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UBC Theses and Dissertations

Influence of incubation salinity and temperature and post-hatching temperature on salinity tolerance… Dueñas, Corazon Echevarria 1981

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INFLUENCE OF INCUBATION SALINITY AND TEMPERATURE AND POST-HATCHING TEMPERATURE ON SALINITY TOLERANCE OF PACIFIC HERRING (Clupea p a l l a s i Valenciennes) LARVAE by CORAZON ECHEVARRIA DUENAS B.Sc., U n i v e r s i t y of San C a r l o s , P h i l i p p i n e s , 1976 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1981 © Corazon E c h e v a r r i a Dueftas, 1981 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Z O O U O G Y  The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e J u W SLA-. 1 3 & l / i n \ ABSTRACT A study was conducted t o det e r m i n e the e f f e c t s of i n c u b a t i o n s a l i n i t y and temperature . and p o s t - h a t c h i n g temperature on s a l i n i t y t o l e r a n c e of P a c i f i c h e r r i n g (Clupea  pa 1 1 a s i V a l e n c i e n n e s ) l a r v a e . C a l o r i m e t r y e x p e r i m e n t s were a l s o conducted on h e r r i n g l a r v a e t h a t were i n c u b a t e d and r e a r e d i n v a r i o u s c o m b i n a t i o n s of s a l i n i t y and t e m p e r a t u r e . In the s a l i n i t y t o l e r a n c e t e s t , h e r r i n g eggs were a r t i f i c i a l l y f e r t i l i z e d i n t h r e e s a l i n i t i e s (13, 21 and 29°/ooS) at 6°C. Newly f e r t i l i z e d eggs were i n c u b a t e d i n the same s a l i n i t i e s as those i n which they were f e r t i l i z e d at two l e v e l s of i n c u b a t i o n temperature (6 and 12°C), making s i x i n c u b a t i o n groups. D u r i n g the peak of the h a t c h i n g p e r i o d , groups of l a r v a e from each i n c u b a t i o n group were exposed t o a s e r i e s of t e s t s a l i n i t i e s , r a n g i n g from 20 t o 55°/ooS a t t h r e e l e v e l s of p o s t -h a t c h i n g t e m p e r a t u r e s (6, 9 and 12°C). S a l i n i t y t o l e r a n c e was measured by d e t e r m i n i n g the m o r t a l i t y of l a r v a e o c c u r r i n g i n each t e s t s a l i n i t y over a p e r i o d of 72 h o u r s . Two approaches were employed: 1) t i m e - m o r t a l i t y , and 2) d o s a g e - m o r t a l i t y . The former p r o v i d e d e s t i m a t e s of the e f f e c t i v e time t o 50% m o r t a l i t y ( E T 5 0 ) , the l a t t e r , the e f f e c t i v e dosage ( s a l i n i t y ) t o 50% m o r t a l i t y ( E D 5 0 ) . The E T 5 0 or E D 5 0 v a l u e s were used as bases f o r comparison of the responses of l a r v a e t o i n c u b a t i o n temperature and s a l i n i t y , and p o s t - h a t c h i n g t e m p e r a t u r e . The r e s u l t s i n d i c a t e t h a t both i n c u b a t i o n s a l i n i t y and temperature e x e r t a s i g n i f i c a n t i n f l u e n c e on the s a l i n i t y t o l e r a n c e of l a r v a e . Eggs i n c u b a t e d i n the h i g h e s t s a l i n i t y (29°/ooS) and lower temperature (6°C) produced l a r v a e most t o l e r a n t t o h i g h e r p o s t -h a t c h i n g s a l i n i t i e s . P o s t - h a t c h i n g temperature a l s o showed a p o s s i b l e i n f l u e n c e , a l t h o u g h the t r e n d s seen were not s t a t i s t i c a l l y s i g n i f i c a n t . S a l i n i t y t o l e r a n c e of l a r v a e h a t c h i n g from the 6°C i n c u b a t i o n t e m p e r a t u r e appeared t o be maximized a t p o s t - h a t c h i n g t e m p e r a t u r e s between 6 and 9°C; whereas, those l a r v a e from the 12°C i n c u b a t i o n temperature showed maximum s a l i n i t y t o l e r a n c e a t 9 to 12°C p o s t - h a t c h i n g t e m p e r a t u r e s . The response. o f . l a r v a e t o s a l i n i t y may be a r e s u l t of an i r r e v e r s i b l e n o n - g e n e t i c a d a p t a t i o n of the embryo t o s a l i n i t y and t e m p e r a t u r e d u r i n g i n c u b a t i o n . In t he c a l o r i m e t r y e x p e r i m e n t s , samples of h e r r i n g l a r v a e were combusted i n a microbomb c a l o r i m e t e r t o det e r m i n e c a l o r i c v a l u e s . These l a r v a e were h a t c h e d and r e a r e d i n the same s i x c o m b i n a t i o n s of i n c u b a t i o n s a l i n i t i e s and tempe r a t u r e s as those used i n t h e main experiment. L a r v a e were sampled a t day 0 (peak of h a t c h i n g ) , 3, 6 and 9. S t a n d a r d l e n g t h s , d r y w e i g h t s , ash-f r e e d r y w e i g h t s and ash c o n t e n t of the l a r v a l samples were a l s o t a k e n . There was a c o n s i d e r a b l e v a r i a b i l i t y i n the d a t a f o r c a l o r i m e t r y of the h e r r i n g l a r v a . In g e n e r a l , l a r v a e from a l l t r e a t m e n t s , a t h a t c h i n g , averaged 6,586 c a l / g a s h - f r e e d r y weight and had a mean ash v a l u e of 8.25%, o b t a i n e d by d i r e c t a s h i n g . Mean s t a n d a r d l e n g t h of l a r v a e a t the peak of h a t c h i n g (day 0) ranged from 7.67 mm (12°C, 29°/ooS) t o 9.93 mm (6°C, 13°/ooS). There was a t r e n d towards l a r g e r l a r v a e ( g r e a t e r s t a n d a r d l e n g t h ) a t lower t e m p e r a t u r e s i n lower s a l i n i t i e s i n a l l ages t e s t e d . Data on dry w e i g h t s show a l i n e a r d e c r ease i n i v l a r v a l (body and y o l k ) weight from day 0 t o day 9. Mean d r y weight of l a r v a a t the peak of h a t c h i n g ranged from 0.142 mg (6°C, 13°/OOS) t o 0.162 mg (12°C, 21°/ooS; 12°C, 29°/ooS). At day 0, the l a r g e s t l a r v a e ( i n terms of dry weight) were found a t h i g h e r t e m p e r a t u r e s i n h i g h e r s a l i n i t i e s , w h i l e a t day 9 (past y o l k - s a c s t a g e ) , the l a r g e s t l a r v a e were found a t lower t e m p e r a t u r e s i n lower s a l i n i t i e s . The same t r e n d was obser v e d i n terms of a s h - f r e e d r y w e i g h t . V TABLE OF CONTENTS A b s t r a c t 1 1 Table of Contents v L i s t of Tables v i L i s t of. F i g u r e s and Photographs v i i Acknowledgments v i i i I n t r o d u c t i o n 1 M a t e r i a l s and Methods 6 1. C o l l e c t i o n of Spawners 6 2. Spawning and F e r t i l i z a t i o n 6 3. Incubation 7 4. S a l i n i t y Tolerance T e s t s of Larvae 15 a) 1979 Experiment ( T i m e - M o r t a l i t y Approach) 16 b) 1980 Experiment (Dosage-Mortality Approach) .... 17 c) S t a t i s t i c a l A n a l y s i s 18 (1 ) 1979 Data 18 (2) 1980 Data 19 5. C a l o r i m e t r y 22 R e s u l t s 29 1. S a l i n i t y Tolerance T e s t s 29 a) 1979 Experiment 29 b) 1980 Experiment 34 2. C a l o r i m e t r y (1979 T r i a l s ) 49 a) Unequal Sample S i z e 49 b) Standard Length of Larvae 49 c) Dry Weight of Larvae 54 d) C a l o r i e Determination 59 (1) Percent Unburned Organic Residue 59 (2) Percent Ash from D i r e c t and I n d i r e c t Ashing Methods 59 (3) C a l o r i c Values of H e r r i n g Larvae 59 D i s c u s s i o n 67 1. E f f e c t of Incubation S a l i n i t y 67 2. E f f e c t of Incubation Temperature 71 3. E f f e c t of Post-Hatching Temperature and Combined E f f e c t s of the Three F a c t o r s 72 4. C a l o r i m e t r y 75 5. E c o l o g i c a l I m p l i c a t i o n s 78 L i t e r a t u r e C i t e d 81 v i LIST OF TABLES Table 1: Range of Length and Weight Measurements Taken i n He r r i n g Spawners 9 Table 2: Experimental Design f o r the S a l i n i t y T olerance T e s t s 14 Table 3: Number of Larvae Sampled f o r C a l o r i m e t r y 24 Table 4: Estimates of E D 5 0 f o r the 1979 Experiments 31 Table 5: Summary of A n a l y s i s of Variance f o r the E D 5 0 Values of the 1979 Experiment 33 Table 6: Estimates of E D 5 0 and T h e i r Mean Values f o r the. 1980 Experiment 36 Table 7: Summary of A n a l y s i s of V a r i a n c e f o r the E D 5 0 Values of the 1980 Experiment 40 Table 8: Comparison of Mean E D 5 0 Values of H e r r i n g Larvae i n R e l a t i o n to Incubation S a l i n i t y and Incubation Temperature 43 Table 9: Table of Means Showing Lengths, Weights and C a l o r i c Values of H e r r i n g Larvae 53 Table 10: Table of Means Showing Dry Weights of P e l l e t s Combusted i n Bomb C a l o r i m e t e r , Percent Unburned Organic Residue and Percent Ash Obtained from D i r e c t and I n d i r e c t Methods 61 Table 11: Summary of A n a l y s i s of Variance f o r the c a l / g Ash-Free Dry Weight of H e r r i n g Larvae 66 v i i LIST OF FIGURES AND PHOTOGRAPHS Fi g u r e 1 : 11 Part a: H e r r i n g Egg Incubator i n Egg Incubation Tank .. 11 Part b: Incubator Showing Four Compartments and Incubation C e l l s 11 Fi g u r e 2: . Dosage-Mortality Graph Showing G r a p h i c a l Method of E s t i m a t i n g E D 5 0 f o r 1980 Experiment 21 F i g u r e 3: T y p i c a l G r a p h i c a l Combustion Record and Standard Method of C a l c u l a t i n g " T o t a l C o r r e c t e d R i s e " 28 Fi g u r e 4: Mean E D 5 0 Values ( C a l c u l a t e d from Three R e p l i c a t e s ) of H e r r i n g Larvae Subjected to the S a l i n i t y Tolerance Test (1980 Experiment) 38 Fi g u r e 5: I n t e r a c t i o n between Incubation S a l i n i t y and Incubation Temperature 45 F i g u r e 6: Comparison of Mean E D 5 0 Values of H e r r i n g Larvae i n R e l a t i o n t o : 48 Part a: C u l t u r e Temperature and Incubation Temperature 48 Part b: C u l t u r e Temperature and Incubation S a l i n i t y ... 48 Fi g u r e 7: Mean (± 1 SD) Standard Length (mm) of Her r i n g Larvae 51 Fi g u r e 8: C a l c u l a t e d Regression L i n e s f o r Mean Dry Weights of H e r r i n g Larvae Used f o r C a l o r i m e t r i c Experiment i n 1979 56 Fi g u r e 9: C a l c u l a t e d Regression L i n e s f o r Mean Ash-Free Dry Weights of He r r i n g Larvae Used f o r C a l o r i m e t r i c Experiment i n 1979 58 Fi g u r e 10: C a l c u l a t e d Regression L i n e s f o r the Mean C a l o r i e s per Larva 64 F i g u r e 11: E s t i m a t i o n of Upper L e t h a l S a l i n i t y L i m i t s of He r r i n g Larvae Exposed to V a r i o u s Combinations of S a l i n i t y and Temperature duri n g Incubation and L a r v a l P e r i o d 74 . v i i i ACKNOWLEDGMENTS I am v e r y much i n d e b t e d t o Dr. D. F. A l d e r d i c e and Dr. D. E. Hay of the P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B.C., f o r t h e i r generous h e l p throughout the v a r i o u s a s p e c t s of t h i s s t u d y . T h e i r v a l u a b l e a d v i c e , encouragement and c r i t i c i s m of the . ma n u s c r i p t a r e g r a t e f u l l y acknowledged. I a l s o w i s h t o thank my academic s u p e r v i s o r , Dr. W. S. Hoar, f o r h i s gui d a n c e , support and c r i t i c i s m of the t h e s i s ; and t o o t h e r members of the a d v i s o r y committee: Dr. H. E. K a s i n s k y , Dr. A. M. P e r k s and Dr. N. J . Wi1imovsky, f o r t h e i r v a l u a b l e s u g g e s t i o n s and c o n s t r u c t i v e c r i t i c i s m of the t h e s i s . My thank s a re a l s o g i v e n t o the s t a f f of the P a c i f i c B i o l o g i c a l S t a t i o n , e s p e c i a l l y t o the members of the P r o d u c t i o n O p t i m i z a t i o n s e c t i o n f o r p r o v i d i n g me w i t h l a b o r a t o r y space and equipment and a s s i s t a n c e i n the e x p e r i m e n t s , and t o the H e r r i n g I n v e s t i g a t i o n f o r s u p p l y i n g me w i t h h e r r i n g spawners. The ex p e r i m e n t s were conducted w i t h c o n s i d e r a b l e a s s i s t a n c e from F. P. J . V e l s e n , J . Jense n , C. Lam, A. Shimozawa, T r u d i e M u l l i n and Barb S m i t h . I am g r a t e f u l t o s e v e r a l f r i e n d s a t U.B.C. and the P a c i f i c B i o l o g i c a l S t a t i o n and my F i l i p i n o f r i e n d s f o r t h e i r a s s i s t a n c e i n v a r i o u s ways d u r i n g the c o u r s e of my graduate s t u d i e s . The graduate study program of t h i s a u t h o r has been f i n a n c i a l l y s u p p o r t e d by the I n t e r n a t i o n a l Development Research C e n t r e (IDRC) of Canada as p a r t of the f i n a n c i a l g r a n t t o the A q u a c u l t u r e Department of South e a s t A s i a n F i s h e r i e s Development C e n t r e (SEAFDEC) i n I l o i l o , P h i l i p p i n e s . I w i s h t o e x p r e s s my g r e a t a p p r e c i a t i o n t o both IDRC and my employer, SEAFDEC, f o r a l l o w i n g me t o pursue t h i s g raduate program. 1 INTRODUCTION P a c i f i c h e r r i n g (Clupea p a 1 1 a s i ) spawn i n the i n t e r t i d a l and s u b t i d a l r e g i o n s of the P a c i f i c Ocean c o a s t a l waters of Mexico, U.S.A. ( C a l i f o r n i a and A l a s k a ) , Canada, USSR, Japan and Korea ( A l d e r d i c e and V e l s e n , 1971; Hourston and Haegele, 1980). In B r i t i s h Columbia w a t e r s , they spawn from F e b r u a r y t o June, w i t h a peak p e r i o d g e n e r a l l y l a s t i n g from e a r l y March t o e a r l y A p r i l . Most h e r r i n g spawn i n waters w i t h s a l i n i t i e s (°/ooS) r a n g i n g from 24 t o 29 °/ooS (7.0 t o 9.9°C) (Humphreys and Haegele, 1976; Haegele and Humphreys, 1977; A l d e r d i c e e_t a l . , 1979). Under n a t u r a l c o n d i t i o n s , the eggs i n c u b a t e a t these s a l i n i t i e s and te m p e r a t u r e s and h a t c h e d l a r v a e a l s o a r e found i n the same s a l i n i t i e s . L a b o r a t o r y e x p e r i m e n t s demonstrate t h a t the i n c u b a t i o n s a l i n i t y range p r o v i d i n g maximum s u r v i v a l of eggs t o h a t c h i n g i s t h a t between 13 and 1 9 ° / O O S (7.6 t o 8.7°C) ( A l d e r d i c e and V e l s e n , 1971). In a f u r t h e r s t u d y , i t was deduced t h a t t h i s same s a l i n i t y range p r o b a b l y p r o v i d e s the l e a s t osmotic s t r e s s t o embryos ( A l d e r d i c e e_t a l . , 1979). Hence, t h e r e seems t o be a s u b s t a n t i a l d i f f e r e n c e between the s a l i n i t i e s p r o v i d i n g the l e a s t osmotic work f o r embryos and maximum s u r v i v a l of embryos t o h a t c h i n g , and the s a l i n i t i e s a t which eggs and l a r v a e a re found i n n a t u r e . Temperature i s c o n s i d e r e d a c o n t r o l l i n g f a c t o r i n most b i o l o g i c a l phenomena. Temperature i n f l u e n c e s m e t a b o l i c r a t e , which c o n t r o l s the r a t e of development i n t e l e o s t eggs. Many o t h e r p r o c e s s e s a r e i n f l u e n c e d i n d i r e c t l y by temperature a c t i n g 2 t h r o u g h m e t a b o l i c r a t e . R e s p i r a t o r y uptake of d i s s o l v e d oxygen and r a t e s of p r o d u c t i o n of m e t a b o l i t e s a re temperature dependent. Temperature n o r m a l l y - i n f l u e n c e s the v i a b i l i t y of development, t h e b i o k i n e t i c range v a r y i n g between s p e c i e s and s t a g e s of development w i t h i n s p e c i e s ( B r e t t , 1970). For P a c i f i c h e r r i n g eggs, t h i s range i s < 4 t o about 14°C or more ( A l d e r d i c e and V e l s e n , 1971). S a l i n i t y i n f l u e n c e s the i n t e r n a l o s m o t i c environment of the t e l e o s t egg. In the ovary of the a d u l t the u n f e r t i l i z e d ovum ap p r o x i m a t e s the b l o o d of the a d u l t i n osmotic c o n c e n t r a t i o n ( H o l l i d a y , 1965; H o l l i d a y and Jones, 1965). At t h i s s t a g e , the v i t e l l i n e membrane i s h i g h l y permeable t o i o n s moving a c r o s s i t . When the egg i s shed, i t tends r a p i d l y t o come i n t o e q u i l i b r i u m w i t h the s u r r o u n d i n g medium ( A l d e r d i c e e_t §_1. , 1979). A f t e r f e r t i l i z a t i o n , which i s almost synchronous w i t h spawning i n most t e l e o s t s , p e r m e a b i l i t y of the v i t e l l i n e membrane d e c r e a s e s r a p i d l y ; by the time the f i r s t few c e l l d i v i s i o n s have o c c u r r e d , the membrane has become v i r t u a l l y impermeable t o movement of i o n s a c r o s s i t . Hence, the spawning medium and i t s s a l i n i t y i n f l u e n c e the osmotic c o n c e n t r a t i o n of the y o l k of the newly f e r t i l i z e d egg, which i s m a i n t a i n e d a f t e r the v i t e l l i n e membrane becomes r e l a t i v e l y impermeable t o i o n movement a c r o s s i t . The os m o t i c c o n c e n t r a t i o n of the egg i n f l u e n c e s development p r o c e s s e s , i n c l u d i n g embryonic growth, a c t i v i t y , water c o n t e n t of the d e v e l o p i n g l a r v a l t i s s u e , r e g u l a t i o n of osmotic c o n c e n t r a t i o n of embryonic t i s s u e f o l l o w i n g b l a s t o p o r e c l o s u r e , and s u r v i v a l t o h a t c h i n g ( A l d e r d i c e e t §_1. , 1979). The c a p a c i t y f o r i o n i c and osmotic r e g u l a t i o n by t e l e o s t 3 eggs and those of o t h e r organisms may be i n f l u e n c e d by te m p e r a t u r e . These r e g u l a t o r y p r o c e s s e s c o u l d f a i l above and below c e r t a i n l i m i t i n g t e m p e r a t u r e s ( T a i t , 1972). On the o t h e r hand, t h e r m a l t o l e r a n c e of an organism may be i n f l u e n c e d by s a l i n i t y . Exposure t o extreme s a l i n i t i e s r e s u l t s i n an osmotic s t r e s s . Such s t r e s s c o u l d s h i f t the range of t h e r m a l t o l e r a n c e , p r o b a b l y due t o a r e d u c t i o n i n m e t a b o l i c e f f i c i e n c y ( K i n n e , 1963; Waugh and G a r s i d e , 1971). Other e n v i r o n m e n t a l f a c t o r s (oxygen, pH, h y d r o s t a t i c p r e s s u r e ) may a l s o a f f e c t the s a l i n i t y and t h e r m a l t o l e r a n c e of an organism, but the e x t e n t of t h e i r i n f l u e n c e i s not w e l l u n d e r s t o o d . The combined e f f e c t s of s a l i n i t y and temperature on t e l e o s t egg development have been noted i n s e v e r a l l a b o r a t o r y e x p e r i m e n t s ( A l d e r d i c e and F o r r e s t e r , 1967, 1971; A l d e r d i c e and V e l s e n , 1971; see re v i e w s by K i n n e , 1964a and Hempel, 1979). Kinne (1964a) mentioned two g e n e r a l t y p e s of s a l i n i t y -t e m p e r a t u r e i n t e r a c t i o n , namely: 1) l o w / h i g h -- h i g h / l o w and 2) low/low -- h i g h / h i g h . In the f i r s t t y p e , an organism has b e t t e r s u r v i v a l i n lower s a l i n i t i e s when a s s o c i a t e d w i t h h i g h e r t e m p e r a t u r e s , and i n h i g h e r s a l i n i t i e s w i t h lower t e m p e r a t u r e s . In t he second t y p e , b e t t e r s u r v i v a l i s a c h i e v e d i n both low s a l i n i t y - low temperature c o m b i n a t i o n s or i n h i g h s a l i n i t y -h i g h t e m p e r a t u r e c o m b i n a t i o n s . Among P a c i f i c h e r r i n g eggs, e v i d e n c e i n d i c a t e s t h a t a low/low -- h i g h / h i g h s a l i n i t y -temperature i n t e r a c t i o n a f f e c t s the development of eggs t o h a t c h i n g ( A l d e r d i c e and V e l s e n , 1971). That i s , eggs i n c u b a t e d i n lower s a l i n i t i e s show maximum s u r v i v a l (maximum h a t c h of v i a b l e l a r v a e ) a t lower t e m p e r a t u r e s , and i n h i g h e r s a l i n i t i e s 4 a t h i g h e r t e m p e r a t u r e s . T h i s f i n d i n g l e a d s t o q u e s t i o n s r e g a r d i n g the s a l i n i t y t o l e r a n c e of h e r r i n g l a r v a e . F i r s t , i f the i n t e r a c t i o n between s a l i n i t y and temperature o c c u r r i n g d u r i n g i n c u b a t i o n were t o c o n t i n u e i n the l a r v a l s t a g e , such an i n t e r a c t i o n would be e c o l o g i c a l l y advantageous, s i n c e most h e r r i n g l a r v a e h a t c h j u s t p r i o r t o or d u r i n g the a n n u a l s p r i n g i n c r e a s e i n t e m p e r a t u r e . Hence, the r i s i n g e n v i r o n m e n t a l temperatures d u r i n g the s p r i n g i n c r e a s e c o u l d f a v o r l a r v a l s u r v i v a l i n the h i g h e r s a l i n i t i e s t o which the l a r v a e a r e exposed i n n a t u r e . A second q u e s t i o n c o ncerns the p o s s i b l e i n f l u e n c e of i n c u b a t i o n s a l i n i t y and temperature on the s a l i n i t y t o l e r a n c e of l a r v a e . I s the s u r v i v a l of l a r v a e i n h i g h e r s a l i n i t i e s a r e s u l t of t h e i r accommodation to h i g h e r s a l i n i t i e s d u r i n g i n c u b a t i o n ? Does i n c u b a t i o n temperature a l s o i n f l u e n c e the s a l i n i t y t o l e r a n c e of the r e s u l t i n g l a r v a e ? The c u r r e n t i n v e s t i g a t i o n examines the e f f e c t s of (1) i n c u b a t i o n s a l i n i t y , (2) i n c u b a t i o n t e m p e r a t u r e , and (3) c u l t u r e ( p o s t - h a t c h i n g ) temperature on the s a l i n i t y t o l e r a n c e of h e r r i n g l a r v a e . The study was d e s i g n e d t o accommodate an e x a m i n a t i o n of the main e f f e c t s and i n t e r a c t i o n s between these t h r e e v a r i a b l e s on s a l i n i t y t o l e r a n c e of l a r v a l h e r r i n g . In a d d i t i o n t o the s a l i n i t y t o l e r a n c e t e s t s , a c a l o r i m e t r i c study was undertaken u s i n g 0- (newly h a t c h e d ) , 3-, 6- and 9-day o l d h e r r i n g l a r v a e . The l a r v a e used were hat c h e d from eggs i n c u b a t e d i n the same s a l i n i t y - t e m p e r a t u r e c o n d i t i o n s as t h o s e used f o r the s a l i n i t y t o l e r a n c e t e s t s . The purpose of t h i s companion s t u d y was t o d e t e r m i n e th e p o s s i b l e i n f l u e n c e of i n c u b a t i o n s a l i n i t y and t e m p e r a t u r e on a s p e c t s of l a r v a l 5 development afte r hatching. 6 MATERIALS AND METHODS A f i r s t e xperiment was conducted i n 1979. Problems w i t h the equipment made i t n e c e s s a r y t o conduct a f u r t h e r s e t of t r i a l s i n 1980. 1. C o l l e c t i o n of Spawners A d u l t h e r r i n g were caught i n Nanoose Bay, Vancouver I s l a n d , B.C. on F e b r u a r y 28, 1979. For the 1980 e x p e r i m e n t , the h e r r i n g spawners were caught on March 3 from the same l o c a l i t y . They were t r a n s p o r t e d t o the P a c i f i c B i o l o g i c a l S t a t i o n , Nanaimo, B.C., and m a i n t a i n e d i n c i r c u l a r t a n k s of a e r a t e d sea water (28-29°/ooS, 8-9°C). 2. Spawning and F e r t i l i z a t i o n In 1979, eggs were f e r t i l i z e d a t t h r e e d i f f e r e n t t i m e s : March 20, A p r i l 12 and A p r i l 20. In 1980, f e r t i l i z a t i o n was c a r r i e d out on one o c c a s i o n , on March 11. The f o l l o w i n g spawning and f e r t i l i z a t i o n p r o c e d u r e s r e f e r t o both 1979 and 1980 e x p e r i m e n t s . P r i o r t o spawning, a c o n s t a n t - t e m p e r a t u r e water b a t h was p r e p a r e d , c o n t a i n i n g 12 beakers f i l l e d w i t h water of s p e c i f i c s a l i n i t y . Three f e r t i l i z a t i o n s a l i n i t i e s were chosen (13, 21, and 29°/ooS) and water of each s a l i n i t y was a s s i g n e d t o f o u r 7 b e a k e r s . Water temperature was m a i n t a i n e d a t 6°C. V a r i o u s numbers of female and male spawners were used on the d i f f e r e n t spawning d a t e s . Length and weight measurements taken b e f o r e spawning are shown i n T a b l e 1. E i g h t ml of m i l t was o b t a i n e d and s t o r e d a t 4°C u n t i l r e q u i r e d . The eggs were e x p r e s s e d onto r e c t a n g u l a r n y l o n s c r e e n s (3.5 x 7.5 cm, 9 f i l a m e n t s / c m ) t o form rows of eggs, one t o two l a y e r s t h i c k . Twenty-four s c r e e n s were p r e p a r e d and two of the e g g - f i l l e d s c r e e n s were s t o r e d i n each beaker of water a t the a p p r o p r i a t e s a l i n i t y (13, 21 or 29°/ooS). When a l l 24 s c r e e n s had been p r e p a r e d , the s t o r e d m i l t was d i l u t e d w i t h sea water (2?°/ooS, 8°C) t o make a 40-ml s u s p e n s i o n of m i l t . Ten drops of the d i l u t e d m i l t (about 0.5 ml) were added t o each beaker, a l l o w i n g a t l e a s t ten minutes f o r f e r t i l i z a t i o n t o o c c u r . The f i n a l d i l u t i o n of the m i l t was 1 drop/15 ml of seawater. The f e r t i l i z e d eggs were r i n s e d b r i e f l y w i t h water of the same s a l i n i t y as the f e r t i l i z a t i o n medium and t r a n s f e r r e d t o the i n c u b a t o r s . 3. I n c u b a t i o n S i x 40-1 i n c u b a t i o n t a n k s were p r e p a r e d . Each tank h e l d one i n c u b a t o r ( F i g u r e 1a, A l d e r d i c e and V e l s e n , 1968). The f o u r compartments or c e l l s of the i n c u b a t o r ( F i g u r e 1b) each h e l d one r e c t a n g u l a r s c r e e n of the f e r t i l i z e d eggs. The f o u r compartments were i n t e r c o n n e c t e d i n s e r i e s by p l a s t i c t u b i n g t h rough which water a t the d e s i r e d s a l i n i t y and temperature was pumped pa s t the eggs a t a p e r f u s i o n ( b u l k ) v e l o c i t y of 500 cm/hr. 8 Table 1 Range of l e n g t h (cm) and weight (g) measurements taken in h e r r i n g spawners used i n the 1979 and 1980 experiments. Spawning and Fert11 I z a t 1 o n Date In c u b a t I o n Number Range Fork Length (cm) Range S t a n d a r d Length (cm) Rang T o t a l Wei e t ght (g) Group ? <? ? •? March 20, 1979 6 and 12* C ( r e p l I c a t e I) 3 5 23.5 -24.7 18.5 -26 .0 2 1 . 5 -22.5 17.0 -23.6 163.4 -191.4 62.2 -210.0 Apr11 12, 1979 12' C ( r e p l I c a t e I I ) 1 2 22.7 22.5 -22.8 21.5 21.1 -21.6 145.7 128.3 -129 .0 A p r i l 20, 1979 6' C ( r e p l I c a t e I I ) 1 3 24.9 20.2 -22.4 23.7 19.2 -20.9 183.2 96.8 -112.1 March 11. 1980 6 and 12*C ( r e p l I c a t e s I , I I . I l l ) 3 5 24.9 -30.8 25.0 -29.2 22.7 -27 .8 22.4 -26.3 146.5 -254.3 117.1 -185.3 T a b l e 1 10 F i g u r e 1a H e r r i n g egg i n c u b a t o r ( A l d e r d i c e and V e l s e n , 1968) i n i n c u b a t i o n t a n k . H e r r i n g egg i n c u b a t i o n c e l l s F i g u r e 1b i n c u b a t o r showing the f o u r compartments and Figure 1b 12 S i x i n c u b a t i o n t e m p e r a t u r e - s a l i n i t y c o m b i n a t i o n s were a s s i g n e d randomly t o the s i x t a n k s (Table 2 ) . The s i x i n c u b a t i o n groups were a c o m b i n a t i o n of two te m p e r a t u r e s (6 and 12°C) and t h r e e s a l i n i t i e s (13, 21 and 29°/ooS). D i f f e r e n t s a l i n i t i e s were p r e p a r e d e i t h e r by d i l u t i n g sea water (28-29°/ooS) w i t h f r e s h water t o p r e p a r e lower s a l i n i t i e s , or by adding a s y n t h e t i c sea s a l t ( R i l a M a r ine Mix, R i l a P r o d u c t s , Teaneck, N.J.) t o sea water t o p r e p a r e h i g h e r s a l i n i t i e s . S a l i n i t y was d e t e r m i n e d by t i t r a t i o n ( S t r i c k l a n d and P a r s o n s , 1968). The mean e x p e r i m e n t a l s a l i n i t y and temperature l e v e l s o b t a i n e d d u r i n g i n c u b a t i o n a r e shown i n T a b l e 2. The eggs were i n c u b a t e d i n the same s a l i n i t i e s as those i n which they were f e r t i l i z e d . Each i n c u b a t i o n tank was a e r a t e d , t o m a i n t a i n d i s s o l v e d oxygen a t s a t u r a t i o n l e v e l s , and c o v e r e d w i t h a p i e c e of g l a s s t o a v o i d e v a p o r a t i o n . In the 1979 e x p e r i m e n t , w h i t e paper t o w e l s h e e t i n g was p l a c e d over the g l a s s c o v e r s t o m a i n t a i n semi-dark c o n d i t i o n s . In the 1980 e x p e r i m e n t , b l a c k p l a s t i c s h e e t i n g was used i n s t e a d of the w h i t e paper t o m a i n t a i n c o n s t a n t dark c o n d i t i o n s . The water i n l e t and o u t l e t tubes of each i n c u b a t o r ( F i g u r e l a ) were r o t a t e d t h rough one p o s i t i o n d a i l y t o d i s t r i b u t e e v e n l y the p o t e n t i a l b i a s r e s u l t i n g from the s e r i e s arrangement of water f l o w t h r o u g h the f o u r i n c u b a t o r c e l l s . E very f o u r days, o n e - h a l f of the tank volume ( t o t a l volume, 40 l i t r e s ) was r e p l a c e d w i t h newly p r e p a r e d water of the same s a l i n i t y and te m p e r a t u r e . P e r c e n t f e r t i l i z a t i o n of the eggs was d e t e r m i n e d by examining s e p a r a t e samples of eggs a d h e r i n g t o and f e r t i l i z e d on g l a s s s l i d e s . The e x p e c t e d time t o 50% h a t c h i n g was c a l c u l a t e d 1 3 Table 2 E x p e r i m e n t a l d e s i g n f o r the s a l i n i t y t o l e r a n c e t e s t s . H o r i z o n t a l b a r s under " L a r v a l Stage" i n d i c a t e a s e r i e s of t e s t s a l i n i t i e s : (1) 20, 25, 30, 35, 40, 45, 50 and 55 °/ooS i n 1979; (2) 25, 30, 35, 40, 45 and 50 °/ooS i n 1980. Numbers i n b r a c k e t s i n d i c a t e 18 d i f f e r e n t t r i a l s (3 i n c u b a t i o n s a l i n i t i e s x 2 i n c u b a t i o n t e m p e r a t u r e s x 3 c u l t u r e t e m p e r a t u r e s ) . In 1979, t h e r e were 2 r e p l i c a t e s per t r i a l , p r o d u c i n g 36 i n d i v i d u a l t e s t s (18 t r i a l s x 2 r e p l i c a t e s ) . In 1980, t h e r e were 3 r e p l i c a t e s per t r i a l , p r o d u c i n g 54 i n d i v i d u a l t e s t s (18 t r i a l s x 3 r e p l i c a t e s ) . Group 1 2 3 4 5 6 INCUBATION Des1gn Temp. C C ) 6 6 6 12 12 12 Sal . C / o o ) 13 21 29 13 21 29 E x p t l . L e v e l + 1 SD Temperature C c ) G.00 ± .05 6.00 ± .01 6.00 + .01 12.00 ± .01 12.01 ± .08 12.02 ± .12 S a l 1 n i t y C / o o ) 13.08 ± .07 20.98 ± .17 28.96 ± .11 13.15 ± .24 20.95 ± .28 28.93 ± .09 LARVAL STAGE C u l t u r e ( P o s t - h a t c h i n g ) Temperature C O ( D _L1L _L7J_ (10)  (13)  (16) (2) AIL (B) (11)  (14)  (17) 12 J 6 1 J 9 L (12)  (15)  (18) T a b l e 2 15 f o r both 6 and 12°C i n c u b a t i o n groups f o l l o w i n g the r e l a t i o n p r o v i d e d by A l d e r d i c e and V e l s e n (1971). Newly ha t c h e d l a r v a e were t r a n s f e r r e d t o l a r v a l t a n k s f o r the s a l i n i t y t o l e r a n c e t e s t s . 4• S a l i n i t y T o l e r a n c e T e s t s of L a r v a e The b a s i c p r o c e d u r e i n the l a r v a l t e s t s was t o expose groups of newly hatched l a r v a e , o b t a i n e d a t the peak p e r i o d of h a t c h i n g from the v a r i o u s i n c u b a t i o n groups, t o a s e r i e s of t e s t s a l i n i t i e s . L a r v a e were t e s t e d a t t h r e e c u l t u r e ( p o s t - h a t c h i n g ) t e m p e r a t u r e s (6, 9 and 12°C) and the m o r t a l i t y o c c u r r i n g i n the d i f f e r e n t t r e a t m e n t s was noted f o r a f i x e d exposure p e r i o d of 72 hours (Table 2 ) . Two t e c h n i q u e s were used i n the s a l i n i t y t o l e r a n c e t r i a l s . The f i r s t , a " t i m e - m o r t a l i t y " a pproach, was used i n 1979. The t i m e - m o r t a l i t y t e c h n i q u e i n v o l v e d an e s t i m a t i o n of the time t o d e a t h of i n d i v i d u a l l a r v a e i n each t e s t s a l i n i t y over the 72-hour p e r i o d . The r e s u l t i n g t i m e - m o r t a l i t y s e r i e s p r o v i d e d an e s t i m a t e of the e f f e c t i v e time t o 50% m o r t a l i t y or median e f f e c t i v e time ( E T 5 0 ) . The second or " d o s a g e - m o r t a l i t y " t e c h n i q u e was used i n 1980. T h i s approach p r o v i d e d an e s t i m a t e of t o t a l l a r v a l m o r t a l i t y o c c u r r i n g i n each t e s t s a l i n i t y a t the end of the 72-hour p e r i o d . The dosage i n the l a t t e r i n s t a n c e i s taken as the t e s t s a l i n i t y . The r e s u l t i n g d o s a g e - m o r t a l i t y s e r i e s p r o v i d e d an e s t i m a t e of the e f f e c t i v e dose t o 50% m o r t a l i t y , or median e f f e c t i v e dose ( E D 5 0 ) , f o r an exposure p e r i o d of 72 h o u r s . 16 a) 1979 Experiment ( T i m e - M o r t a l i t y Approach) The eggs d e v e l o p i n g i n the t h r e e i n c u b a t i o n s a l i n i t i e s a t the same te m p e r a t u r e (6 and 12°C) were ex p e c t e d t o h a t c h on the same day, based on f i n d i n g s from p r e v i o u s e x p e r i m e n t s . Nine 40-1 l a r v a l t a n k s were p r e p a r e d s i m u l t a n e o u s l y t o accommodate the ni n e groups of l a r v a e t o be t e s t e d ( t h r e e i n c u b a t i o n s a l i n i t y groups X t h r e e c u l t u r e t e m p e r a t u r e s ) . The t h r e e c u l t u r e t e m p e r a t u r e s (6, 9 and 12°C) were a s s i g n e d randomly t o the t e m p e r a t u r e - c o n t r o l l e d t a n k s . Each tank c o n t a i n e d an a r r a y of e i g h t 200-ml c u l t u r e d i s h e s t o which e i g h t t e s t s a l i n i t i e s (20, 25, 30, 35, 40, 45, 50 and 55 °/ooS) were a s s i g n e d . The d i s h e s were c o v e r e d i n d i v i d u a l l y w i t h g l a s s c o v e r s t o a v o i d e v a p o r a t i o n . A s e r i e s of t h e s e e i g h t s a l i n i t i e s p r o v i d e d one r e p l i c a t e ( T a b l e 2 ) . The i n i t i a l p l a n was t o conduct the t e s t s w i t h two r e p l i c a t e s (each tank w i t h 16 c u l t u r e d i s h e s ) t e s t e d a t the same t i m e . However, i n the 1979 s e r i e s , the pr o c e d u r e had t o be a l t e r e d as t h e r e were not enough l a r v a e a v a i l a b l e due t o h i g h m o r t a l i t y a t h a t c h i n g caused by t e c h n i c a l problems i n the i n c u b a t i o n system. Moreover, a t w o - r e p l i c a t e s e r i e s f o r a l l n i n e groups, u s i n g the t i m e - m o r t a l i t y approach, produced an e x p e r i m e n t a l a r r a y too l a r g e t o be conducted e x p e r i m e n t a l l y a t one t i m e . C o n s e q u e n t l y , the s a l i n i t y t e s t s were performed a t fo u r d i f f e r e n t t i m e s , r e q u i r i n g f e r t i l i z a t i o n and i n c u b a t i o n of two f u r t h e r b a t c h e s of eggs. D u r i n g the peak of the h a t c h i n g p e r i o d , newly ha t c h e d l a r v a e were t r a n s f e r r e d t o the c u l t u r e d i s h e s . In the f i r s t l a r v a l t e s t (12°C i n c u b a t i o n group, r e p l i c a t e I , 1979), groups of 15 l a r v a e were t e s t e d i n each s a l i n i t y t o l e r a n c e s e r i e s . In 17 subsequent t e s t s , the sample s i z e was reduced t o t e n . In a l l t e s t s , o n l y v i a b l e l a r v a e were used. Larvae were kept i n semi-dark c o n d i t i o n s by p l a c i n g w h i t e paper t o w e l i n g over the g l a s s c o v e r s i n each t a n k . E x a m i n a t i o n of l a r v a l m o r t a l i t y commenced soon a f t e r t r a n s f e r of l a r v a e t o the c u l t u r e d i s h e s and c o n t i n u e d throughout the 72-hour p e r i o d . A l a r v a was c o n s i d e r e d "dead" when i t was g e n e r a l l y i n a c t i v e , the b r a i n and s p i n a l c o r d were opaque and the f i n f o l d s were shrunk and w r i n k l e d . C a r d i a c f u n c t i o n might s t i l l be o b s e r v e d , but the damage t o the l a r v a was a l r e a d y c o n s i d e r e d i r r e v e r s i b l e . Dead l a r v a e were removed a t ever y o b s e r v a t i o n p e r i o d . At the end of 72 h o u r s , the r e m a i n i n g l a r v a e were counted t o check i n d i v i d u a l sample s i z e . b) 1980 Experiment ( D o s a g e - M o r t a l i t y Approach) One day p r i o r t o the e x p e c t e d peak of h a t c h i n g , the n y l o n mesh s c r e e n s and a d h e r i n g eggs were removed from the i n c u b a t o r c e l l s and t r a n s f e r r e d t o c i r c u l a r 100-mm di a m e t e r b a s k e t s . The t r a n s f e r m i n i m i z e d m o r t a l i t y of l a r v a e caused by the upward movement of water i n the i n c u b a t o r c e l l s . The b a s k e t s were kept i n the same i n c u b a t i o n t a n k , a e r a t e d and m a i n t a i n e d at the same s a l i n i t y and temperature of i n c u b a t i o n . N ine l a r v a l t a n k s were p r e p a r e d i n a s i m i l a r manner as i n the 1979 e x p e r i m e n t . S i m i l a r l y , the t h r e e c u l t u r e t e m p e r a t u r e s (6, 9 and 12°C) were a s s i g n e d randomly t o the n i n e t e m p e r a t u r e -c o n t r o l l e d l a r v a l t a n k s . At the peak p e r i o d of h a t c h i n g , groups of 10 l a r v a e were t r a n s f e r r e d t o a s e r i e s of s i x c u l t u r e d i s h e s c o n t a i n i n g waters of s p e c i f i c s a l i n i t y (25, 30, 35, 40, 45 and 50°/ooS). In the 18 1980 experiment, the 20 and 55°/oo t e s t s a l i n i t i e s used i n the 1979 t e s t s were excluded from the s e r i e s . The e x c l u s i o n was co n s i d e r e d reasonable as the two s a l i n i t i e s c o n s i s t e n t l y produced 0 or 100% m o r t a l i t y over a 72-hour p e r i o d i n the 1979 t e s t s . With s i x s a l i n i t i e s , a 3 - r e p l i c a t e s e r i e s was accommodated i n one l a r v a l tank (18 c u l t u r e d i s h e s / t a n k ) , a procedure that i n c r e a s e d the s e n s i t i v i t y of the ensuing a n a l y s e s . The 1 8 dishes i n each tank were covered with a p i e c e of P l e x i g l a s s to avoid e v a p o r a t i o n . The water i n each d i s h was ae r a t e d with a gentle flow of a i r f o r 30 seconds d a i l y throughout the t e s t i n g p e r i o d . The l a r v a e were kept i n constant dark c o n d i t i o n s by p l a c i n g black p l a s t i c sheeting on the g l a s s covers over each tank. M o r t a l i t y of larvae at each t e s t s a l i n i t y was recorded at the end of 72 hours. The same c r i t e r i a f o r d e c l a r i n g a l a r v a "dead" used i n 1979 were a p p l i e d to the.1980 experiment. At the end of the t e s t p e r i o d , the remaining l i v e l a r v a e were counted to check i n d i v i d u a l sample s i z e . c) S t a t i s t i c a l A n a l y s i s (1) 1979 Data. P r o b i t a n a l y s i s was a p p l i e d to the data, f o l l o w i n g B l i s s ' (1937) t i m e - m o r t a l i t y methods. From the v a r i o u s t i m e - m o r t a l i t y curves, the E T 5 0 of each r e p l i c a t e of the 18 t r i a l s was estimated g r a p h i c a l l y . I t was found, however, that some t r i a l s showed tru n c a t e d d i s t r i b u t i o n s , where m o r t a l i t y was no longer observed dur i n g the l a t t e r p a r t of the o b s e r v a t i o n p e r i o d and where the highest cumulative m o r t a l i t y was l e s s than 19 50%. T h i s made i t d i f f i c u l t t o e s t i m a t e the E T 5 0 ' s , . n e c e s s i t a t i n g a change i n approach t o use d o s a g e - m o r t a l i t y a n a l y s i s f o r the 1979 d a t a . D o s a g e - m o r t a l i t y c u r v e s were drawn on p r o b a b i l i t y paper and v a l u e s of E D 5 0 and t h e i r c o r r e s p o n d i n g c o n f i d e n c e l i m i t s were c a l c u l a t e d f o l l o w i n g the maximum l i k e l i h o o d method of e s t i m a t i o n ( B l i s s , 1935). The E D 5 0 ' s formed the bases f o r comparison of the upper l e t h a l s a l i n i t y l i m i t s of the y o l k - s a c l a r v a e . A n a l y s i s of v a r i a n c e (ANOVA) was used t o examine the main e f f e c t s and i n t e r a c t i o n s f o r the d i f f e r e n t t r e a t m e n t s , employing an F - t e s t f o r t e s t s o f s i g n i f i c a n c e . The f o l l o w i n g a n a l y s e s of v a r i a n c e were c a l c u l a t e d : i ) o v e r - a l l ANOVA (3 i n c u b a t i o n s a l i n i t i e s , 2 i n c u b a t i o n t e m p e r a t u r e s , 3 c u l t u r e t e m p e r a t u r e s , 2 r e p l i c a t e s ) i i ) r e p l i c a t e I ( 3 x 2 x 3 ) i i i ) r e p l i c a t e I I ( 3 x 2 x 3 ) (2) 1980 D a t a . The d o s a g e - m o r t a l i t y data o b t a i n e d from the 54 t e s t s were a n a l y z e d u s i n g L i t c h f i e l d and W i l c o x o n ' s (1949) s i m p l i f i e d method f o r e s t i m a t i n g the E D 5 0 . F i g u r e 2 shows an example of the d o s a g e - m o r t a l i t y c u r v e from which E D 5 0 e s t i m a t e s were o b t a i n e d g r a p h i c a l l y . B l i s s ' method p r o v i d e d somewhat more a c c u r a t e r e s u l t s ; however, w i t h L i t c h f i e l d and W i l c o x o n ' s method, the c o n f i d e n c e l i m i t s ( o = 0.05) of the E D 5 0 ' s c o u l d be c a l c u l a t e d and the homogeneity of v a r i a n c e c o u l d be measured u s i n g a x2 t e s t . The f i r s t i t e r a t i o n was a c c e p t e d i f the x 2 t e s t showed t h a t d i f f e r e n c e s between e s t i m a t e s of the parameters o b t a i n e d i n the f i r s t and second i t e r a t i o n s were not 20 F i g u r e 2 D o s a g e - m o r t a l i t y graph showing the g r a p h i c a l method of e s t i m a t i n g E D 5 0 f o r the 1980 e x p e r i m e n t . F a c t o r f o r E D 5 0 ( f E D 5 0 ) i s c a l c u l a t e d f o l l o w i n g the method of L i t c h f i e l d and W i l c o x o n (1949). C o n f i d e n c e l i m i t s of E D 5 0 = E D 5 0 x/+ fED 5 0 . Downward s h o r t , s o l i d arrow i n d i c a t e s 0% observed m o r t a l i t y response. P o i n t w i t h b r a c k e t s i n d i c a t e s "expected" p e r c e n t m o r t a l i t y ( o b t a i n e d from t a b l e s , L i t c h f i e l d and W i l c o x o n , 1949) f o r 0% observ e d m o r t a l i t y . 21 22 s i g n i f i c a n t l y d i f f e r e n t (o = 0.05). I f s i g n i f i c a n t , two more i t e r a t i o n s were made. The i t e r a t i o n w i t h the l e a s t x2 v a l u e , whether s i g n i f i c a n t or n o t , was a c c e p t e d . In. the case of heterogeneous v a r i a n c e , the c o n f i d e n c e l i m i t s of the E D 5 0 were c a l c u l a t e d i n a d i f f e r e n t manner. In those i n s t a n c e s where m o r t a l i t y of l a r v a e i n the s e r i e s of t e s t s a l i n i t i e s was l e s s than 50%, the E D 5 0 was e s t i m a t e d by e x t r a p o l a t i o n by e x t e n d i n g the p r o b i t l i n e u n t i l i t c r o s s e d the 50% response p o i n t . The E D 5 0 ' s were a n a l y z e d by a n a l y s i s of v a r i a n c e f o l l o w i n g the 1979 p r o c e d u r e . In a d d i t i o n t o an o v e r - a l l ANOVA, s e p a r a t e a n a l y s i s was conducted i n each i n c u b a t i o n temperature group t o examine the main e f f e c t s and i n t e r a c t i o n s of i n c u b a t i o n s a l i n i t y and c u l t u r e t emperature a t d i f f e r e n t l e v e l s of i n c u b a t i o n t e m p e r a t u r e . 5. C a l o r i m e t r y In 1979, c a l o r i m e t r y was conducted t o det e r m i n e the c a l o r i c v a l u e s of h e r r i n g l a r v a e hatched from eggs i n c u b a t e d i n the s i x d i f f e r e n t s a l i n i t y - t e m p e r a t u r e c o m b i n a t i o n s ( T a b l e 3 ) . The l a r v a e t e s t e d were 0 (newly h a t c h e d ) , 3, 6 and 9 days o l d . Hatched l a r v a e were m a i n t a i n e d i n the same s a l i n i t y and temperature as those i n which they were i n c u b a t e d . F i f t e e n v i a b l e l a r v a e , numbers p e r m i t t i n g , were sampled from each i n c u b a t i o n group w i t h i n each of two r e p l i c a t e s (Table 3 ) . The s t a n d a r d l e n g t h of l a r v a e , measured from the t i p of the snout t o the end of the n o t o c h o r d , was o b t a i n e d a f t e r a n a e s t h e t i z i n g the l a r v a e w i t h MS-222 ( t r i c a n e m e t h a n e 23 Table 3 Number of l a r v a e sampled f o r c a l o r i m e t r y . Larvae were sampled from each treatment group a t day 0, 3, 6 and 9 and a f t e r h a t c h i n g . (-) = no l a r v a e a v a i l a b l e . 24 I n c u b a t i o n and C u l t u r e C o n d i t i o n s Number of L a r v a e Days A f t e r H a t c h i n g G roup No. Temp. (°C) S a l i n i t y (°/oo) R e p l i c a t e I R e p l i c a t e II 0 3 6 9 0 3 6 9 1 •6 1 3 1 5 15 15 15 1 5 15 1 5 1 5 2 6 21 1 5 15 15 15 1 5 15 1 5 1 5 3 6 29 1 5 1 5 15 15 1 5 1 5 1 5 1 5 4 1 2 1 3 1 5 - - 1 5 1 5 1 5 15 5 1 2 21 1 5 1 5 - 1 5 1 5 1 5 1 5 6 1 2 29 1 5 4 1 5 15 1 5 1 5 T a b l e 3 25 s u l f o n a t e ) . The l a r v a e then were r i n s e d b r i e f l y w i t h d i s t i l l e d water and p l a c e d on r i n g s l i d e s p r e v i o u s l y c o a t e d w i t h a 1% s o l u t i o n of a compound ( S i l i c l a d ) t h a t a l l o w e d the l a r v a e t o be removed i n t a c t l a t e r w i t h o u t damage r e s u l t i n g from a d h e s i o n of the l a r v a e t o the g l a s s s u r f a c e . The a i r - d r i e d l a r v a e were s t o r e d i n a d e s i c c a t o r (over CaSO a) u n t i l the c a l o r i f i c work was performed. C a l o r i f i c a n a l y s i s was conducted 3 t o 4 months a f t e r d e s i c c a t o r - d r y i n g of the l a r v a e . The l a r v a e were removed from the r i n g s l i d e s w i t h a sharp r a z o r b l a d e . The 15 l a r v a e from each r e p l i c a t e of one i n c u b a t i o n group were d i v i d e d i n t o t h r e e groups. The group dry weight of f i v e l a r v a e was taken b e f o r e the f i v e l a r v a e were p e l l e t i z e d . A l l p e l l e t s were weighed b e f o r e and a f t e r b e i n g o v e n - d r i e d a t 105°C f o r 24 h o u r s . A l l w e i g h i n g s were made t o the n e a r e s t 0.01 mg on an e l e c t r o b a l a n c e (Cahn, G2). Combustion of p e l l e t e d l a r v a e was c a r r i e d out u s i n g a P h i l l i p s o n oxygen microbomb c a l o r i m e t e r (Gentry and Wiegert I n s t r u m e n t s , I n c . , A i k e n , S . C ) . Of the t h r e e p e l l e t s made, two were combusted i n the microbomb c a l o r i m e t e r w h i l e one was combusted d i r e c t l y i n a m u f f l e f u r n a c e t o determine ash c o n t e n t by d i r e c t a s h i n g . A s h i n g was done a t 550°C f o r t h r e e h o u r s , as recommended by Cummins and Wuycheck (1971). The c a l o r i m e t e r was connected t o a p o t e n t i o m e t e r ( P h i l l i p s F l a t - B e d Recorder Type PM 8100), which measured t h e v o l t a g e c r e a t e d by the temperature d i f f e r e n c e between the hot j u n c t i o n of the bomb s t a n d and the c o l d r e f e r e n c e j u n c t i o n (aluminum b l o c k ) of the c a l o r i m e t e r . When combustion took p l a c e , the p o t e n t i o m e t e r pen d e f l e c t e d a d i s t a n c e p r o p o r t i o n a l t o the 26 v o l t a g e c r e a t e d by the temperature r i s e o c c u r r i n g as a r e s u l t of p e l l e t c ombustion. The amount of pen d e f l e c t i o n ( r i s e ) p r o v i d e d the b a s i s f o r c a l c u l a t i o n of c a l o r i c v a l u e s . F i g u r e 3 shows a t y p i c a l combustion graph and the s t a n d a r d method of c a l c u l a t i n g " t o t a l c o r r e c t e d r i s e " . W i th the use of a c a l i b r a t i o n e q u a t i o n d e t e r m i n e d e a r l i e r u s i n g b e n z o i c a c i d , the c a l o r i c c o n t e n t c o u l d be c a l c u l a t e d (Dueftas, u n p u b l i s h e d d a t a ) . A l i n e a r r e g r e s s i o n e q u a t i o n was o b t a i n e d , g i v i n g the r e l a t i o n s h i p between t o t a l c o r r e c t e d r i s e and c a l o r i c v a l u e s . C o r r e c t i o n s t o the c a l o r i c c a l c u l a t i o n were a p p l i e d when combustion of the p e l l e t s was i n c o m p l e t e o r when l o s s of l a r v a l p a r t s o c c u r r e d w h i l e p e l l e t s were b e i n g made. The c a l o r i c c o n t e n t of the unburned o r g a n i c m a t e r i a l and of the o r g a n i c weight of the l o s t p a r t s were c a l c u l a t e d p r o p o r t i o n a l l y and added t o the obse r v e d c a l o r i c v a l u e s . C a l c u l a t i o n was based on the l i n e a r r e l a t i o n s h i p between weight of o r g a n i c m a t e r i a l and t h e i r c a l o r i c e q u i v a l e n t s . The a s h - f r e e d r y w e i g h t s of l a r v a e were c a l c u l a t e d d i r e c t l y from the ash v a l u e s o b t a i n e d from d i r e c t a s h i n g i n the m u f f l e f u r n a c e . For c o m p a r i s o n , ash v a l u e s a l s o were o b t a i n e d from the c a l o r i m e t r y r e s i d u e s ( i n d i r e c t a s h i n g ) . C a l o r i c v a l u e s were e x p r e s s e d i n terms of c a l / g d r y w e i g h t , c a l / g a s h - f r e e d r y weight and c a l / l a r v a . A d e t a i l e d d e s c r i p t i o n of the c a l o r i m e t r y p r o c e d u r e i s d e s c r i b e d elsewhere (DuefTas, u n p u b l i s h e d d a t a ) . 27 Figure 3 Typical graphical combustion record and standard method cal c u l a t i n g " t o t a l corrected r i s e " . 2 8 ( 5 ) P o s t - f i r e change _ No. o f l i n e s (mm) Time ( m i n ) (3) R i s e = Peak (mm) (mm) F i r i n g p o i n t ( m m ) ( 2 ) F i r i n g p o i n t Peak (mm) (1) Time f r o m 60% t o peak ( m i n ) ( 7 ) Time t o 60% r i s e ( m i n ) ( 6 ) P r e - f i r e change No. o f l i n e s (mm) Time ( m i n ) ( 4 ) C a l c u l a t i o n : R i s e Peak h e i g h t (mm) = (-) F i r i n g p o i n t (mm) (-) ( 1 ) R i s e (nun) (3) II. Time c o r r e c t i o n A- P r e - f i r e change x Time t o 6 0 % r i s e ( p r e - f i r e c o r r e c t i o n ) P o s t - f i r e change x Time f r o m 6 0 % to peak ( p o s t - f i r e c o r r e c t i o n ) B. (+) ( 4 ) x ( 6 ) ) ( 5 ) x ( 7 ) Time c o r r e c t i o n (mm) ( 8 ) III. W i r e c o r r e c t i o n (mm) = Mean r i s e of 6 d e t e r m i n a t i o n s u s i n g f u s e w i r e only (no sample) = (9) IV. T o t a l c o r r e c t e d r i s e , , R i s e (mm) W i r e c o r r e c t i o n (mm) (-) (3) (9) , + . W i r e c o r r e c t e d r i s e (mm) = ^ + ^ ( 1 0 ) Time c o r r e c t i o n (mm) ( 8 ) T o t a l c o r r e c t e d r i s e (mm) (ID 29 RESULTS 1 . S a l i n i t y T o l e r a n c e T e s t s a) 1979 Experiment The 36 E D 5 0 (median e f f e c t i v e dose) v a l u e s c a l c u l a t e d from the two r e p l i c a t e s of the 18 t r i a l s a r e shown i n Ta b l e 4 . The mean E D 5 0 v a l u e s showed a p a t t e r n i n d i c a t i n g p o t e n t i a l e f f e c t s of the t h r e e f a c t o r s c o n s i d e r e d . The a n a l y s i s of v a r i a n c e (Table 5) shows t h a t a h i g h e r r o r v a r i a n c e may have masked a l l o t h e r p o t e n t i a l e f f e c t s . A n a l y s i s of i n d i v i d u a l r e p l i c a t e s ( T a b l e s 5b and 5 c ) , u s i n g the l i n e a r x l i n e a r x l i n e a r i n t e r a c t i o n term (IS x IT x CT) as a measure of e x p e r i m e n t a l e r r o r , i s i n d i c a t i v e of two t h i n g s : ( 1 ) a s i g n i f i c a n t e f f e c t of i n c u b a t i o n temperature on s a l i n i t y t o l e r a n c e i s s u g g e s t e d , b e i n g common t o both r e p l i c a t e s , and (2) major d i f f e r e n c e s e x i s t between r e p l i c a t e s w i t h r e s p e c t t o i n t e r p r e t a t i o n of the i n f l u e n c e of the o t h e r main e f f e c t s and i n t e r a c t i o n s on s a l i n i t y t o l e r a n c e . In r e p l i c a t e I I (Table 5 c ) , s i g n i f i c a n t d i f f e r e n c e s i n E D 5 0 v a l u e s were found o n l y between i n c u b a t i o n t e m p e r a t u r e s . A l t h o u g h , t h e s e s i g n i f i c a n t d i f f e r e n c e s were found i n each r e p l i c a t e , no attempt was made t o a n a l y z e the data f u r t h e r . The h i g h v a r i a b i l i t y i n the 1979 experiment i s thought t o have a r i s e n as f o l l o w s : 30 T a b l e 4 E s t i m a t e s of E D 5 0 f o r the 1979 ex p e r i m e n t . The two i n i t i a l v a l u e s i n each c e l l a r e the E D 5 0 ' s from each r e p l i c a t e , each shown w i t h i t s c o n f i d e n c e l i m i t s (a=0.05; B l i s s , 1937). The f i n a l v a l u e i n each c e l l i s the mean of the two E D 5 0 ' s ± 1SD. The n o t a t i o n (1) i n d i c a t e s a s i g n i f i c a n t x 2 t e s t f o r homogeneity of v a r i a n c e (c=0.05). 31 Incubat ion E D 5 0 Estimates (°/ooS) Temp. (°C) C u l t u r e Temperature (°C) S a l . (%>o) 6 9 1 2 6 1 3 45.6 ± 1.3 (1) 34.8 ± 2.2 44.2 ± 2.8 37.4 ± 3.0 ( 1 ) 40.2 ± 1.7 33.0 ± 2.1 (1) x=40.2 ± 7.6 40.8 ± 4.8 36.6 ± 5.1 6 21 39.8 ± 1.3 33.7 ± 1.7 37.5 ± 1.6 34.9 ± 2.0 35.3 ± 1.4 31.3 ± 1.7 x=36.8 ± 4.3 36.2 ± 1.8 33.3 ± 2.8 6 29 40.0 ± 1.6 36.5 ± 2.2 36.4 ± 1.2 32.7 ± 1.8 39.9 ± 1.8 41.0 ± 1.8 x=38.2 ± 2.5 34.6 ± 2.6 40.4 ± 0.8 1 2 1 3 36.7- ± 1.7 33.5 ± 2.0 37.9 ± 1.8 38.6 ± 2.0' 36.5 ± 2.0 41.0 ± 2.1 ( 1 ) x=35.1 ± 2 . 3 38.2 ± 0.5 38.8 ± 3.2 12 21 30.3 ± 1.3 43.2 ± 1.2 34.9 ± 2.2 (1) 45.5 ± 1.5 42.5 ± 1.7 ( 1 ) 42.7 ± 1.3 x=36.8 ± 9 . 1 40.2 ± 7.5 42.6 ± 0.1 1 2 29 22.2 ± 2.1 42.1 ± 1.8 ( 1 ) 25.3 ± 2.3 38.8 ± 2.0 30.9 ± 2.0 41.5 ± 2.6 x=32.2 ± 14.1 32. 1 ± 9.6 36.2 ± 7.5 Table 4 3 2 Table 5 Summary of analysis of variance for the ED 5 0 values of the 1979 experiment. A. Overall analysis, B. Replicate I, C : R e p l i c a t e II. * = Sig n i f i c a n t difference (c=0.05), NS = not s i g n i f i c a n t , IS = incubation s a l i n i t y (°/ooS), IT = incubation temperature (°C), CT = culture temperature (°C). A. Overal1 A n a l y s 1 s Source of V a r i a t i o n DF SS MS F ( c a l c ) F ( t a b ) («=0.05) S 1 g n 1 f I c a n c e I n c u b a t i o n S a l i n i t y (IS) 2 46 72 23 . 36 0.65 3.55 NS I n c u b a t i o n Temperature ( I T ) 1 2 83 2.83 0.08 4.41 NS C u l t u r e Temperature (CT) 2 13 15 6.58 0. 18 3 . 55 NS I n t e r a c t Ion 3.55 NS IS x IT 2 121 25 30^31 0.84 IS x CT 4 52 41 13 . 10 0.36 2 .93 NS IT x CT 2 56 60 28.30 0. 78 3.55 NS IS x IT x CT 4 20 46 5.12 0. 14 2.93 NS R e p l 1 c a t I o n 18 651 13 36 . 17 T o t a l 35 964 55 B. R e p l I c a t e I Source of V a r i a t i o n DF SS . MS F ( c a l c ) F ( t a b ) (a=0.05) S i g n i f I c a n c e I n c u b a t i o n S a l i n i t y ( I S ) 2 180 05 90.03 18 . 37 6.94 * I n c u b a t i o n Temperature ( I T ) 1 211 49 211.49 43. 16 7.71 * C u l t u r e Temperature (CT) 2 1 1 10 5 . 55 1.13 6.94 NS IS x IT 2 91 45 45 . 72 9.33 6 .94 * IS x CT 4 40 89 10. 22 2 .09 6.39 NS IT x CT 2 78 63 39.32 8.02 6.94 * E r r o r = IS x IT x CT 4 19 58 4 .90 T o t a l 17 633 19 C. R e p l I c a t e 11 Source of V a r i a t i o n I n c u b a t i o n S a l i n i t y ( I S ) I n c u b a t i o n Temperature ( I T ) C u l t u r e Temperature (CT) IS x IT IS x CT IT x CT E r r o r = IS x IT x CT T o t a l DF 2 1 2 2 4 2 4 17 SS 20.84 147.92 3.80 52.67 53.54 3.23 30.44 312.44 MS 10.42 147.92 1 .90 26.34 13.38 1 .62 7.61 F ( c a l c ) 1 .37 19.44 0.25 3.46 1 .76 0.21 F ( t a b ) (a=0.05) 6.94 7.71 6.94 6.94 6.39 6.94 S i g n i f I c a n c e NS * NS NS NS NS co T a b l e 5 34 (1) The two r e p l i c a t e s were conducted a t d i f f e r e n t t i m e s . Hence, the l a r v a l samples were from d i f f e r e n t b a t c h e s of eggs, f e r t i l i z e d on s e p a r a t e d a t e s and t h e r e was a p e r i o d of 23 days t o one month between f e r t i l i z a t i o n d a t e s . (2) A t e c h n i c a l problem i n the i n c u b a t i n g tank r e s u l t e d i n h i g h m o r t a l i t y of l a r v a e , n e c e s s i t a t i n g a r e d u c t i o n i n sample s i z e and r e p e t i t o n of the experiment f o r r e p l i c a t i o n , i n s t e a d of do i n g the r e p l i c a t e s at the same t i m e . (3) The f i r s t p r o c e d u r e employed f o r the l a r v a l s a l i n i t y t o l e r a n c e t e s t was the t i m e - m o r t a l i t y t e c h n i q u e . T h i s t e c h n i q u e r e q u i r e d f r e q u e n t h a n d l i n g of the c u l t u r e d i s h e s and of the samples, a s t e p which may have c o n t r i b u t e d t o e r r o r . b) 1980 Experiment The 54 E D 5 0 v a l u e s e s t i m a t e d from the t h r e e r e p l i c a t e s of the 1 8 t r i a l s a r e shown i n T a b l e 6. E x t r a p o l a t e d v a l u e s c o n s t i t u t e d 6% of a l l E D 5 0 e s t i m a t e s . Some da t a s e t s a l s o showed s i g n i f i c a n t x 2 v a l u e s i n d i c a t i n g heterogeneous v a r i a n c e . They c o n s t i t u t e d 28% of a l l t r i a l s . The mean E D 5 0 ' s c a l c u l a t e d from the t h r e e r e p l i c a t e s ( T able 6) were p l o t t e d ( F i g u r e 4) t o compare t h e i r v a l u e s i n r e l a t i o n t o the t h r e e main f a c t o r s ( e . g . , i n c u b a t i o n s a l i n i t y , i n c u b a t i o n t e m p e r a t u r e , c u l t u r e t e m p e r a t u r e ) . In the o v e r a l l a n a l y s i s of v a r i a n c e of the E D 5 0 e s t i m a t e s ( T a b l e s 7A, B, and C ) , the f o l l o w i n g main e f f e c t s and i n t e r a c t i o n s were found t o be s i g n i f i c a n t (c=0.05): (1) i n c u b a t i o n s a l i n i t y , (2) i n c u b a t i o n t e m p e r a t u r e , and (3) i n t e r a c t i o n between i n c u b a t i o n s a l i n i t y and i n c u b a t i o n 35 Table 6 Estimates of ED 5 0 and their mean values for the 1980 experiments. The f i r s t three values in each c e l l are the ED 5 0's from the three replicates x/V f E D 5 0 (a=0.05). The f i n a l value in each c e l l i s the mean ED 5 0 ± 1 SD. The notation (l) indicates a s i g n i f i c a n t x 2 test for homogeneity of variance. (2) indicates an extrapolated ED 5 0 value. Incubat i o n EDso E s t i m a t e s x/f, f EDs o and Mean ED>o + 1 SD C / o o S ) Temp. C c ) Sal . ('/oo) C u l t u r e Temperature (*C) 6 9 12 6 13 46.8 x/f 1 . 1 49.0 x/f 1.5 ( 1 ) 46.8 x/-r 1.1 46 . 2 x/-f 1 . 1 44 .5 x/f 1.1 51.5 x/f 1.2 48.5 x/f 1.2 46 .0 x/-r 1.1 50.5 x/f 1.6 ( 1 ) x=47.5 ± 1 . 3 47 .4 ± 3.7 48.3 ± 2.3 6 21 45.8 x/f 1.1 49.5 x/f 1 .2 50.2 x/f 1.2 46.2 x/- 1.2 51.0 x/- 1.2 52 .0 x/-r 1 . 2 43.5 x/f 1.2 45.5 x/f 1.2 50.5 x/f 1.2 x=48.5 ± 2 . 4 49.7 ± 3.1 46.5 ± 3.6 6 29 49.5 x/f 1 .2 51.0 x/f 1.2 51.0 x/f 1 . 2 53 .0 x/-r 1.2 45.5 x/- 1.4 (1) 54.5 x/- 1.5 (1,2) 46.5 x/f 1.2 43.5 x/- 1.5 52.2 x/f 1.4 ( 1 ) x=50.5 ± 0.9 51.0 ± 4.8 47.4 ± 4 .4 12 13 38 .0 x/-r 1 . 1 32.5 x/f 1.2 33.5 x/-r 1.3 (1) 33.5 x/- 1.4 37 .0 x/-r 2.7 ( 1 ) 32.2 x/-r 1.4 ( 1 ) 33.2 x/f 1.6 (1) 43.0 x/f 1.9 (1) 38 . 2 x/-r 1 . 1 x=34.7 + 2.9 34.2 ± 2,5 38.1 + 4.9 12 21 37 .0 x/f 1 . 1 38.5 x/-!- 1 . 1 36.0 x/f 1 . 1 45.5 x/-r 1.4 ( 1 ) 45 .0 x/-r 1.1 34.2 x/-r 1 . 1 36.5 x/f 1 . 2 35.5 x/-r 1 . 1 4 2 . 5 X / T . 1 . 5 (1) x=37.2 ± 1 .3 4 1 . 6 + 6 . 4 38.2 ± 3.8 12 2 9 41.8 x / f - 1 . 1 ( 1 ) 4 4 . 5 x/f 1 . 1 4 4 . 2 x/f 1 . 2 5 0 . 0 x/-r- 1.1 ( 1 ) 4 7 . 5 x/f 1.1 4 9 . 0 x/-:- 1 .2 43.7 x/f 1.7 5 0 . 5 x/f 1 . 2 . ( 2 ) 48.8 x/f 1.1 x=43 .5 + 1 . 5 48.8 + 1.3 47.7 + 3 . 5 CO 01 T a b l e 6 3 7 F i g u r e 4 Mean E D S 0 values ( c a l c u l a t e d from three r e p l i c a t e s ) of h e r r i n g l a r v a e s u b j e c t e d to the s a l i n i t y t o l e r a n c e t e s t (1980 experiment). Numbers above each l i n e show c u l t u r e temperature (°C); those beside each l i n e show i n c u b a t i o n temperature (°C) (See Table 6). 3 8 cn o IT) Q LU < LU 5 4 5 0 T E M P E R A T U R E (°C) INCUBATION 4 6 4 2 3 8 h 3 4 6 ° C C U L T U R E ( P O S T - H A T C H ) I2°C 13 21 2 9 INCUBATION SALINITY ( % o ) 3 9 Table 7 Summary of analysis of variance for the ED 5 0 values of the 1 9 8 0 experiment. A. Overall analysis, B. 6°C incubation group, C. 12°C incubation group. * = Sig n i f i c a n t difference ( c = 0 . 0 5 ) , NS = not s i g n i f i c a n t , IS = incubation s a l i n i t y (°/ooS), IT = incubation temperature (°C), CT = culture temperature (°C). A. Overa11 A n a l y s 1 s Source of V a r i a t i o n DF SS MS F ( c a l c ) F ( t a b ) (a=0.05) S i g n i f i c a n c e I n c u b a t i o n S a l 1 n 1 t y ( I S ) I n c u b a t i o n Temperature ( I T ) C u l t u r e Temperature (CT) I n t e r a c t 1 o n IS x IT IS x CT IT x CT IS x IT x CT Repl 1 c a t 1 o n 2 1 2 2 4 2 4 36 393:65 887.36 30.12 196.02 58.14 42 . 32 16 . 25 406.99 196.82 887.36 15 .06 98 .01 14.53 21.16 4 .06 11.31 17.41 78 .49 1 . 33 8 .67 1 .29 1 .87 0.36 3.26 4.11 3.26 3 . 26 2.63 3. 26 2.63 * NS * NS NS NS T o t a l 53 2.030.84 B. 6'C I n c u b a t i o n Group Source of V a r i a t i o n DF SS MS F ( c a l c ) F ( t a b ) (<j=0.05) S i g n i f Icance I n c u b a t i o n S a l i n i t y ( I S ) C u l t u r e Temperature ( I T ) I n t e r a c t 1 o n IS x CT R e p l i c a t 1 o n 2 2 4 18 17 .08 18.62 21.71 183.52 8.54 9.31 5.43 10.20 0.84 0.91 0.53 3.56 3 . 56 2 .93 NS NS NS T o t a l 53 240.93 C. 12'C I n c u b a t i o n Group Source of V a r i a t i o n DF SS MS F ( c a l c ) F ( t a b ) (o=0.05) S i g n i f I c a n c e . I n c u b a t i o n S a l i n i t y ( I S ) C u l t u r e Temperature ( I T ) I n t e r a c t i o n IS x CT R e p l i c a t i o n 2 2 4 18 572.59 53.82 52.68 223.47 286.29 26.91 13.17 12.42 23.06 2.71 1 .06 3.56 3.56 2.93 * NS NS T o t a l 26 902.56 902.56 o T a b l e 7 41 temperature. The e f f e c t of c u l t u r e temperature was not s t a t i s t i c a l l y s i g n i f i c a n t , nor was i t s i n t e r a c t i o n with the other f a c t o r s . The i n f l u e n c e of i n c u b a t i o n s a l i n i t y on the s a l i n i t y t o l e r a n c e of l a r v a e may be seen by comparing the mean E D 5 0 v a l u e s at the three i n c u b a t i o n s a l i n i t i e s (Table 8, l a s t row). Larvae h a t c h i n g from eggs incubated at 1 3 , 21 and 29°/ooS had mean E D 5 0 values of 41.72, 43.61 and 48.15°/ooS, r e s p e c t i v e l y . These data suggest that the eggs incubated at higher s a l i n i t i e s would produce l a r v a e more t o l e r a n t to higher s a l i n i t i e s . The i n f l u e n c e of i n c u b a t i o n temperature can be seen in a s i m i l a r manner (Table 8, l a s t column). Larvae from the 6 and 12°C i n c u b a t i o n groups had mean E D 5 0 values of 48.54 and 40.44°/ooS, r e s p e c t i v e l y . T h i s suggests that l a r v a e were more t o l e r a n t to higher s a l i n i t i e s when incubated as eggs at lower temperatures (6°C). A n a l y s i s of v a r i a n c e i n d i c a t e s that there i s a s i g n i f i c a n t i n t e r a c t i o n between i n c u b a t i o n s a l i n i t y and i n c u b a t i o n temperature. That i s , s a l i n i t y t o l e r a n c e of the l a r v a e v a r i e d between i n c u b a t i o n temperatures at d i f f e r e n t l e v e l s of i n c u b a t i o n s a l i n i t y (Table 8 ) . T h i s i s i l l u s t r a t e d i n F i g u r e 5, where higher E D 5 0 ' s were ob t a i n e d at higher i n c u b a t i o n s a l i n i t i e s f o r the two i n c u b a t i o n temperature l e v e l s . T h i s i n c r e a s e i n E D 5 0 l e v e l with i n c u b a t i o n s a l i n i t y i s most ev i d e n t i n the 12°C i n c u b a t i o n group. A n a l y s i s of v a r i a n c e conducted on separate incubation-temperature groups (6 and 12°C) produced two d i f f e r e n t r e s u l t s (Tables 7B and 7C): E D 5 0 v a l u e s f o r the s e v e r a l i n c u b a t i o n s a l i n i t i e s were not s i g n i f i c a n t l y d i f f e r e n t 4 2 Table 8 Comparison of mean ED 5 0 values of herring larvae in rel a t i o n to incubation s a l i n i t y and incubation temperature. Both main effects and interaction of these two factors are s i g n i f i c a n t (P < 0 . 0 1 ) . Mean values were calculated from nine ED 5 0 estimates ( 3 culture temperatures x 3 r e p l i c a t e s ) . Incubation Temperature (°C) Mean E D 5 0 (°/ooS) Incubation S a l i n i t y (°/ooS) . 1 3 21 29 Mean 6 12 47.76 35.68 48.24 38.97 49.63 46.67 48.54 40.44 Mean 41.72 ' 43.61 48. 1 5 Table 8 4 4 Figure 5 Interaction between incubation s a l i n i t y and incubation temperature. Additive interaction i s s i g n i f i c a n t ( P < 0 . 0 1 ; See Table 8 ) . I N C U B A T I O N T E M P E R A T U R E ( ° C ) A / / / i •f 6 12 30 13 21 29 INCUBATION SALINITY (%o ) 46 in the 6°C group, but they were s i g n i f i c a n t l y d i f f e r e n t i n the 12°C group. Between i n c u b a t i o n temperatures, l a r v a e from the 6°C group had higher E D 5 0 v a l u e s than those from 12°C group at a l l s a l i n i t y l e v e l s (Figure 5). The g r e a t e s t d i f f e r e n c e was obtained f o r l a r v a e from the 13°/oo i n c u b a t i o n s a l i n i t y group, and the l e a s t from the 29°/oo group. The o v e r l a p p i n g standard d e v i a t i o n s of the 6°C, 29°/ooS and 12°C, 29°/ooS groups ( F i g u r e 5) suggest n o n - s i g n i f i c a n t d i f f e r e n c e s between the two mean E D 5 0 v a l u e s at 29°/ooS. C u l t u r e (post-hatching) temperature does not appear to have a s t a t i s t i c a l l y s i g n i f i c a n t i n f l u e n c e on l a r v a l t o l e r a n c e (Table 7). However, c l o s e r i n s p e c t i o n suggests that there c o u l d be some trends common to both i n c u b a t i o n temperature groups (Figure 6a). Those l a r v a e hatching from eggs incubated at 6°C showed higher s a l i n i t y t o l e r a n c e at c u l t u r e temperatures between 6 and 9°C, while those l a r v a e h a t c h i n g from 12°C i n c u b a t i o n group showed higher s a l i n i t y t o l e r a n c e at c u l t u r e temperatures between 9 and 12°C. In both i n c u b a t i o n temperature groups of 6 and 12°C, the r e s u l t i n g l a r v a e showed high t o l e r a n c e to s a l i n i t y at a c u l t u r e temperature of 9°C. T h i s i s most evident i n l a r v a l groups incubated at higher s a l i n i t i e s ( F i gure 6b). In Fi g u r e 11 of the d i s c u s s i o n s e c t i o n , a s y n t h e s i s of the r e s u l t s i s presented. 47 Figure 6 Comparison of mean E D 5 0 v a l u e s of h e r r i n g l a r v a e i n r e l a t i o n t o : A. C u l t u r e temperature and in c u b a t i o n temperature, B. C u l t u r e temperature and i n c u b a t i o n s a l i n i t y . Mean E D 5 0 values were c a l c u l a t e d from 9 E D 5 0 estimates for A (3 i n c u b a t i o n s a l i n i t i e s x 3 r e p l i c a t e s ) ; 6 E D 5 0 v a l u e s f o r B (2 i n c u b a t i o n temperatures x 3 r e p l i c a t e s ) (see Table 6). — I 1 I I I 1_ 9 12 6 9 12 CULTURE T E M P E R A T U R E ( ° C ) 49 JL. C a l o r i m e t r y (1979 T r i a l s ) a) Unequal Sample S i z e In r e p l i c a t e I, sampling was incomplete because of m o r t a l i t y i n the 12°C l a r v a e (Table 3). In r e p l i c a t e I I , s u f f i c i e n t l a r v a e s u r v i v e d up to day 9 i n a l l treatments. This r e s u l t e d i n unequal sample s i z e s . The mean values (Tables 9, 10, 11) were c a l c u l a t e d , t h e r e f o r e , from d i f f e r e n t sample s i z e s . b) Standard Length of Larvae Larvae hatching from eggs incubated at lower temperature (6°C) were g e n e r a l l y longer than those l a r v a e h a t c h i n g from higher i n c u b a t i o n temperature (12°C) (F i g u r e 7, Table 9). Mean standard l e n g t h at peak of hatching (day 0) ranged from 7.67 mm (12°C, 29°/ooS) to 9.93 mm (6°C, 13°/ooS). Among the 6°C group, mean standard lengths at days 0, 3, 6 and 9 were 9.74 mm, 9.84 mm, 10.36 mm and 10.37 mm, r e s p e c t i v e l y . Among the 12°C group, the lengths of the same age order were: 7.90 mm, 8.97 mm, 9.87 mm and 9.74 mm r e s p e c t i v e l y . The g r e a t e s t d i f f e r e n c e i n l a r v a l l e n g t h between the two temperatures (6 and 12°C) was observed at day 0. The d i f f e r e n c e d e c l i n e d as the l a r v a e i n both groups grew. Larvae reared at 6°C showed a gradual i n c r e a s e i n leng t h , in an approximate l i n e a r manner, from day 0 to day 9 (Figure 7). Larvae reared at 12°C showed more r a p i d growth than those at 6°C. The i n c r e a s e i n l e n g t h from day 0 to day 6 i s a l s o l i n e a r . There i s an i n d i c a t i o n that those l a r v a e at 12°C may have a t t a i n e d t h e i r maximum l e n g t h at day 6, a p e r i o d c o i n c i d i n g with 50 F i g u r e 7 Mean (± 1 SD) standard l e n g t h (mm) of h e r r i n g l a r v a e sampled from the s i x s a l i n i t y - t e m p e r a t u r e treatments at v a r i o u s ages. Larvae were used f o r c a l o r i m e t r i c experiment i n 1979 (see Table 9). Ti ( 6 ° C - I 3 % c S ) e 9 E X I-"2 LU Q tr. < "2 < 9 LL! H _J I L_ To. ( I2°C " I 3 % » S ) _i i 1— To { 6 ° C - 21 % o S ) TP, ( 12°C " 21 %o S ) T, ( 6 ° C - 2 9 % o S ) T f i ( I2°C - 2 9 % . S ) ^ - 5 0 3 6 9 0 3 6 9 0 3 6 9 D A Y S A F T E R H A T C H I N G 10 C O M P O S I T E G R A P H T, T O T 6 T R E A T M E N T ( T ) 13 21 2 9 E T / / • / / r ifi o S °C - - O 13 29 21 6 13 ~ 21 12 29 _ 1 3 0 i 2i d 2 9 ° (1/ cn 52 Table 9 Table of means showing lengths (mm), weights (mg), and c a l o r i c values ( c a l ) of h e r r i n g l a r v a e sampled from the s i x s a l i n i t y - t e m p e r a t u r e treatments at v a r i o u s ages. Larvae were used for c a l o r i m e t r i c experiment i n 1979. In columns 3, 4 and 5, sample s i z e -- 30 l a r v a e , unless otherwise i n d i c a t e d ( b r a c k e t s ) . In columns 6, 7 and 8, sample s i z e -- 4 p e l l e t s , unless otherwise i n d i c a t e d ( b r a c k e t s ) . 1 2 3 4 5 6 7 8 Days A f t e r H a t c h i n g Treatment (Temperature-Sal 1n1 ty C o m b i n a t i o n s ) C C - V o o S ) Mean Standard Length/ L a r v a (mm) Mean Dry Weight'/ L a r v a (ng) ; Mean A s h - f r e e Dry Wt./ L a r v a (mg) Mean C a l o r i e s / L a r v a ( c a l ) Mean C a l o r 1 e s / gram d r y weight ( c a l ) Mean C a l o r i e s / gram a s h -f r e e d r y wt. ( c a l ) 0 (peak of h a t c h i n g ) 1 (6-13) 2 (6-21) 3 (6-29) 4 (12-13) 5 (12-21) 6 ( 12-29) 9.93 9.82 9.46 8 . 20 7.84 7.67 0.142 0. 153 0.158 0. 153 0. 162 O. 162 0.129 0. 139 0. 144 0.140 0.150 0.151 0.853 0.868 0.977 0.915 0.978 1.035 6,032 5,677 6, 150 5,990 6,024 6,377 6,622 6,245 6,736 6,520 6,518 6,875 3 1 2 3 4 5 6 9.81 9.86 9.86 9.20 (15) 8 .66 9.06 (19) 0. 138 0. 138 0.142 0. 139 (15) 0.141 0. 138 (19) 0. 127 0. 124 0.129 0.128 (15) 0.127 0. 128 (19) 0.806 0. 769 0.801 0.820 (2) 0.814 0.667 (3) 5,860 5,548 5,6 10 5,882 (2) 5,770 4,823 (3) 6,364 6,201 6,171 6.392 (2) 6.418 5 , 5 1 2 ( 3 ) 6 (end of y o l k - s a c s t a g e ) 1 2 3 4 5 6 10.40 10.31 10.36 9.95 (15) 9.97 (15) 9.70 (15) 0.137 0. 13.1 0. 134 0. 122 ( 15) 0. 122 (15) 0. 126 (15) 0.123 0.117 0. 1 20 0. 113 (15) 0,112 (15) 0.115 (15) 0 793 0.839 0.859 0.851 (2) 0.706 (2) 0.655 (2) 5,795 6,417 6,416 6.962 (2) 5,812 (2) 5,2 18 (2) 6,426 7,178 7,121 7,656 (2) 6,304 (2) 5,684 (2) 9 1 2 3 4 5 6 10.54 10.23 10.33 9.98 (15) 9.84 (15) 9.41 (15) 0.118 0. 123 0.117 0.101 (15) 0.108 (15) 0.111 (15) 0.106 0.109 0. 105 0.091 (15) 0.099 (15) 0.098 (15) 0.674 0.624 0.645 0.693 (2) 0.552 (2) 0.660 (2) 5,703 5,090 5,449 6,882 (2) 5,108 (2) 5,924 (2) 6.326 5,722 6, 107 7.633 (2) 5,585 (2) 6.756 (2) T a b l e 9 cn 54 the end of the yolk sac stage. At day 9, some l a r v a e of the 12°C group showed a decrease i n l e n g t h . Comparison of lengths of l a r v a e reared at d i f f e r e n t s a l i n i t i e s (13, 21 and 29°/ooS) shows that l a r v a e kept i n lower s a l i n i t i e s were l a r g e r than those i n higher s a l i n i t i e s ( F i g u r e 7, Table 9). T h i s trend i s t r u e i n both temperature groups. c) Dry Weight of Larvae Mean dry weight and a s h - f r e e dry weight per l a r v a are shown in Table 9. Mean dry weight of l a r v a e at the peak of hatching (day 0) ranged from 0.142 mg (6°C, 13°/ooS) to 0.162 mg (12°C, 21°/ooS; 12°C, 29°/ooS). In a s h - f r e e dry weight, the values at day 0 ranged from 0.129 mg (6°C, 13°/ooS) to 0.151 mg (12°C, 29°/ooS). In a l l treatments, there was a l i n e a r decrease i n l a r v a l weight from day 0 to day 9 ( F i g u r e s 8 and 9). A combined p l o t of the s i x l i n e a r r e g r e s s i o n l i n e s (T, to T 6 ) f o r the dry weights of h e r r i n g l a r v a e ( F i g u r e 8) shows that the eggs incubated at the higher temperature (12°C) produced s l i g h t l y h e a v i e r l a r v a e at day 0 than those incubated at the lower temperature (6°C). At day 9, however, the reverse was t r u e . Larvae c u l t u r e d at lower temperature (6°C) had the h i g h e s t dry weight while those c u l t u r e d at higher temperature (12°C) had the l e a s t . Among the 12°C group (Figure 8, T„, T 5 and T 6 ) , the slopes of the three r e g r e s s i o n l i n e s c a l c u l a t e d f o r the three s a l i n i t i e s were s i m i l a r (slope = -0.006 mg/day), which i n d i c a t e s a uniform r a t e of weight decrease. Among the 6°C group (Figure 8, T,, T 2 and T 3 ) , the slopes were not uniform. There was an 55 Fig u r e 8 C a l c u l a t e d r e g r e s s i o n l i n e s f o r mean dry weights (mg) of h e r r i n g l a r v a e used f o r c a l o r i m e t r i c experiment i n 1979. Larvae were sampled from the s i x s a l i n i t y - t e m p e r a t u r e treatments at v a r i o u s ages (see Table 9). In a given r e g r e s s i o n equation, Y = expected dry weight (mg), X = age (days a f t e r h a t c h i n g ) , r = c o r r e l a t i o n c o e f f i c i e n t . Tj ( 6 ° C - I 3 % O S ) y = . 1 4 5 + ( " . 002 x) r = ( - ) 0 . 8 7 9 E 2 or < i o LU > or o < LU .16 .1 4 .12 .10 T 2 ( 6 ° C - 2 l%oS ) y= .151 +• ( - .003x) r = (-) 0 . 9 8 3 T 3 ( 6 ° C - 2 9 % o S ) y = . 1 5 7 +•(- . 0 0 4 x ) r =(-) 0 . 9 9 2 • .14 .12 .10 T 4 ( I2°C " 13 % o S ) y= . I 5 5 t ( " . 0 0 6 x X-- (") 0 . 9 9 6 • T 5 ( I 2 ° C - 21 %oS ) y =. 16I + (- . 0 0 6 x ) r= (-) 0 . 9 9 6 T 6 ( I 2°C - 2 9 % o S ) y-- . I 5 9 +• (" . 0 0 6 x) r = (-) 0 . 9 8 9 0 3 6 9 0 3 6 9 0 DAYS A F T E R HATCHING 3 6 C O M P O S I T E G R A P H T, T O T 6 T R E A T M E N T ( T ) 57 F i g u r e 9 C a l c u l a t e d r e g r e s s i o n l i n e s f o r mean as h - f r e e dry weights (mg) of h e r r i n g l a r v a e used for c a l o r i m e t r i c experiment in 1979. Larvae were sampled from the s i x s a l i n i t y - t e m p e r a t u r e treatments at v a r i o u s ages (see Table 9). In a given r e g r e s s i o n equation, Y = expected a s h - f r e e dry weight (mg), X = age (days a f t e r h a t c h i n g ) , r = c o r r e l a t i o n c o e f f i c i e n t . .16 .14 .1 2 .10 .1 2 .10 .0 8 T , ( 6 ° C - l 3 % o S ) y= .132 + (" . 0 0 2 x) r = (-) 0 . 9 0 0 T 4 (12 ° C - I 3 % O S ) y= . l 4 2 + ( - . 0 0 5 x ) r = (") 0 . 9 9 0 T 2 (6°C- 21 % o S ) y= .137 + ( - . 0 0 3 x) r=(") 0 . 9 8 3 T 5 ( I 2 ° C - 21 % » S ) y = J 4 7 + (- . 0 0 6 x ) r = (-) .991 T 3 ( 6 ° C - 2 9 % o S ) y= J 4 3 + (- . 0 0 4 x ) r= (') 0 . 9 9 6 • T 6 ( 12°C - 2 9 % o S ) y = . l 4 9 + (- . 0 0 6 x ) r = (") 0 . 9 9 4 0 3 6 9 0 3 6 9 0 DAYS A F T E R HATCHING C O M P O S I T E G R A P H T, TO T 6 0 3 6 9 59 i n c r e a s i n g slope i n higher s a l i n i t i e s (e.g., s a l i n i t i e s 13, 21 and 29°/ooS had slopes equal to -0.002, -0.003 and -0.004 mg/day, r e s p e c t i v e l y ) . The v a r y i n g s l o p e s at a c u l t u r e temperature of 6°C show that l a r v a l weight decreases at lower temperatures proceeded at a slower r a t e i n lower s a l i n i t i e s . In terms of a s h - f r e e dry weight, a s i m i l a r r e s u l t was obtained ( F i g u r e 9). d) C a l o r i e Determination (1) Percent Unburned Organic Residue. Incomplete combustion occurred i n a l l p e l l e t samples f i r e d i n the c a l o r i m e t e r . The mean percentage of unburned organic residue per p e l l e t weight i s shown in Table 10. There i s a t r e n d towards higher percentage of unburned or g a n i c residue with smaller p e l l e t s . (2) Percent Ash from D i r e c t and I n d i r e c t Ashing Methods. The percentages of ash estimated from the d i r e c t ashing method g e n e r a l l y were higher than those obtained from the i n d i r e c t method (ashing of c a l o r i m e t r y r e s i d u e ) . Table 10 (column 7) shows d i f f e r e n c e s in ash percentages ranging from 0.43 to 4.66% ( e x c l u d i n g two negative v a l u e s ) . The comparison i s based on d i f f e r e n t sample s i z e s , 1-2 p e l l e t s f o r d i r e c t ashing, 2-4 p e l l e t s f o r i n d i r e c t a s h i n g . (3) C a l o r i c Values of H e r r i n g Larvae. R e s u l t s of t h i s c a l o r i m e t r i c t e s t are presented i n c a l / l a r v a , c a l / g dry weight, and c a l / g a s h - f r e e dry weight (Table 9). During the peak of h a t c h i n g (day 0), the c a l o r i c values of l a r v a e averaged from a l l treatments were: 6,042 c a l / g dry weight, 6,586 c a l / g a s h-free 60 Table 10 Table of means showing dry weights of p e l l e t s (1 p e l l e t = 5 l a r v a e ) combusted in bomb c a l o r i m e t e r , percent unburned organic r e s i d u e , and percent ash obtained from d i r e c t and i n d i r e c t (from residue) methods. Percent values based on p e l l e t dry weight. Sample s i z e — 4 p e l l e t s , u n l e s s otherwise i n d i c a t e d (brackets) (1979 d a t a ) . 1 2 3 4 5 6 7 Days A f t e r H a t c h i n g Treatment (Temperature-Sal 1n1 t y C o m b i n a t i o n s ) C C - '/ooS) Mean Dry Weight Pel l e t (mg) Mean Unburned O r g a n i c Residue (%) Mean Ash ( d i r e c t method) (%) Mean Ash (from r e s 1 due) (%) D1 f f e r e n c e 1n Ash V a l u e s (5-6) • (%) 0 (peak of h a t c h i n g ) 1 (6-13) 2 (6-21) 3 (6-29) 4 (12-13) 5 ( 12-21 ) 6 (12-29) 0.708 0. 739 0.777 0.762 0.809 0. 794 3.51 3.79 3.39 1 . 86 2 .72 2.00 8.88 (2) 9.04 (2) 8.66 (2) 8.14 (2) 7.51 (2) 7.26 (2) 6. 12 7 .94 4 . 45 7.19 5 .43 5 . 30 2 .76 1 . io 4.21 0.95 2.08 1.96 3 1 2 3 4 5 6 0.670 0.692 0.712 0.696 (2) 0. 702 0.676 (2) 2.95 3.60 4 .00 4.04 (2) 5.13 5.00 (3) 7.94 (2) 10.40 (2) 9.07 (2) 7.96 ( 1 ) 10. 12 (2) 7.30 ( 1 ) 8.63 9.51 9 . 10 4.60 (2) 7 .05 6.87 (3) (-) 0.69 0.89 (-) 0.03 3 . 36 3 .07 0.43 6 (end of y o l k - s a c s t a g e ) 1 2 3 4 5 6 0.684 0.655 0.666 0.612 (2) 0.608 (2) 0.628 (2) 3.48 2 .88 3 .59 5.60 (2) 5.94 (2) 5.10 (2) 9.78 (2) 10.59 (2) 9.82 (2) 9.15 (1) 7.79 ( 1 ) 8.24 ( 1 ) 7 .84 7.31 7 .82 6.88 (2) 7.26 (2) 7.01 (2) 1 .94 3.28 2.00 2.27 0.53 1 . 23 9 1 2 3 4 5 6 0.589 0.613 0.574 0.504 (2) 0.540 0.556 4.17 3.12 4 .68 5.17 (2) 5.56 (2) 5.77 (2) 9.84 (2) 11.07 (2 ) 10.78 (2) 9.85 (1) 8.57 (1) 12.31 ( 1) 8.50 9 .95 9.77 5. 19 (2) 7.04 (2) 7.92 (2) 1 . 34 1.12 1 .01 4 .66 1 .53 4.39 T a b l e 10 62 dry weight and 0.938 c a l / l a r v a . At day 9, these values were: 5,693 c a l / g dry weight, 6,355 c a l / g a s h - f r e e dry weight and 0.641 c a l / l a r v a . There was a g e n e r a l p a t t e r n of d e c r e a s i n g mean c a l / l a r v a from day 0 to day 9 i n a l l treatments (Figure 10). T h i s p a t t e r n , however, was not c o n s i s t e n t e i t h e r i n c a l / g dry weight or c a l / g a s h-free dry weight. Unusually high c a l o r i c v alues were obtained f o r day 6 l a r v a e (treatments 2, 3 and 4) and f o r day 9 l a r v a e (treatment 4) (Table 9). The c a l o r i c values at day 6 were much higher than those at day 0 and day 3. The r a t e of c a l o r i c decrease ( c a l / l a r v a ) from day 0 to day 9 v a r i e d between the s i x treatments ( F i g u r e 10). Treatments 1 (6°C-13°/ooS) and 4 (12°C-13°/ooS) showed the l e a s t slopes (-0.018 and -0.021 c a l / l a r v a / d a y , r e s p e c t i v e l y ) , i n d i c a t i n g a r e l a t i v e l y slower r a t e of c a l o r i c decrease; whereas treatments 5 (12°C-21°/ooS) and 6 (12°C-29°/ooS) showed the h i g h e s t slopes (-0.046 and -0.038 c a l / l a r v a / d a y , r e s p e c t i v e l y ) , i n d i c a t i n g a f a s t e r r a t e of c a l o r i c decrease. A n a l y s i s of v a r i a n c e of the c a l / g a s h - f r e e dry weight data (Table 11) shows the f o l l o w i n g s i g n i f i c a n t e f f e c t s (o=0.05): (1) age from day 0 to day 9, and (2) i n t e r a c t i o n between s a l i n i t y , temperature and age. No f u r t h e r s t a t i s t i c a l a n a l y s i s was done on the c a l o r i c data due to unequal sample s i z e s . 63 F i g u r e 10 C a l c u l a t e d r e g r e s s i o n l i n e s f o r the mean c a l o r i e s / l a r v a . H e r r i n g l a r v a e samples were taken from the s i x s a l i n i t y -temperature treatments at v a r i o u s ages (see Table 9). In a given r e g r e s s i o n equation, Y = expected c a l o r i e s / l a r v a , X = age (days a f t e r h a t c h i n g ) , r = c o r r e l a t i o n c o e f f i c i e n t . 0 3 6 9 0 3 6 9 0 3 6 9 0 3 6 9 cn D A Y S A F T E R H A T C H I N G *" 65 Table 11 Summary of a n a l y s i s of v a r i a n c e f o r the c a l / g ash-free dry weight of h e r r i n g l a r v a e . Larvae were sampled at day 0, 3, 6 and 9 a f t e r h a t c h i n g from the s i x s a l i n i t y - t e m p e r a t u r e treatments. * s i g n i f i c a n t d i f f e r e n c e at o=0.05; NS — not s i g n i f i c a n t ; S — i n c u b a t i o n and r e a r i n g s a l i n i t y (°/ooS); T -- i n c u b a t i o n and r e a r i n g temperature (°C); A -- age (days a f t e r hatching) (1979 data) . Source of V a r i a t i o n OF SS MS F ( c a 1 c ) F ( t a b ) («=0.05) S I g n 1 f i c a n c e S a l 1 n 1 t y C / o o ) (S) 2 1 ,426,496.66 713,248 34 1 .805 3. 150 NS Temperature C C ) (T) 1 6,756.08 6,756 08 0.017 4 .001 NS Age (days a f t e r h a t c h i n g ) (A) 3 5,162,769.62 1 ,720,923 21 4.354 2.758 * I n t e r a c t i o n S x T 2 2,414,411.30 1,207,205 65 3 .055 3. 150 NS S x A 6 4,652,145.32 775,357 55 1 .962 2.254 NS T x A 3 2,332,169.81 777,389 94 1 .967 2 . 758 NS S x T x A 6 5,798,450.12 966,408 35 2 .445 2.254 * R e p l I c a t 1 o n 57 22,527,283.26 395,215 .50 T o t a l 80 44,320,482.18 T a b l e 11 • i 67 DISCUSSION T h i s present i n v e s t i g a t i o n examines the hypothesis that s a l i n i t y t o l e r a n c e of yolk sac h e r r i n g l a r v a e i s i n f l u e n c e d by (1) i n c u b a t i o n s a l i n i t y , (2) i n c u b a t i o n temperature, and (3) c u l t u r e (post-hatching) temperature. A c a l o r i m e t r i c experiment was a l s o c a r r i e d out to examine the e f f e c t s of v a r i o u s combinations of s a l i n i t i e s and temperatures on aspects of l a r v a l development. In the s a l i n i t y t o l e r a n c e t e s t employed, only the upper l e t h a l s a l i n i t y l i m i t s or upper i n c i p i e n t l e t h a l s a l i n i t y l e v e l (Fry, 1971) of h e r r i n g l a r v a e was measured. The t e s t f o r the lower l e t h a l s a l i n i t y l i m i t s was not done. R e s u l t s of the s a l i n i t y t o l e r a n c e t e s t show that both i n c u b a t i o n s a l i n i t y and temperature exert a s i g n i f i c a n t e f f e c t on the t o l e r a n c e of l a r v a e to s a l i n i t y . The e f f e c t of c u l t u r e temperature i s l e s s w e l l d e f i n e d . A s y n t h e s i s of these r e s u l t s w i l l be presented in F i g u r e 11. 1 . E f f e c t of Incubation S a l i n i t y S a l i n i t y t o l e r a n c e of l a r v a e was maximized f o r eggs incubated i n higher s a l i n i t i e s . I t appears that the c a p a c i t y of l a r v a e to t o l e r a t e higher s a l i n i t y l e v e l s i n enhanced by the exposure of embryos to higher s a l i n i t i e s d u r i n g i n c u b a t i o n . H e r r i n g eggs and l a r v a e have the c a p a c i t y to osmoregulate 68 (Holliday,.1965; 1969; H o l l i d a y and Jones, 1965). Osmoregulation of the h e r r i n g embryo s t a r t s d u r i n g blastodermal overgrowth and i s f u n c t i o n a l at the completion of b l a s t o p o r e c l o s u r e . T h i s i s true to both A t l a n t i c (Clupea harengus L.) and P a c i f i c h e r r i n g eggs ( H o l l i d a y and Jones, 1965; H o l l i d a y , 1969; A l d e r d i c e e_t a l . , 1979). In order f o r eggs and l a r v a e to s u r v i v e i n sub- or supra-normal s a l i n i t i e s , t h e i r t i s s u e s must t o l e r a t e those osmotic media and r e g u l a t e and r e t u r n t h e i r body f l u i d c o n c e n t r a t i o n to l e v e l s near t h e i r o r i g i n a l v a l u e s ( H o l l i d a y , 1965; B l a x t e r and H o l l i d a y , 1963). If h e r r i n g l a r v a e are exposed to a s a l i n i t y d i f f e r e n t from that experienced d u r i n g i n c u b a t i o n , then they w i l l have to undergo an osmoregulation process in order to s u r v i v e . The s i t e of t h i s r e g u l a t o r y process i s b e l i e v e d to be the epidermal c e l l s t h a t arose from the ectoderm of the embryo ( H o l l i d a y and Jones, 1965). If an egg i s incubated at a higher s a l i n i t y , which i t can t o l e r a t e , i t may a c c l i m a t e i n some way to that s a l i n i t y and i n the process r a i s e i t s r e s i s t a n c e to higher, supranormal s a l i n i t i e s . A c c l i m a t i o n l e a d i n g to an i n c r e a s e in e f f i c i e n c y of r e g u l a t i o n would decrease the energy requirement of l a r v a e (Kinne, 1964b). In t h i s study, 29°/ooS was the h i g h e s t i n c u b a t i o n s a l i n i t y and l a r v a e h a t c h i n g from t h i s medium showed the h i g h e s t t o l e r a n c e to upper extreme s a l i n i t i e s . I t appears that the a c c l i m a t i o n h i s t o r y of l a r v a e p l a y s an important r o l e i n t h e i r f u t u r e responses to other f a c t o r s . Fry (1947) has p o i n t e d t h i s out, s t a t i n g that p r e v i o u s experience of the organism i n r e l a t i o n to the f a c t o r s of the environment under c o n s i d e r a t i o n must always be taken i n t o account. The importance 69 of knowing the a c c l i m a t i o n h i s t o r y may be e x p l a i n e d by c i t i n g some examples. Kinne (1962) provided evidence that the spawning s a l i n i t y of d e s e r t p u p f i s h (Cyprinodon macularius B a i r d and G i r a r d ) had a s i g n i f i c a n t e f f e c t on the f u n c t i o n a l c h a r a c t e r i s t i c s of the o f f s p r i n g . Growth r a t e s and food c o n v e r s i o n e f f i c i e n c y of f i s h h a t c h i n g from eggs that remained i n the spawning s a l i n i t y were higher than i n those f i s h a r i s i n g from eggs t r a n s f e r r e d to another s a l i n i t y l e v e l . These d i f f e r e n c e s p e r s i s t e d beyond the stage of sexual m a t u r i t y . Kinne suggested that t h i s was p r i m a r i l y due to an i r r e v e r s i b l e , f u n c t i o n a l non-genetic a d a p t a t i o n to the spawning s a l i n i t y . In order to understand the concept of an i r r e v e r s i b l e , f u n c t i o n a l non-genetic a d a p t a t i o n , some d e f i n i t i o n of terms, a c c o r d i n g to Kinne (1962, 1964b), are given. Adaptation r e f e r s to "an e c o l o g i c a l phenomenon comprising adjustments of organisms to a l t e r a t i o n s i n the i n t e n s i t y p a t t e r n of v a r i a b l e s i n t h e i r environment, which u l t i m a t e l y r e s u l t i n a r e l a t i v e i n c r e a s e i n t h e i r c a p a c i t y to s u r v i v e , reproduce or compete under the new c o n d i t i o n s . " I t may be genetic or non-genetic i n nature, the former r e f e r s to ge n e t i c changes i n the response mechanism of i n d i v i d u a l s passed on to the next g e n e r a t i o n , the l a t t e r to somatic changes not t r a n s m i t t e d to the next g e n e r a t i o n . Non-gen e t i c a d a p t a t i o n , which i s a l s o c a l l e d a c c l i m a t i z a t i o n , may comprise f u n c t i o n a l and s t r u c t u r a l changes. Examples of f u n c t i o n a l a d a p t a t i o n , as c i t e d by Kinne (1962), i n c l u d e changes i n the r a t e s or e f f i c i e n c e s of metabolism and of a c t i v i t y or changes of h a b i t s , while those of s t r u c t u r a l a d a p t a t i o n i n c l u d e changes of the a r c h i t e c t u r e of c e l l s , t i s s u e s , organs or the 70 whole organism. F u n c t i o n a l non-genetic a d a p t a t i o n c o u l d be r e v e r s i b l e or i r r e v e r s i b l e , the l a t t e r i n v o l v i n g changes in response mechanism which p e r s i s t i n the subsequent l i f e of the i n d i v i d u a l . The above example on Cyprinodon macularius i l l u s t r a t e s that the spawning medium a f f e c t e d the p h y s i o l o g i c a l p r o p e r t i e s of the immediate environment of the embryo and that these induced p r o p e r t i e s tend to p e r s i s t i n l a t e r development at d i f f e r e n t s a l i n i t i e s . T h i s o b s e r v a t i o n conforms, with that of A l d e r d i c e and V e l s e n (1978) on P a c i f i c h e r r i n g eggs. The exposure of eggs to d i f f e r e n t s a l i n i t i e s (0 - 42°/ooS, 6.5°C) for a two and one h a l f hour p e r i o d f o l l o w i n g f e r t i l i z a t i o n had a c o n t i n u i n g i n f l u e n c e on egg development, d e s p i t e the f a c t that they were incubated in i d e n t i c a l c o n d i t i o n s (17°/ooS, 7°C). The authors suggested that the i o n i c and osmotic p r o p e r t i e s of yolk may have been " f i x e d " d u r i n g the two and one h a l f hour exposure p e r i o d which c o i n c i d e d with the shutdown of the v i t e l l i n e membrane p e r m e a b i l i t y a f t e r f e r t i l i z a t i o n . They suspected that an i r r e v e r s i b l e , non-genetic a d a p t a t i o n was a s s o c i a t e d with t h e i r o b s e r v a t i o n . In the case of the h e r r i n g eggs used in t h i s experiment, they were spawned, f e r t i l i z e d and incubated i n a constant l e v e l of a s p e c i f i c s a l i n i t y ; t h a t i s , 13, 21 and 29°/ooS. When h e r r i n g eggs are shed, t h e i r i n t e r n a l osmotic c o n c e n t r a t i o n approaches that of the o u t s i d e environment. They have no osmoregulatory c a p a c i t y s h o r t l y before and a f t e r f e r t i l i z a t i o n ( H o l l i d a y , 1965; H o l l i d a y and Jones, 1965). As suggested by A l d e r d i c e and Velsen (1978), the osmotic and i o n i c p r o p e r t i e s of the eggs may be f i x e d d u r i n g a two and one h a l f hour p e r i o d 7 1 f o l l o w i n g f e r t i l i z a t i o n . I t i s assumed that the osmotic and i o n i c c o n c e n t r a t i o n s experienced by the present eggs d u r i n g spawning and f e r t i l i z a t i o n have i n f l u e n c e d the embryo and the r e s u l t i n g l a r v a e i n t h e i r f u t u r e responses to e x t e r n a l s a l i n i t y . A non-genetic a d a p t a t i o n of the embryos to s a l i n i t y may be a s s o c i a t e d with t h e i r response to the p o s t - h a t c h i n g s a l i n i t i e s . E a r l i e r experiments showed that the s a l i n i t y range apparently p r o v i d i n g the l e a s t osmotic work in P a c i f i c h e r r i n g embryos i s from 1 3 to 1 9 ° / O O S ( A l d e r d i c e et a l . , 1 9 7 9 ) . T h i s s a l i n i t y range pro v i d e s maximum s u r v i v a l of eggs to hatching when a s s o c i a t e d with an i n t e r m e d i a t e temperature of 8 . 3 ° C . At the l a r v a l stage, however, there appears to be d i f f e r e n t s a l i n i t y - t e m p e r a t u r e optima that maximize l a r v a l s u r v i v a l i n d i f f e r e n t s a l i n i t i e s . T h i s o b s e r v a t i o n conforms with that of Costlow e_t a_l. ( 1 9 6 6 ) . They found s e v e r a l s a l i n i t y - t e m p e r a t u r e optima f o r the v a r i o u s stages of z o e a l and megalops l a r v a e of a crab, Rhithropanopeus h a r r i s i i (Gould). 2. E f f e c t of Incubation Temperature Incubation temperature a l s o produced a s i g n i f i c a n t i n f l u e n c e on the s a l i n i t y t o l e r a n c e of l a r v a e . The lower i n c u b a t i o n temperature ( 6 ° C ) was f a v o r a b l e t o . t h e s u r v i v a l of the r e s u l t i n g l a r v a e . Larvae hatching from the lower temperatures may have g r e a t e r yolk r e s e r v e s than those hatching from higher temperatures. Greater y o l k content i s advantageous to the l a r v a e , e s p e c i a l l y when they are s u b j e c t e d to sub- or supra-normal c o n d i t i o n s . E x t r a energy c o u l d be r e q u i r e d i n 72 a c t i v e ion r e g u l a t i o n of t i s s u e s at abnormal s a l i n i t y condi t i o n s . From these r e s u l t s , i t appears that a high i n c u b a t i o n s a l i n i t y and low i n c u b a t i o n temperature enhance the t o l e r a n c e of l a r v a e to higher s a l i n i t i e s . 3. E f f e c t of Post-Hatching Temperature and Combined E f f e c t s of  the Three F a c t o r s S t a t i s t i c a l l y , the e f f e c t of p o s t - h a t c h i n g temperature i s not s i g n i f i c a n t . However, some i n d i c a t i o n s of an i n t e r a c t i o n of t h i s f a c t o r with i n c u b a t i o n s a l i n i t y and temperature can be observed. In F i g u r e 11, s u r f a c e contours drawn f o r the E D 5 0 p o i n t s show the f o l l o w i n g p a t t e r n s : (1) eggs incubated at lower temperature (6°C) and higher s a l i n i t i e s r e s u l t in higher s a l i n i t y t o l e r a n c e at 6 to 9°C p o s t - h a t c h i n g temperatures, (2) eggs incubated at higher temperature (12°C) and higher s a l i n i t i e s r e s u l t i n higher s a l i n i t y t o l e r a n c e at 9 to 12°C p o s t - h a t c h i n g temperatures. As noted, higher s a l i n i t y t o l e r a n c e was observed i n l a r v a e hatching from eggs incubated at lower temperature (6°C). The data, however, are too l i m i t e d to suggest that s a l i n i t y t o l e r a n c e would continue to increase i f the i n c u b a t i o n temperature were f u r t h e r reduced (e.g., < 6°C). There i s another p o s s i b i l i t y that s a l i n i t y t o l e r a n c e of l a r v a e may be maximized i f the eggs were incubated at a temperature near 9°C. When la r v a e h a t c h i n g from 6 and 12°C i n c u b a t i o n groups were t e s t e d at 9°C, they a l l showed higher s a l i n i t y t o l e r a n c e , even s l i g h t l y 73 F i g u r e 11 E s t i m a t i o n of upper l e t h a l s a l i n i t y l i m i t s ( E D 5 0 at 72 hours) of h e r r i n g l a r v a e exposed to v a r i o u s combinations of s a l i n i t y and temperature d u r i n g i n c u b a t i o n and l a r v a l p e r i o d . Each curve i n the two p l o t s approximates the expected E D 5 0 value (°/ooS) at v a r i o u s l e v e l s of i n c u b a t i o n s a l i n i t y , i n c u b a t i o n temperature and c u l t u r e temperature. 75 higher than those l a r v a e t e s t e d at the same temperature as that at i n c u b a t i o n (e.g., 6 -> 6°C; 1 2 —> 12°C). An i n c u b a t i o n of eggs at 9°C and t e s t i n g of the s a l i n i t y t o l e r a n c e of l a r v a e at the same temperature would probably support t h i s second suggestion. To summarize, l a r v a l t o l e r a n c e to upper s a l i n i t y l e v e l s may be maximized at l a r v a l temperatures near 9°C when i n c u b a t i o n o c c u r r e d at 9°C. At lower and higher i n c u b a t i o n temperatures, the optimal l a r v a l temperature may be d i s p l a c e d toward lower and higher temperatures r e s p e c t i v e l y -- that i s in the d i r e c t i o n of the temperature of i n c u b a t i o n . In a d d i t i o n , the absolute l e v e l of s a l i n i t y t o l e r a n c e i s i n c r e a s e d i f the eggs were incubated at higher s a l i n i t i e s . The low/low - high/high s a l i n i t y - t e m p e r a t u r e i n t e r a c t i o n , a s s o c i a t e d with egg s u r v i v a l to hatc h i n g , does not apply i n the s a l i n i t y t o l e r a n c e of h e r r i n g l a r v a e . The in c u b a t i o n s a l i n i t y and temperature play a greater r o l e i n the l a r v a l c a p a c i t y to s u r v i v e in d i f f e r e n t s a l i n i t i e s . 4. C a l o r i m e t r y Newly hatched P a c i f i c h e r r i n g l a r v a e from the s i x s a l i n i t y -temperature combinations show mean c a l o r i c values ranging from 6,245 to 6,875 c a l / g ash-free dry weight (x=6,586 c a l / g a s h - f r e e dry wt.). These values are higher than those obtained from s p r i n g spawning Western B a l t i c Sea h e r r i n g (Clupea harengus). PaffenhSfer and Rosenthal (1968) obtained c a l o r i c values of 5,940 c a l / g organic substance from newly hatched B a l t i c h e r r i n g l a r v a e . Even at day'3 a f t e r h a t c h i n g , the P a c i f i c h e r r i n g l a r v a e 76 s t i l l showed hig h e r c a l o r i c content than the B a l t i c h e r r i n g measured at the same age. The d i f f e r e n c e i n c a l o r i c contents of the l a r v a e may be due to the d i f f e r e n c e i n the o r i g i n a l . e g g s i z e and i n the amount of endogenous supply of energy of the two s p e c i e s . E l d r i d g e et a l . (1977) found P a c i f i c h e r r i n g eggs to c o n t a i n 1.298 c a l / e g g (mean egg weight = 0.216 mg), whereas, Paf f e n h S f e r and Rosenthal (1968) obtained a mean c a l o r i c value of 0.748 c a l / e g g (mean egg weight = 0.170 mg) f o r B a l t i c h e r r i n g eggs. P a c i f i c h e r r i n g eggs tend to c o n t a i n higher c a l o r i c values than t h e i r A t l a n t i c c o u n t e r p a r t s and those of other species ( E l d r i d g e et a l . , 1977). Comparison of c a l o r i c c o ntents per l a r v a obtained i n the c u r r e n t experiment and those by E l d r i d g e et a_l. shows s l i g h t d i f f e r e n c e s ; the values o b t a i n e d by these authors were s l i g h t l y lower. They r e a r e d l a r v a e at 12.5°C, 23°/ooS and o b t a i n e d 0.904 and 0.737 c a l / l a r v a at day 0 and day 3, r e s p e c t i v e l y . In comparison to those l a r v a e reared at 12°C, 21°/ooS of the c u r r e n t experiment, the c a l o r i c values were 0.978 (day 0) and 0.814 (day 3). D i f f e r e n c e s i n the above c a l o r i c v a l u e s c o u l d be due to d i f f e r e n c e s i n c a l o r i m e t r i c techniques. Although, a microbomb c a l o r i m e t e r was used i n both experiments, the two c a l o r i m e t e r s may not be i d e n t i c a l i n t h e i r performance. Furthermore, E l d r i d g e et a l . measured the c a l o r i c contents of the yolk and l a r v a l body s e p a r a t e l y , whereas, i n the present experiment, i n t a c t l a r v a e were used. The c a l o r i c values of l a r v a e reared at d i f f e r e n t s a l i n i t i e s and temperatures show a p o t e n t i a l i n f l u e n c e from the two f a c t o r s . From the present data, i t i s , however, d i f f i c u l t to 77 make v a l i d comparisons because of . the unusually high c a l o r i c values i n some treatments. These anomalous values may have been a r e s u l t of the incomplete combustion of the l a r v a l p e l l e t s . The e f f e c t of s a l i n i t y and temperature on l a r v a l development may be seen by examining the lengths and dry weights of h e r r i n g l a r v a e used i n the c a l o r i m e t r y experiment. Larvae reared i n lower temperatures and s a l i n i t i e s were g e n e r a l l y longer. The i n v e r s e r e l a t i o n s h i p between s a l i n i t y and length of l a r v a e was a l s o observed by other authors. A l d e r d i c e and Velsen (1971) and Dushkina (1973) found t h i s r e l a t i o n s h i p i n P a c i f i c h e r r i n g l a r v a e and H o l l i d a y and B l a x t e r (1960) i n A t l a n t i c h e r r i n g l a r v a e . These authors suggest that the l a r g e s i z e s of la r v a e i n lower s a l i n i t i e s are due to t h e i r g r e a t e r water content. Larvae reared i n higher temperature (12°C) showed a f a s t e r growth r a t e (length) and weight decrease from day 0 to day 9. The weight decrease i s an i n d i c a t i o n of yolk u t i l i z a t i o n . I f separate dry weight measurements of yolk and l a r v a l body t i s s u e had been taken, the growth of l a r v a e c o u l d have been observed through the convers i o n of yolk m a t e r i a l to l a r v a l body t i s s u e . Those l a r v a e from the high temperature - high s a l i n i t y groups (12°C, 29°/ooS) showed g r e a t e r dry weights and as h - f r e e dry weights d u r i n g peak of hatching (day 0) than those from the low temperature - low s a l i n i t y (6°C, 13°/OOS) groups. T h i s suggests that the longer l a r v a e at lower s a l i n i t i e s r e f l e c t a high water content as dry weights at lower s a l i n i t i e s were lower than those at high s a l i n i t i e s . 78 5. E c o l o g i c a l I m p l i c a t i o n s In B r i t i s h Columbia waters, spawning of P a c i f i c h e r r i n g i s observed in s a l i n i t i e s ranging from 8 to 29°/ooS (McMynn and Hoar, 1953; A l d e r d i c e et a l . , 1979), with most of the spawning o c c u r r i n g i n s a l i n i t i e s of 24 to 29°/ooS at 7 to 10°C. Hatched l a r v a e are found i n the v i c i n i t y of the spawning ground (Stevenson, 1962), where they presumably experience the same s a l i n i t i e s as those at i n c u b a t i o n . These s a l i n i t i e s are n o t a b l y higher than the range of s a l i n i t i e s (13 to 19°/OOS) that p r o v i d e s the l e a s t osmotic work f o r embryos. I t was hypothesized that the c a p a c i t y f o r h e r r i n g l a r v a e to t o l e r a t e h i g h environmental s a l i n i t i e s may be i n f l u e n c e d by p o s t - h a t c h i n g temperature. T h i s was based on the l a b o r a t o r y f i n d i n g that a low/low - high/high i n t e r a c t i o n between s a l i n i t y and temperature i n f l u e n c e d egg development to h a t c h i n g ( A l d e r d i c e and V e l s e n , 1971). H e r r i n g l a r v a e hatch d u r i n g the s p r i n g increase i n temperature, and i f a s i m i l a r low/low - h i g h / h i g h s a l i n i t y -temperature i n t e r a c t i o n occurs at the l a r v a l stage, then the r i s e i n temperature during s p r i n g would be e c o l o g i c a l l y advantageous to the l a r v a e . T h i s h y p o t h e s i s , however, was not supported by the r e s u l t s of the c u r r e n t experiment. The r i s i n g temperature may s t i l l be advantageous to the l a r v a e . Food pr o d u c t i o n in the sea w i l l l i k e l y i n c r e a s e at r i s i n g temperatures and the abundance of food c o u l d be b e n e f i c i a l to the l a r v a e . The second hypothesis that the s a l i n i t y and temperature of i n c u b a t i o n c o u l d i n f l u e n c e the s a l i n i t y t o l e r a n c e of l a r v a e i s supported by the r e s u l t s of the present experiment. High 79 i n c u b a t i o n s a l i n i t y and low i n c u b a t i o n temperature favor l a r v a l s u r v i v a l to high s a l i n i t i e s . T h i s c o u l d be of e c o l o g i c a l importance. In nature, the h e r r i n g eggs are spawned, f e r t i l i z e d , and incubated i n high osmotic media. When l a r v a e hatch, they are more l i k e l y to experience the same media of h i g h osmotic c o n c e n t r a t i o n . The exposure of embryos to high s a l i n i t i e s during e a r l y egg development may r e s u l t i n an a d a p t a t i o n by the embryos to the high osmotic c o n d i t i o n . . If t h i s a d a p t a t i o n , which i s b e l i e v e d to be non-genetic and i r r e v e r s i b l e , occurs, then h e r r i n g l a r v a e subjected to the high s a l i n i t i e s i n the environment may be able to s u r v i v e . T h i s non-genetic a d a p t a t i o n to s a l i n i t y i s suggested only as one of the p o s s i b l e mechanisms that the h e r r i n g embryos may u t i l i z e i n nature. The t o l e r a n c e of h e r r i n g l a r v a e to higher s a l i n i t i e s may be i n f l u e n c e d by other environmental f a c t o r s not c o n s i d e r e d i n the experiment, but may perhaps act i n the n a t u r a l environment. The hypothesis r e g a r d i n g the low/low - high/high i n t e r a c t i o n between i n c u b a t i o n temperature and s a l i n i t y suggested f o r egg development ( A l d e r d i c e and V e l s e n , 1971) does not appear to apply to the f o l l o w i n g l a r v a l stage. Greatest r e s i s t a n c e to supranormal s a l i n i t i e s was found i n l a r v a e hatching from eggs incubated at lower (6°C) temperatures and higher (29°/oo) s a l i n i t i e s . Yet, i t i s r e c o g n i z e d that i n a l l i n c u b a t i o n groups t e s t e d , s a l i n i t y t o l e r a n c e of the r e s u l t i n g l a r v a e was g r e a t e s t at s a l i n i t i e s near 20°/oo, the lowest s a l i n i t y examined i n the s e r i e s of p o s t - h a t c h i n g t e s t c o n d i t i o n s . Hence l a r v a e at s a l i n i t i e s of 20°/oo may have been under the lowest osmotic s t r e s s . The i n c r e a s e i n r e s i s t a n c e to 80 higher s a l i n i t i e s noted f o r eggs incubated at the highest s a l i n i t y (29°/oo) t h e r e f o r e may have r e s u l t e d from an a d a p t i v e s h i f t i n the range of s a l i n i t y t o l e r a n c e . Hence, a non-genetic a d a p t a t i o n of the egg and embryo to i n c u b a t i o n s a l i n i t y may be one of s h i f t i n g the range of s a l i n i t y t o l e r a n c e upward with i n c r e a s e d i n c u b a t i o n s a l i n i t y . F u r t h e r examination of t h i s q u e s t i o n would i n v o l v e examination of both lower and higher i n c i p i e n t l e t h a l s a l i n i t y l e v e l s and the manner in which they may vary as suggested by Precht's (1959, 1961, 1967) a n a l y s i s of r e s i s t a n c e a d a p t a t i o n . 81 LITERATURE CITED A l d e r d i c e , D. F. and C. R. F o r r e s t e r . 1967. 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