Biological Control Options for Fly Control in Poultry Facilities

Progress report for LNE19-380

Project Type: Research and Education
Funds awarded in 2019: $224,003.00
Projected End Date: 03/31/2022
Grant Recipient: Pennsylvania State University
Region: Northeast
State: Pennsylvania
Project Leader:
Dr. Erika Machtinger
Pennsylvania State University
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Project Information

Performance Target:

Forty poultry producers, representing ~640,000 birds will adopt an IPM plan for fly control. Twenty will document decreases in fly numbers and 20 will save $200 by reducing chemical pesticide application for summer fly control.

Introduction:

Pennsylvania ranks second in the production of eggs in the US (USDA-NASS 2012) with 9,539-layer hen farms, including 586 farms that are organic/exempt. Despite its major role in the layer industry, it has been over 20 years since poultry arthropod control research has been conducted in this state. High populations of fly pests of poultry facilities present biosecurity risks of national concern. Muscoid flies are competent vectors of >100 pathogens including Salmonella, E. coli. (Mian et al. 2002), Coronavirus, New Castle disease (Malik et al. 2007) and potentially avian influenza virus (Wanaratana et al. 2013, Habibi et al. 2018). Of the more than 9 million foodborne illnesses in the United States, 22% are attributed to poultry (Painter et al. 2013). To offset these pathogen risks, the FDA egg safety rule requires the management and monitoring of flies as part of food safety regulations.

House flies are notorious for developing insecticide resistance. As a result, flies can’t be managed on most poultry farms using the traditional insecticides including organophosphates, carbamates, pyrethroids, diflubenzuron, and cyromazine (Boxler and Campbell 1983, Scott et al. 1999, Plapp 1990, Kaufman et al. 2001, Butler et al. 2007, Kozaki et al. 2009, Memmi 2010, see literature review for more detail). It has been estimated that the poultry industry spends about $22.5 million/annually on insecticides for fly control or $0.07/bird, exclusive of labor costs and other fly management efforts (FDA 2009). Losses due to house flies alone exceed $375 million/year (Geden and Hogsette, 2001). The country’s poultry producers face a pest control crisis with few options to protect animal welfare and human health.

Biological control with biopesticides and parasitoids is a safe and environmentally friendly way to manage flies. Fungi have been shown to have good efficacy, the only commercial mycoinsecticide available for poultry is “balEnceTM.” This product has produced disappointing results in both lab and field trials (Kaufman et al. 2005; Machtinger et al. 2016). A better fungal product is needed that will be as effective as conventional insecticides, and education is required on the use of biological control for fly control.

Poultry producers were surveyed in 2017 and 2018 via distribution through the membership of PennAg, integrators, and social media and responses were collected by mail and surveymonkey.com for a total of 215 responses. The survey was opportunistic and reached out to the poultry community, so a total response rate is not known but represented approximately 2,167,691 birds based on range responses.

Flies were listed by 77.6% (n=167) producers as a major issue. Over a quarter of respondents currently use biological control (26.0%, n=57), validating the need to test products for non-target arthropod safety, and 17.2% have at least tried biopesticides. Many respondents support the development of new biopesticides (69.7% n=150); 22.7% (n=49) responded with “maybe,” depending on cost. Cumulatively, 75.3% of survey takers would use a new biopesticide. However, 14.8% stated that they maybe would use a new biopesticide but would require
more education on safety and use to do so. Most respondents (71.2%; n=153), were willing to take a short course in poultry pest management if it was online. Many had time commitment concerns for in-person education.

To address this missed opportunity, the research proposed will develop a better biopesticide for fly control efficacy and application. The education portion will be developing a short course in poultry fly pest management using an IPM framework and will focus on reducing insecticide applications by educating producers on available biological control options and effective use strategies.

Cooperators

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  • Dr. Christopher Geden (Researcher)
  • Dr. Gregory Martin (Educator)

Research

Hypothesis:

A virulent isolate of Beauveria bassiana can be developed from poultry-specific fungi isolated from house flies in Pennsylvania that will reduce fly kill times to <3 days. This isolate can be formulated as part of a bait and developed in an attract-and-kill/autodissemination device that will protect the fungi from environmental effects and will distribute the fungi in the environment, reducing cost and application time for the producer, while reducing pest fly numbers.

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

Objective 2: Screen isolates for high virulence against house flies and select for greater virulence in new isolates.

Objective 3: Test high-virulence strains for compatibility with beneficial insects

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

Materials and methods:

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

Treatments: Eight layer facilities will be identified in Pennsylvania with fly concerns.

Methods: At least 100 individual flies (house flies, little house flies, black dump flies) will be collected from poultry layer houses for a goal of 1,000 flies minimum. These flies will be held in individual cages by farm in an environmental chamber at 25 oC.Data collection: Dead flies will be removed from cages every 2-3 days, surface sterilized, and placed in 96-well plates. Moisture will be maintained with moistened kim wipes. Plates with spore-laden fly cadavers will be shipped to the USDA, ARS (Geden) laboratory for screening and selection. Cages will be bleached and dried under UV light between farm collections to prevent contamination. No statistical analysis will be performed as it is a discovery-based survey of local isolates of Beauveria bassiana.

Objective 2: Screen isolates for high virulence against house flies and select for greater virulence in new isolates.

Treatments: Spore-laden house fly cadavers collected from poultry facilities in Pennsylvania. As reference standards, we will also test two commercial B. bassiana strains (GHA and HF23) as well as a strain that was originally collected from flies at a dairy farm (L90).

Methods: Individual spore-laden cadavers will be used to start pure B. bassiana colonies by streaking spores on Sabaraud Dextose Agar with antibiotics. Colonies will be propagated on SDA agar with 1% yeast extract. Spores will be harvested from plates mechanically, sieved to remove mycelial clumps, and stored at 34 oC until tested. Strains will be tested by preparing suspensions of 1 and 10 mg of spores per ml of 0.01% Tween, applying 1 ml of these suspensions to disks of filter paper, and allowing the papers to dry in a laminar flow biosafety cabinet. Filter papers will be placed in Petri dishes, and groups of 20, 2- to 4-day old female flies will be placed in the dishes for 24 hours. Flies will be fed and watered via a cotton ball soaked with 10% sucrose placed over a hole in the Petri dish lid. After 24 hours, flies will be transferred to small holding cages and monitored daily for mortality for 7 days. A second replication will be conducted for each strain from a different production batch of spores. We anticipate testing at least 20 strains in this manner, and the time to 50% mortality (LT50) will be calculated for each strain. As reference standards we will also test two commercial B. bassiana strains (GHA and HF23) as well as a strain that was originally collected from flies at a dairy farm (L90). Selections will be conducted on the three most promising (fastest-killing) strains of B. bassiana from the preceding strain screening. To do this, flies will be exposed to 10 mg spores/ml in the manner described previously. Data collection: Mortality from B. bassiana typically follows a bell curve, with the first dead flies appearing on day 4 and most on day 6. We will remove the first flies that die in each set and use them to start a new culture. This process will be repeated for 10 generations, selecting the earliest-dying flies each generation. At the end of the selection we will compare LT50s of selected with unselected strains to determine whether the selection was successful in accelerating the death rate.

Objective 3: Test high-virulence strains for compatibility with beneficial insects

Treatments: Colonies of Muscidifurax raptor, Spalangia cameroni, S. endius (parasitoids), and the predator Carcinops pumilio will be established.

Methods: Natural enemies will be tested in standard glass vial assays at varying fungal concentrations as detailed in Objective 2. Ten 24-h-old females will be placed into vials treated with one of the respective concentrations and a control vial (five glass vials total per replicate). Parasitoids and beetles will be exposed for 24-hours and removed for observation.

Data collection: Cups will be checked on days 4, 6, an 8 for mortality. Cadavers will be observed for sporulation. The experiment will be replicated 5 times. Susceptibility to fungal exposure will be analyzed for each species individually by fitting a generalized linear model for a binomial distribution with a logit link. Least significant difference tests will be used to compare sporulation (proportion) between the control and fungal treatments (?? =0.05).

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

Treatments: Non-nutritive sugars (erythritol and xylitol) will be evaluated laced with Beauveria conidia from the isolates selected from Objective 2. Effective bait will be tested in acrylic designs for use by house flies both with and without B. bassiana, and with sucrose as a positive control.

Methods: Fungal efficacy will be tested in different concentrations in the sugars to determine fly susceptibility, as well as attraction to the bait in the presence of other food items. House flies will be tested for ability to transfer fungal sports from treated individuals to untreated individuals. For these tests, male and female flies will placed in cages with Beauveria – laced sugars for susceptibility tests, and only males for dissemination tests. Bait will be weighed daily to determine consumption. Dead flies will be placed on moistened filter paper in a petri dish and kept for sporulation. For dissemination, after 24-h exposure to sugar with Beauveria, one male will be placed in a cage with 20 females. Dead flies will be removed and processed as above. Three versions an autodissemination device will be constructed from acrylic and plastic materials. The devices will be tested for fly acceptance in varying lengths and widths, as well as orientation. Fly use of the device will initially be tested with sucrose sugar. After a successful design is completed, the successful bait formulation in the above trials will be tested in the prototype structures against a nutritive sugar control.

Data collection: LT50s will be determined in bait trials to evaluate effective combinations and dose. Bait formulations will be tested for longevity and shelf life. Probit analysis will be used to compare LD50 times of fungal concentrations by dose and sugar type. For autodissemination devices, differences in bait use and number of flies will be evaluated with t-test if assumptions of normality are met, and by Mann-Whitney if
assumptions are not met.

Research results and discussion:

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

Results:

During the first collection season (2018), 5,366 filth flies were collected (Table 2).  Of these, 4,009 were house flies, 1,192 were black dump flies (Hydrotea aenescens, Diptera: Muscidae,Wiedemann 1830), and 165 werelittle house flies (Fannia canicularis, Diptera: Muscidae, Linneaus 1761), and other species. From these collections 662 candidates were chosen for further testing and and five isolates were confirmed to be B. bassiana. This gave an overall infection rate of 0.09% .Of these five, four were house flies from a June 8th 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.  The majority of specimens that displayed Beauveria-like mycoses and all of confirmed isolates were from collection sites in Southeastern Pennsylvania. 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.

 

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 due to the fact that one of the locations was no longer accessible due to a new flock.

 

June

July

August

Site

Musca domestica

Hydrotea aenescens

Musca domestica

Hydrotea aenescens

Musca domestica

Hydrotea aenescens

1

89

555

29

99

84

59

2

310

3

196

5

-*

-*

3

546

8

347

0

16

2

4

210

0

109

0

130

0

5

242

0

391

0

364

0

6

210

1

261

3

247

0

7

22

220

55

115

151

122

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 8th, and four were even from the same location.

 

June

July

August

Site

Flies (N)

Flies Infected (N/%)

Flies (N)

Flies Infected (N/%)

Flies (N)

Flies Infected (N/%)

1

653

0

261

0

143

0

2

313

0

210

0

-

-

3

562

0

355

0

16

0

4

210

0

109

0

130

0

5

242

0

391

0

364

0

6

211

4/1.8%

264

0

247

0

7

242

1/0.4%

170

0

273

0

[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 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 indicates 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 fungi 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.

Results: 

Overall, selections as of the 9th generation resulted in a shortening of the average time until death of 3 days, from 7.6 to 4.7, at the end of the 9th generation. Results varied considerably with each generation, however, and seemingly anomalous results in the 10th generation gave the appearance of loss of the faster kill times. This is because the quality of the cadavers varied with each passage. Some batches of cadavers produced sparse blooms with reduced spore loads, with the result that the next generation of flies was exposed to reduced pressure by the B. bassiana. Sadly, this was the case with the cadavers after the 9th selection, which resulted in longer kill times in generation 10. Perhaps the more important lesson from this test was that some strains were consistent high-performers. Strain PSU 5 consistently delivered fast kill rates throughout the test and nearly always produced lushly sporulated cadavers with heavy conidial loads. A close second was strain PSU 1, although this strain’s performance was more variable and resulted in cadavers with lower conidial loads. Final analysis of the selected strains has been postponed due to laboratory closure as a result of the COVID-19 pandemic.

 

Results of selection for faster-killing strains of B. bassiana by exposing flies to cadavers from the earlies-dying cadavers from the previous passage for 10 generations.

Generation

number

Time to 50% mortality after exposure of 200 female flies to 10 cadavers of strain:

 

PSU 1

PSU 2

PSU 3

PSU 4

PSU 5

average

1

5

8

6

>9

5

7.6

2

5

6

5

>9

5

7

3

7

6

5

8

4

6

4

6

6.5

5

5

5

5.5

5

4

5

4

5

5

4.6

6

9

4

6.5

>9

5

7.3

7

8

5

5.5

6

4.5

5.8

8

5

5

5

5

4

4.8

9

5

4.5

4.5

4

5.5

4.7

10

4.5

>9

4.5

>9

6

7.8

 

 

 

 

 

 

 

Strain average

5.85

6.2

5.1

8.5

4.9

 

 

 

 

 

 

 

 

Objective 3: Test high-virulence strains for compatibility with beneficial insects

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. The next highest mean survival time was GHA at 8.28 days, and the lowest was L90 at 5.67 days. There were three parameters compared with the log rank test. The first of these was the strains vs the control, to confirm that an affect 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). In the pair-wise test, every treatment was significantly different from the control, with the rest of the strains demonstrating varying degrees of significant differences from each other.  Looking at the efficacy overall, the ranking was L90> PSU5>PSU4>PSU1>GHA. PSU1 has the lowest mean survival time among all parasitoid species. 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. Whencompared 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. Among the 3 parasitoid species, all strains of B. bassiana had the most effect on S. Cameron i2=55.93 ; DF =5 P=<0.001 )followed by M. raptor( χ2=38.39 ; DF =5, P=<0.001 ), and S. endius 2=11.95 ; DF =5 P=<0.001 )

 

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 no 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.

 

Comparisons

Χ2, p-value 

M. domestica 

S. cameroni 

S. endius 

M. raptor 

Treatment vs. control

428.67, <0.0001

 

55.393, <0.0001

 

16.46, 0.006

 

38.39, <0.0001

GHA vs House fly isolates

 

 

76.85, <0.0001

 

2.15, 0.708

-

 

4.62, 0.327

House fly isolate vs. House fly isolate

 

47.00, <0.0001

-

-

 

Mean survival time for house flies 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 GHA being the least efficient and L90 being the most efficient in killing house flies.

Treatment

Mean survival time (14 days)

Std. Error

Lower Bound

Upper Bound

Control

13.05 a

0.18

12.69

13.41

GHA

8.28 b

0.28

7.73

8.84

PSU1

7.76 b

0.23

7.31

8.21

PSU4

7.04 b,c*

0.24

6.57

7.50

PSU5

6.22 c,d

0.21

5.82

6.62

L90

5.67 d

0.20

5.28

6.06

*Significant value in pairwise comparison between PSU4 and PSU5 is p= 0. 0013, close to significant

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.

Treatment

Mean survival time (14 days)

Std. Error

Lower Bound

Upper Bound

Control

8.57a

0.32

7.94

9.21

GHA

6.09b

0.25

5.62

6.57

PSU1

5.63b

0.23

5.18

6.08

PSU4

5.88b

0.27

5.34

6.41

PSU5

6.13b

0.29

5.56

6.70

L90

5.73b

0.31

5.13

6.33

 

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.

Treatment

Mean survival time (14 days)

Std. Error

Lower Bound

Upper Bound

Control

7.479a

0.299

6.893

8.066

GHA

6.403a b

0.279

5.857

6.949

PSU4

6.151a b

0.269

5.624

6.678

PSU5

6.456 a b

0.311

5.847

7.065

L90

6.911a b

0.339

6.247

7.575

PSU1

6.036 b

0.238

5.570

6.503

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.

Treatment

Mean survival time (14 days)

Std. Error

Lower Bound

Upper Bound

Control

7.872a

0.334

7.217

8.528

GHA

5.355b

0.329

4.710

6.001

L90

5.476b

0.324

4.842

6.110

PSU4

5.706b

0.318

5.083

6.330

PSU5

5.335b

0.330

4.687

5.983

PSU1

5.198b

0.261

4.687

5.710

 

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 in an amount that 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 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 of 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 are 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 the house flies. Male house flies have 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. Which 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 a 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 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, as 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.

In progress but delayed due to the COVID-19 Pandemic

Participation Summary
9 Farmers participating in research

Education

Educational approach:

The proposed educational solution to the problem of pest control on poultry facilities in Pennsylvania is to create a short-course in poultry pest management focused on biological control options and appropriate use for fly control, hosted by Penn State Extension. The outline of the online short course was created and will include

Introduction

Section 1: Damage caused by pest flies in poultry facilities

Section 2: Integrated Pest Management

Section 3: IPM step 1: Pest fly identification

Section 4: IPM step 2: Pest fly life cycles

Section 5: IPM step 3: Monitoring

Section 6: IPM step 4: Action thresholds

Section 7: IPM step 5: Deciding on control methods

Section 8: IPM Control: Cultural Control

Section 9: IPM Control: Physical and Mechanical Control

Section 10: IPM Control: Biological Control

Section 11: IPM Control: Chemical Control

Section 12: IPM: Putting it all together

Section 13: Extra module: Natural Resources professionals

Final thoughts

Resources

Glossary

 

Milestones

Milestone #1 (click to expand/collapse)
What beneficiaries do and learn:

Establish monitoring and record keeping - 20 producers will establish a fly monitoring program or continue a fly monitoring program and will maintain records on fly control expenditures.

Proposed number of farmer beneficiaries who will participate:
20
Proposed Completion Date:
August 31, 2019
Status:
In Progress
Accomplishments:

A survey has been developed to assess producer use of IPM measures to control pest flies. This will correspond to a post-use survey after the short-course has been completed. 

Milestone #2 (click to expand/collapse)
What beneficiaries do and learn:

Pilot Testing - Ten producers will be given the pilot of the completed short-course and feedback will be

Proposed number of farmer beneficiaries who will participate:
10
Proposed Completion Date:
April 30, 2020
Status:
In Progress
Accomplishments:

Due to COVID-19, the production of the short-course was delayed. The course was unable to be filmed because services were shut down. However, Sections 1-4 of the short course have now been filmed and the course is scheduled to be completed in April 2021. 

Milestone #3 (click to expand/collapse)
What beneficiaries do and learn:

Recruitment - At least 500 poultry producers will receive direct mailings, emails, or social media information for registration. Advertisements will be placed with/in poultry organizations and magazines.

Proposed number of farmer beneficiaries who will participate:
500
Proposed Completion Date:
October 31, 2020
Status:
In Progress
Accomplishments:

Due to COVID-19, the production of the short course was delayed. Recruitment is scheduled for early spring 2021

Milestone #4 (click to expand/collapse)
What beneficiaries do and learn:

Pre-training engagement - At least 100 poultry producers will register and complete the short-course (prior to the emergence of most poultry pests). These participants will answer questionnaire questions regarding current practices and take a pre-test to analyze their current
knowledge.

Proposed number of farmer beneficiaries who will participate:
100
Proposed Completion Date:
March 31, 2021
Status:
In Progress
Accomplishments:

Nothing to report

Milestone #5 (click to expand/collapse)
What beneficiaries do and learn:

Learning through the education program - 100 registrants will:
• Work through specific fly pest of poultry modules.
• Gain knowledge on biological information of fly pests of poultry (as described in the educational approach).
• Learn about monitoring methods, and action thresholds/economic thresholds.
• Learn about integrated pest management, and the ways to control fly pests of poultry.
• Develop an IPM plan for their farm for fly pests and learn about methods to evaluate that plan.
• Receive a certificate of completion.

Proposed number of farmer beneficiaries who will participate:
100
Proposed Completion Date:
March 31, 2021
Status:
In Progress
Accomplishments:

Nothing to report

Milestone #6 (click to expand/collapse)
What beneficiaries do and learn:

Support to take follow-up action - 100 poultry producers will submit their IPM plans to the educators at the completion of the course. All 100 will have access to educators will be provided throughout the course.

Proposed number of farmer beneficiaries who will participate:
100
Proposed Completion Date:
March 31, 2021
Status:
In Progress
Accomplishments:

Nothing to report

Milestone #7 (click to expand/collapse)
What beneficiaries do and learn:

At least 100 producers will visit the “Bug Bytes” blog during the first project spring and summer and 200 the second project spring and summer.

Proposed number of farmer beneficiaries who will participate:
100
Proposed Completion Date:
September 30, 2022
Status:
In Progress
Accomplishments:

Nothing to report

Milestone #8 (click to expand/collapse)
What beneficiaries do and learn:

Verification of actions and resulting benefits - 75 producers will respond to verification survey to report actions taken to develop and execute an IPM plan and incorporate biological control for fly management.

Proposed number of farmer beneficiaries who will participate:
75
Proposed Completion Date:
September 30, 2021
Status:
In Progress
Accomplishments:

Nothing to report

Milestone Activities and Participation Summary

Educational activities:

1 Other educational activities: Development of course content for short-course in poultry pest management.
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.