Brenda V. Ball

The incidence and world distribution of honey bee viruses

Honey bee viruses are increasingly of interest to both bee researchers and beekeepers, primarily because of their association with the parasitic mite Varroa jacobsoni. The identification of viruses requires specialized techniques, and reviews of honey bee disease distribution that have been published so far do not contain much information on viruses. The authors, leading experts on bee viruses, have collected together data from many sources to produce this important review.

Fungal Biocontrol of Acari

Mites and ticks are susceptible to pathogenic fungi, and there are opportunities to exploit these micro-organisms for biological control. We have collated records of 58 species of fungi infecting at least 73 species of Acari, either naturally or in experiments. Fungal pathogens have been reported to kill representatives of all three orders of the Actinotrichida (the Astigmata, Oribatida and Prostigmata) and the Ixodida and Mesostigmata in the Anactinotrichida. Most reports concern infections in the Prostigmata, particularly in the families Tetranychidae and Eriophyidae. Two species of Acari-specific pathogens - Hirsutella thompsonii and Neozygites floridana - are important natural regulators of pestiferous eriophyoid and tetranychid mites respectively. Research has been done to understand the factors leading to epizootics of these fungi and to conserve and enhance natural pest control. Hirsutella thompsonii was also developed as the commercial product Mycar for the control of eriophyoid mites on citrus, but was withdrawn from sale in the 1980s, despite some promising effects in the field. Beauveria bassiana , Metarhizium anisopliae, Paecilomyces farinosus, Paecilomyces fumosoroseus and Verticillium lecanii infect ixodid ticks in nature, and B. bassiana and M. anisopliae are being studied as biological control agents of cattle ticks in Africa and South America. Beauveria bassiana also has potential as a mycopesticide of the two-spotted spider mite, Tetranychus urticae . There is scope to develop fungal biocontrol agents against a range of acarine pests, both as stand-alone treatments and for use in integrated pest management. Further research is required to clarify the taxonomic status of fungal pathogens of Acari, to study their ecosystem function, and to develop efficient mass production systems for species of Hirsutella and Neozygites .

Standard methods for virus research in Apis mellifera

Summary Honey bee virus research is an enormously broad area, ranging from subcellular molecular biology through physiology and behaviour, to individual and colony-level symptoms, transmission and epidemiology. The research methods used in virology are therefore equally diverse. This article covers those methods that are very particular to virological research in bees, with numerous cross-referrals to other BEEBOOK papers on more general methods, used in virology as well as other research. At the root of these methods is the realization that viruses at their most primary level inhabit a molecular, subcellular world, which they manipulate and interact with, to produce all higher order phenomena associated with virus infection and disease. Secondly, that viruses operate in an exponential world, while the host operates in a linear world and that much of the understanding and management of viruses hinges on reconciling these fundamental mathematical differences between virus and host. The article concentrates heavily on virus propagation and methods for detection, with minor excursions into surveying, sampling management and background information on the many viruses found in bees.

Acute Paralysis Virus Isolates from Honeybee Colonies Infested with Varroa Jacobsoni

SummaryLarge amounts of acute paralysis virus (APV) were extracted from dead, field-collected samples of European honeybees (Apis mellifera) from Russia and German Federal Republic. Virus isolates were compared to the British type strain. Particles of the three isolates were physically indistinguishable and closely related serologically, and each produced three well-defined protein bands of apparently identical mol wt on SDS Polyacrylamide gels. In Britain APV has never been found to be associated with mortality in nature. In both Russia and Germany the occurrence of APV in dead field bees and brood was associated with infestation of the colonies by the mite Varroa jacobsoni. The possible role of the mite in APV infection of honeybees is discussed.

Honey bee colony collapse and changes in viral prevalence associated with Varroa destructor

Present address: Department of Biological and Environmental Science, University of Sussex, Falmer, Brighton, East Sussex, BN1 9QG, UK. Received 16 March 2009, accepted subject to revision 15 July 2009, accepted for publication 30 November 2009. *Corresponding author: Email: norman.carreck@btinternet.com

Incidence and molecular characterization of viruses found in dying New Zealand honey bee (Apis mellifera) colonies infested with Varroa destructor

The virus status of New Zealand honey bee colonies infested with Varroa destructor was studied from 2001 to 2003. The viruses CBPV, BQCV, SBV, CWV, and KBV were all found during the study, with CWV and KBV the most common, as inferred from serological and protein profile analyses. DWV, SPV and ABPV were not detected in these colonies. CWV was present in the colonies throughout the season, while the appearance of KBV generally coincided with autumn colony collapse when V. destructor populations were large. Inconsistencies between serological analyses and viral capsid protein profiles of the extracts containing CWV and KBV were probably a result of strain differences between the viruses found in New Zealand and those used to generate the diagnostic antisera. The genome of the New Zealand KBV strain was partially sequenced. Phylogenetic and serological analyses showed this strain to be unique and most closely related to Canadian KBV isolates.ZusammenfassungBeim Saugen an adulten Bienen und an Puppen kann die parasitische Milbe Varrao destructor verschiedene Honigbienenviren über Bienenpopulationen hinweg verbreiten. Diese Viren verursachen Infektionen, die zum Tod oder zu Missbildungen bei der infizierten Brut und adulten Bienen führen können, und damit letztendlich zum Absterben der betroffenen Bienenvölker. Vier Bienenvieren konnten bereits mit Völkerverlusten in Europa und den USA in Zusammenhang gebracht werden: das Kaschmir-Bienenvirus (KBV), das Akute-Bienenparalysevirus (ABPV, das Langsame-Paralysevirus (SBV) und das Verkrüppelte-Flügelvirus (DWV). Wir benutzten eine Reihe von Methoden, um nachzuweisen, welche dieser Viren in Milben und absterbenden Völkern in Neuseeland vorkommen. Von 30 im November 2001 etablierten Völker waren im Mai 2002 alle tot, und weitere 13 im November 2002 etablierte Völker starben im August 2003. Bereits erste serologische Untersuchungen wiesen vermehrt auf einen Befall mit dem Trübe-Flügelvirus (CWV) hin (Abb. 1a und 1b). Aber dieses war nicht in allen Fällen mit dem Zusammenbruch jener Völker assoziiert. Mittels einer SDS-PAGE-Analyse von Bienenextrakten konnten wir ein Muster viraler Capsidproteine nachweisen, die dem bekannter KBV-Stämme ähnlich war (Abb. 2a). Dieses Profil stimmte auch mit dem von Bienen überein, die als Folge einer Injektion mit Kopfextrakten lebender Bienen gestorben waren (Abb. 2b). AGID (Agar-Gel-Immunodiffusion) Tests mit steigenden Konzentrationen von Antiseren gegen verschiedene KBV-Stämme ergab positive Reaktionen, was darauf hinwies, dass das Virus ein neuseeländischer KBV-Stamm (KBV-NZ) sein könnte. Das Virus etablierte sich in der Bienenpopulation im späten Sommer und Herbst, als die Milbenpopulation in diesen Völkern hoch war (Abb. 1b). Eine RT-PCR (reverse-Transkriptions-Kettenpolymerasereaktion) Analyse mit Milben aus zusammenbrechenden Völkern (mithilfe der in Tab. I aufgelisteten Primer) detektierte eindeutig ein KBV-Produkt und wies daraufhin, dass die Milben das Virus verbreiteten. Das Genom dieses KBV-NZ Stamms wurde kloniert und partiell sequenziert (Abb. 3) und eine phylogenetische Analyse zeigte, dass es eng verwandt ist mit KBV-Isolaten aus Kanada und den USA (Abb. 4). Eine Direktsequenzierung der RT-PCR-Produkte der Virusextrakte aus 2002–2003 resultierte in zwei eng verwandten Sequenzvarianten (A und B; Abb. 4) des KBV-NZ Stamms. Unterschiede in der Aminosäurensequenz der Strukturproteine zwischen dem KBV-NZ und dem KBV-Pennsylvanien Stamm (Abb. 5) könnten der Grund sein für das unterschiedliche serologische Verhalten dieser Virusisolate. Die Ergebnisse zeigen, dass KBV-Infektionen eine wichtige Rolle spielen und grosse Schäden in Varroa destructor befallenen neuseeländischen Bienenvölkern verursachen. Der Virulenzgrad dieser Infektion deutet an, dass eine hohe Milbenpopulation erforderlich ist, um eine Infektion in dem entprechenden Volk aufrechtzuerhalten. Eine Kontrolle der Milbenpopulation sollte dementprechend ein aussichtsreiches Verfahren sein, um den Ausbruch epidemischer KBV-Infektionen zu verhindern. Weitere Untersuchungen werden notwendig sein, um diese Hypothese zu testen. Interessanterweise fanden wir selbst mittels sensitiver RT-PCR-Methoden kein Verkrüppelte-Flügelvirus in Bienen- und Milbenproben aus Neuseeland.

An Iridovirus from Bees

An iridovirus, Apis iridescent virus (AIV), isolated from sick adult specimens of Apis cerana (Hymenoptera) from Kashmir, closely resembles iridescent viruses from Tipula and Sericesthis spp. (TIV and SIV). However, AIV is only distantly related serologically to TIV and SIV and is even more remotely related to several other similar viruses that were tested in tube precipitation tests with intact particles. AIV multiplies in Apis mellifera, forming cytoplasmic iridescent crystalline aggregates in several tissues, but unlike all the other iridoviruses tested, it failed to multiply in Galleria mellonella.

Prevalence and persistence of deformed wing virus (DWV) in untreated or acaricide-treated Varroa destructor infested honey bee (Apis mellifera) colonies

Summary The ectoparasitic mite Varroa destructor is a serious pest of the honey bee Apis mellifera. The naturally occurring virus known as deformed wing virus (DWV) has long been linked with the collapse of mite infested honey bee colonies. We therefore surveyed the prevalence and persistence of DWV in four heavily infested untreated colonies (Survey 1), and five heavily infested colonies that were treated with an acaricide (Survey 2). The presence of DWV in samples of adult bees, sealed brood and mites was detected using an Enzyme Linked Immunosorbent Assay (ELISA). Twenty individuals of each type were sampled monthly from each colony over the course of the study. During the summer, the proportion of adults, sealed brood and mites in which DWV was detected increased until either the colony died or was treated. When colonies were treated, thus removing mites from the colony, DWV became undetectable in the sealed bee brood at a similar rate to the loss of mites. The speed at which DWV became undetectable in adult workers depended, however, on the season, reflecting differences in life span between adult workers emerging in summer or winter. If treatment was delayed until October, DWV was still detected in adult bees during the winter even in the absence of mites. To reduce the viral load of the colony, therefore, mite treatment should be started no later than the end of August in order to remove the mites before production of the overwintering bees begins.

Prospective Biological Control Agents of Varroa destructor n. sp., an Important Pest of the European Honeybee, Apis mellifera

This paper reviews prospective biological control agents of the varroa mite, Varroa destructor n. sp. (Acari, Mesostigmata). This ectoparasite has caused severe damage to populations of the European honeybee, Apis mellifera , world-wide in recent years. To date, no promising natural enemies of varroa species have been identified on A. mellifera or its original host, Apis cerana . Therefore, biological control will probably require natural enemies from other hosts. The following groups of organisms were reviewed as potential biological control agents: predatory mites, parasitoids and entomopathogens (nematodes, protozoa, viruses, Bacillus thuringiensis , rickettsiae, and fungi). The candidate groups were ranked according to their lethality to Acari, likely ability to operate under the physical conditions of honeybee colonies, ease of targeting, and ease of mass-production. Preferential consideration was given to the natural enemies of Acari that occupy taxonomic groups close to varroa. Entomopathogenic fungi, which kill a wide range of acarine species, were identified as prime candidates for screening against varroa. Bacillus thuringiensi s also requires study, particularly strains producing novel toxins active against non-insect hosts. Entomopathogenic protozoa and nematodes show less potential for varroa control, but nonetheless warrant preliminary investigation. We consider predators, parasitoids, viruses and rickettsiae to have little potential to control varroa. Because the physical conditions within honeybee colonies are similar everywhere, it is very likely that a biological control agent of varroa could be used successfully throughout the world.

Laboratory testing of a mycopesticide on non‐target organisms: the effects of an oil formulation of Metarhizium flavoviride applied to Apis mellifera

A technique was developed to allow ultra‐low volume (ULV) application of an oil formulation of the deuteromycete, Metarhizium flavoviride Gams & Rozsypal, to Apis mellifera (Linnaeus). In the first experiment, application of a dose equivalent to twice the expected field application rate killed 30% of the bees and a twenty‐fold dose killed 87%. In a second experiment, a realistic field dose formulated in oil killed 11% of the bees and a similar dose formulated in water killed 8%. The dose applied effectively killed the target host, the desert locust Schistocerca gregaria (Forskal). Application of a chemical pesticide comprising an organophosphate and two pyrethroids at a dose that was just sub‐lethal to locusts, killed all treated bees. Very few untreated bees died. The results demonstrated the feasibility of safety testing a mycopesticide with bees as a non‐target organism. In addition, it was demonstrated that the mycopesticide currently under development for the control of locusts and grasshoppers was quantifiably less hazardous to bees than the chemical pesticide