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Butterflies are ubiquitous and abundant, occurring in a wide variety of environments that contain diverse microbial communities with varied pathogenic pressures. These pathogens and parasites present a constant threat to organisms, and have led to the evolution of complex and intricate immune responses. Despite strong selection against immunological threats, organisms display great variation in their immune capabilities, both on the genetic and physiological level. Investigating this variation remains challenging, since differences in immune responses might arise from changes in the amount, size or performance of cells or organs. Disentangling these relative contributions is important, as the targets of selection are expected to differ, ranging from immune genes directly related to the phenotype to genes indirectly involved via cell proliferation. This thesis focuses on characterizing the immune system of the butterfly Pieris napi and investigating its remarkable variation across populations by using both phenotypic and genotypic measurements. By integrating RNA-seq with life history measurements, I found that the cost of infection and wounding in the final larval stage carries over the metamorphic boundary in P. napi (Paper II). Using population comparisons, I identified both the action and potential targets of natural selection in wild populations within their respective immune responses (Paper I, III & IV). The immune genes in P. napi show increased genetic variation compared to the rest of the genome, and microevolutionary selection dynamics act on these genes between and among populations (Paper I). I measured the cellular immune responses related to phagocytosis and melanization in common garden reared larvae originating from two allopatric populations (Spain, Sweden) (Paper III & IV). The two populations were found to differ in their blood cell composition, and overall phagocytic capability, driven by the increased phagocytic propensity of each cell type (Paper III). However, genome wide analysis of divergence between these populations found no excess genetic differentiation in genes annotated to phagocytic capacity, suggesting that our observed population differences might arise from genes affecting the activation or transdifferentiation of cells, which currently lack functional annotation. Interestingly, genes involved in glutamine metabolism, which have been linked to immune cell differentiation in mammals, did show divergence between the populations. In addition, the populations also differed in prophenoloxidase activity, a common method for quantifying immune related melanization in insects, along with the abundance of the cell-type (oenocytoids) related to this important immune function (Paper IV). Integrative analysis using both transcriptomic and genomic data revealed that the genes involved in this phenotype showed no significant differentiation between the populations. However, a gene involved with proper trafficking of melanogenic enzymes in vertebrates was found to be highly expressed and highly diverged between the two populations, providing an interesting candidate for future studies. This thesis demonstrates the advantages of integrating several genomic tools with lab experiments to quantify natural variation in the immune system of butterflies. At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript. |
