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.     

Salinity tolerance of stable fly (Diptera: Muscidae)

Effects of salinity on the survival, growth, and development of stable fly, Stomoxys calcitrans (L.), were investigated in the laboratory. Larvae failed to develop to pupation when reared in media containing a salinity of 40 parts per thousand (ppt) sodium chloride (NaCl). Maximum salinity supporting larval development equaled the salinity of seawater (34 ppt); the larval LC90 was 24.2 ppt. Deleterious effects of high salinity decreased as larvae matured. Six-day-old larvae reared at a salinity of 34 ppt weighed 79% less than controls, compared with a 36% difference in 9-d-old larvae; by pupation, the difference was only 24%. Salinity did not influence the duration of larval, pupal, or adult stages. Survival of pupae was unimpaired despite a slight increase in number of pupal deformities, and normal adults emerged. Eggs were highly tolerant to saline. They hatched at salinity concentrations lethal to larvae; greater than 50% hatch occurred even when eggs were maintained at 80 ppt NaCl. Sensitivity of larvae to salinities close to that of seawater might be important for control of stable flies inhabiting marine areas.     

Parasites that attack stable fly and house fly (Diptera: Muscidae) puparia during the winter on dairies in northwestern Florida

Throughout the winter and early spring months, stable fly, Stomoxys calcitrans (L.), and house fly, Musca domestica L., puparia were collected from silage, hay, and manure from six dairies in northwestern Florida and evaluated for parasitism. Of the puparia producing flies or parasites, 23% of the stable flies and 46% of the house flies were parasitized. The predominant parasite observed attacking muscoid flies (76% for stable flies and 58% for house flies) was Spalangia cameroni Perkins. Muscidifurax sp. was recovered from 11 and 36% of the stable fly and house fly pupae, respectively. Other parasite species encountered were Spalangia endius Walker and S. nigroaenea Curtis. Significantly more parasitized fly pupae were collected from silage than from hay residues or manure. Winter and early spring parasite populations in northwestern Florida appear to be present as long as viable fly pupae are available to support the developing parasites.     

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.     

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.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

Comparative efficiency of six stable fly (Diptera: Muscidae) traps

Five adhesive traps and the Nzi cloth-target trap were compared to determine their trapping efficiency and biases for stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae). Two configurations of the BiteFree prototype trap, constructed of polyethylene terephthalate, were most efficient for trapping stable flies, whereas the EZ trap was least efficient. The two Alsynite traps, Broce and Olson, were intermediate to the BiteFree prototype and EZ traps. All adhesive traps collected a ratio of approximately two males for each female. Approximately 50% of the flies collected on the adhesive traps, both male and female, were blood fed, and 20% were vitellogenic. The Nzi trap collected an older component of the stable fly population, 81% blood fed and 62% vitellogenic, but it was much less efficient than the adhesive traps. The effectiveness of the BiteFree prototype trap indicates that materials other than Alsynite are attractive to stable flies.     

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.     

Selection for resistance to imidacloprid in the house fly (Diptera: Muscidae)

The house fly, Musca domestica L. (Diptera: Muscidae), continues to be a primary pest of livestock facilities worldwide. This pest also has shown a propensity for pesticide resistance development when under high selection pressures. In this study the house fly strain FDm was created by a 20% contribution from each of five colonies collected from dairies in Florida with known imidacloprid resistance. The FDm strain was used to evaluate the level ofimidacloprid resistance after five selections near the LC70 value of each selected generation. Overall, the mean selection mortality was 72.7, with males being considerably more susceptible than females. The unselected (F0) FDm strain showed considerable susceptibility to imidacloprid after its creation, compared with the five parental strains. Between 9500 and 14,000 virgin house flies were used in each selection. After the fifth and final selection, a 331-fold increase in imidacloprid resistance at the LC70 was observed over the parental FDm strain. In parallel studies, the FDm strain showed increasing tolerance of the commercial imidacloprid product QuickBayt. These results suggest that livestock producers should use caution when choosing pesticides and consider rotating fly baits, as is encouraged with other pesticide treatment regimes on farms.     

Chronology of permethrin resistance in a southern Illinois population of the horn fly (Diptera: Muscidae) during and after selection by pyrethroid use

From 1985 through 1988, horn flies (Haematobia irritans (L)) collected at the Dixon Springs Agricultural Center (DSAC) in southern Illinois were tested in 22 h bioassays for permethrin resistance with residues on cotton cloths. The LC90 for a susceptible field population collected in June 1985 was 0.19 micrograms/cm2. In comparison, flies collected from pyrethroid-tagged cattle in 1985 and 1986 exhibited 25- to 116-fold resistance to permethrin. A 25-fold level of resistance allowed survival on treated cattle 8 wk after pyrethroid tag application. Flies representing the local background population were collected periodically from an untreated herd 2.4 km from the nearest cattle treated with a pyrethroid; these flies exhibited up to 18-fold resistance. Although pyrethroids were not used on DSAC animals after October 1986, all bioassays done in 1987 and 1988 indicated resistance levels of greater than or equal to 7-fold. The 95% confidence intervals for LC90s from all 1987 bioassays overlapped the confidence interval from the corresponding July 1986 estimate for resistant flies collected from pyrethroid-tagged cattle. Although some decline in resistance was evident in 1988, bioassays done at the end of the season produced resistance ratios of 7.4 and 15.3. Survivorship at a diagnostic dose indicated that resistance frequencies remained at 4-8% throughout 1988. Two years' abstinence from pyrethroid use was insufficient to allow an adequate decline in resistance levels.     

Azadirachtin as a larvicide against the horn fly, stable fly, and house fly (Diptera: Muscidae)

Effects of azadirachtin, a triterpenoid extracted from neem seed, Azadirachta indica A. Juss., were similar to those of insect growth regulators against the immature stages of the born fly, Haematobia irritans (L.), the stable fly, Stomoxys calcitrans (L.), and the house fly, Musca domestica L. When an ethanolic extract of ground seed was blended into cow manure, LC50 and LC90's for larval horn flies were 0.096 and 0.133 ppm azadirachtin, respectively. An emulsifiable concentrate (EC) had an LC50 for larval horn flies of 0.151 ppm and an LC90 of 0.268 ppm. For larval stable flies, the EC formulation had an LC50 of 7.7 ppm and an LC90 of 18.7 ppm azadirachtin in manure. Against larval house flies, the LC50 and LC90 were 10.5 and 20.2 ppm, respectively. When the EC formulation was administered orally to cattle at a rate of greater than or equal to 0.03 mg azadirachtin per kg of body weight per day or when ground neem seed was given as a daily supplement of greater than or equal to 10 mg seed per kg body weight, horn fly development in the manure was almost completely inhibited. In contrast, ground seed mixed in cattle feed at the rate of 100-400 mg seed per kg of body weight per day caused less than 50% inhibition of stable flies in the manure.     

Managing the horn fly (Diptera: Muscidae) using an electric walk-through fly trap

An electric walk-through fly trap was evaluated for the management of the horn fly, Hematobia irritans (L.), on dairy cattle in North Carolina over 2 yr. The trap relies on black lights and electrocution grids to attract and kill flies that are brushed from the cattle passing through. During the first season, horn fly densities were reduced from >1,400 to <200 flies per animal. Horn fly density averaged 269.2 +/- 25.8 on cattle using the walk-through fly trap twice daily, and 400.2 +/- 43.5 on the control group during the first year. The second year, seasonal mean horn fly density was 177.3 +/- 10.8 on cattle using the walk-through fly trap compared with 321.1 +/- 15.8 on the control group. No insecticides were used to control horn flies during this 2-yr study.     

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.     

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.