Science, Faculty of
Zoology, Department of
DSpace
UBCV
Robertson, James Grant
Master of Arts - MA
University of British Columbia
Chum salmon embryonal discs were smeared and the cells stained with acetocarmine (60° C.) for chromosome content. Thirty-eight chromosomes (granular, sigmoid, and j-shaped) were counted, but the number was not considered absolute. The presence of sex chromosomes could not be established. Chromosomes in cells of differentiating gonads showed such excessive clumping that a count could not be made. Sex differentiation was followed in chum salmon. Sections from alevins (three weeks old) showed the gonads to be indifferent. Isolated nests of germ cells appeared which eventually formed a continuous cord (five weeks). At nine weeks the primordial ovary developed oocytes and in the succeeding week formed an endovarial canal. At this time the testis was first recognized (ten weeks). It was a small compact organ containing spermatogonia. Up to the final examination (fifteen weeks) the ovary showed no new structures excepting a prominent vena commites anteriad, while the testis retained its primitive features. The sex of pink and chum salmon fry migrants were shown by dissection. Ultraviolet light and temperature treatment of developing chum, pink, and sockeye embryos did not show that these factors would influence the sex ratio. Temperature data were decidedly inconclusive. An equal distribution of the sexes in pink and chum fry and sockeye smolts was found in data compiled from migrant runs at Port John, B. C. It is suggested that precocious male sockeye mature in the sea. The sex ratios at stages in the life history of the pink salmon are presented to show that there had occurred a natural sea mortality of 40.2 percent.
DETERMINATION AND DIFFERENTIATION OF SEX IN PACIFIC SALMON by JAMES GRANT ROBERTSON (isi Or 4 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF-ARTS i n the Department of Zoolpgy We accept t h i s t h e s i s as conforming to the standard r e q u i r e d from candidates f o r the degree o f MASTER OF ARTS. Members o f the Department o f Zoology THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1951 ABSTRACT Chum salmon embryonal d i s c s were smeared and the c e l l s s t a i n e d with acetocarmine (60° C.) f o r chromosome content. T h i r t y - e i g h t chromosomes ( g r a n u l a r , sigmoid,and j-shaped) were counted, but the number was not c o n s i d e r e d a b s o l u t e . The presence o f sex chromosomes c o u l d not be e s t a b l i s h e d . Chromosomes i n c e l l s o f d i f f e r e n t i a t i n g gonads such showed A e x c e s s i v e clumping t h a t a count could not be made. Sex d i f f e r e n t i a t i o n was f o l l o w e d i n chum salmon. S e c t i o n s from a l e v i n s (three weeks old) showed the gonads to be i n d i f f e r e n t . I s o l a t e d n e s t s o f germ c e l l s appeared which e v e n t u a l l y formed a continuous c o r d ( f i v e weeks). At n ine weeks the p r i m o r d i a l ovary developed oocytes and i n the succeeding week formed an e n d o v a r i a l c a n a l . At t h i s time the t e s t i s was f i r s t r e c o g n i z e d (ten weeks). I t was a s m a l l compact organ c o n t a i n i n g spermatogonia. Up to the f i n a l examination ( f i f t e e n weeks) the ovary showed no new s t r u c t u r e s e x c e p t i n g a prominent vena commites a n t e r i a d , w h ile the t e s t i s r e t a i n e d i t s p r i m i t i v e f e a t u r e s . The sex o f pink and chum salliion f r y migrants were shown by d i s s e c t i o n . U l t r a v i o l e t l i g h t and temperature treatment of developing chum, pink, and sockeye embryos d i d not show: that, these f a c t o r s would i n f l u e n c e the sex r a t i o . Temperature data were d e c i d e d l y i n c o n c l u s i v e . An equal d i s t r i b u t i o n o f the sexes i n pink and chum f r y and sockeye smolts was found i n data compiled from migrant runs a t P o r t John, B. C. I t i s suggested t h a t p r e c o c i o u s male sockeye mature i n the sea. The sex r a t i o s a t stages i n the l i f e h i s t o r y o f the p i n k salmon are presented to show t h a t there had o c c u r r e d a n a t u r a l sea m o r t a l i t y o f 40.2 p e r c e n t . i CONTENTS I . INTRODUCTION, . 1 I I . REVIEW OF THE LITERATURE 3 a) I n t r o d u c t i o n 3 b) The c y t o l o g i c a l and g e n e t i c a l a s p e c t s o f sex d e t e r m i n a t i o n 4 c) The embryologioal aspects o f sex d e t e r -m i n a t i o n and d i f f e r e n t i a t i o n ,. 7 d) The chemical aspects of sex d e t e r m i n a t i o n 12 I I I . MATERIALS AND METHODS l6 S e c t i o n 1. Chromosome s t u d i e s i n chum salmon.... 16 S e c t i o n 2, The h i s t o g e n e s i s o f sex d i f f e r e n t i a -t i o n 18 (a) The h i s t o l o g i c a l method 18 (b) F i e l d technique i n the examination o f young salmon gonads • 18 S e c t i o n 3. A p p l i c a t i o n o f u l t r a v i o l e t l i g h t and 19 temperature on developing salmon...., S e c t i o n 4. Sex r a t i o s o f young salmon a t P o r t John, B. C 24 S e c t i o n 5« Sex r a t i o s o f pi n k salmon i n the f i s h e r y 25 IV. RESULTS 26 S e c t i o n 1. Chromosome s t u d i e s i n the chum salmon 26 S e c t i o n 2, Germ c e l l d i f f e r e n t i a t i o n i n chum salmon 27 (a) The h i s t o l o g i c a l examination...., 27 (b) The f i e l d examination 36 S e c t i o n 3. U l t r a v i o l e t l i g h t and temperature experiments • 40 (a) The e f f e c t of u l t r a v i o l e t r a d i a -t i o n on the sex r a t i o o f develop-i i Ing chum and sockeye salmon 40 (b) The e f f e c t o f temperature on the sex r a t i o s o f chum, sockeye, and pink salmon r o a r i n g s . . 40 S e c t i o n 4. Remarks on the hatch e r y experiments.. 40 (a) General 40 (b) S e l e c t i v e m o r t a l i t y 46 S e c t i o n 5. The f i e l d sex a n a l y s e s . . . 47 (a) The sex r a t i o o f salmon migrants. 47 (b) The sex r a t i o s o f a d u l t p i n k salmon 47 S e c t i o n 6. Remarks on the f i e l d a n a l y s e s 50 V.. DISCUSSION 53 V I . SUMMARY 64 V I I . ACKNOWLEDGMENTS 66 V I I I . ADDENDUM. The gonads i n week of h a t c h i n g 67 i i i LIST OF TABLES AND FIGURES Table I. The source and u t i l i z a t i o n of salmon eggs In the hatchery experiments 1949-50, 1950-51... 20 I I . Sex r a t i o s of sockeye and chum salmon embryos exposed to u l t r a v i o l e t l i g h t 1949-50 42 I I I . Sex r a t i o s of chum and sockeye salmon embryos af t e r temperature treatment 1949-50.. 42 IV. Sex r a t i o s of pink salmon embryos a f t e r tem-^ perature treatment 1950-51 44 V. Sex r a t i o s of chum salmon embryos a f t e r tem-perature treatment 1950-51,... 44 VI. The sex r a t i o s of salmon migrants at port John, B. C, i n 1949 and 1950 48 VII. The sex analyses of salmon retained i n the sea and lake at Port John, B. C. i n 1949 and 1950..... 49 VIII. The sex r a t i o s of pink salmon sampled from the commercial f i s h e r y at Port John, B. C. i n 1950 50 Figure 1. The application of u l t r a v i o l e t l i g h t ( v e r t i -c a l lines) and temperature (horizontal l i n e s ) i n r e l a t i o n to f e r t i l i z a t i o n and d a i l y hatch-ery water temperature i n the 1949-50 experi-ments. The A p r i l to July data i s expressed i n weeks 19 2. Temperature treatment of developing pink salmon embryos i n r e l a t i o n to the hatchery water temperatures ( i n 1950-51). 22 5. Temperature -treatment of developing chum salmon embryos i n r e l a t i o n to the hatchery temperatures ( i n 1950-51). 22 4. Anaphase chromosomes from smeared embryonal c e l l s of chum salmon 26 5. Polar view of metaphase plate from smeared embryonal c e l l s of chum salmon 26 6. Section anteriad showing clumps of chromo-somes i n germ c e l l s of a chum salmon al e v i n 7 weeks o l d 28 i v F i g u r e 7. S e c t i o n a n t e r i a d showing p e r i p h e r a l n u c l e o l i i n oocytes o f a chum salmon a l e v i n 14 weeks • o l d 28 8. S e c t i o n a n t e r i a d showing a nest o f germ c e l l s i n a chum salmon a l e v i n 3 weeks o l d 29 9. Diagram showing the arrangement o f germ c e l l s i n an i n d i f f e r e n t gonad o f chum salmon a l e v i n 3 weeks o l d 29 10. S e c t i o n p o s t e r i a d showing the germ cord o f an a l e v i n 7 weeks o l d 31 11. S e c t i o n a n t e r i a d showing oogonia and young oocytes i n an a l e v i n 9 weeks o l d 31 12. S e c t i o n showing oocytes and an e n d o v a r i a l c a n a l i n the ovary o f an a l e v i n 10 weeks o l d . 32 13. S e c t i o n showing a spermatogonium i n meta-phase i n the t e s t i s o f an a l e v i n 10 weeks o l d . 32 14. S e c t i o n showing oocytes i n pockets of con-n e c t i v e t i s s u e i n an a l e v i n 11 weeks o l d . . . . . 34 15. S e c t i o n a n t e r i a d showing the vena eommites i n the 1ovary of a chum salmon a l e v i n 13 weeks o l d 34 16. S e o t i o n showing the r e l a t i o n o f the o v a r i e s to the body coelom i n an a l e v i n 14 weeks o l d . 33 17. S e c t i o n or.ani.ad:showingthe t e s t i s o f an a l e v i n 14 weeks o l d 36 18. D i s s e c t e d male gonads o f pi n k salmon f r y (33 mm. i n length) along the p e r i p h e r y of the a i r - b l a d d e r . 38 19. E n l a r g e d p o r t i o n of a male gonad w i t h the a i r b l a d d e r i n the background and melanophores to the r i g h t 38 20. D i s s e c t e d female gonads o f a pink salmon f r y (33 mm. i n length) along the p e r i p h e r y of the a i r - b l a d d e r . • 39 21. E n l a r g e d p o r t i o n of a female gonad w i t h the a i r - b l a d d e r and melanophores i n view 39 V Figure 22. The m o r t a l i t i e s r e s u l t i n g from u l t r a v i o l e t treatment of developing chum salmon i n the 1949-50 experiments 41 23. The m o r t a l i t i e s r e s u l t i n g from u l t r a v i o l e t treatment of developing sockeye salmon i n the 1949-50 experiments.... 41 24. The m o r t a l i t i e s r e s u l t i n g from temperature treatment of developing chum salmon i n the 1949-50 experiments 43 25. The m o r t a l i t i e s r e s u l t i n g from temperature treatment of developing sockeye salmon i n the 1949-50 experiments 43 26. The v a r i a t i o n i n the sex r a t i o of pink salmon i n t h e i r period of migration at Port John, B. C. i n the springs of 1949 and 1950..... 50 DETERMINATION AND DIFFERENTIATION QF SEX IN PACIFIC. SALMON BY J . G. ROBERTSON I. INTRODUCTION A marked disproportion between the two sexes of the sockeye salmon (Oncorhynchus nerka) has been frequently noted. Such disproportions have been observed not only i n commercial catches but also at experimental counting weirs and on the spawning grounds. Samples from the g i l l net fi s h e r y at Rivers i n l e t i n 1942 indicated a not infrequent female proportion of as much as 62 percent (Clemens 1943). At Port John, B. G. the 1948 run of "creek 1* sockeye counted through a weir tributary to the lake was comprised of 25f. females (Robertson 1949). I t has been suggested that the sex r a t i o should be considered i n evaluating e f f e c t i v e escapement and e f f i c i e n c y of natural propagation. For example, an enumer-ating weir located at Babine lake,near Hazelton, B. C , passed 522,561 sockeye i n 1947. Of t h i s number the e f f e c t i v e escapement was considered to be 273»000 (147,500 females, 125,500 males) since the remaining f i s h (249,561) were precocious male sockeye (Milne, and Pritchard 1948). At Cultus lake, near Chilllwack, B. C , a deficiency of 2 males among sockeye spawners was suggested as a f a c t o r reducing-smoIt p r o d u c t i o n ( F o e r s t e r 1926). The b a s i s f o r the d i s p r o p o r t i o n a t e sex r a t i o i n salmon has not been c a r e f u l l y a n a l y s e d . Sockeye c a t c h a n a l yses h a v e s u g g e s t e d g i l l net s e l e c t i v i t y and age group (Clemens 1943). Sporadic runs o f p r e c o c i o u s f i s h are p o s s i b l y i n i t i a t e d by oceanographic c o n d i t i o n s (Clemens 1949*). Such suggestions are based on the assumption t h a t the sexes are produced i n equal numbers. A r e c u r r e n t 1:1 sex d i s t r i b u t i o n has not y e t been demonstrated nor are the f a c t o r s which c o n t r o l the d e t e r -m i n a t i o n o f sex i n f i s h c l e a r l y understood (G-oldschmidt 1937; C S ^ I r d s o n 1945). An understanding o f the f a c t o r s which c o n t r o l sex i n young salmon i s e s s e n t i a l b e f o r e i t i s p o s s i b l e to evaluate r e t u r n i n g d i s t r i b u t i o n s . In the pres e n t study an attempt has been made to a) d e s c r i b e the g e n e t i c a l b a s i s f o r sex d e t e r m i n a t i o n i n the genus Oncorhynchus, b) d e s c r i b e the h i s t o g e n e s i s o f the gonad from the i n d i f f e r e n t stage, c) determine the e x t r i n s i c f a c t o r s which may modify sex r a t i o s , d) compile data f o r sex r a t i o o f salmon i n the r e g i o n o f P o r t John, B. C , and e) ev a l u a t e the r o l e o f d i f f e r e n t f a c t o r s i n determining the r a t i o o f the sexes i n the P a c i f i c salmon. *1949 p e r s o n a l communication. 3 I I . REVIEW OF THE LITERATURE a) Introduotion The pioneer work i n sex determination was that of McClung (1901) who hypothesized the r o l e of the accessory chromosomes as sex determinants, and Bridges (1914) who demonstrated sex chromosomes i n Drosophila. Cyt o l o g i c a l demonstration of the sex chromosomes i s d i f f i c u l t and i n the Pisces and Amphibia has not, apparently, been possible (Wickbom 1943; Makino 1947). Cytological evidence exists to show that the male i s the heterogametic sex (x and y Chromosomes) i n the Diptera, Reptiles, and Mammals, while i n the Lepidoptera and Aves i t i s the female that possesses chromosome pairs of unequal mates or an unpaired chromo-some (Crew 1933)• The r o l e of the gonosomes (sex chromo-somes) i n sex determination has been established i n f i s h by breeding experiments (Goldschmidt 1937, 1938; Winge 1922a, 1922b, 1932; Aida 1936; et a l ) . Certain discrepancies i n the X-Y mechanism were apparent and sex research was i n t e n s i f i e d i n r e l a t e d f i e l d s of biology. Theories of the o r i g i n and/or d i f f e r e n t i a t i o n of germ c e l l s wereforward* . (Okkelberg 1921; Geiser 1924; Mrsic 1923; Witschi 1929; Padoa 1939; Nieuwkoop 1944; et a l ) . Biochemists and physiologists made contributions as to the mechanism of the secondary sexual c h a r a c t e r i s t i c s (hormone i s o l a t i o n and injection) and more recently on the primary determiners (Shull 1912; Sh u l l and Ladoff 1916; 4 Uchida 1937; Moewus and Kuhn 1940; Witschi 1942; D»Aneona 1949; Takede 1950; et a l ) . b) The Cytological and G-enetioal Aspects of Sex Determination C y t o l o g i c a l l y the sex chromosomes may sometimes he distinguished from the other chromosomes (autosomes) because of t h e i r precession or succession on the spindle i n the d i v i d i n g o e l l (Crew 1933). The behaviour of these chromosomes „has been mainly elucidated from studies on sex-linked inheritance. In t h i s manner the gonosomes came to be regarded as the sole determiners of the 1:1 sex d i s t r i -bution apparent i n so many animal species. The gonosomes are grouped as to three types: the abraxis (XY females), protenor (XQ males), and normal (XY males). It follows that i f one sex i s homogametic (XX) the other must be heterogametic (X0, XY)• Experimental work Indicates that i n the f i s h Leblstes retioulatus and Aplocheilus l a t i p e s (male : :r,-digamety) males can be produced having the consti t u t i o n XX, YY, as well as digametic females (Winge 1922a,1922fc, 1932; Aida 1936). It i s also claimed that autosomes have been transformed into sex chromosomes, and these into auto-somes i n Leblstes (Winge 1932). In the Cyclostomes the sex chromosomes are thought to be absent (Okkelberg 1921) as they are i n the f i s h Xiphorous s t e l l e r i (Kosswig 1931-41)• This l a b i l e chromosome equipment would suggest the presence of other sex-determining factors. I t was shown that the autosomes were also concerned 5 i n sex d e t e r m i n a t i o n by s u i t a b l e breedings o f D r o s o p h i l a melanogaster i n whioh n o n - d i s j u n c t i o n o f the gonosomes had o c c u r r e d (Bridges 1921, 1922, 1925). The r e s u l t a n t sex i n t e r g r a d e s were shown to be dependent upon the r e l a t i v e p r o p o r t i o n o f the X chromosomes to the autosomes and i n d e -pendent o f the Y chromosome. Thus where three X chromo-somes were balanced a g a i n s t 1 the d i p l o i d number o f autosomes a s o - c a l l e d superfemale was formed; where a s i n g l e X chromo-some was present a normal. male -.would Q c c u r and when two, a female. F u r t h e r , where no X chromosomes were pr e s e n t a supermale r e s u l t e d . Thus p o l y g e n i c f a c t o r s ( s i n c e genes are the determining sex f a c t o r s on chromosomes) a r i s i n g from the chromosome complex were suggested as fundamental to sex de t e r m i n a t i o n i n D r o s o p h i l a . Opinion d i f f e r e d as to the manner i n which the genes induced sex d i f f e r e n t i a t i o n i n o t h e r animals. In the e x t e n s i v e experiments on the gypsy moth Lymantria d i s p a r (Goldschmidt 1937, 1938) the q u e s t i o n o f i n t e r s e x e s ( g e n e t i c c o n s t i t u t i o n a c t s to g i v e the a d u l t a mixture o f male and female c h a r a c t e r s ) and sex r e v e r s a l was e x p l a i n e d by assuming t h a t weak and s t r o n g F f a c t o r s (female-determining) and M f a c t o r s (male-determining) were p r e s e n t . Since t h e i r p o t e n c i e s v a r i e d f o r d i f f e r e n t f e r t i l i z e d eggs the r e s u l t i n g o f f s p r i n g would become a female or male o n l y where the dominance o f one sex tendency over the o t h e r reached a c e r t a i n t h r e s h o l d , the s o - c a l l e d e p i s t a t l c minimum). I f the t h r e s h o l d f e l l below t h i s v a l u e , i n t e r s e x e s would be formed. 6 that Goldschmidt (1938) suggests/the male f a c t o r s i n the gypsy-moth are genes s i t u a t e d i n the X-chromosomes, while the female f a c t o r s are something i n h e r i t e d through the maternal cytoplasm. Sex r e v e r s a l i n the f i s h L e p i s t e s r e t i c u l a t u s (male digamety) i n d i c a t e d t h a t F and M f a c t o r s l o c a t e d i n the autosomes would e s t a b l i s h the sex as feminine o r masculine but were dependent upon which zygote r e c e i v e d the w e p i s t a t i o male-determining gene* l o c a t e d on a l a b i l e Y chromosome (Winge 1932). S t u d i e s on A p l o o h e i l u s l a t i p e s suggested t h a t w h i l e sex genes corresponding to the primary s e x u a l c h a r a c t e r s are d i s t r i b u t e d i n the autosomes they must be s t i m u l a t e d by '"sexual e x c i t e r s " d i f f e r i n g i n l o c a t i o n and potency among the X and Y chromosomes (Aida 1936). The r e s e a r c h o f Kosswig on the f i s h Xiphophorus s t e l l e r i and P l a t y p o e c i l u s maoulatus has not been c o n s u l t e d . However, i t i s i n d i c a t e d t h a t t h i s s c h o o l t h e o r i z e s t h a t sex i s determined.by the " i n t e r p l a y o f autosomal genes, i . e . the s o - c a l l e d T-genes which a c t as polymeric f a c t o r s * (SvSrdson 1945). Kosswig holds t h a t "such t h i n g s e x i s t as phenotypic sex de t e r m i n a t i o n (environmental i n d u c t i o n ) , male and female h e t e r o z y g o s i s w i t h i n the same genus, empty X-chromosomes, female determiners i n the Y, m u l t i p l e auto-somal male determiners, e t c . " (Goldschmidt 1937). In summary, the sex-determining mechanism, among lower animals a t l e a s t , i s extremely l a b i l e . The extent 7 to which the e x t e r n a l f o r c e s may modify the u l t i m a t e sex d i s t r i b u t i o n remains to be co n s i d e r e d . C o l l a t e r a l demon-s t r a t i o n o f an environmental i n f l u e n c e i s e v i n c e d i n the aforementioned r e s e a r c h . The graded i n t e r s e x e s i n Lymantria were a t f i r s t o b t a i n e d by c r o s s i n g g e o g r a p h i c a l l y i s o l a t e d r a c e s , i d e n -t i c a l r e s u l t s were produced "by the a c t i o n o f extreme temperature w i t h i n a pure race 1* (Goldsohmidt 1921, 1922, 192?)• S t u d i e s on L e b i s t e s i n d i c a t e d t h a t there was a seasonal i n f l u e n c e on the sex de t e r m i n a t i o n (Winge 1932). The i n f l u e n c e o f temperature i n the sex d i f f e r e n t i a t i o n o f A p l o o h e l l u a l a t i p e s suggested t h a t h i g h temperature r e a r i n g favoured a dominant male frequency (Aida 193&). L i t t l e c y t o l o g i c a l and g e n e t i c a l r e s e a r c h has been attempted i n the salmonoid f i s h e s . S t u d i e s on the rainbow t r o u t Salmo i r i d e u s (probably i s S. g a i r d n e r i i ) suggested t h a t t h e i r d i p l o i d number (of chromosomes) was 12 (Mrsic 1925)• R e c e n t l y the chromosome morphology o f c e r t a i n salmonid and coregonid s p e c i e s and t h e i r h y b r i d s has been t r e a t e d i n d e t a i l (SvSrdson 1945). I n P a c i f i c salmon the chum 0. k e t a i s s a i d to c o n t a i n 74 chromosomes (Makino 1937). c) E m b r y o l o g i c a l Aspects o f Sex Determination and D i f f e r e n t i a t i o n In the f o r e g o i n g i t was i n d i c a t e d t h a t g e n e r a l l y primary sex det e r m i n a t i o n was dependent upon the balance between two types o f gene i n f l u e n c e (autosomal and gono-8 somal). I t was a l s o seen that the environment c o u l d modify t h e i r normal i n t e r p l a y , thus l e a d i n g to the phenomena o f the i n t e r s e x and dominant male f r e q u e n c i e s . Meanwhile c o n t r i b u t i o n s were b e i n g made i n r e l a t e d r e s e a r c h . A c l a i m to demonstrate f o r the f i r s t time the complete germ c e l l c y c l e i n any v e r t e b r a t e was advanced from s t u d i e s on the brook lamprey (Okkelberg 1921). T h e o r i e s h e l d on the s u b j e c t were comprehensively s t a t e d by Okkelberg and are from t h a t source reviewed. In v e r t e b r a t e s the p r i m o r d i a l germ c e l l s were thought to be d i s c e r n i b l e when the three germ l a y e r s formed. F u r t h e r , the d e f i n i t i v e germ c e l l s were d e r i v e d from these o e l l s , a c o n c l u s i o n demanded by ¥eismann»s theory o f germ plasma c o n t i n u i t y . Others h e l d the germ c e l l s to be pro-ducts o f the germinal e p i t h e l i u m and hence were formed from the entoderm. C o n s e r v a t i s t s b e l i e v e d t h e _ e a r l y segregated p r i m o r d i a l germ c e l l s and the entoderm were the f o r e r u n n e r s o f the germ c e l l s . Okkelberg*s s t u d i e s o f the lamprey induced him to conceive the i d e a t h a t the germ c e l l anlagen were a s s o c i a t e d w i t h the y o l k entoderm and t h a t t h i s anlagen became i n c l u d e d i n the mesoderm on i t s d i f f e r e n t i a t i o n . When the germ c e l l p r i m o r d i a mingled w i t h the mesenchyme d o r s a l to the p e r i -toneum they were regarded as the d e f i n i t i v e germ o e l l s . Moreover, when these c e l l s took up t h e i r p o s i t i o n i n the gonad there c o u l d be d i s t i n g u i s h e d two k i n d s : those .that d i v i d e d r a p i d l y ( k a t a b o l i c ) and those showing growth (anabolic). The former were p o t e n t i a l male and the l a t t e r p o t e n t i a l female c e l l s . The r e l a t i v e proportion of these c e l l s determined whether t h i s juvenile hermaphrodite would become a male or female. Despite advances i n embryological techniques there are s t i l l differences of opinion as to the o r i g i n of the germ c e l l s . Nieuwkoop (1944) from studies i n the Urodeles states "the o r i g i n of the primary germ c e l l s i s probably mesodermal with entodermal inductive action (the entoderm stimulates the mesoderm to form germ c e l l s ) , and that °the paired g e n i t a l ridges (precursor to the germinal epithelium) originate from the coelomic epithelium". This single o r i g i n of the g e n i t a l ridges has been indicated f o r the cyclostomes and teleosts (Anguillidae, Sparidae), but Witschi and Dantschakoff hold to a double o r i g i n (the in t e r r e n a l blastema and the peritoneal wall) i n the Amphibia (D»Aneona 1947, 1949a,1949b)~//V. . The method of o r i g i n i s perhaps important when sex determination i s viewed from the cytoplasm-nucleus point of view since the g e n i t a l ridge provides the somatic substratum of the gonad. It would be implied that t h i s stromatic tissue has hetero-sexual induotive action capable of overriding, or at the l e a s t , influencing, genotypic sex determination. From studies i n the anurans (Witschi 1929a, 1929b) the somatic portion of the undifferentiated gonad was seen to have a c o r t i c a l and medullary portion. Further, the cortex was regarded as the permanent s i t e of the germ c e l l s . 10 Under the inductive action of the gonad substratum the germ c e l l s remained i n the cortex and produced oooytes or else migrated into the rete cords of the medulla and became spermatocytes. Witschi explained that the phenomenon could be attributed to sexual inductors emitted from the germinal epithelium and that suppression of one d i f f e r e n t i a t o r l e f t the other to determine the p a r t i c u l a r sex. Such sex s p e c i f i c substances were c a l l e d medullarin and cortexin, the former being masculinizing and the l a t t e r femininizing. The application of high temperature (32° C.) on tadpoles of Rana sylvatioa transformed the sexually d i f f e r e n t i a t e d females into males i n a sequence of events that helped substantiate the presence of sex s p e c i f i c substances. Un-l i k e the well-known sex hormones these substances were con-sidered to be protein rather than s t e r o i d i n nature (Witschi 1942). Similar inductive action has been admitted for the teleosts Sparus auratus and Anguilla a n g u i l l a , but the sexual d i f f e r e n t i a t o r s have been c a l l e d androgenin and gynogenin (D»Ancona 1947, 1949a, 1949b). D i f f e r e n t i a t i o n i n the two species proceeds d i f f e r e n t l y . In the gonad of Sparus heterosexual areas are topographically separate but not i d e n t i f i a b l e as cortex and medulla. In Anguilla the d i f f e r e n t i a t i o n proceeds from a " t r a n s i t o r y i n t e r s e x u a l i t y " i n which male and female germ o e l l s are intimately mixed over the entire gonad substratum. Somewhat analogous research has been pursued i n the Urodeles 11 by various Japanese investigators. Their methods involve fragmented gonad implantation into undifferentiated or semi-d i f f e r e n t i a t e d larvae and high temperature roarings. They found that despite the presence of grafted t e s t i s continued suppression of the genetical females of the s e m i - d i f f e r e n t i -ated Hynobius retardatu3 could not be guaranteed and that high temperatures (30° C.) induced a dominant male d i s t r i b u -t i o n (Uchida 1937a i . f 1937°), Injection of the f o l l i c u l a r hormone into the l a r v a l gonad of the same species indicated that d i f f e r e n t i a -t i o n "proceeds independently 1* and that "the germ c e l l s become.responsive to the hormone only a f t e r metamorphoses" (Hanaoka 1941a;; s 1941b};;;* I t was found that testosterone proprionate modified sex d i f f e r e n t i a t i o n i n either sex by reducing the number of germ c e l l s . Undifferentiated embryos of the trout S. i r i d e u s g a i r d n e r i l ) have been reared i n f o l l i c u l a r solutions producing only s l i g h t feminization of the male genotype (Padoa 1939). From subsequent rearings (at 17-20° C.) i n the absence of the f o l l i c u l a r hormone Padoa suggested that d i f f e r e n t i a t i o n proceeded from an i n d i f f e r e n t stage into e i t h e r male or female. Such findings contradicted e a r l i e r research i n the rainbow trout (reared at 10-?14° C.) where i t was found that the male passed through an oocyte stage (Mrsic 1923) and confirmed i n l a t e r studies (Mraio 1931). D i s p a r i t i e s i n the method of sex d i f f e r e n t i a t i o n were .attributed by Padoa to the d i f f e r e n t temperature rearings 12: of the trout. Similar protogynous development has been recorded f o r Salmo salar ( F e l i x 1906). Orientation to the sexual apparatus i n teleosts remains to be considered. The following account i s adapted from Goodrich (1930). The recognition of early male and female germ c e l l products i n the teleosts i s made d i f f i c u l t by the nature of the t e s t i c u l a r and o v a r i a l network. In the Amphibia the topographic r e l a t i o n of the rete and sex cords to the Wolffian duct, and the ovigerous cords to the Mullerian duct aids studies of germ c e l l d i f f e r e n t i a t i o n . In the Teleosts such topographical l o c a l i z a t i o n i s not apparent since the t e s t i c u l a r network does not pass through the kidney. Rather the vas efferens i s p r o l i f e r a t e d from the caudal region of the t e s t i s to j o i n the mesonephric duet i n the region of the urogenital sinus. In the ovary the Mullerian duct i s represented by open and/or closed parova-r i a l or endovarial canals which may or may not extend the length of the ovary. In some cases a free ovary i s developed, i t s products being shed to the general ooelom. d) The Chemical Aspects of Sex Determination In addition to the genotypic theories on sex determination i t was indicated that the i n d i f f e r e n t gonad has both male and female Inducing substances. The mani-fe s t a t i o n of one or the other of these substances determines the sex. In lower vertebrates the d i f f e r e n t i a t i o n seems to proceed i n the male or female d i r e c t i o n from a state of 13 j u v e n i l e hermaphroditism. I t was seen t h a t the hormones u s u a l l y a s s o c i a t e d w i t h the secondary sex c h a r a c t e r s do not always g i v e a p o s i t i v e r e a c t i o n on the u n d i f f e r e n t i a t e d gonad. Such d i s p a r i t y may be due to the f o r e i g n o r a n t a g o n i s t i c a c t i o n o f the ambient on the germ c e l l s (Brachet 1950). S t u d i e s on l e s s complex animals have added to our knowledge o f sex determining f a c t o r s . Among those s e l e c t e d was the r o t i f e r Hydatina senta i n which were c o n s i d e r e d osmotic and oxygen f a c t o r s ( S h u l l 1912.; S h u l l and L a d o f f 1916). Here i t was found t h a t by d e c r e a s i n g the osmotic p r e s s u r e o f male-producing females there was a reduced male-p r o d u c t i o n . Conversely, r a i s i n g the osmotic p r e s s u r e i n c r e a s e d male-production. F u r t h e r , i t was shown t h a t s i m i l a r a c t i o n o b t a i n s where the q u a l i t y o f the food i s v a r i e d . Such a d i e t as manure suppressed male p r o d u c t i o n , whereas Euglena or Chlamydomonas f e e d i n g s had the r e v e r s e e f f e c t . The d i f f e r e n t amounts o f a v a i l a b l e oxygen were co n s i d e r e d the d i r e c t i n g f a c t o r s . In v e r t e b r a t e s the r o l e o f r e s p i r a t o r y metabolism i n sex d e t e r m i n a t i o n remains obscure i n view o f the absence o f demonstrated m e t a b o l i c d i f f e r e n c e s p r i o r t o , and a t , the time o f d i f f e r e n t i a t i o n (Brachet 1950). Osmotic p r e s s u r e and food r e l a t i o n s have not been overlo o k e d i n f i s h . Where eggs o f Salmo i r i d e u s were graded as to r i p e n e s s , those t h a t were o v e r - r i p e showed a male frequency. S e l e c t i v e m o r t a l i t y was not a f a c t o r 14 (Mrsic 1 9 2 3 ) • In B e t t a splendens, adverse c o n d i t i o n s o f temperature, food, and l i g h t o f f s e t the normal balance by i n c r e a s i n g the number o f males, and a g a i n s e l e c t i v e m o r t a l i t y was not c o n s i d e r e d a f a c t o r (Eberhardt 1 9 4 3 ) . In the marine copepod T i g r i o p u s japonious i t was found t h a t such developmental e x c i t o r s as potassium c h l o r a t e , d i i o d o t y r o s i n , e t c . i n c r e a s e d male p r o d u c t i o n , while develop-mental i n h i b i t o r s l i k e c h l o r e t o n e , c h l o r a l hydrate e t c . i n c r e a s e d the female frequency (Takeda 1 9 5 0 ) . Temperature r e a r i n g s were a l s o undertaken from which i t was concluded t h a t the seasonal v a r i a t i o n i n the sex r a t i o may be a t t r i b -u t e d to temperature f l u c t u a t i o n s . B i o c h e m i c a l s t u d i e s on the b i s e x u a l f l a g e l l a t e Ghlamydomonas s y n o i c a i n d i c a t e sex i s determined q u a n t i t a -t i v e l y (Moewus 1 9 3 9 , 1941, 1 9 4 3 ; Kuhn, Moewus and Wendt 1 9 3 8 , 1 9 4 0 ) . T h e i r o b s e r v a t i o n s have shown t h a t "gamones", d i f f e r i n g o n l y i n chemical p r o p o r t i o n , determine the moment at which male or female gametes are induced to conjugate. These gamones are s i m i l a r i n e f f e c t to the c i s and t r a n s e s t e r s o f d i m e t h y l c r o c e t i n e . A p p r o p r i a t e mixtures o f these e s t e r s were e f f e c t i v e a t a d i l u t i o n o f 1 p a r t i n 3 3 m i l l i o n . F u r t h e r , they found that chemical substances i d e n t i f i e d as androtermone and gynotermone determined the sex. These were e f f e c t i v e a t c o n c e n t r a t i o n s i n the order o f 1 molecule per c e l l . A s s o c i a t e d with termone a c t i o n were c e l l u l a r enzymes c o n t r o l l e d by genes or perhaps i d e n t i c a l with them. Brachet (1950) sums up the s i t u a t i o n as f o l l o w s : 15 n t h e problem o f sexual d e t e r m i n a t i o n becomes c l e a r o n l y when we in t r o d u c e the fundamental concept o f p o t e n t i a l - b i s e x u a l i t y ....there i s no lon g e r any doubt t h a t sex i s determined by chemical substances or by the r a t i o o f the c o n c e n t r a t i o n o f s p e c i a l chemical substances. The r e a c t i o n s which l e a d to t h e i r formation i n d e f i n i t e q u a n t i t i e s from p r e c u r s o r s must be c a t a l y z e d by enzymes t h a t may be i d e n t i f i e d i n p a r t o r i n whole w i t h genes.—We can see without d i f f i c u l t y t h a t the r e a c t i o n s c o u l d a l s o be m o d i f i e d by changes i n the e x t e r n a l environment such t h a t the optima f o r these enzymes are d i s t i n c t l y d i f f e r e n t ( i n Ghlamydomonas a t any r a t e ) . . . 1 * 16 •III.. MATERIALS AND METHODS SECTION I. CHROMOSOME STUDIES IN CHUM SALMON The chum salmon (0. keta) was s e l e c t e d f o r c y t o -l o g i c a l o b s e r v a t i o n p a r t l y because o f a v a i l a b i l i t y and part-? l y because some e a r l i e r work had been done on t h i s s p e c i e s (Makino 1937). Embryonic d i s c s were d i s s e c t e d from eggs o f seventy-two and n i n e t y - s i x hour chum salmon i n p h y s i o l o g i c a l s a l i n e (one percent sodium c h l o r i d e ) . The d i s c s were f i x e d i n iacetocarmine and smeared on albumen-coated s l i d e s . The chromosomes were then s t a i n e d w i t h acetocarmine proper a t 60° C. f o r two hours a f t e r having been mordanted i n .001% . ammonium i r o n rlv/:. sulphate f o r 30-60 seconds. T h i s technique had been s a t i s f a c t o r i l y used i n oth e r salmonoid f i s h e s (SvSrdson 1943). F i f t e e n s l i d e s prepared i n t h i s way d i d not p r o v i d e good r e s u l t s p r o b a b l y due to the c r i t i c a l squashing procedure (chromosome "fanning") and the h i g h number o f chromosomes. Chromatin content was a l s o s t u d i e d i n germ c e l l s o f chum a l e v i n s and f r y t h a t had been p r e s e r v e d i n Bouin»s p i c r o - a c e t i c f o r m a l i n . T h i s i n v o l v e d t w e n t y - f i v e f i s h c o v e r i n g the p e r i o d A p r i l 4th to June 26th. The head and the r e g i o n p o s t e r i o r to the p e l v l o f i n s were removed and the remaining m a t e r i a l washed i n 70% a l c o h o l o v e r n i g h t . The m a t e r i a l was then passed through dioxan and imbedded i n p a r a f f i n mat (melting p o i n t 53° G.). Subsequently the 17 m a t e r i a l was s e c t i o n e d (10 microns) and the n u c l e a r compo-nents s t a i n e d w i t h Heidenhain»s haemotoxylin or Feulgen s t a i n s . Four f i s h were s t a i n e d w i t h M a l l o r y t s polychrome s t a i n . Where haemotoxylin was used, 40 minutes i n i r o n ammonium r..'-./'. sulphate s u f f i c e d to mordant the m a t e r i a l and the same p e r i o d was used w i t h the s t a i n . A f t e r c r i t i c a l l y d e s t a i n i n g , the s e c t i o n s were c o u n t e r - s t a i n e d w i t h e o s i n chromate s o l u t i o n . More f r e q u e n t l y l i g h t green i n c l o v e o i l was used as the c o u n t e r - s t a i n s i n c e i t seemed to p r o v i d e b e t t e r con-? t r a s t . The s e c t i o n s were c l e a r e d i n x y l o l , flamed, and mounted i n Canada balsam. Feulgen*s chromosome s t a i n was used on s e c t i o n s r e p r e s e n t a t i v e o f the above m a t e r i a l . A f t e r r i n s i n g the s l i d e s f o r one week i n 70$ a l c o h o l to ensure removal o f the aldehyde component o f Bouin*s f i x , the s e c t i o n s were brought down to water ( g l a s s d i s t i l l e d water was used throughout). They were then p l a c e d f o r one minute i n c o l d normal hydro-c h l o r i c a c i d and t r a n s f e r r e d to the d i g e s t e r . In t h i s hot normal h y d r o c h l o r i c a c i d (60° C.) d i g e s t i o n was e x p e r i -m e n t a l l y t r i e d a t i n t e r v a l s from 30 seconds up to 45 minutes. The m a t e r i a l was r i n s e d i n c o l d normal h y d r o c h l o r i c a c i d and p l a c e d i n Feulgen*s b a s i c f u c h s i n f o r 2 to 5 hours. Then the s l i d e s were p l a c e d a t ten-minute i n t e r v a l s i n three s u c c e s s i v e s o l u t i o n s o f Feulgen*s potassium metabisulphate. The m a t e r i a l was dehydrated and c r i t i c a l l y c o u n t e r s t a i n e d w i t h 0.5$ l i g h t green i n c l o v e o i l , c l e a r e d i n x y l o l , flamed, 18 and mounted i n Canada balsam. The chromosomes d i d not take the s t a i n , whioh may be due to the l o n g - s t a n d i n g p e r i o d o f the a l e v i n s i n Bouints s o l u t i o n (8 months), e -.:..? The r e c -ommendation t h a t Flemming's or Zenker»s f i x be used where t h i s s t a i n i s employed ( D a r l i n g t o n afid'/la Cour 1947)is- noted. A Spencer microscope f i t t e d w i t h a 15X o c u l a r and a 97X o i l immersion o b j e c t i v e were used i n examining the m a t e r i a l . SECTION 2. THE HISTOGENESIS OF SEX DIFFERENTIATION a) The H i s t o l o g i c a l Method The p o s t - h a t c h i n g m a t e r i a l a l r e a d y mentioned was used to t r a c e gonad development from the i n d i f f e r e n t s t a g e . b) F i e l d Technique i n the Examination o f Young Salmon Gonads In the a l e v i n specimens examination was made by removal o f the y o l k sac which l e a v e s bare the sparse v i s c e r a l c ontent. In the other stages i n c i s i o n s were made along the l a t e r a l l i n e s , v e n t r a l plane and c i r c u m c i s e d about the p o s t e r i o r o p e r c l e r e g i o n . S k i n and muscle was then removed by f o r c e p s , care being taken not to d i s t o r t i n t e r n a l organs. From the c l o a c a l r e g i o n the i n t e s t i n e was d i s c o n n e c t e d and removed i n an a n t e r i o r l y d i r e c t e d movement at the oesophageal-stomach c o n n e c t i o n . T h i s l e a v e s a macroscopic view o f the a i r - b l a d d e r which i n l a t e r a l aspects may or may not r e v e a l the sex organs. In any case the c o n n e c t i o n between a i r -b l a d d e r and oesophagus i s observable and i t i s i n t h i s r e g i o n t h a t the gonads appear to commence growth. The a i r was b l a d d e r connection :/ pinched o f f w i t h f o r c e p s and a i r 19 Bladder with adhering gonads pulled away from the body. The ~ "" • were contents n / p l a c e d on a glass s l i d e and examined by micro-scope without staining under a magnification of 100 diameters, SECTION 2. APPLICATION OF ULTRAVIOLET LIGHT AND TEMPERA3JDRE ON DEVELOPING SALMON. Sockeye, chum, and pink (0. gorbusoha) salmon were selected f o r u l t r a v i o l e t r a d i a t i o n and temperature treatment, The eggs were taken by the dry method, transported to the University hatchery, and maintained i n troughs with water flows varying from 1-4 gallons per minute (Table I, Figs. 1, 2, 2). 20 19 28 NOV. CHUM CHUM AND SOCKEYE ie 28 DEC 7 7AN1.7 27 16 FEB. e is MAR 28 2 APR-JUL Figure 1. The application of u l t r a v i o l e t l i g h t ( v e r t i c a l lines) and temperature"(horizontal l i n e s ) i n r e l a t i o n to f e r t i l i z a t i o n and d a i l y hatchery water temperature i n the 1949-30 experiments. The A p r i l to J u l y data i s expressed i n weeks. Table I . The source and ut i l i z a t i o n of salmon eggs in the hatchery experiments 1 9 4 9 - 5 0 , 1 9 5 0 - 5 1 . Species Source Date of Spawn No. of eggs Experiment A. (chum) (sockeye) B. (chum) C. Ohum Cultus lake Nov. 1 8 , 1 9 4 9 1 3 ,394 Sockeye Cultus lake Dec. 7, 1949 l6,6l8 Pink Ptsolum river Sept. 30, 1950 14,719 Chum Cultus lake Oct. 2 8 , 1950 9,917 Ultraviolet light treatment in 1949-50 (Fig. 1) 1. 1381 eggs treated with 9 applications of .5 finsen units 2. 1 4 4 1 ti tt n 9 n tr 1.0 ti tt 3. 1575 n ti tut 8 tt tt: 1-5 tt tt 4 . 1337 n Control 5. 1763 tt> treated «• 4 ti »t: .5 tt tt 6. 1859 ti- tt ti- 3 tt tt 1.0 tt it 7. 1759 ll 11 lt 4 tt 11 1.5 11 re 8. 2622 n Control Temperature treatment in 1949-50 (Fig. 1) 9. 1450 egg£ 5 held at 10.4° C. for 14 days 10. 1406 tr * » 14.5 v C tt 11 tl: 11. 2452 it " » . 1 7 . 7 ° c. ti 5 tt 12. 2352 tt » * 23.3 c. tt 2 tl 13. 1237 tt Control 14. 2008 tt held at 10.4° C. for 14 tt 15. 1786 tt " " 14 .5° C « » 1 7 . 7 ° C tr 11 tt 16. 1913 tt tt 5 tt 17. 2908 tt tr tt 2 3 . 3 ° C. it 2 tt 18, 2622 tt Control Table I. Continued. Temperature treatment i n 1950-51 (gigs. 2, 3) (pink) E. (chum) F. 19. 1217 eggs held at 1 . 8 ° e . 4 f o r 41 days 20 2528 « « n 1 2 . 4 ° c . tt 11 tt 21. 1886 it ti 1 4 . 8 ° c . tt 41 n 22. 1290 n » it 1 8 . 1 ° c . n 11 tt 23. 1486 H tt tt: 1 8 . 9 ° c . n 23 tt 24. 1497 It: Control 25. 3679 It w 26. 1136 tt t t c * 27. 968 tt held at 1 . 8 ° for 28 days 28. 944 It n » 1 2 . 4 ° c . n 35 tt 29. 1586 n tt it 1 4 . 8 © c . 2 42 » 30. 1145 n, it tt 1 8 . 9 ° c . tt 9 tt 31. 1294 tt tt n 18 .9° c . tt 25 tt 32. 1575 tt Control 33. 1286 w n 34. 1119 tt » ro Incubation of overripe and green eggs 1950-51 (pink) G. 55. -224 eggs taken from 14 "spent" salmon 36. ovaries " " 1 "green" salmon. *(1.8° C. -1.3 0 ) ( 2.4 On Oct. 28 temperature went to 11.0 C.) 22 W 3 SFPT F i g u r e 2. Temperature treatment o f developing p i n k salmon embryos i n r e l a t i o n to the h a t c h e r y water temperatures ( i n 1950-51). F i g u r e 3; Temperature treatment o f developing chum salmon embryos i n r e l a t i o n to the h a t c h e r y temperatures ( i n 1950-J l ) . ' • 25 Application of u l t r a v i o l e t l i g h t was c a r r i e d out i n the hatchery troughs using a General E l e c t r i c type R. S. sun lamp. The dosages were standardized by the photo-chemical method (Pohle 1916) and applied within the sub-l e t h a l range indicated by previous research ( B e l l and Hoar 1949). For the purpose of temperature treatment thermo-s t a t i c a l l y controlled 100 gallon aquaria were used except where a near zero temperature was required. Here a 5 gallon battery jar was immersed i n a r e f r i g e r a t i o n u n i t . The temperature and i n t e r v a l of treatment were a r b i t r a r i l y selected. Unless otherwise noted temperatures were constant within 1° C. The control l o t s remained at the hatchery water temperature. Both u l t r a v i o l e t and temperature application i n 1949-50 were continued u n t i l death or hatching of the f i s h i n t e r f e r e d with the experiment. Pink salmon were substituted f o r sockeye i n 1950-51 because of the former»s rapid germ c e l l development. In addition some "green" and "overripe" eggs were f e r t i l i z e d (Table I . ) , The f r y were fed on ground beef (or hog) l i v e r mixed with powdered milk (Klim) u n t i l the sexes d i f f e r e n t i -ated. During the l a t t e r part of the 1949-50 experiments f i s h were maintained on a mixture of canned salmon and pablum. Routine hatchery sanitation methods were followed 24 throughout the experiments. S a t u r a t e d potassium permanganate s o l u t i o n was used as a f u n g i c i d e . SECTION 4. SEX RATIOS OE YOUNG SALMON AT PORT JOHN, B. C. The f e a s i b i l i t y o f determining sex i n the a l e v i n s and f r y o f P a c i f i c salmon was e s t a b l i s h e d i n p r e l i m i n a r y s t u d i e s i n 1948. Systematic c o l l e c t i o n o f pink, coho (0_. k i s u t c h ) , and chum f r y as w e l l as sockeye and coho smolts was i n i t i a t e d a t P o r t John, B. C, i n 1949 and continued i n 1950. Sockeye f r y migrants were too s m a l l to d i s t i n g u i s h sex and were r e t a i n e d i n e s p e c i a l l y c o n s t r u c t e d cages (2 f x 5*) a t " s u i t a b l e " depths i n P o r t John l a k e and i n the sea. D i s p r o p o r t i o n s i n the 1949 sex r a t i o of chum f r y l e d to r e t e n t i o n o f a 1950 sample i n the sea. S i m i l a r coho m a t e r i a l was taken f o r t u i t o u s l y . The data were taken i n p r o p o r t i o n to the d a i l y and/or weekly m i g r a t i o n s . F o r pinks the p e r i o d s were between A p r i l 27-May 23 (1949) and A p r i l 15-May 21 (1950); chums were A p r i l 29-May 28 and A p r i l 15-j;une^l4 i n those y e a r s ; s i m i l a r l y sockeye y e a r l i n g s were April'29-May -30 and May 3-June 8. Likewise coho y e a r l i n g s were taken i n 1950, w h i l e f o r the 1949 sockeye and coho f r y and coho y e a r l i n g s , the samples were not r e p r e s e n t a t i v e o f the runs. Kamloops t r o u t (Salmo g a i r d n e r i i kamloops) t h a t had been r e a r e d i n the Summerland h a t c h e r y o f the B. C. Game Commission a t about 10.3° i C. are i n c l u d e d i n these data. Length measurements ( f o r k o f t a i l to t i p o f snout) were o b t a i n e d i n p a r t or whole o f the samples. 2$ SECTION 5. SEX RATIOS OF PINK SALMON IN THE FISHERY In addition to the young salmon taken in fresh water, data on the sex of fish in commercial catches were collected in 1950. The pink salmon was selected for the, purpose because a) i t s invariable two-year cycle, and b) i t s strong-ly marked secondary sex characters make superficial sex determination reliable. Sampling for sex in the commercial fishery was begun at a time when some Port John fish might be expected to comprise part of the catch. The best evidence for this was migration routes used by Port John destined fish, the wlast-minute'» sampling of seine catches (since this lessens interference with the immense Bella Goola river run), the dwindling number of pink salmon taken by g i l l nets, and the appearance of these fish in Port John bay. Thus the total seine catch of four boats was examined August 9 and 10 (the latest dates possible) and g i l l net caught fish were similarly sampled (August 9-31)• 26 IT. RESULTS SECTION 1. CHROMOSOME STUDIES IN THE CHUM SALMON The seventy-two and ninety-six hour embryo smears did not provide a good chromosome count or reveal sex chromosomes. In each smear there were estimated to be two to three hundred c e l l s which frequently.contained"mitotic figures (Fig. 4 ) . The procedure for "fanning" the chromo-somes from t h e i r spindle f i b r e attachment was not adequate. Side views of the metaphase plate s t i l l showed the chromo-somes to be clumped, and i n early anaphase they remained bridged. From polar view there could be distinguished three kinds of chromosomes on the metaphase plate: granular, sigmoid, and j-shaped. Thirty-eight such chromosomes were counted, but thi s count i s not absolute ( F i g . 5)« F i n a l l y , there was no evidence to suggest the presence or absence of sex chromosomes. Anaphase figures i n the smear material showed no precession or succession of ind i v i d u a l chromo-somes to those clumped on the spindle f i b r e s . Figure 4. Anaphase chromo- Figure 5. Polar view of somes from smeared embryonal metaphase plate from smeared c e l l s of chum salmon ( X I 8 5 0 ) . embryonal c e l l s of chum salmon ( X I 8 5 O ) . 27 The s e c t i o n m a t e r i a l from the d i f f e r e n t i a t i n g gonads gave u n s a t i s f a c t o r y r e s u l t s . The chromosomes i n the germ c e l l s were i n d i s t i n g u i s h a b l e m o r p h o l o g i c a l l y and c o u l d not be counted ( F i g . 6). The same c o n d i t i o n p e r s i s t e d i n the spermatogonia. The young oocytes showed condensations of chromatin m a t e r i a l about the p e r i p h e r y o f the n u c l e o -plasm r e m i n i s c e n t o f d i p l o t e n e chromosomes i n m e i o t i c pro-phase. However, these condensations w e r e ^ a c i d o p h i l i c ) to l i g h t green and hence are i d e n t i f i e d as n u c l e o l i ( F i g . 7). SECTION 2. GERM CELL DIFFERENTIATION IN CHUM SALMON a) The H i s t o l o g i c a l Examination 1. Three weeks a f t e r h a t c h i n g : Three a l e v i n s were examined f o r gonad p r i m o r d i a at t h i s time. An elongated s t r u c t u r e appeared i n the connec t i v e t i s s u e between the mesonephros and gut. Cross s e c t i o n s from the a n t e r i o r r e g i o n showed d i s t i n c t n e s t s o f c e l l s i n t h i s s t r u c t u r e . These c e l l s , about 8 i n a group, were 17.6 microns i n diameter. In comparison, the bloo d c e l l s i n the d o r s a l a o r t a were 11.0 microns. The l a r g e c e l l s c o n tained n u c l e i c h a r a c t e r i z e d by a f i n e chromatin network and e a r l y prophase chromosomes ( F i g . 8). By t r a c i n g the s t r u c t u r e c a u d a l l y i t was observed t h a t such c e l l s o c c u r r e d i n packets s e p a r a t e d by p y r i f o r m i n t e r s t i t i a l c e l l s which measured 6.6 microns i n l o n g a x i s . The c e l l s were l e s s numerous i n the p o s t e r i o r s e c t i o n s and there were fewer c e l l s i n a nest ( F i g . 9). The s t r u c t u r e was present i n two of the three 28 F i g u r e 6. S e c t i o n a n t e r i a d showing clumps o f chromosomes i n germ c e l l s o f a chum salmon a l e v i n 7 weeks o l d (X1850). NUCLEOLUS OOCYTES BLOOD CELL F i g u r e 7« S e c t i o n a n t e r i a d showing p e r i p h e r a l n u c l e o l i i n oocytes o f a chum salmon a l e v i n 14 weeks o l d (X14.50). 2* embryos and was i d e n t i f i e d as an i n d i f f e r e n t gonad c o n t a i n i n g germ c e l l s . The absence o f gonads i n s e c t i o n s from the t h i r d a l e v i n was the r e s u l t o f e x p e r i m e n t a l l y cut s a g g i t a l s e c t i o n s . CELL F i g u r e 8. S e c t i o n a n t e r i a d showing a nest o f germ c e l l s i n a chum salmon a l e v i n 3 weeks o l d (X1850). GERM CELL INTERSTITIAL CELL F i g u r e 9. Diagram showing the arrangement of germ c e l l s i n an i n d i f f e r e n t gonad o f chum salmon a l e v i n 3 weeks o l d . 2. l i v e weeks a f t e r h a t c h i n g ( 2 a l e v i n s ) : The gonads at t h i s time showed no f u r t h e r develop-ment from the pr e c e d i n g stage. 3. Seven weeks a f t e r h a t c h i n g (2 a l e v i n s ) : The gonad anlagen were v e r y c l e a r l y d i f f e r e n t i a t e d 30 i n t o two areas. The germ c e l l s formed a cord e n c l o s e d i n a stroma o f connective t i s s u e and i n t e r s t i t i a l c e l l s . C r a n i a d the germ c e l l s were m i t o t i c a l l y a c t i v e and appeared i n l a r g e numbers ( F i g . 6, p. 28), while caudad no a c t i v e d i v i s i o n and few c e l l s o b t ained ( F i g . 10). 4. Nine weeks a f t e r h a t c h i n g (3 a l e v i n s ) : S e c t i o n s showed the gonads to d i f f e r from those a l r e a d y d e s c r i b e d because o f the conspicuous development o f b l o o d c e l l s . One a l e v i n had germ c e l l s i n the a n t e r i o r p o r t i o n o f the gonad t h a t were o f unequal s i z e ( F i g . 11), a f i r s t i n d i c a t i o n o f a u x o c y t o s l s and sex d i f f e r e n t i a t i o n . The s m a l l germ c e l l s may now be regarded as oogonia w h i l e the l a r g e r c e l l s are undoubtedly young oooytes. There was no evidence to suggest the gonads o f the o t h e r embryo would not assume t h i s appearance a t a l a t e r stage. Here the germ c e l l s were equal i n s i z e and the gonads r e t a i n e d t h e i r p r i m i t i v e f e a t u r e s . 5. Ten weeks a f t e r h a t c h i n g (2 a l e v i n s ) : One p a i r o f gonads co n t a i n e d oocytes, some o f which were more mature than those d e s c r i b e d p r e v i o u s l y . Not o n l y were they l a r g e r , but there appeared a v a r i a b l e number o f n u c l e o l i about the i n n e r margin o f the n u c l e a r membrane. A new f e a t u r e o f the ovary was shown by the i n p o c k e t i n g o f the stroma to form the s o - c a l l e d open endovarian c a n a l ( F i g . 12). The o t h e r a l e v i n c o n t a i n e d a s m a l l p r i m i t i v e gonad wi t h d i v i d i n g germ c e l l s . T h i s gonad may be c a l l e d an e a r l y t e s t i s , and i t s germ c e l l s , spermatogonia, because 31 F i g u r e 1 1 . S e c t i o n a n t e r i a d showing oogonia and young oocytes i n an a l e v i n 9 weeks o l d (X1850). 32 F i g u r e 1 2 . S e c t i o n showing oocytes and an e n d o v a r i a l c a n a l i n the ovary o f an a l e v i n 1 0 weeks o l d ( X 1 8 5 0 ) . F i g u r e 13. S e c t i o n showing a spermatogonium i n metaphase i n the t e s t i s o f an a l e v i n 10 weeks o l d (X103O). 33 o f t h e i r f requent mitoses and no tendency to undergo growth ( F i g , 13). T h i s i s the f i r s t stage where the two sexes were c l e a r l y d i s t i n c t . The gonads d i f f e r e d i n s i z e , shape, development o f an e n d o v a r i a l c a n a l , and p a r t i c u l a r l y i n the appearance o f the gonocytes. 6. E l e v e n weeks a f t e r h a t c h i n g (2 a l e v i n s ) : One i n d i v i d u a l was a female whose o v a r i e s con-t a i n e d oocytes i n v a r i o u s stages o f growth as w e l l as n e s t s o f oogonia separated by connective t i s s u e ( F i g . 14). The other f i s h was a male, i t s t e s t e s showing no f u r t h e r d e v e l -opment. 7. T h i r t e e n weeks a f t e r h a t c h i n g (3 a l e v i n s ) : At t h i s time the coelom o f the a l e v i n c o n t a i n e d o n l y remnants o f the y o l k m a t e r i a l (deutoplasm). Two o f the a l e v i n s were females, the o t h e r a male. In the former the o v a r i e s were pe n e t r a t e d a n t e r i a d by a vena commites (an a r t e r y and i t s accompanying vein) ( F i g . 13). T h i s v a s c u l a r i -z a t i o n was n o t a b l y absent i n the t e s t e s . 8. Fourteen weeks a f t e r h a t c h i n g : Of f i v e a l e v i n s examined, one was a female, f o u r were males. The o v a r i e s remained t y p i c a l ( F i g . 16) and the t e s t e s showed no f u r t h e r development ( F i g . 17). 9. F i f t e e n weeks a f t e r h a t c h i n g : A f i n a l examination was made on two a l e v i n s . Both sexes were present and the gonads remained the same as those e a r l i e r d e s c r i b e d . From t h i s study i t i s seen the gonads o f the chum F i g u r e 15. S e c t i o n a n t e r i a d showing the vena commites i n the ovary o f a chum salmon a l e v i n 13 weeks o l d (XI85O)• 35 Figure 16. Section showing the relation of the ovaries to the body coelom in an alevin 14 weeks old (X150). 36 salmon can be d i s t i n g u i s h e d as to sex ten weeks a f t e r h a t c h i n g . Thus they develop a t an e a r l y stage from an i n d i f f e r e n t band o f c e l l s and a t no time i s there evidence f o r a j u v e n i l e hermaphrodite stage. b) The F i e l d Examination I t was found t h a t sex c o u l d be r e a d i l y determined i n pink salmon a t a l a t e a l e v i n stage. However the chum, coho, and sockeye salmon d i f f e r e n t i a t e d a t a l a t e r p e r i o d . The chum salmon showed sex d i f f e r e n c e s (about 20% had oocyte formations) . f o l l o w i n g a b s o r p t i o n o f the y o l k sac (36.6 mm.), but the r a t i o c o u l d not be guaranteed u n t i l a l e n g t h o f 3 8 .1 mm. was a t t a i n e d . I f the diameter o f the 37 germinal epithelium he considered sex i n d i c a t i v e , then the migrating chum f r y could be analysed immediately. This was done f o r the 1930 run. In the sockeye and coho, the f r y could be sexed only a f t e r retention i n the sea. Under these conditions the sockeye d i f f e r e n t i a t e d at 41 mm. i n length, the coho at 34.7 mm* F a i l u r e of the sockeye to survive at d i f f e r e n t depths i n Port John lake i n the summers of 1949 and 1950 prevented a fresh water comparison. The early male reproductive organs (Figs. 18, 19) were macroscopically of constant diameter extending along either side of the air-bladder. They were composed of spermatogonia of dense c e l l content. In contrast the female organs (Figs. 20, 21) were of larger diameter and tapered i n the caudal d i r e c t i o n . Oocyte formation was the i r most notable feature. Adipose tissue o f t e r removed With the air-bladder should not be confused with the ovary. The sex of the sockeye and coho smolt (seaward migrating one and two year o l d salmon) may be determined without resort to magnification. For Kamloops trout of.the same.size, microscopic examination was e s s e n t i a l . 38 F i g u r e 18 . D i s s e c t e d male gonads o f pin k salmon f r y (33mm. i n length) along the p e r i p h e r y o f the a i r - b l a d d e r . (X10). F i g u r e 19. E n l a r g e d p o r t i o n o f a male gonad w i t h the a i r -bladder i n the background and melanophores to the r i g h t . ( X 8 0 ) . 39 F i g u r e 20. D i s s e c t e d female gonads of a pink salmon f r y (33 mm. i n length) along the p e r i p h e r y o f the a i r - b l a d d e r . (HO). OVARY F i g u r e 21. E n l a r g e d p o r t i o n o f a female gonad with the a i r -bladder and melanophores i n view. Note the l a r g e o o c y t e s . (X80). 40 SECTION 3. ULTRAVIOLET LIGHT AND TMCPERATURE EXPERIMENTS a) The E f f e c t of U l t r a v i o l e t Radiation on the Sex Ratio of Developing Chum and Sockeye Salmon The m o r t a l i t i e s accruing from the ap p l i c a t i o n of u l t r a v i o l e t l i g h t on developing chum and sockeye embryos are shown (Figs. 22, 23). The sex r a t i o s of the surviving f i s h along with raw data are shown (Table I I ) . b) The e f f e c t of temperature on the sex r a t i o s of chum, sockeye, and pink salmon rearlngs The mortality r e s u l t i n g from the d i f f e r e n t temperature treatment of developing chum and sockeye salmon i s shown Figs. 24, 2.5. The sex r a t i o s of the surviving f i s h along with data are shown Table I I I . In the 1950-51 experiments few pink and no chum salmon survived the arduous conditions of incubation i n the new hatchery and the temperature treatment. The overripe eggs appeared to have been f e r t i l i z e d as did some of the fre e r eggs i n the green ovaries. The sex r a t i o s and raw data for these f i s h are shown Tables IV, V. SECTION 4. REMARKS ON THE HATCHERY EXPERIMENTS a) General The data as presented showed that s u r v i v a l of the 1949-50 chum and sockeye salmon to the »sex-difference stage* was dependent upon treatment. However, the species 41 2.0 Figure 22, The m o r t a l i t i e s r e s u l t i n g from u l t r a v i o l e t treat' ment of developing chum salmon i n the 1949-50 experiments. T I M E N W E E K S Figure 23. The m o r t a l i t i e s r e s u l t i n g from u l t r a v i o l e t treat-ment of developing sockeye salmon in the 1949-50 experiments. T a b l e II, Sex r a t i o s o f sockeye and chum salmon embryos exposed to u l t r a v i o l e t l i g h t 1949-50. U l t r a v i o l e t dosage Ghum salmon Sockeye salmon C o n t r o l 9x.5f 9xlf 8x1.5f C o n t r o l 4x.5f 5zlf 4x1.5f No. eggs a t Dec 25 1357 1381 1441 1575 2622 1765 1859 1759 No. f r y at J u l 7 156 248 143 58 647 261 6 50 % s u r v i v a l 11.68 17.90 9.91 3.68 24.67 14.80 .52 1.71 Nos. o f sex - F 77 122 27 - M 79 126 67 51 U n d i f f e r e n t i a t e d % o f sex - F 49.3 49.2 53.2 41.5 ? - M 50.7 50.8 46.8 55.5 X v a l u e .05 .06 .57 .27 ro Table III. Sex r a t i o s o f chum and sockeye salmon embryos a f t e r temperature treatment 1949-50. Temperature 01 lum salmon Socl ceve salmon. C o n t r o l 11° c. •1»° C. i r c. 23° C, C o n t r o l l l u C. 14° C. 18° C. 25° C. No. eggs a t 1337 1450 1406 2452 2552 26 22 2008 1786 1915 2908 Dec 25 647 284 No. f r y at 156 5 1 104 0 5 23 0 J u l 7 24.67 14.84 % s u r v i v a l 11.68 .21 .07 4.25 .0G .25 1.29 .00 No. o f F 77 1 0 65 0 . sex M 79 2 1 39 0 * U n d i f f e r e n t i a t e d f. sex F 49.3 33.3 0.00 62.5 0 * 9 M 50.7 66.7 100.00 37.5 0 T d v a l u e .05 .33 1.00 6.50 0 \ 4? Figure 24. The m o r t a l i t i e s r e s u l t i n g from temperature treat-ment of developing chum salmon i n the 1949-50 experiments. 8 12 IS 20 H 28 TIME IN WEEKS Figure 25. The m o r t a l i t i e s r e s u l t i n g from temperature t r e a t -ment of developing sockeye salmon i n the 1949-50 experiments. Table IV. Sex r a t i o s of pink salmon embryos a f t e r temperature treatment 1950-51. Conditions 1.8° G. 12.4° C. 14.8° C. 18.1° c. 18.9° G. "over-r i p e " "green" Control 1 Control 2 Control 5 No, eggs 1217 2528 1886 1290 1486 324 L & R 1497 3679 1136 ovary 64 Alevins 10 2 0 0 1 0 0 4 32 % s u r v i v a l .82 .04 .00 .00 .07 .00 .00 4.27 2.81 Nos. of) F 2 - wm 0 — - 34** 15 17ffitt sex ) M 5 0 - mm 1 mm - 26 1 12 % of sex F 66.6 100 - mm - - 56.6 75.0 58.6 . p M 53,5 mm - - 100 - - 43.4 25.0 41.4 .50 1.00 mm — - — •; - 1.07 1.00 .86 *1 unki lown *^ 4 unki lown AA&3 un-known periments concluded on January 29, excepting 1.8 C. l o t , which was March 6. Table V. Sex r a t i o s o f chum salmon embryos a f t e r temperature treatment 1950-51. Conditions 1.8° G.*** 12.4° C. 14.8° C. 18.9° C 18,90 C # * £ Control 1 Control 2 Control 5* No. eggs Alevins % s u r v i v a l 968 0 .00 944 0 .00 1586 0 .00 1145 0 .00 1294 0 .00 1575 0 .00 1286 0 .00 1119 0 .00 SMareh 2. The two7 chums surviving were not sexually d i f f e r e n t i a t e d . * % a r c h 17. The f i v e surviving chums were not sexually d i f f e r e n t i a t e d . ^ ^ A p r i l 2. Six chums were s t i l l a l i v e . 45 considered i n 1950-51 indicated that other factors operated on the s u r vival rate. These were thought to be plumbing break-downs r e s u l t i n g i n high temperature and water flow variations within the incubating troughs, as well as Saprolegnia blooms (a fungus thought to prevent oxygen intake i n developing eggs). I n i t i a l mortality a r i s i n g from u l t r a v i o l e t treatment were d i f f e r e n t i n the embryos considered (Figs. 22, 25). The differences were thought due to the species of salmon and the stage of development attained at time of i r r a d i a t i o n . The chums were sexually d i f f e r e n t i a t e d June 26 but the high summer temperatures terminated the experiment pre-maturely f o r the sockeye (July 6). Under the conditions of u l t r a v i o l e t r a d i a t i o n and stage of development the chum salmon d i f f e r e n t i a t e d into male and female i n near equal numbers (Table I I ) . Contrary to these findings the sex r a t i o s a r i s i n g from temperature treatment of eggs were d i f f e r e n t , although the high death rate perhaps caused unsatisfactory r e s u l t s . In the absence of temperature tolerance data on de-veloping P a c i f i c salmon, the 1949-50 treatment was f o r t u i t o u s . That no f i s h survived the 23° shocking i s perhaps understand-able. But where so few f i s h withstood treatment at the 11° and 14° centigrade l e v e l s i s not explainable when the return at 18° i s considered (Figs. 24, 25; Table I I I ) . Thus of the sex r a t i o s a r i s i n g from these embryos under temperature treatment, r e s u l t s can only be considered i n the 18° C. chum. In respect to the control group and 46 s t a t i s t i c a l p r o b a b i l i t y there was a s i g n i f i c a n t l y high female frequency ( 6 2 . 5 $ ) . Again sex could not be distinguished i n the sockeye rearings. In 1950-51 the pink and chum salmon eggs treated at , sub-lethal temperatures provided ( i n part or whole) inconclu-sive sex r a t i o data. Three control l o t s of pink salmon d i f f e r e d considerably i n the percent sex r a t i o , the balance leaning i n the female direotion (Tables IV, V). Adverse hatchery conditions operating on the sex mechanism may have been a contributing factor. However, i n view of the d i f f e r e n t numbers of eggs used i t i s admitted that s e l e c t i v e mortality might also have operated. The numbers surviving i n the control groups and those under experiment were considered q u a n t i t a t i v e l y inade-quate. However, f o r the discussion i t Is claimed that a female tendency pr e v a i l s i n the controls (percentages of 5 6 . 6 , 75.0, 58.6) and under treatment (66 .6$ , 100$) (Table IV). b) Selective M o r t a l i t y -Problems of t h i s kind always e l i c i t c r i t i c i s m from the viewpoint of sel e c t i v e mortality. I f the 1949-50 chum data be considered (Table I H ) , i t is evident that r e l a t i v e l y few f i s h survived the u l t r a -v i o l e t treatments. Despite the mortality the sex d i s t r i b u t i o n was c l o s e l y equal. S i m i l a r l y the chum f r y from the control group showed an equal sex r a t i o . In 1950-51 the sex data obtained from pink alevins 47 (controls) showed d i s p a r i t y i n r a t i o (Table 17). That a s l i g h t female tendency was prevalent perhaps j u s t i f i e s the idea of select i v e mortality. However, s i m i l a r researches have shown select i v e mortality to be inoperative (Mrsic 192?; Eberhardt 1943) and indeed never demonstrated i n any early vertebrate development (Brachet 1950). Therefore t h i s factor i s not considered as having biased the r e s u l t s . Where the data are 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 sex r a t i o s were thought affected by e x t r i n s i c conditions, SECTION 5. THE FIELD SEX ANALYSES a) The Sex Ratio of Salmon Migrants The sex r a t i o s of the salmon speoies migrating from the Port John watershed along with raw data are shown Table VI. The sex r a t i o s obtained from salmon f r y that had been held i n c a p t i v i t y i n 1949 and 1950 are shown Table VII. No one sample was s u f f i c i e n t to describe the sex r a t i o of migrating young salmon. Indicative of sampling tech-nique i s that exemplified by pink salmon ( F i g . 26). I t i s shown that the 1949 and 1950 r a t i o s were a l i k e (47.7% and 47.9% respectively) despite fluctuations through the period of sampling. b) Sex Ratios of Adult Pink Salmon The sex r a t i o s of pink salmon, as enumerated from catches of the seiners (68.8% females) and g i l l netters (8.1% females) i n the v i c i n i t y of Port John, are shown i n Table VIII. Table VI, The sex ratios of salmon migrants at Port John, B. C. in 1949 and 1950, Run sampled Sockeye smolts *Kam» loops trout Coho smolts Pink fry Chum fry Coho fry Sockeye fry 1949 Number Sampling level (I f , ) % of sexes F 2 M X 2 No, measured for length Average F length-mm. M 277 1.40 48.4 51.6 0.29 277 84.0 86.0 1950 1950 1950 259 1.69 54.5 45.5 2.06 112 58.0 42.0 2.89 56 1.89 42.9 57.1 1.14 56 111.0 1949 5^ 9 .57 47.7 52.3 0.78 49 31.9 32.2 1950 1949 232 .43 47.9 52.1 0.43 122 23.7 33.9 252 .45 20.4 79.6 122.8 72 26.6 26.4 1950 stream 1949 212 .48 42.9 56.1 2.19 169 56.7 26.5 100 .45 24.0 66.0 10.24 29 S33.2 lake 1950 1949 1950 23 1.15 8.7 91.2 15.70 22 25.0 25,2 15 44 .06 .12 44 26.1 •From Summer land Hatchery. Table V I I . The sex a n a l y s e s o f salmon r e t a i n e d i n the sea and la k e a t P o r t John, B. C. i n 1949 and 1950. P l a c e P o r t John Bay (Sea) P o r t John Lake F r y Sockeye Chum C o n t r o l Chum Coho Sockeye E p i l i m n i o n Thermocline Hypolimnion Year 1949 1950 1950 1950 1949 1950 1949 1950 1949 1950 Number 41 42 174 98 200 51 - 105 - 200 % o f sexes F) 39.1 52.4 52.3 44.9 2 M) 60.9 47.6 47.7 55.1 4 1.89 .09 .27 1.02 P e r i s h e d P e r i s h e d P e r i s h e d X ^ s . 1 0 r) 2.71 2.71 2.71 2.71 Aver, l e n g t h (mm,) 41.3 35.3 28.0 24.6 M) 42.4 55.4 28.2 50 4 5 6 7 8 NUMBER OF SAMPLES II TOTAL Figure 26. The variation in the sex ratio of pink salmon in their period of migration at Port John, B.C., in the springs of 1949 and 1950. Table VIII. The sex ratios of pink salmon sampled from the commercial fishery at Port John, B. C. in 1950. Pink salmon fishery Gillnetters Seiners ^Escapement Number sampled F Percent of sexes M X 2 xZQOPi.lO 211 8 . 1 91.9 148.4 2.71 1740 68 .8 3 2 . 1 244.3 2.71 1857 55 .3 44.7 20.9 2.71 Counted and sexed through Port John fence. Section 6, REMARKS ON THE FIELD ANALYSES Sex differentiation as i t appeared by the "a i r -bladder technique" has been considered (Section 2 (b), page 3 6 ) . In the sex ratio data (Table VI, p. 48) the sockeye smolts were so equally distributed as to sex that no inference 51 c o u l d be made on p r e c o c i o u s male r e t u r n s observed i n these f i s h . In the f r y , a 79.6?, male frequency i n the 1949 chum salmon was the r e s u l t of incomplete sex d i f f e r e n t i a t i o n as shown by r e a r -i n g s conducted i n 1950 (Table V I I ) . The coho f r y were a t the same stage,while no sex d i f f e r e n c e s c o u l d be shown f o r the sock-eye. However, the chum migrants can be sexed when the diameter of the gonad»s stroma i s c o n s i d e r e d . The female*s s t r o m a t i c t i s s u e i s 2 to 5 times wider than that o f the male, Lake r e a r i n g s o f sockeye f r y p r o v i d e d no a d d i t i o n a l i n f o r m a t i o n c o v e r i n g the time o f sex d i f f e r e n t i a t i o n i n t h e i r normal environment (Table V I I ) . Death was considered due to s t a r v a t i o n i n view o f t h e i r l a r g e heads and t h r e a d - l i k e body. However, oth e r sockeye f r y r e a r e d i n the sea d i f f e r e n t i a t e d q u i c k l y ( i n about three weeks) w i t h aeses appearing i n near: equal, number (Table V I I ) . I t f o l l o w s t h a t o f the f o u r s p e c i e s m i g r a t i n g at the f r y stage o n l y the pink and chum salmon p r o v i d e sex r a t i o by the d i s s e c t i o n method. The a d u l t p i n k salmon sampled from the g i l l n e t and seine f i s h e r i e s gave female percentages of 8.1 and 68,8 r e s p e c t i v e l y (Table V I I I ) . A n a l y s i s o f t h i s sex d i s p r o p o r t i o n i s d i f f i c u l t , but some f a c t o r s may be d i f f e r e n t i a l sea m o r t a l i t y , d i f f e r e n t i a l a g i l i t y , s e g r e g a t i o n o f sexes i n the sea, and g i l l net s e l e c t i v i t y . For the g i l l net caught f i s h the use o f l a r g e mesh f a l l n e t s might s e l e c t f i s h having pronounced secondary s e x u a l c h a r a c t e r i s t i c s , v i z . , the males. However s e l e c t i v e the f i s h e r y may orrmay not have 52 been, the escapement to P o r t John showed a female tendency (55.3?o, Table V I I I ) . I f the f i s h e r i e s comprised p a r t o f the P o r t John d e s t i n e d fish,, t h i s r a t i o might have r e s u l t e d from the commercial n e t s and/or d i f f e r e n t i a l sea m o r t a l i t y , p e r - ' haps i n d i c a t i v e o f t h i s statement are 1) the h i g h frequency o f females i n seine-oaught f i s h , and 2) the dominant male c a t c h among the g i l l n e t t e r s f o r three weeks f o l l o w i n g c l o s u r e o f the se i n e f i s h e r y . The sex h i s t o r y o f t h i s p i n k c y c l e (by sampling) was 4f.ijf% females a t the time o f seaward m i g r a t i o n , 68.8 and 8.1 p e r c e n t s through seine and g i l l net f i s h e r i e s , and 55.3% on the spawning grounds. These data w i l l be used subsequently. 53 •V. DISCUSSION In the pre c e d i n g stages an account has been g i v e n o f the sex mechanism i n P a c i f i c salmon along w i t h the sex r a t i o s observed a f t e r temperature treatment and u l t r a v i o l e t r a d i a t i o n o f developing embryos, sex r a t i o data compiled from f i e l d s t u d i e s a t P o r t John, B. C. were a l s o p r e s e n t e d . Since salmon do not l e n d themselves to breed i n g experiments, a c y t o l o g i c a l approach to the problem o f sex deter m i n a t i o n appeared d e s i r a b l e , i f o n l y to c o n f i r m the p r e -v i o u s l y r e c o r d e d chromosome count i n chum salmon (74 chromo-somes, Makino 1937)• From embryonal c e l l s a minimum count o f 38 chromosomes were o b t a i n e d . The chromosomes c o u l d be m o r p h o l o g i c a l l y d i s t i n g u i s h e d as g r a n u l a r , sigmoid, and j -shaped. In the gonad m a t e r i a l a p e r s i s t e n t clumping o f chromo-somes made enumeration i m p o s s i b l e , but a p p a r e n t l y Makino used s i m i l a r m a t e r i a l w i t h s u c c e s s . High chromosome counts observed i n f i s h have made c y t o l o g i c a l demonstration o f the sex chromosomes d i f f i c u l t . In Amphibia, d e s p i t e more f a c i l i t a t i n g m a t e r i a l and number o f chromosomes, there can be shown no mo r p h o l o g i c a l d i f f e r e n c e s t h a t might c h a r a c t e r i z e sex chromosomes. These c o n d i t i o n s i n the brook lamprey suggested t h a t sex chromosomes were absent, and the same s i t u a t i o n c o u l d be expected i n the t e l e o s t s (Okkelberg 1921). The c l a i m to t r a n s f o r m a t i o n of; s©x i :Ohromo^ somes i n t o autosomes, and autosomes i n t o sex chromosomes i n the f i s h L e b i s t e s (Winge 1932) has o h l y a g e n e t ! c a l b a s i s . 54 Cytological studies i n the Anabantidae f a i l e d to reveal sex chromosomes (Svttrdson and Wickbom 1942), as did research i n the Cyprinodontidae (Wickbom 1943). This has l e d Wickbom to con-clude "there i s no c y t o l o g i c a l evidence for the presence of heterochromosomes i n teleosts" but because of sex-linked inheritance they may none-the-less be present. Studies i n the Gasterosteidae and the Urodeles .showed the-same r e s u l t s , v i z . , "the sex chromo.somes are in a very lo.TK..si;ate o f d i f f e r e n t i a t i o n being ent i r e l y - i n d i s t i n g u i s h a b l e from the other, autosomes" (Makino 1947). -To the contrary, there has .been claimed " demonstration of a chromosome fragment i n Coregonus laveretus that "must be regarded as a more or less heterochromatic sex-chromosome" (Svftrdson 1945), However, the^presence of sex chromosomes i n f i s h i s postulated from- gene.ti.cal experiments (Winge 1922a-* 1922b, 1932; Aida 1936; Goldschmidt 1937; et al) although t h e i r r o l e i n sex determination remains obscure, In the present, study there was no evidence to suggest the presence or absence of sex chromosomes. I t may be noted late-prophase figures showed no lagging chromosomes that serve to distinguish sex chromosomes i n some animals. Whether or not sex chromosomes are present i n f i s h i s perhaps of minor importance in- considering the sex-determining mechanism. The heterogametle male o f Lebistes has been bred with the constitution XX, YY and females with XY (Winge 1922a, 1922b, 1932), Similar r e s u l t s have been obtained i n the case of Aplocheilus excepting the claim to produce males of the YY constitution (Aida 1936), Aida would 55 suggest that s e x u a l e x c i t o r s e x i s t on the X and Y chromosomes which s t i m u l a t e autosomal genes to a c t as primary s e x u a l determiners. I t i s a l s o b e l i e v e d t h a t sex i s determined s o l e -l y by autosomal genes which express maleness or femaleness ac c o r d i n g as they are m o d i f i e d by the e x t e r n a l environment, i . e . sex i s determined-.phenotypically (Kosswig 1931-1941). In d i s c u s s i n g germ c e l l s and t h e i r d i f f e r e n t i a t i o n I t i s worthwhile to know t h e i r o r i g i n because of t h e i r p a r t i n h e r e d i t y . In t h i s r e s p e c t i t . i s shown t h a t they can be t r a c e d i n c e r t a i n i n v e r t e b r a t e s such as A s c a r i s from f i r s t cleavage (Wilson-19_24). In v e r t e b r a t e s t h e i r o r i g i n and f a t e l a c k s demonstration, although i t i s - g e n e r a l l y conceded that germ c e l l s do not a r i s e - f r o m the g e r m i n a l e p i t h e l i u m . F o r example, i n the Cyclostomes they are s a i d to' be found i n the gut p r i o r to and concurrent w i t h t h e i r presence i n the gonad (Okkelberg 1921). In the c h i c k , germ c e i l s have been thought l o c a t e d i n the blood stream (Goldsmith 1928), Thus the former evidence suggests an en to dermal o r i g i n , , the l a t t e r mesodermal. Urodelen s t u d i e s i n d i c a t e t h a t the germ c e l l s : m a y be d i f f e r -e n t i a t e d from mesodermal elements by i n d u c t i v e a c t i o n o f the entoderm (N-ieuwkoop 1944). In chum salmon-there i s no i n f o r -mation on e a r l y germ c e l l h i s t o r y . With-regard to germ' c e l l l i n e a g e there can be con-s i d e r e d , the demonstration t h a t j u v e n i l e hermaphroditism o c c u r s i n th.e..lamprey,- and the c o n c e p t i o n -that the same condi-t i o n p r e v a i l s i n the t e l e o s t s (Okkelberg 1921), On an e v o l u t i o n a r y b a s i s such an assumption i s g i v e n support by the 56 occurrence of transit o r y i n t e r s e x u a l i t y i n the anurans (Witschi 1929a) and Urodelans (Hanoako 1941; Uchida 1 9 3 7 ) . Evidence accumulating from genetical and histogenic studies i n the Anabantidae, Cyprinidae, Cyprinodontidae, and Salmoni-dae have perhaps suggested the statement "In a l l l i k e l i h o o d , a l l the teleosts go through a protogynous hermaphroditic stage" [Svfirdson 1945). An independent source finds true hermaphro-ditism occurring among the Sparidae and Serranidae and that immature members of the Anguillidae show coexistence of male and female germ c e l l s (D fAncona 1947, 1949a, 1949b). D^Ancona acknowledges that l i k e the e e l the same condition "seems to be r e a l i z e d also i n Salmonids, Cyprinids, Gyprinodonts and other groups". In the Salmonldae the method of sex d i f f e r e n t i a t i o n appears controversial. For Salmo salar i t was stated that i n the course of d i f f e r e n t i a t i o n a l l the young f i s h passed through a protogynous hermaphrodite phase ( F e l i x 1906). Studies of spermatogenesis i n A t l a n t i c salmon (Jones 1940) would suggest no condition of juvenile-hermaphroditism, although the author does not mention t h i s . In the rainbow trout protogynous development was demonstrated (Mrsie 1923, 1931) as were ova-testes i n f r y developed from l a t e f e r t i l i -zation (Huxley 1 9 2 3 ) . However, there i s recognized a unisex-ua l d i f f e r e n t i a t i o n i n this species which i s attributed to the d i f f e r e n t temperatures at which the trout (Mrsio»s and Padoa»s) had been reared (Padoa 1939). The present studies have shown the chum salmon to 57 c o n t a i n an i n d i f f e r e n t gonad three weeks a f t e r h a t c h i n g . Large germ c e l l s (17.6 microns) were l o c a t e d i n n e s t s separated by-i n t e r s t i t i a l c e l l s (6,6 m i c r o n s ) . The n e s t s had fewer germ c e l l s and showed l e s s f requent m i t o t i c f i g u r e s as the gonad was t r a c e d caudad. These c h a r a c t e r i s t i c s were e s s e n t i a l l y maintained to the n i n t h week. At t h i s time the ovary had en l a r g e d and contained young oocytes, but the ^ t e s t i s " remained i n d i f f e r e n t . The presence o f b l o o d c e l l s was observed i n both s t r u c t u r e s . At the t e n t h week the ovary contained w e l l -developed oocytes and an e n d o v a r i a l c a n a l had appeared. A t e s t i s proper was c o n s i d e r e d d i f f e r e n t i a t e d , i t s s t r u c t u r e b e i n g s m a l l , compact, and c o n t a i n i n g spermatogonia. In the f i v e succeeding weeks no f u r t h e r d i s t i n g u i s h i n g f e a t u r e s were developed i n e i t h e r sex e x c e p t i n g the appearance of a prominent vena commites i n the a n t e r i o r p o r t i o n o f the ovary. I t i s e v i d e n t then t h a t sex d i f f e r e n t i a t i o n i n the chum salmon proceeded i n the male o r female d i r e c t i o n without intermediate hermaphroditism. On t h i s b a s i s much doubt i s oast on Mrsic»s observed protogynous stage i n rainbow t r o u t , while there i s much to f a v o r PadoaVs c l a i m o f u n i s e x u a l d i f f e r e n t i a -t i o n i n t h a t s p e c i e s . However, Padpa found t h a t the t e s t i s was c h a r a c t e r i z e d by the vena commites ( s i n c e i t i s a p r e l u d e to the e f f e r e n t system) while the p r e s e n t study showed these blood v e s s e l s i n the ovary. The chromosome and sex d i f f e r e n t i a t i o n s t u d i e s p r e -sented f o r chum salmon v i d e ut supra do not suggest the r o l e o f the environment as a sex-determinant, The extent to which the i n t e r n a l environment 58 modifies the sex mechanism has been investigated i n other animals. In the urodelans t e s t i c u l a r transplantation into females indicated that the development of ovaries could not be suppressed (Uchida 1937o)« The i n j e c t i o n of f o l l i c u l a r and t e s t i c u l a r hormones showed the germ c e l l s were not responsive or that t h e i r number was reduced (Hanoako 1941a, 1941b). S i m i l a r l y i n teleosts f o l l i c u l a r reared trout produced only s l i g h t feminization of the male genotype (Padoa 1 9 3 9 ) . F i n a l l y , the evolution of topographically separate sexual areas i n the gonad sustratum of f i s h (D»Aneona 1949b) and p a r t i c u l a r l y i n the anurans (Witschi 1929a) have led to the theory that the stroma acts as a bi p o t e n t i a l sex inductor (Witschi 1942; D»Ancpna 1947). Witschi suggests the inductors have a protein constitution rather than s t e r o l as i n sex hormones. The present study makes no contribution to this f i e l d other than to state such topographic l o c a l i z a t i o n of male and female areas does not exis t i n the chum salmon as has also been shown f o r the ee l (D^Ancona 1949). The extent to which the external environment plays a role i n determining the proportions of sex has been investigated i n a d i v e r s i t y o f animal species. Evidence presented i n f i s h and amphibia has indicated that adverse environmental o©nditions p r i o r to f e r t i l i z a t i o n a f f e c t the sex r a t i o . Where eggs are graded through stages o f "green" to "overripe", i t was found the l a t t e r condition favoured a high male frequency i n f i s h 59 (Mrsic 1923). Similar r e s u l t s were found i n the anura (Witschi 1924). These r e s u l t s were also obtained by Huxley (1923), who calculated the probable errors f o r h i s and Mrsic»s claim. The data i s summarized as follows (Brambell 1930): Author Treatment Total M Percent Probable error Huxley Control tt F e r t i l i z e d 4 -7 days l a t e 119 47.90 -3.09 Mrsic ' Control 332 54.52 -1.83 » F e r t i l i z e d 4 - 7 days late 90 47.8 -3.55 n tt 15 « tt 199 39.7 -2.34 ti « 21 n . tt 77 50.6 -3.84 57 66.7 -4.21 An attempt was made to demonstrate these findings f o r pink salmon but general adverse hatchery conditions terminated the experiment prematurely. It may be added that while the "overripe 1* eggs were apparently f e r t i l i z e d , i n the green ovaries only a few peripheral eggs seemed so. The r o l e of other e x t r i n s i c conditions ( l i g h t , temperature, a v a i l a b i l i t y of oxygen, good, etc.) i n modify-ing the sex r a t i o a f t e r f e r t i l i z a t i o n have been investigated. Roarings of Betta splendens under a combination of these conditions showed a high male frequency (Eberhardt 1943). In the present experiment the factor l i g h t was considered i n the 1949-50 chum and sockeye studies. U l t r a v i o l e t dosages varying i n strength and number were applied i n successive developmental periods. Under these conditions u l t r a v i o l e t r a d i a t i o n appeared to have no e f f e c t on the sex r a t i o s obtained on completion of sex d i f f e r e n t i a t i o n i n chum salmon. That there were near equal numbers of males and females appeared to confirm e a r l i e r studies which 6o indicated u l t r a v i o l e t l i g h t was only s u p e r f i c i a l l y detrimental i n this species ( B e l l and Hoar 194°). However, l i g h t has been considered as a factor i n the maturation of the gonads in birds (Rowan 1931), and of the ovaries of the four-spined stickleback (Merriman and Schedl 1941). The equal d i s t r i b u t i o n of the sexes i n chum salmon suggests that t h e - u l t r a v i o l e t r a d i a t i o n did not cause s u f f i c i e n t a g i t a t i o n i n the germ c e l l prlmordia at a c r i t i c a l period i n t h e i r ontogeny. Temperature seems to influence the sex-determining mechanism among d i f f e r e n t animal species, In the inverte-brates, high temperature rearings of the gypsy moth resulted i n dominant male frequencies (Goldschmidt 1938), as was also evident i n the marine copepod, Tigriopus (Takeda 1950). Similar r e s u l t s have been obtained i n the f i s h Aplochellus (Aida 1936) and suggested f o r Betta splendens (Eberhardt 1943), In the Amphibia high male frequencies r e s u l t i n g from temperature application have been shown i n the anurans (Witschi 1929b) and i n the urodelans (Uchida 1937b). In the present experiments an attempt was made to show the e f f e c t of high and low temperature applications on the developing salmon embryos. The 1949-50 r e s u l t s indicated that only the 18° G. treatment was e f f e c t i v e , producing a s i g n i f i c a n t female surplus o f 12.5$. I t could be argued that sel e c t i v e mortality biased the experiment. Support might be obtained from studies i n the f i s h Gambusia, where f i e l d c o l l e c t i o n s of adults suggested a d i f f e r e n t i a l mortal-61 i t y o c c u r r e d r e s u l t i n g i n a h i g h percentage o f females ( G e i s e r 1924). However, the m o r t a l i t y among the 18° G. embryos was not e x c e s s i v e i n r e s p e c t to the c o n t r o l or the embryos t r e a t e d w i t h u l t r a v i o l e t l i g h t , which showed an equal d i s t r i b u t i o n o f the sexes. E x p l a n a t i o n i s made d i f f i c u l t s i n c e the female frequency r e f u t e s the male dominancy found i n o t h e r i n v e s t i g a t i o n s o f t h i s n a t u r e . The 1950 pink c o n t r o l data, w h i l e not s t a t i s t i -c a l l y sound, a l s o showed a female tendency. T h i s might suggest that i n these salmon there e x i s t s a strong female r a c e . Such an h y p o t h e s i s stems from theooceurrences o f s e x u a l r a c e s i n the moth Lymantria (Goldschmidt 1937, 1938), the s u g g e s t i o n t h a t s i m i l a r geographic r a c e s might p o s s i b l y occur f o r Salmo i r i d e u s (Padoa 1939) and the Amphibia demonstration ( W i t s c h i 1930). To the c o n t r a r y i t i s s t a t e d t h a t a 1:1 sex d i s t r i b u t i o n o b t a i n s i n the Salmo s p e c i e s t r u t t a and f o n t i n a l i s (probably i s S a l v e l i n u s ) f o n t l n a l i s ) ( S v t t r d s o n 1945) and the observed 50:50 r a t i o i n pink salmon f r y migrants (Table V I , p. 48) . I t has been suggested t h a t germ c e l l s have a d i f f e r e n t i a l metabolism and t h a t substances i n f l u e n c i n g i t s r a t e are sex-determining (Okkelberg 1921). For c o n s i d e r a t i o n of t h i s aspect there were made a v a i l a b l e well-advanced chum salmon a l e v i n s that had been r e a r e d i n a) t h i o u r e a and t r a n s f e r r e d to t h y r o x i n e , b) i n t h y r o x i n e (Dales 1951)* Sex a n a l y s i s o f the a l e v i n s showed there cc c u r r e d p r e c o c i o u s development of the gonads to f a v o u r a ifsmale- frequency.'---62 This offered evidence i n contradiction to the diiodotyrosin e f f e c t i n stimulating male production i n Tigriopus (Takeda 1950) and the evidence a r i s i n g from temperature and l i g h t rearings i n the vertebrates already discussed. Since sex has been shown indeterminant i n chum salmon u n t i l the yolk sac i s absorbed (dissection method) i t would be suggested o v a r i a l d i f f e r e n t i a t i o n was speeded up as a r e s u l t of the thyroxine. However the ••control* f i s h examined from Dales* material also showed precocious maturation. I t may be added that from the writer»s rearings, developing chum eggs treated at 19° C. f o r 3 days had not d i f f e r e n t i a t e d 6 weeks following the above-mentioned examinations despite the same f e r t i l i z a -tion dates. Neither could any female tendency be shown i n the rearings o f 1949-50 u n t i l the yolk sac was absorbed, nor in the f i e l d data (1949, 1950). There remains to be evaluated the f i e l d data accu-mulated to the present time. Apart from the general consideration of fecundity (Raitt 1934) and the ever-present suggestion that controlled hybridization could increase egg production, there Is the problem of sex r a t i o s i n P a c i f i c salmon. The present migrant analysis Indicates that for sockeye smolts no runs have yet developed where there could be predicted a heavy returning male population of the precocious type. Where these "jack sockeye* are considered, i t i s perhaps more j u s t i f i a b l e to think that l i g h t and other factors speeded t h e i r maturation. Further, i t appeared that t h i s e f f e c t must have been experi-6 5 enced i n the ocean s i n c e the smolt analyses d i d not i n d i c a t e the e a r l y sperm a c t i v i t y suggested i n the male gonads o f the t r o u t examined. F i n a l l y , the sex r a t i o data may p r o v i d e a rough estimate i n computing n a t u r a l sea m o r t a l i t y i n p i n k salmon. I f there i s (a) an e v e n ly d i s t r i b u t e d sea m o r t a l i t y f o r pink salmon and (b) the commercial f i s h e r y sample was made up of the P o r t John salmon (p. 2 5 ) , then i n c o n s i d e r a -t i o n o f data presented (p. 5 2 ) i t may be s t a t e d t h a t a t P o r t John, B. G . 1 . the 1 9 4 9 f r y migrated w i t h a female percentage d e f i c i e n c y of 2 . 3 ; 2 . the 1 9 5 0 a d u l t r e t u r n r e a l i z e d i n the s e i n e and g i l l net f i s h e r i e s a female percentage d e f i -c i e n c y o f 2 3 . 1 ; 3 . the 1 9 5 0 escapement c a r r i e d a female percentage s u r p l u s o f 5 . 3 . I t f o l l o w s t h a t on the b a s i s o f sex p r o p o r t i o n s the females r e a l i z e d a n a t u r a l sea m o r t a l i t y of 2 0 . 1 p e r c e n t . Since iihere was assumed no d i f f e r e n t i a l sea m o r t a l i t y , the n a t u r a l sea m o r t a l i t y of the 1 9 4 8 c y c l e of p i n k salmon i s then 4 0 . 2 % . 64 V I . SUMMARY The g e n e r a l problem o f sex d e t e r m i n a t i o n and d i f f e r e n -t i a t i o n has been oonsldered i n P a e i f i e salmon w i t h the f o l l o w -i n g r e s u l t s : 1. An ab s o l u t e chromosome count o f chum salmon c o u l d not be made-.. T h i r t y - e i g h t chromosomes ( g r a n u l a r , sigmoid, and j-shaped) were counted from smears o f embryonal d i s c s . S e c t i o n e d m a t e r i a l o f d i f f e r e n t i a t i n g gonads gave no f u r t h e r a i d due to e x c e s s i v e clumping o f the chromosomes. The presence o f sex chromosomes cou l d not be e s t a b l i s h e d . 2. Sex d i f f e r e n t i a t i o n was found to be g o n o c h o r i s t i c i n chum salmon. The f i r s t m a t e r i a l s e c t i o n e d ( a l e v i n s three weeks old) showed the gonads to c o n t a i n i s o -l a t e d n e s t s o f germ c e l l s . The number o f c e l l s per ne s t decreased as the g l a n d was t r a c e d caudad. The sexes c o u l d not be determined. L a t e r ( f i v e weeks) frequent mitoses were observed i n the germ c e l l s which now formed a cord through the g l a n d . At nine weeks young oocytes appeared and i n the succeeding week an e n d o v a r i a l c a n a l formed i n the ovary. At t h i s time the t e s t i s was f i r s t r e c o g n i z e d . I t was s m a l l , compact, and had c e l l s i d e n t i f i e d as sperma-t o g o n i a . The f i n a l examination ( f i f t e e n weeks) showed no new s t r u c t u r e s i n the ovary (a vena commites was presen t at t h i r t e e n weeks) or i n the t e s t i s . The sex o f p i n k and chum salmon ( d i s s e c t i o n method) could he determined a t time of f r y m i g r a t i o n , hut sex d i f f e r e n c e s were not e v i d e n t i n coho and sockeye f r y . U l t r a v i o l e t r a d i a t i o n o f developing" chum and sockeye embryos suggested t h i s was not a f a c t o r t h a t would a f f e c t the u l t i m a t e sex r a t i o . Ghum, sockeye, and pink salmon embryos s u b j e c t e d to temperature treatment d i d not p r o v i d e sex r a t i o data t h a t would demonstrate temperature as a s e x - d e t e r -mining f a c t o r . In chum salmon, one experiment (18° C.) gave a s i g n i f i c a n t l y h i g h female frequency. Sex r a t i o data was compiled a t P o r t John, B. C. An equal d i s t r i b u t i o n o f the sexes was o b t a i n e d from pink and chum f r y , and sockeye smolts. On the b a s i s o f these data, i t was suggested t h a t p r e c o c i o u s male sockeye are developed i n the sea. The sex r a t i o s at stages i n the l i f e ' h i s t o r y o f the p i n k salmon were presented to show t h a t there had o c c u r r e d a n a t u r a l sea m o r t a l i t y o f 40.2 p e r c e n t . 66 V I I . AGKNGILEDGMENTS I wish to thank Dr. W. A. Clemens, the Head o f the Department o f Zoology, and Dr. J . L. Hart, the D i r e c t o r o f the P a c i f i c B i o l o g i c a l S t a t i o n , f o r p r o v i d i n g the o p p o r t u n i t y to c a r r y out t h i s work. E s p e c i a l l y am I i n d e b t -ed to Dr. Wm. S. Hoar f o r h i s p a r t i n s u p e r v i s i n g the problem. I wish to thank Dr. A. H. Hutchinson, the Head o f the Botany and B i o l o g y Department, f o r h i s i n s t r u c t i o n i n c e l l u l a r anatomy, s t a i n i n g techniques, and photomicrog-raphy, and Dr. P. Ford f o r v a l u a b l e comments and r e f e r e n c e s to r e c e n t e m b r y o l o g i c a l l i t e r a t u r e . I a l s o wish to thank my w i f e , Irma, f o r the French and German t r a n s l a t i o n s , the i n k work on the graphs, and f o r t y p i n g t h i s t h e s i s . I am g l a d to mention the h e l p g i v e n by my u n i v e r s i t y a s s o c i a t e s , Messrs. R. Hourston, J . Stokes, S. Dales, and M i s s A. R e d l i c h ; and by my f i e l d a s s o c i a t e s , Messrs. J . G. Hunter, A. Andrekson, K. J,. Ross, and P. 0»Shaughnessy. For t h e i r p a r t i n p r o c u r i n g chum, sockeye, and p i n k salmon eggs, I am g r a t e f u l to Mr. T. Stepp o f the I n t e r n a t i o n a l Salmon Commission, and Messrs. W, C a u l f i e l d and H. Meats o f the P a c i f i c B i o l o g i c a l S t a t i o n . F i n a l l y , I a p p r e c i a t e the h e l p o f Messrs. C M o r l e y and J . S. T. Gibson o f the P a c i f i c B i o l o g i c a l S t a t i o n f o r photographing F i g u r e s 1-2 and 18-23. 67 VIII. ADDENDUM Two chum salmon alevins were examined for gonad primordia from material taken through the week of hatch-ing (March 22) • The gonads were present and showed essent-i a l l y the same features as described for the three weeks old material (page 27) with the exception that there were fewer germ c e l l s . it LITERATURE CITED 1. Aida, T. Sex reversal i n Aplocheilus l a t i p e s and a new explanation of sex d i f f e r e n t i a t i o n . Genetics 21: 13o-153. 1936. 2. B e l l , G. M. and W. S. Hoar. Some ef f e c t s of u l t r a v i o l e t radiation oh sockeye salmon eggs and alevins. Can. J. Research, D, 28: 35-43. 1950. 3 . Brachet, J . Chemical embryology. 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Thesis/Dissertation
eng
Zoology
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Graduate
Determination and differentiation of sex in Pacific salmon
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