Method and device for the biological control of flying insects

A method for control and extermination of flying insects, especially the housefly, by infection of the insects with an entomopathogenic fungus, preferably soil-dwelling fungi, by means of an infection chamber. The chamber maintains the spores of a fungus pathogenic to the insects in a viable form, serves as an attractant for the insects, and serves to inoculate the insects with high numbers of spores. The spores attach to the insects and originate germ tubes that penetrate into the insect, resulting in death within three to four days. The chamber design, i.e., shape and color, can be the sole attractants for the insects. Alternatively, food or scents can be used to further enhance the attraction of the insects for the chamber. Although the primary means of infection is by external contact with the fungal growth, the insects may also be infected by contact with each other and by ingestion of the spores.

BACKGROUND OF THE INVENTION 
The present invention is in the field of biological control of insect 
pests, specifically in the area of use of entomopathogenic fungi for the 
control of flying insects. 
Control of the house fly is of major economic importance throughout the 
world because of public health concerns. The fly has the potential to 
mechanically transmit a wide variety of human pathogens, as reviewed by 
Bida Wid, S. P., J. I. Braim and R. M. Matossian, Ann. Trop. Med. 
Parasitol. 72(2): 117-121 (1978). The fly can also be annoying to people, 
livestock and poultry, to the extent that it even decreases time spent by 
animals in feeding, thereby decreasing feed efficiency. 
Because of the economic and public health importance of the house fly, a 
significant amount of effort has been devoted to develop methods to 
control it. The biggest effort has been directed towards chemical 
insecticides, as reviewed by J. G. Scott and D. A. Rutz, J. Econ. Entomol. 
81(3): 804-807 (1988). The use of chemical insecticides has a number of 
serious drawbacks, such as the destruction of non-target biological 
control agents, development of insecticide resistance, harmful levels of 
insecticide residue and environmental pollution. Therefore, it is 
desirable to have less ecologically-disruptive means to control house 
flies. 
New approaches to fly control include the use of parasitoid wasps of 
various genera, as reported by J.D. Mandeville, et al. Can Ent 120: 
153-159 (1988). This method of control reduces the fly population but is 
not adequate in itself to provide satisfactory fly control. 
Insect pathogens are a possible alternative to the common use of highly 
toxic chemical insecticides for the control of insect pests. Fungi are one 
of the promising groups of insect pathogens suitable for use as biological 
agents for the control of insects. 
Fungi are found either as single cell organisms or as multicellular 
colonies. While fungi are eukaryotic and therefore more highly 
differentiated than bacteria, they are less differentiated than higher 
plants. Fungi are incapable of utilizing light as an energy source and 
therefore restricted to a saprophytic or parasitic existence. 
The most common mode of growth and reproduction for fungi is vegetative or 
asexual reproduction which involves sporulation followed by germination of 
the spores. Asexual spores, or conidia, form at the tips and along the 
sides of hyphae, the branching filamentous structures of multicellular 
colonies. In the proper environment, the conidia germinate, become 
enlarged and produce germ tubes. The germ tubes develop, in time, into 
hyphae which in turn form colonies. 
One would expect that pathogens had been extensively considered as 
biological control agents, however, a review of the literature reveals the 
scarcity of pathogens that appear to offer potential to control M. 
domestica. The bulk of scientific literature on associations of pathogens 
with house flies refers to isolated reports of diagnosis of dead flies or 
laboratory studies without practical, short-term applications. 
An extensive review of the literature reveals only isolated cases of fungal 
infection (see, for example, Table 1 in Briggs and Milligan, Bull. World 
Health Organization 58(Supplement): 245-257 (1980); Briggs and Milligan, 
Bull. World Health Organization 55(Supplement): 129-131 (1977)). Most 
reports of fungi associated with flies appear to refer to situations where 
the fungi did not cause patent infections or major predictable collapses 
of fly populations. Therefore, it does not appear as though fungi can be 
practically used for fly control. For example, although the fungi 
Aspergillus niger, A. flavus, A. ustus and Mucor racemosus from pupae or 
adults of M. domestica by Zuberi, et al., Pakistan J. Sci. Ind. Res. 12, 
77-82 (1969) there was no evidence that these fungi were inflicting 
serious pathological effect on the fly populations. 
It is possible to infect adult house flies with fungi under certain 
laboratory conditions, leading to death of the infected flies. For 
example, Aspergillus flavus was pathogenic to M. domestica when the 
insects were fed high concentrations (up to 1.times.10.sup.9) of fungal 
spores, presumably due to toxins in the spores. Mortality after seven days 
of exposure was 57%; mortality was 100% twenty-one days after exposure. 
One hundred percent mortality occurred in flies seven days after they were 
anesthetized and placed in contact with fungal spores, as reported by 
Amonker and Nair, J. Invertebr. Pathol. 7: 513-514 (1965). Dresner, J. 
N.Y. Entomol. Soc. 58: 269-279 (1950), also reported that an isolate of 
the fungus Beauveria bassiana infected adult M. domestica when the insects 
were exposed to a dust of germinating conidia adhered in a nutrient 
medium. The fungus was also infective to flies when the insects were 
exposed to a dish of milk containing fungal conidia. 
D. C. Rizzo conducted studies, reported in J. Invert. Pathol. 30, 127-130 
(1977), on the mortality of flies infected with either Metarhizium 
anisopliae or Beauveria bassiana and determined that the time to death 
after infection was independent of age. Flies were infected by rolling 
them for ten minutes in four-week-old fungal culture slants until they 
were completely exposed to the spores, then maintaining them in humidity 
chambers. As noted by the author, in reference to the infecting fungi, 
"these pathogens have never been reported as having caused mycoses in fly 
populations in nature" at page 127. 
In 1990, however, D. C. Steinkraus, et al., reported in J. Med. Entomology 
27(3), 309-312, that Musca domestica L., infected with Beauveria bassiana 
had been found on dairy farms in New York, although at a prevalence of 
less than 1% (28 out of 31,165). Isolates of the fungi were infective for 
laboratory raised flies, but the low naturally occurring incidence led to 
the conclusion by the authors that "it seems unlikely that these 
infections represent naturally occurring epizootics within house fly 
populations" at page 310. 
These studies have led to the recognition that there is a potential for 
fungal control of insects. However, no one has yet developed a consistent 
and commercially viable way of infecting insects and assuring that the 
fungi are dispersed throughout the breeding populations. For example, with 
reference to house flies, it is clearly impractical, and will make the 
registration of any product with the Environmental Protection Agency in 
the United States very difficult, to disperse conidia on surfaces or 
dishes of nutrient media whenever there is a need to control the fly 
population. 
As of this time, there has been no successful demonstration by others of 
the practical, reliable and economical employment of an entomopathogenic 
fungus for the management and biological control of flying insects such as 
the common housefly. 
It is therefore an object of the present invention to biologically control 
flying insects, especially the housefly, using entomopathogenic fungi. 
It is a further object of the present invention to provide a device for the 
convenient, reliable and economically feasible application of fungi in the 
biological control of flying insects. 
SUMMARY OF THE INVENTION 
A method for control and extermination of flying insects, especially the 
housefly, by infection of the insects with an entomopathogenic fungus by 
means of an infection chamber. The chamber maintains the spores of a 
fungus pathogenic to the insects in a viable form, protecting the fungi 
from the environment (including rain, ultraviolet light and the wind), 
serves as an attractant for the insects, and serves to inoculate the 
insects with high numbers of spores. The fungal culture provides a 
continuous supply of spores over a prolonged period of time, even if 
desiccated. The spores attach to the insects and originate germ tubes that 
penetrate into the insect, which can result in death within three to four 
days. The chamber design, i.e., shape and color, can be the sole 
attractants for the insects. Alternatively, food or scents can be used to 
further enhance the attraction of the insects for the chamber. Although 
the primary means of infection is by external contact, the insects may 
also be infected by contact with each other and by ingestion of the 
spores. 
The two most preferred entomopathogenic fungi are Metarhizium anisopliae 
and Beauveria bassiana, although other fungi can be used which are 
pathogenic when the insect is inoculated via the infection chamber, such 
as Paecilomyces and Verticillium. Examples demonstrate control of Fannia 
canicularis and Musca domestica under laboratory conditions and of Musca 
domestica in chicken coops using chambers containing Metarhizium 
anisopliae. Although exemplified as a method for fly control, the chamber 
can also be used for control of other flying insects that will enter the 
chamber and be infected by the fungus.

DETAILED DESCRIPTION OF THE INVENTION 
Under normal circumstances, flying insects are not exposed to high 
concentrations of spores of soil-dwelling entomopathogenic fungi. The 
primary advantages of the infection chamber are that (1) it concentrates 
an extremely high number of fungal inoculum in a very small space within 
the infection chamber, forcing entering insects into contact with the 
spores which infect and kill the insects, and (2) it contains the fungal 
spores, resulting in minimal exposure of the environment to the pathogenic 
fungi, and protecting the fungus from the environment, thereby increasing 
viability of the culture and minimizing contamination of the fungal 
culture. 
The devices described below provide a convenient, non-toxic and reliable 
method for the administration of entomopathogenic fungi in an economical 
and cost-effective fashion. The small, lightweight infection chambers are 
unobtrusive and are easily placed in locations of heavy insect 
infestation, increasing the efficacy of the device. Because the devices 
provide an environment within which the fungus can flourish over extended 
periods of time, a single device is effective for a longer period of time 
than with other methods, such as spraying, where effectiveness of the 
agent dissipates over a short time. The longevity of the devices also 
decreases the number of applications and maintenance time required for 
effective treatment. Another advantage of the devices is that they are 
constructed of readily available and relatively inexpensive materials, 
which insures an abundant supply of cost-effective devices. 
Although described with reference to flies, especially the common housefly 
M. domestica, the term "flies" is used to refer to any type of flying 
insect which will enter the device and be infected by the entomopathogenic 
fungi. Examples of flying insects include other flies such as the little 
housefly (Fannia canicularis), tsetse fly, Mediterranean fruit fly, and 
Oriental fruit fly, wasps, white flies, and the adult forms of some 
insects, such as the corn rootworm, Diabrotica undecempunctata. 
In a preferred embodiment, the flies are infected by exposure to the fungus 
in small chambers having apertures through which the flies enter and exit. 
A fly enters the chamber either as the result of general exploration or, 
more generally, as the result of being lured inside the device by the 
action of fly attractants (such as food sources, pheremones, or the color 
and shape of the chamber). Once inside the chamber the fly comes in 
contact with the entomopathogenic fungus. The conidia of the pathogen 
attach to the body of the fly. The infected fly leaves the chamber. 
Conidia attached to the fly's integument can be dislodged and may 
contaminate the habitat, thereby exposing additional flies to infectious 
spores. Further, after the fly dies and the fungal mycelium sporulates on 
the body of the insect, other flies can be infected by exposure to the 
conidia produced on the dead insect. 
As diagrammed in FIG. 1, an infection chamber 10 can be constructed using 
standard technology to form a container 12 for fungal culture medium 14 
and a cover 16 for the chamber, having openings 18 allowing insects free 
access to the interior of the chamber. The fungus grows on the medium 14, 
forming mycelia 20 and spores 22. A food attractant 24 is located on the 
interior of the chamber 10, in close proximity to the spores 22. The 
attractant is optionally located on a platform secured to the container 12 
or the cover 16 to avoid direct contact with the fungus, which can serve 
as a landing platform for the flies. The moisture content can be regulated 
by the design of the chamber, for example, by the size and number of 
openings. In the preferred embodiment, the chamber is hung via a hook 28 
in a location most likely to attract flying insects. 
The chamber can be constructed using conventional materials, including 
glass or metal, but is preferably constructed of an extrudable or moldable 
plastic to keep costs to a minimum. The chamber must have openings large 
enough to allow free passage of the insects. The top of the chamber 
preferably fits securely over the bottom, or the chamber is constructed of 
one piece. The location of food attractants and landing platform, if any, 
should be such that the insects are forced into close contact with the 
spores. The chamber can be designed so that the fungus grows on the 
bottom, top and/or sides of the chamber, to maximize infectivity. The 
insects are infected when they contact the fungus in the chamber, or when 
during grooming from spores acquired on their feet. 
Suitable culture media are known which can be used in the chamber. Examples 
of media known to those skilled in the art and which are commercially 
available include potato, dextrose, agar, or rice agar. 
An example of a useful culture medium for Metarhizium and Beauveria 
consists of 1% dextrose, 1% yeast extract, 5% rice flour, 1.5% agar and 
0.5% 5.times. Dubois sporulation salts. The 5.times. Dubois sporulation 
salts consists of 15 g (NH.sub.4).sub.2 SO.sub.4 /1000 ml; 0.30 g 
MgSO.sub.4 7H.sub.2 O/1000 ml; 0.15 g MnSO. H.sub.2 O/1000 ml; 0.0375 g 
CuSO.sub.4 5H.sub.2 O/1000 ml; 0.0375 g ZnSO.sub.4 7H.sub.2 /1000 ml; and 
0.0038 g FeSO.sub.4 7H.sub.2 O/1000 ml. Each salt is completely dissolved 
before the next salt is added and the solution is autoclaved. 
The culture medium is inoculated with spores of the appropriate fungal 
pathogen (inoculation is accomplished by streaking the surface of the 
medium with an inoculating loop carrying fungal spores or by mixing the 
spores with the liquid medium). After seven days of growth at 28.degree. 
C. with 75% relative humidity, the fungus will have produced a thick layer 
of mycelia and conidia that cover the surface of the culture medium. 
Attractants that are useful will be dependent on the type of flying insect 
to be controlled. For example, attractants for flies include fruit, such 
as raisins, pheromones such as the sex pheromone muscalure, described by 
Carlson and Bereza Environ. Entomol. 2, 555-560 (1973), and synthetic 
compounds, such as the feeding attractant Lursect.TM., McClaughlin, 
Gormley and King Co., Minneapolis, Mn. The shape and/or color of the 
chamber, as well as the location of the chamber, can also be used to 
attract flying insects. Three studies conducted on the spatial and 
temporal responses of flies to attractive bait, and the attractiveness and 
formulation of different baits, are reported by Willson and Mulla, in 
Environ. Entomol. 4(3), 395-399 (1975) and 2(5), 815-822 (1973) for Musca 
domestica and by Mulla, et al., Environ. Entomol. 66(5), 1089-1094 (1973). 
At least two species of entomopathogenic fungi have been shown to be 
effective in control of the housefly, Metarhizium anisopliae and Beauveria 
bassiana. Others that should be useful are fungi that are easy to grow on 
artificial media and quickly grow and produce large amounts of conidia. 
Examples include Verticillium and Paecilomyces spp. 
The following non-limiting examples demonstrate the efficacy of the 
infection chambers in controlling flies. In all cases the fly populations 
were significantly reduced by the fungus present in the infection 
chambers. 
EXAMPLE 1 
Infection of Musca domestica with Fungi in Infection Chambers 
House fly pupae were placed in closed cages that had either a fly chamber 
with sporulating fungus (treatment chamber) or a control chamber without 
fungus. Vials containing sugary water, cotton, and powdered milk were 
provided in each cage to assure that the adult flies had an energy source 
and water when they emerged from the pupae. 
After the adult flies emerged, mortality was recorded daily and plotted. 
Selected dead flies from the treatment chamber were surface-sterilized, 
examined under the microscope and found to be infected, and incubated in 
wet chambers to ascertain whether the entomopathogenic fungus that was in 
the treatment cultures would grow from the dead flies. 
Exposure of the adult flies to the chambers containing either the fungi 
Metarhizium anisopliae or Beauveria bassiana resulted in a significant 
reduction in survival of adult house flies as compared to flies exposed to 
chambers without fungus, as shown by FIGS. 2 and 3, respectively. FIG. 2 
summarizes the results of the study where flies were exposed to M. 
anisopliae. 80% of the flies were dead after only five days; almost 100% 
were dead by seven days following exposure to the fungus. 
Formaldehyde-killed fungus did not result in a greater mortality than 
controls exposed to the chambers without fungus. FIG. 3 summarizes the 
results of the study where flies were exposed to B. bassiana. Essentially 
100% of the flies were dead by four days following exposure to the fungus. 
Dead surface-sterilized flies from the treatment chambers where flies were 
exposed to B. bassiana were found to contain fungus inside of the opened 
bodies. Control flies not exposed to the fungus did not contain fungus. 
This demonstrates that the fungus infected the flies and invaded the flies 
internally before they died. 
EXAMPLE 2 
Infection of Fannia canicularis with Fungi in Infection Chambers 
Fly pupae were placed in closed cages. One week after emergence either a 
fly chamber with sporulating fungus (treatment chamber) or a control 
chamber without fungus were added to the cage. Vials containing sugar, 
powdered milk, water and cotton were provided in each cage to assure that 
the adult flies had an energy source and water when they emerged from the 
pupae. Fungi were obtained from the American Type Culture Collection, 
12301 Parklawn Drive, Rockville, Md. 20852, USA, where they are available 
without restriction. 
After the adult flies emerged, mortality was recorded daily and plotted. 
Exposure of the adult flies to the chambers containing either of two 
strains of the fungi Metarhizium anisopliae or Beauveria bassiana resulted 
in a significant reduction in survival of adult house flies as compared to 
flies exposed to chambers without fungus, as shown by FIGS. 4 and 5, 
respectively. FIG. 4 summarizes the results of the study where flies were 
exposed to M. anisopliae strains 62176 and 38249. 80% of the flies were 
dead after only six days; almost 100% were dead by eight days following 
exposure to the fungus. FIG. 5 summarizes the results of the study where 
flies were exposed to B. bassiana strains 24318 and 48585. Essentially 
100% of the flies were dead by four days following exposure to the fungus. 
EXAMPLE 3 
Control of Musca domestica in chicken coops using chambers containing 
Metarhizium anisopliae 
The effectiveness of the chambers containing fungus for control of flies 
under field conditions, in contrast to laboratory conditions, was 
determined using two chicken coops 12'.times.12'.times.6', containing 20 
chambers per coop, fresh chicken and cow manure, and 10,000 M. domestica 
flies. 100 flies were removed per coop four, eight, eleven and fifteen 
days after exposure to the chambers and reared in the laboratory to 
determine mortality. Fifteen paper sheets (8.5".times.11") were placed in 
each coop for counting resting flies. Fifteen 3".times.5" cards were 
placed in each coop for counting fecal and vomit spots as an indicator of 
the number of flies remaining after exposure to the chambers. 
The results, graphically shown in FIG. 6, demonstrate that 100% mortality 
was achieved of all flies collected from the coops having chambers 
containing fungus. The results shown in FIG. 7A of the numbers of resting 
flies indicate a 78% reduction in flies by the fifteenth day. The results 
shown in FIG. 7B of the numbers of vomit spots and feces indicate an 80% 
reduction in flies by the fifteenth day of the study. 
Modifications and variations of the method and device for biological 
control of flying insects using entomopathogenic fungi will be obvious to 
those skilled in the art from the foregoing detailed description. Such 
modifications and variations are intended to come within the scope of the 
appended claims.