HELGO~ER MEERESUNTERSUCHUNGEN Helgol~inder Meeresunters. 38, 201-206 (1984)

Haemoglobin polymorphism in G a d u s m o r h u a : Genotypic differences in haematocrit* J. M o r k & G. S u n d n e s Biological Station; N-7000 Trondheim, Norway

ABSTRACT: Haematocrit (erythrocyte/plasma ratio) values of the three common HbI genotypes were recorded in two samples totalling 149 specimens of Atlantic cod (Gadus morhua L.) caught in the Trondheimsfjord, Norway, during March-April 1984. The haematocrit values were shown to depend on both HbI genotype and sex; the females revealed higher average haematocrit values than males, and among the males the HbI-22 genotypes displayed higher average haematocrit values than the two other HbI genotypes.

INTRODUCTION The h a e m o g l o b i n s of the A t l a n t i c cod (Gadus morhua L.) are polymorphic. The three most c o m m o n p h e n o t y p e s , as r e v e a l e d b y electrophoresis, r e p r e s e n t c o m b i n a t i o n s of two alleles at one a u t o s o m a l locus, d e s i g n a t e d /-/bI b y Sick (1961). T h e s e two a l l e l e s are p r e s e n t i n virtually all c u r r e n t stocks of cod, b u t their relative proportions m a y differ considerably, e v e n b e t w e e n a d j a c e n t stocks ( F r y d e n b e r g et al., 1965; Sick, 1965a, b). Generally, however, there is a c l i n a l r e d u c t i o n i n the f r e q u e n c y of the HbI-I allele with i n c r e a s i n g latitude on both sides of the A t l a n t i c as well as i n the Baltic Sea. K i r p i c h n i k o v (1981) c o n s i d e r e d the clines a l o n g the N o r w e g i a n coast a n d i n the Baltic, a n d c o n c l u d e d that they were p r o b a b l y s u p p o r t e d b y e n v i r o n m e n t a l selection. A possible physiological basis for such a selection was p r o v i d e d b y the study of Karpov & Novikov (1980). T h e i r results from a study of o x y g e n disassociation curves u s i n g erythrocytes from the three most c o m m o n HbIgenotypes s u g g e s t that t e m p e r a t u r e m a y be a p o t e n t selection factor; the HbI-22 m o l e c u l e is b y far the most efficient o x y g e n carrier at low t e m p e r a t u r e s w h i l e the HbI-11 m o l e c u l e has similar a d v a n t a g e s at h i g h e r temperatures. Red cells from the heterozygote HbI-12 (both m o l e c u l e s i n e q u a l proportions) were consistently i n t e r m e d i a t e i n p e r f o r m a n c e (Karpov & Novikov, 1980). Water t e m p e r a t u r e s g e n e r a l l y d e c l i n e South-North on the n o r t h e r n h e m i s p h e r e ; thus the observed South-North i n c r e a s e i n the f r e q u e n c y of the HbI-2 allele m i g h t reflect the better p e r f o r m a n c e of the HbI-22 m o l e c u l e i n cold e n v i r o n m e n t s . S u b s e q u e n t studies o n N o r w e g i a n coastal cod h a v e r e v e a l e d results w h i c h s e e m to be biological m a n i f e s t a t i o n s of a strong e n v i r o n m e n t a l s e l e c t i o n a c t i n g o n cod h a e m o g lobin genotypes. Thus Mork et al. (1983; 1984a, b) reported o b s e r v a t i o n s o n /-/hi genotypic differences i n growth, mortality, age at maturation, a n d w i t h i n - s e a s o n g o n a d " Contribution No. 211, Biological Station, N-7000 Trondheim, Norway © Biologische Anstalt Helgoland, Hamburg

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d e v e l o p m e n t . As j u d g e d from the o b s e r v e d r a n k of g e n o t y p e s in those comparisons, the /-/bI-2-possessing g e n o t y p e s w e r e b e s t fitted for t h e e n v i r o n m e n t on t h e a c t u a l s a m p l i n g locations. A m o n g b o t h m a l e a n d f e m a l e cod the HbI-11 h o m o z y g o t e s w e r e shown to be c o n s i s t e n t l y inferior w i t h r e s p e c t to growth; a m o n g m a l e s the HbI-11 s p e c i m e n s were also inferior w i t h r e s p e c t to a g e for s e x u a l maturation, a n d w i t h i n - s e a s o n g o n a d d e v e l o p m e n t r a t e s IMorK et al., 1983; 19t~4a, D}. As p o i n t e d out b y M o r k et al. (1983} all t h e s e differing traits (cf. above) in the H b I g e n o t y p e s a r e r e l a t e d to g e n e r a l b o d y growth. Recently, w e h a v e e x p a n d e d our routine h a n d l i n g of H b I g e n o t y p e d cod s p e c i m e n s to i n c l u d e m e a s u r e m e n t s of h a e m a t o c r i t ( e r y t h r o c y t e / p l a s m a ratio in blood}, w h i c h can g i v e v a l u a b l e i n f o r m a t i o n on the g e n e r a l p h y s i o l o g i c a l c o n d i t i o n of i n d i v i d u a l s . T h e p r e s e n t p a p e r d e a l s w i t h h a e m a t o c r i t observ a t i o n s in two s a m p l e s of cod c a u g h t d u r i n g M a r c h - A p r i l 1984 in the Trondheimsfjord, Norway. MATERIAL AND METHODS Two s a m p l e s , t a k e n at two different t i m e s a n d locations in the Trondheimsfjord, N o r w a y , w e r e a n a l y z e d . S a m p l e 1, c o n t a i n i n g 58 m a l e a n d 25 f e m a l e cod, w e r e t a k e n b y s h r i m p t r a w l a n d l u r e at 5 5 - 1 0 5 m d e p t h at the m a i n cod s p a w n i n g p l a c e (location 1) in the T r o n d h e i m s f j o r d on M a r c h 27, 1984. This s a m p l e w a s h e t e r o g e n e o u s with r e s p e c t to s e x u a l m a t u r a t i o n , size (27-97 cm), a n d a g e (not r e c o r d e d ; d e d u c e d from size composition}. S a m p l e 2 w a s t a k e n b y s h r i m p t r a w l on A p r i l 27, 1984, in a s h a l l o w (max d e p t h 40 m} s i d e - a r m (location 2) of the m a i n fjord. It c o n s i s t e d of 33 i m m a t u r e s p e c i m e n s of e a c h sex. T h e y w e r e all 1 y e a r of a g e as d e t e r m i n e d b y otolith r e a d i n g s a c c o r d i n g to Rollefsen (1933}. T h e two s a m p l i n g l o c a t i o n s differ c o n s i d e r a b l y w i t h r e s p e c t to h y d r o g r a p h y . Location 1 (Verrabotn) is p a r t of the m a i n fjord with its e s t u a r i n e t y p e of h y d r o g r a p h y i n c l u d i n g r e l a t i v e l y s t a b l e d e e p - w a t e r t e m p e r a t u r e s {6-8 °C} t h r o u g h o u t the y e a r (Jacobson, 1983). Location 2 (Borgenfjord) is q u i t e different. D u e to e x t e n s i v e w a t e r t u r b u l e n c e set u p b y strong t i d a l c u r r e n t s t h r o u g h a n a r r o w a n d s h a l l o w entrance, the w a t e r m a s s e s in this s i d e - f j o r d are n e a r l y m e t a - s t a b l e at all t i m e s of the year; t e m p e r a ture a n d s a l i n i t y d i s p l a y h o m o g e n e o u s v a l u e s from surface to bottom. Thus the cod p r e s e n t t h e r e w i l l e x p e r i e n c e t e m p e r a t u r e s w h i c h n o r m a l l y r a n g e from 0 - 2 °C in w i n t e r to 16-20 °C in s u m m e r (Gulliksen, 1972). T a g g i n g e x p e r i m e n t s (Mork, unpublished} h a v e not i n d i c a t e d a n n u a l m i g r a t i o n s to a n d from l o c a t i o n 2; the cod is p r e s e n t there t h r o u g h o u t the w h o l e y e a r (Denstadli, 1972). Blood s a m p l e s for h a e m a t o c r i t d e t e r m i n a t i o n a n d h a e m o g l o b i n g e n o t y p i n g w e r e o b t a i n e d b y h e a r t p u n c t u r e . H a e m o g l o b i n (HbI) g e n o t y p i n g b y a g a r g e l e l e c t r o p h o r e s i s f o l l o w e d the a n a l y t i c a l p r o c e d u r e s a n d g e n e t i c n o m e n c l a t u r e of Sick (1961). H a e m a t o c tit v a l u e s w e r e m e a s u r e d after c e n t r i f u g i n g w h o l e b l o o d in h e p a r i n i z e d t u b e s for 5 min on a s t a n d a r d micro h a e m a t o c r i t centrifuge. RESULTS AND DISCUSSION Haematocrit values differed significantly between HbI genotypes. Both Hbl-11 and HbI-12 specimens displayed relative anemia compared to the Hbl-22 specimens in our

Haemoglobin

polymorphism

in cod

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s a m p l e s t a k e n i n t h e c o l d s e a s o n of t h e y e a r . T h i s r e s u l t , w h i c h w a s s t a t i s t i c a l l y s i g n i f i c a n t i n S a m p l e 1, w a s c o n f i r m e d b y a d d i t i o n a l s a m p l i n g ( S a m p l e 2). I n b o t h samples, the Hblgenotype effect on haematocrit was detected in males only. In Sample 2 the average haematocrit was higher in females than in males. Details from the analyses of t h e t w o s a m p l e s a r e d i s c u s s e d b e l o w . S a m p l e 1. A s t a t i s t i c a l l y s i g n i f i c a n t h e t e r o g e n e i t y of h a e m a t o c r i t for t h e H b l g e n o t y p e s i n p o o l e d s e x e s ( 1 - f a c t o r a n a l y s i s of v a r i a n c e : F (2,80) ---- 4.799, : P --- 0.011) w a s s h o w n to b e d u e m a i n l y t o t h e g e n o t y p i c v a r i a t i o n of h a e m a t o c r i t i n m a l e s , w h i l e n o h e t e r o g e n e i t y w a s d e t e c t e d i n f e m a l e s ( T a b l e 1). I n m a l e s , N e w m a n - K e u l s t e s t s r e v e a l e d a significant difference between HbI-22 and the other genotypes. There was no detecta b l e e f f e c t of s e x o n h a e m a t o c r i t i n S a m p l e 1. Table 1. Gadus morhua. M e a n haematocrit of H b I genotypes in S a m p l e 1. Results from 1-factor analyses of variance, a n d N e w m a n - K e u l s test procedures for differences b e t w e e n m e a n s are g i v e n

No. of specimens M e a n haematocrit Standard deviation of m e a n

HbI-11

Males HbI-12

I--IbI-22

Females I--IbI-11 HbI-12

HbI-22

18 0.2733 0.0398

28 0.2635 0.0500

12 0.3302 0.0655

5 0.2708 0.0572

5 0.2944 0.0867

15 0.2900 0.0608

Males: F (2,55) = 7.5052, P = 0.0013, a n d HbI-11 = HbI-I2 • HbI-22 Females: F (2,22} = 0.1995, P = 0.8206

S a m p l e 2. A s i n S a m p l e 1, a s i g n i f i c a n t H b I g e n o t y p i c h e t e r o g e n e i t y of m e a n h a e m a t o c r i t i n p o o l e d s e x e s ( 1 - f a c t o r a n a l y s i s of v a r i a n c e : F (2,63) = 3.816, P = 0.027) w a s s h o w n to b e c a u s e d m a i n l y b y t h e m a l e p a r t of t h e s a m p l e ( T a b l e 2). I n m a l e s , a m u l t i p l e r a n g e t e s t ( N e w r n a n - K e u l s ) s h o w e d t h a t t h e m e a n h a e m a t o c r i t of H b I - 1 2 s p e c i m e n s w a s s i g n i f i c a n t l y (P < 0.05) l o w e r t h a n t h a t of H b I - 2 2 s p e c i m e n s , w h i l e t h e m e a n of H b I - l l g e n o t y p e s d i d n o t d i f f e r s i g n i f i c a n t l y f r o m e i t h e r of t h e t w o o t h e r s . I n a d d i t i o n to t h e c o m p o n e n t of t o t a l v a r i a t i o n c o n t r i b u t e d b y H b l g e n o t y p e , t h e r e w a s a l s o a c o m p o n e n t c a u s e d b y s e x ( T a b l e 3). Table 2. Gadus morhua, M e a n haematocrit of H b l genotypes in Sample 2. Results from 1-factor analyses of variance are given. Cf text for results from N e w m a n - K e u l s test for differences b e t w e e n means

No. of specimens M e a n haematocrit Standard deviation of m e a n

HbI-11

Males HbI-12

Hbl-22

Hbl-ll

Females I-Ibl-12

Hbl-22

6 0.3087 0.0323

14 0.2785 0.0514

13 0.3402 0.0453

4 0.3668 0.0544

19 0,3224 0.0566

10 0.3411 0.0335

Males: P (2,30) = 6.002, P = 0.006 Females: F (2,30) = 1.439, P = 0.253

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Table 3. G a d u s morhua. Bffect of H b l g e n o t y p e and sex on haematocrit in Sample 2 {age: 1 year}. Summary of results from 2-factor analysis of variance Source of variation

Sum of squares

d.f.

Mean square

F-value

A HbI genotype B Sex A °B Interaction Within {error)

0.02615 0.01410 0.00778 0.14770

2 I 2 60

0.01308 0,01410 0.00389 0.00246

5.312 5.729 1.581

0.19573

65

Total

Factor A: P = 0.008, Factor B: P = 0.020, A ' B : P = 0.214

Both in n o m i n a l r a n k i n g a n d in t e r m s of t h e s t a t i s t i c a l s i g n i f i c a n c e of t h e H b I genotypic haematocrit differences, Sample 2 confirmed the observations in Sample 1 ( T a b l e s 1 a n d 2). A p p a r e n t l y thus, t h e d i f f e r e n t h y d r o g r a p h i c a l c o n d i t i o n s on t h e two s a m p l i n g l o c a t i o n s d i d n o t c a u s e a c h a n g e in t h e m a i n p a t t e r n of H b l g e n o t y p i c effects o n h a e m a t o c r i t . T h e o v e r a l l , j o i n t r e s u l t in t h e t w o s a m p l e s is t h a t t h e m a l e H b I - 2 2 specimens on average have higher haematocrit than the two other genotype groups. T h i s r e s u l t is i n t e r e s t i n g in l i g h t of t h e fact t h a t s u p e r i o r i t y of t h e m a l e H b I - 2 2 i n d i v i d u a l s h a s b e e n t h e m a i n trait a l s o i n p r e v i o u s i n v e s t i g a t i o n s c o n s i d e r i n g e.g. g r o w t h rates, s e x u a l m a t u r a t i o n , a n d w i t h i n - s e a s o n g o n a d m a t u r a t i o n ( M o r k et al,, 19837 1984a, b). In fact, a c h e c k of t h e m e a n l e n g t h s for H b l g e n o t y p e s in S a m p l e 2 c o n f i r m e d t h o s e p r e v i o u s r e s u l t s in t h a t t h e m a l e H b I - 2 2 g e n o t y p e w a s s u p e r i o r in this s a m p l e , too ( T a b l e 4). Table 4. G a d u s m o r h u a . Mean lengths of H b l g e n o t y p e s (age: 1 year) in Sample 2. Results from 1factor analyses of variance, and Newman-Keuls test procedures for differences between means are given Males

No. of specimens Mean length Standard deviation of m e a n

Females

HbI-11

HbI-12

HbI-22

HbI-1I

HbI-12

Hbl-22

6 13.85 2.75

14 14.08 2.15

13 17.51 3.22

4 14.00 1.91

19 15.54 3.05

10 15.85 3.46

Males: F (2,30) = 6.505, P = 0.005, and I--IbI-11 = H b I - 1 2 4= 147o1-22 Females: F {2,30} = 0.532, P = 0.593

It c o u l d b e t h a t t h e h i g h e r h a e m a t o c r i t v a l u e of m a l e H b I - 2 2 s p e c i m e n s w a s c a u s e d b y a c o v a r i a t i o n b e t w e e n l e n g t h a n d h a e m a t o c r i t ~ i.e. i n c r e a s i n g h a e m a t o c r i t w i t h i n c r e a s i n g l e n g t h . W e t e s t e d this p o s s i b i l i t y b y c o n s i d e r i n g t h e c o r r e l a t i o n c o e f f i c i e n t s (r} b e t w e e n b o d y l e n g t h a n d h a e m a t o c r i t for all 12 c o m b i n a t i o n s of s e x a n d / - / b l g e n o t y p e in t h e t w o s a m p l e s . H o w e v e r , n o n e of t h e s e r - v a l u e s w e r e s i g n i f i c a n t l y d i f f e r e n t from zero. S i n c e n o / - / b l g e n o t y p i c h e t e r o g e n e i t y of h a e m a t o c r i t h a d b e e n o b s e r v e d in f e m a l e s

H a e m o g l o b i n p o l y m o r p h i s m i n cod

205

we were justified i n p o o l i n g HbI groups i n this sex to e n h a n c e test power. D o i n g so in Sample 1, the overall (pooled HbI g e n o t y p e groups) correlation coefficient b e t w e e n l e n g t h a n d haematocrit i n f e m a l e s was --0.159, w h i c h is not s i g n i f i c a n t l y different from zero (t ---- --0.774, df = 23, P ---- 0.447}. A n y w a y , if the " l e n g t h effect" h a d b e e n significant w e w o u l d not h a v e b e e n a b l e to d i s c r i m i n a t e b e t w e e n the effects of a g e a n d l e n g t h i n this sample. This p r o b l e m did not exist i n S a m p l e 2, i n w h i c h the s p e c i m e n s w e r e aged. In this sample the overall (33 s p e c i m e n s ; pooled HbI groups) r - v a l u e for the correlation b e t w e e n l e n g t h a n d h a e m a t o c r i t i n f e m a l e s w a s 0.042 w h i c h is not s i g n i f i c a n t l y different from zero (t = 0.234, df = 31, P ----0.816). C o n s i d e r i n g both m a l e a n d female HbIgroups, the 6 calculated r-values in S a m p l e 2 r a n g e d b e t w e e n - 0 . 0 1 8 (female HbI-11) a n d 0.416 (male HbI-22}. N o n e of them were s i g n i f i c a n t l y different from zero, a n d as tested b y the chi-square p r o c e d u r e of Sokal & Rohlf (1981; p. 588) there was no s i g n i f i c a n t h e t e r o g e n e i t y a m o n g these v a l u e s (X2 = 1.823, df = 5, P = 0.873}. Thus, as far as these test results can express, the h a e m a t o c r i t v a l u e s m a y b e r e g a r d e d as i n d e p e n d e n t of b o d y length, a n d the o b s e r v e d H b I g e n o t y p i c differences i n h a e m a t o c r i t is therefore not likely to be just a side-effect of size differences. Instead, it m a y b e a s s u m e d that i n m a l e s the I--lbi-22 s p e c i m e n s t e n d to have h i g h e r h a e m a t o c r i t t h a n the two other H b I g e n o t y p e s regardless of i n d i v i d u a l size. At p r e s e n t we do not have sufficient i n f o r m a t i o n for d r a w i n g c o n c l u s i o n s on the causal relationships b e h i n d the o b s e r v e d HbI g e n o t y p i c h a e m a t o c r i t differences. Formally, the m a l e HbI-II and HbI-12 g e n o t y p e s suffer from relative a n e m i a c o m p a r e d to the HbI-22 g e n o t y p e s i n the p r e s e n t samples. This could, or could not, be as s i m p l e as a n iron deficiency a n e m i a , b u t the e x p l a n a t i o n m a y b e more complex. If, as seems p l a u s ible, the haematocrit differences are r e l a t e d to the t e m p e r a t u r e - d e p e n d e n t f u n c t i o n a l properties of the h a e m o g l o b i n s , there m i g h t b e a s e a s o n a l (temperature} v a r i a t i o n c o m p o n e n t of c u r r e n t u n k n o w n m a g n i t u d e . Additionally, it is u n c e r t a i n to what d e g r e e haematocrit reflects c u r r e n t or past e n v i r o n m e n t effects since, for e x a m p l e , the t u r n o v e r rate of cod erythrocytes is not k n o w n . O n e s h o u l d also be a w a r e that c o m p e n s a t o r y h a e m o g l o b i n p r o d u c t i o n (cf. h i g h a l t i t u d e a d a p t i o n i n h u m a n s ) m a y t a k e place, a n d t e n d to m a s k causal relationships. Finally: it is k n o w n that the proper structure of the h a e m o g l o b i n m o l e c u l e c a n affect erythrocyte shape, a n d t h e r e b y h a e m a t o c r i t (cf. the sickle-cells i n humans). However, since the /-/bI effect o n h a e m a t o c r i t s e e m s to b e restricted to m a l e s p e c i m e n s i n the p r e s e n t samples, such effects are not i n d i c a t e d a l t h o u g h formally they r e m a i n to b e studied, e.g. microscopically. A l t h o u g h s a m p l i n g area (despite h y d r o g r a p h i c a l differences) did n o t a p p e a r to affect the m a i n results s u b s t a n t i a l l y for the p r e s e n t two s a m p l e s from the s a m e fjord, it seems r e a s o n a b l e to expect that the nature, a n d d e t e c t a b i l i t y of H b l g e n o t y p i c effects o n haematocrit will d e p e n d on the e n v i r o n m e n t to w h i c h cod s p e c i m e n s h a v e b e e n exposed. The effects m i g h t b e q u i t e different i n different r e g i o n s i n the d i s t r i b u t i o n range. The p r e s e n t location 2 displays a n e x t e n s i v e a n n u a l t e m p e r a t u r e cyclus. Probably, a s a m p l i n g p r o g r a m w h i c h covered this location at m u l t i p l e times t h r o u g h the year could indicate w h e t h e r or not season plays a role for the actual effect of H b l g e n o t y p e on haematocrit. It w o u l d also b e d e s i r a b l e to record a d d i t i o n a l p a r a m e t e r s from each b l o o d sample (e.g. cell count, a v e r a g e cell size, s i n g l e cell haematocrit) i n order to study potential c o m p e n s a t o r y effects.

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For the present, we will refrain from extensive speculations on the physiological r e a l i t i e s b e h i n d o u r o b s e r v a t i o n s . T h e m o s t i m p o r t a n t r e s u l t h e r e is t h e a c t u a l d e m o n s t r a t i o n t h a t h a e m o g l o b i n g e n o t y p e c a n a f f e c t i n d i v i d u a l s p h y s i o l o g i c a l l y to t h e d e g r e e t h a t t h e i r h a e m a t o c r i t v a l u e s b e c o m e d i f f e r e n t . It is l i k e l y t h a t t h i s m a y u n d e r c e r t a i n circumstances affect their physical performance and fitness. B o t h w i t h r e s p e c t to t h e s e x u a l d i f f e r e n c e i n m a n i f e s t a t i o n , a n d t h e i n t r i n s i c r a n k of H b I g e n o t y p e s t h e h a e m a t o c r i t r e s u l t s r e s e m b l e s t h e p a t t e r n s o b s e r v e d i n s t u d i e s ol growth rates, age at maturation, and within-season gonad maturation in cod from the s a m e a r e a s ( M o r k e t al., 1983; 1984a, b); t h e H b / e f f e c t is m o s t p r e d o m i n a n t i n m a l e s , a n d (at t h e t i m e of s a m p l e c o l l e c t i o n } t h e m a l e HbI-22 g e n o t y p e s e e m s s u p e r i o r to t h e t w o others. Together with those previous studies, the present results demonstrate the prof o u n d e f f e c t t h a t H b I g e n o t y p e c a n h a v e o n t h e a d a p t i v e p r o p e r t i e s of i n d i v i d u a l cod.

Acknowledgements. We greatly appreciate the w h o l e - h e a r t e d effort by C a p t a i n B, Soreng, and crew a n d assistants H. Hansen, P. Pedersen, a n d H. Folstad on b o a r d R. V. Harry Borthen I during s a m p l e collections. This study was supported b y grants from the N o r w e g i a n Fisheries Research Council (I 309.07), a n d from Nansenfondet,

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