蜜蜂及熊蜂微孢子虫rRNA基因研究

核糖体RNA(rRNA)是一种理想的进化计时器,已广泛地应用于研究微生物的系统发育和进化关系。蜜蜂和熊蜂都被微孢子虫感染,关于蜜蜂和熊蜂微孢子虫rRNA基因研究主要有rRNA基因序列的测定分析、二级结构的研究和用rRNA基因检测诊断微孢子虫等。这些对蜜蜂和熊蜂微孢子虫系统发育的研究和微孢子虫的防治等具有重要的推动作用。     

Chronic Nosema ceranae infection inflicts comprehensive and persistent immunosuppression and accelerated lipid loss in host Apis mellifera honey bees

Nosema ceranae is an intracellular microsporidian parasite of the Asian honey bee Apis cerana and the European honey bee Apis mellifera. Until relatively recently, A. mellifera honey bees were naïve to N. ceranae infection. Symptoms of nosemosis, or Nosema disease, in the infected hosts include immunosuppression, damage to gut epithelium, nutrient and energetic stress, precocious foraging and reduced longevity of infected bees. Links remain unclear between immunosuppression, the symptoms of nutrient and energetic stress, and precocious foraging behavior of hosts. To clarify physiological connections, we inoculated newly emerged A. mellifera adult workers with N. ceranae spores, and over 21?days post inoculation (21?days?pi), gauged infection intensity and quantified expression of genes representing two innate immune pathways, Toll and Imd. Additionally, we measured each host’s whole-body protein, lipids, carbohydrates and quantified respirometric and activity levels. Results show sustained suppression of genes of both humorally regulated immune response pathways after 6?days?pi. At 7?days?pi, elevated protein levels of infected bees may reflect synthesis of antimicrobial peptides from an initial immune response, but the lack of protein gain compared with uninfected bees at 14?days?pi may represent low de novo protein synthesis. Carbohydrate data do not indicate that hosts experience severe metabolic stress related to this nutrient. At 14?days?pi infected honey bees show high respirometric and activity levels, and corresponding lipid loss, suggesting lipids may be used as fuel for increased metabolic demands resulting from infection. Accelerated lipid loss during nurse honey bee behavioral development can have cascading effects on downstream physiology that may lead to precocious foraging, which is a major factor driving colony collapse.     

A multiplex PCR assay to diagnose and quantify Nosema infections in honey bees (Apis mellifera)

Correct identification of the microsporidia, Nosema apis and Nosema ceranae, is key to the study and control of Nosema disease of honey bees (Apis mellifera). A rapid DNA extraction method combined with multiplex PCR to amplify the 16S rRNA gene with species-specific primers was compared with a previously published assay requiring spore-germination buffer and a DNA extraction kit. When the spore germination-extraction kit method was used, 10 or more bees were required to detect the pathogens, whereas the new extraction method made it possible to detect the pathogens in single bees. Approx. 4-8 times better detection of N. ceranae was found with the new method compared to the spore germination-extraction kit method. In addition, the time and cost required to process samples was lower with the proposed method compared to using a kit. Using the new DNA extraction method, a spore quantification procedure was developed using a triplex PCR involving co-amplifying the N. apis and N. ceranae 16S rRNA gene with the ribosomal protein gene, RpS5, from the honey bee. The accuracy of this semi-quantitative PCR was determined by comparing the relative band intensities to the number of spores per bee determined by microscopy for 23 samples, and a high correlation (R² = 0.95) was observed. This method of Nosema spore quantification revealed that spore numbers as low as 100 spores/bee could be detected by PCR. The new semi-quantitative triplex PCR assay is more sensitive, economical, rapid, simple, and reliable than previously published standard PCR-based methods for detection of Nosema and will be useful in laboratories where real-time PCR is not available.     

Eicosanoids act in nodulation reactions to bacterial infections in newly emerged adult honey bees, Apis mellifera, but not in older foragers

Nodulation is the first, and qualitatively predominant, cellular defense reaction to bacterial infections in insects. We tested the hypothesis that eicosanoids also mediate nodulation reactions to bacterial challenge in adults of a social insect, the honey bee, Apis mellifera. Treating newly-emerged experimental bees with the eicosanoid biosynthesis inhibitor, dexamethasone, impaired nodulation reactions to bacterial infections, and the influence of dexamethasone was reversed by treating infected insects with arachidonic acid, an eicosanoid precursor. Several other eicosanoid biosynthesis inhibitors, including the cyclooxygenase inhibitor, indomethacin, and the dual cyclooxygenase/lipoxygenase inhibitor, phenidone, also impaired the ability of experimental honeybees to form nodules in reaction to bacterial challenge. The influence of phenidone on nodulation was expressed in a dose-dependent manner. However, in experiments with older honey bees foragers, similar bacterial challenge did not evoke nodulation reactions. We infer from our results that while eicosanoids mediate cellular immune responses to bacterial infections in newly emerged honey bees, and more broadly, in most insect species, nodulation reactions to bacterial challenge probably do not occur in all phases of insect life cycles.     

Influence of temperature and carbon dioxide concentration on juvenile hormone titre and dependent parameters of adult worker honey bees (Apis mellifera L.)

It is known that juvenile hormone plays an important role in the regulation of labour division and of the different life spans, and that the microclimate of the bee hive is characterized by its high CO₂ concentration and its varying temperature depending on the presence of brood.We have investigated the influence of microclimates characteristic of breeding and broodless areas on the juvenile hormone titre in the haemolymph and whole body extracts, on the corpora allata in vitro activity, on the degradation of juvenile hormone and on the dry weight of the hypopharyngeal glands using bees of known ages. A microclimate of 35°C and 1.5% CO₂, as observed in the breeding area, induces a rapid and pronounced increase in the juvenile hormone titre. On the other hand, this titre remains low in bees kept at 27°C and 1.5% CO₂, a microclimate associated with broodless combs. Rates of juvenile hormone synthesis by corpora allata in vitro were found to be extremely low, even in the presence of farnesenic acid, and not related to the juvenile hormone titre. In vitro incubation of juvenile hormone in haemolymph revealed no degradation while injected juvenile hormone was found to be degraded and taken up by the gut at rates only weakly correlated with the juvenile hormone titre.We propose a hypothetical model for the regulation of the juvenile hormone titre as well as the course of labour division by the varying microclimates observed in the bee hive.     

The inhibiting effect of the queen bee (Apis mellifera L.) foot-print pheromone on the construction of swarming queen cups

Experiments were conducted to determine the rôle of population density of queenright honey bee colonies, and that of the queen bee pheromonal secretions, on the induction and inhibition of swarming queen cup construction during swarming and non-swarming seasons. Construction of queen cups was induced experimentally in overcrowded queenright colonies, during winter, which is a non-swarming season. This construction was induced by high population density of bee workers: above a threshold of 2.3 bee workers/ml there was a relationship between the number of cups constructed and the colony density. During the swarming season a relationship was established between the free volume of a hive (population density) and the number of queen cups constructed: 1.5 cups in a colony that occupied 80,960 ml, compared with 77 cups in a colony hived within a volume of 20, 240 ml. Observations of the queen's movements upon combs in colonies of high and normal population densities showed that in an overcrowded colony the queen bee was almost absent from the bottom edges of the comb, where queen swarming cups and cells are constructed. The tarsal glands of queens are located in the fifth tarsomere and the glandular oily secretion is deposited by the foot-pads upon the combs surface. The rate of secretion by the queen's tarsal glands was about 13 times higher than by those of the workers. A bioassay for testing the inhibitory effects of the queen's glandular extracts on the construction of queen cups was developed. It was based on increasing worker bee population densities, and can be used effectively throughout the year in a subtropical climate.The application of tarsal and mandibular glands' secretion to comb bottom edges in overcrowded colonies (bioassay) caused the inhibition of queen cup construction. None of these two secretions affected construction of these cups when applied separately. We presume that due to colony overcrowding the queen bee is unable to deposit the non-volatile secretions from tarsal glands along the comb edges and that the deficiency of the foot-print pheromone triggers the construction of swarming cups along the non-inhibited areas.     

Differential gene expression of the honey bees Apis mellifera and A. cerana induced by Varroa destructor infection

Varroa destructor mite is currently the most serious threat to the world bee industry. Differences in mite tolerance are reported between two honey bee species Apis mellifera and Apis cerana. Differential gene expression of two honey bee species induced by V. destructor infection was investigated by constructing two suppression subtractive hybridization (SSH) libraries, as first steps toward elucidating molecular mechanisms of Varroa tolerance. From the SSH libraries, we obtained 289 high quality sequences which clustered into 132 unique sequences grouped in 26 contigs and 106 singlets where 49 consisted in A. cerana subtracted library and 83 in A. mellifera. Using BLAST, we found that 85% sequences had counterpart known genes whereas 15% were undescribed. A Gene Ontology analysis classified 51 unique sequences into different functional categories. Eight of these differentially expressed genes, representative of different regulation patterns, were confirmed by qRT-PCR. Upon the mite induction, the differentially expressed genes from both bee species were different, except hex 110 gene, which was up-regulated in A. cerana but down-regulated in A. mellifera, and Npy-r gene, which was down-regulated in both species. In general, most of the differential expression genes were involved in metabolic processes and nerve signaling. The results provide information on the molecular response of these two bee species to Varroa infection.     

Effects at Nearctic north-temperate latitudes of indoor versus outdoor overwintering on the microsporidium Nosema ceranae and western honey bees (Apis mellifera)

In northern temperate climates, western honey bee (Apis mellifera) colonies can be wintered outdoors exposed to ambient conditions, or indoors in a controlled setting. Because very little is known about how this affects the recently-detected microsporidium Nosema ceranae, we investigated effects of indoor versus outdoor overwintering on spring N. ceranae intensity (spores per bee), and on winter and spring colony mortality. For colonies medicated with Fumagilin-B® to control N. ceranae, overwintering treatment did not affect N. ceranae intensity, despite outdoor-wintered colonies having significantly greater mortality. These findings suggest that N. ceranae may not always pose the most significant threat to western honey bees, and that indoor-wintering may ensure that a greater number of colonies are available for honey production and pollination services during the summer.     

Thermoregulation in mixed-species colonies of honeybees (Apis cerana and Apis mellifera)

Apis cerana and Apis mellifera normally display different strategies in cooling hive temperature, raising the question whether they would coordinate their efforts in to achieve stable thermoregulation in mixed colonies. The results show that the normal temperatures in the brood area in mixed colonies are more similar to those of pure A. cerana colonies than pure A. mellifera colonies. Under heat stress, A. cerana workers are more sensitive, and initiate fanning earlier than A. mellifera workers. In mixed colonies, the former become the main force for thermoregulation. When worker bees of both species were fanning together at the entrance, their own species-specific postures were adopted, but due to a significantly smaller number of A. mellifera workers engaged in fanning, the cooling efficiency of mixed colonies were closest to that of pure A. cerana colonies.     

Detection of aerosolized bacterial spores (Bacillus atrophaeus) using free-flying honey bees (Hymenoptera: Apidae) as collectors

An aerosol cloud of Bacillus atrophaeus (previously B. subtilis variety niger) spores, an anthrax surrogate, was created in a large 0.4 ha (1 ac), bee-containing, open-mesh tent. Bees from a B. atrophaeus uncontaminated hive flying through the cloud adsorbed the spores in statistically significant quantities. After removal of the B. atrophaeus contaminated hive and introduction of another B. atrophaeus uncontaminated hive, the bees again were monitored for the next few days for B. atrophaeus spores. B. atrophaeus spores accumulated on the bees bodies following their exposure to the residual B. atrophaeus contamination in the tent. The spore loads on the bees quickly returned to background levels after the hives were removed from the contaminated tent area. It may therefore be practical to use honey bee colonies to monitor foraging areas for disease-causing spores.     

Regulation of hypopharyngeal gland activity and oogenesis in honey bee (Apis mellifera) workers

In the honey bee, vitellogenin has several functions in addition to egg provisioning. Among others, it serves as a precursor to brood food proteins secreted by the hypopharyngeal glands of worker bees. In queenless workers with developing gonads, oogenesis and development of the hypopharyngeal glands are correlated. Here we describe two experiments that explored whether this relationship also exists in non-reproductive workers, and investigated a possible role of ecdysteroid hormones in the regulation of vitellogenin uptake. In the first experiment, the correlation between oocyte length and hypopharyngeal gland development was measured in workers before and after de-queening. In the second experiment, we induced middle-aged bees with resting glands to suddenly initiate brood care behaviour, and measured haemolymph ecdysteroid and vitellogenin titres. A strong positive relationship existed between morphometrical parameters of hypopharyngeal glands and ovaries in both queenless and queenright (functionally sterile) workers. No response of ecdysteroid titres to the addition of brood was detected in experiment 2, but high concentrations were measured in a small group of bees characterised by the possession of oocytes on the brink of yolk incorporation. We conclude that hypopharyngeal glands may belong to a previously described group of reproduction-related traits that are pleiotropically regulated in workers. A possible role for ecdysteroids in honey bee reproduction is discussed.     

Deformed wing virus in western honey bees (Apis mellifera) from Atlantic Canada and the first description of an overtly-infected emerging queen

Deformed wing virus (DWV) in western honey bees (Apis mellifera) often remains asymptomatic in workers and drones, and symptoms have never been described from queens. However, intense infections linked to parasitism by the mite Varroa destructor can cause worker wing deformity and death within 67 h of emergence. Ten workers (eight with deformed wings and two with normal wings) and three drones (two with deformed wings and one with normal wings) from two colonies infected with V. destructor from Nova Scotia, Canada, and two newly-emerged queens (one with deformed wings and one with normal wings) from two colonies infected with V. destructor from Prince Edward Island, Canada, were genetically analyzed for DWV. We detected DWV in all workers and drones, regardless of wing morphology, but only in the deformed-winged queen. This is the first report of DWV from Atlantic Canada and the first detection of a symptomatic queen with DWV from anywhere.     

Effect of primer pheromones and pollen diet on the food producing glands of worker honey bees (Apis mellifera L.)

Cooperative brood care is highly developed in the honey bee such that workers called nurses use their hypopharyngeal and mandibular glands to biosynthesize proteinaceous secretions that are progressively provisioned to larvae. The role that honey bee primer pheromones play in the functional physiology of food producing glands was examined. The combined and separate effects of queen mandibular pheromone (QMP) and brood pheromone (BP) on amount of protein extractable from hypopharyngeal and mandibular glands of workers reared for 12 days with and without pollen diets was measured. In rearing environments with a pollen diet, BP, and QMP + BP pheromone treatments significantly increased extractable protein from both glands. Bees reared with QMP + pollen had amounts of protein extractable from both glands that were not significantly different from control bees (no pheromones, no pollen). Pollen in the diet alone significantly increased amounts of protein extractable from glands versus control. In rearing environments without pollen, QMP + BP had a synergizing effect on amount of protein in both glands. The QMP + BP treatment was the only rearing environment without a pollen diet where protein amounts were significantly greater than the control. The synergizing effect of QMP + BP on extractable mandibular and hypopharyngeal gland protein suggests a highly derived role for the combined effect of these two primer pheromones on honey bee cooperative brood care. Mandibular gland area was significantly and positively correlated with extractable protein. Amounts of extractable protein from both glands declined significantly with age of workers in all treatments. However, treatment significantly affected rate of decline. The adaptive significance of gland protein amounts in response to pheromones and pollen diet are discussed.     

Adaptive spatial biases in nectar deposition in the nests of honey bees

Honey bees store their large supply of honey at the top and, to a lesser extent, along the edges of their nests. Whether this pattern is the result of preferences for where nectar is deposited is unclear. Camazine, in a path breaking study of pattern formation, found evidence that workers deposit nectar at random, while Free and Williams in an earlier study found evidence that bees prefer to deposit nectar into particular types of comb. Here we reexamine this question. We tested three hypotheses, all of which posit that bees have adaptive biases which allow them to deposit nectar directly into the most useful locations. We found that bees have preferences for depositing nectar into cells at the top of the nest, into old vs. new comb, and into interior facing comb vs. comb facing the exterior environment. These preferences may ensure that nectar is placed directly into those regions where it will ultimately be stored. They may also result in an optimal pattern of comb usage with respect to brood rearing. This work, along with the results of previous studies, suggests that comb usage patterns may reflect competing selective pressures and be more complicated than previously thought. Received 17 May 2007; revised 15 June 2007; accepted 3 July 2007.     

The generalization of directional visual stimuli in the honey bee, Apis mellifera

In transfer tests the ability of bees to generalize visual stimuli was tested by using differently inclined stripes and stripe patterns offered on a vertical screen. After having been trained to single stripes or equidistant stripe patterns, which were orientated by α₊ = 45° to the horizontal, the bees had to discriminate between the training direction α₊ and the competition direction α_c = 135° by means of special stripe configurations. These transfer patterns were obtained by varying different stimulus parameters of the original training stripes, for example by (1) reversing contrast between a stripe and the surrounding visual field, (2) changing the ratio of length/width and by this the dimensions of the stripe, and (3) inserting white intervals into the black stripes. In all three test series the bees succeeded in detecting the α₊-direction along a broad range of stimulus variations. As the bees in the transfer tests positively responded to patterns, which on the other side were significantly discriminated from the training pattern (control tests), the information about the direction of the visual cue had been transferred to a new pattern configuration never seen by the bees during the training situation.     

Venom replenishment,as indicated by histamine,in honey bee (Apis mellifera) venom

Venom resynthesis, as indicated by histamine, has been investigated in the venom system of worker honey bees.After depletion of the venom contained in the venom reservoir, by electrical “milking”, histamine resynthesis was most active between 18 and 24 hr after “milking”.The ability of a bee to resynthesise histamine is age dependent. Young bees show some resynthetic ability, two week old bees show maximal ability to replace histamine. This capability is gradually lost as bees age and is virtually absent in 40 day old bees.     

Nosema ceranae has been present in Brazil for more than three decades infecting Africanized honey bees

Until the mid-1990s, the only microsporidium known to infect bees of the genus Apis was Nosema apis. A second species, Nosema ceranae, was first identified in 1996 from Asian honey bees; it is postulated that this parasite was transmitted from the Asian honey bee, Apis cerana, to the European honey bee, Apis mellifera. Currently, N. ceranae is found on all continents and has often been associated with honey bee colony collapse and other reports of high bee losses. Samples of Africanized drones collected in 1979, preserved in alcohol, were analyzed by light microscopy to count spores and were subjected to DNA extraction, after which duplex PCR was conducted. All molecular analyses (triplicate) indicated that the drones were infected with both N. ceranae and N. apis. PCR products were sequenced and matched to sequences reported in the GenBank (Acc. Nos. JQ639316.1 and JQ639301.1). The venation pattern of the wings of these males was compared to those of the current population living in the same area and with the pattern of drones collected in 1968 from Ribeirão Preto, SP, Brazil, from a location close to where African swarms first escaped in 1956. The morphometric results indicated that the population collected in 1979 was significantly different from the current living population, confirming its antiquity. Considering that the use of molecular tools for identifying Nosema species is relatively recent, it is possible that previous reports of infections (which used only light microscopy, without ultrastructural analysis) wrongly identified N. ceranae as N. apis. Although we can conclude that N. ceranae has been affecting Africanized honeybees in Brazil for at least 34 years, the impact of this pathogen remains unclear.     

Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay

The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR-RFLP diagnosis, 29 samples of infected honeybees obtained in 2007-2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant.