The influence of anticoagulant resistance on effective rodent control in the UK1

Widespread use of warfarin, and other multiple-dose anticoagulants, selected populations of warfarin-resistant rodents in the UK and in other countries. The effectiveness of rodent control was dramatically reduced when the resistant animals formed a large proportion of a population. The second-generation anticoagulants were developed to overcome these practical control problems with warfarin and were considered to be effective rodenticides against warfarin-resistant populations. In the UK, however, difenacoum showed reduced efficacy for controlling an established warfarin-resistant Rattus norvegicus population covering a substantial area of farmland in southern England. Continued use of difenacoum selected for difenacoum-resistant animals, so that the rodenticide is now ineffective for controlling rats in three counties. Brodifacoum was subsequently used to control some infestations and there is evidence that these treatments selected for animals with an increased level of resistance to brodifacoum. Anticoagulant resistance in Mus domesticus is less of a practical problem because of the availability of non-anticoagulant rodenticides for the control of this species. There is evidence, however, of bromadiolone resistance in M. domesticus trapped on farms in the UK. Recent investigations of the mechanism of anticoagulant resistance in R. norvegicus have indicated that vitamin K3 (menadione) is an antidote to anticoagulants in resistant animals. This form of the vitamin is included as a dietary supplement in poultry and pig food and would aid the survival, and therefore increase the selection, of resistant animals when anticoagulants are used exclusively.     

Laboratory and field trials of the rodenticide brodifacoum against warfarin-resistant Norway rats

Norway rats (Rattus norvegicus) trapped in a low-income housing area in Raleigh, North Carolina, and on a poultry farm in Garner, North Carolina, manifested high levels of resistance to warfarin in laboratory tests. Of the rats tested, 59.1 and 91.7% from the Raleigh and Garner trap sites, respectively, survived a 6-day no-choice test of a diet containing 0.005 % warfarin. The warfarin-resistant rats were subsequently given a choice of a 0.005% brodifacoum bait and a placebo diet. Of 27 rats, 19 died after a 3-day test. Survivors had consumed significantly less brodifacoum (0.1 < P > 0.05) than those that died, but the survivors also died when subjected to an additional 1-day no-choice test of the brodifacoum bait. Field trials, in which warfarin-resistant rats in four poultry houses were fed with poison bait for 14–16 days with 0.005% brodifacoum in a pelletised bait (‘Talon’), resulted in 87–99 % control being achieved.     

The genetic mechanisms of warfarin resistance in Rattus rattus found in the wild in Japan

Warfarin is commonly used worldwide as a rodenticide. It inhibits blood coagulation by inhibiting vitamin K 2,3-epoxide reductase (VKOR) activity leading to hemorrhage. However, it has been reported that repeated or long-term treatment with warfarin results in resistance emerging in wild rodents. Such resistance may explain why it is difficult to control rodents in many regions in Japan. In this report, we studied mutations in the VKOR gene (including the VKOR complex subunit 1 (VKORC1)), while also analyzing VKOR and clotting factor activity in black rats (Rattus rattus) in order to understand better the mechanism of warfarin resistance in this species.We sequenced the VKORC1 gene from 275 rats living in the wild in Japan. We found several types of novel base substitutions, some of which conferred warfarin resistance.There was no difference in coagulation times between warfarin-sensitive and resistant rats measured under physiological conditions. However, after warfarin administration, no effect was noted in warfarin-resistant rats, although a prolonged coagulation time was noted in warfarin-sensitive rats.We also determined the kinetic differences in hepatic microsomal VKOR-dependent activity between warfarin-resistant and sensitive rats. Warfarin-resistant rats showed 2-3-fold lower V_max/K_m values than did sensitive rats. In addition, we report that resistant rats found in the Tokyo area had a VKOR activity which was poorly inhibited by warfarin.Finally, we conclude that reduced VKOR activity and warfarin resistance in the Japanese black rat might be due to mutations in the VKORC1 gene. However, further study is needed to clarify how such rats can maintain adequate vitamin K-dependent clotting factor levels, while simultaneously exhibiting low VKOR activity and warfarin resistance.     

Revised methodology for a blood-clotting response test for identification of warfarin-resistant Norway rats (Rattus norvegicus)

A blood-clotting response test for warfarin resistance in Norway rats ( Rattus norvegicus ) was developed by our laboratory in the late 1970s, based on knowledge of biochemical mechanisms available at that time. That test used warfarin, its the free base which is not watersoluble, and vitamin K₁ 2,3-epoxide as an antidote for warfarin-resistant rats only. We have modified the above test to use a water-soluble salt of vitamin K₃ (1 mg kgg⁻¹ body weight), which is an antidote in a wider range of warfarin-resistant rat strains than vitamin K₁ 2,3- epoxide, and a water-soluble salt of warfarin (5.4 mg kg⁻¹ body weight). Sodium warfarin and vitamin K₃ are administered by oral gavage, and the blood-clotting response measured 24 h later. In a series of validation tests there were no significant differences between results obtained by either method. The new method, however, uses materials which are commercially available, and eliminates the possibility of misclassifying warfarin-resistant rats as susceptible because vitamin K₁ 2,3-epoxide was not an antidote in those strains. We also present evidence that measuring blood-clotting response to feeding on a vitamin K-free diet for 4 days is not a reliable method for determining the genotype of warfarin-resistant rats.     

Distribution and frequency of VKORC1 sequence variants conferring resistance to anticoagulants in Mus musculus

BACKGROUND: Emerging resistance to anticoagulant rodenticides may significantly impair house mouse (Mus musculus L.) control. As in humans and rats, sequence variants in the gene vitamin K epoxide reductase complex subunit 1 (VKORC1) of house mice are strongly implicated in the responses of mice to anticoagulants. This study gives a first overview of the distribution and frequency of such potentially resistance‐conferring sequence variants in house mice, based on tissue samples from 30 populations in Germany, Switzerland and the Azores. RESULTS: Except for one population from south Germany, sequence variants were found in individuals from all locations sampled (29 out of 30 sites surveyed), with less than 10% of the individuals matching the wild‐type genotype. The most frequent and widespread amino acid substitutions were Leu128Ser, Tyr139Cys and a group of linked sequence changes (Arg12Trp/Ala26Ser/Ala48Thr/Arg61Leu). Where these substitutions occurred as the sole variant, the proportion of homozygous individuals was 72–83%. CONCLUSIONS: An evaluation of published data revealed that the three most frequently found sequence variants are associated with a substantial loss of rodenticide efficacy of first‐generation anticoagulants (e.g. warfarin, coumatetralyl), as well as the second‐generation compound bromadiolone and most probably also difenacoum. Knowledge of the distribution and frequency of resistance‐conferring sequence variants will stimulate their further functional characterisation and facilitate the choice of effective active substances for house mouse control. Copyright © 2011 Society of Chemical Industry     

Warfarin resistance in Rattus losea in Guangdong Province, China

Control of rodent populations is performed worldwide with coumarin derivatives, such as warfarin. After widespread use, their effect has been diminished by the rapid spread of resistant rodents. Warfarin resistance in Rattus loseas in Jiangmen and Zhanjiang City, Guangdong Province, was investigated by lethal feeding tests. Twenty-three of 30 R. loseas trapped in Jiangmen City were assayed as warfarin-resistant individuals, whereas only 1 of 30 rodents in Zhanjiang was resistant. These results emphasize the need for thorough resistance monitoring as a basis for adequate control measures to prevent the use of ineffective rodenticides in Jiangmen City. The resistance mechanism mainly involves VKORC1, the molecular target for coumarin drugs. VKORC1 mRNA expression in wild-caught resistant animals showed no difference compared with that in susceptible individuals. Mutation screening of VKORC1 was carried out and an Arg58Gly mutation was identified as the prevailing type in R. loseas from Jiangmen City, which may constitute the genetic basis of anticoagulation resistance in R. losea in this resistance region.     

Warfarin-based rodenticides: Mode of action and mechanism of resistance

Warfarin and related substances have been in use as rodenticides for fifty years. They act by binding to the enzyme vitamin K 2,3-epoxide reductase, thereby interrupting the cellular recycling of vitamin K. Vitamin K in its hydroquinone form is an essential cofactor for the synthesis of functional prothrombin and related blood-clotting factors. The binding with the reductase is essentially irreversible, indicating these compounds have a prolonged half-life in target tissues; 7–10 days for warfarin and congeners and over 100 days for the second generation rodenticides or ‘superwarfarins’ such as difenacoum, brodi-facoum, and flocoumafen. Rat liver contains 1–2 nmole of enzyme per gram tissue which is a 4–5 fold overcapacity for maintaining effective vitamin K recycling. The use of warfarin as a rat poison has resulted in the natural selection of warfarin-resistant rats. The resistance is inheritable. Two distinct warfarin resistance genotypes, Welsh and Scottish, have been identified, clearly differing in their biochemistry of vitamin K epoxide reductase. In the Welsh strain, resistance arises from an altered enzyme expressing reduced reactivity to warfarin, whereas the reductase from the Scottish strain is as sensitive as the normal enzyme, but the interaction with warfarin is now readily reversible. The altered enzyme leaves the Welsh rat in need of higher dietary vitamin K intake. The superwarfarins cope with the resistance by having their structures firmly bind with the altered enzymes. Reduced sensitivity to warfarin-based rodenticides may also be pharmacokin-etically based, arising from increased warfarin biotransformation. This mechanism may be responsible for resistance to some of the superwarfarins such as difenacoum. A third resistance mechanism may arise from an enhanced capacity to synthesize vitamin K from menadione, a commonly used additive in animal foods on farms. The choice of rodenticide in the case of warfarin resistance should be guided by the underlying mechanism of resistance.     

The susceptibility of the Indian field mouse,Mus booduga gray,to anticoagulant rodenticidal baits

The baseline susceptibility of the Indian field mouse, Mus booduga was established for warfarin, bromadiolone and brodifacoum by means of nochoice feeding tests. The mice were found to be more susceptible to the second generation anticoagulants bromadiolone (0.005 g kg^?1) and brodifacoum (0.002 g kg^?1) than warfarin (0-25 g kg^?1). They required 8–10 days feeding on warfarin (0.25 g kg^?1) diet for complete mortality. A feeding period of 8 days corresponding to 99% mortality was suggested as a checking test for registering future warfarin resistance in suspect samples of M. booduga.     

Diversity and frequencies of genetic mutations involved in insecticide resistance in field populations of the house fly (Musca domestica L.) from China

Insecticides have been extensively used for house fly control in China, with dichlorvos and deltamethrin being widely used. Knowledge about the current status of insecticide resistance and the underlying genetic changes is crucial for developing effective fly control strategies. The susceptibility to dichlorvos and deltamethrin, and the frequencies of genetic mutations involved in insecticide resistance were studied in five field populations of the house fly collected across China. Bioassay results show that flies exhibit 14- to 28-fold resistance to dichlorvos and 41- to 94-fold resistance to deltamethrin, indicating that dichlorvos and deltamethrin resistance are common in house fly populations in China. Molecular analysis reveals that flies from the five various locations carry resistance alleles at multiple loci and have diverse allelic types, different relative frequencies and combinations of each allele. Four non-synonymous single nucleotide polymorphisms (SNPs) (i.e. V260L, G342A/V, F407Y) in acetylcholinesterase (Ace) and two mutations (W251L/S) in a carboxylesterase (MdαE7) were commonly present in the field house flies. The L1014H rather than L1014F mutation in the voltage sensitive sodium channel gene (Vssc) was widely distributed in Chinese house flies. CYP6D1v1, which confers pyrethroid resistance, was found in all the five tested populations in China, although its frequency in house fly from Shandong province was very low. Our results suggest that resistance monitoring and management of house flies should be customized for a given location.     

Population Dynamics of Small Rodents in Farm Buildings and on Arable Land1

Live-capture/recapture data on house mice caught in buildings on a single farm in England over a 1-year period showed that the populations were essentially unstable, being characterized by constant changes in size and composition. Less than 50% of the mice were recovered 8 weeks after they had been first captured. Although there was considerable individual variation in the movements of male and of female mice within particular buildings, movement between isolated populations was rare (6/439 animals; 1.4%). The house mouse was also found to be present in hedgerows near and distant to the farm buildings, albeit in low number. Movements of out-door living mice into farm buildings were not extensive, but could be important in relation to the re-invasion of cleared premises. The most abundant small rodent in the hedgerows was found to be Apodemus sylvaticus (L.).     

An evaluation of the role of oxidative enzymes in Colorado potato beetle resistance to carbamate insecticides

Carbofuran and carbaryl LD₅₀ values were determined with and without piperonyl butoxide pretreatment for a resistant (New Jersey) and two susceptible (Utah and Netherland) populations of Colorado potato beetle larvae. Similar bioassays were conducted with carbofuran for resistant (Rutgers) and susceptible (NAIDM) adult house flies. The degree of resistance development by New Jersey Colorado potato beetles (RR = 848) was greater than that of the laboratory-selected colony of Rutgers house flies (RR = 583). Comparisons of synergist difference calculations including “percentage synergism” (%S), “log percentage synergism” (L%S), and “relative percentage synergism (R%S) for the resistant (R) and the susceptible (S) populations indicated the possibility that monooxygenases and other resistance mechanisms may be involved in Colorado potato beetle resistance to these carbamates. Monooxygenase involvement in resistance of Rutgers house flies was demonstrated in vitro by a 4-fold enhancement of p-nitroanisole O-demethylation over that of NAIDM house flies. O-demethylation of p-nitroanisole could not be demonstrated for potato beetle larvae. Colorado potato beetle resistance was associated with increases in microsomal levels of NADPH-cytochrome c reductase (ca. 2-fold) and NADPH oxidation (1.2-fold). The inability to measure O-demethylation in Colorado potato beetles may have been due to the solubilization of NADPH-cytochrome c reductase during microsomal preparation. Significant differences between resistant and susceptible Colorado potato beetle larvae were not observed in the penetration of [¹⁴C]carbaryl. Excretion of the radiocarbon may have been significantly greater in the resistant New Jersey population, but some of the insecticide may have also rubbed off the cuticle. This increased capacity for excretion, combined with increased levels of monooxygenase enzymes, could account for the high resistance level of this population.     

Absorption, translocation and metabolism of metamitron in Chenopodium album

BACKGROUND: In recent years, common lambsquarters (Chenopodium album L.) populations from sugar beet fields in different European countries have responded as resistant to the as‐triazinone metamitron. The populations have been found to have the same D1 point mutation as known for atrazine‐resistant biotypes (Ser₂₆₄ to Gly). However, pot experiments revealed that metamitron resistance is not as clear‐cut as observed with triazine resistance in the past. The objectives of this study were to clarify the absorption, translocation and metabolic fate of metamitron in C. album. RESULTS: Root absorption and foliar absorption experiments showed minor differences in absorption, translocation and metabolism of metamitron between the susceptible and resistant C. album populations. A rapid metabolism in the C. album populations was observed when metamitron was absorbed by the roots. The primary products of metamitron metabolism were identified as deamino‐metamitron and metamitron‐N‐glucoside. PABA, known to inhibit the deamination of metribuzin, did not alter the metabolism of metamitron, and nor did the cytochrome P450 inhibitor PBO. However, inhibition of metamitron metabolism in the presence of the cytochrome P450 inhibitor ABT was demonstrated. CONCLUSION: Metamitron metabolism in C. album may act as a basic tolerance mechanism, which can be important in circumstances favouring this degradation pathway. Copyright © 2011 Society of Chemical Industry     

Cytochrome P450 mediated pyrethroid resistance in European populations of Meligethes aeneus (Coleoptera: Nitidulidae)

Pollen beetle, Meligethes aeneus (Coleoptera: Nitidulidae) is a major pest on several million hectares in European winter oilseed rape cultivation. Synthetic pyrethroids have been successfully used for many years to keep them under economic damage thresholds. Recently wide-spread resistance development to pyrethroids in pollen beetle populations was described in many European countries, including Germany, France, Poland, Denmark and others. Resistance monitoring is conducted by incubating beetles for 24 h in glass vials coated with different concentrations of lambda-cyhalothrin. Using such an assay format we were able to show cross-resistance to other pyrethroids, such as deltamethrin, cypermethrin, and to a somewhat lower extent bifenthrin, etofenprox and tau-fluvalinate. Here we also investigated in more detail in 27 different populations the biochemical mechanism of pyrethroid resistance. Synergism experiments revealed a high synergistic potential for piperonyl butoxide in vivo, whereas other compounds such as S,S,S-tributylphosphorotrithioate and diethylmaleimide failed to suppress pyrethroid resistance. Incubating microsomal fractions of pollen beetle with deltamethrin and subsequent LC–MS/MS analysis revealed 4-OH-deltamethrin as the major metabolite. Metabolite formation in vitro and pyrethroid resistance in vivo is correlated and inhibition trials with piperonyl butoxide, tebuconazole and aminobenzotriazole suggest the involvement of cytochrome P450′s. Furthermore we were able to show cross-resistance to tau-fluvalinate which is supported by the competitive inhibition of 4-OH-deltamethrin formation by increasing concentrations of tau-fluvalinate in microsomal hydroxylation assays. Although we provided clear experimental evidence for an oxidative mechanism of resistance in numerous populations, other mechanisms might be involved based on the data discussed.     

Resistance to anticoagulant rodenticides in Germany and future strategies to control Rattus norvegicus

Widespread anticoagulant resistance was discovered in populations of the brown rat (Rattus norvegicus Berk.) in an area of roughly 8000 km² in north-west Germany. Resistance testing was performed by feeding tests and/or by measuring blood clotting response after intraperitoneal injection of a sublethal testing solution. A hierarchical resistance system was found with warfarin resistance at the base followed by bromadiolone/coumatetralyl resistance to difenacoum resistance at the top. Warfarin resistance was spread over the whole area with reduced incidence towards the edges. Difenacoum resistance represented the highest level found so far and was restricted to the inner zone of the resistance area where it occurred with a frequency of 6% of all individuals tested. Breeding experiments with bromadiolone-resistant rats showed that expression of the bromadiolone resistance gene differed in the two sexes, suggesting additional sex-linked modifying effects to the resistance gene. Control strategy within the resistance area with respect to prevention of further selection for resistance is discussed.     

The A302S mutation in Rdl that confers resistance to cyclodienes and limited cross-resistance to fipronil is undetectable in field populations of house flies from the USA

Fipronil is a relatively new insecticide with great potential for insect control, however widespread use of cyclodiene insecticides has selected for an A302S mutation in the Rdl (GABA gated chloride channel) allele. This mutation gives resistance to cyclodienes and limited cross-resistance to fipronil. Given the concern over the possible reduction in efficacy and/or lifetime that fipronil might be used for pest control (given the extensive use of cyclodienes in the past), it is important to know the frequency of the A302S Rdl mutation in field populations. To ascertain the relative frequency of the A302S Rdl mutation in house fly populations we used three experimental approaches. First, we attempted to select for fipronil resistance by initially treating 33,100 field collected flies and selecting 14 additional generations. We were unable to produce a highly resistant strain. Second, we directly sequenced field collected flies. Third, we tested field collected house flies with a diagnostic dose of dieldrin and then genotyped the survivors. Out of the 4750 flies tested, there were no Rdl resistance alleles detected. We conclude that the resistant Rdl allele is rare in house flies in the US due to decades without cyclodiene use and a fitness disadvantage (in the absence of cyclodienes) of the 302S Rdl allele. The limited cross-resistance provided by the cyclodiene resistant Rdl allele, combined with the very low frequency of this allele in field populations, suggests that fipronil could be a promising insecticide for house fly control.     

Frequencies and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and field populations of the house fly, Musca domestica L.

Resistance to organophosphate (OP) and/or carbamate insecticides can be due to mutations in the acetylcholinesterase gene (Ace). Genotypes of house fly, Musca domestica L., Ace were determined in twelve laboratory maintained strains (originally from North America, Europe and Asia) and two field collected populations from New York and Florida. There were 15 Ace alleles found and 11 of the alleles coded for a susceptible form of the enzyme (i.e., V260, A316, G342 and F407). Three of the four resistance alleles were previously described, while one is new. Phylogenetic analysis of the alleles suggests multiple origins of the F407Y mutation and multiple origins of the G342A mutation that confer OP resistance. Genotyping of field collected house flies from New York and Florida populations revealed the presence of only one resistance allele, Acev10 (containing the non-synonymous mutations for A342 and Y407). All other alleles detected from the field-collected flies coded for a susceptible AChE. Thus, we were able to categorize individual flies as having homozygous susceptible (Ace^S/Ace^S), homozygous insensitive (Ace^I/Ace^I or Acev10/Acev10) or heterozygous AChE. The frequencies of Ace^S and Ace^I were not different between the NY2002 and FL2002 populations. Both populations were out of Castle-Hardy-Weinberg equilibrium, having an excess of Ace^S/Ace^I individuals and very few Ace^S/Ace^S individuals. Comparison of Ace, Vssc and CYP6D1 genotypes indicates individual house flies commonly have resistance alleles at multiple loci. Comparison of genotype data with bioassays, as well as the use of genotype data in resistance studies is discussed.     

Studies on the chiral isomers of fonofos and fonofos oxon: I. Toxicity and antiesterase activities

The toxicity of the (R)_P and (S)_P chiral isomers and racemates of fonofos and fonofos oxon to insects and white mice were determined. (R)_P-Fonofos and (S)_P-fonofos oxon were 2- to 12-fold more toxic to house flies, mosquito larvae, and mice than were the corresponding enantiomers. The racemates were intermediate in toxicity. Stereoselectivity also was observed in the in vitro inhibition of house fly-head and bovine erythrocyte acetylcholinesterase, horse serum cholinesterase, chymotrypsin, trypsin, and a variety of esterases. In all cases the (S)_P-oxon was a more potent inhibitor than the (R)_P-oxon with k₁ ratios of $\text{(S)}{}_{\mathrm{<mtext>P</mtext>}}\text{(R)}{}_{\mathrm{<mtext>P</mtext>}}$ ranging from 4- to 60-fold. Further, differences in levels of house fly-head, mouse brain, and blood cholinesterase obtained from house flies and mice treated with the enantiomers and racemates of fonofos and fonofos oxon were observed. Differences in toxicity of the enantiomers and racemates to house flies and mice were more closely related to in vivo than to in vitro cholinesterase inhibition.     

Multiple herbicide resistance in littleseed canarygrass (Phalaris minor): A threat to wheat production in India

Littleseed canarygrass (Phalaris minor Retz.), a troublesome weed of wheat in India, has evolved multiple herbicide resistance across three modes of action: photosynthesis at the photosystem II site A, acetyl‐coA carboxylase (ACCase), and acetolactate synthase inhibition. The multiple herbicide‐resistant (MHR) populations had a low level of sulfosulfuron resistance but a high level of resistance to clodinafop and fenoxaprop (ACCase inhibitors). Some of the populations had GR₅₀ (50% growth reduction) values for clodinafop that were 11.7‐fold greater than that of the most susceptible population. The clodinafop‐resistant populations also showed a higher level of cross‐resistance to fenoxaprop (fop group) but a low level of cross‐resistance to pinoxaden (den group). Although clodinafop and pinoxaden are from two different chemical families (fop and den groups), their same site of action is responsible for cross‐resistance behavior. The populations that were resistant to four groups of herbicides (phenylureas, sulfonylurea, aryloxyphenoxypropionate, and phenylpyrazolin) were susceptible to the triazine (metribuzin and terbutryn) and dinitroaniline (pendimethalin) herbicides. The P. minor populations that were resistant to the aryloxyphenoxypropionate and phenylurea herbicides were effectively controlled by the sulfonylurea herbicide, sulfosulfuron. In the fields infested with P. minor that was resistant to clodinafop, a sulfosulfuron application (25 g ha^?1) increased the wheat yield by 99.2% over that achieved using the recommended rate of clodinafop (60 g ha^?1). However, the evolution of multiple resistance against the four groups is a threat to wheat production. To prevent the spread of MHR P. minor populations, as well as the extension of multiple resistance to new chemicals, concerted efforts in developing and implementing a sound, integrated weed management program are needed. The integrated approach, consisting of crop and herbicide rotation with cultural and mechanical weed control tactics, should be considered as a long‐term resistance management strategy that will help to sustain wheat productivity and farmers' income.     

The Knockdown Resistance (kdr) Mutation in Pyrethroid-Resistant German Cockroaches

A point mutation in thepara-homologous sodium channel gene has been shown to be associated with knockdown resistance (kdr) in several insect species including the German cockroach. In this study, we analyzed the genomic organization of the region where thekdrmutation resides and then performed polymerase chain reaction (PCR) and sequencing using genomic DNA as the template to detectkdrmutation in 24 pyrethroid-resistant German cockroach strains, most of which have been collected recently from the field. Thekdrmutation, G to C at nt 2979 resulting in a leucine to phenylalanine amino acid substitution, was detected in 20 strains including 2 strains from overseas (China and Germany). Our results clearly indicate that thekdrmutation is widespread in German cockroach populations. However, the super-kdrmutation detected in super-kdrhouse flies was not found in any of the 4 strains that showed higher levels of knockdown resistance. Little correlation was observed between the presence of thekdrmutation and the level of knockdown resistance, suggesting the existence of multiple resistance mechanisms in many of these strains.     

A simple bioassay for detecting and characterising insecticide resistance

A simple and rapid paralysis assay was developed to detect and characterise knockdown resistance in larvae of the house fly and pink bollworm. Pyrethroidtreated larvae unable to perform a stereotypic curling movement when probed with a warm needle, 10 min (house fly) or 60 min (pink bollworm) after treatment, were considered to be paralysed. Responses in a susceptible strain of house fly were compared with those of the pyrethroid-resistant strains kdr and super-kdr. In this assay, the kdr strain displayed over 28 fold resistance to deltamethrin, and super-kdr larvae were unaffected by doses up to 100μg. These results are in good qualitative agreement with previous studies. The assay detected no significant fenvalerate resistance in pink bollworm larvae collected from the field; this was consistent with mortality bioassays performed on wild adult males. The limitations and potential utility of the paralysis bioassay in resistance screening are discussed.