Apimondia 2015 Abstract book (South Korea)

BHP-036

Sperm viability in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide imidacloprid and the organophosphate acaricide coumaphos

Veeranan Chaimanee1, Jeff Pettis2, Jay Evans2

1Maejo University Phrae Campus, Thailand

2 Bee Research Laboratory, USDA, USA

Honey bee population declines are a global concern. Numerous factors appear to cause the decline including parasites, pathogens, malnutrition and pesticides. Residues of the organophosphate acaricide coumaphos and the neonicotinoid insecticide imidacloprid, widely used to combat Varroa mites and for crop protection in agriculture, respectively, have been detected in wax, pollen and comb samples. Here, we assess the effects of these compounds at different doses on the viability of sperm stored in the honey bee queens’ spermatheca. Our results demonstrate that sub-lethal doses of imidacloprid (0.02 ppm) decreased sperm viability by 50%, 7 days after treatment. Sperm viability was also reduced about 33% in queens treated with high doses of coumaphos (100 ppm). This research clearly demonstrates that chemical exposure can affect sperm viability in queen honey bees.

BHP-037

Natural plant extract (HO21) with effective antimicrobial activity against Paenibacillus larvae is not toxic to Apis mellifera and could be used in beehives to control American Foulbrood disease

Jessica Martínez, Merly deArmas, Germán Leal-Kenal, Marcelo Henriquez, Paulette Conget

Universidad del Desarrollo, Chile

American foulbrood (AFB) is a serious worldwide spreading disease of honeybee (Apis mellifera L) caused by the spore forming bacterium Paenibacillus larvae. In infected hives, spores are the main vectors for the spreading of the disease, which can be found in brood, honey, wax, pollen and hive walls. The curative treatments with antibiotics is usually used in some countries, unfortunately the extensive use of antibiotics leads to an accumulation of residues in beehive products (especially in honey), and this practice has been shown to lead to bacterial resistance. The natural plant extract HO21 with antimicrobial properties has been shown to have an inhibitory effect on the growth of spores and vegetative cells of P. larvae. The aim of this study was to determine the acute oral toxicity of the extract on adult honeybees and also evaluate the effect of chronic oral administration of HO21 on the survival of larvae and adult bees grown in vitro. Toxicity expressed as LD50 showed lethal effect to bees at very high concentrations, 256 g/bee, showing HO21 it is non-toxic. Survival of larvae and honeybees orally treated with HO21 presented similar results to the group without treatment, causing no toxic effects or death

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at the same or superior concentration that exhibits antimicrobial activity (MIC for spores 0,9 g/l), resulting in a survival rate of 85-90% after 5 and 10 days of observation respectively. These results provide initial evidence that HO21 can be administered orally to the colony. HO21 is a potential therapeutic alternative against AFB.

BHP-038

Prevalence of tracheal mite at the beginning of the season in apiaries from Melipilla province, Chile

Patricia Aldea Sánchez, Naomi Durán, Rafael Rodríguez

Universidad Mayor, Chile

Acaropisosis is produced by the endoparasite Acarapis woodi, who affects the respiratory system in honeybees. This parasite invades the trachea, feeding on hemolymph, causing a degeneration of the air conducts. The Chilean reality is unknown but some informal data show a prevalence less than 9%. However it is believed that this mite generated economic losses and sanitary problems especially in wintering season. The aim of this study was to determine the prevalence of Acarapis woodi early in the growing season in the Province of Melipilla and to get some association factors such as professional beekeeper, transhumance and treatments used against Varroa mite. A spatial description of the beehives was done by parasite load on a map of the province. The results indicate that the average prevalence of acaropisosis in Melipilla Province was 14.6% and when it was calculated by commune for the same period move between 25% in María Pinto, 20.8% in Curacaví, 16% in Melipilla, 7.3% in San Pedro and 2.8% in Alhué. It was obtained a positive correlations between the professional level in the beekeeper, a negative correlation between numbers of hives from each beekeeper and no correlations between transhumance and type of treatments used against Varroa mite, on the prevalence of Acarapisosis. We conclude that the prevalence in the province of Melipilla at the beginning of the growing season was higher than expected calculated according to current national records and there is some factors that could affect the prevalence of this disease.

BHP-039

Comparing the level of resistance of varroa mite obteined from apiaries which usually apply synthetic or organic acaricides

Patricia Aldea Sánchez, Ú rsula Videla, Rafael Rodríguez

Universidad Mayor, Chile

Globally, the mite Varroa destructor, has become the most serious threat to the honeybee. Because in Chile have a low diversity of products to treat varroa mite has gained strength. The aim of this study was to determine if the mite Varroa destructor has developed resistance to synthetic acaricides flumethrin, amitraz and coumaphos, reducing its effectiveness to values less than 90%, in central Chile. To get this aim, a sample of 48 colonies from beehives in which usually flumethrin, amitraz coumaphos are applied was taken. As a control group, 48 sampling apiary hives from certified organic or in the process of, to compare the efficacies achieved between one group and another was made. In each sample a set of 200 bees with at least 10 Varroa mites were collected. As a result it was found that the average resistance of the mites present in hives treated with flumethrin was 69.9%, as the apiaries treated with coumaphos and 66.5% for the group treated with amitraz. Resistance levels in the control groups were on average 7.1% in flumethrin, from 6.9% resistance to coumaphos and 6.5% when it was exposed to amitraz. When comparing these values with the chi square test, a significant difference between the apiaries with continuous use of synthetic acaricides products relative to those that only were used organics treatments was

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obtained. The results confirm the presence of Varroa mite resistance to synthetic acaricides commonly used by beekeepers in Chile.

BHP-040

Association between Antimicrobial Activity of Lactic Acid Bacteria from the Intestinal Tract of Apis mellifera and The Floral Species in Chile

Patricia Aldea Sánchez, Patricia Henríquez, Javiera Quintana, Rafael Rodríguez, Annette

Trombert

Universidad Mayor, Chile

When honeybees (Apis mellifera) become foragers, foreign microorganisms are introduced into bees and other nectarivorous through the collection of nectar / pollen and through trophallaxis. The composition of the bee’s microbiota may vary according to the floral species, climate, microbes from other pollinating insects, airborne microorganisms, and the nutritional composition of the nectar and pollen of the species of flora. Lactic acid bacteria are part of this microbiota and their diversity and properties may vary in different conditions such as nutritional composition of nectar and pollen, and the presence of different microorganisms between flowers. These conditions may also trigger an array of different responses from lactic acid bacteria of the same species, like the ability to inhibit different pathogens, the ability to survive and thrive under difficult conditions or to stablish themselves in the digestive tract of Apis mellifera or other insects. Lactic acid bacteria were isolated from beehives located in north-central, central and south regions of Chile. The honeybee colonies were healthy, without commercial pollination activity and synthetic treatments that could alter the normal microbiota. Digestive tracts were extracted, the content spread on plates with MRS agar medium and incubated under capnophilic conditions. The isolates were identified by PCR amplification and sequencing of the 16S rDNA gene. Our aim was to do a correlation between the antimicrobial properties of the strains isolated and the chilean flora were 45.83% of it is endemic, and demonstrate the differences and correlations between strains from different regions of the country.

BHP-041

Porous ceramics for controlling the release of bioactive compounds in combating bee parasites

Uraiwan Intatha1, Sukum Eitssayeam2

1 School of Science, Mae Fah Luang University

2 Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Thailand

Porous ceramics are studied for natural extracts packaging application to control bee mites. Firstly, the amount of starting precursors such as cellulose (pore former) and PVP (surfactant) are varied to find the suitable preparing condition. The ratio of cellulose and PVP are affected by density and porosity of ceramic. The best ceramic condition is cellulose 10 g and PVP 3 cc with 90.54 % porosity. Second, the effects of lemongrass oil on Varroa jacobsoni and Tropilaelaps mercedesae mites are studied in laboratory scale. The % corrected mortality of V.

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jacobsoni mites and T. mercedesae mites were reported 100 %. However, controlled conditions have less that 25 % of corrected mortality. Finally, the effects of lemongrass oil on bees are studied. The lemongrass oil has no effects to bee population. The porous ceramics could be used to release lemongrass oil up to 30 days.

BHP-042

Only Vespa simillima xanthoptera Cameron visits apiaries in Jeju

JongMoon Cho1, Youngseok Jung1, Chuleui Jung2, Dong-soon Kim3, Yoonkyu Lim4

1 College of Veterinary medicine, Jeju National University

2 Andong National University

3 College of applied Life Science, Jeju National University

4 College of Veterinary Medicine, Jeju National University, Republic of Korea

Vespid wasps are harmful predators of honeybee. Species inventory and phonological data is the basic information for management of any pest population. We surveyed the vespid wasp occurrence nearby the experimental apiary (Apis mellifera) of Jeju National University from 2013 fall and 2014 season. Conventional traps were located in 3 different locations with altitudes of 255, 356 and 410m. Total 108 Vespa wasps captured were all identified as Vespa simillima xanthoptera Cameron and most Vespa were captured from apiary in the region of altitude 356m. Lowest Vespid wasps were captured in July 2014, cause of heavy rainfall. average precipitation in July, 2014 is more than 255.5mm in July, 2013. Spring population was significantly bigger (25.6 : 6.7 mm) than that of fall (23.7 : 6.4 mm, length : width, respectively), implying spring population would be mostly overwintered queens. Also, late season survey covering Jeju island also confirmed that Vespa simillima xanthoptera only visits apiaries in Jeju. It is known that there are 10 species of Vespa inhabiting in south Korea. But only Vespa simillima xanthoptera Cameron is found in Jeju. Further study on the inventory and altitudinal distribution is demanded to better understand of the vespa community in Jeju island. Sticking pesticide to vespa and repatriating it is one of methods of pest control. Also, study on enviromental-friendly control of Vespa mandarina is deviced. Application to Vespa simillima xanthoptera Cameron is needed to control Vespa wasps in Jeju island.

BHP-043

Rearing Apis cerana larvae by In vitro method

Yong Soo Choi, Myeong Lyeol Lee, Man Young Lee, Hye Kyung Kim, Kyu Ho Byeon

NAAS, Republic of Korea

Honey bees Apis cerana Farbricius 1793 is one of nine species of sting bees in the world. In Korea A. cerana is native bees. Korean A. cerana is a gentle, less absconding and resistant to cold winter. Behavior of A. cerana is much different with Apis mellifera. In This study, we are introduced beekeeping techniques for A. cerana. And then, explain about in vitro larva rearing technique. An in vitro method to rear worker honey bee (A. mellifera) was applied to rear worker honey bee (A. cerana) larvae to the adult stage. Our study results demonstrate that honey bee larvae A. cerana could be reared in vitro. The onset of adult emergence started on day 17 from grafting.

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Worker bee emergence peak on day 18 and declined thereafter. The average survival rate from larvae to pre-pupae stage was 74.6%. The average survival rates from pre-pupae to adult stage and from larvae to adult stage were 40.7 % and 30.4 % respectively.

BHP-044

Organic Compounds, Minerals and Vitamin C of Codonopsis (Codonopsis lanceolata) Honey Produced in Korea

Weon Ki Paik1, Ae Kyung Kwak 1, Se Jong Oh1, Myeong Lyeol Lee2, Yong Soo Choi2,

Hye Kyung Kim2

1 Division of Life Science & Chemistry, Daejin University

2 Department of Agricultural Biology, RDA, National Academy of Agricultural Science, Republic of Korea

In order to use as a new functional food material, we analyzed the chemical components including the organic compounds, minerals and Vitamin C of codonopsis honey which was produced in South Korea. The condensed rate of methanol extraction in honey was 82.5% and main organic compounds that extract by organic solvents in GC-MS analysis were solaestin, acetidin, heminellitene, propyl carbinol, cicloesano, acetoxyethane, cyclopentane and so on. Also, main aromatic compounds that extract by organic solvents in SPME analysis were hydrazine, dimazin, carbamide, isourea, pseudourea, varioform, tropilidene and many more. As proximate composition, crude ash content was higher than manuka honey (0.24%) by 0.57%, and crude protein was higher than manuka honey(0.23%) by 0.36%, but crude fat was lower content than manuka honey(0.34%) by 0.11%. Free sugar that analyze by HPLC consisted of fructose 36.67%, glucose 27.26%, and total sugars was 63.93%. Minerals by ICP analysis were detected total 17 kinds, K 24.63ppm > Na 5.813ppm > Ca 3.708ppm > Mg 1.127ppm etc. Vitamin C was not detected and the antioxidation test result by DPPH free radical scavenge effect of codonopsis honey was lowered than manuka honey.

BHP-045

Organic Compounds, Minerals and Vitamin C of Hedysarum (Astragalus membranaceus) Honey Produced in Korea

Weon Ki Paik1, Ae Kyung Kwak1, Mun Seok Kim1, Myeong Lyeol Lee2, Yong Soo Choi2,

Hye Kyung Kim2

1 Division of Life Science & Chemistry, Daejin University

2 Department of Agricultural Biology, RDA, National Academy of Agricultural Science, Republic of Korea

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In order to use as a new functional food material, we analyzed the chemical components including the organic compounds, minerals and Vitamin C of hedysarum honey which was produced in South Korea. The condensed rate of methanol extraction in honey was 68% and main organic compounds that extract by organic solvents in GC-MS analysis were solaestin, hemellitol, heminellitene, propanoic acid, propyl carbinol, cyclopropanamine and so on. Also, main aromatic compounds that extract by organic solvents in SPME analysis were adilimoll, hydrazine, dimazin, carbamide, isourea, pseudourea, varioform and many more. As proximate composition, crude ash content was higher than manuka honey (0.24%) by 0.65%, and crude protein was higher than manuka honey (0.23%) by 0.28%, but crude fat was lower content than manuka honey (0.34%) by 0.06%. Free sugar that analyze by HPLC consisted of fructose 33.85%, glucose 26.99%, and total sugars was 60.84%. Minerals by ICP analysis were detected total 17 kinds, K 29.315ppm > Na 6.069ppm > Ca 3.702ppm > Mg 1.125ppm etc. Vitamin C was not detected and the antioxidation test result by DPPH free radical scavenge effect of hedysarum honey was lowered than manuka honey.

BHP-046

Resistance of Varroa destructor to apistan© and bayvarol©

Koumad Salima

ENSA, Morocco

Efficacy of Fluvalinate and Flumethrin molecules against Varroa destructor was evaluated in bee colonies in Central Algeria through field tests using Apistan, Bayvarol, we have used Apiguard© as an alternative treatment. The average efficacy was found to be in the order of 53% and 41%, respectively, far from the real therapeutic value of 99%, probably due to the resistance of Varroa mite populations to Pyrethroid treatment.

BHP-047

Finding new antibiotics through Actinobacteria and other bacteria isolated from soil samples

Tuan Manh Nguyen, Jaisoo Kim

Kyonggi University, Republic of Korea

We have selected the most effective15 strains through more than 2,000 colonies, isolated from soil samples at various areas in South Korea. Those can be effective growth inhibitors against Paenibacillus larvae as an agent of American Foulbrood and common microbial human pathogens such as Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and Aspergillus niger. On the basis of 16S rRNA gene sequence analysis, many were involved in genus Streptomyces, and 6 strains were possible new species, which might produce new antibiotic compounds. Through thin layer chromatography (TLC), the compounds included polar and non-polar ones, which will be purified and developed as various drugs for bee pathogens as well as human pathogens.

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BHP-048

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) Colonies in Mongolia

Khaliunaa Tsevegmid, Selenge Dooshi

Nosema, among the most serious of the bee disease which is believed exist everywhere, however, relatively little is known about the distribution or prevalence of Nosema and its source in bees of Mongolia. To determine the prevalence and potential impact of this pathogen on honey bee colonies in the country, each 10 colonies were sampled from five apiaries. Samples were analyzed microscopically for Nosema spores. Local bees were rarely tested positive by nosema in spring and summer seasons, but most of (56%) just arrived (imported from abroad) bee colonies from more than 2000 km were heavily infected. The prevalence and mean levels of infection will be presented.

BHP-049

A tale of bees, their ectoparasite and vectored viruses

Thomas Gillard, Emily Remnant, Niklas Mather, Madeleine Beekman

Behaviour and Genetics of Social Insects Laboratory, School of Biological Sciences, The University of Sydney,

NSW 2006, Australia

When a pathogen requires its host to remain mobile to be spread to naïve hosts, selection should act against pathogens that immobilise their hosts too quickly. Thus, over evolutionary time, pathogens are predicted to become less virulent. Classic epidemiological theory predicts that virulence increases when pathogens are transmitted via a vector: pathogens can spread even when their presence debilitates the host. Implicit in these predictions is the presence of a trade-off between virulence and replication rate of pathogens; the faster pathogens replicate, the more severe the host effects. We investigate the effect of mode of transmission on evolution of virulence using RNA viruses of honeybees. Many bee viruses are vectored by the ectoparasitic mite Varroa destructor. Originally found on the Asian hive bee Apis cerana, Varroa jumped species to infect the Western honeybee A. mellifera sometime in the 1980s and is now present on all beekeeping continents apart from Australia. A range of positive-sense single-strand RNA viruses of the Dicistroviridae and Iflaviridae families present covert infections in Varroa-naïve colonies of honeybees, with occasional seasonal outbreaks. The introduction of Varroa sees a marked increase in viral titre, a decrease in viral diversity, and colonies in collapse. We use an experimental evolution approach to tease apart the roles of mode of transmission, the vector itself, and differences in bee responses to viral infections in the evolution of virulence of RNA viruses. We performed serial transmission experiments, determined if viral titres increased and tested the virulence of evolved viruses on adult Varroa-naïve bees.

BHP-050

Development of rapid detection methods of Korean sacbrood virus

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Mi-Sun Yoo, Hyun-Ji Seo, Ha-Na Jung, Woo Ram Bae, Hee-Soo Lee, Seung-Won Kang,

Yun Sang Cho

Animal and Plant Quarantine Agency, Republic of Korea

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, Korean Sacbrood virus (KSBV) caused a great loss 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) and a novel micro PCRbased detection method, termed ultra-rapid real-time PCR (URRT-PCR) were applied for rapid detection for korean sacbrood virus (KSBV) from honeybees (Apis cerana) infected with SBV in Korea. The LAMP could be detect the virus in RT-LAMP reactions containing 102copies of pBX-KSBV within 30 min, which was 10 times more sensitive than a RT-PCR assay. The URRT-PCR showed high sensitivities which were able to detect 10 copies in the standard assays. In the application of URRT-PCR detection to a KSBV-infected honeybee, the shortest detection time was 10 min 12 sec, including reverse transcription. In addition, these methods could be distinguished between KSBV and other closelyrelated SBV strains. These rapid methods were rapid molecularbased diagnostic tools and useful tool for the rapid and sensitive diagnosis of KSBV infection of honeybees.

BHP-051

Control of Korean Sacbrood Virus using RNA Interference in Apis cerana

Mi-Sun Yoo, Hyun-Ji Seo, Ha-Na Jung, Woo Ram Bae, Hee-Soo Lee,

Seung-Won Kang, Yun Sang Cho

Animal and Plant Quarantine Agency, Republic of Korea

Sacbrood virus (SBV), a causative pathogen of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Since 2010, the Korean Sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. However, there is no treatment method culture for honeybee viruses including SBV. RNA interference (RNAi) is a genesilencing technology by which small double-stranded RNAs are used to target the degradation of RNA with complementary sequence. In this study, we report on prevention of SBV infection by feeding with double-stranded RNA. SBV sequences corresponding to a segment of structural protein VP1 gene for dsVP1 and a segment of the structural polyprotein open reading frame for dsSBV1 were used for cloning. dsRNA synthesis was carried out according to the protocol of the mMESSAGE mMACHINE T7 kit, it was treated with food. The feeding activity and mortality of larvae were observed every day. Experiments were carried out to examine whether ingestion of dsRNAs of SBV sequences would protect bees from SBV infection. The result indicated that two SBV-derived dsRNAs (dsSBV1 and dsVP1) protected bee larvae from subsequent SBV infection. dsRNA of SBV will be used as an efficient and feasible way of controlling bee viral disease aw well as SBV.

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BHP-052

Expression and purification of SBV antigen for rapid diagnostic method using Immunochromatography assay

Mi-Sun Yoo, Hyun-Ji Seo, Ha-Na Jung, Woo Ram Bae, Hee-Soo Lee, Seung-Won Kang,

Yun Sang Cho

Animal and Plant Quarantine Agency, Republic of Korea

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, the research on Korean Sacbrood virus is practically limited until present. In this study, we investigated the expression and purification of SBV structural protein from Apis cerana for rapid diagnostic method using Immunochromatography assay. The VP1(1,023 bp), VP2 (948 bp) , VP3 (1,068 bp) of SBV from Apis cerana was obtained by RT-PCR. Amplified VP1, VP2, VP2 were constructed into expression vector pET21a by BamHI and XhoI site. Recombinant VP1, VP2, VP2 were successfully overexpressed in E.coli system and purified by FPLC. This purified recombinant VP1, VP2, VP2 will be used for the generation of monoclonal antibody to develop a diagnosis tool for SBV infection in honeybee.

BHP-053

Survey on Honeybee Disease in Apis mellifera and Apis cerana in Korean apiaries, 2014

Mi-Sun Yoo, Hyun-Ji Seo, Ha-Na Jung, Woo Ram Bae, Hee-Soo Lee, Seung-Won Kang,

Yun Sang Cho

Animal and Plant Quarantine Agency, Republic of Korea

The honeybee is a major pollinator of crops. The main species present in Korea are the European honeybee, A. mellifera, and the Asian honeybee. A. cerana. The occurrence and distribution of twelve bee diseases was investigated in A.mellifera and A.cerana in Korea using PCR diagnostic method: american foulbrood (AFB), european foulbrood (EFB), chalkbrood, stonebrood, nosema, sacbrood virus (SBV), acute bee paralysis virus (ABPV), deformed wing virus (DWV), black queen cell virus (BQCV), kashmir bee virus (KBV), chronic bee paralysis virus (CBPV) and Israeli acute paralysis virus (IAPV). Samples were collected from 412 apiaries located in 10 different regions of the country in 2014. BQCV was the most prevalent (identified in 46.32% of samples), follo wed by stonebrood(37.05%) , sacbrood(23.99%) , Nosema(16.15%) , DWV(15.20%) , AFB(10.93%) , IAPV(8.31%), chalkbrood(4.51%), EFB(4.28%), KBV(2.14%) in honey bee samples(adult bee, larvae and comb), respectively.

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BHP-054

Development of a multiplex PCR for rapid diagnosis of viral honey bee disease in Korea

Ha-Na Jung, Mi-SunYoo, Hyun-Ji Seo, Woo Ram Bea, Hee-Soo Lee,

Seung-Won Kang, Yun Sang Cho

Animal and Plant Quarantine Agency, Republic of Korea

According to investigated during the last decade, Virus infections of the honeybee(Apis mellifera) have been increased. In general, honeybee viruses are widespread and most of them continue as inapparent infections. We screened honeybee colonies for the existence of several bee viruses, including sacbrood virus(SBV), black queen cell virus(BQCV), Kashmir bee virus(KBV), Deformed wing virus(DWV), Israeli acute paralysis virus(IAPV) and Acute bee paralysis virus(ABPV), using single target RTPCR. Frequently simultaneous infections with several viruses are diagnosed in seemingly healthy bee colonies. Therefore, we developed a multiplex RT-PCR strategy for the simultaneous detection of six bee viruses. The aim of this study was to establish a multiplex polymerase chain reaction strategy for rapid simultaneous detection of the honeybee viruses commonly infected in Korea. The multiplex RT-PCR strategy would be useful for the observation of honeybee viral diseases in the field. Bee samples used for virus screening were collected from all over the country. Viral RNA was extracted using viral gene-spin viral DNA/RNA extraction kit(Intron). Specific primer for SBV, BQCV, KBV, DWV, IAPV and ABPV were designed based on the published nucleotide sequences. To screening for virus infection in honeybee colonies, brood were tested for the presence of honeybee viruses separately by the uniplex RT-PCR. uniplex PCR assay was performed using Top simple DryMix PCR kit(Enzynomics). Multiplex PCR assay was performed using 2X Master/MultiMAX PCR Kit (Intron). The PCR Products were electrophoresed in 1.5% agarose gel, stained with ethidium bromide, and visualized under UV light. It was cloned in Escherichia coli pGEM-T Easy vector (Promega). The use of broad-range primers enable us to detect a maximum of viral variants ideal to declare honeybee colonies virus. The SBV, BQCV, KBV, DWV, IAPV and ABPV primer sets, when used together in the multiplex reaction, amplified only specific products of the expected sizes of 192, 317, 413, 479 ,725, 900bp, respectively, which could be easily distinguished by agarose gel electrophoresis. And viral clones were used as positive controls in field sample tests. And sequences of each band were confirmed. The main objective of this study was to develop a tool that detects the variants of bee viral species infecting honeybee. We expect to apply of a multiplex RT-PCR assay for offers a significant time and cost-saving advantage, especially when a lot of samples are analyzed.

BHP-055

Artificial infection of Honeybee larvae with Sacbrood virus for virus propagation

Ha-Na Jung, Mi-Sun Yoo, Hyun-Ji Seo, Woo Ram Bae, Hee-Soo Lee,

Seung-Won Kang, Yun Sang Cho

Animal and Plant Quarantine Agency, Republic of Korea

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