Identification of Drosophila species based on 16S rRNA and CO1 gene sequences Mohammad Shamimul Alam, Khandaker Asif Ahmed, Rowshan Ara Begum, and Reza M Shahjahan. Address: Genetics and Molecular biology laboratory, Department of Zoology, University of Dhaka, Dhaka, Bangladesh

INTRODUCTION Drosophila is a genus of fruit flies, which is belongs to the family Drosophilidae (Class: Insecta and Order: Diptera) Identification of Drosophila is an essential part prior to study human homolog genes and their expression in this fly. External or internal or both features of an individual is used in identification and taxonomic study.

DISADVANTAGES OF MORPHOLOGY-BASED IDENTIFICATION Considerable morphological plasticity between same species. Time-consuming and require expertise. Fragile and easily damaged morphological features. Existence of sibling species. Existence of convergent evolution. DNA based analyses became popular among researchers.

WHY DNA BASED METHODS? DNA is an extremely stable and long-lived biological molecule Can be recovered easily from biological material of any kind A variety of methods exist to make the collection and storage of DNA samples simple and efficient. Can be found in all biological tissues or fluids with nucleated cells

Some of the most used DNA based methods are – DNA hybridization, RFLPs (Restriction Fragment Length Polymorphisms), AFLPs (Amplified Fragment Length Polymorphisms), RAPD (Random Amplified Polymorphic DNA), Conventional PCR technique, Real-time PCR technique, Sequencing technique and Microarray technique.

The main focus of the present study was to identify Drosophila species of Dhaka city using sequences of mitochondrial genes, 16S ribosomal RNA (16S) and Cytochrome c oxidase 1 (CO1), in addition to morphological features.

OBJECTIVES Raising isofemale stocks of Drosophila Differentiating various species of Drosophila morphologically Isolating and extracting total genome of the cultured specimen Comparing DNA extraction methods Amplifying mitochondrial genes (16S rRNA and CO1) by polymerase chain reaction (PCR). Identification of Drosophila sp., based on the sequences of mitochondrial genes, such as 16S rRNA and CO1. Studying phylogenetic relationships among different species.

METHODOLOGY Raise Isofemale Taxonomic Collection DNA isolation population Collection Taxonomic identification DNA isolation Sequence analysis DNA sequencing Polymerase chain reaction

FLY COLLECTION Total four Drosophila species was collected and identified based on morphology first. D. ananassae, D. parabipectinata, D. repleta and D. melanogaster

RECORD OF A NEW FLY Besides Drosophila species, a fast moving fly was isolated from yeasted banana bait. According to Encyclopedia of Flora and Fauna of Bangladesh, flybase and existing literatures, this fly has not yet reported from Bangladesh.

DNA ISOLATION Genomic DNA was extracted with typical CTAB DNA extraction protocol and purified using phenol chloroform precipitation In addition, a modified version of CTAB extraction method was used and tested during DNA isolation from two other invertebrate species

Fig. Extracted DNA of Drosophila melanogaster and Megaselia scalaris visualized in 1% agarose gel Fig. Extracted DNA of three Drosophila sp. as visualized in 1% agarose gel. Where (R) denotes use of RNAse

Fig. Extracted DNA of Drosophila sp visualized in 1% agarose gel, where DNA extracted using different protocols Fig. Extracted DNA of Machrobrachium and Aedes albopictus visualized in 1% agarose gel, , where DNA extracted with Modified CTAB Extraction Protocol

POLYMERASE CHAIN REACTION Fragments of 16S and CO1 were amplified by PCR (polymerase chain reaction) using universal primers. 550 nucleotide base pair was amplified in case of 16S and 700 base pair in CO1 A total of seven samples were sequenced.

Fig. PCR products of 16S rRNA and CO1 gene regions from three different Drosophila species and Megaselia scalaris

16S SEQUENCES D. ananassae 16S D. parabipectinata 16S TTTTTTCAAAAACATGTCTTTTTGAATTAAATATAAAGTCTAACCTGCCCACTGAAAATTTTTAAATGGCCGCAGTATTC TGACTGTGCAAAGGTAGCATAATCATTAGTCTTTTAATTGAAGGCTGGAATGAATGGTTGGACGAAATATTAACTGTTTC ATTTAAATTTTTTATAGAATTTTATTTTTTAGTCAAAAAGCTAAAATAAATTTAAAAGACGAGAAGACCCTATAAATCTT TATATTTTATTTATTTTAATTATAAAGATTATTTTAATTTTAATAAAATAAAATATTTTATTGGGGTGATATTAAAATTT AAAAAACTTTTAATTTATAAGAACATTAATTTATGAATTGTTGATCCATTAATAATGATTAAAAAATTAAGTTACTTTAG GGATAACAGCGTAATTTTTTTGGAGAGTTCATATCGATAAAAAAGATTGCGACCTCGATGTTGGATTAAGATATAATTTT GGGTGTAGCCGTTCAAATTTTAAGTCTGTTCGACTTTTAAATTCTTACATGATCTGT D. parabipectinata 16S AAAAACATGTCTTTTTGAATTAAATATAAAGTCTAACCTGCCCACTGAAATTTTTAAATGGCCGCAGTATTCTGACTGTG CAAAGGTAGCATAATCATTAGTCTTTTAATTGAAGGCTGGAATGAATGGTTGGACGAAATATTAACTGTTTCATTTAAAT TTTTTATAGAATTTTATTTTTTAGTCAAAAAGCTAAAATAAATTTAAAAGACGAGAAGACCCTATAAATCTTTATATTTT ATTTATTTTAATTATAAAGATTATTTTAATTTTAATAAAATAAAATATTTTATTGGGGTGATATTAAAATTTAAAAAACT TTTAATTTATTAAAACATTAATTTATGAATTATTGATCCATTAATAATGATTAAAAAATTAAGTTAATTTAGGGATAACA GCGTAATTTTTTTGGAGAGTTCATATCGATAAAAAAGATTGCGACCTCGATGTTGGATTAAGATATAATTTTGGGTGTAG CCGTTCAAATTTTAAGTCTGTTCGACTTTTAAATTCTTACATGATCTGT

D. repleta 16S D. melanogaster 16S M. scalaris 16S AAAAACATGTCTTTTTGAAATTTATTTAAAGTCTAACCTGCCCACTGAAAATTTTTAAATGGCCGCAGTATCCTAACTGT GCAAAGGTAGCATAATCATTAGTCTTTTAATTGAAGGCTGGAATGAATGGTTGGACGAAATATAATCTGTTTCATTTAAA TTTTTTTTAGAATTTTATTTTTTAGTCAAAAAGCTAAAATTTATTTAAAAGACGAGAAGACCCTATAAATCTTTATATTT TATTTATAATAATTAAAAAGATTAAGTTATTTATTATAATGTAAAATATTTTATTGGGGTGATATTAAAATTTAAAAAAC TTTTAATTTTATAAATCATTAATTTATGAGTATTTGATCCATTAATAGTGATTAAAAATTTAAGTTACTTTAGGGATAAC AGCGTAATTTTTTTGGAGAGTTCTTATCGATAAAAAAGATTGCGACCTCGATGTTGGATTAAGATATAATTTTGGGTGTA GCCGTTCAAATTTTAAGTCTGTTCGACTTTTAAATTCTTACATGATCTGAG D. melanogaster 16S CGCCTGTTTTAACAAAAACATGTCTTTTTTGAATTATATATAAAGTCTAACCTGCCCACTGAAAAATTTTAAATGGCCGCAGTATTTTGACTGTGCAAAGGTAGCATAATCATTAGTCTTTTAATTGAAGGCTGGAATGAATGGTTGGACGAAATATTAACTGTTTCATTTAAAATTTTTATAGAATTTTATTTTTTAGTCAAAAAGCTAAAATTTATTTTTAAGACGAGAAGACCCTATAAATCTTTATATTTTTTTTTATTTTAATTATATAGATTAATTAAATTTTAATAAATAAAAATATTTTATTGGGGTGATATTAAAATTTAAAATATTGCTTAAAAATTTTTTAAAAACATAAATTTATGAATATTTGATCCATTAATAATGATTAAAAAATTAAGTTACTTTAGGGATATTAACAGCGTAATTTTTTTGGAGAGTTTTTTTTTTAAAAATAATTAAAAATGTATTTTT M. scalaris 16S ATTTATTGATGGCTGCAGTATCTTAACGTGCAAAGGTGGCATAATCATTAGTCTGAAATTGAGGGCTGGTATGAATGGTT GGATGAAATATTAACTGTTTCATATAAATTTATAATAGAATTTTATTTTTTAGTTATAAAGCTAAAATAATTTAAAAGAC GAGAAGACCCTATAAATCTTTATATATAAGTTATTTTAATTATATAGATAAAAATTAAATTATTATAAATTTATATATTT TATTGGGGTGATATTAAAATTTGTTAAATTTTTAATTCtataataaAACATAAATTAATGAATTATTGATCCATTATTAA TGATTAAGAAATCAAGTTACTTTAGGGATAACAGCGTAATTTTTTTAGAGAGTTcTTTTGATAAAAAAGACTGCCCCCCT CGATGTTTGGATTAAGATACAGTTTTTAGGTGCAGCAGCTTAAACTTAAAGTCTGTTCCGACTTTTTAAATTCTTACATG ATCTGAGTTCCAAACC

CO1 SEQUENCES D. melanogaster CO1 M. scalaris CO1 CCTGGAGCATTAATTGGAGATGATCAAATTTATAAAGTAATTGTAACTGCACATGCTTTTATTATAATTTTTTTTATAGTTATACCTATTATAATTGGTGGATTTTTTAAACTGACTAGTGCCTTTAATATTAGGTGCTCCTGATATAGCATTCCCACGAATAAATAATATAAGATTTTGACTTCTACCTCCTGCTCTTTCTTTACTATTAGTAAGTAGAATAGTTGAAAATGGAGCTGGGACAGGATGAACTGTTTATCCACCTCTATCCGCTGGAATTGCTCATGGTGGAGCTTCAGTTGATTTAGCTATTTTTTCTCTACATTTAGCAGGAATTTCTTCAATTTTAGGAGCTGGAAATTTTATTACAACTGTAATTAATATACGATCAACAGGAATTTCATTAGATCGTATACCTTTATTTGTTTGATCAGGAGTTATTACTGGCTTTATTATTATAATTATCACTTCCAGTACTAGCAGGAGCTATTACTAATATTATTAACAGAATCGAAATTAAATACATCATTTTTTGACCGACCGGGAGGAGGAGATCCTATTTTATACCAACATTTATT M. scalaris CO1 GGGGCCTGAGCTGGGATAGTAGGAACATCTTTAAGTATTATAATTCGAGCTGAATTATGGAACCCTGGTGCTTTAATTGGTGATGATCAAATTTATAAAGAAATTGTTACTGCCCATGCATTTATTATAATTTTTTTTATAGTAATACCTATTATAATAGGAGGATTTGGAATCTGACTAAATCGCCTAATATTAGGGGCACCTGATATGGCCTTTCCACGAATAAATAATATAAGTTTTTGAATACTTCCCCCTTCTCTAACTCTTTTATTAGCAAGAATTATAGTACAAAATGGAGCTGGAACTGGTTGAACAGTTTATCCACCCCTATCTTCTAGAATTGCCCATAGAGGAGCTTCAGTCGATTTAGCAATTTTTTCATTACATCTTGCCGGAATTTCTTCTATTCTTGGAGCAGTAAATTTTATTACTACAATTATTAATATACGATCCACAGGAATTACTTTTGATCGAATACCTTTATTTGTATGAATAGTAGGGAGTACTGCTCTTTTATTATACTTTCACTACCTGGTCTAGCAGGTGCTATTACTATCTATTAACAGATCGAAATTTTAATACATCATTCTTTGACCCGGAAGAAGGGGGAGACCCCATTCTATATCAACATCTATT

SEQUENCE ANALYSIS BLAST search at NCBI confirmed the species identification. Multiple sequence alignments were performed to find out polymorphic sites among sophophora subgenus and Drosophila genus. Nucleotide variation was also observed in D. melanogaster and M. scalaris

Fig. Alignment showing 16S sequence variation among different Drosophila sp. Fig. Alignment showing CO1 sequence variations among different Drosophila sp.

TABLE: A table showing comparative polymorphic site variations among different Drosophila sp. based on 16S and CO1 gene sequences   Variation Name of gene Number of sequences Total number of nucleotide base pairs Percentage of polymorphic sites Percentage of conserved sites Variation in Sophophora subgenus 16S 6 536 3.54% 96.46% CO1 16.04% 83.96% Drosophila genus 13 10.63% 89.37% 27.24% 72.76%

Fig. A bar chart showing polymorphic site variations among different Drosophila sp. based on 16S and CO1 sequences

Fig. A bar chart showing conserved regions among different Drosophila sp. based on 16S and CO1 sequences

Comparative analysis showed CO1 gene sequences have more nucleotide diversity than that found in 16S sequences, though length and number of sequences were same in each case. Percentage of nucleotide variations was high in sophophora subgenus than in Drosophila genus.

PHYLOGENETIC TREE A neighbor joining tree has been constructed using 16S sequences of fourteen different Drosophila species. The tree was constructed using 450 base pair nucleotide of each sequence with bootstrap value of 100 replicons.

Fig. Neighbor joining tree of 14 Drosophila species based on partial sequence of 16S rRNA gene (Bootstrap value = 100).

Complete separation of two subgenus sophophora and drosophila. Each of the subgenus showed monophyly. D. repleta belonging to the repleta species group showed distant relationship with other selected Drosophila flies.

CONCLUSION Presence of more Drosophila and Megaselia species in the area cannot be nullified. So, extensive sampling with expanded area coverage may help identify more of the species. Getting better DNA yield with modified CTAB protocol will greatly facilitate further molecular studies of these species and similar other species in future. Sequence analysis of mitochondrial 16S and CO1 gene fragments in the present study enhanced our understanding about the relationship among different Drosophila species of Bangladesh and some selected species of other parts of the world. Present study could be a beginning toward utilization of huge information available online regarding the fruit flies.