Hemoglobin polymorphism in the Atlantic croaker, Micropogon undulatus

Cump. Biochem. Physlol. Vol. l18A, No. 4, pp. 1419-1428, Copyright 0 1997 Elsevier Science Inc. All nghts reserved.

ISSN 0300.9629/97/$17.00 I’11 SO300-9629(97)00029-7

ELSEVIER

Hemoglobin Polymorphism in the Atlantic Croaker, Micropogon undulatus * and

Daniel A. Shelly

Charlotte P. Mangum

DEPARTMENTOF BIOLOGY, COLLEGE OF WILLIAM & MARY, WILLIAMSBURG, VA 23187-8795,

Ten hemoglobins

ABSTRACT.

gel electrophoresis.

confirmed

a polymorphic

condition,

variation

was unrelated

to body length,

mals. The intraspecific 10 hemoglobins infrequent,

from the teleost Micropogon undulates were separated in native polyacrylamide

In a large sample taken from a tributary of the Chesapeake

(If these hemoglobins

were expressed

in seven different

no single phenotype

Several phenotypes

comprised

differences

dialyzed hemolysates, 1 lBA;4:1419-1428, KEY

WORDS.

reaches

polymorphism,

species,

hemoglobins

its maximum

(Hbs)

0:

among

the teleost

or polymorphism,

salmonid

various

fishes,

in

Hbs (11). a species,

in this group [for

that

rn one salmonid

gradual,

and

Therefore,

they

to reproductive

the

and a clupeoid

continue and

after

was more closely state.

differs

concluded to growth

In his 1986 review,

docu-

of phenotypic

maturation

related

and in

careful

the changes

lles (19)

is ontoge-

in adults

chronology

Specifically,

Wilkens

typic change

found

stages (6). In addition,

indicates

maturation.

the polymorphism

Hb phenotypes

developmental

mentation shifts

species,

with distinct

sexual

is also widespread

mul-

see (8,18,19)].

In several netic,

which

from that

of

are far more is complete. that

pheno-

per se than

Wilkens

several morphs were 1s quite great.

several North Carolina

pheis

stripped of co-factors

Bay morphs. The most variable were also ohserved.

and cooperativity

may he involved

(18) ap-

Address rrprrnt requests to: C. P. Mangum, Department of Biology, College of Willlam & Mary, Williamsburg, VA 23187.8795, U.S.A. Tel. (804) 22l-222612229; Fax (804) 221-6483; E- mail: [email protected]. *Present address: Department of Biological Sciences, Florida State University, Tallahassee, FL 32306, U.S.A. Recewed 24 September 1996; revised 10 February 1997; accepted 26 February 1997.

as well. COMP BICK’HEMPHYSIOI.

Inc.

binding,

are often

form. Molecular

which a species may have as many as 25 different Multiplicity is a prerequisite for variation within reviews,

in pH dependence

fish

peared

a vertebrate

Although

that the variation

animals and, conversely,

suggesting that organic PO, co-factors

found in more than a single molecular tiplicity

banding patterns.

also differed. These differences were not identical to those between

INTRODUCTION

Within

The

between some, but not all, Chesapeake

1997. 0 1997 Elsevier Science Hemoglobin,

ani-

that it is an adult phenomenon.

alone. The 01 binding of hemolysates

a few differences

The 02 binding of red blood cell preparations

the distribution

known in North Grolina

Bay sample. This finding suggests that the polymorphism

than observed in either investigation

property was 02 affinity, although

indicating

phenotypic

differed from those in North Carolina

hy dialysis revealed functional

Bay in Virginia,

whrch was previously

a majority of the sample, indicating

notypes were nor recovered from the Chesapeake more complex

U.S.A.

to regard

ontogenetic. ample

all intraspecific

In his investigation

information

the persistence

indicated

of change

variation

in salmonids

of sprat (18), however, that

it was not.

well into adulthood

as the

Regardless, means

that

data on body size and maturity must be available to decide whether the polymorphism is or is not related to life history. To our knowledge,

this information

adult teleost

than

other

those

is not available

investigated

for any

by Wilkens

and

lles (19). The functional morphism

significance

has been

of Hh multiplicity

the subject

of much

with no clear resolution (8). Several attempts the degree of multiplicity with environmental met with little success. To our knowledge, tion of this kind has not been demonstrated protein

and poly-

discussion,

albeit

to correlate instability

a crude correlafor any multiple

system.

The functional lar structure electrophoretic

consequences

are also uncertain. phenotypes

of the variation Purified

characteristic

in molecu-

Hbs expressing of adults

the

and de-

velopmental stages differ in 0: binding (5,8). The 0; binding of different Hb phenotypes found only in adults, whether related to growth or not, is not known. Differences in the 0: binding of isolated components of multiple Hb systems in two salmonids are well known (2-4,12,17). But, in one investigation, the inbred strain examined was de-

1420

D. A. Shelly and C. P. Mangum

scribed

as monomorphic

in the other, not mentioned

stages the

investigation,

of a teleost.

in our sample

Wilkens

and

Iles

which advice

in

the Oz binding

proved

Micropogon

We a

was

the

(Linnaeus),

available

for both

structural

by

characterized

population,

and

the Atlantic

proved

is sufficiently different

croaker on the

to be correct. great

In

that

ample

phenotypes

were

and functional

MATERIALS

characteriza-

AND

METHODS

Collection and Care of Animals In the summer by otter

of 1992,

trawl from

the Chesapeake additional

we

of all but one morph,

Family Sciaenidae,

which

expressing

investigated

quantitatively

properties

variation

of material

the Hbs in a of life history

to that

natural

of J. Bonaventura,

quantities tion.

distribution

to be rare. We chose

undulatus

this species,

variation

we examined

The

was similar

(19).

polymorphism

examined

in the adult stage;

of intraspecific

(17).

In the present large sample

(3), presumably

the possibility

150 adult

animals

the York River

Bay in Virginia;

83 animals

were collected

estuary,

a tributary

in the summer

of

of 1993, an

were collected.

In 1992, the structural

Hb phenotype

alone

was deter-

mined, using native PAGE. Fish were placed in rapidly running sea water while still on board and then transferred on land to rapidly running ity). Within

sea water (23-28”C,

24 hr of capture,

l&24%0

blood was taken

rinized syringe from a caudal vessel of anesthetized animals occasion, laboratory

and then, however,

in general,

from August

(MS222)

the fish was released.

26 animals

were

to December

tagged,

salin-

into a hepa-

held

and sampled

On one in the several

FIG. 1. (Top) Photographs of the seven Hb phenotypes, designated A-G. Each phenotype is shown in a higher (left of each pair of lanes) and lower (right) Hb concentration. The repetition of phenotype A on each of the four gels allows precise comparison of all phenotypes. (Bottom) Diagrammatic representation of the seven phenotypes, designated A-G. The height of the bands (Hbs l- 10) represents relative densities of Coomassie Blue. The Coomassie Blue-positive material in the top panel, which is not reproduced here, was not heme positive.

Fish Hemoglobin

times.

During

the holding

from 26 to PC, In 1993, both These

period,

whereas

and PO2 (147-149 mined.

1421

Polymorphisms

Torr)

changed

Hb phenotype

animals

the temperature

the salinity

dropped

(19-22%0),

(15). Again,

pH (7.8)

sufficient

very little.

that from other

and OL binding

were deter-

morphs

were tagged and brought

to the labo-

single

ratory, where they were held in vigorously aerated recirculating sea water (2O”C, 2 1%0) for as long as 12 weeks. There was ml mortality

during

the holding

material

numbers

individuals

available

A, B and C) was pooled

individuals

expressing

D, E and F are represented individual

Data

period.

from several

(morphs

in with

the same phenotype; in these

data by only a

each.

Analysis

The frequencies of body lengths of the Hh phenotypes and of each Hb hand were analyzed by contingency tests of indeElectrophoresis

The

pendence,

heparinized

blood

was centrifuged

the serum was discarded. cells (RBCs)

determined

(Sigma Technical sate was dialyzed

1) with

After huhhling native

and with

0.04 mol/l

abandoned.

mmol/heme

EDTA;

however,

hemolysate

Binding

step was 1

specified

with

with CO, non-disunder the conditions

by Hames

and Rickwood

only with Coomassie

of the phenotypes,

Oxygen

it did

to 5 ,ug/,ul ( -0.3

multiple

examples

3,3,5,5,-tetramethyl

Blue. After of each were

henzidine

were obtained

range

30-70X

plots.

Because

tit to the that

values,

iteratively

(0.5

between

0: affmity

the relationship gression

lines.

The

below

Bohr

continned the dam into

between

them.

line tit hy ANCOVA.

of the regression

where

in the as Bohr

lines were

procedure

by subdividing

by a regression

effects

lines were not

differ),

differences

values at a given pH were assessed

of 95% contidence Differences

from contidence

regression

Hill plots

the break points

the slopes (i.e.,

in

these

by Scheffe’s

to be hiphasic,

(20).

the hest fit was obtained

In cases where

from

are reported

the plots appeared

homogeneous

hetween

and identified

also obtained

oxygenation,

data

to Hill plots,

test.

two sets, and it identified

sessed

some cells

from the slopes

Differences

hy ANOVA

comparison

Each set was described

was performed

(7). Most gels were stained for heme

because

hecause

data were transformed

values

values were tested Or affinity

procedure

the same buffer.

the diluted

the reagents

stained mg/ml).

and Hh

the dialysis

Hh was diluted

PAGE

the identification

to RBC volume

statistic

the range 30-7OY0 oxygenation.

within

using the cyanmetHh

the separation,

subsequently

sociating

equal

using the chi-square

memhers.

and cooperativity

analysis;

was electrophoresed

lacked

The O1 equilibrium

red blood

before

Bulletin No. 525). Initially, the hemolyovernight against 0.05 mol/l Tris-HCl

huffer (pH 7.4) containing not improve

and

In both cases, the RBCs were lysed

of cold Hz0

concentration

immediately

the material

a few hours of bleeding. with a volume

speed,

In 1992, the sedimented

were frozen until

in 1993, however,

at high

intervals

hetween intervals

Bohr

around

around effects

from the re-

were

the slopes

as-

c>f the

lines.

RESULTS Hb Phenotypes

Using

the

freshly curves

non-optical

cclllected

were ohtained

Before washed lysed

experiments three

with

cell

(never

times

0.05 mol/l

respiration

frozen)

for both on

dialyzed

dialyzed

in cold

method

material,

the

RBCs

sea water

Tris-maleate

buffer

(pH

0.1 mol/l

KC1 and 5 mmol/l

individuals hemolysates

expressing the same phenotype were available, from them were pooled to yield sufficient mate-

ment

was performed

of morph

the entire

on material

against

Hh phenotypes

among

the

(Fig. 1). As few as 2 and as many

A

B

EDTA.

pH range

If several

investi-

A, B and C. The measurefrom only two individuals

F and one each of morphs

of 0.05 mol/l.

seven

the buffer,

D and E. No individual

of morph G was available at the time. Small volumes of concentrated Tris-maleate buffer stock were added to manipulate pH and to reach a final conccntration

examined

7.5). The

containing

was true of morphs

revealed

were

dialyzed

This

PAGE

(12%0) and

was then

rial for a Bohr plot spanning

Native

233 individuals

hemolysate

gated.

overnight

and

Hbs and RBCs.

Hhs,

filtered

(10)

01 equilibrium

For experiments

on RBCs,

the cells

were washed as ahove and resuspended in 0.05 mol/l Trismaleate buffered physiological saline, prepared from the data for this species reported by Scholnick and Mangum

ABCDEFG MORPH

12345678910 BAND

FIG. 2. (A) The frequency of each morph in the sample (R = 233). (B) The frequency of each Hb band in the sample (D = 233; i.e., the percentages of individuals in which a particular band occurred).

D. A. Shelly and C. P. Mangum

1422

as 8 Hb bands were found in an individual; bands was detected

a total of 10

in the sample as a whole. Almost

material that stained with Coomassie

qualitative;

all

Blue was also positive

for heme; a few bands that stained very faintly with Coomassie Blue but not tetramethyl in Fig. 1. The PAGE

separation

benzidine

no differences

in relative

band density were

noted. The densities of the bands that were expressed in more than one phenotype, however, did differ in the different phenotypes

are not shown

(Fig. 1).

Hb 3 was expressed in all phenotypes,

and Hbs 4 and 10

were expressed in all except F. Hbs 5,8 and 9 were expressed

was highly and precisely repeat-

in all but two phenotypes,

but the other components

of the

able. There was no difference between aliquots of individual

system were expressed in only one (Hbs 1 and 7) or two

samples that were analyzed immediately,

(Hb 2) phenotypes

with no freezing,

and those that were frozen for up to 2 months

and then

Although

thawed, even when the freezing and thawing was repeated several times. Similarly,

any other, it still composed only about half of the sample

between

(Fig. ZA). Morphs B and C were sufficiently frequent that

the frequencies of either the number of bands in an individ-

they are likely to be present in much smaller samples, but

ual (P = 0.989) (P = 0.770)

there was no difference

(Fig. 1).

morph A was more than twice as frequent as

or the frequencies

of the seven morphs

the remaining

in 1992, when the material had been frozen

before PAGE, and in 1993, when it had not. For that reason, the two data sets were combined for presentation Within

four morphs were infrequent

panied by a change in another

(Fig. 2).

The frequency distribution

the group of individuals that expressed a particu-

of body lengths appears to be

fairly normal in the different morphs, with no pronounced

lar phenotype, here called a morph, the variation in compo-

skewness in either direction

distinguish particular morphs. Contingency

I

Hb system appeared to be entirely

(Fig. 3). It certainly does not tests performed

I

morph

A

n=118

morph

I

B

I

n=54

morph

morph

E

n=3

100 morph n=l

75

G

50 25 0

Body FIG.

3. The frequencies

accom-

(Fig. 1).

nents of the multiple

JO ,

or rare. None

of the variation of any one Hb band was consistently

Length

(cm)

of each morph in relation to body length classes.

C

n=39

Fish Hemoglobin

1423

Polymorphisms

on the data for morphs A, B and C, of representatives result, indicate

that the probabilities

not great (0.195-0.342). ception

from different sets of individuals expressing phenotypes

in which the numbers

B or C. Additional

are large enough to yield a meaningful of independence

are

information

data for a given phenotype

on the replicability

A,

of the

was given by ShelIy (16).

In addition, with the possible ex-

of morph G, the frequencies

morphs are not enhanced

in the less common

DIALYZED

investigated

(Fig. 3).

The

0, Binding

points,

the

found in all morphs.

pH value

at which

vary from 7.35 to 7.76. Within

data sets for a particular

In Fig. 4, the different symbols described by a single regres-

of Hb O2

greater in the low end of the range

(Fig. 4), a relationship

critical

changes,

The pH dependence

HEMOLYSATES.

affinity is consistently

at either end of the size range

the

slope

each of the two

morph, the variation

is small (P

values for T’ are cO.01). The slopes of the regression lines describing the data in the lower pH range are heteroge-

sion line in each panel represent data for material prepared

3 C

2 1 1.4

.:

1.0

.:::.

.:r!

:

. IL,_ . .

-_

._

... ‘*....

.;_

‘ye..

0.6

:

‘.

‘.

I

_I :

:-.

is. a

._

0

-i:::...

J

‘.

:.

. ..._:._

0.2

‘.

.I_

: ..

‘.

y

‘.

.E

C

‘.

:‘\:::“: l

g

A

1%

I

‘a.-

._

.’ ... .

3 c

2 1

I

I

1.4

g

..‘.‘..‘. ._ ‘.

1.0

\

B 0.6

I

I

E

0.

F ‘W.6::. ‘. .a-. ..,:.‘&.., ._‘. .,., ‘. ., .I. ‘.‘.‘.‘. ‘. \ .I.: \ ..:e.

‘.‘_ ‘,.‘.‘... ‘.‘_

O....

I?-&$._ 0.2 6.8

7.2

7.6

8.0

I

I

I

6.8

7.2

7.6

....

8.0

I

I

I

I

6.8

7.2

7.6

8.0

PH FIG. 4. The effect of pH on oxygen affinity (I?& and cooperativity (n) of dialyzed hemolysates and RBCs in morphs A-F at 20°C. Squares and triangles show data for hemolysates. Solid lines fitted by regression analysis; dotted lines show 95% confidence intervals. Number of individuals represented in each pooh morph A, q (n = 31, A (n = 8), W (n = 5); morph B, 17 (n= ll),a(n=5),~(n=5);morphC,O(n=6),A(n=3),m(n=6);morphD,[7(n= l);morphE,O(n= 1);morph F, q (n = 2). Hbs (0.93 mmol hemell) in 0.05 molll Tris-maleate buffer containing 100 mmollKC1. Circles show data for RBCs. Numbered of individuals represented in each pool: morph A, 0 (n = 3), 0 (n = 4); morph B, 0 (n = 5); morph C, 0 (II = 3); morph, D 0 (n = 1); morph E, 0 (II = 1); morph F, 0 (II = 1). RBCs were suspended in 0.05 mob1 buffered saline containing 265 mmolll NaCl, 5.6 mmolll KCl, 4.9 mmolll CaCl,, 12.3 mmol/l MgCl,, 2.5 mmolll Na2S04 and 3.4 mmoll 1 NaHC03.

1424

D. A. Shelly and C. P. Mangum

TABLE 1. Results of ANCOVA test for homogeneity of slopes of regression lines describing O2 affinity (PSO) as a function of DH

TABLE 3. Significance of differences between (I’,,) values of stripped Hbs at high pH range Morph

O2 affinity

B

C

D

E

F

1.000 0.030 1.000 0.903 0.007

1.000 0.640 0.023 0.000

1.ooo 0.937 0.128

1.000 0.467

1.ooo

A

Temp Effect

Preparation

Morph

pH Range

Hb Hb RBC RBC Hb Hb RBC RBC Hb Hb RBC RBC

PH

pH and morph

(“C)

p

20 20 20 15 20 20 20 15 20 20 20 15

0.032 0.087 0.000 0.010 0.000 0.000 0.000 0.000 0.030 0.093 0.000 0.005

Low High

Low High

Low High

A 0 C D E F

1.000 0.056

1.000 0.672 0.037 0.000

Results hased on Scheffe’s multi+

comparison test.

the opposite

3). In both

was true (Table

Hb O2 affinitres

are significantly

and F. O2 affinity neous,

whereas

those

for the higher

range

are not

(Table

Throughout

the low pH range, Hb 02 affinity is uniformly

higher

in morph

Above

about pH 7.0, it is higher

C. Although morphs tively

the absolute

A and F appear

in morph

for morph

No other

differences

Because

overlap

the values for in fact, the rela-

around

the few data

those

for morph

at low pH are significant

of the smaller difficult

intervals

2, Fig. 4).

A than in morph

between

to be even greater;

F broadly

low pH, one might

to be more

B (Table

in morph

difference

wide 95% confidence

available

than

A than

numerical expect

to demonstrate

values

differences

(Table

A. 2).

for I’50 at high between

in this range.

TABLE 2. PH ranges in which O2 affinity (P&

them In fact,

than

is also lower in morph

A and C,

in morphs

B than

E

in morphs

C and F. Finally,

we note that the probability

between

A and B in this range is 0.056. The differ-

ence

1).

higher

morphs

morphs

between

morph

whole of the higher confined

F and morphs

morphs

comparisons

revealed

higher pH range. Significant differences to a few at high

(Table

between

are

2, Fig. 4).

(A vs C and B vs D)

no clear differences

pH (Table

the

other differences

of this pH range

Only two of the possible between

A, B and C spans

pH range, whereas

to a portion

of a difference

at all in the

Bohr effects were limited

4). Specifically,

morph

B Hbs

were more pH depenJent than those of morphs E and F. The cooperativity values also differ very little (Table 4). In the lower pH range,

morph

than

E.

those

of morph

C Hbs were more

cooperative

values differ significantly Morph

Morph

B

C

D

E

F

ns = not significant.

Preparation

A

Hhs, high pH Hbs, low pH RBCs, 20°C RBCs, 15°C Hbs, high pH Hbs, low pH RBCs, 20°C RBCs, 15°C Hbs, high pH Hhs, low pH RBCs, 20°C RBCs, 15°C Hbs, high pH Hbs, low pH RBCs, 20°C RBCs, 15°C Hbs, high pH Hbs, low pH RBCs, 20°C RBCs, 15°C

7.6-7.7 6.8-7.4 7.8-8.2 ns ns 7.1-7.6 ns ns 7.7-7.9

B

7.8y8.l 7.8-7.9 7.5-8.2

7.5-7.8 ns ns ns ns ns nb ns 8.0-8.2 ns 7.7-8.2 7.4-8.1 7.6-8.2

7.5Y7.8 ns

8.Oy8.2 ns

7.61SK.I 7.9y8.2

C

7.6-7.9 ns 11s 7.9-8. I 7.8-8.1 ns 7.6-8.1 7.4-8.1 7.5-8.2 ns ns ns

D

E

8.0-8.2 ns 7.5-8.1 7.6-8.1 7.5-8.2

7.5-8.1

7.8E8.1 ns

7.6y8.2 7.5-8.1

Fish Hemoglobin

1425

Polymorphisms

TABLE 4. Bohr factors

(dlogP50/dpH),

correlation

coefficients (ti), estimates of cooperativity

Temperature PH

(“C)

AlogPSOfApH* (295% CI)

Iz

nS0*

n Sample size

c7.45 >7.45

20 20

1.08 ? 0.08 0.28 -c 0.11

0.99 0.84

2.10 ? 0.05 1.94 + 0.07

16 13

20 15 20 20 20 15 20 20 20 15 20 20 20 15 20 20 20 15 20 20 20 15

0.62 0.65 1.01 0.36 0.33 0.61 0.97 0.19 0.60 0.75 1.03 0.29 0.46 0.34 0.91 0.15 0.90 0.79 1.17 0.20 0.64 0.58

0.89 0.94 0.96 0.86 0.96 0.96 0.98 0.60 0.96 0.99 0.98 1.00 0.94 0.76 0.97 0.98 0.99 0.97

1.60 1.59 1.97 1.76 1.65 1.50 2.11 1.79 1.62 1.58 1.88

t 2 z z it z z rt f 2 i-

0.06 0.07 0.03 0.03 0.10 0.10 0.04 0.06 it.08 0.10

7 7 21 21 5 5 15 15 3 3

1.78 1.68 1.87 1.76 1.81 1.74

2 2 z t 2 z

0.179 0.13 17.04 0.12 0.08 0.10 0.174 17.174 0.w 0.179

Morph A

Hhb Hh> RBC:s RB(:s Hhh HbS R ACS RRCS HI% Hh> RBCs RBG HhS Hhh RBc 1s RB( 1s Hhh Hha RBCs RBc 1s Hhk HIY. RR& RR<:s

B

C

D

E

F

c7.45 >7.45

~7.66 >7.66

~7.60 >7.60

~7.46 >7.46

~7.52 >7.52

RBC 0: affinity

RBG.

Root shift is so great)

in this species

that accurate

is so low (i.e., the

Or equilibrium

data were

not obtained

at 20°C and low pH (Fig. 5). Lowering

experimental

temperature,

to 10°C little;

(Fig. 5A),

no further

however, highly

significant

are robust

extended

was noted

heterogeneity

at both

were

least one other

(Table

noted

A relative

remained

to most

Morph between

these

slopes of regression

between

differing

sometimes

two and

in order

2 2 1 1

but one morph (C) at 2O”C, but there are no signiticant values are differences at 15°C (Table 4). Th e cooperativity entirely homogeneous (P 2 0.05).

DISCUSSION Hb PAGE PoZymorphism The present

lines

the other

in the Atlantic

results indicate

vertebrates

Croaker

that the magnitude

in adults of this teleost

of Hb varia-

species

that have been characterized.

in the Chesapeake

be usefully described as the wild-type. Extensive information is available for the life history

and,

morph details

(Table

at

are com-

remained

high

the

species

in the

same sampling

lower

is so frequent

Chesapeake

F, 01

morph

E Hbs

also the present

data, we estimate

in at least

and a maximum

of 81% of our sample

the

pH

range.

only from E and 2). Intermediate

of decreasing

magnitude

individuals; examined which

the remainder here

includes

both sexes mature,

sizes. And

Bay, including

thar a minimum

the

smallest

and

The

yet, the frequency

distribution

and

of mature size range

largest

as well as even smaller

of the

of 74%

consisted

were juveniles.

in

that it can

site as ours (1). On the basis of these

in morph

from all others differing

and

Bay, no morph

than

At least

1). At 2O”C, signiti-

throughout,

of the pH range

1.90 2

1.72 z 1.66 + I .69 2

each

further

others

C was the least distinctive,

only in a portion

data are

that the data

low. The RBCs containing

of, and

0.98

0.94 0.99 0.91

tion is much greater

Hbs, RBC O? affinity

were the m<>st distinctive, a portion

indicating

2), although

plex. Like the stripped in morph

among

0.04 1.69 -t- cl.11

very

exception,

shown.

15 and 20°C (Table

differences

affinity

pH range

Without

lines fit to Bohr plots of these (P for r’
the

2 0.12 2 0.30 -c 0.11 + 0.08 5 0.19 +- 0.10 -+ 0.09 2 0.10 i 0.39 -c 0.13 t 0.42 + 0.49 ? 0.13 k 0.35 5 0.28 ? 0.07 ? 0.13 -c 0.20 k 0.34 z 0.05 z 0.06 + 0.21

(Fig. 5) and then

the workable was made.

in the pH ranges

Significant cant

first to 15°C

attempt

regression

(nso) and sample size (n)

sizes at

and larger

of body sizes was

of the remaining differences, the morphs can be ranked as follows: F, A, L) and B. At 15”C, morph E RBCs remained the most distinctive

not distinctive of morph. The present tindings do not, of course, exclude the possibility of an ontogenetic polymorphism in larval stages or in even smaller juveniles than

(Table 2), and morph C RBCs were intermediate; morphs A, B and F RBCs all differed from only one other.

those in our sample; characterized here

The Bohr effect

of morph

E RBCs differs from that in all

history.

they just mean that the polymorphism is not primarily related to the life

D. A. Shelly and C. P. Mangum

1426

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PH FIG. 5. The effect of pH on oxygen affinity (PSO) and cooperativity (n) of RBCs in morphs A-F at 15°C (0) and 10°C (0, A only). Solid lines fitted by regression analysis; dotted lines enclose 95% confidence intervals. Number of individuals and buf. fered saline as in Fig. 3.

Hb PAGE

Phenotypes

and North

Carolina

Bonaventura

in Chesapeake

Bay

Animals

et al. (3) summarized their unpublished results

for Hb phenotypes

in more than 50 species of fish from the

primarily of North Carolina animals but also including individuals from the same site as ours. And yet, despite the sample size, morph A was not recovered (13,14). investigators

recovered

Instead, these

morphs C and F (and none other

waters surrounding Beaufort, North Carolina. Even though sample sizes were small, it was clear that the Hbs are polymorphic in 11 species. One was M. undulattcs, in which first

reported here), along with phenotypes that we did not observe in our Chesapeake Bay sample. Thus, the available information may suggest that morph A is far less frequent

four (3) and later six (J. Bonaventura, personal communication) Hb phenotypes were found. Diagrams of the banding

in North Carolina than in Chesapeake Bay waters, and F may be more common. On the other hand, the size range (3.6-19.5 cm length)

patterns were kindly communicated to us. Interestingly, only two of the six resemble a phenotype in Chesapeake Bay animals; one (A) was the most frequent in our sample, but the other (F) was found in only a few individuals. Six Hb phenotypes were also reported later by Sullivan and coworkers, for a much larger sample of this species consisting

investigated by Sullivan and co-workers (13,14) overlaps ours (16.0-26.5 cm) very little. None of their Chesapeake Bay animals exceeded 16 cm length, and only a dozen (of almost 500) of their North Carolina animals exceeded 16 cm in length. Thus, unlike ours, the previous results could

Fish Hemoglobin

be due to ontogenetic direct

comparison

of Atlantic graphic

1427

Polymorphisms

change

croakers

harrier

at both

of the present from

(Cape

would be of great communication),

localities.

shorter

Obviously,

results with a large sample

south

of the

Hatteras)

well-known

separating

the

two

expressed

phenotype

sites

as morph

A. After

active.”

A expressed

Finally,

the present

variation and

results

in structural

that

it can

show

that

phenotype

be

at least some

is reflected

detected

at the

physiological

level

phoretic than

(collected

kindly

communicated

02 binding

at 20°C and pH 7.5) for stripped

three of their original

four North

data

phenotypes.

these

The

that sharp.

the other,

three

pheno-

and

however.

holding

temperature

morphs

in salmonids

have been able to resolve pH 7.5 alone. functional PAGE

differences

ferences handing

are not

between

indicate

a necessary

not

others

at

that obvious

concomitant

of

[see also (17)].

here, however,

whereas

ferences

hetween

physiologically

binding

properties

phenotypes

others

the

meaningful

do not. The magnitude

phenotypes

and

are not invariably

A anJ

B differ only

10 and in the presence

dif-

those

related.

of Hbs 3

of Hb 8. And

intact

tetramers

tides, the discrepancy less, it underscores Hb components.

rather

than

is not especially

the interest

in the properties

The O! binding

of isolated

Hbs 3, 8 and 10, as well as the others, interest.

In view of the differences

the RBCs found

in different

individual surprising.

yet

morphs,

During

preparations

of

be of special O1 binding

of

it will also be of inter-

Stability

the O1 binding

experiments,

the same

polymorphism

freezing

eliminate

in adult

and it was always iced bands

three

in each

individuals

the possibility a degradation

that one product

that

of

differences

phenotypes

were

Even though

on the ontogeny

(6), we believe

of

were

than freezing

0: binding these

in RBC O1 binding.

plasticity

freezing, for longer

of Hb

the alternative

should

be systematically

M. undulatus.

of the resolution

of this point,

the considerable

in the Atlantic

croaker,

the present

magnitude

re-

of Hh

and they also show

that it can be accompanied by appreciable functional differences hetween morphs, at both the molecular and cellular levels.

We thank 1. Bonauentura ad S. W. Ross for generously sharing heir unpublished infurmation We also thank G WilItims, P. Grer an& the Trawl Surwy Program of the Virginia Institute of Marine Science for their invaluable assistance in collecting the animals. This inwesttgation was supported by NSF DCB 88-16972 (Physiological Processes).

polypep-

est to investigate the levels of organic POg and the sensitivities of the various Hb components to them.

Phenotypic

Regardless

these

had no effect

of phenotypic

investigated

above

in electro-

above

Nonetheof particular

would

between

phe-

O2

For example,

in the densities

or absence

of dif-

between

the 0: affinity of morph A Hb differs uniformly from that cjf morph B Hb throughout the low pH range. Because we examined

possibility

changes

storage

Nonetheless,

sults clearly demonstrate

in 0; binding can accompany differences in PAGE pattern. It is not yet clear why some morphs differ

functionally

and

and we might

the differences

The two sets of findings

differences

As shown

Bay morphs,

in

expressing

by differences

between

Chesapeake

as morph

by repeatedly

about by some agent other

types were recovered in our sample. Our tindings are at least consistent with his data in that we also found no differences several

held

represents

hemolysates

paralleled

reported

electrophoretic

We cannot

brought

between

of the

identified

phenotype

an experiment, The

of the two phenotypes and thawing,

none

have

was never

phenotypes

not

Unfortunately,

after 47 and 49 days

after prolonged

period.

data indicate no differences in cooperativity at 20°C and pH 7.5 and differences in P50 so slight that they are probably significant.

to change

investigators phenotype

equally

Hbs expressing

Carolina

to un-

that originally

were not successful.

an hour before

during

(RBCs). J. Bonaventura

Efforts

but our material

in 02 binding

appeared

B. In all three cases, the initial

and thawing

Several

of the

B were identified 49 days, one initially

phenotype

replaced.

freezing to Oz Binding

temperatures Two individuals

notype was unchanged after only 20 days. We have no concrete reason to conclude that their tags were lost and incorrectly

Relationship of Hb PAGE Polymorphism

change.

geo-

interest. According to S. Ross (personal none of their North Carolina animals

were “reproductively

period but at declining

dergo a phenotypic

individuals

were sampled repeatedly, and both the PAGE Hb phenotype and the functional properties remained unchanged, as expected. These animals were held in the laboratory under constant conditions. In contrast, 3 of 26 (but not the remaining 23) fish held in running sea water for a somewhat

References 1. Barbieri, L.R. Life history, population dynamics and yield-perrecruit modelling of the Atlantic croaker, Micropogon u&laUS. Ph.D. dissertation. Williamsburg, VA: College of William & Mary; 1993. 2. Binotti, S.; Giovenco, S.; Giardina, B.; Antonini, E., Brunori, M.; Wyman, J. Studies on the functional properties of fish hemoglobins. II. The oxygen equilibrium of isolated hemoglobin components from trout blood. Arch. Biophys. Biochem. 142:274-280;1971. 3. Bonaventura, J.; Bonaventura, C.; Sullivan, B. Hemoglobins and hemocyanins: Comparative aspects of structure and function. J. Exp. Zool. 194:155-174;1975. J.; Bonaventura, C.; Giardina, B.; 4. Brunori, M.; Bonaventura, Bossa, F.; Antonini, E. Hemoglobins from trout: Structural and functional properties. Mol. Cell. Biochem. 1:189- 196; 1973. D.J. Oxygenation characteristics of the 5. Giles, M.A.; Randall, polymorphic hemoglobins of coho salmon (Oncorhynchws ki-

1428

6.

7. 8.

9.

10.

11.

12.

sutch) at different developmental stages. Camp. Biochem. Physiol. 65A:265-271;1980. Giles, M.A.; Vanstone, W.E. Ontogenetic variation in the multiple hemoglobins of the coho salmon (Oncorhynchus kisutch) and effect of environmental factors on their expression. J. Fish Res. Bd. Can. 33:1144-1149;1976. Hames, B.D.; Rickwood, D. Gel electrophoresis of proteins. Washington DC: IRL Press; 1991. Ingermann, R.L. Structure-function relationships of the ectothermic vertebrate hemoglobins. In: Mangum, C.P. (ed). Blood and Tissue Oxygen Carriers. Heidelberg: SpringerVerlag; 1992:41 l-432. luchi, I. Chemical and physiological properties of the larval and adult hemoglobins in rainbow trout, Salmo gairdneri iri&us. Comp. Biochem. Physiol. 44B:1087-1101;1973. Mangum, C.P.; Lykkeboe, G. The influence of inorganic ions and pH on oxygenation properties of the blood in the gastropod mollusc Busycon can&datum. J. Exp. Zool. 207:417-430; 1978. Riggs, A. Properties of fish hemoglobins. In: Hoar, W.S.; Randall, D.J. (eds). Fish Physiology, Vol. V. New York: Academic Press; 1970:209-251. Ronald, A.P.; Tsuyuki, H. The subunit structures and the molecular basis of the multiple hemoglobins of two species of trout, Salmo gairdneri and S. chki clarki. Comp. Biochem. Physiol. 39B:195-202;1971.

D. A. Shelly

and C. P. Mangum

13. Ross, S.W.; Sullivan, B. Population (stock) determination for Atlantic croaker. NOAA Technical Memorandum NMFSSEFC-199. Proceedings of the Stock Identification Workshop 7:109-191;1987. 14. Ross, J.L., Sullivan, J.B., DeVrels, D.A. Assessment of North Carolina commercial fisheries. Ann. Progr. Rept. Proj. 2-386R, No. Carolina Dept. Nat. Resources, Morehead City NC; 1984. 15. Scholnick, D.A.; Mangum, C.P. Sensitivity of hemoglobins to intracellular effecters: Primitive and derived features. J. Exp. Zool. 259:32-42;1991. 16. Shelly, D.A. Functional characterization of the electrophc>, retie hemoglobin polymorphism in the Atlantic croaker (Micropopn unduhus). M.A. Thesis. Williamsburg, Va: College cd William 6r Mary; 1994. 17. Southard, J.N.; Berry, CR.; Farley, T.M. Multiple hemoglo, bins of the cutthroat trout, Salmo darkt. J. Exp. Zoc>l. 239:716;1986. 18. Wilkens, N.P. Ontogeny and evolution of salmonid hemoglobins. Int. Rev. Cytol. 94:269-298;1985. 19. Wilkins, N.P.; Iles, T.D. H aemoglobin polymorphism and its ontogeny in herring (C&ea hnrengus) and sprat (Sprattus sprattus). Camp. Biochem. Physiol. 17:1141-l 158;1966. 20. Yeager, D.P.; Ultsch, G.R. Physiological regulation and conformation: A BASIC program for the determination ofcritical points. Physiol. Zool. 62:888-907;1989.