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The rest are RNA viruses and most have particles 30 nm in diameter, thus resembling many of the viruses familiar to animal and plant pathologists. These small isometric viruses of bees, however, are a very heterogeneous group with characteristics that differentiate them from other morphologically similar virus particles. Knowledge of honey bee viruses has now greatly increased and a further 19 have been identified, including some serologically related strains. Several of the viruses, however, are closely associated with and augment the pathogenicity of one or more of these parasites, and a few depend on the relationship for their infectivity. Similar associations may perhaps be anticipated between the viruses and parasites of other insect species. Until about the middle of the century only one virus disease of honey bees, sacbrood of larvae, had been recognized. The filamentous virus of honey bees has been detected in Apis mellifera from almost every continent.

Summary The mite Varroa destructor has been associated with the collapse of millions of Apis mellifera honey bee colonies world-wide. During the past decade, a large body of research has revealed various interactions between varroa, the honey bee and various viral pathogens. One pathogen in particular, deformed wing virus (DWV), has emerged as the key pathogen involved in colony collapse. As varroa has permanently changed the viral landscape in which honey bees exist, we present a large body of data on the effects of DWV during the initial phase of varroa infestation in the UK during 1998. This provides baseline data for further comparative studies. We carried out DWV transmission studies, and observed the effects of DWV on bee longevity. As the ELISA technique used in these studies had a detection limit of ~10 7 viral particles per bee, only high viral (overt) titres were detected. During the initial phase of varroa establishment, DWV was detected in 0.6% of non-infested sealed brood, but in 89% of sealed brood invaded by varroa. Once DWV was introduced into the bee’s haemolymph via mite feeding on either pupae or adults, an overt virus infection was rapidly produced in 3-4 days. In sealed brood the presence of varroa was fatal for 21% of the brood, caused wing deformity in some emerging adults and significantly reduced longevity as an adult. However, adult bees that became infected after they had emerged, did not develop wing deformity nor show any reduced longevity, but acted as reservoirs of DWV infection.

SummaryCloudy wing virus (CWV) is a little studied small icosahedral virus commonly found in honey bees (Apis mellifera), especially in collapsing colonies infested by Varroa destructor, providing circumstantial evidence that the mite may act as a vector. This study investigated the potential relationship between V. destructor, CWV and honey bees in 29 naturally infested colonies in the UK. We found that CWV was detected in less than half of the colonies, despite all having been infested by V. destructor. In contrast to several other viruses, we also found no evidence that V. destructor is able to vector CWV when feeding, and conclude that if transmitted by the mite, the virus fails to become established as an overt infection in the honey bee. The reasons why CWV has apparently increased in incidence in the UK since the arrival of V. destructor therefore remain unclear.

SummaryThe 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...

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.

We have developed a specific assay for the detection of deformed wing virus (DWV) in Apis mellifera L. and Varroa destructor based on the reverse transcriptase polymerase chain reaction (RT-PCR) technology. Primers were designed from the sequence of a 4700 nucleotides cDNA fragment located in the 3'-end of the DWV genome. This fragment encodes a single open reading frame of 1565 amino acids showing similarity to viral RNA dependent RNA polymerase consensus motif. RT-PCR assays from DWV infected individual mite or bee produced a 395 nucleotide DNA fragment clearly identifiable by agarose gel electrophoresis. The signal in bees having deformed wings was significantly higher than in normal ones. A search for DWV in 40 colonies showed that DWV is broadly distributed in bee colonies and mites. As an average, greater virus prevalence of virus was detected in bees collected in autumn compared to bees collected in spring or during the summer period. deformed wing virus (DWV) / diagnosis / bee virus / Varroa destructor

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 fun...

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, b...

Viruses of the honey bee have been known for a long time; however, recently the attention of scientists and apiculturalists has turned towards the relationship between these viruses and the parasitic miteVarroa jacobsoni. Although clinical symptoms indicated the presence of some of the viruses of bees in Hungary, none have previously been isolated or identified. During July unusual adult bee and brood mortality was observed in some colonies of an apiary in Budapest known to be infested withVarroa jacobsoni. Large amounts of acute paralysis virus (APV) were detected serologically in healthy honey bee pupae killed by the injection of a bacteria-free extract of diseased adult bees. Crystalline arrays of 30 nm particles were seen in ultrathin sections of the tissues of injected pupae and naturally infected adult bees. In spite of the application of acaricide treatments the bee population in several colonies had collapsed by the end of summer and the apiary suffered severe wintering losses.

The role of the parasitic mite Varroa destructor in the transmission of slow bee paralysis virus (SBPV) was investigated. Virus transmission experiments were done in two seasons (July and September...

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.

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.

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

Nucleic acids from 41 strains of Metarhizium anisopliae, obtained from different parts of the world were extracted and examined by electrophoresis. Strong bands of double‐stranded RNA (dsRNA) were detected in two isolates from Brazil, V215 and V291, which had, respectively, 13 and 9 distinct bands ranging in size from ca. 0.75 to 3.5 kb. Icosahedral virus‐like particles (VLPs) (ca. 33 nm in diameter) were observed by transmission electron microscopy in extracts of these isolates. The VLPs and dsRNA were both absent from a clone of the isolate V291 which had been subcultured successively on solid medium. Bioassays against the aphid Myzus persicae showed no detectable difference in virulence between the clone of V291 which contained dsRNA and the clone that did not.

SUMMARYMelissococcus pluton was isolated from Apis mellifera larvae from Brazil, China and the UK. The cultural characteristics and nutritional requirements of these isolates were compared with the UK type isolate, a previously described isolate from Brazil and an isolate from A. cerana from India. All isolates were fastidious in their cultural requirements except for the Brazilian isolates which grew on a wider range of bacteriological media.Polyclonal antisera were prepared against a UK isolate and a Brazilian isolate, and with three previously prepared antisera (two against a North American isolate and one against the A. cerana isolate) were used in tube agglutination tests to study the serological relationships of M. pluton isolates. All isolates were closely related serologically, but could be separated into three groups with respect to their heterologous serological reactivity which reflected the geographical regions in which they originated.

Complete genome sequences were determined for two distinct strains of slow bee paralysis virus (SBPV) of honeybees (Apis mellifera). The SBPV genome is approximately 9.5 kb long and contains a single ORF flanked by 5′- and 3′-UTRs and a naturally polyadenylated 3′ tail, with a genome organization typical of members of the family Iflaviridae. The two strains, labelled ‘Rothamsted’ and ‘Harpenden’, are 83 % identical at the nucleotide level (94 % identical at the amino acid level), although this variation is distributed unevenly over the genome. The two strains were found to co-exist at different proportions in two independently propagated SBPV preparations. The natural prevalence of SBPV for 847 colonies in 162 apiaries across five European countries was

To investigate the role of the parasitic mite Varroa destructorin the transmission of slow paralysis virus (SPV) and deformed wing virus (DWV) and to gain some insight into host-parasite-pathogen i...

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

Deformed wing virus (DWV) induces wing deformation when bees are infected during their pupal development. Field observations and laboratory experiments suggest that the miteVarroa destructoris a vector of the virus. Moreover, it has been stated that DWV replicates within this mite. In order to understand the role ofV. destructorin the transmission of DWV, the objective of this work was to locate the sites of retention and/or replication of DWV within the mite by immunohistochemistry. There was no evidence that DWV was replicating in the mite as no tissues showed specific antibody binding to DWV. Also, there were no specific structures that could be suggested as retention sites. DWV was found only in the midgut lumen ofV. destructorin structures resembling large, dense spheres, which were presumably faecal pellets.

Three known viruses infect honey-bees. Two can cause paralysis and early death of adult bees but only one, chronic bee-paralysis virus, is known to do so in nature. This virus, which has anisometric particles resembling prolate spheroids, is a common cause of sickness of adult bees, but it is very widely distributed as an inapparent infection, especially of the salivary glands of adults, and is probably transmitted when bees feed each other. Susceptibility to paralysis seems determined mainly by hereditary factors. The second virus, acute bee-paralysis virus, which has isometric particles, has been associated with disease only in the laboratory, but it also is very common as an inapparent infection of adult bees. The third virus, which has particles resembling those of acute bee-paralysis virus, causes a disease, sacbrood, of larvae. It can also multiply in adult bees, when it causes no overt symptoms, but accumulates in their hypopharyngeal glands, which secrete food for larvae.

Viruses affecting human and animal populations have been hitting the headlines recently and it has to be said that it has been mostly bad press. These minute particles have a fundamentally simple structure comprising a protein coat enclosing a nucleic acid core, yet this apparently uncomplicated organization conceals a great diversity of highly successful strategies for survival and spread.

SUMMARY Undisturbed honey bee colonies near Rothamsted were commonly infected with a filamentous DNA virus (FV) which was usually associated with Nosema apis in infected adult individuals and occurred most frequently about June. No symptoms were caused by FV, which appeared less harmful than either black queen-ceil virus (BQCV) or bee virus Y (BVY), two isometric RNA viruses also associated with N. apis. Bee virus X, another isometric RNA virus distantly related to BVY, was not associated with N. apis, but prevailed in winter and was then associated significantly with Malpighamoeba mellificae in dead individuals, although it frequently multiplied alone. Results of laboratory experiments supported conclusions made from the field observations about these relationships and their pathology.

Summary The amounts of chronic bee-paralysis virus associate (CPVA) produced in bees injected with purified chronic bee-paralysis virus (CPV) were negatively associated with the amounts, lengths of particles and maximum sedimentation coefficients of CPV. The ratio of the amounts of CPV/CPVA varied widely between individuals, but more CPVA was produced and CPV/CPVA ratios were smaller when CPVA was injected with the CPV preparations than when the latter were injected alone. The multiplication of the RNAs of CPV and of CPVA were also negatively correlated. The nature of the relationship between CPV and CPVA and the packaging arrangement of CPV RNA are discussed.

SummaryIn laboratory tests, paralysis virus spread best between individual adult bees when they were most crowded. Spread was probably by contact of their epidermal cytoplasm, because the virus was readily transmitted when topically applied to freshly broken cuticular hairs. A significant positive regression of paralysis outbreaks on diminishing numbers of colonies in England and Wales is interpreted as a result of relatively increased foraging activity by the remaining colonies which led to less contact between healthy and infected individuals within enzootically infected colonies.

Summary The RNA components of chronic bee-paralysis virus (CPV) and those of chronic bee-paralysis virus associate (CPVA) have been compared. CPV has five single-stranded RNA components, two larger RNAs designated 1 (mol. wt. 1.35 × 106; 4200 nucleotides) and 2 (mol. wt. 0.9 × 106; 2800 nucleotides), and three smaller RNAs designated 3a, 3b and 3c, each with mol. wt. 0.35 × 106 (1100 nucleotides). CPVA has three single-stranded RNA components designated A, B and C, each with mol. wt. 0.35 × 106 (1100 nucleotides). Gel electrophoretic and T1 fingerprint analyses have shown that RNAs 3a, 3b and 3c are very similar to, and probably identical to, RNAs A, B and C respectively, i.e. these three RNAs appear to be encapsidated in both CPV and CPVA particles. A positive correlation was observed between the proportion of RNAs 3a, 3b and 3c in CPV particles and the amount of CPVA (and hence RNAs A, B and C) which had replicated. Fingerprint analysis has shown that RNAs A, B and C are distinct but related and also that about 50% of the sequence of each these three RNAs is homologous with RNA 2. It is therefore possible that, although CPV and CPVA are serologically unrelated, the RNAs of CPVA have evolved from CPV RNA 2.

The lengths of individual symbiotes and the numbers of these per unit area in ultrathin sections of the mycetomes did not differ significantly between strains of Myzus persicae (Sulz.) that were resistant or susceptible to demeton-S-methyl in Britain. This is contrary to observations made in Germany on the symbiotes of similar aphids resistant or susceptible to parathion.

SUMMARY Adult individuals of honey bee colonies in Britain are commonly infected with several small RNA viruses. Black queen cell virus and bee virus Y are the commonest and occur most frequently about June, together with Nosema apis with which both viruses are intimately but independently associated. Bee virus X is less common than bee virus Y and showed no pronounced seasonal fluctuations in local surveys, but was detected mostly in winter and early spring. Sacbrood virus occurred in adults of most local colonies in summer, chronic paralysis virus and cloudy wing particle occurred commonly but without seasonal variations and acute paralysis occurred commonly throughout the active season of bees but does not multiply sufficiently to cause overt disease. Slow paralysis virus was rare.

Summary 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 multiplied 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.

Summary Chronic bee-paralysis virus associate (CPVA) and cloudy wing particle (CWP) are about 17 nm in diam. contain RNA, have single polypeptide species and have indistinguishable buoyant densities in CsCl of about 1.38 g/ml; but they are serologically unrelated, have proteins of different mol. wt. and have significantly different sedimentation coefficients of 41S and 49S respectively. CPVA is significantly and possibly specifically associated with chronic paralysis virus but multiplies, relative to the paralysis virus, by different amounts in different tissues. CWP multiplies apparently independently.

SUMMARY The prevalence of nine honey bee viruses in samples of dead adult bees from Apis mellifera colonies in the Netherlands and Germany infested with the parasitic mite Varroa jacobsoni was compared with virus incidence in uninfested colonies in Britain. In colonies with low mite populations the viruses present and their incidence during the year were similar to the results obtained from British colonies. However, in marked contrast with findings in Britain, acute paralysis virus (APV) was the primary cause of adult bee mortality in German honey bee colonies severely infested with V. jacobsoni. Dead brood from unsealed and sealed infested cells from German colonies with high mite populations also contained much APV. The evidence suggests that V. jacobsoni activates APV replication in adult bees by its feeding behaviour and transmits virus from adult honey bees to pupae. In addition, adult bees, in which APV is multiplying, transmit the virus to unsealed brood in the larval food.

AIMS To investigate the thermal biology of entomopathogenic fungi being examined as potential microbial control agents of Varroa destructor, an ectoparasite of the European honey bee Apis mellifera. METHODS AND RESULTS Colony extension rates were measured at three temperatures (20, 30 and 35 degrees C) for 41 isolates of entomopathogenic fungi. All of the isolates grew at 20 and 30 degrees C but only 11 isolates grew at 35 degrees C. Twenty-two isolates were then selected on the basis of appreciable growth at 30-35 degrees C (the temperature range found within honey bee colonies) and/or infectivity to V. destructor, and their colony extension rates were measured at 10 temperatures (12.5-35 degrees C). This data were then fitted to Schoolfield et al. [J Theor Biol (1981)88:719-731] re-formulation of the Sharpe and DeMichele [J Theor Biol (1977)64:649-670] model of poikilotherm development. Overall, this model accounted for 87.6-93.9% of the data variance. Eleven isolates exhibited growth above 35 degrees C. The optimum temperatures for extension rate ranged from 22.9 to 31.2 degrees C. Only three isolates exhibited temperature optima above 30 degrees C. The super-optimum temperatures (temperature above the optimum at which the colony extension rate was 10% of the maximum rate) ranged from 31.9 to 43.2 degrees C. CONCLUSIONS The thermal requirements of the isolates examined against V. destructor are well matched to the temperatures in the broodless areas of honey bee colonies, and a proportion of isolates, should also be able to function within drone brood areas. SIGNIFICANCE AND IMPACT OF THE STUDY Potential exists for the control of V. destructor with entomopathogenic fungi in honey bee colonies. The methods employed in this study could be utilized in the selection of isolates for microbial control prior to screening for infectivity and could help in predicting the activity of a fungal control agent of V. destructor under fluctuating temperature conditions.

A laboratory bioassay was developed to measure the pathogenicities of isolates of mitosporic fungi to Varroa destructor, an ectoparasite of the European honeybee, Apis mellifera. Forty isolates of entomopathogenic fungi were assessed against V. destructor in a single-dose experiment (conidial concentration 1×108 ml−1) at 25 °C and 100% RH. 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 isolates could infect and kill V. destructor and 26 caused mean times to death of less than 100 h. Control (Tween-treated) mortality was 5% at 7 days post-treatment. Nineteen isolates were also examined for side effects against bees. Caged bees sprayed with conidial suspensions (1×108 ml−1) of seven of these isolates died within 14 days. However, not all mortality could be attributed to fungal infection as confirmed by sporulation; the mortality of control bees was 27%. Nine isolates were selected for further examination against V. destructor at 30 °C and 40% RH to simulate the conditions in bee colonies. Of these, three isolates of M. anisopliae, one of V. lecanii, and one of B. bassiana killed 100% of V. destructor within 7 days at a conidial concentration of 1×108 ml−1. One isolate of M. anisopliae also killed 97% of V. destructor within 7 days at a conidial concentration of 1×106 ml−1. These results indicate that entomopathogenic fungi have potential as microbial control agents of V. destructor in honeybee colonies.