Dynamics and mechanisms of permethrin resistance in a field population of the horn fly, Haematobia irritans irritans

A study was conducted at the Pressler ranch, near Kerrville, Texas, USA between 2002 and 2006 to determine the dynamics and mechanisms of resistance to permethrin in a field population of the horn fly, Haematobia irritans irritans (L.). Changes of resistance to pyrethroid insecticide associated with use of a pour-on formulation of cyfluthrin in 2002 and use of diazinon ear tags in subsequent years were studied using a filter paper bioassay technique and a polymerase chain reaction assay that detects two sodium channel mutations, kdr and super-kdr resistance alleles. A maximum of 294-fold resistance to permethrin was observed in the summer of 2002. A significant decrease in the resistance level was observed in spring 2003, and resistance continued to decline after animals were treated with diazinon ear tags. In response to pyrethroid treatments, the allelic kdr and super-kdr frequency increased from 56.3% to 93.8% and from 7.5% to 43.8%, respectively in 2002, and decreased significantly in 2003 when the pyrethroid insecticide was no longer used to treat animals. Females were found to have a higher allelic super-kdr frequency than males in 2002, while no difference was detected between males and females in the allelic kdr frequency. There was a significant positive correlation between frequencies of the sodium channel mutations and levels of permethrin resistance, suggesting that the sodium channel mutations, kdr and super-kdr , are the major mechanisms of resistance to pyrethroids in this horn fly population. Results of synergist bioassays also indicated possible contributions of two metabolic detoxification mechanisms, the mixed function oxidases (MFO) and glutathione S-transferases (GST). Compared to a horn fly infestation of an untreated herd, treatments with the pyrethroid pour-on formulation failed to control horn flies at the Pressler ranch in 2002. Sustained control of horn flies was achieved with the use of diazinon ear tags in 2003 and subsequent years.     

Susceptibility to diazinon in populations of the horn fly,Haematobia irritans (Diptera: Muscidae), in Central Brazil

From October 2000 to April 2001, insecticide bioassays were conducted in 18 ranches from 10 counties in the states of Mato Grosso and Mato Grosso do Sul, in Central Brazil. Horn flies from wild populations were exposed to diazinon-impregnated filter papers immediately after collection on cattle, and mortality was recorded after 2 h. A high susceptibility to diazinon was observed in all tested populations. The LC50s ranged from 0.15 to 0.64 micro g/cm2, and resistance ratios were always lower than one (ranging 0.1-0.6). Pyrethroid products, most applied by backpack sprayers, have been used since the horn fly entered the region, about 10 years ago. The high susceptibility observed to diazinon indicates that this insecticide (as probably other organophosphate insecticides) represents an useful tool for horn fly control and resistance management, particularly in pyrethroid-resistant populations.     

Genetic diversity and insecticide resistance of Myzus persicae (Hemiptera: Aphididae) populations from tobacco in Chile: evidence for the existence of a single predominant clone

The tobacco-feeding race of Myzus persicae (Sulzer), formerly known as M. nicotianae Blackman, was introduced into Chile during the last decade. In order to evaluate the genetic diversity and insecticide resistance status of Chilean tobacco aphid populations, a field survey was conducted in 35 tobacco fields covering a 300 km latitudinal survey. The populations sampled were characterized using microsatellite markers and morphometric multivariate analysis. Insecticide resistance levels were assessed through a microplate esterase assay and the mutation status of the kdr gene. All samples collected corresponded to the same anholocyclic aphid genotype, and showed morphological variation within the range expected for the tobacco-feeding race of M. persicae. Esterase activity showed the level and variability expected for an R1 clone lacking mutations in the sodium channels (susceptible kdr), thus corresponding to a type slightly resistant to organophosphate and carbamate, and susceptible to pyrethroid insecticides.     

Influence of permethrin, diazinon and ivermectin treatments on insecticide resistance in the horn fly (Diptera: Muscidae)

The history of insecticide resistance in the horn fly, Haematobia irritans, and the relationship between the characteristics of horn fly biology and insecticide use on resistance development is discussed. Colonies of susceptible horn flies were selected for resistance with six insecticide treatment regimens: continuous single use of permethrin, diazinon and ivermectin: permethrin-diazinon (1:2) mixture; and permethrin-diazinon and permethrin-ivermectin rotation (4-month cycle). Under laboratory conditions, resistance developed during generations 21, 31 and 30 to permethrin, diazinon and ivermectin, respectively. The magnitude of resistance ranged from < 3-fold with ivermectin to 1470-fold with permethrin. Field studies demonstrated that use of a single class of insecticidal ear tag during the horn-fly season resulted in product failure within 3-4 years for pyrethroids and organophosphates, respectively. In laboratory studies, use of alternating insecticides or a mixture of insecticides delayed the onset of resistance for up to 12 generations and reduced the magnitude of pyrethroid resistance. In field studies, yearly alternated use of pyrethroids and organophosphates did not slow or reverse pyrethroid resistance (Barros et al., unpublished data), while a 2-year alternated use with organophosphates resulted in partial reversion of pyrethroid resistance. When pyrethroid and organophosphate ear tags were used in a mosaic strategy at two different locations, efficacy of products did not change during a 3-year period.     

Incidence of insecticide resistance alleles in sexually-reproducing populations of the peach-potato aphid Myzus persicae (Hemiptera: Aphididae) from southern France

Intensive chemical treatments have led to the development of a number of insecticide resistance mechanisms in the peach-potato aphid Myzus persicae (Sulzer). Some of these mechanisms are known to be associated with negative pleiotropic effects (resistance costs). Molecular and biochemical methods were used to determine the genotypes or phenotypes associated with four insecticide resistance mechanisms in single aphids from sexually-reproducing populations in southern France. The mechanisms considered were E4 and FE4 carboxylesterase overproduction, modified acetycholinesterase, and kdr and rdl resistance-associated mutations. A new method for determining individual kdr genotypes is presented. Almost all resistant individuals overproduced FE4 carboxylesterase, whereas modified acetylcholinesterase was rare. Both the kdr and rdl resistance mutations were present at high frequencies in French sexually-reproducing populations. The frequencies of insecticide resistance genes were compared before and after sexual reproduction in one peach orchard at Avignon to evaluate the potential impact of selection on the persistence of resistance alleles in the over-wintering phase. The frequencies of the kdr and rdl mutations varied significantly between autumn and spring sampling periods. The frequency of the kdr mutation increased, probably due to pyrethroid treatments at the end of the winter. Conversely, the frequency of the rdl mutation decreased significantly during winter, probably because of a fitness cost associated with this mutation.     

Genetics of organophosphate resistance in field populations of the house fly (Diptera: Muscidae)

The genetics of resistance to the organophosphate insecticide diazinon were investigated in four populations of the house fly, Musca domestica L., collected in the southern United States. Crosses were made between individual females of lines derived from each population and males of a susceptible strain with three recessive mutants on chromosome II. Individual F1 females were crossed to mutant males, and the progenies were scored for resistance to diazinon and for the presence of mutant phenotypes. A major chromosome II gene for resistance to diazinon was present in all populations at an overall frequency of 83%. Map distances between the resistance gene and the mutant aristapedia and between the mutants aristapedia and stubby wing were highly variable in all populations. Recombination among the visible mutants was usually reduced in resistant progenies relative to susceptible progenies. The data suggest that a single major gene for resistance to diazinon was present on chromosome II in all test populations at variable map positions and is usually associated with a chromosome rearrangement, probably an inversion. The results are similar to those obtained earlier with house fly populations selected for resistance to insecticides in the laboratory; therefore, they seem to be characteristic of field and laboratory populations of the house fly. Overall, the data offer an explanation for previous results suggesting the existence of multiple, closely linked genes for metabolic resistance to insecticides on house fly chromosome II.     

Variation in general esterase activity within a population of Haematobia irritans (Diptera: Muscidae).

Control of the horn fly, Hematobia irritans (L.), is generally dependent on chemical insecticides. However, the biology and behavior of the horn fly favors rapid development of insecticide resistance. To prolong the effectiveness of the insecticide option, information is required regarding the mechanisms of insecticide resistance. Metabolic hydrolysis of insecticides by esterases is a detoxification mechanism in many insect species. Measurement of general esterase activity within populations of horn flies may provide a diagnostic tool for resistance management. In this study we evaluated the amount of variation in general esterase activity within female and male horn fly samples from a population that had not been exposed to insecticides for 8 yr. We found considerable variation in general esterase activity within samples of each sex, with females demonstrating the greater variation. The observed variation is thought to be the result of age-structure dynamics within the population. The amount of inherent variation makes it difficult to detect small mean differences between populations, thus limiting the utility of general esterase assays. Thus, effective diagnosis of esterase-mediated resistance mechanisms can only be achieved by the identification of specific detoxification esterases and the design of assays, either biochemical or molecular, for their detection and measurement.     

Genetic architecture in codling moth populations: comparison between microsatellite and insecticide resistance markers

The codling moth, Cydia pomonella, is renowned for developing resistance to insecticides and causing significant economic damage to pome fruits worldwide. In spite of its economic importance, little is known about the patterns of movement of this pest and the effects of insecticide treatment on the population genetic structure. Here, we investigated the genetic structure of the pest in 27 orchards from France, Italy, Armenia and Chile at seven microsatellite loci and two resistance markers [biochemical activity of cytochrome P450 oxidases and proportion of knockdown resistance (kdr) alleles in the sodium channel gene]. According to the microsatellite loci, we detected isolation by distance at the supranational scale but found no evidence of geographical structure among the 24 French orchards, which were mainly structured by the intensity of the insecticide treatments. Similarly, the highest levels of metabolic resistance associated with activity of the cytochrome P450 oxidases were detected in the most treated orchards. The kdr alleles were observed in southern France and Armenia where the pyrethroid insecticides were or have been intensively sprayed. The intensity of the insecticide treatments marginally affected the allelic richness in each orchard, but not the level of inbreeding. These results suggest important and high-distance gene flow among the codling moth populations, which were mainly structured according to the history of insecticide applications. Differences in mutation-migration-drift equilibrium among treated and untreated orchards also suggest that insecticide applications are the main force regulating the local dynamics of codling moth populations.     

First report on co-occurrence knockdown resistance mutations and susceptibility to beta-cypermethrin in Anopheles sinensis from Jiangsu Province, China

The increasing prevalence of insecticide resistance in Anopheles sinensis, a major vector of malaria in Jiangsu province in eastern China, threatens to compromise the successful use of insecticides in malaria control strategies. It is therefore vital to understand the insecticide resistance status of An. sinensis in the region. This study examined the nucleotide diversity of the para-sodium channel and knockdown resistance (kdr) in five field populations of adult An. sinensis mosquitoes collected in Jiangsu province, identifying the L1014F and L1014C substitutions for the first time. Competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) for resistance diagnosis were developed and validated. Comparing the results with direct sequencing revealed that the PCR-RFLP method was more sensitive and specific whereas the cPASA method was more convenient and suitable. The significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014C substitutions in the kdr gene provides a useful molecular marker for monitoring beta-cypermethrin resistance in natural populations of An. sinensis. Our results point to the L1014F substitution as the key mutation associated with beta-cypermethrin resistance. The high resistance and mutation frequency detected in the five populations also suggest cross-resistance with other pyrethroids may occur in An. sinensis, highlighting the need for further surveys to map insecticide resistance in China and the adoption of a rational management of insecticide application for resistance management and mosquito vector control.     

Field Survey of Insecticide Resistance in Haematobia irritans exigua de Meijere (Diptera: Muscidae)

A survey of farms in northern New South Wales and southeastern, central, western and northern Queensland was conducted to determine levels of insecticide resistance in populations of buffalo fly Haematobia irritans exigua. A field bioassay using discriminating concentrations of 10 insecticides commonly used for buffalo fly control was used. Resistance to all synthetic pyrethroids tested (cypermethrin, deltamethrin, cyhalothrin, flumethrin and cyfluthrin) was common and widespread in coastal zones, but was lower in inland zones. In contrast, there was no resistance to the organophosphate diazinon and only low levels of resistance to ethion and chlorfenvinfos. Synergism between piperonyl butoxide and cypermethrin was demonstrated.     

Horn fly (Diptera: Muscidae) insecticide resistance in Kentucky and Arkansas

The effect of previous insecticide use patterns for horn fly control on the susceptibility spectrum of horn fly (Haematobia irritans [L.]) populations from Kentucky and Arkansas is described. Populations of horn flies from both states were tested with three pyrethroids (cyhalothrin, cypermethrin, and permethrin), three organophosphates (diazinon, pirimiphos methyl, and tetrachlorvinphos), and a chlorinated hydrocarbon (methoxychlor). Dose-mortality data indicated insecticide resistance in Arkansas and Kentucky. Two permethrin-resistant horn fly populations in Kentucky that did not have a history of exposure to methoxychlor were cross-resistant to this chlorinated hydrocarbon. Horn fly populations from both states with a history of at least three consecutive years of exposure to various pyrethroid ear tags were subsequently exposed to cattle tagged with cyhalothrin-impregnated ear tags for 15-16 wk. Such exposure resulted in a decrease in susceptibility to this pyrethroid (ranging from approximately 30 to greater than 100-fold) when compared with levels before treatment. Horn fly populations from Arkansas resistant to cyhalothrin (as a result of exposure to cyhalothrin ear tags) were cross-resistant to pirimiphos methyl. Seasonal exposure of an Arkansas and Kentucky horn fly population to cattle with ear tags impregnated with pirimiphos methyl resulted in a significant decrease in susceptibility to this organophosphate.     

Frequency of kdr gene in house fly field populations: correlation of pyrethroid resistance and kdr frequency

The frequency of the L1014 F kdr mutation was determined in 14 field populations of house flies, Musca domestica L., with resistance factors at LD50 for pyrethrin/piperonyl butoxide and bioresmethrin/piperonyl butoxide from 4 to 29 and 2 to 98, respectively. A polymerase chain reaction test for identifying kdr homo- or heterozygote house flies was used to determine the frequency of kdr. The L1014 F allele was found in all populations tested. The frequency of kdr in the field populations was high and varied from 0.46 to 0.99. Eleven of the populations were in Hardy-Weinberg equilibrium, whereas two strains had higher number of heterozygotes than expected, indicating a possible heterozygote advantage. The frequency of kdr was strongly correlated with the reduced mortality observed in the bioassays with pyrethrum and bioresmethrin synergized by piperonyl butoxide. This indicates that kdr is a major mechanism for pyrethroid resistance in these field populations. Five field populations had resistance factors >25 and >10 for bioresmethrin/piperonyl butoxide and pyrethrin/piperonyl butoxide, respectively. The frequencies of kdr in these five populations varied from 0.89 to 0.99. The frequencies of kdr in the field populations showing no or a low level of resistance had frequencies of kdr from 0.46 to 0.75, which indicates that the L1014 F kdr allele is a fully recessive genetic trait in house flies. We have shown that the molecular diagnostic PASA method to determine the resistance phenotypes and the frequency of kdr is a powerful tool, which could be used to get information to make recommendations about pest and resistance management.     

Cross-resistance to insecticides in a malathion-resistant strain of Ceratitis capitata (Diptera: Tephritidae)

Resistance to malathion has been reported in field populations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), in areas of Spain where an intensive use of this insecticide was maintained for several years. The main goal of this study was to determine whether resistance to malathion confers cross-resistance to different types of insecticides. Susceptibility bioassays showed that the malathion-resistant W-4Km strain (176-fold more resistant to malathion than the susceptible C strain) has moderate levels of cross-resistance (three- to 16-fold) to other organophosphates (trichlorphon, diazinon, phosmet and methyl-chlorpyrifos), the carbamate carbaryl, the pyrethroid lambda-cyhalothrin, and the benzoylphenylurea derivative lufenuron, whereas cross-resistance to spinosad was below two-fold. The W-4Km strain was selected with lambda-cyhalothrin to establish the lambda-cyhalothrin-resistant W-1Klamda strain (35-fold resistant to lambda-cyhalothrin). The synergistic activity of the esterase inhibitor DEF with lambda-cyhalothrin and the increase in esterase activity in the W-1Klamda strain suggests that esterases may be involved in the development of resistance to this insecticide. Our results showed that resistance to malathion may confer some degree of cross-resistance to insecticides currently approved for the control of Mediterranean fruit fly in citrus crops (lambda-cyhalothrin, lufenuron, and methyl-chlorpyrifos). Especially relevant is the case of lambda-cyhalothrin, because we have shown that resistance to this insecticide can rapidly evolve to levels that may compromise its effectiveness in the field.     

Spectrum of cross-resistance to insecticides in field samples of the primary sheep blowfly, Lucilia cuprina

Hughes P. B. (1981) Spectrum of cross-resistance to insecticides in field samples of the primary sheep blowfly, Lucilia cuprina. Internationaljournal for Parasitology11:475–479. Resistance to the organophosphorous (OP) insecticide, diazinon, is due to a major gene on chromosome 4. To examine the spectrum of cross-resistance in field populations of Lucilia cuprina, larvae from 41 field samples were exposed to the susceptible discriminating doses of five insecticides. A comparison of the mortalities produced by diazinon to those caused by the other OPs tested (fenthion-ethyl, chlorfenvinphos and coumaphos) indicates that the diazinon resistance gene may confer cross-resistance to other OPs used for blowfly control. However, two samples were found to be resistant to diazinon but susceptible to coumaphos. The OP resistance gene is present at a high frequency in field populations, with a mean frequency of 0–82. Mortalities produced by the carbamate, butacarb, did not relate to those due to diazinon. It seems that a separate mechanism for carbamate resistance is also present in the field. No indication of resistance was found to the insect growth regulator Vetrazin^® in any sample tested.     

Identification of a mutation associated with permethrin resistance in the para-type sodium channel of the stable fly (Diptera: Muscidae)

The insect sodium channel is of particular interest for evaluating resistance to pyrethroids because it is the target molecule for this major class of neurotoxic insecticides. The stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), sodium channel coding sequence representing domains IS6 through IVS6 was isolated, and the sequence encoding domain II was compared among individuals of a laboratory strain selected for resistance to permethrin and the unselected, parental generation. A point mutation resulting in a leucine-to-histidine amino acid change was identified (Leul014His), and its location corresponded with that observed for knockdown resistance (kdr) mutations in other insects. As a result, the allele was designated kdr-his. A molecular assay was developed to assess the frequency of this mutation in genomic DNA of individual stable flies from the laboratory selections, which provided further evidence that the kdr-his allele accounts for the observed level ofpermethrin resistance in the selected strain. The assay was then used to evaluate the frequency of the mutation from five field-collected populations originating from three horse farms near Ocala, FL; one horse farm near Gainesville, FL; and one dairy farm near Hague, FL. Frequency of the kdr-his allele ranged from 0.46 to 0.78, supporting further investigation of allele prevalence throughout the stable fly season and in response to field insecticide application.     

Clonal turnover of MACE-carrying peach-potato aphids (Myzus persicae (Sulzer), Homoptera: Aphididae) colonizing Scotland

Peach-potato aphids, Myzus persicae (Sulzer), collected in Scotland in the years 1995 and 2002-2004 were characterized using four microsatellite loci and three insecticide resistance mechanisms. From 868 samples, 14 multilocus genotypes were defined (designated clones A-N). Five of these (denoted A, B, H, M and N) carried modified acetylcholinesterase (MACE) resistance, the most recent resistance mechanism to have evolved in M. persicae. The current paper shows that the continued presence of MACE aphids is due to turnover, as clones A and B were replaced in field samples by clones H, M and N in later seasons. Thus, insecticide-resistant populations in Scotland can be attributed to multiple waves of rapid clone colonisations and not to the continued presence of stable resistant clones or mutation or sexual recombination in local populations. The MACE clones carried varying levels of the other insecticide resistance mechanisms, kdr and esterase. The presence of these mechanisms could alter the clones success in the field depending on insecticide spraying (positive selection) and resistance fitness costs (negative selection).     

Sequence and transcript expression of the super-kdr locus of the horn fly,Haematobia irritans

In horn flies, Haematobia irritans irritans (Diptera: Muscidae) (Linnaeus, 1758), target site resistance to pyrethroids can be diagnosed by an allele-specific PCR that genotypes individual flies at both the super-kdr (skdr) and the knock down resistance (kdr) associated loci. When this technique uses genomic DNA as template, modifications, such as alternative RNA splicing and RNA editing are not specifically detected. Alternative splicing at the skdr locus has been reported in Dipterans; thus, the genomic DNA-based allele-specific PCR may not accurately reflect the frequency of the skdr mutation in horn fly field populations. To investigate if alternative splicing occurs at the skdr locus of horn flies, genomic DNA and cDNA sequences isolated from two wild populations and two laboratory-reared colonies with varying degrees of pyrethroid resistance were compared. There was no indication of alternative splicing at the super-kdr locus neither in the wild populations nor in the laboratory-reared colonies.