Effects of Chemical Insecticide Residues and Household Surface Type on a Beauveria bassiana-Based Biopesticide (Aprehend®) for Bed Bug Management.

The biopesticide Aprehend, containing spores of the entomopathogenic fungus Beauveria bassiana, is a biological control agent for the management of the common bed bug (Cimex lectularius L.) (Hemiptera: Cimicidae). The spores are applied in strategically placed barriers, which bed bugs walk across as they search for a bloodmeal. Application of chemical insecticides by the general public and professional pest managers is common, which means that Aprehend may be sprayed on existing insecticide residues. We evaluated the effect of chemical residues, of 22 different chemical insecticides on different household surface types. We found that residues from 12 chemical pesticides significantly reduced spore viability measured 5 weeks after application in comparison to the control. However, efficacy of Aprehend, as measured by bed bug mortality and mean survival time after exposure to sprayed surfaces, seven weeks after application was not impacted detrimentally. Furthermore, in some cases, efficacy of old chemical residues was enhanced by the combination of chemical and Aprehend seven weeks after application. Surface type also played a role in the relative efficacy of all products and combinations, particularly as the residues aged.

Biology of Mushroom Phorid Flies, Megaselia halterata (Diptera: Phoridae): Effects of Temperature, Humidity, Crowding, and Compost Stage.

The mushroom phorid fly, Megaselia halterata (Wood), is a common pest of mushroom production in many parts of the world. Due to the reduced availability of conventional insecticides for mushroom production, M. halterata has recently developed into a major pest in the top mushroom-producing county in the United States (Chester County, PA). Mushrooms are grown entirely indoors, and though larval development of M. halterata occurs in the mushroom-growing substrate, adult flies have been captured both inside and outside of the facilities. Here, we investigated three factors that might contribute to their growth and development. 1) The effects of ambient temperature (15-30°C) and relative humidity (RH; 21-98%) on adult M. halterata lifespan, 2) the effect of spawned compost stage (freshly inoculated with spawn vs 14-d spawned compost) on reproductive output, and 3) the effect of population density on reproductive output. The longevity of adult M. halterata increased under cooler temperatures and more humid conditions (>75% RH), which reflect the conditions inside mushroom-growing facilities. Similar numbers of flies emerged from freshly inoculated and 14-d spawned compost, but flies emerged earlier from 14-d spawned compost. The higher the parental fly density, the more offspring emerged from spawned compost, but the positive relationship reached a plateau beyond 40 parental mating pairs per 100 g of compost. Our findings highlight relevant abiotic and biotic factors that may contribute to M. halterata population dynamics.

Applications of Beauveria bassiana (Hypocreales: Cordycipitaceae) to Control Populations of Spotted Lanternfly (Hemiptera: Fulgoridae), in Semi-Natural Landscapes and on Grapevines.

Spotted lanternfly, Lycorma delicatula (White), is an invasive Asian insect that was initially found in Berks County, Pennsylvania, in 2014. As of early 2020, this pest had been found in five more eastern states and it is expected to continue to expand its geographical range. Lycorma delicatula is highly polyphagous but seems to prefer tree-of-heaven, Ailanthus altissima. However, grape growers in Pennsylvania have reported significant damage and loss of vines caused by L. delicatula adults. In fall 2018, two fungal entomopathogens (Beauveria bassiana and Batkoa major) drove localized collapses in L. delicatula populations in Berks County, Pennsylvania. In 2019, we tested applications of a commercialized mycoinsecticide based on B. bassiana strain GHA on L. delicatula populations in a public park in southeastern Pennsylvania. A single application of B. bassiana reduced fourth instar nymphs by 48% after 14 d. Applications of B. bassiana to L. delicatula adults in the same park resulted in 43% mortality after 14 d. Beauveria bassiana spores remained viable on foliage for 5-7 d after spraying. We also conducted semi-field bioassays with B. bassiana GHA (formulated as BoteGHA and Aprehend) and another mycoinsecticide containing Isaria fumosorosea Apopka Strain 97 against L. delicatula adults feeding on potted grapes. All the mycoinsecticides killed ≥90% of adults after 9 d using direct applications. Aprehend killed 99% of adults after 9 d with exposure to residues on sprayed grapes. These data show that fungal entomopathogens can help to suppress populations of L. delicatula in agroecosystems and natural areas.

Persistence and Lethality of a Fungal Biopesticide (Aprehend) Applied to Insecticide-impregnated and Encasement-type Box Spring Covers for Bed Bug Management.

The newly developed fungal biopesticide Aprehend, containing spores of Beauveria bassiana, is the first biological control agent to be incorporated into management programs to control the common bed bug (Cimex lectularius L.) (Hemiptera: Cimicidae). Aprehend is sprayed as barriers where bed bugs are likely to walk and pick up spores as they search for a bloodmeal. A key application target for Aprehend is the box spring, which may be covered by encasement-type or insecticide-impregnated covers. Since some insecticides can reduce the persistence of fungal spores, we tested the efficacy and spore germination percentages of Aprehend when applied to the two types of box spring covers. We found that spore germination was about 11% lower on the permethrin-impregnated ActiveGuard cover than on the encasement-type AllerEase cover. However, bed bugs exposed for 15 min to Aprehend on the two box spring covers suffered similarly high levels of mortality irrespective of the cover material. Thus, there was no inhibitory or additive effect of the ActiveGuard cover on bed bug mortality. Lastly, overall mortality was higher if bed bugs were exposed to Aprehend-treated ActiveGuard than the ActiveGuard cover alone. Our findings indicate that if pest managers are using ActiveGuard covers in combination with Aprehend, best practice would be to use ActiveGuard on mattresses and apply Aprehend directly to the box spring or to a box spring covered by an encasement-type cover.

Susceptibility of insecticide-resistant bed bugs (Cimex lectularius) to infection by fungal biopesticide.

BACKGROUND: Bed bugs are a public health concern, and their incidence is increasing worldwide. Bed bug infestations are notoriously difficult to eradicate, further exacerbated by widespread resistance to pyrethroid and neonicotinoid insecticides. This study evaluated the efficacy of the newly developed fungal biopesticide Aprehend™, containing Beauveria bassiana, against insecticide-resistant bed bugs. RESULTS: Overall mortality for the Harold Harlan (insecticide-susceptible) strain was high (98-100%) following exposure to Aprehend™ or Suspend SC (deltamethrin). The mean survival times (MSTs) for Harold Harlan bed bugs were 5.1 days for Aprehend™ and 4.8 and 3.0 days for the low and high concentrations of Suspend SC respectively. All three strains of pyrethroid-resistant bed bugs were susceptible to infection by B. bassiana, resulting in MSTs of <6 days (median = 4 days) and >94% overall mortality. Conversely, mortality of the three insecticide-resistant strains after exposure to Suspend SC was only 16-40%. CONCLUSION: These results demonstrate that Aprehend™ is equally effective against insecticide-susceptible and insecticide-resistant bed bugs and could provide pest control operators with a promising new tool for control of bed bugs and insecticide resistance management. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Attraction, Oviposition and Larval Survival of the Fungus Gnat, Lycoriella ingenua, on Fungal Species Isolated from Adults, Larvae, and Mushroom Compost.

We previously showed that the females of the mushroom sciarid, Lycoriella ingenua (Dufour, 1839) (Diptera: Sciaridae), one of the most severe pests of the cultivated white button mushroom, Agaricus bisporus (J.E. Lange) Emil J. Imbach (Agaricales: Agaricaceae), are attracted to the mushroom compost that mushrooms are grown on and not to the mushrooms themselves. We also showed that females are attracted to the parasitic green mold, Trichoderma aggressivum. In an attempt to identify what is in the mushroom compost that attracts female L. ingenua, we isolated several species of fungi from adult males and females, third instar larvae, and mushroom compost itself. We then analyzed the attraction of females to these substrates using a static-flow two choice olfactometer, as well as their oviposition tendencies in another type of assay under choice and no-choice conditions. We also assessed the survival of larvae to adulthood when first instar larvae were placed on each of the isolated fungal species. We found that female flies were attracted most to the mycoparasitic green mold, T. aggressivum, to Penicilium citrinum isolated from adult female bodies, and to Scatylidium thermophilium isolated from the mushroom compost. Gravid female flies laid the most eggs on T. aggressivum, Aspergillus flavus isolated from third instar larval frass, Aspergillus fumigatus isolated from adult male bodies, and on P. citrinum. This egg-laying trend remained consistent under no-choice conditions as females aged. First instar larvae developed to adulthood only on S. thermophilium and Chaetomium sp. isolated from mushroom compost, and on P. citrinum. Our results indicate that the volatiles from a suite of different fungal species act in tandem in the natural setting of mushroom compost, with some first attracting gravid female flies and then others causing them to oviposit. The ecological context of these findings is important for creating an optimal strategy for using possible semiochemicals isolated from these fungal species to better monitor and control this pestiferous mushroom fly species.

Malaria mosquitoes attracted by fatal fungus.

Insect-killing fungi such as Beauveria bassiana are being evaluated as possible active ingredients for use in novel biopesticides against mosquito vectors that transmit malaria. Fungal pathogens infect through contact and so applications of spores to surfaces such as walls, nets, or other resting sites provide possible routes to infect mosquitoes in and around domestic dwellings. However, some insects can detect and actively avoid fungal spores to reduce infection risk. If true for mosquitoes, such behavior could render the biopesticide approach ineffective. Here we find that the spores of B. bassiana are highly attractive to females of Anopheles stephensi, a major anopheline mosquito vector of human malaria in Asia. We further find that An. stephensi females are preferentially attracted to dead and dying caterpillars infected with B. bassiana, landing on them and subsequently becoming infected with the fungus. Females are also preferentially attracted to cloth sprayed with oil-formulated B. bassiana spores, with 95% of the attracted females becoming infected after a one-minute visit on the cloth. This is the first report of an insect being attracted to a lethal fungal pathogen. The exact mechanisms involved in this behavior remain unclear. Nonetheless, our results indicate that biopesticidal formulations comprising B. bassiana spores will be conducive to attraction and on-source visitation by malaria vectors.

Conidial acquisition and survivorship of adult Asian longhorned beetles exposed to flat versus shaggy agar fungal bands.

Fungal bands can deliver lethal conidial doses to adult Asian longhorned beetles. Because higher doses result in shorter survival times, developing a method to deliver more conidia to beetles walking across the fungal bands is desirable. We compared fungal bands made using standard flat material to bands made using a shaggy, textured material. The median survival time of adult beetles exposed to shaggy bands was reduced to 10 d versus 18 d for beetles exposed to flat bands. Beetles climbing across shaggy bands acquired 1.83×10(6) conidia per beetle, which was 14.6 times greater than beetles exposed to flat bands.

Comparing fungal band formulations for Asian longhorned beetle biological control.

Experiments were conducted with the fungal entomopathogen Metarhizium brunneum to determine the feasibility of using agar-based fungal bands versus two new types of oil-formulated fungal bands for Asian longhorned beetle management. We investigated conidial retention and survival on three types of bands attached to trees in New York and Pennsylvania: standard polyester fiber agar-based bands containing fungal cultures, and two types of bands made by soaking either polyester fiber or jute burlap with oil-conidia suspensions. Fungal band formulation did not affect the number or viability of conidia on bands over the 2-month test period, although percentage conidial viability decreased significantly with time for all band types. In a laboratory experiment testing the effect of the three band formulations on conidial acquisition and beetle survival, traditional agar-based fungal bands delivered the most conidia to adult beetles and killed higher percentages of beetles significantly faster (median survival time of 27d) than the two oil-formulated materials (36-37d). We also tested the effect of band formulation on conidial acquisition by adult beetles kept individually in cages with a single band for 24h, and significantly more conidia (3-7times) were acquired by beetles from agar-based bands compared to the two oil formulations.

Discriminating fever behavior in house flies.

Fever has generally been shown to benefit infected hosts. However, fever temperatures also carry costs. While endotherms are able to limit fever costs physiologically, the means by which behavioral thermoregulators constrain these costs are less understood. Here we investigated the behavioral fever response of house flies (Musca domestica L.) challenged with different doses of the fungal entomopathogen, Beauveria bassiana. Infected flies invoked a behavioral fever selecting the hottest temperature early in the day and then moving to cooler temperatures as the day progressed. In addition, flies infected with a higher dose of fungus exhibited more intense fever responses. These variable patterns of fever are consistent with the observation that higher fever temperatures had greater impact on fungal growth. The results demonstrate the capacity of insects to modulate the degree and duration of the fever response depending on the severity of the pathogen challenge and in so doing, balance the costs and benefits of fever.

Evaluating the lethal and pre-lethal effects of a range of fungi against adult Anopheles stephensi mosquitoes.

BACKGROUND: Insecticide resistance is seriously undermining efforts to eliminate malaria. In response, research on alternatives to the use of chemical insecticides against adult mosquito vectors has been increasing. Fungal entomopathogens formulated as biopesticides have received much attention and have shown considerable potential. This research has necessarily focused on relatively few fungal isolates in order to 'prove concept'. Further, most attention has been paid to examining fungal virulence (lethality) and not the other properties of fungal infection that might also contribute to reducing transmission potential. Here, a range of fungal isolates were screened to examine variation in virulence and how this relates to additional pre-lethal reductions in feeding propensity. METHODS: The Asian malaria vector, Anopheles stephensi was exposed to 17 different isolates of entomopathogenic fungi belonging to species of Beauveria bassiana, Metarhizium anisopliae, Metarhizium acridum and Isaria farinosus. Each isolate was applied to a test substrate at a standard dose rate of 1×109 spores ml-1 and the mosquitoes exposed for six hours. Subsequently the insects were removed to mesh cages where survival was monitored over the next 14 days. During this incubation period the mosquitoes' propensity to feed was assayed for each isolate by offering a feeding stimulant at the side of the cage and recording the number probing. RESULTS AND CONCLUSIONS: Fungal isolates showed a range of virulence to A. stephensi with some causing >80% mortality within 7 days, while others caused little increase in mortality relative to controls over the study period. Similarly, some isolates had a large impact on feeding propensity, causing >50% pre-lethal reductions in feeding rate, whereas other isolates had very little impact. There was clear correlation between fungal virulence and feeding reduction with virulence explaining nearly 70% of the variation in feeding reduction. However, there were some isolates where either feeding decline was not associated with high virulence, or virulence did not automatically prompt large declines in feeding. These results are discussed in the context of choosing optimum fungal isolates for biopesticide development.

Storage and persistence of a candidate fungal biopesticide for use against adult malaria vectors.

BACKGROUND: New products aimed at augmenting or replacing chemical insecticides must have operational profiles that include both high efficacy in reducing vector numbers and/or blocking parasite transmission and be long lasting following application. Research aimed at developing fungal spores as a biopesticide for vector control have shown considerable potential yet have not been directly assessed for their viability after long-term storage or following application in the field. METHODS: Spores from a single production run of the entomopathogenic fungi Beauveria bassiana were dried and then stored under refrigeration at 7°C. After 585 days these spores were sub-sampled and placed at either 22°C, 26°C or 32°C still sealed in packaging (closed storage) or in open beakers and exposed to the 80% relative humidity of the incubator they were kept in. Samples were subsequently taken from these treatments over a further 165 days to assess viability. Spores from the same production run were also used to test their persistence following application to three different substrates, clay, cement and wood, using a hand held sprayer. The experiments were conducted at two different institutes with one using adult female Anopheles stephensi and the other adult female Anopheles gambiae. Mosquitoes were exposed to the treated substrates for one hour before being removed and their survival monitored for the next 14 days. Assays were performed at monthly intervals over a maximum seven months. RESULTS: Spore storage under refrigeration resulted in no loss of spore viability over more than two years. Spore viability of those samples kept under open and closed storage was highly dependent on the incubation temperature with higher temperatures decreasing viability more rapidly than cooler temperatures. Mosquito survival following exposure was dependent on substrate type. Spore persistence on the clay substrate was greatest achieving 80% population reduction for four months against An. stephensi and for at least five months against Anopheles gambiae. Cement and wood substrates had more variable mortality with the highest spore persistence being two to three months for the two substrates respectively. CONCLUSIONS: Spore shelf-life under refrigeration surpassed the standard two year shelf-life expected of a mosquito control product. Removal to a variety of temperatures under either closed or open storage indicated that samples sent out from refrigeration should be deployed rapidly in control operations to avoid loss of viability. Spore persistence following application onto clay surfaces was comparable to a number of chemical insecticides in common use. Persistence on cement and wood was shorter but in one assay still comparable to some organophosphate and pyrethroid insecticides. Optimized formulations could be expected to improve spore persistence still further.

A preliminary evaluation of the potential of Beauveria bassiana for bed bug control.

Residual biopesticide treatments of Beauveria bassiana were tested against the bed bug Cimex lectularius. An oil formulation of conidia was applied to different substrates. Bed bugs were exposed for 1 h, transferred to an unsprayed environment and monitored for mortality. Separate bioassays evaluated the effect of bed bug strain, sex, life stage, and exposure substrate on mortality. Rapid mortality was observed in all bioassays, with bed bugs exposed to treated jersey knit cotton dying most rapidly. A further assay demonstrated efficient autodissemination of conidia from exposed bed bugs to unexposed bed bugs within artificial harborages.