Multiple virus infections in the honey bee and genome divergence of honey bee viruses

Using uniplex RT-PCR we screened honey bee colonies for the presence of several bee viruses, including black queen cell virus (BQCV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood virus (SBV), and described the detection of mixed virus infections in bees from these colonies. We report for the first time that individual bees can harbor four viruses simultaneously. We also developed a multiplex RT-PCR assay for the simultaneous detection of multiple bee viruses. The feasibility and specificity of the multiplex RT-PCR assay suggests that this assay is an effective tool for simultaneous examination of mixed virus infections in bee colonies and would be useful for the diagnosis and surveillance of honey bee viral diseases in the field and laboratory. Phylogenetic analysis of putative helicase and RNA-dependent RNA polymerase (RdRp) encoded by viruses reveal that DWV and SBV fall into a same clade, whereas KBV and BQCV belong to a distinct lineage with other picorna-like viruses that infect plants, insects and vertebrates. Results from field surveys of these viruses indicate that mixed infections of BQCV, DWV, KBV, and SBV in the honey bee probably arise due to broad geographic distribution of viruses.     

Flight of the honey bee

Summary Using manometric and gas analytical methods oxygen consumption , carbon dioxide production , respiratory quotientRQ, (Fig. 1A-C) and thorax surface temperature difference T _ts (Fig. 3) were determined in single bees. The animals were either sitting in respiratory chambers or were suspended by the scutum, in which case they were resting, walking (turning a small polystyrene ball) or flying in a closed miniature wind tunnel.During resting (sitting in Warburg vessels) at an ambient temperatureT _a=10°C,RQ was 1.01±0.2 (n=905) with variations due to method (Fig. 1D, E).RQ values during walking were determined in single cases. In no case were they significantly different from 1.00. After the first 10 min of flight meanRQ was 1.00±0.04. It was significantly smaller than 1.00 (RQ=0.97) only during the last 5% of long-time flights (mean flight duration 58.8±28.8 min). With the exception of near-exhaustion conditions no signs of fuels other than carbohydrates were found.Metabolic rateP _m was 19.71±21.38 mW g^–1 during resting at 20°CT _a30°C indicating that many resting bees actively thermoregulate at higherT _a. After excluding bees which were actively thermoregulating, by an approximationP _m was 5.65±2.44 mW g^–1 at 20°CT _a30°C. True resting metabolic rate for sitting bees atT _a=10°C was 1.31±0.53 mW g^–1 (Fig. 2A, B).A significant negative correlation was found between relative (specific) oxygen consumption rel and body massM _b at 85 mgM _b150 mg.At 0°CT _ts16.5°C a significant (-0.01) positive correlation was found between and T _ts in single resting bees: T _Ts+0.099, or betweenP _m and T _ts:P _m=1.343 T _ts+0.581 (Fig. 3D) in ml h^–1,P _m in mW,T in °C).During walking (duration 13.15±5.71 min,n=13) at 12.5°CT _a21°C a stable T _ts of 11.41±3.37°C, corresponding to 167 mW g^–1, was reached for 80 to 90% of the walking time (Fig. 4B).During wind tunnel flights of tethered animals the minimal metabolic power measured in exhaustion experiments was 240 mW g^–1. Calculation of factors of increase inP _m is of limited value in poikilotherms, in which true resting conditions are not exactly defined.     

Peroxisomal enzymes in the honey bee midgut

Peroxisomes are ubiquitous organelles that contain catalase (CAT) and an array of inducible enzymes that regulate aspects of lipid, purine, xenobiotic, eicosanoid, and phospholipid metabolism. Although peroxisomes are recognized as essential components in the cellular economy of microorganisms, plants, and mammals, little is known about their specialized functions in insect metabolism. Peroxisomal acyl-CoA oxidase (ACO) is a flavin-linked, H₂O₂-producing enzyme that regulates the β-oxidation of long chain fatty acids. We measured ACO and CAT activity in midgut tissues from worker honey bees, Apis mellifera, of known ages from free-flying colonies. The ACO activity peaked in young worker bees that digest and assimilate nutrients from pollen and from trophallaxis. As the bees aged, ACO activity declined. Conversely, CAT activity increased as the bees aged and reached its highest level in the oldest bees that were assayed. Isolated honey bee midguts were then fractionated using sucrose and Metrizamide (MET) density gradient centrifugation. Organelle-bound CAT activity equilibrated in sucrose at densities between 1.19 and 1.22, which are typical of spherical 1 μm peroxisomes. In the MET gradients, the organelle-bound CAT separated into two distinct fractions. The heavier fraction equilibrated at 1.21 and the lighter fraction at 1.15, a density commonly associated with microperoxisomes. These results support our ultrastructural and cytochemical data and suggest that the diverse functions of regionally specialized midgut epithelial cells lead to a heterogeneous population of peroxisomal organelles. ACO activity confirms the role of midgut peroxisomes in the intermediary metabolism of lipids and the increasing CAT activity suggests that the midgut epithelium may metabolize deleterious pro-oxidants of aerobic metabolism associated with foraging and senescence. © 1996 Wiley-Liss, Inc.     

Senescence in the worker honey bee Apis Mellifera

Honey bees are social insects that exhibit striking caste-specific differences in longevity. Queen honey bees live on average 1-2 years, whereas workers live 2-6 weeks in the summer and about 20 weeks in the winter. It is not clear whether queen-worker differences in longevity are due to intrinsic physiological differences in the rate of senescence, to differential exposure to extrinsic factors such as predation and adverse environmental conditions, or both. To determine if the relatively short lifespan of worker bees involves senescence, we measured age-specific resistance to three different physiological stressors (starvation, thermal, and oxidative stress) while eliminating age-related differences in foraging activity and minimizing age-related differences in energy expenditure. Despite these manipulations, older worker bees were still significantly less resistant to all three stressors than were younger bees. These results indicate that the regulation of worker bee lifespan involves senescence, in addition to extrinsic factors.     

Epidemiology of honey bee parasites

The epidemiology of honey bee parasites has been somewhat neglected, but Lynn Royce and Philippe Rossignol describe their unique characteristics. Indeed, it appears that a parasite of social insects has in essence to adapt to two hosts: first, the individual worker within a colony, the numbers of which grow linearly and second, to the colony itself, the actual reproductive 'organism'. Transmission also has vertical and horizontal components. Analysis of tracheal mite populations in particular suggests that intracolony parasite levels are regulated by the swarming behavior of their hosts. Ironically, current and highly productive methods of honey bee management with movable hives curb swarming and may contribute to increasing the spread and the impact of some parasites. This insight may result in changing management practices to reduce the detrimental effects of bee parasites.     

Hox genes in the honey bee Apis mellifera

Hox genes are known to control the identity of serially repeated structures in arthropods and vertebrates. We analyzed the expression pattern of the Hox genes Deformed (Dfd), Sex combs reduced (Scr), Antennapedia (Antp), and Ultrabithorax/abdominal-A (Ubx/abd-A) from the honey bee Apis mellifera. We also cloned a cDNA with the complete coding region of the Antennapedia gene from Apis. Comparison with Antp proteins from other insect species revealed several regions of homology. The expression patterns of the isolated Hox genes from Apis showed that the original expression patterns of Dfd, Scr, and Antp appear between late blastoderm and early germ band stage in a temporal and spatial sequence. Each of them shows up as a belt, spanning approximately two segment anlagen, Dfd in the anterior gnathal region, Scr in the posterior gnathal and anterior thoracic region, and Antp in the thoracic region. Following expansion of the Antp domain in the abdomen as a gradient towards the posterior, Ubx/abd-A expression appears laterally in the abdomen. During gastrulation and in the germ band stage the domains of strong expression do not overlap any more, but touch each other. After gastrulation the borders of the expression domains partly correlate with parasegment and partly with segment boundaries. Laterally, gaps between the domain of each gene may show no expression of any of the genes examined. Received: 30 August 1999 / Accepted: 28 April 2000     

A filamentous virus of the honey bee

A common and widespread disease of honey bees, Apis mellifera, is caused by an unoccluded, Feulgen-positive, filamentous nuclear virus. Ovoid viral particles seen in diseased bee hemolymph consisted of a folded nucleocapsid within a viral envelope and were 0.40 by 0.10 μm. Virions with unfolded nucleocapsids were about 3060 by 60 nm. The disease was transmissible to bees both per os and by injection, but efforts to infect oriental cockroaches, Blatta orientalis, and the greater wax moth, Galleria mellonella, failed. The disease is apparently the same as that described as a rickettsial disease of European bees.     

Acute paralysis viruses of the honey bee

<正>The alarming decline of honey bee(Apis mellifera)colonies in the last decade drove the attention and research to several pathogens of the honey bee including viruses.Viruses challenge the development of healthy and robust colonies since they manage to prevail in an asymptomatic mode and reemerge in acute infections following external stresses,as well as they are able to infect new healthy colonies(de Miranda J R,et al.,2010a;de Miranda J R,et al.,2010b;Di Prisco G,et al.,2013;Nazzi F,et al.,2012;Yang X L,et al.,2005).     

Filtering mechanism of the honey bee proventriculus

ABSTRACT. The fine structure and function of a honey bee's ( Apis mellifera Linn.) proventriculus were studied by scanning electron microscopy and video-recording. Our observations revealed that the proventriculus is used to engulf pollen and other particles which contaminate the nectar carried into the crop. The four lips are closed and opened, pulled backwards and straightened by the external circular muscles and internal longitudinal muscles. Combs of filiform-hairs (70 μm in length) located on the margins of the lips 'catch' and filter particles from the fluid. By repeated filtering, opening and closing actions of the hairs and lips, particles are filtered and collected in pouches between the ventricular folds to form boluses and are eventually passed into the midgut. In the present experiment, particle sizes ranging from 0.5 to 100 μm in diameter, including dandelion pollen ( Taraxacum officinale Web.), Torula yeast ( Candida utilis Lodder et Kreger-Van Rij), bee disease spores of Nosema apis Zander and Bacillus larvae White, and man-made particles can be filtered by the hairs. Small particles (0.23 μm in diameter) filter through the hair and return back to the fluid. Large particles (100–200 μm in diameter) are caught between the stylets of the mouthparts and are not ingested. These observations suggest that the particle size plays an important role in determining what can be taken by the mouthparts and the proventriculus and what can later be utilized as a food source by the bee. The role of the proventriculus in disease transmission is also discussed.     

Pathogen webs in collapsing honey bee colonies

Recent losses in honey bee colonies are unusual in their severity, geographical distribution, and, in some cases, failure to present recognized characteristics of known disease. Domesticated honey bees face numerous pests and pathogens, tempting hypotheses that colony collapses arise from exposure to new or resurgent pathogens. Here we explore the incidence and abundance of currently known honey bee pathogens in colonies suffering from Colony Collapse Disorder (CCD), otherwise weak colonies, and strong colonies from across the United States. Although pathogen identities differed between the eastern and western United States, there was a greater incidence and abundance of pathogens in CCD colonies. Pathogen loads were highly covariant in CCD but not control hives, suggesting that CCD colonies rapidly become susceptible to a diverse set of pathogens, or that co-infections can act synergistically to produce the rapid depletion of workers that characterizes the disorder. We also tested workers from a CCD-free apiary to confirm that significant positive correlations among pathogen loads can develop at the level of individual bees and not merely as a secondary effect of CCD. This observation and other recent data highlight pathogen interactions as important components of bee disease. Finally, we used deep RNA sequencing to further characterize microbial diversity in CCD and non-CCD hives. We identified novel strains of the recently described Lake Sinai viruses (LSV) and found evidence of a shift in gut bacterial composition that may be a biomarker of CCD. The results are discussed with respect to host-parasite interactions and other environmental stressors of honey bees.     

Physiological consequences of prolonged nursing in the honey bee

Honey bee workers normally produce brood food at an age of 5 to 15 days. However, natural events like swarming or brood diseases may lead to the occurrence of over-aged nurses. Here we investigated the physiological consequences of prolonged nursing for both the nurses and the brood they rear, and tried to separate the effects of chronological age and of task affiliation on some important physiological parameters. Brood was reared in groups of colonies with either a normal age structure or with moderately over-aged workers.The haemolymph concentrations of total protein and vitellogenin, the development of mandibular and hypopharyngeal glands, and the activity of α-glucosidase in the hypopharyngeal glands of nurses from these groups of colonies were compared. Moreover, we used the fertility of young workers reared by normal- and overaged nurses as a bioindicator for the quality of the brood care they had received. It showed that parameters linked to the production of brood food proteins remained stable in over-aged nurses, whereas the development of mandibular glands regressed. Workers reared by over-aged nurses had more ovarioles and showed stronger ovary development under queenless conditions. Our results indicate that while over-aged nurses remain capable of producing brood food, they are not functionally equivalent to young nurses. The partial degeneration of the mandibular glands normally occurring at the end of the nursing period cannot be prevented by prolonged nursing. The distinct phenotype of workers reared by old nurses raises the question of possible age-related specialisations among nurses in colonies with a normal age structure. Received 14 July 2008; revised 18 November 2008; accepted 21 November 2008.