: Glyphodes pyloalis Walker (G. pyl

: Glyphodes pyloalis Walker (G. pyloalis) is a serious pest on mulberry. Due to the increasing pesticide resistance, the development of new and effective environmental methods to controlG. pyloalis is needed. Trehalase is an essential enzyme in trehalose hydrolysis and energy supply, andit has been considered a promising target for insect pest control. However, the specific function oftrehalase in G. pyloalis has not been reported. In this study, two trehalase genes (GpTre1 and GpTre2)were identified from our previous transcriptome database. The functions of the trehalase in chitinmetabolism were studied by injecting larvae with dsRNAs and trehalase inhibitor, Validamycin A.The open reading frames (ORFs) of GpTre1 and GpTre2 were 1,704 bp and 1,869 bp, which encoded567 and 622 amino acid residues, respectively. Both of GpTre1 and GpTre2 were mainly expressedin the head and midgut. The highest expression levels of them were in 5th instar during differentdevelopment stages. Moreover, knockdown both of GpTre1 and GpTre2 by the dsRNAs led to significantly decreased expression of chitin metabolism pathway-related genes, including GpCHSA,GpCDA1, GpCDA2, GpCHT3a, GpCHT7, GpCHSB, GpCHT-h, GpCHT3b, GpPAGM, and GpUAP, andabnormal phenotypes. Furthermore, the trehalase inhibitor, Validamycin A, treatment increased theexpressions of GpTre1 and GpTre2, increased content of trehalose, and decreased the levels of glycogenand glucose. Additionally, the inhibitor caused a significantly increased cumulative mortality of G.pyloalis larvae on the 2nd (16%) to 6th (41.3%) day, and decreased the rate of cumulative pupation(72.3%) compared with the control group (95.6%). After the activities of trehalase were suppressed,the expressions of 6 integument chitin metabolism-related genes decreased significantly at 24 h andincreased at 48 h. The expressions of GpCHSB and GpCHT-h, involved in chitin metabolism pathwayof peritrophic membrane in the midgut, increased at 24 h and 48 h, and there were no changes toGpCHT3b and GpPAGM. These results reveal that GpTre1 and GpTre2 play an essential role in thegrowth of G. pyloalis by affecting chitin metabolism, and this provides useful information for insectpest control in the future.