Dead or alive: deformed wing virus and Varroa destructor reduce the life span of winter honeybees

Elevated winter losses of managed honeybee colonies are a major concern, but the underlying mechanisms remain controversial. Among the suspects are the parasitic mite Varroa destructor, the microsporidian Nosema ceranae, and associated viruses. Here we hypothesize that pathogens reduce the life expectancy of winter bees, thereby constituting a proximate mechanism for colony losses. A monitoring of colonies was performed over 6 months in Switzerland from summer 2007 to winter 2007/2008. Individual dead workers were collected daily and quantitatively analyzed for deformed wing virus (DWV), acute bee paralysis virus (ABPV), N. ceranae, and expression levels of the vitellogenin gene as a biomarker for honeybee longevity. Workers from colonies that failed to survive winter had a reduced life span beginning in late fall, were more likely to be infected with DWV, and had higher DWV loads. Colony levels of infection with the parasitic mite Varroa destructor and individual infections with DWV were also associated with reduced honeybee life expectancy. In sharp contrast, the level of N. ceranae infection was not correlated with longevity. In addition, vitellogenin gene expression was significantly positively correlated with ABPV and N. ceranae loads. The findings strongly suggest that V. destructor and DWV (but neither N. ceranae nor ABPV) reduce the life span of winter bees, thereby constituting a parsimonious possible mechanism for honeybee colony losses.     

Linking evolutionary lineage with parasite and pathogen prevalence in the Iberian honey bee

The recent decline in honey bee colonies observed in both European countries and worldwide is of great interest and concern, although the underlying causes remain poorly understood. In recent years, growing evidence has implicated parasites and pathogens in this decline of both the vitality and number of honey bee colonies. The Iberian Peninsula provides an interesting environment in which to study the occurrence of pathogens and parasites in the host honey bee populations due to the presence of two evolutionary lineages in A. m. iberiensis (Western European [M] or African [A]). Here, we provide the first evidence linking the population structure of the Iberian honey bee with the prevalence of some of its most important parasites and pathogens: the Varroa destructor mite and the microsporidia Nosema apis and Nosema ceranae. Using data collected in two surveys conducted in 2006 and 2010 in 41 Spanish provinces, the evolutionary lineage and the presence of the three parasitic organisms cited above were analyzed in a total of 228 colonies. In 2006 N. apis was found in a significantly higher proportion of M lineage honey bees than in the A lineage. However, in 2010 this situation had changed significantly due to a higher prevalence of N. ceranae. We observed no significant relationships in either year between the distributions of V. destructor or N. ceranae and the evolutionary lineage present in A. m. iberiensis colonies, but the effects of these organisms on the genetic diversity of the honey bee populations need further research.     

Comprehensive Bee Pathogen Screening in Belgium Reveals Crithidia mellificae as a New Contributory Factor to Winter Mortality

Since the last decade, unusually high honey bee colony losses have been reported mainly in North-America and Europe. Here, we report on a comprehensive bee pathogen screening in Belgium covering 363 bee colonies that were screened for 18 known disease-causing pathogens and correlate their incidence in summer with subsequent winter mortality. Our analyses demonstrate that, in addition to Varroa destructor, the presence of the trypanosomatid parasite Crithidia mellificae and the microsporidian parasite Nosema ceranae in summer are also predictive markers of winter mortality, with a negative synergy being observed between the two in terms of their effects on colony mortality. Furthermore, we document the first occurrence of a parasitizing phorid fly in Europe, identify a new fourth strain of Lake Sinai Virus (LSV), and confirm the presence of other little reported pathogens such as Apicystis bombi, Aphid Lethal Paralysis Virus (ALPV), Spiroplasma apis, Spiroplasma melliferum and Varroa destructor Macula-like Virus (VdMLV). Finally, we provide evidence that ALPV and VdMLV replicate in honey bees and show that viruses of the LSV complex and Black Queen Cell Virus tend to non-randomly co-occur together. We also noticed a significant correlation between the number of pathogen species and colony losses. Overall, our results contribute significantly to our understanding of honey bee diseases and the likely causes of their current decline in Europe.     

The effect of induced queen replacement on Nosema spp. infection in honey bee (Apis mellifera iberiensis) colonies

Microsporidiosis of adult honeybees caused by Nosema apis and Nosema ceranae is a common worldwide disease with negative impacts on colony strength and productivity. Few options are available to control the disease at present. The role of the queen in bee population renewal and the replacement of bee losses due to Nosema infection is vital to maintain colony homeostasis. Younger queens have a greater egg laying potential and they produce a greater proportion of uninfected newly eclosed bees to compensate for adult bee losses; hence, a field study was performed to determine the effect of induced queen replacement on Nosema infection in honey bee colonies, focusing on colony strength and honey production. In addition, the impact of long-term Nosema infection of a colony on the ovaries and ventriculus of the queen was evaluated. Queen replacement resulted in a remarkable decrease in the rates of Nosema infection, comparable with that induced by fumagillin treatment. However, detrimental effects on the overall colony state were observed due to the combined effects of stressors such as the queenless condition, lack of brood and high infection rates. The ovaries and ventriculi of queens in infected colonies revealed no signs of Nosema infection and there were no lesions in ovarioles or epithelial ventricular cells.     

Survival and immune response of drones of a Nosemosis tolerant honey bee strain towards N. ceranae infections

Honey bee colonies (Apis mellifera) have been selected for low level of Nosema in Denmark over decades and Nosema is now rarely found in bee colonies from these breeding lines. We compared the immune response of a selected and an unselected honey bee lineage, taking advantage of the haploid males to study its potential impact on the tolerance toward Nosema ceranae, a novel introduced microsporidian pathogen. After artificial infections of the N. ceranae spores, the lineage selected for Nosema tolerance showed a higher N. ceranae spore load, a lower mortality and an up-regulated immune response. The differences in the response of the innate immune system between the selected and unselected lineage were strongest at day six post infection. In particular genes of the Toll pathway were up-regulated in the selected strain, probably is the main immune pathway involved in N. ceranae infection response. After decades of selective breeding for Nosema tolerance in the Danish strain, it appears these bees are tolerant to N. ceranae infections.     

Prospective Large-Scale Field Study Generates Predictive Model Identifying Major Contributors to Colony Losses

Over the last decade, unusually high losses of colonies have been reported by beekeepers across the USA. Multiple factors such as Varroa destructor, bee viruses, Nosema ceranae, weather, beekeeping practices, nutrition, and pesticides have been shown to contribute to colony losses. Here we describe a large-scale controlled trial, in which different bee pathogens, bee population, and weather conditions across winter were monitored at three locations across the USA. In order to minimize influence of various known contributing factors and their interaction, the hives in the study were not treated with antibiotics or miticides. Additionally, the hives were kept at one location and were not exposed to potential stress factors associated with migration. Our results show that a linear association between load of viruses (DWV or IAPV) in Varroa and bees is present at high Varroa infestation levels (>3 mites per 100 bees). The collection of comprehensive data allowed us to draw a predictive model of colony losses and to show that Varroa destructor, along with bee viruses, mainly DWV replication, contributes to approximately 70% of colony losses. This correlation further supports the claim that insufficient control of the virus-vectoring Varroa mite would result in increased hive loss. The predictive model also indicates that a single factor may not be sufficient to trigger colony losses, whereas a combination of stressors appears to impact hive health.     

A cell culture model for Nosema ceranae and Nosema apis allows new insights into the life cycle of these important honey bee-pathogenic microsporidia

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.     

What's in that package? An evaluation of quality of package honey bee (Hymenoptera: Apidae) shipments in the United States

To replace deceased colonies or to increase the colony numbers, beekeepers often purchase honey bees, Apis mellifera L., in a package, which is composed of 909-1,364 g (2-3 lb) of worker bees and a mated queen. Packages are typically produced in warm regions of the United States in spring and shipped throughout the United States to replace colonies that perished during winter. Although the package bee industry is effective in replacing colonies lost in winter, packages also can be an effective means of dispersing diseases, parasites, and undesirable stock to beekeepers throughout the United States. To evaluate the quality of packages, we examined 48 packages representing six lines of bees purchased in the spring 2006. We estimated levels of the parasitic mite Varroa destructor Anderson & Trueman and the percentage of drone (male) honey bees received in packages. We surveyed for presence of the tracheal honey bee mite, Acarapis woodi (Rennie), and a microsporidian parasite, Nosema spp., in the shipped bees. We found significant differences in both the mean Varroa mite per bee ratios (0.004-0.054) and the average percentage of drones (0.04-5.1%) in packages from different producers. We found significant differences in the number of Nosema-infected packages (0.0-75.0%) among the six lines. No packages contained detectable levels ofA. woodi. Considering the observed variability among honey bee packages, beekeepers should be aware of the potential for pest and disease infestations and high drone levels in packages.     

Three QTL in the honey bee Apis mellifera L. suppress reproduction of the parasitic mite Varroa destructor

Varroa destructor is a highly virulent ectoparasitic mite of the honey bee Apis mellifera and a major cause of colony losses for global apiculture. Typically, chemical treatment is essential to control the parasite population in the honey bee colony. Nevertheless a few honey bee populations survive mite infestation without any treatment. We used one such Varroa mite tolerant honey bee lineage from the island of Gotland, Sweden, to identify quantitative trait loci (QTL) controlling reduced mite reproduction. We crossed a queen from this tolerant population with drones from susceptible colonies to rear hybrid queens. Two hybrid queens were used to produce a mapping population of haploid drones. We discriminated drone pupae with and without mite reproduction, and screened the genome for potential QTL using a total of 216 heterozygous microsatellite markers in a bulk segregant analysis. Subsequently, we fine mapped three candidate target regions on chromosomes 4, 7, and 9. Although the individual effect of these three QTL was found to be relatively small, the set of all three had significant impact on suppression of V. destructor reproduction by epistasis. Although it is in principle possible to use these loci for marker-assisted selection, the strong epistatic effects between the three loci complicate selective breeding programs with the Gotland Varroa tolerant honey bee stock.     

蜜蜂病毒学研究进展

蜜蜂是自然界最重要的授粉昆虫,对维护自然生态系统的生物多样性和保持农业生态系统的增产效应发挥着巨大的作用。作为世界第一养蜂大国,中国养蜂业健康发展的意义不仅在于获取大量高品质的蜂产品,更重要的是发挥蜜蜂授粉的农业增产效应,保证我国的粮食安全。和其他动物一样,蜜蜂健康也受到多种病害的威胁,近年来蜜蜂病毒病在世界范围内的广泛流行与传播,是导致世界蜂群持续下降的一个重要原因。蜜蜂病毒长期广泛的以无明显发病症状的低浓度隐性感染方式存在于蜜蜂蜂群中,但多数蜜蜂病毒在特定环境条件下可被激活,在寄主体细胞内快速复制,表现出强烈的致病性,引发致死性蜜蜂病毒病的流行与爆发。蜜蜂病毒病知识的缺乏,以及复杂的蜜蜂病毒鉴定技术使得蜜蜂病毒病难以及时确诊和防治,因此每年在养蜂生产上造成的巨大损失已严重阻碍了我国养蜂业的健康发展。本文将综述这一领域的研究成果和学科发展趋势,为在我国开展蜜蜂病毒学研究提供参考,并介绍国外的一些蜜蜂病毒病诊断方法与防治经验服务于我国养蜂生产实践。     

Honey bees (Apis mellifera) reared in brood combs containing high levels of pesticide residues exhibit increased susceptibility to Nosema (Microsporidia) infection

Nosema ceranae and pesticide exposure can contribute to honey bee health decline. Bees reared from brood comb containing high or low levels of pesticide residues were placed in two common colony environments. One colony was inoculated weekly with N. ceranae spores in sugar syrup and the other colony received sugar syrup only. Worker honey bees were sampled weekly from the treatment and control colonies and analyzed for Nosema spore levels. Regardless of the colony environment (spores+syrup added or syrup only added), a higher proportion of bees reared from the high pesticide residue brood comb became infected with N. ceranae, and at a younger age, compared to those reared in low residue brood combs. These data suggest that developmental exposure to pesticides in brood comb increases the susceptibility of bees to N. ceranae infection.     

How natural infection by Nosema ceranae causes honeybee colony collapse

In recent years, honeybees (Apis mellifera) have been strangely disappearing from their hives, and strong colonies have suddenly become weak and died. The precise aetiology underlying the disappearance of the bees remains a mystery. However, during the same period, Nosema ceranae, a microsporidium of the Asian bee Apis cerana, seems to have colonized A. mellifera, and it's now frequently detected all over the world in both healthy and weak honeybee colonies. For first time, we show that natural N. ceranae infection can cause the sudden collapse of bee colonies, establishing a direct correlation between N. ceranae infection and the death of honeybee colonies under field conditions. Signs of colony weakness were not evident until the queen could no longer replace the loss of the infected bees. The long asymptomatic incubation period can explain the absence of evident symptoms prior to colony collapse. Furthermore, our results demonstrate that healthy colonies near to an infected one can also become infected, and that N. ceranae infection can be controlled with a specific antibiotic, fumagillin. Moreover, the administration of 120 mg of fumagillin has proven to eliminate the infection, but it cannot avoid reinfection after 6 months. We provide Koch's postulates between N. ceranae infection and a syndrome with a long incubation period involving continuous death of adult bees, non-stop brood rearing by the bees and colony loss in winter or early spring despite the presence of sufficient remaining pollen and honey.     

Evidence for emerging parasites and pathogens influencing outbreaks of stress-related diseases like chalkbrood

In agriculture, honey bees play a critical role as commercial pollinators of crop monocultures which depend on insect pollination. Hence, the demise of honey bee colonies in Europe, USA, and Asia caused much concern and initiated many studies and research programmes aiming at elucidating the factors negatively affecting honey bee health and survival. Most of these studies look at individual factors related to colony losses. In contrast, we here present our data on the interaction of pathogens and parasites in honey bee colonies. We performed a longitudinal cohort study over 6 years by closely monitoring 220 honey bee colonies kept in 22 apiaries (ten randomly selected colonies per apiary). Observed winter colony losses varied between 4.8% and 22.4%; lost colonies were replaced to ensure a constant number of monitored colonies over the study period. Data on mite infestation levels, infection with viruses, Nosema apis and Nosema ceranae, and recorded outbreaks of chalkbrood were continuously collected. We now provide statistical evidence (i) that Varroa destructor infestation in summer is related to DWV infections in autumn, (ii) that V. destructor infestation in autumn is related to N. apis infection in the following spring, and most importantly (iii) that chalkbrood outbreaks in summer are related to N. ceranae infection in the preceding spring and to V. destructor infestation in the same season. These highly significant links between emerging parasites/pathogens and established pathogens need further experimental proof but they already illustrate the complexity of the host–pathogen-interactions in honey bee colonies.     

Altered physiology in worker honey bees (Hymenoptera: Apidae) infested with the mite Varroa destructor (Acari: Varroidae): a factor in colony loss during overwintering?

The ectoparasitic mite Varroa destructor (Anderson & Trueman) is the most destructive pest of the honey bee, Apis mellifera L., in Europe and the United States. In temperate zones, the main losses of colonies from the mites occur during colony overwintering. To obtain a deeper knowledge of this phenomenon, we studied the mites' impact on the vitellogenin titer, the total protein stores in the hemolymph, the hemocyte characteristics, and the ecdysteroid titer of adult honey bees. These physiological characteristics are indicators of long-time survival and endocrine function, and we show that they change if bees have been infested by mites during the pupal stage. Compared with noninfested workers, adult bees infested as pupae do not fully develop physiological features typical of long-lived wintering bees. Management procedures designed to kill V. destructor in late autumn may thus fail to prevent losses of colonies because many of the adult bees are no longer able to survive until spring. Beekeepers in temperate climates should therefore combine late autumn management strategies with treatment protocols that keep the mite population at low levels before and during the period when the winter bees emerge.     

蜜蜂瓦螨敏感卫生行为研究进展

蜜蜂具有很高的生态价值和经济价值,对农业生产帮助巨大。然而,狄斯瓦螨Varroa destructor寄生给西方蜜蜂Apis mellifera蜂群造成重大损失,对蜜蜂健康构成严重威胁,因此,狄斯瓦螨的防治变得尤为紧要。虽然化学防治是防治狄斯瓦螨常用且有效措施,但仍存在许多缺点,如造成蜂产品污染、导致蜂螨产生抗药性等。另一方面,培育抗螨蜂种被证明是可持续的狄斯瓦螨防治方法。瓦螨敏感卫生行为(Varroa sensitive hygiene, VSH)是蜜蜂重要的抗螨性状之一。本文从狄斯瓦螨的生活周期、对蜜蜂的危害、蜜蜂抗螨行为、瓦螨敏感卫生行为调控和遗传育种等方面进行综述,为狄斯瓦螨防治和抗螨蜂种选育提供参考。     

Nosema ceranae in age cohorts of the western honey bee (Apis mellifera)

Nosemaceranae intensity (mean spores per bee) and prevalence (proportion of bees infected in a sample) were analyzed in honey bees of known ages. Sealed brood combs from five colonies were removed, emerging bees were marked with paint, released back into their colonies of origin, and collected as recently emerged (0-3 days old), as house bees (8-11 days old), and as foragers (22-25 days old). Fifty bees from each of the five colonies were processed individually at each collection date for the intensity and prevalence of N. ceranae infection. Using PCR and specific primers to differentiate Nosema species, N. ceranae was found to be the only species present during the experiment. At each collection age (recent emergence, house, forager) an additional sample from the inner hive cover (background bees=BG) of each colony was collected to compare the N. ceranae results of this sampling method, commonly used for Nosema spore quantification, to the samples comprised of marked bees of known ages. No recently emerged bees exhibited infection with N. ceranae. One house bee out of the 250 individuals analyzed (prevalence=0.4%) tested positive for N. ceranae, at an infection level of 3.35×10(6) spores. Infection levels were not statistically different between the recently emerged (mean=0 spores/bee) and house bees (mean=1.34×10(4) spores/bee) (P=0.99). Foragers exhibited the highest prevalence (8.3%) and infection intensity (mean=2.38×10(6) spores/bee), with a range of 0-8.72×10(7) spores in individual bees. The average infection level across all foragers was significantly higher than that of recently emerged bees (P=0.01) and house bees (P=0.01). Finally, the prevalence of Nosema in infected bees was found to be positively correlated with the infection intensity in the sample.     

Honeybee viruses in Uruguay

Mortality of honeybees is a serious problem that beekeepers have to face periodically in Uruguay and worldwide. The presence of RNA viruses, in addition to other pathogens may be one of its possible causes. In this work, we detected Chronic bee paralysis virus, Acute bee paralysis virus, Black queen cell virus, Sacbrood virus and Deformed wing virus in samples of Uruguayan honeybees with or without Varroa destructor and Nosema apis. The detection of viruses in different provinces, simultaneous co-infection of colonies by several viruses and the fact that 96% of the samples were infected with one or more virus, indicates they are widely spread in the region.     

Uncapping activity of Apis mellifera L. (Hymenoptera: Apidae) towards worker brood cells infested with the mite Varroa destructor Anderson & Treuman (Mesostigmata: Varroidae)

Varroosis, a disease caused by the mite Varroa destructor Anderson and Treuman has killed hundreds of thousands of Apis mellifera L. colonies in various parts of the world. Nevertheless, the damage caused by this mite varies with the type of bee and climate conditions. Varroa causes little damage to Africanized bee colonies in Brazil, as the infestation rates are relatively stable and low. We evaluated the hygienic behavior (uncapping and removal of brood) of highly hygienic Africanized bees using combs with worker brood cells infested (naturally) and no infested with V. destructor. The daily uncapping rate, measured in eight colonies during six days, was 3.5 fold higher in the combs infested with varroa compared to no infested combs. The results show that the Africanized bees are able to recognise and remove brood cells naturally infested with V. destructor what is an important mechanism for tolerance against varroa.     

Asymptomatic presence of Nosema spp. in Spanish commercial apiaries

Nosemosis is caused by intracellular parasites (Nosema apis and Nosema ceranae) that infect the midgut epithelial cells in adult honey bees. Recent studies relate N. ceranae to Colony Collapse Disorder and there is some suggestion that Nosema spp., especially N. ceranae, induces high mortality in honey bees, a fact that is considered as a serious threat for colony survival. 604 samples of adult honey bees for Nosema spp. analysis were collected from beekeeping colonies across Spain and were analysed using PCR with capillary electrophoresis. We also monitored 77 Andalusian apiaries for 2years; the sampled hives were standard healthy colonies, without any special disease symptoms. We found 100% presence of Nosema spp. in some locations, indicating that this parasite was widespread throughout the country. The two year monitoring indicated that 87% of the hives with Nosema spp. remained viable, with normal honey production and biological development during this period of time. The results of these trials indicated that both N. ceranae and N. apis could be present in these beehives without causing disease symptom and that there is no evidence for the replacement of N. apis by N. ceranae, supporting the hypothesis that nosemosis is not the main reason of the collapse and death of beehives.     

Study of temperature-growth interactions of entomopathogenic fungi with potential for control of Varroa destructor (Acari: Mesostigmata) using a nonlinear model of poikilotherm development

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.