Environ. Entomol. 40(1): 52Ð58 (2011); DOI: /EN09124

Transcription

1 BIOLOGICAL CONTROLÑMICROBIALS Laboratory and Field Evaluation of Formulated Bacillus thuringiensis var. israelensis as a Feed Additive and Using Topical Applications for Control of Musca domestica (Diptera: Muscidae) Larvae in Caged-Poultry Manure L. A. MWAMBURI, 1,2 M. D. LAING, 1 AND R. MILLER 3 Environ. Entomol. 40(1): 52Ð58 (2011); DOI: /EN09124 ABSTRACT Infestations of house ßies, Musca domestica L., are a continual problem around poultry establishments. Acute toxicity of two commercial Bacillus thuringiensis variety israelensis (Bti) formulations (water-dispersible granules and bran formulation) was evaluated against larvae in the laboratory and against natural populations of M. domestica larvae in the Þeld applied in feed to chickens and as topical applications in the poultry houses. Bioassay data showed that susceptibility of M. domestica larvae increased to a given concentration of Bti as the duration of exposure increased. In the laboratory studies, the LC 50 values of Bti for the larvae ranged between 65 and 77.4 g/ml. In the Þeld, a concentration of 10 g Bti/kg of feed resulted in 90% reduction of larvae at 4 wk after treatment. A higher concentration (2 g/liter) of Bti in spray applications was not signiþcantly more effective than the lower concentration of 1 g/liter. Adding Bti to chicken feed is potentially an efþcient measure for the management and control of house ßies in caged-poultry facilities. KEY WORDS manure Bacillus thuringiensis variety israelensis, Musca domestica, biological control, poultry Infestations of house ßy, Musca domestica L., are a continual problem around poultry establishments where caged laying hens are maintained. Accumulation of undisturbed droppings beneath the cages for lengthy periods of time provides an excellent breeding medium for house ßy larvae (Thomas and Skoda 1993). The house ßy is ubiquitous and rapidly infests newly accumulating poultry manure, often reaching enormous numbers. The behavior of this pest is typically synanthropic and, because of its high reproductive rate, it has ability to prosper in a wide range of environments. It possesses a high capacity for dissemination of metaxenic and other types of important diseases, therefore posing a potentially serious health problem. Besides being costly, insecticidal control of the house ßy has many serious drawbacks. If not used properly, insecticides may harm animals and humans, contaminate feed and water, and destroy the biological control agents of ßies. Furthermore, house ßy populations have developed a high level of resistance toward organochlorine and organophosphorous compounds (Keiding 1999, Scott et al. 2000), prompting a 1 Discipline of Plant Pathology, School of Agricultural Sciences and Agribusiness, University of KwaZulu-Natal, Private Bag X01, Scottsville 3201, Pietermaritzburg, South Africa. 2 Corresponding author, lizzymwamburi@hotmail.com. 3 School of Biological and Conservation Sciences, University of KwaZulu-Natal, Private Bag X01, Scottsville 3201, Pietermaritzburg, South Africa. need for more effective solutions. Larviciding and source reduction have a major advantage in that they control ßies before they disperse and transmit disease. Preparations of Bacillus thuringiensis (Bt) Berliner are widely considered as safe and effective pesticides in horticulture and forestry (Kellar and Langenfruch 1993, Navon 1993) and to control mosquitoes and blackßies (Mulla 1990, Becker and Margalit 1993). However, other than Muscabac (Carlberg 1986), to date no commercial preparation of Bt has been released for the control of insect pests of livestock (Gough et al. 2005, Brar et al. 2006). Several isolates of B. thuringiensis are active against house ßy larvae (Johnson et al. 1998) and adults (Indrasith et al. 1992, Hodgman et al. 1993, Zhong et al. 2000). Thuringiensin-containing preparations have also been used to control larvae of M. domestica (Mullens and Rodriguez, 1988; Mullens et al. 1988). Carlberg et al. (1991) reported that nuisance ßies in cattle sheds, slaughter houses, and latrines could be successfully controlled by applying Bt variety thuringiensis to larval breeding sites. Bacillus thuringiensis variety israelensis (Bti) is a gram-positive, spore-forming bacterium. During sporulation it produces protein crystals ( -endotoxins) with insecticidal activity (Höfte and Whiteley 1989). Its mode of action involves ingestion and solubilization of crystals in the gut of the target insect followed by the cleavage of protoxins, activation of toxins, and interaction with the cells of the midgut X/11/0052Ð0058$04.00/ Entomological Society of America

2 February 2011 MWAMBURI ET AL.: EVALUATION OF Bti ON M. domestica 53 epithelium of susceptible larvae (Gill et al. 1992). Studies suggest that these toxins or a combination of unknown components act synergistically (Temeyer and Pinnock 1990) to produce full toxicity, thereby making it difþcult for insect populations to develop resistance to this entomopathogen. These toxins are highly speciþc to their target insect, are safe to humans, vertebrates, and plants, and are completely biodegradable. Therefore, Bti is a viable alternative for the control of insect pests in agriculture and pests that are important vectors of human disease (Bravo et al. 2005, 2007). Safety studies have thus far shown no harmful effects on bees, vertebrates including humans, and most beneþcial insects, even at enormous doses (de Barjac 1978, WHO 1979). In a number of studies, the acute toxicity and pathogenicity of commercial Bt formulations have been evaluated on young bobwhite quail (Colinus virginianus) (Beavers 1991b) and young mallards (Anas platyrhynchus) (Beavers 1991a), when administered orally and daily at high dosages. The Bttreated birds showed no apparent toxic or pathogenic effects by the Bt. In those studies that also evaluated feed consumption and weight gain, the Bt-treated birds showed no effect compared with the nontreated controls. This study reports on the evaluation of two formulations of Bti for M. domestica larvae control under laboratory and Þeld conditions. In the Þeld, Bti was evaluated for control of house ßy larvae in two ways: as a feed-through larvicide to broiler chickens and as a spray application over the manure in poultry houses. Materials and Methods Insect Rearing Adult house ßies were maintained at 28 C, 68 2% RH, with a 12:12 L:D photoperiod. Flies were given a maintenance diet of ad libitum sugar, milk powder, and water and were offered chicken liver as an oviposition medium on a twice weekly basis from the third week after emergence. Larvae for replacement colonies were reared on a diet comprising 50 g active dried yeast (DCL; Clackmannanshire, United Kingdom), 8 g agar (Oxoid; Hampshire, United Kingdom), 250 ml distilled water, and 750 ml ultra-heat-treated full cream milk, prepared by microwaving to boiling point (Johnson et al. 1998). Laboratory Bioassay for Bti Toxicity Against House Fly (M. domestica) Larvae Viable Spores Quantification and Preparation of Spore-Crystal Suspension. Two commercial preparations of Bti (water-dispersible-granules [WDG] and a bran formulation; Plant Health Products, KwaZulu- Natal South Africa) were used. The concentration of viable spores of WDG was estimated. A sample of 1 g of the WDG Bti formulation was suspended in 9 ml sterile water and shaken in an orbital shaker for 30 min. The bacterial suspension was subjected to thermal shock (80 C for 12 min) to kill vegetative cells. After being sequentially diluted, 100- l samples were plated on petri dishes containing nutrient agar and incubated at 28 C for 24 h. The colonies formed were counted and expressed as colony forming units per milliliter. For microscopic observation of morphological features of spores and parasporal bodies, 1-ml samples of Bti obtained from 3- to 4-d-old cultures were incubated in a ßask containing 50 ml trypticase soy broth medium at 30 1 C for 72 h and stained according to Ammons et al. (2002). Approximately 1 ml of the bacterial culture obtained from the ßasks was inoculated into 50 ml nutrient broth (Biolab, Ankara, Turkey) supplemented at 1 ml/liter with mineral salts (Stewart et al. 1981) to aid sporulation in 250-ml ßasks. Liquid cultures were incubated in an orbital shaker at 30 1 C for 72 h. The crystals and spores were harvested by centrifugation at 2,831g for 15 min at 4 C. The pellet was washed twice with cold 0.1 M NaCl and twice with cold water before being resuspending in deionized water to give Þnal concentrations of 1, 10, 50, and 100 g/ml of the crystals and spores. These preparations were stored in small aliquots at 20 C for use in the toxicity assays. Toxicity Assays. Chicken manure from chickens fed with layer mash was collected from poultry houses from the University Research Farm (Ukulinga, Pietermaritzburg, KwaZulu-Natal, South Africa). Three replicates of 50 g manure for each treatment were placed into plastic lunch boxes (120 by 100 by 100 mm) with screened holes in the lids. Aliquots of 1 ml of Bti stock suspension were pipetted evenly over the surface of the manure. Homogenous groups of 30 second-instar larvae were exposed to four different concentrations of the stock solution (1, 10, 50, and 100 g/ml) in triplicates. Three lunch boxes remained untreated for control purposes. The lunch boxes were incubated at 28 C and 60% RH. Larval mortality was quantiþed by counting live larvae remaining every 24 h. Larvae that failed to react to gentle prodding with a glass pipette were considered to be dead (Brown et al. 1998). The larvae were allowed to feed on the compost mixture for 7 d. Larvae in control boxes developed to the third instar and pupal stages. Field Trials Spray and feed trials were run at the same time in separate parts of the same chicken houses. Three replicate sites were randomly selected for each of the trials. All experiments were repeated three times. Spray Trials. The poultry houses at the University Research Farm were used for spray application trials. One week before beginning the spray trial, the manure beneath the poultry houses was completely removed. Three sites of 7 by 1.25 m (8.75 m 2 ) that supported 40 broiler chickens each housed in cages (300 by 400 by 500 mm) were selected. Three similar unsprayed sites were run as controls for comparison. WDG-formulated Bti was applied at 0.11 and 0.23 g/m 2. To prepare the solution for spraying, 1 and2gofbti powder was suspended in 1 liter of water to cover the 8.75 m 2 of

3 54 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 1 Table 1. Percentage mortality of house fly (M. domestica) larvae in manure treated with different concentrations of B. thuringiensis var. israelensis for7d Dose ( g/ml) Mean larval mortality (%) SE a 1d 2d 3d 4d 5d 6d 7d 1 0 0a a a a a a b b b b b b Ð89c Ð33b c c c b d Ð92c c d d c LSD effect b b b b b b Means followed by the same letter within the same day are not signiþcantly different at P a Values are means of three replicates of assays each containing 30 larvae. b SigniÞcant at P ßoor surface (114 ml/m 2 ). The Bti formulation was applied using hydraulic hand sprayers. The Bti applications and house ßy larvae monitoring were carried out at 7-d intervals. Data were analyzed to measure reduction in the ßy larvae numbers in comparison with untreated controls. The trial was run for 6 wk. Feed Trials. For Þeld evaluation experiments, 48 hens that were 72-wk-old commercial broilers (Hyline Brown) were obtained from the University Research Farm. Broilers were evaluated on receipt for signs of disease or other complications that could affect the outcome of the study. After examination, broilers were randomly allocated into 48 hanging pens (440 by 420 by 610 mm) in parallel back-to-back rows of 12 pens per row. Pens were separated by wire-mesh on all sides. Each pen was equipped with a removable feed trough and a nipple drinker. Each pen had a removable tray for individual manure collection. Broilers were housed in an environmentally controlled shed Þtted with fans (21 5 C) and at 16:8 h (L: D) at the University Research Farm. Bti-formulated bran was mixed into the broiler mash (Nutrex KZN; KwaZuluÐNatal, South Africa) in varying quantities (0.1, 0.5, 1.0, 5.0, and 10 g/kg of broiler mash). The control did not contain any Bti-formulated bran. The rations were stored in trash cans lined with plastic bags until fed to the chickens. There were eight broilers per treatment. Feed and drinking water were provided ad libitum. Broilers were fed their respective dietary treatments for 5 wk. House Fly Sampling For the feed trials, excreta from each bird was collected every 7 d. Sampling of house ßy larvae was started a week after the administration of the different feeds to give the broilers time to adjust to the different treatments. House ßy larvae were monitored using a gardenersõ bulb planter (400 ml) to collect two manure cores from each tray/bird/diet. Larvae were extracted using Berlese funnels and enumerated. For the spray trials, 10 samples of 400 ml were taken from each replicate every 7 d during the trial. Statistical Analysis Mortality data for the laboratory assays of the larvae in the treated boxes was corrected against control mortality (Abbott 1925). The corrected mortality was subjected to log-probit regression analysis and median lethal concentration (LC 50 ) associated 95% conþdence intervals were calculated using the GENSTAT program. All percentage mortality data were square root-arcsine percent transformed and used in statistical analysis. Treatments were also compared based on these transformed values. The data were analyzed using repeated-measures analysis of variance (ANOVA). The least signiþcant difference (LSD) method was used to separate and compare means within the treatments. For the Þeld experiments, the percentage reduction of larvae in the treated trays was calculated and corrected to the reduction, if any, in controls using the formula of Mulla et al. (1971). The percentage values were normalized by square root-arcsine transformation. An ANOVA (repeated measures) was performed by taking the percent reduction as the dependent variable and dose as the factor. The LSD method was used to Þnd signiþcant differences between means. Results Evaluation of Toxic Activity and Concentration of Viable Spores. The concentration of viable spores in the WDG Bti formulation, as estimated by the number of colonies, was spores/g. The mean LC 50 was 70.7 g Bti spores/ml with 95% conþdence intervals of 65Ð77.4 g/ml. Mortality data of the laboratory bioassays with the WDG formulation of Bti is presented in Table 1. After 4 d of treatment, there was a sharp decline in M. domestica larval densities in all the treatments. At D4 after treatment, there was at least 50% mortality in three of the dosages (10, 50, and 100 g Bti/ml); the 1 g Bti/ml produced 36% mortality. Although the minimum mortality observed was 42% (1 g Bti/ml) at D5 after treatment, the rest of the dosages; 10, 50, and 100 g/ml produced 58, 67, and 76% larval mortalities, respectively. At D7 after treatment, the highest larval mortality observed was 86%, whereas 10 and 50 g Bti/ml produced 66 and 74% mortality, respectively, and the mortality produced by 1 g Bti/ml was just below 50% (Table 1). There were signiþcant differences in mortality levels of M. domestica larvae between the Bti concentrations (F 58.0; df 3; P 0.001), the time of exposure

4 February 2011 MWAMBURI ET AL.: EVALUATION OF Bti ON M. domestica 55 Table 2. Mean percent reduction of house fly (M. domestica) larvae in manure of broilers sprayed weekly for 6 wk with concentrations (1 or 2 g/liter) of a WDG formulation of B. thuringiensis var. israelensis Bti (g/liter) Percent reduction in no. of larvae SE 1wk 2wk 3wk 4wk 5wk 6wk a a a a a a a a a a a a LSD effect 0.09 NS 0.09 NS 0.12 NS 0.12 NS 0.09 NS 0.07 NS Means followed by the same letter within the same week are not signiþcantly different at P NS, not signiþcant. (F 1072; df 6; P 0.001), and the interaction effects between dosages and days (F 12.05; df 18; P 0.001), indicating a change in the level of larval reduction on posttreatment days with a change in the dosage. SigniÞcant differences between the treatments were apparent after D2 posttreatment (Table 1). There was a dose-dependent relationship in the mortality of larvae (Table 1). Moreover, mortality of larvae exposed to 1Ð100 g Bti/ml differed signiþcantly from the control (P 0.001). The greatest mortality was obtained with the 100 g Bti/ml dose. Indeed, at the 100 g/ml dose, Bti killed 57% of the larvae in 3 d and 87% of the larvae in 7 d, whereas no larval mortalities were recorded in the control treatments because these pupated and completed their life cycles. Spray Trials. The mean number of house ßy larvae collected from the untreated manure core samples in the poultry houses during the 6-wk sampling period was (SE). After 2Ð4 wk of spraying, the density of larvae in the manure was reduced by at least 20% in both the 1 and 2 g/liter dosages. After 4 wk, there was a sharp decline in larval densities at both dosages. Whereas there was a 50% reduction in larvae by 2 g/liter after 5 wk, a larval reduction of just below 50% reduction was obtained with 1 g/liter only after 6 wk. At 6 wk after treatment, 2 g/liter produced 52% mortality, whereas the reduction of larvae by the 1 g/liter treatment was 47% (Table 2). Although the two dosages (1 and 2 g/liter) caused signiþcant reductions in the number of larvae (P 0.05), they did not differ signiþcantly in reducing the number of larvae (F 2.27; df 1; P 0.149). Although there was a signiþcant effect of weeks (F 33.24; df 5; P 0.001), the interaction effect of dosages and week was not signiþcant (F 0.43; df 5; P 0.752). Feed Trials. The mean number of larvae collected from manure of broilers fed with non-bti feed was Continuous feeding any of Þve doses of Bti in the broiler mash resulted in a rapid decline in numbers of house ßy larvae in the manure (Table 3). After 1 wk of feeding chickens with Bti, the larval reduction, as a result of the 0.1, 0.5 and 1.0 g Bti/kg rates, was at least 20%, whereas the larval reduction as a result of the 5 and 10 g Bti/kg feed was 40% (Table 3). Percent reduction in larval numbers was almost doubled after 2 wk of feeding with three dosages (0.1, 0.5, and 1.0 g/kg). After 2 wk of using Bti as a feed additive, the number of larvae was reduced by at least 50% as a result of four of the Þve dosages. By week 3 of feeding, the reduction was at least 70% as a result of the 5 and 10 g Bti/kg doses, whereas at 0.5 and 1.0 g Bti/kg, the reduction was 60%. After 4 wk of treatment, the percent reduction of maggots was 91% at 10 g Bti/kg. In the case of other dosages, 0.1, 0.5, 1.0, and5gbti/kg, the reductions were 62, 75, 78, and 88%, respectively (Table 3). The two main effects, dose and time, were signiþcant (F 13.26; df 4; P and F 73.05; df 3; P 0.001, respectively). However, the interaction effect between them was not signiþcant (F 0.38; df 12; P 0.959). The level of reduction of larvae as a result of dose did not differ signiþcantly in the Þrst week of feeding formulated Bti to chickens but signiþcant differences were apparent between the dosages in subsequent weeks of feeding (Table 3). Table 3. Percentage reduction of house fly (M. domestica) larvae in the manure of broilers fed for 4 wk with five concentrations (g/kg) of a bran formulation of B. thuringiensis var. israelensis Bti (g/kg) Percent reduction of no. of larvae SE 1wk 2wk 3wk 4wk a a a a a ab ab ab ab ab abc bc ab b bc cd b b c d LSD effect 0.24 NS 0.17 a 0.17 a 0.18 b Means followed by the same letter within the same week are not signiþcantly different at P a SigniÞcant at P b SigniÞcant at P NS, not signiþcant.

5 56 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 1 Discussion Our data showed that M. domestica larvae exposed to different spore concentrations (1, 10, 50, and 100 g/ml) of Bti were susceptible to the bacterial toxins. Indeed, 86% of ßy larvae exposed to a concentration of 100 gofbti formulation/ml (10 8 spores/ml of Bti) were dead within 7 d. These results are comparable to those of Lonc et al. (1991). These authors reported a 70% mortality of house ßy larvae as a result of a Bti spore concentrations of /ml. The toxicity of the Bti formulation in this study was not based on international toxicity units, because these were not available. Several authors (Skovmand et al. 2000, de Araújo et al. 2007) found that there is not always a correlation between spore concentration and the toxicity of a formulation and as such the toxicity of the Bti in this study was not based spore concentration. It is also difþcult to directly compare the results of this study with other publications on other Bti formulations, because application rates are expressed in many different ways (de Barjac 1978) and the potencies of various test formulations are different. In the laboratory, all four test dosages produced signiþcant effects toward controlling larval numbers. Mortality of M. domestica larvae increased with concentration of the WDG formulation of Bti as the duration of exposure increased. However, as the exposure duration increased, the amount of Bti required to cause signiþcant mortality decreased, with the sharpest decline of larval numbers occurring after 4Ð5 d of exposure. This could also mean that the Bti reached a lethal concentration more rapidly, but the effect was only temporary. The 100 g Bti/ml concentration killed 50% of the larvae in 3.4 d. The dose of 100 g Bti/ml (with respect to active ingredient) was used to calculate the dose of choice for Þeld application, because 90% control of larvae was achieved in 7 d. Considering that LC 50 values observed in the laboratory are often not enough to cause mortality in the Þeld (de Araújo et al. 2007), a higher dose of 2 g/liter was used for Þeld application, and treatment at this dosage once a week was necessary to cause 90% reduction of ßy larvae, although repeated applications would be more effective in controlling the larval density. However, application of any larvicide twice a week in an operational program may not be feasible or cost effective. Results from the Þeld spray trials with Bti WDG showed that a higher concentration (2 g/liter) was not signiþcantly more effective than the lower concentration (1 g/liter) in causing larval mortalities. Such low application dosages offer the possibility of keeping operational costs low. Even though it would reduce the insecticide cost, the labor would not change. Moreover, both Bti concentrations were equally effective at lowering larval densities in the manure and a reduction of 50% was achieved 6 wk after spraying. Results further showed that, under laboratory conditions, mortality of M. domestica larvae as a result of Bti WDG formulation was higher than in the Þeld. These results indicated that, under static laboratory conditions, it is possible to overestimate the longevity of Bti spores in the ßuctuating Þeld environment (Yousten et al. 1992). The results also suggest that the reputed potency of a product may not be an accurate indicator of its performance in the Þeld, so far as the persistence of the larvicidal activity is concerned (Vilarinhos and Monnerat 2004, de Araújo et al. 2007). This lack of persistence or degree of exposure to target organism of Bti in the Þeld has been observed in other arthropods (Karch et al. 1991, Fillinger et al. 2003, Russell et al. 2003). Several environmental factors may inßuence the effectiveness of Bti formulations in the Þeld (Mulla et al. 1990, Becker et al. 1992), including high temperature (Ignoffo 1992) and high larval densities outside of the bioassay sampled areas (Glare and OÕCallaghan 2000). Exposure of spores to prolonged periods of high temperatures has been reported to result in decreased activity of Bt products, especially in the tropics, where temperatures frequently exceed 30 C (Morris 1983). In addition to possible inactivation of the Bt toxins, high temperatures also reduce feeding by some insects (Han and Bauce 2000). Reduced persistence of Bti in the Þeld may also be attributed to the existence of compounds in chicken litter that may inhibit the growth of some microorganisms (Adams et al. 2002). Moreover, the alkaline ph of chicken manure favors solubility of crystals, making protoxins more prone to degradation (de Araújo et al. 2007). The association of such factors would contribute to reducing the larvicidal activity of Bti products (de Araújo et al. 2007). On the contrary, the prolonged larval control observed in the feeding trials with the bran formulation of Bti suggests that feeding Bti to the chickens might compensate for the partial loss of toxic crystals caused by UV and other factors. Furthermore, 90% reductions of M. domestica larvae were observed with a dose rate of 10 g of bran formulation/kg of standard broiler mash. The bran formulation was approximately 2.5 times as effective as the WDG formulation (on an active ingredient basis), achieving 80% mortality at 4 wk with 1 g/kg and 30% mortality with 1 g/liter, respectively. Such variation in product efþcacy has been reported previously (Brown et al. 2001, Fillinger et al. 2003, Russell et al. 2003) and may be related to formulation characteristics. These observations were similar to those observed by Labib and Rady (2001), who found that adding Bti to chicken food signiþcantly reduced maggot numbers. Gingrich (1965) reported Bt variety thuringiensis Berliner fed to livestock inhibited larval development of the house ßy, M. domestica in feces. Similar observations were reported by Briggs (1960), who observed reductions in ßy development in feces of chickens fed B. thuringiensis spore preparations, Dunn (1960), for cattle, Harvey and Brethour (1960), for chickens and cattle, and Borgatti and Guyer (1963) for the Japanese quail. However, several studies (Larget and Barjac 1981, Vankova 1981) observed little or no effect of Bti on M. domestica. The microbial materials reported used in these tests variously contained soluble metabolites, and spores and/or crystals, from either labora-

6 February 2011 MWAMBURI ET AL.: EVALUATION OF Bti ON M. domestica 57 tory or commercially cultured B. thuringiensis. The strain used was a locally isolated strain of Bti, closely related to the commercial strain of Bti that is sold globally, as conþrmed by AFLP assays (Laing, personal communication). In large-scale programs, for any formulation to be considered for incorporation, it should have residual activity so that the application and thereby operational costs can be minimized. In this study, the bran formulation was shown to be the most effective formulation for use in controlling M. domestica larvae in poultry houses. SpeciÞcally, it is easily applied and lasted for long periods without a reduction in the levels of activity. It also mixed thoroughly in manure compared with surface sprays. The results of our studies on the evaluation of Bti against M. domestica indicate that biological control of M. domestica larvae in poultry facilities may be possible. The highest mortality obtained with the bran formulation of Bti was 91% after 4 wk of feeding 10 g of the formulation/kg of basal diet to chickens. Successful results were also obtained with the WDG Bti formulation, causing M. domestica larvae mortality levels of 53% with 0.23 g/m 2 and 47% with 0.11 g Bti formulation/m 2. Adding Bti to chicken feed may be an effective measure for the management and control of house ßies in large-scale caged-poultry facilities. Acknowledgments We thank the Third World Organization for Women in Science for Þnancial support. References Cited Abbott, W. S A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 18: 265Ð267. Adams, T. T., M. A. Eiteman, and B. M. Hanel Solid state fermentation of broiler litter for production of biocontrol agents. Bioresource Technol. 82: 33Ð41. Ammons, D., J. Rampersad, and A. Khan Usefulness of staining parasporal bodies when screening for Bacillus thuringiensis. J. Invertebr. Pathol. 79: 203Ð204. 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