Organisms inhabiting highly seasonal environments must cope with a wide range of environmentally induced challenges. Many seasonal challenges require extensive physiological modification to survive. In winter, to survive extreme cold and limited resources, insects commonly enter diapause, which is an endogenously derived dormant state associated with minimized cellular processes and low energetic expenditure. Due to the high degree of complexity involved in diapause, substantial cellular regulation is required, of which our understanding primarily derives from the transcriptome via messenger RNA expression dynamics. Here we aim to advance our understanding of diapause by investigating microRNA (miRNA) expression in diapausing and direct developing pupae of the butterfly Pieris napi. We identified coordinated patterns of miRNA expression throughout diapause in both head and abdomen tissues of pupae, and via miRNA target identification, found several expression patterns to be enriched for relevant diapause-related physiological processes. We also identified two candidate miRNAs, miR-14-5p and miR-2a-3p, that are likely involved in diapause progression through the ecdysone synthesis pathway, a critical regulator of diapause termination. miR-14-5p targets phantom, a gene in the ecdysone synthesis pathway, and miR-2a-3p, which has been found to be expressed in response to ecdysone. Together, the expression patterns of these two miRNAs match our current understanding of the timing of hormonal regulation of diapause in P. napi and provide interesting candidates to further explore the mechanistic role of microRNAs in diapause regulation.

Generalist butterflies are characterized by a broad host repertoire that can comprise several families or even different orders of plants. The genetic and physiological mechanisms underlying the use of such a wide host range are still not fully understood. Earlier studies indicate that the consumption of different host plants is associated with host-specific gene expression profiles. It remained, however, unclear if and how larvae can alter these profiles in the case of a changing host environment. Using the polyphagous comma butterfly (Polygonia c-album) we show that larvae can adjust their transcriptional profiles in response to a new host plant. The switch to some of the host plants, however, resulted in a larger transcriptional response and, thus, seems to be more challenging. At a physiological level, no correspondence for these patterns could be found in larval performance. This suggests that a high transcriptional but also phenotypic flexibility are essential for the use of a broad and diverse host range. We furthermore propose that host switch tests in the laboratory followed by transcriptomic investigations can be a valuable tool to examine not only plasticity in host use but also subtle and/or transient trade-offs in the evolution of host plant repertoires. key words: insect-plant association, host plant adaptation, gene expression, phenotypic plasticity

Parasitoid wasps are major causes of mortality of many species, resulting in host immune defences commonly being the target of adaptive evolution, though such targets outside model species are poorly understood. Here we compare the power of different molecular tests of selection to provide such insights in novel species. We combined our understanding of variation in immune defence capacity among three closely related Galerucella leaf beetles with a shared parasitoid wasp, with information on genomic targets of parasitoid attacks from exemplar insect species. Based on this, we predicted that these genomic targets would vary in their evolutionary history across three closely related leaf beetle species, such that genomic targets would experience stronger positive selection in the species with strongest immune response to attack. Codon based tests revealed variation among species in positive selection genome wide, and showed that parasitoid-relevant immune genes experienced more positive selection in the species with the greatest immunocompetence (G. pusilla), while almost no immune genes were under positive selection in the species with the least immunocompetence (G. calmariensis). Genome wide analyses of the haplotype frequency spectrum also identified genes experiencing positive selection across the species, though few were parasitoid-relevant immune genes and no species was particularly enriched for them. Thus, our codon based test, which summarizes all sweep events since the last common ancestor, found results consistent with our a priori hypothesis, providing a series of targets for future functional genomic study.

Parasitoid wasps are major causes of mortality of many species, and therefore traits related to host immune defence are usually favoured by natural selection. One powerful approach to detect functionally important genes under natural selection is through the analysis of directional selection acting upon protein-coding gene sequences across different species. Here, we investigated patterns of positive selection across three closely related leaf beetle species with different immune defence capacity against a shared parasitoid wasp using a Bayesian approach for the McDonald–Kreitman test. Focusing on single-copy orthologs for Coleoptera, as well as on candidate immune related genes, we detected species-specific positive selection on coding regions in each of the closely related Galerucella beetle species. Results indicated that more immune genes had experienced positive selection in the species with the greatest immunocompetence (G. pusilla) against parasitoid wasps, while almost no immune genes were under positive selection in the species with the least immunocompetence (G. calmariensis).

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Mullerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Despite recent studies carried out on ithomiine ecology and genetic structure, no reference genome was available for the tribe. Here, we generated high-quality, chromosome-scale genome assemblies of two Melinaea species, Melinaea marsaeus and Melinaea menophilus, and a draft genome of Ithomia salapia. We obtained genomes with a size ranging from 396 Mb to 503 Mb across the three species and scaffold N50 of 40.5 Mb and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. A detailed annotation of transposable elements and genes was performed, resulting in the identification of 24,341, 31,081 and 31,976 genes in I. salapia, M. marsaeus and M. menophilus, respectively. We used a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models of mimicry, such as Heliconius, and will enable further understanding of the mechanisms of adaptation and the genetic bases underpinning mimicry.