Infected Honey Bees Research Articles

Analysis of the nonstructural and structural polyprotein regions, and complete genome sequences of Israel acute paralysis viruses identified from honeybees (Apis mellifera) in Korea

Phylogenetic trees were constructed for 24 partial nucleotide sequences of the nonstructural polyprotein (ORF1) and structural polyprotein regions (ORF2) of Korean IAPV genotypes, as well as eight previously reported IAPV sequences from various countries. Most of the Korean genotypes formed a distinct cluster, separate from other country genotypes. To investigate this phenomenon in more detail, three complete IAPV genome sequences were identified from different regions in Korea, i.e., Korea1, Korea2, and Korea3. These sequences were aligned with eight previously reported complete genome sequences and various genome regions were compared. The Korean IAPVs were very similar to those from China and Israel, but highly diverged from USA and Australian genotypes. Interestingly, they showed greater variability than the USA and Australian genotypes in ORF1, but highly similar to the Australian genotype in the ORF2 region. Thus, genetic recombination may account for the spatial distance between the Korean IAPV genotypes and those from other countries.

Apoptosis in the pathogenesis of Nosema ceranae (Microsporidia: Nosematidae) in honey bees (Apis mellifera)

Nosema ceranae is a parasite of the epithelial ventricular cells of the honey bee that belongs to the microsporidian phylum, a biological group of single-cell, spore-forming obligate intracellular parasites found in all major animal lineages. The ability of host cells to accommodate a large parasitic burden for several days suggests that these parasites subvert the normal host cells to ensure optimal environmental conditions for growth and development. Once infected, cells can counteract the invasive pathogen by initiating their own death by apoptosis as a defence strategy. To determine whether N. ceranae blocks apoptosis in infected ventricular cells, cell death was assessed in sections of the ventriculum from experimentally infected honey bees using the TUNEL assay and by immunohistochemistry for caspase-3. Ventricular epithelial cells from infected bees were larger than those in the uninfected control bees, and they contained N. ceranae at both mature and immature stages in the cytoplasm. Apoptotic nuclei were only observed in some restricted areas of the ventriculum, whereas apoptosis was typically observed throughout the epithelium in uninfected bees. Indeed, the apoptotic index was higher in uninfected versus infected ventriculi. Our results suggested that N. ceranae prevents apoptosis in epithelial cells of infected ventriculi, a mechanism possible designed to enhance parasite development.

Analysis of the complete genome sequence and capsid region of black queen cell viruses from infected honeybees (Apis mellifera) in Korea

Black queen cell virus (BQCV) infection is one of the most common viral infections in honeybees (Apis mellifera). A phylogenetic tree was constructed for 19 partial nucleotide sequences for the capsid region of South Korean BQCV, which were also compared with 10 previously reported BQCV sequences derived from different countries. The Korean BQCV genomes were highly conserved and showed 97-100% identity. They also showed 92-99% similarity with other country genotypes and showed no significant clustering in the phylogenetic tree. In order to investigate this phenomenon in more detail, the complete genome sequence of the Korean BQCV strain was determined and aligned with those from a South African reference strain and European genotypes, Poland4-6 and Hungary10. A phylogenetic tree was then constructed. The Korean BQCV strain showed a high level of similarity (92%) with Hungary10, but low similarity (86%) with the South African reference genotype. Comparison of the Korean and other sequences across different genome regions revealed that the 5'-UTR, the intergenic region, and the capsid regions of the BQCV genome were highly conserved. ORF1 (a non-structural protein coding region) was more variable than ORF2 (a structural protein coding region). The 5'-proximal third of ORF1 was particularly variable and contained several insertions/deletions. This phenomenon may be explained by intra-molecular recombination between the Korean and other BQCV genotypes; this appeared to have happened more with the South African reference strain than with the European genotypes.

Fine mapping identifies significantly associating markers for resistance to the honey bee brood fungal disease, Chalkbrood

SummaryChalkbrood infection of honey bee (Apis mellifera) brood by the fungus Ascosphaera apis results in fatal encapsulation of susceptible larvae with a mycelial coat. Recent QTL analysis indicates that some level of physiological resistance exists in individual larvae. We performed a fine mapping analysis to define the genetic interval that confers resistance in the larvae and identify the strongest association molecular markers that could by useful for marker assisted selection of the trait. Evaluation of the interval suggests that only two possible genes (single Ig IL-related receptor-like, XM_003251514.1 and juvenile hormone-binding protein, XM_391872.4) are likely to be responsible for the resistance. Both genes are strong physiological candidates and potentially function as modulators of the antifungal-specific innate immunity pathway in insects.

Nosema ceranae has been infecting honey bees Apis mellifera in Italy since at least 1993

(2013). Nosema ceranae has been infecting honey bees Apis mellifera in Italy since at least 1993. Journal of Apicultural Research: Vol. 52, No. 2, pp. 60-61.

Genome sequencing and comparative genomics of honey bee microsporidia, Nosema apis reveal novel insights into host-parasite interactions

BackgroundThe microsporidia parasite Nosema contributes to the steep global decline of honey bees that are critical pollinators of food crops. There are two species of Nosema that have been found to infect honey bees, Nosema apis and N. ceranae. Genome sequencing of N. apis and comparative genome analysis with N. ceranae, a fully sequenced microsporidia species, reveal novel insights into host-parasite interactions underlying the parasite infections.ResultsWe applied the whole-genome shotgun sequencing approach to sequence and assemble the genome of N. apis which has an estimated size of 8.5 Mbp. We predicted 2,771 protein- coding genes and predicted the function of each putative protein using the Gene Ontology. The comparative genomic analysis led to identification of 1,356 orthologs that are conserved between the two Nosema species and genes that are unique characteristics of the individual species, thereby providing a list of virulence factors and new genetic tools for studying host-parasite interactions. We also identified a highly abundant motif in the upstream promoter regions of N. apis genes. This motif is also conserved in N. ceranae and other microsporidia species and likely plays a role in gene regulation across the microsporidia.ConclusionsThe availability of the N. apis genome sequence is a significant addition to the rapidly expanding body of microsprodian genomic data which has been improving our understanding of eukaryotic genome diversity and evolution in a broad sense. The predicted virulent genes and transcriptional regulatory elements are potential targets for innovative therapeutics to break down the life cycle of the parasite.

Genetic and Biochemical Diversity ofPaenibacillus larvaeIsolated from Tunisian Infected Honey Bee Broods

Paenibacillus larvae is the causative agent of American foulbrood (AFB), a virulent disease of honeybee (Apis mellifera) larvae. In Tunisia, AFB has been detected in many beekeeping areas, where it causes important economic losses, but nothing is known about the diversity of the causing agent. Seventy-five isolates of P. larvae, identified by biochemical tests and 16S rRNA gene sequencing, were obtained from fifteen contaminated broods showing typical AFB symptoms, collected in different locations in the northern part of the country. Using BOX-PCR, a distinct profile of P. larvae with respect to related Paenibacillus species was detected which may be useful for its identification. Some P. larvae-specific bands represented novel potential molecular markers for the species. BOX-PCR fingerprints indicated a relatively high intraspecific diversity among the isolates not described previously with several molecular polymorphisms identifying six genotypes on polyacrylamide gel. Polymorphisms were also detected in several biochemical characters (indol production, nitrate reduction, and methyl red and oxidase tests). Contrary to the relatively high intraspecies molecular and phenotypic diversity, the in vivo virulence of three selected P. larvae genotypes did not differ significantly, suggesting that the genotypic/phenotypic differences are neutral or related to ecological aspects other than virulence.

Temporal study of Nosema spp. in a cold climate

In a nationwide Swedish survey, 967 honey bee colonies from 521 beekeepers were sampled in the spring of 2007 and the samples assayed for Nosema spp. infections. Of the 319 positive samples, only 32 samples contained a proportion of N. ceranae DNA in mixed infections with both Nosema spp. above the cut-off point chosen for comparisons of 1%. Only one pure N. ceranae infection was found, with the rest 284 infected samples assayed being pure N. apis infections. In 2009 and 2011, beekeepers or bee inspectors providing N. ceranae mixed positive bee samples in 2007 were again asked to submit samples (2009, n = 96; 2011, n = 83). No trend of an increased proportion of N. ceranae-infected samples could be found. The proportion of N. ceranae DNA in samples with mixed infection did not increase between 2007 and 2011. It is concluded that N. apis is still the dominating Microsporidia infection in honey bees in Sweden and that there is no tendency for one species replacing the other.

Reverse transcription loop-mediated isothermal amplification for sensitive and rapid detection of Korean sacbrood virus

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 10(3) copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees.

Gas chromatography–mass spectrometry metabolite profiling of worker honey bee (Apis mellifera L.) hemolymph for the study of Nosema ceranae infection

Here, we are presenting a gas chromatography–mass spectrometry (GC/MS) approach for the study of infection of the worker honey bee (Apis mellifera L.) by the newly emerged obligate intracellular parasite Nosema ceranae based on metabolite profiling of hemolymph. Because of the severity of the disease, early detection is crucial for its efficient control. Results revealed that the parasite causes a general disturbance of the physiology of the honey bee affecting the mechanisms controlling the mobilization of energy reserves in infected individuals. The imposed nutritional and energetic stress to the host was depicted mainly in the decreased levels of the majority of carbohydrates and amino acids, including metabolites such as fructose, l-proline, and the cryoprotectants sorbitol and glycerol, which are implicated in various biochemical pathways. Interestingly, the level of glucose was detected at significantly higher levels in infected honey bees. Metabolomics analyses were in agreement with those of multiplex quantitative PCR analyses, indicating that it can be used as a complementary tool for the detection and the study of the physiology of the disease.

In vitro Inhibitory Activity of Essential Oil Vapors against Ascosphaera apis

This work evaluates the in vitro inhibitory activity of 70 essential oils (EOs) in the vapor phase for the control of Chalkbrood disease caused by Ascosphaera apis Maassen ex Claussen (Olive et Spiltoir). Two wild strains isolated from infected honey bee colonies together with one standard collection strain were tested by the microatmosphere method. From 70 EOs, 39 exhibited an antifungal effect against A. apis standard and wild strains. The greatest antifungal action was observed for EO vapors from Armoracia rusticana, followed by Thymus vulgaris, Cymbopogon flexosus, Origanum vulgare and Allium sativum. An investigation of chemical composition by GC-MS revealed, that the most active EOs contained allyl isothiocyanate, citral, carvacrol and diallyl sulfides as the main constituents. The chemical composition plays a key role, as activities of different EOs from the same botanical species were different according to their composition.

Infection of honey bees with acute bee paralysis virus does not trigger humoral or cellular immune responses

We have studied the responses of honey bees at different life stages (Apis mellifera) to controlled infection with acute bee paralysis virus and have identified the haemolymph of infected larvae and adult worker bees as the compartment where massive propagation of ABPV occurs. Insects respond with a broad spectrum of induced innate immune reactions to bacterial infections, whereas defence mechanisms based on RNA interference play a major role in antiviral immunity. In this study, we have determined that honey bee larvae and adult workers do not produce a humoral immune reaction upon artificial infection with ABPV, in contrast to control individuals challenged with Escherichia coli. ABPV-infected bees produced neither elevated levels of specific antimicrobial peptides (AMPs), such as hymenoptaecin and defensin, nor any general antimicrobial activity, as revealed by inhibition-zone assays. Additionally, adult bees did not generate melanised nodules upon ABPV infection, an important cellular immune function activated by bacteria and viruses in some insects. Challenge of bees with both ABPV and E. coli showed that innate humoral and cellular immune reactions are induced in mixed infections, albeit at a reduced level.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-012-1223-0) contains supplementary material, which is available to authorized users.

First detection of four viruses in honey bee (Apis mellifera) workers with and without deformed wings and Varroa destructor in Mexico

SummaryLittle information exists in Mexico about the presence of viral diseases in honey bee (Apis mellifera) colonies and their association with Varroa destructor mites. Here we report the first investigation using molecular techniques, of honey bees and varroa mites tested for viruses in Mexico. Samples of varroa mites, worker pupae, and adult bees were collected from five colonies, four of which had a high proportion of workers with deformed wings. Varroa mite infestation rates were determined in worker brood and in adult bees. Samples were analysed by reverse transcription-PCR (RT-PCR) for sacbrood virus (SBV), deformed wing virus (DWV), acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV), chronic bee paralysis virus (CBPV) and Kashmir bee virus (KBV). Of the six viruses surveyed, four were found in adult bees (DWV, IAPV, ABPV and SBV), two in worker brood (DWV and IAPV) and two in varroa mites (DWV and IAPV). Only KBV and CBPV were not detected. This is the first molecular detection of these four viruses infecting honey bees and varroa mites in Mexico. The association of these viruses with V. destructor is discussed.

Critical aspects of the Nosema spp. diagnostic sampling in honey bee (Apis mellifera L.) colonies

Nosemosis is one of the most widespread of the adult honey bee diseases and causes major economic losses to beekeepers. Two microsporidia have been described infecting honey bees worldwide, Nosema apis and Nosema ceranae, whose seasonality and pathology differ markedly. An increasing prevalence of microsporidian infections in honey bees has been observed worldwide during the last years. Because nosemosis has detrimental effects on both strength and productivity of the infected colonies, an accurate and reliable method to evaluate the presence of Nosema in honey bee colonies is needed. In this study a high degree of variability in the detection of microsporidia depending on the random subsample analyzed was found, suggesting that both sample size and the time of collection (month and day of sampling) notably affect the diagnosis.

Dynamics of persistent and acute deformed wing virus infections in honey bees, Apis mellifera.

The dynamics of viruses are critical to our understanding of disease pathogenesis. Using honey bee Deformed wing virus (DWV) as a model, we conducted field and laboratory studies to investigate the roles of abiotic and biotic stress factors as well as host health conditions in dynamics of virus replication in honey bees. The results showed that temperature decline could lead to not only significant decrease in the rate for pupae to emerge as adult bees, but also an increased severity of the virus infection in emerged bees, partly explaining the high levels of winter losses of managed honey bees, Apis mellifera, around the world. By experimentally exposing adult bees with variable levels of parasitic mite Varroa destructor, we showed that the severity of DWV infection was positively correlated with the density and time period of Varroa mite infestation, confirming the role of Varroa mites in virus transmission and activation in honey bees. Further, we showed that host conditions have a significant impact on the outcome of DWV infection as bees that originate from strong colonies resist DWV infection and replication significantly better than bee originating from weak colonies. The information obtained from this study has important implications for enhancing our understanding of host‑pathogen interactions and can be used to develop effective disease control strategies for honey bees.

Genetic and phylogenetic analysis of South Korean sacbrood virus isolates from infected honey bees (Apis cerana)

Sacbrood virus (SBV) is one of the most destructive honey bee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Genetic analysis of SBV infected honey bees (Apis cerana) from five different provinces was carried out based on three nucleotide sequences; one partial structural protein coding sequence and two non-structural protein coding sequences. Sequences amplified by three specific primer pairs were aligned and compared with reference sequences deposited in the GenBank database. Sequence alignments revealed a low level of sequence variation among Korean isolates (≥98.6% nucleotide identity), regardless of the genome regions studied or the geographic origins of the strains. Multiple sequence comparisons indicated that Korean SBV isolates are genetically closely related to Chinese and other Asian strains. Interestingly, the Korean SBV isolates showed a number of unique nucleotides and amino acids that had not been observed in other published strains. Korean and other Asian isolates from the host A. cerana and the UK, European and Japanese strains from the host Apis mellifera showed differences in nucleotide and deduced amino acid identities. This suggests that host-specificity exists among SBV strains isolated from different species. Phylogenetic relatedness between compared sequences was analyzed by MEGA 4.1 software using the neighbor-joining (NJ) method with a boot-strap value of 1000 replicates. Obtained topologies were in agreement with previous studies, in which a distinct group of SBV was formed by UK and European genotypes and another group was comprised of Asian genotypes including strains that originated from China, Japan (japonica), India and Nepal. However, phylogeny based on a partial protein structural coding sequence grouped all Korean SBV isolates identified in A. cerana as a separate cluster. Our findings suggest that further study, including Korean SBV isolated from A. mellifera, is needed.

The growing prevalence of Nosema ceranae in honey bees in Spain, an emerging problem for the last decade

Microsporidiosis caused by infection with Nosema apis or Nosema ceranae has become one of the most widespread diseases of honey bees and can cause important economic losses for beekeepers. Honey can be contaminated by spores of both species and it has been reported as a suitable matrix to study the field prevalence of other honey bee sporulated pathogens. Historical honey sample collections from the CAR laboratory (Centro Apícola Regional) were analyzed by PCR to identify the earliest instance of emergence, and to determine whether the presence of Nosema spp. in honey was linked to the spread of these microsporidia in honey bee apiaries. A total of 240 frozen honey samples were analyzed by PCR and the results compared with rates of Nosema spp. infection in worker bee samples from different years and geographical areas. The presence of Nosema spp. in hive-stored honey from naturally infected honey bee colonies (from an experimental apiary) was also monitored, and although collected honey bees resulted in a more suitable sample to study the presence of microsporidian parasites in the colonies, a high probability of finding Nosema spp. in their hive-stored honey was observed. The first honey sample in which N. ceranae was detected dates back to the year 2000. In subsequent years, the number of samples containing N. ceranae tended to increase, as did the detection of Nosema spp. in adult worker bees. The presence of N. ceranae as early as 2000, long before generalized bee depopulation and colony losses in 2004 may be consistent with a long incubation period for nosemosis type C or related with other unknown factors. The current prevalence of nosemosis, primarily due to N. ceranae, has reached epidemic levels in Spain as confirmed by the analysis of worker honey bees and commercial honey.

Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus

Chinese sacbrood virus (CSBV) has emerged as an important etiologic agent of honeybee infections and is lethal for individual bees, even causing the collapse of entire colonies. Although diagnostic methods for CSBV have been established in many clinical laboratories, application of these methods is largely restricted by the apparatus needed to carry out the reaction and by cost, therefore a simpler and less expensive diagnostic method for CSBV infection is required. In this study a simple and inexpensive system is described that is based on the loop-mediated isothermal amplification (LAMP) assay. The LAMP and the polymerase chain reaction (PCR) methods were compared for their ability to detect CSBV in 31 clinical samples, in purified CSBV-LNQY strains or to be able to discriminate between cDNA samples from other viruses. The detection limit of the LAMP method was 1 pg, showing that LAMP is as sensitive as reverse transcriptase (RT)-PCR for CSBV detection. In addition, no DNA band from other related viruses samples was amplified by either method, suggesting that this LAMP assay is as specific as RT-PCR for CSBV detection. All 31 clinical samples that were LAMP assay-positive were also amplified by RT-PCR, however the LAMP assay was faster, more cost effective, and easier to perform as the target gene amplified rapidly, within 2 h, and only a standard laboratory water bath or heat block was required for the reaction. The results demonstrate clearly that this LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of CSBV infection of bees.

The stability and effectiveness of fumagillin in controlling Nosema ceranae (Microsporidia) infection in honey bees (Apis mellifera) under laboratory and field conditions

Honey bees play an important role in food production (honey, pollen etc.), and their pollinating activity is not only essential to maintain world agriculture production but also to ensure biodiversity in different ecosystems. Nosema ceranae is a highly prevalent worldwide pathogen for honey bees that has been related to colony losses. A commercial formulation that contains fumagillin dicyclohexylamine, Fumidil B®, can control N. ceranae infection. However, the effectiveness of Fumidil B® is affected by several factors, such as storage, treatment preparation, the quantity consumed by bees etc. Indeed, UV exposure (e.g. sunlight) drastically reduces the initial concentration of fumagillin within a few hours, while temperature affects its degradation. Although laboratory tests suggest that a semisolid mixture of honey and powdered sugar is the best option to apply fumagillin, its application in syrup (250 mL per dosage) is more effective for the treatment of infected colonies. The total amount of syrup containing fumagillin ingested by honey bees is a key factor in its efficacy, and it has been found that medicated patties were not fully consumed in field trials. In honey bee colonies, the dose of 120 mg/honey bee colony at the recommended posology is effective against depopulation and colony death due to N. ceranae after 1 year, without residues being detected in honey, although reinfection could be detected 4 months after treatment ended.

Comparison of the energetic stress associated with experimental Nosema ceranae and Nosema apis infection of honeybees (Apis mellifera)

Nosema ceranae is a relatively new and widespread parasite of the western honeybee Apis mellifera that provokes a new form of nosemosis. In comparison to Nosema apis, which has been infecting the honeybee for much longer, N. ceranae seems to have co-evolved less with this host, causing a more virulent disease. Given that N. apis and N. ceranae are obligate intracellular microsporidian parasites, needing host energy to reproduce, energetic stress may be an important factor contributing to the increased virulence observed. Through feeding experiments on caged bees, we show that both mortality and sugar syrup consumption were higher in N. ceranae-infected bees than in N. apis-infected and control bees. The mortality and sugar syrup consumption are also higher in N. apis-infected bees than in controls, but are less than in N. ceranae-infected bees. With both microsporidia, mortality and sugar syrup consumption increased in function of the increasing spore counts administered for infection. The differences in energetic requirements between both Nosema spp. confirm that their metabolic patterns are not the same, which may depend critically on host-parasite interactions and, ultimately, on host pathology. The repercussions of this increased energetic stress may even explain the changes in host behavior due to starvation, lack of thermoregulatory capacity, or higher rates of trophallaxis, which might enhance transmission and bee death.