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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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0
00
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.__
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.
..
._
._
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._
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.
.._‘.. ‘_
‘.
._
..
..
._’
.
._
l
0
l
0
.__.. . ._ . .:: .o
.._
‘_
l
‘.
._
$
l.0
00
_,
_
._ ___ C
I
6. ,,
&..,
ag
B
‘.
. .
‘_
.._,
. .
._
..__
._
. .
._
. .
‘.
:
0.6
F
E l
0
8
l
*
t
0
0..
.f
6.._ ‘...
‘.. .*
ps:1.0
b.
-
‘.
\
0
$ 0.6 0.2
-
6.8
7.2
7.6
8.0
6.8
7.2
7.6
8.0
6.8
7.2
7.6
8.0
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.
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
A 0.00
0
0
00
.
._ ., ..__ Y ‘._O.
1.0
I’..-... ‘I..
y:..
.__
._,
i\;;
4
I
I
‘.
‘..,
‘._;
.
..
._
._
‘.
._
\
.
.._‘.. ‘_
‘.
._
..
..
._’
.
._
l
0
l
0
.__.. . ._ . .:: .o
.._
‘_
l
‘.
._
$
l.0
00
_,
_
._ ___ C
I
6. ,,
&..,
ag
B
‘.
. .
‘_
.._,
. .
._
..__
._
. .
._
. .
‘.
:
0.6
F
E l
0
8
l
*
t
0
0..
.f
6.._ ‘...
‘.. .*
ps:1.0
b.
-
‘.
\
0
$ 0.6 0.2
-
6.8
7.2
7.6
8.0
6.8
7.2
7.6
8.0
6.8
7.2
7.6
8.0
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.
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