Elke Genersch

The German bee monitoring project: a long term study to understand periodically high winter losses of honey bee colonies

The Western honey bee, Apis mellifera, is the most important animal pollinator in agriculture worldwide providing more than 90% of the commercial pollination services. Due to the development in agriculture the demands for honey bee pollination are steadily increasing stressing the pollination capacity of the global managed honey bee population. Hence, the long-term decline of managed honey bee hives in Europe and North-America is of great concern and stimulated intensive research into the possible factors presumably causing honey bee colony collapse. We here present a four-year study involving more than 1200 bee colonies from about 120 apiaries which were monitored for the entire study period. Bee samples were collected twice a year to analyze various pathogenic factors including the ectoparasitic mite Varroa destructor, fungi (Nosema spec., Ascosphaera apis), the bacterium Paenibacillus larvae, and several viruses. Data on environmental factors, beekeeping management practice, and pesticides were also collected. All data were statistically analyzed in respect to the overwintering mortality of the colonies. We can demonstrate for several factors that they are significantly related to the observed winter losses of the monitored honey bee colonies: (i) high varroa infestation level, (ii) infection with deformed wing virus (DWV) and acute bee paralysis virus (ABPV) in autumn, (iii) queen age, and (iv) weakness of the colonies in autumn. No effects could be observed for Nosema spec. or pesticides. The implications of these findings will be discussed.ZusammenfassungDie Honigbiene Apis mellifera ist weltweit der wichtigste Bestäuber in der Landwirtschaft und nach aktuellen Schätzungen wird der globale Bedarf an kommerzieller Bestäubung weiter steigen. Dadurch stellt der seit Jahren zu beobachtende stetige Rückgang der Bienenvölker in Nord-Amerika und Europa ein ernsthaftes Problem für die Landwirtschaft dar. Für die Abnahme der Bienenvölker werden neben wirtschaftlichen Faktoren vor allem periodisch auftretende Völkerverluste verantwortlich gemacht, für die aber eine eindeutige Ursachenanalyse bisher fehlt.Zur Ursachenaufklärung von Winterverlusten führten wir von 2004 bis 2009 ein Monitoringprojekt durch, in dem mehr als 1200 Bienenvölker auf 125 über ganz Deutschland verteilten Bienenständen (Abb. 1) kontinuierlich beprobt und kontrolliert wurden. Die beteiligten „Monitoringimker“ stellten hierfür 10 ihrer Völker zur Verfügung und lieferten Daten zu Honigerträgen, Wanderungen und Ablegerbildung. Mitarbeiter der Bieneninstitute nahmen zweimal im Jahr Bienenproben für Krankheitsuntersuchungen (Nosema spec, Varroa destructor, 4 verschiedene Bienenviren) sowie Bienenbrotproben für Rückstandsuntersuchungen. Die Stärke der Bienenvölker wurde bei der Ein- und Auswinterung bestimmt; als „Überwinterungsverlust“ wurden Völker definiert, die tot waren bzw. nicht genug Bienen für eine erfolgreiche Frühjahrsentwicklung aufwiesen.Die Winterverluste schwankten zwischen 3,5 % und 15,2 % (Abb. 3) mit ungleicher Verteilung innerhalb der beteiligten Imker (Abb. 4). Für die Ursachenanalyse wurden die überlebenden mit den zusammengebrochenen Völkern verglichen. Dabei zeigten sich die größten und hochsignifikanten (P < 0,000001, U-Test) Unterschiede beim Varroabefall der Bienen im Oktober (Tab. III, Abb. 5). Ebenfalls hochsignifikante Unterschiede ergaben sich für die Bienenviren DWV (P < 0,00001) und APBV (P < 0,0039), nicht jedoch für KBV, SBV und den Nosemabefall (Tab. V). Erstaunlicherweise waren Völker mit jungen Königinnen signifikant seltener von Winterverlusten betroffen als mit älteren Königinnen (Tab. VI), während z. B. Beutenmaterial oder Rähmchenmaß keine Rolle spielten.Bei den insgesamt in drei Jahren auf Pestizidrückstände untersuchten 215 Bienenbrotproben wurden insgesamt über 50 Wirkstoffe (von 256) nachgewiesen, die meisten im Spurenbereich. Häufig wurden mehrere Wirkstoffe gefunden und nur etwas mehr als 20 % der Proben waren frei von messbaren Rückständen (Tab. VII). Neonikotinoide wurden nur in einer einzigen Probe nachgewiesen. Es konnte keine Korrelation von Rückstandswerten mit Winterverlusten festgestellt werden. Es gab auch keinen Zusammenhang zwischen der Überwinterung von Bienenvölkern und dem Umfang des zuvor eingetragenen Rapshonigs (Abb. 6).Unser Projekt zeigt, dass der Varroabefall im Herbst (zusammen mit den assoziierten Sekundärinfektionen) eine Hauptursache für Überwinterungsverluste darstellt. Eine konsequente Varroabehandlung und starke Bienenvölker mit jungen Königinnen sind daher die wichtigste Empfehlung, um Winterverlusten vorzubeugen. Ein zusätzlicher Einfluss der übrigen Faktoren kann nicht ausgeschlossen werden, hierfür sind aber modifizierte Versuchsansätze notwendig.

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.

American Foulbrood in honeybees and its causative agent, Paenibacillus larvae

After more than a century of American Foulbrood (AFB) research, this fatal brood infection is still among the most deleterious bee diseases. Its etiological agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Huge progress has been made, especially in the last 20 years, in the understanding of the disease and of the underlying host-pathogen interactions. This review will place these recent developments in the study of American Foulbrood and of P. larvae into the general context of AFB research.

Honey bee pathology: current threats to honey bees and beekeeping

Managed honey bees are the most important commercial pollinators of those crops which depend on animal pollination for reproduction and which account for 35% of the global food production. Hence, they are vital for an economic, sustainable agriculture and for food security. In addition, honey bees also pollinate a variety of wild flowers and, therefore, contribute to the biodiversity of many ecosystems. Honey and other hive products are, at least economically and ecologically rather, by-products of beekeeping. Due to this outstanding role of honey bees, severe and inexplicable honey bee colony losses, which have been reported recently to be steadily increasing, have attracted much attention and stimulated many research activities. Although the phenomenon “decline of honey bees” is far from being finally solved, consensus exists that pests and pathogens are the single most important cause of otherwise inexplicable colony losses. This review will focus on selected bee pathogens and parasites which have been demonstrated to be involved in colony losses in different regions of the world and which, therefore, are considered current threats to honey bees and beekeeping.

Five-year cohort study of Nosema spp. in Germany: does climate shape virulence and assertiveness of Nosema ceranae?

ABSTRACT Nosema ceranae and Nosema apis are two fungal pathogens belonging to the phylum Microsporidia and infecting the European honeybee, Apis mellifera. Recent studies have suggested that N. ceranae is more virulent than N. apis both at the individual insect level and at the colony level. Severe colony losses could be attributed to N. ceranae infections, and an unusual form of nosemosis is caused by this pathogen. In the present study, data from a 5-year cohort study of the prevalence of Nosema spp. in Germany, involving about 220 honeybee colonies and a total of 1,997 samples collected from these colonies each spring and autumn and analyzed via species-specific PCR-restriction fragment length polymorphism (RFLP), are described. Statistical analysis of the data revealed no relation between colony mortality and detectable levels of infection with N. ceranae or N. apis. In addition, N. apis is still more prevalent than N. ceranae in the cohort of the German bee population that was analyzed. A possible explanation for these findings could be the marked decrease in spore germination that was observed after even a short exposure to low temperatures (+4°C) for N. ceranae only. Reduced or inhibited N. ceranae spore germination at low temperatures should hamper the infectivity and spread of this pathogen in climatic regions characterized by a rather cold winter season.

Standard methods for Nosema research

Summary Methods are described for working with Nosema apis and Nosema ceranae in the field and in the laboratory. For fieldwork, different sampling methods are described to determine colony level infections at a given point in time, but also for following the temporal infection dynamics. Suggestions are made for how to standardise field trials for evaluating treatments and disease impact. The laboratory methods described include different means for determining colony level and individual bee infection levels and methods for species determination, including light microscopy, electron microscopy, and molecular methods (PCR). Suggestions are made for how to standardise cage trials, and different inoculation methods for infecting bees are described, including control methods for spore viability. A cell culture system for in vitro rearing of Nosema spp. is described. Finally, how to conduct different types of experiments are described, including infectious dose, dose effects, course of infection and longevity tests.

Deformed wing virus: replication and viral load in mites (Varroa destructor).

Deformed wing virus (DWV) normally causes covert infections but can have devastating effects on bees by inducing morphological deformity or even death when transmitted by the ectoparasitic mite Varroa destructor. In order to determine the role of V. destructor in the development of crippled wings, we analysed individual mites for the presence and replication of DWV. The results supported the correlation between viral replication in mites and morphologically deformed bees. Quantification of viral genome equivalents revealed that mites capable of inducing an overt DWV infection contained 10(10)-10(12) genome equivalents per mite. In contrast, mites which could not induce crippled wings contained a maximum of only 10(8) viral genome equivalents per mite. We conclude that the development of crippled wings not only depends on DWV transmission by V. destructor but also on viral replication in V. destructor and on the DWV titre in the parasitizing mites.

Strain- and Genotype-Specific Differences in Virulence of Paenibacillus larvae subsp. larvae, a Bacterial Pathogen Causing American Foulbrood Disease in Honeybees

ABSTRACT Virulence variations of Paenibacillus larvae subsp. larvae, the causative agent of American foulbrood disease of honeybees, were investigated by analysis of 16 field isolates of this pathogen, belonging to three previously characterized genotypes, as well as the type strain (ATCC 9545) of P. larvae subsp. larvae, with exposure bioassays. We demonstrated that the strain-specific 50% lethal concentrations varied within an order of magnitude and that differences in amount of time for the pathogen to kill 100% of the infected hosts (LT100) correlated with genotype. One genotype killed rather quickly, with a mean LT100 of 7.8 ± 1.7 days postinfection, while the other genotypes acted more slowly, with mean LT100s of 11.2 ± 0.8 and 11.6 ± 0.6 days postinfection.

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.

Fluorescence in situ hybridization (FISH) analysis of the interactions between honeybee larvae and Paenibacillus larvae, the causative agent of American foulbrood of honeybees (Apis mellifera)

American foulbrood (AFB) is a bacterial disease of honeybee larvae caused by the spore-forming bacterium Paenibacillus larvae. Although AFB and its aetiological agent are described now for more than a century, the general and molecular pathogenesis of this notifiable disease is poorly understood. We used fluorescence in situ hybridization (FISH) performed with P. larvae-specific, 16S rRNA-targeted oligonucleotide probes to analyse the early steps in the pathogenesis of American foulbrood. The following chain of events could be demonstrated: (i) the spores germinate in the midgut lumen, (ii) the vegetative bacteria massively proliferate within the midgut before, and (iii) they start to locally breach the epithelium and invade the haemocoel. The paracellular route was shown to be the main mechanism for invasion contrasting earlier hypotheses of phagocytosis of P. larvae. Invasion coincided with the death of the host implicating that the penetration of the midgut epithelium is a critical step determining the time of death.