Molecular Prevalence of Acarapis Mite Infestations in Honey Bees in Korea

Acarapis mites, including Acarapis woodi, Acarapis externus, and Acarapis dorsalis, are parasites of bees which can cause severe damage to the bee industry by destroying colonies and decreasing honey production. All 3 species are prevalent throughout many countries including UK, USA, Iran, Turkey, China, and Japan. Based on previous reports of Acarapis mites occurring in northeast Asia, including China and Japan, we investigated a survey of Acarapis mite infestations in honey bees in Korean apiaries. A total of 99 colonies of Apis mellifera were sampled from 5 provinces. The head and thorax of 20 bees from each colony were removed for DNA extraction. PCR assays were performed with 3 primer sets, including T, A, and K primers. Results indicated that 42.4% (42/99) of samples were Acarapis-positive by PCR assay which were sequenced to identify species. Each sequence showed 92.6-99.3% homology with reference sequences. Based on the homology, the number of colonies infected with A. dorsalis was 32 which showed the highest infection rate among the 3 species, while the number of colonies infected with A. externus and A. woodi was 9 and 1, respectively. However, none of the Acarapis mites were morphologically detected. This result could be explained that all apiaries in the survey used acaricides against bee mites such as Varroa destructor and Tropilaelaps clareae which also affect against Acarapis mites. Based on this study, it is highly probable that Acarapis mites as well as Varroa and Tropilaelaps could be prevalent in Korean apiaries.     

Caste, Sex and Strain of Honey Bees (Apis mellifera) Affect Infestation with Tracheal Mites (Acarapis woodi)*

Worker honey bees from genetic strains selected for being resistant (R) or susceptible (S) to tracheal mites typically show large differences in infestation in field colonies and in bioassays that involve controlled exposure to infested bees. We used bioassays exposing newly emerged individuals to infested workers to compare the propensity for tracheal mites to infest queens, drones and workers from R and S colonies. In tests with queens, newly emerged R and S queens were either simultaneously confined in infested colonies (n = 95 and 87 respectively), or individually caged with groups of 5–20 infested workers (n = 119 and 115 respectively). Mite prevalence (percentage of individuals infested) and abundance (foundress mites per individual) after 4–6 days did not differ between R and S queens. In another test, five newly emerged drones and workers from both an R and an S colony, and a queen of one of the two strains, were caged in each of 38 cages with 20 g of workers infested at 60–96% prevalence. Infestations of the R queens (n = 17) and S queens (n = 19) did not differ significantly, but R workers had half the mite abundance of S workers, while R drones received about a third more migrating mites than S drones. In tests to evaluate possible mechanisms, removal of one mesothoracic leg from R and S workers resulted in 2- to 10-fold increase in mite abundance on the treated side, but excising legs did not affect infestation of the corresponding tracheae in drones. This suggests that differences in infestation between R and S workers, but not drones, are largely determined by their ability to remove mites through autogrooming. If autogrooming is the primary mechanism of colony resistance to tracheal mites, selection for resistance to tracheal mites using infestation of hemizygous drones may be inefficient. *The U.S. Government’s right ot retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

Intensively Cultivated Landscape and Varroa Mite Infestation Are Associated with Reduced Honey Bee Nutritional State

As key pollinators, honey bees are crucial to many natural and agricultural ecosystems. An important factor in the health of honey bees is the availability of diverse floral resources. However, in many parts of the world, high-intensity agriculture could result in a reduction in honey bee forage. Previous studies have investigated how the landscape surrounding honey bee hives affects some aspects of honey bee health, but to our knowledge there have been no investigations of the effects of intensively cultivated landscapes on indicators of individual bee health such as nutritional physiology and pathogen loads. Furthermore, agricultural landscapes in different regions vary greatly in forage and land management, indicating a need for additional information on the relationship between honey bee health and landscape cultivation. Here, we add to this growing body of information by investigating differences in nutritional physiology between honey bees kept in areas of comparatively low and high cultivation in an area generally high agricultural intensity in the Midwestern United States. We focused on bees collected directly before winter, because overwintering stress poses one of the most serious problems for honey bees in temperate climates. We found that honey bees kept in areas of lower cultivation exhibited higher lipid levels than those kept in areas of high cultivation, but this effect was observed only in colonies that were free of Varroa mites. Furthermore, we found that the presence of mites was associated with lower lipid levels and higher titers of deformed wing virus (DWV), as well as a non-significant trend towards higher overwinter losses. Overall, these results show that mite infestation interacts with landscape, obscuring the effects of landscape alone and suggesting that the benefits of improved foraging landscape could be lost without adequate control of mite infestations.     

Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection.     

Resource Partitioning between Highly Eusocial Bees and Possible Impact of the Introduced Africanized Honey Bee on Native Stingless Bees in the Brazilian Atlantic Rainforest

The highly eusocial bee community of the neotropical Atlantic Rainforest was studied at Boracéia Biological Station in the state of São Paulo, Brazil. In this reserve, 17 species of stingless bees and the introduced Africanized honey bee were found, the latter being the most abundant flower visitor. Of all flowering plants, Asteraceae and Myrtaceae were particularly important as resources for bees. Trophic niche overlap between the various species of stingless bees is evident, and it was generally larger within the tribes Meliponini and Trigonini than between members of different tribes. Nevertheless, in the stingless bee community the competitive pressure is rather uniformly spread. The trophic niche of the Africanized honey bee can be positioned between those of Meliponini and Trigonini. Today this introduced species represents the main competitor in this bee community. However, its impact on native stingless bee populations is apparently buffered by mass-flowering trees which are the most important food plants of the indigenous highly eusocial bees.     

Pollen Contamination of Honey by Bees inside the Hive

This paper aims to assess to what degree and in what way the brood chamber affects the pollen content of the honey. Twenty-nine pieces of comb containing only honey were cut from different frames of hives. The percentage of cells in each frame occupied by the brood chamber and the distance between these cells and the cut piece were recorded. A honey sample was extracted from each comb piece, avoiding any contamination with pollen, its sediment examined under the microscope and its botanical constituents identified and counted. The results show that the pollen content of honey was higher in samples from frames containing brood or pollen cells; in these samples the pollen content was positively correlated with the amount of these cells in the frame and the proximity of the honey to them. The proportion of pollen grains from principally nectariferous plants was lower in honeys with a high pollen content.     

Methods for Comparing Nutrients in Beebread Made by Africanized and European Honey Bees and the Effects on Hemolymph Protein Titers

Honey bees obtain nutrients from pollen they collect and store in the hive as beebread. We developed methods to control the pollen source that bees collect and convert to beebread by placing colonies in a specially constructed enclosed flight area. Methods were developed to analyze the protein and amino acid composition of the pollen and beebread. We also describe how consumption of the beebread was measured and methods used to determine adult worker bee hemolymph protein titers after feeding on beebread for 4, 7 and 11 days after emergence. Methods were applied to determine if genotype affects the conversion of pollen to beebread and the rate that bees consume and acquire protein from it. Two subspecies (European and Africanized honey bees; EHB and AHB respectively) were provided with the same pollen source. Based on the developed methods, beebread made by both subspecies had lower protein concentrations and pH values than the pollen. In general, amino acid concentrations in beebread made by either EHB or AHB were similar and occurred at higher levels in beebread than in pollen. Both AHB and EHB consumed significantly more of the beebread made by AHB than by EHB. Though EHB and AHB consumed similar amounts of each type of beebread, hemolymph protein concentrations in AHB were higher than in EHB. Differences in protein acquisition between AHB and EHB might reflect environmental adaptations related to the geographic region where each subspecies evolved. These differences could contribute to the successful establishment of AHB populations in the New World because of the effects on brood rearing and colony growth.     

The Nutrimentary Egg Development of the Mite,Varroa jacobsoni (Acari,Arachnida), an Ectoparasite of Honey Bees

The fine structure of the female gonad of Varroa jacobsoni is described. There are two components: the ovary proper and the so-called lyrate organ. The ovary is the place where oocytes mature, embedded in a supporting tissue composed of two cell types: somacells 1 and somacells 2. The lyrate organ has a nutrimentary function and is comprised of two components: supporting cells and nutritive tissue. The supporting cells are similar to the somacells 2 in that they contain abundant microtubules. The nutritive tissue is an extensive syncytium. It is connected with the oocytes via intercellular bridges, the nutritive cords. Oocytes and nutritive tissue are thought to have derived from common stem cells. From fine structural evidence it is concluded that ribosomes are one of the most important components to be transported via the nutritive cords into the oocytes. However, an increase in number of mitochondria in the middle-stage oocytes may also be a consequence of transport of these organelles from the nutritive tissue into the oocytes. Further characteristics make plausible that the interdependences of oocytes and nutritive tissue are comparable to those found in meroistic ovarioles of insects. The somatic components do not seem to be as important as the follicle cells of insects, however. It is assumed that the evolution of a nutrimentary oogenesis speeds up embryogenesis. Thus, the differentiation of the female gonad of Varroa jacobsoni may have facilitated the species' adaptation to a development completed in a short and limited time within the shelter of the covered brood cell of the bee.     

Overwintering Is Associated with Reduced Expression of Immune Genes and Higher Susceptibility to Virus Infection in Honey Bees

The eusocial honey bee, Apis mellifera, has evolved remarkable abilities to survive extreme seasonal differences in temperature and availability of resources by dividing the worker caste into two groups that differ in physiology and lifespan: summer and winter bees. Most of the recent major losses of managed honey bee colonies occur during the winter, suggesting that winter bees may have compromised immune function and higher susceptibility to diseases. We tested this hypothesis by comparing the expression of eight immune genes and naturally occurring infection levels of deformed wing virus (DWV), one of the most widespread viruses in A. mellifera populations, between summer and winter bees. Possible interactions between immune response and physiological activity were tested by measuring the expression of vitellogenin and methyl farnesoate epoxidase, a gene coding for the last enzyme involved in juvenile hormone biosynthesis. Our data show that high DWV loads in winter bees correlate with reduced expression of genes involved in the cellular immune response and physiological activity and high expression of humoral immune genes involved in antibacterial defense compared with summer bees. This expression pattern could reflect evolutionary adaptations to resist bacterial pathogens and economize energy during the winter under a pathogen landscape with reduced risk of pathogenic viral infections. The outbreak of Varroa destructor infestation could have overcome these adaptations by promoting the transmission of viruses. Our results suggest that reduced cellular immune function during the winter may have increased honey bee’s susceptibility to DWV. These results contribute to our understanding of honey bee colony losses in temperate regions.     

Organic Tracers from Asphalt in Propolis Produced by Urban Honey Bees,Apis mellifera Linn.

Propolis is a gummy material produced by honey bees to protect their hives and currently has drawn the attention of researchers due to its broad clinical use. It has been reported, based only on observations, that honey bees also collect other non-vegetation substances such as paint or asphalt/tar to make propolis. Therefore, propolis samples were collected from bee hives in Riyadh and Al-Bahah, a natural area, Saudi Arabia to determine their compositional characteristics and possible sources of the neutral organic compounds. The samples were extracted with hexane and analyzed by gas chromatography-mass spectrometry. The results showed that the major compounds were n-alkanes, n-alkenes, methyl n-alkanoates, long chain wax esters, triterpenoids and hopanes. The n-alkanes (ranging from C₁₇ to C₄₀) were significant with relative concentrations varying from 23.8 to 56.8% (mean = 44.9+9.4%) of the total extracts. Their odd carbon preference index (CPI) ranged from 3.6 to 7.7, with a maximum concentration at heptacosane indicating inputs from higher plant vegetation wax. The relative concentrations of the n-alkenes varied from 23.8 to 41.19% (mean = 35.6+5.1%), with CPI = 12.4-31.4, range from C₂₅ to C₃₅ and maximum at tritriacontane. Methyl n-alkanoates, ranged from C₁₂ to C₂₆ as acids, with concentrations from 3.11 to 33.2% (mean = 9.6+9.5%). Long chain wax esters and triterpenoids were minor. The main triterpenoids were α- and β-amyrins, amyrones and amyryl acetates. The presence of hopanes in some total extracts (up to 12.5%) indicated that the bees also collected petroleum derivatives from vicinal asphalt and used that as an additional ingredient to make propolis. Therefore, caution should be taken when considering the chemical compositions of propolis as potential sources of natural products for biological and pharmacological applications. Moreover, beekeepers should be aware of the proper source of propolis in the flight range of their bee colonies.     

Static Electricity in the Spatial Orientation and Signaling of Honey Bees

A static electric charge that foraging bees gain by rubbing their body parts against the substrate surface plays a role in social communication of bees. Vibrations of the charged body of a forager with a frequency of approximately 14 Hz indicate the location of the forager bee in a rich variety of bees within a poorly lit nest. The perception of vibrations of the charged ventral body surface of a forager occurs due to the antennal sensory organs of hive mates. An electric charge produced as the foraging bee moves its wings enhances vibrations of the trichoid sensilla, which act as mechanoreceptors. This provides reliable communication between foraging bees and their hive mates.     

The Role of Cuticular Compounds in the Resistance of Honey Bees (Apis Mellifera) to Tracheal Mites (Acarapis Woodi)

This study examined the migration of tracheal mites (Acarapis woodi) into honey bees (Apis mellifera) from different colonies and the relative attraction of mites to hexane extracts from the external body surfaces of young bees. Relative resistance of bees from different colonies initially was assessed with a field bioassay that involved tagging newly emerged bees, pooling them in heavily mite-infested colonies, retrieving them 7 days later, and examining them for tracheal mite prevalence and abundance. For those colonies identified as most resistant and least resistant, cuticular chemicals were extracted in hexane from frozen, newly emerged worker bees. These extracts were presented to individual tracheal mites in pairwise fashion in a laboratory bioassay. The results demonstrated that mites prefer extracts of bees from some colonies more than others, however, no consistent differences were demonstrated. Our inability to predict mite responses to extracts based on our initial assessment of relative resistance indicates that other mechanisms of resistance influence mite success in colonizing new host bees.     

Colony Integration in Honey Bees: Mechanisms of Behavioral Reversion

After confirming that worker honey bees (Apis mellifera) can revert from foraging to brood care, we determined whether juvenile hormone (JH) mediates this form of plasticity in behavioral development and whether worker age and genotype influence the probability of its expression. Measurements of JH titers support the hypothesis that plasticity in honey bee behavioral development is a consequence of modulation of JH by extrinsic factors. Observations of individually marked bees in a colony composed of two phenotypically distinguishable subfamilies revealed that the likelihood of undergoing behavioral reversion was influenced by worker age but not by worker genotype. The effect of worker age on reversion is consistent with a previously formulated model for the regulation of age polyethism in honey bees that predicts that workers of different ages have different response thresholds for task-associated stimuli. The lack of a genotypic effect on reversion is in contrast to results for other forms of behavioral plasticity.     

Nest Defense Behavior in Colonies from Crosses Between Africanized and European Honey Bees (Apis mellifera L.) (Hymenoptera: Apidae)

Honey bee (Apis mellifera L.) colonies with either European or Africanized queens mated to European or Africanized drones alone or in combination were tested for defensive behavior using a breath test. The most defensive colonies were those with European or Africanized queens mated to Africanized drones. In colonies where both European and Africanized patrilines existed, most of the workers participating in nest defense behavior for the first 30 s after a disturbance were of African patrilines. Nest defense behavior appears to be genetically dominant in honey bees.     

Metabolomics Reveals the Origins of Antimicrobial Plant Resins Collected by Honey Bees

The deposition of antimicrobial plant resins in honey bee, Apis mellifera, nests has important physiological benefits. Resin foraging is difficult to approach experimentally because resin composition is highly variable among and between plant families, the environmental and plant-genotypic effects on resins are unknown, and resin foragers are relatively rare and often forage in unobservable tree canopies. Subsequently, little is known about the botanical origins of resins in many regions or the benefits of specific resins to bees. We used metabolomic methods as a type of environmental forensics to track individual resin forager behavior through comparisons of global resin metabolite patterns. The resin from the corbiculae of a single bee was sufficient to identify that resin''s botanical source without prior knowledge of resin composition. Bees from our apiary discriminately foraged for resin from eastern cottonwood (Populus deltoides), and balsam poplar (P. balsamifera) among many available, even closely related, resinous plants. Cottonwood and balsam poplar resin composition did not show significant seasonal or regional changes in composition. Metabolomic analysis of resin from 6 North American Populus spp. and 5 hybrids revealed peaks characteristic to taxonomic nodes within Populus, while antimicrobial analysis revealed that resin from different species varied in inhibition of the bee bacterial pathogen, Paenibacillus larvae. We conclude that honey bees make discrete choices among many resinous plant species, even among closely related species. Bees also maintained fidelity to a single source during a foraging trip. Furthermore, the differential inhibition of P. larvae by Populus spp., thought to be preferential for resin collection in temperate regions, suggests that resins from closely related plant species many have different benefits to bees.     

Structural Conversion from Non-native to Native form of Recombinant Human Epidermal Growth Factor by Brevibacillus choshinensis

Brevibacillus choshinensis (Bacillus brevis) HPD31 is a very efficient producer of recombinant human epidermal growth factor (EGF). The produced EGF is secreted into the medium with high efficiency. However part of the EGF that accumulates in the medium, exists as multimeric forms which are biologically inactive. We found the bacterium has the activity to structurally convert multimeric forms to the monomeric, native ones. Optimal temperature and pH for the conversion were 40°C and pH 9, respectively. The reaction was promoted in the presence of reduced glutathione or cysteine. But the cells which had been sonicated or exposed to moderate heat treatment completely lost the activity. Thus, it was presumed that the activity might be due to the enzyme(s) that catalyze the protein disulfide exchanging reaction, and that they resides on the surface of viable cells.     

Range and Frequency of Africanized Honey Bees in California (USA)

Africanized honey bees entered California in 1994 but few accounts of their northward expansion or their frequency relative to European honey bees have been published. We used mitochondrial markers and morphometric analyses to determine the prevalence of Africanized honeybees in San Diego County and their current northward progress in California west of the Sierra Nevada crest. The northernmost African mitotypes detected were approximately 40 km south of Sacramento in California’s central valley. In San Diego County, 65% of foraging honey bee workers carry African mitochondria and the estimated percentage of Africanized workers using morphological measurements is similar (61%). There was no correlation between mitotype and morphology in San Diego County suggesting Africanized bees result from bidirectional hybridization. Seventy percent of feral hives, but only 13% of managed hives, sampled in San Diego County carried the African mitotype indicating that a large fraction of foraging workers in both urban and rural San Diego County are feral. We also found a single nucleotide polymorphism at the DNA barcode locus COI that distinguishes European and African mitotypes. The utility of this marker was confirmed using 401 georeferenced honey bee sequences from the worldwide Barcode of Life Database. Future censuses can determine whether the current range of the Africanized form is stable, patterns of introgression at nuclear loci, and the environmental factors that may limit the northern range of the Africanized honey bee.     

Social Modulation of Stress Reactivity and Learning in Young Worker Honey Bees

Alarm pheromone and its major component isopentylacetate induce stress-like responses in forager honey bees, impairing their ability to associate odors with a food reward. We investigated whether isopentylacetate exposure decreases appetitive learning also in young worker bees. While isopentylacetate-induced learning deficits were observed in guards and foragers collected from a queen-right colony, learning impairments resulting from exposure to this pheromone could not be detected in bees cleaning cells. As cell cleaners are generally among the youngest workers in the colony, effects of isopentylacetate on learning behavior were examined further using bees of known age. Adult workers were maintained under laboratory conditions from the time of adult emergence. Fifty percent of the bees were exposed to queen mandibular pheromone during this period, whereas control bees were not exposed to this pheromone. Isopentylacetate-induced learning impairments were apparent in young (less than one week old) controls, but not in bees of the same age exposed to queen mandibular pheromone. This study reveals young worker bees can exhibit a stress-like response to alarm pheromone, but isopentylacetate-induced learning impairments in young bees are suppressed by queen mandibular pheromone. While isopentylacetate exposure reduced responses during associative learning (acquisition), it did not affect one-hour memory retrieval.     

Influence of Unrewarded Stimuli on the Classification of Visual Patterns by Honey Bees

Bees were trained to discriminate visual patterns in five experiments. The rewarded pattern (S+), was a 50-mm black disc in all experiments; the unrewarded pattern (S–) was varied. Subsequently bees were given a choice between different stimuli in order to discover what bees learnt about five attributes of the training stimuli. The attributes tested were size, contrast, color, ‘compactness’ vs. ‘dissectedness’ (tests with ring-patterns), and presence or absence of acute points (tests with discs, squares, triangles and stars). The significance of these attributes varied with the particular unrewarded pattern (S–) used in training (Figs. 1, 2). This is interpreted as a modification of the bee's selective attention to certain features during training. The results also indicate a difference in the salience of attributes. Differences in size or outline (presence of acute points) only influenced the bee's preference after a training that specifically required this distinction, while differences in contrast, colour and dissectedness were also significant when the training stimuli did not differ in that respect.     

Host-Parasite Interactions and Purifying Selection in a Microsporidian Parasite of Honey Bees

To clarify the mechanisms of Nosema ceranae parasitism, we deep-sequenced both honey bee host and parasite mRNAs throughout a complete 6-day infection cycle. By time-series analysis, 1122 parasite genes were significantly differently expressed during the reproduction cycle, clustering into 4 expression patterns. We found reactive mitochondrial oxygen species modulator 1 of the host to be significantly down regulated during the entire infection period. Our data support the hypothesis that apoptosis of honey bee cells was suppressed during infection. We further analyzed genome-wide genetic diversity of this parasite by comparing samples collected from the same site in 2007 and 2013. The number of SNP positions per gene and the proportion of non-synonymous substitutions per gene were significantly reduced over this time period, suggesting purifying selection on the parasite genome and supporting the hypothesis that a subset of N. ceranae strains might be dominating infection.