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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.

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Related in: MedlinePlus

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%.
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fig1: 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%.

Mentions: The assay does not amplify mosquito, murine or Plasmodium DNA. It may be used to quantify any of the three fungi tested, although its deliberate lack of specificity will result in counts partially attributable to background fungi present in the natural/experimental environment (see Table 1, Fig. 1). Nevertheless, background counts typically recorded for unchallenged mosquitoes reared within our insectary were minimal: see B. bassiana growth within challenged mosquitoes section below.


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)

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%.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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%.
Mentions: The assay does not amplify mosquito, murine or Plasmodium DNA. It may be used to quantify any of the three fungi tested, although its deliberate lack of specificity will result in counts partially attributable to background fungi present in the natural/experimental environment (see Table 1, Fig. 1). Nevertheless, background counts typically recorded for unchallenged mosquitoes reared within our insectary were minimal: see B. bassiana growth within challenged mosquitoes section below.

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