Microinjection‐enabled gene silencing in first instar larvae of western flower thrips, <i>Frankliniella occidentalis</i> , reveals vital genes for larval survival

The western flower thrips (Frankliniella occidentalis) is a significant agricultural pest, causing severe global yield losses due to extensive feeding damage and the transmission of plant pathogenic viruses. Despite recent advancements in RNA interference (RNAi) in thrips species, its application has been mostly limited to the adult stage. Given the crucial role of first instar larval thrips in acquiring and transmitting orthotospoviruses, achieving gene silencing in these larvae is critical for studying virus entry and acquisition. While thoracic and abdominal injections have proven effective in adult thrips, the low post-injection survival rate hinders their use in larval thrips. This study addresses this challenge by presenting a microinjection methodology to deliver dsRNA into the hemolymph of first instar larval thrips through the coxa, the first proximal segment of the foreleg. This method significantly improved larval survival rate by preventing detrimental damage to the internal tissues. Significant knockdown of V-ATPase-B, cytochrome P450 (CYP3653A2), and apolipophorin-II/I (ApoLp-II/I) transcripts was confirmed after 48 and/or 72 h post injection (hpi), corresponding to the first and second instar larval stages, respectively. Silencing CYP3653A2 or ApoLp-II/I significantly increased larval mortality. These findings demonstrate proof-of-principle of gene silencing and associated silencing phenotype (mortality) for first instar larval thrips and highlight the essential role of CYP3653A2 and ApoLp-II/I in larval vitality. Our RNAi-based tool offers an opportunity to investigate the molecular mechanisms of thrips-orthotospovirus interactions, as the virus must be acquired by young larval thrips for successful transmission to plants, thus presenting potential targets for thrips pest management.