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Beauveria bassianaand Sawdust Bedding for the Management of the House Fly,Musca domestica(Diptera: Muscidae) in Calf Hutches
BIOLOGICAL CONTROL ARTICLE NO.
7, 221–227 (1996)
0087
Beauveria bassiana and Sawdust Bedding for the Management of the House Fly, Musca domestica (Diptera: Muscidae) in Calf Hutches1 D. W. WATSON, D. A. RUTZ,
AND
S. J. LONG
Department of Entomology, Comstock Hall, Cornell University, Ithaca, New York 14853 Received January 22, 1996; accepted May 8, 1996
Beauveria bassiana (Balsamo) Vuillemin was used concurrently with sawdust bedding to manage the house fly, Musca domestica L., in calf hutches on New York dairy farms. Sawdust bedding was compared with straw bedding for larval development. The number of fly larvae recovered from bedding samples was lower in hutches bedded with sawdust (53/liter) than in straw bedding (131/liter). B. bassiana formulated in water and a surfactant (Tween 80) was evaluated for the control of house fly adults. The prevalence of B. bassiana in the adult fly population was significantly greater in hutches sprayed with conidia than in untreated control hutches. Maximum weekly recovery of B. bassiana was 43 and 47% of the collected fly populations at two treatment farms. A second mycosis caused by Entomophthora muscae (Cohn) Fresenius developed to an epizootic in untreated and treated fly populations. Combined mycoses from B. bassiana and E. muscae reached 60 and 54% of the fly population on two of the treatment farms. The prevalence of E. muscae reached 70% in the fly population on the control farm. r 1996 Academic Press, Inc. KEY WORDS: Beauveria bassiana; Entomophthora muscae; house fly; stable fly; biological control; IPM.
INTRODUCTION
Cultural and biological control play important roles for the management of filth flies on the dairy farm. Weaning calves in outdoor hutches stocked with bedding material, feed, and water has become a common dairy husbandry practice (Fig. 1). Therefore, efforts to control flies in this environment are increasingly important (Schmidtmann, 1988). The house fly, Musca domestica L., and the stable fly, Stomoxys calcitrans (L.), readily exploit soiled bedding for oviposition and larval development (Schmidtmann, 1988). Comparative stud-
ies of different bedding materials indicated that larval density is lower in sawdust than in straw and that straw promotes maggot growth (Schmidtmann, 1988; Schmidtmann et al., 1989). The entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin, has potential as a biological control component of an integrated fly management program. Irrespective of its considerable host range, few reports of fly-derived B. bassiana strains exist (Humber, 1992). Steinkraus et al. (1990) reported that B. bassiana infected ,1% of house fly adults under natural conditions in central New York. Despite the low prevalence of disease, strains collected by Steinkraus were determined virulent in subsequent laboratory studies (Watson et al., 1995b). One strain (P89), when formulated in water and a surfactant, induced 99% mortality in house flies (dose 1 3 108 conidia/cm2 ) within 6 days of exposure. To date, few field studies have evaluated entomopathogenic fungi, other than Entomophthora muscae (Cohn) Fresenius, for control of the house fly. E. muscae has been manipulated to induce epizootics in house fly populations (Kramer and Steinkraus, 1987; Geden et al., 1993; and Steinkraus et al., 1993). Unlike E. muscae, B. bassiana can be cultured and harvested from artificial media (Bell, 1974). B. bassiana conidia formulated as a spray or dust would be particularly suited for controlling the house fly because of the tendency for adults to rest on feed bunks and other building surfaces. Newly eclosed house flies often rest on the interior walls of calf hutches (Fig. 1), making the hutch environment well suited to this type of treatment. The objective of this study was to integrate cultural control using sawdust bedding with a spray program using B. bassiana for the management of the house fly in calf hutches. MATERIALS AND METHODS
1
This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by Cornell University.
This study was conducted on three dairy farms (designated B, G, and T). The hutches were constructed
221
1049-9644/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
222
WATSON, RUTZ, AND LONG
FIG. 1. Outdoor calf hutches of molded plastic or fiberglass construction.
of molded plastic or fiberglass (Fig. 1). Two experimental trials were conducted, each lasting 6 weeks. Trial 1 was conducted during July and August, and trial 2 during late August, September, and early October. Each farm had 12 hutches with calves. The alternative bedding study design was patterned after Schmidtmann et al. (1989). Bedding treatments were split on each farm; six hutches were bedded with straw and six hutches were bedded with coarse sawdust. One-liter bedding samples were collected weekly from the inside of each hutch. Bedding samples were placed in Berlese–Tulgren funnels to extract the fly larvae. Extracted larvae were preserved in 70% ethanol until counted. The species composition, i.e., M. domestica or S. calcitrans, was estimated from 25 randomly selected and identified specimens taken from each 1-liter sample. Identification of house fly and stable fly specimens was based on spiracular plate morphology. The B. bassiana strain (P89) selected for use in these experiments was originally isolated from an infected house fly collected at a dairy in central New York
(Steinkraus et al., 1990). Typically, infected house flies die within 7 days of exposure to the conidia (Watson et al., 1995b). B. bassiana cultures were grown on sabouraud–maltose agar supplemented with 1% yeast extract (Difco, 1984). Sporulating cultures (3-week-old) were harvested by brushing the dry conidia from the surface of the agar plate into sterile vials. Conidia were counted with the aid of a hemocytometer to calibrate a dose of 1 3 108 conidia/mg of dry material. Conidia were preserved by freezing at 221°C until used. An aqueous formulation was prepared by mixing 1 3 107 conidia/mg into a solution of water and a surfactant (0.1% dilution of Tween 80, Fisher Scientific, Pittsburgh, PA). B. bassiana conidia were weighed, added to 250 ml of water and Tween 80, and agitated. The resulting suspension was sufficient to cover the inside of the hutch (79,000 cm2 ) with 1 3 107 conidia/cm2. The suspension was applied to the inside surface of the hutch using a handheld Chapin sprayer. Hutches on farm T were treated with B. bassiana during the first trial. Hutches on farms T and G were treated with B. bassiana during the second trial. Conidial spray formulations were applied to the inside of the hutch walls on weeks 0, 2, and 4. As a precaution, a 4-week rest period between trial spray treatments was allowed for B. bassiana to lose its activity, thus reducing carry over (Watson et al., 1995b). Counting the number of fecal and vomit spots on index cards is commonly used to monitor adult house fly populations in dairy barns (Geden et al., 1993; Steinkraus et al., 1993). Unfortunately, calves in hutches tend to lick the card, making accurate counts impossible. Therefore, adult fly populations were monitored weekly by routine sweep net collections from each hutch (Watson and Petersen, 1993). B. bassiana prevalence also was assessed from these collections. Sampling was limited to 3 min/hutch and pooled according to bedding type. Collected flies were held in 3-liter paper cylinders with screened lids. Each container was provisioned with food and water and the flies were held for 1 week for observation. All flies were counted and identified to species, and dead flies were collected and examined for red abdominal coloration indicative of B. bassiana mycosis (Steinkraus et al., 1990). The cause of death was confirmed by placing all cadavers, regardless of coloration, on moist filter paper to initiate fungal bloom. If E. muscae-induced mortality was observed, cadavers were removed and the abdomen examined for B. bassiana. Cadavers with apparent dual infections were recorded. House fly and stable fly larval data were pooled for analysis. Larval data were normalized by using a log (n 1 1) transformation. Disease prevalence data were calculated as the percentage of the collected sample positive for mycosis, then normalized using logit transformations, LN (1 1 %). Analysis of variance was used
223
B. bassiana FOR FLY CONTROL IN CALF HUTCHES
FIG. 2. Average number of fly larvae recovered from 1-liter samples of sawdust or straw bedding collected from calf hutches.
to compare larval density by bedding type and differences between the prevalence of B. bassiana on treated and control farms. Standard error of the means was calculated from individual treatments. Data were analyzed using a general linear model (Minitab, 1993). RESULTS AND DISCUSSION
Calf hutches bedded with sawdust supported significantly fewer house fly and stable fly larvae than those bedded with straw in both trial 1 (df 5 215, F 5 185.35, P # 0.0001) and trial 2 (df 5 191, F 5 12.98, P # 0.0001) (Fig. 2). Overall, sawdust bedding harbored an average 53 fly larvae/liter, compared with 131 larvae/liter for straw bedding. Weekly hutch observations were similar; straw bedding produced more than twice as many maggots as sawdust bedding (Table 1). With treatments pooled from all farms, significant
differences were apparent at weeks 1–4 and 1, 2, and 5 of trials 1 and 2, respectively (Table 1). Although, fewer fly larvae were collected from hutches bedded with sawdust than from those with straw, the species composition was nearly identical. In trial 1, the house fly made up 61 and 62% of the larvae subsampled from sawdust and straw bedding, respectively. Similarly, stable fly larvae composed 37 and 38% of the subsample collected from sawdust and straw bedding, respectively. In trial 2, house fly larvae made up 55 and 54% of the subsample from sawdust and straw bedding, respectively. Stable fly larvae made up 44 and 43% of the subsample from sawdust and straw bedding, respectively. Approximately 2% of the insects collected from the bedding samples were unidentified Coleoptera or other Diptera larvae. A lower fly density in sawdust bedding than in straw bedding confirmed earlier results reported by Schmidtmann (1991). Although sawdust and straw bedding apparently support a variety of microflora that help sustain fly growth and development, the chemical and physical properties of sawdust may be an important limiting factor for fly breeding (Barnard et al., 1995; Schmidtmann and Martin, 1992; Schmidtmann et al., 1993; Watson et al., 1995a). Sawdust tends to be less absorbent than straw bedding (Watson et al., 1995a) and the resulting reduction in moisture content probably influences the growth and development of the fly larvae (Schmidtmann, 1988). Irrespective of the cause, the use of sawdust bedding during the fly breeding season continues to be an effective management practice that limits house fly and stable fly larval density. Bedding type did not influence the distribution of adult flies in the hutches. The stable fly represented a relatively small portion (2.0 to 10.0 flies per hutch) of the overall adult fly collection (Table 2). During trial 1, sweep net collections of house fly adults at farm T averaged 113 flies/hutch compared with 172 and 358 flies/hutch at farms G and B, respectively (Fig. 2). The number of house flies collected at farm B was significantly higher than the number collected at farms G and T: df 5 35, F 5 12.70, P # 0.0001 (Table 2). Trial 2
TABLE 1 Weekly House and Stable Fly Larval Populations from 1-Liter Samples of Calf Hutch Bedding Collected during Two 6-Week Periods Mean 6 SE number of fly larvae collected in week a Trial
Bedding
1
2
3
4
5
6
1
Sawdust Straw Sawdust Straw
39.9 6 24.8a 142.8 6 61.6b 13.4 6 7.8c 86.0 6 41.0d
9.9 6 4.2a 187.5 6 145.2b 85.8 6 60.5c 212.9 6 7.6d
69.1 6 22.5a 236.7 6 93.3b 129.2 6 54.0c 282.8 6 116.7c
32.6 6 8.3a 80.2 6 20.9b 37.4 6 9.3c 96.7 6 22.4c
43.0 6 11.7a 112.7 6 30.6a 25.4 6 9.0c 94.8 6 32.3d
79.1 6 26.9a 132.3 6 25.4a 30.4 6 12.1c 12.9 6 5.0c
2
a
Means in columns followed by different letters are significantly different (n 5 18, P # 0.05).
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WATSON, RUTZ, AND LONG
TABLE 2 House Fly and Stable Fly Adult Populations Collected from Calf Hutches and Prevalence of Entomophthora muscae and Beauveria bassiana during Two 6-Week Trials Mean 6 SE number of adult flies collected per hutch a House fly infected with Trial and farm Trial 1 B G T Trial 2 B G T
Stable fly b
House fly
E. muscae
B. bassiana
2.8 6 1.1a 6.6 6 2.7a 2.0 6 0.9a
358.0 6 51.7a 172.0 6 26.3b 113.3 6 21.9b
56.8 6 16.1a 29.6 6 7.1a 10.1 6 1.9a
1.7 6 0.9a 3.5 6 1.3a 13.8 6 2.8b
6.7 6 3.0a 10.0 6 1.5a 3.5 6 0.8a
137.4 6 33.7a 356.0 6 61.6b 84.6 6 18.0a
14.9 6 3.7a 55.8 6 12.1b 2.9 6 0.8a
6.3 6 3.6a 43.6 6 7.6b 12.1 6 5.0b
a Means in columns followed by different letters are significantly different (n 5 35, P # 0.05). b Stable fly mycosis was ,1%.
sweep net collections of adult flies averaged 356/hutch at farm G followed by 137 and 85 at farms B and T, respectively. The number of house fly adults collected at farm G was significantly higher than the number collected at farms B and T: df 5 35, F 5 12.48, P # 0.0001. B. bassiana was recovered from flies collected from inside the treated hutches. During trial 1, flies captured from farm T treated hutches began to die of B. bassiana within 7 days of collection. The recovery of B. bassiana from farm T flies was low in weeks 1 to 3, but increased to 47 and 44% by weeks 5 and 6, respectively (Fig. 3). The recovery of B. bassiana from flies collected at week 6 at control farms G and B was 6 and ,1%, respectively. B. bassiana was significantly more prevalent among house flies collected from calf hutches on farm T than from hutches on the control farms: df 5 35, F 5 29.90, P # 0.0001 (Table 2). During the second trial, farms T and G received B. bassiana spray treatments. The average number of flies caught was 85 and 356 flies/hutch for farms T and
FIG. 3. Mean number of house fly adults collected and the prevalence of Beauveria bassiana and Entomophthora muscae among flies collected from calf hutches at three dairy farms during trial 1.
B. bassiana FOR FLY CONTROL IN CALF HUTCHES
225
FIG. 4. Mean number of house fly adults collected and the prevalence of Beauveria bassiana and Entomophthora muscae among flies collected from calf hutches at three dairy farms during trial 2.
G, respectively (Fig. 4). The average number of flies caught from control hutches on farm B averaged 137 flies/hutch during the second trial. The prevalence of B. bassiana at farm T was 26% for the first week of the trial but declined throughout the remaining weeks (Fig. 4). Similar results were observed at farm G. The prevalence of B. bassiana in collected flies increased from 6% at the end of the first trial to 43% in week 2 of the second trial. B. bassiana was significantly more prevalent among house flies collected from calf hutches on farms T and G than from hutches on control farm B, where ,5% of the flies were infected: df 5 35, F 5 6.68, P # 0.004 (Table 2). Although hutches at farms G and B were not treated in trial 1 and farm B was not treated in trial 2, B. bassiana was recovered. Steinkraus et al. (1990) observed that a low level of mycosis existed in the fly populations on New York dairies. However, disease prevalence never exceeded 1%, which suggested that the 6% mycosis observed in our study may have been due to contamination despite our efforts to prevent it. A natural epizootic of E. muscae also was observed at
each farm. During the first week of trial 1, the prevalence of E. muscae was ,5% in the collected flies at untreated farms, B and G, and 13% at the B. bassianatreated farm T (Fig. 3). The prevalence of infected flies increased in July and August to 70, 29, and 21% at farms B, G, and T, respectively. Combined mycoses, attributed to B. bassiana and E. muscae, accounted for 54% of the mortality in the house fly populations at farm T during the fifth week of the trial. E. muscae was the primary cause of fly mycoses on farms B and G during the same period. Prevalence of E. muscae was not significantly different among house flies collected from farms B, G, and T: df 5 35, F 5 0.77, P # 0.472. During trial 2, E. muscae prevalence did not exceed 26% (Fig. 4). B. bassiana was more prevalent at farm G than at farms B and T (df 5 35, F 5 7.62, P # 0.002). The proportion of infected flies collected from farms B and T decreased to ,5% by weeks 4, 5, and 6. In contrast, infected fly collections from farm G were higher: df 5 35, F 5 12.48, P # 0.0001 (Table 2), and the prevalence of E. muscae was 15, 15, and 11% during
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WATSON, RUTZ, AND LONG
weeks 4, 5, and 6, respectively (Fig. 4). Brown (1987) reported that the number of available hosts has bearing on the success of an epizootic. The fly populations on farms T and B may have been below the population density threshold necessary to sustain the E. muscae epizootic. Concurrently, the number of fly larvae extracted from the bedding samples was declining, indicating a reduced number of ovipositing females (Fig. 2). Environmental conditions also may have affected fly behavior. New York farmers commonly observe increased adult fly populations in the dairy barn during cooler temperatures typical of late September and October (D. A. Rutz, pers. comm.). As flies seek shelter, they may have emigrated from the hutches to nearby barns. The environmental factors that affect E. muscae epizootics have been discussed in other studies (Watson and Petersen, 1993; Mullens et al., 1987). Typically epizootics occur in the fall of the year and coincide with cool, humid conditions. Although environmental parameters were not measured in our study, it appeared that the factors affecting the decline in E. muscae also were affecting B. bassiana. We expected the frequency of B. bassiana recovery to increase following each hutch treatment, i.e., at weeks 1, 3, and 5. Our results did not reflect the repeated introduction of B. bassiana inoculum into the environment but resembled the naturally occurring E. muscae epizootic. Integrated fly management systems for dairies stress the importance of a manure management program and the use of natural enemies for reducing house fly and stable fly (Rutz and Watson, in press). Before B. bassiana can be acceptable in dairy IPM systems, several factors must be further investigated. We found in a previous study that dry conidial formulations persist much longer on wooden surfaces than do aqueous formulations (Watson et al., 1995b). Most producers use handheld sprayers and are accustomed to liquid formulations. The acceptance of B. bassiana as a microbial insecticide by dairy farmers depends, in part, on the simplicity of application. Therefore, our future studies will focus on formulation improvement. B. bassiana strain P89 has demonstrated potential for controlling the house fly in laboratory studies. This study represents the first experimental introduction of B. bassiana for controlling the house fly on dairy farms. Furthermore, this study demonstrated an effective multifaceted approach to fly management through the combined effects of artificially induced and naturally occurring fungal epizootics and the cultural practice of using sawdust bedding to manage fly populations. ACKNOWLEDGMENTS The authors thank E. T. Schmidtmann and J. J. Petersen for critical reviews of the manuscript. We thank R. Beck of Beck Farms, Gary and Meg Gaige of Gaige Farms, and Dave and Sue Thompson of
Autumn Ridge Farms for their willingness to cooperate in this investigation. We also thank J. Dillis, G. Howser, and T. Kleckner for their assistance in the completion of this project. This work was supported by New York State Integrated Pest Management Grants and Hatch Project 139428.
REFERENCES Barnard, D. R., Harms, R. H., and Sloan, D. R. 1995. Influence of nitrogen, phosphorus, and calcium in poultry manure on survival, growth, and reproduction in house fly (Diptera: Muscidae). Environ. Entomol. 24, 1297–1301. Bell, J. V. 1974. Mycoses. In ‘‘Insect Diseases’’ (G. E. Cantwell, Ed.), Vol. 1, pp. 300. Dekker, New York. Brown, G. C. 1987. Modeling. In ‘‘Epizootiology of Insect Diseases’’ (J. R. Fuxa and Y. Tanada, Eds.), pp. 43–68. Wiley, New York. Difco. 1984. ‘‘Dehydrated Culture Media and Reagents for Microbiology,’’ Difco Manual, 10th ed. Difco Laboratories, Detroit, MI. Geden, C. J., Steinkraus, D. C., and Rutz, D. A. 1993. Evaluation of two methods for release of Entomophthora muscae (Entomophthorales: Entomophthoraceae) to infect house flies (Diptera: Muscidae) on dairy farms. Environ. Entomol. 20, 1201–1208. Humber, R. A. 1992. ‘‘Collection of Entomopathogenic Fungal Cultures: Catalog of Strains, 1992,’’ pp. 177. USDA, ARS, ARS-110. Kramer, J. P., and Steinkraus, D. C. 1987. Experimental induction of the mycosis caused by Entomophthora muscae in a population of house flies (Musca domestica) within a poultry building. J. New York Entomol. Soc. 95, 114–117. Minitab 1993. ‘‘Minitab Reference Manual.,’’ Release 10, pp. 341. Minitab, State College, Pennsylvania. Mullens, B. A., Rodriguez, J. L., and Meyer, J. A. 1987. An epizootiological study of Entomophthora muscae in muscoid fly population on southern California poultry facilities, with emphasis on Musca domestica. Hilgardia 55, 1–41. Rutz, D. A., and Watson, D. W. Parasitoids as a component in an integrated fly management program on dairy farms. In ‘‘MassReared Natural Enemies: Applications and Regulations’’ (R. L. Ridgeway, M. Hoffman, C. S. Glenister, and M. Inscoe, Eds.). Entomological Society of America Monograph, Lanham, MD, in press. Schmidtmann, E. T. 1988. Exploitation of bedding in dairy outdoor calf hutches by immature house and stable flies (Diptera: Muscidae). J. Med. Entomol. 25, 484–488. Schmidtmann, E. T. 1991. Suppressing immature house and stable flies in outdoor calf hutches with sand, gravel, and sawdust bedding. J. Dairy Sci. 74, 3956–3960. Schmidtmann, E. T., Miller, R. W., and Muller, R. 1989. Effect of experimental bedding treatments on the density of immature Musca domestica and Stomoxys calcitrans (Diptera: Muscidae) in outdoor calf hutches. J. Econ. Entomol. 82, 1134–1139. Schmidtmann, E. T., and Martin, P. A. W. 1992. Relationship between selected bacteria and the growth of immature house flies, Musca domestica, in an axenic test system. J. Med. Entomol. 22, 232–235. Schmidtmann, E. T., Watson, D. W., and Martin, P. A. W. 1993. Bacterial control of flies in livestock operations. In ‘‘Pest Management: Biologically Based Technologies’’ (R. D. Lumsden and J. L. Vaughn, Eds.), pp. 47–53. Proceedings of Beltsville Symposium XVIII, American Chemical Society, Washington, D.C. Steinkraus, D. C., Geden, C. J., Rutz, D. A., and Kramer, J. P. 1990. First report of the natural occurrence of Beauveria bassiana (Moniliales: Moniliaceae) in Musca domestica (Diptera: Muscidae). J. Med. Entomol. 27, 309–312. Steinkraus, D. C., Geden, C. J., and Rutz, D. A. 1993. Prevalence of Entomophthora muscae (Cohn) Fresenius (Zygomycetes: Entomophtho-
B. bassiana FOR FLY CONTROL IN CALF HUTCHES raceae) in house flies (Diptera: Muscidae) on dairy farms in New York and induction of epizootics. Biol. Control 3, 93–100. Watson, D. W., and Petersen, J. J. 1993. Seasonal activity of Entomophthora muscae (Zygomycetes: Entomophthorales) in Musca domestica L. (Diptera: Muscidae) with reference to temperature and relative humidity. Biol. Control 3, 182–190.
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Watson, D. W., Schmidtmann, E. T., and Martin, P. A. W. 1995a. Association of Bacillus thuringiensis and other spore-forming bacteria with calf-hutch bedding. Environ. Entomol. 24, 99–104. Watson, D. W., Geden, C. J., Long, S. J., and Rutz, D. A. 1995b. Efficacy of Beauveria bassiana for controlling the house fly and stable fly (Diptera: Muscidae). Biol. Control 5, 405–411.
7, 221–227 (1996)
0087
Beauveria bassiana and Sawdust Bedding for the Management of the House Fly, Musca domestica (Diptera: Muscidae) in Calf Hutches1 D. W. WATSON, D. A. RUTZ,
AND
S. J. LONG
Department of Entomology, Comstock Hall, Cornell University, Ithaca, New York 14853 Received January 22, 1996; accepted May 8, 1996
Beauveria bassiana (Balsamo) Vuillemin was used concurrently with sawdust bedding to manage the house fly, Musca domestica L., in calf hutches on New York dairy farms. Sawdust bedding was compared with straw bedding for larval development. The number of fly larvae recovered from bedding samples was lower in hutches bedded with sawdust (53/liter) than in straw bedding (131/liter). B. bassiana formulated in water and a surfactant (Tween 80) was evaluated for the control of house fly adults. The prevalence of B. bassiana in the adult fly population was significantly greater in hutches sprayed with conidia than in untreated control hutches. Maximum weekly recovery of B. bassiana was 43 and 47% of the collected fly populations at two treatment farms. A second mycosis caused by Entomophthora muscae (Cohn) Fresenius developed to an epizootic in untreated and treated fly populations. Combined mycoses from B. bassiana and E. muscae reached 60 and 54% of the fly population on two of the treatment farms. The prevalence of E. muscae reached 70% in the fly population on the control farm. r 1996 Academic Press, Inc. KEY WORDS: Beauveria bassiana; Entomophthora muscae; house fly; stable fly; biological control; IPM.
INTRODUCTION
Cultural and biological control play important roles for the management of filth flies on the dairy farm. Weaning calves in outdoor hutches stocked with bedding material, feed, and water has become a common dairy husbandry practice (Fig. 1). Therefore, efforts to control flies in this environment are increasingly important (Schmidtmann, 1988). The house fly, Musca domestica L., and the stable fly, Stomoxys calcitrans (L.), readily exploit soiled bedding for oviposition and larval development (Schmidtmann, 1988). Comparative stud-
ies of different bedding materials indicated that larval density is lower in sawdust than in straw and that straw promotes maggot growth (Schmidtmann, 1988; Schmidtmann et al., 1989). The entomopathogenic fungus, Beauveria bassiana (Balsamo) Vuillemin, has potential as a biological control component of an integrated fly management program. Irrespective of its considerable host range, few reports of fly-derived B. bassiana strains exist (Humber, 1992). Steinkraus et al. (1990) reported that B. bassiana infected ,1% of house fly adults under natural conditions in central New York. Despite the low prevalence of disease, strains collected by Steinkraus were determined virulent in subsequent laboratory studies (Watson et al., 1995b). One strain (P89), when formulated in water and a surfactant, induced 99% mortality in house flies (dose 1 3 108 conidia/cm2 ) within 6 days of exposure. To date, few field studies have evaluated entomopathogenic fungi, other than Entomophthora muscae (Cohn) Fresenius, for control of the house fly. E. muscae has been manipulated to induce epizootics in house fly populations (Kramer and Steinkraus, 1987; Geden et al., 1993; and Steinkraus et al., 1993). Unlike E. muscae, B. bassiana can be cultured and harvested from artificial media (Bell, 1974). B. bassiana conidia formulated as a spray or dust would be particularly suited for controlling the house fly because of the tendency for adults to rest on feed bunks and other building surfaces. Newly eclosed house flies often rest on the interior walls of calf hutches (Fig. 1), making the hutch environment well suited to this type of treatment. The objective of this study was to integrate cultural control using sawdust bedding with a spray program using B. bassiana for the management of the house fly in calf hutches. MATERIALS AND METHODS
1
This article reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by Cornell University.
This study was conducted on three dairy farms (designated B, G, and T). The hutches were constructed
221
1049-9644/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
222
WATSON, RUTZ, AND LONG
FIG. 1. Outdoor calf hutches of molded plastic or fiberglass construction.
of molded plastic or fiberglass (Fig. 1). Two experimental trials were conducted, each lasting 6 weeks. Trial 1 was conducted during July and August, and trial 2 during late August, September, and early October. Each farm had 12 hutches with calves. The alternative bedding study design was patterned after Schmidtmann et al. (1989). Bedding treatments were split on each farm; six hutches were bedded with straw and six hutches were bedded with coarse sawdust. One-liter bedding samples were collected weekly from the inside of each hutch. Bedding samples were placed in Berlese–Tulgren funnels to extract the fly larvae. Extracted larvae were preserved in 70% ethanol until counted. The species composition, i.e., M. domestica or S. calcitrans, was estimated from 25 randomly selected and identified specimens taken from each 1-liter sample. Identification of house fly and stable fly specimens was based on spiracular plate morphology. The B. bassiana strain (P89) selected for use in these experiments was originally isolated from an infected house fly collected at a dairy in central New York
(Steinkraus et al., 1990). Typically, infected house flies die within 7 days of exposure to the conidia (Watson et al., 1995b). B. bassiana cultures were grown on sabouraud–maltose agar supplemented with 1% yeast extract (Difco, 1984). Sporulating cultures (3-week-old) were harvested by brushing the dry conidia from the surface of the agar plate into sterile vials. Conidia were counted with the aid of a hemocytometer to calibrate a dose of 1 3 108 conidia/mg of dry material. Conidia were preserved by freezing at 221°C until used. An aqueous formulation was prepared by mixing 1 3 107 conidia/mg into a solution of water and a surfactant (0.1% dilution of Tween 80, Fisher Scientific, Pittsburgh, PA). B. bassiana conidia were weighed, added to 250 ml of water and Tween 80, and agitated. The resulting suspension was sufficient to cover the inside of the hutch (79,000 cm2 ) with 1 3 107 conidia/cm2. The suspension was applied to the inside surface of the hutch using a handheld Chapin sprayer. Hutches on farm T were treated with B. bassiana during the first trial. Hutches on farms T and G were treated with B. bassiana during the second trial. Conidial spray formulations were applied to the inside of the hutch walls on weeks 0, 2, and 4. As a precaution, a 4-week rest period between trial spray treatments was allowed for B. bassiana to lose its activity, thus reducing carry over (Watson et al., 1995b). Counting the number of fecal and vomit spots on index cards is commonly used to monitor adult house fly populations in dairy barns (Geden et al., 1993; Steinkraus et al., 1993). Unfortunately, calves in hutches tend to lick the card, making accurate counts impossible. Therefore, adult fly populations were monitored weekly by routine sweep net collections from each hutch (Watson and Petersen, 1993). B. bassiana prevalence also was assessed from these collections. Sampling was limited to 3 min/hutch and pooled according to bedding type. Collected flies were held in 3-liter paper cylinders with screened lids. Each container was provisioned with food and water and the flies were held for 1 week for observation. All flies were counted and identified to species, and dead flies were collected and examined for red abdominal coloration indicative of B. bassiana mycosis (Steinkraus et al., 1990). The cause of death was confirmed by placing all cadavers, regardless of coloration, on moist filter paper to initiate fungal bloom. If E. muscae-induced mortality was observed, cadavers were removed and the abdomen examined for B. bassiana. Cadavers with apparent dual infections were recorded. House fly and stable fly larval data were pooled for analysis. Larval data were normalized by using a log (n 1 1) transformation. Disease prevalence data were calculated as the percentage of the collected sample positive for mycosis, then normalized using logit transformations, LN (1 1 %). Analysis of variance was used
223
B. bassiana FOR FLY CONTROL IN CALF HUTCHES
FIG. 2. Average number of fly larvae recovered from 1-liter samples of sawdust or straw bedding collected from calf hutches.
to compare larval density by bedding type and differences between the prevalence of B. bassiana on treated and control farms. Standard error of the means was calculated from individual treatments. Data were analyzed using a general linear model (Minitab, 1993). RESULTS AND DISCUSSION
Calf hutches bedded with sawdust supported significantly fewer house fly and stable fly larvae than those bedded with straw in both trial 1 (df 5 215, F 5 185.35, P # 0.0001) and trial 2 (df 5 191, F 5 12.98, P # 0.0001) (Fig. 2). Overall, sawdust bedding harbored an average 53 fly larvae/liter, compared with 131 larvae/liter for straw bedding. Weekly hutch observations were similar; straw bedding produced more than twice as many maggots as sawdust bedding (Table 1). With treatments pooled from all farms, significant
differences were apparent at weeks 1–4 and 1, 2, and 5 of trials 1 and 2, respectively (Table 1). Although, fewer fly larvae were collected from hutches bedded with sawdust than from those with straw, the species composition was nearly identical. In trial 1, the house fly made up 61 and 62% of the larvae subsampled from sawdust and straw bedding, respectively. Similarly, stable fly larvae composed 37 and 38% of the subsample collected from sawdust and straw bedding, respectively. In trial 2, house fly larvae made up 55 and 54% of the subsample from sawdust and straw bedding, respectively. Stable fly larvae made up 44 and 43% of the subsample from sawdust and straw bedding, respectively. Approximately 2% of the insects collected from the bedding samples were unidentified Coleoptera or other Diptera larvae. A lower fly density in sawdust bedding than in straw bedding confirmed earlier results reported by Schmidtmann (1991). Although sawdust and straw bedding apparently support a variety of microflora that help sustain fly growth and development, the chemical and physical properties of sawdust may be an important limiting factor for fly breeding (Barnard et al., 1995; Schmidtmann and Martin, 1992; Schmidtmann et al., 1993; Watson et al., 1995a). Sawdust tends to be less absorbent than straw bedding (Watson et al., 1995a) and the resulting reduction in moisture content probably influences the growth and development of the fly larvae (Schmidtmann, 1988). Irrespective of the cause, the use of sawdust bedding during the fly breeding season continues to be an effective management practice that limits house fly and stable fly larval density. Bedding type did not influence the distribution of adult flies in the hutches. The stable fly represented a relatively small portion (2.0 to 10.0 flies per hutch) of the overall adult fly collection (Table 2). During trial 1, sweep net collections of house fly adults at farm T averaged 113 flies/hutch compared with 172 and 358 flies/hutch at farms G and B, respectively (Fig. 2). The number of house flies collected at farm B was significantly higher than the number collected at farms G and T: df 5 35, F 5 12.70, P # 0.0001 (Table 2). Trial 2
TABLE 1 Weekly House and Stable Fly Larval Populations from 1-Liter Samples of Calf Hutch Bedding Collected during Two 6-Week Periods Mean 6 SE number of fly larvae collected in week a Trial
Bedding
1
2
3
4
5
6
1
Sawdust Straw Sawdust Straw
39.9 6 24.8a 142.8 6 61.6b 13.4 6 7.8c 86.0 6 41.0d
9.9 6 4.2a 187.5 6 145.2b 85.8 6 60.5c 212.9 6 7.6d
69.1 6 22.5a 236.7 6 93.3b 129.2 6 54.0c 282.8 6 116.7c
32.6 6 8.3a 80.2 6 20.9b 37.4 6 9.3c 96.7 6 22.4c
43.0 6 11.7a 112.7 6 30.6a 25.4 6 9.0c 94.8 6 32.3d
79.1 6 26.9a 132.3 6 25.4a 30.4 6 12.1c 12.9 6 5.0c
2
a
Means in columns followed by different letters are significantly different (n 5 18, P # 0.05).
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WATSON, RUTZ, AND LONG
TABLE 2 House Fly and Stable Fly Adult Populations Collected from Calf Hutches and Prevalence of Entomophthora muscae and Beauveria bassiana during Two 6-Week Trials Mean 6 SE number of adult flies collected per hutch a House fly infected with Trial and farm Trial 1 B G T Trial 2 B G T
Stable fly b
House fly
E. muscae
B. bassiana
2.8 6 1.1a 6.6 6 2.7a 2.0 6 0.9a
358.0 6 51.7a 172.0 6 26.3b 113.3 6 21.9b
56.8 6 16.1a 29.6 6 7.1a 10.1 6 1.9a
1.7 6 0.9a 3.5 6 1.3a 13.8 6 2.8b
6.7 6 3.0a 10.0 6 1.5a 3.5 6 0.8a
137.4 6 33.7a 356.0 6 61.6b 84.6 6 18.0a
14.9 6 3.7a 55.8 6 12.1b 2.9 6 0.8a
6.3 6 3.6a 43.6 6 7.6b 12.1 6 5.0b
a Means in columns followed by different letters are significantly different (n 5 35, P # 0.05). b Stable fly mycosis was ,1%.
sweep net collections of adult flies averaged 356/hutch at farm G followed by 137 and 85 at farms B and T, respectively. The number of house fly adults collected at farm G was significantly higher than the number collected at farms B and T: df 5 35, F 5 12.48, P # 0.0001. B. bassiana was recovered from flies collected from inside the treated hutches. During trial 1, flies captured from farm T treated hutches began to die of B. bassiana within 7 days of collection. The recovery of B. bassiana from farm T flies was low in weeks 1 to 3, but increased to 47 and 44% by weeks 5 and 6, respectively (Fig. 3). The recovery of B. bassiana from flies collected at week 6 at control farms G and B was 6 and ,1%, respectively. B. bassiana was significantly more prevalent among house flies collected from calf hutches on farm T than from hutches on the control farms: df 5 35, F 5 29.90, P # 0.0001 (Table 2). During the second trial, farms T and G received B. bassiana spray treatments. The average number of flies caught was 85 and 356 flies/hutch for farms T and
FIG. 3. Mean number of house fly adults collected and the prevalence of Beauveria bassiana and Entomophthora muscae among flies collected from calf hutches at three dairy farms during trial 1.
B. bassiana FOR FLY CONTROL IN CALF HUTCHES
225
FIG. 4. Mean number of house fly adults collected and the prevalence of Beauveria bassiana and Entomophthora muscae among flies collected from calf hutches at three dairy farms during trial 2.
G, respectively (Fig. 4). The average number of flies caught from control hutches on farm B averaged 137 flies/hutch during the second trial. The prevalence of B. bassiana at farm T was 26% for the first week of the trial but declined throughout the remaining weeks (Fig. 4). Similar results were observed at farm G. The prevalence of B. bassiana in collected flies increased from 6% at the end of the first trial to 43% in week 2 of the second trial. B. bassiana was significantly more prevalent among house flies collected from calf hutches on farms T and G than from hutches on control farm B, where ,5% of the flies were infected: df 5 35, F 5 6.68, P # 0.004 (Table 2). Although hutches at farms G and B were not treated in trial 1 and farm B was not treated in trial 2, B. bassiana was recovered. Steinkraus et al. (1990) observed that a low level of mycosis existed in the fly populations on New York dairies. However, disease prevalence never exceeded 1%, which suggested that the 6% mycosis observed in our study may have been due to contamination despite our efforts to prevent it. A natural epizootic of E. muscae also was observed at
each farm. During the first week of trial 1, the prevalence of E. muscae was ,5% in the collected flies at untreated farms, B and G, and 13% at the B. bassianatreated farm T (Fig. 3). The prevalence of infected flies increased in July and August to 70, 29, and 21% at farms B, G, and T, respectively. Combined mycoses, attributed to B. bassiana and E. muscae, accounted for 54% of the mortality in the house fly populations at farm T during the fifth week of the trial. E. muscae was the primary cause of fly mycoses on farms B and G during the same period. Prevalence of E. muscae was not significantly different among house flies collected from farms B, G, and T: df 5 35, F 5 0.77, P # 0.472. During trial 2, E. muscae prevalence did not exceed 26% (Fig. 4). B. bassiana was more prevalent at farm G than at farms B and T (df 5 35, F 5 7.62, P # 0.002). The proportion of infected flies collected from farms B and T decreased to ,5% by weeks 4, 5, and 6. In contrast, infected fly collections from farm G were higher: df 5 35, F 5 12.48, P # 0.0001 (Table 2), and the prevalence of E. muscae was 15, 15, and 11% during
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WATSON, RUTZ, AND LONG
weeks 4, 5, and 6, respectively (Fig. 4). Brown (1987) reported that the number of available hosts has bearing on the success of an epizootic. The fly populations on farms T and B may have been below the population density threshold necessary to sustain the E. muscae epizootic. Concurrently, the number of fly larvae extracted from the bedding samples was declining, indicating a reduced number of ovipositing females (Fig. 2). Environmental conditions also may have affected fly behavior. New York farmers commonly observe increased adult fly populations in the dairy barn during cooler temperatures typical of late September and October (D. A. Rutz, pers. comm.). As flies seek shelter, they may have emigrated from the hutches to nearby barns. The environmental factors that affect E. muscae epizootics have been discussed in other studies (Watson and Petersen, 1993; Mullens et al., 1987). Typically epizootics occur in the fall of the year and coincide with cool, humid conditions. Although environmental parameters were not measured in our study, it appeared that the factors affecting the decline in E. muscae also were affecting B. bassiana. We expected the frequency of B. bassiana recovery to increase following each hutch treatment, i.e., at weeks 1, 3, and 5. Our results did not reflect the repeated introduction of B. bassiana inoculum into the environment but resembled the naturally occurring E. muscae epizootic. Integrated fly management systems for dairies stress the importance of a manure management program and the use of natural enemies for reducing house fly and stable fly (Rutz and Watson, in press). Before B. bassiana can be acceptable in dairy IPM systems, several factors must be further investigated. We found in a previous study that dry conidial formulations persist much longer on wooden surfaces than do aqueous formulations (Watson et al., 1995b). Most producers use handheld sprayers and are accustomed to liquid formulations. The acceptance of B. bassiana as a microbial insecticide by dairy farmers depends, in part, on the simplicity of application. Therefore, our future studies will focus on formulation improvement. B. bassiana strain P89 has demonstrated potential for controlling the house fly in laboratory studies. This study represents the first experimental introduction of B. bassiana for controlling the house fly on dairy farms. Furthermore, this study demonstrated an effective multifaceted approach to fly management through the combined effects of artificially induced and naturally occurring fungal epizootics and the cultural practice of using sawdust bedding to manage fly populations. ACKNOWLEDGMENTS The authors thank E. T. Schmidtmann and J. J. Petersen for critical reviews of the manuscript. We thank R. Beck of Beck Farms, Gary and Meg Gaige of Gaige Farms, and Dave and Sue Thompson of
Autumn Ridge Farms for their willingness to cooperate in this investigation. We also thank J. Dillis, G. Howser, and T. Kleckner for their assistance in the completion of this project. This work was supported by New York State Integrated Pest Management Grants and Hatch Project 139428.
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