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Heterogeneities in immune responsiveness may affect key epidemiological parameters and the dynamics of pathogens. The roles of immunogenetics in these variations remain poorly explored. We analysed the influence of Major histocompatibility complex (Mhc) genes and epigamic traits on the response to phytohaemagglutinin in males from cyclic populations of the montane water vole (Arvicola scherman). Besides, we tested the relevance of lateral scent glands as honest signals of male quality. Our results did not corroborate neither the hypotheses of genome-wide heterozygosity-fitness correlation nor the Mhc heterozygote advantage. We found a negative relationship between Mhc hetetozygosity and response to phytohaemagglutinin, mediated by a specific Mhc homozygous genotype. Our results therefore support the hypothesis of the Arte-Dqa-05 homozygous genotype being a ‘good’ Mhc variant in terms of immunogenetic quality. The development of the scent glands seems to be an honest signal for mate choice as it is negatively correlated with helminth load. The ‘good gene’ hypothesis was not validated as Arte-Dqa-05 homozygous males did not exhibit larger glands. Besides, the negative relationship observed between the size of these glands and the response to phytohaemagglutinin, mainly for Mhc homozygotes, corroborates the immunocompetence handicap hypothesis. The Mhc variants associated with larger glands remain yet to be determined.
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Since the 1990s, ecological immunology has addressed the physiological and molecular bases of variations in immune responsiveness by placing immunity in the context of ecology and adaptation (Schulenburg et al. 2009). Understanding these disparities across individuals or species has major implications in evolutionary ecology as immunocompetence probably represents one of the main components of fitness (Lochmiller and Deerenberg 2000). These variations are also important for zoonosis epidemiology. Heterogeneity in host susceptibility may strongly influence key epidemiological parameters such as parasite intensity, transmission or virulence (e.g. Dowel 2001).
Proximal factors influencing variation in immune defences are multiple, including physiological or environmental factors. They have been largely investigated (for rodents, see Nelson et al. 2002). Besides, the influence of genetics on the intensity of the immune response still remains scarcely explored in natural populations. Major histocompatibility complex (Mhc) genes are relevant candidates to address this question. They encode for glycoproteins that recognize antigens, bind peptides derived from them and present them to lymphocyte T cells (Klein 1986). Therefore, they participate in the initiation of the antigen-specific immune response by inducing communication among different cellular components of the immune system (Klein 1986). In particular, antigen presentation via Mhc class II molecules plays a key role in initiating and maintaining cell-mediated and humoral immune responses.
Few empirical studies have provided evidence for the role of Mhc gene polymorphism in the control of differences in immune responsiveness (e.g. Makhatadze et al. 1995; Apanius et al. 1997; Zhou and Lamont 2003; Kurtz et al. 2004). In natural populations, the role of Mhc alleles was investigated rather than that of Mhc heterozygosity (but see Kurtz et al. 2004 for laboratory experiments; Makhatadze et al. 1995 for human studies). It was thus worthy to assess the effects of Mhc gene heterozygosity on immunocompetence. From a theoretical point of view, Doherty and Zinkernagel (1975) suggested that Mhc heterozygotes should exhibit the highest immunocompetence (the ‘heterozygote advantage’ HA hypothesis), in terms of the recognition and elimination of pathogens, because such individuals could present a broader range of pathogen-derived peptides. Then empirical works rather focused on the associations between Mhc heterozygosity and resistance/tolerance to one or multiple parasites than on associations between Mhc heterozygosity and immune responsiveness (e.g. Froeschke and Sommer 2005; de Eyto et al. 2007; Oliver et al. 2009).
Different patterns of relationships between Mhc gene heterozygosity and the intensity of immune response might be observed.
On one hand, positive associations are expected under the models of genome-wide heterozygosity-fitness correlation (HFC) or Mhc heterozygote advantage (HA, Doherty and Zinkernagel 1975). Under the HFC hypothesis, positive associations between heterozygosity estimated at neutral markers and the intensity of immune response should also be observed. Significant positive correlations between estimates of genome-wide heterozygosity and cell-mediated immune responses have been found in bird species and corroborate this hypothesis (Reid et al. 2003; Hawley et al. 2005; Hale and Briskie 2007). Several mechanisms, including the decline of host immunity with inbreeding, may underlie HFC patterns (O’Brien and Evermann 1988; Coltman et al. 1999; Keller and Waller 2002; Altizer et al. 2003). Conversely, under the HA hypothesis, no expectation can be made on the association between heterozygosity estimated at neutral markers and the intensity of immune response.
On the other hand, associations might be observed between specific Mhc alleles and the intensity of immune response (e.g. Makhatadze et al. 1995; Bonneaud et al. 2005). Such relationships can be expected under ‘good-genes’ models of female mate choice (Trivers 1972; Mays and Hill 2004; Neff and Pitcher 2005). Males bearing ‘good’ alleles, i.e. alleles that increase individual fitness (Andersson 1994) by conferring higher immunocompetence for example, should be preferred. Benefits of such mating are multiple and include direct advantages, i.e. the avoidance of parasitized individuals, as well as indirect ones, i.e. the transmission of these ‘good alleles’ to offsprings (Hamilton and Zuk 1982). Under this scenario, we might expect females to seek mates carrying specific Mhc alleles, locally adapted against prevalent pathogens or associated with higher immune responses. Assuming that mates homozygous for such ‘good’ alleles would be even more favoured (e.g. in Salmo salar, Langefors et al. 2001), we would expect a negative relationship between Mhc gene heterozygosity and the intensity of immune response. This relationship would hence only be driven by those particular ‘good’ Mhc alleles.
Next, studying the relationships between the development of a secondary sexual character and immune responses might provide complementary insights into the mechanisms driving these associations between Mhc gene and immune response.
On one hand, a negative relationship between a secondary sexual trait and immune responses would support the immunocompetence handicap hypothesis (ICHH; Folstad and Karter 1992; Wedekind and Folstad 1994). The ICHH postulates that only males carrying genetic characteristics associated with superior immunocompetence/better disease resistance might afford to allocate more resources to costly ornament traits at the expense of the immune function (Hamilton and Zuk 1982; Folstad and Karter 1992; Wedekind and Folstad 1994). This hypothesis relies on Zahavi’s handicap theory, which proposes that individuals that express male epigamic traits are handicapped by a reduced immune response (Zahavi 1975). In vertebrates, this handicap is linked to testosterone, the primary male sex hormone, which is required for the production of many morphological sexual characters, and has a suppressive effect on the immune system (Zuk 1996).
On the other hand, a positive relationship between an honest secondary sexual trait and immune responses would support the ‘good genes’ hypothesis. Those males carrying ‘good genes’, especially for parasite resistance, would theoretically be able to afford to invest both in immunity and in secondary sexual traits.
The montane water vole Arvicola scherman (Rodentia, Cricetidae, Arvicolinae) is an interesting organism for investigating these hypotheses (HFC, HA, Good genes and ICHH). This rodent exhibits regular 5- to 8-year dynamic cycles (Saucy 1994). It is considered as a pest in Western Europe because outbreaks are associated with extensive damages for agriculture. It is also a reservoir for three important (re)-emerging viral zoonoses in Europe, caused by hantaviruses, orthopoxviruses and arenaviruses (Charbonnel et al. 2008b) and for three agents of priority zoonoses (Echinococcus multilocularis, Leptospirosa sp., Toxoplasma gondi, refs in Giraudoux et al. 2008). Six geographically close localities were sampled during 3 years corresponding to the outbreak and decline phases of A. scherman abundance cycles. Previous population genetic studies have shown that phases of increasing abundance and outbreak during A. scherman cycles were associated with increase in effective size and migration between populations (Bryja et al. 2007). Consequently, these phases were characterized by low spatial and temporal genetic differentiation.
We used the Mhc genotypes characterized at two class II genes (Dqa and Drb) and published in Tollenaere et al. (2008). We estimated immune responsiveness as the magnitude of cell-mediated immune response using a challenge with phytohaemagglutinin. We measured the flank gland, which is considered as a secondary sexual character of voles (Quay 1968; Jannett 1986). Several arguments support the presumption of flank glands being a male honest sexual signalling in montane water voles: these glands regress after castration and develop in response to exogenous testosterone (Stoddart 1972), they go through an annual activity cycle where the maximal output coincides with the breeding season (Stoddart 1972), during which the dominant males defend their territories against intruders. Finally, the chemical composition of these glands varies between social statuses (Stoddart et al. 1975). The glands produce a characteristic odour (Frank 1956). The results of field and laboratory experiments showed that females preferred the odour of dominant males to that of subordinates ones (Evsikov et al. 1994) and that high-ranking males gained reproductive advantage (Evsikov et al. 1997).
Following the reasoning detailed above, we assessed the first prediction of an association between male response to phytohaemagglutinin and Mhc gene heterozygosity, either positive (HFC, HA hypotheses) or negative (‘good genes’ hypothesis and association between Mhc alleles and response to phytohaemagglutinin). Next, we tested the pertinence of scent glands as honest signals of male quality in terms of parasite load. We then investigated the associations between the development of these glands and response to phytohaemagglutinin to analyse the relative influence of the immunocompetence handicap and ‘good genes’ hypotheses.
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This study relies on 150 males sampled between 2003 and 2005 at six localities. Because of low abundance, no voles could be sampled at localities A, B and C in 2005.
Analyses on within sample genetic structure confirmed the absence of kin groups or related individuals that could have biased our results. FIS estimates were low and not significant, as well as relatedness coefficients (Table 1).
Table 1. Within population genetic characteristics estimated over all microsatellites for the different sites and years of sampling. FIS estimates and the associated exact test probability of Hardy–Weinberg equilibrium HW (P) are provided. The relatedness coefficients are estimated following Wang (2002) and the standard error (SE) is computed from a jackknife procedure over all loci.
|Year and site of sampling||Microsatellite FIS||HW (P)||Relatedness coefficient, mean (SE)|
| A||0.065||0.871||−0.0158 (0.0164)|
| B||0.018||0.930||0.0217 (0.0297)|
| C||−0.002||0.509||0.0196 (0.0361)|
| D||0.004||0.463||0.019 (0.031)|
| E||−0.030||0.187||0.0132 (0.0304)|
| F||0.043||0.958||0.0219 (0.0323)|
| A||0.019||0.951||−0.0138 (0.0125)|
| B||0.064||0.975||−0.0195 (0.0128)|
| C7||0.057||0.659||−0.0137 (0.0185)|
| D5||0.028||0.786||−0.0139 (0.0128)|
| E||0.038||0.801||0.0101 (0.0159)|
| F||−0.025||0.398||−0.0091 (0.0140)|
| D||−0.018||0.518||0.0143 (0.0173)|
| E||−0.080||0.015||0.0012 (0.0133)|
| F||0.016||0.779||0.0245 (0.0149)|
An extreme value was identified (PHA = 1.4 cm, next highest values are PHA = 1.28 cm, PHA = 1.26, PHA = 1.22). We decided to remove it as it was highly nonrepresentative (10% more than the next values) and could strongly bias the modelling process. We nevertheless checked that including this point would not have modified our conclusions but only the probabilities associated with our results. After the selection procedure, the final model was defined as follow: PHA∼Drb-dist + Dqa1-het + Sgld + Dqa1-het.Sgld + Age + Year (Table 2). Genome wide heterozygosity had no effect on PHA. A clear Mhc effect was observed, with voles homozygous at Dqa1 having higher levels of response to phytohaemagglutinin than heterozygotes (P = 0.046, Fig. 2). This result was also found for the Drb locus, but in a lesser extent: heterozygotes with highly distant allele sequences had lower response to phytohaemagglutinin than homozygotes or heterozygotes with more similar allele sequences (P = 0.044). A negative association was found between the response to phytohaemagglutinin and the surface of the flank gland. A tendency was observed for the whole dataset, and this was significant when considering Dqa1 homozygotes, as indicated by the significant interaction observed between the surface of the flank gland and the Dqa1 heterozygosity (P = 0.039, Fig. 3). The response to phytohaemagglutinin was also positively correlated with age (P < 10−4) and a temporal effect was detected where responses to phytohaemagglutinin were higher in 2005 than in 2004, in 2004 than in 2003 and in 2005 than in 2003 (all P < 10−4).
Table 2. Summary of the retained terms and coefficients (standard errors and probabilities) of the selected models (AIC = 145.83, % variance = 42.7, F12,130 = 9.83, P < 10−4).
|Dqa1-het||Homozygote vs. heterozygote||−0.0881 (0.0418)||−1.94||0.03|
|Drb-dist|| ||−0.0043 (0.0023)||−2.04||0.04|
|Sgland|| ||−0.0006 (0.0005)||−1.05||0.31|
|Dqa1-het.Sgland|| ||0.0016 (0.0007)||2.08||0.03|
|Age|| ||0.0094 (0.0026)||3.26||<10−4|
|Year||2003 vs. 2004||0.1741 (0.0257)||6.75||<10−4|
|2003 vs. 2005||0.2088 (0.0302)||7.11||<10−4|
|2004 vs. 2005||0.0348 (0.0276)||2.12||<10−4|
Figure 2. Relationship between response to phytohaemagglutinin and Dqa1 heterozygosity (Dqa1-het). The quantile box-plot indicates the mean and its 95% confidence interval. The cross symbol refers to the individual that was considered as an outlier and removed from the analyses.
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Figure 3. Relationship between response to phytohaemagglutinin and the surface of the flank gland for Dqa1 homozygotes (black squares and black line) and Dqa1 heterozygotes (grey triangles and grey line).
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The influence of collinearity between Year and Sgland was assessed by refitting the model without the covariable Year. This did not affect the directionality of the coefficient for Sgland. However, we observed that removing the covariable Year made the variable Sgland become significant (P = 0.032) and decreased the significance level of the P-value coefficient associated with the interaction Dqa1-het.Sgland (P = 0.063). We thus decided to retain both variables Year and Sgland in the selected model.
No significant relationships were observed between response to phytohaemagglutinin and Dqa1 alleles (P > 0.05/5 for all tests). Dqa1 genotypes significantly explained response to phytohaemagglutinin (ANOVA, F14,134 = 2.374, P = 0.005). Using the Tukey–Kramer HSD test, we showed that this result was mediated by the Arte-Dqa-05/Arte-Dqa-05 genotype, which exhibited significantly higher levels of response to phytohaemagglutinin than other Dqa1 genotypes (see Fig. 4).
Figure 4. Relationship between response to phytohaemagglutinin and Dqa1 genotypes. Error bars represent ±1SE of the mean. Stars indicate the five Arte-Dqa genotypes exhibiting significantly lower levels of PHA than the homozygous Arte-Dqa-05 genotype using post-hoc Tukey–Kramer tests.
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Helminth data were available for 131 voles only. Four nematodes (Trichuris arvicolae, Syphacia nigeriana, Aonchotheca sp. and Eucoleus bacillus) and four adult cestodes (Anoplocephaloides dentata, Paranoplocephala gracilis, Paranoplocephala omphalodes and Arostrilepis horrida) have been identified. Helminth richness ranged between 0 and 5. A significant relationship was observed between the surface of the flank gland Sgland and the specific richness in helminth (F4,126 = 3.091, P = 0.018). The gland of unparasitized males was more developed than the gland of males harbouring more helminths.