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.     

Effect of royal jelly on longevity and memory-related traits of Apis mellifera workers

Royal jelly (RJ) is a key factor for honey bee caste determination. The queen bee is fed with RJ by worker bees throughout her life, while the worker bees eat bee bread themselves. This study was designed to explore the effect of nutrient-rich RJ on longevity and learning and memory abilities of workers of the western honey bee Apis mellifera. The newly emerged worker bees were randomly divided into three groups and were fed 50% sucrose solution containing 0%, 10%, and 20% RJ. We found that worker bees fed with 10% and 20% RJ showed significantly improved longevity and higher proboscis extension response success rate compared to bees fed with 50% sucrose containing 0% RJ. Additionally, bees fed with 20% RJ showed significantly higher level of expression of memory related genes (GluRA and Nmdar1) compared to the control group. Furthermore, expression of the Nmdar1 gene of worker bees fed with 10% RJ was also significantly higher than in the control group. These results indicate that RJ has potential effects on the longevity and learning and memory abilities of A. mellifera.     

Enterobacteriaceae islated from honey bees,Apis mellifera,treated with 2,4-D and antibiotics

Klebsiella pneumoniae, Shigella sp., Enterobacter cloacae, Escherichia coli, Enterobacter hafniae, Arizona sp., Enterobacter aerogenes, Serratia liquefaciens, and Citrobacter sp. were isolated from the intestinal contents of honey bees, Apis mellifera, which were obtained either from untreated colonies, from colonies fed the herbicide 2,4-D, or from colonies fed a combination of oxytetracycline and fumagillin. Antibiotics depressed the growth of Enterobacteriaceae; 2,4-D had little effect on the enteric microflora of bees.     

Comparative Sucrose Responsiveness in Apis mellifera and A. cerana Foragers

In the European honey bee, Apis mellifera, pollen foragers have a higher sucrose responsiveness than nectar foragers when tested using a proboscis extension response (PER) assay. In addition, Africanized honey bees have a higher sucrose responsiveness than European honey bees. Based on the biology of the Eastern honey bee, A. cerana, we hypothesized that A. cerana should also have a higher responsiveness to sucrose than A. mellifera. To test this hypothesis, we compared the sucrose thresholds of pollen foragers and nectar foragers in both A. cerana and A. mellifera in Fujian Province, China. Pollen foragers were more responsive to sucrose than nectar foragers in both species, consistent with previous studies. However, contrary to our hypothesis, A. mellifera was more responsive than A. cerana. We also demonstrated that this higher sucrose responsiveness in A. mellifera was not due to differences in the colony environment by co-fostering two species of bees in the same mixed-species colonies. Because A. mellifera foragers were more responsive to sucrose, we predicted that their nectar foragers should bring in less concentrated nectar compared to that of A. cerana. However, we found no differences between the two species. We conclude that A. cerana shows a different pattern in sucrose responsiveness from that of Africanized bees. There may be other mechanisms that enable A. cerana to perform well in areas with sparse nectar resources.     

Fungi isolated from honey bees, Apis mellifera, fed 2,4-D and Antibiotics

Eighteen species of fungi were isolated and identified from the intestines of 388 honey bees, Apis mellifera. Bees fed a combination of oxytetracycline and fumagillin contained fewer fungi than control bees or bees fed 2,4-D. New records of fungi associated with honey bees include Alternaria tenuissima, Cladosporium cladosporoides, Bipolaris sp., Curvularia brachyospora, Penicillium ochro-chloron, Penicillium urticae, and Rhizopus arrhizus.     

Gram-positive cocci from apiarian sources

Frass from the greater wax moth, Galleria mellonella, obtained from feral colonies of honey bees, Apis mellifera; from domesticated (managed) honey bee colonies; and from a laboratory culture of the wax moth was sampled for Gram-positive cocci. One hundred twenty-three of these organisms were isolated and identified. Frass from domesticated colonies yielded only one isolate. Equal numbers of isolates (61) were obtained from frass from feral bee colonies and from the wax moth culture. Catalase-negative cocci were predominant in frass from feral colonies, whereas catalase-positive cocci were the most common isolates from frass from the wax moth culture. Catalase-positive cocci were identified as Staphylococcus epidermidis and Micrococcus sp. Catalase-negative cocci were Streptococcus faecalis var. faecalis and S. faecium. These results are discussed in relation to the rarity of Gram-positive cocci associated with honey bees, pollen, and nectar in Arizona and the frequency of association with honey bees and wax moth frass of bacteria resembling Arthrobacter spp. that appear as Gram-positive cocci during one stage of the life cycle.     

Natural selection for 2,4,5-trichlorophenoxyacetic acid mineralizing bacteria in agent orange contaminated soil

Agent Orange contaminated soils were utilized in direct enrichment culture studies to isolate 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and 2,4-dichlorophenoxyacetic acid (2,4-D) mineralizing bacteria. Two bacterial cultures able to grow at the expense of 2,4,5-T and/or 2,4-D were isolated. The 2,4,5-T degrading culture was a mixed culture containing two bacteria, Burkholderia species strain JR7B2 and Burkholderia species strain JR7B3. JR7B3 was able to metabolize 2,4,5-T as the sole source of carbon and energy, and demonstrated the ability to affect metabolism of 2,4-D to a lesser degree. Strain JR7B3 was able to mineralize 2,4,5-T in pure culture and utilized 2,4,5-T in the presence of 0.01 yeast extract. Subsequent characterization of the 2,4-D degrading culture showed that one bacterium, Burkholderiaspecies strain JRB1, was able to utilize 2,4-D as a sole carbon and energy source in pure culture. Polymerase chain reaction (PCR) experiments utilizing known genetic sequences from other 2,4-D and 2,4,5-T degrading bacteria demonstrated that these organisms contain gene sequences similar to tfdA, B, C, E, and R (Strain JRB1) and the tftA, C, and E genes (Strain JR7B3). Expression analysis confirmed that tftA, C, and E and tfdA, B, and C were transcribed during 2,4,5-T and 2,4-D dependent growth, respectively. The results indicate a strong selective pressure for 2,4,5-T utilizing strains under field condition.     

Nosema ceranae, a new parasite in Thai honeybees

Adult workers of Apis cerana, Apis florea and Apis mellifera from colonies heavily infected with Nosema ceranae were selected for molecular analyses of the parasite. PCR-specific 16S rRNA primers were designed, cloned, sequenced and compared to GenBank entries. The sequenced products corresponded to N. ceranae. We then infected A. cerana with N. ceranae spores isolated from A. florea workers. Newly emerged bees from healthy colonies were fed 10,000, 20,000 and 40,000 spores/bee. There were significant dosage dependent differences in bee infection and survival rates. The ratio of infected cells to non-infected cells increased at 6, 10 and 14 d post infection. In addition, hypopharyngeal glands of bees from the control group had significantly higher protein concentrations than infected groups. Bees infected with 40,000 spores/bee had the lowest protein concentrations. Thus, N. ceranae isolated from A. florea is capable of infecting another bee species, impairing hypopharyngeal gland protein production and reducing bee survival in A. cerana.     

First Detection of the Larval Chalkbrood Disease Pathogen Ascosphaera apis (Ascomycota: Eurotiomycetes: Ascosphaerales) in Adult Bumble Bees

Fungi in the genus Ascosphaera (Ascomycota: Eurotiomycetes: Ascosphaerales) cause chalkbrood disease in larvae of bees. Here, we report the first-ever detection of the fungus in adult bumble bees that were raised in captivity for studies on colony development. Wild queens of Bombus griseocollis, B. nevadensis and B. vosnesenskii were collected and maintained for establishment of nests. Queens that died during rearing or that did not lay eggs within one month of capture were dissected, and tissues were examined microscopically for the presence of pathogens. Filamentous fungi that were detected were plated on artificial media containing broad spectrum antibiotics for isolation and identification. Based on morphological characters, the fungus was identified as Ascosphaera apis (Maasen ex Claussen) Olive and Spiltoir, a species that has been reported earlier only from larvae of the European honey bee, Apis mellifera, the Asian honey bee, Apis cerana, and the carpenter bee Xylocopa californica arizonensis. The identity of the fungus was confirmed using molecular markers and phylogenetic analysis. Ascosphaera apis was detected in queens of all three bumble bee species examined. Of 150 queens dissected, 12 (8%) contained vegetative and reproductive stages of the fungus. Both fungal stages were also detected in two workers collected from colonies with Ascosphaera-infected B. nevadensis queens. In this study, wild bees could have been infected prior to capture for rearing, or, the A. apis infection could have originated via contaminated European honey bee pollen fed to the bumble bees in captivity. Thus, the discovery of A. apis in adult bumble bees in the current study has important implications for commercial production of bumble bee colonies and highlights potential risks to native bees via pathogen spillover from infected bees and infected pollen.     

Viral Infection Affects Sucrose Responsiveness and Homing Ability of Forager Honey Bees,Apis mellifera L.

Honey bee health is mainly affected by Varroa destructor, viruses, Nosema spp., pesticide residues and poor nutrition. Interactions between these proposed factors may be responsible for the colony losses reported worldwide in recent years. In the present study, the effects of a honey bee virus, Israeli acute paralysis virus (IAPV), on the foraging behaviors and homing ability of European honey bees (Apis mellifera L.) were investigated based on proboscis extension response (PER) assays and radio frequency identification (RFID) systems. The pollen forager honey bees originated from colonies that had no detectable level of honey bee viruses and were manually inoculated with IAPV to induce the viral infection. The results showed that IAPV-inoculated honey bees were more responsive to low sucrose solutions compared to that of non-infected foragers. After two days of infection, around 10⁷ copies of IAPV were detected in the heads of these honey bees. The homing ability of IAPV-infected foragers was depressed significantly in comparison to the homing ability of uninfected foragers. The data provided evidence that IAPV infection in the heads may enable the virus to disorder foraging roles of honey bees and to interfere with brain functions that are responsible for learning, navigation, and orientation in the honey bees, thus, making honey bees have a lower response threshold to sucrose and lose their way back to the hive.     

Pyrosequencing analysis of the bacterial communities in the guts of honey bees Apis cerana and Apis mellifera in Korea

The bacterial communities in the guts of the adults and larvae of the Asian honey bee Apis cerana and the European honey bee Apis mellifera were surveyed by pyrosequencing the 16S rRNA genes. Most of the gut bacterial 16S rRNA gene sequences were highly similar to the known honey bee-specific ones and affiliated with Pasteurellaceae or lactic acid bacteria (LAB). The numbers of operational taxonomic units (OTUs, defined at 97% similarity) were lower in the larval guts (6 or 9) than in the adult guts (18 or 20), and the frequencies of Pasteurellaceae-related OTUs were higher in the larval guts while those of LAB-related OTUs in the adult guts. The frequencies of Lactococcus, Bartonella, Spiroplasma, Enterobacteriaceae, and Flavobacteriaceae-related OTUs were much higher in A. cerana guts while Bifidobacterium and Lachnospiraceae-related OTUs were more abundant in A. mellfera guts. The bacterial community structures in the midguts and hindguts of the adult honey bees were not different for A. cerana, but significantly different for A. mellifera. The above results substantiated the previous observation that honey bee guts are dominated by several specific bacterial groups, and also showed that the relative abundances of OTUs could be markedly changed depending on the developmental stage, the location within the gut, and the honey bee species. The possibility of using the gut bacterial community as an indicator of honey bee health was discussed.     

Sterols of organs involved in brood food production and of royal jelly in honey bees

The sterols of the organs (hypopharyngeal and mandibular glands and honey stomachs) involved in worker and queen honey bee brood food production, royal jelly and intact nurse bees were analyzed to obtain information on the selective transfer of specific sterols from one generation to the next. No appreciable increase in the percentage of 24-methylenecholesterol, relative to the total sterols isolated from intact honey bee (Apis mellifera L.), prepupae or adults, was found in the hypopharyngeal or mandibular glands or the honey stomachs from nurse bees reared in colonies fed a chemically-defined diet supplemented with 24-methylenecholesterol. The sterols of these organs contained higher levels of cholesterol than did the sterols of whole body extracts. The other major sterols, sitosterol and isofucosterol, occurred at relative concentrations comparable to whole body extracts. Also, there were higher levels of cholesterol in the sterols from glandular tissues of nurse bees maintained on pollen and sucrose solution than in sterols isolated from intact insects. In a separate study, royal jelly collected over a 6-day period had much higher relative percentages of 24-methylenecholesterol and lower levels of sitosterol and isofucosterol than did the pollen fed to these colonies. The sterols of nurse bees in the latter study had an intermediate concentration of 24-methylenecholesterol. The significance of these findings relative to the unique selective transfer of specific sterols from the diet or from endogenous sterol pools of the nurse bees from generation to generation in the honey bee is discussed.     

Survival and health improvement of Nosema infected Apis florea (Hymenoptera: Apidae) bees after treatment with propolis extract

Nosema ceranae is now considered to be an emerging infectious disease of the European honey bee Apis mellifera. Only one antibiotic, Fumagillin, is commercially available to combat Nosema infections. This antibiotic treatment is banned from use in Europe and elsewhere there is a high probability for antibiotic resistance to develop. We are therefore interested in investigating the effects of a natural propolis extract on its ability to reduce N. ceranae infection loads in the dwarf honey bee, Apis florea, a native honey bee with a range that overlaps with Apis cerana and Apis mellifera that is at risk of infection. Experimentally infected caged bees were fed a treatment consisting of 0%, 50%, or 70% propolis extract. All 50% and 70% propolis treated bees had significantly lower infection loads, and the 50% treated bees had higher survival in comparison to untreated bees. In addition, propolis treated bees had significantly higher haemolymph trehalose levels and hypopharyngeal gland protein content similar to levels of uninfected bees. Propolis ethanolic extract treatment could therefore be considered as a possible viable alternative to Fumagillin to improve bee health. This natural treatment deserves further exploration to develop it as a possible alternative to combat N. ceranae infections distributed around the world.     

Experimental infection of red dwarf honeybee,Apis florea,with Nosema ceranae

This research is the first record of the infection of Apis florea by Nosema ceranae, a newly identified pathogen of honeybee in Thailand which was initially isolated from A. florea workers. Each Nosema free-bee was fed 2 μl of 50% (w/v) sucrose solution containing 0, 10,000 20,000 or 40,000 Nosema spores/bee. The survival rates of treated bees were significantly lower compared to control bees. Infectivity was not statistically different among the three spore concentrations, whereas no infection was found in control bees. Protein content of control bee hypopharyngeal glands 14 days post inoculation (p.i) was significantly higher (21.47 ± 0.17 mg/bee) compared to all treatments. The infection ratio of bees treated with 40,000 spores/bee increased with time after inoculation. These results suggest that N. ceranae has a significant negative effect on honeybee hypopharyngeal gland protein production and contributes to their shortened life span.     

Genes for 2,4,5-Trichlorophenoxyacetic Acid Metabolism in Burkholderia cepacia AC1100: Characterization of the tftC and tftD Genes and Locations of the tft Operons on Multiple Replicons

Burkholderia cepacia AC1100 uses the chlorinated aromatic compound 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as a sole source of carbon and energy. The enzyme which converts the first intermediate in the pathway, 2,4,5-trichlorophenol, to 5-chlorohydroquinone has been purified and consists of two subunits of 58 and 22 kDa, encoded by the tftC and tftD genes (48). A degenerate primer was designed from the N terminus of the 58-kDa polypeptide and used to isolate a clone containing the tftC and tftD genes from a genomic library of AC1100. The derived amino acid sequences of tftC and tftD show significant homology to the two-component monooxygenases HadA of Burkholderia pickettii, HpaBC of Escherichia coli, and HpaAH of Klebsiella pneumonia. Expression of the tftC and tftD genes appeared to be induced when they were grown in the presence of 2,4,5-T, as shown by RNA slot blot and primer extension analyses. Three sets of cloned tft genes were used as probes to explore the genomic organization of the pathway. Pulsed-field gel electrophoresis analyses of whole chromosomes of B. cepacia AC1100 demonstrated that the genome is comprised of five replicons of 4.0, 2.7, 0.53, 0.34, and 0.15 Mbp, designated I to V, respectively. The tft genes are located on the smaller replicons: the tftAB cluster is on replicon IV, tftEFGH is on replicon III, and copies of the tftC and the tftCD operons are found on both replicons III and IV. When cells were grown in the absence of 2,4,5-T, the genes were lost at high frequency by chromosomal deletions and rearrangements to produce 2,4,5-T-negative mutants. In one mutant, the tftA and tftB genes translocated from one replicon to another, with the concomitant loss of tftEFGH and one copy of tftCD.     

Seed set in Guayule (Parthenium argentatum,asteraceae) in relation to insect pollination

Guayule (Tarthenium argentatum, Asteraceae) is one of two major plant species grown for natural rubber. Studies were conducted to determine the effect of honey bee (Apis mellifera) pollination and season on seed set and total seed yield/ha. The experiments involved four pollination treatments: plants caged with bees; plants caged without bees; plants open pollinated (uncovered); and plants individually covered. Seeds were harvested monthly July–September 1984, and May–September 1985. Plots with bees produced at least 150% more seeds than plots without bees, and there were no qualitative differences in the seed weights among treatments. Highest seed yield was in May and September. Results indicate that (1) insect pollination in guayule increases seed yield and (2) fewer seeds are produced in the warmest months.     

Characterization of gut bacterial flora of Apis mellifera from north-west Pakistan

Gut microbiota has been recognized to play a beneficial role in honey bees (Apis mellifera). Present study was designed to characterize the gut bacterial flora of honey bees in north-west Pakistan. Total 150 aerobic and facultative anaerobic bacteria from guts of 45 worker bees were characterized using biochemical assays and 16S rDNA sequencing followed by bioinformatics analysis. The gut isolates were classified into three bacterial phyla of Firmicutes (60%), Proteobacteria (26%) and Actinobacteria (14%). Most of the isolates belonged to genera and families of Staphylococcus, Bacillus, Enterococcus, Ochrobactrum, Sphingomonas, Ralstonia, Enterobacteriaceae, Corynebacterium and Micrococcineae. Many of these bacteria were tolerant to acidic environments and fermented sugars, hence considered beneficial gut inhabitants and involved the maintenance of a healthy microbiota. However, several opportunistic commensals that proliferate in the hive environment including members Staphylococcus haemolyticus group and Sphingomonas paucimobilis were also identified. This is the first report on bee gut microbiota from north-west Pakistan geographically situated at the crossroads of Indian subcontinent and central Asia.     

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.     

The Honey Bee Parasite Nosema ceranae: Transmissible via Food Exchange?

Nosema ceranae, a newly introduced parasite of the honey bee, Apis mellifera, is contributing to worldwide colony losses. Other Nosema species, such as N. apis, tend to be associated with increased defecation and spread via a fecal-oral pathway, but because N. ceranae does not induce defecation, it may instead be spread via an oral-oral pathway. Cages that separated older infected bees from young uninfected bees were used to test whether N. ceranae can be spread during food exchange. When cages were separated by one screen, food could be passed between the older bees and the young bees, but when separated by two screens, food could not be passed between the two cages. Young uninfected bees were also kept isolated in cages, as a solitary control. After 4 days of exposure to the older bees, and 10 days to incubate infections, young bees were more likely to be infected in the 1-Screen Test treatment vs. the 2-Screen Test treatment (P = 0.0097). Young bees fed by older bees showed a 13-fold increase in mean infection level relative to young bees not fed by older bees (1-Screen Test 40.8%; 2-Screen Test 3.4%; Solo Control 2.8%). Although fecal-oral transmission is still possible in this experimental design, oral-oral infectivity could help explain the rapid spread of N. ceranae worldwide.     

Phylogenetic analysis of Starmerella apis in honey bees (Apis mellifera)

Honey bees are among the most effective pollinators that promote plant reproduction. Bees are highly active in the pollen collection season, which can lead to the transmission of selected pathogens between colonies. The clade Starmerella comprises yeasts that are isolated mainly from bees and their environment. When visiting plants, bees can come into contact with Starmerella spp. The aim of this study was to determine the prevalence and phylogenetic position of S. apis in bee colonies. Bee colonies were collected from nine apiaries in three regions. Ten colonies were sampled randomly from each apiary, and pooled samples were collected from the central part of the hive in each colony. A total of 90 (100%) bee colonies from nine apiaries were examined. Starmerella apis was detected in 31 (34.44%) samples, but related species were not identified. The 18S rRNA amplicon sequences of S. apis were compatible with the GenBank sequences of Starmerella spp. from India, Japan, Syria, Thailand, and the USA. The amplicon sequences of S. apis were also 99.06% homologous with the sequences deposited in GenBank under accession numbers JX515988 and NG067631 .This is the first study to perform a phylogenetic analysis of S. apis in Polish honey bees.