Dusky (dy) is required for cytoskeletal reorganization during wing morphogenesis in Drosophila melanogaster, but which genes participate together with dy for wing morphogenesis has remained unclear. In Tribolium castaneum, dy is highly expressed at the late embryonic stage. Tissue-specific expression analysis indicated high expression levels of dy in the epidermis, head and fat body of late-stage larvae. RNA interference (RNAi) targeting dy significantly decreased adult wing size and caused improper folding of the elytra. Meanwhile, dy knockdown reduced the transcription of four-jointed (fj) and forked (f). Our results show that fj RNAi reduces adult wing size and that silencing f results in abnormal wing folding in T. castaneum. Interestingly, knocking down fj and f simultaneously phenocopies dy RNAi, suggesting that dy probably acts upstream of fj and f to regulate wing morphogenesis in T. castaneum.

C‐type lectins (CTLs) are a superfamily of proteins found in almost all vertebrates and invertebrates. They play an important role in innate immune defences, development and epidermal structure. Here, a CTL with one carbohydrate‐recognition domain containing a highly conserved Gln‐Pro‐Asp (QPD) motif was identified in Tribolium castaneum and given the name TcCTL5. Spatiotemporal analyses showed that Tcctl5 was highly expressed in the late pupa stage and mainly existed in the central nervous system and haemolymph. The transcript level of Tcctl5 was prominently induced after bacterial infection. Recombinant TcCTL5 proteins (rTcCTL5) were found to bind to lipopolysaccharide, peptidoglycan and tested bacteria and induce microbial agglutination in the presence of Ca2+. Interestingly, when Tcctl5 was knocked down, the transcript level of antimicrobial peptides (AMPs) (attacin1, defensins3, coleoptericin1 and cecropins3) was prominently downregulated after induction with Gram‐negative Escherichia coli. More interestingly, Tcctl5 was knocked down, leading to increased mortality and loss of locomotor activity, which exhibited less travel distances among early adults. These results demonstrate that Tcctl5 plays an important role in the innate immune reaction and the movement of T. castaneum. Thus, it may represent an alternative molecular target for pest control and thus reduce the use of pesticides in agricultural production.

Latrophilin (LPH) is known as an adhesion G-protein-coupled receptor which involved in multiple physiological processes in organisms. Previous studies showed that lph not only involved the susceptibility to anticholinesterase insecticides but also affected fecundity in Tribolium castaneum. However, its regulatory mechanisms in these biological processes are still not clear. Here, we identified two potential downstream carboxylesterase (cce) genes of Tclph, esterase4 and esterase6, and further characterized their interactions with Tclph. After treatment of T. castaneum larvae with carbofuran or dichlorvos insecticides, the transcript levels of Tcest4 and Tcest6 were significantly induced from 12 to 72 h. RNAi against Tcest4 or Tcest6 led to the higher mortality compared with the controls after the insecticides treatment, suggesting that these two genes play a vital role in detoxification of insecticides in T. castaneum. Furthermore, with insecticides exposure to Tclph knockdown beetles, the expression of Tcest4 was upregulated but Tcest6 was downregulated, indicating that beetles existed a compensatory response against the insecticides. Additionally, RNAi of Tcest6 resulted in 43% reductions in female egg laying and completely inhibited egg hatching, which showed the similar phenotype as that of Tclph knockdown. These results indicated that Tclph affected fecundity by positively regulating Tcest6 expression. Our findings will provide a new insight into the molecular mechanisms of Tclph involved in physiological functions in T. castaneum.