Spores of Beauveria bassiana stored under ideal conditions of low moisture content and low temperature survived with no loss of viability for over two years. Assuming access to cold storage, this baseline viability meets the WHO pesticide shelf-life criterion
. Once removed from refrigeration spore viability declined in a temperature dependent manner with high temperatures resulting in a faster rate of mortality than cooler temperatures. Surprisingly, under open storage, where the spore powder was exposed to high humidity in the incubators, survival was better than when spores were protected from humidity. Why this was so is unclear as maintaining low moisture content should improve spore stability
. Nonetheless, these results suggest the need for relatively rapid distribution and use of product once taken out of some sort of long-term stockpile. Such an approach is similar to standard practice for the mosquito larval biocontrol agent, Bacillus thuringiensis[45–47]. Moreover, certain commercial preparations of B. bassiana used in agriculture exhibit shelf lives of 12 months or more at room temperature
[33, 34, 48], so there is scope for more product development. Further, data from other storage studies suggest that the viability of spores under variable temperature conditions (i.e. not refrigeration) might depend on the absolute age of the spores
. The current experiments used spores that had already been in storage for 585 days. Taking spores from storage at perhaps three or six months after production could potentially alter viability/persistence downstream. Assuming there is some sort of trade-off, optimizing time spent in storage vs time available for distribution and use will be an important factor in supply chain management.
Spore persistence following application varied considerably between substrates and assays. With the An. stephensi assays the control mortality was higher and much more variable than expected, making interpretation of results slightly difficult on some occasions. However, effective persistence was clearly demonstrated up to 4 months on clay substrates (and this could be extended to five months if mosquitoes contacted the treated substrates more than once). Persistence on cement and wood were less good, but 80% morality was still achieved up to months 2 and 3, respectively. Persistence of 2–4 months is within the range of existing chemical insecticides approved for use in IRS by the WHO
[36, 37]. Indeed, the fungus performed considerably better on the clay and concrete substrates than a basic formulation of Lambda-cyhalothrin, which essentially had no impact from month 1 (or even day 1 on clay). Performance of the chemical was much better on wood. These results are consistent with previous observations that porous substrates such as clay can interfere with the pick-up of chemical insecticides
[37, 49, 50].
Control survival in the An. gambiae assays was much more stable, leaving treatment effects unambiguous. On clay tiles the fungal spray residue remained effective for between 5–7 months (the pattern of the mortality data suggest the month 6 exposure would have achieved the 80% cut off, but unfortunately the month 6 samples were not available). On wood and cement tiles, on the other hand, while there was some significant mortality relative to controls for up to 3 months, mortality of 80% was never achieved even from day 1.
The reasons for the differences in persistence and efficacy of fungal spray residues between substrates are unclear and we have no satisfactory explanation as to why spray applications on both wood and cement produced such different results between assays. Physical removal of spores from the substrates by mosquitoes could have occurred but did not obviously contribute to the patterns of decline we saw (i.e. the substrates that remained viable longest actually had the greatest number of repeat exposures). Cement is strongly alkaline and it is possible that this impacted spore survival relative to clay. High pH has been shown to affect persistence of chemical active ingredients
. The poorer effective persistence on wood was slightly more surprising as we had expected it to be a relatively inert, non-absorptive substrate. A previous study examining long-term survival of spores of the current fungal isolate sprayed on glass slides (also inert and non porous), reported a half-life >3 months
. It is possible, therefore, that the commercial plywood we used had some sort of chemical treatment that affected spore viability. Our attempts to verify with the seller that the plywood was completely free of antifungal chemicals were unsuccessful. There are no recommendations in the WHOPES guidelines for exactly what sort of substrates to test beyond wood, clay and cement. Further studies examining different types of these basic substrates would be worthwhile.
Overall, the results of the current study are extremely encouraging. Fungal spores can be produced and stored without loss of viability for >2 years. The fungus is a living organism and once removed from storage, spores are relatively sensitive to temperature but there appears scope for further product development work to improve stability, as well as options for appropriate supply chain management similar to that used for other biologicals. Once sprayed, simple oil formulations can persist as long as some of the standard chemical insecticides. Performance on clay was especially good, which is encouraging as the porous properties of clay/mud are challenging for chemical insecticides. Performance on the other substrates was more variable, but no more so than the basic wettable powder formulation of Lambda-cyhalothrin, which is one of the most widely used chemicals in IRS
[52, 53]). More advanced formulations of Lambda-cyhalothrin, such as capsulated suspensions, have been developed and shown to enhance performance in the field
[52, 53]. It is highly likely that with equivalent research effort, novel formulations of fungal pathogens (including microencapsulation
[54–56]) could also be developed to further improve shelf life and persistence. Furthermore, in line with WHO protocols, assessments in the current study focused on mortality effects alone. While conventional chemicals based on fast-acting neurotoxins do result in rapid death, slower speed of kill (i.e. high mortality by day 14 when mosquitoes could conceivably transmit malaria) is sufficient to provide good malaria control assuming good product coverage see
[24–26]. Moreover, it has been demonstrated that sub- or pre-lethal effects of fungal infection can add to mortality to reduce transmission potential further
[6, 7, 23]. As such, it is probable that levels of persistence reported here are underestimates of overall effective persistence.