Elucidation of the serosal cuticle machinery in the beetle Tribolium by RNA sequencing and functional analysis of Knickkopf1, Retroactive and Laccase2

• The insect egg is known to be protected against dehydration by the serosal cuticle.
• Knickkopf1, Retroactive and Laccase2 are involved in structuring the serosal cuticle.
• TcKnk-1, TcRtv or TcLac-2 knockdown reduces survival of eggs at dry conditions.
• Many cuticle genes show serosa specific expression.
• The Tribolium egg provides a model for the study of cuticle synthesis.

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.

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