Insect cuticle is an extracellular matrix formed primarily from two different biopolymers, chitin and protein. During each molt cycle, a new cuticle is deposited simultaneously with degradation of the old cuticle by molting fluid cuticle degrading-enzymes, including epidermal chitinases (CHTs). Insect CHTs, belonging to family 18 glycosylhydrolase (GH18), have been classified into at least eleven subgroups based on phylogenetic analyses, and group I (CHT5) and group II (CHT10) epidermal CHTs present in molting fluid. In this study we report the physiological function of MaCHT5 and MaCHT10 in the Japanese pine sawyer, Monochamus alternatus. RNAi for either MaCHT5 or MaCHT10 resulted in larval-pupal and pupal-adult molting defects, in which the insects were unable to shed completely their old cuticle and died entrapped in their exuviae. Furthermore, TEM analysis revealed a failure of degradation of the old cuticle in both MaCHT5- and MaCHT10-deficient pharate adults. In the old pupal cuticle, the chitinous horizontal laminar and vertical pore canal essentially remained intact in the endocuticular layer. These results indicate that both CHTs are required for turnover of the chitinous old cuticle, which is critical for completion of insect molting. We also discuss the possible function of two spliced variants of MaCHT10, MaCHT10a and MaCHT10b.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in crystalline polysaccharides including chitin and cellulose. The recent discovery of LPMO family proteins in many insect species suggests that they presumably play a role in chitin degradation in the cuticle/exoskeleton, tracheae and peritrophic matrix during insect development. Insect LPMOs belong to auxiliary activity family 15 (AA15/LPMO15) and have been classified into at least four groups based on phylogenetic analysis. In this study, we identified, characterized and investigated the physiological functions of group I LPMO15 (MaLPMO15-1 and PhLPMO15-1) in two longhorn beetle species, Monochamus alternatus and Psacothea hilaris. In both species, depletion of LPMO15-1 transcripts in last instar larvae by RNAi had no effect on subsequent larval-pupal molting and the resulting pupae developed normally. However, adverse effects on their development were observed during the pupal-adult molting period. The pharate adults were unable to shed their old pupal cuticle and died entrapped in their exuviae probably due to a failure of degradation of the chitin in their old cuticle, which is critical for completion of the insect molting and continuous growth.