Vitamin K requirement and reproduction in bromadiolone-resistant Norway rats

BACKGROUND: Nucleotide polymorphisms in the VKORC1 gene can be linked to anticoagulant rodenticide resistance in Norway rats (Rattus norvegicus Berkenhout). This provides a fitness advantage to rats exposed to anticoagulant actives, but may also cause fitness costs. The vitamin K requirement and reproductive parameters of bromadiolone‐resistant rats (Westphalian resistant strain; VKOR variant Tyr139Cys) and bromadiolone‐susceptible Norway rats were compared. RESULTS: At vitamin K deficiency, blood clotting times increased in all homozygous resistant males within 8 days and in 80% of homozygous resistant females within 15 days. There was little effect on blood clotting in heterozygous males and no effect in heterozygous females and VKOR wild‐type individuals. Litter size was about 20% higher in sensitive pairs compared with resistant pairs. Testes growth, male gonad weight, sperm motility and testis cell concentration were unaffected by the mutation. CONCLUSIONS: The VKOR variant Tyr139Cys causes considerable physiological cost in Norway rats in terms of vitamin K requirement and reproduction. This may affect the distribution and spread of resistant individuals in the wild. Decreased litter size of resistant parents seems to be due to lowered female reproductive performance, as there was no significant effect of the mutation on any aspects of male reproduction considered, but this requires further study. Copyright © 2011 Society of Chemical Industry     

Differential expression of cytochrome P450 genes between bromadiolone-resistant and anticoagulant-susceptible Norway rats: a possible role for pharmacokinetics in bromadiolone resistance

BACKGROUND: Anticoagulant resistance in Norway rats, Rattus norvegicus (Berk.), has been suggested to be conferred by mutations in the VKORC1 gene, encoding the target protein of anticoagulant rodenticides. Other factors, e.g. pharmacokinetics, may also contribute to resistance, however. To examine the involvement of pharmacokinetics in bromadiolone resistance in male and female rats, liver expression profiles of seven cytochrome P450 genes from a Danish bromadiolone-resistant rat strain (with an Y139C-VKORC1 mutation) were compared with profiles from an anticoagulant-susceptible strain. RESULTS: In the presence of bromadiolone, the Cyp2e1, Cyp2c13, Cyp3a2 and Cyp3a3 genes were significantly overexpressed, while Cyp2c12 expression was suppressed in resistant female rats compared with susceptible females. Relative to susceptible males, resistant males showed significant overexpression of the Cyp2a1, Cyp2e1, Cyp3a2 and Cyp3a3 genes. On exposure to bromadiolone, females had higher Cyp2e1 expression than males, which possibly explains why female rats are generally more tolerant to anticoagulants than male rats. CONCLUSION: Results suggest that bromadiolone resistance in a Danish strain of Norway rats involves enhanced anticoagulant metabolism catalysed by cytochrome P450-2e1, -3a2 and -3a3. This pharmacokinetically based bromadiolone resistance is to some extent sex differentiated, as female resistance furthermore seems to involve overexpression of cytochrome P450-2c13 and suppression of P450-2c12, whereas male resistance appears to involve P450-2a1 overexpression.     

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.     

Vitamin K requirement in Danish anticoagulant-resistant Norway rats (Rattus norvegicus)

Resistance to warfarin has been connected to an increase in dietary requirement for vitamin K in British strains of the Norway rat, Rattus norvegicus (Berk). This study examines vitamin K requirement of Danish anticoagulant-resistant Norway rats using a vitamin K deficient feeding test. Wild bromadiolone-resistant rats sampled from different localities in Denmark and rats from bromadiolone-resistant and susceptible laboratory strains were fed on a vitamin K deficient diet over a maximum period of 15 days. Development of vitamin K deficiency, measured as reduced blood-clotting capacity, took place in 43% of the Danish resistant rats and was independent of sex, treatment with supplementary vitamin K3 and sampling locality. Development of deficiency was slower for resistant rats that were supplemented with vitamin K3 prior to the feeding test, suggesting storage of the vitamin K in a vitamin body pool. Intraperitoneal administration of vitamin K1 revealed that 80 microg vitamin K1 kg(-1) bodyweight was sufficient to restore normal blood clotting activity in deficient rats, while 60 microg vitamin K1 kg(-1) bodyweight was insufficient. We conclude that vitamin K requirement is moderately increased in Danish homozygous resistant rats whereas heterozygous resistant rats only have a minor increase in vitamin K requirement compared with susceptible rats. We found no indication of different resistance types being present in our test material since vitamin K requirement was not different between rats from separate sampling localities.     

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

Adult, 20-week-old, rats from a Danish bromadiolone-resistant strain of rats (Rattus norvegicus) over-express the cytochrome P450 genes Cyp2e1, Cyp3a2 and Cyp3a3 upon bromadiolone exposure. Furthermore, adult female rats of this strain over-express the Cyp2c13 gene and suppress Cyp2c12, while males over-express the Cyp2a1 gene. The altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared expression profiles from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant rats. The P450-3a2 and -3a3 isoforms were proposed to be of higher importance in adult male resistance than in pre-pubertal resistance. In contrast, the P450-2c13 and -3a2 isoforms were proposed to be more important in sexual immature female resistance, while the P450-2e1 and -3a3 isoforms were suggested to play a more significant role in adult female resistance.     

Involvement of hepatic xenobiotic related genes in bromadiolone resistance in wild Norway rats, Rattus norvegicus (Berk.)

To examine the role of xenobiotic relevant genes in bromadiolone resistance in wild Norway rats (Rattus norvegicus) we compared the constitutive liver gene expression and expression upon bromadiolone administration in bromadiolone resistant and anticoagulant susceptible female rats using a LNA microarray and quantitative PCR. Resistant rats showed significantly higher constitutive expression of the cytochrome P450 genes Cyp2c13 and Cyp3a2 and lower expression of Cyp2e1 and Gpox1 compared to the susceptible rats. The Cyp1a2, Cyp2c13, Cyp2e1, Cyp3a2 and Cyp3a3 genes were significantly higher expressed in resistant than susceptible rats upon bromadiolone exposure. To establish how bromadiolone affected xenobiotic gene expression in the two strains we compared bromadiolone expression profiles to saline profiles of both strains. Bromadiolone mediated significant up-regulation of Cyp2e1 and Cyp3a3 expression in the resistant rats whereas the rodenticide conferred down-regulation of Cyp2e1, Cyp3a3 and Gpox1 and induction of Cyp2c12 expression in susceptible rats. Cyp2c13 and Cyp3a2 expression were markedly suppressed in both strains upon treatment. This suggests that xenobiotic relevant enzymes play a role in bromadiolone resistance in the Norway rat. A high constitutive expression of Cyp2c13 and Cyp3a2 and induction of Cyp1a2, Cyp2e1 and Cyp3a3 expression during bromadiolone exposure may increase the resistance to bromadiolone presumably by facilitating increased detoxification and decreased liver injury.     

Bromadiolone,a New Anticoagulant Rodenticide1

A search for more effective anticoagulant rodenticides has brought about the development of a new compound, coded LM-637, and temporarily named bromadiolone. Results of efficacy tests on 10 rodent species are discussed in detail. The compound was found to be a very potent and highly palatable anticoagulant which is capable of achieving 100% mortality in Norway rats, Rattus norvegicus (Berkenhout), with 1-day feeding. Efficacy was demonstrated on all 3 commensal rodents and several important agricultural rodent pests native to the United States. A relatively few feedings (1 to 5 days) of bait containing 0.005 % bromadiolone produced death in most species tested. These rodenticidal properties make bromadiolone a most promising control agent.     

Resistance tests and field trials with bromadiolone for the control of Norway rats (Rattus norvegicus) on farms in Westphalia, Germany

BACKGROUND: The aim of this study was to determine the incidence and the level of resistance to bromadiolone among rats on farms suspected of being foci of resistance by using the international normalised ratio (INR)‐based blood clotting response (BCR) test. Whether the level of reduced susceptibility constitutes ‘practical resistance’ was subsequently determined in field trials. RESULTS: The 2.5 multiple of the ED₅₀ baseline was used to test for the incidence of resistance, and higher multiples in the range of the suspected resistance factor were used to investigate the degree of resistance. The ED₅₀ values of bromadiolone in resistant rats were confirmed in the range 4.70–7.05 mg kg^?1 for males and 4.62–6.61 mg kg^?1 for females. Variations within these ranges appeared between farms. According to the BCR resistance tests, 50–100% of rats were classified as resistant prior to the field trials; 29–100% of rats survived the treatments. CONCLUSION: BCR tests based on the use of the INR and baselines are suitable for determining the incidence and for assessing the level of resistance in populations of Norway rats. The majority of rats of the Westphalian resistant strain, characterised by the Y139C marker in VKOR, are resistant to bromadiolone under practical control conditions. Copyright © 2011 Society of Chemical Industry     

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.     

Bromadiolone susceptibility in wild and laboratory Mus musculus L.(house mice) in Buenos Aires,Argentina

BACKGROUND: Rodents are major pests in many agricultural systems, where they can cause significant economic losses and involve a sanitary risk. The application of anticoagulant rodenticides for rodent control has showed a decrease in effectiveness through time because of the development of resistant populations and the development of aversion behaviour. The goal of the present study was to test the susceptibility to bromadiolone and the existence of anticoagulant resistance in Mus musculus L. (house mouse) in Argentina. We conducted a feeding test with wild animals captured in poultry farms and a laboratory strain that were fed with bromadiolone bait. RESULTS: Three animals of the field experimental group survived the 21 days study period, while for laboratory animals mortality was 100%. Control field animals which were fed without anticoagulant showed 100% survival. CONCLUSION: We found evidence of the presence of anticoagulant resistant M. musculus L. in the study area. Feeding behaviour may have contributed to increasing the time of survival, and may be a mechanism that allows metabolic clearance of the bromadiolone. Under field conditions control with anticoagulants would be less effective because animals have alternative food. Copyright © 2009 Society of Chemical Industry     

Multiple forms of carboxylesterase from Leptinotarsa decemlineata haemolymph associated with permethrin resistance

For the first time, it has been unequivocally shown that multiple-feed second-generation anticoagulant rodenticides were ineffective against a population of rats in N.W. Berkshire, UK because of an unusually high prevalence and high degree of resistance. Use of the non-anticoagulant rodenticide calciferol led to a substantial reduction in the population, although primary poisoning of small birds appeared to be greater than with anticoagulant baits. There was strong evidence that many of the surviving rats had developed an aversion towards calciferol-treated bait. A reduction in the degree of anticoagulant resistance in the population was evident after a period of 17 months without anticoagulant use. The long-term strategy to manage the resistant population should integrate non-anticoagulant and anticoagulant rodenticide use to take advantage of possible pleiotropic costs of resistance.     

The impact of resistance on the use of second-generation anticoagulants against rats on farms in Southern England

Resistance amongst rats to second-generation anticoagulants, notably difenacoum, has been recognised since the 1970s. Although initially considered to be of practical significance, doubts were subsequently expressed that the degree of resistance was sufficient to explain ineffective rodenticide treatments. Research published elsewhere demonstrated the overriding importance of poor poisoned bait consumption, caused by the influence of certain ecological factors on rat behaviour, in reducing the effectiveness of second-generation anticoagulant treatments on farms in southern England. In this paper we consider, however, the subtle effects of resistance to anticoagulants on treatment outcome. The prevalence of resistance was increased amongst survivors of treatments and bait consumption by resistant survivors was higher than amongst susceptible survivors. The overall prevalence and degree of resistance to second-generation anticoagulants does not, however, currently represent a practical problem. Nevertheless, selection favouring increased resistance remains a long-term threat to the effectiveness of both difenacoum and bromadiolone.     

伽马辐照对智利小植绥螨生殖及性别决定的影响

辐照可导致染色体变异,是研究生殖机理的常用辅助方法之一。本研究对不同生理状况下智利小植绥螨雌螨、雄螨、不同发育阶段卵进行不同剂量的伽马辐照,探索其后代发育与繁殖情况,以期解释智利小植绥螨性别决定机制及生殖机理。研究结果表明,在160~640 Gy辐照剂量下,辐照的未交配的雄成螨与正常的未交配的雌成螨交配,F1代中只有雄成螨且仅1/3可育。在45~90 Gy辐照剂量下,辐照的未交配的雌成螨与正常的未交配的雄成螨交配,其子代雌性比随着辐照剂量的增加而减小,且至90 Gy处理下无子代完成胚胎发育。辐照交配后产卵前的雌成螨,其子代雌性比间没有显著差异（77.5%~85.0%）。辐照卵龄0~12 h的卵,没有卵发育至成螨;辐照卵龄12~24 h的卵,其中发育至成螨的全为雌螨;辐照卵龄24~48 h的卵,其中发育至成螨的雌性比随着辐照剂量的增加具有增加的趋势。试验结果推测出父系染色体组参与形成配子且对性别起决定作用;父系染色体组异质化的时间可能集中于卵壳形成后至卵期12 h之间。本试验为智利小植绥螨父系染色体组参与性别决定的机制做出了新的诠释,对深入研究性别决定的机理具有重要的意义。     

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     

Cytochrome P450 monooxygenase-mediated neonicotinoid resistance in the house fly Musca domestica L

Neonicotinoids play an essential role in the control of house flies Musca domestica. The development of neonicotinoid resistance was found in two field populations. 766b was 130- and 140-fold resistant to imidacloprid and 17- and 28-fold resistant to thiamethoxam in males and females, respectively. 791a was 22- and 20-fold resistant to imidacloprid and 9- and 23-fold resistant to thiamethoxam in males and females, respectively. Imidacloprid selection of 791a increased imidacloprid resistance to 75- and 150-fold in males and females, respectively, whereas selection with thiamethoxam had minimum impact. Neonicotinoid resistance was in all cases suppressed by PBO. The cytochrome P450 genes CYP6A1, CYP6D1 and CYP6D3 were constitutively over-expressed in resistant strains and CYP6D1 and CYP6D3 differentially expressed between sexes. The highest level of CYP6A1 expression was observed in both gender of the imidacloprid-selected strain after neonicotinoid exposure. CYP6D1 expression was increased after neonicotinoid exposure in resistant males. CYP6D3 expression was induced in both sexes upon neonicotinoid exposure but significantly higher in females.     

Brodifacoum is effective against Norway rats (Rattus norvegicus) in a tyrosine139cysteine focus of anticoagulant resistance in Westphalia,Germany

BACKGROUND: The tyrosine to cysteine amino acid substitution at location 139 of the vkorc1 protein (i.e. tyrosine139cysteine or Y139C) is the most widespread anticoagulant resistance mutation in Norway rats (Rattus norvegicus Berk.) in Europe. Field trials were conducted to determine the incidence of the Y139C mutation at two rat‐infested farms in Westphalia, Germany, and to estimate the practical efficacy against them of applications, using a pulsed baiting treatment regime, of a proprietary bait (Klerat^?) containing 0.005% brodifacoum. RESULTS: DNA analysis for the Y139C mutation showed that resistant rats were prevalent at the two farms, with an incidence of 80.0 and 78.6% respectively. Applications of brodifacoum bait achieved results of 99.2 and 100.0% control at the two farms, when measured by census baiting, although the treatment was somewhat prolonged at one site, possibly owing to the abundance of attractive alternative food. CONCLUSION: The study showed that 0.005% brodifacoum bait is fully effective against Norway rats possessing the Y139C mutation at the Münsterland focus and is likely to be so elsewhere in Europe where this mutation is found. The pulsed baiting regime reduced to relatively low levels the quantity of bait required to control these two substantial resistant Norway rat infestations. Previous studies had shown much larger quantities of bromadiolone and difenacoum baits used in largely ineffective treatments against Y139C resistant rats in the Münsterland. These results should be considered when making decisions about the use of anticoagulants against resistant Norway rats and their potential environmental impacts. Copyright © 2012 Society of Chemical Industry     

3种抗凝血类杀鼠剂在喀什农区防治害鼠效果及对家禽的安全性评价

为确定适于防治南疆农区鼠害的抗凝血类杀鼠剂, 在喀什地区疏勒县采用夹捕法、食饵法、粉迹法评估了杀鼠醚、溴敌隆、溴鼠灵的防治效果, 并采用灌胃法给药测试3种杀鼠剂对家鸡Gallus domestiaus和鸽子Columba livia的安全性。结果表明, 连续投放毒饵10 d后, 与溴敌隆防治区相比, 杀鼠醚防治区的阳性粉块和无毒小麦取食量的下降幅度均无显著差异, 但无毒小麦取食量下降的农户比例更高;且防治后褐家鼠比例更低, 防治期间毒饵消耗量更高。连续单独投放溴敌隆30 d的灭鼠效果可达90.9%, 但10 d溴敌隆与20 d溴鼠灵联合防治防效仅为65.9%。灌胃法测试表明, 对当地家禽类的毒性从低到高依次为杀鼠醚、溴敌隆和溴鼠灵。因此, 建议优选毒性较低的杀鼠醚或溴敌隆用于南疆农区鼠害防治。     

Fitness costs of laboratory-selected imidacloprid resistance in the brown planthopper, Nilaparvata lugens Stål

Imidacloprid has been used as a key insecticide to control the brown planthopper, Nilaparvata lugens Stål, for several years, but no obvious resistance has been identified in field populations as yet. To evaluate the risk, a field population was collected and selected with imidacloprid in the laboratory. After 37‐generation selection a strain with 250‐fold resistance had been successfully achieved. Fitness analysis by constructing life tables demonstrated that resistant hoppers had obvious disadvantages in their reproduction. The fitness of highly resistant hoppers had decreased dramatically (0.169 and 0.104) to only one‐fifth to one‐tenth of that of the susceptible strain. Hence it was concluded that the brown planthopper had the potential to develop high resistance to imidacloprid but that the lower fitness of resistant hoppers could result in a quick recovery of sensitivity when the population did not come into contact with imidacloprid. This means that a reasonable resistance management programme with less imidacloprid use may efficiently delay or even stop resistance development. Copyright © 2006 Society of Chemical Industry     

Preference of female rats for the odours of non-parasitised males: the smell of good genes?

Many animals obtain reliable information about potential mates, including whether they are parasitised or not, mostly from olfactory cues in urine. Previous experiments with rodents have shown that females can detect parasites in males that are potentially transmissible during copulation, so that females can directly avoid infection by discriminating against parasitised males. Here, using choice tests, we examine whether female rats can distinguish males infected with the tapeworm Hymenolepis diminuta Rudolphi, 1819, a parasite with a complex life cycle and thus not directly transmissible among rats. Female rats tended to spend more time investigating the urine of non-parasitised males than that of parasitised males. The magnitude of the parasite burden in the infected males had no effect on the females' preference for the non-parasitised males. We also found that parasitised males had lover testosterone levels in their blood than non-parasitised males. These results suggest that females use cues in male urine reflecting either the presence of the parasite and/or lower testosterone levels to avoid parasitised males and possibly secure resistance genes for their offspring.     

Impact of sub-lethal residues of azinphos-methyl on the pheromone-communication systems of insecticide-susceptible and insecticide-resistant obliquebanded leafrollers Choristoneura rosaceana (Lepidoptera: Tortricidae)

The effects of sub-lethal residues of azinphos-methyl on pheromone production, calling, female attractiveness and the ability of males to locate sources of natural and synthetic pheromone were compared in azinphos-methyl-susceptible (susceptible) and azinphos-methyl-resistant (resistant) obliquebanded leafrollers, Choristoneura rosaceana (Harris). The amount of pheromone in susceptible females was reduced by 29-33% after exposure to azinphos-methyl; this treatment did not affect the pheromone content of resistant females. Azinphos-methyl-treated resistant females contained 39-43% less pheromone than azinphos-methyl-treated susceptible females. Resistant females that were not treated with azinphos-methyl contained 35-56% less pheromone than susceptible females that were not treated with insecticide. The incidence of calling was reduced by 67-100% in azinphos-methyl-treated susceptible females; the incidence of calling by resistant females was not affected by exposure to azinphos-methyl. The incidence of calling by azinphos-methyl-treated susceptible females was 58-100% lower than that of azinphos-methyl-treated resistant females. There was no difference in the incidence of calling between susceptible and resistant females that had not been treated with insecticide. In a flight tunnel, treatment with insecticide reduced the attractiveness of susceptible females by 38%; treatment with insecticide did not affect the attractiveness of resistant females. There was no difference in the proportion of males attracted to susceptible and resistant females that had, or had not been treated with insecticide. In an apple orchard, the attractiveness of susceptible and resistant females treated with azinphos-methyl was reduced by 84 and 12%, respectively. The proportion of males attracted to azinphos-methyl-treated susceptible females was 58% lower than the proportion attracted to azinphos-methyl-treated resistant females, whereas, if females were not treated with insecticide, the proportion attracted to resistant females was 57% lower than the proportion attracted to susceptible females. In a flight tunnel, azinphos-methyl did not affect the ability of susceptible or resistant males to locate a source of pheromone gland extract. Likewise, in an apple orchard, the insecticide treatment had no effect on the ability of susceptible or resistant males to locate a source of synthetic pheromone. In a flight tunnel, there was no difference in the proportion of azinphos-methyl-treated susceptible and resistant males locating a source of pheromone gland extract; however, in the orchard, 39% fewer azinphos-methyl-treated resistant males located a source of synthetic pheromone than azinphos-methyl-treated susceptible males. A similar proportion of susceptible and resistant males that had not been treated with insecticide located a source of pheromone gland extract in the flight tunnel, but in the orchard, the proportion of resistant males not treated with azinphos-methyl that located the source of synthetic pheromone was 32% lower than the proportion of susceptible males not treated with this insecticide. The implications of the differences in the effect of sub-lethal residues of azinphos-methyl on the pheromone communication system of susceptible and resistant moths are discussed in relation to the theory of the development of insecticide resistance, the detection of resistance in feral populations of moths using sex pheromone-baited traps, and the control of moths using sex pheromone-mediated mating disruption.