Immune function of the serosa in a hemimetabolous insect egg. Oncopeltus fasciatus

The unparalleled success of the insects comprising more than a million species has long stood out to evolutionary biologists. A much overlooked evolutionary innovation of the insects is the serosa, an extraembryonic epithelium that covers yolk and embryo in their eggs. We have shown that this epithelium provides innate immune protection to eggs of the beetle Tribolium castaneum. It remained elusive, however, if this innate immune competence evolved in the Tribolium lineage, or is ancestral to all insects. Here, we expand our studies to the bug Oncopeltus fasciatus that belongs to the basal main group of insects, the Hemimetabola. RNA sequencing reveals an extensive transcriptional response upon infection of the egg with Gram-positive and Gram-negative bacteria. We demonstrate the antimicrobial activity of upregulated peptides using in vitro bacterial growth inhibition assays, and describe two novel families of AMPs called Serosins and Ovicins. By qPCR, we determine that eggs become immune responsive when the serosa develops. Finally, in situ hybridizations show that transcripts of upregulated peptides are located in the serosal cells and not in the underlying embryo. We conclude that the serosa protects the O. fasciatus embryo against pathogens. This first evidence from hemimetabolous insect eggs suggests that immune competence is an ancestral property of the serosa. The evolutionary origin of the serosa with its immune function might have been one of the factors that facilitated the spectacular success of the insects. Overall design: In total 9 samples are analysed retrieved from 48-64h old Oncopeltus fasciatus eggs (100 eggs per sample, collected at 25 degrees Celsius). We used a control (Naive) in which eggs were left untreated, a sterile injury (Sterile) in which eggs were pricked with a sterile tungsten needle and a septic injury (Septic) in which eggs were infected with a mixture of E.coli and M.luteus bacteria using a tungsten needle dipped in a bacterial suspension. For all 3 treatments, 3 replicates were obtained, leading to a total of 9 samples.