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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. REFERENCES . Arnqvist G, Wooster D. 1995. Meta analysis: synthesizing research finding in ecology and evolution. Trends i n Ecology and Evolution 10: 236-240. Bell G. 1982. 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