Kevin Gorman

Over-expression of cytochrome P450 CYP6CM1 is associated with high resistance to imidacloprid in the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae).

The two most damaging biotypes of Bemisia tabaci, B and Q, have both evolved strong resistance to the neonicotinoid insecticide imidacloprid. The major mechanism in all samples investigated so far appeared to be enhanced detoxification by cytochrome P450s monooxygenases (P450s). In this study, a polymerase chain reaction (PCR) technology using degenerate primers based on conserved P450 helix I and heme-binding regions was employed to identify P450 cDNA sequences in B. tabaci that might be involved in imidacloprid resistance. Eleven distinct P450 cDNA sequences were isolated and classified as members of the CYP4 or CYP6 families. The mRNA expression levels of all 11 genes were compared by real-time quantitative RT-PCR across nine B and Q field-derived strains of B. tabaci showing strong resistance, moderate resistance or susceptibility to imidacloprid. We found that constitutive over-expression (up to approximately 17-fold) of a single P450 gene, CYP6CM1, was tightly related to imidacloprid resistance in both the B and Q biotypes. Next, we identified three single-nucleotide polymorphic (SNP) markers in the intron region of CYP6CM1 that discriminate between the resistant and susceptible Q-biotype CYP6CM1 alleles (r-Q and s-Q, respectively), and used a heterogeneous strain to test for association between r-Q and resistance. While survivors of a low imidacloprid dose carried both the r-Q and s-Q alleles, approximately 95% of the survivors of a high imidacloprid dose carried only the r-Q allele. Together with previous evidence, the results reported here identify enhanced activity of P450s as the major mechanism of imidacloprid resistance in B. tabaci, and the CYP6CM1 gene as a leading target for DNA-based screening for resistance to imidacloprid and possibly other neonicotinoids in field populations.

Biotype status and genetic polymorphism of the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) in Greece: mitochondrial DNA and microsatellites

The genetic polymorphism and the biotype identity of the tobacco whitefly Bemisia tabaci (Gennadius) have been studied in population samples taken from different localities within Greece from cultivated plants growing in greenhouses or in open environments and from non-cultivated plants. Two different approaches were used: sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene and genotyping using microsatellite markers. Analyses of the mtCOI sequences revealed a high homogeneity between the Greek samples which clustered together with Q biotype samples that had been collected from other countries. When genetic polymorphism was examined using six microsatellite markers, the Greek samples, which were all characterized as Q biotype were significantly differentiated from each other and clustered into at least two distinct genetic populations. Moreover, based on the fixed differences revealed by the mtCOI comparison of known B. tabaci biotype sequences, two diagnostic tests for discriminating between Q and B and non-Q/non-B biotypes were developed. Implementation of these diagnostic tools allowed an absence of the B biotype and presence of the Q biotype in the Greek samples to be determined.

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7‐ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real‐time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40‐fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid.

Neonicotinoid resistance in rice brown planthopper, Nilaparvata lugens.

BACKGROUND Rice brown planthopper, Nilaparvata lugens Stål, is a primary insect pest of cultivated rice, and effective control is essential for economical crop production. Resistance to neonicotinoid insecticides, in particular imidacloprid, has been reported as an increasing constraint in recent years. In order to investigate the extent of resistance, 24 samples of N. lugens were collected from China, India, Indonesia, Malaysia, Thailand and Vietnam during 2005 and 2006. Their responses to two diagnostic doses of imidacloprid (corresponding approximately to the LC(95) and 5 x LC(95) of a susceptible strain) were examined. RESULTS Ten of the 12 samples collected during 2005 were found to be susceptible to imidacloprid, but two late-season samples from India showed reduced mortality at both diagnostic doses. All 13 strains collected in 2006 showed reduced mortality at both doses when compared with the susceptible strain. Dose-response lines showed resistance in one of the most resistant field strains to be approximately 100-fold compared with the susceptible standard. CONCLUSION The data demonstrate the development and spread of neonicotinoid resistance in N. lugens in Asia and support reports of reduced field efficacy of imidacloprid.

Incidence and characterisation of resistance to neonicotinoid insecticides and pymetrozine in the greenhouse whitefly, Trialeurodes vaporariorum Westwood (Hemiptera: Aleyrodidae)

BACKGROUND Trialeurodes vaporariorum (Westwood), also known as the greenhouse whitefly, is a serious pest of protected vegetable and ornamental crops in most temperate regions of the world. Neonicotinoid insecticides are used widely to control this species, although resistance has been reported and may be becoming widespread. RESULTS Mortality rates of UK and European strains of T. vaporariorum to a range of neonicotinoids and pymetrozine, a compound with a different mode of action, were calculated, and significant resistance was found in some of those strains. A strong association was found between neonicotinoids and pymetrozine, and reciprocal selection experiments confirmed this finding. Expression of resistance to the neonicotinoid imidacloprid and pymetrozine was age specific, and resistance in nymphs did not compromise recommended application rates. CONCLUSION This study indicates strong parallels in the phenotypic characteristics of neonicotinoid resistance in T. vaporariorum and the tobacco whitefly Bemisia tabaci Gennadius, suggesting possible parallels in the underlying mechanisms.

Age-specific expression of resistance to a neonicotinoid insecticide in the whitefly Bemisia tabaci

Neonicotinoid insecticides retain a crucial role within many chemical and integrated control strategies for the tobacco whitefly, Bemisia tabaci Gennadius, in spite of the establishment of potent and widespread resistance in many areas. Metabolic resistance mechanisms mediated by overexpression of P450-dependent monooxygenases have been implicated in neonicotinoid resistance in the two most prevalent B. tabaci biotypes. Further characterisation of resistance to the neonicotinoid imidacloprid in populations of both these B- and Q-types is reported.Expression of resistance to imidacloprid was age specific in B- and Q-type strains of B. tabaci. The highest observed resistance ratio at LC(50) expressed in prepupal nymphs was 13, compared with at least 580 in their adult counterparts. For all strains, resistance expressed in immatures was not sufficiently potent to compromise recommended imidacloprid application rates.Targeting neonicotinoids towards immature life stages of B. tabaci may circumvent the protection conferred by current mechanisms of resistance, simultaneously reducing the selection pressures imposed. However, such tactics may enhance the expression of existing resistance mechanisms in immatures, or promote the establishment of novel ones expressed in all life stages.

Cross-resistance relationships of the sulfoximine insecticide sulfoxaflor with neonicotinoids and other insecticides in the whiteflies Bemisia tabaci and Trialeurodes vaporariorum.

BACKGROUND Insecticides are important tools for managing damaging insect pests. Compounds that are effective against pests such as the whiteflies Bemisia tabaci and Trialeurodes vaporariorum, which show resistance to a range of insecticidal modes of action (MOA), have particular value as components of resistance management programmes. The sulfoximine insecticides are chemically unique as the first to incorporate a sulfoximine functional group. Sulfoxaflor is the first sulfoximine compound under commercial development for the control of sap-feeding insects. Its cross-resistance relationships were investigated by comparing the responses of field-collected strains with those of insecticide-susceptible laboratory strains of B. tabaci and T. vaporariorum. RESULTS Sulfoxaflor exhibited very low (less than threefold) resistance ratios (RR) when tested against strains of B. tabaci that produced RR of up to 1000-fold to imidacloprid and cross-resistance to other neonicotinoid insecticides. Similarly, sulfoxaflor was not cross-resistant in a strain of B. tabaci exhibiting resistance to a pyrethroid (deltamethrin) and an organophosphate (profenophos). No cross-resistance was observed between sulfoxaflor and imidacloprid in T. vaporariorum. One population of the three field strains tested showed slightly reduced susceptibility to sufloxaflor with an RR of 4.17. By comparison, this same population exhibited an RR of more than 23.8-fold for imidacloprid relative to the susceptible population. CONCLUSION In spite of sharing a target site with neonicotinoids (the nicotinic acetylcholine receptor), sulfoxaflor was largely unaffected by existing cases of neonicotinoid resistance in B. tabaci and T. vaporariorum. Neonicotinoid resistance mechanisms in these whitefly species are known to be primarily based on enhanced detoxification of insecticide. This lack of cross-resistance indicates that sulfoxaflor is a valuable new tool for the management of sap-feeding pests already resistant to established insecticide groups.

Over-Expression of a Cytochrome P450 Is Associated with Resistance to Pyriproxyfen in the Greenhouse Whitefly Trialeurodes vaporariorum

Background The juvenile hormone mimic, pyriproxyfen is a suppressor of insect embryogenesis and development, and is effective at controlling pests such as the greenhouse whitefly Trialeurodes vaporariorum (Westwood) which are resistant to other chemical classes of insecticides. Although there are reports of insects evolving resistance to pyriproxyfen, the underlying resistance mechanism(s) are poorly understood. Results Bioassays against eggs of a German (TV8) population of T. vaporariorum revealed a moderate level (21-fold) of resistance to pyriproxyfen. This is the first time that pyriproxyfen resistance has been confirmed in this species. Sequential selection of TV8 rapidly generated a strain (TV8pyrsel) displaying a much higher resistance ratio (>4000-fold). The enzyme inhibitor piperonyl butoxide (PBO) suppressed this increased resistance, indicating that it was primarily mediated via metabolic detoxification. Microarray analysis identified a number of significantly over-expressed genes in TV8pyrsel as candidates for a role in resistance including cytochrome-P450 dependent monooxygenases (P450s). Quantitative PCR highlighted a single P450 gene (CYP4G61) that was highly over-expressed (81.7-fold) in TV8pyrsel. Conclusion Over-expression of a single cytochrome P450 gene (CYP4G61) has emerged as a strong candidate for causing the enhanced resistance phenotype. Further work is needed to confirm the role of the encoded P450 enzyme CYP4G61 in detoxifying pyriproxyfen.

Characterisation of Neonicotinoid and Pymetrozine Resistance in Strains of Bemisia tabaci (Hemiptera: Aleyrodidae) from China

Abstract Four strains of the Q biotype and one of the B biotype of the whitefly Bemisia tabaci collected from China were characterised for resistance to four neonicotinoid insecticides and pymetrozine. Q biotype strains showed moderate to strong resistance to imidacloprid, thiamethoxam and acetamiprid, but little or no cross-resistance to dinotefuron. Resistance to neonicotinoids was consistently associated with resistance to pymetrozine, despite the latter having a distinct (though unresolved) mode of action. The single B biotype strain proved largely susceptible to all the insecticides investigated. Resistance in the Q biotype strains was associated with over-expression of a cytochrome P450 monooxygenase gene, CYP6CM1 , whose substrate specificity presumably accounts for the observed cross-resistance profiles.

Field‐evolved resistance to imidacloprid and ethiprole in populations of brown planthopper Nilaparvata lugens collected from across South and East Asia

Abstract BACKGROUND We report on the status of imidacloprid and ethiprole resistance in Nilaparvata lugens Stål collected from across South and East Asia over the period 2005–2012. RESULTS A resistance survey found that field populations had developed up to 220‐fold resistance to imidacloprid and 223‐fold resistance to ethiprole, and that many of the strains collected showed high levels of resistance to both insecticides. We also found that the cytochrome P450 CYP6ER1 was significantly overexpressed in 12 imidacloprid‐resistant populations tested when compared with a laboratory susceptible strain, with fold changes ranging from ten‐ to 90‐fold. In contrast, another cytochrome P450 CYP6AY1, also implicated in imidacloprid resistance, was underexpressed in ten of the populations and only significantly overexpressed (3.5‐fold) in a single population from India compared with the same susceptible strain. Further selection of two of the imidacloprid‐resistant field strains correlated with an approximate threefold increase in expression of CYP6ER1. CONCLUSIONS We conclude that overexpression of CYP6ER1 is associated with field‐evolved resistance to imidacloprid in brown planthopper populations in five countries in South and East Asia.