ABSTRACT
Maconellicoccus hirsutus is a highly polyphagous insect pest, posing a substantial threat to various crop sp., especially in the tropical and sub-tropical regions of the world. While extensive physiological and biological studies have been conducted on this pest, the lack of genetic information has hindered our understanding of the molecular mechanisms underlying its growth, development, and xenobiotic metabolism. The Cytochrome P450 gene, a member of the CYP gene superfamily ubiquitous in living organisms is associated with growth, development, and the metabolism of both endogenous and exogenous substances, contributing to the insect's adaptability in diverse environments. To elucidate the specific role of the CYP450 gene family in M. hirsutus which has remained largely unexplored, a de novo transcriptome assembly of the pink mealybug was constructed. A total of 120 proteins were annotated as CYP450 genes through homology search of the predicted protein sequences across different databases. Phylogenetic studies resulted in categorizing 120 CYP450 genes into four CYP clans. A total of 22 CYP450 families and 30 subfamilies were categorized, with CYP6 forming the dominant family. The study also revealed five genes (Halloween genes) associated with the insect hormone biosynthesis pathway. Further, the expression of ten selected CYP450 genes was studied using qRT-PCR across crawler, nymph, and adult stages, and identified genes that were expressed at specific stages of the insects. Thus, the findings of this study reveal the expression dynamics and possible function of the CYP450 gene family in the growth, development, and adaptive strategies of M. hirsutus which can be further functionally validated.
ABSTRACT
Insecticide resistance is a global concern and requires immediate attention to manage dreadful insect pests. One of the resistance mechanisms adopted by insects is through ATP-binding cassette (ABC) transporter proteins. These proteins rapidly transport and eliminate the insecticidal molecules across the lipid membranes (Phase III detoxification mechanism). In the present study, we investigated the potential role of ABC transporter genes in imparting insecticide resistance in field-collected insecticide resistant larvae of eggplant shoot and fruit borer (Leucinodes orbonalis; Crambidae: Lepidoptera). Dose-mortality bioassays against five insecticidal molecules revealed moderate to high levels of insecticide resistance (32.2. to 134.1-fold). Thirty-one genes encoding ABC transporter proteins were mined from the transcriptome resources of L. orbonalis. They were classified under eight sub-families (ABCA to ABCH). Phylogenetic analysis indicated ABCG is the most divergent, composed of nine genes as compared to many other insects. Transcriptome analysis of the insecticide resistant and susceptible strains of L. orbonalis revealed differential expression of 13 ABC transporter genes. The altered expression of these genes was further validated using qRT-PCR. The knockdown studies indicated the involvement of ABCD1 and ABCG2 genes in chlorantraniliprole resistance in the insecticide-resistant strain of L. orbonalis. This study unveils the additional mechanisms employed by L. orbonalis in resisting insecticide toxicity through accelerated excretion mode. These ABCD and ABCG family genes could be candidate targets in developing genome-assisted pest management strategies in the future.
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.