We sampled 302 water voles for parasite screening and 367 for population genetic analyses. In 2005, the three sites located in the north of the study area had such low population densities that it was not possible to trap any animals over a period of several days.
We tested for antibodies against PUUV, CPXV and LCMV. Seroprevalence results are shown in Table 1. Two coccidian species were observed in the brain (Frenkelia microti and Frenkelia glareoli), four nematodes (T. arvicolae, S. nigeriana, Aonchotheca sp. and Eucoleus bacillus) and four adult cestodes (Anoplocephaloides dentata, Paranoplocephala gracilis, Paranoplocephala omphalodes and Arostrilepis cf horrida) were detected in the digestive tract. Two larval cestodes were observed in the liver (Echinococcus multilocularis and Taenia taeniaeformis) and one in the body cavity (Taenia crassiceps). Detailed prevalence values are shown in Table 1. Individual helminth richness ranged between 0 and 5 and total parasite richness between 0 and 7. As the helminths E. bacillus, T. crassiceps and A. dentata were each found in only one individual (Table 1), we could not include these species in the association analyses.
Table 1. List of viruses and macroparasites identified in the 302 water voles screened.
| Orthopox virus||Cowpox virus||CPXV||60||19.60|
| Arenavirus||Lymphocytic choriomeningitis virus||LCMV||12||3.92|
| Sarcocystidae||Frenkelia glareoli||Cocc-Fg||44||14.38|
| Nematoda||Syphacia nigeriana||Nem-Sn||85||27.77|
| Cestoda larvae||Ecchinococcus multilocularis||Cest-Em||26||8.49|
| Cestoda adults||Anoplocephaloides dentata||Cest-Ad*||1||0.33|
|Arostrilepis cf horrida||Cest-Ah||27||8.82|
We identified five alleles at DQA1 and 12 alleles at DRB in the 367 water voles screened for DNA analyses. All these alleles have previously been detected, cloned and sequenced in water voles (Bryja et al., 2006, 2007). Two DRB alleles (Arte-DRB-12 and Arte-DRB-14) were found at very low frequencies (< 0.01) and so could not be included in association analyses.
Global genotypic differentiation over years and localities was low (FST = 0.017, P < 0.001), especially compared with other A. sherman studies in closed localities (FST estimates ranged between 0.015 and 0.037; Berthier et al., 2006; Bryja et al., 2007).
Co-inertia analysis was first carried out for the 302 individuals screened for both the 19 genetic (five DQA1 alleles, 10 DRB alleles, DQA1, DRB and microsatellite heterozygosity, presence of DQA2) and 15 parasitological variables (three viruses, two coccidias, eight helminth species, helminth and total parasite richness). The binary variable reflecting individual microsatellite heterozygosity was obtained using the proportion of microsatellite locus being heterozygote. This variable thus makes out as ‘high level of heterozygosity’ the individuals having at least eight heterozygous microsatellites out of nine (21% of the individuals). As the choice of the threshold may influence the results, we also performed this analysis considering as ‘highly heterozygote’ the individuals presenting at least six heterozygous loci (76.8% of the individuals).
The CA based on the genetic table (Fig. 2) revealed that the two first axes accounted for 26.1% of the total variance (14.0% F1 and 12.1% F2). The first axis (F1) is structured by the alleles Arte-DQA-06 and Arte-DRB-09 opposed to Arte-DQA-07, Arte-DRB-16 and Arte-DRB-10. The second axis (F2) mainly opposes Arte-DRB-11 to Arte-DRB-13 (and Arte-DRB-15 to a lesser extent). Neither heterozygosity nor duplication makes a major contribution to the structure of this data set. The first two dimensions of the PCA performed on the parasitological table (Fig. 3) accounted for 29.4% of the total variance (18.4% F1 and 11.0% F2). The first axis is structured by the two parasite richness indices: Rp and Rh. The second axis opposes Orthopox virus (CPXV) and Arenavirus (LCMV) and F. glareoli (Cocc-Fg) to the two nematodes located in the caecum, T. arvicolae (Nem-Ta) and S. nigeriana (Nem-Sn).
Figure 2. Correspondence analysis (CA) of the genetic table including 19 variables and 302 voles. The variables DQA1 and DRB alleles coded by name, heterozygosity of DQA1 (Het-DQA1), DRB (Het-DRB) and microsatellites (Het-MS) and DQA duplication (Dupl) are projected on the F1 × F2 map. Variables reflecting genetic diversity are shown in bold and those structuring the data set are underlined using solid (F1) or dotted (F2) lines.
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Figure 3. Principal component analysis (PCA) of the parasitological table including 15 variables and 302 individuals. The variables are projected on the F1 × F2 correlation circle. Nem-, Cest- and Cocc- correspond to nematodes, cestodes and coccidia respectively. The next two letters refer to the code used to identify species (see Table 1). Viruses are coded using CPXV for Orthopox virus, LCMV for Lymphocytic choriomeningitis virus and PUUV for Hantavirus (undetermined species). Rh and Rp correspond to helminth and total parasite richness respectively. Variables structuring the data are underlined: with solid lines for variables structuring F1 and dotted lines for those structuring F2.
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We found no overall relationship between the genetic and parasitological matrices (global co-inertia = 0.112, P = 0.84), although this does not exclude the existence of particular associations. Similar results were obtained when this ACO was carried out with each MHC gene independently (DQA1: P = 0.170 and DRB: P = 0.328).
The two first axes of the ACO accounted for 46.3% of the variance shared between the two matrices (31.2% F1 and 15.1% F2). The genetic variables were plotted on the F1 × F2 ACO factor map (Fig. 4a), and four alleles were identified as principally responsible for co-inertia. All were alleles of the DRB gene (F1: Arte-DRB-11, Arte-DRB-15 and F2: Arte-DRB-07, Arte-DRB-11, Arte-DRB-16). The heterozygosity of DQA1, DRB and microsatellites, and the presence of DQA2, were located very close to the origin on the co-inertia factor maps (Fig. 4a), indicating the absence of linkage between these four variables and parasitism. When considering as ‘high level of heterozygosity’ the individuals having at least six heterozygote loci, similar results were observed, confirming that microsatellite heterozygosity is not associated with parasitism. Richness indices, the nematode T. arvicolae and, to a lesser extent, the two viruses CPXV and LCMV, were all involved in co-inertia (Fig. 4b), and were previously found to structure parasitological data in the PCA. The cestodes P. omphalodes and A. horrida, which were not identified as important in the PCA, strongly structured the co-inertia (first and second axes respectively).
Figure 4. Co-inertia analysis (ACO) of the genetic and parasitological matrices. Projection on common ACO axes of (a) the genetic variables and (b) the parasitological variables, encoded as in Figs 2 and 3 respectively. Variables located in a given direction relative to the origin may be considered to be positively associated (susceptibility), whereas those located in the opposite direction may be considered to be negatively associated (resistance). Associations between genetic and parasite variables visually detected on the F1 axis are underlined with solid lines, whereas those detected on the F2 axis are underlined with dotted lines. Associations shown to be significant by cross-validation are surrounded. Variables reflecting genetic diversity are indicated in bold.
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Superimposing the genetic and parasitological variables on this ACO factor map revealed positive (negative) associations on the first axis between Arte-DRB-11 (Arte-DRB-15) and richness indices, P. omphalodes and T. arvicolae. Another positive association detected on both axes concerned Arte-DRB-11 and A. horrida. The associations detected on the second axis were less important, due to the smaller proportion of the variance accounted for by this axis. Associations were observed between the two viruses CPXV and LCMV and the Arte-DRB-16 and Arte-DRB-10 (positive associations) and Arte-DRB-07 and Arte-DRB-11 (negative) alleles.
We randomly split the data set into two independent data sets, to highlight nonrandom genetic/parasitological associations (Supplementary material). We only confirmed the positive (negative) associations between Arte-DRB-11 and richness indices (Rh and to a lesser extent Rp) and T. arvicolae and the negative association between Arte-DRB-15 and these factors. The ACO analyses performed with the DRB locus only also revealed this strong opposition between Arte-DRB-11 and Arte-DRB-15 (Fig. 5). Individuals carrying Arte-DRB-11 are 1.5 and 6.2 times more likely than other individuals to be infected with at least three parasites or with high loads of T. arvicolae respectively (Table 2). Alternatively, individuals with the Arte-DRB-15 allele have a lower risk of infection with at least three helminths or parasites (by factors of 0.3 and 0.5 respectively). None of the individuals carrying Arte-DRB-15 was infected with high loads of T. arvicolae (Table 2). Note that T. arvicolae intensity was significantly correlated with parasite richness (Spearman, S = 3.1 × 106, P < 10−8, ρ = 0.326) and helminth richness (S = 2.5 × 106, P < 10−8, ρ = 0.456). When removing T. arvicolae from the richness estimates (new variables Rp-T, and Rh-T), these relationships disappeared (Rp-T: S = 5.0 × 106, P = 0.14, and Rh-T: S = 4.5 × 106, P = 0.81). We also performed ACO using Rp-T and Rh-T instead of Rp and Rh. These two variables and T. arvicolae abundance still appeared associated with the alleles Arte-DRB-11 and Arte-DRB-15 (data not shown). However, only the associations with the nematode could be confirmed through cross-validation.
Figure 5. Associations between DRB and parasitological variables projected on the F1 axis of the co-inertia analysis (ACO) based on DRB only. This first axis accounts for 33.1% of the total variance. Grey circles indicate positive associations and white squares, negative associations. The size of the symbol indicates the strength of the association. Variables involved in previously confirmed associations are surrounded.
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Table 2. Correspondence table between alleles and parasitological variables selected by co-inertia analyses.
|Helminth richness Rh|
| ≥ 3||8||36||1||43|
| 1 or 2||28||168||12||184|
| RR (Rh ≥ 3)||1.498|| ||0.282|| |
|Parasite richness Rp|
| ≥ 3||14||64||3||75|
| 1 or 2||22||170||13||179|
| RR (Rp ≥ 3)||1.475|| ||0.485|| |
|Trichuris arvicolae intensity, Nem-Ta|
| ≥ 6||3||3||0||6|
| RR (Nem-Ta ≥ 6)||6.25|| ||0|| |
The other associations found with the whole data set were not confirmed by cross-validation method. Some associations were not observed, regardless of the subset of data considered. This was the case for the association between Arte-DRB-11 (positive) or Arte-DRB-15 (negative) and P. omphalodes, or Arte-DRB-10 (positive) and LCMV or CPXV. Other associations were observed in only one of the two data sets. This was true for the positive associations between Arte-DRB-07 or Arte-DRB-11 and A. horrida (data set 2), and for the positive associations between Arte-DRB-16 and LCMV or CPXV (data set 1).
We performed 815 tests to analyse the linkage disequilibrium between genes (55 pairs of loci × 15 populations minus 10 pairs of loci as one microsatellite was monomorphic in one population). After FDR correction, four tests (0.48%) remained significant. If these remaining significant comparisons were stochastic (random) events, 0.48% of the 55 pairs of loci would correspond to 0.26 cases. Nevertheless, three of the four significant comparisons included DQA1 and DRB, suggesting weak linkage between these two genes.
Significant deviation from mutation–drift equilibrium (Table 3) was detected for DQA1 locus in five populations after Bonferroni correction (all 15 populations had P < 0.05). For DRB locus, only one population stayed significant after Bonferroni correction (four had P < 0.05). Considering microsatellite loci, the mean number of population exhibiting deviation from mutation–drift equilibrium (P < 0.05) was 3.4, ranging from zero (AT3) to eight (AT25). However, P-values were closed to 0.05 (Table 3) and no microsatellite locus differed significantly from mutation–drift equilibrium after Bonferroni correction, whatever the population considered. In this way, DQA1 locus, but not DRB locus, strongly differed from neutral microsatellites in allelic frequency.
Table 3. Detection of overdominance and balancing selection in water vole populations: six sampling sites followed for 3 years.
|Year||Site||N||Heterozygote excess (FIS)||Mutation–drift neutrality tests|
|DQA1||DRB||9 MS||DQA1||DRB||Significant microsatellite loci|
|2003||A||30||−0.077 (0.150) ||−0.110 (0.140) ||0.065 (0.871)||0.009||0.231||AT9 (P = 0.018) AT25 (P = 0.030) AT19 (P = 0.048)|
|B||30||−0.095 (0.249) ||−0.004 (0.497) ||0.018 (0.930)||0.020||0.002||AT13 (P = 0.017) AT22 (P = 0.015) AT9 (P = 0.028) AT25 (P = 0.028)|
|C||26||0.224 (0.980)||0.022 (0.476)||−0.002 (0.509)||0.005||0.145||AT2 (P = 0.026) AT9 (P = 0.034)|
|D||27||0.132 (0.878)||0.120 (0.868)||0.004 (0.463)||0.000||0.034||AT13 (P = 0.047) AT25 (P = 0.029)|
|E||25||0.166 (0.810)||0.006 (0.435)||−0.030 (0.187)||0.024||0.100||AT13 (P = 0.024) AT19 (P = 0.032)|
|F||28||−0.095 (0.316)||−0.107 (0.143)||0.043 (0.958)||0.002||0.152||AT24 (P = 0.029)|
|2004||A||20||0.215 (0.875)||0.164 (0.937)||0.019 (0.951)||0.047||0.114||AT9 (P = 0.005) AT25 (P = 0.005)|
|B||22||0.103 (0.774)||0.079 (0.930)||0.064 (0.975)||0.041||0.183||AT13 (P = 0.018) AT19 (P = 0.010)|
|C||22||0.103 (0.844)||−0.077 (0.187)||0.057 (0.659)||0.001||0.134||AT9 (P = 0.021) AT19 (P = 0.025)|
|D||23||0.215 (0.980)||0.025 (0.511)||0.028 (0.786)||0.017||0.006|| |
|E||21||0.341 (0.997)||−0.128 (0.090) ||0.038 (0.801)||0.009||0.144||AT23 (P = 0.016) AT25 (P = 0.022) AT19 (P = 0.040)|
|F||22||0.091 (0.714)||−0.101 (0.094)||−0.025 (0.398)||0.003||0.004||AT23 (P = 0.048)|
|2005||D||23||0.174 (0.866)||−0.093 (0.243)||−0.018 (0.518)||0.008||0.447||AT13 (P = 0.043) AT25 (P = 0.028)|
|E||24||0.157 (0.959)||0.044 (0.640)||−0.080 (0.015) ||0.006||0.097||AT23 (P = 0.033) AT13 (P = 0.029) AT25 (P = 0.014)|
|F||24||0.343 (0.998)||−0.129 (0.085)||0.016 (0.779)||0.002||0.163||AT25 (P = 0.008) AT19 (P = 0.037)|
Whatever the gene considered, no population deviated from Hardy–Weinberg equilibrium, revealing an absence of significant heterozygosity excess (Table 3).