Exploring RNAi Technology for Management of Stored Grain Beetle: Tribolium castaneum

Julie Zhu Zhu

Tribolium castaneum is one of the most significant stored-grain insect pests internationally. Phosphine is the only fumigant used commercially to control T. castaneum in stored grain, and this pest has developed resistance to phosphine in many places. Thus, there is an urgent requirement for alternative strategies to control T. castaneum. RNA interference (RNAi) is an endogenous pathway in many organisms, including insects, that identifies and degrades specific RNA species through nucleoprotein molecules induced by double-stranded RNA (dsRNA) molecules with high sequence identity to the target transcript. As RNAi acts post-transcriptionally, it can be triggered by the presence of both endogenous and exogenously supplied dsRNA molecules in the cell. Introduction of transcript-specific dsRNA via microinjection to the insect body has been shown to trigger RNAi and down-regulate endogenous gene expression. The deliberate introduction of dsRNA molecules to degrade endogenous transcripts that encode vital proteins in biochemical pathways in T. castaneum is, therefore, a potential means of controlling this pest. Several hurdles must be overcome for this potential control strategy to be commercially applicable. One is identification of target transcripts, another is the means of delivery of dsRNA (microinjection is clearly inappropriate in the field), and another is stability of the dsRNA molecules, which are highly labile. This research project aims to address these hurdles and discuss potential strategies by which RNAi can be harnessed as an alternative control strategy for T. castaneum. I developed a method of orally introducing dsRNA molecules to larval T. castaneum infused into flour-based feeding discs. qRT-PCR results suggest that target genes dre4, Sec61a, Prosa-PA and Snf7 showing the highest transcript level at the larvae stages but with almost no expression at pupae and adult stages except for gene Snf7. Thus, second-instar larvae were the efficient stage to perform RNAi oral feeding essay. I tested the effects of different dsRNA formats against several T. castaneum genes that are essential for insect growth, development and fecundity. Targeting these genes did not induce significant mortality in T. castaneum, except for the target gene Prosa-PA, which resulted to 22% mortality at the 1 μg/mg treatment. However, oral delivery of dsdre4 delayed the development period from larvae to pupae, with the number of days from larvae to pupae being prolonged by three days after feeding of dsdre4. Furthermore, treatment with dsRNA (1μg/mg) targeting dre4 or Prosα-PA caused a significant reduction in offspring reproduction (p < 0.05), with the silencing of Prosa-PA causing about 50% reduction compared to insects treated with GFP dsRNA. Lipids are essential for energy homeostasis during insect development, reproduction and hormone synthesis. Fatty acids and cuticular lipids are major components of lipids, and changes to lipid metabolism can negatively-impact fecundity. To investigate the impact of gene knockdown on metabolic pathways in T. castaneum, a new method, Direct Immersion Solid-Phase Microextraction coupled with GC-MS, was used to analyse lipid compositions in RNAi treated samples. Eight features were identified from adult T. castaneum, including three major fatty acids: palmitic, oleic and octadecanoic acid and five long chain hydrocarbons (CHCs). MetaboAnalys analysis showed that the abundance of palmitic fatty acid was increased significantly (6.2-fold) after silencing of the proteasome gene Rpn7 in female T. castaneum. Proteasome gene Rpn7 gene silencing also increased the levels of two CHCs, 13-methylheptacosane and 3-methylheptacosane, dsRpn7 feeding resulted in a 3.3-fold increase in 13-Methylheptacosane and a 5.5-fold increase in 3-Methylheptacosane. No significant differences were observed between GFP and RNAi-treated groups in male insects. This result revealed that proteasome gene Rpn7 is a potential target gene that may act on regulating palmitic acid metabolism to impact reproduction in T. castaneum. To achieve high efficacy of delivery of exogenous dsRNA via oral delivery, I compared the RNAi-induction efficacy of three different dsRNA formats, including ‘traditional’ (open-ended) dsRNAs, hairpin RNA (hpWT) and G-U base-paired hairpin RNAs (hpGU) against the beetle. My experiments revealed that hpWT molecules induced stronger silencing of the selected target genes dre4 and Prosa-PA. Particularly, hpGU caused the highest increase in larvae mortality and the most persistent effect on offspring reproduction. hpWT[Prosa-PA] and hpGU[Prosa-PA] induced 18% and 27% mortality respectively compared with dsProsa-PA at 30 days, and the number of newly hatched larvae per adult female was reduced by 15%, 35% and 50% respectively for dsProsa-PA, hpWT[Prosa-PA] and hpGU[Prosa-PA]. Northern blot hybridization showed that feeding of hpWT and hpGU resulted in much higher levels of RNAi molecule accumulation in T. castaneum larvae than feeding of dsRNA, a strong band was detected from insects that had fed on both hpWT and hpGU-containing flour discs suggesting more efficient hpWT uptake via oral feeding. In addition, hpWT molecules induced more effective downregulation of the Prosa-PA (30-67%) target gene in T. castaneum larvae than dsRNA. This hpRNAs-induced gene downregulation correlated with significant reduction in body weight and reproduction of T. castaneum. This result suggested that hpRNAs, particularly hpGU, can be efficiently up taken by T. castaneum via oral feeding, and can induce significant RNAi effects on insect mortality and reproduction. The results of this research suggest that oral delivery of dsRNA has a potential in management of T. castaneum and providing a potential tool for the control of insect pests.

Exploring RNAi Technology for Management of Stored Grain Beetle: Tribolium castaneum

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