RNAi Screening in Spodoptera frugiperda.

RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line.

Analysis of resistance to Cry1Ac in field-collected pink bollworm, Pectinophora gossypiella (Lepidoptera:Gelechiidae), populations.

High survivorship of pink bollworrm, Pectinophora gossypiella in bolls of Bollgard® cotton hybrids and resistance to Cry1Ac protein, expressed in Bollgard cotton were reported in field-populations collected from the state of Gujarat (western India) in 2010. We have found Cry1Ac-resistance in pink bollworm populations sourced from Bollgard and non-Bt cotton fields in the adjoining states of Maharashtra and Madhya Pradesh in Central India. Further, we observed reduced binding of labeled Cry1Ac protein to receptors localized on the brush-border membrane of pink bollworm larval strains with high tolerance to Cry1Ac. These strains were sourced from Bollgard and conventional cotton fields. A pooled Cry1Ac-resistant strain, further selected on Cry1Ac diet also showed significantly reduced binding to Cry1Ac protein. The reduced binding of Cry1Ac to receptors could be an underlying mechanism for the observed resistance in pink bollworm populations feeding on Bollgard hybrids.

Effect of gamma radiation on Phenoloxidase pathway, antioxidant defense mechanism in Helicoverpa armigera (Lepidoptera: Noctuidae) and its implication in inherited sterility towards pest suppression.

PURPOSE: To investigate age-correlated radiosensitivity in highly radioresistant lepidopteran pest, Helicoverpa armigera, upon exposure to ionizing radiation and to examine the irradiation impact on stress-molecular responses in F1 (first-filial) progeny of irradiated (100 Gy) male moths in relation to its reproductive behavior. MATERIALS AND METHODS: Efficacy of sub-lethal gamma radiation was evaluated on two markedly apart ontogenic stages, neonates and adult moths. Differential growth, reproductive behavior and stress-indicating molecular responses were examined upto F1 progeny of sub-sterilized moths. Free-radical scavenging enzymes, superoxide dismutase (SOD), catalase (CAT) and Phenoloxidase cascade enzymes, pro-phenoloxidase (PPO), its activating enzyme (PPAE) were studied in irradiated and irradiated plus microbial challenge regimen (dual-stress) by Real-time RT-PCR (reverse-transcription-polymerase-chain-reaction). RESULTS: An inverse correlation of radiosensitivity with developmental age of insect was observed. F1 sterility was higher than parent sterility. F1 progeny exhibited protraction in development and decreased survival upon irradiation. Sex ratio in F1 progeny was skewed towards males. PPO, PPAE, SOD and CAT transcripts were downregulated upon neonate irradiation resulting in enhanced vulnerability of larvae to incidental microbial challenge. These transcripts were upregulated in F1 progeny of sub-sterilized male moths (100 Gy) upon dual-stress. CONCLUSIONS: Irradiation impact on stress-indicating molecular responses in F1 progeny is correlated with its reproductive performance. These observations will permit defining regimen having pragmatic viability of 'F1 sterility technique' for pest suppression. Gamma dose of 100 Gy would ensure balance between induced sterility of males and their field competitiveness. These parameters would facilitate integration of biocontrol strategy with parabiological 'Sterile Insect Release Technique'.

Characterization of a Chitin-Binding Protein from Bacillus thuringiensis HD-1.

Strains of Bacillus thuringiensis produce insecticidal proteins. These strains have been isolated from diverse ecological niches, such as soil, phylloplane, insect cadavers and grain dust. To effectively propagate, these strains produce a range of molecules that facilitate its multiplication in a competing environment. In this report, we have examined synthesis of a chitin-binding protein and evaluated its effect on fungi encountered in environment and its interaction with insecticidal proteins synthesized by B. thuringiensis. The gene encoding chitin-binding protein has been cloned and expressed. The purified protein has been demonstrated to interact with Cry insecticidal protein, Cry1Ac by Circular Dichrosim spectroscopy (CD) and in vitro pull down assays. The chitin-binding protein potentiates insecticidal activity of bacillar insecticidal protein, Cry1Ac. Further, chitin-binding protein was fungistatic against several soil fungi. The chitin binding protein is expressed in spore mother cell and deposited along with insecticidal protein, Cry1Ac. It interacts with Cry1Ac to potentiate its insecticidal activity and facilitate propagation of Bacillus strain in environment by inhibiting growth of certain fungi.

Development associated profiling of chitinase and microRNA of Helicoverpa armigera identified chitinase repressive microRNA.

Expression of chitinase is developmentally regulated in insects in consonance with their molting process. During the larval-larval metamorphosis in Helicoverpa armigera, chitinase gene expression varies from high to negligible. In the five-day metamorphic course of fifth-instar larvae, chitinase transcript is least abundant on third day and maximal on fifth day. MicroRNA library prepared from these highest and lowest chitinase-expressing larval stages resulted in isolation of several miRNAs. In silico analysis of sequenced miRNAs revealed three miRNAs having sequence similarity to 3'UTR of chitinase. Gene-targeted specific action of these miRNAs, was investigated by luciferase reporter having 3'UTR of chitinase. Only one of three miRNAs, miR-24, inhibited luciferase expression. Further, a day-wise in vivo quantification of miR-24 in fifth-instar larvae revealed a negative correlation with corresponding chitinase transcript abundance. The force-feeding of synthetic miR-24 induced significant morphological aberrations accompanied with arrest of molting. These miR-24 force-fed larvae revealed significantly reduced chitinase transcript abundance.

Interaction of Bacillus thuringiensis vegetative insecticidal protein with ribosomal S2 protein triggers larvicidal activity in Spodoptera frugiperda.

Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.

Expression, purification, and characterization of pro-phenoloxidase-activating serine protease from Spodoptera litura.

One of the important trigger molecules for innate immunity is a serine protease that activates zymogen phenol oxidase (PPO). Central to wound healing response is the activation of phenol oxidase zymogen. Molecular characterization of phenol oxidase has been recently reported by us. Here, we report isolation, cloning, expression, and purification of prophenol oxidase activating enzyme 1 (slppae1) from polyphagous pest, Spodoptera litura. SLPPAE1 is induced within 6 h of physical injury. The structural features of the mature polypeptide are reminiscent of other lepidopteran PPAE in having a signal peptide, propeptide, and catalytically active polypeptide. The cDNA has been expressed in Sf21 cells using baculovirus expression vector. Fractionation of expressing Sf21 cells revealed its expression in the membranes. The recombinant protein was solubilized from membranes and purified by Ni-NTA affinity chromatography. The purified enzyme is catalytically active on chromogenic substrate, activates recombinantly expressed prophenol oxidase (PPO) of S. litura, and is sensitive to inhibition by aprotenin. N-terminal sequencing of processed phenol oxidase revealed 11 kDa propeptide instead of in-silico predicted 6 kDa polypeptide.