Mikio Yoshiyama

Bacteria in the gut of Japanese honeybee, Apis cerana japonica, and their antagonistic effect against Paenibacillus larvae, the causal agent of American foulbrood.

We assessed the complexity of bacterial communities occurring in the digestive tract of the Japanese honeybee, Apis cerana japonica, using histological and 16S rRNA gene sequence analyzes. Both Gram-positive and -negative bacteria were observed, and the number of gut bacteria was higher in old larvae compared with young larvae. A total of 35 clones were obtained by a culture-dependent method, and 16S rRNA gene sequence analysis revealed that the bacterial population in the gut of Japanese honeybee was diverse, including the phyla firmicutes, actinobacteria, and alpha-, beta-, and gammaproteobacteria. Further investigation by in vitro inhibition assays was carried out to determine the ability of an isolate to inhibit Paenibacillus larvae, the causal agent of American foulbrood. Out of 35 isolates, seven showed strong inhibitory activity against P. larvae. Most of the antagonistic bacteria belonged to Bacillus species, suggesting that the bacterial isolates obtained in this study appear to be potential candidates for the biological control of P. larvae.

Infestation of Japanese Native Honey Bees by Tracheal Mite and Virus from Non-native European Honey Bees in Japan

Invasion of alien species has been shown to cause detrimental effects on habitats of native species. Insect pollinators represent such examples; the introduction of commercial bumble bee species for crop pollination has resulted in competition for an ecological niche with native species, genetic disturbance caused by mating with native species, and pathogen spillover to native species. The European honey bee, Apis mellifera, was first introduced into Japan for apiculture in 1877, and queen bees have been imported from several countries for many years. However, its effects on Japanese native honey bee, Apis cerana japonica, have never been addressed. We thus conducted the survey of honey bee viruses and Acarapis mites using both A. mellifera and A. c. japonica colonies to examine their infestation in native and non-native honey bee species in Japan. Honey bee viruses, Deformed wing virus (DWV), Black queen cell virus (BQCV), Israeli acute paralysis virus (IAPV), and Sacbrood virus (SBV), were found in both A. mellifera and A. c. japonica colonies; however, the infection frequency of viruses in A. c. japonica was lower than that in A. mellifera colonies. Based on the phylogenies of DWV, BQCV, and SBV isolates from A. mellifera and A. c. japonica, DWV and BQCV may infect both honey bee species; meanwhile, SBV has a clear species barrier. For the first time in Japan, tracheal mite (Acarapis woodi) was specifically found in the dead honey bees from collapsing A. c. japonica colonies. This paper thus provides further evidence that tracheal-mite-infested honey bee colonies can die during cool winters with no other disease present. These results demonstrate the infestation of native honey bees by parasite and pathogens of non-native honey bees that are traded globally.

Diversity of Melissococcus plutonius from Honeybee Larvae in Japan and Experimental Reproduction of European Foulbrood with Cultured Atypical Isolates

European foulbrood (EFB) is an important infectious disease of honeybee larvae, but its pathogenic mechanisms are still poorly understood. The causative agent, Melissococcus plutonius, is a fastidious organism, and microaerophilic to anaerobic conditions and the addition of potassium phosphate to culture media are required for growth. Although M. plutonius is believed to be remarkably homologous, in addition to M. plutonius isolates with typical cultural characteristics, M. plutonius-like organisms, with characteristics seemingly different from those of typical M. plutonius, have often been isolated from diseased larvae with clinical signs of EFB in Japan. Cultural and biochemical characterization of 14 M. plutonius and 19 M. plutonius-like strain/isolates revealed that, unlike typical M. plutonius strain/isolates, M. plutonius-like isolates were not fastidious, and the addition of potassium phosphate was not required for normal growth. Moreover, only M. plutonius-like isolates, but not typical M. plutonius strain/isolates, grew anaerobically on sodium phosphate-supplemented medium and aerobically on some potassium salt-supplemented media, were positive for β-glucosidase activity, hydrolyzed esculin, and produced acid from L-arabinose, D-cellobiose, and salicin. Despite the phenotypic differences, 16S rRNA gene sequence analysis and DNA-DNA hybridization demonstrated that M. plutonius-like organisms were taxonomically identical to M. plutonius. However, by pulsed-field gel electrophoresis analysis, these typical and atypical (M. plutonius-like) isolates were separately grouped into two genetically distinct clusters. Although M. plutonius is known to lose virulence quickly when cultured artificially, experimental infection of representative isolates showed that atypical M. plutonius maintained the ability to cause EFB in honeybee larvae even after cultured in vitro in laboratory media. Because the rapid decrease of virulence in cultured M. plutonius was a major impediment to elucidation of the pathogenesis of EFB, atypical M. plutonius discovered in this study will be a breakthrough in EFB research.

Distribution of Nosema ceranae in the European honeybee, Apis mellifera in Japan

The microsporidian species, Nosema apis and Nosema ceranae are both known to infect the European honeybee, Apis mellifera. Nosema disease has a global distribution and is responsible for considerable economic losses among apiculturists. In this study, 336 honeybee samples from 18 different prefectures in Japan were examined for the presence of N. apis and N. ceranae using a PCR technique. Although N. ceranae was not detected in most of the apiaries surveyed, the parasite was detected at three of the sites examined. Further, N. ceranae appears to be patchily distributed across Japan and no apparent geographic difference was observed among the areas surveyed. In addition, the apparent absence of N. apis suggests that N. ceranae may be displacing N. apis in A. mellifera in Japan. Partial SSU rRNA gene sequence analysis revealed the possible existence of two N. ceranae groups from different geographic regions in Japan. It seems likely that these microsporidian parasites were introduced into Japan through the importation of either contaminated honeybee-related products or infected queens. This study confirmed that PCR detection is effective for indicating the presence of this pathogen in seemingly healthy colonies. It is therefore hoped that the results presented here will improve our understanding of the epidemiology of Nosema disease so that effective controls can be implemented.

Molecular detection of protozoan parasites infecting Apis mellifera colonies in Japan

The role of protozoan parasites in honey bee health and distribution in the world is not well understood. Therefore, we carried out a molecular survey for the presence of Crithidia mellificae and Apicystis bombi in the colonies of both non-native Apis mellifera and native Apis cerana japonica in Japan. We found that A. mellifera, but not A. c. japonica, colonies are parasitized with C. mellificae and A. bombi. Their absence in A. c. japonica colonies indicates that A. mellifera is their native host. Nevertheless, the prevalence in A. mellifera colonies is low compared with other pathogens such as viruses and Nosema microsporidia. Japanese C. mellificae isolates share well-conserved nuclear-encoded gene sequences with Swiss and US isolates. We have found two Japanese haplotypes (A and B) with two nucleotide differences in the kinetoplast-encoded cytochrome b sequence. The haplotype A is identical to Swiss isolate. These results demonstrate that C. mellificae and A. bombi distribute in Asia, Oceania, Europe, and South and North Americas.

The habitat disruption induces immune-suppression and oxidative stress in honey bees

The honey bee is a major insect used for pollination of many commercial crops worldwide. Although the use of honey bees for pollination can disrupt the habitat, the effects on their physiology have never been determined. Recently, honey bee colonies have often collapsed when introduced in greenhouses for pollination in Japan. Thus, suppressing colony collapses and maintaining the number of worker bees in the colonies is essential for successful long-term pollination in greenhouses and recycling of honey bee colonies. To understand the physiological states of honey bees used for long-term pollination in greenhouses, we characterized their gene expression profiles by microarray. We found that the greenhouse environment changes the gene expression profiles and induces immune-suppression and oxidative stress in honey bees. In fact, the increase of the number of Nosema microsporidia and protein carbonyl content was observed in honey bees during pollination in greenhouses. Thus, honey bee colonies are likely to collapse during pollination in greenhouses when heavily infested with pathogens. Degradation of honey bee habitat by changing the outside environment of the colony, during pollination services for example, imposes negative impacts on honey bees. Thus, worldwide use of honey bees for crop pollination in general could be one of reasons for the decline of managed honey bee colonies.

Inhibition of Paenibacillus larvae by lactic acid bacteria isolated from fermented materials

We evaluated the potential application of lactic acid bacteria (LAB) isolated from fermented feeds and foods for use as probiotics against Paenibacillus larvae, the causal agent of American foulbrood (AFB) in vitro. We also assessed the ability of LAB to induce the expression of antimicrobial peptide genes in vivo. Screening of the 208 LAB isolated from fermented feeds and foods revealed that nine strains inhibited the in vitro growth of P. larvae. The LAB strains were identified by 16S rRNA gene sequencing as Enterococcus sp., Weissella sp. and Lactobacillus sp. These strains were screened for their abilities of immune activation in honeybees by real-time RT-PCR using antimicrobial peptide genes as markers. After oral administration of several of the screened LAB to larvae and adults, the transcription levels of antimicrobial peptide genes, such as abaecin, defensin and hymenoptaecin, were found to increase significantly. These findings suggested that selected LAB stimulate the innate immune response in honeybees, which may be useful for preventing bacterial diseases in honeybees. This is the first report to characterize the probiotic effects of LAB isolated from fermented feeds and foods in honeybees.

Characterization of bifidobacteria in the digestive tract of the Japanese honeybee, Apis cerana japonica

Bifidobacteria were isolated from the intestinal tract of the Japanese honeybee, Apis cerana japonica, and investigated for potential application as a probiotic agent against Melissococcus plutonius, the causal agent of European foulbrood (EFB), based on the findings of in vitro inhibition assays. A total of 11 bifidobacteria strains (designated as AcjBF1-AcjBF11) were isolated using a culture-dependent method and their 16S rRNA gene sequences were analyzed. The AcjBF isolates belonged to three distinct bifidobacterial phylotypes that were similar to those found in the European honeybee, Apis mellifera. Although the Japanese and European honeybees are distinct species with different traits and habits, the observation that they share highly similar bifidobacterial phylotypes suggests that bifidobacteria are conserved among honeybee species. Despite having extremely high 16S rRNA gene sequence similarities, the AcjBF isolates had markedly different carbohydrate fermentation profiles. In addition, in vitro growth inhibition assays revealed that the cell-free supernatants of all AcjBF isolates exhibited antagonistic effects on M. plutonius growth. These results indicate that the bifidobacteria isolated from the gut of Japanese honeybee could potentially be employed as a new biological agent to control EFB.

PCR-based detection of a tracheal mite of the honey bee Acarapis woodi.

The effects of the tracheal mite Acarapis woodi on the health of honey bees have been neglected since the prevalence of Varroa mites to Apis mellifera colonies. However, tracheal mite infestation of honey bee colonies still occurs worldwide and could impose negative impact on apiculture. The detection of A. woodi requires the dissection of honey bees followed by microscopic observation of the tracheal sacs. We thus developed PCR methods to detect A. woodi. These methods facilitate rapid and sensitive detection of A. woodi in many honey bee samples for epidemiologic surveys.

Development of duplex PCR assay for detection and differentiation of typical and atypical Melissococcus plutonius strains.

ABSTRACT Melissococcus plutonius is the causative agent of an important honeybee disease, European foulbrood (EFB). In addition to M. plutonius strains with typical characteristics (typical M. plutonius), we recently reported the presence of atypical M. plutonius, which are phenotypically and genetically distinguished from typical M. plutonius. Because typical and atypical M. plutonius may have different pathogenic mechanisms, differentiation of these two types is very important for diagnosis and more effective control of EFB. In this study, therefore, a duplex PCR assay was developed to detect and differentiate typical and atypical M. plutonius rapidly and easily. On the basis of the results of comparative genomic analyses, we selected Na+/H+ antiporter gene and Fur family transcriptional regulator gene as targets for detection of typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex PCR system using the designed primers successfully detected and differentiated all typical and atypical M. plutonius strain/isolates tested, while no product was generated from any other bacterial strains/isolates used in this study, including those isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of chromosome/reaction for both types, and it could detect typical and atypical M. plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR diagnosed mixed infections with both M. plutonius types more precisely than standard culture methods. These results indicate that the duplex PCR assay developed in this study is extremely useful for precise diagnosis and epidemiological study of EFB.