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International Journal of Acarology

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Mycoflora and fungal vector capacity of the parasitic mite Varroa destructor (Mesostigmata: Varroidae) in honey bee (Hymenoptera: Apidae) colonies

Joshua B. Benoita; Jay A. Yodera; Diana Sammatarob; Lawrence W. Zettlerc a Department of Biology, Wittenberg University, Springfield, OH, USA b Carl Hayden Honey Bee Research Center, United States Department of Agriculture, Agricultural Research Service, Tucson, AZ, USA c Department of Biology, The Illinois College, Jacksonville, IL, USA

To cite this Article Benoit, Joshua B. , Yoder, Jay A. , Sammataro, Diana and Zettler, Lawrence W.(2004) 'Mycoflora and

fungal vector capacity of the parasitic mite Varroa destructor (Mesostigmata: Varroidae) in honey bee (Hymenoptera: Apidae) colonies', International Journal of Acarology, 30: 2, 103 — 106 To link to this Article: DOI: 10.1080/01647950408684376 URL:

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Joshua B. Benoit, 1 Jay A. Yoder, 1 Diana Sammataro, 2 and Lawrence W. Zettler 3

1. Department of Biology, Wittenberg University, Springfield, 0H45501, USA, e-mail: [email protected]; 2. Carl Hayden Honey Bee Research Center, United States Department of Agriculture, * Agricultural Research Service, Tucson, AZ 85719, USA; 3. Department of Biology, The Illinois College, Jacksonville, IL 62650, USA.

ABSTRACT - Female adults of the honey bee mite, Varroa destructor, have fimgi on their surfaces, and have the potential to disperse fungal spores (conidia) throughout the bee colony. Fungi present are typical of those associated with bees, their combs and provisions, and are common filamentous, soil saprophytes (listed in order of relative abundance): Aspergillus, Penicillium, Fusarium, Trichoderma, Alternaria, Rhizopus and Mucor. These fungi have no biological control implications (i.e., entomopathogenic) because none were recovered intemally within the mites; absence of fungi internally also implies that this mite is not a fungivore. Our isolation of Aspergillus flavus, agent of stonebrood disease in honey bees, implicates Varroa as a potential fungal vector of stonebrood disease. Key words - Acari, Varroidae, Varroa, parasitic mite, honey bee, fungi, USA.

INTRODUCTION In recent years, the honey bee parasitic mite Varroa destructor Anderson and Trueman (Mesostigmata: Varroidae; ex: V.jacobsoni Oudemans) has displayed increased resistance to acaricides, resulting in serious economic losses to the honey bee (Apis mellifera L.) industry worldwide. Efforts to control V. destructor have been extreme (Bruce and Needham, 1996; Yoder et al., 1999; Yoder and Sammataro, 2003) and generally unsuccessful, prompting the development of alternative methods of control within the framework of integrative pest management (Sammataro et al., 2000). One possible strategy gaining attention is to include entomopathogenic fungi for Varroa control (Kanga et al., 2002; Davidson et al., 2003). Fungi are found in association with honey bee colonies where they persist on nectar, pollen, in colony debris, and inside bees themselves. Some fungi have been shown to be beneficial to the colony by preserving stored pollen and bee bread, whereas others are pathogenic and contribute to colony losses (e.g., chalkbrood via Ascosphaera apis, stonebrood via Aspergillus flavus) (Gilliam and

Vandenberg, 1997). Recent work has been carried out to determine if Varroa is capable of harboring pathogenic microorganisms, including A. apis (Liu, 1996), viruses (Kleespies et al., 2000), and bacteria (Kanbar et al., 2002; de Rycke et al., 2002). The use of entomopathogenic fungi, however, is regarded as problematic because many strains are not selective to the insects they infect, resulting in their limited use in biological control (Roy and Pell, 2000). This is especially true of V. destructor, whose development and life cycle are closely tied to its bee host through parasitism and phoresy (Sammataro et al., 2000). Thus, it is conceivable that entomopathogenic fungi intended for Varroa control could result in reductions of bee populations when applied to a bee colony. Once established, a fungus prevalent in one bee colony may have the potential to spread to other colonies via asexual spores (conidia) carried by adults (Glifiski and Buczek, 2003). One positive feature of entomopathogenic fungi is that many species are most virulent towards their original host, and less so towards distant arthropod relatives (Goettel et al., 1990, Hajek and Butler, 2000). Even so, the use of non-specific fungi aimed at Varroa control, namely species of Beauveria, Hirsutella and Metar-

*Mention of a proprietary product does not constitute an endorsement or use of the product by the U.S.D.A.

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hizium, has shown their pathogenicity towards A. mellifera (Shaw et al., 2002; Peng et al., 2002). The discovery and use of fungal pathogens lethal to Varroa and not to bees would, therefore, be invaluable. Recently, an isolation procedure modified for plants was adapted for use in arthropods (Yoder et al., 2003c) including V. destructor, which permitted the recovery of the internal mycoflora. In this study, the internal and external mycoflora of the bee mite in three states (Arizona, Florida and Maine) is described to: (1) explore what types of fungi may be present internally that are pathogenic to mites, and (2) determine whether V. destructor harbors fungi externally that have the potential to spread throughout the bee colony. MATERIALS AND METHODS Field collections of V. destructor were conducted from December 2002 - May 2003 from mite-infested bee colonies maintained in Arizona (AZ), Florida (FL) and Maine (ME). Only female adults were used in the experiment and the age of the mites was not known. Mites were handled by means of sterile forceps, and all storage materials were sterilized before use. No fungi were visible macroscopicatly or microscopically (100x) on the mites and all mites appeared healthy. External fungi were isolated using standard aseptic techniques as described by Currah et al. (1997) and Yoder et al. (2003c). Briefly, mites (in lots of three) were shaken twice for 1 min in 1 ml fresh deionized (DI) water in capped vials, transferred to a 9 cm i.d. Petri dish, and covered with potato dextrose agar (PDA, Difco, Fisher Scientific, St. Louis, MO). The plate was swirled gently to separate the mites and the agar was allowed to cool. Plates were incubated at 22-24°C for 7 d with daily observations for fungal growth. Hyphal tips visible under a dissection microscope were excised using a sterile scalpel, and subcultured onto PDA. Plates were incubated in darkness at 22-24°C until cultural characteristics (e.g., conidiophore) were evident (= 2 wks) that permitted identification using Barnett (1960). Internal fungi (i.e., parasitic and facultative parasitic forms) were isolated similarly, except mites were first surface sterilized by shaking in a mixture of DI water: ethanol: 5.25% NaOC1 (18:1:1, v/v/v) prior to DI water rinses, and the body was dissected into halves with a sterile scalpel before addition of PDA. This surface sterilization pretreatment was employed to strip the mite surface of external fungi that might have been present. To test the effectiveness of our sterilization technique, a separate subgroup of 15 surface-sterilized mites were plated onto PDA, but no traces of fimgi were detected after 10 d of incubation at 22-24°C (data not presented). Dissected portions of another set of surface-sterilized mites were then immersed in molten PDA and incubated 1 wk until hyphal


tips were visible originating from internal body sections. Permanent cultures of fungi are stored in the Wittenberg Microfungus Collection (specimens number WMC 10554 -10563). One hundred mites were taken by random sampling from each population, AZ, FL and ME. Forty of the mites were used for external analysis. The remaining 60 mites were used for internal analysis, which were halved making 120 body sections. RESULTS AND DISCUSSION

A listing of fungal components from adult female V. destructor is presented in Table 1. Fungi present on external bee mite surfaces included seven species of deuteromycetes (= imperfect fungi) in five genera: Alternaria sp., Aspergillus spp. (2), Fusarium sp., Penicillium spp. (2), and Trichoderma sp. Two genera of zygomycetes were also recovered: Mucor sp. and Rhizopussp. Aspergillus and Penicillium species were recovered most frequently in all of the samples from the three states (AZ, FL, ME), with both genera being equally common. No fungi were recovered internally from any of the mites, ruling out the possibility that V. destructor exists as an occasional fungivore, or harbors pathogenic strains. The fungal taxa recovered from V. destructor in this study support the findings of Batra et al. (1973) and Glifiski and Buczek (2003) who reported on fungi common in beehives. Gilliam and Vandenberg (1997) also reported that Aspergillus and Penicillium were the two most prevalent genera in the honey bee colony. Within the colony, fungi (e.g., Penicillium, Aspergillus, Mucor, Trichoderma) have been isolated from numerous substrates including dead adult bees, combs, bee food, floral nectar, honey, and pollen provisions (Batra et al., 1973). As common opportunistic saprophytes that produce copious conidia, the occurrence of these fungal taxa on various substrates - including mites representing three states (AZ, FL, ME) - is not surprising. Given that the hive microhabitat is universally stable with respect to temperature and relative humidity (Yoder et al., 1999), such conditions likely favor a small number of prolific fungal species, especially anamorphs (= asexual stages). Under such conditions, bees apparently tolerate, and perhaps even regulate their fungal co-habitants to some extent. Upon death of bee larvae and prepupae, species of Aspergiltus, Penicillium and Mucor have been known to become secondary invaders (Glifiski and Buczek, 2003), eventually overgrowing the cells and forming fungal masses (mycelia), also known as "fungal flower" within the brood ceils (Batra et al., 1973). On occasion, some fungi (e.g., Fusarium, Penicillium, Rhizopus, Aspergillus) negatively impact the hive by spoiling provisions, leading to natural population declines due to a reduced food supply (Batra et al., 1973). To date, none of these

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Table 1. Catalog of the mycoflora of honey bee mite, Varroa destructor, from Arizona (AZ), Florida (FL) and Maine (ME), USA.

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Fungi Deuteromycota Alternaria sp.

Aspergillus flavus Aspergillus niger Fusarium sp. Penicillium glabrum Penicillium sp. Trichoderma sp.

No. isolates from mite surface* AZ FL ME










6 0

6 2

8 0

20 2





6 4

4 0

7 0

17 4

2 0 24

0 4 23

4 6 77

Zygomycota Mucor sp. 2 Rhizopus sp. 2 Total 30 * Nil in mite body contents (internal)

fungi have been shown to pose a serious threat to the honey bee colony as a whole, possibly because of well-established behavioral and immunoregulatory mechanisms (Glifiski and Buczek, 2003). The exception is Aspergillus flavus, which is suspected of causing epidemics among honey bees (Batra et al., 1973). This species is widely regarded as the causative agent of stonebrood disease in A. mellifera larvae, known to attack and damage the brood by producing a neuropathic aflatoxin (Batra et al., 1973; Glifiski and Buczek, 2003). Presumably, the switch to parasitism by this otherwise harmless saprophyte (= facultative parasite) is triggered by some environmental cue, or perhaps high incidence of the fungus, or both (modified from Yoder et al., 2003a and b). Our inability to detect A. flavus intemally in V. destructor suggests that this fungus is not a parasite of the mite itself. The presence of A. flavus on external mite surfaces implies that K destructor serves as an additional, albeit minimal, source of fungal inoculnm within the hive as a resident on the adult bee. Of considerable significance, however, is the possibility that A. flavus would gain access into new brood cells via conidia carried on mites that venture out into the hive in search of a new bee host. As such, V. destructor, in addition to causing heavy losses to the honey bee industry by direct infestation (Sammataro et al., 2000), may also promote the spread of stonebrood disease, as first proposed by Liu (1996). For the first time, our study provides evidence for this possibility. Our revelation that A. flavus conidia are carried readily on Varroa begs the question of whether there is an increased incidence of bee death attributed to this fungus. Stonebrood is a rare disease considered to be of minor im-

portance (Gilliam and Vandenberg, 1997). However, A.

flavus not only produces toxins fatal to bee larvae, but has also been reported to kill adult bees on occasion (Batra et al., 1973; Glifiski and Buczek, 2003), and may contribute to colony losses, at least in theory. Future studies are encouraged to explore Varroa's potential as a fungal vector, and to isolate additional fungal isolates that may be of significance to both V. destructor and A. mellifera. ACKNOWLEDGEMENT

Funding for this research was provided by grants from Sigma Xi and the Wittenberg CRGO to JBB. REFERENCES

Barnett, H. L. 1960. Illustrated genera of imperfect fungi. Burgess, Minneapolis, MN. Batra, L. R., S. W. T. Batra and G. E. Bohart. 1973. The mycoflora of domesticated and wild bees (Apoidea). Mycopathol. Mycol. Appl. 49: 13-44. Bruce, W. A. and G. R. Needham. 1996. Effect of temperature and humidity on the water balance of Varroa jacobsoni (Acari: Varroidae), pp. 157-159. In: Needham, G. R. D., J. Horn and R. Mitchell (Eds.). Acarology IX: Proceedings. Ohio Biological Survey, Columbus, OH. Currah, R. S., L. W. Zettler and T. M. McInnis. 1997. Epulorhiza inquilina sp. nov. from Platanthera (Orchidaceae) and key to Epulorhiza species. Mycotaxon. 61: 335-342. Davidson, G., K. Phelps, K. D. Sunderland, J. K. Pell, B. V. Ball, K. E. Shaw and D. Chandler. 2003. Study

Downloaded By: [National Center for Animal Health] At: 21:37 9 June 2010


Benoit et al.

of temperature-growth interactions of entomopathogenic fungi with potential for control of Varroa destructor (Acari: Mesostigmata) using a nonlinear model of poikilotherm development. J. Appl. Microbiol. 94: 816-825. de Rycke, P. H., J. J. Joubert, S. H. Hosseinian and F. J. Jacobs. 2002. The possible role of Varroa destructor in the spreading of American foulbrood among apiaries. Exp. Appl. Acarol. 27:313-318. Gilliam, M. and J. D. Vandenberg. 1997. Fungi, pp. 79-112. In: Morse, R. A.and P. K. Flottum (Eds.). Honey Bee Pests, Predators and Diseases. A.I. Root Co., Medina, OH. Glifiski, Z. and K. Buczek. 2003. Response of the Apoidea to fungal infections. Apicata. 38: 183-189. Goettel, M. S., T. J. Poprawski, J. D. Vandenberg, Z. Li and D. W. Roberts. 1990. Safety to nontarget invertebrates of fungal biocontrol agents, pp. 209-231. In: Land, M., L. A. Lacey and E. W. Davidson (Eds.). Safety of Microbial in Insecticides. CRC Press, Boca Raton, FL. Hajek, A. E. and L. Butler. 2000. Predicting the host range of entomopathogenic fungi, pp. 263-276. In: Follett, P. A. and J. J. Duan (Eds.). Nontarget Effects of Biological Control. Kluwer Academic, The Netherlands. Kanbar, G., W. Engles and G. Winkelmann. 2002. Do Varroa destructor mites transfer European foulbrood (Melissococcus pluton)? Apidologie 33: 487-88. Kanga, L. H. B., R. R. James and D. G. Boucias. 2002. Hirsutella thompsonii and Metarhizuim anisopliae as potential microbial control agents of Varroa destructor, a honey bee parasite. J. Invert. Pathol. 81: 175-184. Kleespies, F. G., J. Radtke and K. Bienefeld. 2000. Virus-like particles found in the ectoparasitic bee mites Varroa jacobsoni Oudemans. J. Invert. Pathol. 15: 87-90. Liu, T. P. 1996. Varroa mites as carriers of honey bee chalkbrood. Amer. Bee J. 138: 655. Peng, C. Y. S., Z. Xinsheng and H. K. Kaya. 2002. Virulence and site o f infection of the fungus, Hirsutalla


thompsonii, to honey bee ectoparasitic mite, Varroa destructor. J. Invert. Pathol. 81: 185-195. Roy, H. E. and J. K. Pell. 2000. Interaction between entomopathogenic fimgi and other natural enemies: Implications for biological control. Bio. Sci. Tech. 10: 737-752. Sammataro, D., U. Gerson and G. R. Needham. 2000. Parasitic mites of honey bees: Life history, implications, and impact. Ann. Rev. Entomol. 45: 519-548. Shaw, K. E., G. Davidson, S. J. Clark, B. V. Ball, J. K. Pell, D. Chandler and K. D. Sutherland. 2002. Laboratory bioassays to assess the pathogenicity of mitosporie fungi to Varroa destructor (Acari: Mesostigmata), an ectoparasitic mite of the honey bee, Apis mellifera. Biol. Control 24: 266-276. Yoder, J. A. and D. Sammataro. 2003. Potential to control Varroa mites (Acari: Varroidae) using chemical ecology. Internat. J. Acarol. 29: 139-143. Yoder, J. A., D. Sammataro, J. A. Peterson, G. R. Needham and W. A. Bruce. 1999. Water requirements of adult females of the honey bee parasitic mite, Varroajacobsoni (Acari: Varroidae) and implications for control. Intemat. J. Aearol. 25: 329-335. Yoder, J. A., J. B. Benoit and L. W. Zettler. 2003a. Effects of salt and temperature on the growth rate of a tick-associated fungus, Scopulariopsis brevicaulis Bainier (Deuteromycota). Intemat. J. Acarol. 29: 265-269. Yoder, J. A., J. B. Benoit and L. W. Zettler. 2003b. Moisture requirements of a soil imperfect fungus, Scopulariopsis brevicaulis Bainer, in relation to its tick host. Interuat. J. Acarol. 29: 271-277. Yoder, J. A., P. E. Hanson, L. W. Zettler, J. B. Benoit, F. Ghisays and K. A. Piskin. 2003c. Internal and external mycoflora of the American dog tick, Dermacentor variabitis, and its ecological implications. Appl. Environ. Microbiol. 69: 4994-4996.