Infectious disease represents an emerging threat to natural populations, particularly when hosts are more susceptible to novel parasites (allopatric) than to parasites from the local area (sympatric). This pattern could arise through evolutionary processes (host populations become adapted to their local parasites and genetically differentiated from other populations at immune-related loci) and/or through ecological interactions (host individuals develop resistance to local parasites through previous exposure). The relative importance of these candidate mechanisms remains unclear. In jawed vertebrates, genes of the major histocompatibility complex (MHC) play a fundamental role in immunity and are compelling candidates for spatially varying selection. We recently showed that song sparrows (Melospiza melodia) are more susceptible to allopatric than to sympatric strains of malaria (Plasmodium). In the current study, to determine whether population differences at MHC explain this pattern, we characterized the peptide-binding regions of MHC (classes I and II) of birds that did or did not become infected in the previous experiment. We recovered up to 4 alleles per individual at class I, implying at least 2 loci, and up to 26 alleles per individual at class II, implying at least 13 loci. Individuals with more class I alleles were less likely to become infected by Plasmodium, consistent with parasite-mediated balancing selection. However, we found no evidence for population genetic differentiation at either class of MHC, based on 36 individuals sequenced. Resistance to sympatric parasites previously described for this system likely stems from individuals’ prior immune experience, not from population differentiation and locally protective alleles at MHC.
We characterized MHC genotypes of 36 song sparrows previously used in a cross-infection experiment (Sarquis-Adamson and MacDougall-Shackleton 2016). Subjects in the cross-infection study were captured at their summer breeding grounds in Ontario, Canada during late summer and early fall (July–October). Nineteen birds were captured at a site in eastern Ontario (44°38′38.77″N, 76°20′4.86″W), and 17 at a site 440 km away in western Ontario (43°00′34.00″N, 81°16′52.50″W). Song sparrows are abundant in both these locations, and neither site was physically isolated from species-suitable breeding habitat (old fields and wetlands), thus we assume that neither population is currently isolated from other surrounding populations. We collected a small blood sample from each bird’s brachial vein for genetic analysis, and then housed the birds indoors in individual cages with ad libitum access to food and water under ambient photoperiod.
Details of the cross-infection experiment are available elsewhere (Sarquis-Adamson and MacDougall-Shackleton 2016), but in brief, we used nested PCR to amplify and sequence hematozoan mitochondrial DNA (Hellgren et al. 2004) and identified lineages of Plasmodium that were apparently confined only to the eastern or only to the western site. Lineage P-SOSP9 (GenBank accession #KT19635; 99% sequence identity to morphospecies P. relictum) was found only in birds from the eastern site, and lineage P-SOSP10 (GenBank accession #KT19636; 99% sequence identity to morphospecies P. homopolare) was found only in birds from the western site. Additional screening of over 300 song sparrows at and around the eastern site confirmed the absence of P-SOSP10 from this location, although comparable screening at and around the western site to confirm the absence of P-SOSP9 was not conducted (Sarquis-Adamson and MacDougall-Shackleton 2016). Although several other lineages of Plasmodium were detected, these occurred at both the eastern and western sites. We thus used P-SOSP9 and P-SOSP10 as our eastern and western lineages, respectively. DNA sequence divergence between these 2 experimental lineages was 8%. All of the other lineages detected within experimental subjects upon capture showed at least 5.2% sequence divergence to both experimental lineages (GenBank accession #KT193627-193634 and KT19637; Sarquis-Adamson and MacDougall-Shackleton 2016).
We inoculated a suspension of whole blood from an eastern bird infected with P-SOSP9 into 2 previously uninfected “amplifiers” (i.e., birds inoculated with parasites, allowed to develop an acute infection, then used to inoculate experimental subjects) from the eastern site. Similarly, 2 previously uninfected amplifiers from the western site were inoculated with a suspension of whole blood from a western bird infected with P-SOSP10 (details in Sarquis-Adamson and MacDougall-Shackleton 2016). At 18 days postinoculation, all 4 amplifiers had infectious stages of Plasmodium (asexual meronts) detectable in peripheral blood (eastern average = western average = 2.0 parasites per 10000 erythrocytes, scored by microscopic examination of thin-film blood smears). Blood from amplifiers was then mixed with buffer and used to inoculate birds in the experimental groups, such that all experimental groups received the same dose of Plasmodium. Experimental birds comprised 4 groups of 6 birds each (i.e., eastern birds inoculated with their sympatric lineage P-SOSP9, eastern birds inoculated with their allopatric lineage P-SOSP10, western birds inoculated with their allopatric lineage P-SOSP9, western birds inoculated with their sympatric lineage P-SOSP10).