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Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera1

Published online by Cambridge University Press:  02 April 2012

Karen L. Burgher-MacLellan ,
Geoffrey R. Williams ,
Dave Shutler ,
Kenna MacKenzie  and
Richard E.L. Rogers
Karen L. Burgher-MacLellan*
Affiliation:
Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, Nova Scotia, Canada B4N 1J5
Geoffrey R. Williams
Affiliation:
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1, and Department of Biology, Acadia University, Wolfville, Nova Scotia, Canada B4P 2R6
Dave Shutler
Affiliation:
Department of Biology, Acadia University, Wolfville, Nova Scotia, Canada B4P 2R6
Kenna MacKenzie
Affiliation:
Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, Nova Scotia, Canada B4N 1J5
Richard E.L. Rogers
Affiliation:
Wildwood Laboratoriess Inc., Kentville, Nova Scotia, Canada B4N 3Z1
*
2 Corresponding author (e-mail: [email protected]).
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Abstract

Honey bees, Apis mellifera (L.) (Hymenoptera: Apidae), are parasitized by the microsporidians Nosema apis (Zander) and Nosema ceranae (Fries). Molecular techniques are commonly used to differentiate between these parasites because light microscopy is inadequate. Our objectives were to (i) adapt the previously published duplex polymerase chain reaction (PCR) targeting the 16S rRNA gene of N. apis (321APIS-FOR, 321APIS-REV) and N. ceranae (218MITOC-FOR, 218MITOC-REV) using qualitative real-time PCR assay with SYBR® Green I dye (R-T PCR) and DNA melting-curve analysis, and (ii) determine whether the two Nosema species can be detected simultaneously in honey bees. Total spore counts and purified total genomic DNA were obtained from 37 bee samples (19 individual workers and 18 pooled samples of 15 workers) collected in Nova Scotia, Prince Edward Island, and Newfoundland, Canada. Overall, the prevalence of Nosema species was 86.5% (32/37 samples of bee DNA), based on conventional PCR and the optimized R-T PCR assay. The melting-curve analysis showed three groups of curve profiles that could determine the prevalence of N. apis, N. ceranae, and co-infection (21.9%, 56.2%, and 21.9%, respectively). The duplex R-T PCR assay was efficient, specific, and more sensitive than duplex conventional PCR because co-infection was identified in 5.4% (n = 2) more samples. Sequencing of R-T PCR products confirmed the results of the melting-curve analysis. Duplex R-T PCR with melting-curve analysis is a sensitive and rapid method of detecting N. apis, N. ceranae, and co-infection in honey bees.

Résumé

Les abeilles domestiques, Apis mellifera (L.) (Hymenoptera: Apidae) sont parasitées par les microsporidies Nosema apis (Zander) et N. ceranae (Fries). Parce que la microscopie optique est inadéquate, on utilise couramment des méthodes moléculaires pour distinguer ces parasites. Nos objectifs sont 1) d'adapter la méthode déjà publiée de la réaction de PCR (amplification en chaîne par polymérase) duplex qui cible le gène 16S de l'ARNr de N. apis (321APIS-FOR et 321APIS-REV) et de N. ceranae (218MITOC-FOR et 218MITOC-REV) à l'aide d'un test qualitatif au vert de SYBR I en temps réel avec une analyse de la courbe de fusion de l'ADN (R-T PCR) et 2) de voir s’il est possible de détecter simultanément les deux espèces de Nosema chez les abeilles. Nous avons obtenu les dénombrements de spores et l'ADN génomique total purifié dans 37 échantillons d'abeilles (19 ouvrières individuelles et 18 échantillons collectifs de 15 ouvrières) récoltés en Nouvelle-Écosse, à l'Île-du-Prince-Édouard et à Terre-Neuve, Canada. La prévalence globale de Nosema est de 86,5 % (32/37 échantillons d'ADN d'abeilles) d'après les analyses de PCR conventionnelle et de R-T PCR optimisée. L’analyse de la courbe de fusion révèle l'existence de trois groupes de profils de courbes qui permettent d'identifier les prévalences de N. apis, de N. ceranae et de co-infections (respectivement 21,9 %, 56,2 % et 21,9 %). Le test de la R-T PCR duplex est efficace, spécifique et plus sensible que la PCR duplex ordinaire parce que la co-infection a pu être décelée dans 5,4 % (n=2) plus d'échantillons. Le séquençage des produits de la R-T PCR confirme les résultats de l'analyse de la courbe de fusion. La PCR duplex au vert SYBR I en temps réel avec une analyse de la courbe de fusion est une méthode sensible et rapide de détection de N. apis, de N. ceranae et des co-infections chez les abeilles.

[Traduit par la Rédaction]

Type
Articles
Information
The Canadian Entomologist , Volume 142 , Issue 3 , June 2010 , pp. 271 - 283
Copyright
Copyright © Entomological Society of Canada 2010

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Footnotes

1

Contribution No. 2365 from the Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, Kentville, Nova Scotia.

References

Berry, O., and Sarre, S.D. 2007. Gel-free species identification using melt-curve analysis. Molecular Ecology Notes, 7: 1–4. doi:10.1111/j.1471-8286.2006.01541.x.CrossRefGoogle Scholar
Burgher-MacLellan, K.L., Gaul, S., Mackenzie, K., and Vincent, C. 2009. The use of real-time PCR to identify blueberry maggot (Diptera: Tephritidae, Rhagoletis mendax) from other Rhagoletis species and in lowbush blueberry fruit (Vaccinium angustifolium). Acta Horticulturae International Society of Horticultural Sciences, 810: 265–274.CrossRefGoogle Scholar
Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., et al. 2009. The MIQE guidelines: minimum information for the publication of quantitative real-time PCR experiments. Clinical Chemistry, 55: 611–622. PMID:19246619 doi:10.1373/clinchem.2008.112797.CrossRefGoogle ScholarPubMed
Cantwell, G.E. 1970. Standard methods for counting Nosema spores. American Bee Journal, 110: 222–223.Google Scholar
Cavalier-Smith, T. 1987. Eukaryotes with no mitochondria. Nature (London), 326: 332–333. PMID:3561476 doi:10.1038/326332a0.CrossRefGoogle ScholarPubMed
Chen, Y., Evans, J.D., Smith, I.B., and Pettis, J.S. 2008. Nosema ceranae is a long-present and widespread microsporidian infection of the European honey bee (Apis mellifera) in the United States. Journal of Invertebrate Pathology, 97: 186–188. PMID:17880997 doi:10.1016/j.jip.2007.07.010.CrossRefGoogle ScholarPubMed
Chen, Y., Evans, Y.D., Zhou, L., Boncristiani, H., Kimura, K., Xiao, T., Litkowski, A.M., and Pettis, J.S. 2009. Asymmetrical coexistence of Nosema ceranae and N. apis in honey bees. Journal of Invertebrate Pathology, 101: 204–209. PMID:19467238 doi:10.1016/j.jip.2009.05.012.CrossRefGoogle Scholar
Fries, I. 1993. Nosema apis: a parasite in the honey bee colony. Bee World, 74: 5–19.CrossRefGoogle Scholar
Fries, I., Feng, F., da Silva, A., Slemenda, S.B., and Pieniazek, N.J. 1996. Nosema ceranae n. sp. (Microspora, Nosematidae): morphological and molecular characterization of a microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). European Journal of Protistology, 32: 356–365.CrossRefGoogle Scholar
Hebert, P.D.M., Cywinska, K., Ball, S.L., and deWaard, J.R. 2003. Biological identification through DNA bar codes. Proceedings of the Royal Society of London B, Biological Sciences, 270: 313–321. doi:10.1098/rspb.2002.2218.CrossRefGoogle Scholar
Higes, M., Martín, R., and Meana, A. 2006. Nosema ceranae, a new microsporidian parasite in honey bees in Europe. Journal of Invertebrate Pathology, 92: 93–95. PMID:16574143 doi:10.1016/j.jip.2006.02.005.CrossRefGoogle ScholarPubMed
Higes, M., García-Palencia, P., Martín-Hernández, R., and Meana, A. 2007. Experimental infection of Apis mellifera honeybees with Nosema ceranae (Microsporidia). Journal of Invertebrate Pathology, 94: 211–217. PMID:17217954 doi:10.1016/j.jip.2006.11.001.CrossRefGoogle ScholarPubMed
Higes, M., Martín-Hernández, R., Botías, C., Garrido Bailón, E., González-Porto, A.V., Barrios, L., et al. 2008. How natural infection by Nosema ceranae causes honeybee colony collapse. Environmental Microbiology, 10: 2659–2669. PMID:18647336 doi:10.1111/j.1462-2920.2008.01687.x.CrossRefGoogle ScholarPubMed
Higes, M., Martín-Hernández, R., Garrido Bailón, E., Botías, C., and Meana, A. 2009. The presence of Nosema ceranae (Microsporidia) in North African honey bees (Apis mellifera intermissa). Journal of Apicultural Research, 48: 217–219. doi:10.3896/IBRA.1.48.3.12.CrossRefGoogle Scholar
Huang, W.F., Jiang, J.H., and Wang, C.H. 2007. A Nosema ceranae isolate from the honey bee Apis mellifera. Apidologie, 38: 30–37. doi:10.1051/apido:2006054.CrossRefGoogle Scholar
Invitrogen Corporation. 2008. Real-time PCR: from theory to practice. Invitrogen Corporation, Carlsbad, California, United States of America.Google Scholar
Klee, J., Besana, A.M., Genersch, E., Gisder, S., Nanetti, A., Tam, D.Q., et al. 2007. Widespread dispersal of the microsporidian Nosema ceranae, an emergent pathogen of the western honey bee, Apis mellifera. Journal of Invertebrate Pathology, 96: 1–10. PMID:17428493 doi:10.1016/j.jip.2007.02.014.CrossRefGoogle ScholarPubMed
Martín-Hernández, R., Meana, A., Prieto, L., Salvador, A.M., Garrido-Bailon, E., and Higes, M. 2007. Outcome of colonization of Apis mellifera by Nosema ceranae. Applied and Environmental Microbiology, 73: 6331–6338. PMID:17675417 doi:10.1128/AEM.00270-07.CrossRefGoogle ScholarPubMed
Martín-Hernández, R., Meana, A., García-Palenci, P., Marín, P., Botías, C., Garrido-Bailón, E., et al. 2009. Effect of temperature on the biotic potential of honeybee microsporidia. Applied and Environmental Microbiology. 75: 2554–2557. PMID: 19233948 doi:10.1128/AEM.02908-08.CrossRefGoogle ScholarPubMed
Paxton, R.J., Klee, J., Korpela, S., and Fries, I. 2007. Nosema ceranae has infected Apis mellifera in Europe since at least 1998 and may be more virulent than Nosema apis. Apidologie, 38: 558–565. doi:10.1051/apido:2007037.CrossRefGoogle Scholar
Ririe, K.M., Rasmussen, R.P., and Wittwer, C.T. 1997. Product differentiation by analysis of DNA melting curves during polymerase chain reaction. Analytical Biochemistry, 245: 154–160. PMID:9056205 doi:10.1006/abio.1996.9916.CrossRefGoogle ScholarPubMed
Rogers, R.E.L., and Williams, G.R. 2007. Monitoring Nosema disease in honey bee colonies. Bee Culture, 135: 19–21.Google Scholar
Williams, G.R., Shafer, A.B.A., Rogers, R.E.L., Shutler, D., and Stewart, D.T. 2008 a. First detection of Nosema ceranae, a microsporidian parasite of European honey bees (Apis mellifera), in Canada and central U.S.A. Journal of Invertebrate Pathology, 97: 189–192. PMID:17897670 doi:10.1016/j.jip.2007.08.005.CrossRefGoogle Scholar
Williams, G.R., Sampson, M.A., Shutler, D., and Rogers, R.E.L. 2008 b. Does fumagillin control the recently detected invasive parasite Nosema ceranae in western honey bees (Apis mellifera)? Journal of Invertebrate Pathology, 99: 342–344. PMID:18550078 doi:10.1016/j.jip.2008.04.005.CrossRefGoogle ScholarPubMed
Williams, G.R., Shutler, D., Little, C.M., Burger-MacLellan, K.L., and Rogers, R.E.L. 2010. The microsporidian Nosema ceranae, the antibiotic Fumagilin-B®, and western honey bee (Apis mellifera) colony strength. Apidologie. In press. doi:10.1051/apido/20100230.CrossRefGoogle Scholar
Wolk, D., Sturbaum, G., Hoffman, R., Sterling, C., and Marshall, M. 2008. Molecular methods for microsporidia detection: use of an inhibitor control with real-time PCR. Report No. 91185, AWWA Research Foundation, IWA Publishing, London.Google Scholar
Yu, D.J., Chen, Z.L., Zhang, R.J., and Yin, W.Y. 2005. Real-time qualitative PCR for the inspection and identification of Bactrocera philippinensis and Bactrocera occipitalis (Diptera: Tephritidae) using SYBR green assay. The Raffles Bulletin of Zoology, 53: 73–78.Google Scholar