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

Bell AS, Blanford S, Jenkins N, Thomas MB, Read AF - J. Invertebr. Pathol. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH93JT, UK. asb15@psu.edu

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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2666797&req=5

fig5: 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.

Mentions: Fig. 5 shows the daily cumulative percentage survival for mosquitoes of each challenge dose.


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

Bell AS, Blanford S, Jenkins N, Thomas MB, Read AF - J. Invertebr. Pathol. (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666797&req=5

fig5: 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.
Mentions: Fig. 5 shows the daily cumulative percentage survival for mosquitoes of each challenge dose.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH93JT, UK. asb15@psu.edu

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.

Show MeSH
Related in: MedlinePlus