Surveillance of European honeybee subspecies distribution in Chiang Mai, Thailand

Nowadays the most common subspecies of western honeybee or European honey bee (Apis mellifera) have 28 subspecies around the world. The subspecies are divided into four major branches were confirmed by mitochondrial DNA analysis. African subspecies belong to branch A, north western European subspecies branch M, south western European subspecies branch C and Middle Eastern subspecies branch O. The different sub species of European honeybees have different characterization factors such as tolerances of environmental temperature changes, specific diseases resistant, defence behaviour and other economically important Traits. In this studied we collected the samples from 50 colonies of Apis mellifera from January to March 2017 at Chiang Mai, Thailand and used PCR-RFLP to identify mitochondrial lineages and subspecies of European Honeybees. There have three different subspecies of Apis mellifera such as Thai A1 Thai A2 and Thai B in Chiang Mai. Province, Thailand. Thai A group are similar match to C or east European lineage A.m. ligustica and A.m. carnica and Thai B group are similar to the match of O or Middle Eastern lineage, A.m. syriaca and A.m. lamarckii. The Haplotype group of ThaiA1 (38% of colonies) and group ThaiA2 (52%) Haplotype group ThaiB (10%).

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Introduction

In Thailand, there are four native species of honey bees: Apis andreniformis, Apis cerana, Apis dorsata and Apis florea (Oldroyd and Wongsiri, 2006) [17] . Thailand is a tropical country it has various plantation and several kinds of flora are blooming in all seasons. Especially in the north of Thailand, have plenty of nectar resources and suitable climate for beekeeping (Oldroyd and Wongsiri, 2006, Chantawannakul, de Guzman, Li, and Williams, 2015) [17,4] . There are four native species of honey bees: Apis andreniformis, Apis cerana, Apis dorsata and Apis florea (Oldroyd and Wongsiri, 2006) [17] . Today beekeeping in Thailand have more than 500,000 of European honey bee colonies and 1,000 beekeepers all over the country (Suwannapong, Benbow, and Nieh, 2011) [25] . Apis mellifera L. was introduced to Chulalongkorn University, Bangkok Thailand in the early 1940s and 1953 Introduced to Kasetsart University (Seanbualuang, P., 2012) [26] . At 1970s there were imported large numbers of A. mellifera from Taiwan to Lampoon and Chiang Mai in northern Thailand for commercial purposes (Wongsiri et al., 1995) [27] . Beekeeping for Apis mellifera were spread out in northern Thailand, especially Chiang Mai province, Thailand. Beekeepers distributions in northern Thailand 7 provinces such as Chiang Mai, Chiang Rai, Phra, Lamphun, Lampang, Phayao, and Nan. In the Northern Thailand have 66% of total honey bee colonies in Thailand and 52% of total beekeepers (Suwannapong, G., et al. 2011) [25] . There are 28 subspecies have been described from A. mellifera's native range in Europe, Africa and the Middle East, based primarily on morphometric variation (Ruttner, 1988; Engel, 1999) [20] . The different sub species of European honey bees have different characterization factors such as tolerances of environmental temperature changes, specific diseases resistant, defence behaviour and other economically important Traits (Ruttner, 1988) [20] . The Studies of A. mellifera mitochondrial DNA have at least 4 major mitochondrial lineages such as M or West European, C or Eastern Mediterranean, A or African, (Hall and Muralidharan, 1989; Arias and Sheppard, 1996) [15,2] and O or Middle Eastern lineage (Franck et al., 2000) [12] . A possible fifth lineage, Y, has been described from Ethiopia (Franck et al., 2001) [13] . Until 2006 the ancestry of A. mellifera in Thailand is unknown, then the matrilineal origins of Apis mellifera were recorded as 5 rear and 3 major subspecies of Apis mellifera by PCR-RFLP and direct DNA sequencing (Suppasat. T., et al., 2007) [24] . The RFLP (Restriction Fragment Length Polymorphisms) is the revealed by the digesting of the whole entire mitochondrial genome with restriction endonucleases. PCR-RFLP is more convenient than RFLP because it is revealed within a specific PCR-amplified region. The most popular PCR amplification of the non-coding region located between the tRNAleu and COII genes by digestion with the Dra restriction enzyme, commonly known as Dra I test. It is widely used in Honey bees maternal identification Clarke et al., 2002; Pinto et al., 2004) [5,18] . In this research to clarify the maternal ancestry of A. mellifera in Chiang Mai Provence, Thailand used PCR-RFLP analysis of four regions of mtDNA restricted with the 6-base restriction enzymes Dra I and a 4base restriction enzyme, Hinf I.

Materials and Methods

Materials and Methods

Collections

Apis mellifera samples were collected between January to March 2017 from 50 colonies from 5 different distinct in Chiang Mai Thailand. All samples were kept in 95% ethanol and stored at -20 •C until extraction. Because it is helpful in phylogeographical studies, easy to understand in natural hybrid zone, detecting of new species from foreign queen's introduction and tracking on temporal changed of honey bee maternal compositions (Cánovas et [3,16,5,13,12,7,11] . PCR-based assays, colony identification using the DraI test can be performed in a small-sized laboratory equipped with basic equipment. The DraI test was adopted as the standard for honey bee maternal identification, for several reasons. Because it has proven to be the most powerful and informative. And COI-COII/DraI variation has been widely documented across natural and introduced honey bee ranges. The combination of length and restriction-site polymorphisms produced by the DraI test has resolved over 100 haplotypes (Franck et [13,3] . In this research to determine the RFLP pattern, the digested products of each samples are electrophoresed in 1.5% Agarose gel at 110V for 90 min.

PCR-RFLP analysis

PCR-RFLP analysis

Result

Restriction enzyme digestion of PCR products are summarized in amplification of tRNAleu-COII revealed fragments of two sizes. Digestion of this fragment with Dra I produced a single pattern and Hinf I have two restriction patterns found. According to recent research got the amplification of tRNAleu-COII revealed fragments of two sizes such as 573 bp and 837p. The smaller fragment was defined as "ThaiA", which is the typical of the C lineage. The digestion of this fragment with Hinf I revealed two restriction patterns which defined as "ThaiA2" and the larger fragment were defined as "ThaiB". When the compared of recent research the result of the European Honey Bee Sub species distribution in Chiang Mai Province have three different subspecies such as "Thai A1", "Thai A2" and "Thai B". Thai a group are similar match to C or east European lineage A.m. ligustica and A.m. carnica and Thai B group are the match similar with O or Middle Eastern lineage, A.m. syriaca and A.m. lamarckii. The Haplotype group of ThaiA1 (38% of colonies) and group ThaiA2 (52%) Haplotype group ThaiB (10%). [27] . From 1970, the European honeybees are distributed in Chiang Mai Provence. Suppasat. T, et al., 2007 [24] , studied matrilineal origins of European Honeybees Apis mellifera in Thailand. The three subspecies of A. mellifera were distributed around the country. Although the bee colonies were increased more than two times the honey production and honeybee product production are didn't increase too much. Thai European Honey Bee sub species can be resistant for climate changes and local diseases problems, but the production and breeding are not so good like European honeybees. To the best quality of beekeeping in Thailand the keeping bees are needed to upgrade the best honey production quality bees, the worker bees foraging activities and the best breeding quality of queen.

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Percentage of European Honey Bees subspecies distribution

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Percentage of European Honey Bee Subspecies distribution in

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PCR-RFLP patterns of Dra I and Hinf I enzymes digestion at

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