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.

Recent reports on a steady decline of honeybee colonies in several parts of the world caused great concern. There is a consensus that pathogens are among the key players in this alarming demise of the most important commercial pollinator. One of the pathogens heavily implicated in colony losses is deformed wing virus (DWV). Overt DWV infections manifested as deformed-wing syndrome started to become a threat to honeybees only in the wake of the ectoparasitic mite Varroa destructor, which horizontally transmits DWV. However, a direct causal link between the virus and the symptom 'wing deformity' has not been established yet. To evaluate the impact of different horizontal transmission routes, and especially the role of the mite in the development of overt DWV infections, we performed laboratory infection assays with pupae and adult bees. We could demonstrate that pupae injected with DWV dose-dependently developed overt infections characterized by deformed wings in adult bees, suggesting that DWV, if transmitted to pupae by the mite, is the causative agent of the deformed-wing syndrome. The OID 50 (overt infection dosage) was approximately 2500 genome equivalents. Injecting more than 1¾10 7 DWV genome equivalents into adult bees also resulted in overt infections while the same viral dosage fed to adult bees only resulted in covert infections. Therefore, both infection of adult bees through DWVtransmitting phoretic mites and infection of nurse bees through their cannibalizing DWV-infected pupae might represent possible horizontal transmission routes of DWV. INTRODUCTIONManaged honeybees are indispensable for profitable and sustainable agriculture and for many non-agricultural ecosystems because of the extraordinary pollination service they provide. Hence, reports on a steady decline in honeybee populations in Europe, USA and Japan in the recent past (for a review see vanEngelsdorp & Meixner, 2010) caused great concern and several studies were initiated to identify the underlying cause. From these studies it became more and more evident that the key players in most of the 'inexplicable' colony losses are pathogens (Ratnieks & Carreck, 2010). Especially, the ectoparasitic mite Varroa destructor in concert with certain pathogenic bee viruses played a major role in the observed colony collapses (Cox-Foster et al., 2007;Genersch et al., 2010; Guzmán-Novoa et al., 2010;Highfield et al., 2009;Siede et al., 2008;vanEngelsdorp, 2008;vanEngelsdorp et al., 2009).One of the viruses heavily implicated in colony losses is Deformed wing virus (DWV), a plus-stranded RNA virus belonging to the genus Iflaviridae (Lanzi et al., 2006). DWV normally causes covert infections in honeybees (de Miranda & Fries, 2008;Hails et al., 2008;Yue et al., 2007). Overt DWV infections characterized by the occurrence of visible disease symptoms (deformed wings, bloated and shortened abdomen, and miscolouring) are associated with V. destructor infestation (Ball & Allen, 1988;Bowen-Walker et al., 1999;Martin, 2001;Martin et al., 1998; Santillán-Galicia et...

Summary The health of the Western honey bee is threatened by a global epidemic of deformed wing virus (DWV) infections driven by the ectoparasitic mite Varroa destructor acting as mechanical and biological virus vector. Three different variants of DWV, DWV‐A, ‐B and ‐C exist. Virulence differences between these variants and their relation to V. destructor are still controversially discussed. We performed laboratory experiments to analyze the virulence of DWV directly isolated from crippled bees (DWVP0) or after one additional passage in bee pupae (DWVP1). We demonstrated that DWVP0 was more virulent than DWVP1 for pupae, when pupal mortality was taken as virulence marker, and for adult bees, when neurotropism and cognitive impairment were taken as virulence markers. Phylogenetic analysis supported that DWV exists as quasispecies and showed that DWVP0 clustered with DWV‐B and DWVP1 with DWV‐A when the phylogeny was based on the master sequences of the RNA‐dependent RNA polymerase but not so when it was based on the VP3 region master sequences. We propose that switching of DWV between the bee and the mite host is accompanied by changes in viral sequence, tissue tropism and virulence and that the RNA‐dependent RNA polymerase is involved in determining host range and virulence.

The population of managed honey bees has been dramatically declining in the recent past in many regions of the world. Consensus now seems to be that pathogens and parasites (e.g. the ectoparasitic mite Varroa destructor, the microsporidium Nosema ceranae and viruses) play a major role in this demise. However, little is known about host-pathogen interactions for bee pathogens and attempts to develop novel strategies to combat bee diseases have been hampered by this gap in our knowledge. One reason for this dire situation is the complete lack of cell cultures for the propagation and study of bee pathogens. Here we present a cell culture model for two honey bee-pathogenic microsporidian species, Nosema apis and N. ceranae. Our cell culture system is based on a lepidopteran cell line, which proved to be susceptible to infection by both N. ceranae and N. apis and enabled us to illustrate the entire life cycle of these microsporidia. We observed hitherto undescribed spindle-shaped meronts and confirmed our findings in infected bees. Our cell culture model provides a previously unavailable means to explore the nature of interactions between the honey bee and its pathogen complex at a mechanistic level and will allow the development of novel treatment strategies.

SummaryCell culture techniques are indispensable in most, if not all life science disciplines to date. Wherever appropriate cell culture models are lacking, scientific development is hampered. Unfortunately this has been and still is the case in honey bee research, because permanent honey bee cell lines have not so far been established. To overcome this hurdle, protocols for the cultivation of primary honey bee cells and of non-permanent honey bee cell lines have been developed. In addition, heterologous cell culture models for honey bee pathogens based on non-Apis insect cell lines have recently been developed. To further advance this progress and to encourage bee scientists to enter the field of cell biology based research, here we present protocols for the cultivation of honey bee primary cells and non-permanent cell lines, as well as hints for the cultivation of permanent insect cell lines suitable for honey bee research. Métodos estándar para cultivos celulares en Apis mellifera ResumenLas técnicas de cultivo celular son indispensables en la mayoría, si no en todas las disciplinas de ciencias de la vida hasta la fecha. Siempre que se carezca de modelos de cultivo celular apropiados, el desarrollo científico se ve obstaculizado. Desafortunadamente, esto ha sido y todavía es el caso de la investigación en la abeja de la miel, ya que hasta ahora, no se han establecido líneas celulares permanentes de abeja de la miel. Para superar este obstáculo, se han desarrollado protocolos para el cultivo de células primarias de la abeja de la miel y líneas celulares no permanentes de abejas. Además, también se han desarrollado recientemente modelos heterólogos de cultivo celular para patógenos de las abejas melíferas basados en líneas celulares de insectos que no pertenecen al género Apis. Para avanzar en este progreso y alentar a los científicos apícolas a entrar en el campo de la investigación basada en la biología celular, presentamos aquí los protocolos para el cultivo de células primarias de abejas y líneas celulares no permanentes, así como consejos para el cultivo de líneas celulares de insectos permanentes adecuadas para la investigación en la abeja de la miel.