Joachim R. de Miranda

Deformed wing virus.

Deformed wing virus (DWV; Iflaviridae) is one of many viruses infecting honeybees and one of the most heavily investigated due to its close association with honeybee colony collapse induced by Varroadestructor. In the absence of V.destructor DWV infection does not result in visible symptoms or any apparent negative impact on host fitness. However, for reasons that are still not fully understood, the transmission of DWV by V.destructor to the developing pupae causes clinical symptoms, including pupal death and adult bees emerging with deformed wings, a bloated, shortened abdomen and discolouration. These bees are not viable and die soon after emergence. In this review we will summarize the historical and recent data on DWV and its relatives, covering the genetics, pathobiology, and transmission of this important viral honeybee pathogen, and discuss these within the wider theoretical concepts relating to the genetic variability and population structure of RNA viruses, the evolution of virulence and the development of disease symptoms.

Molecular and Biological Characterization of Deformed Wing Virus of Honeybees (Apis mellifera L.)

ABSTRACT Deformed wing virus (DWV) of honeybees (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. The virus was purified from diseased insects, and its genome was cloned and sequenced. The genomic RNA of DWV is 10,140 nucleotides in length and contains a single large open reading frame encoding a 328-kDa polyprotein. The coding sequence is flanked by a 1,144-nucleotide 5′ nontranslated leader sequence and a 317-nucleotide 3′ nontranslated region, followed by a poly(A) tail. The three major structural proteins, VP1 (44 kDa), VP2 (32 kDa), and VP3 (28 kDa), were identified, and their genes were mapped to the N-terminal section of the polyprotein. The C-terminal part of the polyprotein contains sequence motifs typical of well-characterized picornavirus nonstructural proteins: an RNA helicase, a chymotrypsin-like 3C protease, and an RNA-dependent RNA polymerase. The genome organization, capsid morphology, and sequence comparison data indicate that DWV is a member of the recently established genus Iflavirus.

The Acute bee paralysis virus–Kashmir bee virus–Israeli acute paralysis virus complex

Acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV) are part of a complex of closely related viruses from the Family Dicistroviridae. These viruses have a widespread prevalence in honey bee (Apis mellifera) colonies and a predominantly sub-clinical etiology that contrasts sharply with the extremely virulent pathology encountered at elevated titres, either artificially induced or encountered naturally. These viruses are frequently implicated in honey bee colony losses, especially when the colonies are infested with the parasitic mite Varroa destructor. Here we review the historical and recent literature of this virus complex, covering history and origins; the geographic, host and tissue distribution; pathology and transmission; genetics and variation; diagnostics, and discuss these within the context of the molecular and biological similarities and differences between the viruses. We also briefly discuss three recent developments relating specifically to IAPV, concerning its association with Colony Collapse Disorder, treatment of IAPV infection with siRNA and possible honey bee resistance to IAPV.

Bees under stress: sublethal doses of a neonicotinoid pesticide and pathogens interact to elevate honey bee mortality across the life cycle

Microbial pathogens are thought to have a profound impact on insect populations. Honey bees are suffering from elevated colony losses in the northern hemisphere possibly because of a variety of emergent microbial pathogens, with which pesticides may interact to exacerbate their impacts. To reveal such potential interactions, we administered at sublethal and field realistic doses one neonicotinoid pesticide (thiacloprid) and two common microbial pathogens, the invasive microsporidian Nosema ceranae and black queen cell virus (BQCV), individually to larval and adult honey bees in the laboratory. Through fully crossed experiments in which treatments were administered singly or in combination, we found an additive interaction between BQCV and thiacloprid on host larval survival likely because the pesticide significantly elevated viral loads. In adult bees, two synergistic interactions increased individual mortality: between N. ceranae and BQCV, and between N. ceranae and thiacloprid. The combination of two pathogens had a more profound effect on elevating adult mortality than N. ceranae plus thiacloprid. Common microbial pathogens appear to be major threats to honey bees, while sublethal doses of pesticide may enhance their deleterious effects on honey bee larvae and adults. It remains an open question as to whether these interactions can affect colony survival.

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.

Varroa destructor : research avenues towards sustainable control

Summary Pollination by honey bees plays a key role in the functioning of ecosystems and optimisation of agricultural yields. Severe honey bee colony losses worldwide have raised concerns about the sustainability of these pollination services. In many cases, bee mortality appears to be the product of many interacting factors, but there is a growing consensus that the ectoparasitic mite Varroa destructor plays the role of the major predisposing liability. We argue that the fight against this mite should be a priority for future honey bee health research. We highlight the lack of efficient control methods currently available against the parasite and discuss the need for new approaches. Gaps in our knowledge of the biology and epidemiology of the mite are identified and a research road map towards sustainable control is drawn. Innovative and challenging approaches are suggested in order to stimulate research efforts and ensure that honey bees will be able to sustainably fulfil their role in the ecosystem.

Acaricide Treatment Affects Viral Dynamics in Varroa destructor-Infested Honey Bee Colonies via both Host Physiology and Mite Control

ABSTRACT Honey bee (Apis mellifera) colonies are declining, and a number of stressors have been identified that affect, alone or in combination, the health of honey bees. The ectoparasitic mite Varroa destructor, honey bee viruses that are often closely associated with the mite, and pesticides used to control the mite population form a complex system of stressors that may affect honey bee health in different ways. During an acaricide treatment using Apistan (plastic strips coated with tau-fluvalinate), we analyzed the infection dynamics of deformed wing virus (DWV), sacbrood virus (SBV), and black queen cell virus (BQCV) in adult bees, mite-infested pupae, their associated Varroa mites, and uninfested pupae, comparing these to similar samples from untreated control colonies. Titers of DWV increased initially with the onset of the acaricide application and then slightly decreased progressively coinciding with the removal of the Varroa mite infestation. This initial increase in DWV titers suggests a physiological effect of tau-fluvalinate on the hosts susceptibility to viral infection. DWV titers in adult bees and uninfested pupae remained higher in treated colonies than in untreated colonies. The titers of SBV and BQCV did not show any direct relationship with mite infestation and showed a variety of possible effects of the acaricide treatment. The results indicate that other factors besides Varroa mite infestation may be important to the development and maintenance of damaging DWV titers in colonies. Possible biochemical explanations for the observed synergistic effects between tau-fluvalinate and virus infections are discussed.

Statistical guidelines for Apis mellifera research

Summary In this article we provide guidelines on statistical design and analysis of data for all kinds of honey bee research. Guidelines and selection of different methods presented are, at least partly, based on experience. This article can be used: to identify the most suitable analysis for the type of data collected; to optimise ones experimental design based on the experimental factors to be investigated, samples to be analysed, and the type of data produced; to determine how, where, and when to sample bees from colonies; or just to inspire. Also included are guidelines on presentation and reporting of data, as well as where to find help and which types of software could be useful.

Research strategies to improve honeybee health in Europe

Understanding the fundaments of colony losses and improving the status of colony health will require cross-cutting research initiatives including honeybee pathology, chemistry, genetics and apicultural extension. The 7th framework of the European Union requested research to empirically and experimentally fill knowledge gaps on honeybee pests and diseases, including ’Colony Collapse Disorder’ and the impact of parasites, pathogens and pesticides on honeybee mortality. The interactions among these drivers of colony loss will be studied in different European regions, using experimental model systems including selected parasites (e.g. Nosema and Varroa mites), viruses (Deformed Wing Virus, Black Queen Cell Virus, Israeli Acute Paralysis Virus) and model pesticides (thiacloprid, τ-fluvalinate). Transcriptome analyses will be used to explore host-pathogen-pesticide interactions and identify novel genes for disease resistance. Special attention will be given to sublethal and chronic exposure to pesticides and will screen how apicultural practices affect colony health. Novel diagnostic screening methods and sustainable concepts for disease prevention will be developed resulting in new treatments and selection tools for resistant stock. Research initiatives will be linked to various national and international ongoing European, North- and South-American colony health monitoring and research programs, to ensure a global transfer of results to apicultural practice in the world community of beekeepers.ZusammenfassungDie letzten Jahrzehnte waren durch einen konstanten Rückgang von Bienenvölkern in den Mitgliedstaaten der EU gekennzeichnet (cf. Abb. 1). Insbesondere dramatische und unkontrollierbare Völkerverluste entwickelten sich zu einer akuten Insolvenz — Bedrohung für Imkereibetriebe. Nach wie vor sind die Ursachen dieser großen, flächendeckenden Völkerverluste auf nationaler Ebene unklar und daher sind zielgerichtete kausale Therapien nicht möglich. Oft wurden daher unnötige, prophylaktisch medikamentöse Behandlungen durchgeführt, um regionale Völkerbestände zu sichern. Dies hat bislang jedoch noch nicht zu einer nachhaltigen Bekämpfung von Bienenkrankheiten geführt, allerdings regelmäßig zur Kontamination des Honigs.Die Forschungspolitik der EU zielt daher darauf ab, die Honigbelastung zu reduzieren, die Rassevielfalt europäischer Honigbienen zu erhalten, Völkerverluste zu vermeiden und die Bedeutung der Interaktionen zwischen Parasiten, Pathogenen und Pestiziden für die Koloniegesundheit zu verstehen. Gerade die Kombination verschiedener Faktoren wird als ein besonderes Problem gesehen. Eine einzelnes Pathogen mag für die Kolonie harmlos sein, aber in Kombination mit anderen zum Zusammenbruch des Volkes führen.Das Forschungsnetzwerk BEE DOC (Bees in EuropE and the Decline Of Honeybee Colonies) wird sich deshalb mit den Interaktionen zwischen Parasiten, Pathogenen und Pestiziden beschäftigen. In Anbetracht der großen Zahl von Pathogenen und Pestiziden ist es allerdings nicht realisierbar, alle möglichen Interaktionen experimentell zu testen. Es ist daher notwendig, sich in Experimenten auf wenige ausgewählte Modellsysteme von besonderer Bedeutung zu beschränken. Im BEE DOC Netzwerk sind dies V. destructor, Nosema spp., häufige assoziierte Viren, und die häufig genutzten Pestizide Thiacloprid und τ-Fluvalinat. Die Forschungsaktivitäten müssen auch die Untersuchung der genetischen und genomischen Kontrolle von Krankheitsresistenz beinhalten. Oligonukleotid DNA-Chips die das gesamte Genom der Honigbiene abdecken sind dabei von besonderem Nutzen. Zusätzlich sollen neue Resistenzgene mit Hilfe von haploiden Drohnen gefunden werden. Antibiotische Substanzen, die entweder von den Bienen selbst erzeugt oder von Pflanzen gesammelt werden sollen auf ihre Wirksamkeit bei der Bekämpfung von Bienenkrankheiten untersucht werden. Gerade sekundäre Metabolite von Pflanzensubstanzen, die von der Honigbeine enzymatisch verändert wurden um eine höhere Wirksamkeit zu erhalten, sind von besonderem Interesse. Neue diagnostische Verfahren, die in der Forschung, im Routine — screening und auf dem Bienenstand eingesetzt werden können müssen entwickelt werden, um rechtzeitig Erkrankungen bei den Bienenvölkern diagnostizieren zu können bevor diese zusammenbrechen.Der Erfolg dieser Forschungsarbeiten wird stark von Koordinierung des Monitoring und der Forschung sowie von der Implementierung der Ergebnisse in die imkerliche Praxis abhängig sein. Das COLOSS Netzwerk (Cost Action) ist hierfür in den nächsten Jahren ein hervorragendes Werkzeug. In ihm sind über 150 Mitglieder aus 39 Ländern vertreten, die die nationalen Forschungsprojekte zur Bienengesundheit koordinieren und aufeinander abstimmen. Nur wenn es gelingt, die Forschungsergebnisse in der Imkerei umzusetzen, werden wir Fortschritte bei der nachhaltigen Prävention von Völkerverlusten auf einer europa- und weltweiten Ebene erzielen können.

On the Front Line: Quantitative Virus Dynamics in Honeybee (Apis mellifera L.) Colonies along a New Expansion Front of the Parasite Varroa destructor

Over the past fifty years, annual honeybee (Apis mellifera) colony losses have been steadily increasing worldwide. These losses have occurred in parallel with the global spread of the honeybee parasite Varroa destructor. Indeed, Varroa mite infestations are considered to be a key explanatory factor for the widespread increase in annual honeybee colony mortality. The host-parasite relationship between honeybees and Varroa is complicated by the mites close association with a range of honeybee viral pathogens. The 10-year history of the expanding front of Varroa infestation in New Zealand offered a rare opportunity to assess the dynamic quantitative and qualitative changes in honeybee viral landscapes in response to the arrival, spread and level of Varroa infestation. We studied the impact of de novo infestation of bee colonies by Varroa on the prevalence and titres of seven well-characterised honeybee viruses in both bees and mites, using a large-scale molecular ecology approach. We also examined the effect of the number of years since Varroa arrival on honeybee and mite viral titres. The dynamic shifts in the viral titres of black queen cell virus and Kashmir bee virus mirrored the patterns of change in Varroa infestation rates along the Varroa expansion front. The deformed wing virus (DWV) titres in bees continued to increase with Varroa infestation history, despite dropping infestation rates, which could be linked to increasing DWV titres in the mites. This suggests that the DWV titres in mites, perhaps boosted by virus replication, may be a major factor in maintaining the DWV epidemic after initial establishment. Both positive and negative associations were identified for several pairs of viruses, in response to the arrival of Varroa. These findings provide important new insights into the role of the parasitic mite Varroa destructor in influencing the viral landscape that affects honeybee colonies.