Mutations in the voltage-gated sodium channel gene associated with deltamethrin resistance in commercially sourced Phytoseiulus persimilis.

The implementation of Integrated Pest Management in current agricultural practice is a convenient and very effective strategy to keep pest populations under control. The use of biological control agents, such as Phytoseiulus persimilis, is key for the success of such an approach. This predatory mite is widely used as it is very effective for controlling Tetranychus urticae, one of the most devastating crop pests. Here, we identify several mutations located in the voltage-gated sodium channel (VGSC) of commercially sourced P. persimilis that correlate with a reduced susceptibility to the pyrethroid deltamethrin. We found that the mites sourced from two different biocontrol product companies have intrinsic genotypic differences that correlate with their phenotype when tested with different concentrations of deltamethrin. Mites from Syngenta Bioline, carrying the mutations M918L and A1536T, were able to survive deltamethrin concentrations of up to 10 ppm, while the mites from Koppert Biological Systems, with the combination M918L, L925V and S1539T, survived treatment with 40 ppm. All of the point mutations identified in the predatory mite samples are located in a particular region of the VGSC, previously proposed as the binding site for this family of pesticides and identified as a 'hot spot' for resistance.

Susceptibility of cat fleas (Siphonaptera: Pulicidae) to fipronil and imidacloprid using adult and larval bioassays.

The monitoring of the susceptibility offleas to insecticides has typically been conducted by exposing adults on treated surfaces. Other methods such as topical applications of insecticides to adults and larval bioassays on treated rearing media have been developed. Unfortunately, baseline responses of susceptible strains of cat flea, Ctenocephalides felis (Bouchè), except for imidacloprid, have not been determined for all on-animal therapies and new classes of chemistry now being used. However, the relationship between adult and larval bioassays of fleas has not been previously investigated. The adult and larval bioassays of fipronil and imidacloprid were compared for both field-collected isolates and laboratory strains. Adult topical bioassays of fipronil and imidacloprid to laboratory strains and field-collected isolates demonstrated that LD50s of fipronil and imidacloprid ranged from 0.11 to 0.40 nanograms per flea and 0.02 to 0.18 nanograms per flea, respectively. Resistance ratios for fipronil and imidacloprid ranged from 0.11 to 2.21. Based on the larval bioassay published for imidacloprid, a larval bioassay was established for fipronil and reported in this article. The ranges of the LC50s of fipronil and imidacloprid in the larval rearing media were 0.07-0.16 and 0.11-0.21 ppm, respectively. Resistance ratios for adult and larval bioassays ranged from 0.11 to 2.2 and 0.58 to 1.75, respectively. Both adult and larval bioassays provided similar patterns for fipronil and imidacloprid. Although the adult bioassays permitted a more precise dosage applied, the larval bioassays allowed for testing isolates without the need to maintain on synthetic or natural hosts.

Resistance to spiromesifen in Trialeurodes vaporariorum is associated with a single amino acid replacement in its target enzyme acetyl-coenzyme A carboxylase.

Spiromesifen is a novel insecticide and is classed as a tetronic acid derivative. It targets the insects' acetyl-coenzyme A carboxylase (ACCase) enzyme, causing a reduction in lipid biosynthesis. At the time of this publication, there are no reports of resistance to this class of insecticides in insects although resistance has been observed in several mite species. The greenhouse whitefly Trialeurodes vaporariorum (Westwood) is a serious pest of protected vegetable and ornamental crops in temperate regions of the world and spiromesifen is widely used in its control. Mortality rates of UK and European populations of T. vaporariorum to spiromesifen were calculated and up to 26-fold resistance was found. We therefore sought to examine the molecular mechanism underlying spiromesifen resistance in this important pest. Pre-treatment with piperonyl butoxide did not synergize spiromesifen, suggesting a target-site resistance mechanism. The full length ACCase gene was sequenced for a range of T. vaporariorum strains and a strong association was found between spiromesifen resistance and a glutamic acid substitution with lysine in position 645 (E645K) of this gene. A TaqMan allelic discrimination assay confirmed these findings. Although this resistance is not considered sufficient to compromise the field performance of spiromesifen, this association of E645K with resistance is the first report of a potential target site mechanism affecting an ACCase inhibitor in an arthropod species.

The re-emergence of the bed bug as a nuisance pest: implications of resistance to the pyrethroid insecticides.

A global resurgence of bed bugs (Hemiptera: Cimicidae) has led to renewed scientific interest in these insects. The current bed bug upsurge appears to have started almost synchronously in the late 1990 s in Europe, the U.S.A. and Australia. Several factors have led to this situation, with resistance to applied insecticides making a significant contribution. With a growing number of insecticides (DDT, carbamates, organophosphates etc.) being no longer available as a result of regulatory restrictions, the mainstay chemistry used for bed bug control over the past few decades has been the pyrethroid insecticides. With reports of increasing tolerance to pyrethroids leading to control failures on the rise, containing and eradicating bed bugs is proving to be a difficult task. Consequently, several recent studies have focused on determining the mode of action of pyrethroid resistance in bed bug populations sourced from different locations. Correct identification of the factor(s) responsible for the increasing resistance is critical to the development of effective management strategies, which need to be based, wherever possible, on firm scientific evidence. Here we review the literature on this topic, highlighting the mechanisms thought to be involved and the problems currently faced by pest control professionals in dealing with a developing pandemic.

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.

Identification of ion channel genes in the Acyrthosiphon pisum genome.

Aphids are major pests of crops, causing hundreds of millions of dollars worth of damage annually. Ion channel proteins are often the targets of modern insecticides and mutations in ion channel genes can lead to resistance to many leading classes of insecticides. The sequencing of the pea aphid, Acyrthosiphon pisum, genome has now allowed detailed in silico analysis of the aphid ion channels. The study has revealed significant differences in the composition of the ion channel families between the aphid and other insects. For example A. pisum does not appear to contain a homologue of the nACh receptor alpha 5 gene whilst the calcium channel beta subunit has been duplicated. These variations could result in differences in function or sensitivity to insecticides. The genome sequence will allow the study of aphid ion channels to be accelerated, leading to a better understanding of the function of these economically important channels. The potential for identifying novel insecticide targets within the aphid is now a step closer.

Identification of pyrethroid resistance associated mutations in the para sodium channel of the two-spotted spider mite Tetranychus urticae (Acari: Tetranychidae).

We investigated pyrethroid resistance mechanisms in Tetranychus urticae strains from Greece. Combined bioassay, biochemical and synergistic data indicated that although P450 mono-oxygenase activities were associated with the trait, target site insensitivity was the major resistance component. A 3.3 kb cDNA fragment of the T. urticae para sodium channel gene encompassing segment 4 of domain II to segment 6 of domain IV was obtained by a degenerate PCR strategy. The T. urticae sequence showed highest identity (56%) to the scabies mite, Sarcoptes scabiei, and was phylogenetically classified within the divergent group of Arachnida. Comparison of resistant and susceptible strains identified the point mutation F1538I in segment 6 of domain III, which is known to confer strong resistance to pyrethroids, along with a second mutation (A1215D) in the intracellular linker connecting domains II and III with an unknown role. Three transcripts were identified corresponding to the k and l alternative exons. The mode of inheritance of resistance was confirmed as incompletely recessive, which is consistent with a target site mechanism for pyrethroids.

Abrupt Climate Change in an Oscillating World.

The notion that small changes can have large consequences in the climate or ecosystems has become popular as the concept of tipping points. Typically, tipping points are thought to arise from a loss of stability of an equilibrium when external conditions are slowly varied. However, this appealingly simple view puts us on the wrong foot for understanding a range of abrupt transitions in the climate or ecosystems because complex environmental systems are never in equilibrium. In particular, they are forced by diurnal variations, the seasons, Milankovitch cycles and internal climate oscillations. Here we show how abrupt and sometimes even irreversible change may be evoked by even small shifts in the amplitude or time scale of such environmental oscillations. By using model simulations and reconciling evidence from previous studies we illustrate how these phenomena can be relevant for ecosystems and elements of the climate system including terrestrial ecosystems, Arctic sea ice and monsoons. Although the systems we address are very different and span a broad range of time scales, the phenomena can be understood in a common framework that can help clarify and unify the interpretation of abrupt shifts in the Earth system.

Mutations in DIIS5 and the DIIS4-S5 linker of Drosophila melanogaster sodium channel define binding domains for pyrethroids and DDT.

Mutations in the DIIS4-S5 linker and DIIS5 have identified hotspots of pyrethroid and DDT interaction with the Drosophila para sodium channel. Wild-type and mutant channels were expressed in Xenopus oocytes and subjected to voltage-clamp analysis. Substitutions L914I, M918T, L925I, T929I and C933A decreased deltamethrin potency, M918T, L925I and T929I decreased permethrin potency and T929I, L925I and I936V decreased fenfluthrin potency. DDT potency was unaffected by M918T, but abolished by T929I and reduced by L925I, L932F and I936V, suggesting that DIIS5 contains at least part of the DDT binding domain. The data support a computer model of pyrethroid and DDT binding.

Heteroduplex DNA correction in Saccharomyces cerevisiae is mismatch specific and requires functional PMS genes.

In vitro-constructed heteroduplex DNAs with defined mismatches were corrected in Saccharomyces cerevisiae cells with efficiencies that were dependent on the mismatch. Single-nucleotide loops were repaired very efficiently; the base/base mismatches G/T, A/C, G/G, A/G, G/A, A/A, T/T, T/C, and C/T were repaired with a high to intermediate efficiency. The mismatch C/C and a 38-nucleotide loop were corrected with low efficiency. This substrate specificity pattern resembles that found in Escherichia coli and Streptococcus pneumoniae, suggesting an evolutionary relationship of DNA mismatch repair in pro- and eucaryotes. Repair of the listed mismatches was severely impaired in the putative S. cerevisiae DNA mismatch repair mutants pms1 and pms2. Low-efficiency repair also characterized pms3 strains, except that correction of single-nucleotide loops occurred with an efficiency close to that of PMS wild-type strains. A close correlation was found between the repair efficiencies determined in this study and the observed postmeiotic segregation frequencies of alleles with known DNA sequence. This suggests an involvement of DNA mismatch repair in recombination and gene conversion in S. cerevisiae.

Molecular cloning of the barley seed protein CMd: a variant member of the alpha-amylase/trypsin inhibitor family of cereals.

The nucleotide and deduced amino-acid sequences of a cDNA clone encoding the barley seed protein CMd are described. The sequence is homologous with those of a family of inhibitors of alpha-amylase and trypsin, except for two short insertions. The longest of these (14 residues) is at the junction between the three proposed ancestral regions that comprise this family of proteins, and has limited identity with alpha-amylases of bacterial origin.

Sensitivity of the Drosophila para sodium channel to DDT is not lowered by the super-kdr mutation M918T on the IIS4-S5 linker that profoundly reduces sensitivity to permethrin and deltamethrin.

DDT inhibits Na channel inactivation and deactivation, promotes Na channel activation and reduces the resting potential of Xenopus oocytes expressing the Drosophila para Na channel. These changes are only marginally influenced by the single mutation M918T (super-kdr) but are reduced approximately 10-fold by either the single mutation L1014F (kdr) or the double mutation L1014F+M918T, both of which confer resistance to the pyrethroids permethrin and deltamethrin. We conclude that DDT binds either to or in the region of L1014 on IIS6 but only weakly to M918 on the IIS4-S5 linker, which is part of a high-affinity binding site for permethrin and deltamethrin.

Activation of Drosophila sodium channels promotes modification by deltamethrin. Reductions in affinity caused by knock-down resistance mutations.

kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4-S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by <10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action.

Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of pms1-1 and pms1-2.

The pms1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked his4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in pms1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered.

Molecular cloning, characterisation and mRNA expression of the ryanodine receptor from the peach-potato aphid, Myzus persicae.

The peach potato aphid, Myzus persicae, is one of the most important agricultural pests of temperate climates. It is mainly controlled through the judicious application of insecticides; however, over time, aphids have developed resistance to many insecticidal classes. The recent introduction of synthetic diamide insecticides, with a novel mode of action, potentially offers new tools to control aphid populations. These diamides act on the ryanodine receptor (RyR), a large endoplasmic calcium release channel. In this study we have cloned cDNAs encoding the complete open reading frame of the RyR from M. persicae. The open reading frame is 15,306 base pairs long and encodes a protein of 5101 amino acids. The aphid RyR shares many of the features of other insect and vertebrate RyRs, including a highly conserved transmembrane region. However, unlike the other RyRs characterised to date, the M. persicae channel does not display alternative splicing at any stage of its developmental cycle, so it cannot generate functional variants of the channel.

Functional analysis of a rat sodium channel carrying a mutation for insect knock-down resistance (kdr) to pyrethroids.

Pyrethroid insensitivity in resistant (kdr) insects has been correlated with a leucine to phenylalanine replacement in the S6 transmembrane segment of domain II of the axonal sodium channel alpha(para)-subunit. An alpha-subunit of rat brain type II sodium channel containing this mutation has been expressed and its sensitivity to permethrin compared with that of the wild-type channel. The steady-state activation curve of the mutant was shifted 14 mV in the depolarizing direction. We propose that an equivalent shift of the sodium current activation curve in kdr insects could account for their low sensitivity to permethrin toxicity.

A single amino acid change makes a rat neuronal sodium channel highly sensitive to pyrethroid insecticides.

Two amino acid substitutions in a housefly sodium channel, L1014F in domain IIS6 and M918T in the IIS4-S5 linker, have been identified in kdr and super-kdr pyrethroid-resistant phenotypes, respectively. Unlike their native insect counterparts, mammalian sodium channels are only weakly sensitive to pyrethroids. Do the sodium channels of mammal and pyrethroid-resistant housefly share similar structural characteristics that account for their low pyrethroid sensitivities? We report here that substitution of isoleucine for methionine at position 874 (equivalent to the super-kdr site 918 in the housefly) in the rat IIA alpha-subunit causes a 100-fold increase in sensitivity.

Mutations in the Bemisia tabaci para sodium channel gene associated with resistance to a pyrethroid plus organophosphate mixture.

The voltage-gated sodium channel is the primary target site of pyrethroid insecticides. In some insects, super knockdown resistance (super-kdr) to pyrethroids is caused by point mutations in the linker fragment between transmembrane segments 4 and 5 of the para-type sodium channel protein domain II (IIS4-5). Here, we identify two mutations in the IIS4-5 linker of the para-type sodium channel of the whitefly, BEMISIA TABACI: methionine to valine at position 918 (M918V) and leucine to isoleucine at position 925 (L925I). Although each mutation was isolated independently from strains >100-fold resistant to a pyrethroid (fenpropathrin) plus organophosphate (acephate) mixture, only L925I was associated with resistance in strains derived from the field in 2000 and 2001. The L925I mutation occurred in all individuals from nine different field collections that survived exposure to a discriminating concentration of fenpropathrin plus acephate. Linkage analysis of hemizygous male progeny of unmated heterozygous F1 females (L925Ixwild-type) shows that the observed resistance is tightly linked to the voltage-gated sodium channel locus. The results provide a molecular tool for better understanding, monitoring and managing pyrethroid resistance in B. tabaci.

Cloning, heterologous expression and co-assembly of Mpbeta1, a nicotinic acetylcholine receptor subunit from the aphid Myzus persicae.

Nicotinic acetylcholine receptors (nAChRs) play a major role in excitatory synaptic transmission in insects and are also the target site for chloronicotinyl insecticides such as imidacloprid. Here we report the cloning and characterization of a novel nAChR beta subunit, Mpbeta1, from the aphid Myzus persicae, an economically important pest species. Sequence analysis has identified an open reading frame of 509 amino acids with features typical of nAChR subunits. The Mpbeta1 gene is expressed as a single major transcript of 4.6 kb, considerably larger than the predicted length of the Mpbeta1 open reading frame (1527 bp). By heterologous expression in Drosophila S2 cells, the Mpbeta1 subunit has been shown to co-assemble with the previously cloned nAChR subunits Mpalpha1 and Mpalpha2. In contrast, no co-assembly of Mpbeta1 could be detected with either Mpalpha3 or Mpalpha4. With the aim of gaining a clearer insight into the influence of subunit composition upon assembly, the ability of M. persicae nAChR subunits to co-assemble with vertebrate nAChR subunits has also been examined.

Cyclosporine A upregulates interleukin-6 gene expression in human gingiva: possible mechanism for gingival overgrowth.

Cyclosporine A (CsA) is a widely used immunosuppressant for transplant patients and is also used for the treatment of a wide variety of systemic diseases with immunologic components. A prominent side effect of CsA administration is gingival overgrowth. It has been postulated that CsA alters fibroblast activity through effects on various cytokines such as the interleukins, however, as yet, data concerning the molecular mechanisms involved in connective tissue proliferation are still preliminary in nature. The purpose of this study was to evaluate interleukin-6 (IL-6) gene expression in gingival tissues of patients receiving CsA therapy and exhibiting gingival overgrowth. Radioimmunoassay (RIA) demonstrated a significant difference in tissue levels of IL-6 as mean +/- SEM. IL-6 content in CsA-stimulated tissue was 184.3 +/- 30.2 ng/mg total protein versus 23.3 +/- 6.5 ng/mg total protein in control tissue. In situ hybridization indicated that overgrown gingival tissues from patients taking CsA had a significantly higher content of IL-6 mRNA when compared to control tissues. Expressing IL-6 mRNA levels as silver grains/cell, CsA-stimulated tissue had 166.9 +/- 12.0 grains of IL-6 mRNA/cell while control tissue had 12.8 +/- 3.0 grains of IL-6 mRNA/cell. These results demonstrate that CsA therapy results in increased levels of IL-6 protein and IL-6 mRNA in overgrown human gingival tissues. This is the first report of CsA-upregulated IL-6 gene expression in vivo, and may explain in part the molecular mechanisms responsible for CsA-induced gingival overgrowth.