Characteristic field symptoms comprising honeybee hairless-black syndrome induced in the laboratory by a virus

All the characteristic symptoms comprising hairless-black syndrome found in the field were reproduced in the laboratory by transferring virus-fed adult honeybees, Apis mellifera, to cages containing healthy bees. The experimental, virus-fed bees showed high mortality and various combinations of other symptoms, such as paralysis, withdrawal from the cluster, trembling, subjection to attack by healthy bees, and the hairless-black condition. Control bees, treated identically except not fed virus, displayed none of these symptoms. Virus-fed bees transmitted the disease to other bees; control bees did not. Suspensions made from experimental bees contained virus; suspensions made from control bees did not. It is concluded that the virus is the causal agent of hairless-black syndrome.     

The fate and effect of hairs removed from honeybees with hairless-black syndrome

Honeybees, Apis mellifera, attacking other bees exhibiting hairless-black syndrome were found to have ingested bee hairs probably as a result of the attack. Experimental samples of bees were fed bee hairs and virus isolated from sick bees both separately and in combination. Control samples, samples fed hair, samples fed virus, and samples fed both hairs and virus averaged 8, 7, 37, and 70% mortality, respectively. Presence of hairs enhanced the effect of the virus.     

Distribution of <Emphasis Type="Italic">Paenibacillus larvae</Emphasis> Spores Among Adult Honey Bees (<Emphasis Type="Italic">Apis mellifera</Emphasis>) and the Relationship with Clinical Symptoms of American Foulbrood

Knowledge of the distribution of Paenibacillus larvae spores, the causative agent of American foulbrood (AFB), among individual adult honey bees is crucial for determining the appropriate number of adult bees to include in apiary composite samples when screening for diseased colonies. To study spore distribution at the individual bee level, 500 honey bees were collected from different parts of eight clinically diseased colonies and individually analyzed for P. larvae. From the brood chamber and from the super, bees were randomly collected and individually put in Eppendorf vials. The samples were frozen as soon as possible after collection. Concurrently with sampling, each colony was visually inspected for clinical symptoms of AFB. The number of clinically diseased cells in the colony was visually estimated. All samples were cultured in the laboratory for P. larvae. The results demonstrate that the spores are not randomly distributed among the bees; some bees have much higher spore loads than others. It is also clear that as the proportion of contaminated bees increase, the number of spores from each positive bee also increases. The data also demonstrated a relationship between the number of clinically diseased cells and the proportion of positive bees in individual colonies. This relationship was used to develop a mathematical formula for estimating the minimum number of bees in a sample to detect clinical disease. The formula takes into account the size of the apiary and the degree of certainty with which one aims to discover clinical symptoms. Calculations using the formula suggest that adult bee samples at the colony level will detect light AFB infections with a high probability. However, the skewed spore distribution of the adult bees makes composite sampling at the apiary level more problematic, if the aim of the sampling is to locate lightly infected individual colonies within apiaries. The results suggest that false-negative culturing results from composite samples of adult bees from individual colonies with clinical symptoms of AFB are highly improbable. However, if single colonies have light infections in large apiaries, the dilution effect from uncontaminated bees from healthy colonies on the positive bees from diseased colonies may yield false-negative results at the apiary level.     

Selection for resistance and susceptibility to hairless-black syndrome in the honeybee.

A two-way selection experiment for resistance and susceptibility to hairless-black syndrome, a probable viral disease in the honeybee, was carried out for four generations. From the parental generation of 15 colonies (reared from three basic stock colonies) tested for disease resistance, one-fifth were selected to start a resistant line and one-fifth to start a susceptible one. Thereafter these two groups were kept genetically separate and, from each tested generation, an average of about 22% of the best colonies were used as parents of the next generation.Bacteria-free inoculum, which was fed to challenge these bees, was prepared by macerating and centrifuging specific numbers of bees showing symptoms of hairless-black syndrome and collected from a number of colonies in our apiaries.Resistant and susceptible lines did not differ significantly in the first selected generation. In the second, third, and fourth generations, the two lines diverged increasingly, and in each generation, they differed statistically at the 1% level of probability.     

Ascosphaera aggregata contamination on alfalfa leafcutting bees in a loose cell incubation system

The alfalfa leafcutting bee, a solitary bee used to pollinate alfalfa seed crops, is seriously affected by chalkbrood, a larval disease caused by the fungus Ascosphaera aggregata. One attempt to control the disease includes removing nests from the nesting boards (the “loose cell” system). We report here that adults emerging from the loose cells are heavily contaminated with A. aggregata spores. The contamination levels are not as high as previously reported for bees emerging directly from the boards, but they are still a likely focus for disease spread and may need to be targeted in chalkbrood control strategies.     

感染囊幼病的工蜂咽下腺细胞的超微结构的变化

前言 中华蜜蜂囊幼病是我国养蜂业的一种重要病毒病,发病率很高,有的地区造成大量的幼虫死亡,给我国养蜂生产带来一定的危害,为了对防治病害提供科学的依据,我们首先对该病病原进行了分离、提纯及电子显微镜的研究等工作。发现该病毒对幼虫和成蜂,尤其对工蜂体内各个器官都有程度不同的影响。在幼虫发病期症状特别明显,病幼虫身体松软多水,其表皮容易破裂,当悬挂幼虫时其幼虫末端积聚有透明的液滴,在巢房内幼虫头部尖并变成黑色,头部稍微向上抬起呈船形。死后的幼虫干涸变为褐色的外壳留于巢房内,当感染的幼虫全部封盖后,巢房盖的中央有一小孔,这些异常的变化都是囊幼病的典型症状。幼虫症状虽很明显,但工蜂感染病毒后在外部形态上却没有明显     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Yeasts isolated from honey bees, Apis mellifera, fed 2,4-D and antibiotics

Yeasts belonging to seven species were isolated and identified from the intestines of 388 adult worker honey bees, Apis mellifera. Torulopsis magnoliae, Candida parapsilosis, and Torulopsis grabrata were found in bee guts most frequently. The intestines of bees from colonies fed a combination of Terramycin and Fumidil B contained few or no yeasts. More guts of bees from colonies fed 2,4-D contained yeasts than those examined from bees from control colonies.     

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.     

Oxalic acid: a prospective tool for reducing <Emphasis Type="Italic">Varroa</Emphasis> mite populations in package bees

Numerous studies have investigated using oxalic acid (OA) to control Varroa mites in honey bee colonies. In contrast, techniques for treating package bees with OA have not been investigated. The goal of this study was to develop a protocol for using OA to reduce mite infestation in package bees. We made 97 mini packages of Varroa-infested adult bees. Each package contained 1,613 ± 18 bees and 92 ± 3 mites, and represented an experimental unit. We prepared a 2.8% solution of OA by mixing 35 g OA with 1 l of sugar water (sugar:water = 1:1; w:w). Eight treatments were assigned to the packages based on previous laboratory bioassays that characterized the acute contact toxicity of OA to mites and bees. We administered the treatments by spraying the OA solution directly on the bees through the mesh screen cage using a pressurized air brush and quantified mite and bee mortality over a 10-day period. Our results support applying an optimum volume of 3.0 ml of a 2.8% OA solution per 1,000 bees to packages for effective mite control with minimal adult bee mortality. The outcome of our research provides beekeepers and package bee shippers guidance for using OA to reduce mite populations in package bees.     

The occurrence of Melissococcus plutonius in healthy colonies of Apis mellifera and the efficacy of European foulbrood control measures

European foulbrood (EFB) persists in England and Wales despite current treatment methods, all of which include feeding honey bee colonies with the antibiotic oxytetracycline (OTC). A large-scale field experiment was conducted to monitor a husbandry-based method, using comb replacement (known as Shook swarm), as a drug free EFB control option. The understanding of EFB epidemiology is limited, with little information on the presence of Melissococcus plutonius in disease free colonies. Additional samples were collected from diseased and disease free apiaries to identify symptomless infection. EFB reoccurrence was not significantly different between OTC and husbandry methods and real-time PCR data demonstrated that fewer Shook swarm treated colonies contained M. plutonius carryover to the Spring following treatment. Asymptomatic colonies from diseased apiaries showed an increased risk of testing positive for M. plutonius compared to asymptomatic colonies from disease free apiaries. The probability of a sample being symptomatic increased when a greater quantity of M. plutonius was detected in adult bees and larvae. The possibility of treating EFB as an apiary disease rather than a colony disease and the implications of a control strategy without antibiotics are discussed.     

Olfactory and behavioral response thresholds to odors of diseased brood differ between hygienic and non-hygienic honey bees (Apis mellifera L.)

Through the use of proboscis-extension reflex conditioning, we demonstrate that honey bees (Apis mellifera L.) bred for hygienic behavior (a behavioral mechanism of disease resistance) are able to discriminate between odors of healthy and diseased brood at a lower stimulus level than bees from a non-hygienic line. Electroantennogram recordings confirmed that hygienic bees exhibit increased olfactory sensitivity to low concentrations of the odor of chalkbrood infected pupae (a fungal disease caused by Ascosphaera apis). Three-week-old hygienic bees were able to discriminate between the brood odors significantly better than three-week old non-hygienic bees. However, the differential performance in brood odor discrimination was primarily genetically based, not a direct result of age, experience, or the temporary behavioral state of the bee. Lower stimulus thresholds for both the olfactory and behavioral responses of hygienic bees may facilitate their ability to detect, uncap and remove diseased brood rapidly from the nest. In contrast, non-hygienic bees, possessing higher response thresholds, may not be able to detect diseased brood as easily. Our results provide an example of how physiological and behavioral differences between the hygienic and non-hygienic honey bee lines, operating at the level of the individual, could produce colony-specific behavioral phenotypes.     

Regional distribution of Paenibacillus larvae subspecies larvae, the causative organism of American foulbrood, in honey bee colonies of the Western United States

We examined honey bee, Apis mellifera L., colonies pollinating almonds in California during February 2003 for Paenibacillus larvae subsp. Larvae, the causative organism of the virulent brood disease American foulbrood. Colonies originating from the Rocky Mountain area and California had significantly higher numbers (P < 0.05) of bacterial colony-forming units (CFUs) (408 and 324 per 30 adult bees, respectively) than colonies from the upper Midwest (1.28). Colonies from the northwestern, central, and southwestern United States had intermediate CFU or bacterial colony levels. Operations positive for P. larvae larvae were relatively uniform at approximately 70-80%, and no regional significant differences were found. Percentages of colonies with high CFUs (> or = 400 per 30 bees) differed significantly, with those from the Rocky Mountain region having 8.73% compared with those of the upper Midwest with 0%. The significance of CFU levels was evaluated by inoculating healthy colonies with diseased immatures and sampling adult bees. The number of CFUs detected per diseased immature was conservatively estimated to be approximately 399 CFUs per 30 adult bees. We defined this spore level as 1 disease equivalent. Based on this, 3.86% colonies in our survey had 1 or more disease equivalent number of P. larvae larvae CFUs. Operations with high P. larvae larvae spore levels in their colonies will likely observe American foulbrood if prophylaxis is not practiced diligently.     

Inspection and feeding of larvae by worker honey bees (Hymenoptera: Apidae): Effect of starvation and food quantity

Honey bee larvae are frequently inspected and, sometimes, provided with food by adult workers, but the stimuli that elicit the important task of food provisioning have never been investigated. Larvae with their food experimentally deprived received more frequent inspection and feeding visits from nurse bees than normally fed larvae, suggesting that there could be a hunger signal. Food-deprived larvae with artificially supplied larval food received the same rate of feeding visits from nurse bees as did normally fed larvae but still received more inspection visits. These results suggest that stimuli eliciting feeding are different from those for inspection. They also support the hypothesis that worker bees deposit food in a larval cell only when the quantity of food is below a certain minimum threshold that is perceived during larval inspections. A model is presented regarding the stimuli from larvae that result in worker feeding behavior.     

Is the number of antennal plate organs (sensilla placodea) greater in hygienic than in non-hygienic Africanized honey bees?

Hygienic behavior is a desirable trait in honey bees (Apis mellifera L.), as hygienic bees quickly remove diseased brood, interrupting the infectious cycle. Hygienic lines of honey bees appear to be more sensitive to the odors of dead and diseased honey bee brood, and Africanized honey bees are generally more hygienic than are European honey bees. We compared the number of sensilla placodea, antennal sensory structures involved in the perception of odor, in 10 bees from each of six hygienic and four non-hygienic colonies of Africanized honey bees. The sensilla placodea of three of the terminal segments (flagellomeres) of the right antenna of each bee were counted with a scanning electron microscope. There were no significant differences in the mean numbers of sensilla placodea between the hygienic and non-hygienic bees, though the variance was higher in the hygienic group. Flagellomere 4 had significantly more sensilla placodea than flagellomeres 6 and 8. However, there was no significant difference between the other two flagellomeres. As hygienic bees are capable of identifying dead, injured, or infested brood inside a capped brood cell, sensilla placodea probably have an important role in enabling worker bees to sense sick brood. However, we did not find greater numbers of this sensory structure in the antennae of hygienic, compared to non-hygienic Africanized honey bees.     

A new threat to honey bees, the parasitic phorid fly Apocephalus borealis

Honey bee colonies are subject to numerous pathogens and parasites. Interaction among multiple pathogens and parasites is the proposed cause for Colony Collapse Disorder (CCD), a syndrome characterized by worker bees abandoning their hive. Here we provide the first documentation that the phorid fly Apocephalus borealis, previously known to parasitize bumble bees, also infects and eventually kills honey bees and may pose an emerging threat to North American apiculture. Parasitized honey bees show hive abandonment behavior, leaving their hives at night and dying shortly thereafter. On average, seven days later up to 13 phorid larvae emerge from each dead bee and pupate away from the bee. Using DNA barcoding, we confirmed that phorids that emerged from honey bees and bumble bees were the same species. Microarray analyses of honey bees from infected hives revealed that these bees are often infected with deformed wing virus and Nosema ceranae. Larvae and adult phorids also tested positive for these pathogens, implicating the fly as a potential vector or reservoir of these honey bee pathogens. Phorid parasitism may affect hive viability since 77% of sites sampled in the San Francisco Bay Area were infected by the fly and microarray analyses detected phorids in commercial hives in South Dakota and California's Central Valley. Understanding details of phorid infection may shed light on similar hive abandonment behaviors seen in CCD.     

The prevalence of pathogens in honey bee (Apis mellifera) colonies infested with the parasitic mite Varroa jacobsoni

The prevalence of nine honey bee viruses in samples of dead adult bees from Apis mellifera colonies in the Netherlands and Germany infested with the parasitic mite Varroa jacobsoni was compared with virus incidence in uninfested colonies in Britain. In colonies with low mite populations the viruses present and their incidence during the year were similar to the results obtained from British colonies. However, in marked contrast with findings in Britain, acute paralysis virus (APV) was the primary cause of adult bee mortality in German honey bee colonies severely infested with V. jacobsoni. Dead brood from unsealed and sealed infested cells from German colonies with high mite populations also contained much APV. The evidence suggests that V. jacobsoni activates APV replication in adult bees by its feeding behaviour and transmits virus from adult honey bees to pupae. In addition, adult bees, in which APV is multiplying, transmit the virus to unsealed brood in the larval food.     

A spiroplasma of serogroup IV causes a May-disease-like disorder of honeybees in Southwestern France

Honeybees affected by a disorder resembling the classical May disease in southwestern France contained numerous helical, motile organisms in their digestive tracts and hemolymph. Two strains of the organism (B31 and B39) were cultured and triply cloned in the BSR spiroplasma medium. The electrophoretic patterns of spiroplasmal proteins in 1 - and 2-dimensional polyacrylamide gels were similar to those of group IV spiroplasmas F1 and F2, cultured previously from flower surfaces in France. The organism could be introduced into adult bees by injection or food ingestion at various stages after emergence. Agent administered by either route multiplied to high titers in the hemolymph and killed the bees. Both multiplication and the induced lethal effect of the agent could be prevented by tetracycline but not penicillin. Spiroplasmas that were nearly identical to the B31 and B39 strains were also recovered from the surface of flowers collected within the area visited by the bees from the diseased hives.     

Ultrastructural differences of neurosecretion granules in the corpora cardiaca of the honeybee with and without infection by Nosema apis

Axons of corpora cardiaca in the Nosema infected honeybees contained numerous tightly packed neurosecretion granules. These granules invariably possessed high electron densities. Neurosecretion granules of similar size were also observed in the axons of corpora cardiaca in the healthy honeybees. But these granules were shown to possess various electron densities. Image analysis revealed that some of these granules possessed an electron dense core, and the electron densities decreased gradually from the core, to the fringe of the granule. Some granules appeared to be completely broken down into fine granular substances, while others appeared to be completely broken down into finer particles. It is suggested that in the healthy honeybees, the releasing of neurosecretion from the corpora cardiaca had taken place normally, whereas in the diseased bee, the releasing mechanism had been blocked. The blockage is probably due to the infection.     

Nurse bees support the physiological development of young bees (Apis mellifera L.)

Newly emerged worker honeybees (focal bees) were caged individually for 8 days either isolated or together with one companion bee of known age (2–30 days) taken from a colony. The companion bee was replaced every 2nd day. After 8 days, various parameters were investigated in the focal bees as indicators of the level of development. Focal bees which had been caged with 6-day-old companion bees were better developed than isolated focal bees, newly emerged bees, or focal bees caged with almost all other ages of companion bees. They had hypopharyngeal glands that were larger and contained more protein, their thoraces had a higher protein content, and they had a higher rate of proteolytic activity in the midgut. Although the focal bees were supplied with pollen as well as honey, they consumed only small amounts of pollen. We attribute their better development to their having been fed worker jelly by the accompanying companion bees. The 6-day-old companion bees consumed high quantities of pollen and spent more time (18.7 ± 11.85 s/h) feeding focal bees than 12-day-old bees (6.5 ± 4.09 s/h) or foragers (no feeding of focal bees). The results show that even under such artificial conditions, the exchange of food (trophallaxis) promotes the development of young honeybee workers. Accepted: 26 February 1999