[media] Entomopathogenic fungi show great promise as pesticides in terms of their relatively high target specificity, low non-target toxicity, and low residual effects in agricultural fields and the environment. However, they also frequently have characteristics that limit their use, especially concerning tolerances to temperature, ultraviolet radiation, or other abiotic factors. The devastating ectoparasite of honey bees, Varroa destructor, is susceptible to entomopathogenic fungi, but the relatively warm temperatures inside honey bee hives have prevented these fungi from becoming effective control measures.
Using a combination of traditional selection and directed evolution techniques developed for this system, new strains of Metarhizium brunneum were created that survived, germinated, and grew better at bee hive temperatures (35℃).
Field tests with full-sized honey bee colonies confirmed that the new strain JH1078 is more virulent against Varroa mites and controls the pest comparable to current treatments.
These results indicate that entomopathogenic fungi are evolutionarily labile and capable of playing a larger role in modern pest management practices.
…the primary mode of action for Varroa control is highly likely through mitospore adhesion and germination on the mite exoskeleton, followed by hyphal penetration through the exoskeleton and proliferation throughout internal tissues of the mite.
Mites from this initial field trial were collected off sticky cards, surface sterilized, and plated on agar. Metarhizium that grew out of infected mites were subcultured and used as the starting population for a directed evolution process we designed to induce thermotolerance. The fungus was subjected to repetitive cycles of growth and reproduction under stressful conditions at increasing temperatures (Figure 2a). The stressful conditions were either oxidative stress and mild mutagenicity induced by hydrogen peroxide treatments or nutritional stress induced by growth on minimal media agar amended with or without chitin. Spores exposed to nutritional stress are better able to withstand UV-stress and heat stress and exhibit increased infectivity7,58. There is, however, a tradeoff for fungi grown in nutritionally deficient media; hyphal development is slowed, and mitospore production is decreased59. With each repeated cycle the mitospore population was admixed, and the incubator temperature was gradually increased from the ideal growth temperatures for the starting F52 strain (27℃) to the temperature found in honey bee hives (35℃).
The last generation of spores resulting from the directed evolution process was then used as the starting population for repetitive rounds of field selection. The mitospores were germinated on malt extract agar (MEA) plates, allowed to grow and to produce another generation of mitospores, and then the agar disc was inverted onto the top bars of the frames of comb in full-sized outdoor honey bee colonies (Figure 2b). A new apiary, designated as the stationary apiary, was established using full-sized colonies started from “two-pound packages” (0.91 kg of bees taken from a common population), with a total of 48 colonies being allocated for repeated treatment with either Metarhizium or uninoculated agar discs as controls. The first round of treatment after the directed evolution procedure did not result in high levels of infection in the mites; we were able to reculture living Metarhizium from 3.38% of mites collected off of sticky cards (Figure 3a), indicating that a low number of mites were killed by the fungus. This low number was not unexpected, as many of the genetic changes acquired during the directed evolution process would not be favorable for virulence in living hosts under field conditions. Additionally, repeated subculturing on artificial media is known to decrease virulence in as little as 20 subcultures60. Living fungus that was recultured from the infected mites was then grown to sporulation, and the subsequent generation was used to treat the same population of hives again (Figure 3a). After a single generation of selection through Varroa hosts, this treatment resulted in 49.9% of mites dying from mycosis. The process of harvesting mitospores from dead mites, growing another generation, and treating the colony again was repeated two additional times that field season. The final treatment exhibited extended efficacy, lasting up to 5 weeks post treatment (Figure 3a), indicating increased tolerance to bee hive conditions. No negative effects were detected and the colonies in the treatment and control groups went into winter with similar bee population estimates (t-test p = 0.72; see Supplementary Figure S1 online).
[All told, Han and Naeger counted more than 27,000 dead mites over the course of their experiments. “When you close your eyes, you still see little Varroa”, Han says…Further tests are needed to demonstrate the treatment’s efficacy, says Scott McArt, an entomologist at Cornell University. Mite populations tend to proliferate later in the year than when the study was conducted, he notes, so the fungus would need to be tested against higher numbers of mites to prove its worth. Another question is cost. The biopesticide will likely be more expensive than oxalic acid, Han and Naeger say, and it is more time-consuming and complicated to use than common chemical pesticides. But the fungus is likely safer for hives. Bees can fall sick or die if concentrations of oxalic acid are too high, and other chemical miticides can cause reproductive problems in the pollinators. Han and her colleagues are continuing to develop more effective strains of the fungus and reduce their costs. “I think this is going to be a long process”, she says. But if they succeed, it would be a “really big advance”, McArt says. “There are a ton of beekeepers who do not want to put pesticides in their hives.”]