Biological Control Options for Fly Control in Poultry Facilities

Objective 1: Identify new strains of B. bassiana collected from poultry facilities.

During the first collection season (2018), 5,366 filth flies were collected.  Of these, 4,009 were house flies, 1,192 were black dump flies (Hydrotea aenescens, Diptera: Muscidae,Wiedemann 1830), and 165 were little house flies (Fannia canicularis, Diptera: Muscidae, Linneaus 1761). The remainder were other species that were not identified (Table 1). From these collections, 662 fungal candidates were chosen for further testing and five isolates were confirmed to be B. bassiana. This gave an overall infection rate of 0.09%. Of these five isolates, four were house flies from a June 8 collection in Marietta, PA (0.01 % infection in house flies) and one was a black dump fly from a collection in Manheim, PA (0.08% infection in black dump flies) on the same day.  Most specimens that displayed Beauveria-like mycoses and all of the confirmed isolates were from collection sites in Southeastern Pennsylvania (Table 2).

Samples from 2019 were held in the same environmental chamber as the previous year. Unfortunately, a separate part of this overall project was added shortly after the season started, which involved rearing large numbers of house fly immatures in this chamber. There is a good amount of ammonia produced by the combination of house fly larvae and the media they are reared in and placing them in a warm enclosed space caused the ammonia levels to become very high. I suspect that these high ammonia levels prevented any fungus from sporulating, as no cadavers were salvageable, and no data was recorded for the summer of 2019.

Table 1: Total number of Musca domestica and Hydrotea aenescens caught at layer facilities in Central and Southeastern Pennsylvania in the summer of 2018. June had the highest number of collections for both groups, with August having the least. This may be partly because one of the locations was no longer accessible due to a new flock.

Table 2: Total number of flies from all species caught at layer facilities in Central and Southeastern Pennsylvania in the summer of 2018, with accompanying infection rates by Beauveria bassiana. Of the 5,366 samples recovered, only five were confirmed to be infected with B. bassiana¹. Of those five, all of them were caught on the same day, June 8^th, and four were even from the same location.

[1] House flies were exposed to the isolates collected, and any that demonstrated the ability to cause mortality in house flies were selected for further PCR testing. This is how the isolates were confirmed to be B. bassiana.

Discussion

Biological control using entomopathogenic fungi such as B. bassiana could become a valuable part of pest fly IPM. This potential could be further improved by using strains that are isolated from the target pest itself (Maurer et al. 1997). The discovery of naturally occurring strains of B. bassiana on house flies at layer poultry facilities in Pennsylvania is a promising step for developing more effective biological control strategies using this pathogen.

While B. bassiana has been reported to occur in house fly populations (Steinkraus et al. 1990) rates of natural infection appear to be very low (Siri et al. 2005). Only house flies that displayed outward signs of infection, visible fungal growth on the cadaver, were sent for further screening so it is likely that some cases of B. bassiana infection were missed due to lack of sporulation. The infection rate in the current study, 0.09%, was in line with other studies (Steinkraus et al. 1990, Steenberg et al. 2001, Skovgård and Steenberg 2002, Siri et al. 2005), but those studies have a wide range, from as low as 0.06%,(Steinkraus et al. 1990) or as high as 1.45% (Siri et al. 2005). The percentage was calculated from the 5 strains confirmed to be able to kill flies in preliminary testing and then confirmed by PCR. The reason for this typically low infection rate may be certain environmental factors or differences between host populations. Given the low natural infection rates demonstrated in the literature, paired with the difficulties associated with applying the fungi to poultry facilities, it is encouraging to have recovered as much as we did to demonstrate that natural strains can and do occur in these environments.

Considering the low infection rates among house flies presented here and in accompanying literature, questions arise as to the reservoir for the pathogen. With such low numbers in house flies, it may be possible that they may not wholly be the source for the spread of B. bassiana amongst themselves. If the case is that the infection is not being brought in by house flies from the outside, and the rest of the arthropods present in the manure do not have a great range, it is possible that the fungus is continuing its life cycle in the poultry house in another way. In the future it may be interesting to look at beetles in poultry houses for the presence of B. bassiana, with both Carcinops pumilio and Alphitobius diaperinus as possible vectors. Since B. bassiana can be produced in large amounts commercially on substances like corn or other grains (Karanja et al. 2004, Latifian et al. 2014), the viability of spores grown on chicken feed (pre- and post-digestion) should be researched.

Some of the difficulties associated with B. bassiana growth in poultry houses are related to the high ammonia levels produced by the accumulated manure (Acharya, Rajotte, et al. 2015b). Ultraviolet light can also kill B. bassiana conidia (Daoust and Pereirn 1986, Inglis et al. 1995), which may be an issue in smaller, open-sided poultry houses. Perhaps the main deterrent for  B. bassiana efficiency  in poultry houses is the specific level of humidity required for it to function most efficiently (Walstad et al. 1970, Gillespie and Crawford 1986). Traditionally, manure pits are kept as dry as possible to prevent house fly development. The commercial product balEnce™ recommends applying large amounts of aqueous spray as part of its application. Not only is this a much higher demand on time than a traditional pesticide application, but the increased moisture in the manure leads to higher fly populations. In addition to the issues it has remaining viable in the environment it is applied to, balEnce™ takes 6 days to kill fly pests, giving the flies enough time to breed and start another generation. This suggests that the strain used in balEnce™ is not virulent enough to be used in this context. Future research will have to focus on delivering the fungus in a way that shelters it from the deleterious effects of the manure while also allowing for normal business operations, and on finding an isolate with increased virulence.

Looking at the recovery rate from individual farms, there is an interesting trend that may indicate the degree to which house flies are responsible for transmitting the fungus between each other. As B. bassiana is associated with soil (Hajek, 1999; Keller & Zimmerman,1989; Meyling & Eilenberg, 2007) and can be further spread through mechanisms like wind and rain, more open systems might be expected to have a higher incidence of B. bassiana infections. However, the majority of B. bassiana isolates recovered were from a caged-layer facility with deep manure pits that had solid walls except for the exhaust fans embedded in them (site 6). Conversely, at three of the other collection sites (1, 2, and 3) the ends and sometimes the sides of the manure pit walls had screens in place, which allowed air and rainwater into the manure pit, but none of the confirmed B. bassiana samples came from these locations. Perhaps the increase in ultraviolet light from natural sunlight, or the use of lime at these facilities prevented B. bassiana occurrence.  Additional research is needed to see how the different environmental conditions and designs of layer facilities could affect entomopathogenic fungi. Another factor that may explain this is that these three locations had noticeably lower house fly populations. This would support the idea that the house flies are mainly picking up these infections through contact with other house flies rather than contact with the pathogen in the environment (García-Munguía et al. 2015)

Fungi are diverse in the ecosystem and many species other than the target B. bassiana or M. anisopliae may be recovered. Site 5 had the second highest number of flies captured and of cadavers showing fungal growth yet did not yield any confirmed B. bassiana cadavers. This is especially interesting as Site 6 which was the source of nearly all B. bassiana strains recovered was less than 4 km from site 5. A possible explanation for the lack of B. bassiana could be the high prevalence of Entomophthora muscae at this location. Flies observed on location at site 5 and those brought back to the lab during the late summer months exhibited behavioral symptoms and the mass mortality associated with E. muscae infection (Watson et al. 1993). It’s possible that the presence of this fungus affects the house fly behavior in a way that makes them less likely to encounter B. bassiana, or that infection with E. muscae prevents further infection with B. bassiana. Future studies could look into how co-infection with this common entomopathogenic fungus affects the lethality of B. bassiana.

While the current B. bassiana product labeled for use on poultry has had disappointing returns (Weeks et al. 2017), other strains such as GHA have proven more efficient at killing house flies in comparison (Machtinger et al. 2016; Weeks et al. 2017). At high enough concentrations, GHA has been reported to have a mean survival time of 5-6 d (Anderson et al. 2011; Acharya, Rajotte, et al. 2015a; Weeks et al. 2017) which is faster than the 6-8 d survival range of HF23. If a more virulent strain can be developed it may lead to more effective fly control on poultry facilities using entomopathogenic fungi. These strains could also be tested for other characteristics such as high spore production, performance at higher temperatures, ammonia tolerance, and other factors which would make them well suited for development as a biological control tool. Efficacy of the recovered B. bassiana strains from this project will be evaluated in future studies

Objective 2: Screen isolates for high virulence against house flies and select for greater virulence in new isolates and Objective 3: Test high-virulence strains for compatibility with beneficial insects (**Because objectives 2 and 3 were screened together, results are reported together herein). 

Results

Due to differences in mycoses between strains, and between species, overall mortality was used as a metric instead of visible mycoses on cadavers. All B. bassiana treated house flies had a significantly lower mean survival time than the negative control, which had a mean survival time of 13.05 days (Table 3).

Table 3: Mean survival time for house flies over a 14-day period after exposure to treatments.  All strains were significantly different than the control, with GHA being the least efficient and L90 being the most efficient in killing house flies. 

*Mean survival time with the same letters in did not differ significantly at the α of p <0.001. Significant value in pairwise comparison between PSU4 and PSU5 is p= 0. 0013, close to significant. 

The next highest mean survival time was GHA at 8.28 days, and the lowest was L90 at 5.67 days. The survival curve for all strains is shown in Figure 1. There were three parameters compared with the log-rank test. The first of these was the strains vs the control, to confirm that an effect was taking place. This measure was significant (χ²=428.67; DF=5; P=<0.001). The next grouping that was compared was all the treatment strains against each other, to see if there was a difference in the efficacy of strains isolated from muscoid flies to GHA, which was not isolated from a muscoid fly. These results were also significant (χ²=76.85; DF=4; P=<0.001). The last comparison was between all the fungi isolated from house flies, which also demonstrated a significant difference (χ²=47, DF=3, P=<0.001). Looking at the efficacy overall, the ranking was L90> PSU5>PSU4>PSU1>GHA. 

Among the 3 parasitoid species, all strains of B. bassiana had the most effect on S. cameroni (χ²=55.93 ; DF =5 P=<0.001 )followed by M. raptor( χ²=38.39 ; DF =5, P=<0.001 ), and S. endius (χ²=11.95 ; DF =5 P=<0.001 ) (Table 4).

Table 4: Comparisons of the significant values of the test isolates on house flies and three species of parasitoid. Comparisons of different groups were pooled over strata with a log-rank test. Measurements were taken until a significant value was no longer reached, suggesting any difference between the strains compared. There was a significant difference in house flies between the treatments and the control, between the treatments, and between the treatments isolated specifically from house flies. For S. cameroni and M. raptor, there were only significant differences between the control and the treatments. For S. endius, there was no difference.

PSU1 has the lowest mean survival time among all parasitoid species (Tables 5,6,7). However, highest mean survival time was variable between the three species ranging from PSU5 for S. cameroni, L90 for S. endius, and PSU 4 for M. raptor. When compared again with the log rank test to compare the differences between just the isolate treatments, no significant difference was found for any of the species (S. cameroni , χ² =2.15; DF =4; P=<0.001 ) (M. raptor , χ² =4.63 ; DF =4; P=<0.001 ) meaning that there was little difference in effect between B. bassiana isolates in parasitoids. In considering these results, data taken from over 14d may not be an appropriate measure for these parasitoids as the end of their natural life cycle falls within the treatment time range.

Table 5: Mean survival time for Spalangia cameroni over a 14-day period after exposure to treatments. Pairs of means with the same letters did not differ significantly at the α of p <0.001. All strains were significantly different than the control, with PSU1 being the least efficient and PSU5 being the most efficient in killing S. cameroni, but the difference between these strains was not significant.

Table 6: Mean survival time for Spalangia endius over a 14-day period after exposure to treatments. Pairs of means with the same letters did not differ significantly at the α of p <0.001. The only strain that was significantly different from the control at an α of p<0.001, was PSU1, with p= 0.0075 in a pairwise test against the control. The rest of the strains did not differ significantly from each other.

Table 7: Mean survival time for Muscidifurax raptor over a 14-day period after exposure to treatments. Pairs of means with the same letters did not differ significantly at the α of p <0.001. All isolates were significantly different from the control, but not from each other.

Discussion

Of all the strains of B. bassiana tested, many were shown to have different traits that would be attractive for use as biological control and these traits varied between strains. For example, while PSU 1 had the second highest mean survival time, overall it killed more flies than PSU 4, a strain with a lower mean survival time. Kill rate of PSU 1 was comparable to the more efficient PSU5 and L90. All strains that were originally isolated from muscid flies performed better than GHA, a strain that was isolated from a Chrysomelid beetle. This is interesting, as GHA has been used for over a decade as a commercial application, and has been found to be effective for other insect orders (Ludwig and Oetting 2002, Liu and Bauer 2008). This supports the suggestion that certain strains of B. bassiana show a degree of host specificity (Fargues and Remaudiere 1977, Steinkraus et al. 1990, Viaud et al. 1996, Maurer et al. 1997, Vestergaard et al. 2003).

In house flies, the mortality rate for strains was lower than expected, especially for L90 (Geden et al. 1995, Watson et al. 1995, Weeks et al. 2017, Burgess et al. 2018b, Johnson et al. 2019) and GHA (Anderson et al. 2011) which has yielded high mortality rates in previous studies. The reason for this may have been the delivery method which the test used to expose the house flies to the spores. In theory, only the tarsi of the fly were in contact with the treatment the entire time, with the mouthparts occasionally coming into contact through feeding and grooming behavior. Previous studies suggest that many of the spores present would be removed through grooming or not be effective after germination due to an immune response (Butt et al. 1988, Gillespie et al. 2000). Other attachment points such as the eyes, abdomen, or the base of setae are more effective in initiating infection (Hasaballah et al. 2017). Other studies have exposed the entire surface of the insect to a fungal suspension to determine efficacy (Mwamburi et al. (2010), however, the methods herein were in line with what a fly would encounter in the field. 

Another possible explanation for the lower mean mortality and longer survival times than reported in previous studies may have been due to the formulation used to apply the treatment. Both Weeks et al. 2017 and Anderson 2011 exposed their flies to filter papers in a similar design to this experiment, except that they used oil formulations instead of water and a non-ionic surfactant. Other studies involving house flies have also yielded higher mortality at a faster rate, as well as longer reports of viability when using an oil-based formulation (Acharya, Rajotte, et al. 2015a, 2015b, Acharya, Seliga, et al. 2015). Non-ionic surfactants, while often helpful in improving the stability of the spore suspension, may have detrimental effects on the fungus, while the introduction of oil may stimulate germination (Luz and Batagin 2005). In addition to this, the introduction of oil to a formulation may improve the spore’s adherence to the insect cuticle. Another important factor in B. bassiana virulence is the dose that the insect is exposed to (Watson et al. 1995, Anderson et al. 2011).  All the experiments that yielded higher mean mortality and shorter survival times used a dose of 1x10⁹ spores/ml, the same as this bioassay, or lower even. But if the oil formulations allow for more spores to remain in contact with the insect cuticle than just Tween80 and water, then the effective dose for these experiments is much higher.

In terms of parasitoids, the effective dose of these treatments may also explain why the isolates had less of an effect on them than on the house flies. Male house flies have been shown to be more resistant to B. bassiana exposure than females (Acharya, Rajotte, et al. 2015a). While this may be due to different immune responses between males and females, it may also relate to the fact that females are slightly larger than males. A larger body size means a larger surface area that can pick up more spores. Parasitoid wasps are many times smaller than house flies so they may be getting a reduced dose of spores. Other experiments have also demonstrated reduced effects on parasitoids when directly exposed to B. bassiana (Geden et al. 1995, Lecuona et al. 2007), with one of them only achieving higher mortality when exposing the parasitoids a second time, increasing the effective dose.

Efficacy differences among B. bassiana strains from previous research may have been a result of house fly susceptibility differences by population, perhaps related to immune response. The flies used in this study were caught in poultry houses the year prior, and therefore would better reflect the population that would be found in a field setting than a colony that has been lab reared for many years (Bryant and Meffert 1998). House flies do exhibit an immune response when infected with B. bassiana (Mishra et al. 2015). With possible repeated exposure in the field, these “wild” populations may be better equipped to handle a B. bassiana infection than a colony that has no history of exposure (Dubovskiy et al. 2013). This immune response could be carried in future generations, even without exposure (Trauer and Hilker 2013), which could explain the lower rates of infection from this study. Considering how insular the poultry layer facility ecosystem is, this effect would also have serious considerations for biological control. Certain strains may perform better in certain populations, and those populations could differ from region to region or farm to farm.

While results for the house flies showed considerable variation, the results for all three species of parasitoids were less clear cut. This ultimately may be a good indicator for the use of B. bassiana as part of an IPM program, as we chose these species because they are often incorporated into management programs. For S. cameroni and M. raptor, the difference between the control and the treatment groups was lower than the difference found in the flies, and S. endius had hardly any significant difference at all. Lecuona et al. 2007 also exposed S. endius to B. bassiana with very minimal effects for single dosing, so the interaction between B. bassiana and S. endius, in particular, may be an interesting area for future study. Overall, these lesser effects imply that both tactics could possibly be used together more effectively in an IPM program. This lesser effect is especially beneficial to M. raptor, the species that is more commonly released in poultry facilities (Kaufman et al. 2000). Muscidifurax raptor would be more likely to be exposed to the current commercial application technique of B. bassiana as it spends more time in the upper layers of the manure than the two Spalangia spp. (Geden 2002).

It was not shown that there are any further differences between strains in terms of their effects on parasitoids. Parasitoid mortality was measured over 14 d for all species. Given how different the two life histories of the groups are, further studies may be done with shorter observation ranges to measure fungal efficacy, to better reflect their shorter lifespans. The adult lifespan of both Spalangia genera tested is three weeks while Muscidifurax raptor's is only two, so a mortality measurement over 14 d may not be necessary and a smaller window of time may give better insights to the differences in effectiveness between strains for parasitoids if any exist.

While B. bassiana genotypes do display host specificity (Fargues and Remaudiere 1977, Maurer et al. 1997, Vestergaard et al. 2003), the strains isolated and found primarily in one host can be effective for a range of different species (Butt et al. 2009, Wraight et al. 2010). This suggests that perhaps instead of being well suited to the host, perhaps the specificity arises from the strain being well suited to the environment in which that host is normally found (Uma Devi et al. 2008). Future studies could replicate different conditions found in a poultry house, such as high ammonia levels and high temperatures, to see how they affect lethality in house flies. The increased temperature, in particular, would be an interesting variable to study. In the field house flies can exhibit a form of the fevering behavior seen in which results in lowering the virulence of the B. bassiana infection (Anderson et al. 2013a, Anderson et al. 2013b). Finding a strain that is particularly tolerant of high temperatures would be useful in developing a biological control tool.

Overall, the B. bassiana strains isolated from house flies killed greater numbers of flies than the negative control and GHA. In parasitoids, all strains had a more limited effect than was observed in the house flies, except for in S. endius, in which there was no effect. The susceptibility of these house flies to the treatments and the lack thereof in all parasitoid species is a good indicator of the usefulness of field-collected strains of B. bassiana and their use as a biological control tool. Given that the strains each demonstrated different traits in their infection of house flies, further research should be done to see the extent of each of these traits and if they could be useful for biological control (Gillespie and Claydon 1989, Hajek and St Leger 1994, Kershaw et al. 1999, Anderson et al. 2011).

Objective 4: To develop a novel auto-dissemination method microbial control of house flies.

Results

Sugar feeding trials with house flies were started in 2019. Erythritol and sucrose were used as sugars as xylitol is toxic to birds. Sugar is consumed at a higher rate than erythritol, but erythritol seems to have a lethal effect on flies.

A pattern emerged through the first trials where most treatments with erythritol and the B. bassiana isolates resulted in greater morality than the sucrose with B. bassiana isolates. This was not the case with PSU 5 and L90.  Infectivity of house flies was over twice as high in the erythritol with B. bassiana over the sucrose with B. bassiana, however (Figure 2). This may suggest that there is some component of the erythritol that synergizes with the fungus for greater efficacy.

These trials were not completed as expected. The University required that our fly rearing room be moved on two separate occasions because of incompatibility with other building residents. To make these moves, we had to decrease our rearing scale. The second move occasion required renovations to the room which delayed increasing fly numbers. In addition, because of University policies related to COVID-19, laboratory experiments were significantly reduced throughout 2020 when the MSc student hired to complete this work was available. In addition to these challenges, the Beauveria samples collected proved challenging to rear and propagate and were difficult to combine with the sugars. This slowed the progress of additional replicates. Because of these operational and time constraints, only experiments that the methodology had been confirmed and the outcome positive were completed.

Regardless, these initial results were promising and new research on non-nutritive sugars has shown even more promise as a bait control for flies. These combinations should be explored in the future.

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