Cyromazine resistance in the house fly (Diptera: Muscidae): genetics and cross-resistance to diflubenzuron

Larvae of a house fly, Musca domestica L., strain collected in a chicken house near Pittsburg, Tex, after a control failure with the poultry feedthrough insecticide cyromazine showed 6.5-fold resistance to cyromazine and 10-fold resistance to diflubenzuron. Adults of the strain showed high levels of resistance to carbaryl, DDT, and diazinon; moderate resistance to cypermethrin and permethrin; and low resistance to dieldrin. In contrast, no resistance to cyromazine was observed in eight laboratory house fly strains with resistance to four groups of conventional insecticides. When the genetics of cyromazine resistance was investigated in crosses to susceptible strains with visible mutant markers, results indicated cyromazine resistance was incompletely dominant over susceptibility and the resistance gene was on chromosome V. The same or a closely linked gene conferred resistance to diflubenzuron. A strain containing only chromosome V from the original resistant strain was resistant to cyromazine and diflubenzuron, but not to other insecticides except for low level resistance to DDT and carbaryl. Resistance to the latter insecticides appeared to be due to a linked, but distinct, gene. Therefore, resistance to cyromazine and probably diflubenzuron appears to be genetically distinct from other types of insecticide resistance.     

Larvicide resistance in Musca domestica (Diptera: Muscidae) populations in Denmark and establishment of resistant laboratory strains

We determined the toxicity of the two IGRs, diflubenzuron and cyromazine, in this survey of resistance in Danish field populations of Musca domestica (L.). We observed resistance toward diflubenzuron and for the first time in Denmark and we found field populations with some resistance to cyromazine. Eleven of the twenty-one field populations had larvae surviving a diagnostic dose of 1.6 times of susceptible LC95 of diflubenzuron and two of these populations had larvae surviving 6.1 times of LC95. Eight of the twenty-one field populations had larvae surviving 2.2 times of susceptible LC95 of cyromazine and one population had larvae surviving 4.4 times of LC95. A fivefold cyromazine resistant strain was established after selection with cyromazine. It was 3-, 5-, and 90-fold resistant to diflubenzuron, triflumuron, and methoprene, respectively. Two diflubenzuron resistant strains (120- and 86-fold, respectively) were established. They showed a high level of resistance to triflumuron (1000- and 200-fold, respectively), and moderate resistance to methoprene (73- and 50-fold, respectively). Both were susceptible to cyromazine. This study shows that by applying the recommendations of previous resistance risk assessments, severe control failures and detrimental development of a high level of resistance have been avoided. The development of resistance has not been completely avoided, but has not developed to a level of biological or economic importance.     

The genetic bases of high-level resistance to diflubenzuron and low-level resistance to cyromazine in a field strain of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

Abstract  Several genes on chromosomes IV and VI have a significant influence on high-level resistance to diflubenzuron in a strain of the Australian sheep blowfly from Tara, Queensland. Low-level resistance to cyromazine in the same strain is due to genes on these chromosomes with a gene (gene complex) in the sv marker region of chromosome IV being particularly important. For both insecticides, genetic background influences resistance status. If the results of the Tara strain prove typical for those of other populations, resistance to diflubenzuron in the Australian sheep blowfly has potentially significant consequences for woolgrowers.     

Genetics and fitness costs of cyromazine resistance in the house fly (Diptera: Muscidae)

The genetic basis of cyromazine resistance was investigated in the house fly, Musca domestica L. The ED-R strain, which was collected in Mississippi and selected further in the laboratory, was 116.5-fold resistant compared with the laboratory susceptible strain, OR-S. The SEL strain, which was created by crossing ED-R with OR-S followed by three cycles of reselection and backcrossing to OR-S, was 84.7-fold resistant relative to the susceptible strain. Mortality data from reciprocal crosses of resistant and susceptible flies indicated that resistance was autosomal and not influenced by maternal effects. The relative position of probit lines from the parental strains and reciprocal crosses showed that resistance was expressed as an incompletely dominant trait with D = 0.30 and 0.32 for ED-R and SEL, respectively. To determine the number of genes involved, models of one, two, three, four, and five loci were used to compare observed and expected mortality of F1ED-R x susceptible backcross. Resistance was best described by a polygenic model of three loci when equal and additive effects of loci were assumed. Another approach, which was based on phenotypic variances, showed that nE, or the minimum number of freely segregating genetic factors for ED-R, equaled 3.07. ED-R showed greater reductions in fitness compared with SEL independent of the presence or absence of sublethal concentrations of cyromazine. These data suggested that reduced fitness was not due to deleterious pleiotropic effects of the resistance genes themselves but arose from other loci in the ED-R genotype.     

Putative polymerase chain reaction markers for insecticide resistance in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine and abamectin

In this study, random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to identify polymorphic genomic DNA that would discriminate among cyromazine-resistant, abamectin-resistant, and susceptible Liriomyza trifolii (Burgess) (Diptera: Agromyzidae) leafminers. Using a reference strain that was susceptible to both cyromazine and abamectin, and a cyromazine-resistant strain and an abamectin-resistant strain, 400 oligonucleotides were assayed using RAPD-PCR. We found that two oligonucleotides, B10 and G16, amplified unique bands in the cyromazine-resistant strain but not in the reference or abamectin-resistant strains. Three oligonucleotides, K04, J13, and I02, showed polymorphisms unique to the abamectin-resistant strain but not in the reference or cyromazine-resistant strain. Leaf dip bioassays and RAPD-PCR were performed on two additional reference strains, seven strains from commercial ornamental production greenhouses, and one field strain. The two reference strains were negative for the resistance-correlated oligonucleotides. Of the seven strains from ornamental greenhouses, leaf dip bioassays showed that five had some level of resistance to both abamectin and cyromazine, whereas two were susceptible. The field strain was susceptible to both cyromazine and abamectin. In RAPD-DNA analyses, the five strains with abamectin resistance were positive for the three abamectin resistance-correlated oligonucleotides K04, J13, and I02. In the cases of cyromazine resistance, the five strains with cyromazine resistance were positive for the two cyromazine resistance-correlated oligonucleotides B10 and G16. The field strain and two greenhouse strains that were susceptible in leaf dip bioassays were negative for all three abamectin resistance-correlated oligonucleotides. The field strain and one greenhouse strain were negative for the two cyromazine resistance-correlated oligonucleotides; however, one greenhouse strain that was susceptible to cyromazine in leaf dip bioassay tested positive for one of the cyromazine resistance-correlated oligonucleotides. This method can be used to quickly identify cyromazine resistance, abamectin resistance, or both in leafminers, enabling a grower to choose an effective insecticide for leafminer control in a timely manner.     

Predicting insecticide resistance: mutagenesis, selection and response

Strategies to manage resistance to a particular insecticide have usually been devised after resistance has evolved. If it were possible to predict likely resistance mechanisms to novel insecticides before they evolved in the field, it might be feasible to have programmes that manage susceptibility. With this approach in mind, single-gene variants of the Australian sheep blowfly, Lucilia cuprina, resistant to dieldrin, diazinon and malathion, were selected in the laboratory after mutagenesis of susceptible strains. The genetic and molecular bases of resistance in these variants were identical to those that had previously evolved in natural populations. Given this predictive capacity for known resistances, the approach was extended to anticipate possible mechanisms of resistance to cyromazine, an insecticide to which L. cuprina populations remain susceptible after almost 20 years of exposure. Analysis of the laboratory-generated resistant variants provides an explanation for this observation. The variants show low levels of resistance and a selective advantage over susceptibles for only a limited concentration range. These results are discussed in the context of the choice of insecticides for control purposes and of delivery strategies to minimize the evolution of resistance.     

Development and stability of insecticide resistance in the leafminer Liriomyza trifolii (Diptera: Agromyzidae) to cyromazine, abamectin, and spinosad

Three populations of the leafminer, Liriomyza trifolii (Burgess), were collected from commercial ornamental production greenhouses in the United States and tested for susceptibility to three commercial insecticides. A leaf dip bioassay of leaves containing young (1-2-d-old) larvae was used. Based on larval mortality and compared with a susceptible laboratory reference colony, the three strains varied in spectrum and level of resistance to the insecticides. CA-1, collected from Gerbera daisy, was moderately resistant to cyromazine (18.1-fold) and abamectin (22.0-fold), but highly resistant to spinosad (> 188-fold). CA-2, collected from chrysanthemums, was not resistant to abamectin, had a low level of resistance to cyromazine (8.2-fold), but was extremely resistant to spinosad (1,192-fold). GA-1, collected from chrysanthemums, had very low levels of resistance to cyromazine (5.4-fold) and spinosad (1.9-fold) but was moderately resistant to abamectin (30.6-fold). When reared in the absence of insecticide selection pressure, all three strains reverted to approximately the level of the reference strain. The CA-1 strain reverted in nine generations to cyromazine; however, the lowest levels of abamectin and spinosad resistance reverted to was 3.1-fold at F8 and 3.2 at the F10, respectively. The CA-2 strain reverted in five generations to both cyromazine and spinosad. GA-1 reverted in five generations to abamectin. Based on the results, resistance to these three insecticides was unstable. Additionally, there was no cross-resistance among these three insecticides.     

Abamectin resistance in strains of vegetable leafminer,Liriomyza sativae (Diptera: Agromyzidae) is linked to elevated glutathione S‐transferase activity

Abamectin resistance was selected in the vegetable leafminer, Liriomyza sativae (Blanchard) (Diptera: Agromyzidae) under laboratory conditions, and cross‐resistance patterns and possible resistance mechanisms in the abamectin‐resistant strains (AL‐R, AF‐R) were investigated. Compared with the susceptible strain (SS), strain AL‐R displayed 39‐fold resistance to abamectin after 20 selection cycles during 25 generations, and strain AF‐R exhibited 59‐fold resistance to abamectin after 16 selection cycles during 22 generations. No cross‐resistance to cyromazine was found in both abamectin‐resistant strains. However, we failed to select for cyromazine resistance in L. sativae under laboratory conditions by conducting 17 selection cycles during 22 generations. However, moderate levels of cross‐resistance to abamectin (6–9 fold) were observed in strains which received cyromazine treatments. Biochemical analysis showed that glutathione S‐transferase (GST) activity in both abamectin‐resistant strains (AL‐R, AF‐R) was significantly higher than in the susceptible strain (SS), suggesting metabolically driven resistance to abamectinin L. sativae. Recommendations of mixtures or rotation of cyromazine and abamectin should be considered carefully, as consecutive cyromazine treatments may select for low‐level cross‐resistance to abamectin.     

Selection of dieldrin-resistant strains of Lucilia cuprina (Diptera: Calliphoridae) after ethyl methanesulfonate mutagenesis of a susceptible strain.

After ethyl methanesulfonate (EMS) mutagenesis of a susceptible strain (SWT), selective screening of Lucilia cuprina (Wiedemann) resulted in four strains that were resistant to the insecticide dieldrin. Concentrations used for selection were greater than LC99 of susceptible phenotypes. No resistant variants were screened from the standard laboratory strain (SWT) not treated with EMS. The resistance phenotypes of the four resistant strains were similar to each other and to that of a field-selected resistant strain. The genetic basis of resistance is monogenic in all strains and the data are consistent with the same locus, Rdl, determining resistance status in each strain. The Rdl locus maps to chromosome V, approximately 3.5 map units distal to the Sut locus. Dieldrin resistance may be caused by less effective blocking of insect neuronal GABA receptors by the chemical in resistant strains. The data indicate that the evolution of resistance to an insecticide in the field may be constrained by a limited number of genetical and biochemical options if a monogenic response is selected for and that the spontaneous mutation rate to the Rdl allele is less than 1 in 10(6) in the laboratory.     

Field evaluation of resistance to pyriproxyfen in Bemisia tabaci (B biotype)

We determined effects of aerial sprays of the insect growth regulator pyriproxyfen on sweetpotato whitefly, Bemisia tabaci (Gennadius) (B biotype), in Arizona cotton (Gossypium spp.) fields. We measured survival for males and females from a susceptible strain and a laboratory-selected resistant strain, as well as for hybrid female progeny from crosses between the strains. Insects were exposed directly to pyriproxyfen sprays in the field or indirectly in the laboratory by rearing them on sprayed leaves collected from the field. In all tests, survival was higher for the resistant strain than the susceptible strain, but did not differ between sexes in each strain. Survival to the adult stage did not differ between eggs and nymphs directly exposed to sprays. For susceptible and hybrid individuals, survival was lower on leaves collected the day of spraying than on leaves collected 2 wk after spraying. In contrast, survival of resistant individuals did not differ based on the timing of exposure. Dominance of resistance to pyriproxyfen depended on the type of exposure. Resistance was partially or completely dominant in direct exposure bioassays and on leaves collected 2 wk after spraying (h > 0.6). Resistance was partially recessive on leaves collected the day of spraying (mean h = 0.34). Rapid evolution of resistance to pyriproxyfen could occur if individuals in field populations with traits similar to those of the laboratory-selected strain examined here were treated intensively with this insecticide.     

Inheritance of resistance to pyriproxyfen in Bemisia tabaci (Hemiptera: Aleyrodidae) males and females (B biotype)

We evaluated effects of the insect growth regulator pyriproxyfen on Bemisia tabaci (Gennadius) (B biotype) (Hemiptera: Aleyrodidae) males and females in laboratory bioassays. Insects were treated with pyriproxyfen as either eggs or nymphs. In all tests, the LC50 for a laboratory-selected resistant strain was at least 620 times greater than for an unselected susceptible strain. When insects were treated as eggs, survival did not differ between males and females of either strain. When insects were treated as nymphs, survival did not differ between susceptible males and susceptible females, but resistant males had higher mortality than resistant females. The dominance of resistance decreased as pyriproxyfen concentration increased. Resistance was partially or completely dominant at the lowest concentration tested and completely recessive at the highest concentration tested. Hybrid female progeny from reciprocal crosses between the susceptible and resistant strains responded alike in bioassays; thus, maternal effects were not evident. Rapid evolution of resistance to pyriproxyfen could occur if individuals in field populations had resistance with traits similar to those of the laboratory-selected strain examined here.     

Pleiotropy of insecticide resistance in the codling moth, Cydia pomonella

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), has developed resistance to various insecticides. Relative fitness of one susceptible strain (Sv) and two strains selected for resistance to diflubenzuron (Rt) and deltamethrin (Rv), respectively, was measured in the absence of insecticide selection pressure. Mating rate, fecundity, fertility, developmental time, fifth instar weight, and adult longevity were compared. Both resistant strains were less fecund and fertile, developed more slowly, weighed less, and had shorter life-spans than the susceptible strain. These results indicate that biological constraints are associated with insecticide resistance in the codling moth. We also found that fitness estimates of the Rv strain did not differ statistically from those of the Rt strain. Enhanced mixed-function oxidase and glutathione-S-transferase activities have been shown to be involved in insecticide resistance in both Rt and Rv strains. This suggests that the fitness cost described in both resistant strains was mainly associated to metabolic resistance. The impact of such deleterious pleiotropy of insecticide resistance in C. pomonella in terms of resistance management in the field is discussed.     

Insecticidal resistance and cross-resistance in populations of Cydia pomonella (Lepidoptera: Tortricidae) in central Europe

Insecticide bioassays were used to investigate resistance of Cydia pomonella (L.) (Lepidoptera: Tortricidae) to insecticides with various types of active ingredients. The efficacy baselines of selected insect growth regulators (fenoxycarb), insect growth inhibitors (diflubenzuron and teflubenzuron), organophoshorous insecticides (phosalone), and neonicotinoids (thiacloprid) against the eggs and first and fifth instars of sensitive laboratory strains of codling moth were determined. According to concentration-mortality baseline, 50% lethality concentration values and 90% lethality concentration values were determined for all the tested insecticides. The lethal concentration ratio quantified the relation between the efficacy of selected insecticides against fifth instars found by topical application and against first instars found by diet-treated bioassay. No difference was detected when the efficacy of technical grade diflubenzuron diluted in tetrahydrofuran and diflubenzuron in the formulated product Dimilin 48 SC diluted in water was compared. However, just before the application of insecticide, the integument of larvae must be treated with acetone. Two bioassays were used to monitor the resistance of codling moths collected in 2003-2005 in two apple (Malus spp.) orchards with different intensities of chemical control. Resistance ratios (RRs) to the tested insecticides were determined for both field populations of codling moth. For the population of codling moth from a commercial apple orchard in Velké Bílovice, cross-resistance to fenoxycarb, teflubenzuron, and phosalone was detected after the topical application of insecticides to fifth instars. The population of codling moth from Prague-Ruzyne was slightly resistant to phosalone and teflubenzuron. No resistance to diflubenzuron was detected in either tested population.     

Volatiles from Merino fleece evoke antennal and behavioural responses in the Australian sheep blow fly Lucilia cuprina

To identify flystrike‐related volatile compounds in wool from Merino sheep, the attractiveness of wool to Lucilia cuprina Wiedmann (Diptera: Calliphoridae) was examined. First, a selection of wool samples guided by previous knowledge of sheep lines, predicted to be more susceptible or more resistant to flystrike, was tested. The attractiveness of the 10 samples selected was not associated with field susceptibility: two samples from the more resistant line were identified as most attractive and two samples from the more susceptible line were identified as least attractive, based on the behavioural assays with gravid flies. Comparison of the headspace volatiles of these samples, using solid phase microextraction and gas chromatography‐mass spectrometry‐electroantennographic detection, revealed octanal and nonanal to be present in the attractive wool samples that elicited responses from the fly antenna. Furthermore, the two compounds were not present in wool that was least attractive to L. cuprina. In laboratory bioassays, octanal and nonanal evoked antennal and behavioural responses in gravid L. cuprina, thus confirming their potential role as semiochemicals responsible for attracting L. cuprina to Merino sheep.     

High stability and no fitness costs of the resistance of codling moth to Cydia pomonella granulovirus (CpGV-M)

Resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV-M) was previously observed in field populations of codling moth (CM, C. pomonella) in South-West Germany. Incidental observations in a laboratory reared field colony (CpR) indicated that this resistance is rather stable, even in genetically heterogeneous CM colonies consisting of both susceptible and resistant individuals. To test this hypothesis, the resistance level of CpR that was 1000times less susceptible to CpGV-M was followed for more than 60 generations of rearing. Even without virus selection pressure, the high level of resistance, expressed as median lethal concentration, remained stable for more than 30 generations and declined only by a factor of 10 after 60 generations. When cohorts of the F32 and F56 generations of the same colony were selected to CpGV-M for five and two generations, respectively, the resistance level increased to factor of >1,000,000 compared to a susceptible control colony. Laboratory reared colonies of CpR, did not exhibit any measurable fitness costs under laboratory conditions in terms of fecundity and fertility. Resistance testing of seven selected codling moth field populations collected between 2003 and 2008 in commercial orchards in Germany that were repeatedly sprayed with CpGV products gave evidence of different levels of resistance and a more than 20-fold increase of the resistance in 1-3years when selection by CpGV-M was continued. A maximum 1,000,000-fold level of resistance to CpGV-M that could be induced in the laboratory under virus pressure had been also observed in one field population. The high stability of resistance observed in the genetically heterogenous colony CpR indicates that resistance to CpGV-M is not very costly.     

Development of endosulfan tolerant strain of an egg parasitoid Trichogramma chilonis Ishii (Hymenoptera: Trichogrammatidae)

A strain of T. chilonis, an egg parasitoid of lepidopteran pests tolerant to the most commonly used cyclodiene insecticide--endosulfan was developed in the laboratory. Tolerance to endosulfan was induced by exposing adult parasitoids sequentially from a sub-lethal concentration (0.004%) to the field recommended concentration (0.09%). The strain acquired tolerance to the insecticide after 341 generation of continuous exposure with LC50 values of 1074.96 ppm as compared to LC50 of (70.91 ppm) in susceptible strain. The genetical study showed that F1 crosses exhibited a semi-dominant response to endosulfan with degree of dominance value (D) of 0.58. The resistant factor of tolerant strain was 15.1 folds and of F1 cross were 8.53 folds over susceptible strain. Under net house conditions, the tolerant strain parasitised 56% Helicoverpa armigera eggs on potted cotton plants immediately after an insecticide spray, compared to 3% by the susceptible strain. High percentage survival of the immature stages of the tolerant strain proved their ability to withstand the insecticide load. Breakdown of insecticide tolerance in the strain occurred after four generations in absence of insecticide load. Use of the tolerant strain as a component of bio-intensive IPM in various crops where insecticide use is higher is discussed.     

Resistance to a nuclear polyhedrosis virus in the light-brown apple moth Epiphyas postvittana (Lepidoptera: Tortricidae)

Measurements were made of the relative susceptibility to a nuclear polyhedrosis virus of three populations of light-brown apple moth Epiphyas postvittana: a resistant laboratory strain (CAN), a susceptible laboratory strain (BAR), and a field population. CAN was found to be 50 times more resistant than BAR and 160 times more resistant than the field line. Experiments on hybrid crosses of resistant and susceptible strains showed that resistance is genetically determined. This serves as a warning of the possible selection of virus-resistant strains of insect pests, where viral insecticides are being used in the field.     

Insecticide-insensitive acetylcholinesterase from a laboratory selected and a field strain of housefly (Musca domestica) (L.)

1. Acetylcholinesterase from the heads of a strain of houseflies selected for resistance to the carbamate insecticide methomyl, and from a methomyl-resistant field strain was found to be less sensitive to inhibition by methomyl than that from a susceptible strain. 2. The enzyme from resistant insects was also more tolerant to malaoxon, dichlorvos and bomyl but not to azamethiphos. 3. The decrease in sensitivity to inhibition appeared to be due to an increase in affinity for substrate.     

Biochemical genetics of altered acetylcholinesterase resistance to insecticides in the house fly

Resistance to the organophosphate insecticide tetrachlorvinphos was examined in a house fly (Musca domestica L.) strain with an altered acetylcholinesterase (AChE) of decreased sensitivity to inhibition by the insecticide. Genetic tests showed that both resistance and the altered AChE were controlled by semidominant gene(s) on chromosome II. The gene for resistance was five crossover units from the mutant marker stubby wing (stw). A house fly strain was prepared in which resistance was introduced in to a susceptible stw strain by recombination. Biochemical assays revealed that the altered AChE was introduced along with resistance. Assays of the AChE of resistant and susceptible stw strains by two independent methods showed that the enzyme from resistant flies was 30 times more slowly inhibited by tetrachlorvinphos than the enzyme from susceptible flies.This work was supported in part by NIH Grant ES 00901.Technical Article 13340, Texas Agricultural Experiment Station.     

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.