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 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 by RNAi resulted in a lethal pupal-adult molting defect. The insects were unable to shed their old pupal cuticle and died entrapped in their exuviae. Furthermore, TEM analysis revealed a failure of degradation of the chitinous procuticle layer of their old cuticle, retaining intact horizontal laminae and vertical pore canals containing perpendicularly oriented chitin fibers (pore canal fiber, PCF) in their core. These results indicate that MaLPMO15-1 and PhLPMO15-1 are required for turnover of the chitinous old cuticle, which is critical for insect molting.

Insect cuticular extracellular matrices (ECM) including the eggshell and exoskeleton play vital roles in protecting them from natural environmental stresses. However, these chitinous ECMs must be degraded at least in part during embryonic and post-embyonic molting periods to accommodate continuous growth all the way to the adult stage. In this study we investigated the functions of groups I and II chitinases, TcCHT5 and TcCHT10, in turnover of the eggshell and cuticle in Tribolium castaneum. RNAi and TEM analyses revealed that TcCHT10 is required for digestion of chitin in the serosal cuticle for embryo hatching as well as in the old cuticle during post-embryonic molts including larval-pupal and pupal-adult metamorphosis. However, although TcCHT5 is apparently involved in these vital physiological events, TcCHT10 could substitute for TcCHT5 except during the pupal-adult molting when both enzymes are indispensable to degrade chitin in the old pupal cuticle.

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.

Structural cuticular proteins (CPs) and the liner polysaccharide, chitin, are the primary components of insect cuticle or exoskeleton. A large number of insect CP family proteins are divided into several distinct subfamilies defined by the presence of specific amino acid sequence motifs. One of these subfamilies is composed of Cuticular Protein Analogous to Peritrophins (CPAPs), containing one (CPAP1s) or three (CPAP3s) type-2 chitin-binding domains. In this study, we report a novel function of TcCPAP1-C from Tribolium castaneum in movement of legs. RNAi for TcCPAP1-C at larval stage has no effect on insect molting, growth and development. However, the resulting adults exhibit impaired leg movement, in which internal tendon cuticles are ruptured near the femur-tibia joint. The exoskeletal cuticle, hemiadherens junctions, microtubule array, myotendinous junctions and muscle fibers exhibit normal morphology before the tendon breakage. These results indicate functional specialization of TcCPAP1-C in structural integrity of the internal tendon cuticle, and loss of function of TcCPAP1-C caused breakage of the tendon cuticle, resulting in defective limb movement and locomotion.

Insect eggshell and cuticle/exoskeleton play vital roles in protecting them from natural environmental stresses. However, these chitinous cuticular extracellular matrices must be degraded at least in part during embryo hatching and molting/ecdysis periods to accommodate continuous growth all the way to the adult stage. In this study, we investigated the functional importance of groups I and II chitinases, TcCHT5 and TcCHT10, in the turnover of chitinous cuticle during both embryonic and post-embryonic development in Tribolium castaneum. RNAi and TEM analyses revealed that TcCHT10 is required for digestion of chitin in the serosal cuticle for embryo hatching as well as in the old cuticle during post-embryonic molts including larval-pupal and pupal-adult metamorphosis. TcCHT10 appears to be able to substitute for TcCHT5 in all these vital physiological events except for the pupal-adult molting in which TcCHT5 is indispensable for complete digestion of chitin in the old pupal cuticle.