TY - JOUR
AU - Geden, Christopher, J
AB - Abstract The house fly, Musca domestica L., is a global pest of public health and agricultural importance. The efficacy of conventional management has been waning due to increasing insecticide resistance. A potential management tool is the entomopathogenic fungus, Beauveria bassiana Vuillemin (Hypocreales: Cordycipitaceae) (strain L90), although time-to-death is slower than desired by potential users. This research investigated the effectiveness of three gram-negative bacteria (Pseudomonas protegens Ramette (Psuedomonadales: Pseudomonadaceae) pf-5, Photorhabdus temperata Fischer-Le Saux (Enterobacteriales: Enterobacteriaceae) NC19, and Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae) DB11) on house fly mortality when topically applied, compared to B. bassiana. Each pathogen’s virulence was measured by injection into adult female house flies or by topical applications to their thorax. All bacterial strains were highly virulent after injection with 1 × 104 colony forming units (cfu), causing fly mortality within 24 h. Beauveria bassiana resulted in high mortality, 3 d postinjection at the high dose of 1 × 104 conidia/µl. Mortality due to topical treatments of P. temperata and S. marcescens was low even at the highest dose of 1 × 106 cfu/µl. Mortality after topical treatments with P. protegens was evident 4 d after application of 1 × 106 cfu/µl. Mortality from B. bassiana was low at 4 d but increased at 5 d. These results imply that P. protegens holds great potential as a biological control agent for incorporation into an integrated pest management program against adult house flies. Pseudomonas protegens, Photorhabdus temperata, Serratia marcescens, biocontrol, entomopathogen The house fly, Musca domestica L., has a close, cosmopolitan relationship with humans, domestic animals and livestock, and consequently poses both public health and agricultural concerns (West 1951, Nayduch and Burrus 2017). House flies serve as mechanical vectors of many pathogenic organisms including bacteria, viruses, protozoa, and helminths to humans and other animals (Sasaki et al. 2000, Nayduch and Burrus 2017). Conventional chemical insecticides have traditionally been the standard management tactic to control M. domestica. However, sustainable successful fly control with specific insecticide active ingredients have been limited because flies readily develop resistance to new insecticide classes, often within a few years of their commercialization (Kozaki et al. 2009, Kaufman et al. 2010, Memmi 2010, Scott et al. 2013, Shah et al. 2015). As insecticide resistance has become a recurrent problem often leaving fly populations unchecked, there is a need for alternative tools to reduce fly populations to acceptable levels. Sanitation of developmental sites and biological control maintenance are important noninsecticidal fly management tools. The search for effective biological control agents for house flies has been intensive for decades, with only minimal success. Most of these efforts have concentrated on the immature life stages, where predators attack egg and larval stages, and parasitoids attack fly pupae (reviewed in Rutz and Patterson 1990, Machtinger and Geden 2018). Adult flies are susceptible to microbial infections, but this approach has received comparatively less attention. Most microbial control research has been completed with entomopathogenic fungi, with limited regards on bacterial pathogens, especially on their mortality effects on adult house flies (Weeks et al. 2018). Beauveria bassiana Vuillemin (Hypocreales: Cordycipitaceae) is an anamorphic entomopathogenic fungal pathogen that is environmentally ubiquitous. It has been reported to infect hundreds of insect species in nature (Lipa 1963), and has been documented to infect a variety of fly species (Steinkraus et al. 1990, Geden et al. 1995, Skovgӓrd and Steenberg 2002). One of the major limitations of B. bassiana for fly control is the delay in death of the host. When exposed to B. bassiana, the average time-to-death for house flies is approximately 6 d, with mortality rates affected somewhat by isolate and delivery method (Geden et al. 1995, Lecuona et al. 2005, Mwamburi et al. 2010). Because female house flies oviposit 4 to 5 d post-emergence, B. bassiana does not break their life cycle. However, sublethal effects of fungal infection likely limit her potential reproductive capacity (Acharya et al. 2015). Photorhabdus temperata Fischer-Le Saux et al. (Enterobacteriales: Enterobacteriaceae) is a gram-negative, bioluminescent, motile, entomopathogenic bacterium belonging to the family Enterobacteriaceae (Wollenberg et al. 2016). This bacterium has a symbiotic relationship with heterorhabditid entomopathogenic nematodes (EPNs). The nematode actively seeks out insect hosts while the bacterium resides in the gut of the nematode protected from environmental harm (Hurst et al. 2015). Once the EPN releases the bacterium into the hemocoel of the insect host, bacterial virulence factors such as proteases, chitinases, and toxins act quickly to cause host death (Duchaud et al. 2003). Together, the nematodes and their symbiotic bacteria are pathogenic to a wide range of insects (Duchaud et al. 2003). The use of Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae) is a potential alternative to chemical insecticides in some instances (Pineda-Castellanos et al. 2015). Serratia marcescens is well known for its mobility and capacity to secrete a multitude of virulence factors, such as proteases and chitinases, making it a promising pathogen for biological control of targeted insects (Grimont et al. 1979). This bacterium has been reported to suppress insect host immunity by manipulating immunosurveillance cells (Ishii et al. 2014, Stout 2015). A recent study described the protease serralysin as one of the causes of high fatality in larvae of Phyllophaga blanchardi (Arrow) (Coleoptera: Melolonthidae) when orally inoculated with S. marcescens (Pineda-Castellanos et al. 2015). Pseudomonas spp. are gram-negative bacteria in the family Pseudomonadaceae with more than 100 distinguished species (Palleroni 2003). One member of the genus, Pseudomonas protegens Ramette et al. (Psuedomonadales: Pseudomonadaceae), is capable of producing a range of plant defending products such as antibiotic metabolites, chitinases, exoproteases, and an insect toxin, FitD, that contributes to the deterrence of insect and fungal plant invaders (Cronin et al. 1997, Ellis et al. 2000, Loper et al. 2016). One study determined that the components contributing to oral toxicity of P. protegens in Drosophila melanogaster Macquart (Diptera: Drosophilidae) were a macrolide, rhizoxin, and a secreted chitinase (Loper et al. 2016). Given that bacterial biological agents have received little attention for house fly control, the goal of this study was to examine the effects of three gram-negative bacterial species and strains (Photorhabdus temperata NC19, Pseudomonas protegens pf-5, and Serratia marcescens DB11) on adult house fly mortality as alternative biological control agents with decreased time-to-death. The objectives of this study were to 1) compare the effects of injecting the selected bacterial pathogens and B. bassiana into the hemocoel of adult house flies; 2) identify a suitable carrier for topical applications that would keep all the pathogens in a viable state; and, 3) determine the effects of all four pathogens when applied topically to adult house flies. Methods House Fly Rearing and Handling The M. domestica Orlando Normal strain that has been reared at the Center for Medical, Agricultural and Veterinary Entomology, United States Department of Agriculture, Agricultural Research Service (Gainesville, FL) since 1958 was utilized for this study. Flies were maintained at 28°C in wire mesh cages (37.5 × 37.5 × 37.5 cm), and fed a diet consisting of dried milk, granulated sugar, and powdered egg yolk 8:8:1 mixture by volume. House flies (2–4 d old) were removed from rearing cages using a vacuum aspirator and placed at −20°C for 10 min to lightly sedate them for injection and topical treatments. Each replicate consisted of 20 female flies. After treatment, each experimental cohort of flies was placed into a screen-covered 500 ml plastic container (Instawares, Kennesaw, GA) containing 50 mg of diet and a 30 ml plastic container with water and a lid with a dental wick (to prevent flies from drowning), and held at 29°C. Entomopathogens All fungal and bacterial strains used in this study are classified as biosafety level one (BSL1), and therefore, they are well-characterized agents that do not cause disease in healthy humans. Beauveria bassiana strain L90, was isolated from house flies collected in upstate New York, and is known to be virulent to house flies (Geden et al. 1995). Beauveria bassiana conidia were preserved in 10% glycerol at −80°C at the University of Florida Institute of Food and Agricultural Sciences (UF/IFAS) Entomology and Nematology Department (Gainesville, FL) prior to culturing. Serratia marcescens strain Db11 is a streptomycin-resistant mutant of strain Db10 (Caenorhabditis Genetics Center, University of Minnesota) isolated from a dying D. melanogaster (Iguchi et al. 2014). Photorhabdus temperata strain NC19 was provided by Byron Adams, Brigham Young University (Provo, UT), has been genetically sequenced, and is predicted to contain many encoded insecticidal toxins (Duchaud et al. 2003). Pseudomonas protegens strain Pf-5 (purchased from American Type Culture Collection, Manassas, VA) was formally classified as P. fluorescens Pf-5 but was reclassified as P. protegens. Escherichia coli strain DH5α was purchased from Invitrogen (Carlsbad, CA). Bacterial and Fungal Cultivation For routine cultivation, all bacteria were maintained on Luria Bertani (LB broth, Fisher BioReagents, Pittsburgh, PA) agar plates. After inoculation, plates were incubated at 28°C, overnight and then placed at 4°C until use. Prior to use in experiments, bacteria were propagated, plates were removed from 4°C, and the respective bacterial colony forming units (cfu) were picked with a sterile loop and placed into a 10-ml conical tube containing 3 ml of LB broth to start an ‘overnight culture’. The overnight culture then was placed in a controlled environment shaker (New Brunswick Scientific, Edison, NJ) at 250 rpm and 28°C, for 15 h, overnight. The culture was transferred into a 50 ml glass flask in a 1:20 dilution with LB broth and grown to an optical density of 0.5 measured using a spectrophotometer (Biochrom LKB Ultrospec II; Cambridge, United Kingdom) at an absorbance of 600 nm (OD600). To quantify the bacterial concentrations in broth, 10-fold dilutions of a culture grown to an OD of 0.5, 100 µl of each dilution was plated and incubated at 26°C for 24 h. From these plates, bacterial enumeration was determined by selecting a plate that had between 30 and 300 visible cfu and recording the dilution concentration of that plate. Pseudomonas protegens at 0.5 OD600 measured 1.5 × 108 cfu/ml, S. marcescens at 3.0 × 108 cfu/ml, P. temperata at 2.1 × 108 cfu/ml. Escherichia coli grows rapidly and was measured at 0.2 OD600 at 1 × 108 cfu/ml. For routine cultivation, B. bassiana was grown on Sabouraud’s dextrose agar with yeast extract (SDY; 2% glucose, 1% peptone, 0.5% yeast extract; pH 7.0) at room temperature (23°C) for 7 d to obtain heavily sporulating cultures. After 1 wk, the plates were opened slightly and dried in a sterile laminar flow cabinet for an additional week. After drying, conidia were scraped from each plate with a sterile small metal spatula and stored at 4°C in a sterile glass vial. Prior to use in experiments, vials were removed from 4°C storage and conidial concentration was determined as follows: 1) an aliquot (10 mg) of dried harvested conidia was suspended into 0.1% Tween 20 (Sigma-Aldrich, Saint Louis, MO) and distilled water and then, 2) 10 µl aliquots of this suspension were collected and conidia were counted using an automatic cell counter (Cellometer Vision HSL; Nexcelom Bioscience LLC, Lawrence, MA) to determine the conidial concentration per ml. Injections Bacterial cells were cultured until OD600 = 0.5, and appropriate dilutions were made to acquire the following concentrations: 1 × 107, 1 × 106, 1 × 105, and 1 × 104 cfu/ml. Each dilution was centrifuged for 1 min at 13,200 rpm to produce a pellet, which was resuspended in 1× phosphate buffered saline (PBS). For fungal preparation, dried B. bassiana conidia (10 mg) were weighed and placed in a sterile 1.5 ml tube. Conidia were resuspended in 1 ml of 0.1% Tween 20 (in sterile H20) and conidial counts were completed. After removing sedated flies from the freezer, they were placed on a small laboratory chill table (BioQuip Products, Inc. Rancho Dominguez, CA) for continuous sedation. Each of the 20 female flies per replicate was injected using a microinjector (Nanomite, Harvard Apparatus, Holliston, MA) with a 1 cc syringe and a 28-gauge needle. The microinjector was set to release 1 µl into each fly. The injection site was the thoracic mesopleuron adjacent to the wing base (Lietze et al. 2012). We injected 20 female flies per pathogen and concentration, and the experiment was replicated on three separate occasions using different batches of pathogens and flies (total, 60 flies per treatment). Fly mortality was monitored for 4 d for the bacterial species. Flies exposed to Beauveria bassiana treatments were observed for an additional 24 h compared to other pathogens due to its delayed time-to-death when applied topically. In each replicate there were two controls. The first control was 20 female flies injected with 1 µl 1× PBS, and the second was 20 female flies injected with 1 µl Escherichia coli strain DH5α in the following doses: 1 × 104, 1 × 103, 1 × 102, and 1 × 101 cfu diluted in 1× PBS. This strain of E. coli is known to be avirulent for house flies at these dosages (unpublished data) and was included as a control for injection with bacteria. Surfactants Kinetic (Aquatrols, Paulsboro, NJ) is a nonionic, synthetic surfactant used as a spreader sticker for pesticides and fungicides onto plants. CapSil (Aquatrols, Paulsboro, NJ) is another nonionic surfactant that is an organo-silicone used as a spreader sticker. DyneAmic (Helena, Collierville, TN) is a surfactant containing mostly fatty acids that is used to enhance the spread of insecticides on waxy plant surfaces. Induce (Helena, Collierville, TN) is a nonionic blend containing high amounts of fatty acids; it aids in the adherence of insecticide onto plants and is known for its resistance to wash-off. Various dilutions of each surfactant were made in distilled water and mixed thoroughly. Three concentrations were tested for each: minimum, medium, and maximum, based on the range of recommended dilution rates listed by the manufacturer. Each surfactant was diluted in sterile distilled water according to the label instructions to prepare 10 ml of solution. For CapSil and Kinetic, the minimum, medium, and maximum concentrations were 0.01, 0.10, and 1.00%, respectively. For DyneAmic the concentrations were 0.04, 0.40, and 4.00%, and for Induce they were 0.03, 0.30, and 3.00%. Tween 80 also was included as a control and tested at 0.01, 0.10, and 1.00%. Results of the assays prompted us to conduct a second test evaluating a concentration between 0.1 and 1.0% for Capsil and Kinetic. In this test, these surfactants were diluted to concentrations of 0.50%, with Tween at 0.01% included as a control for fly mortality, as there was no mortality at this concentration in the previous assays. Application of Surfactants to House Flies Utilizing a P-2 pipette (Gilson PIPETMAN, Atlanta, GA), a 1 µl drop was topically applied to the anterior thorax of each female fly of an experimental group with each concentration of the surfactant. All topical treatments were replicated on four separate occasions using five flies per treatment per replicate (20 flies per treatment). Two measurements were recorded; the fly cuticle coverage or spreadability onto the house fly thorax for each surfactant and fly mortality. Spreadability was determined subjectively by making direct observations of the dispersion of the applied droplet immediately after application. A scale of 1 to 3 was used, where a score of 1 indicated poor spreading (i.e., product bubbled or beaded off), a score of 2 indicated a medium spread, in that the product spread out but some bubbling and beading occurred, and a score of 3 indicated a rapid and even dispersion over the thorax. Once flies were treated and the surfactant had spread, they were placed in a 500 ml plastic container with food and water as described previously. Fly mortality was observed at 1 h after topical application and then every 24 h for 3 d. Viability Bioassay for Pathogens Based on the results from the fly spreadability/mortality bioassay, three surfactant solutions were chosen for viability testing with bacteria and fungi, CapSil 0.1%, CapSil 0.5% and Kinetic 0.5%. Bacterial strains were grown overnight and 1 ml of stationary phase culture containing 1 × 108 cfu/ml was moved the next day to a sterile microcentrifuge tube. The tube was centrifuged for 1 min at 13,200 rpm to form a pellet, the supernatant was removed, and the pellet was resuspended in the respective surfactant. Once each bacterial strain was suspended in the surfactants mentioned above, the high concentration (1 × 108 cfu/ml) was diluted in its respective surfactant to 1 × 104, 1 × 103, and 1 × 102 cfu/ml, inoculated onto an LB plate in 10 µl spots, and held at 25°C (time point 0). The dilutions were re-plated after 24 h (time point 1). As a control, bacterial strains were suspended in 1× PBS and cultured using the same plating timeline as the cultures that were held in the surfactants. Each plate was observed for bacterial viability to determine survival in the surfactants. On each plate, the number of cfu’s were counted and recorded from spots that produced 3 to 30 cfu’s and from this number the antibacterial action of the surfactants was determined. For tests with B. bassiana, 1 mg of dried conidia was weighed and suspended in each surfactant to make a stock suspension of 1 × 109 conidia/ml, then diluted to 1 × 104, 1 × 103, and 1 × 102 conidia/ml. Initial assays with B. bassiana using 1× PBS as controls were problematic because conidia could not be kept in suspension long enough to pipette 10 µl aliquots reliably for spot plating. Therefore, for this species, 0.1% Tween 80 was used as a control to compare with the other surfactants. An aliquot of 10 µl of each suspension was plated onto SDY agar (time point 0 equals 1 h), and then plated again at 24 h (time point 1) post-suspension. Colony forming units were counted as with the three bacteria species. Dose-Response Topical Applications With Individual Pathogens Bacteria and fungi were tested by topical application on the thorax of individual flies. Each bacterial pathogen pellet was rinsed 2 times with 1× PBS to remove any possible toxins in the supernatant. Serial dilutions were made of each pathogen in 0.5% CapSil (Aquatrols, Paulsboro, NJ) mixed with 1× PBS, to prepare suspensions containing 1 × 103 to 1 × 106 cfu/µl. One microliter of each suspension was pipetted onto the anterior thorax of 20 female house flies, The experiment was repeated on three separate occasions with different batches of pathogens and flies for a total of 60 flies per pathogen and concentration. CapSil 0.5% with no entomopathogen was applied to an equal number of flies as a control. Statistical Analysis Application of Surfactants to House Flies For the comparison of surfactant spreadability scores, a Wilcoxon Mann Whitney test was done using the NPARONEWAY procedure of the Statistical Analysis System (SAS), version 9.4 (SAS Institute, Cary, NC). Fly mortality at time point 0 (1 h) and 1 (24 h) was assessed using the general linear models procedure (Proc GLM) of SAS, and means were separated using the Means/Tukey statement of Proc GLM. Injections and Application of Individual Pathogens to House Flies For all topical application bioassays, comparisons of fly mortality between time points were analyzed using a linear mixed model fitted with the effects of treatment, time and the interaction using repeated measures analysis of variance (ANOVA) through the Mixed Procedure as implemented in SAS (Proc Mixed), version 9.4 (SAS Institute). Residual terms were modeled by considering an autoregressive order one error structure and the degrees of freedom were adjusted using the Kenward-Roger method. Adjusted treatment means were compared using Tukey’s honest significant difference tests at α = 0.05. For topical applications of pathogens, Tukey’s tests were done at time points 3, 5, and 7 d after treatment. Viability Bioassay for Pathogens The number of cfu’s for each pathogen and surfactant combination was tested for normality. A linear mixed model was fitted with the effects of treatment, time, and the interaction and analyzed using repeated measures ANOVA using the Mixed Procedure as implemented in SAS (Proc Mixed), version 9.4 (SAS Institute). Residual terms were modeled by considering an autoregressive order one error structure and the degrees of freedom were adjusted using the Kenward-Roger method. LSMEANS statements were used to obtain the adjusted means for the effects of treatment, time and the interaction, which were compared using Tukey’s honest significant difference separation test method at P < 0.05. Results Accumulative Fly Mortality From Injections All bacterial pathogens at the highest doses caused significantly greater mortality than the controls, but at different time points. Photorhabdus temperata NC19 caused 38.3% mortality at the lowest dose (1 × 101 cfu) 48 h postinjection, whereas the highest dose (1 × 104 cfu) caused approximately 98% mortality after only 24 h (Table 1). Serratia marcescens DB11 caused half (13.9%) the mortality at the lowest dose compared to P. temperata, and 90% mortality at the highest dose at 24 h. Pseudomonas protegens pf-5 at the lowest dose caused <10% mortality initially (24 h), but mortality increased to 78% after 48 h. Mortality at the highest dose of P. protegens pf-5 was 100% at 24 h postinjection. Mortality from B. bassiana at the lowest dose was <5% after 24 h and only reached ca. 15% after 96 h. At the highest dose of B. bassiana, an increase in fly mortality was observed (82%) at 72 h postinjection when compared to 48 h (<15%). Mortality due to B. bassiana at 96 h was similar to 72-h mortality at the high dose (Table 1). Table 1. Cumulative mortality of female house flies, Musca domestica L., after receiving 1-µl injections of bacteria or fungi Treatment Dose (cfu) Mean (SE) mortality (%) at hours (h) postinjection* 24 h 48 h 72 h 96 h Photorhabdus temperata 1 × 101 0.0 (0.0)a 38.3 (13.4)a 46.7 (18.0)a NA 1 × 102 1.7 (1.3)a 88.3 (5.6)b 90.0 (4.4)b NA 1 × 103 15.0 (8.0)a 85.0 (8.0)bc 100.0 (0.0)b NA 1 × 104 97.0 (2.6)b 97.0 (2.6)c 97.0 (2.6)b NA Serratia marcescens 1 × 101 1.7 (1.3)a 13.9 (3.1)a 13.9 (0.0)a NA 1 × 102 17.2 (4.2)a 62.2 (2.2)b 74.4 (4.4)b NA 1 × 103 52.2 (11.9)b 95.6 (9.3)b 100.0 (3.4)b NA 1 × 104 90.0 (7.7)b 93.3 (2.6)b 93.3 (0.0)b NA Pseudomonas protegens 1 × 101 8.3 (4.6)a 77.8 (4.8)b 95.0 (2.2)b NA 1 × 102 7.8 (3.1)a 90.0 (7.7)b 93.3 (5.2)b NA 1 × 103 94.4 (2.2)b 100.0 (0.0)b 100.0 (0.0)b NA 1 × 104 100.0 (0.0)b 100.0 (0.0)b 100.0 (0.0)b NA Bacteria control (1× PBS) NA 0.0 (0.0) a 1.7 (1.6) a 1.7 (1.6) a NA Control (E. coli) 1 × 104 3.0 (2.3)a 5.0 (3.5)a 5.0 (3.5)a NA Beauveria bassiana 1 × 101 2.5 (1.5)a 10.0 (3.1)a 10.0 (3.1)a 15.0 (3.1)a 1 × 102 2.5 (1.5)a 2.5 (1.5)a 2.5 (1.5)a 17.5 (7.9)a 1 × 103 0.0 (0.0)a 10.0 (0.0)a 22.5 (1.5)a 45 (7.9)ab 1 × 104 5.0 (0.0)a 12.5 (1.5)a 82.5 (7.9)b 90.0 (3.1)b B. bassiana control (Tween 80, 0.1%) NA 2.5 (1.5)a 5.0 (1.5)a 5.0 (1.5)a 5.0 (1.5)a Treatment Dose (cfu) Mean (SE) mortality (%) at hours (h) postinjection* 24 h 48 h 72 h 96 h Photorhabdus temperata 1 × 101 0.0 (0.0)a 38.3 (13.4)a 46.7 (18.0)a NA 1 × 102 1.7 (1.3)a 88.3 (5.6)b 90.0 (4.4)b NA 1 × 103 15.0 (8.0)a 85.0 (8.0)bc 100.0 (0.0)b NA 1 × 104 97.0 (2.6)b 97.0 (2.6)c 97.0 (2.6)b NA Serratia marcescens 1 × 101 1.7 (1.3)a 13.9 (3.1)a 13.9 (0.0)a NA 1 × 102 17.2 (4.2)a 62.2 (2.2)b 74.4 (4.4)b NA 1 × 103 52.2 (11.9)b 95.6 (9.3)b 100.0 (3.4)b NA 1 × 104 90.0 (7.7)b 93.3 (2.6)b 93.3 (0.0)b NA Pseudomonas protegens 1 × 101 8.3 (4.6)a 77.8 (4.8)b 95.0 (2.2)b NA 1 × 102 7.8 (3.1)a 90.0 (7.7)b 93.3 (5.2)b NA 1 × 103 94.4 (2.2)b 100.0 (0.0)b 100.0 (0.0)b NA 1 × 104 100.0 (0.0)b 100.0 (0.0)b 100.0 (0.0)b NA Bacteria control (1× PBS) NA 0.0 (0.0) a 1.7 (1.6) a 1.7 (1.6) a NA Control (E. coli) 1 × 104 3.0 (2.3)a 5.0 (3.5)a 5.0 (3.5)a NA Beauveria bassiana 1 × 101 2.5 (1.5)a 10.0 (3.1)a 10.0 (3.1)a 15.0 (3.1)a 1 × 102 2.5 (1.5)a 2.5 (1.5)a 2.5 (1.5)a 17.5 (7.9)a 1 × 103 0.0 (0.0)a 10.0 (0.0)a 22.5 (1.5)a 45 (7.9)ab 1 × 104 5.0 (0.0)a 12.5 (1.5)a 82.5 (7.9)b 90.0 (3.1)b B. bassiana control (Tween 80, 0.1%) NA 2.5 (1.5)a 5.0 (1.5)a 5.0 (1.5)a 5.0 (1.5)a Pathogens, Beauveria bassiana and three species of bacteria, were administered at four doses from 1 × 101 to 1 × 104 cfu. Controls were either 1 µl injections of 1× PBS alone, or 1 × 104 cfu of an avirulent strain of Escherichia coli. NA = not applicable; only B. bassiana was monitored on the fourth day, controls without pathogens did not have doses. *Means within columns followed by the same letter are not significantly different (P > 0.05, Tukey’s honest significant difference tests). View Large Table 1. Cumulative mortality of female house flies, Musca domestica L., after receiving 1-µl injections of bacteria or fungi Treatment Dose (cfu) Mean (SE) mortality (%) at hours (h) postinjection* 24 h 48 h 72 h 96 h Photorhabdus temperata 1 × 101 0.0 (0.0)a 38.3 (13.4)a 46.7 (18.0)a NA 1 × 102 1.7 (1.3)a 88.3 (5.6)b 90.0 (4.4)b NA 1 × 103 15.0 (8.0)a 85.0 (8.0)bc 100.0 (0.0)b NA 1 × 104 97.0 (2.6)b 97.0 (2.6)c 97.0 (2.6)b NA Serratia marcescens 1 × 101 1.7 (1.3)a 13.9 (3.1)a 13.9 (0.0)a NA 1 × 102 17.2 (4.2)a 62.2 (2.2)b 74.4 (4.4)b NA 1 × 103 52.2 (11.9)b 95.6 (9.3)b 100.0 (3.4)b NA 1 × 104 90.0 (7.7)b 93.3 (2.6)b 93.3 (0.0)b NA Pseudomonas protegens 1 × 101 8.3 (4.6)a 77.8 (4.8)b 95.0 (2.2)b NA 1 × 102 7.8 (3.1)a 90.0 (7.7)b 93.3 (5.2)b NA 1 × 103 94.4 (2.2)b 100.0 (0.0)b 100.0 (0.0)b NA 1 × 104 100.0 (0.0)b 100.0 (0.0)b 100.0 (0.0)b NA Bacteria control (1× PBS) NA 0.0 (0.0) a 1.7 (1.6) a 1.7 (1.6) a NA Control (E. coli) 1 × 104 3.0 (2.3)a 5.0 (3.5)a 5.0 (3.5)a NA Beauveria bassiana 1 × 101 2.5 (1.5)a 10.0 (3.1)a 10.0 (3.1)a 15.0 (3.1)a 1 × 102 2.5 (1.5)a 2.5 (1.5)a 2.5 (1.5)a 17.5 (7.9)a 1 × 103 0.0 (0.0)a 10.0 (0.0)a 22.5 (1.5)a 45 (7.9)ab 1 × 104 5.0 (0.0)a 12.5 (1.5)a 82.5 (7.9)b 90.0 (3.1)b B. bassiana control (Tween 80, 0.1%) NA 2.5 (1.5)a 5.0 (1.5)a 5.0 (1.5)a 5.0 (1.5)a Treatment Dose (cfu) Mean (SE) mortality (%) at hours (h) postinjection* 24 h 48 h 72 h 96 h Photorhabdus temperata 1 × 101 0.0 (0.0)a 38.3 (13.4)a 46.7 (18.0)a NA 1 × 102 1.7 (1.3)a 88.3 (5.6)b 90.0 (4.4)b NA 1 × 103 15.0 (8.0)a 85.0 (8.0)bc 100.0 (0.0)b NA 1 × 104 97.0 (2.6)b 97.0 (2.6)c 97.0 (2.6)b NA Serratia marcescens 1 × 101 1.7 (1.3)a 13.9 (3.1)a 13.9 (0.0)a NA 1 × 102 17.2 (4.2)a 62.2 (2.2)b 74.4 (4.4)b NA 1 × 103 52.2 (11.9)b 95.6 (9.3)b 100.0 (3.4)b NA 1 × 104 90.0 (7.7)b 93.3 (2.6)b 93.3 (0.0)b NA Pseudomonas protegens 1 × 101 8.3 (4.6)a 77.8 (4.8)b 95.0 (2.2)b NA 1 × 102 7.8 (3.1)a 90.0 (7.7)b 93.3 (5.2)b NA 1 × 103 94.4 (2.2)b 100.0 (0.0)b 100.0 (0.0)b NA 1 × 104 100.0 (0.0)b 100.0 (0.0)b 100.0 (0.0)b NA Bacteria control (1× PBS) NA 0.0 (0.0) a 1.7 (1.6) a 1.7 (1.6) a NA Control (E. coli) 1 × 104 3.0 (2.3)a 5.0 (3.5)a 5.0 (3.5)a NA Beauveria bassiana 1 × 101 2.5 (1.5)a 10.0 (3.1)a 10.0 (3.1)a 15.0 (3.1)a 1 × 102 2.5 (1.5)a 2.5 (1.5)a 2.5 (1.5)a 17.5 (7.9)a 1 × 103 0.0 (0.0)a 10.0 (0.0)a 22.5 (1.5)a 45 (7.9)ab 1 × 104 5.0 (0.0)a 12.5 (1.5)a 82.5 (7.9)b 90.0 (3.1)b B. bassiana control (Tween 80, 0.1%) NA 2.5 (1.5)a 5.0 (1.5)a 5.0 (1.5)a 5.0 (1.5)a Pathogens, Beauveria bassiana and three species of bacteria, were administered at four doses from 1 × 101 to 1 × 104 cfu. Controls were either 1 µl injections of 1× PBS alone, or 1 × 104 cfu of an avirulent strain of Escherichia coli. NA = not applicable; only B. bassiana was monitored on the fourth day, controls without pathogens did not have doses. *Means within columns followed by the same letter are not significantly different (P > 0.05, Tukey’s honest significant difference tests). View Large Application of Surfactants to Flies Spreadability scores on fly thoraces varied significantly among the products and concentrations tested (Kruskal-Wallis Chi-Square = 45.9; df = 14; P < 0.01). The highest scores (all flies scoring 3 out of 3 on the scale) were observed for Kinetic at 1.0% and CapSil at 0.1 and 1.0%, although these did not differ significantly from DyneAmic at 4.0% (mean score 2.50), Induce at 3.0% (2.50), or Kinetic at 0.01 or 0.10% (2.25 and 2.00, respectively) (Fig. 1). Tween 80 had the lowest score (1.75) of the five products tested at their highest application concentrations, and did not differ significantly in spreadability over the range of concentrations examined (0.01–1.00%). A concentration response was seen with the other four surfactants, with all of them having significantly higher spreadability scores at the highest concentration compared with the lowest. Fig. 1. View largeDownload slide Spreadability scores for surfactants at different concentrations after application to female house fly, Musca domestica, L. thoraces. Possible scores ranged from one (i.e., surfactant beaded up and rolled off the fly) to three (i.e., rapid and even dispersion of the droplet). Bars represent mean ± standard errors; letters above bars indicate significance, bars with the same letter are not significantly different (Tukey’s honest significant difference tests at P < 0.05). Fig. 1. View largeDownload slide Spreadability scores for surfactants at different concentrations after application to female house fly, Musca domestica, L. thoraces. Possible scores ranged from one (i.e., surfactant beaded up and rolled off the fly) to three (i.e., rapid and even dispersion of the droplet). Bars represent mean ± standard errors; letters above bars indicate significance, bars with the same letter are not significantly different (Tukey’s honest significant difference tests at P < 0.05). Fly mortality immediately after treatment with surfactants was significantly higher for CapSil at 1.0% (45%) than any other surfactant except Kinetic at 1.0% (35%) and DyneAmic at 4.0% (15%) (Overall ANOVA F = 3.60, df = 14, 45, P < 0.01) (Table 2). Initial mortality was ≤5% for all of the other surfactants and was zero for many of the lower concentrations of each surfactant. There was slight mortality 24 h after treatment (Table 2). No additional mortality was observed 48–72 h after treatment. Spreadability scores for Capsil and Kinetic at 0.5% were 3.0 (a score of 3 for every fly tested), which was equal to the scores of the same surfactants at 1.0%. Table 2. Cumulative mortality of female house flies, Musca domestica, L., after receiving topical applications of different surfactants (CapSil, DyneAmic, Induce, Kinetic, and Tween 80) diluted in phosphate buffered saline at concentrations depending upon the products label rate Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 1.00% 45 (9.7)a 50 (9.7)a 50 (9.7)a 50 (9.7)a DyneAmic 0.04% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.40% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 4.00% 15 (4.9)ab 25 (4.9)ab 25 (4.9)ab 25 (4.9)ab Induce 0.03% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.30% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 3.00% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b Kinetic 0.01% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 35 (8.8)a 35 (8.8)a 35 (8.8)a 35 (8.8)a Tween 80 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 1.00% 45 (9.7)a 50 (9.7)a 50 (9.7)a 50 (9.7)a DyneAmic 0.04% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.40% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 4.00% 15 (4.9)ab 25 (4.9)ab 25 (4.9)ab 25 (4.9)ab Induce 0.03% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.30% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 3.00% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b Kinetic 0.01% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 35 (8.8)a 35 (8.8)a 35 (8.8)a 35 (8.8)a Tween 80 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b *Means within columns followed by the same letter are not significantly different (P > 0.05, two-way ANOVA with repeated measures). View Large Table 2. Cumulative mortality of female house flies, Musca domestica, L., after receiving topical applications of different surfactants (CapSil, DyneAmic, Induce, Kinetic, and Tween 80) diluted in phosphate buffered saline at concentrations depending upon the products label rate Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 1.00% 45 (9.7)a 50 (9.7)a 50 (9.7)a 50 (9.7)a DyneAmic 0.04% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.40% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 4.00% 15 (4.9)ab 25 (4.9)ab 25 (4.9)ab 25 (4.9)ab Induce 0.03% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.30% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 3.00% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b Kinetic 0.01% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 35 (8.8)a 35 (8.8)a 35 (8.8)a 35 (8.8)a Tween 80 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 1.00% 45 (9.7)a 50 (9.7)a 50 (9.7)a 50 (9.7)a DyneAmic 0.04% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.40% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 4.00% 15 (4.9)ab 25 (4.9)ab 25 (4.9)ab 25 (4.9)ab Induce 0.03% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.30% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 3.00% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b Kinetic 0.01% 5 (2.5)b 5 (2.5)b 5 (2.5)b 5 (2.5)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 35 (8.8)a 35 (8.8)a 35 (8.8)a 35 (8.8)a Tween 80 0.01% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 0.10% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b 1.00% 0 (0.0)b 0 (0.0)b 0 (0.0)b 0 (0.0)b *Means within columns followed by the same letter are not significantly different (P > 0.05, two-way ANOVA with repeated measures). View Large Viability Bioassay for Pathogens. Viability for all four pathogens was high in all solutions, and there was no significant difference between the initial time point and 24 h later (Table 3). Colony forming units of P. temperata NC19 increased marginally during the 24-h holding period, and growth was significantly higher in Capsil 0.5% than in Kinetic 0.5%. Colony forming unit counts of S. marcescens DB11 and P. protegens pf-5 generally increased two- to fourfold after being held for 24 h in the suspensions, and there was no significant surfactant effect on viability for either species (Table 3). The only instance where significant growth was not observed was S. marcescens held in Kinetic 0.5%. In contrast, there were no significant time or surfactant effects for B. bassiana, with similar conidia counts at 1 h and at 24 h post-suspension for all of the surfactants tested (Table 3). Table 3. Cumulative mortality of female house flies, Musca domestica, L., after receiving topical applications of select surfactants and specific concentrations: CapSil at 0.1% and 0.5%, Kinetic at 0.1%, and Tween 80 at 0.01% in 1× PBS Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.33 (0.33)a 0.3 (0.30)a 0.50% 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.80)a Kinetic 0.50% 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.80)a Tween 80 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.33 (0.33)a 0.3 (0.30)a 0.50% 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.80)a Kinetic 0.50% 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.80)a Tween 80 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a *Means within columns followed by the same letter are not significantly different (P > 0.05, two-way ANOVA with repeated measures). View Large Table 3. Cumulative mortality of female house flies, Musca domestica, L., after receiving topical applications of select surfactants and specific concentrations: CapSil at 0.1% and 0.5%, Kinetic at 0.1%, and Tween 80 at 0.01% in 1× PBS Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.33 (0.33)a 0.3 (0.30)a 0.50% 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.80)a Kinetic 0.50% 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.80)a Tween 80 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a Mean (SE) mortality (%) at time after treatment* Treatment 1 h 24 h 48 h 72 h CapSil 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.33 (0.33)a 0.3 (0.30)a 0.50% 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.77)a 10.0 (5.80)a Kinetic 0.50% 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.82)a 13.3 (8.80)a Tween 80 0.01% 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a 0.0 (0.00)a *Means within columns followed by the same letter are not significantly different (P > 0.05, two-way ANOVA with repeated measures). View Large Topical Applications of Individual Pathogens A dose-response relationship for mortality was observed for all four pathogens when applied topically, although the relationship was stronger with some pathogens than with others (Figs. 2–5). Mortality due to P. temperata NC19 and S. marcescens DB11 were low at all doses assessed and reached 22% mortality on day 7 at the highest dose of 1 × 106 cfu (Figs. 2 and 3). Pseudomonas protegens pf-5 had the highest topical virulence of the three bacterial pathogens (Fig. 4). Mortality at 1 × 106 cfu was 49% 2 d after application and reached 60% by day 4. Mortality at the two lower doses (1 × 103 and 1 × 104 cfu) did not reach 35% through the duration of the test. All doses of B. bassiana killed <20% of the flies until day 5, when mortality at 1 × 105 and 1 × 106 conidia increased to 40 and 68%, respectively (Fig. 5). Mortality increased until day 7, reaching 70 and 90% at 1 × 105 and 1 × 106 conidia, respectively. Fig. 2. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Photorhabdus temperata (Pt) colony forming units. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard errors. Fig. 2. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Photorhabdus temperata (Pt) colony forming units. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard errors. Fig. 3. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Serratia marcescens (Sm) colony forming units. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard errors. Fig. 3. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Serratia marcescens (Sm) colony forming units. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard errors. Fig. 4. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Pseudomonas protegens (Pp) colony forming units. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard errors. Fig. 4. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Pseudomonas protegens (Pp) colony forming units. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard errors. Fig. 5. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Beauveria bassiana (Bb) conidia. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard error. Fig. 5. View largeDownload slide Mortality of adult house flies, Musca domestica, L. for 7 d after topical application of 1 µl of 0.5% CapSil containing 1 × 103 through 1 × 106Beauveria bassiana (Bb) conidia. Control = CapSil 0.5% alone. Points on the line represent mean percentage mortality ± standard error. Discussion Entomopathogenic fungi long have been studied for their potential as biological control agents for arthropod pests (Butt et al. 2001, Shah and Pell 2003, Lacey et al. 2015). Although at least 700 entomopathogenic species have been identified (Sandhu et al. 2012) and they are present in almost all insect ecosystems (Lacey et al. 2015), only a select few have been developed as microbial insecticides. Beauveria bassiana is one of the most widely studied species and accounts for over one-third of the 171 commercial myco-biocontrol products available globally (Sandhu et al. 2012). However, B. bassiana is slow acting, potentially allowing flies to reproduce before they are killed by the fungus. Typically, bacteria are faster acting than fungi once they gain entry to the host. Therefore, the goal of our study was to examine whether bacterial pathogens would demonstrate accelerated pathogen-induced mortality, hypothesizing that if the bacteria could gain access through the cuticle to the hemocoel they would cause rapid death from sepsis. Testing this hypothesis first requires identifying suitable bacteria and a surfactant that would not be inimical to either fungi or bacteria. The three gram-negative bacterial strains evaluated are known to be pathogenic for insects but the effect they would have on house flies when applied topically, because of the complexities of penetrating through the exoskeleton, was not known. Serratia marcescens is a common laboratory contaminant and natural bioinsecticide (Grimont et al. 1979, Flyg et al. 1980, Pineda-Castellanos et al. 2015). It inhabits soil and water and some strains are pathogenic to insects, whereas others are pathogenic to humans and other mammals. It displays virulence in approximately 70 species of insects including wasps, termites, grasshoppers, and flies (Grimont et al. 1979). Photorhabdus temperata is a bioluminescent, motile, entomopathogenic bacterium that has a symbiotic relationship with entomopathogenic heterorhabditid nematodes. Pseudomonas protegens is known as a plant-protecting species because it excretes toxic metabolites that deter pathogenic plant invaders (Cronin et al. 1997, Ellis et al. 2000, Loper et al. 2016). In our study, all three bacterial species were highly virulent for adult house flies when injected into the hemocoel, suggesting that all were acceptable candidates for evaluation as pathogenic synergists with B. bassiana. At high doses of P. protegens, flies appeared lethargic and hyperphagic within several hours postinjection. Aside from this, flies injected with bacteria showed no visible symptoms of infection. The next step was to find a surfactant that could be used to apply these house fly pathogens to the cuticle for topical application. When treating flies with bacterial pathogens for contact or premise treatments, the minimum requirement was determining a surfactant that would ensure viability of the organisms. It also was important for testing and evaluation purposes that the surfactant itself has low toxicity for the house fly. We chose to evaluate nonionic surfactants, a group that includes spreader stickers, wetting agents, and detergents. They are composed of hydrophobic and hydrophilic components that aid in breaking the surface tension of water (van Os et al. 1993). Because of these properties, they have a variety of uses and are currently in many industrial products. However, their chemical nature varies depending on the intended use of the products (van Os et al. 1993). For this study, our primary focus was on horticultural wetting agents and spreader stickers because of their known safety profiles and availability (reviewed in Krogh et al. 2003). These products allow the preparation of aqueous solutions of otherwise-insoluble active ingredients and aid in adherence to the hydrophobic surfaces of plant foliage. The latter was an attractive trait for the application on the house fly thorax, which also is highly hydrophobic. Moreover, some nonionic surfactants already are known to be compatible with B. bassiana conidia (Prasad 1993, Polar et al. 2005, Mishra et al. 2013). In the spreadability bioassay, there were numerous promising candidate surfactants, with all five products scoring well at higher concentrations for their ability to provide even dispersion over the dorsal thoracic cuticle of the house fly (Fig. 1). Several of these, however, resulted in >10% mortality immediately after treatment (Table 2). It is uncertain whether this mortality was due to toxicity of the solutions or whether the surfactants interfered with gas exchange through the thoracic spiracles. Spiracle coverage was difficult to observe with these materials because they do not leave a visible residue on the fly after drying. The spreadability scores for Tween 80 were surprisingly low, considering that this surfactant is commonly used in laboratory bioassays with B. bassiana (Mishra et al. 2013, Immediato et al. 2015, Andreadis et al. 2016). It is possible that the hydrophobicity of the house fly cuticle did not interact well with the hydrophilic polyoxyethylene groups of Tween 80. CapSil and Kinetic were chosen for use in subsequent experiments because of their high spreadability scores, after adjusting the concentrations to prevent unacceptable fly mortality without compromising high spreadability. Beauveria bassiana conidia can tolerate a wide range of environments, and there is a great deal of information available on methods to formulate them for insecticidal application (Wraight et al. 2001). In contrast, less work has been done in formulating gram-negative bacterial strains for field use. The only current gram-negative pathogen developed as a live microbial insecticide is Serratia entomophila, which is produced commercially for control of grass-feeding larvae(Jackson 2007). Gram-negative bacteria generally have a narrower range of tolerance for their environment than B. bassiana conidia, and it was unknown whether the bacterial species chosen for study in this project would survive in any surfactant. All of the surfactants tested supported viability of all four pathogens (Tables 3 and 4). No reduction in cfu counts were observed after 24 h suspension in all surfactants. Indeed, there was an increase in bacterial (but not B. bassiana) cfu after 24 h. This was most likely due to cell division occurring during the incubation period. The surfactants differed little in their effects on viability, except for P. protegens in CapSil where significantly higher bacterial growth was observed after 24 h post-suspension than in at least one other surfactant, e.g., Kinetic. Table 4. Viability of fungal and bacterial pathogens following exposure to surfactants (i.e., CapSil, and Kinetic) at different concentrations Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 h 24 h Photorhabdus temperata  CapSil 0.1% 10.0 (1.5)a 13.0 (4.0)ab  CapSil 0.5% 11.0 (2.3)a 18.3 (5.8)b  Kinetic 0.5% 6.3 (0.9)a 5.0 (2.5)a  Control (1× PBS) 6.7 (2.2)a 13.3 (0.9)ab Serratia marcescens  CapSil 0.1% 16.0 (1.0)a 58.3 (11.5)ab  CapSil 0.5% 20.3 (2.2)a 50.0 (7.6)b  Kinetic 0.5% 26.7 (11.7)a 19.3 (3.2)a  Control (1× PBS) 11.3 (3.9)a 32.3 (5.8)ab Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 hr 24 hr Pseudomonas protegens  CapSil 0.1% 13.3 (1.9)a 38.3 (4.3)a  CapSil 0.5% 13.3 (2.6)a 36.0 (1.2)a  Kinetic 0.5% 15.3 (1.3)a 28.7 (4.7)a  Control (1× PBS) 11.0 (1.2)a 44.3 (20.1)a Beauveria bassiana  CapSil 0.1% 3.8 (1.2 a 4.6 (1.8)a  CapSil 0.5% 3.6 (1.2)a 3.6 (0.9)a  Kinetic 0.5% 3.4 (1.1)a 3.2 (1.1)a  Control (0.1% Tween 80) 3.6 (0.6)a 1.6 (0.4)a Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 h 24 h Photorhabdus temperata  CapSil 0.1% 10.0 (1.5)a 13.0 (4.0)ab  CapSil 0.5% 11.0 (2.3)a 18.3 (5.8)b  Kinetic 0.5% 6.3 (0.9)a 5.0 (2.5)a  Control (1× PBS) 6.7 (2.2)a 13.3 (0.9)ab Serratia marcescens  CapSil 0.1% 16.0 (1.0)a 58.3 (11.5)ab  CapSil 0.5% 20.3 (2.2)a 50.0 (7.6)b  Kinetic 0.5% 26.7 (11.7)a 19.3 (3.2)a  Control (1× PBS) 11.3 (3.9)a 32.3 (5.8)ab Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 hr 24 hr Pseudomonas protegens  CapSil 0.1% 13.3 (1.9)a 38.3 (4.3)a  CapSil 0.5% 13.3 (2.6)a 36.0 (1.2)a  Kinetic 0.5% 15.3 (1.3)a 28.7 (4.7)a  Control (1× PBS) 11.0 (1.2)a 44.3 (20.1)a Beauveria bassiana  CapSil 0.1% 3.8 (1.2 a 4.6 (1.8)a  CapSil 0.5% 3.6 (1.2)a 3.6 (0.9)a  Kinetic 0.5% 3.4 (1.1)a 3.2 (1.1)a  Control (0.1% Tween 80) 3.6 (0.6)a 1.6 (0.4)a Colony forming units of Beauveria bassiana, Photorhabdus temperata, Pseudomonas protegens, and Serratia marcescens 1 and 24 h post-suspension in the surfactants. Controls consisted of 1 × PBS for bacteria and Tween 80 for fungi. *Means within columns and species followed by the same letter are not significantly different (P > 0.05, two-way ANOVA with repeated measures). View Large Table 4. Viability of fungal and bacterial pathogens following exposure to surfactants (i.e., CapSil, and Kinetic) at different concentrations Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 h 24 h Photorhabdus temperata  CapSil 0.1% 10.0 (1.5)a 13.0 (4.0)ab  CapSil 0.5% 11.0 (2.3)a 18.3 (5.8)b  Kinetic 0.5% 6.3 (0.9)a 5.0 (2.5)a  Control (1× PBS) 6.7 (2.2)a 13.3 (0.9)ab Serratia marcescens  CapSil 0.1% 16.0 (1.0)a 58.3 (11.5)ab  CapSil 0.5% 20.3 (2.2)a 50.0 (7.6)b  Kinetic 0.5% 26.7 (11.7)a 19.3 (3.2)a  Control (1× PBS) 11.3 (3.9)a 32.3 (5.8)ab Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 hr 24 hr Pseudomonas protegens  CapSil 0.1% 13.3 (1.9)a 38.3 (4.3)a  CapSil 0.5% 13.3 (2.6)a 36.0 (1.2)a  Kinetic 0.5% 15.3 (1.3)a 28.7 (4.7)a  Control (1× PBS) 11.0 (1.2)a 44.3 (20.1)a Beauveria bassiana  CapSil 0.1% 3.8 (1.2 a 4.6 (1.8)a  CapSil 0.5% 3.6 (1.2)a 3.6 (0.9)a  Kinetic 0.5% 3.4 (1.1)a 3.2 (1.1)a  Control (0.1% Tween 80) 3.6 (0.6)a 1.6 (0.4)a Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 h 24 h Photorhabdus temperata  CapSil 0.1% 10.0 (1.5)a 13.0 (4.0)ab  CapSil 0.5% 11.0 (2.3)a 18.3 (5.8)b  Kinetic 0.5% 6.3 (0.9)a 5.0 (2.5)a  Control (1× PBS) 6.7 (2.2)a 13.3 (0.9)ab Serratia marcescens  CapSil 0.1% 16.0 (1.0)a 58.3 (11.5)ab  CapSil 0.5% 20.3 (2.2)a 50.0 (7.6)b  Kinetic 0.5% 26.7 (11.7)a 19.3 (3.2)a  Control (1× PBS) 11.3 (3.9)a 32.3 (5.8)ab Mean (SE) colony forming units at 1 and 24 h post-suspension* Treatment 1 hr 24 hr Pseudomonas protegens  CapSil 0.1% 13.3 (1.9)a 38.3 (4.3)a  CapSil 0.5% 13.3 (2.6)a 36.0 (1.2)a  Kinetic 0.5% 15.3 (1.3)a 28.7 (4.7)a  Control (1× PBS) 11.0 (1.2)a 44.3 (20.1)a Beauveria bassiana  CapSil 0.1% 3.8 (1.2 a 4.6 (1.8)a  CapSil 0.5% 3.6 (1.2)a 3.6 (0.9)a  Kinetic 0.5% 3.4 (1.1)a 3.2 (1.1)a  Control (0.1% Tween 80) 3.6 (0.6)a 1.6 (0.4)a Colony forming units of Beauveria bassiana, Photorhabdus temperata, Pseudomonas protegens, and Serratia marcescens 1 and 24 h post-suspension in the surfactants. Controls consisted of 1 × PBS for bacteria and Tween 80 for fungi. *Means within columns and species followed by the same letter are not significantly different (P > 0.05, two-way ANOVA with repeated measures). View Large Fly mortality after topical applications of the bacteria was greatly reduced when compared with injections. For example, mortality was <26% when P. temperata and S. marcescens were applied topically, even after treatment with 1 × 106 cfu. In contrast, topical application of P. protegens resulted in surprisingly high (ca. 50%) house fly mortality after only 48 h (Fig. 4). Because P. protegens is motile (Song et al. 2016), topical mortality may have been partially due to bacteria entering the fly through natural openings such as the spiracles, anus, or mouth. Another factor could be the toxins associated with this species, especially FitD (for P. fluorescens insecticidal toxin) (Péchy-Tarr et al. 2008), which might have been responsible for high fly mortality. Rangel et al. (2016) observed that P. protegens strain pf-5 exhibited significant oral toxicity against D. melanogaster and attributed the mortality to FitD. During the initial topical bioassays with the species we observed unacceptably high mortality as soon as 1 h after treatment (results not presented), and found that a second rinse and suspension in PBS was needed to remove extracellular materials that presumably included the toxin. To our knowledge, this is the first report of P. protegens causing lethal infections in house flies. Further research with this species and associated toxins could lead to its development as a biological control agent for the house fly. In summary, P. temperata, S. marcescens, and P. protegens were all highly virulent when injected into the fly, could be formulated with nonionic surfactants, and caused low to moderate mortality when applied topically. The results with P. protegens suggest that this species warrants further evaluation as a potential microbial insecticide for house flies. Future work with this species should include assays for oral toxicity in adult flies, potential use as a larvicide, and impacts on beneficial arthropods in livestock and poultry systems. In addition, our results will facilitate combining these bacterial pathogens with B. bassiana to assess the efficacy of pathogen combinations. Acknowledgments We thank Roxie White for assisting with bioassays. References Cited Acharya , N. , E. G. Rajotte , N. E. Jenkins , and M. B. Thomas . 2015 . Potential for biocontrol of house flies, Musca domestica, using fungal biopesticides . Biocontrol Sci. Techn . 25 : 513 – 524 . Google Scholar Crossref Search ADS Andreadis , S. , K. Cloonan , G. S. Bellicanta , and N. E. Jenkins . 2016 . Efficacy of Beauveria bassiana formulations against the fungus gnat Lycoriella ingenua . Bio Control . 103 : 165–171. Butt , T. M. , C. Jackson , and N. Magan . 2001 . Fungi as biocontrol agents: progress, problems and potential . CABI , Wallingford, United Kingdom . Cronin , D. , T. Moënne-Loccoz , A. Fenton , C. Dunne , D. N. Dowling , and F. O’Gara . 1997 . Ecological interaction of a biocontrol Pseudomonas fluorescens strain producing 2,4-diacetylphloroglucinol with the soft rot potato pathogen Erwinia carotovora subsp. atroseptica . FEMS Microbiol. Ecol . 23 : 95 – 106 . Google Scholar Crossref Search ADS Duchaud , E. , C. Rusniok , L. Frangeul , C. Buchrieser , A. Givaudan , S. Taourit , S. Bocs , C. Boursaux-Eude , M. Chandler , J. F. Charles , et al. 2003 . The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens . Nat. Biotechnol . 21 : 1307 – 1313 . Google Scholar Crossref Search ADS PubMed Ellis , R. J. , T. M. Timms-Wilson , and M. J. Bailey . 2000 . Identification of conserved traits in fluorescent pseudomonads with antifungal activity . Environ. Microbiol . 2 : 274 – 284 . Google Scholar Crossref Search ADS PubMed Flyg , C. , K. Kenne , and H. G. Boman . 1980 . Insect pathogenic properties of Serratia marcescens: phage-resistant mutants with a decreased resistance to Cecropia immunity and a decreased virulence to Drosophila . J. Gen. Microbiol . 120 : 173 – 181 . Google Scholar PubMed Geden , C. J. , D. C. Steinkraus , and D. A. Rutz . 1995 . Virulence of different isolates and formulations of Beauveria bassiana for house flies and the parasitoid Muscidifurax raptor . Biol. Control 5 : 615 – 621 . Google Scholar Crossref Search ADS Grimont , P. A. D. , F. Grimont , and O. Lysenko . 1979 . Species and biotype identification of Serratia strains associated with insects . Curr. Microbiol . 2 : 139 – 142 . Google Scholar Crossref Search ADS Hurst , S. , IV , H. Rowedder , B. Michaels , H. Bullock , R. Jackobeck , F. Abebe-Akele , U. Durakovic , J. Gately , E. Janicki , and L. S. Tisa . 2015 . Elucidation of the Photorhabdus temperata genome and generation of a transposon mutant library to identify motility mutants altered in pathogenesis . J. Bacteriol . 197 : 2201 – 2216 . Google Scholar Crossref Search ADS PubMed Iguchi , A. , Y. Nagaya , E. Pradel , T. Ooka , Y. Ogura , K. Katsura , K. Kurokawa , K. Oshima , M. Hattori , J. Parkhill , et al. 2014 . Genome evolution and plasticity of Serratia marcescens, an important multidrug-resistant nosocomial pathogen . Genome Biol. Evol . 6 : 2096 – 2110 . Google Scholar Crossref Search ADS PubMed Immediato , D. , A. Camarda , R. Iatta , M. R. Puttilli , R. A. Ramos , G. Di Paola , A. Giangaspero , D. Otranto , and C. Cafarchia . 2015 . Laboratory evaluation of a native strain of Beauveria bassiana for controlling Dermanyssus gallinae (De Geer, 1778) (Acari: Dermanyssidae) . Vet. Parasitol . 212 : 478 – 482 . Google Scholar Crossref Search ADS PubMed Ishii , K. , T. Adachi , H. Hamamoto , and K. Sekimizu . 2014 . Serratia marcescens suppresses host cellular immunity via the production of an adhesion-inhibitory factor against immunosurveillance cells . J. Biol. Chem . 289 : 5876 – 5888 . Google Scholar Crossref Search ADS PubMed Jackson , T. A . 2007 . A novel bacterium for control of grass grub, pp. 160–168 . In C. M. S. Goettel and G. Lazarovits (eds.), Biological control: a global perspective . CABI , Wallingford, United Kingdom . Kaufman , P. E. , S. C. Nunez , R. S. Mann , C. J. Geden , and M. E. Scharf . 2010 . Nicotinoid and pyrethroid insecticide resistance in houseflies (Diptera: Muscidae) collected from Florida dairies . Pest Manag. Sci . 66 : 290 – 294 . Google Scholar Crossref Search ADS PubMed Krogh , K. A. , B. Halling-Sørensen , B. B. Mogensen , and K. V. Vejrup . 2003 . Environmental properties and effects of nonionic surfactant adjuvants in pesticides: a review . Chemosphere . 50 : 871 – 901 . Google Scholar Crossref Search ADS PubMed Kozaki , T. , S. G. Brady , J. G. Scott . 2009 . Frequencies and evolution of organophosphate insensitive acetylcholinesterase alleles in laboratory and field populations of the house fly, Musca domestica L . Pestic Biochem Physiol . 95 : 6 – 11 . Google Scholar Crossref Search ADS Lacey , L. A. , D. Grzywacz , D. I. Shapiro-Ilan , R. Frutos , M. Brownbridge , and M. S. Goettel . 2015 . Insect pathogens as biological control agents: back to the future . J. Invertebr. Pathol . 132 : 1 – 41 . Google Scholar Crossref Search ADS PubMed Lecuona , R. E. , M. Turica , F. Tarocco , and D. C. Crespo . 2005 . Microbial control of Musca domestica (Diptera: Muscidae) with selected strains of Beauveria bassiana . J. Med. Entomol . 42 : 332 – 336 . Google Scholar Crossref Search ADS PubMed Lietze , V. U. , C. J. Geden , M. A. Doyle , and D. G. Boucias . 2012 . Disease dynamics and persistence of Musca domestica salivary gland hypertrophy virus infections in laboratory house fly (Musca domestica) populations . Appl. Environ. Microbiol . 78 : 311 – 317 . Google Scholar Crossref Search ADS PubMed Lipa , J. J . 1963 . Polish analytical bibliography of insect pathology part I. Diseases and microbial control of noxious insects . Prace Naukowe, Instytutu Ochrony Roślin . 5 : 4 – 102 . Loper , J. E. , M. D. Henkels , L. I. Rangel , M. H. Olcott , F. L. Walker , K. L. Bond , T. A. Kidarsa , C. N. Hesse , B. Sneh , V. O. Stockwell , et al. 2016 . Rhizoxin analogs, orfamide A and chitinase production contribute to the toxicity of Pseudomonas protegens strain Pf-5 to Drosophila melanogaster . Environ. Microbiol . 18 : 3509 – 3521 . Google Scholar Crossref Search ADS PubMed Machtinger , E. T. , and C. J. Geden . 2018 . Biological control of livestock pests: parasitoids, pp. 3562–3567 . In C. Garros , J. Bouyer , W. Takken , and R. C. Smallegange (eds.), Ecology and control of vector-borne diseases, volume 5. Prevention and control of pests and vector-borne diseases in the livestock industry . Wageningen Academic Publishers , Wageningen, the Netherlands (in press). Memmi , B. K . 2010 . Mortality and knockdown effects of imidacloprid and methomyl in house fly (Musca domestica L., Diptera: Muscidae) populations . J. Vector Ecol . 35 : 144 – 148 . Google Scholar Crossref Search ADS PubMed Mishra , S. , P. Kumar , and A. Malik . 2013 . Preparation, characterization, and insecticidal activity evaluation of three different formulations of Beauveria bassiana against Musca domestica . Parasitol. Res . 112 : 3485 – 3495 . Google Scholar Crossref Search ADS PubMed Mwamburi , L. A. , M. D. Laing , and R. M. Miller . 2010 . Laboratory screening of insecticidal activities of Beauveria bassiana and Paecilomyces lilacinus against larval and adult house fly (Musca domestica L.) . African Entomol . 18 : 38 – 46 . Google Scholar Crossref Search ADS Nayduch , D. , and R. G. Burrus . 2017 . Flourishing in filth: house fly–microbe interactions across life history . Ann. Entomol. Soc. Am . 110 : 6 – 18 . Google Scholar Crossref Search ADS van Os , N. M. , J. R. Haak , and L. A. M. Rupert . 1993 . Physico-chemical properties of selected anionic, cationic and nonionic surfactants , pp. 608 . Elsevier , Amsterdam, Netherlands . Palleroni , N. J . 2003 . Prokaryote taxonomy of the 20th century and the impact of studies on the genus Pseudomonas: a personal view . Microbiology . 149 : 1 – 7 . Google Scholar Crossref Search ADS PubMed Péchy-Tarr , M. , D. J. Bruck , M. Maurhofer , E. Fischer , C. Vogne , M. D. Henkels , K. M. Donahue , J. Grunder , J. E. Loper , and C. Keel . 2008 . Molecular analysis of a novel gene cluster encoding an insect toxin in plant-associated strains of Pseudomonas fluorescens . Environ. Microbiol . 10 : 2368 – 2386 . Google Scholar Crossref Search ADS PubMed Pineda-Castellanos , M. L. , Z. Rodríguez-Segura , F. J. Villalobos , L. Hernández , L. Lina , and M. E. Nuñez-Valdez . 2015 . Pathogenicity of isolates of Serratia marcescens towards larvae of the scarab Phyllophaga blanchardi (Coleoptera) . Pathogens . 4 : 210 – 228 . Google Scholar Crossref Search ADS PubMed Polar , P. , M. T. Kairo , D. Moore , R. Pegram , and S. A. John . 2005 . Comparison of water, oils and emulsifiable adjuvant oils as formulating agents for Metarhizium anisopliae for use in control of Boophilus microplus . Mycopathologia 160 : 151 – 157 . Google Scholar Crossref Search ADS PubMed Prasad , R . 1993 . Role of adjuvants in modifying the efficacy of a bioherbicide on forest species: compatibility studies under laboratory conditions . Pestic. Sci . 38 : 273 – 275 . Rutz , D. A. , and R. S. Patterson . 1990 . Biocontrol of arthropods affecting livestock and poultry . Westview Press , Boulder, CO . Rangel , L. I. , M. D. Henkels , B. T. Shaffer , F. L. Walker , E. W. II. David , V. O. Stockwell , D. Bruck , B. J. Taylor , and J. E. Loper . 2016 . Characterization of toxin complex gene clusters and insect toxicity of bacteria representing four subgroups of Pseudomonas fluorescens . PLoS one . 11 : e0161120 . doi: https://doi.org/10.1371/journal.pone.0161120 Google Scholar Crossref Search ADS PubMed Sandhu , S. S. , A. K. Sharma , V. Beniwal , G. Goel , P. Batra , A. Kumar , S. Jaglan , A. K. Sharma , and S. Malhotra . 2012 . Myco-biocontrol of insect pests: factors involved, mechanism, and regulation . J. Pathog . 2012 : 126819 . Google Scholar PubMed Sasaki , T. , M. Kobayashi , and N. Agui . 2000 . Epidemiological potential of excretion and regurgitation by Musca domestica (Diptera: Muscidae) in the dissemination of Escherichia coli O157: H7 to food . J. Med. Entomol . 37 : 945 – 949 . Google Scholar Crossref Search ADS PubMed Scott , J. G. , C. A. Leichter , F. D. Rinkevich , F. D. Rinkevihc , S. A. Harris , C. Su , L. C. Aberegg , R. Moon , C. J. Geden , A. C. Gerry , et al. 2013 . Insecticide resistance in house flies from the United States: resistance levels and frequency of pyrethroid resistance alleles . Pestic. Biochem. Physiol . 107 : 377 – 384 . Google Scholar Crossref Search ADS PubMed Shah , P. A. , and J. K. Pell . 2003 . Entomopathogenic fungi as biological control agents . Appl. Microbiol. Biotechnol . 61 : 413 – 423 . Google Scholar Crossref Search ADS PubMed Shah , R. M. , N. Abbas , S. A. Shad , and A. A. Sial . 2015 . Selection, resistance risk assessment, and reversion toward susceptibility of pyriproxyfen in Musca domestica L . Parasitol. Res . 114 : 487 – 494 . Google Scholar Crossref Search ADS PubMed Skovgärd , H. , and T. Steenberg . 2002 . Activity of pupal parasitoids of the stable fly Stomoxys calcitrans and prevalence of entomopathogenic fungi in the stable fly and the house fly, Musca domestica . BioControl . 47 : 45 – 60 . Google Scholar Crossref Search ADS Steinkraus , D. C. , C. J. Geden , and D. A. Rutz . 1990 . First report of the natural occurrence of Beauveria bassiana (Moniliales: Moniliaceae) in Musca domestica (Diptera: Muscidae) . J. Med. Entomol . 27 : 309 – 312 . Google Scholar Crossref Search ADS Song , C. , T. A. Kidarsa , J. E. van de Mortel , J. E. Loper , and J. M. Raaijmakers . 2016 . Living on the edge: emergence of spontaneous gac mutations in Pseudomonas protegens during swarming motility . Environ. Microbiol . 18 : 3453 – 3465 . Google Scholar Crossref Search ADS PubMed Stout , M. J . 2015 . Insect pathogenic bacteria in integrated pest management . Insects . 6 : 352 – 367 . Google Scholar Crossref Search ADS PubMed Weeks , E. N. I. , E. T. Machtinger , C. J. Geden , and D. Leemon . 2018 . Biological control of livestock pests: entomopathogens . In C. Garros , J. Bouyer , W. Takken , and R. C. Smallegange (eds.), Pests and vector-borne diseases in the livestock industry, pp. 1142–1147 . Wageningen Academic Publishers , Wageningen, the Netherlands . West , L. S . 1951 . The house fly . Comstock Publishing , Ithaca, NY . Wollenberg , A. C. , T. Jagdish , G. Slough , M. E. Hoinville , and M. S. Wollenberg . 2016 . Death becomes them: bacterial community dynamics and stilbene antibiotic production in Galleria mellonella cadavers kills by Heterorhabditis/Photorhabdus . Appl. Environ. Microbiol . 82: AEM-01211 . doi: https://doi.org/10.1128/AEM.01211-16 Wraight , S. P. , M. A. Jackson , and S. L. de Kock . 2001 . Production, stabilization and formulation of fungal biocontrol agents, p. 253 . In T. M. Butt , C. Jackson , and N. Magan (eds.), Fungi as biocontrol agents: progress, problems and potential . CABI , Wallingford, United Kingdom . Published by Oxford University Press on behalf of Entomological Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.
TI - Mortality Effects of Three Bacterial Pathogens and Beauveria bassiana When Topically Applied or Injected Into House Flies (Diptera: Muscidae)
JF - Journal of Medical Entomology
DO - 10.1093/jme/tjy218
DA - 2019-04-16
UR - https://www.deepdyve.com/lp/oxford-university-press/mortality-effects-of-three-bacterial-pathogens-and-beauveria-bassiana-BlYZ0kgSWM
SP - 774
VL - 56
IS - 3
DP - DeepDyve
ER -