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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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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.
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fig2: 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.

Mentions: Actual conidial densities present on challenge pots are shown in Table 2. These differed 2.2-fold between the low and medium doses (in theory should be 2-fold) and 13.2-fold between the medium and high doses (in theory 5-fold). Nevertheless, conidial acquisition by mosquitoes exposed to the high dose was proportionately less than at the other two doses and there was a 1.9-fold difference in conidial pick-up between the low (ca. 1 × 104) and medium (ca. 2 × 104) doses and a 3.3-fold difference between the medium and high (ca. 6 × 104) doses (see also Figs. 2 and 3). These differences in pick-up were significant between challenge doses (F2,57 = 27.2, p < 0.001). Fungal burdens on/within mosquitoes exposed to the two lower challenge doses did not differ significantly from each other at any subsequent sample point (F1,39 < 3.27, p > 0.08), whereas mean numbers of fungal genomes were significantly greater from mosquitoes exposed to the high dose at all sample points (F1,39 > 15.1, p < 0.002). By day 2 post-exposure mosquitoes from all challenge groups had significantly reduced burdens compared to their respective pick-up densities (F1,39 > 15.1, p < 0.001; see Fig. 2). Mean numbers of genomes then increased in all treatment groups between days 2 and 3 post-exposure, although not significantly so for any dose. Burdens increased significantly across all doses from day 3 to day 4 post-challenge (F1,39 > 12.2, p < 0.001) and continued to rise through to the end of the monitoring period at day 6 post-exposure (no mosquitoes remained alive in the high dose treatment on day 6), but there were no further significant increases in burdens. Fresh cadavers (collected at day 5 post-exposure) yielded significantly greater mean burdens than the respective mean densities from live mosquitoes (F1,38 > 4.8, p < 0.035).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: 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.
Mentions: Actual conidial densities present on challenge pots are shown in Table 2. These differed 2.2-fold between the low and medium doses (in theory should be 2-fold) and 13.2-fold between the medium and high doses (in theory 5-fold). Nevertheless, conidial acquisition by mosquitoes exposed to the high dose was proportionately less than at the other two doses and there was a 1.9-fold difference in conidial pick-up between the low (ca. 1 × 104) and medium (ca. 2 × 104) doses and a 3.3-fold difference between the medium and high (ca. 6 × 104) doses (see also Figs. 2 and 3). These differences in pick-up were significant between challenge doses (F2,57 = 27.2, p < 0.001). Fungal burdens on/within mosquitoes exposed to the two lower challenge doses did not differ significantly from each other at any subsequent sample point (F1,39 < 3.27, p > 0.08), whereas mean numbers of fungal genomes were significantly greater from mosquitoes exposed to the high dose at all sample points (F1,39 > 15.1, p < 0.002). By day 2 post-exposure mosquitoes from all challenge groups had significantly reduced burdens compared to their respective pick-up densities (F1,39 > 15.1, p < 0.001; see Fig. 2). Mean numbers of genomes then increased in all treatment groups between days 2 and 3 post-exposure, although not significantly so for any dose. Burdens increased significantly across all doses from day 3 to day 4 post-challenge (F1,39 > 12.2, p < 0.001) and continued to rise through to the end of the monitoring period at day 6 post-exposure (no mosquitoes remained alive in the high dose treatment on day 6), but there were no further significant increases in burdens. Fresh cadavers (collected at day 5 post-exposure) yielded significantly greater mean burdens than the respective mean densities from live mosquitoes (F1,38 > 4.8, p < 0.035).

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