The results of this study show how certain formulations can improve the ability of entomopathogenic fungus spores to spread over a water surface as well as increase their persistence. The results also show that better spreading and persistence leads to an enhanced efficacy of fungal spores. The study also demonstrates that both M. anisopliae and B. bassiana caused a high impact on the survival of An. gambiae s.s. larvae under field conditions, when formulated in Shellsol T.
Anopheles stephensi and An. gambiae larvae were found to be equally susceptible to unformulated M. anisopliae and B. bassiana spores . This suggests that these fungi are likely to also affect other anopheline vector species.
Formulating fungal spores with Tween 80 and wheat flour was found to be unsuitable. Spores formulated with Tween 80 did not spread over the water surface, the primary feeding site of anopheline larvae, but sunk to the bottom [25, 28]. Surfactants are known to impair attachment of the spore to the host so even if the spores were spread on the water surface they would not have been effective against anopheline larvae [20, 42]. Wheat flour, although due to its organic nature could have served as a bait, did not spread the fungal spores over the water surface . The wheat flour clumped together and sunk.
Powdered pepper and Ondina oil caused 100% mortality in anopheline larvae even without the fungal spores. Extracts of fruits of the Piperaceae family have been shown to be toxic for Aedes aegypti L. larvae , but the exact toxicity mechanism remains unclear. Although fungal spores were effectively spread with white pepper, pepper was considered an unsuitable carrier due to its own toxic effect on the anopheline larvae. Ondina oil, in the amount tested (200 μl), formed an oily layer over the water surface causing the larvae to suffocate. As compared to ShellSol T, Ondina oil is denser and evaporates less. This may explain the difference in the mortality observed with Ondina oil and ShellSol T controls. The amount of Ondina oil tested could not be reduced as, in that case, it was not possible to make a homogeneous suspension with the fungal spores.
Dry unformulated M. anisopliae and B. bassiana spores lost their pathogenicity five days after being applied to the water surface as the survival of larvae exposed to the fungal spores five days after application was similar to that of the controls. Similar results were shown in a study by Alves et al. (2002), where M. anisopliae caused no mortality in Cx. quinquefasciatus Say larvae introduced four days after the spores were applied . This is in contrast to Pereira et al. (2009), who found M. anisopliae spores to cause 50% mortality in Ae. aegypti larvae exposed to fungal spores that were applied ten days previously . The studies mentioned here were carried out in controlled climate conditions (25-27°C) in the laboratory. In field conditions the spores are more likely to lose their pathogenicity in less time due to exposure to hight temperatures and UV-radiations. This may explain why unformulated fungal spores did not cause any significant reduction in pupation in the field bioassays, where the water surface temperatures were measured to be as high as 38.8°C. The measured (water surface) temperatures agree with those reported by Paaijmans et al. (2008) for similar sized water-bodies and are known to exhibit high daily fluctuations .
When the larvae were exposed to fungal spores on the same day as the spores were applied, unformulated spores and spores formulated in WaterSavr or Shellsol T caused larval mortality over the next few days. However, only fungal spores formulated in ShellSol T caused significantly higher mortality in larvae introduced seven days after the fungal spores had been applied. Fungal spores formulated in ShellSol T remained pathogenic possibly because ShellSol T prevented spores from absorbing the amount of moisture required to stimulate germination [21, 31]. ShellSol T was also considered a good carrier of fungal spores in other studies [31, 45]. WaterSavr, on the other hand, did not protect fungal spores.
ShellSol T was the only formulation that we tested in the field as the laboratory results showed high persistence of pathogenicity in the fungal spores formulated only with this product. Unformulated M. anisopliae and B. bassiana did not suppress the larval population effectively in the field. In contrast to the situation in the laboratory, the spores were exposed to sunlight, rain and fluctuating temperatures in the field which might have reduced spore survival. By contrast, only 10-20% of the larvae treated with spores formulated in ShellSol T, developed into pupae. Both M. anisopliae and B. bassiana spores were found to be equally effective when formulated in ShellSol T. Oil formulations are known to improve spore survival, improve fungal efficacy against insects and reduce spore sensitivity to UV radiation [31, 45].
In the field residual effect of formulated spores could not be tested after a certain number of days because the plastic containers began to harbour Culex larvae and thus had to be drained. The presence of Culex larvae is an indication that ovipositing female Culex mosquitoes were not repelled by the fungus treatment. It is disadvantageous for a larval control agent to have an oviposition-repellent effect because in that case ovipositing mosquito females are forced to seek and deposit their eggs at alternative untreated sites. This means that the control agent only targets the existing larval population and needs to be reapplied after the site has been inhabited again. Studies specifically designed to establish the response of ovipositing anopheline female mosquitoes to fungal spores and the residual effect of fungal spore treatment are required for a better understanding. Oil-formulated M. anisopliae spores have been shown to have an increased ovicidal activity in case of Ae. aegypti eggs . This might be an added advantage if anopheline eggs are also affected by M. anisopliae spores similar to the Ae. aegypti eggs.
Pathogenicity of control agents in the field is generally lower than that in the laboratory settings . In the field bioassays, therefore, a higher dose (20 mg/450 cm2) of fungal spores was also tested together with the dose tested in the laboratory (10 mg/441 cm2). The laboratory dose, however, showed similar efficacy in the field by reducing pupation similar to the higher dose. Therefore doses lower than used in the current study should be evaluated to establish the lowest effective amount of fungal spores required to treat a certain area.
ShellSol T was a candidate carrier that not only facilitated the application of spores but also improved their efficacy by providing maximum chance for contact (spreading the spores on the water surface) with the larvae and increasing spore persistence. The fungal spores readily suspend in ShellSol T with a slight agitation. This is advantageous as the spores can be conveniently mixed in ShellSol T, on the spot, which means that during transport and storage only the bio-active agent would have to be kept at low temperatures rather than the whole mixture. This can reduce the cooling space requirement as ShellSol T itself is a stable product and has no particular storage demands. It has been shown that the percentage germination of dry spores is generally higher than that of oil-formulated spores when stored at the same temperature for the same number of days [; unpublished data]. The fungal spores Metarhizium flavoviride had a germination rate of 80% when stored at 30°C for 90 days as compared to 90% when stored dry under similar environmental conditions . In this context, it seems more efficient to store fungal spores separately and only mix them with the oil-component shortly before application.
The results of this study show the necessity of a good formulation for fungal spores when these are to be utilised in the field. The efficacy of unformulated (dry) spores was so low in the field situation that their application, as such, is not justified. While ShellSol T-formulated spores were highly effective in killing anopheline larvae in the field an important point to consider is the potential increased risk to the non-target organisms due to their improved persistence and/or undesirable properties of the solvent [33, 48–50]. ShellSol T has a low toxicity effect on fish, aquatic invertebrates and microorganisms at concentration higher than 1 g/liter . Considering the volume of ShellSol T that we tested (200-230 μl on 1 L of water), the concentration of ShellSol T was 0.15 g/L which is nearly seven times lower than the lowest lethal concentration. ShellSol T evaporates and therefore is less likely to remain in the aquatic habitats. Detailed safety studies, however, are necessary to have a better understanding of any adverse effect ShellSol T might have on the environment and non-target organisms, at the required doses.
Besides formulation, it is very important to identify the best delivery method (where, when and how) to fully utilize the entomopathogenic potential of M. anisopliae and B. bassiana spores. Frequency of re-application has to be determined based on the residual effect of formulated spores in the field. The feasibility of applying formulated spores at artificial breeding sites, baited to attract ovipositing females, is also worth testing . A good delivery system will reduce the chances of non-target organisms coming into contact with fungal spores.