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Biological Journal of the Linnean Society (2001),72: 401407. With 1 figure
doi:10.1006/bij1.2000.0507, available online at httpj//www.idealibrary.com on
1D f
@
Difference in parasite load and nonspecific immune
reaction between sexual and gynogenetic forms of
Carassius auratus
@
HIROSHI HAKOYAMA*
Department of Biology, Faculty of Science, Kyushu University, CREST Japan Science and Technology
Corporation (JST), Fukuoka 812-8581, Japan
TEIICHI NISHIMURA, NAOTO MATSUBARA and KEI’ICHIROH IGUCHI
National Research Institute
of
Fisheries Science, 1088, Komaki, Ueda 386-0031, Japan
Received 2 November 1999; accepted for publication 2 November 2000
For coexistence, the sexual form in sexual/asexual complexes needs short-term advantages that can compensate
for the two-fold disadvantage of sex. Higher mortality in the asexual form due t o a higher parasite load will provide
an advantage to the sexual form. In Lake Suwa, Japan, the parasite load (Metagonnimus sp.; Trematoda) of triploid
gynogenetic females of Carassius auratus was significantly higher than that of diploid sexual females. In a n
immunoassay using healthy wild fish that were conditioned for 1month in laboratory tanks, the nitroblue tetrazolium
(NBT) immune reaction of sexual females was significantly higher than that of gynogenetic females. The NBT
activity indicates the abundance of oxygen radicals from phagocytes, and hence the level of immune activity of the
phagocytes. We suggest that the higher parasite load of the gynogenetic form is in part due to the lower immune
activity of the phagocytes (nonspecific immune reaction) in the gynogenetic form compared to the sexual form.
0 2001 The Linnean Society of London
ADDITIONAL KEYWORDS: Carassius auratus - coexistence - gynogenesis - nonspecific immune reaction parasite load.
INTRODUCTION
Gynogenetit/sexual complexes persist (Dawley, 1989;
Vrijenhoek et al., 1989; Vrijenhoek, 1994) despite the
fact that the asexual gynogenetic form has the twofold advantage of not producing males, and therefore
has potentially a two-fold growth rate (see the twofold cost of sex; Williams, 1975; Maynard Smith, 1978;
Bell, 1982). The all-female gynogenetic form is asexual
but requires males of the sexual form for insemination.
All else being equal, a population of a gynogenetic
form will outcompete the sexual form from which it
gets sperm, and therefore finally, both the gynogenetic
* Corresponding author. Present address: Hokkaido National
Fisheries Research Institute, Kasurakoi 116, Kushiro 085-0802,
Japan. E-mail: [email protected]
00244066/01/030401+ 07 $35.00/0
and sexual forms will become extinct. For their coexistence, the sexual form needs short-term advantages that compensate for the two-fold
disadvantage of producing males (Maynard Smith,
1978). I n addition, some negative frequency-dependence in reproductive success or other fitness components (apostatic selection, see Endler, 1991) is
needed for the stability of the coexistence (Moore &
McKay, 1971; Moore, 1975, 1976).
One of the factors for the coexistence is the mate
preference of males for conspecific females over asexual
females, which increases the relative success of the
sexual populations, producing a short-term advantage
for sexual females and the negative frequency-dependence in reproductive success (McKay, 1971; Moore
& McKay, 1971; Moore, 1975; Lmyning & Kirkendall,
1996). In Poeciliopsis (McKay, 1971; Moore & McKay,
1971), when sexual individuals predominated, males
401
0 2001 The Linnean Society of London
402
H. HAKOYAMA E T A L .
established dominance hierarchies and a few dominant
males monopolized and mated with conspecific females
and subordinate males mated with asexual females.
In contrast, when asexual females predominated, dominance hierarchies disappeared and most males mated
with conspecificfemales. Theoretically, this frequencydependent process may result in a dynamic equilibrium
between the asexual and sexual forms (Moore &
McKay, 1971; Moore, 1975, 1976).
The role of parasites in the gynogenetic complex is
also potentially important. If the immunity differs
genetically between the sexual and asexual hosts, and
if the mortality of the asexual host is higher than that
of the sexual host, the two-fold cost of sex will be
reduced. The hosts’ (1) nonspecific immune reaction
and (2) specific immune reaction cause different effects
on the dynamics of the gynogenetic complex; only the
latter may provide the negative density-dependence
regulation on the gynogenetic complex.
The lower nonspecific immune reaction such as cynorexia of the phagocytes of the gynogenetic form
may cause a higher mortality of the gynogenetic form.
However, if a single host reacts to all parasites nonspecifically by an immune performance, the difference
in the nonspecific immune reaction (mortality) between
the gynogenetic and sexual form is independent of the
relative frequency of the two forms in the gynogenetic
complex.
On the other hand, the difference in the specific immune reaction such as the MHC (major histocompatibility complex) may cause a negative densitydependencemortality in the gynogenetic complex. As it
is not feasible that a single host can resist all the different kinds of parasites genetically(because of the genetic individuality in the specific immune reaction), nor
is one parasite strain able to infect all the potential
hosts, selection will favour parasites that can exploit
the most common host phenotype. If parasites disproportionately attack the commonest phenotype in a
gynogeneticcomplex as a consequenceof the host-parasite coevolution,rare host phenotypeswill be more likely
to be less affected by parasitism. In this case, the sexual
host has an advantage to reduce offspring with rare
phenotypes by recombination, resulting in the coexistence of sexual and gynogenetic forms and polymorphism in clone lineages in a frequencydependent manner. This hypothesis is a variation of the
Red Queen hypothesis, which explains the maintenance
of sexual reproduction in populations (Jaenike, 1978;
Bell, 1982; Hamilton, 1982). Some studies have confirmed the predictions of the Red Queen hypothesis
(Lively, 1987;Lively, Craddock & Vrijenhoek, 1990;Moritz et al., 1991; Dybdahl &Lively, 1998; Lively & Dybdahl, 2000). The Red Queen hypothesis predicts that
the parasite loads of the sexual form are lower than
those of the more common clone (Lively et al., 1990).
Further, if clone strains are not so diverse, on average,
the asexual form will also be more intensely infected
than the sexual form. The Red Queen hypothesis has no
prediction for the difference in the nonspecific immune
reaction between the sexual and asexual forms.
In this study, we examined (1) the parasite load and
(2) the immune activity of the phagocytes (nonspecific
immune responses) of a gynogenetic complex of Carassius auratus in Lake Suwa, Japan.
METHODS
STUDY SYSTEM
The all-female Japanese crucian carp, Carassius auratus langsdorfii Temminck et Schlegel, 1846 (‘ginbuna’ in Japanese) is a triploid and tetraploid
gynogenetic fish (Kobayasi, Kawashima & Takeuchi,
1970; Kobayasi, 1971, 1976; Kobayasi, Nakano &
Nakamura, 1977). C. a. langsdorfii is widely distributed and coexists with diploid sexual C. a. subspp.
in freshwater lakes and rivers of Japan (Liu et al.,
1980; Kobayasi, 1982). The tetraploid asexual form is
infrequent (Kobayasi, 1982; Hakoyama, Matsubara &
Iguchi, 2001). Hybridgenesis (Schultz, 1969; Dawley,
1989; Vrijenhoek, 1994) and is not known in this
system. Individuals of the asexuaysexual complexes of
Carassius usually school, show no agonistic behaviour,
and spawn at night in a group during the mating
season (Hakoyama & Iguchi, 2001).The mating system
of Carassius is promiscuous, and males show no agonistic behaviour and no significant mate preference
for conspecific females during group mating (Hakoyama & Iguchi, 2000). Mate preference is unlikely t o
be a strong frequency-dependentforce maintaining the
coexistence of asexual/sexual complexes of Japanese
crucian carp (Hakoyama & Iguchi, 2001). No frequency-dependent reproductive success has been reported in the asexuaysexual complexes of Carassius.
One of the parasites of Carassius is Metagonnimus sp.
(Order: Digenea; Class: Trematoda). M. takahashii
Takashi, 1929 parasitizes several freshwater fishes
including Carassius and produces black spots on the
epidermis of the fish (Egusa, 1978).Generally,primary
intermediate hosts of the trematodes of Digenea are
snails, secondary intermediate hosts are fishes and
tertiary hosts are mammals (Egusa, 1978). When an
individual of Metagonnimus sp. parasitizes a fish, it
enters the metacercaria stage and stays in a tunica
formed on the epidermis of fish, and the tunica looks
like a black spot because of the black excreta in the
tunica (Egusa, 1978). A single Metagonnimus sp.
makes a single black spot. Metagonnimus sp. has no
clear negative effect on fish (Egusa, 1978).
SAMPLE COLLECTION AND PARASITE MEASUREMENT
We sampled several hundred gynogenetic C. a. langsdorfii and the sibling sexual C. a. bulgeri Temminck
PARASITE LOAD AND NONSPECIFIC IMMUNE REACTION
et Schlegel, 1846 from the Funato River, a tributary
of Lake Suwa (12.91km') in Japan on 22 April, 7 May
and 5 June, 1997 (see Hakoyama et al., 2000 for
details). The number of fish measured (range = 58-159)
was large enough t o determine parasite load within
the population. We measured the standard length (SL)
and body weight of the fish and identified the sex
under anaesthesia (100 ppm 2-phenoxyethanol). All
fish were mature, and sex was easily discernible by
gently pushing the gonopore; males issue sperm and
females eggs. Gynogenetic C. a. langsdorfii and sexual
C. a. bii%eri are morphologically distinguishable based
on the difference in the body coloration (silver and
brown) and body depth (high and low) (Nakamura,
1969). We identified triploid asexual fish and diploid
sexual fish based on the erythrocyte size following the
procedure of Sezaki, Kobayasi & Nakamura (1977) and
Onozato et al. (1983). The erythrocyte size becomes
larger with polyploidy, and the critical size between
diploid and triploid is 15 pm in mean major diameter
of the erythrocytes and that between the triploid and
tetraploid is 19pm (Onozato, Torisawa & Kusama,
1983). We took blood samples by withdrawing blood
from the caudal vein, made blood smear samples and
measured the major diameter of the erythrocytes using
a light microscope (a magnification of 400 diameters).
Diploid and triploid fish identified by the mean erythrocyte size morphologically corresponded t o C. a. burgeri and C . a. langsdorfii. In this complex, gynogenetic
forms were infrequent (c.20Yo of total fish; see Hakoyama et al., 2000 for details). We counted the number
of black spots on the tail fin. In some fish, we took
metacercaria individuals out of the tunica using a
stereomicroscope (a magnification of 50-100 diameters), and identified them as Metagonnimus sp. To
examine the difference in parasite load between the
sexual and asexual forms, we compared the number
of black spots (the number of metacercaria larvae)
between the gynogenetic females and sexual females.
We did not use males in the analysis in order to remove
any sexual difference in the parasite load.
NITROBLUE TETRAZOLIUM TEST
To examine the baseline immune response of healthy
fish, we used conditioned wild fish sampled on 5 June
for the nitroblue tetrazolium (NBT) test (Rook et al.,
1985; Siwicki & Anderson, 1993). NBT is reduced t o
formazan in the reaction with the oxygen radicals
from neutrophils and monocytes (Siwicki & Anderson,
1993). Before the NBT assay, sexual and gynogenetic
fish (c.30 fish each) were separately reared for one
month in two 2501 stock tanks. First, we filled the
stock tanks using medicated water (1% NaCl and 5 mg/
L oxolinic acid; Green F Gold Liquid, Japan Animal
Druggery Co., Katushika-ku, Tokyo, Japan) for one
403
day, and then maintained the tanks under a constant
temperature (17 & 1OC) using a running water system
(c. 10 1 per hour flow of pumped ground water). Nourishment was from assorted feed (Koi 2P, Synthetic
Feed Laboratory, Takasaki, Gunma, Japan). During
the maintenance period, black spots on the fins disappeared in all fish. We took samples by withdrawing
blood from the caudal vein from 12 sexual diploid
females and 12 gynogenetic 3n females. We used syringes that had been rinsed in heparin (150 unitdml).
Standard length (SL) of the sampled fish was about
10-12cm, and there was no significant difference in
the SL (Mann-Whitney U-test, z = - 0.52, D0.05) and
condition factor (body weight/SL3)between the sexual
and asexual fish (Manr-Whitney U-test, z= -0.87,
B0.05). We placed 0.1 ml of the blood into a microtiter
plate well, than added an equal amount of 0.2% NBT
solution, and incubated it for 30min at room temperature (c. 20°C). We took out 0.05 ml of the NBTblood cell suspension and added it t o a glass tube
containing 1.0 ml N,N-dimethyl formamide (DMF)
(Wako Pure Chemical Industries, Chuo-ku, Osaka,
Japan), then centrifuged the glass tube for 5min at
3000g. The supernatant was then read in a spectrophotometer at 540 nm in 1.0ml cuvettes. Values of
the extinction were transposed according to a standard
curve into mg of NBT/l ml of blood (extinction reading
x 4 =mg NBT formatdl ml blood Siwicki & Anderson,
1993).
DATA ANALYSIS
To synthesize and compare the statistical relationship
between two variables from independent statistical
units (samples or statistical blocks), we used a metaanalysis (Hedges & Olkin, 1985; Rosenthal, 1991;
Arnqvist & Wooster, 1995). Meta-analysis facilitates
(1) estimation of the overall level of significance and
(2) testing the statistical homogeneity of the effect
sizes between statistical units (Rosenthal, 1991: 5961). First, for each unit, we calculated accurate onetailed form P values with a fixed direction of effect
and the corresponding standard normal deviate 2 s .
Second, we estimated the overall standard normal
deviate as
and the corresponding overall significance level Paverall
by the two-tailed form (Rosenthal, 1991: 85), where 2,
is the standard normal deviate of unit i, and K is the
number of statistical units. We estimated the effect
size (Pearson's r) of each statistical unit as r=Z/,/N,
where N is the sample size. Statistical heterogeneity
404 H. HAKOYAMA ET AL.
.~
in effect sizes r among units was tested using the
standard procedure of calculating a x2 statistic as
sf: 6.76 k 6.06
50
L-1
a
40
30
(Rosenthall, 1991: 73-74), where Zr, is the Fisher Zr
corresponding to r,, and Zr is the weighted mean Zr,
K
20
10
0
K
Zr = x ( N L- 3)Zr, z ( N c- 3).
*=1
1-
9
8
10
12
11
1
The X2 has K - 1 degrees of freedom. As benchmarks,
effect sizes of r= 0.2-0.4 are considered a s weak effects,
0.4-0.7 as moderate effects, and 0.7-0.9 as strong
effects (Sprithall, 1982). We used two-tailed tests in
all the statistical analyses.
*
100
*
sf 4.38 5.00
RESULTS
FIELD PARASITE LOAD
The number of metacercaria larvae on gynogenetic
females was significantly larger than that on sexual
females (Mann-Whitney U-tests, z=4.55, 2.93 and
4.75, P<0.0001,P=0.0034 and P<O.OOOl for 22 April,
7 May and 5 June; P,,,,,,,<0.0001; Fig. l), the magnitude
of the overall weighted effect size
was roverall
=
0.42 (a moderate effect), and there was no significant
heterogeneity in effect size among samples (x%+~
=
3.79, P=0.16). Variance in the number of larvae on
gynogenetic females was significantly larger than that
of sexual females (F-tests, FIz6,31 = 0.077, F47, = 0.016
and FIT;,47=0.177,P values <0.0001 for 22 April, 7
May and 5 June; P,,,,,l,<O.OOO1; Fig. l), ro',,,,,ll
=0.58 (a
moderate effect), and there was no significant heterogeneity in effect size among samples
1.63, P =
0.44). Standard length of the sexual females did not
differ from that of gynogenetic females (MannWhitney U-test, z=1.31, 0.68 and 0.15, P=O.19, 0.50
and 0.15 for 22 April, 7 May and 5 June; Poveroll=
0.11; Fig. l),ro,,,,l,=0.08, and there was no significant
heterogeneity in effect size among samples (x&, =
0.35, P=0.84). There was no significant correlation
between the standard length and number of larvae
(Pearson's r = -0.11, 0.18, -0.16, -0.41, 0.049 and
0.24; n= 127, 32,48, 10,48 and 20; P=0.24,0.33,0.29,
0.24, 0.74 and 0.31 for the sexual and gynogenetic
females on 22 April, sexual and gynogenetic females
on 7 May and sexual and gynogenetic females on 5
= 0.05,
June, respectively; Po,erall
=0.60; Fig. l),
and effect size r did not differ among 6 blocks
=
4.60, P=0.48). The absence of a significant correlation
indicates that the standard length is not a n adequate
(~z+~=
,
1
,
1
1
L__
m i
.
25
20
-5
0
.=
. I
1
I
0
,
'
8
9
10
I
12
11
Standard length (em)
Figure 1. Relationship between the number of encysted
trematode larvae (metacercariae) on the fail fin of female
Japanese crucian carp and the standard length (cm)measured on (A) 22 April, (B) 7 May and (C) 5 June. Mean
( fSD) number of larvae is shown for both (m) gynogenetic
(gf) and (0)
sexual (sf) females.
covariate to examine the difference in parasite load
between sexual and gynogenetic females.
NBT ASSAY
The NBT activity of sexual diploid females
(1.51 f0.35 mg NBT formatdl ml of blood; mean SD)
was significantly higher than that of gynogenetic triploid females (0.98 0.16) (Mann-Whitney U-test,
*
*
PARASITE LOAD AND NONSPECIFIC IMMUNE REACTION
Nsexual
= 12, Nasexual
= 12, z= 3.54, P= 0.0004), indicating
that the immune activity of phagocytes such as neutrophils and monocytes of the sexual females was greater
than that of the gynogenetic females. The magnitude
of the effect size was r=0.72 (a strong effect).
DISCUSSION
In the present study, we found a higher parasite load
in gynogenetic females compared to sexual females in
a gynogenetic complex of Carassius auratus. This
result is consistent with both the prediction of the
lower nonspecific immune activity in the gynogenetic
form and the Red Queen hypothesis. Since the NBT
test indicates that the immune activity of phagocytes
was higher in sexual females than in gynogenetic
females, the difference in the parasite load can most
probably be attributed to the difference in the nonspecific immune reaction (cynorexia). The lower activity of phagocytes in the gynogenetic form is likely
t o lead to a higher mortality of the gynogenetic form,
and may contribute to compensating for the disadvantage of sex (Williams, 1975; Maynard Smith,
1978; Bell, 1982). Although the Red Queen hypothesis
also predicts this result, the specific immune reaction
(antigen-antibody reaction) t o the parasite Metagonnimus sp. has not been examined in the present
study. To examine the antigen-antibody reaction, we
need t o inoculate the antigen into the host fish (see
Ebert & Hamilton, 1996; Lively & Dybdhal, 2000).
Due to the effects of inbreeding of the sexual population
(Lively et aE., 1990),time-lags in frequency-dependence
of the host-parasite arms race (Dybdhal & Lively,
1998) and dispersion of hosts and parasites among
local habitats (Vernon et al., 1996; Judson, 1997), the
simple prediction from the Red Queen hypothesis may
be not consistent in natural populations. Long-term
research focusing on these factors is the next step to
confirm the assumptions and predictions of the Red
Queen model in the Carassius complex.
Variance in the number of parasites of gynogenetic
females was larger than that of sexual females. According t o the Red Queen hypothesis (see Lively et
al., 1990; Ebert & Hamilton, 1996), genetic variation
among clone lineages possibly explains this result. In
Lake Suwa, multiple clones exist (Murayama et al.,
1984).If so, since our data was a random sample of the
natural population without distinguishing the clone
lineages, the parasite load of the dominant clone
lineages is likely t o be higher than that of the sexual
form, but that of some infrequent clones might not be
necessarily higher than that of the sexual form. By
using the geneticmarker (see Vrijenhoek, 1993;Vernon
et al., 1996), we will be able t o examine the difference
in the susceptibility among clones and the variance in
the susceptibility within a clone lineage. Note that
405
the variance in susceptibility of genetically uniform
individuals of a clone lineage will be smaller than that
of sexual individuals which have genetic variance for
susceptibility (Lively et al., 1990).
The mechanism by which higher activity of phagocytes occurs in the sexual form is not known, but
might be explained by: (1) the accumulation of deleterious mutations, (2) heterosis and (3) the enlarged
body cell by polyploidy. In general, sexual forms have
long-term advantages to counter the accumulation of
deleterious mutations (‘Muller’sratchet’, Muller, 1964;
see a review in Vrijenhoek, 1994).
A clone of hybrid origin may show heterosis by which
the fitness of the clone becomes higher or lower than
that of the parental species (Ladle, 1992; see also
Craddock, Vrijenhoek & Lively, 1993 and Ladle, 1993).
Therefore, for example, perturbations of the immune
system during hybridization (Ladle, 1992) may cause
the lower activity of phagocytes in the asexual form.
In general, two types of origin are known for polyploid
asexual clones,hybridization of sexual lineages (Vrijenhoek, 1993) or emergence from a diploid sexual
lineage (Innes & Herbert, 1988; Dybdhal & Lively,
1995); however, the origin of C. a. langsdorfii is not
known.
Though polyploidization enlarges the size of the
cells (Sezaki et al., 1977; Onozato et al., 1983), the
body size of diploid and polyploid vertebrates at
comparative stages of development is almost the
same (Fankhauser, 1945). This fact depends on the
compensation by a decrease of the number of cells
and a change of the shape of cells (Fankhauser,
1945). The enlarged body cell size by polyploidy and
the compensation will affect the physiological activity
in which cell size and cell number may play a role
(Swarup, 1959). For example, the oxygen consumption
of triploid Gastemsteus aculeatus was lower than
that of diploid individuals, suggesting a general fall
in activity in triploids (Swarup, 1959). It may also
give the advese effect in brain and immunologic
mechanism, etc. If the ‘cell size’ hypothesis is true,
the disadvantage in the polyploid asexual form may
be a common phenomenon, because most of the
asexual invertebrates involve polyploidy (Dwaley,
1989; Vrijenhoek et al., 1989).
ACKNOWLEDGEMENTS
We thank members of Natl. Inst. Fish Sci., Ueda
Station. Y. Iwasa, B. Wood and the anonymous referees
provided useful comments on the manuscript. This
work has been supported by CREST (Core Research
for Evolutional Science and Technology) of Japan Science and Technology Corporation (JST) (Principal investigator J. Nakanishi). Partial financial support was
406 H. HAKOYAMA ET AL.
provided by the J a p a n Ministry of Agriculture, For-
estry and Fisheries.
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