RESULTS

The pathogenicity of 6 strains of B. bassiana and 4 strains of M. anisopliae was different at 3 and 7 days after exposure (p = 0.05) (Table 1, 2). Although all B. bassiana strains caused mortality in A. stephensi larvae and their pathogenicity differed with that of the control group, Bb 429C and Bb 796C were, respectively, the most virulent strains with 65% and 63% mortality at 3 days post-exposure (Table 2). Larval mortality rates, due to B. bassiana, increased in the following days and reached 100% in Bb 429C and Bb 796C on the seventh day.

Table 1:	Fungal isolates used in bioassays against Anopheles stephensi larvae

Table 2:	Mortality means and lethal time values (after 7 days) of early instar larvae of A. stephensi exposed to 108 conidia mL-1 of B. bassiana and M. anisopliae

Table 3:	Mortality percentage Means (±SE) of A. stephensi exposed to of M. anisopliae Iran 1018C
*Dissimilar letters in each column indicate significant differences

The lethal times of A. stephensi larvae by these two strains were 2.29 and 2.53 days for LT₅₀ and 4.34 and 4.34 days for LT₉₀. There were no significant differences between the lethal times of Bb 429C and Bb 796C because their confidence limits completely overlapped. Other B. bassiana strains also caused high mortality in the early larval instars but it took long time. In other words, the highest mortality rates in the lowest lethal times were related to Bb 429C and Bb 796C (Table 2). The pathogenicity of M. anisopliae strains were also different and Ma 1018C was the most virulent strain due to its higher mortality at a shorter lethal time (60 and 97% mortality at 3 and 7 days) (Table 2). The differences in lethal times and the eventual mortality rates of Ma 437C, Ma 1018C and Ma DEMI 001, however, do not appear to be significant (Table 2).

In the second bioassay test, M. anisopliae Iran 1018C caused varying mortality rates in the early larval instars of A. stephensi at different concentrations and times (Table 3). Mortality rates increased with increased conidia concentrations in Ma 1018C from 10⁷ to 5x10⁸ conidia mL^-1 but it decreased in the concentration of 10⁹ conidia mL^-1 (Fig. 1).

Fig. 1:	Mortality percentage means (±SE) of A. stephensi exposed to different concentrations of M. anisopliae Iran 1018C after 72 h

Fig. 2:	Lethal time values (day) of M. anisopliae 1018C against early instar larvae of A. stephensi

There were no significant differences in concentrations of 5x10⁷, 10⁸ and 5x10⁸ conidia mL^-1 (p-value = 0.05) and they, respectively killed 99, 97 and 99% of larva on the fourth day. Comparison of lethal time values of Ma 1018C at five concentrations cleared no uniform increase or decrease trend as the lethal time decreased from 10⁷ to 5x10⁷ and increased again in 10⁸ to 10⁹ conidia mL^-1. The lowest lethal times were related to the concentration of 5x10⁷ as they were 0.63 and 1.93 days for LT₅₀ and LT₉₀, respectively (Fig. 2). The mortality rate in this concentration, however, was equal to the mortality rate observed in 5x10⁸ conidia mL^-1 (99%) but the lethal time values in the concentration of 5x10⁷ conidia mL^-1 were lower than the previous one. We were unable to determine the lethal concentrations (LC₅₀ and LC₉₀) probably because the mortality rates showed no significant difference and were all above 50%.

DISCUSSION

Fungal diseases in insects are common and widespread and can decimate their populations in spectacular epizootics. Nearly all insect orders are susceptible to fungal diseases, including dipterans. Fungal pathogens such as Lagenidium, Coelomomyces and Culicinomyces are known to affect mosquito populations and have been studied extensively. There are, however, many other fungi, such as Beauveria and Metarhizium that infect and kill mosquitoes at the larval and/or adult stage (Scholte et al., 2004). Problems such as mass production, storage conditions and the high dosage required to kill mosquitoes were reasons that stopped researchers from applying these microbial agents as alternatives for chemical insecticides.

To enhance the potential of this microbial control agent, improvements should be made to develop new culture media, economic storage conditions that can prolong the stability of infective stages and to develop formulations that increase the likelihood of contact with target species, such as flotation in Anopheles habitats. Certain formulations can improve the ability of entomopathogenic fungus spores to spread over a water surface as well as increase their persistence and can lead to an enhanced efficacy of fungal spores (Bukhari et al., 2011). Successive subculturing on artificial media often results in a decrease in virulence. The virulence of attenuated isolates can often be regained, however, by passing through an appropriate host. The effects of culture history on virulence poses a special problem if bioassays are used to compare virulence among isolates obtained from various sources and culture collections. In an attempt to address this problem, each isolate can first be passed through an insect host prior to culture on artificial media and use in bioassays (Butt and Goettel, 2000). For this purpose, the larval stage of the housefly, Musca domestica L. was selected for passage of the selected fungal strains in this study and the new cultures were used in screening tests against A. stephensi larvae.

Many previous studies demonstrated the efficacy of Beauveria and Metarhizium against adults of Anopheles spp. (Scholte et al., 2006; Kannan et al., 2008; Mnyone et al., 2010; Mouatcho, 2010; Farenhorst et al., 2007) but there is little research (Alves et al., 2002; Bukhari et al., 2011; Prasad and Veerwal, 2010) about the use of these agents to control larvae in their water habitats. The results of our study detected that almost all the selected Iranian isolates of B. bassiana and M. anisopliae were pathogenic against first and second instar larvae of the A. stephensi strain Chabahar and larval mortality was considerable at 4-7 days after exposure. The pathogenicity of these entomopathogenous fungi against larvae of A. stephensi and A. gambiae was also reported by Bukhari et al. (2011). Our results also confirmed those of (Prasad and Veerwal, 2010) who observed 23-61% mortality in A. stephensi larvae using B. bassiana conidia. In another study, the mortality rate of Culex quin quefaciatus larvae was reported as 0-90% at 5 days after using M. anisoplaie (Alves et al., 2002; Lacey et al., 1988). This study also confirms our observations that these fungi are pathogenesous against mosquito larvae in their water habitat. Additionally, all the selected Iranian strains of B. bassiana and M. anisopliae were virulent against the housefly, M. domestica, as they caused 28-100% mortality in larvae and adults (Sharififard et al., 2011).

We selected the M. anisoplaie Iran 1018C strain for further evaluation because it caused a high mortality in a lower lethal time in preliminary screening tests. Larval mortality rates did not show a proportional increase or decrease with an increase in spore concentrations, as the mortality rate was lowest at the highest concentration of 10⁹ conidia mL^-1. At a certain concentration, however, it increased with time. Fungal spores are hydrophobic and they clump together into masses when applied over the water surface without a suitable surfactant, so spore feeding by larvae or spore attaching to larval cuticle will be reduced (Prasad and Veerwal, 2010). We used Tween-80 as a surfactant and since the spores of Metarhizium do not clump together like or as much as Beauveria, the decreasing rate of mortality in the highest concentration could be the result of clumping or due to the sedimentation rate of spores. In higher concentrations these two reasons can result to avoid spore attachment to the larval cuticle. All at all, M. anisopliae has several characteristics that make it interesting as a microbial mosquito control agent. It causes high mortality of mosquito larvae in laboratory populations, the fungus can be grown in massive amounts on inexpensive artificial media and conidia can be stored easily. Moreover, its failure to germinate in the mosquito environment until actual exposure to a host and its resulting persistence in the environment as well as the fact that its effect is not limited to periods of host molting (as for B. bassiana) make this fungus a very promising control agent. B. bassiana conidia germinate in mosquito habitats even when not in contact with larvae. This limitation along with the high dosage needed is serious drawbacks for mosquito larval control (Scholte et al., 2004). Our results were obtained under controlled conditions and on a small scale. It is necessary, however, to evaluate the table M. anisoplaie and B. bassiana under field conditions and on a large scale. It is also necessary to evaluate other formulations of the selected isolate such as conidia-dust formulation and oil formulation against A. stephensi larvae.

ACKNOWLEDGMENTS

The authors would like to thank deputy of research of Shiraz University of Medical Sciences (SUMS) for financial sponsoring of the project, School of Health, Jundishapur University of Medical Science for providing laboratory facilities and Pasture Institute of Iran for providing susceptible strain of Anopheles stephensi. We also thank the Plant Protection Institute of Iran for providing the fungal isolates. This study is part of an M.Sc. Thesis emanated from a research plan (No. 91-6098 dated June 2012) carried out under the auspices of SUMS by the second coauthor (RaheleVeys-Behbahani) in Medical Entomology dated June 2013.