RNA Interference of Four Genes in Adult Bactrocera dorsalis by Feeding Their dsRNAs

BACKGROUND: RNA interference (RNAi) is a powerful method to inhibit gene expression in a sequence specific manner. Recently silencing the target gene through feeding has been successfully carried out in many insect species. METHODOLOGY/PRINCIPAL FINDINGS: Escherichia coli strain HT115 was genetically engineered to express dsRNA targeting genes that encode ribosomal protein Rpl19, V type ATPase D subunit, the fatty acid elongase Noa and a small GTPase Rab11. qRT-PCR showed that mRNA level of four target genes was reduced compared to ds-egfp control by feeding either engineered bacteria or dsRNAs. The maximum down-regulation of each gene varied from 35% to 100%. Tissue specific examination indicated that RNAi could be observed not only in midgut but also in other tissues like the ovary, nervous system and fat body. Silencing of rab11 through ingestion of dsRNA killed 20% of adult flies. Egg production was affected through feeding ds-noa and ds-rab11 compared to ds-egfp group. Adult flies were continuously fed with dsRNA and bacteria expressing dsRNA for 14 days and up-regulations of target genes were observed during this process. The transcripts of noa showed up-regulation compared to ds-egfp control group in four tissues on day 7 after continuous feeding either dsRNA or engineered bacteria. The maximum over-expression is 21 times compared to ds-egfp control group. Up-regulation of rab11 mRNA level could be observed in testes on day 7 after continuous bacteria treatment and in midgut on day 2 after ds-rab11 treatment. This phenomenon could also be observed in rpl19 groups. CONCLUSIONS: Our results suggested that it is feasible to silence genes by feeding dsRNA and bacteria expressing dsRNA in Bactrocera dorsalis. Additionally the over-expression of the target gene after continuously feeding dsRNA or bacteria was observed

Tobacco Rattle Virus Vector: A Rapid and Transient Means of Silencing Manduca sexta Genes by Plant Mediated RNA Interference

Background: RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant’s lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata’s dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Methodology/Principal Findings: Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for tw

Down Regulation of a Gene for Cadherin, but Not Alkaline Phosphatase, Associated with Cry1Ab Resistance in the Sugarcane Borer Diatraea saccharalis

The sugarcane borer, Diatraea saccharalis, is a major target pest of transgenic corn expressing Bacillus thuringiensis (Bt) proteins (i.e., Cry1Ab) in South America and the mid-southern region of the United States. Evolution of insecticide resistance in such target pests is a major threat to the durability of transgenic Bt crops. Understanding the pests' resistance mechanisms will facilitate development of effective strategies for delaying or countering resistance. Alterations in expression of cadherin- and alkaline phosphatase (ALP) have been associated with Bt resistance in several species of pest insects. In this study, neither the activity nor gene regulation of ALP was associated with Cry1Ab resistance in D. saccharalis. Total ALP enzymatic activity was similar between Cry1Ab-susceptible (Cry1Ab-SS) and -resistant (Cry1Ab-RR) strains of D. saccharalis. In addition, expression levels of three ALP genes were also similar between Cry1Ab-SS and -RR, and cDNA sequences did not differ between susceptible and resistant larvae. In contrast, altered expression of a midgut cadherin (DsCAD1) was associated with the Cry1Ab resistance. Whereas cDNA sequences of DsCAD1 were identical between the two strains, the transcript abundance of DsCAD1 was significantly lower in Cry1Ab-RR. To verify the involvement of DsCAD1 in susceptibility to Cry1Ab, RNA interference (RNAi) was employed to knock-down DsCAD1 expression in the susceptible larvae. Down-regulation of DsCAD1 expression by RNAi was functionally correlated with a decrease in Cry1Ab susceptibility. These results suggest that down-regulation of DsCAD1 is associated with resistance to Cry1Ab in D. saccharalis

A sensory appendage protein protects malaria vectors from pyrethroids

Pyrethroid-impregnated bednets have driven significant reductions in malaria morbidity and mortality in Africa since the beginning of the century 1. The intense selection pressure exerted by bednets has precipitated widespread and escalating pyrethroid resistance in African Anopheles populations, threatening to reverse gains made by malaria control 2. Here we show that a leg-enriched sensory appendage protein, SAP2, confers pyrethroid resistance in Anopheles gambiae. SAP2 expression is elevated in insecticide-resistant populations and is further induced upon mosquito contact with pyrethroids. SAP2 silencing fully restores mosquito mortality, whilst its overexpression results in increased resistance, likely due to the high-affinity SAP2 binding to pyrethroid insecticides. Mining of genome sequence data reveals a selective sweep near the SAP2 locus in three West African countries, with the observed increase in haplotype associated SNPs mirroring increasing resistance reported in Burkina Faso. Our study identifies a new insecticide resistance mechanism that is likely highly relevant to malaria control efforts