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Mycoflora and fungal vector capacity of the parasitic Varroa destructor (: 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: http://dx.doi.org/10.1080/01647950408684376

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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. Benoit, 1 Jay A. Yoder, 1 Diana Sammataro, 2 and Lawrence W. Zettler3 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 ; 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 - , Varroidae, Varroa, parasitic mite, honey bee, fungi, USA.

INTRODUCTION Vandenberg, 1997). Recent work has been carried out to determine if Varroa is capable of harboring pathogenic In recent years, the honey bee parasitic mite Varroa microorganisms, including A. apis (Liu, 1996), viruses destructor Anderson and Trueman (Mesostigmata: (Kleespies et al., 2000), and bacteria (Kanbar et al., 2002; Varroidae; ex: V.jacobsoni Oudemans) has displayed in- de Rycke et al., 2002). The use of entomopathogenic creased resistance to acaricides, resulting in serious eco- fungi, however, is regarded as problematic because many nomic losses to the honey bee (Apis mellifera L.) industry strains are not selective to the insects they infect, resulting

Downloaded By: [National Center for Animal Health] At: 21:37 9 June 2010 worldwide. Efforts to control V. destructor have been ex- in their limited use in biological control (Roy and Pell, treme (Bruce and Needham, 1996; Yoder et al., 1999; 2000). This is especially true of V. destructor, whose de- Yoder and Sammataro, 2003) and generally unsuccessful, velopment and life cycle are closely tied to its bee host prompting the development of alternative methods of through parasitism and phoresy (Sammataro et al., 2000). control within the framework of integrative pest manage- Thus, it is conceivable that entomopathogenic fungi in- ment (Sammataro et al., 2000). One possible strategy tended for Varroa control could result in reductions of gaining attention is to include entomopathogenic fungi bee populations when applied to a bee colony. Once es- for Varroa control (Kanga et al., 2002; Davidson et al., tablished, a fungus prevalent in one bee colony may have 2003). the potential to spread to other colonies via asexual spores Fungi are found in association with honey bee colo- (conidia) carried by adults (Glifiski and Buczek, 2003). nies where they persist on nectar, pollen, in colony debris, One positive feature of entomopathogenic fungi is that and inside bees themselves. Some fungi have been shown many species are most virulent towards their original to be beneficial to the colony by preserving stored pollen host, and less so towards distant relatives and bee bread, whereas others are pathogenic and contrib- (Goettel et al., 1990, Hajek and Butler, 2000). Even so, ute to colony losses (e.g., chalkbrood via Ascosphaera the use of non-specific fungi aimed at Varroa control, apis, stonebrood via Aspergillus flavus) (Gilliam and 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. 104 Benoit et al. 2004

hizium, has shown their pathogenicity towards A. tips were visible originating from internal body sections. mellifera (Shaw et al., 2002; Peng et al., 2002). The dis- Permanent cultures of fungi are stored in the Wittenberg covery and use of fungal pathogens lethal to Varroa and Microfungus Collection (specimens number WMC 10554 not to bees would, therefore, be invaluable. -10563). Recently, an isolation procedure modified for plants One hundred mites were taken by random sampling was adapted for use in (Yoder et al., 2003c) from each population, AZ, FL and ME. Forty of the mites including V. destructor, which permitted the recovery of were used for external analysis. The remaining 60 mites the internal mycoflora. In this study, the internal and ex- were used for internal analysis, which were halved mak- ternal mycoflora of the bee mite in three states (Arizona, ing 120 body sections. Florida and Maine) is described to: (1) explore what types of fungi may be present internally that are pathogenic to RESULTS AND DISCUSSION mites, and (2) determine whether V. destructor harbors fungi externally that have the potential to spread through- A listing of fungal components from adult female V. out the bee colony. destructor is presented in Table 1. Fungi present on ex- ternal bee mite surfaces included seven species of MATERIALS AND METHODS deuteromycetes (= imperfect fungi) in five genera: Alternaria sp., Aspergillus spp. (2), Fusarium sp., Field collections of V. destructor were conducted Penicillium spp. (2), and Trichoderma sp. Two genera of from December 2002 - May 2003 from mite-infested bee zygomycetes were also recovered: Mucor sp. and Rhizo- colonies maintained in Arizona (AZ), Florida (FL) and pussp. Aspergillus and Penicillium species were recov- Maine (ME). Only female adults were used in the experi- ered most frequently in all of the samples from the three ment and the age of the mites was not known. Mites were states (AZ, FL, ME), with both genera being equally com- handled by means of sterile forceps, and all storage mate- mon. No fungi were recovered internally from any of the rials were sterilized before use. No fungi were visible mites, ruling out the possibility that V. destructor exists as macroscopicatly or microscopically (100x) on the mites an occasional fungivore, or harbors pathogenic strains. and all mites appeared healthy. The fungal taxa recovered from V. destructor in this External fungi were isolated using standard aseptic study support the findings of Batra et al. (1973) and techniques as described by Currah et al. (1997) and Yoder Glifiski and Buczek (2003) who reported on fungi com- et al. (2003c). Briefly, mites (in lots of three) were mon in beehives. Gilliam and Vandenberg (1997) also re- shaken twice for 1 min in 1 ml fresh deionized (DI) water ported that Aspergillus and Penicillium were the two most in capped vials, transferred to a 9 cm i.d. Petri dish, and prevalent genera in the honey bee colony. Within the col- covered with potato dextrose agar (PDA, Difco, Fisher ony, fungi (e.g., Penicillium, Aspergillus, Mucor, Scientific, St. Louis, MO). The plate was swirled gently Trichoderma) have been isolated from numerous sub- to separate the mites and the agar was allowed to cool. strates including dead adult bees, combs, bee food, floral Plates were incubated at 22-24°C for 7 d with daily obser- nectar, honey, and pollen provisions (Batra et al., 1973). vations for fungal growth. Hyphal tips visible under a As common opportunistic saprophytes that produce copi-

Downloaded By: [National Center for Animal Health] At: 21:37 9 June 2010 dissection microscope were excised using a sterile scal- ous conidia, the occurrence of these fungal taxa on vari- pel, and subcultured onto PDA. Plates were incubated in ous substrates - including mites representing three states darkness at 22-24°C until cultural characteristics (e.g., (AZ, FL, ME) - is not surprising. Given that the hive conidiophore) were evident (= 2 wks) that permitted iden- microhabitat is universally stable with respect to tempera- tification using Barnett (1960). ture and relative humidity (Yoder et al., 1999), such con- Internal fungi (i.e., parasitic and facultative parasitic ditions likely favor a small number of prolific fungal spe- forms) were isolated similarly, except mites were first cies, especially anamorphs (= asexual stages). Under surface sterilized by shaking in a mixture of DI water: eth- such conditions, bees apparently tolerate, and perhaps anol: 5.25% NaOC1 (18:1:1, v/v/v) prior to DI water even regulate their fungal co-habitants to some extent. rinses, and the body was dissected into halves with a ster- Upon death of bee larvae and prepupae, species of ile scalpel before addition of PDA. This surface steriliza- Aspergiltus, Penicillium and Mucor have been known to tion pretreatment was employed to strip the mite surface become secondary invaders (Glifiski and Buczek, 2003), of external fungi that might have been present. To test the eventually overgrowing the cells and forming fungal effectiveness of our sterilization technique, a separate masses (mycelia), also known as "fungal flower" within subgroup of 15 surface-sterilized mites were plated onto the brood ceils (Batra et al., 1973). On occasion, some PDA, but no traces of fimgi were detected after 10 d of in- fungi (e.g., Fusarium, Penicillium, Rhizopus, Asper- cubation at 22-24°C (data not presented). Dissected por- gillus) negatively impact the hive by spoiling provisions, tions of another set of surface-sterilized mites were then leading to natural population declines due to a reduced immersed in molten PDA and incubated 1 wk until hyphal food supply (Batra et al., 1973). To date, none of these Vol. 30, No. 2 Internat. J. Acarol. 105

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

No. isolates from mite surface* Fungi AZ FL ME Total Deuteromycota Alternaria sp. 0 0 3 3 Aspergillus flavus 7 7 0 14 Aspergillus niger 6 6 8 20 Fusarium sp. 0 2 0 2 Penicillium glabrum 3 3 I 7 Penicillium sp. 6 4 7 17 Trichoderma sp. 4 0 0 4 Zygomycota Mucor sp. 2 2 0 4 Rhizopus sp. 2 0 4 6 Total 30 24 23 77 * Nil in mite body contents (internal)

fungi have been shown to pose a serious threat to the portance (Gilliam and Vandenberg, 1997). However, A. honey bee colony as a whole, possibly because of well-es- flavus not only produces toxins fatal to bee larvae, but has tablished behavioral and immunoregulatory mechanisms also been reported to kill adult bees on occasion (Batra et (Glifiski and Buczek, 2003). al., 1973; Glifiski and Buczek, 2003), and may contribute The exception is Aspergillus flavus, which is sus- to colony losses, at least in theory. Future studies are en- pected of causing epidemics among honey bees (Batra et couraged to explore Varroa's potential as a fungal vector, al., 1973). This species is widely regarded as the caus- and to isolate additional fungal isolates that may be of ative agent of stonebrood disease in A. mellifera larvae, significance to both V. destructor and A. mellifera. known to attack and damage the brood by producing a neuropathic aflatoxin (Batra et al., 1973; Glifiski and ACKNOWLEDGEMENT Buczek, 2003). Presumably, the switch to parasitism by this otherwise harmless saprophyte (= facultative para- Funding for this research was provided by grants site) is triggered by some environmental cue, or perhaps from Sigma Xi and the Wittenberg CRGO to JBB. high incidence of the fungus, or both (modified from

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