Insecticide resistance in Trialeurodes vaporariorum populations and novel diagnostics for kdr mutations.

BACKGROUND: Neonicotinoids, pyrethroids and ketoenols are currently used for the control of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). In this study, insecticide resistance status and mechanisms were investigated using classical bioassays and molecular techniques. RESULTS: Dose-response bioassays were performed on 19 Greek populations, among the 35 different whitefly populations used for the whole analysis. Resistance factors scaled up to 207-, 4657- and 59-fold for imidacloprid, bifenthrin and spiromesifen, respectively. Molecular assays were used to investigate the frequency of known resistance mutations. A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for detecting the pyrethroid-resistant alleles r1 (mutation L925I) and r2 (mutation T929I) of the para-type voltage-gated sodium channel gene (VGSC). Both alleles were present at high frequencies (on average 65% and 33%, respectively) in 14 populations from Greece. The M918 L pyrethroid resistance mutation was not detected in any of the Greek populations. Sequencing and a Taqman allelic discrimination were used to monitor the frequency of the mutation E645K of the acetyl-coenzyme A carboxylase gene (ACC) recently linked to spiromesifen resistance. This mutation was detected in 20 of the 24 populations examined in ∼38% frequency among the 433 individuals tested. However, its association with the spiromesifen resistance phenotype was not confirmed in the Greek populations. Finally, two homologues of the CYP6CM1 Bemisia tabaci P450, the known neonicotinoid metabolizer, were found upregulated in two T. vaporariorum neonicotinoid-resistant populations; they were both functionally expressed in Escherichia coli, but the recombinant proteins encoded did not metabolize those neonicotinoid insecticides tested. CONCLUSION: The development of simple diagnostics and their use alongside classical and molecular techniques for the early detection of resistant populations are of great importance for pest management strategies. The practical implications of our results are discussed in light of whitefly control. © 2017 Society of Chemical Industry.

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

BACKGROUND: Bemisia 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. RESULTS: We 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. CONCLUSION: Our 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.

Low levels of mitochondrial DNA and symbiont diversity in the worldwide agricultural pest, the greenhouse whitefly Trialeurodes vaporariorum (Hemiptera: Aleyrodidae).

Trialeurodes vaporariorum, the greenhouse whitefly, is a cosmopolitan agricultural pest. Little is known about the genetic diversity of T. vaporariorum and the bacterial symbionts associated with this species. Here, we undertook a large phylogeographic study by investigating both the mitochondrial (mt) diversity and the infection status of 38 T. vaporariorum collections from 18 countries around the world. Genetic diversity of T. vaporariorum was studied by analyzing sequence data from the mt cytochrome oxidase I, cytochrome b, and NADH dehydrogenase subunit 5 genes. Maximum-likelihood (ML) phylogeny reconstruction delineated 2 clades characterized by limited sequence divergence: one clade comprised samples only from the Northern hemisphere whereas the other comprised samples from a broader geographical range. The presence of secondary symbionts was determined by PCR using primers specific for Hamiltonella, Rickettsia, Arsenophonus, Cardinium, Wolbachia, and Fritschea. Most individuals examined harbored at least one secondary endosymbiont, and Arsenophonus was detected in almost all male and female individuals. Wolbachia was present at a much lower frequency, and Cardinium was detected in only a few individuals from Greece. Rickettsia, Hamiltonella, and Fritschea were not found. Additionally, we set out to further analyze Arsenophonus diversity by multilocus sequence typing analysis; however, the Arsenophonus sequences did not exhibit any polymorphism. Our results revealed remarkably low diversity in both mtDNA and symbionts in this worldwide agricultural pest, contrasting sharply with that of the ecologically similar Bemisia tabaci.