Functions of ion transport peptide and ion transport peptide-like in the red flour beetle Tribolium castaneum

Abstract Ion transport peptide (ITP) and ITP-like (ITPL) are highly conserved neuropeptides in insects and crustaceans. We investigated the alternatively spliced variants of ITP/ITPL in Tribolium castaneum to understand their functions. We identified three alternatively spliced transcripts named itp , itpl-1 , and itpl-2 . Expression patterns of the splice variants investigated by exon-specific in situ hybridization were somewhat different from those previously reported in other insect species. Most importantly, we found for the first time that itpl-1 transcripts are abundantly expressed in the midgut at the late larval stage, showing an expression pattern similar to that of the crustacean hyperglycemic hormone (CHH) in the crab Carcinus maenas . CHH was shown to function by increasing the body volume through fluid absorption, resulting in breakage of the outer shell at the time of molt. Exon-specific RNA interference (RNAi) was designed to distinguish between itp and itpl-1 , but we were unable to design a dsRNA uniquely targeting or uniquely excluding itpl-2 ; therefore, RNAi targeting was limited to either itp / itpl-2 or itpl-1 / itpl-2. For dsRNA injections in the larval stages, either RNAi led to gradually increasing mortality in the larval and pupal stages, with 100% cumulative mortality at the time of eclosion or shortly afterward. Developmental deficiencies in the adult tarsal segments were observed after RNAi suppressing either itp / itpl-2 or itpl-1 / itpl-2 . After dsRNA injections at the pupal stage, the most striking observation was a significant reduction in egg numbers (8% of control) and reduced survival of the offspring (5%) in RNAi targeting itpl-1 / itpl-2 , while a milder degree of the same phenotype was observed in that targeting itp / itpl-2 .