Horizontally transferred genes as RNA interference targets for aphid and whitefly control

**Background:** RNA interference (RNAi)-based technologies are emerging as a novel strategy for agricultural pest control. Horizontally transferred genes (HTGs)—genes acquired from viruses, bacteria, fungi, or plants—are often unique to specific insect families or genera, making them attractive targets for RNAi with minimal off-target effects on beneficial insects. This study explores HTGs as RNAi targets for controlling aphids (Myzus persicae) and whiteflies (Bemisia tabaci).

**Methods:** The authors sequenced and assembled a chromosome-scale genome of a red, tobacco-adapted M. persicae strain (USDA-Red) using PacBio long-read, Illumina short-read, and Hi-C sequencing, yielding a 383.0 Mb assembly with 27,430 predicted protein-coding genes. HTGs were identified by comparing protein sequences against UniProt databases (archaea, bacteria, fungi, plants, metazoa, other eukaryotes) using an h-index threshold ≥30 and bitscore ≥100, validated by phylogenetic analysis. Thirty HTGs were identified in M. persicae. For functional testing, dsRNA fragments (150–300 bp) targeting selected HTGs were cloned into a tobacco rattle virus (TRV)-based VIGS vector and transiently expressed in Nicotiana benthamiana via Agrobacterium infiltration. Aphid bioassays used 25 newborn nymphs per cage on VIGS plants, with survival scored every 24 h for 5 days and reproduction measured over 7–8 days. Whitefly bioassays used asat2-1 mutant N. benthamiana, with 10 adult whiteflies per cage and survival assessed after 7 days. Ladybug (Coccinella septempunctata) larvae were fed aphids from VIGS plants and tracked for 7 days. Gene expression was measured by qRT-PCR using the 2−ΔΔCT method.

**Key Results:** Silencing bacterial-origin HTGs (amidase, ATP binding protein, ldcA) significantly reduced aphid survival (P < 0.05) as early as 24 h post-feeding, with effects persisting through 120 h. Silencing five plant-origin HTGs (encoding ankyrin repeat proteins) reduced aphid survival by 30–40% after 120 h (P < 0.05). For fungal-origin Tor and CarRP genes, expression was reduced by approximately 50% (P < 0.05), and progeny production over 8 days was significantly reduced compared to controls (P < 0.05, Tukey's HSD). No significant effects on aphid reproduction were observed for bacterial- or plant-origin HTGs. Silencing the viral-origin cdtB gene unexpectedly up-regulated its expression and did not affect aphid survival. In whiteflies, VIGS targeting squalene synthase (SQS) and diaminopimelate decarboxylase (DAPDC) reduced survival after 7 days on asat2-1 mutant N. benthamiana (P < 0.05). Importantly, tri-trophic persistence of dsRNA was confirmed: dsGFP was detected in both C. septempunctata larvae and Cryptolaemus montrouzieri adults after feeding on aphids from dsGFP plants. Despite this, no negative effects on ladybug survival were observed across all VIGS treatments.

**Clinical Implications:** This study provides proof-of-concept that HTGs are effective and species-specific targets for RNAi-based control of hemipteran pests. The absence of negative effects on ladybug predators supports the safety of this approach. The authors propose that HTGs should be further explored as targets for plant genetic engineering to control aphid and whitefly populations in agricultural settings, potentially reducing reliance on chemical pesticides.