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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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Spore distribution. Photo of a piece of proofing paper surface sprayed (left) or coated (right) with fungus formulation (zoomed in 4 ×). Equal volumes of a 5 × 109 spores/ml formulation (20% Ondina/80% Shellsol) were applied per surface area using the spray method described by Bell et al [35] or the optimized coating method. Spots on the left piece represent the spray droplets. The white background is not visible on the coated piece (right) because it is covered fully with the spore layer.
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Figure 7: Spore distribution. Photo of a piece of proofing paper surface sprayed (left) or coated (right) with fungus formulation (zoomed in 4 ×). Equal volumes of a 5 × 109 spores/ml formulation (20% Ondina/80% Shellsol) were applied per surface area using the spray method described by Bell et al [35] or the optimized coating method. Spots on the left piece represent the spray droplets. The white background is not visible on the coated piece (right) because it is covered fully with the spore layer.

Mentions: K-bar coating provided a simple and consistent method for coating layers of fungal spores onto paper substrates. By applying exact suspension volumes of known concentration onto a pre-determined substrate surface, the effective end-concentration of spores per unit surface area could be determined. The precision of the coating method could be somewhat affected by variations due to the manual use of the pipette and small proportions of formulation being coated outside the treatment surface boundaries or remaining on the K-bar as residue. These variations are, however, considered negligent compared to spraying, which has been reported to lose more than 90% of the application volume due to vaporization and bounce-off [35]. When applying the same volume per surface area, much more spores ended up on a coated paper compared with a sprayed paper, which is illustrated by its darker colour in Figure 7. The homogeneity of spore layers after application could not be quantified. Though fluorescent dyes may be used to improve visualisation of suspended spores [38], it was now not possible to quantify the number of spores with a microscope after application onto the paper. Therefore, the uniformity of spore coatings could only be determined visually. When using high Metarhizium concentrations, the K-bar deposited relatively homogeneous, non-clumping layers, where spraying would result in a more patchy distribution (Figure 7). The use of novel techniques such as quantitative PCR [35] may be used to quantify the spore layer of a coated paper and to determine the application efficacy and homogeneity of the coating method more precisely and allow more direct comparisons with spraying.


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

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

Spore distribution. Photo of a piece of proofing paper surface sprayed (left) or coated (right) with fungus formulation (zoomed in 4 ×). Equal volumes of a 5 × 109 spores/ml formulation (20% Ondina/80% Shellsol) were applied per surface area using the spray method described by Bell et al [35] or the optimized coating method. Spots on the left piece represent the spray droplets. The white background is not visible on the coated piece (right) because it is covered fully with the spore layer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Spore distribution. Photo of a piece of proofing paper surface sprayed (left) or coated (right) with fungus formulation (zoomed in 4 ×). Equal volumes of a 5 × 109 spores/ml formulation (20% Ondina/80% Shellsol) were applied per surface area using the spray method described by Bell et al [35] or the optimized coating method. Spots on the left piece represent the spray droplets. The white background is not visible on the coated piece (right) because it is covered fully with the spore layer.
Mentions: K-bar coating provided a simple and consistent method for coating layers of fungal spores onto paper substrates. By applying exact suspension volumes of known concentration onto a pre-determined substrate surface, the effective end-concentration of spores per unit surface area could be determined. The precision of the coating method could be somewhat affected by variations due to the manual use of the pipette and small proportions of formulation being coated outside the treatment surface boundaries or remaining on the K-bar as residue. These variations are, however, considered negligent compared to spraying, which has been reported to lose more than 90% of the application volume due to vaporization and bounce-off [35]. When applying the same volume per surface area, much more spores ended up on a coated paper compared with a sprayed paper, which is illustrated by its darker colour in Figure 7. The homogeneity of spore layers after application could not be quantified. Though fluorescent dyes may be used to improve visualisation of suspended spores [38], it was now not possible to quantify the number of spores with a microscope after application onto the paper. Therefore, the uniformity of spore coatings could only be determined visually. When using high Metarhizium concentrations, the K-bar deposited relatively homogeneous, non-clumping layers, where spraying would result in a more patchy distribution (Figure 7). The use of novel techniques such as quantitative PCR [35] may be used to quantify the spore layer of a coated paper and to determine the application efficacy and homogeneity of the coating method more precisely and allow more direct comparisons with spraying.

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