Original article

Virus-like particles in the tracheal woodi (Rennie)

TP Liu

University of California, Department of Entomology, Davis, CA 95616, USA

(Received 8 August 1990; accepted 10 March 1991)

Summary — Tracheal mite infected honey were collected from Scotland and from California. The lyzed cells of the body cavity of tracheal in the honey bees collected from Scotland har- boured numerous 27 nm isometric virus-like particles. These virus-like particles were formed in a crystalline array and were arranged in a hexagonal pattern. No virus-like particles were observed in the tracheal mites of the honey bees collected from California. The significance of these observa- tions in relation to the honey tracheal mite resistance is discussed. / / virus-like particle / electron microscopy

INTRODUCTION aware of environmental pollution and food safety issues associated with nearly 50 yr The continuing range expansion of tra- of synthetic and organic pesticide use. cheal mite Acarapis woodi (Rennie) (Aca- Chemical residue problems will certainly ri, ) infestation in Canada affect the marketability of honey which is and the United States has prompted ex- harvested from bee hives treated with periments in order to find chemical means pesticides. An attempt has also been of controlling this damaging honey bee made to import British honey bees in or- disease (Guzman-Novoa and Zozaya- der to introduce natural tracheal mite re- Rubio, 1984; Anonymous, 1986; Gruszka, sistance to the North American stocks 1987; Wilson et al, 1988; Eischen et al, (Morse, 1990). Breeding tracheal mite re- 1989). Chemical control of the tracheal sistant honey bees is certainly the most mite is very attractive because of its rela- desirable approach to reduce the damage tively low cost application and reliability. caused by tracheal mite infestation. Bio- Unfortunately, consumers are increasingly logical control of tracheal mite infestation,

* This work was performed during sabbatical leave. ** Present address and affiliated institution: Box 29, Agriculture Canada Research Station, Beaver- lodge, Alberta,Canada TOH OCO may be one of the other attractive alterna- ed, tissues were extensively lyzed, and tives. Because biological control is based these mites appeared to be dead before on organism interaction, it involves ecolog- fixation. Residual membranes and large la- ical studies of factors that regulate or af- mellar bodies were also observed (figs 1- fect interaction. Attempts have been made 3). All of the virus-like particles were during the last 5 yr to find organisms which packed in crystalline arrays in a hexagonal have the potential of becoming biological arrangement (figs 1, 3-4), while scattered control agents in tracheal mites, or are as- single particles were rarely observed. sociated with tracheal mites. This paper These particles, exclusively found within describes the ultrastructural features of vi- the mites, were isometric and measured rus-like particles found in the lyzed tissue 27 nM in diameter, and were not embed- of some tracheal mites. ded in protein matrices. None of them had been observed in the tracheal lumen. In 2 of the mites, the entire body cavity ap- MATERIALS AND METHODS peared to be filled with these virus-like crystalline inclusions (fig 2). At higher mag- nification the consisted of a dense Tracheal mite infected honey bees were collect- particles ed from Buckham, Scotland during the summer core ringed by an electron-dense coat (fig of 1985. Infected honey bees were fixed in 8% 4). EM grade glutaraldehyde (Polyscience, War- rington, PA) before being shipped to Canada. were also collected Specimens during 1988- DISCUSSION 1989 from Vacaville, CA. Tracheae with heavy mite infestation were dissected in saline and transferred to vials 4% containing glutaralde- These virus-like particles found in the tra- hyde in 0.2 M phosphate buffer, pH 7.4 (Liu et cheal mites bear a close resemblance to al, 1989). Specimens were fixed overnight at 4 °C, rinsed in the buffer, postfixed in 1% osmi- some of the honey bee viruses (Bailey, um tetroxide in the same buffer, dehydrated in a 1976, 1981; Bailey et al, 1980, 1981). The graded ethanol series, and embedded in Poly- identification of the small isometric virus- bed 812 (Polyscience, Warrington, PA). Silver- like particles is handicapped by the lack of gray sections were cut on a Sorval MT2 ultrami- fresh tracheal mites infected by this virus. crotome with glass knives, and mounted on Ultrastructural appearance of these 400-mesh copper grids. Sections were stained parti- with uranyl acetate followed by lead citrate then cles described in the present study resem- examined and photographed with a Philips EM bles several insect picornaviruses, includ- 410 or 400 electron microscope operated at an ing the P virus of Drosophila melanogaster accelerating voltage of 80 Kv. (Teninges and Plus, 1972), and the grass- hopper crystalline array virus (Henry and Oma, 1973). These small viruses typically RESULTS form cytoplasmic crystalline arrays. This type of virus has been described in many Of 51 tracheal mites from 17 Scotland invertebrates (Longworth, 1978). Picorna- honey bees examined, 3 mites from 2 indi- viruses are single-stranded RNA viruses, vidual bees harboured virus-like particles. ranging in size from 22-30 nm (Matthews, Of 75 tracheal mites from 27 California 1982). Many small honey bee RNA viruses honey bees examined, no virus-like parti- in Britain, such as black queen cell, acute cles were found. In all of the tracheal mites paralysis and Kashmir viruses, have a di- from which virus-like particles were detect- mension of 30 nm. Viruses Y and X are

35 nm in size. The very common cloudy honey bee viruses ingested by the tracheal wing particle virus is 17 nm in size (Bailey, mites. 1981). The size of these honey bee virus Viruses of any kind have not previously particles is either larger of smaller than the been described in the tracheal mite. Be- virus particles observed in the tracheal cause these virus-like particle were found mites. Due to a lack of thin-section elec- in the extensively lyzed cells, it could indi- tron microscope studies, it is impossible to cate that they are lethal to the tracheal make a direct comparison between the mites.These particles were found among honey bee viruses and the virus-like parti- cellular debris of the lyzed cells, which is cles in the tracheal mites, concerning the an indication of the multiplication that took size of the virus particles and the pattern of place in the cells and accumulated in the cytoplasmic crystalline arrays. In addition, cytoplasm. All of these virus-like particles the large number of virus crystals found in were found in the mites from honey bees the tracheal mites is always associated collected in Scotland. None have been ob- with cellular debris, which is an indication served in the mites from the honey bees that these particles replicated themselves collected in California. Because of the within the cells of tracheal mites. There- sample size the extent of the infection in fore, it is unlikely that these particles are the tracheal mite population within the cotland honey bees it not certain. Since ont été réalisées au microtome, colorées à these virus-like particles have not been ob- l’acétate d’uranyle et au citrate de plomb, served in the tracheal mites from the Cali- puis examinées en microscopie électroni- fornia honey bees, it is quite possible that que (Philips EL 400) à 80 kV. Les Acarapis the British honey bees are more resistant des abeilles venant d’Écosse renfermaient to tracheal mite infestation due to the pres- dans les cellules lysées des particules de ence of a viral desease in the tracheal type viral. Ces particules isométriques de mite population. The tracheal mite popula- 27 nm se présentaient sous forme cristalli- tion in the British honey bees may be sub- sée dans une disposition hexagonale. Il n’y jected to viral disease regulation. An ex- avait pas de particules de type viral chez tensive survey is underway to determine les Acarapis des abeilles provenant de Ca- the incidence and pathogenicity of these lifornie. II est possible que ces abeilles virus-like particles, in order to demonstrate soient plus résistantes à A woodi à cause the above postulation. de la présence d’une virose chez ce der- nier.

ACKNOWLEDGMENTS Acarapis woodi / particule de type viral / microscopie électronique The author wishes to thank members of the Bee Biology Facilities, University of California, Davis Campus, for their hospitality and to M Mobus, Zusammenfassug — Virus-ähnliche North Scotland Agricultural College, for collect- Partikel bei der Tracheenmilbe Acarapis in Scotland. This work was ing specimens sup- woodi. Von Tracheenmilbe befallene Ho- ported by the work transfer program, Agriculture Canada. nigbienen wurden 1985 in Aberdeen, Schottland, und 1988/1989 in Vaccaville, Kalifornien, gesammelt. Tracheen von mit Milben schwer befallenen *Bienen wurden Résumé — Des de viral particules type in und in chez l’acarien Acarapis woodi (Rennie). Insekten-Salzlösung präpariert Gläschen mit 4% in 0.2 M Des abeilles (Apis mellifera L) parasitées Glutaradehyd 7.4, Die par l’acarien des trachées, woodi Phosphatpuffer, pH übertragen. Acarapis mit Milben besetzten Tracheen wurden (Rennie) (, Tarsonemidae), ont été über Nacht bei 4 °C in dieser prélevées à Aberdeen (Écosse) en 1985 Fixierlösung belassen, dann mit demselben Puffer et à Vacaville (Californie) en 1988-1989. ge- und anschießend in 1% Osmiumte- Les trachées des abeilles fortement para- spült troxid in demselben Puffer für 1 h nachfi- sitées ont été disséquées dans du liquide xiert. Die wurden dann stufen- physiologique et transférées dans des ré- Präparate weise in in Po- cipients contenant 4% de glutaraldéhyde Äthylalkohol dehydriert und dans un tampon phosphate à 0,2 mol.l-1, lybed eingebettet. pH 7,4. Les trachées renfermant des aca- Mittels Glasmesser hergestellte Dünn- riens ont été laissées dans ce milieu toute schliffe wurden mit Uranylazetat und Bleizi- une nuit à 4 °C puis rincées dans le même trat gefärbt und unter einem Philips EM tampon et fixées au tétroxyde d’osmium à 400 Elektronenmikroskop bei einer Be- 1% dans le même tampon durant 1 h. schleunigungs-Spannung von 80 kv unter- Elles ont été ensuite déshydratées pro- sucht. Einige der Tracheenmilben aus gressivement par l’alcool éthylique et mon- Bienen, die in Schottland gesammelt tées dans du Polybed. Des coupes minces worden waren, enthielten Virus-ähnliche Partikel in aufgelösten Zellen (Fig. 1, 3, 4). ng honey bees. II. An apiary test. Apidologie Diese 27 nM messende isometrischen 20, 41-51 Virus-ähnlichen Partikel befanden sich in Gruszka J (1987) Honey bee tracheal mites: are kristalliner Anordnung mit hexagonalem they harmful ? Am Bee J 127, 653 Muster. In den Milben, die aus in Kalifor- Guzman-Novoa E, Zozaya-Rubio A (1984) The nien gesammelten Bienen stammten, effects of chemotherapy on the level of infes- tation and of in colonies of wurden keine Virus-ähnlichen Partikel ge- production honey honey bees with acariosis. Am Bee J 124, funden. Es wird vermutet, daß ein Zusam- 669-672 zwischen diesem Befund und menhang JE, Oma EA Ultrastructure of the der erhöhten der Bienen Henry (1973) Milbenanfälligkeit replication of the grasshopper crystalline ar- in Kalifornien bestehen könnte. ray virus in Schistocera americana compared with other picornaviruses. J Invertebr Pathol Honigbiene / Acarapis woodi / Virus- 21, 273-281 ähnliche Partikel / Elektronen- Liu TP, Mobus B, Braybrook G (1989) Fine Mikroskopie structure of hypopharyngeal glands from hon- eybees with and without infestation by the tracheal mite, Acarapis woodi (Rennie). Res 85-92 REFERENCES J Apic 28, Longworth JF (1978) Small isometric viruses of invertebrates. Adv Virus Res 23, 103-157 Anonymous (1986) Manitoba quarantines tra- cheal mite infested colonies. Am Bee J 126, Matthews REF (1982) Classification and nomen- 589 clature of viruses. Fourth report of the Inter- national Committee on of Viruses. Bailey L (1976) Viruses attacking the honey Intervirology 17, 1-200 bee. Adv Virus Res 20, 271-304 Morse RA (1990) Importation of British queen Bailey L (1981) Honey Bee Pathology. Academ- honey bees into the United States. Am Bee J ic Press, London 130, 106-120 Bailey L, Ball BV, Carpenter JM, Woods RD Teninges D, Plus N (1972) P virus of Drosophila Small virus-like in (1980) particles honey melanogaster, as a new picornavirus. J Gen bees associated with chronic paralysis virus Virol 16, 103-109 and with a previously undescribed disease. J Gen Virol 46, 149-155 Wilson WT, Moffett JO, Cox RL, Maki DL, Rich- ardson H, Rivera R (1988) Menthol treatment Bailey L, Ball BV, Perry JN (1981) The preva- for Acarapis woodi control in Apis mellifera lence of viruses of honey bees in Britain. Ann and the resulting residues in honey. In: Afri- Appl Biol 97, 109-118 canized Honey Bees and Bee Mites (Need- Eischen FA, Cardoso-Tames D, Dieta A, Ware ham GR, Page RE, Delfinado-Baker M, Bow- GO (1989) Cymiazole, a systemic acaricide man CE, eds) John Wiley and Sons, NY, that controls Acarapis woodi (Rennie) infest- 535-540