Gill Transcriptome Analysis Revealed the Difference in Gene Expression Between Freshwater and Seawater Acclimated Guppy (Poecilia reticulata).

Guppy (Poecilia reticulata) can adapt to a wide range of salinity changes. To investigate the gene expression changes in the guppy exposed to seawater, we characterized its gill transcriptome using RNA sequencing. Experimental fish were exposed to salinity increase from 0 to 30‰ within 4 days, while control fish were cultured in freshwater (0‰ salinity). Seven days after salinity exposure, the gills were sampled and the mortality within 2 weeks was recorded. No significant difference in the cumulative mortality at the second week was found between the two groups. Transcriptomic analysis identified 3477 differentially expressed genes (DEGs), including 1067 upregulated and 2410 downregulated genes. These DEGs were enriched in several biological processes, including ion transport, ion homeostasis, ATP biosynthetic process, metabolic process, and immune system process. Oxidative phosphorylation was the most activated pathway. DEGs involved in the pathway "endoplasmic reticulum (ER)-mediated phagocytosis," "starch and sucrose metabolism," and "steroid biosynthesis" were mainly downregulated; chemokines and interleukins involved in "cytokine-cytokine receptor interaction" were differentially expressed. The present results suggested that oxidative phosphorylation had essential roles in osmoregulation in the gills of seawater acclimated guppy, during which the decline in the expression of genes encoding V-ATPases and calreticulin had a negative effect on the phagocytosis and immune response. Besides, several metabolic processes including "starch and sucrose metabolism" and "steroid biosynthesis" were affected. This study elucidates transcriptomic changes in osmotic regulation, metabolism, and immunity in seawater acclimated guppy.

Molecular insights into post-mating immune response in a fish with internal fertilization.

The tight connection between immunity and reproduction has been studied for decades. However, basic knowledge at the molecular level of the effect of mating on immune function is still lacking in many taxa. Determining whether and how the immune system is engaged after mating is a crucial step in understanding post-mating mechanisms of reproduction and sexual selection. Here, we study the transcriptional changes in immunity-related genes caused by the ejaculate in the female reproductive tract using a model species for sexual selection studies, the guppy Poecilia reticulata. To study changes triggered by the ejaculate only, rather than caused by mating, we used artificial inseminations to transfer ejaculate into females. We then compared gene expression in the reproductive tract (gonoduct and ovary) of females artificially inseminated either with ejaculate or with a control solution, after 1 hr and after 6 hr. Overall, contact with ejaculate caused short-term changes in the expression of immune-related genes in the female reproductive tract, with a complex pattern of up- and down-regulation of immune-related pathways, but with clear indication of a marked down-regulation of the immune system shortly after ejaculate contact. This suggests a link between immune function and processes occurring between mating and fertilization in this species.

Non-specific immunity promotion in response to garlic extract supplemented diets in female Guppy (Poecilia reticulata).

In the present study, the effect of aqueous garlic (Allium sativum) extract on skin mucus immune parameters of Poecilia reticulata was evaluated. A total of 240 P. reticulate juveniles, weighing 0.013 ± 0.001 g, were randomly stocked in 12 experimental glass tanks at a density of 30 fish per tank. The fish were fed with diets supplemented with garlic extract at the concentrations of 0, 0.10, 0.15, and 0.20 ml kg-1 of diet three times a day at a ratio of 2.5% of their body weight for 80 days. Results of the study showed that, lysozyme activity increased significantly in the skin mucus of garlic-treated Guppy (P < 0.05). Values of ACH50 significantly increased (P < 0.05) in fish by increasing concentration of garlic extract from 0 to 0.15 ml kg-1 in diet. ACH50 also showed a significant declining trend (P < 0.05) by increasing concentration of garlic extract from 0.15 to 0.20 ml kg-1. Total Ig content was enhanced significantly in 0.15 and 0.20 ml kg-1 treatments as well. ALP activity significantly increased in garlic-fed treatments compared to the control. Inclusion of garlic extract in diet was found to have no significant effect on final body weight and weight gain. Results revealed that, administration of 0.15 mL of garlic extract per kg feed is suggested to obtain optimal skin mucus immunity in P. reticulata.

Analyzing complement activity in the serum and body homogenates of different fish species, using rabbit and sheep red blood cells.

Alternative complement activity was determined in whole body homogenates (WBHs) and serum samples of different fish species, by measuring the amount of sample that induces 50% hemolysis of red blood cells using the ACH50 assay (Alternative Complement pathway Hemolytic activity). Values of ACH50 obtained for serum samples were about two-fold higher when using rabbit red blood cells (RRBC), as compared to sheep red blood cells (SRBC). The increase in ACH50 when using RRBCs for WBH samples was 28, 7 and 4 folds for guppy, molly and zebrafish, respectively. Large variability in complement activity was evident between fish species for both serum and WBHs. Evaluating the effect of freeze-thaw cycles on complement revealed significant reduction in complement activity in all tested samples. Loss of activity following three freeze-thaw cycles amounted to 48-59% when serum was tested and over 95% loss in activity for WBH. To our knowledge, this is the first study where fish WBHs were used for assaying complement activity. Our results support the suitability of this method in evaluating complement activity in small fish species or larvae, where blood cannot be obtained, as long as samples can be tested upon first thawing.

Evolutionary genetics of immunological supertypes reveals two faces of the Red Queen.

Red Queen host-parasite co-evolution can drive adaptations of immune genes by positive selection that erodes genetic variation (Red Queen arms race) or results in a balanced polymorphism (Red Queen dynamics) and long-term preservation of genetic variation (trans-species polymorphism). These two Red Queen processes are opposite extremes of the co-evolutionary spectrum. Here we show that both Red Queen processes can operate simultaneously by analysing the major histocompatibility complex (MHC) in guppies (Poecilia reticulata and P. obscura) and swamp guppies (Micropoecilia picta). Sub-functionalisation of MHC alleles into 'supertypes' explains how polymorphisms persist during rapid host-parasite co-evolution. Simulations show the maintenance of supertypes as balanced polymorphisms, consistent with Red Queen dynamics, whereas alleles within supertypes are subject to positive selection in a Red Queen arms race. Building on the divergent allele advantage hypothesis, we show that functional aspects of allelic diversity help to elucidate the evolution of polymorphic genes involved in Red Queen co-evolution.

Selection for brain size impairs innate, but not adaptive immune responses.

Both the brain and the immune system are energetically demanding organs, and when natural selection favours increased investment into one, then the size or performance of the other should be reduced. While comparative analyses have attempted to test this potential evolutionary trade-off, the results remain inconclusive. To test this hypothesis, we compared the tissue graft rejection (an assay for measuring innate and acquired immune responses) in guppies (Poecilia reticulata) artificially selected for large and small relative brain size. Individual scales were transplanted between pairs of fish, creating reciprocal allografts, and the rejection reaction was scored over 8 days (before acquired immunity develops). Acquired immune responses were tested two weeks later, when the same pairs of fish received a second set of allografts and were scored again. Compared with large-brained animals, small-brained animals of both sexes mounted a significantly stronger rejection response to the first allograft. The rejection response to the second set of allografts did not differ between large- and small-brained fish. Our results show that selection for large brain size reduced innate immune responses to an allograft, which supports the hypothesis that there is a selective trade-off between investing into brain size and innate immunity.

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection.

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non-stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 µg per mg protein) compared with CI-, NI- and I-treated groups (94.5, 64 and 62.3 µg mg(-1), respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re-infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3-75%, higher than in the non-exposed control (40.6% mortality).

Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure.

Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre-eminent system for the study of selective pressures that arise from host-pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population-genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.

Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA).

Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse anti-guppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10 µL) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund's adjuvant (15,000 Tetrahymena/fish in total a volume of 20 µL). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from pathogen challenge by immersion. The results suggest a direct correlation between the anti-Tetrahymena Ab response and fish resistance to i.p.-injected Tetrahymena, but not to infection by immersion preceded by cold stress.

Selection from parasites favours immunogenetic diversity but not divergence among locally adapted host populations.

The unprecedented polymorphism in the major histocompatibility complex (MHC) genes is thought to be maintained by balancing selection from parasites. However, do parasites also drive divergence at MHC loci between host populations, or do the effects of balancing selection maintain similarities among populations? We examined MHC variation in populations of the livebearing fish Poecilia mexicana and characterized their parasite communities. Poecilia mexicana populations in the Cueva del Azufre system are locally adapted to darkness and the presence of toxic hydrogen sulphide, representing highly divergent ecotypes or incipient species. Parasite communities differed significantly across populations, and populations with higher parasite loads had higher levels of diversity at class II MHC genes. However, despite different parasite communities, marked divergence in adaptive traits and in neutral genetic markers, we found MHC alleles to be remarkably similar among host populations. Our findings indicate that balancing selection from parasites maintains immunogenetic diversity of hosts, but this process does not promote MHC divergence in this system. On the contrary, we suggest that balancing selection on immunogenetic loci may outweigh divergent selection causing divergence, thereby hindering host divergence and speciation. Our findings support the hypothesis that balancing selection maintains MHC similarities among lineages during and after speciation (trans-species evolution).

The effects of elevated temperature on the sexual traits, immunology and survivorship of a tropical ectotherm.

In 2007, the Intergovernmental Panel on Climate Change projected an average global air temperature increase of 1.1-6.4°C by the end of the 21st century. Although the tropics are predicted to experience less extreme temperature increases than regions of higher latitude, tropical ectotherms live close to their thermal limits, and are thus particularly vulnerable to increases in temperature. In this study, we examined how predicted patterns of global warming will affect survival and sexual traits in the Trinidadian guppy (Poecilia reticulata). Guppies were exposed from birth to one of four temperature treatments: 23, 25 (control), 28 or 30°C. We measured brood survival and, at sexual maturity, male ornamentation, sperm traits and immune response. Our results show that increases in temperature result in guppies that have shorter, slower sperm but that there is an optimum temperature for ornamental hue at 28°C. Given the importance of sperm quality for reproduction, these results suggest population viability could be affected by warming. However, we found no difference in brood survival or immune response to a novel antigen across the treatments, indicating that survival may not be as vulnerable as previously thought. Overall, our data suggest that male sexual traits, and in particular sperm performance, are more sensitive than survival to a warming environment.

Inference of selection based on temporal genetic differentiation in the study of highly polymorphic multigene families.

The co-evolutionary arms race between host immune genes and parasite virulence genes is known as Red Queen dynamics. Temporal fluctuations in allele frequencies, or the 'turnover' of alleles at immune genes, are concordant with predictions of the Red Queen hypothesis. Such observations are often taken as evidence of host-parasite co-evolution. Here, we use computer simulations of the Major Histocompatibility Complex (MHC) of guppies (Poecilia reticulata) to study the turnover rate of alleles (temporal genetic differentiation, G'(ST)). Temporal fluctuations in MHC allele frequencies can be ≥≤order of magnitude larger than changes observed at neutral loci. Although such large fluctuations in the MHC are consistent with Red Queen dynamics, simulations show that other demographic and population genetic processes can account for this observation, these include: (1) overdominant selection, (2) fluctuating population size within a metapopulation, and (3) the number of novel MHC alleles introduced by immigrants when there are multiple duplicated genes. Synergy between these forces combined with migration rate and the effective population size can drive the rapid turnover in MHC alleles. We posit that rapid allelic turnover is an inherent property of highly polymorphic multigene families and that it cannot be taken as evidence of Red Queen dynamics. Furthermore, combining temporal samples in spatial F(ST) outlier analysis may obscure the signal of selection.

Major histocompatibility complex variability in the clonal Amazon molly, Poecilia formosa: is copy number less important than genotype?

The evolution of sex is still a major unsolved puzzle in biology. One of the most promising theoretical models to answer this question is the Red Queen hypothesis. The Red Queen hypothesis proposes a fast adaptation of pathogens to common genotypes and therefore a negative frequency-dependent selection against common genotypes. Clonal organisms should be especially endangered when co-occurring with closely related sexual species. In this context, major histocompatibility (MHC) genes have been discussed to be auspicious candidates that could provide the genetic basis on which selection for immune competence could act. In this study, we investigated MHC variability in a clonal teleost fish: the Amazon molly, Poecilia formosa. The Amazon molly is an ideal candidate to test the Red Queen hypothesis as it is a clonal species but co-occurs with a closely related sexual species and should therefore be especially susceptible to pathogen infection. We found that allele numbers did in general not differ between sexual and clonal 'species' but that genotypic variability is reduced in the clonally reproducing fish, especially in the polyploids. We conclude that in clonal organisms, genotype frequency might be more important for immune competence than MHC allele number. Amazon mollies and their co-occurring parental species clearly fulfil a prerequisite of the Red Queen hypothesis and should therefore provide an ideal system to experimentally test this basic principle probably underlying the evolution of sex.

Localization of tyrosine hydroxylase immunoreactive neurons in the forebrain of the guppy Poecilia reticulata.

The current study reports for the first time the distribution of tyrosine hydroxylase immunoreactive (TH-ir) neurons in the forebrain of the guppy Poecilia reticulata. Numerous small TH-ir neurons were observed in the olfactory bulbs, located mainly in the periphery of the bulbs. The TH-ir telencephalic neurons are localized in the ventral telencephalic area where they are grouped in three distinct nuclei (Vv,Vd and Vp) composed of a small number of cells forming a continuous strip. The largest number of forebrain TH-ir neurons was observed in the diencephalon where both small and larger neurons are present. Diencephalic TH-ir neurons are subdivided in large nuclei located in the preoptic region (nSC, nPOp and nPOm), the thalamus (nDM), the pretectal region (nPPv and nAP), the hypothalamus (nPP and nRP) and the posterior tuberculum (nPT). Many diencephalic nuclei are distributed in periventricular regions and no TH-ir cells were observed in the paraventricular organ. A comparative analysis indicates that the present observations are consistent with the general pattern of TH-ir neurons distribution reported for the forebrain of other teleosts, but with some interspecies variability present, mainly in the diencephalon. This paper also provides valuable neuroanatomical information for P. reticulata, a teleost frequently used in toxicological tests, for future studies investigating the effects of environmental pollutants on the catecholaminergic system.

Polymorphic MHC loci in an asexual fish, the amazon molly (Poecilia formosa; Poeciliidae).

Genes of the major histocompatibility complex (MHC) encode molecules that control immune recognition and are highly polymorphic in most vertebrates. The remarkable polymorphisms at MHC loci may be maintained by selection from parasites, sexual selection, or both. If asexual species show equal (or higher) levels of polymorphisms at MHC loci as sexual ones, this would mean that sexual selection is not necessary to explain the high levels of diversity at MHC loci. In this study, we surveyed the MHC diversity of the asexual amazon molly (Poecilia formosa) and one of its sexual ancestors, the sailfin molly (P. latipinna), which lives in the same habitat. We found that the asexual molly has polymorphic MHC loci despite its clonal reproduction, yet not as polymorphic as the sexual species. Although the nucleotide diversity was similar between the asexual and sexual species, the sexual species exhibited a greater genotypic diversity compared to the asexual one from the same habitats. Within-genome diversity was similar for MHC class I loci, but for class IIB, the sexual species had higher diversity compared to the asexual--despite the hybrid origins and higher levels of heterozygosity at microsatellite loci in the asexual species. The level of positive selection appears to be similar between the two species, which suggests that these polymorphisms are maintained by selection. Thus, our findings do not allow us to rule out the sexual selection hypothesis for the evolution of MHC diversity, and although the sexual fish has higher levels of MHC-diversity compared to the asexual species, this may be due to differences in demography, parasites, or other factors, rather than sexual selection.

Gyro-scope: an individual-based computer model to forecast gyrodactylid infections on fish hosts.

Individual-based computer models (IBM) feature prominently in current theoretical ecology but have only been applied in a small number of parasitological studies. Here we designed an IBM to simulate the infection dynamics of gyrodactylid parasites and immune defence of naïve hosts (i.e. fish previously not exposed to these parasites). We compared the results of the model with empirical data from guppies (Poecilia reticulata) infected with Gyrodactylus parasites. The laboratory experiments on guppies showed that larger fish acquired a heavier parasite load at the peak of the infection. The survival probability declined with increased body size and no fish survived a parasite load of 80 or more worms in this experiment (i.e. lethal load). The model was a good predictor of the Gyrodactylus infection dynamics of guppies and the model output was congruent with previously published data on Gyrodactylus salaris infections of salmon (Salmo salar). Computer simulations indicated that the infections persisted longer on larger hosts and that the parasite load increased exponentially with the body size of the host. Simulations furthermore predicted that the parasite load of fish with a standard length in excess of 17mm (i.e. the size of adult guppies) reached a lethal load. This suggests that in the conditions of the experiment, the immune defence of naïve guppies can offer moderate protection against gyrodactylid infections to juveniles, but not to naïve adult guppies. The model is a useful tool to forecast the development of gyrodactylid infections on single hosts and make predictions about optimal life history strategies of parasites.

Balancing selection, random genetic drift, and genetic variation at the major histocompatibility complex in two wild populations of guppies (Poecilia reticulata).

Our understanding of the evolution of genes of the major histocompatibility complex (MHC) is rapidly increasing, but there are still enigmatic questions remaining, particularly regarding the maintenance of high levels of MHC polymorphisms in small, isolated populations. Here, we analyze the genetic variation at eight microsatellite loci and sequence variation at exon 2 of the MHC class IIB (DAB) genes in two wild populations of the Trinidadian guppy, Poecilia reticulata. We compare the genetic variation of a small (Ne, 100) and relatively isolated upland population to that of its much larger (Ne approximately 2400) downstream counterpart. As predicted, microsatellite diversity in the upland population is significantly lower and highly differentiated from the population further downstream. Surprisingly, however, these guppy populations are not differentiated by MHC genetic variation and show very similar levels of allelic richness. Computer simulations indicate that the observed level of genetic variation can be maintained with overdominant selection acting at three DAB loci. The selection coefficients differ dramatically between the upland (s > or = 0.2) and lowland (s < or = 0.01) populations. Parasitological analysis on wild-caught fish shows that parasite load is significantly higher on upland than on lowland fish, which suggests that large differences in selection intensity may indeed exist between populations. Based on the infection intensity, a substantial proportion of the upland fish would have suffered direct or indirect fitness consequences as a result of their high parasite loads. Selection by parasites plays a particularly important role in the evolution of guppies in the upland habitat, which has resulted in high levels of MHC diversity being maintained in this population despite considerable genetic drift.

Sex-specific effects of carotenoid intake on the immunological response to allografts in guppies (Poecilia reticulata).

Rarely are the evolutionary origins of mate preferences known, but, recently, the preference of female guppies (Poecilia reticulata) for males with carotenoid-based sexual coloration has been linked to a sensory bias that may have originally evolved for detecting carotenoid-rich fruits. If carotenoids enhance the immune systems of these fishes, as has been suggested for other species, this could explain the origin of the attraction to orange fruits as well as the maintenance of the female preference for orange males. We used the classic immunological technique of tissue grafting to assay a component of the immune response of guppies raised on two different dietary levels of carotenoids. Individual scales were transplanted between pairs of unrelated fishes, creating reciprocal allografts. Transplanted scales were scored on a six-point rejection scale every day for 10 days. Five days later, the same pairs of fishes received a second set of allografts and were scored again. Compared with low-carotenoid-diet males, high-carotenoid-diet males mounted a significantly stronger rejection response to the second allograft but not to the first allograft. High-carotenoid-diet females, however, showed no improvement in graft rejection compared with low-carotenoid-diet females. To our knowledge, this is the first experimental evidence for sex-specific effects of carotenoid consumption on the immune system of a species with carotenoid-based sexual coloration. These results are consistent with the hypothesis that the mate preference for carotenoid coloration is maintained by the benefits to females of choosing healthy mates, but they cast doubt on the idea that the benefits of carotenoid consumption, per se, could account for the origin of the preference. The sex-specificity of carotenoid effects on allograft rejection in guppies provides indirect support for the general hypothesis that males pay an immunological cost for sexual ornamentation.

Sex-specific effects of carotenoid intake on the immunological response to allografts in guppies (Poecilia reticulata)

Rarely are the evolutionary origins of mate preferences known, but, recently, the preference of female guppies (Poecilia reticulata) for males with carotenoid-based sexual coloration has been linked to a sensory bias that may have originally evolved for detecting carotenoid-rich fruits. If carotenoids enhance the immune systems of these fishes, as has been suggested for other species, this could explain the origin of the attraction to orange fruits as well as the maintenance of the female preference for orange males. We used the classic immunological technique of tissue grafting to assay a component of the immune response of guppies raised on two different dietary levels of carotenoids. Individual scales were transplanted between pairs of unrelated fishes, creating reciprocal allografts. Transplanted scales were scored on a six-point rejection scale every day for 10 days. Five days later, the same pairs of fishes received a second set of allografts and were scored again. Compared with low-carotenoid-diet males, high-carotenoid-diet males mounted a significantly stronger rejection response to the second allograft but not to the first allograft. High-carotenoid-diet females, however, showed no improvement in graft rejection compared with low-carotenoid-diet females. To our knowledge, this is the first experimental evidence for sex-specific effects of carotenoid consumption on the immune system of a species with carotenoid-based sexual coloration. These results are consistent with the hypothesis that the mate preference for carotenoid coloration is maintained by the benefits to females of choosing healthy mates, but they cast doubt on the idea that the benefits of carotenoid consumption, per se, could account for the origin of the preference. The sex-specificity of carotenoid effects on allograft rejection in guppies provides indirect support for the general hypothesis that males pay an immunological cost for sexual ornamentation.

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes.

In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked.