| Summary: | Rapid cold hardening (RCH) is known to rapidly enhance the cold tolerance of insects. Trehalose has been demonstrated to be a cryoprotectant in <i>Lissorhoptrus oryzophilus</i>, an important invasive pest of rice in China. Trehalose synthesis mainly occurs through the Trehalose-6-phosphate synthase (TPS)/trehalose-6-phosphate phosphatase (TPP) pathway in insects. In this study, the <i>TPS</i> gene from <i>L. oryzophilus</i> (<i>LoTPS</i>) was cloned and characterized for the first time. Its expression and trehalose content changes elicited by RCH were investigated. Our results revealed that RCH not only increased the survival rate of adults but also upregulated the expression level of <i>LoTPS</i> and increased the trehalose content under low temperature. We hypothesized that upregulated <i>LoTPS</i> promoted trehalose synthesis and accumulation to protect adults from low-temperature damage. To further verify the function of the <i>LoTPS</i> gene, we employed RNA interference (RNAi) technology. Our findings showed that RCH efficiency disappeared and the survival rate did not increase when the adults were fed dsRNA of <i>LoTPS</i>. Additionally, inhibiting <i>LoTPS</i> expression resulted in no significant difference in trehalose content between the RCH and non-RCH treatments. Furthermore, the expression patterns of trehalose transporter (<i>TRET</i>) and trehalase (<i>TRE</i>) were also affected. Collectively, these results indicate the critical role of <i>LoTPS</i> in <i>L. oryzophilus</i> cold resistance after RCH induction. <i>LoTPS</i> can enhance survival ability by regulating trehalose metabolism. These findings contribute to further understanding the role of <i>TPS</i> in insect cold resistance and the invasiveness of <i>L. oryzophilus</i>. Moreover, RNAi of <i>LoTPS</i> opens up possibilities for novel control strategies against <i>L. oryzophilus</i> in the future.
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