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.

Fig. 1

Assay amplification plots and standard curves. Left hand panels show real-time qPCR amplification plots: each set of parallel lines indicate a 10-fold dilution in DNA sample (known standards – for fungal assays from 10⁸ to 10² conidia) from which the standard curves are derived (right hand panels) by plotting the cycle at which each standard enters into log-linear amplification (crosses the machine-determined threshold: solid horizontal line) against the known number of conidia present in that standard. Conidial numbers in unknown samples are determined from where their amplification plot crosses the threshold and is read from the standard curve. Non-amplified samples fail to generate an amplification plot and cross the threshold. A standard curve with a slope of −3.32 represents an assay with a hypothetical PCR efficiency of 100%.

Fig. 2

Number of Beauveria bassiana conidial units on/within mosquitoes with respect to time post-challenge. Symbols represent mean log₁₀ number of conidial units at each sample time point for each challenge dose and vertical lines ±1SE.

Fig. 3

Number of Beauveria bassiana conidial units on/within mosquitoes with respect to time post-challenge. Solid bars represent the number of mosquitoes with a particular burden; dashed lines numerical means and horizontal lines (numbers in brackets) ±1SE.

Fig. 4

Relative burdens of visually-infected and visually-uninfected mosquitoes dissected on day 5 post-exposure. Symbols represent counts from individual mosquitoes; horizontal bars means and heavy horizontal bars ±1SE.

Fig. 5

Mean cumulative daily percent survival of Anopheles stephensi exposed to either low (5 × 10⁸ spores/ml⁻¹), medium (1 × 10⁹ spores/ml⁻¹) or high (5 × 10⁹ spores/ml⁻¹) formulations of Beauveria bassiana (see text for further application details). Survival was estimated as the number dying on a particular day as a percent of those alive at the end of the previous day. Survival does not reach zero as mosquitoes remaining alive on their last recorded survival day were sampled for PCR analysis. Mean and SEM are taken from two replicated cages per dose regime.