Queen Cell Research Articles

Molecular detection and phylogenetic assessment of six honeybee viruses in Apis mellifera L. colonies in Bulgaria.

Honey bee colonies suffer from various pathogens, including honey bee viruses. About 24 viruses have been reported so far. However, six of them are considered to cause severe infection which inflicts heavy losses on beekeeping. The aim of this study was to investigate incidence of six honey bee viruses: deformed wing virus (DWV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), sacbrood virus (SBV), kashmir bee virus (KBV), and black queen cell virus (BQCV) by a reverse transcription polymerase chain reaction (RT-PCR). A total of 250 adult honey bee samples were obtained from 50 colonies from eight apiaries situated in three different parts of the country (South, North and West Bulgaria). The results showed the highest prevalence of DWV followed by SBV and ABPV, and one case of BQCV. A comparison with homology sequences available in GenBank was performed by phylogenetic analysis, and phylogenetic relationships were discussed in the context of newly described genotypes in the uninvestigated South Eastern region of Europe. In conclusion, the present study has been the first to provide sequencing data and phylogenetics analyses of some honey bee viruses in Bulgaria.

DETECTION OF HONEY BEE VIRUSES ON THE TERRITORY OF UKRAINE

A survey of honey bee viruses on the territory of Ukraine has been conducted forthe first time in 2016-2017 years. The samples of adult bees, affected combs andmites were collected from apiaries in two regions (Kyiv and Cherkasy). Detailedstudying of the bee samples revealed following morphological changes: deformedwings, saccular brood, blackened pistil, changing of the body color. Virus-likeparticles were observed under TEM after purification of bee samples.Symptomless samples were also analyzed for the presence of bee viruses by RTPCR.According to results of RT-PCR, we concluded that the following virusescirculate on the territory of Ukraine: Acute Bee Paralysis Virus (ABPV), KashmirBee Virus (KBV), Israeli Acute Paralysis Virus (IAPV), Black Queen Cell Virus(BQCV), Sacbrood Bee Virus (SBV) Chronic bee paralysis virus (CBPV). Takentogether, these data indicate that bee virus infections occur in bee populations onthe territory of Ukraine. Despite the lack of symptoms viruses are often present incolonies and environmental factors might result disease outbreaks that lead to theactivation of viral replication in bees.

Seasonal Effects and the Impact of In-Hive Pesticide Treatments on Parasite, Pathogens, and Health of Honey Bees.

Honey bee, Apis mellifera (L.; Hymenoptera: Apidae), populations are in decline and their losses pose a serious threat for crop pollination and food production. The specific causes of these losses are believed to be multifactorial. Pesticides, parasites and pathogens, and nutritional deficiencies have been implicated in the losses due to their ability to exert energetic stress on bees. While our understanding of the role of these factors in honey bee colony losses has improved, there is still a lack of knowledge of how they impact the immune system of the honey bee. In this study, honey bee colonies were exposed to Fumagilin-B, Apistan (tau-fluvalinate), and chlorothalonil at field realistic levels. No significant effects of the antibiotic and two pesticides were observed on the levels of varroa mite, Nosema ceranae (Fries; Microsporidia: Nosematidae), black queen cell virus, deformed wing virus, or immunity as measured by phenoloxidase and glucose oxidase activity. Any effects on the parasites, pathogens, and immunity we observed appear to be due mainly to seasonal changes within the honey bee colonies. The results suggest that Fumagilin-B, Apistan, and chlorothalonil do not significantly impact the health of honey bee colonies, based on the factors analyzed and the concentration of chemicals tested.

Gamma irradiation inactivates honey bee fungal, microsporidian, and viral pathogens and parasites.

Managed honey bee (Apis mellifera) populations are currently facing unsustainable losses due to a variety of factors. Colonies are challenged with brood pathogens, such as the fungal agent of chalkbrood disease, the microsporidian gut parasite Nosema spp., and several viruses. These pathogens may be transmitted horizontally from worker to worker, vertically from queen to egg and via vectors like the parasitic mite, Varroa destructor. Despite the fact that these pathogens are widespread and often harbored in wax comb that is reused from year to year and transferred across beekeeping operations, few, if any, universal treatments exist for their control. In order to mitigate some of these biological threats to honey bees and to allow for more sustainable reuse of equipment, investigations into techniques for the sterilization of hive equipment and comb are of particular significance. Here, we investigated the potential of gamma irradiation for inactivation of the fungal pathogen Ascosphaera apis, the microsporidian Nosema ceranae and three honey bee viruses (Deformed wing virus [DWV], Black queen cell virus [BQCV], and Chronic bee paralysis virus [CBPV]), focusing on the infectivity of these pathogens post-irradiation. Results indicate that gamma irradiation can effectively inactivate A. apis, N. ceranae, and DWV. Partial inactivation was noted for BQCV and CBPV, but this did not reduce effects on mortality at the tested, relatively high doses. These findings highlight the importance of studying infection rate and symptom development post-treatment and not simply rate or quantity detected. These findings suggest that gamma irradiation may function as a broad treatment to help mitigate colony losses and the spread of pathogens through the exchange of comb across colonies, but raises the question why some viruses appear to be unaffected. These results provide the basis for subsequent studies on benefits of irradiation of used comb for colony health and productivity.

First detection of bee viruses in hoverfly (syrphid) pollinators.

Global declines of insect pollinators jeopardize the delivery of pollination services in both agricultural and natural ecosystems. The importance of infectious diseases has been documented in honeybees, but there is little information on the extent to which these diseases are shared with other pollinator orders. Here, we establish for the first time the presence of three important bee viruses in hoverfly pollinators (Diptera: Syrphidae): black queen cell virus (BQCV), sacbrood virus (SBV) and deformed wing virus strain B (DWV-B). These viruses were detected in two Eristalis species, which are behavioural and morphological bee mimics and share a foraging niche with honeybees. Nucleotide sequences of viruses isolated from the Eristalis species and Apis mellifera were up to 99 and 100% identical for the two viruses, suggesting that these pathogens are being shared freely between bees and hoverflies. Interestingly, while replicative intermediates (negative strand virus) were not detected in the hoverflies, viral titres of SBV were similar to those found in A. mellifera. These results suggest that syrphid pollinators may play an important but previously unexplored role in pollinator disease dynamics.

Effect of colony strength, queen cell cups spacing on the graft acceptance and consumption of sugar syrup on quality rearing queens in Apis mellifera

Effect of colony strength, queen cell cups spacing on the graft acceptance and consumption of sugar syrup on quality rearing queens in <i>Apis mellifera</i>

Phylogenetic analysis of black queen cell virus and deformed wing virus in honeybee colonies infected by mites in Van, Eastern Turkey

This study aimed to determine the presence and prevalence of viral and parasitic infections causing high rates of colony loss in honey bee colonies in Van province, eastern Turkey. Twenty-six different apiaries were collected from five counties in Van province. These samples were tested by Reverse-Transcriptase PCR (RT-PCR) for acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), black queen cell virus (BQCV) and deformed wing virus (DWV). Selected positives were sequenced, phylogenetically analyzed and investigated in terms of Varroa. DWV and BQCV were identified in 69.23% (18/26) and 88.46% (23/26) of the bees respectively whereas ABPV and CBPV were not detected in the sampled apiaries. Results of the phylogenetic analysis of DWV and BQCV sequences showed 94–100% similarity to DWV and BQCV isolates obtained from Genbank. Prevalence of varroasis was 89% (23/26) in Van. The obtained samples were identified as V. Varroa destructor by morphological investigation. The study showed that viral and parasitic agents commonly infect honeybees in Van province, with high prevalence rates for BQCV and DWV. There was also a high degree of conservation of DWV and BQCV sequences distinct from DWV and BQCV isolates from other geographical regions. These findings, including current prevalence and phylogenetic analysis data for DWV, BQCV and varroazis in honeybees, are useful for future studies. .

Co-occurrence of RNA viruses in Tasmanian-introduced bumble bees (Bombus terrestris) and honey bees (Apis mellifera)

A number of bee RNA viruses, including Deformed wing virus (DWV), are so far unreported from Australia. These viruses can be introduced together with imported live honey bees (Apis mellifera) and their products, with other bee species, and bee parasites. Given that bee viruses have a profound impact on bee health, it is surprising that since the introduction of bumble bees (Bombus terrestris) onto Tasmania in 1992 from New Zealand, no work has been done to investigate which RNA viruses are associated with these bees. Consequently, we investigate the current prevalence of RNA viruses in B. terrestris and A. mellifera collected in south-eastern Tasmania. We did not find DWV in either A. mellifera and B. terrestris. However, both bee species shared Kashmir bee virus (KBV) and Sacbrood virus (SBV), but Black queen cell virus (BQCV) was detected only in A. mellifera. This reinforces the importance of ongoing strong regulation of the anthropogenic movement of live bees and their products.

Prevalence of common honey bee pathogens at selected apiaries in Kenya, 2013/2014

AbstractThe present study was part of a larger surveillance effort to identify the determinants of African honey bee health, and, particularly, to detect honey bee pathogens across Kenya, where 160 colonies were examined from 32 apiaries (five colonies/apiary). From each colony, 20 individual foragers, nurse bees, worker pupae, and drone pupae were sampled separately. These were organized as 30 foragers, 32 nurse bees, 28 worker pupae, and 10 drone pupae pools. Nucleic acid was extracted from the pooled homogenates and tested using a panel of 18 different (RT-)PCR methods targeted at detectingPaenibacillus larvae,Melissococcus plutonius,Ascophaera apis,Aspergillusspp.,Nosema ceranae, N. apis,Deformed wing virus(DWV),Varroa destructor virus 1(VDV 1),Acute bee paralysis virus(ABPV),Sacbrood virus(SBV),Israeli acute paralysis virus(IAPV),Black queen cell virus(BQCV),Chronic bee paralysis virus(CBPV), andKashmir bee virus. All amplified bands were sequenced and compared to the GenBank database. VDV 1 was the most abundant virus at 50% prevalence in the 100 bee pools. It was closely followed by DWV at 44%. The others were BQCV (36%), SBV (14%), IAPV (9%), ABPV (8%), andN. ceranae(5%). The pathogens co-existed within apiaries. VDV 1 was present in 66% of the apiaries, DWV in 69%, BQCV in 69%, SBV in 28%, IAPV in 22%, ABPV in 19%, andN. ceranaein 13%. The study concludes that these pathogens should be incorporated in honey bee disease surveillance activities in the region.

Viral and intestinal diseases detected in Apis mellifera in Central and Southern Chile

The honeybee (Apis mellifera L.) is an important pollinator worldwide. Beekeepers have experienced an increasing loss of colonies in recent years and pathologies including viruses and intestinal parasites such as Nosema ceranae are among those held responsible for this decline. The aim of this study was to determine the prevalence and distribution, using the real time PCR method, of six honeybee viruses including Deformed Wing Virus (DWV), Black Queen Cell Virus (BQCV), Sacbrood Virus (SBV), Acute Bee Paralysis Virus (ABPV), Chronic Bee Paralysis Virus (CBPV) and Israeli Acute Paralysis Virus (IAPV) in addition to three intestinal parasites, N. ceranae, N. apis and Lotmaria passim in Chilean beehives distributed between the Metropolitan Region and La Araucania Region. Among the viruses, BQCV was the most prevalent in the analyzed hives, ranging from 72% to 100%, N. ceranae was detected between 19% to 53% and L. passim showed infections ranging from 45% to 73%. Similarly, BQCV (96%) and N. ceranae (42%) were more prevalent in spring-summer whilst DWV (37%) and L. passim (68%) were more frequents in autumn-winter seasons. Furthermore, mixed infections were also detected, wherein viruses were found along with L. passim or viruses with N. ceranae and L. passim, which proved to be the most prevalent. Of the 467 hives analyzed, 97.2% were infected with one or more of the studied pathogens, indicating that they are widely spread in Chile.

A molecular epidemiological study of black queen cell virus in honeybees (Apis mellifera) of Turkey: the first genetic characterization and phylogenetic analysis of field viruses

Black queen cell virus (BQCV) is one of the most common honeybee pathogens causing queen brood deaths. The 63 apiaries were sampled between 2007 and 2013 from four different ecogeographic regions in Turkey to estimate BQCV molecular structural characteristics. The BQCV positivity was 47.6%. The 25 local Black queen cell viruses (TrBQCVs) were molecularly characterized and investigated for their genetic relationship with previous records. The identity of the helicase gene among the TrBQCVs was 92–98%, whereas the similarity ranged from 37 to 85% in comparison with the intercontinental records. The identity of the partial capsid gene among the TrBQCVs was 91–100%, and the similarity rate varied from 86 to 97, 88–96, 90–97 and 89–99% in comparison with the Asian, African, American and European counterparts, respectively. The four nonsynonymous substitutions on the partial capsid protein suggest a predicted genotype that is specific among TrBQCVs.

Absence of deformed wing virus and Varroa destructor in Australia provides unique perspectives on honeybee viral landscapes and colony losses

Honeybee (Apis mellifera) health is threatened globally by the complex interaction of multiple stressors, including the parasitic mite Varroa destructor and a number of pathogenic viruses. Australia provides a unique opportunity to study this pathogenic viral landscape in the absence of V. destructor. We analysed 1,240A. mellifera colonies across Australia by reverse transcription-polymerase chain reaction (RT-PCR) and next-generation sequencing (NGS). Five viruses were prevalent: black queen cell virus (BQCV), sacbrood virus (SBV), Israeli acute paralysis virus (IAPV) and the Lake Sinai viruses (LSV1 and LSV2), of which the latter three were detected for the first time in Australia. We also showed several viruses were absent in our sampling, including deformed wing virus (DWV) and slow bee paralysis virus (SBPV). Our findings highlight that viruses can be highly prevalent in A. mellifera populations independently of V. destructor. Placing these results in an international context, our results support the hypothesis that the co-pathogenic interaction of V. destructor and DWV is a key driver of increased colony losses, but additional stressors such as pesticides, poor nutrition, etc. may enable more severe and frequent colony losses to occur.

Single-stranded RNA viruses infecting the invasive Argentine ant, Linepithema humile

Social insects host a diversity of viruses. We examined New Zealand populations of the globally widely distributed invasive Argentine ant (Linepithema humile) for RNA viruses. We used metatranscriptomic analysis, which identified six potential novel viruses in the Dicistroviridae family. Of these, three contigs were confirmed by Sanger sequencing as Linepithema humile virus-1 (LHUV-1), a novel strain of Kashmir bee virus (KBV) and Black queen cell virus (BQCV), while the others were chimeric or misassembled sequences. We extended the known sequence of LHUV-1 to confirm its placement in the Dicistroviridae and categorised its relationship to closest relatives, which were all viruses infecting Hymenoptera. We examined further for known viruses by mapping our metatranscriptomic sequences to all viral genomes, and confirmed KBV, BQCV, LHUV-1 and Deformed wing virus (DWV) presence using qRT-PCR. Viral replication was confirmed for DWV, KBV and LHUV-1. Viral titers in ants were higher in the presence of honey bee hives. Argentine ants appear to host a range of’ honey bee’ pathogens in addition to a virus currently described only from this invasive ant. The role of these viruses in the population dynamics of the ant remain to be determined, but offer potential targets for biocontrol approaches.

Relationship Between Number of Grafted Queen Cell Cups and Amount of Produced Royal Jelly in Honey Bee Colonies Apis mellifera L.

Relationship Between Number of Grafted Queen Cell Cups and Amount of Produced Royal Jelly in Honey Bee Colonies Apis mellifera L.

The first comprehensive molecular detection of six honey bee viruses in Iran in 2015-2016

At least 18 viruses have been reported in the honey bee (Apis mellifera L.). However, severe diseases in honey bees are mainly caused by six viruses, and these are the most important in beekeeping. These viruses include: deformed wing virus (DWV), acute bee paralysis virus (ABPV), chronic bee paralysis virus (CBPV), sacbrood virus (SBV), kashmir bee virus (KBV), and black queen cell virus (BQCV). In this study, we evaluated 89 Iranian honey bee apiaries (during the period 2015-2016) suffering from symptoms of depopulation, sudden collapse, paralysis, or dark coloring, by employing reverse transcription-PCR. Samples were collected from four regions (Mazandaran, Hormozgan, Kurdistan, and Khorasan Razavi) of Iran. Of the 89 apiaries examined, 16 (17.97%), three (3.37%), and three (3.37%) were infected by DWV, ABPV, and CBPV, respectively. The study results for the other viruses (SBV, KBV, and BQCV) were negative. The present study evaluated the presence of the six most important honey bee viruses in bee colonies with suspected infections, and identified remarkable differences in the distribution patterns of the viruses in different geographic regions of Iran.

Replication of honey bee-associated RNA viruses across multiple bee species in apple orchards of Georgia, Germany and Kyrgyzstan

The essential ecosystem service of pollination is provided largely by insects, which are considered threatened by diverse biotic and abiotic global change pressures. RNA viruses are one such pressure, and have risen in prominence as a major threat for honey bees (Apis mellifera) and global apiculture, as well as a risk factor for other bee species through pathogen spill-over between managed honey bees and sympatric wild pollinator communities. Yet despite their potential role in global bee decline, the prevalence of honey bee-associated RNA viruses in wild bees is poorly known from both geographic and taxonomic perspectives. We screened members of pollinator communities (honey bees, bumble bees and other wild bees belonging to four families) collected from apple orchards in Georgia, Germany and Kyrgyzstan for six common honey bee-associated RNA virus complexes encompassing nine virus targets. The Deformed wing virus complex (DWV genotypes A and B) had the highest prevalence across all localities and host species and was the only virus complex found in wild bee species belonging to all four studied families. Based on amplification of negative-strand viral RNA, we found evidence for viral replication in wild bee species of DWV-A/DWV-B (hosts: Andrena haemorrhoa and several Bombus spp.) and Black queen cell virus (hosts: Anthophora plumipes, several Bombus spp., Osmia bicornis and Xylocopa spp.). Viral amplicon sequences revealed that DWV-A and DWV-B are regionally distinct but identical in two or more bee species at any one site, suggesting virus is shared amongst sympatric bee taxa. This study demonstrates that honey bee associated RNA viruses are geographically and taxonomically widespread, likely infective in wild bee species, and shared across bee taxa.

Prevalence of honey bee (Apis mellifera) viruses in temperate and subtropical regions from Argentina

In Argentina, bee virus studies are still incipient, and there are no studies regarding the climatic effect. The aim of this study was to assess and compare the presence of honeybee viruses in different climatic regions from Argentina. A total of 385 colonies distributed in five Argentinean eco-regions were examined to evaluate the percentage of infestation with Varroa destructor and the presence of seven virus species (Deformed wing virus, DWV; Acute bee paralysis virus, ABPV; Chronic bee paralysis virus, CBPV; Black queen cell virus, BQCV; Kashmer bee virus, KBV; Israeli acute bee paralysis virus, IAPV; and Sacbrood bee virus, SBV) after honey yield. Two viruses, KBV and IAPV, were not detected. The other five viruses were found in different prevalences: DWV (35%), ABPV (21.5%), BQCV (8.0%), CBPV (2.2%), and SBV (1.1%). We found double and triple viral associations in approximately 25% of the sampled colonies. The mean V. destructor infestation in the colonies prior to the acaricide treatment was 7.12%±8.7%. The knowledge of the prevalence of these viruses in the region and their relation with the mite and other possible influencing factors is important for preventing colony losses. Further studies are necessary to identify the risk factors associated with virus presence and its relationship with other pathogens such as V. destructor.

Risk factors for the presence of Deformed wing virus and Acute bee paralysis virus under temperate and subtropical climate in Argentinian bee colonies

Beekeepers all across the world are suffering important losses of their colonies, and the parasitic mites Varroa destructor and Nosema sp, as well as several bee viruses, are being pointed out as the possible causes of these losses, generally associated with environmental and management factors. The objective of the present study was to evaluate the presence of seven virus species (Deformed wing virus –DWV-, Acute bee paralysis virus –ABPV-, Chronic bee paralysis virus –CBPV-, Black queen cell virus –BQCV-, Kashmir bee virus –KBV-, Israeli acute bee paralysis virus –IAPV-, and Sacbrood bee virus –SBV), as well as the prevalence of Nosema sp. and Varroa destructor, and their possible associated factors, under temperate and subtropical climate conditions in Argentinean colonies. A total of 385 colonies distributed in five Argentinean eco-regions were examined after honey harvest. The final multivariable model revealed only one variable associated with the presence of DWV and two with the presence of ABPV. The apiary random effect was significant in both cases (P=0.018; P=0.006, respectively). Colonies with a Varroa infestation rate >3% showed higher presence of DWV than colonies with <3% of Varroa infestation level (OR=1.91; 95% CI: 1.02–3.57; P<0.044). The same pattern was observed for the presence of ABPV (OR=2.23; 95% CI: 1.04–4.77; P<0.039). Also, colonies where replacement of old combs was not a common practice had higher presence of ABPV (OR=6.02; 95% CI: 1.16–31.25; P<0.033). Regardless of the location of the colonies, virus presence was strongly associated with V. destructor level. Therefore, all the factors that directly or indirectly influence the levels of mites will be also influencing the presence of the viruses.

Virion Structure of Black Queen Cell Virus, a Common Honeybee Pathogen.

ABSTRACTViral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales. Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae. The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

An Inert Pesticide Adjuvant Synergizes Viral Pathogenicity and Mortality in Honey Bee Larvae

Honey bees are highly valued for their pollination services in agricultural settings, and recent declines in managed populations have caused concern. Colony losses following a major pollination event in the United States, almond pollination, have been characterized by brood mortality with specific symptoms, followed by eventual colony loss weeks later. In this study, we demonstrate that these symptoms can be produced by chronically exposing brood to both an organosilicone surfactant adjuvant (OSS) commonly used on many agricultural crops including wine grapes, tree nuts and tree fruits and exogenous viral pathogens by simulating a horizontal transmission event. Observed synergistic mortality occurred during the larval-pupal molt. Using q-PCR techniques to measure gene expression and viral levels in larvae taken prior to observed mortality at metamorphosis, we found that exposure to OSS and exogenous virus resulted in significantly heightened Black Queen Cell Virus (BQCV) titers and lower expression of a Toll 7-like-receptor associated with autophagic viral defense (Am18w). These results demonstrate that organosilicone spray adjuvants that are considered biologically inert potentiate viral pathogenicity in honey bee larvae, and guidelines for OSS use may be warranted.