The Partial Mitochondrial Sequence of the Old World Stingless Bee, Tetragonula Pagdeni (2013) Journal of Genetics, Pp

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Thummajitsakul, S., Silprasit, K., Klinbunga, S., Sittipraneed, S. The partial mitochondrial sequence of the Old World stingless bee, Tetragonula pagdeni (2013) Journal of Genetics, pp. 1-5. Article in Press.

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The partial mitochondrial sequence of the Old World stingless Citation Network bee, Tetragonula pagdeni

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Keywords All Times Cited Counts Author Keywords: stingless bees; mitochondrial DNA gene order; long PCR; Tetragonula; Tetragonula 0 in All Databases pagdeni 0 in Web of Science Core Collection KeyWords Plus: APIS-MELLIFERA; MOLECULAR PHYLOGENY; GENE ORGANIZATION; DNA; 0 in BIOSIS Citation Index APIDAE; EVOLUTIONARY; HYMENOPTERA; MELIPONINI; ORIGIN; GENOME 0 in Chinese Science Citation Database Author Information 0 in Data Citation Index Reprint Address: Thummajitsakul, S (reprint author) 0 in SciELO Citation Index Srinakharinwirot Univ Ongkharak, Fac Hlth Sci, Nakhon Nayok 26120, Thailand.

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RESEARCH NOTE

The partial mitochondrial sequence of the Old World stingless bee, Tetragonula pagdeni

SIRIKUL THUMMAJITSAKUL1,2∗, KUN SILPRASIT3, SIRAWUT KLINBUNGA4 and SIRIPORN SITTIPRANEED2

1Faculty of Health Science, Srinakharinwirot University Ongkharak, Nakhon-Nayok 26120, Thailand 2Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand 3Faculty of Environmental Culture and Ecotourism, Srinakharinwirot University, Nakhon-Nayok 26120, Thailand 4Aquatic Molecular Genetics and Biotechnology, National Science Center for Genetic Engineering and Biotechnology (NSTDA), 113 Paholyothin Road, Klong 1, Klong Luang, Pathum Thani 12120, Thailand

[Thummajitsakul S., Silprasit K., Klinbunga S. and Sittipraneed S. 2013 The partial mitochondrial sequence of the Old World stingless bee, Tetragonula pagdeni. J. Genet. 92, xx–xx]

Introduction 2001). Besides, the nearly complete mtDNA sequence has been recently determined in stingless bee, Melipona bicolor The partial mitochondrial sequence of Tetragonula pagdeni (Silvestre et al. 2008). However, the information of mtDNA was examined using long PCR technique and subsequently sequences from Tetragonula species is very limited, only cloned, and sequenced using primer walking strategy. The few mitochondrial genes such as 16S rRNA gene reported objective of this study was to characterize T. pagdeni mito- in genetic variability study (Costa et al. 2003; Rasmussen chondrial DNA sequence, focussing on gene order. The and Cameron 2007; Thummajitsakul et al. 2011). T. pagdeni partial mitochondrial sequence of T. pagdeni obtained in Schwarz is widely distributed in Indo-Malayan/Australasian this study represents approximately 69.2% of the Meliponini and Neotropical regions (Michener 2000) and is one of mtDNA. The protein-coding genes have a bias towards the most common indigenous stingless bees in Thailand AT-rich codons. Codon usage and amino acid sequences do (Sakagami 1978). not deviate substantially from those reported for other stingless bees or honeybees. Similar to other Meliponini species (Arias et al. 2006), the mitochondrial genome of T. pagdeni lacks the COI–COII intergenic region. Materials and methods In past, mitochondrial DNA was widely employed to Sample and DNA extraction resolve population structure, phylogeography and phylogenetic relationships at various taxonomic levels (Kmiec et al. Individuals of T. pagdeni collected from a single colony were 2006). Generally, animal mtDNA exhibits a high evolution preserved in 95% ethanol and stored at 4◦C until required. rate compared to that of the nuclear DNA and very conserved Total genomic DNA was extracted from each entire bee using gene order comprising two rRNA genes, 22 tRNA genes, 13 phenol–chloroform extraction and ethanol precipitation fol- protein-coding genes, as well as a noncoding control region lowing Thummajitsakul et al. (2011). (D-loop) (Wolstenholme 1992). The mtDNA has been extensively used in honeybee, Apis mellifera, to investigate natu- ral range origin, and genetic polymorphisms based on PCR- PCR and sequencing RFLP method (Smith and Brown 1990). For stingless bees, the mitochondrial genome of several Meliponine species has In our previous studies, the mtDNA regions for cytb, COI, been determined through RFLP analysis (Francisco et al. 16S rRNA and ATP(6, 8) + COIII genes of T. pagdeni were ampliﬁed by using primer pairs; cyt-b-F/R, COI-F/R, LR13107-F/LR12647-R and ATPS6-F/tRNA-ASP-R, respectively (table 1). On the basis of each sequence por- tion, three sets of primer pairs LR12647-R + COI2494, ∗ For correspondence. E-mail: [email protected]. LR12677 + cytb10729 and COIII9821 + cytb5031 were Keywords. stingless bees; mitochondrial DNA gene order; long PCR; Tetragonula; Tetragonula pagdeni.

Journal of Genetics, Vol. 92, No. 2, August 2013 Sirikul Thummajitsakul et al.

Table 1. Sequences of primers used in PCR reactions of T. pagdeni mtDNA.

mtDNA genes Primer Sequence

ATPase (6,8) ATPS6-F 5-AAG ATA TAT GGA AAT AAG CT-3 tRNA-ASP-R 5-ATA AAA TAA CGT CAA AAT GTC A-3 COI COI-F 5-ATAATTATTGTTGCTGATGTA-3 COI-R 5-CTA TTC ATA TAA CTG GAA TTT C-3 Cytb cyt b-F 5-TTC ACT ATA TTA TAA AAG ATG TAA GTT C-3 cyt b-R 5-GGC AAA AAG AAA ATA TCA TTC AGG-3 16S rRNA LR13107-F 5-TGG CTG CAG TAT AAC TGA CTG TAC AAA GG-3 LR12647-R 5-GAA ACC AAT CTG ACT TAC GTC GAT TTG A-3 COI, COII, ATPase8, ATPase6, COIII, LR12647-R 5-GAAACCAATCTGACTTACGTCGATTTGA-3 ND3, 12S, 16S rRNA COI2494 5-CGAGCATATTTTACATCAGCAACAAT-3 Cytb, ND6, ND5, ND4, ND4L LR12677 5-GTTCAAATCGACGTAAGTCAGATTGGTTTC-3 cytb10729 5-AGCTCCTCAAAATGATATTTGTCCTCATGG-3 COIII, ND3, 12S, 16S rRNA, ND1, cytb COIII9821 5-TTAACGATAGAGTTTACGGGTCAAT-3 cytb5031 5-AGCTACAGCATTTCTTGGGTATGTAC-3

designed on each gene for the 16S/COI, 16S/cytb and were performed by alignment with those of M. bicolor and cytb/COIII regions, respectively (table 1). A. mellifera using the BLASTX algorithm (NCBI). Transfer PCR was carried out with 30 cycles of a 25 μL reac- RNA and ribosomal RNA sequences were identified by eye tion volume containing 4.75 μL of sterile distilled H2O, comparison with homologues of A. mellifera, M. bicolor and 2.5 μLof10× LA PCR buffer II (Takara Bio, Shiga, Japan), Bombus ignites. Phylogenetic analysis using cyt-b mtDNA 4.0 μLofdNTP(2.5mM),5.0μL of each primer (0.4 μM), sequence among these bees was shown by neighbour-joining 0.25 μL of 1.25 unit Takara LA TaqTM (Takara), and 1.0 μL method in MEGA ver. 5.10 (Tamura et al. 2011) (figure 1). of template containing approximately 5 ng DNA. The PCR condition was used with denaturation at 98◦C for 10 s and annealing and extension combined at the same tempera- Results and discussion ture (60◦C) for 10 min. The PCR products were verified on a 1.0% agarose gel and visualized by ethidium bro- The total size of stingless bees mtDNA has been estimated mide staining via ultraviolet transillumination. Each purified in M. bicolor species by RFLP analysis and sequencing PCR product was cloned into pGEMR -T easy vectors as approximately 18,500 bp and 14,422 bp (Silvestre (Promega, Madison, USA) and subsequently sequenced by et al. 2008), respectively. In our study, the partial mtDNA primer walking using BigDye terminator cycling condi- sequence of T. pagdeni obtained was 12,802 bp or 69.2% of tions on a 3730xl automatic sequencer (Sequencing Service, the total mtDNA genome of Meliponini species. The incom- Macrogen, Seoul, Korea). All sequences were analysed and plete mtDNA sequence contained 11 complete genes, a par- compared by the homology search to assure the correct frag- tial COI gene, both rRNA genes and 12 tRNA genes. Each ments using BLASTN (nucleotide similarity) available at protein-coding gene in T. pagdeni was similar in length http://www.ncbi.nlm.nih.gov. and nucleotides to their counterpart genes in other stingless bees (M. bicolor) and honey bees (A. melliferra)(table 2). The four overlapping regions, ND4/ND5, cytb/tRNA-Ser Sequence analysis (S2), ND1/tRNA-Leu (L1)andATP8/ATP6, were observed Each coding region was identified by searching for open (figure 2). Some of the overlapped genes have been reported reading frames, including start and stop codons. The com- for M. bicolor mtDNA by Silvestre et al. (2008)suchasND1 parisons of nucleotide or amino acid sequences of T. pagdeni and tRNA-Leu (L1) shared six nucleotides; ATP6 and ATP8 shared 10 nucleotides. Moreover, 14 noncoding regions were observed with total intergenic region of 419 bp. In honeybee A. mellifera, the number of noncoding nucleotides, exclud- ing the COI–COII intergenic and control region is 618 bp (Crozier and Crozier 1993). The noncoding region found in mtDNA sequences of T. pagdeni showed no significant similarities with any regions of the mtDNA of M. bicolor. The intergenic region Figure 1. Phylogenetic analysis of cyt-b mtDNA genes among between COI and COII genes of A. mellifera mtDNA is the bees was conducted using neighbour-joining method, using known as hypervariable region. This intergenic region has Triatoma dimidiata as outgroup (AF301594). been widely studied in A. mellifera, and size polymorphism

Journal of Genetics, Vol. 92, No. 2, August 2013 Partial mitochondrial sequence of Tetragonula pagdeni

Table 2. Size, start codon and stop codon comparisons of protein-coding genes between T. pagdeni (Tp), M. bicolor (Mb) and A. mellifera (Am).

% Nucleotide similarity Length (bp) Start codon Stop codon between two species

Gene Tp Mb Am Tp Mb Am Tp Mb Am Tp and Mb Tp and Am

ND4L 300 279 264 ATA ATA ATT TAA TAA TAA 69% 68% ND4 1392 1323 1344 ATG ATT ATA TAG TAA TAA 78% 71% ND5 1665 1647 1665 ATC ATT ATT TAA TAA TAA 76% 70% ND6 522 540 504 ATT ATT ATT TAA TAA TAA 71% 69% cytb 1089 1050 1152 ATT ATT ATG TAA TAA TAA 79% 74% ND1 933 930 918 ATA ATA ATT TAA TAA TAA 76% 72% ND3 351 354 354 ATA ATA ATA TAA TAA TAA 71% 0% COIII 780 780 777 ATG ATG ATG TAG TAA TAA 70% 67% ATPase6 687 684 681 ATG ATG ATG TAG TAA TAA 69% 66% ATPase8 168 168 159 ATT ATT ATT TAA TAA TAA 72% 73% COII 627 678 676 ATT ATT ATT TAG TAA T 76% 71%

has been reported (from 200 to 650 bp) among subspecies advantage of AT bias could be explained by one hypoth- (Garnery et al. 1992,Francket al. 1998). It has also been esis that the DNA polymerase could use those bases in a referred as a possible second origin of mtDNA replication more efficient way during mtDNA replication (Clary and and transcription (Cornuet et al. 1991). Our results reveal that Wolstenholme 1985) because the energetic cost to break the COI–COII intergenic region was absent in T. pagdeni,as A–T links is lower compared to G–C links. The A + T-rich has been previously reported in the case of M. bicolor and at region of A. melifera is located between the 12S rRNA least 16 other Meliponini species (Arias et al. 2006). and tRNA-Glu genes (Crozier and Crozier 1993). It con- The A + T contents of each protein-coding gene in tains several short repeating sequences (6–13 bp) with vary- T. pagdeni were high, ranging from 66 to 87% (table 3), sim- ing copy numbers (two-to-four copies), scattered through ilar to M. bicolor (87%, Silvestre et al. 2008)andA. mel- the whole region and a polythymidine stretch. This poly- lifera (85%, Crozier and Crozier 1993). The highly biased thymidine stretch is reported to be a transcription control or A + T content has been known as a major characteristic the initiation of replication (Zhang and Hewitt 1997). For of the mitochondrial genome of M. bicolor and A. mellif- stingless bee M. bicolor,theA+ T-rich region could era (Crozier and Crozier 1993; Silvestre et al. 2008). The not be sequenced because of difficulties in amplification

Figure 2. Overlapping the three sequenced fragments (5855, 4879 and 5089 bp, respectively) obtained from long PCR ampliﬁcation with LR12647-R + COI2494, COIII9821 + cytb5031 and LR12677 + cytb10729, respectively and sequenced by primer walking.

Journal of Genetics, Vol. 92, No. 2, August 2013 Sirikul Thummajitsakul et al.

Table 3. Presentation of base compositions (%) in each gene of failure can be explained by the existence of tandem repeats, T. pagdeni mtDNA. heteroplasmy and great length variation at intraspecific and interspecific levels (Zhang and Hewitt 1997), including par- Gene Length A + T content (%) tial duplications that commonly occur in insect mtDNA. All ND4L 300 87 protein-coding genes of T. pagdeni indicated slight differ- ND4 1392 83 ences when compared to M. bicolor and A. mellifera.The ND5 1665 83 nucleotide sequence of T. pagdeni is more similar to M. ND6 522 86 bicolor than A. mellifera. cytb 1089 77 The 12S and 16S rRNA genes of T. pagdeni mtDNA were ND1 933 83 ND3 351 66 estimated to be 762 and 1351 bp, respectively, and showed COIII 780 68 83% and 78% similarity compared to the partial 12S (437 bp) ATPase6 687 66 complete 16S rRNA (1354 bp) nucleotide sequence genes ATPase8 168 80 of M. bicolor, respectively. The srRNA and lrRNA genes of COII 627 69 T. pagdeni were located between the tRNA-Leu (1) and 16S rRNA 1351 77 12S rRNA 762 76 tRNA-Val gene and between the tRNA-Val and tRNA-Gln tRNA-Pro 67 84 genes, respectively (figure 3). The order of 12S rRNA, 16S tRNA-Phe 67 85 rRNA, ND1, cytb and ND6 genes of T. pagdeni mtDNA was tRNA-Asn 68 82 different from those of M. bicolor (Silvestre et al. 2008). tRNA-Thr 67 79 All tRNA sequences had 61–81 nucleotides with 12 to 29% tRNA-Ser(2) 67 85 + + tRNA-Leu(1) 69 83 G C and 70.77 to 88% A T (data not shown). The anti- tRNA-Val 68 88 codon nucleotides were identical to those commonly found tRNA-Gln 68 79 for the corresponding tRNA genes in A. mellifera, M. bicolor tRNA-Arg 61 85 and B. ignitus mtDNA. However, tRNA-Gln found in T. pag- tRNA-Glu 65 71 deni mtDNA resembled those in A. mellifera and B. ignitus tRNA-Asp 81 78 tRNA-Leu(2) 65 75 mtDNA, but not seen in M. bicolor mtDNA (Crozier and Crozier 1993; Silvestre et al. 2008). The tRNA-Gln gene was located between 12S and tRNA-Arg (figure 2). In conclusion, the nearly complete mitochondrial genome (Silvestre et al. 2008), but it was described by its size of T. pagdeni may provide critical information for future using RFLP analysis. It was estimated in size approxi- mtDNA analyses on biology, ecology and evolution at mately 3300 bp, about 2.5-kb longer than in A. mellifera intraspecific and interspecific levels of other Tetragonula (Crozier and Crozier 1993). In this study, because we were species. Studies of mtDNA gene organization illustrate an not able to amplify the entire mtDNA of T. pagdeni,the expedited rate in hymenopterans (Dowton et al. 2002). A + T-rich region, ND2 and 10 tRNA genes were not found The mitochondrial gene order could be explained by on the sequenced mtDNA fragment. The DNA amplification several mechanisms. One of the most extensively-accepted

Figure 3. Gene organization of the mtDNA from T. pagdeni (A), M. bicolor (B) (Silvestre et al. 2008)andA. melliferra (Crozier and Crozier 1993). All tRNA genes were indicated by the amino acid they encode: I (tRNAIle), A (tRNAAla), K (tRNALys), M (tRNAMet), W (tRNATrp), Y (tRNATyr), L2 (tRNALeu(UUR)), D (tRNAAsp), G (tRNAGly), R (tRNAArg), N (tRNAAsn), E (tRNAGlu), F (tRNAPhe), H (tRNAHis), T (tRNAThr), P (tRNAPro), S2 (tRNASer(UCN)), L1 (tRNALeu(CUN)) and V (tRNAVal).

Journal of Genetics, Vol. 92, No. 2, August 2013 Partial mitochondrial sequence of Tetragonula pagdeni mechanisms is tandem duplication of gene regions as a result (Apidae: Meliponini): RFLP and restriction maps. Apidologie 32, of slipped-strand mispairing followed by gene deletions 323–332. (Levinson and Gutman 1987; Macey et al. 1998). Garnery L., Cornuet J. M. and Solignac M. 1992 Evolutionary history of the honey bee Apis mellifera inferred from mitochondrial DNA analysis. Mol. Ecol. 1, 145–154. Acknowledgements Kmiec B., Woloszynska M. and Janska H. 2006 Heteroplasmy as a common state of mitochondrial genetic information in plants and We thank Department of Biochemistry, Faculty of science, animals. Curr. Genet. 50, 149–159. Chulalongkorn University and Aquatic Molecular Genetics and Levinson G. and Gutman G. A. 1987 Slipped-strand mispairing: a Biotechnology Laboratory, National Center for Genetic Engineer- major mechanism for DNA sequence evolution. Mol. Biol. Evol. ing and Biotechnology (BIOTEC), NSTDA, for providing facilities 4, 203–221. required for the experiments. Macey J. R., Schulte J. A., Larson A. and Papenfuss T. J. 1998 Tandem duplication via light-strand synthesis may provide a precursor for mitochondrial genomic rearrangement. Mol. Biol. Evol. 15, 71–75. References Michener C. D. 2000 The bees of the world. Johns Hopkins Univer- sity Press, Baltimore, USA. Arias M. C., Brito R. M., Francisco F. O., Moretto G., de Oliveira Rasmussen C. and Cameron S. A. 2007 A molecular phylogeny of F. F., Silvestre D. and Sheppard W. S. 2006 Molecular markers as the Old World stingless bees (Hymenoptera: Apidae: Meliponini) a tool for population and evolutionary studies of stingless bees. and the non-monophyly of the large genus Trigona. Syst. Apidologie 37, 259–274. Entomol. 32, 26–39. Clary D. O. and Wolstenholme D. R. 1985 The mitochondrial Sakagami S. F. 1978 Tetragonula stingless bees of the continental DNA molecule of Drosophila yakuba: nucleotide sequence, gene Asia and Sri Lanka. J. Fac. Sci. Hokkaido Univ. Ser. VI Zool. 21, organization, and genetic code. J. Mol. Evol. 22, 252–171. 165–247. Costa M. A., Del Lama M. A., Melo G. A. R. and Sheppard W. S. Silvestre D., Dowton M. and Arias M. C. 2008 The mitochondrial 2003 Molecular phylogeny of the stingless bees (Apidae, Apinae, genome of the stingless bee Melipona bicolor (Hymenoptera, Meliponini) inferred from mitochondrial 16S rDNA sequences. Apidae, Meliponini): Sequence, gene organization and a unique Apidologie 34, 73–84. tRNA translocation event conserved across the tribe Meliponini. Cornuet J. M., Garnery L. and Solignac M. 1991 Putative ori- Genet. Mol. Biol. 31, 451–460. gin and function of the intergenic region between COI and Smith D. R. and Brown W. M. 1990 Restriction endonuclease COII of Apis mellifera L. mitochondrial DNA. Genetics 1128, cleavage site and length polymorphism in mitochondrial DNA of 393–403. Apis mellifera and A. m. carnica (Hymenoptera: Apidae). Ann. Crozier R. H. and Crozier Y. C. 1993 The mitochondrial genome of Entomol. Soc. Am. 83, 81–88. the honeybee Apis mellifera: Complete sequence and the genome Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar organization. Genetics 133, 97–117. S. 2011 MEGA5: Molecular evolutionary genetics analysis using Dowton M., Belshaw R., Austin A. D. and Quicke D. L. 2002 maximum likelihood, evolutionary distance, and maximum par- Simultaneous molecular and morphological analysis of Braconid simony methods. Mol. Biol. Evol. 28, 2731–2739. relationships (Insecta, Hymenoptera, Braconidae) indicates inde- Thummajitsakul S., Klinbunga S. and Sittipraneed S. 2011 Genetic pendent mt-tRNA gene inversions within a single wasp family. differentiation of the stingless bee Tetragonula pagdeni in J. Mol. Evol. 54, 210–226. Thailand using SSCP analysis of a large subunit of mitochondrial Franck P., Garnery L., Solignac M. and Cornuet J. M. 1998 The ribosomal DNA. Biochem. Genet. 49, 499–510. origin of west European subspecies of honeybees (Apis mellif- Wolstenholme D. R. 1992 Animal mitochondrial DNA: structure era) new insights from microsatellite and mitochondrial data. and evolution. Int. Rev. Cytol. 141, 173–216. Evolution 52, 1119–1134. Zhang D. and Hewitt G. M. 1997 Insect mitochondrial control Francisco F. O., Silvestre D. and Arias M. C. 2001 Mito- region: A review of its structure, evolution and usefulness in chondrial DNA characterization of ﬁve species of Plebeia evolutionary studies. Biochem. Syst. Ecol. 25, 99–120.

Received 6 November 2012, in ﬁnal revised form 8 February 2013; accepted 22 February 2013 Published on the Web: 26 June 2013

Journal of Genetics, Vol. 92, No. 2, August 2013