DOI: 10.2478/JAS-2020-0018 J. APIC. SCI. VOL. 64 NO. 2 2020J. APIC. SCI. Vol. 64 No. 2 2020 Original Article HRM ANALYSIS OF SPERMATHECAL CONTENTS TO DETERMINE THE ORIGIN OF DRONES THAT INSEMINATED BEE QUEENS Yasin Kahya* Ankara University, Turkey *corresponding author: [email protected] Received: 13 November 2019; accepted: 30 June 2020 Abstract Europe, Africa and the Middle East have several original subspecies of the western (Apis mellifera L.), each with distinctive characteristics. These subspecies are the product of natural selection in their native range. Nevertheless, anthropogenic impacts such as migratory and use of non-native queens result in an admixture of these subspecies and their ecotypes. I aimed to develop a SNP-based method to detect whether queen honey bees were mated with drones from foreign subspecies. For this purpose, Caucasian and Italian queens and drones were reared. Each queen was instrumentally inseminated with mixed semen collected from Caucasian (4 µl) and Italian drones (4 µl). The spermathecae of queens were dissected out after the onset of oviposition. The DNA was extracted from each spermatheca and from the thoraces of Caucasian and Italian drones. Seven regions on mtDNA that were isolated from drones were sequenced to determine the SNPs, enabling the discrimination of Caucasian sperm from Italian in spermathecal contents. Based on one SNP (11606. bp, T/C) residing on the Cytb gene, a specific primer was designed to be used in High Resolution Melting (HRM) analysis. HRM analysis indicated that heteroduplex peak profiles were present in all spermathecal contents of instrumentally inseminated queens. The results provide proof of the concept that the presence of likely non-native mitochondrial lineages can be detected by HRM analysis based on the SNP genotyping of spermathecal contents.

Keywords: honey bee, HRM analysis, mtDNA, SNP, spermatheca

INTRODUCTION bee (A. m. carnica), European Dark bee (A. m. mellifera), (A. m. ligustica) and (Apis mellifera L.) has a Caucasian bee (A. m. caucasica), are of current wide biodiversity and many of its subspecies economic importance, and the most common have adapted to the habitats of Africa, Asia subspecies breeding/produced all over the and Europe. The subspecies have differen- world. Since honey bees adapt to the particular tiated in terms of morphological, molecular, geographic area and conditions in which they behavioral and physiological characteristics, and live, each subpopulation has potential value with to date approximately thirty western honey bee regard to their unique genetic and phenotypic subspecies have been described (Meixner et traits, and it is important to protect them (Rúa al., 2011; Ruttner, 1988; Sheppard et al., 1997; et al., 2013). Sheppard & Meixner, 2003; Chen et al., 2016). Such basic reproductive characteristics of Analyses showed that the morphological charac- honey bees as haplodiploid sex determination teristics of western honey bee subspecies were mechanism, mating of the queens with more than distributed into five morpho-genetic lineages: one (polyandry) at one or several nuptial Tropical Africa (A), North and Western Europe flights, drones dying immediately after their (M), South-Eastern Europe (C), Near East (O) and single mating (monogamy) and storing live sperm Yemenitica (Y) (Kauhausen-Keller, Ruttner, & for fertilization in spermatheca during their life Keller, 1997; Ruttner, Tassencourt, & Louveaux, (Winston, 1987; Baer, 2005) make it difficult to 1978; Ruttner, 1988). conserve genetically distinct subpopulations, but Four western honey bee subspecies, Carniolan the most important are polyandry and in-flight 241 Kahya HRM analysis for non-native mating control mating at unpredictable drone congregation one copy of nuclear DNA as opposed to multiple areas (Baudry et al., 1998; Koeniger et al., 2005). copies of mtDNA (Meusel & Moritz, 1993), this Honey bee queens mate with between seven initial work focused on an mtDNA marker. After and eighteen drones (average ten drones) during SNP determination, queen bees were instrumen- one or several nuptial flights (Woyke, 1962). tally inseminated with the semen mixture of two Estimated mating frequencies in various studies subspecies. Then, isolated DNA from the sper- range from 6.5 (Taber, 1954) to 41.3 (Kraus et mathecal content was genotypically identified al., 2004). by Real-Time PCR-HRM analysis based on mtDNA The conservation of honey bee genetic sources SNP marker. are possible through the controlled mating within closed populations or through instrumental MATERIAL AND METHODS insemination to prevent introgression between subpopulations. Besides the main challenges of This research was carried out at the Honey Bee honey bee reproductive biology, anthropogenic and Beekeeping Laboratory, Department of and beekeeping activities including migratory Science, Faculty of Agriculture, Ankara beekeeping, use of foreign queens and selective University. Field studies were done in 2013 and breeding also affect genetic resources by laboratory tests in 2014. In the spring season, increasing genetic diversity or mixing honey bee one Caucasian (A. m. caucasica) and one Italian (A. populations (Harpur et al., 2012, 2013; Rúa et m. ligustica) colony with a strong bee population al., 2009 and 2013). The combination of these were chosen and given to empty drone comb factors increases the degeneration (or speed within a plastic cage for drone of the genetic admixture) of endemic honey rearing. The queen of the colony was confined bee subspecies and ecotypes with foreign to the plastic cage on the drone comb and was subspecies. The loss of valuable traits along with provided for egg laying into the empty drone the degeneration of native honeybee populations cells. Drones were observed to emerge from poses a threat to sustainable beekeeping and sealed cells and were marked with paint (Edding agriculture. Therefore, successful conservation 751) on the thorax. They were kept in a large strategies are under development to prevent drone banking colony for the sexual maturation introgression between native and foreign honey period (10-12 days of age). The marked drones bee populations (Rúa et al., 2009). were used for semen collection and DNA Practical molecular genetic tools should be sequencing. developed to mating control and determine The queen bees were reared through grafting. whether there is gene flow from foreign sources Upon reaching sexual maturity (6 days of age), in honey bee populations (Zayed, 2009). In this they were instrumentally inseminated with 8 µl study, a simple and fast method is suggested semen mixture of Caucasian and Italian drones for conservation approaches of native honey (1:1) in order to obtain heterospermic spermath- bee subspecies. A SNP-based method, Real-Time ecal content. A Gilmont micrometer connected to PCR-HRM, was implemented to determine the a Harbo syringe was used for measuring semen genetic sources of the spermathecal contents of volume. Instrumentally inseminated queens honey bee queens. For this purpose, an mtDNA were grabbed after egg laying in the mating SNP found to vary across two subspecies boxes and kept in ethyl alcohol (96%, -20ºC) (Caucasian and Italian) was identified. An until the dissection process. DNA was isolated mtDNA SNP was chosen due to concerns about via a kit (Invitrogen, PureLink® Genomic DNA kit) analytical sensitivity during HRM analysis, from both thorax of drones and spermathecal though the future integration of additional contents of queens. Sperms in the spermathe- nuclear markers would constitute an advance cae of instrumentally inseminated queens (25 by enabling a more complete characterization of queen bees) were dissected under the stereo lineage introgression. Since sperm cells includes microscope (Leica, Z16 APO). The concentration

242 J. APIC. SCI. Vol. 64 No. 2 2020 and purity of isolated DNA were measured by Real-Time Thermal Cycler (36 wells). PCR prior to Nanodrop Spectrophotometer (ND 2000), and HRM analysis was set up in 20 µl total volume. DNA integrity was tested by running with 1% Each reaction contained a mixture of Roche- agarose gel electrophoresis. DNA samples were LightCycler® 480 High Resolution Melting Master, diluted to 7 ng/µl concentration with ddH2O (1X), forward and reverse primer (0.2 µM), MgCl2 before PCR. (2.0 mM) and DNA (14 ng/reaction) isolated from The positions of SNPs on mtDNA were spermathecal contents. The conditions of the determined by DNA sequencing in order to Real-Time thermal cycler were initial denatura- distinguish the Caucasian drones from the tion at 94°C for 10 min., 50 cycles of denatura- Italian drones. Eight Caucasian and six Italian tion at 94°C for 10 sec., annealing at 59°C for drones were used for DNA sequencing. The 15 sec., extension at 72°C 20 sec. And the final SNPs were searched on seven mtDNA region. extension at 72°C for 2 min. After PCR, the HRM Sequences of the forward and reverse primer analysis was performed at 470 nm (excitation) were for amplifying mtDNA regions; 5-GGCA- and 510 nm (emission) wavelength channel. GAATAAGTGCATTG-3 and 5-CAATATCATTGAT- Before the HRM analysis, PCR products were GACC-3 (Garnery et al., 1993) for tRNAleu-COΙI, kept to ensure heteroduplex formation 94ºC for 5-TTTTGTACCTTTTGTATCAGGGTTG-3 and 1 min., at 40ºC for 1 min. Melting curves were 5-CTATAGGGTCTTATCGTCCC-3 (Hall & Smith, then generated by increasing the tempera- 1991), for LrRNA, 5-TTAAGATCCCCAGGAT- tures in steps 0.1ºC for two sec between 65 and CATG-3 and 5-TGCAAATACTGCACCTATTG-3 (Hall 75ºC. Besides the HRM analysis of DNA isolated & Smith, 1991) for COΙ, 5-TATGTACTACCATGAG- from the spermathecal contents, we examined GACAAATATC-3 and 5-ATTACACCTCCTAATT- the DNA obtained from Caucasian and Italian TATTAGGAAT-3 (Crozier, Koulianos, & Crozier, drone thoraces artificially mixed at different 1991) for Cytb, 5-TGATAAAAGAAATATTTTGA-3 proportions (100% Caucasian, 75% Caucasian and 5-TGAAACTATTATATAAATTG-3 (Arias & + 25% Italian, 50% Caucasian + 50% Italian, Sheppard, 1996) for ND2, 5- CAACATCGAGGTCG- 25% Caucasian + 75% Italian, 100% Italian) for CAAACATC-3 and 5-GTACCTTTTGTATCAGGGTT- sensitivity of HRM analysis and the melting GA-3 (Nielsen, Page, & Crosland, 1994) for 16s curve behaviors obtained from HRM analysis rRNA, 5-TCGAAATGAATAGGATACAG-3 and (homoduplex/heteroduplex). 5-GGTTGAGATGGTTTAGGATT-3 (Bouga et The sequence results of the Caucasian and al., 2005) for ND5. Each PCR was performed Italian drones were visualized through FinchTV in 30 µl volume containing dNTP (0.167 mM, (1.4.0) and the base sequences were aligned Thermo Scientific dNTP Set), forward and with Bioedit (Hall, 1999). Raw data of Real-Time reverse primers (0.167 µM, IDT), Taq Buffer (1x PCR-HRM analyses was obtained from Thermo Scientific Fermentas), Taq polymerase Rotorgene Q (2.1.0) software. The raw HRM (1 U, Thermo Scientific Fermentas) and 30 ng/ data was transferred to the Gnumeric (1.12.35) reaction DNA. The thermal cycling parameters software, geometric means were calculated of PCR amplification were thirty-five cycles at from three points and melting curve graphics 94ºC for 1 min., at 50-55ºC for 1 min., at 72ºC were generated. for 3 min. and at 72ºC for 15 min. After SNPs were found on mtDNA regions RESULTS of Caucasian and Italian drones, a new primer (5-TGAATTTGAGGTGGATTTTCA-3 and 5-CCAA- One SNP (T/C 11606 bp, Cau/Lig) was found to GAGGATTAGATGATCCAG-3) was designed discriminate the Caucasian and Italian drones. for the region where the SNP (T/C 11606 bp, Fig. 1 shows the aligned nucleotide sequence of Cau/Lig) were found in the Cytb region of the the mtDNA cytb region in two drone genotypes. drones for use in HRM analyses. HRM analysis The image of PCR products (143 bp) which was performed on the Qiagen Rotor-Gene Q contain detected SNP on the mtDNA Cytb region

243 Kahya HRM analysis for non-native mating control

Fig. 1. Cytb nucleotide sequence of Caucasian and Italian drones (7-10 Caucasian drones, 12-14 Italian drones).

Fig. 2. Agarose gel image of mtDNA Cytb (143 bp) region (L: 100-3000 bp). is shown in Fig. 2. curves of pure Caucasian and Italian samples Isolated DNA from Caucasian and Italian drones (homozygotes) showed melting curves with one was used as pure (navy blue: 100% Caucasian, peak (homozygous). Fig. 3 shows the different claret red: 100% Italian) and as mixed in different melting curves of the pure Caucasian (navy blue) proportions (pink: 75% Caucasian + 25% Italian, and Italian (claret red) DNA fragments amplified yellow: 50% Caucasian + 50% Italian, light by qPCR. A difference of about 1°C was observed blue: 25% Caucasian + % 75 Italian) to test the at the peak of the melting curves with one peak analytical sensitivity of HRM analyses. HRM between pure Caucasian (navy blue) and Italian analysis was found to be applicable for detecting (claret red) DNA fragments. Caucasian alleles the presence of foreign drone DNA. The melting consisted of a T nucleotide at position 11606

Fig. 3. Melting curves of pure and mixed Caucasian and Italian DNA samples (Cau; T, Caucasian, It; C, Italian).

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Fig. 4. HRM derivative curves of spermathecal contents (blue arrow shows early peaks due to heterozygosity). in mtDNA leading to fragments with lower DISCUSSION melting temperature (69°C), while the Italian alleles had C nucleotide, leading to fragments In this study, a Real-Time PCR-HRM analysis was with higher melt peak (70°C). Due to imperfect developed for targeting a genetic marker for binding, all mixed samples of two genotypes the rapid discrimination of two drone genotypes (heterozygotes) showed another early peak in in spermathecal contents in order to determine the melting curves (heteropolymers) regardless mating with drones or foreign maternal ancestry. of the mixture proportion (Fig. 3). The results of Thus, it has been tested whether HRM analysis HRM analysis clearly showed promise for deter- could be used as a genomic tool to determine mining genotypes in spermatheca depending on gene flow to native honey bee populations from the melting curve shape. foreign subspecies. HRM analysis is widely used The HRM analysis of isolated DNA from sper- in heteroduplex analysis, SNP analysis, mutation mathecal contents of queen was successful screening and DNA fingerprint analysis. This in detecting the described SNP diversity of method in which the melting thermodynamics mtDNA associated with Caucasian and Italian of the double helix structure is monitored with genotypes. The spermathecal contents of the help of intercalating fluorescent dyes which twenty-five queen bees were analyzed with the can bound between double-stranded DNA. Real-Time HRM method. The derivative curves The HRM analysis of all DNA samples isolated obtained from the raw data in the HRM analysis from the spermathecae were heteroduplexed. of the isolated DNA from the spermathecal The melting curves of spermathecal contents contents are presented in Fig. 4. All spermathe- were presented without any normalization cal DNA isolates of queens inseminated with the procedure. HRM analysis results can be evaluated Caucasian and Italian sperm mixtures showed successfully without applying normalization an early peak in the curves (blue arrow shows to melting curves data (Druml & Cichna-Markl, early peaks due to heterozygosity). 2014). In this research, all the queens were in- strumentally inseminated with 50% Caucasian + 50% Italian sperm mixture (8 µl). Also, artificial

245 Kahya HRM analysis for non-native mating control mixtures of isolated DNA from Caucasian and bee populations is important not only in terms Italian drone thoraces were studied for tests of of biology but also in determining whether the HRM analysis. However, the sensitivity of the practices of breeding and conservation genetics HRM analysis was not tested under the 25:75% are being done effectively. The monitoring DNA mixture (Fig. 3). Although the results may of gene flow from foreign genetic sources to change according to the amplicon length and native honeybee populations depends on the SNP class, heteroduplex structure is known to identification of markers at the molecular level. be detected in DNA mixture ratios as low as In this research one SNP on mtDNA was used 1-5% by HRM analysis (Carillo et al., 2011; Do & as a marker. The results showed that a genetic Dobrovic, 2009; Ganopoulos et al., 2012; Krypuy mixture from foreign sources to native honey et al., 2007). bee populations could be quickly determined Nucleotide variation within the mitochondrial based on SNP genotyping with Real-Time HRM genome is frequently used in the identifica- analysis in spermathecae. tion of subspecies and linage of honey bees Future works would benefit from nuclear (Collet, Arias, & Lama, 2007; Cornuet & Garnery, markers as well as a broader set of markers. 1991; Smith et al., 1997; Solorzano et al., 2009). In this context, published genomes and SNP AT-rich mtDNA (~ 80%) (Crozier & Crozier, 1993) or indel (insertion/deletion) variant libraries of limits the success of primer design for HRM honey bee subspecies will be important. With analysis (tm = ~ 60°C and fragment length additional genomic data, this method can be <150 bp). Additionally, the class of the SNP to extended for screening additional genotypes, be studied should be considered, because HRM although HRM analysis typically requires an analysis shows higher success in Class 1 SNP individual PCR reaction per marker for such mismatches (T/C and A/G) that change more the genotyping. The use of more DNA regions for stability of the DNA chain (Liew, 2004; Ramirez each genotype comparison will allow the power et al., 2010). of the HRM genotyping method to be increased One of the critical points in this research is and the method may be made available for DNA isolation from the contents of spermathe- screening the success of honey bee conserva- ca, which is a small organ (~1 mm3). The small tion breeding efforts. spermathecal content is a risk for obtaining sufficient quantity and quality of DNA to carry ACKNOWLEDGEMENTS out molecular genetic analyses, but it was possible to obtain DNA in quantity and quality I would like to thank Dr. H. Vasfi Gençer, Dr. that would allow the execution of the studies Aykut Özkul, Dr. E. Doruk Engin and Dr. Rodney (10-30 ng/µl). Microsatellites were used in Richardson for help in improving the contents of past works that focused on the identification this article and laboratory assays. This research of drone subspecies that inseminated honey was supported partially by TÜBİTAK (The bee queens, (Oleksa et al., 2013). An additional Scientific and Technological Research Council of study was conducted to develop a SNP panel Turkey, Grant No: TOVAG- 112O007). for the detection of Africanization in honey bees (Chapman et al., 2017). While this assay REFERENCES is commendable, our understanding of honey bee population genetics should be substan- Arias, M. C., & Sheppard, W. S. (1996). Molecular phy- tially improved in order to apply this analysis logenetics of honey bee subspecies (Apis mellifera type to the lineages of interest in this work. An L.) inferred from mitochondrial DNA sequence. Mo- HRM-based analysis may be more feasible in lecular Phylogenetics and Evolution, 5(3), 557-566. some cases as well given potential limitations on https://doi.org/10.1006/mpev.1996.0050 available laboratory technology and resources. Controlling the colony-level genetics of honey Baer, B. (2005). Sexual selection in Apis bees.

246 J. APIC. SCI. Vol. 64 No. 2 2020

Apidologie, 36(2), 187-200. https://doi.org/10.1051/ Crozier, Y. C., Koulianos, S., & Crozier, R. H. (1991). An apido:2005013 improved test for Africanized honeybee mitochon- drial DNA. Experientia, 47(9), 968-969. Baudry, E., Solignac, M., Garnery, L., Gries, M., Cornuet, J.-M., Koeniger, N. (1998). Relatedness among hon- Do, H., & Dobrovic, A. (2009). Limited copy number eybees (Apis mellifera) of a drone congregation. Pro- - high resolution melting (LCN-HRM) enables the ceedings of the Royal Society B: Biological Sciences, detection and identification by sequencing of low 265(1409), 2009-2014. https://doi.org/10.1098/ level mutations in cancer biopsies. Molecular Cancer, rspb.1998.0533 8, 82. https://doi.org/10.1186/1476-4598-8-82

Bouga, M., Harizanis, P. C., Kilias, G., & Alahiotis, S. Druml, B., & Cichna-Markl, M. (2014). High resolution (2005). Genetic divergence and phylogenetic rela- melting (HRM) analysis of DNA - Its role and poten- tionships of honey bee Apis mellifera (: tial in food analysis. Food Chemistry, 158, 245-254. ) populations from Greece and Cyprus using https://doi.org/10.1016/j.foodchem.2014.02.111 PCR - RFLP analysis of three mtDNA segments. Apidologie, 36(3), 335-344. https://doi.org/10.1051/ Ganopoulos, I., Madesis, P., Darzentas, N., Argiriou, A., apido:2005021 Tsaftaris, A. (2012). Barcode High Resolution Melting (Bar-HRM) analysis for detection and quantification Carillo, S., Henry, L., Lippert, E., Girodon, F., Guiraud, of PDO “Fava Santorinis” (Lathyrus clymenum) adul- I., Richard, C., ... Lavabre-Bertrand, T. (2011). Nested terants. Food Chemistry, 133(2), 505-512. https:// High-Resolution Melting Curve Analysis. The Jour- doi.org/10.1016/j.foodchem.2012.01.015 nal of Molecular Diagnostics : JMD, 13(3), 263-270. https://doi.org/10.1016/j.jmoldx.2010.12.002 Garnery, L., Solignac, M., Celebrano, G., & Cornuet, J.-M. (1993). A simple test using restricted PCR-amplified Chapman, N. C., Bourgeois, A. L., Beaman, L. D., Lim, mitochondrial DNA to study the genetic structure J., Harpur, B. A., Zayed, A., ...Oldroyd, B. P. (2017). An of Apis mellifera L. Experientia, 49(11), 1016-1021. abbreviated SNP panel for ancestry assignment of https://doi.org/10.1007/BF02125651 honeybees (Apis mellifera). Apidologie, 48(6), 776- 783. https://doi.org/10.1007/s13592-017-0522-6 Hall, H. G., & Smith, D. R. (1991). Distinguishing Afri- can and European honeybee matrilines using ampli- Chen, C., Liu, Z., Pan, Q., Chen, X., Wang, H., Guo, H., fied mitochondrial DNA.Proceedings of the National ... Shi, W. (2016). Genomic Analyses Reveal Demo- Academy of Sciences of the United States of Amer- graphic History and Temperate Adaptation of the ica, 88(10), 4548-4552. Newly Discovered Honey Bee Subspecies Apis mel- lifera sinisxinyuan n. Ssp. Molecular Biology and Evo- Hall, T. (1999). BioEdit: a user-friendly biological se- lution, 33(5), 1337-1348. https://doi.org/10.1093/ quence alignment editor and analysis program for molbev/msw017 Windows 95/98/NT. Nucleic Acids Symposium Se- ries, 41, 95-98. Collet, T., Arias, M. C., & Lama, M. A. D. (2007). 16S mtDNA variation in Apis mellifera detected by Harpur, B. A., Minaei, S., Kent, C. F., & Zayed, A. PCR-RFLP. Apidologie, 38(1), 47-54. https://doi. (2012). Management increases genetic diversity org/10.1051/apido:2006056 of honey bees via admixture. Molecular Ecology, 21(18), 4414-4421. https://doi.org/10.1111/j.1365- Cornuet, J. M., & Garnery, L. (1991). Mitochondrial DNA 294X.2012.05614.x variability in honeybees and its phylogeographic im- plications. Apidologie, 22(6), 627-642. https://doi. Harpur, B. A., Minaei, S., Kent, C. F., & Zayed, A. (2013). org/10.1051/apido:19910606 Admixture increases diversity in managed honey bees: Reply to De la Rúa et al. (2013). Molecular

247 Kahya HRM analysis for non-native mating control

Ecology, 22(12) , 3211-3215. ht t p s : //d o i . o rg /10.1111/ for a fourth lineage of Apis mellifera mtDNA. Journal mec.12332 of Heredity, 91(1), 42-46.

Kauhausen-Keller, D., Ruttner, F., & Keller, R. (1997). Ramirez, M. V., Cowart, K. C., Campbell, P. J., Morlock, Morphometric studies on the microtaxonomy of the G. P., Sikes, D., Winchell, J. M., Posey, J. E. (2010). Rapid species Apis mellifera L. Apidologie, 28(5), 295-307. Detection of Multidrug-Resistant Mycobacterium https://doi.org/10.1051/apido:19970506 tuberculosis by Use of Real-Time PCR and High- Resolution Melt Analysis. Journal of Clinical Microbi- Koeniger, N., Koeniger, G., Gries, M., & Tingek, S. ology, 48(11), 4003-4009. https://doi.org/10.1128/ (2005). Drone competition at drone congregation JCM.00812-10 areas in four Apis species. Apidologie, 36( 2) , 211- 221. https://doi.org/10.1051/apido:2005011 Rúa, P. De la, Jaffé, R., Dall’Olio, R., Muñoz, I., Serra- no, J. (2009). Biodiversity, conservation and current Kraus, F. B., Neumann, P., van Praagh, J., & Moritz, R. threats to European honeybees. Apidologie, 40(3), F. A. (2004). Sperm limitation and the evolution of 263-284. https://doi.org/10.1051/apido/2009027 extreme polyandry in honeybees (Apis mellifera L.). Behavioral Ecology and Sociobiology, 55(5), 494- Rúa, P. De la, Jaffé, R., Muñoz, I., Serrano, J., Moritz, R. 501. https://doi.org/10.1007/s00265-003-0706-0 F. A., Kraus, F. B. (2013). Conserving genetic diversity in the honeybee: Comments on Harpur et al. (2012). Krypuy, M., Ahmed, A. A., Etemadmoghadam, D., Hy- Molecular Ecology, 22(12), 3208-3210. https://doi. land, S. J., deFazio, A., Fox, S. B., ... Dobrovic, A., (2007). o rg /10.1111/m e c .12333 High resolution melting for mutation scanning of TP53exons 5-8. BMC Cancer, 7(1), 168. https://doi. Ruttner, F., Tassencourt, L., & Louveaux, J. (1978). org/10.1186/1471-2407-7-168 Biometrical-statistical analysis of the geographic variability of Apis mellifera L. I. Material and Methods. Liew, M. (2004). Genotyping of Single-Nucleotide Apidologie, 9(4), 363-381. https://doi.org/10.1051/ Polymorphisms by High-Resolution Melting of Small apido:19780408 Amplicons. Clinical Chemistry, 50( 7 ) , 1156 -116 4. ht t- ps://doi.org/10.1373/clinchem.2004.032136 Ruttner, F. (1988). Biogeography and Taxonomy of Honeybees. Springer-Verlag Berlin Heidelberg Meixner, M. D., Leta, M. A., Koeniger, N., & Fuchs, S. GmbH. (2011). The honey bees of Ethiopia represent a new subspecies of Apis mellifera-Apis mellifera simen- Sheppard, W. S., Arias, M. C., Grech, A., & Meixner, M. sis n. ssp. Apidologie, 42(3), 425-437. https://doi. D. (1997). Apis mellifera ruttneri, a new honey bee org/10.1007/s13592-011-0007-y subspecies from Malta. Apidologie, 28(5), 287-293. https://doi.org/10.1051/apido:19970505 Meusel, M. S., & Moritz, R. F. A. (1993). Transfer of paternal mitochondrial DNA during fertilization Sheppard, W. S., & Meixner, M. D. (2003). Apis mel- of honeybee (Apis mellifera L.) eggs. Current Ge- lifera pomonella, a new honey bee subspecies from netics, 24(6), 539-543. https://doi.org/10.1007/ Central Asia. Apidologie, 34(4), 367-375. https://doi. BF00351719 org/10.1051/apido:2003037

Nielsen, D., Page, R. E., & Crosland, M. W. (1994). Clinal Smith, D. R., Slaymaker, A., Palmer, M., & Kaftanoğlu, O. variation and selection of MDH allozymes in honey (1997). Turkish honey bees belong to the east Medi- bee populations. Experientia, 50(9), 867-871. terranean mitochondrial lineage. Apidologie, 28(5), 269-274. https://doi.org/10.1051/apido:19970503 Palmer, M. R., Smith, D. R., & Kaftanoğlu, O. (2000). Turkish honeybees: Genetic variation and evidence

248 J. APIC. SCI. Vol. 64 No. 2 2020

Solorzano, C. D., Szalanski, A. L., Kence, M., McKern, Winston, M. L. (1987). The biology of the honey bee. J. A., Austin, J. W., Kence, A. (2009). Phylogeography Harvard University Press; Cambridge, Massachu- and Population Genetics of Honey Bees (Apis setts, London, UK., 281. mellifera) From Turkey Based on COI-COII Sequence Data. Sociobiology, 53(1), 237-246. Woyke, J. (1962). Natural and Artificial Insemination of Queen Honeybees. Bee World, 43(1), 21-25. htt- Taber, S. (1954). The Frequency of Multiple Mating ps://doi.org/10.1080/0005772X.1962.11096922 of Queen Honey Bees. Journal of Economic Ento- mology, 47(6), 995-998. https://doi.org/10.1093/ Zayed, A. (2009). Bee genetics and conservation. jee/47.6.995 Apidologie, 40(3), 237-262. https://doi.org/10.1051/ apido/2009026 Oleksa, A., Wilde, J., Tofilski, A., & Chybicki, I. J. (2013). Partial reproductive isolation between European subspecies of honey bees. Apidologie, 44(5), 611- 619. https://doi.org/10.1007/s13592-013-0212-y

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