Implications of temperature on sex ratio and fecundity in Amblyseius orientalis (Acari: Phytoseiidae): evidence from sex-determination gene expression.

Global agricultural pest control urgently requires sustainable alternatives to chemical pesticides. This underscores the crucial role of biological control using natural enemies, such as Amblyseius orientalis, in managing pests in citrus orchards. Enhancing its biocontrol efficacy hinges on optimizing reproductive potential, primarily influenced by female proportion. While temperature is a known environmental modulator of arthropod sex ratio and reproduction, the molecular mechanisms linking temperature to the sex-determination cascade in A. orientalis remain poorly understood. Addressing this gap, our field and laboratory experiments consistently revealed an optimal reproductive temperature of 25 °C, which maximized female proportion, fecundity, and egg hatchability. We identified three sex-determination-related genes (AoTRA2, AoDSX2, and AoBTB2) with conserved functional domains. RNA interference (RNAi) uncovered a cascade regulation: silencing AoTRA2 downregulated AoDSX2 and AoBTB2; AoDSX2 silencing affected AoBTB2; and AoBTB2 silencing exhibited feedback inhibition. Importantly, silencing any of these genes reduced fecundity, female offspring, and impaired egg development, leading to decreased AoVg and AoVgR expression. Furthermore, temperature treatment experiments demonstrated that at 25 °C, the expression levels of AoTRA2 and AoDSX2 peaked, AoVg was significantly upregulated, coinciding with optimal egg development. These findings reveal that temperature modulates the sex-determination cascade to regulate the reproduction and sex ratio of A. orientalis, providing a molecular framework for understanding temperature-dependent sex determination and optimizing mass-rearing protocols. Given that female-biased predatory mite populations generally exhibit enhanced biocontrol potential due to their predatory and reproductive roles, temperature manipulation may represent a viable strategy for improving the production and deployment of this species in integrated pest management.