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

ABSTRACT: 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.

SUBMITTER: Bell AS

PROVIDER: S-EPMC2666797 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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

Bell Andrew S AS Blanford Simon S Jenkins Nina N Thomas Matthew B MB Read Andrew F AF

Journal of invertebrate pathology 20090301 3

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 IMI3915 ...[more]

PMID: 19320043

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Project description:The effectiveness of conventional malaria vector control is being threatened by the spread of insecticide resistance. One promising alternative to chemicals is the use of naturally-occurring insect-killing fungi. Numerous laboratory studies have shown that isolates of fungal pathogens such as Beauveria bassiana can infect and kill adult mosquitoes, including those resistant to chemical insecticides.Unlike chemical insecticides, fungi may take up to a week or more to kill mosquitoes following exposure. This slow kill speed can still reduce malaria transmission because the malaria parasite itself takes at least eight days to complete its development within the mosquito. However, both fungal virulence and parasite development rate are strongly temperature-dependent, so it is possible that biopesticide efficacy could vary across different transmission environments.We examined the virulence of a candidate fungal isolate against two key malaria vectors at temperatures from 10-34 °C. Regardless of temperature, the fungus killed more than 90% of exposed mosquitoes within the predicted duration of the malarial extrinsic incubation period, a result that was robust to realistic diurnal temperature variation.We then incorporated temperature sensitivities of a suite of mosquito, parasite and fungus life-history traits that are important determinants of malaria transmission into a stage-structured malaria transmission model. The model predicted that, at achievable daily fungal infection rates, fungal biopesticides have the potential to deliver substantial reductions in the density of malaria-infectious mosquitoes across all temperatures representative of malaria transmission environments.Synthesis and applications. Our study combines empirical data and theoretical modelling to prospectively evaluate the potential of fungal biopesticides to control adult malaria vectors. Our results suggest that Beauveria bassiana could be a potent tool for malaria control and support further development of fungal biopesticides to manage infectious disease vectors.

Dataset Information

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

Publications

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

Similar Datasets

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