Knocking down chitin synthase 2 by RNAi is lethal to the cotton boll weevil

The cotton boll weevil (Anthonomus grandis) is the most destructive cotton insect pest in Brazil. The endophytic habit of this insect makes difficult its chemical control. Chitin synthase (CHS) is an integral membrane glycosyltransferase that is essential for chitin chain polymerization and deposition in insect chitinous structures, such as the peritrophic membrane (PM). Because it is not present in plants or vertebrates, CHS can be considered a promising target for eco-friendly biotechnological approaches, such as RNA interference (RNAi)-mediated gene silencing. Considering the relevance of CHS genes in the chitin biosynthetic pathway in insects, we report here the molecular cloning of the full-length CHS2 cDNA from the cotton boll weevil, and its functional validation via RNAi. The AgraCHS2 cDNA sequence is 4,869 bp, with a 4,446 bp open reading frame that encodes a predicted protein with 1,482 amino acid residues. Predicted protein has high similarity (53 to 78%) with other insects CHS. Moreover, only one copy is present in A. grandis genome. Transcriptional analysis showed that AgraCHS2 transcripts are restricted to the insect midgut at the third-larval instar and adult stages, which are considered the main feeding stages. RNAi-mediated knockdown of the AgraCHS2 affected A. grandis development, resulting in oviposition reduction of 93% and leading to 100% adult mortality. These data, in addition to the observation of PM severe disorganization in the midgut after AgraCHS2 knock-down, suggest AgraCHS2 as a promising target for developing RNAi-based biotechnological alternatives to specifically control the cotton boll weevil.