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Pyriproxyfen and House Flies (Diptera: Muscidae): Effects of Direct Exposure and Autodissemination to Larval Habitats

Christopher J. Geden, Gregor J. Devine
DOI: http://dx.doi.org/10.1603/ME11226 606-613 First published online: 1 May 2012


Pyriproxyfen is an insect growth regulator with juvenile hormone-like activity that has potential uses for dipterans that are difficult to manage with conventional insecticides, such as house flies (Musca domestica L.). The objectives of this study were to determine the efficacy of this insect growth regulator against house flies using variety of delivery systems and target life stages, including an evaluation of the potential for autodissemination by female flies to larval development sites. Adult female house flies exposed to filter paper (3.75% active ingredient) or sugar treated with pyriproxyfen (0.01–0.1%) produced significantly fewer F1 pupae than untreated flies. Adult emergence from pupae was unaffected. In contrast, treatment of larval rearing medium with 0.35 ml/cm2 of a 12 mg pyriproxyfen/liter preparation had no effect on the number of pupae developing from eggs but markedly inhibited adult emergence from those pupae. There was little difference in susceptibility between an insecticide-susceptible and a wild strain of house fly. The LC50 for inhibiting fly emergence of dust formulations in diatomaceous earth incorporating commercial pyriproxyfen products ranged from 8 to 26 mg/liter, with little difference among products. Compared with untreated flies, significantly fewer pupae were produced at concentrations >0.5% and no adults were produced at concentrations >0.05% pyriproxyfen. When gravid females were exposed for 1 h to treated fabric (6 mg pyriproxyfen/cm2) and allowed to oviposit in rearing media containing eggs, sufficient pyriproxyfen was autodisseminated to reduce adult emergence from those eggs by >99%. Intermittent contact with treated fabric over 2 d reduced adult emergence by 63–76%.

  • house fly
  • Musca domestica
  • pyriproxyfen
  • autodissemination

House flies (Musca domestica L.) remain a major agricultural and public health pest throughout the world. Efforts to control them are compromised by their ability to develop resistance to new chemical control agents. Resistance to organophosphate, carbamate, and pyrethroid insecticides was observed soon after their introduction (Boxler and Campbell 1983, Plapp 1984, Scott and Georghiou 1986, Scott et al. 1989, Kaufman et al. 2001, Butler et al. 2007, Kozaki et al. 2009). Resistance to spinosad was documented almost immediately after market entry (Shono and Scott 2003, Deacutis et al. 2006) and the neonicotinoid imidacloprid, which provided such superior performance that it quickly dominated the fly control market in the early 2000s, is now facing product failure (Kaufman et al. 2010a,b; Memmi 2010).

Most research on chemical control of the house fly has concentrated on the adult stage using space sprays, residual treatments, and baits. Few materials have been developed that target the immatures. The insect growth regulator (IGR) cyromazine was used in the 1980s, but excessive reliance on this material as a feed-through product in the poultry industry led to overwhelming resistance in just a few years (Bloomcamp et al. 1987, Sheppard et al. 1990, Sheni and Plapp, 1990).

Pyriproxyfen mimics juvenile hormone, inhibiting pupal-adult metamorphosis (Invest and Lucas 2008, Seng et al. 2008). It has high activity against immature dipterans including house flies (Hatakoshi et al. 1987, Kawada et al. 1987, Bull and Meola 1994a). House flies are several orders of magnitude more susceptible to pyriproxyfen than are the parasitoids that attack the fly in the pupal stage (Shono et al. 1993). This suggests that pyriproxfen would be an attractive tool for use in integrated pest management (IPM) programs for flies that include the use of parasitoids (Geden et al. 1992, Crespo et al. 1998). Recently it has been shown that pyriproxyfen can be disseminated to the aquatic habitats of mosquitoes by the adult females themselves; both in the laboratory (Gaugler et al. 2011) and field (Devine et al. 2009). This prompted a reexamination of its activity against house flies. In this study we examine 1) the effects of pyriproxyfen on the F1 progeny of females exposed to treated surfaces and baits, 2) impact of different formulations as surface treatments, and 3) the use of adult female house flies to autodisseminate pyriproxfen to larval development sites.

Materials and Methods

Insect Rearing and Test Conditions.

Unless otherwise stated, house flies were from an insecticide-susceptible colony established in the late 1950s from flies collected near Orlando, FL. In some tests, flies from the Center for Medical, Agricultural and Veterinary Entomology (CMAVE) wild-type colony were used. This colony is restarted periodically from flies collected from local dairy farms (Gilchrist County, FL) and is maintained so that the colony is <6 generations removed from the field. These populations are moderately resistant to many of the insecticides commonly used for fly control such as permethrin and imidacloprid (Deacutis et al. 2006, Rinkevich et al. 2007, Kaufman et al. 2010a). The colonies are maintained using standard rearing procedures (Hogsette 1992). All assays were conducted at 27°C, ≈70% RH and constant (24-h) light conditions. Liquid, emulsifiable concentrates of pyriproxyfen were used: Nyguard IGR (10% active ingredient [a.i.]), Knack (11.23% a.i.), and Sumilarv (2% a.i.). These formulations were produced by MGK, Minneapolis, MN; Valent Biosciences Co., Walnut Creek, CA; and Sumitomo Chemical Australia, Chatswood, NSW, Australia, respectively.

Impact on the F1 Progeny of Exposed Females.

Filter paper disks were treated with 1 ml of pyriproxyfen (Nyguard IGR) diluted in distilled water to make six solutions in a fivefold dilution series ranging from 3.75 to 0.0012% a.i. Control papers were treated with distilled water alone. Papers were allowed to dry and groups of 10 female, 1-d-old flies were exposed for 24 h in petri dishes lined with treated paper. Flies were provided with 10% sucrose on cotton balls placed over a hole in the lid of the dishes. After 24 h, the exposed females were transferred to untreated cages with 10 untreated male flies. Five female flies were chosen randomly from each cage on day 7 and allowed to oviposit for 24 h on a 30 cm3 ball of spent larval rearing medium (medium that had been used previously to rear fly larvae). This oviposition substrate and eggs were then transferred to a cup containing ≈400 cm3 of fresh larval rearing medium. The pupae that developed were separated from the medium 7 d after transfer, counted, and held in cups at 27°C for adult emergence. Three sets of flies were tested for each concentration per test date, and the experiment was repeated on three test dates with different cohorts of flies.

In a second contact method, which allowed flies to display more natural resting behaviors, groups of 10 male and 10 female 1-d-old flies were placed in 17 × 17 × 17 cm cages and provided with food and water. A disk of pyriproxyfen-treated filter paper (diameter, 9 cm) was suspended over the food supply; control cages received water-treated papers. Flies visited and rested on the suspended papers repeatedly. After 7 d, five females were removed from each cage and allowed to oviposit for 24 h. As in the previous test, pupae were collected, counted, and held for emergence. Treatment effects on pupal and adult production in both tests were analyzed by one-way analysis of variance (ANOVA) using the General Linear Models Procedure (PROC GLM) of the Statistical Analysis System (SAS Institute 1992), and differences among means were compared using the Means/Tukey statement of PROC GLM.

In tests that exposed females to treated food baits, pyriproxyfen was diluted in acetone and mixed with sugar to give, after evaporation, a series of five threefold dilutions from 0.001 to 0.1% wt:wt pyriproxyfen. Sugar treated with acetone but without pyriproxyfen served as controls. Groups of 10 male and 10 female 1-d-old flies were given water and the treated sugar in small cages for 3 d, after which they were given standard food for a further 4 d. On day 7, five female flies were removed from each cage and allowed to oviposit for 24 h as before; pupae were collected, counted and held for emergence as in the contact assays, and data analyzed by one-way ANOVA as before.

Evaluation of Pyriproxyfen Formulations as Surface Treatments.

Eggs of an insecticide-susceptible laboratory strain (500 eggs) or the wild-type strain (350 eggs) were placed on 400 cm3 of larval medium in 500-cm3 cups (surface area, 86 cm2) and lightly covered with ≈5 cm3 of medium. Fewer eggs were used for the wild-type strain because it showed elongated development times at egg loads of >350 in this volume of medium. The surface of the medium was evenly treated with 3 ml of pyriproxyfen (Nyguard IGR) diluted in distilled water to make six solutions in a fivefold dilution series ranging from 3.75 to 0.0012% a.i. Pupae were separated 9 d later, counted, and held for adult emergence; data were analyzed with ANOVA as in previous tests.

In a second test, dust formulations were made by mixing diatomaceous earth powder (Safer Brand Ant and Crawling Insect Killer, 78% SiO2, Woodstream, Lititz, PA) with three liquid pyriproxyfen concentrates: Nyguard IGR, Knack, and Sumilarv. After drying, these preparations contained between 0.0005% (5 μg/g) and 0.5% pyriproxyfen (wt:wt). Five hundred eggs of susceptible flies were pipetted onto 86 cm2 of rearing medium (volume, 450 cm3) and lightly covered with medium. The surface of the medium was then treated evenly with 0.5 g of dust. Controls were treated with 0.5 g of dust containing no pyriproxyfen. Pupae were separated 9 d later, counted, and held for adult emergence.

For all evaluations, three sets of fly eggs were used to test each concentration and the control. The entire experiment was repeated three times. The results were subject to probit analyses (PROC PROBIT of SAS) to identify differences in the inhibition of adult fly emergence. The pyriproxifen concentration (LC50) that resulted in 50% suppression of emergence of adult flies was calculated after adjusting for control mortality by Abbott's formula.

Autodissemination by Female Flies.

Groups of five 6-d-old gravid females were gently shaken for 30 s in a 60-ml cup with 0.5 g of pyriproxyfen dust containing 0, 0.05, 0.5, 2.5, 5, and 10% (wt:wt) pyriproxyfen (Nyguard IGR) in diatomaceous earth. Flies were chilled after the exposure, removed from the cups, tapped lightly to remove any excess or caked dust, and transferred (in groups of five) to screen-covered 500-cm3 cups containing 400 cm3 of fly larval rearing medium. Flies were allowed to oviposit for 24 h then removed. Pupae were collected 7 d later, counted, and held until emergence as in previous tests, and data were analyzed by one-way ANOVA as before.

In the next tests, dusts containing 0, 0.05, 0.5, and 5% pyriproxyfen were prepared from both the Nyguard and Knack liquid concentrates using diatomaceous earth as before and applied to cotton fabric. Cotton muslin (104 cm2) was used to line the sides of 60-ml plastic cups, 0.5 g of dust was added, and the cups were covered and shaken for 30 s. Excess dust was removed from the fabric by inverting the cup and tapping it lightly. This resulted in an application rate of 6 mg of dust/cm2.

Groups of five 5-d-old gravid female flies were exposed to the treated fabric in two ways. In the first test (forced contact), flies were chilled and placed on the bottom of the cup. After recovering, the flies were allowed to walk on the treated inner lining of the cup for 1 h then transferred to cages with larval rearing medium as before. In a second test, the bottom was cut from the treated cup that was then placed over the food dish in a cage. This arrangement created a sub-conical cylindrical treated surface surrounding the food. Flies made repeated contacts with pyriproxyfen as they alighted on their way to and from the food, as well as from contact with dust that fell onto the food surface. After 2 d the treated fabric was removed and rearing medium was placed in the cages for oviposition.

In both methods, the rearing medium was removed from the cages after 24 h and held to monitor pupal and adult production as before (n = 3 replications of three cages for each product and concentration). Data on pupal and adult production were analyzed by two-way ANOVA using dust concentration and product (Nyguard vs. Knack) as fixed main effects, plus the concentration-product interaction.

Two additional tests were conducted to determine whether reductions in adult production seen in the F1 generation resulted from the autodissemination of material by the parent and not from direct impacts of pyriproxyfen on fecundity or fertility. The first of these tests was designed to examine the immediate impact of treatment on the number and viability of eggs produced. In this test, groups of 50 6-d-old gravid females were shaken with dusts containing 0, 0.5, or 5% pyriproxyfen. Dusted flies were transferred to cages, provided with food and water, and allowed to oviposit for 6 h on an oviposition pad made by wrapping a ball of spent larval rearing medium in black cloth. These eggs were rinsed from the oviposition pads and counted. One set of 200 eggs from each cage was then held on moist blotting paper and assessed for fertility by counting unhatched eggs after 24 h. Another set of 200 eggs was placed on larval rearing medium and allowed to develop normally. Pupae were collected and counted after 7 d and held for adult emergence. Three sets of 50 flies were tested for each concentration. Treatment effects on egg deposition and survivorship to larval, pupal, and adult stages were analyzed by one-way ANOVA.

The second test was designed to examine the effect of treating adult flies on the development of immatures from untreated flies. In this test, groups of five 6-d-old gravid females were first dusted as above and transferred to cages with food and water (n = 3 cages per concentration). These flies were presented with cups of 400 cm3 of fly larval rearing medium that also contained 500 eggs that had been collected from untreated flies and pipetted onto the medium surface 1 h previously. Treated flies were allowed to oviposit in (and transfer pyriproxyfen to) that medium for 12 h, after which the medium was removed. Additional fresh medium was added 3 d later if crowding levels warranted, and pupal production and adult emergence was determined and analyzed as in previous tests. Not included in the experimental design and analysis were additional cups that were set up for quality control to determine fly production from cups that received only the 500 untreated eggs or the 12-h oviposition event by five gravid females.


Impact on the F1 Progeny of Exposed Females.

When young adult female flies were exposed to pyriproxyfen in forced-contact assays there was no significant effect on the number of pupae or adults produced (Table 1). However, when female flies were allowed to make voluntary contacts with treated papers for 7 d, there was a significant effect on pupal production at the higher rate of 3.75% pyriproxyfen (38% of control productivity) but little effect on adult emergence success (80–90% of control emergence; Table 1).

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Table 1.

Female flies that were allowed to feed on treated sugar (0.01 and 0.33% pyriproxyfen) for 3 d produced ≈35% as many pupae as the controls. Pupal production at the higher concentration of 0.1% pyriproxifen was only 14% of the controls (Table 2). Again, adult emergence success from the pupae that did develop was ≈90% across all baited sugar treatments.

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Table 2.

Evaluation of Pyriproxyfen Formulations as Surface Treatments.

When pyriproxyfen was applied as an aqueous solution to rearing medium containing eggs, there was no effect on the numbers of pupae produced at any dose for either the insecticide-susceptible or wild colony flies (Table 3). In contrast, adult emergence was almost 100% inhibited at all doses. Pyriproxyfen applied as dusts to rearing media containing eggs at the rate of 0.5 g/86 cm2 was also effective at inhibiting adult emergence. Dusts prepared from three commercial concentrates had similar effects on adult emergence, with LC50s of 8–26 mg/liter (Table 4).

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Table 3.
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Table 4.

Autodissemination by Female Flies.

Gravid females that were shaken with pyriproxyfen (Nyguard) dust at concentrations >0.05% produced eggs that did not yield any adults. At pyriproxyfen concentrations >0.5%, pupal production was also affected (Fig. 1).

Fig. 1.

Mean (SE) pupal and adult progeny produced by groups of five gravid female house flies after being shaken in a container with pyriproxyfen dust. Means on bars with the same letters (pupae) are not significantly different (ANOVA F = Tukey's method, P ≤ 0.05; overall ANOVA F5,12 = 18.73). ANOVA was not performed on adult data because no flies were produced in four of the treatments (zero variance).

When flies were confined on treated fabric for 1 h (forced tarsal contact), pupal production was significantly affected by the concentration of the dusts (F3,16 = 5.77; P < 0.01) but not by the product (Nyguard vs. Knack) (F1,16 = 1.64; P > 0.05) or the concentration-product interaction (F3,16 = 1.94; P > 0.05; Table 5). Effects on pupal production were restricted to 30% reductions in pupal numbers in flies treated with the highest concentration of dust prepared from Nyguard. Adult production was significantly affected by concentration (F3,16 = 48.59; P < 0.01) but not product (F1,16 = 0.41; P > 0.05) or the interaction (F3,16 = 3.10; P > 0.05). The strongest effect on adult production was observed in flies treated with dust made with Knack at 5%, where treatments produced just 8% of the flies seen in the controls.

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Table 5.

When gravid females were allowed to make intermittent contact with treated fabric over 2 d there was no effect on pupal production because of product (Nyguard vs. Knack, F1,16 = 0.47; P > 0.05), concentration (F3,16 = 2.92; P > 0.05), or the interaction of product and concentration (F3,16 = 1.44; P > 0.05; Table 5). Adult production was significantly affected by concentration (F3,16 = 37.03; P < 0.01) but not product (F1,16 = 1.43; P > 0.05) or the interaction (F3,16 = 1.01; P > 0.05). Adult production among flies treated at the highest concentration was 24 and 36% of control values for Nyguard and Knack, respectively.

The fecundity of gravid female flies was unaffected by treatment with pyriproxyfen dust. Treated and untreated flies laid 154–180 eggs per female during a 6-h oviposition window after treatment (Table 6). Eggs from treated flies were as viable as controls (overall hatch rate, 92–97%), and there were no significant differences in survival to the pupal or adult stage of progeny from treated flies that were reared in untreated medium (Table 6).

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Table 6.

However, when gravid females dusted with the higher treatment concentration (5% a.i.) were allowed to disseminate pyriproxyfen to media already containing the eggs of untreated flies, almost no adult flies emerged (Table 7). There was no significant effect on adult emergence at the lower concentration (0.5% a.i.), or on pupal production at either concentration.

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Table 7.


Pyriproxyfen is an IGR whose activity mimics that of juvenile hormone and has its primary effect on the target by blocking development from the pupal to adult stages, although embyrogenic and reproductive effects are known as well (Invest and Lucas 2008). Mosquitoes are highly sensitive to this IGR, and long-lasting control of species of Culex, Aedes, and Anopheles has been reported after treatment of larval habitats (Lee 2002, Yapabandara and Curtis 2004, Seccacini et al. 2008). Pyriproxyfen is compatible with other control agents, and combinations with Bacillus thuringiensis, permethrin, and spinosad have been used successfully, with evidence of strong synergism in the latter combination (Lee et al. 2005, Darriet and Corbel 2006, Lucia et al. 2009, Darriett et al. 2010).

Comparatively, little is known about the activity of pyriproxyfen on muscoid flies, although its activity against house flies was recognized at the time of its discovery (Hatakoshi et al. 1987; Kawada et al. 1987, 1992). In one of these initial reports, LC50s of 30–270 μg/liter were observed when the IGR was incorporated into larval rearing medium or poultry manure (Hatakoshi et al. 1987). In another study, lower LC50s (31–48 μg/liter) were observed for larvae in treated rearing medium than for larvae dipped in the same solutions (Kawada et al. 1987). Bull and Meola (1994a) observed that flies exposed to glass treated with up to 142.9 μg/cm2 of pyriproxyfen for 5 d produced significantly fewer pupae after exposure than untreated flies. These flies were confined in glass jars so that contact was essentially continuous throughout the 5-d exposure. The same authors found that stable flies (Stomoxys calcitrans L.) were also sensitive to pyriproxyfen (Bull and Meola 1994b). In our tests, a 1-h exposure of adults to treated filter paper did not result in reduction of F1 pupal production at any dose (Table 1). However, flies that made frequent contact with treated papers suspended in their rearing cages produced fewer pupae, although only at the high rate of 3.75% a.i. Presumably, flies in the latter group acquired a cumulatively higher dose over time than did flies that were confined on a treated surface for a short time. Adult emergence from surviving pupae was similar to emergence from untreated controls. This was also observed by Bull and Meola (1994a). A much stronger effect was observed in flies that ingested pyriproxyfen-treated sugar for 3d after emergence. Both pupal and adult production in the F1 were affected when the parent flies were fed 0.01% a.i. IGR solutions (Table 2). Only pupal production, and not fecundity, egg, or larval mortality was measured in these tests. Therefore, the exact stage at which effects were manifest is unknown, but Bull and Meola (1994a) reported that the primary cause of reduced pupal production was because of embryogenic effects and nonviable eggs. Although such direct effects are interesting, the rates required to treat adults with sufficient material to have substantial embryogenic impacts on F1 progeny are quite high.

Treatment of larval rearing medium with dust formulations made from liquid commercial pyriproxyfen products resulted in LC50s of 8–26 μg/g (Table 4). These rates appear at first glance to be several orders of magnitude higher than those reported by Bull and Meola (1994a) (8 μg/liter), but in the latter study the dose was expressed as the concentration of pyriproxyfen in water that was incorporated into dry ingredients to prepare the diet. In contrast, our doses reflect the concentration used in a dust that was applied to already-prepared diet at a rate of 0.5 g/assay cup. Our results are comparable to the earlier studies after accounting for these differences in approach. Taken together, the data indicate that surface treatment with 0.03–0.35 μg/cm2 is required to ensure inhibition of adult house fly emergence.

The status of cross-resistance to pyriproxyfen is uncertain. Tolerance to pyriproxyfen has been noted in a number of strains resistant to other insecticides (Kawada et al. 1987, Bull and Meola 1994a, Londershausen et al. 1996). In our tests, a few wild-type flies emerged from medium treated at the lowest dose (12 mg/liter), no flies from either strain emerged from medium treated with higher concentrations. House flies are notorious for their ability to develop resistance to insecticides and IGRs, especially when used in continuous delivery systems (Sheppard et al. 1990). Were pyriproxyfen ever to be used ubiquitously and continuously [i.e., incorporation into animal feeds to provide continuous treatment of manure (Miller and Miller 1994)], it would likely result in the rapid development of resistance problems. Therefore, pyriproxyfen must be used as part of integrated resistance and pest management programs.

Perhaps the most exciting potential for pyriproxyfen use lies in the autodissemination approach, in which adult females treat themselves at bait stations then transfer the IGR to oviposition sites. This approach was first suggested for mosquitoes by Itoh (1994) in laboratory tests with Aedes aegypti L., and later by Chism and Apperson (2003) for Ae. albopictus (Skuse) and Ae. triseriatus (Say). Devine et al. (2009) demonstrated the feasibility of the autodissemination method in the field, using treat-and-disseminate stations to treat gravid Ae. aegypti that then carried pyriproxyfen to larval breeding sites (cemetery vases) in a Peruvian cemetery. Simulation models indicate that this approach might even be effective against malaria vectors (Devine and Killeen 2010), and a new formulation and autodissemination station was described recently for use against Ae. albopictus (Gaugler et al. 2011).

Can a parallel approach be developed for muscoid flies? Hargrove and Langeley (1990, 1993) observed significant reductions in tsetse populations in Zimbabwe after deployment of traps or visual targets for adult flies that were treated with pyriproxyfen at 2 mg/cm2 of surface area. In the tests reported in Fig. 1 and Table 5 we exposed gravid females briefly to dusts and then allowed them to oviposit, resulting in reduced emergence of adult progeny. This was unlikely to be the result of direct embryogenic effects on females, as these were small and required long exposure times (Tables 1 and 2). In further tests we showed unequivocally that the treatment of gravid females had no immediate impact on fecundity or fertility of treated flies (Table 6) but that the eggs of untreated flies failed to develop to the adult stage when their rearing substrate was visited by treated flies during oviposition (Table 7). For these reasons, reductions in fly production in the autodissemination tests can be attributed to the morphogenic effects of the fly-transferred pyriproxyfen on fly immatures in the oviposition or larval development medium. Results presented in Fig. 1 and Table 7 demonstrate that house flies are capable of carrying a large enough quantity of pyriproxyfen to transfer a sufficient amount to block fly development at the site where eggs are deposited. Male flies could also transfer material during visits to larval habitats, and treated males might even facilitate the venereal transfer of pyriproxyfen to females during mating and impact parental fecundity and fertility. This has been shown in mosquitoes (Gaugler et al. 2011), house flies (Kawada et al. 1992), and tsetse flies (Langely et al. 1990). Our results with self-treatment methods involving brief and intermittent visits to treated fabric were encouraging, but high doses were necessary (Table 5). A high-potency formulation that would adhere readily to the fly at the time of pickup, remain adhered after acquisition, and detach on contact with manure would be ideal. Further research is needed to identify those formulations and to design exposure tools and autodissemination station designs that might make this approach an operational option for house flies.


We thank M. Doyle and H. Mckeithen for providing flies and assisting with the bioassays. This research was supported in part by the Deployed War Fighter Protection Research Program of the Armed Forces Pest Management Board.

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References Cited

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