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High-resolution (mtDNA) melting analysis for simple and efficient characterization of Africanized honey bee Apis mellifera (Hymenoptera:Apidae)

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Abstract

Analysis of the mtDNA variation in Apis mellifera L. has allowed distinguishing subspecies and evolutionary lineages by means of different molecular methods; from RFLP, to PCR-RFLP and direct sequencing. Likewise, geometric morphometrics (GM) has been used to distinguish Africanized honey bees with a high degree of consistency with studies using molecular information. High-resolution fusion analysis (HRM) allows one to quickly identify sequence polymorphisms by comparing DNA melting curves in short amplicons generated by real-time PCR (qPCR). The objective of this work was to implement the HRM technique in the diagnosis of Africanization of colonies of A. mellifera from Argentina, using GM as a validation method. DNA was extracted from 60 A. mellifera colonies for mitotype identification. Samples were initially analyzed by HRM, through qPCRs of two regions (485 bp/385 bp) of the mitochondrial cytochrome b gene (cytb). This technique was then optimizing to amplify a smaller PCR product (207 bp) for the HRM diagnosis for the Africanization of colonies. Of the 60 colony samples analyzed, 41 were classified as colonies of European origin whereas 19 revealed African origin. All the samples classified by HRM were correctly validated by GM, demonstrating that this technique could be implemented for a rapid identification of African mitotypes in Apis mellifera samples.

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References

  • Baylac M, Garnery L, Tharavy D et al (2008) ApiClass, an automatic wing morphometric expert system for honeybee identification http://apiclass.mnhn.fr

  • Collet T, Ferreira K, Arias M et al (2006) Genetic structure of Africanized honeybee populations (Apis mellifera L.) from Brazil and Uruguay viewed through mitochondrial DNA COI–COII patterns. Heredity 97:329–335

    Article  CAS  Google Scholar 

  • Crozier YC, Koulianos S, Crozier RH (1991) An improved test for Africanized honeybee mitochondrial DNA. Cell Mol Life Sci 47(9):968–969

    Article  CAS  Google Scholar 

  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9(8):772

    Article  CAS  Google Scholar 

  • De La Rúa P, Hernandez-Garcia R, Jiménez Y, Galián J, Serrano J (2005) Biodiversity of Apis mellifera iberica (Hymenoptera: Apidae) from northeastern Spain assessed by mitochondrial analysis. Insect Syst Evol 36:21–28

    Article  Google Scholar 

  • Diniz MN, Soares AEE, Sheppard WS, Del Lama MA (2003) Genetic structure of honey bee populations from southern Brazil and Uruguay. Genet Mol Biol 26:47–52

    Article  CAS  Google Scholar 

  • Dryden IL, Mardia KV (2016) Statistical shape analysis: with applications in R. Wiley

  • Erali M, Voelkerding KV, Wittwer CT (2008) High resolution melting applications for clinical laboratory medicine. Exp Mol Pathol 85:50–58

    Article  CAS  Google Scholar 

  • Franck P, Garnery L, Loiseau A, Oldroyd BP, Hepburn HR, Solignac M, Cornuet JM (2001) Genetic diversity of the honeybee in Africa: microsatellite and mitochondrial data. Heredity 86(4):420–430

    Article  CAS  Google Scholar 

  • Francoy TM, Prado PRR, Gonçalves LS, Da Fontoura Costa L, De Jong D (2006) Morphometric differences in a single wing cell can discriminate Apis mellifera racial types. Apidologie 37:91–97

    Article  Google Scholar 

  • Francoy TM, Bezerra-Laure MAF, Jong DD, Wittmann D, Drauschke M, Muller S, Steinhage V, Goncalves LS (2008) Identification of Africanized honeybee through wing Morphometrics: two fast and efficient procedures. Apidologie 39:488–494

    Article  Google Scholar 

  • Garnery L, Solignac M, Celebrano G, Cornuet JM (1993) A simple test using restricted PCR-amplified mitochondrial DNA to study the genetic structure of Apis mellifera L. Experientia 49(11):1016–1021

    Article  CAS  Google Scholar 

  • Guzman-Novoa E, Morfin N, De la Mora A, Macías-Macías JO, Tapia-González JM, Contreras-Escareño F et al (2020) The process and outcome of the africanization of honey bees in Mexico: lessons and future directions. Front Ecol Evol 8:404

    Article  Google Scholar 

  • Hall HG, Muralidharan K (1989) Evidence from mitochondrial DNA that African honey bees spread as continuous maternal lineages. Nature 339:211–213

    Article  CAS  Google Scholar 

  • Hall HG, Smith DR (1991) Distinguishing African and European honeybee matrilines using amplified mitochondrial DNA. Proc Natl Acad Sci 88:4548–4552

    Article  CAS  Google Scholar 

  • Harpur BA, Minaei S, Kent CF, y Zayed A (2012) Management increases genetic diversity of honey bees via admixture. Mol Ecol 21:4414–4421

    Article  Google Scholar 

  • Kandemir I, Özkan A, Fuchs S (2011) Reevaluation of honey bee (Apis mellifera) microtaxonomy: a geometric morphometric approach. Apidologie 42:618–627

    Article  Google Scholar 

  • Kerr WE, Leon Del Rio S, Barrionuevo MD (1982) The southern limits of the distribution of the Africanized honey bee in South America. Am Bee J 121:196–198

    Google Scholar 

  • Klingenberg CP (2011) MorphoJ: an integrated software package for geometric morphometrics. Mol Ecol Resour 11:353–357

    Article  Google Scholar 

  • Klingenberg CP, Monteiro LR (2005) Distances and directions in multidimensional shape spaces: implications for morphometric applications. Syst Biol 54:678–688

    Article  Google Scholar 

  • Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874

    Article  CAS  Google Scholar 

  • Leclercq G, Gengler N, Francis F (2018) How human reshaped diversity in honey bees (Apis mellifera L.): a review. Entomologie Faunistique-Faunistic Entomology

  • Marin MS, Quintana S, Leunda MR et al (2016) A new method for simultaneous detection and discrimination of Bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) using real time PCR with high resolution melting (HRM) analysis. J Virol Methods 227:14–22

    Article  CAS  Google Scholar 

  • Meixner MD, Pinto MA, Bouga M et al (2013) Standard methods for characterising subspecies and ecotypes of Apis mellifera. J Apic Res 52:1–28

    Article  Google Scholar 

  • Miguel I, Baylac M, Iriondo M, Manzano C, Garnery L, Estonba A (2011) Both geometric morphometric and microsatellite data consistently support the differentiation of the Apis mellifera M evolutionary branch. Apidologie 42:150–161

    Article  Google Scholar 

  • Nielsen DI, Ebert PR, Hunt GJ et al (1999) Identification of Africanized honey bees (Hymenoptera: Apidae) incorporating morphometrics and an improved polymerase chain reaction mitotyping procedure. Ann Entomol Soc Am 92:167–174

    Article  Google Scholar 

  • Pinto MA, Johnston JS, Rubink WL et al (2003) Identification of Africanized honey bee (Hymenoptera: Apidae) mitochondrial DNA: validation of a rapid polymerase chain reaction-based assay. Ann Entomol Soc Am 96:679–684

    Article  CAS  Google Scholar 

  • Pinto MA, Rubink WL, Patton JC, Coulson RN, Johnston JS (2005) Africanization in the United States:replacement of feral European honeybees (Apis mellifera L.) by an African hybrid swarm. Genetics 170:1653–1665

    Article  CAS  Google Scholar 

  • Pinto MA, Muñoz I, Chávez-Galarza J, De La Rua P (2012) The Atlantic side of the Iberian peninsula: a hot-spot of novel African honey bee maternal diversity. Apidologie 43:663–673

    Article  Google Scholar 

  • Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics

  • Rohlf FJ (2010) tpsDig v2. 16. Free software available. http://morphometrics.org/morphmet.html. Accessed 22 June 2011

  • Rubio JLU, Novoa EG, Hunt GJ, Benítez AC, Rubio JAZ (2003) Efecto de la africanización sobre la producción de miel, comportamiento defensivo y tamaño de las abejas melíferas (Apis mellifera L.) en el altiplano mexicano. Vet Méx 34(1):47–59

    Google Scholar 

  • Santos GB, Espínola SM, Ferreira HB et al (2013) Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach. Parasites vectors 6:1–5

    Article  Google Scholar 

  • Scott Schneider S, DeGrandi-Hoffman G, Smith DR (2004) The African honey bee: factors contributing to a successful biological invasion. Annu Rev Entomol 49(1):351–376

    Article  Google Scholar 

  • Sheppard WS, Rinderer TE, Garnery L, Shimanuki H (1999) Analysis of Africanized honey bee mitochondrial DNA reveals further diversity of origin. Genet Mol Biol 22(1):73–75

    Article  Google Scholar 

  • Smith DR, Taylor OR, Brown WL (1989) Neotropical Africanized honey bees have African mitochondrial DNA. Nature 339:213–215

    Article  CAS  Google Scholar 

  • Soares S, Grazina L, Mafra I, Costa J, Pinto MA, Oliveira MBP, y Amaral JS (2019) Towards honey authentication: Differentiation of Apis mellifera subspecies in European honeys based on mitochondrial DNA markers. Food Chem 283:294–301

    Article  CAS  Google Scholar 

  • Szalanski AL, McKern JA (2007) Multiplex PCR-RFLP diagnostics of the Africanized honey bee (Hymenoptera: Apidae). Sociobiology 50:939–946

    Google Scholar 

  • Taylor OR (1988) Ecology and economic impact of African and Africanized honey bees. In: Needham GR, Page RE, Delfinado-Baker M, Bowman CE (eds) Africanized honey bees and bee mites. Ellis Horwood, Chichester, pp 29–41

    Google Scholar 

  • Tofilsky A (2008) Using geometric morphometrics and standard morphometry to discriminant three honeybee subspecies. Apidologie 38:538–563

    Google Scholar 

  • Tucker EJ, Huynh BL (2014) Genotyping by high-resolution melting analysis. In: Crop breeding. Springer, pp 59–66

  • Viscosi V, Cardini A (2011) Leaf morphology, taxonomy and geometric morphometrics: a simplified protocol for beginners. PLoS ONE 6:e25630

    Article  CAS  Google Scholar 

  • Vossen RH (2017) Genotyping DNA variants with high-resolution melting analysis. In: Genotyping. Springer, pp 17–28

  • Whitfield CW, Behura SK, Berlocher SH, Clark AG, Johnston JS, Sheppard WS, Tsutsui ND (2006) Thrice out of Africa: ancient and recent expansions of the honey bee, Apis mellifera. Science 314(5799):642–645

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the beekeepers who collected and forwarded samples from different localities as well as Dr.Tiago Mauricio Francoy, who provided us with the wings images from pure subspecies used in Geometric morphometric analysis. This research was supported by the CIAS and UNMdP. We appreciate the contribution of the anonymous reviewers and editorial team who helped improve this manuscript.

Funding

Financial support of projects of UNMdP EXA 2016-2017, PID 035/2016 and Project PIT-AP-BA 2016 Comicion de investigaciones científicas (CIC).

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Authors and Affiliations

  1. Instituto de Investigaciones en Producción Sanidad y Ambiente (IIPROSAM), CONICET-UNMdP, Centro de Asociación Simple CIC PBA, Mar del Plata, Argentina

    Leonardo P. Porrini, Constanza Brasesco, Matias Maggi, Martín J. Eguaras & Silvina Quintana

  2. Centro de Investigación en Abejas Sociales, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina

    Leonardo P. Porrini, Constanza Brasesco, Matias Maggi, Martín J. Eguaras & Silvina Quintana

  3. Área Biología Molecular Instituto de Análisis Fares Taie, Mar del Plata, Argentina

    Silvina Quintana

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  1. Leonardo P. Porrini
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  2. Constanza Brasesco
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  3. Matias Maggi
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  5. Silvina Quintana
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Correspondence to Leonardo P. Porrini.

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Porrini, L.P., Brasesco, C., Maggi, M. et al. High-resolution (mtDNA) melting analysis for simple and efficient characterization of Africanized honey bee Apis mellifera (Hymenoptera:Apidae). Genetica 149, 343–350 (2021). https://doi.org/10.1007/s10709-021-00139-1

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