Nina E. Jenkins

Real-time quantitative PCR for analysis of candidate fungal biopesticides against malaria: Technique validation and first applications

Recent research has indicated that fungal biopesticides could augment existing malaria vector control tools. Here we present a set of methodologies to monitor the in vivo kinetics of entomopathogenic fungi in Anopheles in the presence or absence of malaria parasites using quantitative real-time PCR. Three qPCR assays were successfully developed for counting fungal genomes: “specific” assays capable of distinguishing two well characterized fungal entomopathogens Beauveria bassiana isolate IMI391510 and Metarhizium anisopliae var. acridum isolate IMI330189, both of which have previously been shown to be virulent to Anopheles mosquitoes, and a “generic” fungal assay for determining any fungal burden. A fourth assay to Plasmodium chabaudi enabled quantification of co-infecting malarial parasites. All qPCR assays provide sensitive, target-specific, and robust quantification over a linear range of greater than five orders of magnitude (seven orders of magnitude for the fungal assays). B. bassiana growth within mosquitoes exposed to three different conidial challenge doses was monitored using the B. bassiana-specific assay and represents the first description of entomopathogenic fungal replication within an insect host. This revealed that, irrespective of challenge dose, after several days of relatively little replication, a sudden on-set of substantial nuclear division occurs, accompanied by physical fungal growth (hyphae) within the mosquito haemocoel shortly before death. Exposure to higher densities of conidia resulted in significantly greater pick-up by mosquitoes and to elevated fungal burdens at each time point sampled. High fungal burdens, comparable to those identified in cadavers, were attained more rapidly and mortalities occurred earlier post-exposure with increasing challenge dose. The lines of research made possible by the qPCR assays described here will contribute to optimization of fungal biopesticides against malaria and other vector-borne diseases.

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.

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.

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

BackgroundNew 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.MethodsSpores 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.ResultsSpore 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.ConclusionsSpore 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.

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

BackgroundInsecticide 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.MethodsThe 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 conclusionsFungal 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.

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.

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.

Potential for biocontrol of house flies, Musca domestica, using fungal biopesticides

Chemical control of house flies in poultry production facilities is becoming increasingly difficult due to insecticide resistance and regulatory constraints. Biopesticides based on entomopathogenic fungi could provide an alternative approach. Here we evaluated population control potential of two fungal pathogens, Beauveria bassiana and Metarhizium anisopliae. Cohorts of adult flies were established in large plastic boxes in the laboratory and were exposed to residues of oil-formulated fungal conidia sprayed on strips of plastic sheeting attached to the box walls. Exposure to the biopesticide barrier treatments caused 100% mortality in adult populations within 8–16 days, depending on the fungal species. In contrast, control flies survived until 96–110 days. Additionally, fungal infections caused 13–20% reduction in egg viability and >70% reduction in fecundity of flies prior to death. The combined lethal and pre-lethal impacts resulted in 21- to 26-fold reduction in basic reproductive rate in the fungus-exposed populations relative to controls. Based on these promising proof-of-principle results, further research is currently under way to determine the feasibility of developing a biopesticide product for operational use.

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.

Persistence and efficacy of a Beauveria bassiana biopesticide against the house fly, Musca domestica, on typical structural substrates of poultry houses

Entomopathogenic fungi, such as Beauveria bassiana, offer potential for use as biopesticides for control of house flies in poultry production facilities. This study evaluates persistence and efficacy of oil-formulated B. bassiana conidia against adult house flies on a range of structural substrates commonly found in poultry houses. Exposure of flies to fungal-treated surfaces produced high levels of infection leading up to 100% mortality in 6–10 days. However, the infectivity of the spray residues declined rapidly within 1 or 2 weeks following repeated fly exposures. Investigations showed that, in the absence of flies, conidia remained viable on test surfaces for up to 3 months regardless of substrate type, application method or fungal production batch. Rather, it was the presence of flies themselves that was responsible for reducing persistence. The exact mechanisms remain unclear but involve a combination of physical removal and chemical deactivation, with decay rates increasing at higher fly densities. While the rapid decay could pose a challenge for operational use, the results suggest it might be possible to tailor treatment frequencies to fly densities with, for example, weekly applications at high fly densities and longer intervals when populations decline. Further research is needed to determine persistence in semifield and field settings and to quantify the influence of fly densities under natural exposure conditions.