Frequency Of Resistance Alleles Research Articles

Bactrocera oleae (Diptera: Tephritidae) organophosphate resistance alleles in Iberia: Recent expansion and variable frequencies

The olive fly, Bactrocera oleae (Rossi, 1790) (Diptera: Tephritidae), is the most important pest of olive trees globally, caus- ing losses that, in the absence of control measures, can exceed 90% of the crop. In the Mediterranean basin, where the overwhelming majority of production is concentrated (~ 98%), organophosphate insecticides (OPs) have been the main tool for B. oleae control for the last four decades, leading to the development of resistance to these compounds. Mutations of the Ace gene, which codes for ace- tylcholinesterase, the target enzyme of OPs and other insecticides, have been identified as the underlying cause, with studies reporting mid to very high frequencies of resistance alleles in several countries. Interestingly, no resistance alleles were detected in Portugal, at the Western end of the Mediterranean basin. As the original study was done almost a decade ago and did not include many samples, we decided to re-evaluate the situation, by analysing a larger number of individuals from multiple locations in Western and Southern Iberia (Portugal and Spain). In our present study, resistance-associated Ace alleles were found to have become widespread in both regions, but with highly varying frequencies. Together with the observed haplotype distributions, this finding is consistent with previous sug- gestions of a recent, selection-driven spread and has implications for the importance of Ace mutations in organophosphate resistance in the field as well as the importance of gene flow between Mediterranean populations of B. oleae.

Emergence and early evolution of fungicide resistance in North American populations of Zymoseptoria tritici

Although fungicide resistance in crop pathogens is a global threat to food production, surprisingly little is known about the evolutionary processes associated with the emergence and spread of fungicide resistance. Early stages in the evolution of fungicide resistance were evaluated using the wheat pathogen Zymoseptoria tritici, taking advantage of an isolate collection spanning 20 years in Oregon, USA, and including two sites with differing intensity of fungicide use. Sequences of the mitochondrial cytb protein conferring single‐mutation resistance to QoI fungicides and the nuclear CYP51 gene implicated in multiple‐mutation resistance to azole fungicides were analysed. Mutations associated with resistance to both fungicides were absent in the 1992 isolates, but frequent in the 2012 collection, with higher frequencies of resistance alleles found at the field site with more intensive fungicide use. Results suggest that the QoI resistance evolved independently in several lineages, and resulted in significant mitochondrial genome bottlenecks. In contrast, the CYP51 gene showed signatures of diversifying selection and intragenic recombination among three phylogenetic clades. The findings support a recent emergence of resistance to the two fungicide classes in Oregon, facilitated by selection for mutations in the associated resistance genes.

Molecular Prevalence, Phylogenetic Characterization and Benzimidazole Resistance of Haemonchus contortus from Sheep

This study was conducted to determine the molecular prevalence and characterization of Haemonchus contortus from sheep along with the benzimidazole (BZ) resistance in H. contortus populations. Fecal samples were collected from a total of 300 sheep in research area and analyzed by fecal flotation. qPCR assays were utilized on trichostrongylid egg positive samples in order to identify H. contortus. Phylogenetic analyses were performed on ribosomal ITS-2 and mt-COI gene regions. BZ sensitive and resistant allele frequencies were determined by qPCR along with sequence analyses of the s-tubulin isotype 1 gene for single nucleotide polimorphizms (SNPs). H. contortus was identified in 36 (24.8%) out of 145 trichostrongylid egg positive samples. H. contortus isolates showed 100.0% identity to each other and 0.1% difference with the isolates available in the GenBank based on phylogenetic analyses of ITS-2 gene while mt-COI analyses of the isolates exhibited a mean of 97.4±0.5% identity to each other and 5.5±0.8% difference with the isolates in GenBank. BZ sensitive and resistant allele frequencies in H. contortus populations were determined as 87.1%±16.2 and 12.9%±16.2, respectively. SNPs were detected only in the codon 200 of the sequenced isolates belong to resistant allele. This study provides the first data on molecular prevalence, phylogenetic characterization and BZ resistance in H. contortus populations from sheep in Turkey. (Less)

Managing resistance to chemical treatments in stored products pests

This review focuses on key trends in resistance to chemical treatments in stored product pests, and advances in resistance management, with an emphasis on resistance to the fumigant phosphine. Findings: Phosphine resistance continues to be a major concern. In particular, phosphine resistance in Cryptolestes ferrugineus has emerged as a serious issue, with some populations exhibiting the strongest level detected so far for this fumigant. In response, a 'quick knock down test' has been established to deliver industry and scientists 'same day' advice on the resistance status of field samples; sulfuryl fluoride is being developed as a 'resistance breaker' and phosphine dosages are being revised to manage this problem. There has been major progress in identifying the genes responsible for phosphine resistance and the development of molecular resistance diagnostics for key pests. Several studies on Rhyzopertha dominica have demonstrated that molecular screening can be used to determine the frequency of resistance alleles in samples collected from farm storages. Despite on-going research in several pests, there is no definitive answer to the question of whether there is a fitness cost associated phosphine resistance, with some studies showing a clear cost and others none. Evidence continues to emerge of resistance to grain protectants, including the juvenile hormone analogue methoprene. The development and adoption of spinosad, as a next generation 'green' treatment, and the use of protectant combinations provides opportunities to counter the problem of protectant resistance.Directions for future research: A uniform set of protocols should be developed for phosphine resistance detection for all major species. It should combine 'quick tests' and molecular diagnostics to be adopted internationally. Research is required on the establishment of a decision making system that integrates newly developed grain protectants and fumigants, other alternative control methods, as well as an accurate and rapid resistance detection system for early warning of the emergence of new resistances.

Toxicodynamic mechanisms and monitoring of acaricide resistance in the two-spotted spider mite

The two-spotted spider (Tetranychus urticae) is one of the most serious pests world-wide and has developed resistance to many types of acaricides. Various mutations on acaricide target site genes have been determined to be responsible for toxicodynamic resistance, and the genotyping and frequency prediction of these mutations can be employed as an alternative resistance monitoring strategy. A quantitative sequencing (QS) protocol was reported as a population-based genotyping technique, and applied for the determination of resistance allele frequencies in T. urticae field populations. In addition, a modified glass vial bioassay method (residual contact vial bioassay, RCV) was implemented as a rapid on-site resistance monitoring tool. The QS protocol, together with the RCV, would greatly facilitate monitoring of T. urticae resistance. Recent completion of T. urticae genome analysis should facilitate the identification of additional resistance genetic markers that can be employed for molecular resistance monitoring.

Cry1F resistance in fall armyworm Spodoptera frugiperda: single gene versus pyramided Bt maize.

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F2 screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012–2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda.

Detection of genetically isolated entities within the Mediterranean species of Bemisia tabaci: new insights into the systematics of this worldwide pest.

The taxonomy of the species complex Bemisia tabaci, a serious agricultural pest worldwide, is not well resolved yet, even though species delimitation is critical for designing effective control strategies. Based on a threshold of 3.5% mitochondrial (mtCOI) sequence divergence, recent studies have identified 28 putative species. Among them, mitochondrial variability associated with particular symbiotic compositions (=cytotypes) can be observed, as in MED, which raises the question of whether it is a single or a complex of biological species. Using microsatellites, an investigation was made of the genetic relatedness of Q1 and ASL cytotypes that belong to MED. Samples of the two cytotypes were collected in West Africa where they live in sympatry on the same hosts. Genotyping revealed a high level of differentiation, without evidence of gene flow. Moreover, they differed highly in frequencies of resistance alleles to insecticides, which were much higher in Q1 than in ASL. Q1 and ASL are sufficiently reproductively isolated for the introgression of neutral alleles to be prevented, suggesting that they are actually different species. This indicates that nuclear genetic differentiation must be investigated within groups with less than 3.5% mtCOI divergence in order to elucidate the taxonomy of B. tabaci at a finer level. Overall, these data provide important information for pest management.

Discovery and development of a new semiochemical biopesticide for cotton pest management: assessment of extract effects on the cotton pest Helicoverpa spp.

AbstractThe Australian cotton production system is dominated by transgenic Bt (Bollgard II®) cotton varieties, Gossypium hirsutum L. (Malvaceae). These provide good control of lepidopteran pests (Helicoverpa spp.; Noctuidae) but are not effective against sucking pests. Recent trends in the frequency of resistance alleles in Helicoverpa spp. indicate the threat of pest resistance. Therefore, new technologies are required to manage the development of resistance and the resurgence of sucking pests. Consequently, the study was aimed to develop a semiochemical product able to deter feeding and oviposition of Helicoverpa spp., as well as cause direct mortality to the pest on transgenic and non‐transgenic crops. Plants including sorghum, sweet corn, pigeon pea, and lucerne (used as refuge crops in cotton), various cotton genotypes (MHR11, Lumein, and Sicala VII), and the native plant Clitoria ternatea L. (Fabaceae) were cultivated within cotton crops and in cages, and assessed for the occurrence of oviposition by Helicoverpa spp. and survival of the pest larvae. The results indicated that pigeon pea, cotton, sorghum, and sweet corn were most preferred for Helicoverpa spp. oviposition and feeding, whereas C. ternatea was least preferred. We used a solid phase extraction technique to obtain six fractions of an extract of C. ternatea, which we assessed for bioactivity against Helicoverpa spp. We found that fractions 2, 3, 4, and 6 had oviposition and feeding deterrence as well as direct toxicity to Helicoverpa spp. Clitoria ternatea fractions 2, 3, 4, and 6 have been developed into a product (Sero‐X®) that is effective against Helicoverpa spp. and sucking pests, and is in an advanced stage of commercialisation.

Pollen‐mediated transfer of herbicide resistance in Echinochloa crus‐galli

Pollen-mediated gene flow (PMGF) can facilitate the dispersal and spread of herbicide resistance from one weed population to another within an agricultural landscape. The aim of this study was to quantify the extent of PMGF in Echinochloa crus-galli (barnyardgrass), an important herbicide-resistant weed species in the United States and across the world. Gene flow declined exponentially with distance, and the double exponential decay model predicted an average gene flow of 5.6% when the pollen donor and recipient plants were at a close distance of 0.25 m from each other (12.5% at 0 m). Gene flow declined by 90% at 0.9 m from the pollen source, yet gene flow was detected as far as 50 m (the farthest distance studied). The farthest gene flow occurred in directions of the fastest wind events, but mean gene flow levels were similar among the directions. Results indicate that long-distance, landscape-scale PMGF is unlikely in barnyardgrass, but gene flow is likely to occur between adjacent fields at levels greater than initial frequencies of resistance alleles in natural, unselected populations. Thus, any resistance management strategy should consider the likelihood that PMGF can contribute to the spread of herbicide resistance between production fields.

Quantification of the Pirimicarb Resistance Allele Frequency in Pooled Cotton Aphid (Aphis gossypii Glover) Samples by TaqMan SNP Genotyping Assay

BackgroundPesticide resistance monitoring is a crucial part to achieving sustainable integrated pest management (IPM) in agricultural production systems. Monitoring of resistance in arthropod populations is initially performed by bioassay, a method that detects a phenotypic response to pesticides. Molecular diagnostic assays, offering speed and cost improvements, can be developed when the causative mutation for resistance has been identified. However, improvements to throughput are limited as genotyping methods cannot be accurately applied to pooled DNA. Quantifying an allele frequency from pooled DNA would allow faster and cheaper monitoring of pesticide resistance.Methodology/Principal FindingsWe demonstrate a new method to quantify a resistance allele frequency (RAF) from pooled insects via TaqMan assay by using raw fluorescence data to calculate the transformed fluorescence ratio k’ at the inflexion point based on a four parameter sigmoid curve. Our results show that k’ is reproducible and highly correlated with RAF (r >0.99). We also demonstrate that k’ has a non-linear relationship with RAF and that five standard points are sufficient to build a prediction model. Additionally, we identified a non-linear relationship between runs for k’, allowing the combination of samples across multiple runs in a single analysis.Conclusions/SignificanceThe transformed fluorescence ratio (k') method can be used to monitor pesticide resistance in IPM and to accurately quantify allele frequency from pooled samples. We have determined that five standards (0.0, 0.2, 0.5, 0.8, and 1.0) are sufficient for accurate prediction and are statistically-equivalent to the 13 standard points used experimentally

Knockdown resistance allele frequencies in North American head louse (Anoplura: Pediculidae) populations.

The study examines the extent and frequency of a knockdown-type resistance allele (kdr type) in North American populations of human head lice. Lice were collected from 32 locations in Canada and the United States. DNA was extracted from individual lice and used to determine their zygosity using the serial invasive signal amplification technique to detect the kdr-type T917I (TI) mutation, which is most responsible for nerve insensitivity that results in the kdr phenotype and permethrin resistance. Previously sampled sites were resampled to determine if the frequency of the TI mutation was changing. The TI frequency was also reevaluated using a quantitative sequencing method on pooled DNA samples from selected sites to validate this population genotyping method. Genotyping substantiated that TI occurs at high levels in North American lice (88.4%). Overall, the TI frequency in U.S. lice was 84.4% from 1999 to 2009, increased to 99.6% from 2007 to 2009, and was 97.1% in Canadian lice in 2008. Genotyping results using the serial invasive signal amplification reaction (99.54%) and quantitative sequencing (99.45%) techniques were highly correlated. Thus, the frequencies of TI in North American head louse populations were found to be uniformly high, which may be due to the high selection pressure from the intensive and widespread use of the pyrethrins- or pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins- or permethrin-based products in Canada and the United States. Alternative approaches to treatment of head lice infestations are critically needed.

Fitness costs ofCry1Fresistance in fall armyworm,Spodoptera frugiperda

AbstractTransgenic corn,Zea maysL., expressing theBacillus thuringiensisBerliner (Bt) protein Cry1F has been registered forSpodoptera frugiperda(J. E. Smith) control since 2003 in theUSA. Unexpected damage to Cry1F corn was reported in 2006 in Puerto Rico, and Cry1F resistance inS. frugiperdafrom Puerto Rico was documented. The study of fitness costs associated with insect resistance toBtinsecticidal proteins is important for understanding resistance evolution and for evaluating resistance management practices used to mitigate resistance to transgenic corn. Currently, no studies have addressed the fitness costs associated with Cry1F resistance inS. frugiperda. In this study, susceptible and resistant strains with similar genetic background and their reciprocal crosses were used to estimate Cry1F resistance fitness costs. Comparisons between life‐history traits and population growth rates of homozygous susceptible, heterozygous and homozygous resistantS. frugiperdawere used to determine whether the resistance is associated with fitness costs. Major fitness costs were not apparent in either heterozygotes or homozygous resistant insects. However, there was a slight indication of hybrid vigour in the heterozygotes. Additionally, two lines in which the frequency of the resistant alleles was fixed at 0.5 were followed for seven generations, after which the frequency of resistant alleles slightly decreased in both lines. The lack of strong fitness costs associated with Cry1F resistance inS. frugiperdaindicates that initial allele frequencies may be higher than expected in field populations and will tend to remain stable in field populations in the absence of selection pressure (e.g. Puerto Rico).

Determination of acaricide resistance allele frequencies in field populations of Tetranychus urticae using quantitative sequencing

Resistances to monocrotophos, fenpropathrin and abamectin in Tetranychus urticae are primarily conferred by reduced sensitivities of respective target sites [i.e., acetylcholinesterase (TuAChE), voltage-sensitive sodium channel (TuVSSC) and glutamate-gated chloride channel (TuGluCl)], which are due to point mutations (G228S and F439W in TuAChE; L1022V in TuVSSC; G323D in TuGluCl). As a population-based genotyping technique, a quantitative sequencing (QS) protocol was developed for the determination of the resistance-associated mutation frequencies in T. urticae. Standard prediction equations revealed high correlation coefficients (r2=0.993–0.999), demonstrating that the resistant nucleotide signal ratio is highly proportional to the resistance allele frequencies. The lower and higher detection limits for the four resistance mutations were 3.7–13.1% (7.8±3.3%) and 89.4–97.3% (93.3±3.2%), respectively, suggesting that QS can be employed as a preliminary monitoring tool for the detection of resistance allele frequencies, which ranged approximately 7.8–93.3% at the 95% confidence level. The QS was successfully employed for the determination of resistance allele frequencies in 26 T. urticae populations. The two TuAChE mutations responsible for monocrotophos resistance were almost saturated in most field populations. The TuVSSC L1022V mutation tentatively associated with fenpropathrin resistance was also found in 9 field populations. However, the TuGluCl G323D mutation conferring abamectin resistance was found only in one field population, suggesting that abamectin resistance is not yet widespread. The QS protocol, as an alternative to traditional bioassays, will greatly facilitate resistance monitoring of T. urticae.

Estimating the frequency of Cry1F resistance in field populations of the European corn borer (Lepidoptera: Crambidae)

Transgenic corn hybrids that express toxins from Bacillus thuringiensis (Bt) have suppressed European corn borer populations and reduced the pest status of this insect throughout much of the US corn belt. A major assumption of the high-dose/refuge strategy proposed for insect resistance management and Bt corn is that the frequency of resistance alleles is low so that resistant pests surviving exposure to Bt corn will be rare. The frequency of resistance to the Cry1F Bt toxin was estimated using two different screening tools and compared with annual susceptibility monitoring based on diagnostic bioassays and LC50 and EC50 determinations. An F1 screening approach where field-collected individuals were mated to a resistant laboratory strain and progeny were assayed to determine genotype revealed that resistance alleles could be recovered even during the first year of commercially available Cry1F corn (2003). Estimates of frequency from 2003-2005 and 2006-2008 indicated that, although allele frequency was higher than theoretical assumptions (0.0286 and 0.0253 respectively), there was no indication that the frequency was increasing. Similar estimates in 2008 and 2009 using an F2 screening approach confirmed the presence of non-rare resistance alleles (frequency ≈ 0.0093 and 0.0142 for 2008 and 2009, respectively). The results of both screening methods were in general agreement with the observed mortality in diagnostic bioassays and LC50 and EC50 determinations. These results are consistent with previous modeling results, suggesting that the high-dose/refuge strategy that is in place for Bt corn may be effective in delaying resistance evolution even when a relatively high frequency of resistance alleles exists.

Insecticide resistance in house flies from the United States: Resistance levels and frequency of pyrethroid resistance alleles

Although insecticide resistance is a widespread problem for most insect pests, frequently the assessment of resistance occurs over a limited geographic range. Herein, we report the first widespread survey of insecticide resistance in the USA ever undertaken for the house fly, Musca domestica, a major pest in animal production facilities. The levels of resistance to six different insecticides were determined (using discriminating concentration bioassays) in 10 collections of house flies from dairies in nine different states. In addition, the frequencies of Vssc and CYP6D1 alleles that confer resistance to pyrethroid insecticides were determined for each fly population. Levels of resistance to the six insecticides varied among states and insecticides. Resistance to permethrin was highest overall and most consistent across the states. Resistance to methomyl was relatively consistent, with 65–91% survival in nine of the ten collections. In contrast, resistance to cyfluthrin and pyrethrins + piperonyl butoxide varied considerably (2.9–76% survival). Resistance to imidacloprid was overall modest and showed no signs of increasing relative to collections made in 2004, despite increasing use of this insecticide. The frequency of Vssc alleles that confer pyrethroid resistance was variable between locations. The highest frequencies of kdr, kdr-his and super-kdr were found in Minnesota, North Carolina and Kansas, respectively. In contrast, the New Mexico population had the highest frequency (0.67) of the susceptible allele. The implications of these results to resistance management and to the understanding of the evolution of insecticide resistance are discussed.

Modeling the Evolution of Glyphosate Resistance in Barnyardgrass (Echinochloa crus-galli) in Cotton-Based Production Systems of the Midsouthern United States

Glyphosate-resistant (GR) weeds have been a prime challenge to the sustainability of GR cotton-based production systems of the midsouthern United States. Barnyardgrass is known to be a high-risk species for evolving herbicide resistance, and a simulation model was developed for understanding the likelihood of glyphosate resistance evolution in this species in cotton-based systems. Under a worst-case scenario of five glyphosate applications in monoculture GR cotton, the model predicts resistance evolution in about 9 yr of continuous glyphosate use, with about 47% risk by year 15. A unique insight from this model is that management in response to GR Palmer amaranth in this system (a reactive response) provided a proactive means to greatly reduce the risks of glyphosate resistance evolution in barnyardgrass. Subsequent model analysis revealed that the risk of resistance is high in fields characterized by high barnyardgrass seedbank levels, seedling emergence, and seed production per square meter, whereas the risk is low in fields with high levels of postdispersal seed loss and annual seedbank loss. The initial frequency of resistance alleles was a high determinant of resistance evolution (e.g., 47% risk at year 15 at an initial frequency of 5e−8vs. 4% risk at 5e−10). Monte Carlo simulations were performed to understand the influence of various glyphosate use patterns and production practices in reducing the rate and risk of glyphosate resistance evolution in barnyardgrass. Early planting and interrow cultivation are useful tools. Crop rotation is effective, but the diversity of weed management options practiced in the rotational crop is more important. Diversifying weed management options is the key, yet application timing and the choice of management option is critical. Model analyses illustrate the relative effectiveness of a number of diversified glyphosate use strategies in preventing resistance evolution and preserving the long-term utility of glyphosate in midsouthern U.S. cotton-based production systems.

Inheritance of Cry1F resistance, cross-resistance and frequency of resistant alleles inSpodoptera frugiperda(Lepidoptera: Noctuidae)

Transgenic maize, Zea maize L., expressing the Cry1F protein from Bacillus thuringiensis has been registered for Spodoptera frugiperda (J. E. Smith) control since 2003. Unexpected damage to Cry1F maize was reported in 2006 in Puerto Rico and Cry1F resistance in S. frugiperda was documented. The inheritance of Cry1F resistance was characterized in a S. frugiperda resistant strain originating from Puerto Rico, which displayed >289-fold resistance to purified Cry1F. Concentration-response bioassays of reciprocal crosses of resistant and susceptible parental populations indicated that resistance is recessive and autosomal. Bioassays of the backcross of the F1 generation crossed with the resistant parental strain suggest that a single locus is responsible for resistance. In addition, cross-resistance to Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba, Cry2Aa and Vip3Aa was assessed in the Cry1F-resistant strain. There was no significant cross-resistance to Cry1Aa, Cry1Ba and Cry2Aa, although only limited effects were observed in the susceptible strain. Vip3Aa was highly effective against susceptible and resistant insects indicating no cross-resistance with Cry1F. In contrast, low levels of cross-resistance were observed for both Cry1Ab and Cry1Ac. Because the resistance is recessive and conferred by a single locus, an F1 screening assay was used to measure the frequency of Cry1F-resistant alleles from populations of Florida and Texas in 2010 and 2011. A total frequency of resistant alleles of 0.13 and 0.02 was found for Florida and Texas populations, respectively, indicating resistant alleles could be found in US populations, although there have been no reports of reduced efficacy of Cry1F-expressing plants.

Assessment of benzimidazole resistance in Haemonchus contortus in sheep flocks in Ontario, Canada: Comparison of detection methods for drug resistance

In 2011, a field study was conducted to assess drug resistance of gastro-intestinal nematodes in sheep flocks in Ontario, Canada. Benzimidazole resistance in Haemonchus contortus was assessed by genetic analysis of eggs; measurement of resistant allele percentages at codons 167, 198 and 200 in the β-tubulin gene was determined on pools of H. contortus eggs using pyrosequencing. Susceptibility to benzimidazoles in gastro-intestinal nematodes was also determined using a Faecal Egg Count Reduction Test (FECRT) and a Larval Development Assay (LDA). In total, 16 farms were assessed with the genetic test. Based on resistant allele frequencies, all of the farms (16/16) tested had benzimidazole resistance in H. contortus; the overall percentage of benzimidazole-resistant H. contortus (estimated prior to treatment using the Hardy–Weinberg formula) was 68.5%. The FECRT and LDA were performed on 11 and 13 farms, respectively. Resistance to fenbendazole was detected on 100% (11/11) of the farms where the FECRT was performed. The LDA revealed the presence of thiabendazole resistance in H. contortus in 92% (12/13) of the farms. Estimated percentages of resistant parasites in H. contortus populations obtained with the two biological tests and the genetic test were compared. The results of the genetic test were in agreement with the biological tests and confirmed that benzimidazole resistance in H. contortus is present in Ontario sheep flocks. Differences between the different methods of drug resistance detection are discussed in terms of cost, time and sampling.

Insect resistance to Bt crops: lessons from the first billion acres

Evolution of resistance in pests can reduce the effectiveness of insecticidal proteins from Bacillus thuringiensis (Bt) produced by transgenic crops. We analyzed results of 77 studies from five continents reporting field monitoring data for resistance to Bt crops, empirical evaluation of factors affecting resistance or both. Although most pest populations remained susceptible, reduced efficacy of Bt crops caused by field-evolved resistance has been reported now for some populations of 5 of 13 major pest species examined, compared with resistant populations of only one pest species in 2005. Field outcomes support theoretical predictions that factors delaying resistance include recessive inheritance of resistance, low initial frequency of resistance alleles, abundant refuges of non-Bt host plants and two-toxin Bt crops deployed separately from one-toxin Bt crops. The results imply that proactive evaluation of the inheritance and initial frequency of resistance are useful for predicting the risk of resistance and improving strategies to sustain the effectiveness of Bt crops.

Determining changes in the distribution and abundance of a Rhyzopertha dominica phosphine resistance allele in farm grain storages using a DNA marker

The lesser grain borer, Rhyzopertha dominica (F.), is a highly destructive pest of stored grain that is strongly resistant to the fumigant phosphine (PH3 ). Phosphine resistance is due to genetic variants at the rph2 locus that alter the function of the dihydrolipoamide dehydrogenase (DLD) gene. This discovery now enables direct detection of resistance variants at the rph2 locus in field populations. A genotype assay was developed for direct detection of changes in distribution and frequency of a phosphine resistance allele in field populations of R. dominica. Beetles were collected from ten farms in south-east Queensland in 2006 and resampled in 2011. Resistance allele frequency increased in the period from 2006 to 2011 on organic farms with no history of phosphine use, implying that migration of phosphine-resistant R. dominica had occurred from nearby storages. Increasing resistance allele frequencies on organic farms suggest local movement of beetles and dispersal of insects from areas where phosphine has been used. This research also highlighted for the first time the utility of a genetic DNA marker in accurate and rapid determination of the distribution of phosphine-resistant insects in the grain value chain. Extending this research over larger landscapes would help in identifying resistance problems and enable timely pest management decisions.