Pesticides, in particular insecticides, can be very beneficial but have also been found to have harmful side effects on non-target insects. Butterflies play an important role in ecosystems, are well monitored and are recognised as good indicators of environmental health. The amount of information already known about butterfly ecology and the increased availability of genomes make them a very valuable model for the study of non-target effects of pesticide usage. The effects of pesticides are not simply linear, but complex through their interactions with a large variety of biotic and abiotic factors. Furthermore, these effects manifest themselves at a variety of levels, from the molecular to metapopulation level. Research should therefore aim to dissect these complex effects at a number of levels, but as we discuss in this review, this is seldom if ever done in butterflies. We suggest that in order dissect the complex effects of pesticides on butterflies we need to integrate detailed molecular studies, including characterising sequence variability of relevant target genes, with more classical evolutionary ecology; from direct toxicity tests on individual larvae in the laboratory to field studies that consider the potentiation of pesticides by ecologically relevant environmental biotic and abiotic stressors. Such integration would better inform population-level responses across broad geographical scales and provide more in-depth information about the non-target impacts of pesticides.

Wnt signaling pathways are involved in many important cellular processes including proliferation and differentiation. Wnt ligands are released by source cells and signal to target cells by binding to the Frizzled receptor family and triggering changes in downstream target gene expression. Wnt signaling appeared at the base of metazoans and there was an early expansion in the repertoire of Wnt ligands to the 13 known subfamilies. However, little is known about functionality of these ligands in many animal lineages. Understanding the roles of these important signaling molecules in a wider range of animals is crucial to understand the regulation and evolution of cell fate during development and how this can lead to diversification. Here, we analyzed the Wnt repertoire among lepidopterans, where the embryological functionality of these ligands is understudied compared to other insect orders. To be able to explore Wnt gene roles during butterfly embryogenesis we first established a staging system for the butterfly model, Bicyclus anynana, and assayed the expression pattern of all eight lepidopteran Wnt genes during early butterfly development. We detected expression of Wnt1, Wnt10, and WntA in several expression domains, such as segmental stripes as well as expression of Wnt7 in the nervous system and Wnt11 in several head structures. Overall, our study provides, a basis for future research into butterfly embryogenesis and much needed new insights into the potential roles of Wnt genes in specifying cell fate in these animals as well as how this compares to other animals.

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Insects have been extraordinary successful in colonizing terrestrial habitats and this success is partly due to a protective cuticle that mainly contains chitin and proteins. The cuticle has been well studied in larvae and adults, but little attention has been paid to the cuticle of the egg. This cuticle is secreted by the serosa, an extraembryonic epithelium that surrounds the yolk and embryo in all insect eggs, but was lost in the Schizophoran flies to which Drosophila belongs. We therefore set out to investigate serosal cuticle formation and function in a beetle (Tribolium castaneum) using RNAi-mediated knockdown of three candidate genes known to structure chitin in the adult cuticle, and we aimed to identify other serosal cuticle genes using RNA sequencing. Knockdown of Knickkopf (TcKnk-1) or Retroactive (TcRtv) affects the laminar structure of the serosal cuticle, as revealed by Transmission Electron Microscopy in knockdown eggs. In the absence of this laminar structure, significantly fewer eggs survive at low humidity compared to wild-type eggs. Survival in dry conditions is also adversely affected when cross-linking among proteins and chitin is prevented by Laccase2 (TcLac-2) RNAi. Finally, we compare the transcriptomes of wild-type eggs to serosa-less eggs and find serosa-biased expression of 21 cuticle-related genes including structural components, chitin deacetylases and chitinases. Our data indicate that the serosal cuticle utilizes the same machinery for structuring the cuticle as adults. We demonstrate that the structure of the cuticle is crucial for desiccation resistance, and we put forward the serosal cuticle of Tribolium as an excellent model to study the ecological properties of the insect cuticle.