Requirement of group I lytic polysaccharide monooxygenase for turnover of chitinous cuticle during moulting in two forest pest beetles, <i>Monochamus alternatus</i> and <i>Psacothea hilaris</i>

Lytic polysaccharide monooxygenases (LPMOs) that are capable of oxidative cleavage of glycosidic bonds in crystalline polysaccharides including chitin and cellulose are widely distributed among organisms. Insect LPMOs belong to auxiliary activity family 15 (LPMO15/AA15) and have been classified further into at least four subgroups. However, unlike LPMOs from microorganisms and viruses, their physiological functions in insects have not been well studied. In the present work, we investigated the functions of two group I LPMO15s, MaLPMO15-1 and PhLPMO15-1, in chitinous cuticle turnover during moulting of two important forest pest longhorn beetles-the Japanese pine sawyer beetle, Monochamus alternatus, and the yellow spotted longicorn beetle, Psacothea hilaris. Real-time qPCR showed a similar pattern of expression of MaLPMO15-1 and PhLPMO15-1 during late stages of development with high levels present at young pharate pupal and young pupal stages and declining thereafter. Injection of double-stranded RNA (dsRNA) for MaLPMO15-1 (dsMaLPMO15-1) or PhLPMO15-1 (dsPhLPMO15-1) into last instar larvae of M. alternatus and P. hilaris, respectively, did not affect subsequent larval-pupal moulting and the resulting pupae exhibited normal development. However, the pupae were unable to eclose to the adult stage and died entrapped inside their old pupal cuticle. TEM analysis revealed that, unlike the respective dsRNA for the enhanced green fluorescent protein (dsEGFP)-treated control insects, both dsMaLPMO15-1- and dsPhLPMO15-1-treated pharate adults failed to degrade the endocuticular layer of their pupal cuticle in which the horizontal chitinous laminae remained largely intact. These results demonstrate that the group I LPMO15-1 enzymes play a role in pupal cuticle chitin turnover, which is critical for moulting to the adult. Because LPMO15-1 is highly conserved among many insect species, this gene/enzyme is a potential target for the control of populations of both M. alternatus and P. hilaris as well as other pest insect species.