Bee Buzz Box March 2025 Smite the Mite Part IV – In Search of the All Mitey Sledge Hammer Fungal Predators

Alan Wade

Canberra Region Beekeepers

Howl ye; for the day of the Lord is at hand;It shall come as a destruction from the Almighty.

Isiah 13:6

At the turn of the millennium, Chandler and coworkers (2001) set out to scope potential varroa biological control agents. They began their review with a candid observation:

...no promising natural enemies of varroa species have been identified on Apis mellifera or its original host, Apis cerana.

They then identified the full gamut of potential biological control agents: predatory mites, parasitoids and entomopathogens – nematodes, protozoa, viruses, Bacillus thuringiensis, rickettsiae, and fungi. Of these they were of the view that predators, parasitoids, viruses and rickettsiae had little mite control potential, but posited that:

Entomopathogenic fungi appear to have most potential, followed by Bacillus thuringiensis, protozoa and nematodes.

In a slightly earlier review, Chandler and coworkers (2000) collated records of fifty eight species of fungi that infect at least seventy three species of the Acari (mites and ticks), either naturally or experimentally. These entomopathogenic fungi (insect pathogens) kill or seriously disable insects and, by extension, many other invertebrates including mites. Pathogenic conidial fungi are spore-forming, their spores germinating and directly penetrating the exoskeleton of invertebrates including insects, spiders and mites. Hyphae that develop from these spores then invade and destroy internal tissue and organs.

Fungal pathogens have been reported to kill representatives of the two super orders of mites, the Acariformes (orders Astigmata, Oribatida and Prostigmata) and the Parasitiformes (orders Ixodida, the ticks, and Mesostigmata). The honey bee parasites Varroa, Euvarroa and Tropilaelaps are members of the Mesostigmata order so might be controlled by any of a number of fungal pathogens.

Since natural populations of Varroa are predator and seemingly almost pathogen free, introducing fungi to control these predatory mites would seem to be an obvious endeavour. However their potential to also have a deleterious effect on the army of phytophagous honey bee colony mites, those that perform the essential house cleaning role of consuming hive debris, also warrants consideration.

To put the bee parasites Varroa, Euvarroa and Tropilaelaps into the honey bee colony mite perspective, Warrit and Lekprayoon (2011) note that:

Parasitic mites represent only a minor fraction of the diversity of Acari associations with honeybees. Most Acari found in the nests of honeybees usually have a saprophagous lifestyle and feed on fungus-infected debris in the hives, dead bees and sometimes pollen (kleptophages).

Many pathogenic fungi target a broad spectrum of invertebrates but have limited impact on some insect orders including the hymenoptera to which all the bees belong. As we saw in our search for new natural products, we want agents that kill mites and that have minimal impact on honey bees.

An increasing number of fungi have found to be effective in killing Varroa in vitro, that is under controlled laboratory conditions. However hive conditions of low humidity and elevated temperature has resulted in few of these fungi surviving. Most past attempts to employ them in the hive – in vivo – to control mites consequentially failed.

Here it is worth noting that apart from the fungus Beauveria bassiana used to protect vegetable pests and endo and ecto-arbuscular mycorrhizal fungi used as seed coatings, along with a few bacterial biocontrol agents such as Bacillus thuringiensis, few biocontrol agents are currently employed in the Australian horticulture (Hort Innovation, 2018). The range entomopathogenic fungi employed in global agriculture is somewhat larger (Sharma et al., 2023): Of the important genera, including Beauveria, Hirsutella, Lecanicillium, Metarhizium and Isaria, many are marketed to target specific insect, mite or nematodes and have minimal or no impact on beneficial organisms such as honey bees. The USDA’s Fernando Vega and an international team of coworkers (Vega et al., 2009) provide a lucid overview of the ecology of entemopathogenic fungi outlining limits to employ them for pest management.

Heat tolerant fungi

In investigating the potential to target honey bee brood parasites mycologists sensibly set out to select heat tolerant fungal strains (Davidson et al., 2003) those that survive brood nest temperatures. In practice some have been found to work briefly inside hives but have not persisted. A few have been shown to be effective varroa knockdown agents but require repeat dosing to chase down emerging mites.

Whether such fungi can be further engineered to survive and proliferate in honey bee colonies and eventually resolve the mite problem is a far more open question. For now they have the advantage over chemical miticides, both natural and synthetic, in not leaving residues or harmful breakdown products.

Hamiduzzaman and coworkers (2012) summarise this well in stating that:

The ideal varroa control treatment should be environmentally friendly (i.e., limited non-target effects), varroa-selective (i.e., kills varroa at doses that are relatively harmless to bees) and should leave little to no residues in honey and wax.

The Hamiduzzaman group go on to suggest that entemopathogenic fungi could infect varroa, be non-toxic to humans, could be readily cultured and do occur naturally. There will likely also be regulatory roadblocks well articulated by Strasser, Vey and Butt (2000):

Entomopathogenic fungi are promising alternatives to chemical insecticides. However, a major hurdle concerning the registration of these fungi as plant protection [here mite control] agents is the possible toxicity of secreted metabolites, especially secondary metabolites.

This said they lead on to state that:

So far, there have been no reports of entomogenous fungi or their toxins having any negative effects on humans and non-target organisms even though they have been deployed extensively in specific crop production systems.

Shaw and coworkers (2002), investigating a suite of promising fungal pathogens, reported that:

Forty isolates of entomopathogenic fungi were assessed against Varroa destructor in a single-dose experiment (employing conidial – asexual fungal spore – concentrations of 1×10⁸ per millilitre at 25 ⁰C and 100% relative humidity). The fungal species were Verticillium lecanii (nine isolates), Hirsutella spp. (16 isolates), Paecilomyces spp. (three isolates), Beauveria bassiana (four isolates), Metarhizium spp. (six isolates), and Tolypocladium spp. (two isolates). All forty isolates could infect and kill Varroa destructor and twenty six caused mean times to death of less than 100 hours.

Of these, three isolates of Metarhizium anisopliae, one of Verticillium lecanii, and one of Beauveria bassiana killed 100% of Varroa destructor within seven days at a conidial concentration of 1×10⁸ per millilitre while one isolate of Metarhizium anisopliae also killed 97% of Varroa destructor within seven days at a [one hundred fold lower] conidial concentration of 1×10⁶ per millilitre.

Other early researchers made progress in finding other promising fungal candidates: Peng and coworkers (2002) targeted Hirsutella thompsonii; Rodríguez and coworkers (Rodríguez, Gerding, France and Ceballos, 2009) also evaluated the performance of Beauveria bassiana and Metarhiziumanisopliae, finding that they were pathogenic to varroa mites under hive-like conditions, showing also that Clonostachys rosea was rather less pathogenic.

Let’s examine each of these and other fungal mite pathogens (Table 1) focusing on where research has led in assessing their potential to control Varroa mites.

Table 1 Promising candidate fungi for Varroa destructor control.* Laboratory testing** Field testing with some control

Aspergillus spp.

A number of Aspergillus species have been found in association with honey bees (Becchimanzi and Nicoletti, 2022), Aspergillus flavus and less commonly Aspergillus fumigatus being responsible for the relatively uncommon stonebrood disease. There is little evidence that Aspergillus species infect mites though a number of authors make reference to a Russian study by Chernov (1981) that showed that Aspergillus and Beauveria cultures caused mortality of Varroa in the laboratory at 26 ⁰C.

Beauveria bassiana, Metarhizium anisopliae and Metarhizium brunneum

Beauveria species are typically associated with insects typically presenting as an off white conidial spore growth (Figure 1). Beauveria bassiana has been developed as a biological insect pesticide andis naturally associated with Varroa destructor.

Figure 1 Beauveria bassiana infection of a cicada. Doctor's Creek walking track, Reefton, Victoria (Leithhead, 2000).

Rodríguez, Gerding and France (2009) cultivated Beauveria bassiana and Metarhizium anisopliae on a dextrose-agar medium at 30 and 35 ºC. Of the few isolates that grew at 35 ºC, the most effective was Metarhizium anisopliae effecting an 85% Varroa destructor mortality whereas the Beauveria bassiana isolates achieved only 20-30% mortality over six days. Metarhizium anisopliae fungi had been successfully employed to control plagues locusts (Figure 2).

Figure 2 Locust killed by Metarhizium anisopliae strain CGMCC 3.18023 (Zhuoer Chemistry Co., 2023).

Both Steenberg, Kryger and Holst (2010) and García-Fernández and coworkers (2008), as well as Meikle and coworkers (2007), report on finding of Beauveria bassiana naturally associated withVarroa destructor.

While all isolates of these fungi grew well and at a linear rate at 30 ºC, relatively few isolates survived at the higher temperature. Innoculums of the strains surviving at 35 ⁰C, suspensions containing 1×10⁷ conidia per milliltre, were employed to test for their miticidal efficacy. In a followup study (Rodríguez, M., Gerding, France and Ceballos (2009), their most effective isolate Metarhizium anisopliae var. anisopliae Qu-M845 reduced hive mite infestation in both autumn and spring. These findings align with those of Kanga, Jones and James (2003). Later Kanga, Jones and Gracia (2006) applied strips coated with a Metarhizium culture to brood nests successfully achieving the same effective control of varroa as that achieved using τ-fluvalinate.

Meikle and coworkers (2007) treated honey bees with cultivated Beauveria bassiana isolated from Varroa destructor in two separate field trials. They found that not only did the level of infection of mites increase in treated hives but that mite numbers also increased in some untreated hives, an effect they attributed to cross infection by drifting bees. While the study failed to reduce colony mortality attributable to mite infection, increases in pathogen levels in hives and in bee cadavers outside hives suggests progress in efforts to use this entomopathogen to control varroa.

Hamiduzzaman, Sinia, Guzman-Novoa and Goodwin (2012) from The University of Guelph confirmed that Beauveria bassiana and Metarhizium anisopliae were highly pathogenic to varroa mites. Several isolates caused more than 90% mite mortality in laboratory trials, while Clonostachys rosea showed more limited pathogenicity none resulting in a more than 60% mite kill. These researchers also signal that the adverse effects of entomopathogens on mites also extends to some impact on bees, particularly on bee brood.

Alice Sinia undertook an expansive mite kill study on strains of Metarhizium anisopliae, Clonostachys rosea and Beauveria bassiana finding that Metarhizium anisopliae was the most pathogenic towards Varroa destructor and least lethal to honey bees. She concluded that:

Based on results of laboratory evaluations, the positive attributes of Metarhizium anisopliae UAMH 9198 (i.e. its high toxicity to Varroa, moderate toxicity to bees and the ability germinate, grow and sporulate at temperatures of 25 – 30 ºC) makes it the best candidate of the isolates evaluated for testing as a potential bio-control agent against Varroa destructor.

In a more recent study Jenifer Han and coworkers (2021) conducted field trials on a new strain of Metarhizium brunneum selected to survive, germinate and grow well at 35 °C in the laboratory. Their field tests on honey bee colonies demonstrated that their new strain (JH1078) was effective against varroa mites and controls the pest at a level comparable to that achieved by oxalic acid dribble treatment.

A lucid note reviewing this study, penned by Scott Weybright from Washington State University, opined that this finding demonstrates that pathogenic fungi are evolutionarily labile and that pathogenic fungi are capable of playing an important role in Varroa management practice.

Hirsutella thompsonii

A general review of the Hirsutella genus by Reddy and coworkers (2020) note that three species (of about seventy) have been employed to control a wide range of crop and animal pests signalling their potential for control of varroa. While Hirsutella rostrata has been cited as a potential miticide, only Hirsutella thompsonii had been trialled for varroa control.

Of nine Hirsutella thompsonii USDA isolates, Peng, Zhou and Kaya (2002) found that only three sporulating Hirsutella thompsonii cultures, ARSEF 257, 1947 and 332 successfully infected varroa mites. Varroa half life mortalities ranged from about two to four days. Recycling spores passing through varroa resulted shortened mite half lives of isolates 1947 and 332 but had no effect on isolate 257. None of these isolates had any honey bee life stage impact. Their potential to work effectively under hive conditions has not been reported on.

Future of fungal pathogens for Varroa control

The pathogenic fungi we report on here are well established bio-pesticides employed in agricultural settings. As off the shelf products, however, none would effect any control of honey bee mite parasites. Those that have been successfully deployed have come from research agency laboratory trials of selected fungal strains. For now they only exist as rare and perhaps difficult to maintain isolates.

The steps needed to extensively field test and mass produce them represent barriers to their deployment and use. Cost-wise they would need to match proven arachnicides and natural product remedies already in use.

As they now stand current formulations of pathogenic fungi are only useful as quick mite knockdown agents, an attribute they share with thymol and the organic acids. This means that unless pathogenic fungi can be further selected to survive and replicate at least for some weeks, repeat treatments will be needed. For now it seems that entomopathogenic fungi will join the queue as part of any ongoing integrated pest management strategy.

In this Smite the Mite series we have attempted to sketch the many schemes beekeepers have adopted to control varroa and to provide an insight into evolving control measures. Varroa and Tropilaelaps mites will not go away, so it remains to be seen whether we can devise effective means to assist bees to survive and thrive or whether bees will simply have to find their own solutions in a rapidly changing world.

Readings

Becchimanzi, A. and Nicoletti, R. (2022). Aspergillus-bees: A dynamic symbiotic association.Frontiers in Microbiology 13:968963. https://pmc.ncbi.nlm.nih.gov/articles/PMC9489833/

Chandler, D., Davidson, G., Pell, J.K., Ball, B.V., Shaw, K. and Sunderland, K.D. (2000). Fungal biocontrol of Acari. Biocontrol Science and Technology 10(4):357-384. https://doi.org/10.1080/09583150050114972

Chandler, D., Sunderland, K.D., Ball, B.V., Davison, G., (2001). Prospective biological control agents of Varroa destructor n. sp., an important pest of the European honey bee, Apis mellifera.Biocontrol Science and Technology 11(4):429-448. https://doi:org/10.1080/09583150120067472 https://www.wellesu.com/10.1080/09583150120067472

Chernov, K.S. (1981). Transmission of mycoses, an aspect of Varroa infestations. Byulleten’ Vsesoyuznogo Instituta Eksperimental’noi Veterinarii 41:59-60. https://www.cabidigitallibrary.org/doi/full/10.5555/19840215950

Davidson, G., Phelps, K., Sunderland, K.D., Pell, J.K., Ball, B.V., Shaw, K.E. and Chandler, D. (2003). Study of temperature-growth interactions of entomopathogenic fungi with potential for control of Varroa destructor (Acari: Mesostigmata) using a nonlinear model of poikilotherm development. Journal of Applied Microbiology 94(5):816-825. https://repository.rothamsted.ac.uk/download/3c4fcd7c6dfdbce3965f605bcf7f8a6ba8aa2e0fd425aae713d593105f3b1726/150742/Davidson-2003-Study-of-temperature-growth-interac.pdf

García-Fernández, P., Santiago-Álvarez, C. and Quesada-Moraga, E. (2008). Pathogenicity and thermal biology of mitosporic fungi as potential microbial control agents of Varroa destructor (Acari: Mesostigmata), an ectoparasitic mite of honey bee, Apis mellifera (Hymenoptera: Apidae). Apidologie 39(6):662-673. https://www.apidologie.org/articles/apido/pdf/2008/06/m08009.pdf

Hamiduzzaman, M.M., Sinia, A., Guzman-Novoa, E. and Goodwin, P.H. (2012). Entomopathogenic fungi as potential biocontrol agents of the ecto-parasitic mite, Varroa destructor, and their effect on the immune response of honey bees (Apis mellifera L.). Journal of Invertebrate Pathology 111(3):237-243. https://www.wellesu.com/10.1016/j.jip.2012.09.001

Han, J.O., Naeger, N.L., Hopkins, B.K., Sumerlin, D., Stamets, P.E., Carris, L.M. and Sheppard, W.S. (2021). Directed evolution of Metarhizium fungus improves its biocontrol efficacy against Varroa mites in honey bee colonies. Scientific Reports 11:10582. https://doi:10.1038/s41598-021-89811-2

Hort Innovation (January 2018). Biopesticides in Australia. https://www.horticulture.com.au/globalassets/hort-innovation/resource-assets/vg15010-biopesticides-in-australia-fact-sheet.pdf

Kanga, L.H.B., Jones, W.A. and Gracia, C. (2006). Efficacy of strips coated with Metarhizium anisopliae for control of Varroa destructor (Acari: Varroidae) in honey bee colonies in Texas and Florida. Experimental and Applied Acarology 40(3-4):249-258. https://doi:10.1007/s10493-006-9033-2

Kanga, L.H.B., Jones, W.A. and James, R.R. (2003). Field trials using the fungal pathogen, Metarhizium anisopliae (Deuteromycetes: Hyphomycetes) to control the ectoparasitic mite, Varroa destructor (Acari: Varroidae) in honey bee, Apis mellifera (Hymenoptera: Apidae) colonies. Journal of Economic Entomology 96(4):1091-1099. https://doi:10.1603/0022-0493-96.4.1091

Leithhead, W. (2000). Bill Leithhead's fungi pages Beauveria bassiana. https://www.elfram.com/fungi/fungipics_b.html#beabas_a

Meikle, W.G., Mercadier, G., Holst, N., Nansen, C. and Girod, V. (2007). Duration and spread of an entomopathogenic fungus, Beauveria bassiana (Deuteromycota: Hyphomycetes), used to treat Varroa mites (Acari: Varroidae) in honey bee (Hymenoptera: Apidae) hives. Journal of Economic Entomology 100(1):1-10. https://sci-hub.usualwant.com/10.1603/0022-0493(2007)100%5B1:DASOAE%5D2.0.CO;2

Peng, C.Y., Zhou, X. and Kaya, H.K. (2002). Virulence and site of infection of the fungus, Hirsutella thompsonii, to the honey bee ectoparasitic mite, Varroa destructor. Journal of invertebrate Pathology 81(3):185-195. https://doi:10.1016/s0022-2011(02)00188-x

Reddy, N., Mahesh, G., Priya, M., Singh, R.U.S. and Manjunatha, L. (2020). Hirsutella. Chapter 43,pp.817-831 in Beneficial microbes in agro-ecology. Academic Press. https://www.wellesu.com/10.1016/B978-0-12-823414-3.00043-5

Rodríguez, M., Gerding, M. and France, A. (2009). Selection of entomopathogenic fungi to control Varroa destructor (Acari: Varroidae). Chilean Journal of Agricultural Research 69(4):534-540. https://www.scielo.cl/pdf/chiljar/v69n4/AT08.pdf

Rodríguez, M., Gerding, M., France, A. and Ceballos, R. (2009). Evaluation of Metarhizium anisopliae var. anisopliae Qu-M845 isolate to control Varroa destructor (Acari: Varroidae) in laboratory and field trials. Chilean Journal of Agricultural Research 69(4):541-547. https://oes.chileanjar.cl/files/V69_I4_2009_ENG_MartaRodriguez.pdf

Sharma, A., Sharma, S. and Yadav, P.K. (2023). Entomopathogenic fungi and their relevance in sustainable agriculture: A review. Cogent Food and Agriculture 9(1):2180857. https://www.tandfonline.com/doi/pdf/10.1080/23311932.2023.2180857

Shaw, K.E., Davidson, G., Clark, S.J., Ball, B.V., Pell, J.K., Chandler, D. and Sunderland, K.D. (2002). Laboratory bioassays to assess the pathogenicity of mitosporic fungi of Varroa destructor(Acari: Mesostigmata), an ectoparasitic mite of honey bee Apis mellifera. Biological Control 24(3):266-276. https://doi:org/10.1016/S1049-9644(02)00029-4

Sinia, A. (2013). Evaluation of the fungi Beauveria bassiana, Metarhizium anisopliae, and Clonostachys rosea as bio-control agents against the honey bee parasitic mite, Varroa destructor(Doctoral dissertation, University of Guelph). https://atrium.lib.uoguelph.ca/server/api/core/bitstreams/0edeec92-fe3e-48db-ad89-6672855d7d01/content

Steenberg, T., Kryger, P. and Holst, N. (2010). A scientific note on the fungus Beauveria bassianainfecting Varroa destructor in worker brood cells in honey bee hives. Apidologie 41(1):127-128. https://www.apidologie.org/articles/apido/pdf/2010/01/m09030.pdf https://www.wellesu.com/10.1051/apido/2009057

Strasser, H., Vey, A. and Butt, T.M. (2000). Are there any risks in using entomopathogenic fungi for pest control, with particular reference to the bioactive metabolites of Metarhizium, Tolypocladiumand Beauveria species? Biocontrol Science and Technology 10(6):717-735. https://www.wellesu.com/10.1080/09583150020011690

Vega, F.E., Goettel, M.S., Blackwell, M., Chandler, D., Jackson, M.A., Keller, S., Koike, M., Maniania, N.K., Monzón, A., Ownley, B.H. and Pell, J.K. (2009). Fungal entomopathogens: new insights on their ecology. Fungal Ecology 2(4):149-159. https://wrap.warwick.ac.uk/id/eprint/2161/1/WRAP_Chandler_0380313-hr-071009-vega_et_al_-_funeco-d-09-00016_-_april_24.pdf

Warrit, N. and Lekprayoon, C. (2011). Asian honeybee mites in Honeybees of Asia. (Hepburn, H.R. and Radloff, S.E. [eds]), Chapter 16, p.359. Springer-Verlag Berlin Heidelberg. https://www.researchgate.net/publication/226164342_Asian_Honeybee_Mites

Zhuoer Chemistry Co., Limited (2023). Locust killer Metarhizium anisopliae powder, strain CGMCC 3.18023 https://www.zhuoerchem.com/locust-killer-metarhizium-anisopliae-powder-product/