ABSTRACT
BACKGROUND: Maconellicoccus hirsutus is a destructive pest which causes severe losses of agricultural and horticultural crops. For the management of M. hirsutus, many insecticides have been used and it has been exposed to insufficient dosage or uneven spray coverage which resulted in the development of insecticide resistance. Xenobiotic metabolism can be better understood with the help of gene expression studies by unveiling the underlying molecular mechanisms. The qRT-PCR is the simplest method to analyse gene expression, however, it highly relies on suitable reference genes concerning the different experimental conditions. METHODS AND RESULTS: We evaluated the stability of five reference genes in two sets of experimental conditions viz. developmental stages (nymphs and adults) and agrochemical stress (GA3 and Buprofezin sprayed) against M. hirsutus, using different softwares-NormFinder, geNorm, BestKeeper, and RefFinder. The study revealed that ATP51a and GAPDH can be used as reference genes for gene expression studies when exposed to Gibberellic acid. Additionally, the study revealed that the ideal pair of reference genes for data validation in M. hirsutus treated with Buprofezin was GAPDH and ß-tubulin. The ideal reference gene combination for various developmental stages was found to be 28S and Actin. CONCLUSION: According to the study, GAPDH can be utilized as a reliable reference gene in the agrochemical (GA3 and Buprofezin) exposure set. The genes can be utilized as a suitable reference for qRT-PCR gene expression studies of xenobiotic metabolism to understand the underlying molecular mechanism, which will help further to design suitable management strategies.
Subject(s)
Hemiptera , Thiadiazines , Animals , Hemiptera/genetics , Xenobiotics , Real-Time Polymerase Chain Reaction , Gene Expression Profiling/methods , Reference StandardsABSTRACT
The aphid lion, Chrysoperla zastrowi sillemi (Neuroptera: Chrysopidae) is a highly effective beneficial predator of many agricultural pests and has developed resistance to several insecticides. Understanding the molecular mechanism of insecticide resistance in the predators is crucial for its effective application in IPM programs. Therefore, transcriptomes of imidacloprid-resistant and susceptible strains have been assessed using RNA-seq. Cytochrome P450 is one of the important gene families involved in xenobiotic metabolism. Hence, our study focused on the CYP gene family where mining, nomenclature, and phylogenetic analysis revealed a total of 95 unique CYP genes with considerable expansion in CYP3 and CYP4 clans. Further, differential gene expression (DGE) analysis revealed ten CYP genes from CYP3 and CYP4 clans to be differentially expressed, out of which nine genes (CYP4419A1, CYP4XK1, CYP4416A10, CYP4416A-fragment8, CYP6YL1, CYP6YH6, CYP9GK-fragment16, CYP9GN2, CYP9GK6) were downregulated and one (CYP9GK3) was upregulated in the resistant strain as compared to the susceptible strain. Expression validation by quantitative real-time PCR (qRT-PCR) is consistent with the DGE results. The expansion and differential expression of CYP genes may be an indicator of the capacity of the predator to detoxify a particular group of insecticides.
ABSTRACT
Diamondback moth, Plutella xylostella is a serious pest of cruciferous vegetables and causes substantial economic loss all over the world. This study was undertaken to decipher the molecular mechanisms involved in the field evolved insecticide resistance in P. xylostella upon exposure to spinosad. To do so, spinosad-resistant and susceptible larval populations were subjected to transcriptome analysis using Illumina paired-end sequencing. De novo assembly was generated from raw reads of both the samples which resulted in the identification of 41,205 unigenes. Functional annotation and digital gene expression analysis were carried out to determine the differentially expressed genes. 1,348 unigenes were found to have a significant differential expression in the resistant population. Several genes involved in insecticide resistance like CYP P450, GSTs, small heat shock protein, and UDP glycosyltransferase were found to be up-regulated while genes related to mitochondrial energy metabolism and cuticular processes were down-regulated. Further, gene mining and phylogenetic analysis of two important gene families namely, CYP and GSTs were performed and the results revealed that these genes could play a major role in the development of field evolved spinosad resistance in P. xylostella by gene duplication and differential gene expression.
