far as the fixation of pectoral rays are concerned, effects of increasing temperature, thyroxine and increased light appeared to be identical. A l l three factors apparently increased the metabolism of the embryo and fry and brought about a decrease in the mean pectoral ray counts of the samples. In other words, the biochemical processes involved in the fixation of pectoral rays are different than those for vertebrae and responded to the alteration of metabolism in an identical manner, except that in thyroxine this was much more pronounced. In contrast to the above, low temperature, low light conditions, thiourea, dinitrophenol, and urethan increased the mean pectoral ray counts by influencing the metabolism in a similar manner. Though the response of pectoral rays was consistent for most genotypes, in some no effect was produced by the treatments. From the data, however, i t may be concluded that i t is possible to bring about similar alteration in a single meristic character by duplicating the effects of low and high temperature upon metabolism with other environmental agents that alters the energetics of a developing embryo in a similar manner. -169-Other meristic series: The above conclusions cannot, however, be applied wholly to the dorsal, anal and total caudal f i n ray counts obtained in the majority of the experiments. In sustained temperature, dorsal ray counts of different genotype followed , a somewhat similar pattern, but anal and caudal ray counts differed widely. This makes i t d i f f i c u l t to compare the effect of temperature on,these characters with those obtained in thyroxine, thiourea, dinitrophenol and urethan. Results obtained by rearing larvae in :thyroxine and thiourea solution indicate that by stimulating the metabolism with thyroxine, dorsal, anal and caudal f i n ray counts can be reduced whereas inhibition induced by thiourea increases anal rays and tends to increase dorsal rays. Decrease of total caudal rays in both thyroxine and thiourea solutions i s comparable to the results obtained in some genotype where caudal ray counts showed an inverted V-shaped relationship to temperature with lowest mean counts at both highest and lowest temperatures. Decrease or increase of metabolism by both low or high temperature and thiourea or thyroxine perhaps affect the enzyme systems in an identical manner thereby producing similar or closely similar effects on the ultimate expression of the number of caudal f i n rays. The effect of dinitrophenol on anal and caudal f i n rays was similar to the effect of high temperature on that genotype (genotype Y). Dawson (1938) suggested similarity between the effects produced by dinitrophenol, high temperature and thyroxine on the embryo of frog, Rana pipiens. As pointed out earlier,dinintrophenol resembled the action of low (or high) temperature and thiourea in i t s -170-effect on vertebrae, but resembled the action of low temperature and thiourea in i t s effect on pectoral rays. Although i t is d i f f i c u l t to visualize how dinitrophenol can simulate effects of both thyroxine and thiourea at the same time, i t may be pointed out that differentiation of the different meristic series i s a very complex process and manifest-ation of each series i s probably controlled by different enzyme systems which become activated at different stages of development. Dinitrophenol alters the energetics of the growing embryo and the alteration may have completely different effect on these different reaction systems. Conclusion; The expression of meristic characters are apparently dependent upon the metabolic activity and therefore on the energetics of the growing embryo. At an intermediate temperature metabolism is high, and low vertebral counts are produced. At higher temperatures metabolism becomes uneconomic and the control breaks down as reflected by a reverse effect on the number of vertebrae. Similarly, in low temperature also uneconomic metabolism occurs which produces the same effect on vertebrae as high but unfavourable temperature. Pectoral rays, on the other hand, are reduced progressively with increase in temperature. Temperature affects a l l the metabolic processes and acts as a general controlling factor. Thyroxine and thiourea, on the other hand, probably alters some aspects of metabolism only. Thyroxine probably increases metabolism in some of i t s aspects and also lowers meristic series in general, whereas thiourea lowers metabolism and also tends to increase the meristic characters. Dinitrophenol reduces supply of energy necessary for growth or differentiation by uncoupling phosphorylation thereby i n c r e a s i n g the number of vertebrae. Urethan acts also in a similar manner. -171-Salinity upsets the energetics of the developing embryo by forcing the expenditure of energy for osmotic or ionic regulation. Comparable meristic increases resulting from unfavourable conditions (low oxygen concentration or high carbon dioxide concentration) were shown in sea trout by Taning (1952). Thyroxine duplicates the effect of high temperature and photoperiod in i t s effect on certain characters whereas thiourea produces the effect of low temperature. Dinitrophenol, urethan and salinity parallels the effect of low or high (unfavourable) on p e c t o r a l r a y s , temperature in regard to vertrebrae whereas/the effects of dinitrophenol and urethan are identical to the effects of thiourea and low temperature. Since metabolism is the sum of total of a l l enyzme ac t i v i t i e s , i t would be worth while to isolate the eneyme system or systems for each meristic series and study the effects of the environment upon each enzyme system separately. Certain factors which do not bear directly on the theme of metabolism had to be tested in the course of these experiments and these are discussed briefly below. Effect of yolk diameter: Taning (1952) mentioned that in sea trout, no relationship was found between the egg size and the number of vertebrae. No relationship between yolk diameter and different meristic series in steelhead trout Salmo gairdneri was also found (Lindsey, 1962). The data in medaka also failed to show any influence of the yolk diameter on the number of vertebrae and the f i n rays. With respect to Vertebral count, this result does not agree with Garside and Fry (1959), who suggested that below a c r i t i c a l size, yolk diameters become a -172-limiting factor and prevents the formation of the normal myomere complement. But no such relationship was evident in the present case, although the eggs of medaka are small and would be lower than the c r i t i c a l size of Garside and Fry. On the contrary, the resultant counts obtained in reciprocal crossings indicated that the characters tended to follow the genetic makeup of the parents regardless of yolk size. Possibility of an association between length of individual f i s h within the sample and meristic series was looked for in one of the replicates with eggs of genotype F and J and crosses thereof. There was no correlation between size and any of the characters studied i.e. vertebrae, pectoral, anal, dorsal and caudal rays, except anal rays of the sample from the cross between J<j> and Fc? . In other words meristic differences between the individuals were not the result of differences in egg size. Fixation of meristic characters; With a series of transfer experiments, Taning (1946) established that the determination of vertebrae in the sea trout begins during the gastrulation period and a sensitive period for this character prevails during 145-165 day degrees of development. Transfers during this sensitive period may bring about wide variations in the vertebral count as a result of the temperature shock. (Taning, 1950). Lindsey (1954) demonstrated in the paradise f i s h that sensitive period for the abdominal and caudal vertebrae are different and the abdominal vertebrae are fixed earlier in development. He also pointed out that emergence from the egg i s not a criterion for cessation of environmental influence on the fixation of meristic characters. Using light as the environmental factor, Canagaratnam (1959) found that the sensitive period for the vertebrae in sockeye salmon extended from 142 to 300 day degrees which co r r e s p o n d e d -173-to the stage of development when optic cup and otic capsule have appeared to the stage when the notochord was bent up at the t a i l and eyes became distinct. As the correspondence between 0 ° C and the lowest tem-perature at which development takes place is not known, the use of day degrees to reflect developmental process is somewhat arbitrary and questionable. This may account in part for the differences in stages of development at comparable day degrees in two sets of transfers shown in Figure II. Day degrees (calculated assuming biological zero as equal to 0 ° C ) have been used here only to represent the results of transfers made from both directions of temperature on the same scale. Results of the present experiment demonstrate that the temperature-sensitive period for vertebrae in medaka extends from approximately 40 to 120 day degrees (corresponding to the embryonic -afile-let,-I stage to the stage when pectoral buds have appeared: 48 to 144 hours in 2 0 ° C ) . This result is more or less similar to the findings of Tuning (1946) in terms of the stages of embryonic development. Results ons, the effect of temperature shock are, however, not in agreement. It has been hypothesized that consistently fewer elements form when the embryos in their sensitive period for a particular meristic series are transferred from low to high temperature (Barlow, 1961). But the mean vertebral counts of the transfer lots in this experiment suggest that this i s not always true. Similar exception to the generalization of Barlow (1961) was also demonstrated by Orska (1957: in vertebral count) and Lindsey (1954: in anal rays). -174-Although the number of pectoral rays are not yet fin a l l y fixed even after hatching (as demonstrated in experiment Xld: rearing of larvae in thyroxine and thiourea after hatching), the sensitive period for this character seemed to commence quite early in the embryonic l i f e , i.e. shortly after the pectoral f i n bud has appeared. During the early part of the sensitive period, the number of rays increased in spite of the temperature shocks. This i s another instance where Barlow's generalization (consistent lower number of element in low to high temperature transfer shocks) i s not applicable. Experiment Xld also demonstrated that the final number of anal, dorsal and total caudal rays are s t i l l alterable after hatching. But the results of the transfers from low to high temperature suggest that formation of anal rays commences quite early and in the earliest part of the sensitive period, temperature shock results in a significant decrease in the mean anal ray count (see the result of samples transferred at the end of 96 and 120 hours of development in the lower temperature). Lindsey (1954) found that mean,'anal count in paradise fi s h showed a significant increase when transferred from low to high temperature after 22 days development in the former. Though no transfer corresponding to the transfer for paradish f i s h was made for medaka, i t may be postulated that the anal rays are decreased or increased by temperature shock during the sensitive period depending onlthe stage of differentiation (bio-chemical and biophysical) reached at the time shock i s applied. From the data on dorsal and caudal rays, i t can only be suggested that the sensitive period probably commences in the former at a quite later stage in development (approximately 220 dayddegrees) while in the latter, this period coincides with the stage of development reached at 100 day degrees. -175-Unfortunately, the mean counts of anal, dorsal and caudal rays of low and high temperature controls were not different and as such, a satisfactory interpretation of the variations obtained in the transferred lots i s d i f f i c u l t . The mechanism through which temperature transfer affects the meristic character is probably the alteration of metabolic pattern. In his work with sea trout, Marckmann (1958) found that the rate of respiratory metabolism of the eggs transferred permanently to high temperature changes and becomes adapted to the rate of the eggs reared continuously in the higher temperature. The metabolic rate of eggs transferred from high to low temperature, on the other hand, continued to be somewhat lower than that of the lot reared continuously in the lower temperature. In other words, development of the lot transferred to lower temperature was delayed compared to the control lot in that temperature. Information on hatching time in the present experiment, however, does not reveal any clear pattern in regard to alteration of metabolism. Effect of extraneous factors; (Battle 1944) found that developing eggs of threespined sticklebacks, k i l l i f i s h and fourbearded rockling, Enchelyopus cimbrius were sensitive to mechanical shocks. This sensitivity was most acute in the early cleavage and blastula stages and decreased gradually with progress in gastrulation and epiboly. Similar results were also described for cod eggs by Rollefsen (1930, 1932, cited in Battle, 1944). Application of mechanical shock by dropping the eggs resulted in abnormality in the notochord. In the present experiments, eggs of medaka were shaken daily beginning from f e r t i l i z a t i o n . -176-No abnormality in tne vertebral column was found in treated individuals nor any significant alteration in vertebral counts or other meristic characters except anal rays. Anal rays appear late i n the ontogeny and the variation (.decrease in the shaken lots) found in the treated lots was probably not the result of shaking. Similar variation in the anal counts but in no other series was observed when two or three lots of eggs from the same parent were reared separately under identical conditions. The source of this variation was not clear. Retarding etrects of low levelsrof dissolved oxygen upon developing embryos have been described tor many species of fis h , Seymour 1956, 1959: Garside 1959, 1960). This effect increases at higher temperature (Garside, 1959). Further, the effect tends to be stronger in stagnant water, as gaseous exchange on the egg surface i s less efficient than in water with movement (Kinne and Kinne 1962). But the hatching time of the eggs of medaka reared in bottles without any aeration at a high temperature does not indicate any lowering of developmental rate. In terms of incubation period and hatching time, the lot in non-aerated bottle required less than half of the time required for the control lot (in aerated bottle) to complete 507. hatching. This accelerated hatching may be due to lowered oxygen av a i l a b i l i t y in water of the non-aerated bottle. Condition of asphyxia was found to induce hatching of salmon eggs 5 to 7 days in advance of the due period (Trifonova, 1937). Milkman (1954) reported that hatching of the eggs of Fundulus can be delayed indefinitely by putting them in sea water with high oxygen tension. Hatching time does not really reflect the rate -177-of development. Mean vertebral count of the lot in basket (water aerated and gradually replaced in the surrounding bath) did not di f f e r from lots in aerated water or in nonaerated water in bottles. Variation of mean vertebral count of medaka cannot, therefore, be attributed to a variation in the oxygen level in water. Wide variation in the i n i t i a l number of eggs used in any particular treatment or the quality of eggs obtained from the same parent on different days did not produce any change in the mean vertebral, pectoral and dorsal ray counts. Anal and caudal ray counts, however, responded differently. The mean anal and caudal ray counts of the lot from smallest number of eggs (25 eggs) was greater as compared to the mean counts of samples with higher density. The difference in anal ray became significant when the egg density was increased by four or eight times. The caudal ray counts were altered in some instances where the density was doubled only. Contrary to the above, in successive days eggs experiment, the anal and caudal ray counts were higher in lots with lesser density. This is not attributable to the difference in the time of rearing of the two lots as the lot with higher mean anal and caudal rays was reared for lesser time than the lots having lower mean counts. Other possible factors contributing to this variation could be social interaction or "space factor" (Vladykov 1934) involving the accumulation of hormones, waste products and other solutes in confined quarters. In the remaining ort experiment/extraneous factors, no difference in anal and caudal ray counts of the control and treatment lots of each genotype occurred although the density of the corresponding populations was somewhat different. From this, i t appears that density has to be beyond a certain level before any effect becomes apparent on the anal and caudal rays. -178-Other extraneous factors like altering the medium with malachite green or pricking the chorion also had no influence on meristic characters. The decrease in the anal count in the samples from pricked eggs was caused by some unknown factor. In summary, extraneous factors (malachite green treatment of eggs, quality of eggs of different dates from same parent, nature of rearing containers and slight alteration in aeration, egg density, shaking of eggs, pricking the chorion of eggs) do not affect the number of vertebrae, pectoral rays and dorsal rays, whereas the fixation of anal and caudal rays i s complicated by some factors that could not be isolated by these experiments Selective mortality; Some experimental studies with different environmental factors (Taning 1952, Lindsey 1962 and Molander and Molander-Swedmark 1957 with temperature; Canagaratnam 1959 with light) showed that the influence of selective mortality on meristic v a r i a b i l i t y could not be excluded, except in lining's (1944) experiment on sea trout. Heuts (1947) demonstrated that in sticklebacks selective mortality with respect to number of lateral plates occurred before hatching. In the present series of experiment, mortality before hatching and up to preservation was lower in general, and almost insignificant in many cases (e.g. genotypes U, W, X and Y in experiment IX; genotypes S, N and reciprocal cross thereof in experiment VII; genotypes S, V and Y in experiment XLa). Significant variation in one or more meristic series in different treatment as demonstrated in the genotypes cited above cannot -179-therefore be attributed to the effect of selective mortality. Results obtained from genotypes showing higher mortality in some experiments were identical to those from genotypes with low mortality,(e.g. vertebral count in higher concentration of thyroxine of genotype V and P). From these and other results presented, i t may be concluded that meristic variations are not a by-product of selective mortality amongst f i s h with different genetically controlled meristic counts, but are the direct outcome of phenotypic alteration by the treatments. -180-LITERATURE CITED Barlow, G. W., 1961. Causes and significance of morphological variation in fishes. Systematic Zoology 10: 105-117. Battle, H. I., 1944. Effects of dropping on the subsequent hatching of Teleostean ova. J. Fish. Res. Bd. Can. 6:252-256. Battle, H. I. and K. K. Hisaoka. 1952. Effects of ethyl carbamate (urethan) on the early development of the teleost (Brachydenio rerio). Cancer Res., 12(5):334-340. Blaxter, J. H. S. 1957. Herring rearing-II. The effect of temperature and other factors on myotome counts. Scottish Home Department, Marine Research 1:1-16. Bodine, J. H. and L. R. Fitzgerald.lt 1948. The action of ethyl carbamate (urethane) on the respiration of active and blocked embryonic c e l l s . 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Respiratory responses of normal and castrated goldfish to teleost and mammalian hormones. J. Exp. Zool. 91:391-404. Heilbrunn, L. V. 1955. An outline of general physiology. 3rd edition. W. B. Saunders Co., Philadelphia. Hempel, G. and J. H. S. Blaxter. 1961. Einfluss von Temperature und Salzgehalt auf Myomerenzahl und Korpergrosse von Herringsiavven. Z. Naturforsch. 16b (3):227-228. Heuts, M. J. 1947. Experimental studies on adaptive evolution in Gasterosteus  aculeatus L. Evolution, 1:89-102. Heuts, M. J. 1949. Racial divergence in f i n ray variation patterns in Gasterosteus aculeatus. J. Genetics 49:183-191. -182-Hickman, Jr. CP. 1959. The osmoregulatory role of the t-hyroid gland in the starry flounder, Platichthys stellatus. Can Zool. 37; 997-1060 Hoar, W.S. 1951. "Hormones in fi s h " , in "Some aspects of the physiology of f i s h " . Univ. Toronto Biol.Ser.(59) Pub. Ontario Fish, Res.Lab. (71): 1-51 pp. Hollister, G. 1934. Clearing and dyeing of fish for bone study. Zoologica, 12 (10): 89-101 Itazawa, Y. 1959. Influence of temperature on the number of vertebrae in fi s h . Nature, 183: 1408 - 1409 Kinne, 0. and Kinne Eva M. 1962. Rates of development in embryos of a cyprinodont f i s h exposed to different temperature - salinity - oxygen combinations. Can. J. Zool. 40 (2): 231 - 253 L i l l i e , R.S. 1941. The action of various anaesthetics in suppressing c e l l division in sea-urchin eggs. J. Biol . Chem., 17: 121-140 Lindsey, C.C. 1954. Temperature controlled meristic variation in the paradise f i s h Macropodus opercularis (L) Can. J. Zool. 32: 87-98 Lindsey, C.C. 1958. Modification of meristic variation by light duration in kokanee, Oncorhynchus nerka. Copeia 1958 2: 134-136 Lindsey, C.C. 1962. Experimental study of meristic variation in a population of threespine sticklebacks, Gasterosteus aculeatus. Can. J. Zool., 40 (2)5 271-312. Lyubitskaya, A.I. 1956. Influence of different parts of the visible spectrum regions on the developmental stages of embryos and larvae of the fishes. Zool. Jour., 35 (12): 1873-1886 (In Russian - English abstract). Lyubitskaya, A.I. 1961. Action of visible light, ultra-violet rays and temperature on body metamery in fishes. Part I. Zool. Jour. 40 (3): 397-407 (In Russian - English abstract). Magnuson, John J. 1961. An analysis of aggressive behaviour, growth, and competition for food and space in medaka (Oryzias latipes) - Pisces, Cyprinodontidae, Ph.D. thesis. Univ. of British Columbia. Marckmann, K. 1954. Is there any correlation between metabolism and number of vertebrae (and other meristic characters) in the sea trout (Salmo trutta trutta L). Meddelelser fra Danmarks Fiskeri-Og Havunderstigelser Ny Serie. Bind I, Nr.3. -183-Marckmann K. 1958. The influence of temperature on the respiratory metabolism during the development of the sea trout. Meddeleser fra Danmarks Fiskeri-Og Havunders^gelser. Ny Serie Bind II Nr.21 McHugh, J.L. 1954. The influence of light on the number of vertebrae in the grunion. Leuresthes tenuis. Copeia 19.54 (1): 23-25 Milkman, R. 1954. Controlled observation of hatching in Fundulus heteroelitus. Biol. Bull. 107: 300 Molander, A.R. and Molander-Swedmark M. 1957. Experimental investigations on variation in plaice (Pleuronectes platessa Linne). Inst.Marine Research Lysekil, Ser.Biol.Rept.No.7. Nakano, E. 1953. Respiration during maturation and at fe r t i l i z a t i o n of fis h eggs. Embryologia 2: 21-51 Oppenheimer, J. 1937. The normal stages of Fundulus 'heteroeli tus Anat. Rec. 68: 1-8. Orska, J. 1956. The influence of temperature on the development of the skeleton in teleosts. Zool. Poloniae 7 (3): 271-326 Osborn, P.E. 1951. Some experimental on the use of thiouracil as an aid in holding and transporting f i s h . Progr. Fish-Cut., 13: 75-79 Pickford G.E. and J.W. Atz. 1957. The physiology of the pituitaryy gland of fishes. New York Zoological Society, New York Rugh, R. 1962. Experimental embryology. Burgess publishing Co. Minneapolis 15, Minnesota. Schmidt, Johs, 1917. Racial investigations. II. Comptes-rendus dus travaux du Laboratoire Carlsberg. 14 (1): 11-17 Schmidt, Johs, 1919. Racial investigations II. Experiments with Lebistes reticulatus Peters (Regan). C.R. Lab. Carlsberg. 14 (5): 1-7 Schmidt Johs. 1920. Racial investigations. V. Experimental investigation with Zoarces viviparus L. C.R. Lab. Carlsberg. 14 (9): 1-14 Schmidt Johs. 1921. Racial investigations VII. Annual fluctuations of racial characters in Zoarces viviparus L. C.R. Lab. Carlsberg. 14 (15): 19-23 Schnakenbeck, W. 1931. *Zum Rassenproblem bei den Fischen' J. Cons. Int. Explor. Mer. 6: 28-40 -184-Seymour, A.H. 1956. Effects of temperature upon young chinook salmon. Ph.D. Thesis. Univ. of Washington Seymour, A.H. 1959. Effects of temperature upon the formation of vertebrae and f i n rays in young chinook salmon. 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Effects of 2,4-Dinitrophenol on the early development of the teleost, Oryzias Latipes. Biol. Bull. 76: 162-170 Vladykov, V.D. 1934. Environmental and taxonomic characters of fishes. Trans. Roy. Can.Inst. 20 (1): 99-140 Yamamoto T. 1936. Shrinkage amd permeability of the chorion of Oryzias egg with special reference to the reversal of selective permeability. J. Fac. Sci. Tokyo Imp.Univ. IV. (4): 249-261 Yamamoto, T. 1952. Action of lipoid solventscon the unfertilized eggs of the medaka (Oryzias-latipes) Annot. Zool. Japen, 24: 74-82 -185-Appendix I . Frequency d i s t r i b u t i o n fo t o t a l E f f e c t of sustained temperature, vertebrae i n experiment IX: • Temp (°C) To t a l vertebrae 29 30 31 32 Number Mean Remarks (1) Parent: A o 20 14 2 16 31.13 Higher than 26°C (P<.01). 22°(a) 2 8 10 30.80 22°(b) 7 18 25 '30.72 24° 16 16 1 33 30.54 0 26 4 2 6 30.33 Not d i f f e r e n t from 28° and 32°C (P>.05). 28° 10 18 28 30.64 30° 2 4 6 30.66 32°(a) 6 5 11 30.45 32°(b) 7 3 10 30.30 (2) Parent: B o 22 2 12 1 15 30.93 23° 19 6 25 30.24 Lower than 22°C (P<.01). 32° 9 7 16 30.44 Not d i f f e r e n t from 28°C (P>.05). -186-Appendix I . Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment IX: contd. E f f e c t of sustained temperature. Temp <°C) T o t a l vertebrae 29 30 31 32 Number Mean Remark s (3) Parent: C 20°(a) 3 10 13 30.77 Reared e n t i r e l y i n 20°C. 20°(b) 9 16 1 26 30.69 Hatched and reared i n 26°C a f t e r 15-19 days i n 20°C. 20°(c) 2 6 1 9 30.89 Reared i n 26°C a f t e r hatching. 22° 1 7 8 1 17 30.53 24°(a) 4 35 10 49 30.12 24°(b) 3 20 3 26 30.00 26°(a) 5 38 12 55 30.12 26°(b) 2 21 3 26 30.04 30° 6 23 29 29.79 32° 4 23 1 28 29.89 (4) Parent: D 22° 11 11 31.00 24°(a) 1 10 11 30.91 24°(b) 1 9 10 30.90 24°(c) 2 4 6 30.67 Not d i f f e r e n t from 24°(a) of ( b ) . 26° 10 10 20 30.50 32° 2 7 9 30.78 -187-Appendix I continued. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment IX: E f f e c t of sustained temperature. T o t a l vertebrae 29 30 31 32 Number Mean Remarks Parent: E 20° 9 27 36 30.75 24° 13 3 16 30.19 Lower than 20°C (P<.01). 28° 15 6 21 30.29 32° 5 5 10 30.50 (6) Parent: G 20° 4 4 31.00 22° 3 5 8 30.62 24° 10 19 29 30.65 26° 4 30 34 30.88 28° 5 10 15 30.67 o 32 8 25 33 30.76 (7) Parent: H 20° 3 54 8 65 31.09 Reared i n 24°C a f t e r hatching. 22° 6 45 1 52 30.90 24° 17 46 63 30.73 26° 3 32 1 36 30.94 28° 6 20 26 30.77 30° 15 55 1 71 30.77 32° 21 103 2 126 30.85 -188-Appenclix I continued. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment IX: E f f e c t of sustained temperature. Temp <°C) T o t a l vertebrae 29 30 31 32 Number Mean Remarks (3) Parent :: I 20° 1 13 14 30.93 Reared i n 24°C a f t e r hatching. o 22 10 23 33 30.70 24° 31 30 61 30.49 26° 15 13 28 30.46 Lower than 20°C (P<.01). 28° 20 34 54 30.63 30° 1 23 51 75 30.67 32° 5 55 60 30.91 Higher than 26°C (P<.01). 34° 3 4 7 31.57 Reared i n 32°C a f t e r hatching. (9) Parent: K ' 0 20 1 30 3 34 31.06 Higher than 28°C (P<.01). 0 22 5 22 1 28 30.86 24° 1 25 19 1 46 30.43 26° 18 9 27 30.33 28° 17 5 22 30.22 30° 33 13 46 30.28 32°(a) 1 11 8 20 30.35 Not d i f f e r e n t from 28°C (P>.05). 32°(b) 1 16 11 28 30.36 Not d i f f e r e n t from 28°C (P>.05). -189-Appendix I continued. Frequency distribution of total vertebrae in experiment IX: Effect of temperature. Temp Total vertebrae Number Mean Remarks ( C) 29 30 31 32 (10) Parent: Q - o -24 17 13 30 30.43 28° 11 16 27 30.59 Not different from 24° and 32°C. 32° 1 7 8 16 30.44 (11) Parent: R 20° 8 6 14 31.43 22° 1 31 4 36 31.08 26° 19 54 1 74 30.76 28° 13 28 41 30.68 30° 1 13 14 1 29 30.51 32° 1 9 9 19 30.42 34° 9 8 1 18 30.56 Reared in 32°C after hatching. (12) Parent : U 20° 1 22 13 36 31.33 24°(a) 2 27 1 30 30.97 24°(b) 3 30 3 36 31.00 28° 7 41 3 51 30.92 30° 7 22 1 30 30.80 32° 20 50 1 71 30.73 Lower than 20°C (P<.01). -190-Appendix I continued. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment IX: E f f e c t of sustained temperature. Temp (°C) T o t a l vertebrae 29 30 31 32 Number Mean Remarks (13) Parent: W 22° 4 41 5 50 31.02 26° 15 35 50 30.70 Lower than 22°C (P<.01). 30° 33 17 50 30.34 Lower than 26°C (P<.01). 32° 35 15 50 30.30 34° 7 2 9 30.22 Lower than 22° (P<.01). (14) Parent: X 22°" 6 46 14 66 31.12 26° 13 36 4 53 30.83 30° 30 39 69 30.56 Lower than 22°C (P<.01). 32° 27 39 66 30.59 34° 11 28 2 41 30.78 Tends to be higher than 30°C (P<.05) Lower than 22°C (P<.01). (15) Parent: Y 20° 37 54 91 31.59 (P<.01) Higher than 24°, 26°, 30°, & 32°C i . 24° 5 79 6 90 31.01 26° 8 71 8 87 31.00 o 30 13 79 3 95 30.89 32° 17 42 5 64 30.81 34°(a) 12 4 16 31.25 34°(b) 1 3 4 31.75 a+b+c=Mean=> 31.34 i s higher than 32°(P<.01) and lower than 20°(P<.01) and lower than 20°(P . 0 1 ) . 34°(c) 6 3 9 31.33 -191-Appendix I I . Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment IX: E f f e c t of sustained temperature. Tgmp Pec tora1 ( C) 9 10 11 rays 12 13 Number Mean Remarks (1) Parent: A o 20 11 14 25 11.56 22° 33 22 1 56 11.43 24° 3 19 10 32 11.22 0 28 24 4 28 11.14 32° 13 1 14 11.07 (2) Parent: B • 0 22 1 12 2 15 11.07 28° 7 18 25 10.7.2 32° 13 3 16 10.19 Lower than 20°C (P<..01). (3) Parent: C 20° 11 2 13 12.15 Reared e n t i r e l y i n 20°C. 22° 1 7 9 17 11.47 24°(a) 1 16 9 26 11.31 24°(b) 1 27 20 48 11.39 26° 2 35 18 55 11.29 30° 4 25 29 10.86 32° 2 5 19 2 28 10.75 Lower than 20°C and 22°C (P<.01). -192-Appendix I I . continued. Frequency d i s t r i b u t i o n fo p e c t o r a l rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) Pe c t o r a l 9 10 11 rays 12 13 Number Mean Remarks 0) Parent: D 22° 4 7 11 11.64 24° 1 18 6 25 11.20 26° 4 13 2 19 10.89 Not d i f f e r e n t from 32°C (P>.05). 32° 1 5 3 9 11.22 (5) Parent: E 20° 3 17 15 35 11.34 Hatched by temp, shock and reared i n 24°C a f t e r hatching. 24° 14 1 15 11.07 28° 1 15 5 21 11.19 32° 8 1 9 11.11 (6) Parent: G 20° 4 4 12.00 Hatched and reared i n 24° a f t e r hatching. 22° 4 2 6 11.33 24° 18 11 29 11.38 26° 5 24 2 :. 31 10.90 28° 1 13 14 10.93 32° 1 23 9 33 11.24 -193-Appendix II continued. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) Pec t o r a l 9 10 11 rays 12 13 Number Mean Remarks (7) Parent: H 20° 20 32 2 54 11.67 Hatched by temp, shock and reared i n 24°C a f t e r hatching. 20° 3 6 1 10 11.80 Reared e n t i r e l y i n 20°C. 22° 19 31 2 52 11.67 24° 4 28 7 39 11.08 26° 2 28 3 33 11.03 28° 2 19 5 26 11.11 30° 9 43 2 54 10.87 32° 31 85 4 120 10.77 (8) Parent: I 20° 17 11 28 11.39 Reared i n 24° a f t e r hatching. 22° 1 36 25 2 64 11.44 24° 3 86 8 97 11.05 26° 6 46 4 56 10.96 o 23 14 93 1 108 10.88 30° 2 55 85 142 10.58 32° 47 12 59 10.20 -194-Appendix I I continued. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) 9 Pe c t o r a l 10 11 rays 12 13 Number Mean Remarks (9) Parent: K 20° 40 22 4 66 11.45 22° 1 2 34 19 56 11.27 24° 29 7 36 11.19 26° 2 47 3 52 11.02 28° 5 35 4 44 10.98 30° 9 75 7 *92 10.95 * 1 f i s h w i t h 8 rays. 32° 1 35 57 3 96 10.64 (10) Parent: q 24° 6 41 11 58 11.08 28° 8 38 5 51 10.94 32° 7 24 1 32 10.81 (11) Parent: R 20° 1 23 4 28 12.11 Reared i n 24°C a f t e r hatching. 22° 1 22 49 72 11.67 26° 3 88 57 148 11.37 28° 3 23 14 40 11.30 30° 9 43 5 57 10.93 32° 1 24 13 38 10.32 Lower than 22°C (P<.01). -195-Appendix II continued. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) Pec t o r a l 9 10 11 rays 12 13 14 Number Mean Remarks (12) Parent: U 20° 2 50 20 72 12.25 0 24 27 91 14 132 11.90 Two samples combined; lower than 20°C (P<.01). 28° 2 65 33 2 102 11.34 30° 23 7 30 11.23 32° 10 121 11 142 11.00 (13) Parent: W 22° 5 87 8 100 12.03 26° 42 56 2 100 11.60 Lower than 22°C (P<.01). 30° 22 77 1 100 10.79 Lower than 26°C (P<.01). 32° 39 59 2 100 10.63 (14) Parent: X 22° 8 101 23 132 12.11 26° 32 67 7 106 11.76 Lower than 22°C (P<r.01). 30° 1 59 77 1 138 11.56 32° 4 100 26 130 11.17 Lower than 30°C (P<r.01). 34° 16 64 2 82 10.83 Lower than 32°C (P<.01). (15) Parent: Y o 24 41 136 3 180 12.79 26° 63 106 3 172 12.65 30° 3 140 46 1 190 12.24 Lower than 26°C (P<.01). 32° 28 96 4 128 11.81 Lower than 30°C (P<.01). 34° 13 9 22 11.40 Lower than 32°C (P<.01); F i n ray: of f i s h reared e n t i r e l y i n 34°C. -196-Appendix I I I . Frequency d i s t r i b u t i o n of anal rays i n experiment IX: E f f e c t of sustained temperature. Temp Anal rays Number Mean Remarks (°C) 17 18 19 20 21 22 (1) Parent : A 20° 4 9 3 16 17.94 o Reared i n 24 C a f t e r hatching. o 22 9 17 6 1 33 17.97 Tends to be lower than 28°C (P<.02). 0 24 6 21 5 1 33 18.03 26° 1 2 3 6 18.33 28° 2 14 11 1 28 18.43 30° 2 3 1 6 17.83 32° 1 4 4 2 11 18.64 Tends to be higher than 22°C (P<.02). (2) Parent : B 22° 3 11 1 15 18.87 28° 1 12 9 3 25 19.56 Higher than 22°C (P<.01). 32° 3 3 8 2 16 19.56 Higher than 22°C (P<.01). (3) Parent : C 20° 1 8 4 13 19.25 Reared e n t i r e l y i n 20°C. 22° 11 5 1 17 19.41 Lower than 30°C (P<.01). 24°(a) 5 25 15 3 48 19.33 24°(b) 2 13 11 26 19.35 26°(a) 2 14 28 11 55 19.87 26°(b) 3 16 7 26 19.15 30° 3 18 8 29 20.17 Higher than 20°C (P<:01). 32° 1 7 16 4 28 19.82 Tends to be higher than 20°C (P<.02). -197-Appendix I I I . Frequency d i s t r i b u t i o n of anal rays i n experiment IX: E f f e c t of sustained temperature. Temp Anal (°C) 17 18 19 rays 20 21 n o Number Mean .Remarks (4) Parent: D 22° 1 8 2 11 19.09 Lower than 32°C (P<.01). 24°(a) 1 5 4 1 11 19.45 24°(b) 7 1 2 10 19.50 24°(c) 1 3 2 6 19.17 Not d i f f e r e n t from 24°a or c. 26° 11 8 1 20 19.50 o 32 1 4 3 1 9 20.44 Higher than both 24°a and c. (5) Parent: E 20° 7 26 2 35 18.86 o Reared i n 24 C a f t e r hatching, 24° 4 11 1 16 18.81 28° 2 12 7 21 19.24 Tends to be higher than 24°C (P<.05;>.02). 32° 5 5 10 18.50 Tends to be lower than 24°C (P<-.05;>.02). Lower than 28°C (P<.01). . Anal rays 15 16 17 18 19 20 21 (6) Parent: G 22° 1 1 6 8 17.62 24° 6 21 2 29 17.86 26° 1 2 11 18 1 33 18.48 Higher than 22°C (P<.01). 28° 3 9 3 15 18.00 Not d i f f e r e n t from 26°C. 32° 12 17 4 33 18.76 Higher than 22°C (P<.01). -198-Appendix I I I continued. Frequency d i s t r i b u t i o n of anal rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) 15 16 Anal 17 rays 18 19 20 21 Number Mean Remarks (7) Parent: 20° 1 H 3 5 1 10 16.60 Reared e n t i r e l y i n 20°C. 22° 5 36 11 52 17.12 Higher than 20°C (P<.01). 24° 1 28 26 2 57 17.51 26° 2 14 15 2 33 17.48 28° 16 10 26 18.38 Higher than 24° & 22°C (P<.01 30° 5 40 23 1 69 18.30 32° 12 63 45 6 126 18.36 (8) Parent: I 22° 11 17 5 33 17.82 24° 4 31 25 60 18.35 Higher than 22°C (P<.01). 26° 4 8 14 2 28 18.50 28° 3 21 28 2 54 18.54 30° 18 42 13 1 74 18.92 Higher than 24°C (P<.01). 32° 3 19 4 26 19.04 Higher than 24°C (P<.01). (9) Parent: K 22° 1 10 12 5 28 18.75 24° 1 15 22 8 46 18.80 26° 10 14 3 27 18.74 28° 1 10 10 1 22 19.50 Higher than 22°C (P<.01). 30° 2 20 20 4 46 19.57 32°(a) 1 5 7 7 20 20.00 Higher than 22°C (P<.01). 32°(b) 5 10 10 3 28 19.39 Lower than 3 2 ° ( a ) (P<;.05). Higher than 22°C (P<.01). -199-Appendix I I I continued. Frequency d i s t r i b u t i o n of anal rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) Anal rays 15 16 17 18 19 20 21 Number Mean Remarks (10) Parent: Q 24° ~ 1 3 15 10 1 30 19.23 28° 11 11 4 26 19.73 Tends to be higher than 24°C (P<.05). 32° 5 8 3 16 18.88 Lower than 28°C (P<.01). Not d i f f e r e n t from 24°C. Anal 17 18 19 rays 20 21 22 (11) Parent: R 22° 2 9 17 6 2 36 19.92 Lower than 32°C (P<.01). 26° 4 14 48 7 1 74 19.82 28° 11 19 11 41 20.00 30° 2 9 14 3 *29 19.48 *1 f i s h w i t h 15 r a y s . Not d i f f e r e n t from 22°C (P; 32° 2 6 8 2 1 19 18.68 Tends to be lower than 30°C (P<.02). (12) Parent: U -20° 2 9 18 5 2 36 19.89 24°(a) 5 16 9 30 20.13 Tends to be higher than 24°C ( b ) . 24°(b) 11 22 3 36 19.78 28° 14 22 13 2 51 20.06 30° 1 9 17 3 30 19.73 32° 3 19 35 11 3 71 19.89 -200-Appenclix I I I continued. Frequency d i s t r i b u t i o n of anal rays i n experiment IX: E f f e c t of sustained temperature. Temp C°C) Anal 17 18 19 rays 20 21 22 Number Mean Remarks (13) Parent: W 22° 7 29 12 1 1 50 18.20 26° 9 31 10 50 18.02 -, 30° 7 29 14 50 18.14 32° 11 24 14 1 50 18.10 (14) Parent: X 22° 9 30 23 3 1 66 19.35 26° 22 16 28 6 1 53 19.77 Higher than 22°C (P<.01). 30° 1 14 38 12 4 69 20.06 32° 4 17 34 10 1 66 19.80 34° 2 12 17 10 41 19.85 Higher than 22°C (P<.01). (15) Parent: Y * 24° 21 50 18 1 90 18.99 26° 2 28 44 12 1 87 18.79 30° 39 52 4 95 18.63 32° 5 36 19 4 64 18.34 34° 4 5 2 11 17.81 Lower than 24°C (P<.01). Tends to be lower than 32°C (P<.05). -201-Appendix IV. Frequency d i s t r i b u t i o n of d o r s a l rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) Dorsal rays 5 6 7 8 Number Mean Remarks (1) Parent: A 22° 2 30 1 33 5.97 24° 30 3 33 6.09 28° 27 1 28 6.04 32° 14 14 6.00 (2) Parent: B 22° 12 2 14 6.14 28° 19 6 25 6.24 32° 16 16 6.00 (3) Parent: C 20° 10 3 13 6.23 Reared e n t i r e l y i n 20°C. 22° 11 6 17 6.35 24°(a) 19 28 47 6.60 24°(b) 12 14 26 6.54 26° 15 39 1 55 6.74 30° 9 20 29 6.69 32° 16 12 28 6.43 (4) Parent: D 22° 10 1 11 6.09 24° 15 12 27 6.44 26° 13 7 20 6.35 32° 7 2 9 6.22 -202-Appendix IV. continued. Frequency d i s t r i b u t i o n of d o r s a l rays i n experiment IX: E f f e c t of sustained temperature. Temp <°C) 5 Dorsal rays 6 7 8 Number Mean Remark s (5) Parent: E 20° 19 17 36 6.47 Reared i n 24°C a f t e r hatching. 24° 12 4 16 6.25 28° 14 7 21 6.33 32° 9 9 6.00 (6) Parent: G 22° 1 6 7 5.86 24° 26 3 29 6.10 26° 27 6 33 6.18 o 28 15 15 6.00 32° 20 11 2 33 6.45 (7) Parent: H 22° 1 51 52 5.98 o 24 5 50 4 59 5.98 26° 1 32 33 5.99 28° 26 26 6.00 30° 65 65 6.00 32° 1 124 125 5.99 -203-Appendix IV continued. Frequency d i s t r i b u t i o n of d o r s a l rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) Dorsal rays 5 . 6 7 8 Number Mean Remarks (3) Parent: I 22° 32 32 6.00 24° 42 15 57 6.26 26° 24 4 28 6.14 28° 46 8 54 6.14 30° 52 22 74 6.32 32° 41 7 48 6.15 (9) Parent: K 22° 25 3 28 6.10 24° 28 8 36 6.22 26° 21 6 27 6.22 o 28 17 5 22 6.23 30° 39 7 46 6.15 32° 41 7 48 6.15 (10) Parent: q 24° 20 9 29 6.31 28° 17 9 26 6.35 32° 13 3 16 6.19 -204-Appendix IV continued. Frequency d i s t r i b u t i o n of d o r s a l rays i n experiment IX: E f f e c t of sustained temperature. Temp (°C) 5 Dorsal rays . 6 7 8 Number Mean Remarks (11) Parent: R 22° 2 20 14 36 6.33 26° 30 43 1 74 6.60 28° 14 27 41 6.66 30° 20 9 29 6.31 32° 14 5 19 6.26 (12) Parent: U 20° 1 31 4 36 6.08 24° 59 7 66 6.10 28° 38 13 51 6.25 30° 25 5 30 6.17 32° 53 18 71 6.25 (13) Parent: VI 22° 39 11 50 6.22 26° 41 9 50 6.18 30° 33 16 1 50 6.36 32° 41 9 50 6.18 -205-Appendix IV continued. Frequency E f f e c t of d i s t r i b u t i o n of do r s a l rays i n experiment IX sustained temperature. Temp (°C) Dorsal rays 5 6 7 8 Number Mean Remarks (14) Parent: X 22° 59 7 66 6,10 26° 38 15 53 6.28 30° 58 11 69 6.16 32° 60 6 66 6.09 34° 40 1 41 6.02 (15) Parent: Y 24° 68 22 90 6.24 26° 1 64 22 87 6.24 30° 76 18 1 95 6.21 32° 2 57 5 54 6.05 34° 7 7 6.00 -206-Appendix V. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment IX. E f f e c t of temperature. Temp T o t a l caudal rays Number Mean Remarks (°C) 19 20 21 22 23 24 25 26 CD Parent: A 22° 1 1 3 4 12 4 4 29 22.83 Lower than 28°C (P<.01) 24° 5 18 3 1 32 23.15 28° 1 9 13 5 28 23.79 Not d i f f e r e n t from 32°C (P>.05). Not d i f f e r e n t from 22°C 32° 1 5 5 11 23.36 (P>.05). (2) Parent: B 22° 1 3 6 5 15 23.00 Lower than 28°C (P<.01) 23° 2 6 10 7 25 23.88 32° 2 2 7 1 2 2 16 23.31 Not d i f f e r e n t from 28° or 22°C ( P X 0 5 ) . (3) Parent: C 22° 2 8 4 3 17 22.47 24°(a) 14 13 18 3 48 22.21 24°(b) 1 5 6 11 3 26 22.35 26°(a) 8 22 18 5 2 55 23.47 26°(b) 1 6 8 10 1 26 23.15 Not d i f f e r e n t from 26°(a) (P>.05). 30° 1 7 14 7 29 22.93 Not d i f f e r e n t from 22 C (P>.05). 32° 2 7 9 9 1 23 23.00 (4) Parent: D 22° 4 1 4 2 11 24.36 _ , o Not d i f f e r e n t from 24 4 7 8 7 1 27 23.78 22°C (P>.05). 26° 2 3 12 2 -20 23.90 *1 f i s h w i t h 27 rays. 32° 1 5 2 1 9 24.22 -207-Appendix V "continued. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment IX: E f f e c t of temperature. Temp (°C) 19 To t a l caudal 20 21 22 23 rays 24 25 26 Number Mean Remark s (5) Parent : E 24° 4 4 5 3 16 22.44 28° 2 4 8 6 *21 22.67 * 1 w i t h 18 r a y s . 0 32 1 2 4 2 9 23.78 Higher than 24° and 28° (P<.01). (6) Parent: G 22° 1 1 3 2 7 22.86 Not d i f f e r e n t from 24°C (P>.05). 24° 2 13 10 3 1 29 23.58 26° 1 2 2 12 14 1 1 33 23.30 28° 1 2 4 6 2 15 23.40 Not d i f f e r e n t from 32°C (P>.05). 32° 1 2 5 17 8 33 23.88 Tends to be higher than 26°C (P<.05). (7) Parent : H 22° 3 3 6 26 10 3 1 52 22.96 24° 7 11 29 3 50 22.96 26° 7 3 21 2 33 22.54 Not d i f f e r e n t from 22°C (P>.05). 28° 4 3 9 6 4 26 23,11 30° 1 4 13 20 18 3 2 61 23.48 Tends to be higher than 22°C (P<.05). 32° 1 14 21 26 44 11 1 118 23.14 Tends to be lower than 30°C (P<..05). (8) Parent: I -208-Appendix V continued. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment IX: E f f e c t of temperature. Temp (°C) 19 20 To t a l caudal 21 22 23 rays 24 25 26 Number Mean Remarks (8) Parent: I 22° 1 8 13 6 2 1 31 23.13 24° 6 10 30 9 55 22.76 26° 1 2 11 7 1 28 22.75 28° 5 7 17 22 3 54 23.20 30° 1 11 13 25 17 3 70 22.78 32° 6 10 7 6 29 22.45 (9) Parent: ! 22° 3 5 15 4 1 28 22.82 24° 4 11 13 7 1 36 22.72 26° 2 4 14 7 27 22.93 28° 4 4 8 6 22 22.73 30° 1 3 8 11 19 4 46 23.21 32°(a) 5 7 6 2 20 22.25 32°(b) 3 5 11 9 28 22.93 Tends to be higher than 32°(a). (10) Parent: Q 24° 1 11 10 7 29 22.76 28° 3 5 5 11 3 27 23.22 Not d i f f e r e n t from 24°C (P>.05). o 32 1 2 3 8 2 16 23.44 Tends to be higher than 24°C (P<.05). -209-Appendix V continued. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment IX: E f f e c t of temperature. Temp (°C) 19 20 Tot a l 21 caudal 22 23 rays 24 25 26 Number Mean Remarks (11) Parent: R 22° 12 12 9 3 36 22.08 26° 1 22 15 28 7 1 74 22.23 2S° 1 10 9 16 4 1 41 22.37 30° 3 3 14 6 3 29 23.10 Higher than 28°C (P<.01). o 32 2 2 10 2 3 19 , .„ Tends to be higher than 2 J , i V ( P < . 0 5 ) . (12) Parent: U 20° 1 7 20 6 2 36 23.08 24°(a) 2 11 14 3 30 23.60 24°(b) 1 11 12 9 2 1 36 23.03 Tends to be lower than 24°(a) (P<.05). 28° 1 4 23 21 1 *51 23.41 -I w i t h 27 r a y s . 30° 1 7 13 8 29 22.97 Tends to be lower than 28 C (P<.05). 32° . 2 3 34 13 14 71 23.56 Higher than 30°C (P<.01). (13) Parent: W 22° 2 1 27 9 11 50 21.54 26° 8 13 21 7 1 50 22.60 Higher than 22°C (P<.01). 30° 9 10 23 6 1 *50 22.64 "1 w i t h 27 ra y s . 32° 2 11 9 23 4 1 50 22.40 -210-Appendix V continued. Frequency d i s t r i b u t i o n of t o t a l caudal rays i n experiment IX: E f f e c t of temperature. Temp Tot a l caudal rays Number Mean Remarks ( ° c ) 19 20 21 22 23 24 25 26 (14) Parent: X 22° 14 21 5 3 *66 20.38 "T w i t h 18 r a y s . 26° 1 12 18 15 5 1 *53 21,21 , v 1 w i t h 18 r a y s . Higher than 22°C (P<.01) 30° 6 22 21 17 3 69 21.84 Higher than 26°C (P<.01). 32° 1 1 12 22 25 4 1 66 22.51 Higher than 30°C (P<.01). 34° 3 11 8 14 3 2 41 22.22 Not d i f f e r e n t from 32°C (P>.05). (15) Parent: Y 24° 2 5 35 33 11 3 1 90 21.67 26° 5 5 32 29 13 3 87 21.56 30° 9 17 42 19 6 93 22.96 Higher than 26° (P<.01). 32° 1 5 11 24 16 6 1 64 23.10 Higher than 25°C (P<.01). Not d i f f e r e n t from 30°C. 34° 4 3 3 1 11 22.18 Tends to be lower than 32°C (P<.05). -211-Appendix VI. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment X I : E f f e c t of thyroxine and thiourea. (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp Tot a l vertebrae Number Mean Remark s (°C) 29 30 31 32 (1) Parent: G .0025% thiou r e a 24° 2 18 1 21 30.95 .005% 24° 4 29 1 34 30.91 .01% " 24° 1 23 3 27 31.07 .02% 0 24 1 14 1 16 31.00 .04% '» 24° 11 1 12 31.08 .05% " 24° 1 16 17 30.94 (2) Parent: M Fresh water con- 24° 1 11 3 15 30.13 t r o l 0.1 PPM thyroxine 0 24 26 20 46 30.43 Tends to be higher than c o n t r o l (P=.05) 0.2 PPM thyroxine 24° 10 6 16 30.37 0.4 PFM thyroxine 24° 22 7 29 30.24 (3) Parent: 0 Fresh water 24° 16 39 55 30.73 c o n t r o l 0.2 PPM thyroxine 24° 2 6 8 30.75 0.4 PPM " 24° 4 16 20 30.80 0.8 PPM " 24° 10 5 15 30.33 Lower than c o n t r o l (P<.01). 1.6 PPM " 24° 6 2 8 30.25 Lower than c o n t r o l (P<.01). .017= t h i o u r e a 24° 5 14 19 30.74 .02% " 24° 5 16 21 30.76 .04% " 24° 6 18 24 30.75 -212-Appendix VI continued. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment X I : E f f e c t of thyroxine and t h i o u r e a , (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp T o t a l vertebrae Number Mean Remarks (°C) 29 30 31 32 (4) Parent: P Fresh water 24° 4 19 23 30.83 c o n t r o l 0.2 PPM thyroxine 24° 6 17 23 30.74 0.4 PPM " 0 24 12 8 20 30.40 Lower than c o n t r o l (P<.01). 0.8 PPM " 0 24 2 12 1 15 30.93 Not d i f f e r e n t from c o n t r o l (P>.05). .01% t h i o u r e a 0 24 7 22 1 30 30.80 .02% " 24° 3 14 17 30.82 .04% " 24° 2 13 15 30.86 (5) Parent: Q Fresh water 24° 16 10 26 30.38 c o n t r o l 0.8 PPM thyroxine 24° 15 6 21 30.29 .02% thio u r e a 24° 10 10 20 30.50 (6) Parent: S Fresh x^ater 24° 25 6 31 31.19 c o n t r o l 0.2 PPM thyroxine 24° 17 27 44 31.61 Higher than c o n t r o l (P<.01). 0.4 PPM " 24° 17 41 58 31.71 Higher than c o n t r o l (P^.01). 0.8 PPM " 24° 14 16 30 31.53 Higher'than c o n t r o l (P<.01). 1.6 PPM " 24° 9 13 22 31.59 Higher than c o n t r o l (P<.01). .01% 24° 14 16 30 31.53 Higher -than c o n t r o l (P^.01). .02% " 24° 21 9 30 31.30 Not d i f f e r e n t from c o n t r o l (P>.05). .04% " 24° 19 11 30 31.37 -213-Appendix VI. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment XI: continued. E f f e c t of thyroxine and t h i o u r e a . (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp <°C) • Tot a l • 29 30 vertebrae 31 32 Number Mean Remarks (7) Parent: V Fresh water c o n t r o l 26° 2 63 5 70 31.04 0.4 PPM thyroxine 26° 11 36 5 52 30.88 Tends to be lower than c o n t r o l (P<.05; > .02). 0.3 PPM " 26° 8 51 4 63 30.94 Not d i f f e r e n t from con-t r o l (P>.05). 3.2 PPM " 26° 16 19 1 36 30.57 489 h r s . i n thyroxine & hatched i n water; Lower than c o n t r o l (P<.01). .05% thio u r e a 26° 5 49 6 60 31.02 Not d i f f e r e n t from c o n t r o l (P>.05). (8) Parent: Y Fresh water c o n t r o l 26° 12 54 4 70 30.89 0.4 PPM thyroxine 26° 4 46 50 30.92 0.8 PPM " 26° 2 42 6 50 31.08 Tends to be higher than c o n t r o l (P<.02). .027, t h i o u r e a 26° 1 38 3 42 31.05 Not d i f f e r e n t from c o n t r o l (P=.05). (b) Eggs f e r t i l i z e d and reared i n the s o l u t i o n s up to hatching. (1) ' Parent: Y Fresh water con-t r o l 26° 12 54 4 70 30.89 0.8 PPM thyroxine 26° 6 62 9 77 31.04 Tends to be higher than c o n t r o l (P<f.05; > .02). .027, thiourea 26° 2 45 3 50 31.02 Not d i f f e r e n t from c o n t r o l (P>.05). -214-Appendix VI continued. Frequency d i s t r i b u t i o n of t o t a l vertebrae i n experiment XI: E f f e c t of thyroxine and t h i o u r e a . (b) Eggs f e r t i l i z e d and reared i n the s o l u t i o n s up to hatching. Treatment Temp (°C) Total vertebrae 29 30 31 32 Number Mean Remarks (2) Parent: a Fresh water c o n t r o l 30° 21 12 33 30.36 0.8 PPM thyroxine 30° 5 21 6 1 33 30.09 Not d i f f e r e n t from c o n t r o l (P>.05). .04% thio u r e a 30° 7 25 32 30.78 Higher than c o n t r o l (P<.01). (3) Parent: b Fresh water c o n t r o l 26° 1 42 3 46 31.04 0.8 PPM thyroxine 26° 6 19 2 27 30.85 Not d i f f e r e n t from c o n t r o l (P>.05). .04% thiourea 26° 1 14 15 30.93 Not d i f f e r e n t from c o n t r o l (P>.05). (c) Chorion p r i c k e d eggs hatching. reared : Ln the s o l u t i o n s up to Parent-: V Fresh water c o n t r o l 26° 25 4 29 31.14 3.2 PPM thyroxine 26° 1 3 17 1 22 30.81 Tends to be lower than c o n t r o l (P<.02; > .01). .02% t h i o u r e a 26° 4 16 1 21 30.86 Tends to be lower than c o n t r o l (P=.02); Not d i f f e r e n t from .05% l o t i n expt. X l a ( V I I ) . (d) Larvae reared i n the s o l u t i o n s a f t e r hatching. Parent: V Fresh water c o n t r o l 26° 4 29 2 35 30.94 3,..2" PPM thyroxine 26° 6 19 2 27 30.85 .05% thiourea 26° 4 28 2 34 30.94 -215-Appendix V I I . Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment X I : E f f e c t of thyroxine and th i o u r e a . (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp (°C) 6 7 P e c t o r a l rays 8 9 10 11 12 13 Number Mean Remark: (1) Parent: G .0025% thiou r e a 24° 16 4 20 11.20 .005% " 24° 25 9 34 11.26 .01% " 24° 1 17 7 25 11.24 .02% " 24° 10 5 15 11.33 .04% " 24° 10 2 12 11.17 .05% " 24° 8 9 17 11.53 *! (2) Parent: M Fresh water c o n t r o l 24° 2 13 15 10.37 0.1 PPM thyroxine 24° 3 34 1 38 10.95 0.2 PPM " 24° 5 11 16 10.69 * 2 0.4 PPM " 24° 1 12 16 29 10.51 * 3 Notes: 1. Tends to be higher than .0025% (P<.05). 2. Not d i f f e r e n t from c o n t r o l (P>.05). 3. Tends to be lower than control0> oos). -216-Appendix VII continued. Frequency d i s t r i b u t i o n of p e c t o r a l rays i n experiment X I : E f f e c t of thyroxine and t h i o u r e a , (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp P e c t o r a l rays Number Mean Remarks (°C) 6 7 8 9 10 11 12 13 (3) Parent: 0 Fresh water 24° 27 68 2 97 11.74 c o n t r o l 0.2 PPM thyroxine24° 6 2 8 11.25 ** 0.4 PPM " 24° 3 8 9 20 2 11.30 * 0.8 PPM " 2 4 ° 1 2 6 6 15 9.13 1.6 PPM 24° 1 1 2 2 2 8 8.37 .01% thiourea 24° 1 6 12 19 11.58 * .02% 24^ 7 14 21 11.67 .04% 24 5 18 24 11.83 (4) Parent: P Fresh water c o n t r o l 24° 18 5 23 11.22 0.2 PPM thyrox-i n e 0 24 16 7 23 11.30 0.4 PPM thyroxine24° 1 4 8 5 2 20 9.15 0.8 PPM " 24° 4 10 1 15 9.80 v.-5 .01% thiourea 24° 1 24 5 30 11.13 .02% " 24° 14 3 17 11.18 .04% " 24° 1 12 2 15 11.07 *6 Note: 1. Lower than c o n t r o l (P<.01). 2. Lower than c o n t r o l (P<.01). 3. Not d i f f e r e n t from c o n t r o l (P>.05). 4. Lower than c o n t r o l (P^.01)." 5. Lower than c o n t r o l (P<.01); Higher than .4PPM (P<.01). 6. Not d i f f e r e n t from c o n t r o l (P>.05). -217-Appendix V I I continued. Frequency d i s t r i b u t i o n of P e c t o r a l rays i n experiment XI: E f f e c t of thyroxine and t h i o u r e a , (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp (°C) 6 7 P e c t o r a l rays 8 9 10 11 12 13 Number Mean Remark! (5) Parent: Q Fresh water 24° 4 18 2 24 10.91 0.8 PPM thyroxine 24° 5 14 2 21 10.85 .027, t h i o u r e a 24° 3 11 6 20 11.15 (6) Parent: S Fresh water 24° 6 19 25 11.76 0.2 PPM thyroxine 24° 22 20 2 44 11.54 0.4 PPM " 24° 40 18 58 11.31 1 0.8 PPM " o 24 2 10 16 2 30 10.60 * 2 1.6 PPM " 24° 6 13 2 1 , 22 8.91 .017= th i o u r e a 24° 10 18 2 30 11.73 .027= " 24° 6 23 1 30 11.83 .047= " 24° 12 18 30 11.60 (7) Parent: V Fresh water 26° 8 113 19 140 11.08 0.4 PPM thyroxine 26° 6 78 20 104 11.13 0.8 PPM " 26° 4 71 51 126 10.37 *3 3.2 PPM " 26° 1 22 27 19 3 72 8.04 ..A .057= th i o u r e a o 26 3 65 49 3 120 11.43 *5 Note: 1. Lower than c o n t r o l (P<.01). 2. Lower than c o n t r o l (P<.01). 3. Lower than c o n t r o l (P<.01). 4. 489 h r s . i n thyroxine and hatched i n water. 5. Higher than c o n t r o l (P<.01). -218-Appendix V I I continued. Frequency d i s t r i b u t i o n of P e c t o r a l rays i n experiment X I : E f f e c t of thyroxine and t h i o u r e a , (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp P e c t o r a l rays Number Mean Remarks (°C) 6 7 8 9 10 11 12 13 14 (8) Parent: Y Fresh water c o n t r o l 26° 63 77 140 12.55 0.4 PPM thyroxine 26° 54 46 100 12.46 0.8 PPM " 26° 1 84 15 100 12.14 , a .02% thiourea 2 6 ° 28 53 2 83 12.69 ...2 (b) Eggs f e r t i l i z e d and reared i n the s o l u t i o n s up to hatching. (1) Parent: Y Fresh water c o n t r o l 26° 63 77 140 12.55 0.8 PPM thyroxine 26° 1 53 99 1 154 12.65 v^3 .02% thiourea 26° 28 71 1 100 12.73 (2) Parent: a Fresh water c o n t r o l 30° 2 45 19 66 11.26 0.8 FPM thyroxine 30° 17 45 4 66 10.80 .5 w .04% thio u r e a 0 30 1 40 22 1 64 11.36 6 •)'.-(3) Parent: b Fresh water c o n t r o l 26° 21 66 5 92 11.83 0.8 PPM thyroxine 26° 13 37 4 54 10.83 7 .04% thiourea 26° 2 23 5 30 12.10 . . . 8 Note: 1. Lower than c o n t r o l (P<.01). 5. Lower than c o n t r o l (P<.01). 2. Tends to be higher than c o n t r o l 6. Not d i f f e r e n t from c o n t r o l (P>.05). (P<.05; >.02). 7. Lower than c o n t r o l (P<.01). 3. Not d i f f e r e n t from c o n t r o l (P>.05). 4. Higher than c o n t r o l (P<.01). 8- Tends to be higher than control^-") -219-Appendix V I I continued. Frequency d i s t r i b u t i o n f o p e c t o r a l rays i n experiment XI: E f f e c t of thyroxine and t h i o u r e a , (c) Chorion p r i c k e d eggs reared i n the s o l u t i o n up to -hatching. Treatment Temp P e c t o r a l rays Number Mean Remarks (°C) 5 6 7 8 9 10 11 12 13 Parent: V Fresh water 26 2 42 14 58 11.21 c o n t r o l 3.2 PPM thyroxine 26° 10 18 12 4 44 9.23 * l .047. thiourea 26° 15 26 1 42 11.67 ,2 (d) Larvae reared i n the s o l u t i o n s a f t e r hatching. Parent: V Fresh water c o n t r o l 26° 6 52 12 70 11.09 3.2 PPM thyroxine 26° 23 28 3 54 5.63 *3 .05% thiourea 26° 1 21 45 1 68 11.68 4 Note : 1. 2. Lower than c o n t r o l (P<.01); 475 hours i n thyroxine and hatched i n water; higher than 3.2 PPM i n expt. XI a ( V I I ) . Higher than c o n t r o l (P<.01). 3. Lower than 3.2 PPM means i n expt. XI a ( V I I ) , and XIc. 4. Higher than c o n t r o l (P<.01). -220-Appendix V I I I . Frequency d i s t r i b u t i o n of anal rays i n experiment X I : E f f e c t of thyroxine and t h i o u r e a . (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment TemD (°C) 17 18 Anal 19 rays 20 21 22 Number Mean Remarks (1) Parent: G .0025% thiourea 24° 1 11 8 1 21 18.43 .005% f? 24° 1 18 15 34 18.41 .01% it 24° 3 9 14 1 27 18.48 .02% tt 24° 1 12 2 1 16 18.19 .04% »? 24° 1 3 7 1 12 18.67 .05% tt 24° 1 13 3 17 18.12 (2) Parent: M Fresh water 24° 5 10 15 19.67 c o n t r o l 0.1 PPM thyroxine 24° 1 12 21 8 2 44 19.95 -v1 0.2 PPM " 24° 1 5 7 3 16 19.75 0.4 PPM " 24° 4 14 9 1 1 29 19.34 ,2 Note: 1. Not d i f f e r e n t from c o n t r o l (P>.05). 2. Not d i f f e r e n t from c o n t r o l (P>.05). -221-Appendix V I I I continued. Frequency d i s t r i b u t i o n of anal rays i n experiment X I : E f f e c t of thyroxine and t h i o u r e a , (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. ^ Treatment Temp (°c) 17 18 Anal 19 rays 20 21 22 Number Mean Remarks (3) Parent: 0 Fresh water c o n t r o l 24° 6 28 15 6 55 19.38 0.2 PPM thyroxine 24° 2 3 2 1 8 19.25 0.4 PPM " 24° 6 10 3 1 20 19.95 ** 0.8 PPM " 24° 7 4 3 1 15 18.87 ,2 1.6 PPM " 24° 1 5 2 8 19.00 3 .01% thiourea 0 24 6 12 1 19 19.74 .02% " 24° 1 2 14 4 21 20.00 .04% " 24° 1 8 1 1 1 3 24 19.88 * 6 (4) Parent: P Fresh water c o n t r o l 24° 3 13 6 1 23 19.22 0.2 PPM thyroxine 24° 4 16 3 23 18.96 .J 0.4 PPM " 24° 13 6 1 20 18.40 *s 0.8 PPM " 24° 9 5 1 15 18.47 .01% thio u r e a o 24 1 7 18 4 30 18.33 .02% 24° > 2 12 2 1 17 19.12 .04% " 24° 3 8 4 15 19.07 6. Not d i f f e r e n t from c o n t r o l (P>.05). 7. 8. Lower than c o n t r o l (P<.01). 9. Note: 1. Tends to be higher than c o n t r o l (P<.02; >.01). 2. Not d i f f e r e n t from c o n t r o l (P=.05). 3. Not d i f f e r e n t from c o n t r o l (P>.05). 4. 5. Higher than c o n t r o l (P^.01). -222-Appendix V I I I continued. Frequency d i s t r i b u t i o n of anal rays i n experiemtn X I : E f f e c t of thyroxine and t h i o u r e a , (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment Temp Anal rays Number Mean Remarks ( C) 17 18 19 20 21 22 (5) Parent: Q Fresh water c o n t r o l 0 24 5 10 9 2 26 19.31 0.8 PPM thyroxine 24° 1 8 7 5 2 21 18.7e" Tends to be ) lower than c o n t r o l (P<.05). .02% thiourea 24° 5 3 5 2 20 19.20 Treatment Temp CC) 16 17 18 Anal 19 rays 20 21 22 23 Number Mean 1 Remarks (6) Parent: S Fresh water c o n t r o l 24° 5 17 7 2 31 20.19 0.2 PPM thyroxine 24° 10 23 10 1 44 20.04 0.4 PPM " 24° 13 26 16 3 58 20.16 0.8 PPM " 24° 4 13 12 1 30 20.33 1.6 PPM " 24° 8 10 3 1 22 19.86 .017, t h i o u r e a 24° 6 10 9 4 1 30 20.47 .02% " 24° 1 3 16 8 2 30 21.23 * 1 .04% " 24° 4 16 7 3 30 21.30 *2 *1. Higher than c o n t r o l (P<.01) *2. Higher than c o n t r o l (P<.01) -223 Appendix V I I I (Cont'd). Frequency d i s t r i b u t i o n of anal rays i n experiment X I : E f f e c t of thyroxine and t h i o u r e a , (a) F e r t i l i z e d eggs reared i n the s o l u t i o n s up to hatching. Treatment TemD (°C)16 17 Anal 18 19 rays 20 21 22 Number Mean Remarks (7) Parent: V Fresh Water ( c o n t r o l ) 26° S 34 26 2 70 19.31 0.4 PPM thyroxine 26° 4 14 26 7 1 52 18.75 *1 0.8 PPM " 26° 1 20 34 7 1 63 18.79 *2 3.2 PPM " 26° 1 9 19 6 1 36 17.92 *3 .05 "L t h i o u r e a 26° ' 1 13 27 IS 1 60 19.08 *1. Lower than c o n t r o l (P<.01). *2. Lower than c o n t r o l (P<.01). -3. Lower than c o n t r o l (P<.01); 489 h r s . i n thyroxine and hatched i n water. (8) Parent: Y Fresh Water ( c o n t r o l ) 26° 12 18 37 12 1 70 18.89 0.4 PPM thyroxine 26° 9 32 8 1 50 19.02 0.8 PPM " 25° 10 32 7 1 50 18.98 .02 % thiourea 26° 10 21 11 42 19.02 -224-Appendix V I I I (Cont'd). Frequency d i s t r i b u t i o n of anal rays i n experiment X I : E f f e c t of thyroxine and th i o u r e a . (b) Eggs f e r t i l i z e d and reared i n the s o l u t i o n s up to hatching. Treatment TeniD (°C) 16 17 Anal 18 rays 19 20 21 22 Number Mean Remarks (1) Parent: Y Fresh Water (Control) 26° 2 18 37 12 1 70 18.89 0.8 PPM thyroxine 26° 1 13 38 22 2 1 77 19.18 *1 .027= thiourea 26° 10 26 13 1 50 19.10 *2 *l. Tends to be higher "2. Not d i f f e r e n t from than c o n t r o l (P<.05). c o n t r o l (P>.05). (2!) Parent: a Fresh Water (Control) 30° 4 16 11 2 33 17.37 0.8 PPM thyroxine 30° 4 16 10 3 33 17.37 .047= thiourea 30° 2 5 15 9 1 32 18.06 -I "1. Tends to be higher than c o n t r o l (P<.02). ('•3! ) Parent: b Fresh Water (Control) 26° 19 19 6 2 46 19.80 0.8 PPM thyroxine 26° 2 5 17 1 2 27 18.85 *1 .047= thiourea 26° 1 1 8 3 2 15 19.27 *2 "1. Lower than c o n t r o l (P<.01). -2. Not d i f f e r e n t from c o n t r o l (P>.05). -225-Appendix VIII (Cont'd). Frequency distribution of anal rays in experiment XI Effect of thyroxine and thiourea, (c) Chorion pricked eggs reared in the solutions up to hatching. Treatment Temp Anal (°C) 15 16 17 rays 18 19 20 21 Number Mean Remarks Parent: V Fresh Water _ (Control) 26 U 4 13 5 4 3 29 18.62 3.2 PPM thyroxine 26° 1 10 3 2 16 18.37 *1 .027. thiourea 26° 3 13 5 21 19.09 *2 *1. Not different from control (P>.05). *2. Not different from control (P>.05). (d) Larvae reared in the solutions after hatching. Parent: V Fresh water (Control) 26° 7 15 12 1 35 19.20 3.2 PPM thyroxine 26° 8 9 7 2 1 27 16.22 .057. thiourea 26° 4 10 12 8 34 19.71 n *1. Tends to be higher than control (P<.02). -226-Appendix IX. Frequency distribution of dorsal rays in experiment XI: Effect of thyroxine and thiourea (a) Fertilized eggs reared in the solutions up to hatching Treatment Temp (°C) Dorsal 5 6 rays 7 8 Number Mean Remarks (1) Parent: G .0025% thiourea 24° 18 3 21 6.14 .005% " 24° 25 9 34 6.26 .01% " 24° 22 4 26 6.15 .02% " 24° 12 4 16 6.25 .04% ..." 24° 4 8 12 6.67 .05% " 24° 13 4 17 6.23 (2) Parent: M Fresh Water (Control) 24° 9 6 15 6.40 0.1 PPM thyroxine 24° 16 29 45 6.64 0.2 PPM " 24° 7 9 16 6.56 0.4 PPM " 24° 15 13 1 29 6.51 (3) Parent: 0 Fresh Water (Control) 24° 37 18 55 6.33 0.1 PPM thyroxine 24° 4 4 8 6.50 0.4 PPM " 24° 10 9 1 20 6.55 0.8 PPM " 24° 9 6 15 6.40 -227-Appendix IX. (Cont'd). Frequency distribution of dorsal rays in experiment XI:: Effect of thyroxine and thiourea (a) Fertilized eggs reared in the solutions up to hatching Treatment Temp (°C) Dorsal rays 5 6 7 8 Number Mean Remarks (3) Parent: 0 (Cont'd) 1.6 PPM thyroxine 24° 5 3 8 6.37 .01% thiourea 24° 5 14 19 6.74 *1 .02% •» 24° 8 13 21 6.62 #2 .04% " 24° 4 19 1 24 6.88 *3 (4) Parent: P Fresh Water (Control) .24°. 1 20 2 23 6.04 0.2 PPM thyroxine 24° 20 3 23 6.13 0.4 PPM " 24° 15 5 20 6.25 0.8 PPM " 24° 15 15 6.00 .017. thiourea 24° 1 20 9 30 6.27 .02% » 24° 10 7 17 6.41 *4 .04% 24° 11 4 15 6.23 *1. Higher than control (P<.01). *2. Not different from control. *3. Higher than control (P<.01). *4. Higher than control (P<.01). -228-Appendix IX (Cont'd). Frequency distribution of dorsal rays in experiment XI: Effect of thyroxine and thiourea (a) Fertilized eggs reared in the solutions up to hatching Treatment Temp (°C) Dorsal rays 5 6 7 8 Number Mean Remarks (5) Parent: Q Fresh Water (Control) 24° 22 4 26 6.15 0.8 PPM thyroxine 24° 19 2 21 6.09 .02% thiourea 24° 1 13 6 20 6.25 (6) Parent: S Fresh Water (Control) 24° 21 10 31 6.32 0.2 PPM thyroxine 24° 30 14 44 6.32 0.4 PPM " 24° 49 9 58 6.16 0.8 PPM n 24° 22 8 30 6.27 1.6 PPM " 24° 18 4 22 6.18 .01% thiourea 24° 24 6 30 6.20 .02% M 24° 17 13 30 6.43 .04% " 24° 18 12 30 6.40 (7) Parent: V Fresh Water (Control) 26° 4 53 13 70 6.13 0.4 PPM thyroxine 26° 2 42 8 52 6.11 -229-Appendix IX (Cont'd). Frequency distribution of dorsal rays in experiment XI: Effect of thyroxine and thiourea (a) Fertilized eggs reared in the solutions up to hatching Treatment Temp (°C) Dorsal rays 5 6 7 8 Number Mean Remarks • (7) Parent: V (Cont'd) 0.8 PPM thyroxine 26° 4 53 6 63 6.03 3.2 PPM " 26° 29 7 36 6.19 .057. thiourea 26° 1 45 13 1 60 6.23 (8) Parent: Y Fresh Water 0 (Control) 26 50 20 70 6.29 0.4 PPM thyroxine 26° 33 17 50 6.34 0.8 PPM " 26° 28 22 50 6.44 .027. thiourea 26° 19 23 42 6.55 *1 *1. Higher than control (P<.01). (b) Eggs f e r t i l i z e d and reared in the solutions up to hatching (1) Parent: Y Fresh Water (Control) 26° 50 20 70 6.29 0.8 PPM thyroxine 26° 44 31 2 77 6.45 .027. thiourea 26° 30 20 50 6.40 -230-Appendix IX (Cont'd). Frequency distribution of dorsal rays in experiment XI Effect of thyroxine and thiourea (b) Eggs f e r t i l i z e d and reared in the solutions up to hatching Treatment Temp <°C) Dorsal rays 5 6 7 8 Number Mean Remarks (2) Parent: & Fresh Water (Control) 30° 1 32 33 5.97 0 .8 PPM thyroxine 30° 1 30 2 33 6.03 .04% thiourea 30° 27 5 32 6.16 (3) Parent: £ Fresh Water (Control) 26° 1 33 11 1 46 6.26 0.8 PPM thyroxine 26° 1 24 2 27 6.04 .04% thiourea 26° 14 1 15 6.07 (c) Chorion pricked eggs reared in the solutions up to hatching Parent: V Fresh Water (Control) 26° 6 16 7 29 6.03 3.2 PPM thyroxine 26° 18 3 21 6.14 .02% thiourea 26° 13 8 21 6.38 Parent: V Fresh Water (Control) (d) Larvae reared in the solutions after hatching 26° 3 28 4 35 6.03 -231-Appendix IX (Cont'd). Frequency distribution of dorsal rays in experiment XI: (d) Larvae reared in the solutions after hatching A Parent: V (Cont'd) Temp (°C) Dorsal rays 5 6 7 8 Number Mean Remarks 3.2 PPM thyroxine 26° 13 13 1 27 5.56 *1 .057. thiourea 26° 2 22 10 34 6.24 *1. Lower than control (P<.01). -232-Appendix X. Frequency distribution of total caudal rays in experiment XI: Effect of thyroxine and thiourea. (a) Fertilized eggs reared in the solutions up to hatching Treatment Temp (°C) 19 Total Caudal 20 21 22 rays 23 24 25 26 No. Mean Remarks (1) Parent: G .0025% thiourea 24° 1 11 8 1 21 23.38 .005% • 24° 8 21 5 34 23.91 .01% " 24° 2 5 8 8 2 25 23.12 .02% " 24° 1 1 8 6 16 23.19 .04% " 24° 1 7 3 1 12 23.25 .05% H 24° 1 4 10 2 17 23.76 (2) Parent: M Fresh Water (Control) 24° 6 5 4 15 23.87 0.1 PPM thyroxine 24° 1 2 16 14 8 2 43 23.72 0.2 PPM " 24° 2 8 6 16 23.25 *1 0.4 PPM " 24° 2 7 14 6 29 22.83 *2 *1. Tends *2. Lower to be than i lower control than control (P<.01). (P<.05). (3) Parent: 0 Fresh Water (Control) 24° 1 14 20 17 1 ?54 23.13 *1 *1. One f i s h with 27 rays. -233-Appendix X. (Cont'd). Frequency distribution of total caudal rays in experiment XI: Effect of thyroxine and thiourea. (a) Fertilized eggs reared in the solutions up to hatching Treatment (3) Parent: 0 (Cont'd) Temp (°C) 19 Total Caudal 20 21 22 rays 23 24 25 26 No. Mean Remarks 0.2 PPM thyrozine 24° 4 2 1 1 8 23.75 0.4 PPM " 24° 14 6 20 23.30 0.8 PPM " 24° J2 S 4 1 15 23.27 1.6 PPM H 24° 1 1 4 2 8 22.75 .01% thiourea 24° 1 4 7 6 1 19 23.10 .02% " 24° 1 11 6 3 21 23.52 .04% 24° 1 11 9 3 24 23.58 (4) Parent: P Fresh Water (Control) 24° 3 11 9 23 24.26 0.2xPPM thyroxine 24° 2 6 10 5 23 23.78 *1 0.4 PPM " 24° 3 7 8 2 20 23.45 *2 0.8 PPM n 24° 2 4 6 3 15 23.67 *3 .01% thiourea 24° 1 1 3 6 16 3 30 23.43 *4 .02% " 24° 6 9 2 17 23.76 *5 *1. Tends to be lower than control (P<.05). *2. Lower than control (P.<0J>)-*3. Tends to be lower than control (P<.05). *4. Lower than control (P<.01). *5. Tends to be lower than control (P<.05). -234-Appendix X. (Cont'd). Frequency distribution of total caudal rays in experiment XI: Effect of thyroxine and thiourea, (a) Fertilized eggs reared in the solutions up to hatching Treatment Temp Total Caudal rays No. Mean Remarks (°C) 19 20 21 22 23 24 25 26 (4) Parent: P (Cont'd) .047. thiourea 24° 1 5 6 3 15 23.73 *1 *1. Tends to be lower than control (P^.05). (5) Parent: Q Fresh Water (Control) 24° 1 13 10 2 25 23.50 0.8 PPM thyroxine 24° 9 12 21 23.57 .027. thiourea o 24 2 1 7 6 3 1 20 23.50 (6) Parent: S Fresh Water (Control) 24° 1 13 10 4 2 30 22.81 0.2 PPM thyroxine 24° 3 18 12 9 2 44 22.75 0.4 PPM " 24° 8 15 16 17 1 1 58 22.84 0.8 PPM " 24° 4 11 9 4 1 1 30 22.67 1.6 PPM " 24° 3 8 9 1 1 22 22.50 .017. thiourea 24° 4 13 8 3 1 29 22.48 .02% " 24° 1 9 10 7 3 30 23.07 .047. " o 24 1 4 6 10 8 1 30 22.77 ,-235-Appendix X. (Cont'd).. Frequency distribution of total caudal rays in experiment XI: Effect of thyroxine and thiourea, (a) Fertilized eggs reared in the solutions up to hatching -• Treatment Temp (°C) Total 19 20 21 Caudal rays 22 23 24 25 26 No. Mean Remarks (7) Parent: V Fresh Water (Control) 26° 5 8 41 13 2 69 23.50 0.4 PPM thyroxine 26° 3 13 24 8 4 52 22.96 *1 0.8 PPM " o 26 6 12 34 10 1 63 22.82 #2 3.2 PPM " 26° 1 9 6 19 1 36 22.28 *3 .057. thiourea o 26 2 12 26 14 5 1 60 23.17 *4 *1. Lower than control (P<1.01). *2. Lower than control (P<.01). *3. Lower than control (P<.01); 489 hrs. in thyroxine and hatched in water. *4. Tends to be lower than control (P<.05). (8) Parent: Y Fresh Water (Control) 26° 1 2 23 23 16 5 70 21.94 0.4 PPM thyroxine 26° 1 8 16 17 6 2 50 22.50 *1 0.8 PPM " 26° 1 13 19 12 5 50 21.16 *2 .027. thiourea 26° 1 11 20 8 2 42 21.97 *1. Higher than control (P<.01). *2. Not different from control_(P^.05). , -236-Appendix X. (Cont'd). Frequency distribution of total caudal rays in experiment XI: Effect of thyroxine and thiourea. (b) Eggs f e r t i l i z e d arid reared hatching in the solutions up to Treatment Temp (°C) 19 Total Caudal rays 20 21 22 23 24 25 No. 26 Mean Remarks (1) Parent: Y Fresh Water (Control) 26° 1 2 23 23 16 5 70 21.94 0.8 PPM thyroxine 26° 1 25 17 26 7 *77 22.11 *1 .027. thiourea 26° 1 11 21 14 3 50 22.12 *1. One with 18 rays. (2) Parent: a Fresh Water (Control) 30° 3 15 10 4 1 33 23.54 0.8 PPM thyroxine 30° 2 2 13 5 11 33 23.63 .047. thiourea 0 30 2 16 5 6 2 *32 23.72 *1 • *1. One with 27 rays. (2) Parent: b Fresh Water (Control) 26° 1 3 19 10 13 46 21.67 0.8 PPM thyroxine 26° 1 2 17 3 4 27 21.26 .047. thiourea 26° 1 11 1 2 15 21.27 -237-Appendix X. (Cont'd). Frequency distribution of total caudal rays in experiment XI: Effect of thyroxine and thiourea (c) Chorion pricked eggs reared in the solution up to hatching Treatment Temp Total Caudal rays No. Mean Remarks (°C) 19 20 21 22 23 24 25 26 Parent: V Fresh Water (Control) 26 5 10 12 2 29 22.38 3.2 PPM thyroxine 26° 1 1 6 4 6 3 *22 21.79 *1 .02% thiourea 26° 2 6 9 4 21 22.71 *1. One with 12 rays. Not different from control *2. Not different from control (P>.05). (P>.05). (d) Larvae reared in the solutions after hatching Treatment Temp (°C) 13 Total Caudal 14 15 16 17 18 19 Rays 20 21 22 23 24 No. Mean Rema Parent: V Fresh Water (Control) 26° 5 11 12 6 35 22.40 3.2 PPM . thyroxine 26°; 1 1 5 4 6 1 3 2 4 27 17.63 .05% thiourea 26° 1 5 17 3 4 2 33 21.24 *1 *1. Lower than control (P<.01). 

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