Dissecting the Lethal Mechanism of Silencing TcSP3 in Tetranychus cinnabarinus Eggs Reveals a Biosafety-Favorable RNAi Target

Serine proteases are essential for development and physiology in arthropods. We previously showed that RNAi-mediated silencing of the serine protease gene TcSP3 induces embryonic lethality in the carmine spider mite, Tetranychus cinnabarinus, but the underlying mechanism was unclear. Here, we adopted an integrated strategy—transcriptome sequencing, RT-qPCR validation, and gene-specific RNAi—to pursue two distinct but complementary objectives: (i) to map the downstream transcriptional response to TcSP3 silencing and thereby identify candidate RNAi targets and (ii) to functionally interrogate the leading candidate, TcHKB, in order to infer the lethal mechanism. We additionally performed a preliminary evaluation of the environmental biosafety of the resulting candidate dsTcHKB for RNAi-based mite management. TcSP3 silencing altered 495 genes (464 upregulated and 31 downregulated). Six downregulated candidate genes were validated; among these, silencing TcHKB resulted in the highest egg mortality (53.57%), accompanied by malformation and incomplete development, whereas silencing TcbHLH and Tcngn-1 caused 34.99% and 22.93% mortality. Sequence homology and domain prediction indicate that TcHKB encodes a Sp5-like transcription factor of the Sp/KLF family, suggesting that TcHKB may indirectly affect developmental signaling. The downregulation of TcHKB is therefore proposed as a possible, yet unverified, explanation for the observed embryonic lethality; direct measurements of Sp5, β-catenin, or Wnt pathway activity are still required to confirm this link. For translational application, we also conducted a preliminary biosafety evaluation of dsTcHKB. Under our tested conditions, sequence alignment showed no extensive homology between dsTcHKB and genes of the predatory mite N. barkeri, and direct bioassays detected no adverse effects on its survival, hatchability, or development; the dsRNA additionally degraded almost completely on cowpea leaves within three days. These initial observations address two specific risk aspects—environmental dissipation on cowpea leaves and non-target effects on N. barkeri—but do not yet establish general safety. This study suggests a plausible mechanism for TcSP3/TcHKB-mediated lethality and identifies TcHKB as a candidate RNAi target warranting further biosafety assessment.