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Osmotic and ionic regulation in embryos, alevins and fry of the five species of Pacific salmon Weisbart, Melvin 1967

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The University of British Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY • of MELVIN WEISBART B.Sc, University of Toronto, 1961 M.A., University of Toronto, 1963 THURSDAY, APRIL 27, 1967 AT 3;30 P.M. IN ROOM 3332, BIOLOGICAL SCIENCES BUILDING External Examiner: F.G.T. Holliday Department of Biology University of St i r l i n g , Scotland Research Supervisor: W. S. Hoar COMMITTEE IN CHARGE Chairman: G. H. N. Towers P. A. Dehnel J. E. Phillips C. V. Finnegan W. S. Hoar D. McPhail E. B. Tregunna OSMOTIC AND IONIC REGULATION IN EMBRYOS, ALEVINS AND FRY OF THE FIVE SPECIES OF PACIFIC SALMON ABSTRACT The purpose of t h i s study was to examine the physiological basis of., the differences i n the early l i f e h i s t o r i e s of pink Oncorhynchus gorbuscha, chum 0; keta, coho 0. kisutch, chinook '0. tshawytscha and sockeye salmon 0. nerka. To t h i s end the. follow-ing working hypotheses were tested: ( i ) the embryo, alevin and f r y of pink and chum salmon are euryhaline, whereas the same l i f e stages of coho, chinook and sockeye are stenohaline and ( i i ) the euryhalinity of pink and chum i s due not to high tissue tolerance but to their a b i l i t y to regulate the osmotic and io n i c concentrations i n thei r blood, whereas the stenohalin-i t y of coho, chinook and sockeye stems from the i r i n -a b i l i t y to osmoregulate and ion regulate. The LD5Q values and the osmoregulatory data i n -dicated that pink and chum embryos are not euryhaline as hypothesized but l i k e coho, chinook and sockeye embryos are stenohaline. However, pink and chum embryos showed s i g n i f i c a n t l y greater s a l i n i t y r e s i s -tance and osmoregulatory a b i l i t y than embryos of the other species.. Contrary to the working hypothesis, a l l five species of alevins were found to be stenohaline; again, pink and chum showed greater s a l i n i t y resistance and osmo-regulatory a b i l i t i e s than coho and sockeye alevins. Although chinook alevins survived considerably longer in 31«8 o/oo sea water than coho and sockeye alevins their ability to osmoregulate and ion regulate was not significantly different from that of coho and sockeye alevins,. It was concluded, therefore, that the greater salinity resistance of chinook alevins was due to high tissue tolerance. The data obtained from fry supported the working hypotheses* As in the case of alevins, chinook fry, due to higher tissue tolerance, manifested greater salinity resistance than coho and sockeye. When the data for a l l species of embryos and fry were grouped and compared to the alevins taken as a group, the LD50 values of embryos and fry were found to be significantly greater than those of the alevins^ The blood osmotic concentrations of embryos and fry exposed for the same duration in 31v8 0/00 sea water were significantly lower than the blood osmotic.con-centrations for alevins. It was concluded, therefore,., that embryos and fry have greater salinity resistance than alevins because of the greater osmoregulatory ability of embryos and fry. On the basis of these results as well as informa-tion obtained from the literature, speculations were made on the phylogenetic relations within the genus Oncorhynchus. GRADUATE STUDIES Field of Study: Zoology Biology of Fishes Marine Field Course Comparative Invertebrate Embryology Introduction to Synoptic Oceanography G. L. Pickard Introduction to Chemical Oceanography , E. V. G r i l l . C. C. Lindsey P. A. Dehnel C. V. Finnegan PUBLICATION . Weisbart, M. and Fenwick, J.C.1966. Effect of pinealectomy on osmotic and ionic regulation in the goldfish. Carassius auratus Amer. Z00I.-6: 562 O S M O T I C AND I O N I C R E G U L A T I O N I N E M B R Y O S , A L E V I N S AND F R Y OF T H E F I V E S P E C I E S OF P A C I F I C S A L M O N by MELVIN WEISBART B.Sc, University of Toronto, 1961 i M.A., University of Toronto, 1963 A THESIS SUBMITTED IN PARTIAL FULFILMENT.OF THE REQUIREMENTS FOR.THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l i 1967 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y aval]able f o r reference and study, 1 f u r t h e r agree that permission-for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of Zoology The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date April 28, 1967 ABSTRACT The major purpose of this study was to examine the physiological basis of the differences in the early l i f e histories of pink Oncorhynchus gorbuscha, chum 0_. keta, coho 0_. kisutch, chinook 0_. tshawytscha and sockeye 0. nerka. To this end, the following working hypotheses were tested: (i) the embryo, alevin and fry of pink and chum salmon are euryhaline, whereas the same.life stages of coho, chinook and sockeye are stenohaline and ( i i ) the euryhalinity of pink and chum is due not to high tissue tolerance but to their a b i l i t y to regulate the osmotic and ionic concentrations in their blood, whereas the stenohalinity of coho, chinook and sockeye stems from their i n a b i l i t y to osmoregulate and ion regulate. The results did not completely support these hypotheses. The LD^Q values and the osmoregulatory data obtained from embryos indicated that pink and chum are not euryhaline as hypothesized but like coho, chinook and sockeye embryos are stenohaline. However, pink and chum embryos showed significantly greater salinity resistance and osmoregulatory a b i l i t y than embryos of the other species. The data obtained from alevins also did not support the working hypothesis for alevins of a l l five species were found to be stenohaline. But, as in the case for embryos, pink and chum alevins showed greater salin i t y resistance and osmoregulatory a b i l i t i e s than coho and sockeye alevins. This a b i l i t y of pink and chum was correlated with better ionic regulation of sodium and chloride. Although chinook alevins survived considerably longer in 31.8 °/oo sea water than coho and sockeye alevins , their a b i l i t y to osmoregulate and ion regulate - i i -was not very different from that of coho and sockeye alevins. It was concluded, therefore, that the greater salin i t y resistance of chinook alevins was due to high tissue tolerance. Contrary to the results with embryos and alevins, the data obtained from fry supported the working hypotheses. The results showed that pink and chum were euryhaline and that this condition was due to their a b i l i t y to.osmoregulate and control the levels of sodium and chloride in the blood. Coho, chinook and sockeye fry were found to be stenohaline and were unable to regulate the osmotic and ionic concentrations in their blood. As in the case of alevins, chinook fry, due to higher tissue tolerance, manifested greater salinity resistance than coho and sockeye. When the data for a l l species of embryos and fry were grouped and compared to the alevins taken as a group, the LD^Q values of embryos and fry were found to be significantly greater than those of the alevins . The blood osmotic concentrations of embryos and fry exposed for the same duration in 31.8 °/oo sea water were significantly lower than the blood osmotic concentrations for alevins. It was included, therefore, that embryos and fry have greater salini t y resistance than alevins because of the greater osmoregulatory a b i l i t y of embryos and fry. On the basis of these results as well as information obtained from the literature, speculations were made on the phylogenetic relations within the genus Oncorhynchus. - i i i -TABLE OF CONTENTS PAGE LIST OF TABLES v LIST OF FIGURES v i i ACKNOWLEDGMENTS v i i i INTRODUCTION...... 1 MATERIALS AND METHODS 4 Sources and Maintenance of Fish.... 4 • Transfer Experiments 4-Collection of Blood 7 Analyses of Blood 8 Stat i s t i c a l Methods , 10 RESULTS 12 A. Embryos 12 LD C„ Values 12 bu Serum Osmotic Concentrations 15 B. Alevins 19 LD C„ Values 19 ou Serum Osmotic Concentrations 22 Serum Na+ Concentrations 26 Serum Cl Concentrations 29 Serum K+ Concentrations 31 C. Fry 36 LD C„ Values- 36 Plasma Osmotic Concentrations , 36 Plasma Na+ Concentrations 40 - iv -PAGE : Plasma Cl Concentrations.. 44 Plasma Mg + + Concentrations 47 D. Comparison of Embryo, Alevin and Fry.Data 51 LD 5 Q Values 51 Osmotic Concentrations 51 E. Relationship Between Osmotic and Ionic Concentrations 58 DISCUSSION 61 A. Examination of Working Hypotheses 61 B. Mechanisms of Osmotic and Ionic Regulation in Embryos 62 C. Mechanisms of Osmotic and Ionic-Regulation in Alevins 67 D. Mechanisms of Osmotic and Ionic Regulation in Fry 69 E. Comparison of Mechanisms of Osmotic and Ionic ;Regulation in.Embryos, Alevins and Fry 72 F. Phylogenetic Interrelations in Oncorhynchus 76 LITERATURE CITED 82 APPENDIX 86 LIST OF TABLES TABLE PAGE I General information on the f i s h 5 II Dilutions used in analysing Na+, K+ and Mg + + by flame photometry 9 III LD50 (hr) of embryos transferred directly from fresh water to 31.8°/00 sea water 13 IV Analysis of variance of Embryo LD50 values 14 V Analysis of variance of the osmotic concentrations of the five species of embryos :exposed to 0, 4, 8, 12 and 24 hr of sea water 17 VI k^50 (hr) of alevins transferred from fresh water directly into 31.8 °/oo sea water 20 VII Analysis of variance of the LD5Q values of the five species of alevins transferred from fresh water directly into 31.8 °/oo sea water 21 VIII Analysis of variance of the serum osmotic concentration of the.five species of alevins exposed to 0, 4-, 8 and 12 hr of sea water 24 IX Analysis of variance of the serum Na+ concentrations of the five species of alevins exposed to 0, 4, 8 and 12 hr of sea water 28 X. Analysis of variance of the serum Cl concentrations of the five species of alevins exposed to 0,4,8 and 12 hr of sea water 32 XL Analysis of variance of the serum K+ concentrations of the five species of alevins exposed to 0, 4, 8 and 12 hr of sea water 35 XII- LD50 (hr) of 1964-65 fry transferred directly from . . fresh water to 31.8°/oo sea water.. 37 XIII Analysis of variance of plasma osmotic concentrations of the five species of fry exposed to 0, 4, 8 and 12 hr of sea water 39 +• XIV Analysis of variance of plasma Na concentrations.of the five species of fry exposed to 0, 4, 8 and 12 hr sea water .43 - v i -TABLE PAGE XV Analysis of variance.of plasma Cl concentrations of the five species of fry exposed to 0, 4, 8 and 12 hr of sea water 46 XVI Analysis of variance of plasma Mg + + concentrations of the .five species of fry exposed to 0, 4, 8 and 12 hr sea water 49 XVII L D 5 Q (hr) of embryos, alevins and fry of :Pacific salmon transferred directly from fresh water to 31.8 °/oo sea water 52 XVIII Analysis of variance of blood osmotic concentrations of the five species of Pacific salmon,embryos, alevins and fry exposed.to 0, 4, 8 and 12 hr sea water 56 XIX Blood osmotic concentrations (milliosmoles) of fi s h in fresh water and in sea water 71, XX Approximate LDgg and LDj_oo (days) values of chinook alevins in 50%, 75% and.100% sea water as calculated from the-data of Rutter (1902) 74. XXI Comparison of Pacific salmon. Presence of a character is indicated by,+ mark while i t s absence. by 0. Intensity of activity i s rated according to the number of-+ marks. 78 - v i i -LIST OF FIGURES. FIGURE PAGE 1 Average weekly temperatures in which 1964-65-66 embryos, alevins and fry were incubated 6 2 Serum osmotic concentrations of Pacific salmon embryos plotted against exposure times to 31.8°/oo sea water... .... .16 3 Serum osmotic concentrations of Pacific salmon.alevins plotted against exposure times to .31.8 °/oo sea water 23 4 Serum Na+ concentrations • of Pacific salmon alevins plotted against exposure times to 31.8 °/oo sea water. 27 5 Serum Cl, concentrations of Pacific salmon alevins plotted against exposure times,to 31.8°/oo sea water 30 6 Serum K+ concentrations of Pacific salmon alevins plotted against exposure times to 31.8°/oo sea water 33 7 Plasma osmotic concentrations.of Pacific salmon fry plotted against.exposure times to 31.8 °/oo sea water 38 8 Plasma Na+ concentrations, of Pacific salmon fry plotted against exposure times to 31.8°/oo sea water 41 9 Plasma Cl concentrations of Pacific salmon fry plotted against. exposure . times to 31.8°/oo sea water 45 10 Plasma Mg"1"1" concentrations of Pacific salmon fry plotted against exposure, times to 31.8°/oo sea water... 48 11 The blood osmotic concentration of Pacific salmon embryos., (E), alevins (A) and fry ( F ) exposed to 0, 4, 8 and.12 hr of 31.8 °/oo sea water. Each triangle is the result of joining the mean blood osmotic concentrations of embryos, alevins and fry exposed for the same duration to sea water 54 12 Osmotic, sodium and chloride concentrations of Pacific, salmon alevins plotted against exposure time to 31.8°/oo,sea water 59 13 Osmotic sodium and chloride concentrations of Pacific salmon fry plotted against exposure , time to 31.8°/oo sea water 60 14- Phylogenetic relationships of Oncorhynchus , 79 - v i i i -ACKNOWLEDGMENTS I would like to express my sincere appreciation to my supervisor Dr. William S. Hoar who suggested this problem and who .guided the investigation and the writing of the .manuscript. Dr. Paul.A. Dehnel, Dr. Cyr i l V. Finnegan and Dr. John E. Phillips read the .manuscript c r i t i c a l l y and made several helpful suggestions'during the investigation. Dr..James T. McFadden helped in the design of the experiments and the s t a t i s t i c a l methods. Their efforts are gratefully acknowledged. During the .investigations I had many f r u i t f u l discussions with Dr. Peter H. Johansen, Mr. Toong Jin Lam, Dr. Mizuho Ogawa, Mr. James C. Fenwick and Mr. Satendra Pandey.. These discussions shall be long remembered. Mr. Alan Handley, Mr. Kees W. Hoogendyk and Mr. Eugene McCullock greatly fac i l i t a t e d the day to day requirements of this investigation. Their help i s appreciated. The author acknowledges the contributions of fis h and fish-keeping f a c i l i t i e s made,by the following organizations: Department of .Fisheries, Olympia, Washington; Department of Fisheries, Vancouver, B.C.; Biological. Station of the Fisheries Research Board of,Canada, Nanaimo, B.C.; International North Pacific Salmon Commission, Cultus Lake, B.C.; British Columbia Fish and Game Branch, Abbotsford, B.C. Finally, I would like to express my appreciation to my wife, Marilyn, for her unfailing support and encouragement and her financial assistance. - 1 -INTRODUCTION Pacific salmon are.migratory fish which spend a year or more in,the ocean and then migrate in.the f a l l usually to.the freshwater system in which they hatched. The eggs are la i d in a nest and are covered with gravel after f e r t i l i z a t i o n . Hatching occurs after several months of development at which time the yolk-laden embryos are referred to as alevins; after yolk-sac absorption, they are referred to as fry. The six species of Pacific salmon a l l belong to the genus Oncorhynchus. Pink 0. gorbuscha, chum 0. keta, coho 0. kisutch, chinook 0..tshawytscha,and sockeye 0. nerka are found along the American and Asiatic coasts :of the North Pacific Ocean as well as in•the Arctic Ocean (Nikol'skii, 1961). The masou. 0. masou, however, occur exclusively along the Asiatic coast (Nikol'skii, 1961). The'five North American .species show several similarities during their l i f e histories. As young fry their appearance is such that the recognition of:each species can be made accurately only by•an expert. In the ocean several species have been caught in a single ..net haul and during the f a l l spawning migration several species have been observed spawning very close together (Neave, 1958). In addition, Brett (1952) has reported very similar preferred temperatures and ultimate,incipient lethal temperatures for the five species . Although there are these similarities in morphology, ecology and physiology, important differences.exist among the five North American species of Pacific salmon. Pink and chum salmon frequently spawn in close.proximity to the sea (Clemens and Wilby, 1961) or in the intertidal,zone,in which the eggs are subjected to sea water for part of the day (Skud, 1 9 5 4 ) . In addition, pink and chum embryos which, soon ;after hatching, return to .the sea'-as. alevins or fry, .lack the.,cryptic parr marks'associated with l i f e in. fresh-'water. These circumstances-contrast sharply-with the greater distance from: the-sea travelled;by :many chinook' and "sockeye populations during the , spawning-migration, the'longer-residency in fresh water of many coho, chinook and.sockeye fry and the cryptic , parr marks of these salmon as ;alevins and fry. These differences, along with :the ethological differences described by. Hoar ( 1 9 5 8 ) and those described by Neave ( 1 9 5 8 ) , support the hypothesis that Pacific- salmon have evolved towards-'a more marine l i f e . It was speculated that examination of.the physiological.basis of these.differences in the .early l i f e stages might .bring further evidence to bear on,the theory. To.this end, the-following working hypotheses were tested:. (i) ' the embryo, alevin and fry of pink and chum salmon.are euryhaline, whereas the same l i f e stages of coho, chinook and sockeye are stenohaline and ( i i ) the euryhalinity of pink and chum is due not to high ••• tissue tolerance but to their a b i l i t y to regulate the .:. : osmotic and-ionic-concentrations in their blood, whereas the stenohalinity of coho, chinook and sockeye stems from their i n a b i l i t y to osmoregulate and ion regulate. To test these hypotheses, groups of fi s h were transferred from.fresh water into 31.8 °/oo sea. water and the time ,of death of the median f i s h (LDJ-Q) determined. Since-,Gordon ( 1 9 5 9 ) and Parry ( 1 9 6 1 ) have shown that a large portion of the osmotic concentration in the blood can be.attributed to sodium and chloride,-the" concentrations of these two ions along with - 3 -the .melting points were measured.in the plasma or serum of f i s h maintained in fresh .water or. of f i s h transferred abruptly into 31.8°/oo sea water. As supplementary information, the potassium and magnesium concentrations.in serum or plasma, also were determined. Because of the short .period i n which material was available, large • numbers'of animals could not be used to test the working hypotheses. Therefore, the data; was analyzed by orthogonal.contrasts, in which pink and chum results were-compared, in most cases, as a group, to the coho, chinook and sockeye results .taken as a group. When the differences between the two groups were large, often individual species differences also were observed. - 4 -MATERIALS AND METHODS Sources and Maintenance of Fish The experiments have been carried out on hatchery reared embryos, alevins and fry of .the five species of Pacific salmon obtained from various sources during the 1964-65 and 1965-66 spawning seasons, as outlined in Table I. Eggs from each species were maintained in flowing fresh water in separate incubation tanks (with an overflow standpipe at one end) measuring 110 X 35 X 30 cm. Under these conditions embryos and alevins were kept in darkness but with the approach of complete yolk absorption, late.alevins were exposed to natural photoperiod and fed finely chopped frozen brine shrimp. Fry were fed frozen brine shrimp and wet food preparation consisting of 65% beef l i v e r , 21% canned salmon, 8% Pablum (Mead Johnson of Canada Ltd., Toronto), 6% Brewer's yeast and 5 g of table salt per 300 g of mixture. The average weekly water temperatures in which the 1964-65 and 1965-66 material were incubated are shown in Figure 1. Transfer Experiments The basic design of a l l experiments was to transfer eggs, alevins or fry directly from fresh water to sea water. A l l f i s h used in such experiments were moved from the incubation tanks to glass dishes 21 cm in,diameter and 2 l i t r e s in capacity. The dishes were immersed in a 183 X 61 cm trough maintained at 9° ± 1°C for embryos and alevins and 11° ± 1°C for fry. The dishes were, f i l l e d with fresh water and were supplied with air which had been washed previously in water. After about 24 hr, the fi s h were transferred to glass dishes f i l l e d with sea water which had a salinity of. 31.8°/oo and TABLE I General information on the fi s h . Species Chinook Pink Sockeye Chum Coho Sockeye Sockeye Chinook Pink Coho Chum Stage of Development When.Incubation was Begun at University a few hours after f e r t i l i z a t i o n eyed eyed. eyed eyed eyed. alevins. eyed: a few hours after f e r t i l i z a t i o n Source eyed eyed Samish State Salmon. Hatchery, Washington Bear River, B.C. Cultus Lake, B.C. Qualicum River, B.C. Hunts Creek, B.C. Nanika Hatchery,. B^C„ Lakelse Lake, B.C. Skykomish State Hatchery, Washington Sweltzer Creek, B.C. Qualicum River, B.C. Qualicum River, B.C. Date Fertilized October 13, 1964 October 22, 1964 September-.11, 1965 September 1, 1965 October 19, 1965 Date Hatched December 18, 1964 January 1, 1965 March 17, 1965 April 13, 1965 April 19, 1965 November 11, 1965 December 10, 1965 January 7, 1966 en I March 16, 1966 December 11, 1965 March 20, 1966 - 6 -FIGURE 1 Average weekly temperatures in which 1964-65-66 embryos, alevins and fry were incubated. o o o o p o o o o o o o o (H 6 <> ® <o ui « ci O aaviioiiNBo saiaoad - 7 -the following ionic concentrations: Na+ K+ Ca + + Mg + + C l " SO^"" mEq/1 433.0 10.1- 25.6 97.9 497.2 68.6 This sea water was made by the addition of salts to brackish water (27°/oo) from Burrard Inlet. A l l animals placed in the glass dishes f i l l e d with either sea water or fresh water were kept in total darkness except during sampling of blood. Under these conditions, f i s h were transferred directly from fresh water to 31.8 °/oo sea water and the time to death of the median fis h (LDC.) determined. ou In conjunction with these experiments, the osmolal, Na+, K+, Mg + + and Cl concentrations of the serum or plasma were measured from f i s h exposed to sea water for 0, 4, 8, 12 and sometimes 24, 45 and 48 hr. Collection of-Blood Each embryo was freed from i t s chorion by tearing open the chorion with a pair of fine watchmaker's forceps. The embryo was then quickly dried on cellucotton and i t s epidermis was removed near the caudal peduncle. There an incision was made with the forceps and the blood collected (approximately 0.001 ml) in a small glass capillary tube (#40090, Central Scientific Co., Vancouver) connected to rubber tubing through which mouth suction could be applied. The capillary tube containing blood was plugged at one end with paraffin and centrifuged at approximately 550 X g for 2 min to obtain serum. A group of six to eight alevins was removed from fresh water or sea water, quickly dried .on paper towelling and cellucotton, and their blood, collectively sampled (approximately 0.002 ml per animal) into a cold (0°C) glass tube (#9530 Pyrex, Corning Glass Co., N.Y.) by mouth suction. Bloo;d was taken near the caudal peduncle of the alevins and serum prepared in a - 8 -manner similar to that for embryos. While the tube was spinning in.the centrifuge, the collection of blood was repeated with another group of six to eight alevins. When sufficient blood had been obtained, the serum, which was separated from the cells in each tube, was pooled into one tube and. subsamples of serum obtained for measurement of each blood parameter. In the case of fry, blood was obtained by severing the caudal ''peduncle and permitting the bipod to run down.a heparinized capillary tube. The tubes were sealed with paraffin wax. and centrifuged for 2 min at 550 X g. Plasma and cells were separated and subsamples taken as in the case of serum from alevins. Analyses of Blood Melting points of serum and plasma were determined with a Melting Point Determination Apparatus designed by Ramsay and Brown (1955). Small samples of serum or plasma were drawn.into fine glass capillary tubes (#40090, Central Scientific Co. , Vancouver). These samples were enclosed by paraffin o i l to prevent evaporation and were kept on dry ice unt i l analysed. Melting points (C°) of blood were determined on the day of collection and the values converted to milliosmoles. Concentrations of the cations Na+ and K+ were analysed by emission flame photometry using a Unicam SP900A Spectrophotometer (Unicam Instruments Ltd., Cambridge, England). Table II indicates the dilutions used in determining these ions. The dilutions were made in polyethylene vials (10. ml) using Drummond Microcaps (Drummond • Scientific Co., Broomall, Pa.). The vials were stored at -20°C prior to analysis; Care was taken to analyse samples only when they had reached room temperatures. As a check on the quantitative accuracy of these cation determinations, dilutions,of Harleco - 9 -TABLE II Dilutions used in analysing Na+, K+ and Mg++by flame photometry. Volume of Ion Serum or Plasma Dissolved In Dilution Na + 0.001.ml 5 ml of H20 5000 X K+ 0.002 ml 3 ml of 100 ppm Na+ 1500 X Mg + + 0.005 ml 3 ml of 8 ppm Na+ 600 X - 10 -Serum Control (#64098 Harlman - Leddon Co., Philadelphia, Pa;) were also analysed by flame photometry. Chloride concentrations were measured potentiometrically on 1 microlitre (0.001 ml) serum or plasma samples diluted in 20 microlitres of 50% acetic acid following the method of Ramsay et a l . (1955). A Radiometer pH Meter 25SE (Radiometer, Copenhagen, Denmark), and.a Misco Vibrating Stirrer (#1385 Microchemical Specialities Co., Berkeley, California) with the single modification of a 60 ohm resistor between the transformer and solenoid proved very suitable. S t a t i s t i c a l Methods The material used in the experiments about to be described are available for only a short period each year. Because of this and the time required to perform microtechniques, only three replicates for each species were used in measuring the blood.parameters at each age and at each exposure time to sea water. Two-; and three-way analyses of variance were carried out on the data. Each analysis was considered to be of the Model I type .and therefore F tests were run with the error mean square as denominator (Steel and Torrie, 1960). Tukey's w, which was determined from each analysis of variance, permitted a l l comparisons of treatment means. Because of the relative insensitivity of Tukey's w, orthogonal contrasts were also performed. However, orthogonal contrasts do not permit a l l possible questions to be asked. Therefore data were lumped so as to test the working hypotheses. For example, the serum osmotic concentrations of pink and chum embryos at 0, 4, 8 and 12 hr sea water have been taken as the group, pink/chum and compared to the coho/chinook/sockeye group. If these two groups are s t a t i s t i c a l l y different from each other and members within each group are not s t a t i s t i c a l l y different from other.members - 11 -in the group, then pink and chum are different from coho, chinook and sockeye. Regression equations were calculated for each species at each age and 2 for each parameter measured. If the curvilinear equation Y = A + BX + CX f i t the data better than the straight-line equation Y= A + BX, the curvilinear was used in describing the regression. The regression equations are included in the appendix. Student's t-tests were considered to be two tailed. Unless stated otherwise, the term "significantly different" has a probability level less than or equal to 0.05. - 1 2 -RESULTS A. Embryos . LD r. Values . 50 Two weeks prior to hatching five embryos with intact chorions and five dechorionated embryos from each species ( 1 9 6 5 - 6 6 progeny) were transferred from fresh water to 3 1 . 8 ^ 0 0 sea water and theLD^^ determined for each species. The high salinity of the sea water proved to be a lethal factor in a l l cases. The analysis of variance,of the LD 5 Q values in Table III, indicates that there, is at least one,significant difference among the five species (Table IV). Tukey's w of 42 hr calculated from this analysis of variance indicates that pink and chum LDJJQ values are significantly higher than ,coho. and sockeye values. In addition, orthogonal contrasts,.also calculated from the analysis of variance in Table III, reveal that pink/chum* LD,_Q values are significantly higher than chinook values. Since the LD,_0 value for pink is not significantly different from chum, pink and chum have significantly higher LD^^ values than chinook. The presence of the chorion results i n increased salin i t y resistance on the part of a l l embryos, (P < 0 . 0 5 ) . This increased salinity resistance cannot be attributed to impermeability of the chorion for the peri v i t e l l i n e f l u i d of eggs immersed 4-.hr or more in 31.8 °/oo sea water is slightly hyperosmotic with the external environment (P < 0 . 0 5 ) . * Pink/chum refers to pink and chum data treated as a unit. For. example, i f pink/chum are significantly different from chinook, i t does not necessarily follow that chum are.significantly different"from chinook. - 13 -TABLE III, LD (hr) of embryos transferred directly from fresh water to 31.8°/oo 50 sea water. Pink Chum Coho Chinook Sockeye Normal 102 118 47 81 52 Dechorionated 95 94 33 65 50 - 14 -TABLE IV Analysis of variance of Embryo LD-gQ values. Source 1 d f 2 SS3 MS^  F 5 F A n r ( 4 , 0 . uo Species 4 6618.5 1654.6 15.3 5.19 Error 5 541.5 108.3 Since F = 15.3 is greater than.5.19, the null hypothesis of no difference.among the five species of embryos.must be rejected. 1 source of variation 2 degrees of freedom 3 sum of squares 4 mean.sum of squares 5 F test - 15 -Serum Osmotic Concentrations The greater salinity resistance of pink and chum embryos may be the result of a greater a b i l i t y of their tissues to function in a wide range of sa l i n i t i e s or be the result of greater osmoregulatory a b i l i t i e s . To evaluate these p o s s i b i l i t i e s , embryos of each species were transferred directly from fresh water to 31.8 °/oo sea water. Blood was sampled from embryos immersed for 0, 4, 8, 12, 24, 45 and 48 hr in sea water and the melting points of the serums determined. The results of this experiment are shown in Figure 2. Every point i s the mean of three determinations and each determination was obtained from one embryo. When salmon embryos are abruptly transferred from fresh water to 31.8 °/oo sea water, a rapid rise in the osmotic concentration of the serum occurs. In fact, the largest increase takes place in the pinks which are smaller than the chum, coho and chinook. At 12 hr, the osmotic concentration of the serum has levelled in the pink, chum and chinook but continues to increase in the coho and sockeye. At 24 hr, differences among the five species widen considerably; after 24 hr the differences, between pink and chum on the one hand and chinook on the other, increase s t i l l further. Tukey's w was calculated from the analysis of variance (Table V) of the five species exposed to 0, 4, 8, 12 and 24 hr of sea water. If the difference between any two treatment means exceeds the calculated value of Tukey's w, in this case 120 mOsm, then this difference is significant. On this basis, no significant differences occur among the five species in 0, 4, 8 and 12 hr exposure to sea water. However at 24 hr, the osmotic concentration of sockeye and coho embryo serum is significantly different from that of pink and chum embryos. Also, chinook, at this time, are significantly different from sockeye. - 16 -FIGURE 2 Serum osmotic concentrations of Pacific salmon embryos plotted against exposure times to 31.8°/oo sea water. - 17 -TABLE.V Analysis of variance of the osmotic concentrations of the five species of embryos exposed to 0, 4, 8, 12 and 24 hr of.sea water. Source Species Exposure Interaction Error df 4 4 16 50 SS 34,457 228,745 124,051 72,178 MS 8,614 57,186 7,753 1,444 5.965* 39.602* 5.369* * Highly significant (P < 0.005). Therefore, the null hypotheses of no difference among the five species of embryos, of no difference among the five exposures and of no significant interaction between species and exposures must be rejected. - 18 -Orthogonal contrasts, calculated from the same analysis of variance used in deriving Tukey's w, point to more subtle differences among the five species than are indicated by Tukey's w. The difference in the osmotic concentration of pink/chum serums between 0 and 24 hr exposure to sea water is significantly different from the difference of chinook serums between 0 and 24 hr sea water (Table I, Appendix). However, the difference between 0 and 24 hr for coho is not-significantly different from the difference for sockeye (Table II, Appendix). The coho, chinook and sockeye data f i t straight-line regressions.better than curvilinear, whereas the pink and chum data are more accurately described by curvilinear regressions (Table III, Appendix). In summary, the higher salinity resistance of pink and chum embryos has been correlated with their greater a b i l i t y to.osmoregulate and the lower salinity resistance of coho, chinook and sockeye embryos correlated with their inferior a b i l i t y to.osmoregulate. - 19 -B. Alevins L D 5 Q Values Nine alevins from each species ( 1 9 6 5 - 6 6 progeny) were placed in 31.8°/oo sea water and the LE^Q values determined. These alevins were approximately 2 weeks post hatching. Table VI indicates that pink alevins, in both years, had a higher resistance to the lethal factor than the chum, coho, chinook and sockeye alevins. When the experiment was repeated with alevins exposed to 10 °/oo sea water for 2 days followed by 20 °/oo for a subsequent 2 days, the LD^Q values of these alevins from a l l five species in 31.8 °/oo sea water were, the same as for alevins transferred directly from fresh water to 31.8°/oo sea water. Tukey's w of 43 hr, calculated from the analysis of variance shown in Table VII, indicates that pink LD^Q values are significantly higher than coho and sockeye. Orthogonal contrasts further reveal that pink LD^g values are higher than chum/chinook values and that chum are not significantly different from chinook. If, instead of comparing LD^Q values of pink and chum/chinook, the latter group had been compared to coho/sockeye, the LD^Q values of chum/chinook would have been found significantly greater than those of coho/sockeye. However, once the former, comparison has been made, the latter is not orthogonal. Therefore, i t is not possible to show that coho/sockeye LD,.. values are significantly lower than chum/chinook. Nevertheless, 50 inasmuch as i t was possible to obtain samples of blood for chum and chinook alevins exposed to 24 hr of sea water while i t was not possible to do so for coho and sockeye because of considerable mortality, the evidence points.to greater salinity resistance of chum and chinook over that of sockeye.and coho. Therefore, the LD values of chum and chinook are probably intermediate TABLE VI LD_. (hr) of alevins transferred from fresh water directly into bu 31.8 °/oo sea water. Pink. Chum 'Coho Chinook Sockeye 1964 - 65 *, 85 45 <24 22 <22 1965 - 66 ** 69 56 26 49 27 * cessation of opercular movements ** cessation of blood flow in v i t e l l i n e vein - 21 -TABLE VII Analysis of variance of the LD^0 values of the five species of alevins transferred from fresh water directly into 31.8°/oo sea water. Source df SS MS F F 0 n c(4,5) Species 4 3,867.0 966.75 8.5 5.19 Error 5 568.5 113.7 Since F = 8.5 is greater than 5.19, the null hypothesis of no difference among the five species must be rejected. - 22 -between that for pink alevins on the one hand and coho and sockeye on the other hand. The greater salin i t y resistance of pink alevins may be the result of a greater a b i l i t y of their'tissues..tovfunetion in a."wide,:range of s a l i n i t i e s , or, as in the case of pink and chum embryos, greater osmoregulatory a b i l i t y . Tp evaluate these p o s s i b i l i t i e s , alevins (1965-66) of each species were transferred directly from fresh water to 31.8 °/oo sea water. Blood was sampled from these alevins immersed in sea water 0, 4, 8, 12 and 24 hr and the melting points and concentrations of Na+, K+ and Cl of the pooled serum were determined. In Figures 3, 4, 5 and 6 which display the results of these measurements, every point i s the mean of three determinations and each determination was made from the pooled blood of 20 to 30 individuals. Serum Osmotic Concentrations The osmotic concentrations of serums determined in this experiment are shown in Figure 3. As in the case of the embryos, a large increase in serum osmotic concentrations occurs within the f i r s t 4 hr of exposure to sea water. The serum osmotic concentration of pink alevins decreases between 4 and 12 hr exposure to sea water, while the serum osmotic concentration of chum alevins decreases between 4 and 8 hr. In contrast, serum osmotic concentrations of sockeye and chinook alevins continue to rise rapidly as the exposure time to sea water increases. Although serum osmotic concentrations of coho alevins show no levelling as observed for pink and chum, the coho, nevertheless, do not show the same rapid rise as sockeye and chinook. On the basis of Tukey's w of 59 mOsm, calculated from the analysis of variance of the five species exposed to 0, 4, 8 and 12 hr of sea water (Table VIII), no significant differences occur among the alevins in fresh water. At FIGURE 3 Serum osmotic concentrations of Pacific salmon alevins plotted against exposure times to 31.8 °/oo sea water. ALEVINS 3 2 0 L— 1 — I I i O 4 8 12 24 Hours - 24 -TABLE VIII Analysis of variance of the serum osmotic concentrations of the five species of alevins exposed to 0, 4, 8 and 12 hr of sea water. . Source df SS MS F Species 4 20,004 5,001 13. .96 Exposure 3 124,261 41,420 . 115, .63 Interaction 12 35,760 2,980 8, .32 Error 40 14,328 358.2 F 0 . 0 0 5 ( ^ 0 ) 4 - 3 7  F0.005 ( 3' 4 0 ) 4 - 9 8  F 0 . 0 0 5 ( 1 2 ' 4 0 ) 2 ' 9 5 Therefore Species, Exposure and Interaction are highly significant. - 25 -4 hr exposure to sea water, pinks show a significantly higher serum osmotic concentration than coho alevins.- At 8 hr, no significant differences are found and at 12 hr, the serum osmotic concentrations of pinks are significantly lower from that in coho, chinook and sockeye, whereas chum, at this time, is significantly different from chinook and sockeye but not from pink and coho. By the Student's t-test, the serum osmotic concentration of pink and chum alevins at 24 hr is significantly different from chinook. Orthogonal contrasts show that the difference in response to sea water between pink/chum and coho as measured by serum osmotic concentrations at 0, 4, 8 and 12 hr is not significant. Similarly, there is no significant difference in response between pink and chum alevins, and no significant difference between chinook and sockeye alevins. However, the interactions between species and exposure times are more interesting. The difference in serum osmotic concentrations of pink/chiim in fresh water and at 12 hr in sea water is significantly different (P < 0.01) from the difference observed for coho alevins (Table IV, Appendix). Similarly, chinook alevins are significantly different (P < 0.01) from sockeye alevins (Table V, Appendix) whereas pinks are not significantly different from chums (Table VI, Appendix). Except.for the pinks, the data of the four other species individually f i t straight-line regressions better than curvilinear (Table VII, Appendix). The slope of the chum regression line i s considerably less than that for the coho, chinook and sockeye regressions. In summary, the resistance of chum/chinook alevins to high s a l i n i t i e s is intermediate between pink alevins on the one hand and coho/sockeye on the other. Nevertheless, pink and chum have very similar osmoregulatory a b i l i t i e s whereas chinook a b i l i t i e s d i f f e r significantly from pink and chum. - 26 -Although chinook survive longer in sea water than coho and sockeye alevins, the osmoregulatory abilities of chinook are not considerably greater than those of coho and sockeye. Therefore, the ability of chinook to survive longer in sea water than coho and sockeye is not due to osmoregulation but to high tissue tolerance. Serum Na+ Concentrations The measurements of serum Na+ concentrations are shown in Figure 4. A rapid rise in Na+ concentration occurs for a l l species within the first 4 hr of exposure to sea water. Both pink and chum salmon alevins show + decreased serum Na concentrations between 4 and 8 hr and increased concentrations between 8 and 12 hr. However, coho, chinook and sockeye, show increased levels of serum Na+ throughout the first 12 hr in sea water, although coho do not manifest the same rapid rise as sockeye and chinook. Beyond 12 hr, the serum Na+ concentrations in pink, chum and chinook are increasing at about the same rate. On the basis of Tukey's w of 31 mEq/1 Na+, calculated from the analysis of variance of the five-species exposed to 0, 4, 8 and 12 hr of sea water (Table IX), pink alevins in fresh water are significantly different from chum alevins in fresh water. At 4 hr in sea water, no significant differences are found among the five species. At 8 hr, pinks are significantly different from chinook and sockeye but are not significantly different from coho (0.10 <P >0.05). However, chums at 8 hr are not significantly different from any of the other four species. At 12 hr, pink and chum are significantly different from chinook and sockeye but not from coho. In addition, coho are significantly different from chinook and sockeye. Thus coho, on the basis of Tukey's w, arte not significantly different from pink and chum at any exposure - 27 -FIGURE 4 Serum Na concentrations of Pacific salmon alevins plotted against exposure times to 31.8°/oo sea water. Na* ALEVINS • | | O I -L — I — J ' O 4 8 12 24 Hours - 28 -TABLE IX Analysis of variance of the serum Na+ concentrations of the five species of alevins exposed to 0, 4, 8 and 12 hr of sea water. Source df SS MS . F Species 4 12,209 3,052 30.5 F o . 0 0 5 ( ^ 0 ) 4. .37 Exposure 3 28,054 9,351 93.5 F o . 0 0 5 ( 3 ^ 0 ) 4, .98 Interaction 12 2,480 202 2.02 F o > Q 5 ( 1 2 , 4 0 ) 2. .00 Error 40 4,012 100 Therefore Species and Exposure are highly significant and Interaction is just significant at 0.05 level. - 29 -time. But orthogonal contrasts, which are more sensitive than Tukey's w, show pink/chum significantly different from coho when the levels of serum Na+ are compared collectively at 0, 4, 8 and 12 hr. Since pinks are significantly different from chums, we may not conclude that either one is significantly different from coho but that both taken together are different (see footnote, page 12). The interactions between species and exposure times also have been analysed by orthogonal contrasts. The difference between pink/chum serum Na+ levels at 4 and 8 hr is significantly different from the difference in coho Na+ levels (Table VIII, Appendix). This.same comparison of serum Na+ levels at 4 and 8 hr is not significantly different between pink and chum (Table IX, Appendix) but the difference between 0 and 12 hr is significantly different (Table X, Appendix). The difference in serum Na+ levels of coho alevins between 0 and 12 hr exposure is not significantly different from the difference observed for pink/chum (Table XI, Appendix). For a l l five species , linear regressions, f i t the blood Na+ data better than do curvilinear regressions but the slopes of pink and chum regressions are less than those for coho, chinook and sockeye (Table XII, Appendix). In summary, the a b i l i t y of pink and chum alevins to regulate their serum Na+ concentrations,is greater than that of chinook and sockeye. The ab i l i t y of coho alevins in this regard is intermediate between these two groups. Serum Cl Concentrations The measurements of serum Cl concentrations are shown in Figure 5. Serum Cl levels rise rapidly for a l l species in the f i r s t 4 hr of exposure to sea water. Between 4 and 12 hr, the serum Cl levels in pink and chum - 30 -FIGURE 5 Serum Cl concentrations of Pacific salmon alevins plotted against exposure times to 31.8 °/oo sea water. - 31 -alevins do not change,appreciably while coho, chinook.and sockeye show increasing levels. Beyond 12 hr, Cl levels in pink and chinook increase at about the same rate, while chum show l i t t l e increase. On the basis of Tukey's w of 16 mEq/1 Cl , calculated from the analysis of variance of the five species exposed to 0, 4, 8 and 12 hr of sea water (Table X), several significant differences occur in fresh water: pink alevins are significantly different from chum, coho and chinook but not sockeye (0.1 < P > 0.05). At 4 hr of exposure to sea water, pink alevins are significantly different from coho, chinook and sockeye but not from chum. Chum alevins at 4 hr are also significantly different from chinook but not from sockeye.(0.1 < P > 0.05) or coho. At 8 hr, pink and.chum are both significantly different from coho, chinook and sockeye; coho are significantly different from chinook and sockeye; but pink alevins are not significantly different from chum. Similarly, at .12 hr, pink and chum are significantly different from the other three species, but not from one another. Coho and sockeye are significantly different from chinook but not from one another. At 24 hr, pink and chum are significantly different from chinook alevins. Curvilinear regressions f i t the pink and sockeye data better than linear regressions, while the reverse holds for chum, coho and chinook (Table XIII, Appendix). In summary, the a b i l i t y of pink and chum alevins to regulate their.Cl concentrations is greater than that of coho, chinook and sockeye. Serum K+ Concentrations The results of serum K+ measurements are shown in Figure 6. Serum K+ levels f a l l rapidly in the f i r s t 4 hr of exposure to sea water for a l l species except.chinook. Between 4 and 8 hr a l l species except chinook show - 32 -TABLE X Analysis of variance of the serum Cl. concentrations of the five species of alevins exposed to 0, 4, 8 and 12 hr of sea water. Source Species Exposure Interaction Error Therefore.Species, Exposure and Interaction are highly significant. df SS MS F 4 . 14,056 3,514 135 F o > 0 Q 5 ( 4 , 4 0 ) 4.37 3 22,776 7,592 292 F o _ 0 Q 5 ( 3 , 4 0 ) 4.98 12 3,904 325 12 F o W 1 2 , 4 0 ) 2 " 9 5 40 1,051 26 - 33 -FIGURE 6 Serum K concentrations of Pacific salmon alevins plotted against exposure times to 31.8°/oo sea water. K* ALEVINS Hours - 34 -a rapid rise in serum K levels bringing them near freshwater levels. Between 8 and 12 hr a l l species except coho show increases in serum K+ levels. Between 12 and 24 hr K+ levels in chum and chinook diverge considerably.from those of pink alevins. However, an analysis of variance of the ,data for the five species at 0, 4, 8 and 12 hr (Table XI), show no significant differences among the five species, among the four exposures and between the interaction of, species and exposures. - 35 -TABLE XI Analysis of variance of the serum K+ concentrations of the five species of alevins exposed to 0, 4, 8 and 12 hr of sea water. Source df SS MS F Species. 4 1,279 319.7 1.76 F 0 > 0 5 ( 4 , 4 0 ) 2.61 Exposure 3 872 290.7 1.60 F o Q 5 ( 3 , 4 0 ) 2.84 Interaction 12 547 45.6 .25 F o Q 5 ( 1 2 , 4 0 ) 2.00 Error 40 7,263 181.6 Therefore there are no significant differences. - 36 -C. Fry LD _ Values oU Approximately five months after hatching, nine fry from each species (1964-65 progeny) were transferred from fresh water to 31.8°/oo sea water and the LD determined for each species. The sea water proved lethal for coho, chinook and sockeye fry but not pink and chum (Table XII). In conjunction with the LD determinations, fry of each species were. 50 transferred directly from fresh water to 31.8 °/oo sea water and the blood was sampled following immersion for 0, 4, 8, 12, 24 and 48 hr. Subsamples were taken from the plasma obtained from individual fish and the melting points and concentrations of Na+, Mg + + and Cl were determined. In Figures 7, 8, 9 and 10, which display the results of these measurements, every point is the mean of three determinations and each determination was made from the blood of one individual. Plasma Osmotic.Concentrations The measurements of plasma osmotic concentrations are shown in Figure 7. Similar to the results of embryos and alevins, a large increase in plasma osmotic concentrations of fry occurs.within the f i r s t 4 hr of exposure to sea.water. Between 4 and 12 hr exposure to sea water, the plasma osmotic concentrations of pink and chum fry do not change appreciably whereas coho, chinook and sockeye fry show increasing levels. Between 12 and 24 hr pink and.chum manifest declining plasma osmotic concentrations; and between 24 and 48 hr essentially no change in pink or chum levels occurs. On the basis of Tukey's w of 45 mOsm, calculated from the analysis of variance of the five species exposed to 0, 4, 8 and 12 hr of sea water (Table XIII), i t may be concluded that no significant differences occur - 37 -TABLE XII LDJ_Q (hr) of 1964-65 fry transferred directly from fresh water to 31.8 °/oo sea water. Pink Chum Coho Chinook Sockeye > 336 > 336 32 120 18 - 38 -FIGURE 7 Plasma osmotic concentrations of Pacific salmon fry plotted against exposure times. to 31.8°/oo sea water. 3 0 0 ' - —1 . I' . , L 1| L_ O 4 8 12 24 48 Hou rs - 39 -TABLE XIII Analysis of variance of plasma osmotic concentrations of the five species of fry exposed to 0, 4, 8 and 12 hr of sea water. Source df SS MS F Species 4 21,301 5,325 .3 25.60 Fo ( 0 0 5(4,40) 4.37 Exposure 3 49,314 16,438 .0 79.01 Fo < Q 0 5(3,40) 4.98 Interaction 12 11,726 977 .15 4.70 Fo > 0 0 5(12,40) 2.95 Error 40 8,321.6 208 .04 Therefore Species, Exposure and Interaction are highly significant. - 40 -among the f i v e species i n fresh water. At 4 hr exposure to sea water, pinks show a s i g n i f i c a n t l y lower plasma osmotic concentration than sockeye. At 8 hr, pink and chum l e v e l s are s i g n i f i c a n t l y lower than coho and sockeye. And f i n a l l y , at 12 hr of exposure, pink f r y are s i g n i f i c a n t l y d i f f e r e n t from coho, chinook and sockeye while chum are s i g n i f i c a n t l y d i f f e r e n t from sockeye but not from coho (0.10 < P > 0.5). Orthogonal contrasts show that the differ e n c e i n response to sea water between pink/chum and coho/chinook/sockeye as measured by plasma osmotic concentrations at 0/4/8/12 hr i s s i g n i f i c a n t . However, the differ e n c e between pink and chum f r y i s highly s i g n i f i c a n t (P < 0.005). S i m i l a r l y coho/chinook respond s i g n i f i c a n t l y d i f f e r e n t l y from sockeye but coho f r y do not d i f f e r from chinook. In ad d i t i o n , the differ e n c e i n plasma osmotic concentration of chum at 8 and 12 hr exposure i s not s i g n i f i c a n t l y d i f f e r e n t from the same difference f o r chinook (Table XIV, Appendix). The pink and chinook data f i t s t r a i g h t - l i n e regressions better than c u r v i l i n e a r ; the slope of the pink regression l i n e i s considerably less than that of the chinook (Table XV, Appendix). In contrast, the data of coho and sockeye f r y are better described by c u r v i l i n e a r regressions. Chum data cannot be f i t t e d to eit h e r the s t r a i g h t l i n e or the c u r v i l i n e a r regression. In summary, pink and chum f r y manifest an a b i l i t y to osmoregulate which i s greater than that shown by sockeye. The a b i l i t i e s of coho and chinook f r y to osmoregulate are intermediate. These ca p a c i t i e s c o r r e l a t e with the tolerance of sea water by pink and chum f r y and the greater s a l i n i t y resistance of coho and chinook over that of sockeye. Plasma Na' Concentrations + The. measurements of plasma Na concentrations .are shown i n Figure 8. A - 41 -FIGURE 8 Plasma Na concentrations of Pacific salmon fry plotted against exposure times to 31.8°/ sea water. - H2 -rapid rise in Na concentration occurs for a l l species within the f i r s t 4 hr of exposure to sea water. Between 4 and 12 hr both pink and chum fry show decreased levels of Na+ whereas coho, chinook and sockeye manifest increased levels. Between 12 and 24 hr only small changes occur in pink and chum fry levels. On the basis of Tukey's w of 34 mEq/1, calculated from the analysis of variance of the five species exposed to 0, 4, 8 and 12 hr of sea water (Table XIV), no significant differences are present among the five species at 0, 4 and 8 hr sea water. However, at 12 hr, pink and chum fry have plasma Na+ levels which are significantly lower than coho levels but are not significantly different from sockeye levels (0.01 < P > 0.05). Thus, chinook, on the basis of Tukey's w is not significantly different from pink and chum at any exposure time. But orthogonal contrasts, which are more sensitive than Tukey's w, show pink/chum significantly different from coho/chinook/sockeye when the levels of plasma Na+ are compared collectively at 0, 4, 8 and 12 hr. When pink and chum are compared collectively at 0, 4, 8 and 12 hr, they are not significantly different from each other. Similarly, coho are.not significantly different from sockeye.and coho/sockeye are not significantly different from chinook. Therefore, pink and chum are significantly different from coho,; chinook and sockeye. The chum and sockeye.data f i t curvilinear regressions better than straight-line regressions; coho and fry data are better described by straight-line regressions; pink data cannot be f i t t e d to either the straight-line or the curvilinear regression (Table XVI, Appendix). In summary, the a b i l i t i e s of pink and chum fry to regulate their plasma Na+ concentrations i s greater than the a b i l i t i e s of coho, chinook and sockeye. - 43 -TABLE XIV Analysis of variance of plasma Na+ concentrations of the five species of fry exposed to 0, 4, 8 and 12 hr of sea water. Source df Species 4 Exposure 3 Interaction 12 Error 40 SS MS F 2,718.4 679.61 5.53 F A A„(4,40) 4.37 10,787 3,595.8 29.27 F n A A C(3,40) 4.98 U . UOo 4,499.1 374.93 3.05 F Q 0 Q 5 (12,40) 2.95 4,914.0 112.85 Therefore Species,. Exposure and Interaction are highly significant. - i+H -Plasma Cl Concentrations The measurements of plasma Cl concentrations are shown in Figure 9. A rapid rise in plasma Cl concentrations occurs for a l l species and particularly Sockeye.within the f i r s t 4 hr of exposure to sea water. Between 4 and 8 hr, pink, chum and sockeye plasma Cl levels do not change appreciably, while coho and chinook Cl levels continue to increase. Between 8 and 12 hr, pink and chum Cl levels decrease and coho, chinook and sockeye Cl levels increase. At 24 and 48 hr, pink and chum plasma Cl appear to.have stabilized at levels slightly higher than fresh water values. On the basis of Tukey'sw of 21 mEq/1, calculated from the analysis of variance of the five species exposed to 0, 4, 8 and 12 hr of sea water (Table XV), no significant differences occur in the plasma Cl levels among the five species at 0 and 4 hr sea water. At 8 hr sea water, pink plasma Cl levels are significantly lower than coho levels. At 12 hr, pink levels are significantly lower than coho, chinook.and sockeye, whereas chum levels are significantly lower than coho but.not from any of the other species. Orthogonal contrasts show that pink/chum are significantly different from coho/chinook/sockeye as measured by plasma Cl concentrations.at 0/4/8/12 hr in sea water. Since.pink plasma Cl levels are not significantly different from those of chum at 0/4/8/12 hr, nor are coho/sockeye levels significantly different from chinook levels or coho.levels from sockeye levels, the plasma Cl concentrations in pink and chum are significantly different from those in coho, chinook and sockeye at 0/4/8/12 hr in sea water. Curvilinear regressions f i t the pink, chum and sockeye data better than linear regressions, whereas the reverse holds for coho and'chinook: (Table - 45 -FIGURE 9 Plasma Cl concentrations of Pacific salmon fry-plotted against exposure times to 31.8°/oo sea water. O 4 8 12 24 48 Hours - 46 -TABLE XV Analysis of variance of plasma Cl concentrations of the five species of fry exposed to 0, 4, 8 and 12 hr of sea water. Source df SS MS. . F Species 4 2,416.4 604.11 12.96 F n n n c(4,40) 4.37 U . DUO Exposure 3 5,337.5 1,779.2 38.16 F A n c(3,40) 4.98 0 . UUo Interaction 12 1,594.3 132.86 2.85 F Q (12,40) 2.66 Error 40 1,864.7 46.618 Therefore Species and Exposure are highly significant and Interaction i s significant. - 47 -XVII, Appendix). Plasma Mg + + Concentrations The measurements of plasma Mg + + concentrations are shown in Figure 10. A very large increase in plasma Mg + + concentration occurs for a l l species within the f i r s t 4 hr in sea water. Between 4 and 8 hr pink, chum and chinook plasma Mg + + levels decline while coho and sockeye levels r i s e . Between 8 and 12 hr, pink and coho levels decline while chum, chinook and sockeye levels increase. Between 12 and 48 hr, pink plasma Mg + + levels increase slightly while between 12 and 24 hr, chum levels decrease considerably and after 24 hr increase moderately. On the basis of Tukey's w of 2.92 mEq/1, calculated from the analysis of variance of the five species exposed to 0, 4, 8 and 12 hr of sea water (Table XVI), no significant differences are present among the five species at 0, 4, 8 and 12 hr with the single exception of the sockeye plasma Mg + + concentration at 12 hr which i s significantly higher than the plasma Mg + + concentrations of pink, chum and chinook fry in fresh water. Orthogonal contrasts show that the difference between plasma Mg + + concentrations of pink fry and chum/coho/chinook fry at 0/4/8/12 hr is highly significant (P < 0.005). However, the difference between chum and coho/chinook at.0/4/8/12 hr is not significant nor is the difference between coho and chinook. There is no significant interaction between species and exposure times (Table XVI). The data for pink fry f i t a curvilinear regression better than a straight-line whereas the reverse is true for sockeye data (Table XVIII, Appendix). The chum, coho and chinook data cannot be fi t t e d to the - 48 -FIGURE 10 Plasma Mg concentrations of Pacific salmon fry-plotted against exposure times to 31.8 °/oo sea water. 5-..5Q h 2 0 0 Hours TABLE XVI Analysis of variance of plasma Mg concentrations of the five species of fry exposed to 0, 4, 8 and 12 hr sea water. Source df SS MS F Species 4 14.785 3.6963 4.14 Fo > Q 1(4,40) 3.! 33 Exposure 3 30.052 10.017 11.23 Fo < 0 0 5(3,40) 4 .98 Interaction 12 14.238 1.1865 1.33 Fo > 1 Q(12,40) 1 .71 Error 40 35.676 .89190 Therefore Species i s significant, Exposure is highly significant and Interaction is not significant. - 50 -curvilinear or the straight-line regressions. In summary, pink fry can regulate the Mg + + concentration in their plasma whereas sockeye fry do not manifest this a b i l i t y . The data for chum, coho and chinook are inconclusive. - 51 -P.. Comparison of Embryo, Alevin and.Fry Data LD r n Values ou The salinity resistance times which have.been outlined previously for each species at each age are brought together in Table XVII for purposes of comparison. The five species, as shown.by their LD^^ values, f a l l ' i n t o three groups. Pink and chum at a i l three ages are considerably more resistant to sea water than the coho and sockeye at.the same ages.. The salin i t y resistance of chinook is intermediate between these.two groups.. In addition, embryos .and fry o f , a l l five species (except sockeye) manifest greater salinity resistance than their respective alevins. Because the criterion for death of embryos and alevins was the • arrestment of blood flow in the v i t e l l i n e veins while the criterion for death of fry was. the cessation of opercular movements, the salinity resistance of fry must be ,compared to that of embryos with some reservation. Since ; opercular movements are more rapidly inhibited by high s a l i n i t i e s than is. heart beat, the L D , . Q values of fry would be.higher than those in Table XVII i f the cessation of blood flow had been used as the criterion of death in fry. Therefore, the survival of coho,and sockeye fry may: not be less than but equal to or greater than their respective embryos. However, without" doubt, pink and chum fry survive considerably longer in 31.8 °/oo sea water than pink and chum embryos and alevins; in addition, chinook fry survive longer than chinook embryos. Osmotic Concentrations. The question that arises from the L D ^ Q data is whether the measurements of blood osmotic concentration correlate with the differences . outlined-.. Before answering this .question, i t must be noted that the method of TABLE X V I I L D 5 Q (hr) of embryos, alevins and fry of Pacific salmon transferred directly from fresh water to 31.8 °/op • sea water. Embryos Alevins Fry 3 Pink 102 69 > 336 Chum 118 56 > 336 Coho 47 26 32 Chinook 81 49 120 Sockeye 52 27 18 1 2 3 1965-66 embryos 1965-66 alevins 1964-65 fry - 53 -collecting blood was different for a l l three ages. As a result a test was made to determine i f the blood collecting technique influenced the osmotic concentrations. When fry were sampled by the embryo collecting method (serum obtained) and by the usual fry method (plasma obtained) and the osmotic concentrations determined, no significant differences were observed between the two techniques. The^  reverse experiment, that i s , comparing embryo and fry blood collecting methods on embryos was not possible. However, when blood from fry was collected by the alevin method (pooled serum obtained) and the fry method, the former resulted in osmotic concentrations which were significantly higher than the latter. The mean value obtained by the alevin method was 333 mOsm and the mean by the fry method was 320 mOsm. It was not possible to use the alevin blood collecting method for fry because of more rapid clotting in fry and.the reverse was impossible because of hemolysis and clotting, both of which were not abetted by heparin. Therefore, for comparative purposes, osmotic concentrations of embryo serums and fry plasma.have not been altered but osmotic concentrations of pooled alevin serums have been decreased by 13 mOsm. On this basis, the. blood osmotic concentrations of the five species of Oncorhynchus, at four exposure times to sea water and at three .ages are compared in Figure 11. For any one species, blood osmotic concentrations of alevins are higher than those.of embryos or fry at a l l exposures. However, within any one of the five species, the blood osmotic concentrations of alevins at 12 hr in sea water not only are higher than those of embryos and fry but by amounts which are greater than the difference,between alevins and embryos or fry in fresh water. Osmotic concentrations of pink and chum fry are considerably below both embryo and alevin levels at 4, 8 and.12 hr but only slightly below - 54 -FIGURE 11 The blood osmotic concentration of Pacific salmon embryos (E), alevins (A) and fry (F) exposed to 0, 4, 8 and 12 hr of 31.8°/oo sea water. Each triangle is the result of joining the mean blood osmotic concentrations of embryos, alevins and fry exposed for the same duration to sea water. - 55 -embryo levels in fresh water. With Tukey's w of 56 mOsm, calculated from the, analysis of variance of the five species exposed to 0, 4, 8 and 12 hr of sea water as- embryos, alevins and fry (Table XVIII), the following conclusions may be made: (1) The serum osmotic concentrations of pink, chum and coho embryos at 0, 4, 8 and 12- hr in sea water are not significantly different from those of the respective alevins, whereas.chinook embryos at 8 hr are significantly different from chinook alevins at 8 hr, and sockeye embryos at 4 and 12 hr are significantly different from sockeye alevins at 4 and 12 hr. (2) The serum osmotic concentrations of chum, coho, chinook and sockeye alevins at 12 hr sea water are significantly different from the plasma osmotic, concentrations of the respective fry at 12 hr; in addition, pink alevins at 4 and 8 hr are significantly different from pink fry at 4 and 8 hr, and chinook alevins at 8 hr are significantly different from chinook-fry at 8 hr. (3) The serum osmotic concentration of pink embryos at 4 hr is significantly different from the plasma osmotic concentration of pink fry at 4 hr; otherwise no other significant differences are observed between embryos and fry of the same species. Broader comparisons have also been made using orthogonal contrasts. The1 blood osmotic concentrations of pink/chum salmon as embryos/alevins/fry at 0/4/8/12 hr in sea water are: highly, •significantly"different from coho/chinobk/ sockeye osmotic concentrations as embryos/alevins/fry at 0/4/8/12 hr (P < 0.005). Similarly, the difference between coho/chinook and sockeye is highly significant (P < 0.005), but pink are not - significantly different from chum, and coho are not significantly different from chinook. The interaction between species and ages has also been analysed by - 56 -TABLE XVIII Analysis of variance of blood osmotic concentrations of the five species of Pacific salmon embryos, alevins and fry exposed to 0, 4, 8 and 12 hr sea water. Source df SS MS F Table F o Species 4 28,566 7,141 .6 25 .89 3 .92 Age 2 80,500 40,250 145 .92 5 .54 Exposure 3. 213,240 71^080 257 .68 4 .50 Species X Age 8 14,220 1,777 .6 6 .44 2 .93 Species X Exposure 12 40,754 3,396 .1 12 .31 2 .54 Age X Exposure 6 12,902 2,150 .3 7' .80 3 .28 Species X Age X Exposure 24 19,431 809 .62 2 .94 2 .09 Error 12Q 33,101 275 .84 .005 Therefore a l l main effects and a l l interactions are highly significant. - 57 -orthogonal contrasts. The difference between embryo and fry osmotic concentrations of pink/chum is highly significant (P < 0.005) from the difference between embryo and fry of coho/chinook/sockeye (Table XIX, Appendix). Similarly, pink are not significantly different from chum (Table XX, Appendix); coho/chinook are not significantly different from sockeye (Table XXI, Appendix) and coho are not significantly different from chinook (Table XXII, Appendix). Therefore, the differences in blood osmotic concentrations between embryos and fry of pink and chum are significantly different from those of coho, chinook and sockeye. The difference between embryos/fry and alevins of pink/chum is significantly different from the difference between embryos/fry and alevins of coho/chinook/sockeye (Table XXIII, Appendix). However, the difference between embryos/fry and alevins of pink is not significant from that,of chum (Table XXIV, Appendix). Similarly, coho/chinook is not significantly different from sockeye-(Table XXV, Appendix), but the difference between coho and chinook is highly significant (P < 0.005; Table XXVI, Appendix). Returning to more general comparison, orthogonal contrasts indicate that embryos/fry of pink/chum/coho/chinook/sockeye at 0/4/8/12 hr are highly significantly different from alevins (P < 0.005) and that embryos are significantly different from fry. And f i n a l l y , the three-way interaction (Table XVIII) has been analysed by orthogonal contrasts and the following interaction ,found to be significant (P < 0.01). The difference between 0 and 12 hr in sea water for embryos/fry vs alevins of pink/chum is significantly different from the difference between 0 and 12 hr for embryos/fry vs alevins of coho/chinook/sockeye (Table XXVII,. Appendix). - 58 -Therefore, embryos and fry in sea water have.lower blood osmotic concentrations than alevins in sea water. Since the embryos.and fry (except sockeye) survive longer in•sea water than their respective alevins, the results on blood osmotic concentrations correlate positively with the LD^A values. Therefore, embryos and fry probably survive longer in sea water than alevins due to greater osmoregulatory a b i l i t i e s . E... Relationship Between Osmotic and Ionic Concentrations The contributions that Na+ and Cl concentrations make to the serum and plasma osmotic concentrations are shown in Figures 12 and 13 respectively. It is assumed that Na+ and Cl in serum or plasma are in ideal solutions,in which one milliequivilent per l i t r e of Na+ or Cl has an osmotic concentration of one milliosmole. In alevin serum, Na+ and Cl contribute 63 to 82% of the osmotic concentration which is significantly lower than the 80 to 90% in fry plasma (P < 0.005). In addition, sodium-to-chloride ratios in alevin serum average.1.22 which is significantly different from the same ratio for fry plasma which averages 1.35 (P < 0.005). No significant differences were observed in these ratios between alevins in fresh water and alevins in sea water; similar results were obtained with fry. FIGURE 12 Osmotic, sodium and chloride concentrations of Pacific salmon alevins plotted against exposure time to 31.8 °/oo sea water. ALEVINS O I I I I I t i f i i i t | | i l l 1 _ J I I I l 0 4 8 12 24 0 4 8 12 24 O 4 8 12 O 4 8 12 24 0 4 8 12 Hours - 60 -FIGURE 13 Osmotic sodium and chloride concentrations of Pacific salmon fry plotted against exposure time to 31.8 °/oo sea water. SOO 4 0 0 2 0 0 • OO FRY Pink Chum Coho Chinook Sockeye mOsm 3 0 0 CNa+1 Cci-3 i i i I I I i i i I I i i I I » « ' • 0 4 8 12 O 4 8 12 0 4 8 12 0 4 8 12 O 4 8 12 Hours - 61 -DISCUSSION A. Examination of Working Hypotheses Differences in ecology and behaviour among the five species of Pacific salmon are particularly.evident in the early l i f e stages (Neave, 1958; Hoar, 1958). Based on this fact, i t was postulated that (i) the embryo, alevin and fry of pink and chum salmon are euryhaline, whereas.the same l i f e stages of coho, chinook and sockeye are stenohaline and ( i i ) the euryhalinity of pink and chum is due not to high tissue tolerance but to their a b i l i t y to regulate the osmotic and ionic concentrations in their blood, whereas the stenohalinity of coho, chinook.and sockeye stems from their inability to osmoregulate and ion regulate. The LD^Q values and the osmoregulatory data obtained from embryos indicate that pink and chum are not euryhaline as hypothesized; that i s , they cannot survive in a wide range of s a l i n i t i e s . Therefore, embryos of pink and chum, like coho, chinook and sockeye, are stenohaline. However, the LD^A values and osmotic concentrations of pink and chum embryos.indicate greater salini t y resistance and osmoregulatory a b i l i t y than embryos of the other species. The data obtained from alevins also does not support the working hypotheses for alevins of a l l five species are stenohaline. But, as in the case for embryos, pink and chum alevins have greater salinity resistance, and osmoregulatory a b i l i t i e s than coho and sockeye alevins. This a b i l i t y of pink and chum has been correlated with better ionic regulation of•sodium and chloride. Although chinook alevins survived considerably longer in 31.8°/oo sea water than.coho and sockeye alevins, their a b i l i t y to.osmoregulate and - 62 -ion regulate is not very different from that of coho and sockeye alevins. Therefore, the greater salinity resistance of chinook alevins is due to high tissue tolerance. Their organs are able to function in a wider range of osmotic and ionic concentrations of the milieu interieur than i s the case for coho and sockeye. Contrary to the results with embryos and alevins, the data obtained from fry supports the working hypotheses. The data indicate that pink and chum fry are euryhaline and that this condition is due to their a b i l i t y to osmoregulate and control the levels of sodium and chloride in the blood. Coho, chinook and sockeye fry are stenohaline and are unable to regulate the osmotic and ionic concentrations in their blood. As in the case of alevins, chinook fry, due to higher tissue tolerance, manifest greater salinity resistance than coho and sockeye. In summary, the working hypotheses have been shown to be only partially correct. Pink and chum fry are euryhaline whereas embryos and alevins of a l l five species are stenohaline. However, pink and chum embryos :and alevins manifest greater a b i l i t i e s to regulate the osmotic and ionic concentrations in their blood than coho, chinook and sockeye. The a b i l i t i e s of pink and chum fry to osmoregulate and ion regulate support .the hypothesis that their euryhalinity is not due to high tissue tolerance. Although chinook alevins and fry are stenohaline and therefore•support the hypothesis, they survive longer.in sea water than coho and sockeye. The greater survival is not the result of better osmoregulation but is due to higher tissue tolerance. B. Mechanisms of Osmotic and Ionic. Regulation in Embryos Coelomic salmonid eggs, which are slightly hypo-osmotic to the maternal blood (Hayes, 1949), are permeable to water (Zotin, 1965), but after. - 63 -f e r t i l i z a t i o n , no net gain or loss of water occurs in normally developing eggs. Kao et_. a l . (1954) believes that high pressure of the perivitelline f l u i d formed at f e r t i l i z a t i o n may cause reduction in the.size of pores in the v i t e l l i n e membrane and thereby reduce permeability to water, whereas Zotin (1965) believes that "eggs (teleost) contain water under special conditions and that as a result i t possesses properties somewhat differing from those of water in more ' usual physicochemical systems". Ferti l i z a t i o n of salmonid eggs is possible in dilute sea water but not in high concentrations. Rockwell (1956) found that a salinity of 18 °/oo or less does not inhibit f e r t i l i z a t i o n of pink and chum eggs. In fact, dilute salt water prolonged the period of egg and sperm f e r t i l i t y (Rutter, 1902; E l l i s and Jones, 1939; Rucker, 1949). This enhancement of f e r t i l i t y is: .not:due.to. ions but the osmotic conditions of the dilute solutions ( E l l i s and Jones, 1939). Just after entrance of a spermatozoan'into the micropyle'of the ovum, cortical alveoli break down releasing a colloid which imbibes water between the v i t e l l i n e membrane and the chorion thereby forming the periv i t e l l i n e ' space.(Bogucki, 1930; Hayes and Armstrong, 1942; Zotin, 1958). As a result of this uptake of water, there is a 20% increase in weight-of Salmo salar eggs (Hayes and Armstrong, 1942). The net uptake of water in Salmo salar is complete soon after f e r t i l i z a t i o n while hardening of the chorion reaches i t s peak much later (Hayes and Armstrong, 1942; Zotin, 1958). . While Na,.K, Ca, Sr and Mg ions inhibit the release of cortical alveoli and also the release of hardening enzyme (for chorion) at least one of these ions is necessary in the f i r s t 5 min after f e r t i l i z a t i o n for the hardening enzyme to function. The hardening enzyme is not present in the cortical alveoli (Zotin, 1958) but i t s normal activity may require some of the ions' probably released from the - 64 -breakdown of the cortical.alveoli. Although 0.1 N NaCl blocks release of cortical alveoli and hardening enzyme (Zotin, 1958), normal development of soft,eggs in fresh water is possible (Rockwell, 1956). The views of Kao et_ a l . (1954) and Zotin (1965) have already been outlined as explanations of the fact that soon after f e r t i l i z a t i o n 100% of the total volume of salmonid embryos is osmotically inactive. However, this does not preclude the possibility of water movement into or out'of salmonid embryos for at least four different phenomena of water movement exist and have been defined by Zotin (1965): (1) entry or exit of water as determined by osmosis (2) water renewal without net change in water content of embryo (3) unilateral water transport dictated by needs of the embryo (4) redistribution of water within the embryo Krogh and Ussing (1937) showed that soon after f e r t i l i z a t i o n , penetration of heavy water .ceased and did not resume unt i l after formation, of the eye. These results are in agreement with those of Svetlov (1929) who showed that the freezing point depression of the trout yolk did not-change during development and those of Gray (1932) who showed no change in trout egg weight in hypertonic solutions. Later, Hayes and Armstrong (1942) observed no change in wet weight of embryos and no change in weight of water in eggs up to hatching and Busnel et_ a l . (1946) found that the percent water of developing Salmo salar up to hatching remains constant. These results suggest that the penetration of.heavy water for a short period after f e r t i l i z a t i o n is indicative of. Zotin's Type (2) water movement, that i s , water renewal. In addition, the duration of this phenomenon after f e r t i l i z a t i o n is species-specific for while chinook and rainbow trout eggs lose the a b i l i t y to exchange - 65 -water soon after f e r t i l i z a t i o n , eggs of Salmo salar require as long as ten days (Zotin, 1965). One is therefore l e f t with the impression from these results that the salmonid egg from f e r t i l i z a t i o n to hatching (excluding the periv i t e l l i n e space) does not take up net amounts of water. Winnicki (1958) and Domural (1961) however, have shown that trout eggs cannot develop normally in a waterless environment for under such conditions a prolongation of embryonic development occurs. These results may be due to an accumulation of metabolites, to limiting oxygen concentrations or, as Winnicki (1958)and Domurat (1961) have argued, to a need for water from the external medium during the process of morphogenesis. Since there is no net change in water content of the embryo, the water requirement, i f Winnicki and Domurat are correct, must be of Zotin's Type (2). In addition to water renewal, salmonid embryos obtain water from the yolk. The water content of the yolk of Salmo salar eggs decreased from 63.5%.at f e r t i l i z a t i o n to 59% at hatching (Hayes and Armstrong, 194-2), even though, as stated previously; the freezing point depression of the . yolk remains constant throughout embryonic development (Svetlov,' 1929):. 1 i Although eyed salmonid embryos may renew their,water and obtain water from their yolk, they do not make net gains or losses of water. Busnel . et_ al_. (1946) found that the percentage of water in Salmo salar embryos remains around 63 to 64% when the eggs, near hatching, are immersed in fresh water, 10°/oo, 15°/oo, 20 °/oo or 25°/oo saline water despite the fact the osmotic concentration of the milieu interieur increases from 318 to 712 milliosmoles. Therefore, since no net•water gains are made by eggs before hatching (Hayes and Armstrong, 1942), the embryos must be capable of making - 66 -net gains or losses of salts but not water. This conclusion is supported by-Hayes (1949) in that he observed net losses of phosphorus and potassium from the salmon egg before hatching. Finally, pink and chum embryos, about to hatch, are not prevented from doing so by s a l i n i t i e s up to 29.67°/oo; in addition, brackish water tends to accelerate hatching and improve the percentage of successful hatching (Rockwell, 1956). Enhanced and accelerated hatching in brackish water is probably due to the ionic strength effect of such solutions on the hatching enzyme, chorionase, and the more.alkaline conditions in the pe r i v i t e l l i n e f l u i d which would increase the activity of the enzyme (Kaighn, 1964). If the above interpretations are correct, then embryos of Oncorhynchus near hatching (2 weeks) can take up or lose net amounts of ions, but not water, and the greater a b i l i t y of pink and chum embryos to osmoregulate must be due to their a b i l i t y to ion regulate^ If the differences among the five species of Pacific salmon were due to the surface-to-volume ratios, then sockeye embryos should have the least resistance to sea water and be the poorest osmoregulator while the reverse should hold for chinook embryos. My results agree with the contention for sockeye but not for chinook. In fact, although pink eggs are smaller than chinook or chum eggs, pink embryos osmoregulate better than chinook and as well as chum. Size, then, at the interspecific level is not as important as some other properties, but the other, characters involved can only be conjectured at this point. The roles of the incompletely differentiated alimentary canal in extracting ions and water from sea water, and of the pronephros and g i l l s in secreting excess ions into the environment have not as yet been studied; nor has the role of the epidermis. - 67 -C. Mechanisms of Osmotic and Ionic Regulation in Alevins While the evidence indicates that the salmonid embryo can make a net gain or loss of salts, but not water, the situation after hatching in the salmonid alevins is not as clear. Hayes and Armstrong (1942 and Busnel et a l . (1946) have reported increased hydration of Salmo salar alevins in fresh water which Hayes and Armstrong showed was due to a net uptake of water, although the alevin was also obtaining water from the yolk. In addition, Rockwell (1956), Busnel et. a l . (1946) and Wilier and Trahms (1942) reported dehydration of pink and chum, Atlantic salmon and brown trout alevins, respectively in sea water. Rockwell (1956) argued that because the salinity-time curves are smooth, a single cause of death was probable and that dehydration "with salt penetration possibly playing a minor role" was the main cause. However, as observed in my experiments and those 'of Busnal et. a l . (1946) for Salmo salar, the osmotic concentration of the blood or body fluids of the alevins increases shortly after hatching. Parry (1961) reports that the osmotic concentration of Salmo trutta eggs is considerably less than that of parr when both are maintained in fresh water. In addition, Hayes (1949) and his co-workers have observed a considerable loss of phosphorus and a smaller loss of potassium from Salmo salar alevins as well as no net gain or loss of calcium and sodium before hatching, but a considerable gain of these two ions after hatching. The evidence, therefore, suggests that osmoregulation in salmonid alevins is due to both water and ion regulation whereas in the salmonid embryo ion regulation alone is important in regulating the osmotic concentration of the body fluid s . If the above conclusions are valid then the greater a b i l i t y of pink and chum alevins to osmoregulate may be due to either superior water or ion - 68 -regulation or both. As in the case of embryos, the surface-to-volume ratio phenomenon does not appear to be important interspecifically, for while sockeye alevins are considerably smaller than chinook alevins, their poor osmoregulatory a b i l i t i e s are only slightly worse than that of chinook alevins. The concentrations of sodium and chloride in the serum, assuming an ideal solution, contribute 63 to 82% of the osmotic concentration. To my knowledge there are no data available from other species at this stage with which comparisons could be made. However, i t is known that in older f i s h sodium and chloride ions make up a large part of the osmotic concentration of the blood (Gordon, 1959; Conte and Wagner, 1965; Conte et_ a l . , 1966). Correlated positively with the greater a b i l i t y of pink and chum alevins to osmoregulate is their greater ability: to regulate the levels of sodium and chloride ions in the blood. The mechanisms which account for these differences in ionic and osmotic regulation among the five species of Oncorhynchus probably include the alimentary canal, kidney, g i l l s and skin. With respect to the skin, i t is of interest to note that Holliday and Blaxter (1960) have shown that herring larvae (alevins) have a wider salinity tolerance than adults despite the fact the larvae lack kidneys and g i l l s . In this regard, Holliday and Jones (1965) have postulated that the epidermis of the larvae has the a b i l i t y to osmoregulate. However, more recently, Jones et_ a l . (1966) have observed no ultrastructural differences in the epidermis from larvae reared in various s a l i n i t i e s . Preliminary observations that I have made indicate that Pacific salmon embryo and alevin skin is richly endowed with mucous cells (unpublished). Parry (1960) who has also observed that newly hatched Salmo salar alevins have an actively secreting mucous epithelium, believes that "the mucus produced cannot by i t s e l f maintain an osmotic barrier". - 69 -However, Munz and McFarland (1964) have suggested that.the slime produced by hagfish aids in cation regulation in that the slime contains high concentrations of calcium, magnesium and potassium. The low osmotic, sodium and chloride concentrations of pink alevins in fresh.water may be indicative of the electrolyte depletion which Fontaine (.1960) and his co-workers have postulated as stimulating seaward migration. The low chloride levels which have also been observed by Callamand and Fontaine (1940) for eel, Kubo (1955) for Oncorhynchus masou smolts and Fontaine (1951) for Salmo.salar suggests that chloremia,is characteristic of anadromous- f i s h . Houston (1959) however, puts forth the view that "the 'dimineralization' of pre-migrants may be considered more plausibly to be a correlative of parr-smolt transformation rather than as an i n i t i a t o r of downstream migration". The drop in chloride levels may more li k e l y be the result of the i n i t i a t i o n of chloride c e l l secretory activity as suggested by the work of Hoar (unpublished) and Nishida (1953) (both.cited by Black, 1957). However, chloride secretory activity of the acidophil•cells found in fi s h g i l l s is far from universally accepted (Parry, 1966), although recent unpublished findings of F. P. Conte (personal communication) strongly support the • chloride c e l l theory. D. Mechanisms of Osmotic,and.Ionic Regulation In Fry When salmon fry are five months old they possess many of the definitive characteristics which are.either lacking or incompletely developed in the embryos and early alevins. In particular, the opisthonephros is well formed, the alimentary canal is more highly differentiated and the number of chloride secretory cells may be greater. The results indicate that pink and chum fry are.able to survive a direct - 70 -transfer from fresh water to sea water while the coho, chinook and sockeye cannot, although the chinook do show greater salinity resistance than coho and sockeye. Correlated positively with these results are the osmotic and ionic concentrations of the plasma. The pink and chum fry manifest an adjustive phase in osmoregulatory adaptation of about 24 hr in which the osmotic and ionic concentrations rise rapidly and then begin to return to near freshwater levels and a regulatory phase in which osmotic and ionic levels appear to be maintained slightly higher than in fresh water (Figures 7, 8 and 9). These results are in agreement with the findings of Black (1951) for chum fry but contrast sharply with Parry's (1960) results. She observed that nine month old parr of Salmo gairdneri, S_. salar and S_. trutta in 17°/oo sea water have adjustive phases of 150, 200 and 300 hr respectively. The adjustive phase of five month old pink and chum fry, which is therefore considerably shorter than for nine month old species of Salmo, indicates the greater osmoregulatory a b i l i t i e s of these Pacific salmon. Pink and chum fry results also support the view that euryhaline fi s h in the regulatory phase of seawater adaptation maintain plasma osmotic concentrations slightly hyperosmotic to freshwater individuals (Table XIX). With regard to the coho, chinook and sockeye fry the results indicate the lack of a b i l i t y to reach the regulatory phase. These fry are unable to survive in sea water and their poor osmotic and ionic regulatory a b i l i t i e s support this fact. The chinook which have a considerably higher salin i t y resistance than coho and sockeye also do not, however, manifest considerably greater osmotic and ionic regulatory a b i l i t y . The results for coho fry are in agreement with those of Conte et_ a l . (1966) who observed very poor survival of 5 - 6 cm coho fry approximately five months old and those of - 71 T TABLE XIX Blood osmotic concentrations (milliosmoles) of fis h in fresh water and in sea water. Species F. W. Blood mOsm S. W. Blood mOsm Reference; Oncorhynchus gorbuscha 313 333 Present study Oncorhynchus keta 330 343 Present study Anguilla.anguilla 346 428 Parry, 1966 Gf.sterosteus aculeatus 290 340 Lange and Fugelli, 1965 Pleuronectes flesus 304 364 Lange and Fugelli, 1965 Salvelinus alpinus 328 431 Parry, 1966 Platichthys stellatus- 306 380 Parry, 1966 Salmo salar 328 344 Parry, 1966 Salmo trutta 326 356 Gordon, 1959 - 72 -Black ( 1 9 5 1 ) who observed high mortality in coho fry transferred to 27 - 31°/oo sea water which she correlated with high body chloride concentrations at 24 hr. E. Comparison of Mechanisms of Osmotic and Ionic Regulation in Embryos,  Alevins and Fry When the osmotic concentrations and LD^Q values are compared among the five species at three ages, several significant observations can be made. The alevins of a l l five species have LD_„ values about half those of their respective embryos. This observation is contrary to the generally held belief that osmotic and ionic regulatory a b i l i t y increases with age. These results are explained by the probable fact that embryos can take up.net amounts of ions but not take up or lose net amounts of water, whereas alevins can take up both net amounts of ions and water. It may in fact be argued that the net loss of water and the net gain of ions proceed at approximately equal rates or have equal lethal effects since two of these factors cause the median death to occur about twice as fast as one factor. In addition to this difference, the pronephros of the late embryo may be better able to handle the osmotic and ionic problem in sea water than the mesonephros of the early alevin. The notion of increasing sali n i t y tolerance with age is well established in the literature, but evidence for i t between late embryos and early alevins is lacking. In fact, the existing evidence for alevins is contradictory. Moore (1900), for example, observed that resistance times of Salmo trutta larvae (alevins) in saline solutions did not increase with age as expected and Rockwell (1956) found that in 32°/oo sea water, pink alevins, two days after hatching, have an LD 5 Q of 216 hr but an LD^Q of 84 hr 40 days after hatching. Although herring larvae lack g i l l s and kidneys, the larvae manifest a wider salinity tolerance than adults (Hol-liday and Blaxter, 1960). - 73 -Similarly, Parry (1960) found that Salmo salar alevins one week after hatching had an LDC_. of 8 hr in 100% sea water, but at six weeks the LD.^ was 0.5 hr, bU bu whereas Auvergnat and Secondat (1941) observed greater osmoregulatory a b i l i t i e s in late S_. salar alevins than in early alevins. And f i n a l l y , the work.of Rutter (1902), as shown in Table XX, indicates that while the time to 100% mortality increases with age, LD^, calculated from his results, decreases or remains the same for chinook alevins 6, 12 and 19 days after hatching. We, therefore, have the anomaly of increasing salinity resistance by some members of a population but decreasing or unchanging salinity resistance by the median individual of that population. The increase in LD^^Q values with increasing age may be due to rapid growth of only a few individuals while decreasing LD<_Q values may be the result of the inherent periodicity of salinity physiology present in salmonidae as postulated by Hoar (1965). However, i t is doubtful that the phenomenon of decreased alevin salin i t y resistance over that of embryos can be explained by this hypothesis, since the experiments: on: Pacific salmon were run from December 1965 to April 1966 (Table I ) . There is considerable evidence for increased tolerance of post-alevin fis h with increasing age (Black, 1951; Parry, 1958, 1960; Mclnerney, 1963 as cited by Hoar, 1965; Conte and Wagner, 1965 and Conte et_ a l . , 1966). The work of Parry (1960), Conte and Wagner (1965) and Conte et_ a l . (1966) has clearly shown that the increase in salin i t y tolerance is not due to chronological age per se but to increased size. Conte and Wagner have suggested that there is a c r i t i c a l size, 14 - 15 cm at which steelhead trout become tolerant of sea water. This c r i t i c a l size appears to be 12 - 15 cm for Salmo salar (Parry, 1960) and 7 - 8 cm for Oncorhynchus kisutch (Conte et a l . , 1966) and less than 6 cm for Oncorhynchus keta and 0_. gorbuscha but - 74 -TABLE,XX Approximate LD and LD (days) values of chinook alevins in 50%, 75% and 100% sea water as calculated from the data of Rutter (1902) • 50% S .W. 75% S.W. 100% S.W. Age* L D 5 0 L D i o o L D 5 0 L D 100 L D 5 0 . L D 100 6- - 4 2 3 1 2 12 5 8 1 4 1 2 19 2 12 * days after hatching - 75 -greater than 7 cm for Oncorhynchus tschawytscha (personal observations). If, the chloride secretory.cell theory is correct, there should be increasing numbers of acidophil cells in the g i l l s of larger fi s h (Parry, 1966). In fact, Threadgold and Houston (1964-) have shown that there.is an increase.in activity of these cells during heightened plasma electrolyte levels. Hoar (unpublished) and Nishida (1953) (both cited by Black, 1957) observed the d»=': -^lopment of acidophil cells (chloride secretory cells) before seaward migration of coho and masou respectively. The development and functional . capacity of these cells may be determined for each species at a particular size and account for some of the differences in osmoregulatory a b i l i t y observed in the five species of Pacific salmon. Although the a b i l i t y of the acidophil cells of the g i l l s to secrete chloride actively against a concentration gradient has not been absolutely established (Strauss, 1963; Parry, 1966), the evidence of Threadgold and Houston (1964), Philpott (1965) and F. P. Conte (personal communication) strongly support.the theory. The gut, as well, must play some role.in absorbing water and ions from the sea water (House and Green, 1965; Aull, 1966) which marine fi s h drink (Black, 1957). However, there is no evidence that this adaptation is dependent on the size or age of the f i s h . The increase in blood osmotic concentration of freshwater alevins over that of.freshwater embryos has been observed for a l l chum, coho, chinook and sockeye but not pink salmon. This difference may l i e in the fact that the chloride secretory cells may have developed early in the pinks as previously suggested. The phenomenon of increased blood osmotic concentration has been discussed. It has also been observed in embryos of S_. salar (Busnel et a l . , 1946), of S. trutta (Parry, 1961) as well as in chick embryos (Howard, 1957). - 76 -The fact that sodium-to-chloride ratios in fresh water and sea water are the same is contrary to the results of Houston (1959) who observed lower ratios in sea water f i s h . However, the average sodium-to-chloride ratio ,of 1.22 for alevin serum l i e s within the range 1.18 to 1.28,that Gordon (1959) observed for sexually mature sea trout S_. trutta while the .average ratio of 1.35 for fry plasma lie s outside this range. This difference may.be indicative of :he increased a b i l i t y of fry to .excrete chloride rather than increased concentrations.of other anions such as bicarbonate. It has previously been shown that longer.survival of embryos and fry than alevins in 31.8 °/oo sea water was probably due to greater osmoregulatory a b i l i t i e s of embryos and fry. On the basis of the foregoing discussion, this situation can be,explained further. The greater osmoregulatory a b i l i t i e s of embryos is probably due to the fact that embryos take up net amounts -; of ions • but not water whereas alevins can take up both.net amounts of water and ions. In the case of fry, their greater osmoregulatory a b i l i t y is due to increased size and possibly to the presence of chloride cells in the g i l l s as well as a more highly differentiated gut. F. Phylogenetic Interralations In Oncorhynchus It was speculated that testing of .the working hypotheses would provide further information on,the phylogenetic relations within the genus Oncorhynchus. Such speculation has been based previously on behavioural, ecological.and morphological studies (Hoar, 1958; Hikita, 1962). To these studies may be added the following: Jones (1963) observed species specific differences in electrophoretic blood protein patterns of adult salmon;.Baggerman (1960) reported differences in salinity preferences of young salmon,fry; Mclnerney (1963) found,species differences in the salinity tolerances of summer fry - 77 -and Tsuyuki and Roberts (1966) observed species-specificity in electrophoretic myogen patterns of Oncorhynchus. These studies and those of Foerster (1935), Brett (1952) and Kobayasi (1963) are included in Table XXI. It i s assumed that the ancestral oncorhynchid may have been a steelhead trout-like descendant of Salmo (Neave, 1958), which had already acquired an obligatory marine l i f e . This ancestral oncorhynchid was a stream or river dweller much like the masou and coho are today. From such a habitat, • evolution may have taken place in two other directions, that i s , towards a more marine l i f e (Neave, 1958) and towards a more freshwater existence. It is suggested that both directions have been exploited. The electrophoretic myogen patterns of pink- and sockeye salmon are very similar, but are most unlike those of coho and masou. While pink embryos are better osmoregulators than coho embryos, sockeye embryos are far worse. In other characteristics such as osmoregulation by alevins, schooling behaviour, elongate bodies as fry and numerous gill-rakers, pink and sockeye salmon are at- considerable variance with coho. Pink and sockeye, therefore, appear to represent the two extremes so far reached in the evolution of Oncorhynchus. On the one hand, pink embryos, alevins and fry show the greatest adaptation to marine l i f e while, sockeye embryos, alevins and fry on the other hand show the greatest adaptation to freshwater l i f e . The presence of intertidal spawning populations of pink salmon contrasts sharply with the incipient kokanee species of sockeye and i t s lacustrine habit. On this basis, the evidence in Table XXI has been used to draw a family tree of the genus Oncorhynchus (Figure 14). The masou, coho and chinook share common characteristics such as. parr marks and deep bodies as fry whereas the pink, chum and sockeye share different common features such as TABLE XXI Comparison of Pacific salmon. Presence of a character is indicated by + mark while i t s absence by 0. Intensity of activity is rated according to the number of + marks. Character Pink Chum Sockeye Coho Chinook Masou Reference . Salinity Resistance of Embryos ++++ ++++ ++ ++ +++ Present Study Osmoregulation by Embryos +++ +++ 0 + ++ Present Study Salinity Resistance of Alevins +++ ++ + + ++ Present Study Osmoregulation by Alevins ++ ++ 0 + 0 Present Study Na+ Regulation by Alevins ++ ++ 0 + 0 Present Study Cl- Regulation by Alevins ++ ++ + + 0 Present Study Salinity Resistance of Fry * * + + ++++ Present Study Osmoregulation by Fry ++++ ++++ + ++. ++ Present Study Na+ Regulation by Fry ++++ ++++ + + ++ Present Study Cl- Regulation by Fry ++++ ++++ + + ++ Present Study Intertidal Spawning + + 0 0 0 Rockwell, 1956 Salinity Tolerance of Summer Fry ++++ ++++ ++ + ++ Mclnerney, 1963 Salt Preference of Young Fry +++ +++ +++ + Baggerman, 1960 Similarity of Myogens to 0 + 0 +++ ++ ++++ Tsuyuki and S. gairdneri Roberts, 1966 River Residents 0 0 0 + Hoar, 1958 Lake Residents 0 0 ++ + Hoar, 1958 Fry Prefer Sea Water ++ ++ + 0 Hoar, 1958 Fry Hide Under Stones + ++ +++ +++ Hoar, 1958 Nipping Behaviour 0 + ++ +++. Hoar, 1958 Terr i t o r i a l Behaviour 0 0 + ++ Hoar, 1958 Schooling Behaviour +++ ++ +++ + Hoar, 1958 Smolt Transformation 0 0 + + + Hoar, 1958 Increased Activity at Night + + + 0 Hoar, 1958 Parr Marks: Alevins 0 + +++ +++ +++ +++ Hikita, 1962 Parr Marks: Fry 0 + +++ +++ +++ +++ Hikita, 1962 Fry Body Elongate + + + 0 0 0 Hikita, 1962 Number of G i l l Rakers + 0 + 0 0 0 Hikita, 1962 Number of Pyloric Caeca + + 0 0 + Hikita, 1962 Blood Proteins ++ + + + 0 Jones, 1963 Hybridization ++ ++ ++ + Foerster, 1935 Hybridization ++++ ++++ Kobayasi, 1963 High Temperature Tolerance + + ++ +++ +++ Brett, 1952 * Pink and chum fry do not resist sea water, they tolerate i t . - 79 -FIGURE 14 Phylogenetic relationships of Oncorhynchus Salmo Masou Coho Chinook Sockeye Chum Pink - 80 -schooling behaviour, electrophoretic myogen patterns and elongate bodies as fry. Therefore, the pink-chum-sockeye group has been shown as diverging from the masou-coho-chinook group. Within the masou-coho-chinook group, the salinity resistance, osmotic and ionic regulation of fry, salinity tolerance of young fry, smolt transformation, high temperature tolerance and electrophoretic myogen patterns suggest that coho and chinook are very closely related but that chinook are somewhat more highly evolved. In addition, the electrophoretic myogen patterns of masou are more similar to Salmo gairdneri than are coho and chinook patterns. Separation within the pink-chum-sockeye group is more d i f f i c u l t for Foerster (1935), Kobayasi (1963) and Jones (1963) observed great similarities among this group. The data on osmotic and ionic regulation indicate that sockeye embryos, alevins and fry are more poorly adapted for marine l i f e than are coho•and chinook. These results di f f e r from those'of Hunter (cited by Hoar, 1958) who found that sockeye fry could be readily adapted to dilute sea water and from i t s earliest stages, was much more resistant to sea water than coho. Mclnerney (1963) also observed greater salinity tolerance in sockeye fry over that of coho fry. In addition, Baggerman (1960) has shown that the salt water preference of sockeye fry is maintained from April u n t i l the end of August (except in June, when no preference was shown) and that in September the preference changed to fresh water. If this were the case for my sockeye, i t seems probable that as alevins they would have preferred sea water. However, because of their poor salin i t y resistance, a preference for sea water seems unlikely. Therefore, on the basis of physiological c r i t e r i a , including salinity resistance as observed in the present study and high - 81 -temperature tolerance as reported by Brett (1952), sockeye i s shown in Figure 14 as diverging earlier than chum from the pink-chum-sockeye group. This arrangement, however, does not agree with the evidence of Tsuyuki and Roberts (1966) who observed that the electrophoretic myogen patterns of pink salmon are more similar to those of sockeye than those of chum salmon. This discrepancy cannot be explained at this time. Because pink salmon manifest the greatest adaptation to marine l i f e among the Pacific salmon and, therefore, the greatest divergence from the steelhead trout-like ancester, they are accorded the position on the tree furthest from the masou. Figure 14, thus, derived in part from the physiological data obtained in the present research, agrees with the phylogenetic speculations of Hoar (1958). Therefore, the results of testing :the working hypotheses have provided further information on the phylogenetic relations within the genus•Oncorhynchus.. - 82 -LITERATURE CITED Aull, F. 1966. ..Absorption of f l u i d from isolated intestine of the toadfish, Opsanus tau. .Comp.Biochem.Physiol. 17:867-870. Auvergnat, R. and'Secondatj M. 1941. Influence des variations,de salinite'' sur l a pression osmotique des alevins vesicules de saumon migrateur (Salmo salar L.). Bull.Inst.oceanogr.Monaco.805.7p. Baggerman, B. 1960. Salinity preference, thyroid activity and the seaward migration of four species of Pacific salmon (Oncorhynchus). J.Fish.Res'.Bd.Can. 17:295-322. Black, V.S. 1951. Changes in body chloride, density and water content of chum (Oncorhynchus keta) and coho '(0_. kisutch) salmon fry when transferred from fresh water to sea water. J.Fish.Res.Bd.Can. 8:164-177. Black, V.S. 1957. Excretion and osmoregulation, p.163-205. In M.E;Brown (ed.) Physiology of fishes. Academic Press Inc., New York. Bogucki, M. 1930. Recherches sur la permeabilite'des membranes et sur la pression osmotique des oeufs salmonides. Protoplasma 9:345-369. Brett, J.R. 1952. Temperature tolerance in young Pacific salmon, genus Oncorhynchus. J.Fish.Res.Bd.Can. 9:265-323. Busnel, R.G., Drilhon, A. and Raffy, A. 1946. Recherches sur la physiologie. des salmonides. Bull.Inst.oceanogr.Monaco.893.23p. Callamand, 0. and Fontaine, M. 1940. La chloremie de.1'Anguille femelle au cours de son developpement. C.R.Acad.Sci., Paris 211:298. Clemens, W.A. and Wilby, G.V. 1961. Fishes of the Pacific coast of Canada. . Second edition Fish.Res.Bd.Can.Bull.68.443p. Conte, F.P. and Wagner, H.H. 1965. Development of osmotic and ionic regulation in juvenile steelhead trout Salmo gairdneri. Comp.Biochem.Physiol. 14:603-620. Conte, F.P., Wagner, H.H. , Fessler, J. and Gnose, C. 1966. Development of : osmotic and ionic regulation in juvenile coho salmon (Oncorhynchus  kisutch. 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Physical changes in the constituent, parts of developing eggs. Canad.J.Res. D.20:99-114. Hikita, T. 1962. Ecological and morphological studies of the genus Oncorhynchus (Salmonidae) with particular consideration on phylogeny. Sci.Rep.Hokkaido Salm.Hatch. 17:1-97. Hoar, W.S. 1958. The evolution of migratory behaviour among Juvenile salmon of the genus Oncorhynchus• J.Fish.Res.Bd.Can. 15:391-428. Hoar, W.S. 1965. -The endocrine system as a chemical link between the organism and i t s environment. Trans.roy.Soc.Can.(Ill.) 3:175-200. Holliday, F.G.T. and Blaxter, J.H.S. 1960. The effects of salin i t y on the developing eggs and larvae of the herring. J.Mar.biol.Ass.U.K. 39:591-603. Holliday, F.G.T. and Jones, M.P. 1965. Osmotic regulation in the embryo of the herring (Clupea harengus). J.Mar.biol.Ass.U.K. 45:305-311. House, CR. and Green, K. 1965. Ion and water transport in isolated intestine of the marine.teleost, Cottus scorpius. J.exp.Biol. 42:177-189. Houston, A.H. 1959. Osmoregulatory adaptation of the steelhead trout (Salmo  gairdnerii Richardson) to sea water. Canad.J.Zool. 37:729-748. Howard, E. 1957. Ontogenetic changes in the freezing point and sodium and potassium content of the subgerminal f l u i d and blood plasma of the chick embryo. J.cell.comp.Physiol. 50:451-470. Jones, H. 1963. A comparative study of the blood proteins of five species of Oncorhynchus using starch-gel electrophoresis. M.Sc. Thesis, Univ.B.C. 70p. - 84 -Jones, M.P. 1966. The .ultrastructure of the epidermis,.of larvaeof the herring (Clupea harengus) in relation to the rearing sa l i n i t y . J.Mar.biol.Ass.U.K. 46:-235-240. Kaighn, M.E. 1964. A biochemical study,of the hatching process in Fundulus. heteroclitus. Developm.Biol. 9:56-80. Kao, C.Y., Chambers, R. and Chambers, E.L. 1954. Internal hydrostatic pressure of the Fundulus egg. II Permeability of'.the chorion. J.cell.comp.Physiol. 44:447-461. Kobayasi, H. 1963. Some.cytological observations on f e r t i l i z a t i o n in, the cross, between the dog-salmon,and the pink-salmon. International J.Cytol. 28:365-375. Krogh,.A. and Ussing, H.H. 1937i A note on the permeability of•trout eggs to, D20 and H20. J.exp.Biol. 14:35-37. Kubo, T. 1955. Changes in some characteristics of blood of smolt.of Oncorhynchus masou during seaward.migration. Bull.Fac;Fish. Hokkaido. 6:201-207. Lange, R. and Fugelli, K. 1965. The osmotic .adjustment in the euryhaline tel^osts, the flounder, Pleuronectes flesus :L. and the ,three-spinedT 'tickleback Gasterosteus aculeatus ;L. Comp.Biochem.Physiol. 15:283-292. Mclnerney, J*E. 1963., Salinity preference - an orientation mechanism in salmon migration. Ph.D."Thesis. Univ.B.C. 63 p. Moore, A. 1900. Further evidence of.the poisonous.effects of a pure NaCl solution. Amer.J.Physiol. 4:386-396. Munz, F.W., McFarland, W.N. 1964. Regulatory function of a primitive kidney. Comp.Biochem.Physiol. 13:381-400. Neave, F. 1958. The origin and.speciation of -Oncorhynchus. Trans;roy.Soc.Can.(V.) 52:25-39. Nikol'skii, G.V. 1961. Special ichthyology. Israel Program for Scientific Translations Ltd., Jerusalem. 538 p. Nishida, ti'. 1953. The cyto-histological. observations . on the- gland c e l l of, the branchial epidermis with the comparison of;two types.of Oncorhynchus masou, land-locked and sea-run forms v Sci.Rep.Hokkaido Salm.Hatch. 8:33-37. Parry, G. 1958. Size and osmoregulation in fishes. Nature, Lend. 181:1218-1219. Parry, G. 1960. . The development of salinity tolerance in the salmon, Salmo  salar L. and some related species. J.exp.Biol. 37:425-434. - 85 -Parry, G. 1961. Osmotic and ionic changes in blood and muscle of migrating salmonids. J.exp.Biol. 38:411-427. Parry, G. 1966. Osmotic adaptation in fishes. Biol.Rev. 41:392-444. Philpott, C.W. .196.5. Halide localization in the teleost chloride c e l l and i t s identification by selected area electron diffraction. Protoplasma 60:7-23. Ramsay, J.A. and Brown., R.H.J. 1955. Simplified apparatus and procedure for freezing-point determinations upon small volumes of f l u i d . J..sci.Instrum. 32:372-375. Rockwell., J. Jr. 1956. Some effects of sea water and temperature on the embryos of the Pacific salmon, Oncorhynchus gorbuscha (Walbaum) and Oncorhynchus keta (Walbaum). Ph.D. Thesis. Univ. Washington. 416 p. Rucker, R.R. 1949. Facts and f i c t i o n in spawntaking: Addenda. Progr.Fish Cult. 11:75-77. Rutter, C. 1902. Natural history of the quinnat salmon. A report of investigations in the Sacramento River 1896-1901. Bull.U.S.Fish. 22:65-142. SkucL, B.E-. 1954. .-Salinity gradients in the intertidal zone of an Alaskan pink salmon stream. Fish.Bull., U.S. 56:177-185. Steel, R.G.D. and-Torrie, J.H. 1960. Principles and procedures of s t a t i s t i c s . McGraw-Hill Book Co., Toronto. 481 p. Strauss, L.P. 1963. A study of the fine structure of the so-called chloride c e l l in the . g i l l of the guppy Lebistes reticulatus P. 'Physiol.Zool. 36:183-198. •Svetlov, P. 1929. Entwicklungsphysiologische beobachtungen.an forelleneiern. Roux Arch.EntwMech.Organ. 114:771-785. Threadgold, L.T. and Houston, A.H. 1964. An electron microscope study of the "chloride c e l l " of Salmo salar L. Exp.Cell Res. 34:1-23. Tsuyuki, H. and Roberts, E. 1966. Inter-Species Relationships within the .genus Oncorhynchus based on biochemical systematics. J.Fish.Res.Bd.Can. 23:101-107. Wilier, A. and Trahms, O.K. 1942. Versuche uber den einsatz von salmonidenbrut in meerwasser. Z.Fisch. 40:5-40. Winnicki, A. 1958. Waterless environment as a factor causing a prolongation of embryonic development of the brook trout (Salmo fontinalis Mitch.). Zool.Polon. 9:131-139. Zotin, A. 1965. The uptake and movement.of water in embryos. Symp.Soc.exp.Biol. 19:365-384. - 86 -APPENDIX TABLE I Orthogonal.contrast integers.for the comparison of the difference in serum osmotic concentrations of pink/chum in,0 and 24 hr sea water against the difference in chinook levels at 0 and 24 hr sea water. Pink Chum Coho Chinook Sockeye. 0 +1 +1 0 -2 Q 4 0 0 0 0 0 8 0 0 0 G 0 12 0 0 0 0 0 24 -1 -1 0 + 2 0 TABLE II • Orthogonal contrast integers for the comparison of the difference : in serum osmotic concentrations.of coho in.O and.24 hr.sea water against the difference in sockeye levels at 0 and 24 hr sea water. Pink Chum Coho Chinook Sockeye. 0 Q 0 +1 : 0 -1 4 0 0 0 • 0 0 8 0 0 0 0 0 12 0 0 0 0 0 24 0 0 -1 0 +1 TABLE III Regression equations of serum osmotic concentrations of embryos plotted against exposure time in 31.8°/oo sea water. P values indicate the significance of the regression slopes. Species Regression Equation P Pink Y.= 347.14 + 7.23X -0.22X 0.05 Chum Y = 343.33 + 7.34X -0.22X2 0.01 Coho Y= 310.30 + 10.55X 0.005 Chinook Y = 342.01 + 5.09X• 0.005 Sockeye ' Y.= 309.14 + 13.23X 0.005 TABLE IV Orthogonal contrast integers for the comparison of the difference in serum osmotic concentrations.of pink/chum in 0 and 12 hr sea water against.the difference in coho levels at 0 and 12 hr sea water. Pink Chum : Coho; Chinook Sockeye 0 +1 +1 -2 0 0 4 0 0 0 0 0 . 8 0 0 0 0 0 12 -1 -1 + 2 0 0 TABLE V Orthogonal contrast integers for the comparison of the .difference in serum osmotic . concentrations of chinook in 0 and 12 hr sea water against.the difference in sockeye levels at 0 and 12 hr sea water. . ' Pink Chum Coho Chinook • Sockeye 0 0 0 0 +1 -1 4 0 0 0 0 0 • 8 0 0 0 0 0 12. i 0 0 0 -1 +1 TABLE VI Orthogonal contrast integers for the comparison of the difference-, in serum osmotic concentrations,of pink in 0 and 12 hr sea water against the difference.in chum levels at 0 and 12.hr sea water. Pink Chum Coho Chinook Sockeye 0 + 1 - 1 0 0 0 4 0 0 , 0 0 0 8 0 0 0 0 0 12 -1 +1 0 0 0 - 88 -TABLE VII Regression equations.of serum osmotic concentration of alevins plotted against exposure time in 31.8°/oo,sea water. P values indicate the significance of the regression slopes. Species Regression Equation P Pink Y = 338.97 + 33.24X -2.34X 0.005 Chum Y = 388.47 + 4.58X 0.025 Coho Y = 353.23 + 10.64X 0.005 Chinook Y = 37'4.80 .+ 11.24X 0.005 Sockeye Y = 359.57 + 16.02X 0.005 TABLE VIII Orthogonal contrast integers for the .comparison of the difference in serum Na+ concentrations of pink/chum alevins in 4 , and 8 hr. sea water against the difference in coho alevin levels at 4 and 8 hr sea water. Pink Chum Coho. Chinook Sockeye 0 0 0 0 Q 0 4 -1 -1 +2 0 0 8- +1 +1 -2 0 0 12 0 0 0 0 0 TABLE IX Orthogonal contrast integers for the comparison of the .difference in serum Na+ concentrations of pink alevins in 4 and 8 hr sea water against the difference.in chum alevin levels at 4 and .8 hr sea water. Pink Chum Coho. Chinook Sockeye 0 0 0 0 0 0 4 - 1 + 1 0 0 0 8 + 1 - 1 0 0 0 12 0 0 0 0 0 - 89 -TABLE X Orthogonal contrast integers for the comparison of the difference in serum Na+ concentrations of pink alevins in 0 and 12 hr sea water against the difference in chum alevin levels at 0 and 12 hr sea water. Pink Chum Coho Chinook. Sockeye 0. +1 -1 0 0 0 4 0 0 0 0 0 8 0 0 0 0 0 12 -1 +1 0 . 0 0 TABLE,XI Orthogonal contrast integers for the comparison of the .difference in serum Na+ concentrations of• pink/chum alevins in 0 and 12 hr sea water against the difference in coho alevins at 0 !and 12..hr sea water. Pink Chum Coho Chinook Sockeye 0 +1 +1 -2 0 ! 0 4 0 0 0 0 ' 0 8 0 '0 0 0 . • 0 12 -1 -1 + 2 0 0 TABLE XII Regression equations, of serum Na+ concentrations of alevins plotted,against exposure time in 31.8°/oo sea water. P values indicate the .significance of the regression slopes. Species Regression Equation, P Pink Y = 136.75 + 2.46X 0.005 Chum Y = 155.23 + 1.85X 0.005 Coho Y =146.97 + 3.99X 0.005 Chinook Y = 145.53 + 6.59X 0.005 Sockeye. , Y = 141.40 + 6.89X 0.005 - 90 -TABLE XIII Regression equations of serum Cl- concentrations of alevins plotted against-exposure time in 31.8 °/oo sea water. P vali indicate the significance of the regression;slopes. Species Regression.Equation Pink Y = 91.67 + 9.63X -0.55X 0.005 Chum Y. = 120.30+ 1.70X 0.005 Coho Y = 121.97 + 4 . 0 8 X 0 . 0 0 5 Chinook Y = 125.90 + 5.98X 0.005 Sockeye Y = 107.10 + 12.44X -0.55X 0.005 TABLE XIV Orthogonal contrast integers for the comparison of the difference. in plasma osmotic concentrations of chum fry in 8 and 12 hr sea : water against the difference in chinook levels at 8 and 12 hr sea water. Pink Chum Coho Chinook Sockeye 0 0 0 0 0 0 4 0 0 0 0 0 8 0 + 1 0 -1 0 12 0 - 1 , 0 +1 0 TABLE XV Regression equations of plasma osmotic concentrations of fry plotted against exposure time in.31.8G/oo sea water. P values indicate,the significance of the regression slopes, Species Regression Equation P Pink Y = 319.93 + 3.11X 0.025 Chum No- significant regression n Coho ' Y =313.07 + 18.56X -0.88X 0.005 Chinook Y = 326.07 + 6.73X 0.005 Sockeye Y = 320.85 + 22.59X -1.11X 0.005 - 91 -TABLE XVI Regression equations of plasma Na+ concentrations'of fry plotted against exposure time in 31.8°/oo sea water. P values indicate the significance of the regression slopes. S£ e c i e s Regression Equation Pink Chum Coho Chinook Sockeye No significant regression . Y = 161.42 T 6.19X -0.48X2 Y - 156 40 + 4.52X Y = 164.37 + 2.30X Y = 146.78 + 12.45X -0.68X' 0.05 0.005 0.005 0.01 TABLE .XVII . Regression equations of plasma Cl-,concentrations of fry plotted against exposure time in 31.8°/oo sea water. P-values indicate the significance of the regression slopes.' Species Regression Equation P Fink Y = 110.42 + 5.48X -0.41X2. 0.005 Chum Y = 118.43 + 4.36X -0;30X 0.05 Coho Y = 116.23 + 3.31X 0.005 Chinook Y = 120.50 + 2.00X 0.005 Sockeye Y = 117.37 + 7.30X -0.42X 0.05 TABLE,XVIII Regression equations of plasma Mg++ concentrations of fry plotted against,exposure time in 31.8°/oo sea.water. P values indicate the significance of the regression slopes. Species Regression Equation . . P -,; Pink Y =2.529 + 0.2884X -0.0210X2 0.05 • Sockeye Y = 3.2828 + 0.2134X 0.005 Chum, coho and chinook data cannot be fi t t e d to either the straight line or curvilinear regressions - 92 -TABLE XIX Orthogonal contrast integers for the comparison of the difference between embryo and fry osmotic concentrations of pink/chum against-the difference,of embryo and fry levels of coho/chinook/sockeye. Pink Chum Coho Chinook Sockeye Embryo + 3 + 3 -2 -2 -2 Alevin 0 0 0 0 0 Fry -3 -3 +2 + 2 + 2 TABLE XX. Orthogonal contrast integers for the comparison of the difference between embryo and fry osmotic concentrations of pink against:the difference of embryo ' and fry levels of chum, Pink Chum Coho Chinook' Sockeye Embryo -1 +1 0 0 0 Alevin 0 0 0 0 0 Fry +1 . -1 0 0 0 TABLE XXI Orthogonal contrast integers for the comparison of the difference between embryo and fry osmotic concentrations of coho/chinook against .the difference of embryo and fry levels of sockeye. Pink Chum Coho Chinook Sockeye Embryo 0 0 - 1 -1 +2 Alevin 0 0 0 0 0 Fry 0 0. +1 +1 -2 - 93 -TABLE XXII • Orthogonal contrast integers for the comparison of the difference between embryo.and fry osmotic concentrations of coho against:the difference of embryo and fry levels of chinook. Pink Chum Coho Chinook Sockeye Embryo 0 0 -1 + 1 0 Alevin 0 0 0 0 0 Fry 0 0 +1 -1 0 TABLE XXIII Orthogonal contrast integers for the comparison of the difference between embryo/fry and alevin osmotic concentrations of pink/chum against the difference of embryo/fry and alevin levels of coho/chinook/sockeye. Pink Chum Coho Chinook Sockeye Emb r y o + 3 + 3 -2 -2 -2 •/in -6 -6 +4 +4 +4 Fry + 3 + 3 -2 -2 -2 TABLE XXIV Orthogonal contrast integers for the comparison of the difference between embryo/fry and alevin osmotic concentrations of pink against the difference of embryo/fry and alevin levels of chum. Pink Chum Coho Chinook Sockeye Embryo +1 -1 0 0 0 . Alevin -2 + 2 0 0 0 Fry +1 -1 0 0 0 - 94 -TABLE XXV  Orthogonal contrast integers for the comparison of the difference between embryo/fry and alevin osmotic concentrations of coho/chinook against the difference of embryo/fry and alevin levels of sockeye. Pink Chum Coho Chinook Sockeye Embryo 0 0 + 1 +1 -2 Alevin 0 0 - 2 -2 +4 Fry 0 0 + 1 +1 -2 TABLE XXVI Orthogonal contrast integers for the comparison of the difference between embryo/fry and alevin osmotic concentrations of coho against the difference of embryo/fry and alevin levels of chinook. Pink Chum Coho Chinook Sockeye Embryo 0 0 +1 -1 0 Alevin 0 0 - 2 + 2 0 Fry 0 0 + 1 - 1 0 TABLE XXVII Orthogonal contrast integers for the comparison of the difference between embryos/fry vs alevin osmotic concentrations of pink/chum in 0 and 12 hr sea water against the difference between embryos/fry vs alevin levels of coho/chinook/sockeye in 0 and 12 hr sea water. Pink Chum Coho Chinook Sockeye E 1 A 2 F 3 E A F E A F E A F E A F 0 + 3 -6 + 3 + 3 -6 + 3 -2 +4 -2 -2 +4 -2 -2 +4 -2 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12 -3 + 6 -3 -3 + 6 -3 + 2 -4 + 2 + 2 -4 +2 + 2 -4 + 2 embryo alevin fry 

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