Innexin genes function in the development, metamorphosis, and reproduction of Tribolium castaneum

Innexins are the main components of gap junctions in invertebrates. However, the evolution and function of these in insects remain elusive. Phylogenetic analysis indicated that insect innexin genes originated from one ancestral gene, and they were divided into 6 clusters. Totally, 8 innexin (inx) genes were identified from Tribolium castaneum. These genes were highly expressed in different stages, including inx1 and inx7 in late pupae, inx2, inx3, and inx7-like2 in early pupae, shaking-B and inx7-like1 at postembryonic stages, and inx4/5/6 at early embryos and late adults. Tissue expression analysis indicated they had high transcription in distinct tissues, containing inx1 and inx7-like1 in the wing and gut of late pupae, inx7 in the gut and central nervous system of late pupae, shaking-B in most tissues except wings of late pupae, inx2 and inx3 in all tissues of early pupae, inx7-like2 in the gut and fat body of early pupae, and inx4/5/6 in the ovary of late adults. RNA interference (RNAi) experiments revealed that inx2 and inx4/5/6 were required for metamorphosis, fertility, and ovarian development, inx3 functioned during eclosion, inx1, inx7, and inx7-like1 played important roles in embryonic development, and shaking-B was critical for adult survival. Furthermore, silencing inx2, inx4/5/6, inx7, or shaking-B downregulated the expression of vitellogenin (Vg) and Vg receptor in female adults. These results indicated that innexin genes had crucial roles in the metamorphosis, fertility, and embryonic development of insects and provided the theoretical basis for developing inx2, inx3, and inx4/5/6 as potential targets of RNAi to control pests.