Hive Debris Research Articles

Megaselia scalaris and Senotainia tricuspis Infesting Apis mellifera: Detection by Quantitative PCR, Genotyping, and Involvement in the Transmission of Microbial Pathogens.

The Megaselia scalaris and Senotainia tricuspis parasitoid flies of the honeybee Apis mellifera were found to infest apiaries of different European and Mediterranean countries but their prevalence and impact on apiary health are little known. Therefore, in this study, quantitative PCR (qPCR)-based methods were developed for their rapid detection directly in hive matrices. The newly developed qPCR assays were targeted at the mitochondrial cytochrome oxidase subunit I (COI) gene for the M. scalaris and the cytochrome B (cytB) gene for the S. tricuspis. The tests were preliminarily applied to 64 samples of adult honeybees and hive debris collected in the Abruzzo and Molise regions, Central Italy, and the Republic of Kosovo showing that both flies occur in the two countries and more frequently in Italy. The positive apiaries in Italy were re-sampled by capturing viable forager bees and isolating emerging flies to carry out the genotyping and analyses aimed at defining if these flies can transmit honeybee pathogens. Genotyping based on the COI and cytB gene sequencing for M. scalaris and S. tricuspis, respectively, identified one S. tricuspis genotype and diverse genotypes of M. scalaris highly similar to those from distant countries. Some fly isolates harbored the DNA or RNA of honeybee microbial pathogens Paenibacillus larvae, deformed wing viruses A and B (DWVA and B), black queen cell virus (BQCV), chronic paralysis virus (CBPV), and Nosema ceranae. The results indicated that these parasites should be efficiently controlled in apiaries by using rapid detection methods to facilitate the large screening studies and early detection.

The Diagnostic Value of qPCR Quantification of Paenibacillus larvae in Hive Debris and Adult Bees for Predicting the Onset of American Foulbrood.

American foulbrood (AFB) is a serious infectious disease of honeybees (Apis mellifera) caused by Paenibacillus larvae. Increased P. larvae count in hive-related material is associated with an increased risk of AFB. Here, we quantified P. larvae cells in 106 adult bee and 97 hive debris samples using quantitative PCR (qPCR); 66/106 adult bee and 66/97 hive debris samples were collected simultaneously from the same bee colony (paired-sample design). The corresponding bee colonies were also examined for the presence of AFB clinical signs. A binary logistic regression model to distinguish between AFB-affected and unaffected honeybee colonies showed a strong diagnostic accuracy of both sample types for predicting the onset of AFB based on P. larvae counts determined by qPCR. The colonies with a P. larvae count greater than 4.5 log cells/adult bee or 7.3 log cells/mL hive debris had a 50% probability of being clinically affected and were categorized as high-risk. The AFB-unaffected colonies had significantly lower P. larvae counts than the AFB-affected colonies, but the latter did not differ significantly in P. larvae counts in relation to the severity of clinical signs. Both bee-related sample types had a high diagnostic value for predicting disease outcome based on P. larvae counts. These results improve the understanding of the relationship between P. larvae counts and AFB occurrence, which is essential for early detection of high-risk colonies.

Selection and Multiplexing of Reverse Transcription-Quantitative PCR Tests Targeting Relevant Honeybee Viral Pathogens.

Verifying the inclusivity of molecular detection methods gives indications about the reliability of viral infection diagnosis because of the tendency of viral pathogens to undergo sequence variation. This study was aimed at selecting inclusive probes based on reverse transcription-quantitative PCR (RT-qPCR) assays for the diagnosis of the most widespread and detrimental viruses infecting honeybees, namely the acute bee paralysis virus (ABPV), the black queen cell virus (BQCV), the chronic paralysis bee virus (CBPV), the deformed wing virus variants A (DWVA) and B (DWVB), and the sacbrood virus (SBV). Therefore, previously described detection methods were re-evaluated in silico for their specificity and inclusivity. Based on this evaluation, selected methods were modified, or new ones were designed and tested in duplex RT-qPCR reactions. The limits of detection (LODs), effect of multiplexing on sensitivity and the viral RNA quantification potential in bees and hive debris were assessed. This study made available diagnostic assays able to detect an increased number of virus variants compared with previously described tests and two viral pathogens in a single PCR reaction.

A Comprehensive Analysis of Beekeeping Risks and Validation of Biosecurity Measures against Major Infectious Diseases in Apis mellifera in Europe

Background/Objectives: In a collaborative effort, FAO, the International Federation of Beekeepers’ Association “Apimondia”, the Appalachian State University, and the Istituto Zooprofilattico Sperimentale del Lazio e Toscana (IZSLT), the FAO Reference Centre for Animal Health and Food Security Discipline Apiculture, health, and biosecurity, designed an online international survey (“Knowledge, Awareness and Practice”-KAP) as a risk assessment tool to validate biosecurity measures able to prevent and control the main honeybee (Apis mellifera subsp.) infectious diseases. Methods: The KAP survey was disseminated through various channels for 14 months (January 2019–March 2020). Beekeepers’ knowledge on nosemosis, American foulbrood (AFB), and European foulbrood (EFB) management was assessed. Beekeepers’ attitudes were investigated by asking them to provide a score called “Acceptance by beekeepers”. Results: Of the 410 respondents, the majority of them (68.3%, n = 252) were from Europe. European beekeepers (n = 252) considered “Highly acceptable” the following practices: removing combs that show signs of dysentery and feeding colonies for nosemosis; the quick identification and management of affected hives for American foulbrood and European foulbrood. Instead, the following practices were considered as not acceptable by beekeepers: for nosemosis, the use of antibiotics and sampling of hive debris for early detection; for American foulbrood and European foulbrood, the shook swarm, the destruction of the whole apiary, as well as their treatment with antibiotics. Conclusion: This study demonstrated the validity of the present “Knowledge, Awareness and Practice” (KAP) survey to validate the best practices for the main infectious honeybee diseases, considering the pragmatic point of view of beekeepers. Sustainable practices for disease control are preferred by the European beekeepers. While having an adequate knowledge base for the early detection of the investigated honeybee diseases, European beekeepers tend to be aware of the health status of their hives, embracing advanced, practice-oriented training. Authors highlight that the selection of practices and their validation should be revised on a regular basis, to be adapted in a timely manner to the current infectious honeybee disease situation, in order to build up a resilient and effective stock of practices.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)—American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I–IV (strain DSM 7030—ERIC I, DSM 25430—ERIC II, LMG 16252—ERIC III, DSM 3615—ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Can the examination of different types of hive samples be a non-invasive method for detection and quantification of viruses in honey bee (Apis mellifera L.) colonies?

Honey bee viruses have been shown to negatively affect the vigour and longevity of European honey bees (Apis mellifera L). In the present work, beehive materials were tested for their potential to serve as non-invasive samples for honey bee virus detection. Honey, pollen, hive debris, hive grid smears and forager honey bees were collected from 24 hives at four locations in the Czech Republic. Deformed wing virus (DWV), acute bee paralysis virus (ABPV), sacbrood virus (SBV) and black queen cell virus (BQCV) were detected using a reverse transcription PCR (RT-PCR) and real-time quantitative RT-PCR and the results for bees and alternative materials compared. All forager bee samples contained DWV, BQCV and SBV and 54.2% had ABPV. When comparing beehive materials to bees, the most promising results were obtained from honey and pollen samples, with BQCV and SBV detected in all honey samples and ABPV in 12.5%. Detection of SBV was achieved in 91.6% of pollen samples, detection of BQCV in 87.5% and detection of DWW in 75%. The results for debris and smears were less consistent with the viral profile of the forager samples. The best candidate materials for honey bee virus detection in a non-invasive technique are honey and pollen.

Holobiont Urbanism: sampling urban beehives reveals cities’ metagenomes

BackgroundOver half of the world’s population lives in urban areas with, according to the United Nations, nearly 70% expected to live in cities by 2050. Our cities are built by and for humans, but are also complex, adaptive biological systems involving a diversity of other living species. The majority of these species are invisible and constitute the city’s microbiome. Our design decisions for the built environment shape these invisible populations, and as inhabitants we interact with them on a constant basis. A growing body of evidence shows us that human health and well-being are dependent on these interactions. Indeed, multicellular organisms owe meaningful aspects of their development and phenotype to interactions with the microorganisms—bacteria or fungi—with which they live in continual exchange and symbiosis. Therefore, it is meaningful to establish microbial maps of the cities we inhabit. While the processing and sequencing of environmental microbiome samples can be high-throughput, gathering samples is still labor and time intensive, and can require mobilizing large numbers of volunteers to get a snapshot of the microbial landscape of a city.ResultsHere we postulate that honeybees may be effective collaborators in gathering samples of urban microbiota, as they forage daily within a 2-mile radius of their hive. We describe the results of a pilot study conducted with three rooftop beehives in Brooklyn, NY, where we evaluated the potential of various hive materials (honey, debris, hive swabs, bee bodies) to reveal information as to the surrounding metagenomic landscape, and where we conclude that the bee debris are the richest substrate. Based on these results, we profiled 4 additional cities through collected hive debris: Sydney, Melbourne, Venice and Tokyo. We show that each city displays a unique metagenomic profile as seen by honeybees. These profiles yield information relevant to hive health such as known bee symbionts and pathogens. Additionally, we show that this method can be used for human pathogen surveillance, with a proof-of-concept example in which we recover the majority of virulence factor genes for Rickettsia felis, a pathogen known to be responsible for “cat scratch fever”.ConclusionsWe show that this method yields information relevant to hive health and human health, providing a strategy to monitor environmental microbiomes on a city scale. Here we present the results of this study, and discuss them in terms of architectural implications, as well as the potential of this method for epidemic surveillance.

Testing of the Efficacy of Bee Probiotic Lactobacilli Under In Vivo Conditions

Abstract American foulbrood is amongst the most dangerous diseases of the bee-family affecting many honeybee colonies worldwide. In countries of European Union, based on veterinary legislation, the bee colonies tested positive to American foulbrood are eradicated with high economic losses. It is therefore necessary to look for effective prevention, especially by the using of natural ingredients such as probiotics. In this study, we used lactobacilli isolated from digestive tracts of adult healthy honey bees and selected based on their good probiotic properties and ability to inhibit the growth of Paenibacillus larvae. These isolates were identified as Lactobacillus brevis and Lactobacillus plantarum. Night cultures of both strains were used for the preparation of probiotic suspensions and pollen was selected as an appropriate carrier for application of probiotic lacto-bacilli to the hives. Half a litre of pollen suspension was prepared for each hive. The suspension for experimental hives contained probiotic lactobacilli in concentration of 107–108 CFU.ml−1. Bees in control hives received pollen suspension without addition of probiotic lacto-bacilli. The suspensions were supplied to bees three times, once a week. Before and during administration of probiotics, samples of honey bees from each hive were taken every week and numbers of lactobacilli, P. larvae, enterobacteria and coliform bacteria were determined in their digestive tracts. Four weeks after the first administration of probiotic-pollen solution the numbers of lactobacilli were increased approximately by 0.5 log. Before starting this experiment, P. larvae (approx. 107 CFU.ml−1) were detected in digestive tracts of honey bees. In the experimental group after 3 weeks and in the control group after 4 weeks, no viable counts of P. larvae were found. The numbers of enterobacteria, coliform bacteria and Bacillus sp. decreased in both groups. During the experiment the health and condition of the hives were monitored. In addition, also monitored were: the development of the bee colony, the number of dead bees, the amount of hive debris, the aggressiveness of the bees, and the amount of honey spun; later during the autumn treatment also the fall of Varroa jacobsoni was noted. In the experimental and control groups, we recorded a decrease in the amount of hive debris. Only in the experimental group was a slightly above-average development of the bee colony, slightly above-average honey yields and a 70 % lower drop of Varroa mites. Based on these results, we assumed that the probiotic-pollen solution had a positive influence on the composition of microbiota in bee digestive tracts and it can increase resistance to P. larvae. It also had a positive effect on the health and condition of the bee colony. Probiotic-free pollen solution showed similar but weaker effects.

A Comparison of Different Matrices for the Laboratory Diagnosis of the Epizootic American Foulbrood of Honey Bees.

American Foulbrood (AFB) of honey bees caused by the spore-forming bacterium Paenibacillus larvae is a notifiable epizootic in most countries. Authorities often consider a rigorous eradication policy the only sustainable control measure. However, early diagnosis of infected but not yet diseased colonies opens up the possibility of ridding these colonies of P. larvae spores by the shook swarm method, thus preventing colony destruction by AFB or official control orders. Therefore, surveillance of bee colonies for P. larvae infection followed by appropriate sanitary measures is a very important intervention to control AFB. For the detection of P. larvae spores in infected colonies, samples of brood comb honey, adult bees, or hive debris are commonly used. We here present our results from a comparative study on the suitability of these matrices in reliably and correctly detecting P. larvae spores contained in these matrices. Based on the sensitivity and limit of detection of P. larvae spores in samples from hive debris, adult bees, and brood comb honey, we conclude that the latter two are equally well-suited for AFB surveillance programs. Hive debris samples should only be used when it is not possible to collect honey or adult bee samples from brood combs.

Molecular Detection of Acarapis woodi Using Hive Debris as Innovative and Non-Invasive Matrix

Acarapis woodi is a pathogen affecting honey bees health worldwide. Its prevalence may be underestimated due to the time-consuming traditional method for its diagnosis and the attitude in focusing the attention only onto Varroa destructor. New PCR techniques have allowed for the verification of the presence of A. woodi in 44 samples of honey bees and 11 samples of hive debris collected from 17 apiaries by the veterinary services of the Latium region (Central Italy). Overall, 9.1% of adult honey bee samples (all belonging to one apiary) and 6.3% of hive debris samples (belonging to 6 apiaries) were positive in an end point PCR and presence of the pathogen was confirmed through Sanger sequencing. Results demonstrated the potential underestimation of A. woodi occurrence in Italian apiaries and reported the first detection of A. woodi in hive debris samples.

Novel TaqMan PCR Assay for the Quantification of Paenibacillus larvae Spores in Bee-Related Samples

Simple SummaryAmerican foulbrood (AFB) is the most severe bacterial disease of honeybees, caused by Paenibacillus larvae. Larvae become infected by ingesting food contaminated with P. larvae spores, which are extremely resistant and can remain infectious for decades. Burning affected colonies is widely used to prevent further spread of the disease. The presence of P. larvae spores in bee-related samples is associated with an increased risk of developing clinical symptoms, and spore counts can be used for early detection of at-risk colonies, which should then undergo thorough clinical examination. Because quantification of P. larvae spores by plate counting is time-consuming and unreliable, due to poor and inconsistent germination, molecular quantification is more suitable. To overcome the limitations of available quantification methods, we developed a quantitative PCR (qPCR) assay for reliable quantification of P. larvae that also performs well at low spore counts. The assay was validated for honey and hive debris samples but can be extended to other sample types. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies, both for honey and hive debris samples. By comparing plate and qPCR counts, the germination rate of P. larvae spores was found to be low and inconsistent.Paenibacillus larvae is the causative agent of American foulbrood (AFB), a devastating disease of honeybees. P. larvae spore counts in bee-related samples correlate with the presence of AFB symptoms and may, therefore, be used to identify at-risk colonies. Here, we constructed a TaqMan-based real-time PCR (qPCR) assay targeting a single-copy chromosomal metalloproteinase gene for reliable quantification of P. larvae. The assay was calibrated using digital PCR (dPCR) to allow absolute quantification of P. larvae spores in honey and hive debris samples. The limits of detection and quantification were 8 and 58 spores/g for honey and 188 and 707 spores/mL for hive debris, respectively. To assess the association between AFB clinical symptoms and spore counts, we quantified spores in honey and hive debris samples originating from honeybee colonies with known severity of clinical symptoms. Spore counts in AFB-positive colonies were significantly higher than those in asymptomatic colonies but did not differ significantly with regard to the severity of clinical symptoms. For honey, the average spore germination rate was 0.52% (range = 0.04–6.05%), indicating poor and inconsistent in vitro germination. The newly developed qPCR assay allows reliable detection and quantification of P. larvae in honey and hive debris samples but can also be extended to other sample types.

New Matrixes to Diagnose Aethina tumida Presence at Apiary Level

Abstract Aethina tumida (Coleoptera: Nitidulidae - Small Hive Beetle - SHB), is a parasite of honey bee colonies that causes the notifiable disease called aethinosis. In 2014, SHB was detected in Southern Italy, where it is still present (Calabria region). As part of surveillance activities, official diagnosis of the disease is performed by veterinary services via visual inspection of single hives in the apiaries. New outbreaks can be eradicated and the spread of SHBs limited by early detection of new introductions. We report an alternative protocol for the diagnosis of SHB through swabs and hive debris analysis by PCR. This was tested in three apiaries through the evaluation of different SHB infestation levels with a hive inspection method. This approach for sampling, followed by biomolecular methods, was fast and useful in unfavorable conditions (bad weather, aggressiveness, robbing), could be integrated in the official diagnosis procedures and may act as pre-clinic indicator.

Upconversion-Linked Immunoassay for the Diagnosis of Honeybee Disease American Foulbrood

American foulbrood (AFB) caused by the bacterium Paenibacillus larvae is the most destructive disease of the honeybee brood. Therefore, rapid and sensitive detection methods are required to limit spreading of this pathogen, which has a major impact on agriculture and biodiversity. While P. larvae is typically detected by microbial cultivation or polymerase chain reaction, antibody-based detection represents a viable alternative. Here, we prepared an antibody specific for P. larvae and used it for the development of an upconversion-linked immunosorbent assay (ULISA). Photon-upconversion nanoparticles (UCNP) were conjugated to streptavidin via a PEG-linker using copper-catalyzed click chemistry to replace the enzyme label in conventional enzyme-linked immunosorbent assay (ELISA). The ULISA showed low cross-reactivity and provided a limit of detection of 2.9 × 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> CFU/mL, representing a 22-fold improvement compared to the ELISA. This level is within the bacterial loads present in honeybee larvae during an AFB infection. The assay was successfully applied to the analysis of spiked samples of bees, larvae, and hive debris.

Microbial communities associated with honey bees in Brazil and in the United States

Honey bee colony losses worldwide call for a more in-depth understanding of the pathogenic and mutualistic components of the honey bee microbiota and their relation with the environment. In this descriptive study, we characterized the yeast and bacterial communities that arise from six substrates associated with honey bees: corbicular pollen, beebread, hive debris, intestinal contents, body surface of nurses and forager bees, comparing two different landscapes, Minas Gerais, Brazil and Maryland, United States. The sampling of five hives in Brazil and four in the USA yielded 217 yeast and 284 bacterial isolates. Whereas the yeast community, accounted for 47 species from 29 genera, was dominated in Brazil by Aureobasidiumsp. and Candida orthopsilosis, the major yeast recovered from the USA was Debaryomyces hansenii. The bacterial community was more diverse, encompassing 65 species distributed across 31 genera. Overall, most isolates belonged to Firmicutes, genus Bacillus. Among LAB, species from Lactobacillus were the most prevalent. Cluster analysis evidenced high structuration of the microbial communities, with two distinguished microbial groups between Brazil and the United States. In general, the higher difference among sites and substrates were dependents on the turnover effect (~ 93% of the beta diversity), with a more pronounced effect of nestedness (~ 28%) observed from Brazil microbiota change. The relative abundance of yeasts and bacteria also showed the dissimilarity of the microbial communities between both environments. These results provide a comprehensive view of microorganisms associated with A. mellifera, highlighting the importance of the environment in the establishment of the microbiota associated with honey bees.

Melissococcus plutonius Can Be Effectively and Economically Detected Using Hive Debris and Conventional PCR

Simple SummaryLaboratory diagnostics of the presence of Melissococcus plutonius is necessary for the confirmation of European foulbrood (EFB) in honey bee colonies. Seeking EFB positive colonies is based on inspections performed by beekeepers or authorized inspectors. Bee brood, adult bees, or honey are usually sampled and confirmed by PCR. Here, we tested a new concept of searching suspected colonies by simple and effective collecting hive debris. Conventional PCR detection of M. plutonius in hive debris gives comparable sensitivity and specificity as for adults or honey. The main advantage of testing hive debris combines its effectiveness and costs compared to the sampling of adult bees or honey. Easy and fast collection of hive debris samples in areas with EFB outbreaks could fasten disease early detection and elimination.European foulbrood (EFB) is an infectious disease of honey bees caused by the bacterium Melissococcus plutonius. A method for DNA isolation and conventional PCR diagnosis was developed using hive debris, which was non-invasively collected on paper sheets placed on the bottom boards of hives. Field trials utilized 23 honey bee colonies with clinically positive symptoms and 21 colonies without symptoms. Bayes statistics were applied to calculate the comparable parameters for EFB diagnostics when using honey, hive debris, or samples of adult bees. The reliability of the conventional PCR was 100% at 6.7 × 103 Colony Forming Unit of M. plutonius in 1 g of debris. The sensitivity of the method for the sampled honey, hive debris, and adult bees was 0.867, 0.714, and 1.000, respectively. The specificity for the tested matrices was 0.842, 0.800, and 0.833. The predictive values for the positive tests from selected populations with 52% prevalence were 0.813, 0.833, and 0.842, and the real accuracies were 0.853, 0.750, and 0.912, for the honey, hive debris, and adult bees, respectively. It was concluded that hive debris can effectively be utilized to non-invasively monitor EFB in honey bee colonies.

Rapid identification of the invasive Small hive beetle (Aethina tumida) using LAMP.

Small hive beetle (SHB), Aethina tumida is an invasive pest of managed honey bees and has invaded Europe from Africa. The main risk point identified for incursions of this pest into the UK is imports of package bees and queens. Surveillance of this pest, carried out by the National Bee Unit, involves monitoring imports of live bees and routine screening of sentinel apiaries around the UK through collection of hive debris for visual inspection of pests. Currently, no molecular methods are used for screening hive debris for this pest in the UK. A new invasion of this pest would likely occur at low levels and might present with partial specimens broken up in the hive or eggs from the beetle, difficult to identify by visual inspection. Therefore, we sought to develop a fast and sensitive method for detecting SHB in hive debris by developing a loop-mediated isothermal amplification (LAMP) assay. The LAMP assay was able to detect the SHB in 19 to 25.37 min and did not cross-react with any non-target species tested. The assay was sensitive and could detect 12pg of DNA and it was able to detect less than 1 mg of tissue in a 30 g complex matrix of honey bee hive debris. The assay could successfully amplify from crude extracts of partial tissue specimens. This tool will allow rapid field screening of suspect beetle specimens and laboratory screening of hive debris for a notifiable exotic pest of honey bees. © 2020 Crown copyright. Pest Management Science © 2020 Society of Chemical Industry.

봉변에서 특이 유전자 검출법에 의한 봉군 내 꿀벌가시응애류 (Tropilaelaps)의 정량적 검출

Rapid detection of Tropilaelaps, an external parasite of honeybees that lead to malformation of honeybee or colony collapse disorder, is becoming important. But it is very difficult to find with the naked eye of Tropilaelaps. In this study, we have developed a method to detect the specific gene of Tropilaelaps from the hive debris and to know the number of Tropilaelaps in the hive through Tropilaelaps-specific quantitative detection. Tropilaelaps-specific gene amplified in DNA extracted from hive debris by consecutive PCR (1st detection, 2nd nested PCR). It could detect 10SUP1/SUP molecules level of Tropilaelapsspecific gene and confirm the amplification of the Tropilaelaps-specific gene. It was possible to accurately quantify the number of Tropilaelaps from the hive debris sample, which is difficult to discriminate the presence of Tropilaelaps visually, through Tropilaelaps-specific detection. Under the microscope, Tropilaelaps was collected and quantitative detection of Tropilaelaps-specific genes was performed. It was possible to quantify the number of Tropilaelaps present in the hive through the molecules of the quantified Tropilaelaps-specific genes. We suggest that hive debris can represent as a micro-environment to hive and show that it can be a simpler and more accurate sample than using a parasitic host honeybee. We expect that hive debris should facilitate the monitoring of Tropilaelaps in hive.

Evaluation of Quantitative PCR (qPCR) Paenibacillus larvae Targeted Assays and Definition of Optimal Conditions for Its Detection/Quantification in Honey and Hive Debris.

The application of quantitative PCR (qPCR) as a routine method to detect and enumerate Paenibacillus larvae in honey and hive debris could greatly speed up the estimation of prevalence and outbreak risk of the American foulbrood (AFB) disease of Apis mellifera. However, none of the qPCR tests described so far has been officially proposed as a standard procedure for P. larvae detection and enumeration for surveillance purposes. Therefore, in this study, inclusivity, exclusivity and sensitivity of detection of P. larvae spores directly in samples of honey and hive debris were re-evaluated for the previously published qPCR methods. To this aim, recently acquired P. larvae sequence data were considered to assess inclusivity in silico and more appropriate non-target species were used to verify exclusivity experimentally. This led to the modification of a previously described method by shortening the forward primer, designing a new reverse primer and using more stringent amplification conditions. The new test allowed the detection of P. larvae spores in honey and hive debris down to 1 CFU/g. The qPCR test optimized in this study proved suitable for quantification and also for identification of field P. larvae strains and real contaminated samples. Therefore, it is proposed for reliable detection and quantification of P. larvae in honey and hive debris, thus circumventing the disadvantages of late AFB diagnosis based on clinical symptoms and possible underestimation of spore numbers that is the main drawback of culture-dependent procedures.

봉변을 이용한 꿀벌 바이러스성 병원체의 다중 초고속 PCR법

봉변을 이용한 꿀벌 바이러스성 병원체의 다중 초고속 PCR법

Bee Counters: Measuring a nest's occupation by its capacitance [Resources_Hands On

Bees are important: they pollinate dozens of crops, including almonds, cacao, and coffee. While there has been a lot of attention paid to Western honeybees owing to colony collapse disorder, this specific disease and others like it are really measurable only once a colony collapses. And in any case, honeybees are not the only important bee pollinators. What we need is the ability to measure and monitor bee activity as it happens. · Historically, such monitoring was the purview of undergraduates armed with clipboards. More recently, optical sensors have allowed for the automatic detection of bees entering and exiting the hive. But placing optical sensors in a habitat of pollen, mud, and other hive debris drastically degrades their effectiveness. What if there was a better way? · A solution suggested itself when the two of us--a field applications engineer for Analog Devices and an amateur bee enthusiast--were working together on a previous project that involved capacitive sensing. Teachman (the bee enthusiast) commented to Perrault (the applications engineer) that the sensitivity of the AD7746 capacitance-to-digital conversion chip was better than he had expected, and wondered, Do you think we could measure bees with this? All else being equal, the capacitance between two electrodes depends on the dielectric constant of the substance between them. Air has a dielectric constant of roughly 1, while water comes in at around 80. As living cells are mostly water, a bee should have a detectable dielectric signature. Intrigued by the idea, we developed a custom sensor setup to measure just that.