Transcription analysis of neonicotinoid resistance in Mediterranean (MED) populations of B. tabaci reveal novel cytochrome P450s, but no nAChR mutations associated with the phenotype

Abstract

BackgroundBemisia tabaci is one of the most damaging agricultural pests world-wide. Although its control is based on insecticides, B. tabaci has developed resistance against almost all classes of insecticides, including neonicotinoids.ResultsWe employed an RNA-seq approach to generate genome wide expression data and identify genes associated with neonicotinoid resistance in Mediterranean (MED) B. tabaci (Q1 biotype). Twelve libraries from insecticide resistant and susceptible whitefly populations were sequenced on an Illumina Next-generation sequencing platform, and genomic sequence information of approximately 73 Gbp was generated.A reference transcriptome was built by de novo assembly and functionally annotated. A total of 146 P450s, 18 GSTs and 23 CCEs enzymes (unigenes) potentially involved in the detoxification of xenobiotics were identified, along with 78 contigs encoding putative target proteins of six different insecticide classes. Ten unigenes encoding nicotinic Acetylcholine Receptors (nAChR), the target of neoinicotinoids, were identified and phylogenetically classified. No nAChR polymorphism potentially related with the resistant phenotypes, was observed among the studied strains.DE analysis revealed that among the 550 differentially (logFC > 1) over-transcribed unigenes, 52 detoxification enzymes were over expressed including unigenes with orthologues in P450s, GSTs, CCE and UDP-glucuronosyltransferases.Eight P450 unigenes belonging to clades CYP2, CYP3 and CYP4 were highly up-regulated (logFC > 2) including CYP6CM1, a gene already known to confer imidacloprid resistance in B. tabaci. Using quantitative qPCRs, a larger screening of field MED B. tabaci from Crete with known neonicotinoid phenotype was performed to associate expression levels of P450s with resistance levels. Expression levels of five P450s, including CYP6CM1, were found associated with neonicotinoid resistance. However, a significant correlation was found only in CYP303 and CYP6CX3, with imidacloprid and acetamiprid respectively.ConclusionOur work has generated new toxicological data and genomic resources which will significantly enrich the available dataset and substantially facilitate the molecular studies in MED B. tabaci. No evidence of target site neonicotinoid resistance has been found. Eight P450 unigenes, including CYP6CM1, were found significantly over-expressed in resistant B. tabaci. This study suggests at least two novel P450s (CYP303 and CYP6CX3) as candidates for their functional characterization as detoxification mechanisms of neonicotinoid resistance in B. tabaci.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2161-5) contains supplementary material, which is available to authorized users.

Use of the synergist piperonyl butoxide can slow the development of alpha‐cypermethrin resistance in the whitefly Bemisia tabaci

“…This suggests that CYP6CM1 does not explain the difference in resistance levels of these two lines, and that other genes may be involved. Beyond CYP6CM1 other candidate P450 genes have been implicated in neonicotinoid resistance in B. tabaci in previous studies, ie CYP6CX1, CYP6CX3, CYP6CX4, CYP6DZ7 and CYP4C64 (Yang et al, 2013;Ilias et al, 2015); however, none of these was upregulated by thiacloprid selection in our study. No other obvious candidate genes were identified as downregulated in the thiacloprid 1 PBO line compared to the thiacloprid selected line that might explain the greater level of resistance observed in the latter.…”

“…3, Table S6) with the pronounced overexpression of CYP6CM1 confirmed in the thiacloprid selected lines. The overexpression of several P450s has been implicated in neonicotinoid resistance in B. tabaci in previous studies (Karunker et al, 2008;Wang et al, 2009;Yang et al, 2013;Ilias et al, 2015); however, limited work has been done to explore if qualitative changes are also involved in resistance. To explore if qualitative changes in candidate P450s underlie the resistance in the lines under thiacloprid (1/-PBO) selection a Single Nucleotide Polymorphism (SNP) analysis was carried out for CYP6CM1, CYP6CX1, CYP6CX3, CYP6CX4, CYP6DZ7 and CYP4C64 (Table S7).…”

The draft genome of whitefly Bemisia tabaci MEAM1, a global crop pest, provides novel insights into virus transmission, host adaptation, and insecticide resistance

“…Multiple transcriptome data are available, addressing the phylogenetic relationship and transcriptome sequence divergence of different B. tabaci species [19, 20], responses to a begomovirus [21], insecticide resistance [22], development and organ-specific patterns of gene expression [23–25], and the interactions with symbiotic bacteria required by the insect [26]. However, a fully sequenced B. tabaci genome is still greatly needed for further resolution of the species complex conundrum.…”

Identification of glutathione S‐transferases in Bemisia tabaci (Hemiptera: Aleyrodidae) and evidence that GSTd7 helps explain the difference in insecticide susceptibility between B. tabaci Middle East‐Minor Asia 1 and Mediterranean

“…The first GST reported for any B. tabaci species was a Sigma class GST in B. tabaci MEAM1 (Rauch & Nauen, ). Transcriptome sequencing analyses, like Illumina and Roche 454 pyrosequencing, subsequently identified additional putative GSTs with ESTs and unigenes in B. tabaci ; these included 18 GSTs in B. tabaci MED (Ilias et al ., ), 14 GSTs in B. tabaci MED (Wang et al ., ) and 20 GSTs in MEAM1. (Xie et al ., ).…”