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Background Resistance to diamide insecticides in Lepidoptera is known to be caused primarily by amino acid changes on the ryanodine receptor (RyR). Recently, two new target site mutations, G4946V and I4790M, have emerged in populations of diamondback moth, Plutella xylostella, as well as in other lepidopteran species, and both mutations have been shown empirically to decrease diamide efficacy. Here, we quantify the impact of the I4790M mutation on diamide activation of the receptor, as compared to alterations at the G4946 locus. Results I4790M when introduced into P. xylostella RyR expressed in an insect derived Sf9 cell line was found to mediate just a 10-fold reduction in chlorantraniliprole efficacy (compared to a 104-fold and 146-fold reduction for G4946E and G4946V variants, respectively), whilst in the field its presence is associated with a ≥ 150-fold reduction. I4790M mediated resistance to flubendiamide was estimated to be >24-fold. When the entire coding sequence of P. xylostella RyR was integrated into Drosophila melanogaster, the I4790M variant conferred ~4.4-fold resistance to chlorantraniliprole and 22-fold resistance to flubendiamide in the 3rd instar larvae, confirming that it imparts only a moderate level of resistance to diamide insecticides. Whereas the I4790M substitution appears to bear no fitness costs in terms of the flies' reproductive capacity, when assessed in a non-competitive environment, it does however have potentially major impacts on mobility at both the larval and adult stage. Conclusions I4790M imparts only a moderate level of resistance to diamide insecticides and potentially confers significant fitness costs to the insect. This article is protected by copyright. All rights reserved.

Mitochondria are intracellular organelles responsible for cellular respiration with one of their major roles in the production of energy in the form of ATP. Activities with increased energetic demand are especially dependent on efficient ATP production, hence sufficient mitochondrial function is fundamental. In bees, flight muscle and the brain have particularly high densities of mitochondria to facilitate the substantial ATP production required for flight activity and neuronal signalling. Neonicotinoids are systemic synthetic insecticides that are widely utilised against crop herbivores but have been reported to cause, by unknown mechanisms, mitochondrial dysfunction, decreasing cognitive function and flight activity among pollinating bees. Here we explore, using high-resolution respirometry, how the neonicotinoid imidacloprid may affect oxidative phosphorylation in the brain and flight muscle of the buff-tailed bumblebee, Bombus terrestris. We find that acute exposure increases routine oxygen consumption in the flight muscle of worker bees. This provides a candidate explanation for prior reports of early declines in flight activity following acute exposure. We further find that imidacloprid increases the maximum electron transport capacity in the brain, with a trend towards increased overall oxygen consumption. However, intra-individual variability is high, limiting the extent to which apparent effects of imidacloprid on brain mitochondria are shown conclusively. Overall, our results highlight the necessity to examine tissue-specific effects of imidacloprid on respiration and energy production.

Many non-model organisms lack reference genomes and the sequencing and de novo assembly of an organism's transcriptome is an affordable means by which to characterize the coding component of its genome. Despite the advances that have made this possible, assembling a transcriptome without a known reference usually results in a collection of full-length and partial gene transcripts. The downstream analysis of genes represented as partial transcripts then often requires further experimental work in the laboratory in order to obtain full- length sequences. We have explored whether partial transcripts, encoding genes of interest present in de novo assembled transcriptomes of a model and non-model insect species, could be further extended by iterative mapping against the raw transcriptome sequencing reads. Partial sequences encoding cytochrome P450s and carboxyl/cholinesterase were used in this analysis because they are large multigene families and exhibit significant variation in expression. We present an effective method to improve the continuity of partial transcripts in silico that, in the absence of a reference genome, maybe a quick and cost-effective alternative to their extension by laboratory experimentation. Our approach resulted in the successful extension of incompletely assembled transcripts, often to full length. We experimentally validated these results \textit{in silico} and using real-time PCR and sequencing.

The cabbage stem flea beetle, Psylliodes chrysocephala L. is a major pest of winter oilseed rape in several European countries. Traditionally, neonicotinoid and pyrethroid insecticides have been widely used for control of P. chrysocephala, but in recent years, following the withdrawal of neonicotinoid insecticide seed treatments, control failures have occurred due to an over reliance on pyrethroids. In line with previous surveys, UK populations of P. chrysocephala were found to exhibit high levels of resistance to the pyrethroid lambda-cyhalothrin. This resistance was suppressed by pre-treatment with the cytochrome P450 inhibitor PBO under laboratory conditions, suggesting that the resistance has a strong metabolic component. The L1014F (kdr) mutation in the voltage-gated sodium channel, which confers relatively low levels (10–20 fold) of resistance to pyrethroids, was also found to be widespread across the UK regions sampled, whereas the L925I (s-kdr) mutation was much less common. The current survey also suggests that higher levels of pyrethroid resistance have spread to the North and West of England, and that resistance levels continue to remain high in the South East., Highlights • UK populations of cabbage stem flea beetle exhibit high levels of resistance to pyrethroid insecticides. • Pyrethroid resistance in UK populations is largely the result of increased metabolism. • Resistance has advanced to the North and West of England. • Resistance levels are highest in Yorkshire and the Humber and the South East of the UK.

There is an on-going need to develop new insecticides that are not compromised by resistance and that have improved environmental profiles. However, the cost of developing novel compounds has increased significantly over the last two decades. This is in part due to increased regulatory requirements, including the need to screen both pest and pollinator insect species to ensure that pre-existing resistance will not hamper the efficacy of a new insecticide via cross-resistance, or adversely affect non-target insect species. To add to this problem the collection and maintenance of toxicologically relevant pest and pollinator species and strains is costly and often difficult. Here we present Fly-Tox, a panel of publicly available transgenic Drosophila melanogaster lines each containing one or more pest or pollinator P450 genes that have been previously shown to metabolise insecticides. We describe the range of ways these tools can be used, including in predictive screens to avoid pre-existing cross-resistance, to identify potential resistance-breaking inhibitors, in the initial assessment of potential insecticide toxicity to bee pollinators, and identifying harmful pesticide-pesticide interactions., Graphical abstract Unlabelled Image, Highlights • Drosophila melanogaster is a powerful model system to study cytochrome P450-mediated insecticide tolerance. • Fly-tox comprises a panel of transgenic D. melanogaster lines expressing P450s from insect pest and pollinator species. • Fly-tox can be used in numerous ways to characterise the role of pest/pollinator P450s in insecticide metabolism. • We describe the applications of Fly-Tox and provide detailed protocols on its use. • The benefits and limitations of the system are discussed.

Diamide insecticides selectively acting on insect ryanodine receptors (RyR) were launched to the market more than 10 years ago, particularly targeted for the control of lepidopteran pest species in diverse agronomic and horticultural cropping systems. They are now globally registered in many countries and provide reliable control levels in most settings. However, their frequent application, due to alternative mode of action chemistries often not providing sufficient levels of control, has resulted in the selection of diamide resistance in some of the world’s most destructive lepidopteran species, including populations of diamondback moth, tomato leafminer, rice stem borer and more recently beet armyworm. High levels of diamide resistance, compromising diamide efficacy at recommended field rates, has been shown to be conferred by RyR target-site mutations affecting diamide binding. The present work reviews the global status of diamide insecticide resistance in lepidopteran pests, with special reference to RyR target-site alterations. Furthermore, we discuss principles enabling the prediction of the impact and spread of diamide resistance, based on population genetics and associated fitness costs as influenced by the known target-site mutations recently described. In this context, we reiterate calls by the Insecticide Resistance Action Committee to implement effective diamide insecticide resistance management by following a three-step strategy of resistance identification, tracking and prediction according to the protocols discussed in this article.

BackgroundThe glasshouse whitefly,Trialeurodes vaporariorum, is a damaging crop pest and an invasive generalist capable of feeding on a broad range of host plants. As such this species has evolved mechanisms to circumvent the wide spectrum of anti-herbivore allelochemicals produced by its host range.T. vaporariorumhas also demonstrated a remarkable ability to evolve resistance to many of the synthetic insecticides used for control.ResultsTo gain insight into the molecular mechanisms that underpin the polyphagy ofT. vaporariorumand its resistance to natural and synthetic xenobiotics, we sequenced and assembled a reference genome for this species. Curation of genes putatively involved in the detoxification of natural and synthetic xenobiotics revealed a marked reduction in specific gene families between this species and another generalist whitefly,Bemisia tabaci. Transcriptome profiling ofT. vaporariorumupon transfer to a range of different host plants revealed profound differences in the transcriptional response to more or less challenging hosts. Large scale changes in gene expression (> 20% of genes) were observed during adaptation to challenging hosts with a range of genes involved in gene regulation, signalling, and detoxification differentially expressed. Remarkably, these changes in gene expression were associated with significant shifts in the tolerance of host-adaptedT. vaporariorumlines to natural and synthetic insecticides.ConclusionsOur findings provide further insights into the ability of polyphagous insects to extensively reprogram gene expression during host adaptation and illustrate the potential implications of this on their sensitivity to synthetic insecticides.

We report the discovery that Bombus terrestris audax (Buff-tailed bumblebee) locomotor trajectories adhere to a speed-curvature power law relationship which has previously been found in humans, non-human primates and Drosophila larval trajectories. No previous study has reported such a finding in adult insect locomotion. We used behavioural tracking to study walking Bombus terrestris in an arena under different training environments. Trajectories analysed from this tracking show the speed-curvature power law holds robustly at the population level, displaying an exponent close to two-thirds. This exponent corroborates previous findings in human movement patterns, but differs from the three-quarter exponent reported for Drosophila larval locomotion. There are conflicting hypotheses for the principal origin of these speed-curvature laws, ranging from the role of central planning to kinematic and muscular skeletal constraints. Our findings substantiate the latter idea that dynamic power-law effects are robust, differing only through kinematic constraints due to locomotive method. Our research supports the notion that these laws are present in a greater range of species than previously thought, even in the bumblebee. Such power laws may provide optimal behavioural templates for organisms, delivering a potential analytical tool to study deviations from this template. Our results suggest that curvature and angular speed are constrained geometrically, and independently of the muscles and nerves of the performing body.

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone – an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.

Voltage-gated sodium channels (VGSCs) are a major target site for the action of pyrethroid insecticides and resistance to pyrethroids has been ascribed to mutations in the VGSC gene. VGSCs in insects are encoded by only one gene and their structural and functional diversity results from posttranscriptional modification, particularly, alternative splicing. Using whole cell patch clamping of neurons from pyrethroid susceptible (wild-type) and resistant strains (s-kdr) of housefly, Musca domestica, we have shown that the V50 for activation and steady state inactivation of sodium currents (INa+) is significantly depolarised in s-kdr neurons compared with wild-type and that 10 nM deltamethrin significantly hyperpolarised both of these parameters in the neurons from susceptible but not s-kdr houseflies. Similarly, tail currents were more sensitive to deltamethrin in wild-type neurons (EC15 14.5 nM) than s-kdr (EC15 133 nM). We also found that in both strains, INa+ are of two types: a strongly inactivating (to 6.8% of peak) current, and a more persistent (to 17.1% of peak) current. Analysis of tail currents showed that the persistent current in both strains (wild-type EC15 5.84 nM) was more sensitive to deltamethrin than was the inactivating type (wild-type EC15 35.1 nM). It has been shown previously, that the presence of exon l in the Drosophila melanogaster VGSC gives rise to a more persistent INa+ than does the alternative splice variant containing exon k and we used PCR with housefly head cDNA to confirm the presence of the housefly orthologues of splice variants k and l. Their effect on deltamethrin sensitivity was determined by examining INa+ in Xenopus oocytes expressing either the k or l variants of the Drosophila para VGSC. Analysis of tail currents, in the presence of various concentrations of deltamethrin, showed that the l splice variant was significantly more sensitive (EC50 42 nM) than the k splice variant (EC50 866 nM). We conclude that in addition to the presence of point mutations, target site resistance to pyrethroids may involve the differential expression of splice variants.

Voltage-gated sodium channels (VGSC) are transmembrane proteins that generate an action potential in excitable cells and play an essential role in neuronal signaling. Since VGSCs play a crucial role in nerve transmission they have become primary targets for a broad range of commercial insecticides. RNA interference (RNAi) is a valuable reverse genetics tool used in functional genomics, but recently, it has also shown promise as a novel agent that could be used to control agricultural insect pests. In this study, we targeted the VGSC (MpNav) gene in the peach-potato aphid Myzus persicae, by oral feeding of artificial diets mixed with dsRNAs. Knock-down of MpNav gene expression caused up to 65% mortality in 3rd instar nymphs. Moreover, significantly lower fecundity and longevity was observed in adult aphids that had been fed with dsMpNav solution at the nymphal stage. Analysis of gene expression by qRT-PCR indicated that the aphid mortality rates and the lowered fecundity and longevity were attributable to the down-regulation of MpNav by RNAi. Taken together, our results show that MpNav is a viable candidate target gene for the development of an RNAi-based bio-aphicide.

Recent work has shown that two bumblebee (Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency. The genomic location and complete ORF of CYP9Q6 was corroborated by PCR and its metabolic activity characterised in vitro by expression in an insect cell line. CYP9Q6 metabolises both thiacloprid and acetamiprid more rapidly than the previously reported CYP9Q4 and CYP9Q5. We further demonstrate a direct, in vivo correlation between the expression of the CYP9Q6 enzyme in transgenic Drosophila melanogaster and an increased tolerance to thiacloprid and acetamiprid. We conclude that CYP9Q6 is an efficient metaboliser of N-cyanoamidine neonicotinoids and likely plays a key role in the high tolerance of B. terrestris to these insecticides., Graphical abstract Image 1, Highlights • A cytochrome P450 gene CYP9Q6 is identified as a potential N-cyanoamidine neonicotinoid metaboliser in Bombus terrestris. • CYP9Q6 efficiently metabolized N-cyanoamidine neonicotinoid insecticides when expressed in vitro in insect cell lines. • Transgenic D. melanogaster expressing CYP9Q6 in vivo had increased tolerance to N-cyanoamidine neonicotinoids. • Amino acid substitutions in the P450 active site may be important determinants of binding and metabolism of these compounds.

The pyrethroid insecticides are a very successful group of compounds that have been used extensively for the control of arthropod pests of agricultural crops and vectors of animal and human disease. Unfortunately, this has led to the development of resistance to the compounds in many species. The mode of action of pyrethroids is known to be via interactions with the voltage-gated sodium channel. Understanding how binding to the channel is affected by amino acid substitutions that give rise to resistance has helped to elucidate the mode of action of the compounds and the molecular basis of their selectivity for insects vs mammals and between insects and other arthropods. Modelling of the channel/pyrethroid interactions, coupled with the ability to express mutant channels in oocytes and study function, has led to knowledge of both how the channels function and potentially how to design novel insecticides with greater species selectivity.

Background: The pyrethroid insecticides are a very successful group of compounds that target invertebrate voltage-gated sodium channels and are widely used in the control of insects, ticks and mites. It is well established that some pyrethroids are good insecticides whereas others are more effective as acaricides. This species specificity is advantageous for controlling particular pest(s) in the presence of another non-target invertebrate, for example controlling the Varroa mite in honey bee colonies. Results: We applied in-silico techniques to compare the voltage-gated sodium channels of insects versus ticks and mites and their interactions with a range of pyrethroids and DDT analogues. We identified a single amino acid difference within the pyrethroid binding pocket of ticks/mites that may have significant impact on the effectiveness of pyrethroids as acaricides. Other individual amino acid differences within the binding pocket in distinct tick and mite species may provide a basis for future acaricidal selectivity. Conclusions: 3D modelling of the pyrethroid/DDT receptor site has led to a new hypothesis to explain the preferential binding of acaricidal pyrethroids to the sodium channels of ticks/mites. This is important for understanding pyrethroid selectivity and the potential effects of mutations that can give rise to resistance to pyrethroids in commercially-important pest species.

We discovered the A301S mutation in the RDL GABA-gated chloride channel of fiprole resistant rice brown planthopper, Nilaparvata lugens populations by DNA sequencing and SNP calling via RNASeq. Ethiprole selection of two field N. lugens populations resulted in strong resistance to both ethiprole and fipronil and resulted in fixation of the A301S mutation, as well as the emergence of another mutation, Q359E in one of the selected strains. To analyse the roles of these mutations in resistance to phenylpyrazoles, three Rdl constructs: wild type, A301S and A301S + Q359E were expressed in Xenopus laevis oocytes and assessed for their sensitivity to ethiprole and fipronil using two-electrode voltage-clamp electrophysiology. Neither of the mutant Rdl subtypes significantly reduced the antagonistic action of fipronil, however there was a significant reduction in response to ethiprole in the two mutated subtypes compared with the wild type. Bioassays with a Drosophila melanogaster strain carrying the A301S mutation showed strong resistance to ethiprole but not fipronil compared to a strain without this mutation, thus further supporting a causal role for the A301S mutation in resistance to ethiprole. Homology modelling of the N. lugens RDL channel did not suggest implications of Q359E for fiprole binding in contrast to A301S located in transmembrane domain M2 forming the channel pore. Synergist bioassays provided no evidence of a role for cytochrome P450s in N. lugens resistance to fipronil and the molecular basis of resistance to this compound remains unknown. In summary this study provides strong evidence that target-site resistance underlies widespread ethiprole resistance in N. lugens populations., Graphical abstract Image 1, Highlights • Ethiprole selection of N. lugens increases resistance to ethiprole and fipronil. • Ethiprole selection increases frequency of the RDL A301S and a new mutation, Q359E. • The A301S mutation, but not Q359E, confers significant resistance to ethiprole. • Neither A301S or Q359E confer significant resistance to fipronil.

Background Development and evaluation of new insect pest management tools is critical for overcoming over-reliance upon, and growing resistance to, synthetic, biological and plant-expressed insecticides. For transgenic crops expressing insecticidal proteins from the bacterium Bacillus thuringiensis (‘Bt crops’) emergence of resistance is slowed by maintaining a proportion of the crop as non-Bt varieties, which produce pest insects unselected for resistance. While this strategy has been largely successful, multiple cases of Bt resistance have now been reported. One new approach to pest management is the use of genetically engineered insects to suppress populations of their own species. Models suggest that released insects carrying male-selecting (MS) transgenes would be effective agents of direct, species-specific pest management by preventing survival of female progeny, and simultaneously provide an alternative insecticide resistance management strategy by introgression of susceptibility alleles into target populations. We developed a MS strain of the diamondback moth, Plutella xylostella, a serious global pest of crucifers. MS-strain larvae are reared as normal with dietary tetracycline, but, when reared without tetracycline or on host plants, only males will survive to adulthood. We used this strain in glasshouse-cages to study the effect of MS male P. xylostella releases on target pest population size and spread of Bt resistance in these populations. Results Introductions of MS-engineered P. xylostella males into wild-type populations led to rapid pest population decline, and then elimination. In separate experiments on broccoli plants, relatively low-level releases of MS males in combination with broccoli expressing Cry1Ac (Bt broccoli) suppressed population growth and delayed the spread of Bt resistance. Higher rates of MS male releases in the absence of Bt broccoli were also able to suppress P. xylostella populations, whereas either low-level MS male releases or Bt broccoli alone did not. Conclusions These results support theoretical modeling, indicating that MS-engineered insects can provide a powerful pest population suppressing effect, and could effectively augment current Bt resistance management strategies. We conclude that, subject to field confirmation, MS insects offer an effective and versatile control option against P. xylostella and potentially other pests, and may reduce reliance on and protect insecticide-based approaches, including Bt crops.

Highlights • Aphids have a unique heterodimeric voltage-gated sodium channel. • The aphid channel has an atypical ion-selectivity filter (DENS rather than DEKA). • The channel’s novel selectivity filter may result in a loss of sodium selectivity. • This is the only identifiable voltage-gated sodium channel in aphid genome(s). • This channel has most likely arisen by gene fission or gene duplication., We describe the identification in aphids of a unique heterodimeric voltage-gated sodium channel which has an atypical ion selectivity filter and, unusually for insect channels, is highly insensitive to tetrodotoxin. We demonstrate that this channel has most likely arisen by adaptation (gene fission or duplication) of an invertebrate ancestral mono(hetero)meric channel. This is the only identifiable voltage-gated sodium channel homologue in the aphid genome(s), and the channel’s novel selectivity filter motif (DENS instead of the usual DEKA found in other eukaryotes) may result in a loss of sodium selectivity, as indicated experimentally in mutagenised Drosophila channels.

Diamides, such as flubendiamide and chlorantraniliprole, belong to a new chemical class of insecticides that act as conformation-sensitive activators of insect ryanodine receptors (RyRs). Both compounds are registered for use against lepidopteran species such as the diamondback moth, Plutella xylostella, a notorious global pest of cruciferous crops. Recently acquired resistance to diamide insecticides in this species is thought to be due to a target-site mutation conferring an amino acid substitution (G4946E), located within the trans-membrane domain of the RyR, though the exact role of this mutation has not yet been fully determined. To address this we have cloned a full-length cDNA encoding the P. xylostella RyR and established clonal Sf9 cell lines stably expressing either the wildtype RyR or the G4946E variant, in order to test the sensitivity to flubendiamide and chlorantraniliprole on the recombinant receptor. We report that the efficacy of both diamides was dramatically reduced in clonal Sf9 cells stably expressing the G4946E modified RyR, providing clear functional evidence that the G4946E RyR mutation impairs diamide insecticide binding.

Cultured oat (Avena sativa L. — naked form) aleurone protoplasts were employed as a model system for following changes which accompany the development of vacuoles during in-vitro incubation. Over a 5-d period, the aleurone grains progressively grew and fused to form a large central vacuole and the volume of the protoplasts increased sevenfold. The growth of the vacuole was accompanied by a progressive acidification of the vacuolar sap. Vacuolation was inhibited by high concentrations of mannitol and by cycloheximide and cordycepin applied at various times during the incubation period. Neither cycloheximide nor cordycepin affected the initial phases of vacuolation but cycloheximide retarded subsequent stages, particularly if added early in the incubation. Cordycepin inhibited only the later stages of vacuolation. Radiolabelling studies identified at least three novel microsomal proteins, with relative molecular masses of approximately 34, 47 and 48 kDa, which appeared during vacuolation and whose synthesis was markedly affected by these inhibitors.

The Varroa mite, Varroa destructor, is an important pest of honeybees and has played a prominent role in the decline in bee colony numbers over recent years. Although pyrethroids such as tau-fluvalinate and flumethrin can be highly effective in removing the mites from hives, their intensive use has led to many reports of resistance. To investigate the mechanism of resistance in UK Varroa samples, the transmembrane domain regions of the V. destructor voltage-gated sodium channel (the main target site for pyrethroids) were PCR amplified and sequenced from pyrethroid treated/untreated mites collected at several locations in Central/Southern England. A novel amino acid substitution, L925V, was identified that maps to a known hot spot for resistance within the domain IIS5 helix of the channel protein; a region that has also been proposed to form part of the pyrethroid binding site. Using a high throughput diagnostic assay capable of detecting the mutation in individual mites, the L925V substitution was found to correlate well with resistance, being present in all mites that had survived tau-fluvalinate treatment but in only 8 % of control, untreated samples. The potential for using this assay to detect and manage resistance in Varroa-infected hives is discussed.

Knockdown resistance (kdr) in insects, caused by inherited nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene, is a major threat to the efficacy of pyrethroid insecticides. Classic kdr, resulting from an L1014F substitution in the VGSC is now present in numerous pest species. Two other substitutions at the L1014 locus have also been reported, L1014S and L1014H. Here we have used expression of L1014 modified Drosophila para VGSCs in Xenopus oocytes with two-electrode voltage clamp to characterise all three mutations. The mutations L1014F and L1014H caused significant depolarizing shifts in the half activation voltage (V(50,act)) from -17.3 mV (wild-type) to -13.1 and -13.5 mV respectively, whereas L1014S caused no shift in V(50,act) but its currents decayed significantly faster than wild-type channels. Treatment of the wild-type channel with deltamethrin (≥ 1 nM), permethrin (≥ 30 nM) or DDT (≥ 1 μM) resulted in hyperpolarizing shifts in V(50,act). Deltamethrin, permethrin and DDT also produced "tail currents" with EC₅₀s of 0.043, 0.40 and 65 μM and maximum modifications of 837, 325 and 7% respectively. L1014F provided a high level of resistance against all insecticides for both measured parameters. L1014H most effectively combated deltamethrin induced tail currents while L1014S strongly resisted the large DDT induced shifts in V(50,act). We conclude that L1014H and L1014S may have arisen through heavy exposure to specific pyrethroids and DDT respectively.

DDT and the pyrethroid class of insecticides target voltage-gated sodium channels. Their binding stabilises the channel open state, inducing prolonged tail currents associated with insect paralysis (‘knockdown’) and death. Target-site mutations conferring resistance, while a challenge for pest control programs, have provided valuable information on the location of the elusive insecticide-binding site. Homology modelling of the housefly sodium channel and automated ligand docking studies have identified a binding site consistent with resistance-associated mutagenesis data, structure-activity relationships of insecticides and their state-dependent binding activity [O'Reilly et al (2006) Biochem. J. 396:255-263]. The putative binding site, delimited by the domain II S4-S5 linker, S5 & S6 helices and domain III S6 helix, interfaces the lipid bilayer and is therefore accessible to lipid-soluble insecticide ligands. The model is supported by recent experimental results from voltage-clamp electrophysiology studies on mutant fruitfly sodium channels [Usherwood et al (2007) FEBS Letters 581:5485-5492]. The mutation T929I, predicted to inhibit DDT binding through steric hindrance, abolishes the effects of DDT on channel activity. M918, a residue predicted to form a binding contact with pyrethroids but not DDT, decreased deltamethrin potency without effecting DDT potency when mutated. We have developed methods based on circular dichroism spectroscopy to identify ligand binding to a non-insect channel system, namely the voltage-gated sodium channel NaChBac from Bacillus halodurans [Nurani et al (2008) Biochemistry 31:8114-8121], which will be used as a further test of the binding of various insecticides as predicted by the model. (Supported by grants from the BBSRC)

Single channel analysis was used to compare the electrophysiological properties of wild-type (WT) and I874M mutant (M874) rat Na(V)1.2 channels expressed in Xenopus oocytes and their modulation by the pyrethroid deltamethrin. In the absence of pyrethroid, histograms of channel open times were best-fit by single exponentials. The open time constants at -40 mV for WT (0.53+/-0.05 ms) and M874 (0.65+/-0.08 ms) channels were significantly different and both decreased with depolarisation. At > or =100 nM deltamethrin, WT open time histograms at -40 mV were best-fit by two exponentials (time constants, 0.49+/-0.03 ms (tau(o,fast,WT)) and 5.2+/-0.5 ms (tau(o,slow,WT)). The population of long-duration openings and tau(o,slow,WT) increased when the concentration of deltamethrin was raised, but tau(o,fast,WT) was unaffected. Qualitatively similar results were obtained for the M874 channel, but with > or =10 nM deltamethrin. Deltamethrin also caused a negative shift in the relationships between channel opening probability (P(op)) and membrane potential and first latency and membrane potential, suggesting that the pyrethroid binds to the closed channel. Selectivity for Na was increased by the pyrethroid (10 microM, WT; 1 microM, mutant).

Naturally derived insecticides such as pyrethrum and man-made insecticides such as DDT and the synthetic pyrethroids act on the voltage-gated sodium channel proteins found in insect nerve-cell membranes. The correct functioning of these channels is essential for the normal transmission of nerve impulses, and this process is disrupted by binding of the insecticides, leading to paralysis and eventual death. Some insect pest populations have evolved modifications of the sodium channel protein that inhibit the binding of the insecticide and result in the insect developing resistance. This perspective outlines the current understanding of the molecular processes underlying target-site resistance to these insecticides (termed kdr and super-kdr), and how this knowledge may in future contribute to the design of novel insecticidal compounds. (c) 2008 Society of Chemical Industry

The buff-tailed bumblebee, Bombus terrestris audax is an important pollinator within both landscape ecosystems and agricultural crops. During their lifetime bumblebees are regularly challenged by various environmental stressors including insecticides. Historically the honey bee (Apis mellifera spp.) has been used as an ‘indicator’ species for ‘standard’ ecotoxicological testing, but it has been suggested that it is not always a good proxy for other eusocial or solitary bees. To investigate this, the susceptibility of B. terrestris to selected pesticides within the neonicotinoid, pyrethroid and organophosphate classes was examined using acute insecticide bioassays. Acute oral and topical LD50 values for B. terrestris against these insecticides were broadly consistent with published results for A. mellifera. For the neonicotinoids, imidacloprid was highly toxic, but thiacloprid and acetamiprid were practically non-toxic. For pyrethroids, deltamethrin was highly toxic, but tau-fluvalinate only slightly toxic. For the organophosphates, chlorpyrifos was highly toxic, but coumaphos practically non-toxic. Bioassays using insecticides with common synergists enhanced the sensitivity of B. terrestris to several insecticides, suggesting detoxification enzymes may provide a level of protection against these compounds. The sensitivity of B. terrestris to compounds within three different insecticide classes is similar to that reported for honey bees, with marked variation in sensitivity to different insecticides within the same insecticide class observed in both species. This finding highlights the need to consider each compound within an insecticide class in isolation rather than extrapolating between different insecticides in the same class or sharing the same mode of action., Graphical abstract Unlabelled Image, Highlights • Acute toxicity of popular insecticides to Bombus terrestris audax was assessed. • Compounds from 3 major classes were tested in topical and oral bioassays. • Bioassays with different synergists were done for each compound. • Results show stark differential toxicity of insecticides within the same class.

Highlights • Baseline susceptibility of Plutella xylostella to diamide insecticides collated. • Instances of diamide resistance in P. xylostella summarized. • Diamide insecticide specificity for the insect ryanodine receptor highlighted. • Efforts to isolate and characterize the P. xylostella ryanodine receptor described. • Molecular mechanisms of diamide resistance in P. xylostella discussed., Graphical abstract, Diamide insecticides, such as flubendiamide and chlorantraniliprole, are a new class of insecticide with a novel mode of action, selectively activating the insect ryanodine receptor (RyR). They are particularly active against lepidopteran pests of cruciferous vegetable crops, including the diamondback moth, Plutella xylostella. However, within a relatively short period following their commercialisation, a comparatively large number of control failures have been reported in the field. In this review we summarise the current body of knowledge regarding the molecular mechanisms of diamide resistance in P. xylostella. Resistant phenotypes collected from different countries can often be linked to specific target-site mutation(s) in the ryanodine receptors’ transmembrane domain. Metabolic mechanisms of resistance have also been proposed. Rapid resistance development is probably a consequence of over-reliance on this one class of chemistry for diamondback moth control.

The evolution of resistance to drugs and pesticides poses a major threat to human health and food security. Neonicotinoids are highly effective insecticides used to control agricultural pests. They target the insect nicotinic acetylcholine receptor and mutations of the receptor that confer resistance have been slow to develop, with only one field‐evolved mutation being reported to date. This is an arginine‐to‐threonine substitution at position 81 of the nAChR_β1 subunit in neonicotinoid‐resistant aphids. To validate the role of R81T in neonicotinoid resistance and to test whether it may confer any significant fitness costs to insects, CRISPR/Cas9 was used to introduce an analogous mutation in the genome of Drosophila melanogaster. Flies carrying R81T showed an increased tolerance (resistance) to neonicotinoid insecticides, accompanied by a significant reduction in fitness. In comparison, flies carrying a deletion of the whole nAChR_α6 subunit, the target site of spinosyns, showed an increased tolerance to this class of insecticides but presented almost no fitness deficits.