Background: Although native to North America, the invasion of the aphid-like grape phylloxeraDaktulosphaira vitifoliaeacross the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North AmericanVitisspecies as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome ofD. vitifoliaeas a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture.Results: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world.Conclusions: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture.

Insect pest control by RNA interference (RNAi)-mediated gene expression knockdown can be undermined by many factors, including small sequence differences between double-stranded RNA (dsRNA) and the target gene. It can also be compromised by effects that are independent of the dsRNA sequence on non-target organisms (known as sequence-non-specific effects). This study investigated the species-specificity of RNAi in plant sap-feeding hemipteran pests. We first demonstrated sequence-non-specific suppression of aphid feeding by dsRNA at dietary concentrations ≥0.5 µg µL⁻¹. Then we quantified the expression of NUC (nuclease) genes in insects administered homologous dsRNA (with perfect sequence identity to the target species) or heterologous dsRNA (generated against a related gene of non-identical sequence in a different insect species). For the aphids Acyrthosiphon pisum and Myzus persicae, significantly reduced NUC expression was obtained with the homologous but not heterologous dsRNA at 0.2 µg µL⁻¹, despite high dsNUC sequence identity. Follow-up experiments demonstrated significantly reduced expression of NUC genes in the whitefly Bemisia tabaci and mealybug Planococcus maritimus administered homologous dsNUCs, but not heterologous aphid dsNUCs. Our demonstration of inefficient expression knockdown by heterologous dsRNA in these insects suggests that maximal dsRNA sequence identity is required for RNAi targeting of related pest species, and that heterologous dsRNAs at appropriate concentrations may not be a major risk to non-target sap-feeding hemipterans.

The grape mealybug Pseudococcus maritimus (Ehrhorn, 1900) (Hemiptera: Pseudococcidae) is a significant pest of grapevines (Vitis spp.) and a vector of disease-causing grape viruses, linked to its feeding on phloem sap. The management of this pest is constrained by the lack of naturally occurring resistance traits in Vitis. Here, we obtained proof of concept that RNA interference (RNAi) using double-stranded RNA (dsRNA) molecules against essential genes for phloem sap feeding can depress insect survival. The genes of interest code for an aquaporin (AQP) and a sucrase (SUC) that are required for osmoregulation in related phloem sap-feeding hemipteran insects (aphids and whiteflies). In parallel, we investigated the grape mealybug genes coding non-specific nucleases (NUC), which reduce RNAi efficacy by degrading administered dsRNA. Homologs of AQP and SUC with experimentally validated function in aphids, together with NUC, were identified in the published transcriptome of the citrus mealybug Planococcus citri by phylogenetic analysis, and sequences of the candidate genes were obtained for Ps. maritimus by PCR with degenerate primers. Using this first sequence information for Ps. maritimus, dsRNA was prepared and administered to the insects via an artificial diet. The treatment comprising dsRNA against AQP, SUC and NUC significantly increased insect mortality over three days, relative to dsRNA-free controls. The dsRNA constructs for AQP and NUC were predicted, from sequence analysis to have some activity against other mealybugs, but none of the three dsRNA constructs have predicted activity against aphids. This study provides the basis to develop in planta RNAi strategies against Ps. maritimus and other mealybug pests of grapevines.