Silencing of ecdysone receptor, insect intestinal mucin and sericotropin genes by bacterially produced double‐stranded RNA affects larval growth and development in Plutella xylostella and Helicoverpa armigera

Abstract

RNA interference mediated gene silencing, which is triggered by double-stranded RNA (dsRNA), has become a important tool for functional genomics studies in various systems, including insects. Bacterially produced dsRNA employs the use of a bacterial strain lacking in RNaseIII activity and harbouring a vector with dual T7 promoter sites, which allow the production of intact dsRNA molecules. Here, we report an assessment of the functional relevance of the ecdysone receptor, insect intestinal mucin and sericotropin genes through silencing by dsRNA in two lepidopteran insect pests, Helicoverpa armigera and Plutella xylostella, both of which cause serious crop losses. Oral feeding of dsRNA led to significant reduction in transcripts of the target insect genes, which caused significant larval mortality with various moulting anomalies and an overall developmental delay. We also found a significant decrease in reproductive potential in female moths, with a drop in egg laying and compromised egg hatching from treated larvae as compared to controls. dsRNA was stable in the insect gut and was efficiently processed into small interfering RNAs (siRNAs), thus accounting for the phenotypes observed in the present work. The study revealed the importance of these genes in core insect processes, which are essential for insect development and survival.

Tritrophic metabolism of plant chemical defenses and its effects on herbivore and predator performance

“…Previous studies on Heliothis virescens revealed that exogenous dsRNA is not only readily degraded, but also lacks intracellular transport in larvae, reducing the efficiency of RNAi (Shukla et al, 2016). P. xylostella , on the other hand, has been successfully silenced by feeding droplets of dsRNA (Bautista et al, 2009) and bacterially produced dsRNA (Israni and Rajam, 2017). However, as a result of the absence of RNA-dependent RNA polymerases in insects, continuous feeding is required to maintain gene silencing (Scott et al, 2013).…”

Detoxification of plant defensive glucosinolates by an herbivorous caterpillar is beneficial to its endoparasitic wasp

“…We quantified ecdysone receptor ( EcR ) gene transcripts in nonparasitized and parasitized P. xylostella larvae (five replicates per treatment), and D. semiclausum symbiotic polydnavirus (PDV)‐related gene transcripts, namely vankyrin1 , vankyrin2 and viral innexin1 , in parasitized P. xylostella larvae (10 replicates per treatment). EcR is induced by the insect hormone ecdysone to control larval development and pupation (Israni & Rajam, 2017), while vankyrin1 , vankyrin2 and viral innexin1 are three of the best‐studied PDV‐related gene transcripts (Etebari et al., 2011). For these measurements, fourth‐instar larvae were individually pooled into TRIzol reagent (Invitrogen, Waltham, MA, USA) in 1.5 ml Eppendorf tubes, and then kept at 4°C before RNA isolation.…”

Prospects, challenges and current status of RNAi through insect feeding

“…In addition, another study observed that feeding of dsRNA expressing live bacteria to larvae and adults of Plutella xylostella and H. armigera insects significantly compromised larval molting and mortality up to 50%. Treatment in adults resulted in 68% inhibition of oviposition and egg hatching, but neonates from surviving eggs showed no sign of transcript inhibition, indicating that application of live bacteria might affect the metamorphosis process . On the other hand, a study performed with S. exigua used both live and heat‐killed transgenic E. coli cells expressing β1 integrin dsRNA.…”

“…Treatment in adults resulted in 68% inhibition of oviposition and egg hatching, but neonates from surviving eggs showed no sign of transcript inhibition, indicating that application of live bacteria might affect the metamorphosis process. 104 On the other hand, a study performed with S. exigua used both live and heat-killed transgenic E. coli cells expressing 1 integrin dsRNA. There was no difference in mortality rate (30-35%) with treatment using either live or heat-killed bacteria.…”