Target-site Resistance Mechanisms Research Articles

Target-site resistance to pyrethroid insecticides in German populations of the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae)

Cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) is a major pest of winter oilseed rape in several European countries particularly attacking young emerging plants in autumn. Over the last several decades, pyrethroid insecticides have been foliarly applied to control flea beetle outbreaks. Recent control failures in northern Germany suggested pyrethroid resistance development in cabbage stem flea beetles, which were confirmed by resistance monitoring bioassays using lambda-cyhalothrin in an adult vial test. The purpose of this study was to investigate the presence of polymorphisms in the para-type voltage-gated sodium channel gene of P. chrysocephala known to be involved in knock-down resistance (kdr). By using a degenerate primer approach we PCR amplified part of the para-type sodium channel gene and identified in resistant flea beetles a single nucleotide polymorphism resulting in an L1014F (kdr) mutation within domain IIS6 of the channel protein, known as one of the chief pyrethroid target-site resistance mechanisms in several other pest insects. Twenty populations including four archived museum samples collected between 1945 and 1958 were analyzed using a newly developed pyrosequencing diagnostic assay. The assay revealed a kdr allele frequency of 90–100% in those flea beetle populations expressing high-level cross-resistance in discriminating dose bioassays against different pyrethroids such as lambda-cyhalothrin, tau-fluvalinate, etofenprox and bifenthrin. The presence of target-site resistance to pyrethroids in cabbage stem flea beetle is extremely worrying considering the lack of effective alternative modes of action to control this pest in Germany and other European countries, and is likely to result in major control problems once it expands to other geographies. The striking fact that cabbage stem flea beetle is next to pollen beetle, Meligethes aeneus the second coleopteran pest in European winter oilseed rape resisting pyrethroid treatments by expressing a target-site mutation, underpins the importance of diversity in available chemistry for resistance management tactics based on mode of action rotation in order to guarantee sustainable winter oilseed rape cultivation in Europe.

Population genetics structure of glyphosate‐resistant Johnsongrass (Sorghum halepense L. Pers) does not support a single origin of the resistance

Single sequence repeats (SSR) developed for Sorghum bicolor were used to characterize the genetic distance of 46 different Sorghum halepense (Johnsongrass) accessions from Argentina some of which have evolved toward glyphosate resistance. Since Johnsongrass is an allotetraploid and only one subgenome is homologous to cultivated sorghum, some SSR loci amplified up to two alleles while others (presumably more conserved loci) amplified up to four alleles. Twelve SSR providing information of 24 loci representative of Johnsongrass genome were selected for genetic distance characterization. All of them were highly polymorphic, which was evidenced by the number of different alleles found in the samples studied, in some of them up to 20. UPGMA and Mantel analysis showed that Johnsongrass glyphosate-resistant accessions that belong to different geographic regions do not share similar genetic backgrounds. In contrast, they show closer similarity to their neighboring susceptible counterparts. Discriminant Analysis of Principal Components using the clusters identified by K-means support the lack of a clear pattern of association among samples and resistance status or province of origin. Consequently, these results do not support a single genetic origin of glyphosate resistance. Nucleotide sequencing of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from glyphosate-resistant and susceptible accessions collected from different geographic origins showed that none presented expected mutations in aminoacid positions 101 and 106 which are diagnostic of target-site resistance mechanism.

Synergistic Effects of Atrazine and Mesotrione on Susceptible and Resistant Wild Radish (Raphanus raphanistrum) Populations and the Potential for Overcoming Resistance to Triazine Herbicides

The synergistic interaction between mesotrione, a hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicide, and atrazine, a photosystem II (PS II)-inhibiting herbicide, has been identified in the control of several weed species. A series of dose–response studies examined the synergistic effect of these herbicides on a susceptible (S) wild radish population. The potential for this interaction to overcome target-sitepsbA gene-based atrazine resistance in a resistant (R) wild radish population was also investigated. Control of S wild radish with atrazine was enhanced by up to 40% when low rates (1.0 to 1.5 g ha−1) of mesotrione were applied in combination. This synergistic response was demonstrated across a range of atrazine–mesotrione rate combinations on this S wild radish population. Further, the efficacy of 1.5 g ha−1mesotrione increased control of the R population by a further 60% when applied in combination with 400 g ha−1of atrazine. This result clearly demonstrated the synergistic interaction of these herbicides in overcoming the target-site resistance mechanism. The mechanism responsible for the observed synergistic interaction between mesotrione and atrazine remains unknown. However, it is speculated that an alternate atrazine binding site may be responsible. Regardless of the biochemical nature of this interaction, evidence from whole-plant bioassays clearly demonstrated that synergistic herbicide combinations improve herbicide efficiency, with lower application rates required to control weed populations. This, combined with the potential to overcomepsbA gene-based triazine resistance, and, thereby, regain the use of these herbicides, will result in more sustainable herbicide use.

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.

Resistance mechanism to acetyl coenzyme A carboxylase inhibiting herbicides in Phalaris paradoxa collected in Mexican wheat fields

In this study, we describe the molecular, physiological and agronomic aspects involved in the resistance to acetyl coenzyme A carboxylase inhibiting herbicides (ACCase) observed in one biotype of Phalaris paradoxa from Mexico. Dose–response Assays: The herbicide rate inhibiting plant growth of each biotype by 50% with respect to the untreated control, ED50. Enzyme purification and ACCase assays to determine herbicide rate inhibiting the enzyme of each biotype by 50% with respect to the untreated control, I50. Absorption and Translocation Assays with [14C]diclofop-methyl. Metabolism of diclofop-methyl and its metabolites were identified by thin-layer chromatography. Study of target site resistance mechanism at enzyme and molecular levels. In this work, it has been studied the whole-plant response of Phalaris paradoxa biotypes from Mexico resistant (R) and susceptible (S) to ACCase-inhibiting herbicides: aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ), and the mechanism behind their resistance were studied. To analyse the resistance mechanism, the enzyme ACCase activity was investigated. Results from biochemical assays indicated a target-site resistance as the cause of reduced susceptibility to ACCase inhibitors. The absorption, translocation and metabolism were similar between R and S biotypes. A point mutation never described before was detected within the triplet of glycine at the amino acid position 2096 (referring to EMBL accession no. AJ310767) and resulted in the triplet of serine. This new mutation could explain the loss of affinity for the ACCase-inhibiting herbicides. We found a new mutation, which had never been described before. This mutation was detected within the triplet of glycine at the amino acid position 2096. This new mutation confers cross-resistance to three different chemical groups of ACCase-inhibiting herbicides.

Identifying the presence of neonicotinoidresistant peach‐potato aphid (Myzus persicae) in the peach‐growing regions of southern France and northern Spain

The neonicotinoid class of insecticides is a key component of pest management strategies used by stone fruit producers in Europe. Neonicotinoids are currently one of the most important tools for control of the peach-potato aphid (Myzus persicae). Overreliance on neonicotinoids has led to the development of resistance through a combination of metabolic and target-site resistance mechanisms in individual aphids. A resistance monitoring project was conducted by Syngenta in 2010 to determine the resistance status of M. persicae populations collected from France and Spain, and to determine the frequency of the target-site mutation in those populations. Resistance monitoring suggests that resistance to neonicotinoids is relatively widespread in populations of M. persicae collected from peach orchards in the Languedoc-Roussillon, Provence-Alpes-Cote d'Azur and Rhone-Alpes regions of France, and resistance can be associated with the frequency of the target-site mutation (R81T). The R81T mutation in its heterozygous form is also present in Spanish populations and is associated with neonicotinoid resistance. The widespread nature of neonicotinoid resistance in southern France and the potential for resistance development in northern Spain highlight the need for a coordinated management strategy employing insecticides with different modes of action to reduce the selection pressure with neonicotinoids.

Exploring Mechanisms of Resistance to Respiratory Inhibitors in Field Strains of Botrytis cinerea , the Causal Agent of Gray Mold

Respiratory inhibitors are among the fungicides most widely used for disease control on crops. Most are strobilurins and carboxamides, inhibiting the cytochrome b of mitochondrial complex III and the succinate dehydrogenase of mitochondrial complex II, respectively. A few years after the approval of these inhibitors for use on grapevines, field isolates of Botrytis cinerea, the causal agent of gray mold, resistant to one or both of these classes of fungicide were recovered in France and Germany. However, little was known about the mechanisms underlying this resistance in field populations of this fungus. Such knowledge could facilitate resistance risk assessment. The aim of this study was to investigate the mechanisms of resistance occurring in B. cinerea populations. Highly specific resistance to strobilurins was correlated with a single mutation of the cytb target gene. Changes in its intronic structure may also have occurred due to an evolutionary process controlling selection for resistance. Specific resistance to carboxamides was identified for six phenotypes, with various patterns of resistance levels and cross-resistance. Several mutations specific to B. cinerea were identified within the sdhB and sdhD genes encoding the iron-sulfur protein and an anchor protein of the succinate dehydrogenase complex. Another as-yet-uncharacterized mechanism of resistance was also recorded. In addition to target site resistance mechanisms, multidrug resistance, linked to the overexpression of membrane transporters, was identified in strains with low to moderate resistance to several respiratory inhibitors. This diversity of resistance mechanisms makes resistance management difficult and must be taken into account when developing strategies for Botrytis control.

Exploring the molecular basis of insecticide resistance in the dengue vector Aedes aegypti: a case study in Martinique Island (French West Indies).

BackgroundThe yellow fever mosquito Aedes aegypti is a major vector of dengue and hemorrhagic fevers, causing up to 100 million dengue infections every year. As there is still no medicine and efficient vaccine available, vector control largely based on insecticide treatments remains the only method to reduce dengue virus transmission. Unfortunately, vector control programs are facing operational challenges with mosquitoes becoming resistant to commonly used insecticides. Resistance of Ae. aegypti to chemical insecticides has been reported worldwide and the underlying molecular mechanisms, including the identification of enzymes involved in insecticide detoxification are not completely understood.ResultsThe present paper investigates the molecular basis of insecticide resistance in a population of Ae. aegypti collected in Martinique (French West Indies). Bioassays with insecticides on adults and larvae revealed high levels of resistance to organophosphate and pyrethroid insecticides. Molecular screening for common insecticide target-site mutations showed a high frequency (71%) of the sodium channel 'knock down resistance' (kdr) mutation. Exposing mosquitoes to detoxification enzymes inhibitors prior to bioassays induced a significant increased susceptibility of mosquitoes to insecticides, revealing the presence of metabolic-based resistance mechanisms. This trend was biochemically confirmed by significant elevated activities of cytochrome P450 monooxygenases, glutathione S-transferases and carboxylesterases at both larval and adult stages. Utilization of the microarray Aedes Detox Chip containing probes for all members of detoxification and other insecticide resistance-related enzymes revealed the significant constitutive over-transcription of multiple detoxification genes at both larval and adult stages. The over-transcription of detoxification genes in the resistant strain was confirmed by using real-time quantitative RT-PCR.ConclusionThese results suggest that the high level of insecticide resistance found in Ae. aegypti mosquitoes from Martinique island is the consequence of both target-site and metabolic based resistance mechanisms. Insecticide resistance levels and associated mechanisms are discussed in relation with the environmental context of Martinique Island. These finding have important implications for dengue vector control in Martinique and emphasizes the need to develop new tools and strategies for maintaining an effective control of Aedes mosquito populations worldwide.

Resistance of Aedes aegypti (Diptera: Culicidae) in 2006 to Pyrethroid Insecticides in Indonesia and its Association with Oxidase and Esterase Levels

Three strains of Aedes aegypti collected in 2006 from three major cities in Indonesia, i.e., Bandung, Palembang and Surabaya, were tested to determine their resistance status to pyrethroid (permethrin and deltamethrin) and also the resistance mechanisms regarding three detoxifying enzymes, i.e., oxidase, esterase A and esterase B. The resistance level was expressed as Resistance Ratio (RR) compared to the susceptible VCRU strain. Results of this study showed that Ae. aegypti in Indonesia has developed tolerance to permethrin and deltamethrin, except in the Bandung strain, which was resistant to permethrin and deltametherin with RR90 79.3 and 23.7, respectively. The study also suggested that detoxifying enzymes (oxidase, esterase A and esterase B) apparently were involved in the development of resistance in Bandung strain as indicated by the high level of activity of those enzymes in Bandung strain compared to other more susceptible strains. Although there is a possibility that other mechanisms, such as a target-site resistance mechanism, in Bandung strain were also involved.

Acetylcholinesterase mutation in an insecticide-resistant population of the codling moth Cydia pomonella (L.)

Two strains of Cydia pomonella (L.) (Lepidoptera: Tortricidae) were selected in the lab by exposure to increasing concentrations of diflubenzuron (Rdfb strain) or azinphos-methyl (Raz strain). Insecticide bioassays showed that the adults of the Rdfb strain exhibited a 2.6-fold and a 7.7-fold resistance ratio to azinphos-methyl and carbaryl, respectively compared to a susceptible strain (S) whereas the adults of the Raz strain exhibited a 6.7-fold resistance ratio to azinphos-methyl and a 130-fold resistance ratio to carbaryl. In the Raz strain, a target site resistance mechanism was suggested by the inhibition of acetylcholinesterase (AChE) activity. In fact the ki values did not discriminate the S and Rdfb strains, while the Raz strain exhibited a 1.7-fold and a 14-fold increase in ki value compared to the S strain for azinphos-methyl oxon and carbaryl, respectively. To verify this hypothesis, two cloned AChE cDNAs sequences (named cydpom-ace2 e cydpom-ace1) were compared between the susceptible and the resistant strains. No difference in the deduced amino acid sequence was found in cydpom-ace2 (orthologous to the Drosophila melanogaster AChE). In the putative cydpom-ace1 (paralogous to the Drosophila AChE), a single amino acid substitution F399V was exclusively present in the Raz strain. The F399 lined the active site of the enzyme and the F399V substitution likely could influence the accessibility of different types of inhibitors to the catalytic site of the insensitive cydpom-ace1.

Insecticide Resistance inAnopheles funestus(Diptera: Culicidae) from Mozambique

Malaria control in southern Mozambique is currently by indoor residual carbamate insecticide treatment, with pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles funestus s.s. to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 19 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. funestus from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A high level of pyrethroid resistance was detected in An. funestus populations in southern Mozambique. An. funestus outside Maputo province were still susceptible to pyrethroids. An. funestus from six localities also were resistant to carbamate insecticides propoxur and bendiocarb. Both pyrethroid and carbamate resistance occurred in five of these six localities. Mosquitoes from five of the localities with elevated p450 estimates, compared with the insecticide-susceptible Durban strain, were pyrethroid-resistant. The only exception to this trend was Mozal, which had elevated p450 estimates but full pyrethroid susceptibility by bioassay. The lack of cross-resistance between pyrethroids and DDT in Mozambican An. funestus suggests that a kdr-type target site resistance mechanism has not been selected. Low levels of insecticide-insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in all populations tested. The high level of metabolically based pyrethroid resistance has implications for current malaria control programs in Mozambique.

Insecticide Resistance in <I>Anopheles arabiensis</I> and <I>Anopheles gambiae</I> from Mozambique

Malaria control in the southern part of Mozambique is currently by indoor residual spraying with a carbamate insecticide and by pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles gambiae s.s. and Anopheles arabiensis Patton to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 17 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. gambiae s.s. from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A low level of pyrethroid resistance was detected in populations in southern Mozambique. Populations outside Maputo province were still susceptible to pyrethroids. Low level resistance to the carbamate propoxur also was detected in An. arabiensis from two localities. Mosquitoes from five of the localities had elevated p450 estimates, compared with the insecticide susceptible Durban strain. The lack of cross-resistance between pyrethroids and DDT in Mozambican populations suggests that a kdr-type target site resistance mechanism has not been selected. Increased frequencies of insecticide insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in 16 of the populations tested. Although vector control with bendiocarb is not being compromised by the presence of the acetylcholinesterase mechanism alone, the high level of insensitive acetylcholinesterase unless sensibly managed may have long-term implications for malaria control programs in Mozambique.

Insecticide Resistance in <I>Anopheles funestus</I> (Diptera: Culicidae) from Mozambique

Malaria control in southern Mozambique is currently by indoor residual carbamate insecticide treatment, with pyrethroid-treated bed-nets distributed to pregnant women and children under five in northern Mozambique. The susceptibility of Anopheles funestus s.s. to pyrethroid, carbamate, organochlorine, and organophosphorus insecticides was determined by World Health Organization adult mosquito susceptibility tests at 19 localities in Mozambique, from March 2000 to July 2002. Biochemical assays were carried out on mosquitoes from the same families to detect shifts in the quantity or activity of enzyme families involved in insecticide detoxification. An. funestus from all localities remained fully susceptible to DDT and the organophosphorus insecticide malathion. A high level of pyrethroid resistance was detected in An. funestus populations in southern Mozambique. An. funestus outside Maputo province were still susceptible to pyrethroids. An. funestus from six localities also were resistant to carbamate insecticides propoxur and bendiocarb. Both pyrethroid and carbamate resistance occurred in five of these six localities. Mosquitoes from five of the localities with elevated p450 estimates, compared with the insecticide-susceptible Durban strain, were pyrethroid-resistant. The only exception to this trend was Mozal, which had elevated p450 estimates but full pyrethroid susceptibility by bioassay. The lack of cross-resistance between pyrethroids and DDT in Mozambican An. funestus suggests that a kdr-type target site resistance mechanism has not been selected. Low levels of insecticide-insensitive acetylcholinesterase, the target site for carbamates and organophosphates, were found in all populations tested. The high level of metabolically based pyrethroid resistance has implications for current malaria control programs in Mozambique.

Cross-resistance potential of fipronil in Musca domestica.

The toxicity of fipronil to insecticide-susceptible houseflies and the cross-resistance potential of fipronil were determined for six insecticide-resistant laboratory housefly strains by topical application and feeding bioassay. The insecticide-resistant strains represented different levels and patterns of resistance to pyrethroids, organophosphates, carbamates and organochlorines. Five strains were almost susceptible to fipronil in feeding bioassay with resistance factors at LC50 between 0.36 and 3.0. Four of these strains were almost susceptible to topically applied fipronil (resistance factors at LD50 were 0.55, 0.83, 3.3 and 2.5, respectively), whereas one strain was 13-fold resistant to topically applied fipronil. A highly gamma-HCH-resistant strain, 17e, was 430-fold resistant to fipronil in topical application bioassay and 23-fold resistant in feeding bioassay at LD50/LC50. We also tested the toxicity of fipronil in a feeding bioassay and gamma-HCH in topical application bioassay on thirteen housefly field populations. Eleven of the field populations had resistance factors for fipronil ranging from 0.98 to 2.4 at LC50, whereas two populations were 4.0- and 4.6-fold resistant to fipronil. The resistance level to gamma-HCH at LD50 in the field populations ranged from 1.8- to 8.1-fold. The two strains showing fipronil resistance were 3.4- and 8.1-fold resistant to gamma-HCH. Fipronil and gamma-HCH toxicities were positively correlated in the field populations. Biochemical assays of esterase, glutathione S-transferase and cytochrome P450 monooxygenase indicated that the low fipronil resistance observed in laboratory and field strains could be caused by elevated detoxification or be due to a target-site resistance mechanism with cross-resistance to gamma-HCH.

Intensification of insecticide resistance in UK field populations of the peach-potato aphid, Myzus persicae (Hemiptera: Aphididae) in 1996

AbstractThe well-established carboxylesterase-based resistance to insecticides in Myzus persicae Sulzer has recently been accentuated by the appearance of aphids with a modified acetylcholinesterase (MACE) insensitive to pirimicarb and the novel aphicide, triazamate. This target site resistance mechanism was found in M. persicae from crops in the UK for the first time in 1996, together with especially large proportions of aphids with R2 and R3 levels of carboxylesterases, a combination that was associated with serious insecticide failures. This paper describes the incidence of both mechanisms and discusses the implications for future recommendations for aphid control in the UK.

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI

Molecular studies of knockdown resistance to pyrethroids: cloning of domain II sodium channel gene sequences from insects

Knockdown resistance (kdr) is a target-site resistance mechanism that confers nerve insensitivity to DDT and pyrethroid insecticides. In the housefly, Musca domestica, molecular cloning of the para-type sodium channel gene has revealed two amino acid mutations that are associated with kdr and super-kdr resistance phenotypes. Both mutations are located in the domain II region of the channel; Leu1014 to Phe in the hydrophobic segment IIS6 and Met918 to Thr in the IIS4-IIS5 linker. To investigate whether these mutations also occur in other insects, we have designed degenerate primers based on conserved sequences in the domain II region of the sodium channel and used these to PCR amplify this region from insecticide-susceptible strains of eight diverse insect species representing four different insect Orders: Helicoverpa armigera, Plutella xylostella, Spodoptera littoralis (Lepidoptera), Blattella germanica (Dictyoptera), Tribolium castaneum (Coleoptera), Myzus persicae, Aphis gossypii and Phorodon humuli (Hemiptera). The primers amplified closely related para-type sodium channel sequences from each insect with a minimum of 85% amino acid identity between species. All of the sequences contained ‘susceptible’ Leu and Met residues at the positions associated with kdr and super-kdr resistance in the housefly. Recent results detailing the presence of a kdr-type Leu to Phe mutation in pyrethroid-resistant strains of two important agricultural pests, P. xylostella and M. persicae, are discussed. ©1997 SCI

Spirosultam LY219048: A new chemical class of insecticide acting upon the GABA receptor/chloride ionophore complex

AbstractThis study assessed the toxicity and mode of action of a new experimental insecticide, LY219048 in insects and mammals. LY219048 produced rapid convulsions in mice and had LD50 values of 0.7 mg kg−1 and 4 mg kg−1 after intracerebral and intraperitoneal injection, respectively. In initial screens against insects, LY219048 showed low activity against the German cockroach (Blatella germanica L.). Lethality from dietary exposure required one to two weeks, even at concentrations as high as 10000 mg kg−1 (LC50 = 485 mg kg−1). In contrast, it had an LC50 value of 8.3 mg kg−1 against insecticide‐susceptible Drosophila melanogaster (Meig.) when synergized with piperonyl butoxide. Significant resistance to LY219048 (> 12‐fold) was detected in a cyclodiene‐resistant strain of D. melanogaster possessing an altered target site resistance mechanism. This finding suggested that LY219048 blocked the 4‐aminobutyric acid (GABA)‐gated chloride channel in a manner similar to that of the cyclodienes. In physiological studies in larval D. melanogaster central neurons, LY219048 antagonized the reduction of firing caused by 1 mM GABA. Dose‐response experiments showed that the ED50 for blocking inhibition under these conditions was c. 1 μ. Studies of 36CI uptake into bovine brain synaptosomes found that LY219048 was a potent antagonist. At 10 μ it completely blocked chloride flux stimulated by 50 μM GABA. LY219048 competitively displaced [3H]TBOB binding from bovine brain membranes, with an IC50 of 42 nM, which was comparable to values determined for TBPS (35 nM) and picrotoxinin (267 nM). There was little or no displacement (<25%) of [3H]flunitrazepam or [3H]muscimol binding by 10 μM LY219048. Taken together, these results provide strong evidence that this new chemical class of insecticide manifests its acute toxicity by blocking the GABA‐gated chloride channel.

R-factor trimethoprim resistance mechanism: An insusceptible target site

R-factor R388 increases the resistance of Escherichia coli to trimethoprim by 10,000 fold, and mediates the synthesis of an addional dihydrofolate reductase that is less susceptible to trimethoprim by a similar order of magnitude. The dihydrofolate reductase conferred by the R-factor was of a larger molecular weight than the wild-type enzyme and exhibited a different pattern of response to trimethoprim inhibition. This is thought to be the first example of an R-factor conferring an altered target site mechanism of resistance to a chemotherapeutic agent.