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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  P A C I F I C 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  o f San C a r l o s ,  Philippines,  1976  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE  FACULTY OF GRADUATE STUDIES Department  We a c c e p t to  THE  this  of  Zoology  thesis  the required  as  standard  UNIVERSITY OF BRITISH July  © Corazon  conforming  COLUMBIA  1981  Echevarria  Dueftas,  1981  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t  the L i b r a r y s h a l l make  it  and  f r e e l y a v a i l a b l e for reference  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be  department o r by h i s o r her  granted by  the head o f  representatives.  my  It i s  understood t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain  s h a l l not  be  allowed without my  permission.  Department of  Z O O U O G Y  The U n i v e r s i t y o f B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date  /in \  J u W  SLA-.  13  & l  Columbia  written  ABSTRACT  A  study  was  incubation  salinity  temperature  on  pa 1 1 a s i  conducted and  salinity  to  t e m p e r a t u r e . and tolerance  on  herring  effects  In  the  artificially 6°C.  salinity  post-hatching  of P a c i f i c  herring  (Clupea  salinities  tolerance  fertilized  test,  eggs  as those i n which  incubation  also  and t e m p e r a t u r e . herring  f e r t i l i z e d i n three s a l i n i t i e s  Newly  of  l a r v a e t h a t were i n c u b a t e d a n d 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  of  the  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  conducted  at  determine  temperature  were  eggs  were  ( 1 3 , 21 a n d 29°/ooS)  incubated  in  t h e same  t h e y were f e r t i l i z e d a t two  levels  (6 a n d 12°C), m a k i n g s i x i n c u b a t i o n  g r o u p s . D u r i n g t h e peak o f t h e h a t c h i n g p e r i o d , g r o u p s o f l a r v a e from each  i n c u b a t i o n g r o u p were e x p o s e d  salinities,  a  series  of  test  r a n g i n g f r o m 20 t o 55°/ooS a t t h r e e l e v e l s o f p o s t -  hatching temperatures measured  by  each t e s t  salinity  were  to  ( 6 , 9 a n d 12°C).  determining  employed:  the  Salinity  mortality  over a p e r i o d of  72  tolerance  was  of l a r v a e o c c u r r i n g i n hours.  Two  approaches  1) t i m e - m o r t a l i t y , a n d 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 t h e e f f e c t i v e time t o 50% m o r t a l i t y (ET  5 0  ) , the l a t t e r ,  mortality  (ED  comparison and  5 0  ) . The E T  effective 5 0  or E D  5 0  dosage  (salinity)  that  significant  and  post-hatching  both incubation  to  50%  v a l u e s were u s e d a s b a s e s f o r  of the responses of l a r v a e t o i n c u b a t i o n  salinity,  indicate  the  temperature.  salinity  i n f l u e n c e on t h e s a l i n i t y  temperature  The  results  and t e m p e r a t u r e e x e r t a  t o l e r a n c e of l a r v a e .  Eggs  incubated  in  the  temperature  (6°C) p r o d u c e d  hatching  salinities.  possible  influence,  statistically from  highest  salinity  (29°/ooS)  l a r v a e most t o l e r a n t  Post-hatching although  significant.  trends  salinity  the  12°C  post-  a l s o showed a were  not  tolerance of larvae hatching  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 from  to higher  seen  t h e 6°C i n c u b a t i o n t e m p e r a t u r e a p p e a r e d  larvae  lower  temperature  the  Salinity  and  and  incubation  t o be m a x i m i z e d a t  9°C;  whereas,  temperature  those  showed maximum  t o l e r a n c e a t 9 t o 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 .  response.  of.  larvae  to  salinity  may  be  i r r e v e r s i b l e non-genetic adaptation of the  a  result  embryo  The  of  to  an  salinity  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  the  c a l o r i m e t r y e x p e r i m e n t s , samples  were c o m b u s t e d values.  i n a microbomb c a l o r i m e t e r  These  larvae  were  combinations of incubation used of  to  salinities  and t e m p e r a t u r e s as  h a t c h i n g ) , 3, 6 a n d 9. S t a n d a r d l e n g t h s ,  There  was  a  considerable  c a l o r i m e t r y of the h e r r i n g treatments,  at  larva.  hatching,  averaged  Mean  (day 0) r a n g e d 13°/ooS).  6,586 8.25%,  ash-  were a l s o  i n the data f o r  larvae cal/g  from a l l  ash-free dry  obtained  by  direct  s t a n d a r d l e n g t h o f l a r v a e a t t h e peak o f h a t c h i n g f r o m 7.67 mm  There  was  a  (12°C, 29°/ooS)  trend  towards  standard l e n g t h ) a t lower temperatures ages  weights,  samples  variability  those  a t d a y 0 (peak  dry  In general,  w e i g h t a n d h a d a mean a s h v a l u e o f ashing.  caloric  h a t c h e d a n d r e a r e d i n t h e same s i x  i n t h e m a i n e x p e r i m e n t . L a r v a e were s a m p l e d  taken.  larvae  determine  f r e e d r y w e i g h t s and a s h c o n t e n t o f t h e l a r v a l  all  of h e r r i n g  in  to  larger lower  9.93  mm  larvae  (6°C, (greater  salinities  t e s t e d . D a t a on d r y w e i g h t s show a l i n e a r  in  decrease i n  iv  larval  (body a n d y o l k ) w e i g h t  weight (6°C, day  of  larva  at  higher yolk-sac  larvae  temperatures stage),  to  day  t h e peak o f h a t c h i n g r a n g e d  13°/OOS) t o 0.162 mg  0, t h e l a r g e s t  from day 0  (12°C, 21°/ooS;  Mean  dry  f r o m 0.142 mg 29°/ooS).  At  ( i n t e r m s o f d r y w e i g h t ) were f o u n d a t  i n higher s a l i n i t i e s ,  the  12°C,  9.  largest  temperatures i n lower s a l i n i t i e s . terms of a s h - f r e e d r y weight.  larvae  w h i l e a t day 9  were  found  at  (past lower  The same t r e n d was o b s e r v e d i n  V  TABLE OF  CONTENTS  11  Abstract Table  of C o n t e n t s  List  of T a b l e s  List  of. F i g u r e s and  v vi Photographs  vii  Acknowledgments  viii  Introduction  1  M a t e r i a l s and Methods 1. C o l l e c t i o n o f Spawners 2. Spawning and F e r t i l i z a t i o n 3. I n c u b a t i o n 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 a) 1979 E x p e r i m e n t ( T i m e - M o r t a l i t y Approach) b) 1980 E x p e r i m e n t ( D o s a g e - M o r t a l i t y Approach) c) S t a t i s t i c a l A n a l y s i s (1 ) 1979 D a t a (2) 1980 D a t a 5. C a l o r i m e t r y  6 6 6 7 15 16 17 18 18 19 22  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 E x p e r i m e n t b) 1980 E x p e r i m e n t 2. C a l o r i m e t r y (1979 T r i a l s ) a) U n e q u a l Sample S i z e b) S t a n d a r d L e n g t h of L a r v a e c ) Dry W e i g h t o f L a r v a e d) C a l o r i e D e t e r m i n a t i o n (1) P e r c e n t Unburned O r g a n i c R e s i d u e (2) P e r c e n t Ash from D i r e c t and I n d i r e c t Methods (3) 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 Discussion 1. E f f e c t of I n c u b a t i o n S a l i n i t y 2. E f f e c t of I n c u b a t i o n T e m p e r a t u r e 3. E f f e c t of Post-Hatching Temperature E f f e c t s of the Three F a c t o r s 4. C a l o r i m e t r y 5. E c o l o g i c a l I m p l i c a t i o n s Literature  Cited  ....  29 29 29 34 49 49 49 54 59 59 Ashing 59 59 67 67 71  and  Combined 72 75 78 81  vi  L I S T OF TABLES  T a b l e 1: Range o f L e n g t h H e r r i n g Spawners T a b l e 2: Tests  Experimental  Table  3:  Number  Table  4:  Estimates  T a b l e 5: Values  in 9  Design  for  the S a l i n i t y  Tolerance 14  of Larvae of E D  Sampled  5 0  of  ED  for Calorimetry  5 0  Variance  for  T a b l e 9: Table o f Means Showing C a l o r i c Values of H e r r i n g Larvae  ED  5 0  33  Variance  for  f o r the. 36  the  ED  5 0  40  o f Mean E D Values Incubation Salinity 5 0  31  the  and T h e i r Mean V a l u e s  Summary o f A n a l y s i s o f o f t h e 1980 E x p e r i m e n t  T a b l e 8: Comparison in Relation to Temperature  24  f o r t h e 1979 E x p e r i m e n t s  Summary o f A n a l y s i s o f o f t h e 1979 E x p e r i m e n t  T a b l e 6: Estimates 1980 E x p e r i m e n t T a b l e 7: Values  and Weight Measurements T a k e n  of H e r r i n g L a r v a e and Incubation 43  Lengths,  Weights  and 53  T a b l e 10: T a b l e o f Means Showing D r y W e i g h t s of Pellets Combusted i n Bomb C a l o r i m e t e r , P e r c e n t U n b u r n e d O r g a n i c Residue and Percent Ash Obtained from Direct and I n d i r e c t Methods 61 T a b l e 11: Summary of A n a l y s i s of V a r i a n c e A s h - F r e e D r y Weight o f H e r r i n g L a r v a e  f o r the c a l / g 66  vii  L I S T OF FIGURES AND PHOTOGRAPHS  Figure 1: P a r t a : H e r r i n g Egg I n c u b a t o r P a r t b: I n c u b a t o r Showing Incubation C e l l s  11 i n Egg I n c u b a t i o n Tank .. 11 Four Compartments and 11  F i g u r e 2: . D o s a g e - M o r t a l i t y G r a p h Showing G r a p h i c a l of E s t i m a t i n g E D f o r 1980 E x p e r i m e n t  Method 21  5 0  F i g u r e 3: T y p i c a l G r a p h i c a l C o m b u s t i o n R e c o r d a n d S t a n d a r d Method o f 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 F i g u r e 4: Mean ED Values (Calculated from Three Replicates) of H e r r i n g Larvae Subjected t o the S a l i n i t y T o l e r a n c e T e s t (1980 E x p e r i m e n t ) 38 5 0  F i g u r e 5: I n t e r a c t i o n between I n c u b a t i o n Temperature  Incubation  Salinity  and 45  F i g u r e 6: C o m p a r i s o n of Mean E D V a l u e s of H e r r i n g L a r v a e in Relation t o : 48 P a r t a: C u l t u r e Temperature and I n c u b a t i o n Temperature 48 P a r t b: C u l t u r e T e m p e r a t u r e and I n c u b a t i o n S a l i n i t y ... 48 5 0  F i g u r e 7: Larvae  Mean  (±  1  SD) S t a n d a r d L e n g t h  (mm) o f H e r r i n g 51  F i g 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 D r y W e i g h t s 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 56 F i g u r e 9: C a l c u l a t e d Regression Lines Dry W e i g h t s of H e r r i n g Larvae Used E x p e r i m e n t i n 1979 Regression  f o r Mean A s h - F r e e for Calorimetric  Lines  58  F i g u r e 10: Calories  Calculated per Larva  for  the  Mean  F i g u r e 11: Herring Salinity Period  E s t i m a t i o n o f Upper L e t h a l S a l i n i t y L i m i t s of Larvae Exposed to Various C o m b i n a t i o n s of and Temperature d u r i n g Incubation and L a r v a l  64  74  .vii i  ACKNOWLEDGMENTS  I  am  v e r y much i n d e b t e d  E. Hay o f t h e P a c i f i c  t o D r . D. F. A l d e r d i c e a n d D r . D.  Biological  help  Station,  throughout  generous  study.  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  I a l s o wish  other  Dr.  A. M. P e r k s  of t h i s of the .  t o t h a n k my  academic  supervisor,  and c r i t i c i s m  Dr.  W.  S.  of t h e t h e s i s ; and  members o f t h e a d v i s o r y c o m m i t t e e : D r . H. E. K a s i n s k y ,  suggestions My  aspects  acknowledged.  Hoar, f o r h i s guidance, support to  various  B.C., f o r  their  manuscript a r e g r a t e f u l l y  the  Nanaimo,  Wi1imovsky,  and c o n s t r u c t i v e c r i t i c i s m  thanks  Biological  a n d D r . N. J .  are also  given  Station, especially  Optimization  for their  of t h e t h e s i s .  to  the  staff  me  with  and t o t h e  herring  e x p e r i m e n t s were c o n d u c t e d w i t h c o n s i d e r a b l e P. J . V e l s e n ,  Production  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 t h e e x p e r i m e n t s , f o r supplying  of t h e P a c i f i c  t o t h e members o f t h e  s e c t i o n f o r p r o v i d i n g me w i t h  Investigation  valuable  Herring  spawners.  a s s i s t a n c e from  The F.  J . J e n s e n , C. Lam, A. S h i m o z a w a , T r u d i e M u l l i n a n d  Barb Smith. I  am g r a t e f u l  Biological  t o s e v e r a l f r i e n d s a t U.B.C. a n d t h e P a c i f i c  S t a t i o n a n d my F i l i p i n o  i n v a r i o u s ways d u r i n g The  graduate  financially Centre  Aquaculture  study  supported  (IDRC)  of  the course  friends f o rtheir  assistance  o f my g r a d u a t e s t u d i e s .  program  of  this  author  has  by t h e I n t e r n a t i o n a l D e v e l o p m e n t  Canada a s p a r t o f t h e f i n a n c i a l  Department of Southeast A s i a n  Fisheries  been  Research  grant  t o the  Development  Centre  (SEAFDEC)  great appreciation  in  Iloilo,  Philippines.  t o b o t h IDRC a n d my  a l l o w i n g me t o p u r s u e  t h i s graduate  I w i s h t o e x p r e s s my  employer,  program.  SEAFDEC,  for  1  INTRODUCTION  Pacific and  herring  subtidal  regions  M e x i c o , U.S.A. Korea  ( C l u p e a p a 1 1 a s i ) spawn of  the  Pacific  ( C a l i f o r n i a and A l a s k a ) ,  the  intertidal  Ocean c o a s t a l w a t e r s o f C a n a d a , USSR, J a p a n  ( A l d e r d i c e a n d V e l s e n , 1971; H o u r s t o n a n d H a e g e l e ,  I n B r i t i s h C o l u m b i a w a t e r s , t h e y spawn f r o m with  a  April.  peak p e r i o d Most h e r r i n g  spawn  waters  with  1976; H a e g e l e a n d Humphreys, 1977;  salinities  29  natural  °/ooS  (7.0  conditions,  and  1980).  to  from e a r l y March  Haegele,  Under  to  in  February  lasting  from  1979).  24  generally  ranging  the  in  June,  to early  salinities  (°/ooS)  t o 9.9°C) (Humphreys a n d  the  Alderdice  e_t  al. ,  eggs i n c u b a t e a t t h e s e  a n d t e 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 f o u n d i n  same s a l i n i t i e s .  incubation  salinity  hatching  is  Laboratory experiments demonstrate range p r o v i d i n g  ( A l d e r d i c e a n d V e l s e n , 1 9 7 1 ) . I n a f u r t h e r s t u d y , i t was  deduced  osmotic seems  same  salinity  13  and  range  19°/OOS  probably  (7.6  to  8.7°C)  this  between  maximum s u r v i v a l o f eggs to  that  that  that the  provides  the  least  s t r e s s t o embryos ( A l d e r d i c e e_t a l . , 1 9 7 9 ) . H e n c e , t h e r e to  providing survival  be  a  the of  substantial least  embryos  osmotic to  d i f f e r e n c e between t h e s a l i n i t i e s work  hatching,  f o r embryos  and  and t h e s a l i n i t i e s  maximum a t which  eggs a n d l a r v a e a r e f o u n d i n n a t u r e . Temperature biological  i s considered  phenomena.  a  Temperature  controlling influences  which c o n t r o l s t h e r a t e of development other  processes are influenced  in  indirectly  factor  in  most  metabolic rate,  teleost  eggs.  by t e m p e r a t u r e  Many acting  2  through 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 and  rates  dependent.  of  production  Temperature  development,  the  and V e l s e n ,  are the  oxygen  temperature  viability  of  range v a r y i n g between s p e c i e s and  within  species  (Brett,  range i s < 4 t o about  1970). For  14°C o r more  Pacific  (Alderdice  1971).  Salinity teleost  metabolites  normally- influences  biokinetic  s t a g e s of development h e r r i n g eggs, t h i s  of  dissolved  i n f l u e n c e s the i n t e r n a l osmotic environment of the  egg.  In  the  ovary of the adult the u n f e r t i l i z e d  a p p r o x i m a t e s t h e b l o o d of t h e (Holliday,  1965;  adult  in  osmotic  ovum  concentration  H o l l i d a y and Jones, 1965). At t h i s s t a g e , t h e  v i t e l l i n e membrane i s h i g h l y p e r m e a b l e  t o i o n s moving a c r o s s i t .  When t h e egg i s s h e d , i t t e n d s r a p i d l y  t o come i n t o  with  the  surrounding  fertilization, teleosts, rapidly; the  medium  (Alderdice  e_t §_1. , 1 9 7 9 ) .  which i s almost synchronous w i t h spawning  permeability  of  the  by t h e t i m e t h e f i r s t  membrane  has  become  vitelline  few c e l l  virtually  impermeable  spawning  medium  Hence, t h e  influence  the  osmotic  fertilized  egg, which i s m a i n t a i n e d a f t e r  becomes r e l a t i v e l y  concentration  impermeable  concentration  membrane  of  egg  developing  c o n c e n t r a t i o n of embryonic and s u r v i v a l The  to hatching  capacity  for  tissue  across  influences  of  tissue,  i t s salinity  the v i t e l l i n e  i n c l u d i n g embryonic growth, a c t i v i t y , larval  occurred,  of t h e y o l k of t h e newly  processes, the  i n most  t o movement o f  and  t o i o n movement the  After  decreases  d i v i s i o n s have  ions across i t .  osmotic  equilibrium  i t . The  development  water  regulation  membrane  of  following blastopore  content osmotic closure,  ( A l d e r d i c e e t §_1. , 1 9 7 9 ) . ionic  a n d o s m o t i c r e g u l a t i o n by t e l e o s t  3  eggs  and  those  of  other  temperature.  These  below c e r t a i n  limiting  hand,  thermal  salinity. stress.  regulatory  of  reduction  1963;  Garside,  (oxygen, and  and  pH, h y d r o s t a t i c  thermal  influence The egg  experiments  i n metabolic 1971).  Other  o f an o r g a n i s m ,  have  been  Kinne  (1964a)  t h e second  salinity high  -  hatching  type, better  viable  that  interaction  larvae)  tolerance,  factors  the  salinity  but the e x t e n t of t h e i r  a  several  laboratory  1967, 1971; A l d e r d i c e a n d and  Hempel,  types  of  t y p e , an o r g a n i s m  when  associated w i t h lower  i s achieved  1979).  salinity2)  has b e t t e r  with  higher  temperatures. in  both  low  combinations or i n h i g h s a l i n i t y Among  low/low  affects  the  Pacific --  maximum  development  survival  herring  high/high  and V e l s e n , 1971). That show  on t e l e o s t  1) l o w / h i g h -- h i g h / l o w a n d  combinations.  lower s a l i n i t i e s  osmotic  environmental  1964a  survival  temperature  (Alderdice  an  (Kinne,  in  and i n h i g h e r s a l i n i t i e s  low  other  efficiency  general  namely:  salinities  indicates  temperature  in  lower  temperature  evidence  two  -- h i g h / h i g h . I n t h e f i r s t  temperatures, In  mentioned  interaction,  in  in  and temperature  noted  1971; s e e r e v i e w s by K i n n e ,  survival  the  understood.  Velsen,  low/low  results  ( A l d e r d i c e and F o r r e s t e r ,  temperature  by  above and  t h e range of t h e r m a l  combined e f f e c t s o f s a l i n i t y  development  1 9 7 2 ) . On  p r e s s u r e ) may a l s o a f f e c t  tolerance  i s not w e l l  influenced  o r g a n i s m may be i n f l u e n c e d by  t o extreme s a l i n i t i e s  p r o b a b l y due t o a  be  could f a i l  (Tait,  an  Such s t r e s s c o u l d s h i f t  Waugh  may  processes  temperatures  tolerance  Exposure  organisms  eggs,  salinity of  -  eggs  to  i s , eggs i n c u b a t e d (maximum  a t lower t e m p e r a t u r e s , and i n h i g h e r  hatch  of  salinities  4  at  higher  temperatures.  regarding the  the  salinity  interaction  during  herring  would  in  i n the  exposed  in  salinity The  second  Is  tolerance  l a r v a e . The  study  was  salinity  tolerance  incubated used  for  the  companion study incubation  which  the  of  t e m p e r a t u r e on  examines  the  salinity  was  salinity  to  higher  salinities  also  influence  the  effects  t e m p e r a t u r e , and salinity  0-  of  (1)  (3)  culture  of  herring  tolerance  examination  of  variables  herring. tolerance  tests, a calorimetric  (newly hatched),  larvae  tolerance determine  and  salinity  in  used  3-,  6-  were h a t c h e d  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 salinity  possible  the  larvae  are  r e s u l t i n g larvae?  larval  The  larval  larvae  c o n c e r n s the  of  most spring  favor  i n t e r a c t i o n s between these t h r e e  larvae.  i n the  could  temperature  incubation  undertaken using  herring  annual  an  environmental  d e s i g n e d t o accommodate an  In a d d i t i o n to the was  the  such  since  accommodation to h i g h e r  of t h e  (2)  to  survival  t e m p e r a t u r e on  main e f f e c t s and  old  the  investigation  salinity,  (post-hatching)  and  Does i n c u b a t i o n  current  incubation  increase  occurring  stage,  rising  question  salinity  a r e s u l t of t h e i r  incubation?  questions  advantageous,  the  salinities  A  larval  t o or d u r i n g  spring  higher  larvae.  to  temperature  i n the  Hence,  the  incubation  of  salinities during  just prior  nature.  i n f l u e n c e of  study  and  ecologically  during  survival  on  salinity  temperature.  temperatures  tolerance  be  hatch  leads  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  between  larvae  increase  the  finding  i n c u b a t i o n were t o c o n t i n u e  interaction  the  This  tests. the  temperature  The  possible on  and  9-day  from eggs as  those  p u r p o s e of influence  aspects  of  this of  larval  5  development a f t e r  hatching.  6  MATERIALS AND METHODS  A first  e x p e r i m e n t was c o n d u c t e d  equipment  made  in  1979.  Problems  i t necessary t o conduct a f u r t h e r  with  the  s e t of t r i a l s  i n 1980.  1. C o l l e c t i o n  o f Spawners  A d u l t h e r r i n g were c a u g h t  i n Nanoose B a y , V a n c o u v e r  Island,  B.C. on F e b r u a r y 2 8 , 1979. F o r t h e 1980 e x p e r i m e n t , t h e  herring  spawners  were  caught  on  were t r a n s p o r t e d t o t h e  March  3 f r o m t h e same l o c a l i t y .  Pacific  B.C., a n d m a i n t a i n e d i n c i r c u l a r  Biological  Station,  They  Nanaimo,  tanks of a e r a t e d sea water (28-  29°/ooS, 8-9°C).  2. S p a w n i n g In March  and  1979,  20, A p r i l  Fertilization eggs  were  f e r t i l i z e d at three d i f f e r e n t times:  12 a n d A p r i l  20.  In  c a r r i e d o u t on one o c c a s i o n , on M a r c h and  fertilization  procedures  refer  1980,  fertilization  11. The f o l l o w i n g to  both  1979  was  spawning and  1980  experiments. Prior prepared, salinity. and  t o spawning, containing Three  29°/ooS)  a constant-temperature 12  beakers f i l l e d  fertilization and  water  salinities  water  bath  was  w i t h water of s p e c i f i c were  of each s a l i n i t y  chosen  (13, 21,  was a s s i g n e d t o f o u r  7  b e a k e r s . Water t e m p e r a t u r e was  maintained at  6°C.  V a r i o u s numbers o f f e m a l e and m a l e s p a w n e r s the  different  spawning  taken b e f o r e spawning Eight  ml  r e q u i r e d . The ( 3 . 5 x 7.5 layers  of  was  obtained  Twenty-four  salinity  8°C)  at  (13,  salinity  ( a b o u t 0.5  least  dilution  fertilized  until  ml)  ten minutes  were  two  diluted  were  with  added  to  for f e r t i l i z a t i o n  1 drop/15  ml  briefly  with  rinsed  as the f e r t i l i z a t i o n  at  the  21 o r 29°/ooS). When a l l 24 s c r e e n s  o f t h e m i l t was  eggs  to  water  sea  t o make a 40-ml s u s p e n s i o n of m i l t . Ten  diluted milt  final  s t o r e d a t 4°C  i n each beaker of  had been p r e p a r e d , t h e s t o r e d m i l t was  allowing  and  s c r e e n s were p r e p a r e d and two o f t h e  s c r e e n s were s t o r e d  appropriate  the  1.  9 f i l a m e n t s / c m ) t o f o r m rows o f e g g s , one  thick.  (2?°/ooS,  on  L e n g t h and w e i g h t m e a s u r e m e n t s  a r e shown i n T a b l e  milt  used  e g g s were e x p r e s s e d o n t o r e c t a n g u l a r n y l o n s c r e e n s  cm,  egg-filled  dates.  were  medium  and  of  each  water  d r o p s of beaker,  to occur. seawater.  The The  w a t e r o f t h e same  transferred  to  the  incubators.  3. I n c u b a t i o n Six  40-1  incubator  i n c u b a t i o n t a n k s were p r e p a r e d . E a c h t a n k h e l d  (Figure  1a,  compartments or c e l l s rectangular  the  at  the  and  of the i n c u b a t o r  s c r e e n of t h e f e r t i l i z e d  were i n t e r c o n n e c t e d water  Alderdice  V e l s e n , 1 9 6 8 ) . The (Figure  e g g s . The  i n s e r i e s by p l a s t i c  desired  eggs a t a p e r f u s i o n  salinity  and  tubing  o f 500  one  compartments  through  t e m p e r a t u r e was  (bulk) v e l o c i t y  four  1b) e a c h h e l d four  one  cm/hr.  which  pumped p a s t  8  Table 1 Range of l e n g t h (cm) and w e i g h t (g) measurements taken h e r r i n g spawners u s e d i n t h e 1979 and 1980 e x p e r i m e n t s .  in  Number  Fork  Range Length  (cm)  Range Standard Length  (cm)  Total  Rang e Wei g h t  t  (g)  Spawning and Fert11 Izat1on Date  IncubatIon Group  March 1979  20,  6 a n d 12* C (replIcate I)  3  5  23.5 24.7  18.5 26 .0  21.522.5  17.0 23.6  163.4 191.4  62.2 210.0  Apr11 1979  12,  12' C (replIcate II)  1  2  22.7  22.5 22.8  21.5  21.1 21.6  145.7  128.3 129 .0  April 1979  20,  6' C (replIcate II)  1  3  24.9  20.2 22.4  23.7  19.2 20.9  183.2  96.8 112.1  March 1980  11.  6 a n d 12*C (replIcates I,II.Ill)  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  Table  1  ?  <?  ?  •?  -  10  Figure H e r r i n g egg incubation tank.  incubator  (Alderdice  Figure Herring egg incubation c e l l s  1a  incubator  and  Velsen,  1968)  in  four compartments  and  1b  showing  the  Figure  1b  12  Six  incubation  a s s i g n e d randomly groups  were  prepared e i t h e r  salt water  (6 a n d 12°C) a n d  ( 1 3 , 21 a n d 29°/ooS). D i f f e r e n t by d i l u t i n g  sea water  Marine Mix, R i l a  to  were  t o t h e s i x t a n k s ( T a b l e 2 ) . The s i x i n c u b a t i o n  t o p r e p a r e lower s a l i n i t i e s , (Rila  combinations  a c o m b i n a t i o n o f two t e m p e r a t u r e s  three s a l i n i t i e s  water  temperature-salinity  salinities  (28-29°/ooS)  fresh  or by adding a s y n t h e t i c sea  Products,  prepare higher s a l i n i t i e s .  Teaneck, Salinity  N.J.)  to  sea  was d e t e r m i n e d by  titration  ( S t r i c k l a n d a n d P a r s o n s , 1 9 6 8 ) . The mean  salinity  and  temperature  with  were  experimental  levels obtained during incubation are  shown i n T a b l e 2. The  e g g s were i n c u b a t e d i n t h e same s a l i n i t i e s  w h i c h t h e y were f e r t i l i z e d .  Each  piece  maintain  t o w e l s h e e t i n g was p l a c e d o v e r t h e g l a s s  semi-dark  conditions.  s h e e t i n g was u s e d  In  (Figure  inlet  each  incubator  daily  t o d i s t r i b u t e evenly the p o t e n t i a l  s e r i e s arrangement Every  four  v o l u m e , 40 l i t r e s ) same s a l i n i t y Percent examining glass  with  to  and  to maintain  outlet  tubes  of  l a ) were r o t a t e d t h r o u g h one p o s i t i o n  of water days,  flow  bias resulting  through  one-half  of  was r e p l a c e d w i t h n e w l y  the  four  from t h e incubator  t h e tank volume  (total  prepared water of the  and t e m p e r a t u r e . fertilization  s e p a r a t e samples  slides.  covers  t h e 1980 e x p e r i m e n t , b l a c k  i n s t e a d of t h e w h i t e paper  c o n s t a n t d a r k c o n d i t i o n s . The w a t e r  cells.  l e v e l s , and c o v e r e d  o f g l a s s t o a v o i d e v a p o r a t i o n . I n t h e 1979 e x p e r i m e n t ,  w h i t e paper  plastic  in  i n c u b a t i o n t a n k 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 a  as those  of  the  eggs  was  determined  by  o f e g g s a d h e r i n g t o a n d f e r t i l i z e d on  The e x p e c t e d t i m e t o 5 0 % h a t c h i n g was c a l c u l a t e d  13  Table 2 Experimental design f o r the s a l i n i t y tolerance tests. Horizontal bars 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 ) 2 0 , 2 5 , 3 0 , 3 5 , 4 0 , 4 5 , 50 a n d 55 °/ooS i n 1979; (2) 2 5 , 3 0 , 3 5 , 40, 45 a n d 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 trials (3 i n c u b a t i o n salinities x 2 incubation temperatures x 3 culture t e m p e r a t u r e s ) . I n 1979, t h e r e were 2 r e p l i c a t e s p e r 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 ) . I n 1980, t h e r e were 3 r e p l i c a t e s p e r 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 ) .  LARVAL  INCUBATION  Exptl.  Des1gn  Level  +  Culture  1 SD  STAGE  ( P o s t - h a t c h i n g ) Temperature  Group Temp.  CC)  Sal . C/oo)  Temperature Cc)  12  Sal1nity C/oo)  1  6  13  G.00  ±  .05  13.08  ±  .07  (D  (2)  2  6  21  6.00  ±  .01  20.98  ±  .17  _L1L  AIL  J 6 1  3  6  29  6.00  +  .01  28.96  ±  .11  _L7J_  (B)  J9L  4  12  13  12.00 ±  .01  13.15 ±  .24  (10)  (11)  (12)  5  12  21  12.01 ±  .08  20.95 ±  .28  (13)  (14)  (15)  6  12  29  12.02 ±  .12  28.93 ±  .09  (16)  (17)  (18)  Table  2  C O  15  for  b o t h 6 and  provided  4•  incubation  by A l d e r d i c e  Newly the  12°C  hatched  and  The  T o l e r a n c e T e s t s of  of  hatching  salinities.  different  Two The  ( 6 , 9 and  a  incubation  individual  estimate  of  12°C)  and  time  technique  was  total  end  of  larvae  to  expose  a t t h e peak p e r i o d  culture  of  test  (post-hatching)  the m o r t a l i t y o c c u r r i n g  in  (ET ).  of  the 72  as  the  series provided or  test an  i n each t e s t s a l i n i t y over the  72-  time  50%  second  or  hours.  effective  m o r t a l i t y or  "dosage-mortality"  latter  resulting  the dose  effective (ED  an  median  an  estimate  i n each t e s t s a l i n i t y at  The of  provided  approach provided  dosage i n the  salinity.  median  series  to  This  of t h e  The to  The  estimate  u s e d i n 1979.  estimation  mortality occurring  72-hour p e r i o d .  72  a p p r o a c h , was  trials.  time  The  5 0  u s e d i n 1980.  larval  mortality,  s a l i n i t y tolerance  time-mortality  effective  the  of  was  noted f o r a f i x e d exposure p e r i o d  resulting  the  effective  of  tests  g r o u p s , t o a s e r i e s of  t e c h n i q u e i n v o l v e d an  h o u r p e r i o d . The  period  larval  "time-mortality"  time-mortality  taken  the  t e c h n i q u e s were u s e d i n t h e  of  for  2).  first,  death  various  t r e a t m e n t s was  (Table  tanks  Larvae  L a r v a e were t e s t e d a t t h r e e  temperatures  relation  (1971).  newly hatched l a r v a e , o b t a i n e d  from the  the  tests.  basic procedure in  groups  hours  Velsen  following  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  s a l i n i t y tolerance  Salinity  groups  5 0  ),  instance  the is  dosage-mortality dose f o r an  to  50%  exposure  16  a ) 1979 E x p e r i m e n t  ( T i m e - M o r t a l i t y Approach)  The e g g s d e v e l o p i n g the  same t e m p e r a t u r e  i n the three  incubation  salinities  (6 a n d 12°C) were e x p e c t e d  t o h a t c h on t h e  same d a y , b a s e d on f i n d i n g s f r o m p r e v i o u s e x p e r i m e n t s . larval  t a n k s were p r e p a r e d  nine  groups  groups  X  of  three  temperatures  simultaneously  25,  to  accommodate  culture  culture  (6,  9  and  temperatures). 12°C)  were  The  three  assigned  randomly t o the  contained  an  individually  e v a p o r a t i o n . A s e r i e s of these replicate  (Table  with  glass  eight  salinities  2 ) . The i n i t i a l  w i t h two r e p l i c a t e s  array  of  s a l i n i t i e s (20,  3 0 , 3 5 , 40, 4 5 , 50 a n d 55 °/ooS) were a s s i g n e d . covered  the  salinity  200-ml c u l t u r e d i s h e s t o w h i c h e i g h t t e s t  were  N i n e 40-1  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  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 eight  at  The  covers  dishes  to  avoid  provided  one  p l a n was t o c o n d u c t t h e t e s t 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  t h e same t i m e . H o w e v e r , i n t h e 1979 s e r i e s , t h e p r o c e d u r e  had t o  be a l t e r e d a s t h e r e were n o t enough l a r v a e a v a i l a b l e due t o h i g h mortality at incubation groups,  caused  using  time.  the  During  time-mortality  Consequently,  the  times, requiring  two f u r t h e r b a t c h e s  problems  test  approach,  the  salinity  i n the  f o r a l l nine produced  an  experimentally  at  t e s t s were p e r f o r m e d a t  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  of eggs.  peak  of  the  l a r v a e were t r a n s f e r r e d t o t h e  of  technical  a r r a y t o o l a r g e t o be c o n d u c t e d  four d i f f e r e n t  larval  by  system. Moreover, a t w o - r e p l i c a t e s e r i e s  experimental one  hatching  hatching culture  p e r i o d , newly dishes.  (12°C i n c u b a t i o n g r o u p , r e p l i c a t e  15 l a r v a e were t e s t e d i n e a c h s a l i n i t y  In  the  hatched first  I , 1979), groups  tolerance  series.  In  17  subsequent  tests,  the  tests, only viable dark  soon  i n each after  by p l a c i n g w h i t e p a p e r  transfer  of  were  larvae  the  mortality culture  semi-  commenced dishes  t h e 7 2 - h o u r p e r i o d . A l a r v a was  when i t was g e n e r a l l y opaque  to  in  t o w e l i n g over the g l a s s  tank. E x a m i n a t i o n of l a r v a l  continued throughout "dead"  s i z e was r e d u c e d t o t e n . I n a l l  l a r v a e were u s e d . L a r v a e were k e p t  conditions  covers  sample  and  considered  i n a c t i v e , t h e b r a i n and s p i n a l c o r d  a n d t h e f i n f o l d s were s h r u n k a n d w r i n k l e d . C a r d i a c  f u n c t i o n might  still  be o b s e r v e d , b u t t h e damage  to  the  larva  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 every  o b s e r v a t i o n p e r i o d . A t t h e end o f 72 h o u r s , t h e r e m a i n i n g  l a r v a e were c o u n t e d  t o check  b) 1980 E x p e r i m e n t One day p r i o r mesh  screens  t o t h e e x p e c t e d peak o f h a t c h i n g ,  the  nylon  a n d a d h e r i n g e g g s were removed f r o m t h e i n c u b a t o r  minimized  movement o f w a t e r in  sample s i z e .  ( D o s a g e - M o r t a l i t y Approach)  c e l l s and t r a n s f e r r e d transfer  individual  to circular mortality  of  100-mm d i a m e t e r larvae  i n the incubator c e l l s .  baskets.  The  c a u s e d by t h e u p w a r d The b a s k e t s were  kept  t h e same i n c u b a t i o n t a n k , a e r a t e d a n d m a i n t a i n e d a t t h e same  s a l i n i t y and temperature o f i n c u b a t i o n . Nine l a r v a l the  1979  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  experiment. S i m i l a r l y ,  the three culture  ( 6 , 9 a n d 12°C) were a s s i g n e d r a n d o m l y controlled At  larval  the  transferred of  specific  peak  t o the nine  as  in  temperatures temperature-  tanks. p e r i o d of h a t c h i n g , groups  o f 10 l a r v a e  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 salinity  were  waters  ( 2 5 , 3 0 , 3 5 , 4 0 , 45 a n d 50°/ooS). I n t h e  18  1980  experiment,  1979  tests  were  considered 0 With  six in  procedure  that  analyses.  The  with  throughout dark  one  larval  increased 18 dishes  a  over  end  of  c)  72  Bliss'  time-mortality  some  longer  period  the  flow  of  i n each  air  30  for  The l a r v a e  was  plastic  a  piece  dish  in  sheeting  a  ensuing  seconds  were k e p t  salinity  h o u r s . The same c r i t e r i a  period,  was daily  constant  on t h e g l a s s  was  recorded  for declaring a larva  t o the.1980 e x p e r i m e n t . A t  the remaining  at  live  l a r v a e were  the  counted  size.  Analysis  (1937) t i m e - m o r t a l i t y curves,  was e s t i m a t e d trials  of  The water  1979 D a t a . P r o b i t a n a l y s i s was  following  trials  evaporation.  i n d i v i d u a l sample  (1)  series  (18 c u l t u r e d i s h e s / t a n k ) ,  sensitivity  i n 1979 were a p p l i e d  Statistical  i n t h e 1979  tank.  the t e s t  t o check  tank  was  consistently  3-replicate  of l a r v a e a t each t e s t  of  "dead" used end  salinities  a  by p l a c i n g b l a c k  each  i n the  i n e a c h tank were c o v e r e d w i t h  the t e s t i n g p e r i o d .  Mortality the  two  the  gentle  conditions  covers  the  used  t h e s e r i e s . The e x c l u s i o n  salinities,  P l e x i g l a s s to avoid  aerated  no  as  from  salinities  o r 100% m o r t a l i t y o v e r a 72-hour p e r i o d  accommodated  of  excluded  reasonable  produced tests.  t h e 20 and 5 5 ° / o o t e s t  the  ET  5 0  applied  observed during  of  and where t h e h i g h e s t  data,  e a c h r e p l i c a t e o f t h e 18 found,  distributions,  the l a t t e r  the  methods. From t h e v a r i o u s  g r a p h i c a l l y . I t was  showed t r u n c a t e d  to  part  however,  that  where m o r t a l i t y was of  the  observation  c u m u l a t i v e m o r t a l i t y was l e s s  than  19  50%.  This  made  necessitating  it  a  change  a n a l y s i s f o r the on  difficult  1979  in  limits  estimate  approach  to  v a l u e s of E D  were  calculated  lethal  ED 's  formed  5 0  salinity  variance  limits  (ANOVA)  interactions  of  was  tests  of  were  calculated:  the  used  5 0  dosage-mortality were  their  following  drawn  corresponding the  maximum  1935).  yolk-sac  to  examine  treatments,  s i g n i f i c a n c e . The  o v e r - a l l ANOVA  ET 's,.  b a s e s f o r c o m p a r i s o n of t h e  f o r the d i f f e r e n t  for  i)  the  the  curves  and  5 0  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 , The  use  data. Dosage-mortality  p r o b a b i l i t y p a p e r and  confidence  to  larvae. the  Analysis  of  main e f f e c t s  and  employing  an  f o l l o w i n g a n a l y s e s of  (3 i n c u b a t i o n s a l i n i t i e s , temperatures,  upper  2  F-test variance  incubation  3 c u l t u r e temperatures,  2  replicates) ii)  replicate  I  ( 3 x 2 x 3 )  iii)  replicate  II  ( 3 x 2 x 3 )  (2) 54  1980  Data.  The  t e s t s were a n a l y z e d  simplified  method  dosage-mortality data  using L i t c h f i e l d  for  estimating  the E D  e x a m p l e of t h e d o s a g e - m o r t a l i t y c u r v e were  obtained graphically.  accurate  results;  method,  using a x showed obtained  however,  the c o n f i d e n c e  c a l c u l a t e d and 2  in  limits  first  differences the  first  and  5 0  estimates  somewhat more  and  Wilcoxon's 5 0  could  accepted  estimates  second  (1949)  of t h e E D ' s c o u l d  variance was  the  . F i g u r e 2 shows an  Litchfield  iteration between  5 0  from which E D  ( o = 0.05)  from  Wilcoxon's  method p r o v i d e d  with  t h e h o m o g e n e i t y of  t e s t . The  that  Bliss'  and  obtained  be  measured  i f the  of t h e  iterations  be  x  2  test  parameters were  not  20  Figure  2  Dosage-mortality graph showing the g r a p h i c a l method o f estimating ED f o r t h e 1980 e x p e r i m e n t . F a c t o r f o r E D (fED ) i s c a l c u l a t e d f o l l o w i n g t h e method o f L i t c h f i e l d and W i l c o x o n (1949). Confidence limits of E D = ED x/+ f E D . Downward s h o r t , s o l i d arrow i n d i c a t e s 0% o b s e r v e d mortality response. Point with brackets indicates "expected" percent mortality ( 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% observed m o r t a l i t y . 5 0  5 0  5 0  5 0  50  5 0  21  22  significantly  different  (o  =  0.05). I f s i g n i f i c a n t ,  i t e r a t i o n s were made. The i t e r a t i o n whether  significant  or  heterogeneous variance, calculated  in  a  than the  50%,  The the  the ED  probit  line  n o t , was  manner.  was e s t i m a t e d  5 0  until  i tcrossed  5 0  x  2  value,  In. t h e  case of  least  l i m i t s of the In  5 0  were  those instances  where  i n t h e s e r i e s of t e s t  E D ' s were a n a l y z e d  1979  the  accepted.  the confidence  different  m o r t a l i t y of l a r v a e  with  two more  ED  salinities  was  by e x t r a p o l a t i o n by e x t e n d i n g t h e 50% r e s p o n s e  point.  by a n a l y s i s o f v a r i a n c e  following  p r o c e d u r e . I n a d d i t i o n t o a n 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 c o n d u c t e d  i n each i n c u b a t i o n  temperature  group  examine t h e 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 and  less  culture  temperature  at  different  levels  of  to  salinity incubation  temperature.  5.  Calorimetry In  values  1979, c a l o r i m e t r y of h e r r i n g  different larvae Hatched  was c o n d u c t e d t o d e t e r m i n e t h e c a l o r i c  l a r v a e h a t c h e d from eggs i n c u b a t e d  salinity-temperature  tested  were  larvae  0  were  combinations  (newly  hatched),  maintained  in  Fifteen  viable  from each i n c u b a t i o n 3). snout  The s t a n d a r d to  the  anaesthetizing  larvae,  same  salinity  of  the  The  and  incubated. p e r m i t t i n g , were s a m p l e d  g r o u p w i t h i n e a c h o f two r e p l i c a t e s  length  end  numbers  3).  3, 6 a n d 9 d a y s o l d .  the  t e m p e r a t u r e a s t h o s e i n w h i c h t h e y were  (Table  i n the s i x  (Table  of l a r v a e , measured from t h e t i p of t h e the  notochord,  larvae  with  was MS-222  obtained  after  (tricanemethane  23  Table 3 Number o f larvae sampled for calorimetry. Larvae were s a m p l e d f r o m e a c h t r e a t m e n t g r o u p 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  Incubation Culture  Number o f L a r v a e  and  Group  Temp.  Salinity  No.  (°C)  (°/oo)  After Hatching  Days  Conditions  Replicate  I  3  6  9  15 15 15  0  1 2 3  •6 6 6  1 3 21 29  1 5 15 15 1 5 15 15 1 5 1 5 15  4 5 6  1 2 1 2 1 2  1 3 21 29  1 5 1 5 1 5 1 5 4  Table  3  -  Replicate 0  3  6  II 9  1 5 15 1 5 1 5 1 5 15 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 1 5 15 1 5 1 5 1 5 1 5 1 5 15 1 5 1 5  25  s u l f o n a t e ) . The l a r v a e t h e n were r i n s e d b r i e f l y water  and  placed  on  ring  the  intact  l a t e r without  larvae to the  stored  glass  in a desiccator  distilled  s l i d e s p r e v i o u s l y coated  s o l u t i o n o f a compound ( S i l i c l a d ) removed  with  that allowed  the larvae to  be  damage r e s u l t i n g f r o m a d h e s i o n o f  surface.  (over  w i t h a 1%  The  air-dried  CaSO ) u n t i l  larvae  the c a l o r i f i c  a  were  work was  performed. Calorific  analysis  was  conducted  3  to  d e s i c c a t o r - d r y i n g o f t h e l a r v a e . The l a r v a e the  ring  slides  with  being  oven-dried  made t o t h e n e a r e s t Combustion Phillipson  from  The 15 l a r v a e into  from three  l a r v a e was t a k e n b e f o r e t h e  of  pelleted  microbomb  Inc., Aiken,  were c o m b u s t e d combusted  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  0.01 mg on an e l e c t r o b a l a n c e  oxygen  Instruments,  in  directly  d i r e c t ashing.  the  larvae  The  i n a muffle  (Gentry  out using a and  calorimeter  furnace  while  created  by  Wiegert  one  8100),  was  t o determine ash content hours,  as  (1971).  was c o n n e c t e d t o a p o t e n t i o m e t e r  R e c o r d e r Type PM  which  measured  the  (Phillips voltage  t h e t e m p e r a t u r e d i f f e r e n c e between t h e h o t j u n c t i o n  t h e bomb s t a n d  potentiometer  carried  A s h i n g was done a t 550°C f o r t h r e e  calorimeter  of  (Cahn, G2).  S . C ) . Of t h e t h r e e p e l l e t s made, two  microbomb  Flat-Bed  block)  was  calorimeter  recommended by Cummins a n d Wuycheck  of  removed  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 w e i g h e d b e f o r e a n d  after  by  months a f t e r  g r o u p were d i v i d e d  g r o u p s . The g r o u p d r y w e i g h t o f f i v e five  were  a sharp razor blade.  e a c h r e p l i c a t e o f one i n c u b a t i o n  4  the  and  the  cold  calorimeter.  pen  deflected  reference  When a  junction  combustion  distance  (aluminum  took p l a c e , the  proportional  to the  26  voltage created pellet  by t h e t e m p e r a t u r e r i s e o c c u r r i n g a s a r e s u l t o f  c o m b u s t i o n . The amount o f pen d e f l e c t i o n ( r i s e )  the b a s i s f o r c a l c u l a t i o n typical "total  values. Figure  combustion graph and t h e s t a n d a r d  calculated  equation  using benzoic  (Dueftas,  was o b t a i n e d ,  corrected  rise  and  giving  or  and  the  combustion  organic  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  of  of  of  ash-free ash For  organic  obtained  comparison,  calorimetry  total  weight added  of  to  pellets while  unburned  the  observed  from d i r e c t a s h i n g  in  ash  were  terms  values  also  (indirect  ashing).  of  dry  cal/g  elsewhere  organic were  caloric  r e l a t i o n s h i p between e q u i v a l e n t s . The  the  muffle  obtained Caloric  from the furnace. from  values  the were  weight, c a l / g ash-free dry  w e i g h t 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 o f t h e procedure i s described  was  pellets  the l o s t parts  m a t e r i a l and t h e i r c a l o r i c  residues in  could  regression  d r y w e i g h t s o f 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  values  expressed  the  the  v a l u e s . C a l c u l a t i o n was b a s e d on t h e l i n e a r weight  content  r e l a t i o n s h i p between  when l o s s o f l a r v a l p a r t s o c c u r r e d  of  equation  data). A linear  were b e i n g made. The c a l o r i c c o n t e n t material  a  c a l o r i c values. 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 incomplete  the  shows  calibration  a c i d , the c a l o r i c  unpublished  3  method o f c a l c u l a t i n g  c o r r e c t e d r i s e " . W i t h t h e use of a  determined e a r l i e r be  of c a l o r i c  provided  (DuefTas, u n p u b l i s h e d  calorimetry data).  27  Figure  3  Typical g r a p h i c a l combustion record and c a l c u l a t i n g " t o t a l corrected r i s e " .  standard  method  28  (5)  Post-fire  change  _ No. o f l i n e s (mm) Time ( m i n )  (3) R i s e = (mm)  Peak (mm)  Firing  point  ( m m )  Peak  (mm)  (1)  T i m e f r o m 60% t o p e a k (2) F i r i n g  Time t o 60% r i s e  point  Pre-fire  (min)  (6)  (min)  (4)  change  No. o f l i n e s (mm) Time ( m i n ) Calculation: Rise (-)  II.  III.  P e a k h e i g h t (mm) = Firing point (mm) R i s e (nun)  (-)  (1)  (3)  Time c o r r e c t i o n A- P r e - f i r e c h a n g e x T i m e t o 6 0 % r i s e (pre-fire correction) change x Time f r o m 6 0 % B. P o s t - f i r e to peak ( p o s t - f i r e correction) T i m e c o r r e c t i o n (mm)  (4)  x (6)  ( + )) (5)  x (7) (8)  of 6 d e t e r m i n a t i o n s  W i r e c o r r e c t i o n (mm) = M e a n r i s e  using  only (no sample) IV.  Total corrected rise , ,Rise (mm) W i r e c o r r e c t i o n (mm) , . W i r e c o r r e c t e d r i s e (mm) = T i m e c o r r e c t i o n (mm) Total corrected rise (mm) +  (-)  (3) (9)  ^ ^(10) (8) +  (ID  fuse  =  wire  (9)  (7)  29  RESULTS  1. Salinity  a)  Tests  1979 E x p e r i m e n t The  the  Tolerance  36 E D  two  mean 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  replicates  o f t h e 18 t r i a l s  4 . The  a r e shown i n T a b l e  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  5 0  from  potential  effects  of t h e 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 5)  shows  that  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 and x  5c), using the linear  x linear  two  things: (1) a significant  on  salinity  tolerance  replicates  x linear  I T x CT) a s a m e a s u r e o f e x p e r i m e n t a l e f f e c t of  i s suggested,  with  respect  to  main  effects  and  interpretation interactions  being  on  salinity  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 .  The  the data  high v a r i a b i l i t y  have a r i s e n a s f o l l o w s :  temperature to  both  replicates  of the i n f l u e n c e of the other  were f o u n d  made t o a n a l y z e  term ( I S  common  between  I I (Table 5c), s i g n i f i c a n t  was  5b  i s i n d i c a t i v e of  incubation  replicate  d i f f e r e n c e s were f o u n d  (Tables  interaction  error,  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  significant  (Table  tolerance.  differences in ED  5 0  values  Although,  i n each r e p l i c a t e ,  no  In  these attempt  further.  i n t h e 1979 e x p e r i m e n t  is  thought  to  30  Table 4 Estimates of E D f o r t h e 1979 e x p e r i m e n t . The two initial values i n each cell a r e t h e E D ' 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 limits (a=0.05; B l i s s , 1 9 3 7 ) . The final value i n e a c h c e l l i s t h e mean o f t h e two E D ' 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 t e s t f o r h o m o g e n e i t y of v a r i a n c e ( c = 0 . 0 5 ) . 5 0  5 0  5 0  2  31  ED  Incubat ion  5 0  Estimates  (°/ooS)  C u l t u r e T e m p e r a t u r e (°C) Temp. (°C)  Sal. (%>o)  1 3  6  44.2 37.4  ± 2.8 ± 3.0  40.2 ( 1 ) 33.0  40.8  ± 4.8  36.6  ± 5.1  ± 1.3 ± 1.7  37.5 34.9  ± 1.6 ± 2.0  35.3 31.3  ± 1.4 ± 1.7  x=36.8 ± 4.3  36.2  ± 1.8  33.3  ± 2.8  ± 1.6 ± 2.2  36.4 32.7  ± 1.2 ± 1.8  39.9 41.0  ± 1.8 ± 1.8  x=38.2 ± 2.5  34.6  ± 2.6  40.4  ± 0.8  36.7- ± 1.7 33.5 ± 2.0  37.9 38.6  ± 1.8 ± 2.0'  36.5 41.0  ± 2.0 ± 2.1 ( 1 )  x=35.1 ± 2 . 3  38.2  ± 0.5  38.8  ± 3.2  ± 1.3 ± 1.2  34.9 45.5  ± 2.2 (1) ± 1.5  42.5 42.7  ± 1.7 ± 1.3  x=36.8 ± 9 . 1  40.2  ± 7.5  42.6  ± 0.1  25.3 ( 1 ) 38.8  ± 2.3 ± 2.0  30.9 41.5  ± 2.0 ± 2.6  32. 1 ± 9.6  36.2  ± 7.5  45.6 34.8  ± 1.3 (1) ± 2.2  x=40.2 ± 7.6  21  6  29  6  1 3  1 2  21  12  29  1 2  39.8 33.7  40.0 36.5  30.3 43.2  22.2 42.1  ± 2.1 ± 1.8  x=32.2 ± 14.1  Table  4  1 2  9  6  ± 1.7 ± 2.1 (1)  ( 1 )  32  Table 5 Summary of a n a l y s i s of variance f o r the E D values of the 1979 experiment. A. O v e r a l l analysis, B. R e p l i c a t e I, : e p l i c a t e I I . * = S i g n i f i c a n t d i f f e r e n c e (c=0.05), NS = not significant, IS = incubation s a l i n i t y (°/ooS), IT = i n c u b a t i o n temperature (°C), CT = c u l t u r e temperature (°C). 5 0  C  R  A. O v e r a l 1  Source of  Variation  Incubation S a l i n i t y (IS) Incubation Temperature ( I T ) C u l t u r e Temperature (CT) I n t e r a c t Ion IS x IT IS x CT IT x CT IS x IT x CT  DF  2 1 2  Analys1s  MS  SS  F(calc)  F(tab) («=0.05)  S1gn1fIcance  46 72 2 83 13 15  23 . 36 2.83 6.58  0.65 0.08 0. 18  3.55 4.41 3 . 55  NS NS NS  0.84 0.36 0. 78 0. 14  3.55 2 .93 3.55 2.93  NS NS NS NS  2 4 2 4  121 52 56 20  25 41 60 46  30^31 13 . 10 28.30 5.12  Repl1catIon  18  651  13  36 . 17  Total  35  964 55  B. R e p l I c a t e I  Source of  Variation  Incubation S a l i n i t y (IS) Incubation 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  DF  2 1 2 2 4 2 4 17  Total  .  SS  180 211 1 1 91 40 78 19  05 49 10 45 89 63 58  633  19  90.03 211.49 5 . 55 45 . 72 10. 22 39.32 4 .90  C. R e p l I c a t e  Source of  Variation  Incubation S a l i n i t y (IS) Incubation 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 Total  Table  5  DF  SS  4  20.84 147.92 3.80 52.67 53.54 3.23 30.44  17  312.44  2 1 2  2 4 2  MS  F(calc)  18 . 37 43. 16 1.13 9.33 2 .09 8.02  F(tab) (a=0.05) 6.94 7.71 6.94 6 .94 6.39 6.94  SignifIcance * * NS * NS *  11  MS  10.42 147.92 1 .90  26.34 13.38 1 .62 7.61  F(calc)  F(tab) (a=0.05)  1 .37 19.44 0.25  6.94 7.71 6.94  0.21  6.39 6.94  3.46 1 .76  6.94  SignifIcance  NS *  NS NS NS NS co  34  (1)  The  two  r e p l i c a t e s were c o n d u c t e d  Hence, t h e l a r v a l fertilized  on  at different  s a m p l e s were f r o m d i f f e r e n t b a t c h e s  separate dates  of  resulted  in  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 t h e experiment doing  eggs,  dates.  (2) A t e c h n i c a l p r o b l e m i n t h e i n c u b a t i n g t a n k mortality  of  a n d t h e r e was a p e r i o d o f 23 d a y s  t o one month between f e r t i l i z a t i o n  high  times.  forreplication,  i n s t e a d of  t h e r e p l i c a t e s a t t h e same t i m e .  (3) The f i r s t tolerance required  procedure  employed  f o r the  larval  t e s t was t h e t i m e - m o r t a l i t y t e c h n i q u e . frequent h a n d l i n g of the  culture  salinity  This  dishes  technique  and  of  the  s a m p l e s , a s t e p w h i c h may have c o n t r i b u t e d t o e r r o r .  b)  1980 E x p e r i m e n t The  the  18  54  ED  trials  constituted  5 0  values estimated  are  shown  6% o f a l l E D  significant  x  2  5 0  values  in  the  replicates  (e.g.,  incubation  6.  Extrapolated  values  s e t s a l s o showed  i n d i c a t i n g h e t e r o g e n e o u s v a r i a n c e . They  28% of a l l t r i a l s .  compare t h e i r v a l u e s  Table  e s t i m a t e s . Some d a t a  constituted three  from t h e t h r e e r e p l i c a t e s of  (Table  in  The mean E D 6)  relation  salinity,  were to  5 0  's  plotted  the  incubation  calculated  from  ( F i g u r e 4) t o  three  main  temperature,  factors culture  temperature). In t h e 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 t h e (Tables  7A,  interactions incubation interaction  B, were  and  C),  found  salinity, between  to (2)  the be  following significant  incubation  incubation  ED  main  5 0  effects  (c=0.05):  temperature,  salinity  estimates  and  and  and (1) (3)  incubation  35  Table 6 Estimates of E D and t h e i r mean values f o r the 1980 experiments. The f i r s t three values i n each c e l l are the E D ' s from the three r e p l i c a t e s x/V f E D (a=0.05). The f i n a l value i n each c e l l i s the mean E D ± 1 SD. The n o t a t i o n ( l ) i n d i c a t e s a s i g n i f i c a n t x t e s t f o r homogeneity of v a r i a n c e . (2) i n d i c a t e s an e x t r a p o l a t e d E D value. 5 0  5 0  5 0  5 0  2  5 0  Incubat ion  EDso  Estimates  x/f,  Culture Sal . ('/oo)  Temp. Cc)  13  6  21  29  13  45.8 x/f 49.5 x/f 50.2 x/f  49.5 x/f 51.0 x/f 51.0 x/f  1.1 1 .2 1.2  1 .2  1.2 1. 2  21  12  37 .0 x/f 1. 1 38.5 x/-!- 1 . 1 36.0 x/f 1 . 1 x=37.2  29  12  6  ±  1 .3  41.8  x/f-1.1  44.5  x/f x/f  44.2  x=43.5  Table  + 2.9  1.1 1.2  + 1.5  (1)  C/ooS)  1 SD  12 48.5 x/f 46 .0 x/-r 50.5 x/f  1.2 1.1 1.6 ( 1 )  48.3  ± 2.3  46.2 x / - 1.2 51.0 x / - 1.2 52 .0 x/-r 1 . 2  43.5 45.5 50.5  x/f  49.7  46.5  ± 3.6  53 .0 x/-r 1.2 45.5 x / - 1.4 ( 1 ) 54.5 x / - 1.5 ( 1 , 2 )  46.5 43.5 52.2  x/f  51.0 ±  47.4  ± 4 .4  47 .4 ±  38 .0 x/-r 1 . 1 32.5 x/f 1.2 33.5 x/-r 1.3 ( 1 ) x=34.7  +  (*C)  46 . 2 x/-f 1 . 1 46.8 x/f 1. 1 1.1 49.0 x/f 1.5 ( 1 ) 44 .5 x/f 51.5 x/f 1.2 46.8 x/-r 1.1  x=50.5 ± 0.9  12  ED>o  9  x=48.5 ± 2 . 4  6  Temperature  6  x=47.5 ± 1 . 3  6  f EDs o a n d Mean  3.7  ± 3.1  4.8  1.2 1.2 1.2  x/f x/f  x/x/f  1.2 1.5 1.4 ( 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  34.2  38.1  + 4.9  ± 2,5  45.5 x/-r 1.4 ( 1 ) 45 .0 x/-r 1.1 34.2 x/-r 1 . 1  36.5 35.5  x/f  42.5  X / T . 1 . 5  41.6+6.4  38.2  ±  50.0  1.2  x/-r 1 . 1  x/-r- 1.1 ( 1 ) x/f 1.1 x/-:- 1 .2  43.7  x/f  47.5 49.0  50.5  x/f  48.8  x/f  48.8  + 1.3  47.7  +  (1)  3.8 1.7 1.2.(2)  1.1 3.5  CO  01  37  Figure 4 Mean ED values (calculated from t h r e e 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). Numbers above e a c h l i n e show c u l t u r e t e m p e r a t u r e (°C); t h o s e b e s i d e e a c h l i n e show incubation temperature (°C) (See T a b l e 6 ) . S 0  38  T E M P E R A T U R E (°C) 54  CULTURE  INCUBATION  (POST-HATCH)  50  6°C  cn o  4  6  IT) Q LU  42 < LU  38  h  I2°C  34  13  INCUBATION  21  SALINITY  29  (%o)  39  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 values of the 1 9 8 0 experiment. A. O v e r a l l a n a l y s i s , B. 6 ° C incubation group, C. 1 2 ° C i n c u b a t i o n group. * = S i g n i f i c a n t d i f f e r e n c e ( c = 0 . 0 5 ) , NS = not s i g n i f i c a n t , IS = incubation salinity (°/ooS), IT = i n c u b a t i o n temperature (°C), CT = c u l t u r e temperature (°C). 5 0  A. O v e r a 1 1  Source of  Variation  Incubation Sal1n1ty (IS) Incubation Temperature ( I T ) C u l t u r e Temperature (CT) Interact1on IS x IT IS x CT IT x CT IS x IT x CT  MS  SS  DF  Analys1s  196.82 887.36 15 .06  2 1 2  393:65 887.36 30.12  2 4 2 4  196.02 58.14 42 . 32 16 . 25  98 .01 14.53 21.16 4 .06 11.31  Repl1cat1on  36  406.99  Total  53  2.030.84  B. 6'C  Incubation  F(calc)  17.41 78 .49 1 . 33 8 .67 1 .29 1 .87 0.36  F(tab) (a=0.05)  Significance  3.26 4.11 3.26  NS  3 . 26 2.63 3. 26 2.63  NS NS NS  *  *  Group  F(calc)  F(tab)  S igni f Icance  SS  MS  2 2  17 .08 18.62  8.54 9.31  0.84 0.91  3.56 3 . 56  NS NS  4  21.71  5.43  0.53  2 .93  NS  Replicat1on  18  183.52  10.20  Total  53  240.93  Source of  Variation  Incubation S a l i n i t y (IS) C u l t u r e Temperature (IT) Interact1on IS x CT  DF  C.  Source of  Variation  DF  12'C  SS  Incubation  MS  (<j=0.05)  Group  F(calc)  F(tab)  2 2  572.59 53.82  286.29 26.91  23.06 2.71  3.56 3.56  4  52.68  13.17  1 .06  2.93  Replicat ion  18  223.47  12.42  Total  26  902.56  902.56  Incubation S a l i n i t y (IS) C u l t u r e Temperature ( I T ) Interact ion IS x CT  Table  7  SignifIcance.  (o=0.05) * NS NS  o  41  temperature.  The  statistically other  effect  of  significant,  culture  nor  was  temperature  its  interaction  not  with  the  factors. The  influence  tolerance values  of  at  mean  ED  the  three  incubation be  seen  salinity  by  incubation  of  suggest  that  the  13,  at  41.72, 43.61  and  eggs  on  21  influence  similar 12°C  manner  (Table  incubation  40.44°/ooS, tolerant  of 8,  last  groups  had  and  to  higher  temperatures  of v a r i a n c e  interaction  between  temperature.  That  where  higher  increase the  in E D  separate different several  ED  for  12°C  salinities  values  5 0  be  from  seen  the of  larvae  when i n c u b a t e d  5 0  5 0  i n d i c a t e s that  as  salinity  (Table  's  the  two  level  incubation  with  in a  6  and  48.54  and  were  more  eggs a t  lower  is a  of  the  different  is illustrated  incubation incubation  at  7B  salinities  groups and  salinity  (6 and  were not  incubation larvae varied levels  ED  5 0  5,  incubation levels.  i s most  12°C)  of  in Figure  higher  temperature  7C):  significant  and  group. A n a l y s i s of v a r i a n c e  (Tables  incubation  at  obtained  incubation-temperature results  tolerance  8). This  were  there  salinity  temperatures  salinity  salinities  ED  incubation  is,  incubation  incubation  had  (6°C).  Analysis  between  row).  5 0  salinities.  suggests that  salinities  last  29°/ooS  temperature can  mean  ED  at higher  column). Larvae  respectively. This  8,  mean  48.15°/ooS, r e s p e c t i v e l y .  incubated  incubation  salinity  the  (Table  would p r o d u c e l a r v a e more t o l e r a n t t o h i g h e r The  the  comparing  salinities  from eggs i n c u b a t e d  values  5 0  These data  of  l a r v a e may  Larvae hatching  in  was  This  evident  conducted produced  values  significantly  for  on two the  different  42  Table 8 Comparison of mean E D values of h e r r i n g l a r v a e i n r e l a t i o n to 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. Both main effects and interaction of these two f a c t o r s are s i g n i f i c a n t (P < 0 . 0 1 ) . Mean values were c a l c u l a t e d from nine ED estimates ( 3 c u l t u r e temperatures x 3 r e p l i c a t e s ) . 5 0  5 0  Mean E D Incubation Temperature (°C) 6 12 Mean Table 8  5 0  Incubation .  1  3  21  (°/ooS) Salinity 29  47.76 35.68  48.24 38.97  49.63 46.67  41.72 '  43.61  48. 1 5  (°/ooS) Mean 48.54 40.44  44  Figure 5 Interaction between incubation salinity and incubation temperature. A d d i t i v e i n t e r a c t i o n i s s i g n i f i c a n t (P<0.01; See Table 8 ) .  INCUBATION TEMPERATURE  •f 6  /  30  13 INCUBATION  21 SALINITY  /  /  12  A  i  29 (%o )  (°C)  46  in  t h e 6°C g r o u p ,  12°C  group.  group  for  higher  levels  larvae  least of  Between  had  salinity  from  b u t t h e y were s i g n i f i c a n t l y incubation ED  (Figure  from  t h e 6 ° C , 29°/ooS  non-significant  than those  5 ) . The g r e a t e s t  t h e 13°/oo  t h e 29°/oo  temperatures,  values  5 0  incubation  group.  different larvae  from  from  d i f f e r e n c e was  The o v e r l a p p i n g  t h e 6°C  12°C g r o u p  salinity  and 12°C, 29°/ooS  i n the  ata l l  obtained  group,  and  the  standard  deviations  (Figure  5) s u g g e s t  groups  d i f f e r e n c e s between t h e two mean E D  values  5 0  at  29°/ooS. Culture  (post-hatching)  a statistically 7).  significant  However, c l o s e r  common t o b o t h  Those  larvae  at c u l t u r e temperatures  those  higher  salinity  tolerance  12°C.  In  incubation  resulting  larvae  temperature incubated discussion  larvae  both  of  incubated  hatching  from  groups  between  groups  6  to s a l i n i t y  9°C.  is  evident  salinities  section, a synthesis  most  (Figure  and  group  at a  in larval  6 b ) . In F i g u r e  of the r e s u l t s  higher 9°C, showed 9  and  o f 6 and 12°C, t h e  tolerance  This  be some  (Figure 6a).  a t c u l t u r e t e m p e r a t u r e s between  showed h i g h  at higher  could  (Table  a t 6°C showed  12°C i n c u b a t i o n  temperature  t o have  tolerance  there  temperature  from eggs  while  not appear  i n f l u e n c e on l a r v a l  incubation  hatching  tolerance  does  inspection suggests that  trends  salinity  temperature  culture groups  11 of  i s presented.  the  47  Figure  6  Comparison o f mean ED values of h e r r i n g larvae i n r e l a t i o n t o : A. C u l t u r e t e m p e r a t u r e and i n c u b a t i o n temperature, B. C u l t u r e t e m p e r a t u r e and i n c u b a t i o n s a l i n i t y . Mean E D values were calculated from 9 ED estimates f o r A (3 i n c u b a t i o n salinities x 3 replicates); 6 ED values f o r B (2 incubation temperatures x 3 r e p l i c a t e s ) (see T a b l e 6 ) . 5 0  5 0  5 0  5 0  —I  9  CULTURE  I  1  12  TEMPERATURE  I  I  1_  6  9  12  (°C)  49  JL. C a l o r i m e t r y  a)  Unequal In  Sample  Size  replicate  mortality  in  sufficient resulted 11)  (1979 T r i a l s )  the  I,  sampling  12°C  larvae  larvae  survived  i n unequal  sample  (6°C)  were  higher  incubation  standard  sizes.  from eggs  generally  length  longer  temperature  In  from d i f f e r e n t  of  replicate II,  The mean v a l u e s  mean  standard  mm,  10.36 mm  the  lengths  length day  between  0.  grew.  lengths  (Tables  sample  This  9, 10,  sizes.  The Larvae  reared  (Figure  difference reared  linear  declined  7, T a b l e  from  9).  from  Mean  7.67 mm group,  were: 7.90 mm,  12°C  9.84  group,  8.97 mm,  difference  in  9.87 larval  (6 a n d 12°C) was o b s e r v e d a t as the l a r v a e  i n both  increase  groups  i n length,  manner, from day 0 t o day 9 ( F i g u r e 7 ) .  in length  maximum  larvae hatching  r e s p e c t i v e l y . Among t h e  a t 12°C showed more r a p i d  i s an i n d i c a t i o n  temperature  (day 0) r a n g e d  a t 6°C showed a g r a d u a l  There  lower  (6°C, 1 3 ° / o o S ) . Among t h e 6°C  t h e two t e m p e r a t u r e s  The i n c r e a s e  their  those  r e s p e c t i v e l y . The g r e a t e s t  6°C.  attained  (12°C)  o f t h e same age o r d e r  an a p p r o x i m a t e  Larvae  than  at  a t d a y s 0, 3, 6 and 9 were 9.74 mm,  and 10.37 mm,  and 9.74 mm  incubated  a t peak o f h a t c h i n g  (12°C, 2 9 ° / o o S ) t o 9.93 mm  in  3).  because  Standard Length of Larvae Larvae hatching  mm  (Table  incomplete  up t o day 9 i n a l l t r e a t m e n t s .  were c a l c u l a t e d , t h e r e f o r e ,  b)  was  that length  growth  than  those  from day 0 t o day 6 i s a l s o those  larvae  at  a t day 6, a p e r i o d  12°C  at  linear.  may  coinciding  have with  50  Figure  7  Mean (± 1 SD) standard length (mm) of herring larvae sampled from t h e s i x s a l i n i t y - t e m p e r a t u r e t r e a t m e n t s a t v a r i o u s a g e s . L a r v a e were u s e d f o r c a l o r i m e t r i c e x p e r i m e n t i n 1979 (see Table 9).  Ti  To  (6°C- I3%c S )  { 6 ° C - 21 % o S )  T,  COMPOSITE  (6°C - 2 9 %o S )  T, T O T  e  H  9  GRAPH  6  TREATMENT  oS  E  - - O 13 29 21 13 21  X I"2  10  LU  Q  tr.  <  To.  _J( I2°CI  (T)  13 21 29ET  L_  " I3%» S )  TP,  ( 1 2 ° C " 21 % o S )  T  f i  ( I2°C - 2 9 % . S )  / / •  29  °C  6 ~ 12 _  / / r  ifi  1 3 0(1/ i 2i d  ^ - 5  "2  < 9 LL!  29°  0  _i 3  6  i  9  1—  0 DAYS  3 AFTER  6  9  0  3  6  9  HATCHING  cn  52  Table  9  Table of means showing lengths (mm), w e i g h t s (mg), and c a l o r i c v a l u e s ( c a l ) of h e r r i n g larvae sampled from the six salinity-temperature treatments at various a g e s . L a r v a e were u s e d f o r c a l o r i m e t r i c e x p e r i m e n t i n 1979. In columns 3, 4 and 5, sample s i z e -- 30 l a r v a e , u n l e s s o t h e r w i s e i n d i c a t e d ( b r a c k e t s ) . In columns 6, 7 and 8, sample size -4 pellets, unless otherwise i n d i c a t e d (brackets).  1  2  Days A f t e r Hatching  Treatment (TemperatureS a l 1n1 t y Combinations)  CC  - VooS)  6  7  8 Mean Calories/ gram a s h free dry wt. ( c a l )  Mean Dry Weight'/ Larva (ng) ;  Mean Ash-free D r y Wt./ Larva (mg)  Mean Calories/ Larva (cal)  Mean Calor1es/ gram d r y weight (cal )  9.93 9.82 9.46  0.142 0. 153 0.158  0.129 0. 139 0. 144  0.853 0.868 0.977  6,032 5,677 6, 150  6,622 6,245 6,736  4 5 6  (12-13) (12-21) ( 12-29)  8 . 20 7.84 7.67  0. 153 0. 162 O. 162  0.140 0.150 0.151  0.915 0.978 1.035  5,990 6,024 6,377  6,520 6,518 6,875  1 2 3  9.81 9.86 9.86  0. 138 0. 138 0.142  0. 127 0. 124 0.129  0.806 0. 769 0.801  5,860 5,548 5,6 10  6,364 6,201 6,171  4 5 6  9.20 ( 1 5 ) 8 .66 9.06 ( 1 9 )  0. 139 ( 1 5 ) 0.141 0. 138 ( 1 9 )  0.128 ( 1 5 ) 0.127 0. 128 ( 1 9 )  0.820 ( 2 ) 0.814 0.667 ( 3 )  5,882 ( 2 ) 5,770 4,823 ( 3 )  6.392 ( 2 ) 6.418 5,512(3)  0.137 0. 13.1 0. 134  0.123 0.117 0. 1 20  0 793 0.839 0.859  5,795 6,417 6,416  6,426 7,178 7,121  0. 122 ( 15) 0. 122 ( 1 5 ) 0. 126 ( 1 5 )  0. 113 ( 1 5 ) 0,112 ( 1 5 ) 0.115 ( 1 5 )  0.851 ( 2 ) 0.706 ( 2 ) 0.655 ( 2 )  6.962 ( 2 ) 5,812 ( 2 ) 5,2 18 ( 2 )  7,656 ( 2 ) 6,304 ( 2 ) 5,684 ( 2 )  0.118 0. 123 0.117  0.106 0.109 0. 105  0.674 0.624 0.645  5,703 5,090 5,449  6.326 5,722 6, 107  0.101 ( 1 5 ) 0.108 ( 1 5 ) 0.111 ( 1 5 )  0.091 ( 1 5 ) 0.099 ( 1 5 ) 0.098 ( 1 5 )  0.693 ( 2 ) 0.552 ( 2 ) 0.660 ( 2 )  6,882 ( 2 ) 5,108 ( 2 ) 5,924 ( 2 )  7.633 ( 2 ) 5,585 ( 2 ) 6.756 ( 2 )  1 2 3 4 5 6 1 2 3 9 4 5 6  Table  5  (6-13) (6-21) (6-29)  3  6 ( e n d of yolk-sac stage)  Mean Standard Length/ Larva (mm)  4  1 2 3 0 (peak o f hatching)  3  10.40 10.31 10.36 9.95 ( 1 5 ) 9.97 ( 1 5 ) 9.70 ( 1 5 ) 10.54 10.23 10.33 9.98 ( 1 5 ) 9.84 ( 1 5 ) 9.41 ( 1 5 )  9  cn  54  t h e end group  of t h e y o l k  sac s t a g e . At day  showed a d e c r e a s e Comparison  salinities  of  (13, 21  salinities  were  c ) Dry Weight  Table  (day  0)  9.  21°/ooS; day  0 ranged  larval plot  from  12°C,  29°/ooS).  from  of  0.129  larvae  kept  at  the  larvae  12°C  different in  lower  in higher s a l i n i t i e s  i n both temperature  mg  of  larvae  (6°C,  mg  (Figure  groups.  per  larva  13°/ooS)  there  t o 0.162  0  a t lower  was  lines  (Figure  8)  temperature than  day  those 9,  mg  the v a l u e s a t  0.151  mg  a linear  9). A  combined  f o r the dry  6  (12°C)  (12°C,  decrease in  (T, t o T ) shows  (12°C,  that  the  produced  incubated  at  eggs  slightly  the  lower  however, t h e r e v e r s e was  temperature  while those c u l t u r e d  to  ( F i g u r e s 8 and  regression  higher  At  9  a r e shown  a t t h e peak o f h a t c h i n g  13°/ooS)  0 t o day  larvae  a t day  (6°C).  (6°C,  treatments,  herring  Larvae c u l t u r e d weight  0.142  from day  temperature  at  a s h - f r e e dry weight  the s i x l i n e a r  incubated heavier  and  a l l  weight  weights  i s true  reared  2 9 ° / o o S ) . In a s h - f r e e d r y w e i g h t ,  In  of  shows t h a t  Mean d r y w e i g h t  ranged  of t h e  Larvae  Mean d r y w e i g h t in  larvae  than those  trend  of  of  29°/ooS)  larger  7, T a b l e 9 ) . T h i s  some l a r v a e  in length.  lengths  and  9,  (6°C) had  the  at higher temperature  true.  highest (12°C)  dry  had  the  least. Among of  the  the  12°C  three  salinities a  uniform  8,  T,,  T  2  group  regression  were s i m i l a r rate and  (Figure lines  (slope  of w e i g h t  8,  5  calculated  and  T ) , the  for  6  the  = -0.006 mg/day), w h i c h  d e c r e a s e . Among t h e 6°C  T ) , t h e s l o p e s were n o t 3  T„, T  uniform.  three  indicates  group There  slopes  (Figure was  an  55  Figure  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 d r y weights (mg) of herring l a r v a e u s e d f o r c a l o r i m e t r i c e x p e r i m e n t i n 1979. L a r v a e were sampled from t h e six salinity-temperature treatments at various a g e s ( s e e T a b l e 9 ) . In a g i v e n r e g r e s s i o n e q u a t i o n , Y = e x p e c t e d d r y w e i g h t (mg), X = age ( d a y s after hatching), r = correlation coefficient.  Tj  ( 6 ° C - I3%OS ) y = . 1 4 5 + ( " . 002  T  2  x)  r=(-)0.879  ( 6 ° C - 2l%oS ) y= .151 +• ( - . 0 0 3 x ) r = (-) 0 . 9 8 3  T  (6°C- 29%o S )  3  y = . 1 5 7 +•(r =(-)  .004x)  0.992  .1 4  •  .12 E  2 or <  .10  i o LU  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 % o S ) y =. 16I + (- . 0 0 6 x ) (-) 0 . 9 9 6  T  6  r=  ( I2°C - 2 9 % o S) y-- . I 5 9 +• (" . 0 0 6 x) 0 . 989  r = (-)  > or o  <  LU  .14  .12  .10  0  3  6  9  0 DAYS  3  6  AFTER  9 HATCHING  0  3  6  T, T O T TREATMENT  •  .16  COMPOSITE  GRAPH 6  (T)  57  Figure  9  Calculated regression l i n e s f o r mean a s h - f r e e d r y w e i g h t s (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 e x p e r i m e n t i n 1979. L a r v a e 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 t r e a t m e n t s a t v a r i o u s ages ( s e e T a b l e 9 ) . In a g i v e n r e g r e s s i o n e q u a t i o n , Y = expected ash-free dry weight (mg), X = age (days after hatching), r = c o r r e l a t i o n c o e f f i c i e n t .  T,  (6°C- l 3 % o S) y= .132 + (" . 0 0 2 x) r = (-) 0 . 9 0 0  .16  T  2  (6°C- 21 % o S ) y= . 1 3 7 + ( - . 0 0 3 x) r=(") 0 . 9 8 3  T  3  (6°C-29%oS) y= J 4 3 + (- . 0 0 4 x ) r= (') 0 . 9 9 6  COMPOSITE  GRAPH  T, T O T  6  .14  .1 2  • .10  T  4  (12 ° C - I 3 % O S )  T  5  y = J 4 7 + (- . 0 0 6 x )  y= . l 4 2 + ( - . 0 0 5 x ) r = (")  ( I 2 ° C - 21 % » S ) r = (-)  0.990  T  ( 12°C - 2 9 % o S )  6  y = . l 4 9 + (- . 0 0 6 x ) r = (") 0 . 9 9 4  .991  .1 2  .10  .0 8  0  3  6  9  0 DAYS  3  6  AFTER  9 HATCHING  0  0  3  6  9  59  increasing and  slope  2 9 ° / o o S had  mg/day,  in higher slopes  equal  respectively).  temperature of  6°C  salinities to  The  show t h a t  -0.002,  varying  larval  terms  obtained  d)  of  (Figure  Calorie (1)  Residue.  Incomplete  samples  There  residue Ash  with  of a s h  of  pellets  (3) C a l o r i c calorimetric and  cal/g  hatching treatments  smaller  estimated  than  result  was  is  percentage  of  M e t h o d s.  obtained  from  the  residue).  Table  10  from  0.43  ranging The  pellets  of H e r r i n g  ashing  comparison  for  Larvae.  are  presented  ash-free  dry  weight  (Table  caloric  values  were:  weight  I n d i r e c t Ashing  values). 1-2  The  direct  method indirect  (column to  7)  4.66%  i s based ashing,  on 2-4  ashing.  Values  0 ) , the  pellet  direct  test  (day  calorimeter.  pellets.  percentages  sizes,  combustion  from t h e  those  calorimetry  for indirect  the  r e s i d u e per  from D i r e c t and  negative  sample  in  i s a t r e n d towards h i g h e r  shows d i f f e r e n c e s i n a s h two  fired  unburned o r g a n i c  were h i g h e r  (ashing  different  lower  salinities.  Unburned O r g a n i c  percentages  (excluding  culture at  a similar  10.  (2) P e r c e n t  method  decreases  i n lower  -0.004  a  weight,  of  in Table  generally  at  dry  in a l l pellet  unburned o r g a n i c  The  and  21  Determination  mean p e r c e n t a g e shown  slopes  13,  9).  Percent  occurred  ash-free  rate  salinities  -0.003  weight  temperatures proceeded at a slower In  (e.g.,  6,042  cal/g  Results  in c a l / l a r v a ,  dry  9). of  cal/g  During  of dry  the  larvae averaged  weight,  6,586 c a l / g  this weight,  peak  of  from a l l ash-free  60  Table  10  T a b l e of means showing d r y w e i g h t s of p e l l e t s (1 p e l l e t = 5 larvae) c o m b u s t e d i n bomb c a l o r i m e t e r , p e r c e n t u n b u r n e d o r g a n i c r e s i d u e , and p e r c e n t a s h o b t a i n e d from d i r e c t and i n d i r e c t (from r e s i d u e ) methods. P e r c e n t v a l u e s b a s e d on pellet dry weight. Sample size — 4 p e l l e t s , unless otherwise i n d i c a t e d (brackets) (1979 d a t a ) .  Days A f t e r Hatching  0 (peak o f hatching)  10  Mean A s h (direct method)  Mean A s h (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) • (%)  Mean D r y Weight Pel l e t (mg)  Mean Unburned Organic Residue (%)  1 (6-13) 2 (6-21) 3 (6-29)  0.708 0. 739 0.777  3.51 3.79 3.39  8.88 ( 2 ) 9.04 ( 2 ) 8.66 ( 2 )  6. 12 7 .94 4 . 45  2 .76 1 . io 4.21  4 5 6  0.762 0.809 0. 794  1 . 86 2 .72 2.00  8.14 7.51 7.26  (2) (2) (2)  7.19 5 .43 5 . 30  0.95 2.08 1.96  1 2 3  0.670 0.692 0.712  2.95 3.60 4 .00  7.94 10.40 9.07  (2) (2) (2)  8.63 9.51 9 . 10  4 5 6  0.696 ( 2 ) 0. 702 0.676 ( 2 )  4.04 ( 2 ) 5.13 5.00 ( 3 )  7.96 ( 1 ) 10. 12 ( 2 ) 7.30 ( 1 )  4.60 ( 2 ) 7 .05 6.87 ( 3 )  3 . 36 3 .07 0.43  1 2 3  0.684 0.655 0.666  3.48 2 .88 3 .59  9.78 10.59 9.82  7 .84 7.31 7 .82  1 .94 3.28 2.00  4 5 6  0.612 ( 2 ) 0.608 ( 2 ) 0.628 ( 2 )  5.60 ( 2 ) 5.94 ( 2 ) 5.10 ( 2 )  6.88 ( 2 ) 7.26 ( 2 ) 7.01 ( 2 )  2.27 0.53 1 . 23  1 2 3  0.589 0.613 0.574  4.17 3.12 4 .68  9.84 11.07 10.78  (2) (2 ) (2)  8.50 9 .95 9.77  1 . 34 1.12 1 .01  4 5 6  0.504 ( 2 ) 0.540 0.556  5.17 ( 2 ) 5.56 ( 2 ) 5.77 ( 2 )  9.85 8.57 12.31  (1) (1) ( 1)  5. 19 ( 2 ) 7.04 ( 2 ) 7.92 ( 2 )  4 .66 1 .53 4.39  9  Table  7  6  5  Treatment (TemperatureS a l 1n1 t y Combinations) C C - '/ooS)  3  6 (end of yolk-sac stage)  4  3  2  1  (12-13) ( 12-21 ) (12-29)  (%)  (%)  (2) (2) (2)  9.15 ( 1 ) 7.79 ( 1 ) 8.24 ( 1 )  (-)  0.69 0.89 (-) 0.03  62  dry  w e i g h t and  5,693  cal/g  0.641  0.938 c a l / l a r v a . dry  cal/larva.  cal/larva  weight,  day  0  t o day  or  values and  cal/g  ash-free  were o b t a i n e d  f o r day  a t day  9 larvae  9 v a r i e d between t h e (6°C-13°/ooS) 0.018  and  relatively  and  r a t e of c a l o r i c  faster  r a t e of c a l o r i c  Analysis  age  from  shows t h e  t e m p e r a t u r e and No data  due  (Figure  cal/g  dry  high  caloric  3  and  10).  4)  values  3.  f r o m day  0 to  day  Treatments least  1  slopes  (-  indicating  a  whereas t r e a t m e n t s highest  respectively),  slopes  indicating  5 (a  decrease. of  the  cal/g  ash-free  following significant  0 t o day  10).  day  respectively), decrease;  mean  caloric  0 and  (cal/larva)  cal/larva/day,  of v a r i a n c e  day  a t day  and  (Figure  2,  9 ) . The  ( 1 2 ° C - 2 9 ° / o o S ) showed t h e  and  11)  those  slower  -0.038  (treatments  (Table  decrease  weight  in  ( 1 2 ° C - 1 3 ° / o o S ) showed t h e  0.046  (Table  than  4)  cal/larva/day,  6  dry  were:  p a t t e r n of d e c r e a s i n g  6 larvae  -0.021  ( 1 2 ° C - 2 1 ° / o o S ) and  ash-free  weight. Unusually  six treatments 4  values  consistent either  (treatment  r a t e of c a l o r i c  these  9 i n a l l treatments  dry  f o r day  6 were much h i g h e r  The  not  9,  cal/g  a general  T h i s p a t t e r n , however, was weight  day  6,355  T h e r e was  from  At  9,  and  (2)  dry  effects  interaction  weight (o=0.05):  between  data (1)  salinity,  age.  further s t a t i s t i c a l to unequal  sample  a n a l y s i s was sizes.  done  on  the  caloric  63  Figure  10  Calculated regression lines f o r t h e mean c a l o r i e s / l a r v a . Herring larvae samples were taken from the six salinityt e m p e r a t u r e t r e a t m e n t s a t v a r i o u s a g e s (see T a b l e 9 ) . In a g i v e n regression e q u a t i o n , Y = e x p e c t e d c a l o r i e s / l a r v a , X = age (days after hatching), r = correlation coefficient.  0  3  6  9  0 DAYS  3  6  AFTER  9 HATCHING  0  3  6  9  0  3  6  9  cn *"  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 a s h - f r e e dry w e i g h t of h e r r i n g l a r v a e . L a r v a e were sampled a t 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 a t o=0.05; NS — not s i g n i f i c a n t ; S — incubation and rearing salinity ( ° / o o S ) ; T -- i n c u b a t i o n and r e a r i n g t e m p e r a t u r e ( ° C ) ; A -- age ( d a y s a f t e r hatching) (1979 data) .  Source of Variation  OF  SS  MS  SIgn1ficance  F(ca1c)  F(tab) («=0.05)  1 .805  3. 150  NS  Sal1n1ty C/oo) (S)  2  Temperature CC) (T)  1  6,756.08  6,756 08  0.017  4 .001  NS  3  5,162,769.62  1 ,720,923 21  4.354  2.758  *  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  *  ReplIcat1on  57  22,527,283.26  Total  80  44,320,482.18  Age ( d a y s after hatching) (A)  1 ,426,496.66  713,248  34  Interact ion  Table  • i  11  395,215 .50  67  DISCUSSION  This salinity (1)  present tolerance  incubation  culture was  investigation of y o l k  salinity,  (post-hatching)  also  carried  combinations  sac  of  examines  herring  (2)  larvae  incubation  temperature. A  out  to  salinities  examine  and  the  hypothesis  is  influenced  by  t e m p e r a t u r e , and  (3)  calorimetric the  effects  temperatures  that  on  experiment of  various  aspects  of  larval  only  the  upper  development. In  the  lethal  salinity  salinity  (Fry,  1971)  lower  well  or  tolerance  was  a  significant  The  effect of  lethal  measured.  show t h a t  exert  employed,  incipient  test  to s a l i n i t y .  not  both  test  done.  on  of c u l t u r e  the  will  the  of  the  salinity  be  and  tolerance  temperature  results  level  for  Results  incubation  effect  these  The  salinity  is  of  less  presented  in  11.  1 . Effect  of  Incubation  Salinity incubated larvae  upper  limits  defined. A synthesis  Figure  test  l a r v a e was  salinity  temperature larvae  limits  of h e r r i n g  lethal  salinity  tolerance  Salinity  tolerance  in higher  of  salinities.  to t o l e r a t e higher  e x p o s u r e of Herring  and  was  maximized  I t appears that  salinity  embryos t o h i g h e r eggs  larvae  levels  salinities  l a r v a e have the  in  the  for  capacity  enhanced  during capacity  eggs  by  of the  incubation. to  osmoregulate  68  (Holliday,.1965; of  the  is  herring  1969;  at  the  al.,  (Holliday  and  1979). In  osmotic  blastodermal  of  and  concentration  to  1965;  Blaxter  to a  salinity  then  they w i l l to  believed the  have t o  t o be  the  way  salinity, to  resistance leading  to  to  study,  larvae  upper e x t r e m e It  29°/ooS  from t h i s  important  role  (1947) has  pointed  consideration  this that  and  in  the  the  are  of  process  larvae  highest  medium showed  in  process  from t h e egg  is  ectoderm  is  incubated  acclimate  process  of  exposed  incubation,  salinities.  efficiency  fluid  (Holliday,  regulatory arose  those  raise  in its  Acclimation  regulation  would  (Kinne,  1964b).  In  incubation  salinity  and  the  highest  tolerance  to  salinities.  appears that  organism  larvae  t o l e r a t e , i t may  was  or  sub-  values  i t can  requirement  in  body  1 9 6 5 ) . I f an  in  e_t  osmoregulation  supranormal  increase  hatching  an of  salinity  energy  their  is  Alderdice  tolerate  Jones,  which  higher,  an  d e c r e a s e the this  that  herring  experienced during  epidermal c e l l s  ( H o l l i d a y and  Pacific  must  1963). I f h e r r i n g  site  This  1969;  original  and  closure.  to survive  return  their  undergo  The  and  tissues  and  near  Holliday,  survive.  at a higher  larvae  d i f f e r e n t from t h a t  embryo  some  regulate  overgrowth  blastopore  Holliday,  their  levels  and  1965;  f o r eggs and  salinities,  media  order  Jones,  order  supra-normal  the  completion  1965). O s m o r e g u l a t i o n  t o b o t h A t l a n t i c ( C l u p e a h a r e n g u s L.)  eggs  of  Jones,  embryo s t a r t s d u r i n g  functional  true  H o l l i d a y and  the  acclimation  in their this  in relation  future  out, to  must a l w a y s be  h i s t o r y of  responses to other  s t a t i n g that the  larvae  f a c t o r s of  taken  previous the  plays  factors. experience  environment  i n t o a c c o u n t . The  an Fry of  under  importance  69  of  knowing  the  acclimation history  some e x a m p l e s . K i n n e salinity  of  Girard)  had  desert  pupfish  a  significant  characteristics conversion in  of  the  efficiency  t h e spawning  from  (1962) p r o v i d e d  eggs  that  functional  non-genetic  In o r d e r functional  to to  offspring. hatching  to  this  was  non-genetic t o Kinne  "an e c o l o g i c a l alterations  the  concept  the i n t e n s i t y  capacity  conditions." former  somatic genetic  to  passed  changes  genetic  adaptation,  functional  functional  adaptation,  pattern  the rates or e f f i c i e n c e s  changes  of h a b i t s , while  Kinne  irreversible, salinity. irreversible,  in a relative  of terms, refers  increase  o r compete under  non-genetic  in  in  t h e new  nature,  the  i n t h e r e s p o n s e mechanism o f  called  structural by K i n n e  of metabolism  those  maturity.  of v a r i a b l e s i n t h e i r  t o t h e next  as c i t e d  These  Adaptation  transmitted  and  level.  definition  generation,  i s also  remained arising  an  next  which  comprise  in  changes  on t o t h e not  or  food  adjustments of organisms  s u r v i v e , reproduce  I t may be g e n e t i c  refers  individuals  to  and  fish  an  of  1964b), a r e g i v e n .  environment, which u l t i m a t e l y r e s u l t their  to  some  functional  that  i n those  due  spawning B a i r d and  rates  of s e x u a l  phenomenon c o m p r i s i n g in  the  t o t h e spawning  adaptation,  (1962,  the  salinity  primarily  to understand  on  than  by c i t i n g  macularius  from eggs  another  adaptation  that  Growth  p e r s i s t e d beyond t h e s t a g e  suggested  according  effect  were h i g h e r  transferred  differences  evidence  (Cyprinodon  of f i s h  salinity  may be e x p l a i n e d  latter  generation.  acclimatization, changes. (1962), and  of s t r u c t u r a l  changes of the a r c h i t e c t u r e of c e l l s ,  the  Examples  Nonmay of  i n c l u d e changes of  activity  adaptation  tissues,  to  organs  or  include or  the  70  whole  organism.  reversible response  or  Functional  non-genetic  irreversible,  mechanism  the  latter  which p e r s i s t i n t h e  adaptation  could  be  involving  changes  in  subsequent  life  of  the  individual. The the  above example on  s p a w n i n g medium a f f e c t e d t h e  immediate  environment  properties  tend  salinities. Velsen  This  egg  during with  Pacific  following  and  the the  herring  the  and  (17°/ooS,  one  the  They  adaptation  associated  the  of  a  herring  specific  with  the  approaches  that  osmoregulatory (Holliday, Alderdice the  eggs may  of  be  and  that  yolk  authors may  an  the  induced  of  and  and  eggs one  half  incubated  suggested  have  been which  irreversible,  to  influence in that  "fixed" coincided  membrane p e r m e a b i l i t y  after  non-genetic  observation.  eggs u s e d  in this  experiment,  incubated  in a constant  is,  21  13,  and  and  Jones,  (1978), the  osmotic and  concentration They  after 1965). As  and  ionic  one  half  level  2 9 ° / o o S . When  osmotic  environment.  and  a two  of  different  exposure  t h e y were  The  s h o r t l y before  fixed during  at  continuing  internal  outside  Holliday  Velsen  a  that  their  their  the  capacity  1965; and  shed,  these  that  of A l d e r d i c e  exposure p e r i o d  herring  salinity;  eggs a r e  of  that  t h e y were spawned, f e r t i l i z e d of  had  vitelline  suspected  case  that  6.5°C) f o r a two  7°C).  h a l f hour  shutdown of  was  e g g s . The  fact  osmotic p r o p e r t i e s  two  and  development  fertilization  fertilization.  In  later  (0 - 4 2 ° / o o S ,  conditions  ionic  embryo  conforms, w i t h t h a t  development, d e s p i t e  identical the  on  illustrates  physiological properties  the  observation  salinities  hour p e r i o d  of  to p e r s i s t in  (1978)  different  on  Cyprinodon macularius  have  no  fertilization suggested  by  properties  of  hour  period  71  following ionic  fertilization.  concentrations  spawning  and  resulting  larvae  adaptation  Earlier  their  salinity  range p r o v i d e s  the  of  the  associated larval  stage,  embryos  that  an  intermediate there  different  This observation  optima crab,  a_l. ( 1 9 6 6 ) .  for the  various  of  influence  on  incubation  the  from h i g h e r the  salinity  larvae.  be  salinities. range herring  of  of  eggs  to  temperature to  of  hatching 8.3°C.  be  larval  conforms  This  At  different survival  with  that  in of  several salinity-temperature  z o e a l and  m e g a l o p s l a r v a e of  a  (Gould).  also  (6°C)  was  Larvae  Greater  especially  supra-normal c o n d i t i o n s .  produced  tolerance  a  significant  larvae.  The  favorable to.the  reserves  than  energy  are  is  the those  be  of  lower hatching  advantageous  subjected  could  lower  survival  from  yolk content  when t h e y  Extra  of  hatching  have g r e a t e r y o l k  temperatures.  larvae,  may  Temperature  temperature  may  the  salinity.  Pacific  maximize  found  temperature  resulting  temperatures  to  They  stages  Incubation  Incubation  the  that  Rhithropanopeus h a r r i s i i  2. E f f e c t  embryo and  salinity  in  appears  optima  e_t  work  during  salinity  the  and  ( A l d e r d i c e et a l . , 1 9 7 9 ) .  19°/OOS  salinity-temperature  Costlow  eggs  post-hatching  osmotic  however,  salinities.  to  maximum s u r v i v a l  with  osmotic  present  i n f l u e n c e d the  showed  least  to  13  the  future responses to e x t e r n a l  p r o v i d i n g the  is  that  the  response t o the  embryos  when  by  have  experiments  from  assumed  experienced  in their  with  apparently  is  fertilization  A non-genetic associated  It  t o sub-  required  or in  72  active  ion  regulation  of  tissues  at  abnormal  salinity  condi t ions. From t h e s e salinity larvae  3.  and  low  significant. factor  observed. points  with  salinity  salinities  (6°C)  11,  of  higher  at  at  in  higher  for  the  result  post-hatching  and  tolerance  at  5 0  lower higher  temperatures,  (12°C)  be  ED  at  in  is of  t e m p e r a t u r e can  eggs i n c u b a t e d  salinity  from  eggs are  salinity  t o l e r a n c e was  incubated too  at  were f u r t h e r r e d u c e d  possibility  that  eggs hatching  continue  salinity  were  to  to  from 6 and  a l l showed h i g h e r  12°C  suggest  increase (e.g.,  tolerance  incubated  observed  lower temperature  limited  temperature  they  (1)  interaction  drawn  temperature  would  9°C,  E f f e c t s of  (2)  higher 9 to  12°C  temperatures.  however,  larvae  of  temperature  an  and  salinities  6 t o 9°C  higher  noted, higher  the  incubation tolerance  Combined  contours  tolerance  if  the  post-hatching  salinity  surface  and  result  post-hatching  effect  incubation  tolerance  hatching  high  enhance  T e m p e r a t u r e and  following patterns:  incubated  data,  the  In F i g u r e  temperature  As  temperature  a  However, some i n d i c a t i o n s of  show t h e  eggs  that  Factors  Statistically,  this  appears  salinities.  of P o s t - H a t c h i n g  Three  not  it  incubation  to higher  Effect  the  results,  at  of  if  < 6°C).  larvae  (6°C).  The  salinity  the  incubation  There  i s another  l a r v a e may  a temperature  incubation  salinity  that  in  be near  maximized 9°C.  When  g r o u p s were t e s t e d a t  tolerance,  even  slightly  73  Figure  11  Estimation of upper lethal salinity limits (ED a t 72 h o u r s ) of h e r r i n g l a r v a e exposed to various combinations of salinity 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 . E a c h c u r v e i n t h e two p l o t s a p p r o x i m a t e s t h e e x p e c t e d E D value (°/ooS) a t v a r i o u s l e v e l s of incubation salinity, incubation t e m p e r a t u r e and c u l t u r e t e m p e r a t u r e . 5 0  5 0  75  higher at  than  those  incubation  eggs  at  the  larvae  (e.g.,  t e s t e d a t t h e same t e m p e r a t u r e  6 -> 6 ° C ; 1 2 — >  12°C).  9°C and t e s t i n g o f t h e s a l i n i t y  same  temperature  would  probably  An  as t h a t  incubation  tolerance support  of  of l a r v a e a t this  second  suggestion. To be  maximized  occurred the  of  larval  at  larval  larval  temperature salinity  tolerance  The low/low  apply  the  4.  temperatures,  toward  lower and  i s i n the d i r e c t i o n of absolute  level  i f t h e eggs were i n c u b a t e d  - high/high  tolerance  and t e m p e r a t u r e  to survive  salinity-temperature  of  play  in different  at  herring a greater  does  not  larvae.  The  role  in  the  salinities.  Calorimetry Newly h a t c h e d  temperature  Pacific  combinations  herring  w t . ) . These v a l u e s  spring  spawning  PaffenhSfer 5,940  are  Western  and R o s e n t h a l  c a l / g organic  larvae  from t h e s i x s a l i n i t y -  show mean c a l o r i c  6,245 t o 6,875 c a l / g a s h - f r e e dry  incubation  may  incubation  egg s u r v i v a l t o h a t c h i n g ,  salinity  salinity  capacity  with  levels  9°C when  In a d d i t i o n , t h e  i s increased  associated  incubation  higher  salinity  near  r e s p e c t i v e l y -- t h a t  interaction,  larval  temperatures  of i n c u b a t i o n .  salinities.  in  t o upper  t e m p e r a t u r e may be d i s p l a c e d  temperatures  higher  tolerance  a t 9 ° C . A t l o w e r and  optimal  higher the  summarize,  d r y weight higher  Baltic (1968)  substance  values  (x=6,586 c a l / g  than  those  Sea h e r r i n g obtained  caloric  the P a c i f i c  from  ash-free  obtained  (Clupea  from newly h a t c h e d  l a r v a e . Even a t day'3 a f t e r h a t c h i n g ,  ranging  from  harengus). values  Baltic herring  of  herring larvae  76  still  showed h i g h e r c a l o r i c  measured  at the  content  same age.  l a r v a e may  and  i n t h e amount of endogenous s u p p l y  contain  Eldridge 1.298  and  eggs. P a c i f i c their  due  0.737  larvae at  cal/larva to those  at  day  and  0.216  mg)  mg),  two  whereas,  for Baltic  those  value  herring  caloric  of  values  other  larvae the  Furthermore,  may  species  and  reared caloric  in  day  at  larval  intact  caloric  3,  the  show  were  present  slightly  obtained  0.904  respectively. 21°/ooS  of  in  values could  be  the  Although,  experiments, in  the and  techniques.  both  In  0)  their caloric  s e p a r a t e l y , whereas,  the  a two  performance. contents i n the  of  present  used.  l a r v a e reared at d i f f e r e n t  a  slight  v a l u e s were 0.978 (day  identical  l a r v a e were  v a l u e s of  temperatures From  body  i n the  a _ l . shows  12°C,  calorimetric  not  obtained  these authors  E l d r i d g e et a l . measured and  experiment,  by  et  3 ) . D i f f e r e n c e s i n t h e above c a l o r i c  differences  yolk  larva  Eldridge  0  be  factors.  the  h e r r i n g eggs t o  12.5°C, 23°/ooS and  calorimeters  and  =  of  to contain higher  the v a l u e s o b t a i n e d  used  The  Pacific  = 0.170  by  microbomb c a l o r i m e t e r was  the  energy  c o n t e n t s per  those  experiment,  (day to  weight  caloric and  They r e a r e d  comparison  0.814  of  of  size  (1968) o b t a i n e d a mean c a l o r i c  counterparts  of  experiment  current  contents  e t a l . , 1977).  differences;  and  herring  i n the o r i g i n a l . e g g  weight  h e r r i n g eggs t e n d  Comparison  lower.  (mean egg  Baltic  in c a l o r i c  a l . (1977) f o u n d  (mean egg  Atlantic  (Eldridge  current  et  Rosenthal  0.748 c a l / e g g  than  to the d i f f e r e n c e  cal/egg  PaffenhSfer of  due  the  difference  the  species.  be  The  than  potential data,  influence  from  salinities the  i t i s , however, d i f f i c u l t  two to  77  make v a l i d values  comparisons  of . the  i n some t r e a t m e n t s . T h e s e  a result  effect  development  in  longer. larvae  salinity  may be seen  herring  reared  of  larvae lower  The i n v e r s e was a l s o  (1971)  and  herring  larvae  herring larvae  and  on  salinities  were  salinity  larvae.  (1973) f o u n d t h i s  Holliday  These  i n lower  and  are  Blaxter  due  generally  and l e n g t h o f and  relationship (1960)  authors suggest that  salinities  larval  experiment. Larvae  o b s e r v e d by o t h e r a u t h o r s . A l d e r d i c e  and  been  t h e l e n g t h s and d r y w e i g h t s  r e l a t i o n s h i p between  Dushkina  caloric  pellets.  temperature  i n the c a l o r i m e t r y  temperatures  high  of the l a r v a l  and  by e x a m i n i n g  used  unusually  anomalous v a l u e s may have  of t h e i n c o m p l e t e c o m b u s t i o n  The  of  because  to  Velsen  in Pacific  in  Atlantic  the large  s i z e s of  their  greater  water  (12°C)  showed a  faster  content. Larvae growth The  rate  reared  i n higher temperature  ( l e n g t h ) and w e i g h t  weight  decrease  separate d r y weight had  been  through  taken,  dry low  (12°C,  t h e growth  temperature that  h i g h water  of larvae  of yolk m a t e r i a l  from t h e  29°/ooS)  weights d u r i n g  suggests  than  larvae  an i n d i c a t i o n  measurements o f y o l k  the conversion  Those groups  is  d e c r e a s e from day 0  high  low  the longer  showed g r e a t e r  salinity larvae  could  salinities.  -  9.  body high  tissue observed  tissue. salinity  d r y w e i g h t s and a s h - f r e e  ( d a y 0) t h a n (6°C,  a t lower  c o n t e n t as d r y weights a t lower  those a t high  body  have been  to larval  day  utilization. If  and l a r v a l  temperature  peak o f h a t c h i n g -  of y o l k  to  t h o s e from  13°/OOS)  groups.  salinities salinities  the This  reflect were  a  lower  78  5.  Ecological In  Implications  British  observed Hoar,  in s a l i n i t i e s  1953;  occurring larvae  Columbia  waters,  ranging  of  24  found  in  the  than  the  the  range  of  capacity salinities  temperature.  This  - high/high  influenced 1971).  egg  Herring  temperature in  supported  by  temperature production  in  temperatures the  hatch  the  at  spring  to  be  sea the  the  will  abundance of  and  -  high/high larval be  food  in  salinitythen  the  ecologically  however, was  the  increase could  a  Velsen,  increase  stage,  experiment.  likely  high  temperature  spring  to  that  f i n d i n g that  the  advantageous  notably  post-hatching  and  hypothesis,  current  are  same  tolerate  by  would  the  hypothesized  (Alderdice  the  ground  19°/OOS)  laboratory  low/low  larvae. This  still  and  the  during  occurs  r e s u l t s of  may  larvae  to h a t c h i n g  during  the  the  to  influenced  on  similar  temperature  Hatched  spawning  i n t e r a c t i o n between s a l i n i t y  if a  to  be  based  larvae  10°C.  experience  (13  and  spawning  These s a l i n i t i e s  herring  may  interaction  advantageous  the  is  the  work f o r embryos. I t was  development  t e m p e r a t u r e , and  rise  was  of  salinities  for  environmental  7 to  presumably  incubation.  l e a s t osmotic  the  29°/ooS at  vicinity  as  those at  to  herring  (McMynn  in s a l i n i t i e s  salinities  low/low  29°/ooS  a l . , 1979), w i t h most of  1962), where t h e y  that  to  et  (Stevenson,  provides  from 8  Pacific  Alderdice  are  higher  spawning o f  be  The  not  rising  larvae. at  Food  rising  b e n e f i c i a l to  larvae. The  second h y p o t h e s i s  incubation supported  could by  the  that  the  salinity  influence  the  salinity  results  of  the  and  temperature  tolerance  present  of  of  larvae  experiment.  is  High  79  incubation  salinity  survival  to  high  importance. and  likely  concentration. early to  egg  believed  to  that  the  herring  experience  The  non-genetic subjected be  not  to apply to  (29°/oo)  salinity  the  regarding  lowest  the  may  be  i n the  during embryos  which  occurs,  The  is then  in  the  adaptation  possible  in nature.  are  osmotic  the  salinities  the  incubation  development to  the  mechanisms t o l e r a n c e of  i n f l u e n c e d by  other  e x p e r i m e n t , but  at  in  temperature  ( A l d e r d i c e and  may  at  lower Yet,  the  -  high/high  and  salinity  Velsen,  larval  salinities  was  found  (6°C)  tolerance  series  near of  salinities  s t r e s s . The  of  1971)  stage.  does  Greatest in  larvae  temperatures  i t i s recognized  salinities  larvae at  osmotic  low/low  following  incubated  greatest  Hence  by  adaptation,  of  considered  groups t e s t e d , s a l i n i t y  conditions. under  one  salinities  salinities.  examined  high  salinities  adaptation  high  utilize  supranormal  from eggs  was  of  they  n a t u r a l environment.  f o r egg  incubation  fertilized,  irreversible,  the  o n l y as  f a c t o r s not  suggested  hatching  spawned,  media  this  and  to  to higher  between  resistance  ecological  able to s u r v i v e . This non-genetic  hypothesis  appear  i n an  c o n d i t i o n . . If  i n the  interaction  larvae  result  h e r r i n g embryos may  The  same  larval  of  e x p o s u r e of embryos t o h i g h  i s suggested  larvae  higher  h e r r i n g eggs a r e  be  the  may  perhaps act  could  to  be  environmental  This  favor  m e d i a . When l a r v a e h a t c h ,  larvae  salinity  i n c u b a t i o n temperature  i n high osmotic  osmotic  environment to  the  d e v e l o p m e n t may  the h i g h  herring  low  salinities.  In n a t u r e ,  incubated  more  and  of  that  the  20°/oo,  and in a l l  resulting the  lowest  post-hatching  test  20°/oo may  increase in  have been  resistance  to  80  higher  salinities  salinity shift  in  of  for  eggs  (29°/oo) t h e r e f o r e may the  adaptation one  noted  of  range  shifting  increased  have r e s u l t e d  of s a l i n i t y  t h e egg  and  the  incubation  range  of  salinity.  non-genetic  examination  lethal  salinity  l e v e l s and Precht's  may  Further  incipient  resistance adaptation.  adaptive  t o l e r a n c e upward  examination  by  an  salinity  involve  suggested  from  the h i g h e s t  embryo t o i n c u b a t i o n s a l i n i t y  would  v a r y as  at  t o l e r a n c e . 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