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A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays.

Farenhorst M, Knols BG - Malar. J. (2010)

Bottom Line: Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity.Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival.Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Entomology, Wageningen University and Research Centre, PO Box 8031, 6700 EH, Wageningen, The Netherlands. marit.farenhorst@wur.nl

ABSTRACT

Background: Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared.

Methods: Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes.

Results: K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time.

Conclusions: K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.

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Dose response curves. Cumulative daily proportional survival of 40 female An. gambiae s.s. exposed for 3 hrs to control papers (black) or proofing papers manually coated with 109-1012 spores/m2 of M. anisopliae (red) or B. bassiana (blue).
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Figure 5: Dose response curves. Cumulative daily proportional survival of 40 female An. gambiae s.s. exposed for 3 hrs to control papers (black) or proofing papers manually coated with 109-1012 spores/m2 of M. anisopliae (red) or B. bassiana (blue).

Mentions: Dose-response curves were obtained for both M. anisopliae and B. bassiana by coating 10-fold dilutions of the same stock suspensions with the K-Hand Coater, resulting in end-concentrations ranging between 109 and 1012 viable spores/m2. For both fungi, all tested doses reduced survival significantly compared with control mosquitoes (P < 0.001) (Figure 5). Mosquito survival data show a consistent dose-dependent increase in fungal virulence, with 109 spores/m2 causing the smallest reduction in mosquito survival and 1012 spores/m2 the largest (Figure 5). Infectivity data also showed a dose-dependent increase for fungal infection. For Metarhizium, 19, 37, 76 and 95% of the mosquitoes showed fungal infection after death when exposed to respectively 109, 1010, 1011 and 1012 spores/m2. For Beauveria, this was 23, 49, 84 and 90%.


A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays.

Farenhorst M, Knols BG - Malar. J. (2010)

Dose response curves. Cumulative daily proportional survival of 40 female An. gambiae s.s. exposed for 3 hrs to control papers (black) or proofing papers manually coated with 109-1012 spores/m2 of M. anisopliae (red) or B. bassiana (blue).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2820041&req=5

Figure 5: Dose response curves. Cumulative daily proportional survival of 40 female An. gambiae s.s. exposed for 3 hrs to control papers (black) or proofing papers manually coated with 109-1012 spores/m2 of M. anisopliae (red) or B. bassiana (blue).
Mentions: Dose-response curves were obtained for both M. anisopliae and B. bassiana by coating 10-fold dilutions of the same stock suspensions with the K-Hand Coater, resulting in end-concentrations ranging between 109 and 1012 viable spores/m2. For both fungi, all tested doses reduced survival significantly compared with control mosquitoes (P < 0.001) (Figure 5). Mosquito survival data show a consistent dose-dependent increase in fungal virulence, with 109 spores/m2 causing the smallest reduction in mosquito survival and 1012 spores/m2 the largest (Figure 5). Infectivity data also showed a dose-dependent increase for fungal infection. For Metarhizium, 19, 37, 76 and 95% of the mosquitoes showed fungal infection after death when exposed to respectively 109, 1010, 1011 and 1012 spores/m2. For Beauveria, this was 23, 49, 84 and 90%.

Bottom Line: Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity.Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival.Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Entomology, Wageningen University and Research Centre, PO Box 8031, 6700 EH, Wageningen, The Netherlands. marit.farenhorst@wur.nl

ABSTRACT

Background: Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared.

Methods: Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes.

Results: K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time.

Conclusions: K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.

Show MeSH
Related in: MedlinePlus