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.     

拟除虫菊酯抗性家蝇的交互抗药性研究

拟除虫菊酯的广泛使用使家蝇普遍产生了抗药性,为有效的控制家蝇的危害,需要了解家蝇对轮换或新杀虫剂的交互抗性状况。作者用点滴法测定了两个实验室汰选的拟除虫菊酯抗性家蝇品系对几种杀虫剂的交互抗性,结果表明:二氯苯醚菊酯和溴氰菊酯之间存在较高程度的交互抗药性;拟除虫菊酯抗性较高的家蝇对作用机制不同的新农药(多杀菌素、氟虫腈)表现较低程度的交互抗性。     

Susceptibility of spinosad in Musca domestica (Diptera: Muscidae) field populations

The toxicity of spinosad was determined in one susceptible and five insecticide-resistant laboratory strains of house fly, Musca domestica L. Spinosad was relatively slow-acting, but highly toxic to house flies. In a feeding bioassay, spinosad LC50 at 72 h was 0.51 microg of spinosad per gram of sugar, making it 6.3- and 3.5-fold more toxic to house flies compared with azamethiphos and methomyl, respectively. In topical application bioassay, the LD50 at 48 h of spinosad in susceptible house flies was 40 ng per 20 mg of house fly, making spinosad less toxic than the pyrethroid bioresmethrin synergized by piperonyl butoxide and the organophosphate dimethoate. The insecticide-resistant laboratory strains had resistance factors to spinosad at LC50 in feeding bioassay from 1.5 to 5.5 and at LD50 in topical application bioassay from 2.5 to 4.7, indicating that in house fly cross-resistance to the major insecticide classes will not initially be of major concern for the use of spinosad for house fly control. The toxicity of spinosad was also evaluated against 31 field populations of house flies collected from livestock farms across Denmark. The field populations were 2.2- to 7.5-fold resistant to spinosad at 72 h in feeding bioassay, but based on steep slopes in the bioassay and the limited variation of spinosad toxicity against the various field populations, we consider the field populations to be spinosad-susceptible. We propose a diagnostic dose of 12 microg of spinosad per gram of sugar in feeding bioassay with impregnated sugar for determination of resistant house flies, which is 10x the LC95 of the susceptible strain WHO and approximately = 2x the LD95 of the field populations. Spinosad showed no substantial cross-resistance to the pyrethroid bioresmethrin synergized by piperonyl butoxide, the anticholinesterases dimethoate, azamethiphos, methomyl, and spinosad in house fly field populations.     

Susceptibility of black soldier fly (Diptera: Stratiomyidae) larvae and adults to four insecticides

Dosage-mortality regressions were determined for black soldier fly, Hermetia illucens (L.), larvae fed cyromazine or pyriproxifen treated media. Cyromazine LC50 for larvae dying before becoming prepupae ranged from 0.25 to 0.28 ppm with dosage-mortality regression slopes between 5.79 and 12.04. Cyromazine LC50s for larvae dying before emergence ranged from 0.13 to 0.19 ppm with dosage-mortality regression slopes between 3.94 and 7.69. Pyriproxifen dosage-mortality regressions were not generated for larvae failing to become prepupae since <32% mortality was recorded at the highest concentration of 1,857 ppm. LC50s for larvae failing to become adults ranged from 0.10 to 0.12 ppm with dosage mortality-regression slopes between 1.67 and 2.32. Lambda-cyhalothrin and permethrin dosage-mortality regressions were determined for wild adult black soldier flies and house flies, Musca domestica L., and for susceptible house flies. Our results indicate that the wild house fly, unlike the black soldier fly, population was highly resistant to each of these pyrethroids. Regression slopes for black soldier flies exposed to lambda-cyhalothrin were twice as steep as those determined for the wild house fly strain. Accordingly, LC50s for the black soldier fly and susceptible house fly were 10- to 30-fold lower than those determined for wild house flies. The differential sensitivity between wild black soldier flies and house flies might be due to behavioral differences. Adult house flies usually remain in animal facilities with the possibility of every adult receiving pesticide exposure, while black soldier fly adults are typically present only during emergence and oviposition thereby limiting their exposure.     

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.     

北京地区家蝇对拟除虫菊酯的抗药性与α-乙酸萘酯(α-NA)酯酶的关系

 通过1988年以来对北京地区家蝇(Musca domestica L.)抗药性的研究表明,北京朝阳、回龙观、宣武、景陵地区的家蝇种群与海口种群LD_(50)比较,对溴氰菊酯的抗性为5.4—17.2倍,对二氯苯醚菊酯的抗性为2460—3080倍。不同抗性水平的家蝇种群中,α-NA确酶活性大于0.5(A600值(0.01头·15分))的个体频率与其溴氰菊酯和二氯苯醚菊酯的抗性程度具有显著相关(相关系数分别为0.94和0.98)。抗性程度较高的宣武种群平均酯酶活性为280μmol/(mg蛋白质·分),是海口敏感种群的10.5倍。海口种群α-NA酯酶的米氏常数值(Km)为4.49mol/L,分别是朝阳、回龙观、宣武种群的4.7、4.2和2.3倍,说明抗性种群α-NA酯酶对底物的亲合力高于相对敏感的海口种群。     

Thiamethoxam Resistance in the House Fly,Musca domestica L.: Current Status,Resistance Selection,Cross-Resistance Potential and Possible Biochemical Mechanisms

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC₅₀ ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅰ:水解代谢

水解代谢在家蝇对二氯苯醚菊酯抗性中起重要作用。正常家蝇和抗性家蝇酯酶在对SV₁、SV₂等抑制剂的敏感性和电泳性质上存在着差异。抗性家蝇酯酶水解二氯苯醚菊酯的活性较高,水解乙酸-α-萘酯的活性相对比正常家蝇要低。SV₁及其在体内的代谢产物SV₂在离体和活体情况下对家蝇酯酶都有明显的抑制作用。SV₁和SV₂抑制相同的酯酶电泳条带,但SV₁的抑制作用相对小一些。SV_t对酯酶的抑制是它在家蝇体内对二氯苯醚菊酯增效的机理之一。     

Susceptibility of insecticide-susceptible and wild house flies (Diptera: Muscidae) to abamectin on whitewashed and unpainted wood

Unpainted plywood panels treated with 0.1% abamectin (avermectin B1) provided greater than 90% control of house flies, Musca domestica L., susceptible to insecticides for 4 wk and greater than 70% control for 7 wk compared with 46-92% control observed with permethrin at the same time and rate of application. Efficacy of abamectin on whitewashed panels was similar to that observed on unpainted panels, whereas permethrin was ineffective on whitewashed panels at all rates tested (range, 0.001-0.1%) at all intervals after treatment. Bioassays of newly colonized house flies resistant to permethrin indicated that wild populations may be cross-resistant to abamectin.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅱ:氧化代谢

氧化代谢的增强是引起家蝇对二氯苯醚菊酯产生抗性的因素之一.抗性家蝇多功能氧化酶的萘羟化活性、对二氯苯醚菊酯的氧化代谢能力和微粒体细胞色素P₄₅₀含量分别是正常家蝇的2、1.48、1.33倍.正常家蝇和抗性家蝇细胞色素P₄₅₀在对增效磷(SV₁)和氧化胡椒基丁醚(Pb)的敏感性上也存在着差异.SV₁与多功能氧化酶专一性抑制剂Pb一样,对该酶系催化的萘羟化活性及二氯苯醚菊酯的氧化代谢有明显的抑制作用,这种抑制作用是SV₁在家蝇体内对二氯苯醚菊酯增效的机理之一.SV₁对氧化代谢的抑制与它和微粒体细胞色素P₄₅₀相互作用形成非活性复合体有关.     

Dynamics of insecticide resistance alleles in house fly populations from New York and Florida

The frequency of insecticide-resistance alleles for two genes (Vssc1 and CYP6D1) was studied in field collected populations of house flies from two different climates. While the frequency of these resistance alleles in flies at dairies from four states has recently been reported, there is no information on the relative change of these allele frequencies over time. House flies were collected during the 2003-2004 season from New York and Florida before the first application of permethrin, during the middle of the field season, after the final application, and again the following spring (following months without permethrin use). Bioassay results indicated that homozygous susceptible and extremely resistant flies were rare, while moderately and highly resistant individuals were relatively common at all times in both states. The frequency of resistance alleles at the New York dairy rose during the season and declined over the winter, suggesting an overwintering fitness cost associated with these alleles. The super-kdr allele was detected for the first time in North America at the end of 2003. In Florida the frequency of the resistance alleles did not increase during the spray season or decrease during the winter, suggesting there is substantial immigration of susceptible alleles to the Florida dairy and no overwintering fitness cost associated with resistance alleles in this climate. Resistance to permethrin correlated well with the frequency of the Vssc1 and CYP6D1 resistance alleles in flies from New York, but not as well in the population from Florida. This suggests there may be a new resistance mechanism or allele evolving in Florida.     

Evaluation of resistance to permethrin,cypermethrin and deltamethrin in different populations of Musca domestica (L.), collected from the Iranian dairy cattle farms

The house fly, Musca domestica (Linnaeus) (Diptera: Muscidae), is a major pest for human and livestock health and is also resistant to different insecticides. Herein, six M. domestica populations were collected, five of them from industrial cattle farms and the Koohrang population from a remote area as a susceptible population. The resistance/susceptibility of populations to three pyrethroids was evaluated. High levels of permethrin resistance were observed in all field populations and the resistance ratios (RRs) were estimated to vary from 52- to 129-fold. Resistant populations also exhibited resistance to other pyrethroids (cypermethrin and deltamethrin), with RRs ranging between 45- and 180-fold. According to synergistic (piperonyl butoxide, diethyl maleate and triphenyl phosphate) and enzymatic assays, resistant populations exhibited multiple resistance phenotypes. Cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and carboxylesterases (CarEs) were found to be involved in pyrethroid resistance in Isfahan population, P450s and GSTs in Mobarake population and CarEs detoxified pyrethroids in Natanz and Alavijeh populations. As substitution of Leucine (CTT) with Phenylalanine (TTT) at position 1014 of the voltage sensitive sodium channel (VSSC) gene is the most common mutation conferring resistance to pyrethroids in M. domestica, we sequenced a partial fragment of IIS6 and L1014F mutation was detected in all resistant populations. The present study provides valuable information for early detection of pyrethroid resistance and developing resistance management strategies in the house fly populations.     

Stable fly, house fly (Diptera: Muscidae), and other nuisance fly development in poultry litter associated with horticultural crop production

Poultry litter usage in horticultural crop production is a contributor to nuisance fly populations, in particular stable flies (Stomoxys calcitrans L.) and house flies (Musca domestica L.). Extrapolation of adult emergence data suggests that approximately 1.5 million house flies and 0.2 million stable flies are emerging on average from every hectare of poultry litter applied as a preplant fertilizer for vegetable production in Perth, Western Australia. To a lesser extent, sideband applications to established crops may allow for the development of 0.5 million house flies and 45,000 stable flies per hectare. However, up to 1 million house flies, 0.45 million lesser house flies, Fannia cannicularis L., and 11,000 stable flies per hectare may be produced from surface dressings of poultry litter associated with turf production. Other nuisance flies present in poultry litter included the false stable fly, Muscina stabulans (Fallén), bluebodied blowfly, Calliphora dubia Hardy, black carrion fly, Hydrotaea rostrata Robineau-Desvoidy, Australian sheep blowfly, Lucilia cuprina Wiedemann, and flesh flies (Sarcophagidae). Only house flies developed in poultry litter for the first 4 d after application in the field. Stable flies were not present in poultry litter until 4-7 d after application, and were the only fly species developing in litter > 9 d after application.     

Bioassay for detecting active site insensitivity in horn fly (Diptera: Muscidae) larvae

A bioassay was used to detect active site insensitivity (knock-down resistance [kdr]) in pyrethroid resistant larvae of the horn fly, Haematobia irritans (L.). The larvae of the resistant population had KD50's 42.0-, 28.1- and 29.2-fold greater to permethrin, fenvalerate and lambda-cyhalothrin, respectively, compared with the susceptible population. In filter paper bioassays, resistant adult horn flies were 17 to 39.1 times less susceptible to the pyrethroids than susceptible adults at LC50. These results further document active site insensitivity as the major mechanism of pyrethroid resistance in the horn fly.     

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.     

Co-up-regulation of three P450 genes in response to permethrin exposure in permethrin resistant house flies, Musca domestica

Background

Insects may use various biochemical pathways to enable them to tolerate the lethal action of insecticides. For example, increased cytochrome P450 detoxification is known to play an important role in many insect species. Both constitutively increased expression (overexpression) and induction of P450s are thought to be responsible for increased levels of detoxification of insecticides. However, unlike constitutively overexpressed P450 genes, whose expression association with insecticide resistance has been extensively studied, the induction of P450s is less well characterized in insecticide resistance. The current study focuses on the characterization of individual P450 genes that are induced in response to permethrin treatment in permethrin resistant house flies.

Results

The expression of 3 P450 genes, CYP4D4v2, CYP4G2, and CYP6A38, was co-up-regulated by permethrin treatment in permethrin resistant ALHF house flies in a time and dose-dependent manner. Comparison of the deduced protein sequences of these three P450s from resistant ALHF and susceptible aabys and CS house flies revealed identical protein sequences. Genetic linkage analysis located CYP4D4v2 and CYP6A38 on autosome 5, corresponding to the linkage of P450-mediated resistance in ALHF, whereas CYP4G2 was located on autosome 3, where the major insecticide resistance factor(s) for ALHF had been mapped but no P450 genes reported prior to this study.

Conclusion

Our study provides the first direct evidence that multiple P450 genes are co-up-regulated in permethrin resistant house flies through the induction mechanism, which increases overall expression levels of P450 genes in resistant house flies. Taken together with the significant induction of CYP4D4v2, CYP4G2, and CYP6A38 expression by permethrin only in permethrin resistant house flies and the correlation of the linkage of the genes with resistance and/or P450-mediated resistance in resistant ALHF house flies, this study sheds new light on the functional importance of P450 genes in response to insecticide treatment, detoxification of insecticides, the adaptation of insects to their environment, and the evolution of insecticide resistance.     

Assessing permethrin resistance in the stable fly (Diptera: Muscidae) in Florida by using laboratory selections and field evaluations

Insecticide resistance in the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae),has been demonstrated previously, but mostly with insecticides that are no longer used, such as the organochlorines. Resistance to commonly used pyrethroids has been evaluated twice, but only in the midwestern United States. Stable fly susceptibility to a commonly used pyrethroid, permethrin, was determined in Florida to assess the possibility of resistance development. Diagnostic concentration evaluations of three stable fly field strains demonstrated a maximum of 57 and 21% survival to permethrin residues of 3x and 10x the LC99 of a susceptible strain, respectively. Stable flies from an equine facility with no reported insecticide use demonstrated approximately 20% survival with a 3x diagnostic concentration. Despite a distance of 91-km between field collection sites, survival profiles of field-collected stable fly strains were similar. Although an established stable fly colony collected from a local dairy previously expressed low level resistance to permethrin residues, five generations of laboratory permethrin selection increased resistance 15-fold.     

Autosomal interactions and mechanisms of pyrethroid resistance in house flies, Musca domestica

Five BC? lines and 16 house fly mass-cross homozygous lines were generated from crosses of the pyrethroid resistant ALHF (wild-type) and susceptible aabys (bearing recessive morphological markers on each of five autosomes) strains. Each of the resulting homozygous lines had different combinations of autosomes from the resistant ALHF strain. Levels of resistance to permethrin were measured for each line to determine the autosomal linkage, interaction and, possibly, regulation in pyrethroid resistance of house flies. Results indicated that factors on autosome 4 are not involved in the development of resistance in house flies, while factors on autosomes 1, 2, 3 and 5 play important roles in pyrethroid resistance. The sodium channel gene has been mapped on autosome 3 and multiple cytochrome P450 genes overexpressed in resistant ALHF house flies have been genetically mapped on autosome 5, suggesting that P450 mediated detoxification and sodium channel-mediated target site insensitivity located on autosomes 3 and 5, respectively, are major factors related to resistance development in house flies. However, neither the factors on autosome 3 or 5 alone, nor the factors from both autosomes 3 and 5 combined could confer high levels of resistance to pyrethroid. In addition, strong synergistic effects on resistance was obtained when autosomes 1 and 2 interact with autosome 3 and/or 5, suggesting that the trans factors on autosomes 1 and 2 may interact with factors on autosomes 3 and 5, therefore, playing regulatory roles in the development of sodium channel insensitivity- and P450 detoxification-mediated resistance.     

Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions

The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.     

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.