Evangelia Morou

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

Background Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations. Methodology/Principal Findings We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically. Conclusions/Significance The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.

Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance

The neonicotinoid imidacloprid is one of the most important insecticides worldwide. It is used extensively against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), an insect pest of eminent importance globally, which was also the first pest to develop high levels of resistance against imidacloprid and other neonicotinoids in the field. Recent reports indicated that in both the B and Q biotypes of B. tabaci, the resistant phenotype is associated with over-expression of the cytochrome P450 gene CYP6CM1. In this study, molecular docking and dynamic simulations were used to analyze interactions of imidacloprid with the biotype Q variant of the CYP6CM1 enzyme (CYP6CM1vQ). The binding mode with the lowest energy in the enzyme active site, the key amino acids involved (i.e. Phe-130 and Phe-226), and the putative hydroxylation site (lowest distance to carbon 5 of the imidazolidine ring system of imidacloprid) were predicted. Heterologous expression of the CYP6CM1vQ confirmed the accuracy of our predictions and demonstrated that the enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form (K(cat) = 3.2 pmol/min/pmol P450, K(m) = 36 microM). The data identify CYP6CM1vQ as a principle target for inhibitor design, aimed at inactivating insecticide-metabolizing P450s in natural insect pest populations.

Current status of insecticide resistance in Q biotype Bemisia tabaci populations from Crete

BACKGROUND A major problem of crop protection in Crete, Greece, is the control of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) with chemical insecticides owing to the rapid development of resistance. The aim of this study was to investigate the establishment of resistance and the underlying mechanisms to major insecticide classes with classical bioassays and known biochemical resistance markers. RESULTS During a 2005-2007 survey, 53 Q biotype populations were collected. Application history records showed extensive use of neonicotinoids, organophosphates, carbamates and pyrethroids. High resistance levels were identified in the majority of populations (>80%) for imidacloprid (RF: 38-1958x) and alpha-cypermethrin (RF: 30-600x). Low resistance levels (RF < 12) were observed for pirimiphos-methyl. A strong correlation between resistance to imidacloprid and the number of applications with neonicotinoids was observed. Significant correlations were observed between COE and P450-dependent monoxygenase activity with resistance to alpha-cypermethrin and imidacloprid respectively. A propoxur-based AChE diagnostic test indicated that iAChE was widespread in most populations. Resistance levels for alpha-cypermethrin were increased when compared with a previous survey (2002-2003). Differentiation of LC(50) values between localities was observed for imidacloprid only. CONCLUSION Bemisia tabaci resistance evolved differently in each of the three insecticides studied. Imidacloprid resistance seems less established and less persistent than alpha-cypermethrin resistance. The low resistance levels for pirimiphos-methyl suggest absence of cross-resistance with other organophosphates or carbamates used.

Abamectin is metabolized by CYP392A16, a cytochrome P450 associated with high levels of acaricide resistance in Tetranychus urticae

Abamectin is one of the most important insecticides worldwide. It is used against major agricultural pests and insects of public health importance, as well as against endoparasites in animal health. Abamectin has been used successfully for the control of the spider mite Tetranychus urticae, a major agricultural pest with global distribution, an extremely diverse host range, and a remarkable ability to develop resistance against insecticides including abamectin. Target site resistance mutations may explain a large part of resistance, although genetic evidence and transcriptomic data indicated that additional mechanisms may also be implicated in the abamectin resistant phenotype. To investigate a functional link between cytochrome P450-mediated metabolism and abamectin resistance, we recombinantly expressed three cytochrome P450s (CYP392A16, CYP392D8 and CYP392D10) that have been associated with high levels of abamectin resistance in a resistant T. urticae strain isolated from Greece. CYP392A16 was expressed predominately in its P450 form however, both CYP392D8 and CYP392D10 were expressed predominately as P420, despite optimization efforts on expression conditions. CYP392A16 catalyses the hydroxylation of abamectin (Kcat=0.54 pmol/min/pmol P450; Km=45.9 μM), resulting in a substantially less toxic compound as confirmed by bioassays with the partially purified metabolite. However, CYP392A16 did not metabolize hexythiazox, clofentezine and bifenthrin, active ingredients that also showed reduced toxicity in the abamectin resistant strain. Among a number of fluorescent and luminescent substrates screened, Luciferin-ME EGE was preferentially metabolized by CYP392A16, and it may be a potential diagnostic probe for metabolic resistance detection and monitoring.

Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates

BACKGROUND In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos-methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos-methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR-RLFP assay developed in this study.

Assessment of the Bemisia tabaci CYP6CM1vQ transcript and protein levels in laboratory and field-derived imidacloprid-resistant insects and cross-metabolism potential of the recombinant enzyme

Abstract  Over‐expression of the cytochrome P450 CYP6CM1 gene has been associated with imidacloprid resistance in a number of Q and B biotype Bemisia tabaci laboratory strains from distinct geographical origins worldwide. We recently demonstrated that the Q biotype version of the CYP6CM1 protein (CYP6CM1vQ) is capable of metabolizing imidacloprid. Here, we show that the levels of BtCYP6CM1vQ were also elevated in laboratory‐resistant strains and field‐derived populations, with variable imidacloprid resistance levels, collected in Crete. High levels of CYP6CM1vQ transcripts were also determined in survivors of a heterogeneous field population, after exposure to discriminating imidacloprid dosage. Using peptide antibody‐based detection assays, we demonstrated that in line with transcriptional data, the CYP6CM1vQ protein levels were higher in imidacloprid‐resistant insects, which further implicates the gene as the causal factor of resistance. Finally, assessment of the cross‐metabolism potential of CYP6CM1vQ against additional neonicotinoid molecules used for B. tabaci control revealed that clothianidin and thiacloprid, but not acetamiprid or thiamethoxam, are metabolized by the recombinant enzyme in vitro.

Efficacy of the pyrethroid alpha-cypermethrin against Bactrocera oleae populations from Greece, and improved diagnostic for an iAChE mutation

BACKGROUND The most important pest of olive orchards worldwide is the olive fruit fly Bactrocera oleae (Gmelin). Its control in Greece has been based on organophosphates (OPs), but their intense use has led to the development of resistance. A test previously developed to monitor the trait may not be as robust as originally thought. The pyrethroid alpha-cypermethrin has recently been registered for bait sprays, as an alternative to OPs. RESULTS The susceptibility of 20 B. oleae populations to alpha-cypermethrin was examined. Variation was observed in their response, with LD(50) ranging from 0.14 to 3.28 ng insect(-1) and resistance factors from 2.3 to 54.7. Resistance mechanisms were investigated. Cytochrome P450 monoxygenase activities showed an association with resistance. Sequences in the domain IIS4-IIS6 of the B. oleae para-type sodium channel were also analysed, but no resistance-associated mutations were identified. Finally, a novel diagnostic assay able to reliably monitor the frequency of the iAChE G488S resistance mutation was developed. CONCLUSION This is the first attempt to evaluate the efficacy of alpha-cypermethrin against B. oleae from Greece. Data showed that it can be used effectively, but also highlighted the importance of continuous monitoring. The IIS4-IIS6 sodium channel region is the default area in which to look for resistance mutations if target-site resistance to pyrethroids arises. The application of the novel iAChE molecular diagnostic may facilitate the introduction of pyrethroids alongside OPs currently in use.

Insecticide resistance in Bemisia tabaci from Cyprus

Abstract  A comprehensive study on the Bemisia tabaci (biotype B) resistance to neonicotinoid insecticides imidacloprid, acetamiprid and thiamethoxam, and pyrethroid bifenthrin was conducted in Cyprus. The resistance level to eight field‐collected B. tabaci populations was investigated. The activities of enzymes involved in metabolic detoxification and the frequencies of pyrethroid and organophosphates target site resistance mutations were determined. Moderate to high levels of resistance were detected for imidacloprid (resistance factor [RF] 77–392) and thiamethoxam (RF 50–164) while low resistance levels were observed for acetamiprid (RF 7–12). Uniform responses by the Cypriot whiteflies could be observed against all neonicotinoid insecticides. No cross‐resistance between the neonicotinoids was detected as well as no association with the activity of the P450 microsomal oxidases. Only imidacloprid resistance correlated with carboxylesterase activity. Low to extremely high resistance was observed for insecticide bifenthrin (RF 49–1  243) which was associated with the frequency of the resistant allele in the sodium channel gene but not with the activity of the detoxification enzymes. Finally, the F331W mutation in the acetylcholinesterase enzyme ace1 gene was fixed in all B. tabaci populations from Cyprus.

Resurgence of the cotton bollworm Helicoverpa armigera in northern Greece associated with insecticide resistance

Helicoverpa armigera has been controlled effectively with chemical insecticides in the major cotton crop production areas of northern Greece for many years. However, a resurgence of the pest was observed in 2010, which significantly affected crop production. During a 4‐year survey (2007–2010), we examined the insecticide resistance status of H. armigera populations from two major and representative cotton production areas in northern Greece against seven insecticides (chlorpyrifos, diazinon, methomyl, alpha‐cypermethrin, cypermethrin, gamma‐cyhalothrin and endosulfan). Full dose‐response bioassays on third instar larvae were performed by topical application. Lethal doses at 50% were estimated by probit analysis and resistance factors (RF) were calculated, compared to a susceptible laboratory reference strain. Resistance levels were relatively moderate until 2009, with resistance ratios below 10‐fold for organophosphates and carbamates and up to 16‐fold for the pyrethroid alpha‐cypermethrin. However, resistance rose to 46‐ and 81‐fold for chlorpyrifos and alpha‐cypermethrin, respectively in 2010, when the resurgence of the pest was observed. None of the known pyrethroid resistance mutations were found in the pyrethroid‐resistant insects. The possible association between resistance and H. armigera resurgence in Greece is discussed.

Transcriptome Profiling and Genetic Study Reveal Amplified Carboxylesterase Genes Implicated in Temephos Resistance, in the Asian Tiger Mosquito Aedes albopictus

Background The control of Aedes albopictus, a major vector for viral diseases, such as dengue fever and chikungunya, has been largely reliant on the use of the larvicide temephos for many decades. This insecticide remains a primary control tool for several countries and it is a potential reliable reserve, for emergency epidemics or new invasion cases, in regions such as Europe which have banned its use. Resistance to temephos has been detected in some regions, but the mechanism responsible for the trait has not been investigated. Principal findings Temephos resistance was identified in an Aedes albopictus population isolated from Greece, and subsequently selected in the laboratory for a few generations. Biochemical assays suggested the association of elevated carboxylesterases (CCE), but not target site resistance (altered AChE), with this phenotype. Illumina transcriptomic analysis revealed the up-regulation of three transcripts encoding CCE genes in the temephos resistant strain. CCEae3a and CCEae6a showed the most striking up-regulation (27- and 12-folds respectively, compared to the reference susceptible strain); these genes have been previously shown to be involved in temephos resistance also in Ae. aegypti. Gene amplification was associated with elevated transcription levels of both CCEae6a and CCEae3a genes. Genetic crosses confirmed the genetic link between CCEae6a and CCEae3a amplification and temephos resistance, by demonstrating a strong association between survival to temephos exposure and gene copy numbers in the F2 generation. Other transcripts, encoding cytochrome P450s, UDP-glycosyltransferases (UGTs), cuticle and lipid biosynthesis proteins, were upregulated in resistant mosquitoes, indicating that the co-evolution of multiple mechanisms might contribute to resistance. Significance The identification of specific genes associated with insecticide resistance in Ae. albopictus for the first time is an important pre-requirement for insecticide resistance management. The genomic resources that were produced will be useful to the community, to study relevant aspects of Ae. albopictus biology.