Rukhsana Kokkadan and Sebastian Chempakassery Devasia, Phylogeny and Molecular of Family (Insecta: ) Derived from Mitochondrial DNA Sequence Analysis, Int.J.Curr.Biotechnol., 2015, 3(5):12-16.

International Journal of Current Biotechnology ISSN: 2321 - 8371 Journal Homepage : http://ijcb.mainspringer.com

Phylogeny and Molecular Taxonomy of Family Trichogrammatidae (Insecta: Hymenoptera) Derived from Mitochondrial DNA Sequence Analysis

Rukhsana Kokkadan and Sebastian Chempakassery Devasia*

Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala, India.

ARTICLE INF O ABSTRACT

Family Trichogrammatidae include egg parasitoid of different pest and the Article History: present study reveals molecular barcoding and phylogeny analysis of seven species Received 5 May 2015 in Trichogrammatidae family. The adult Trichogrammatids gave excellent control of Received in revised form 13 May 2015 egg of many insect pests. We have PCR amplified and sequenced the partial fragment Accepted 25 May 2015 of mitochondrial cytochrome oxidase subunit I (COI) gene of these seven species Available online 30 May 2015 isolated from Kerala and its phylogenetic status was studied. The partial COI mtDNA sequences of Trichogrammatoidea genus are found in same clade. Trichgrammatoidea armigera voucher CUTA01 (KP 994542) and Trichgrammatoidea armigera voucher CUTA02 (KP 994541) have no difference which were collected from different areas. Key words: Trichogrammatidae, DNA barcoding, The results obtained can be used for the accurate taxonomic identification and phy- molecular phylogeny, COI gene. logenetic status analysis of this family. This study explores the phylogeny of the different trichogrammatids and confirms the existence of their common ancestor through molecular evidence.

Introduction markers. The genetic diversity and phylogeny of Opisina The family Trichogrammatidae is one of the most widely arenosella (Rukhsana and Sebastian, 2014) and distributed and biologically diverse group of Hyblaeidae family (Chandrasekhar et al., 2008) are also Hymenoptera. The members of this group are minute and studied. The focus of the current study was to decipher are easily recognized by their three segmented tarsi (Salma the systematic position and phylogeny of family and Shoeba, 2014). The trichogrammatids fauna is Trichogrammatidae using mitochondrial COI gene represented by 151 species in 31 genera (Noyes, 2012). sequences. Trichogrammatids are economically important group because of these groups are egg parasitoid of different Materials and Methods insect pest (Yousuf and Shafee, 1987). The taxonomic Sample collection and DNA extraction study of Trichogrammatids is using morphology such as The seven specimen vouchers of six species were collected from the different areas of Kerala, modifications in antennal, wing, male genital characters - ° are laborious, time consuming and very difficult. India. The were stored at 20 C until the DNA was extracted. The genomic DNA from the adults was isolated The eggs of these species have a little elongated and using GeNei Ultrapure Mammalian Genomic DNA Prep sometimes slightly expanded central with both ends Kit (GeNei, Bangalore) as per the manufacturer’s smoothly rounded (Flanders, 1937) or there may be a instruction. peduncle at one end (Silvestri, 1916; Bakkendorf, 1934). The first instar larva is usually one of two types, either PCR amplification and DNA sequencing sacciform or mymariform. The mature larva is robust, The mitochondrial cytochrome oxidase subunit I (COI) distinctly segmented. Pupation takes place within the genes of these species were amplified using the forward remains of the host egg, the adult parasitoid emerging primer with DNA sequence 5'- GGTCAACAAATCATA by biting a hole in the chorion of the egg. AAGATATTGG-3' and reverse primer with DNA sequence 5'- TAAACTTCAGGGTGACCAAAAAATCA-3'. The Using mitochondrial cytochrome oxidase subunit I (COI) PCR reaction mixture consisted of 2 ng of genomic DNA, gene sequences depicted the phylogeny of many insects. 1µl each forward and reverse primers at a concentration The information about phylogenetic relationships of the of 2.5 µM, 2.5 µl of dNTPs (2mM), 2.5 µl of 10X reaction Family Trichogrammatidae is not readily available for the buffer 2µl of Taq polymerase (1U/µl) and 11.8 µl H2O. lack of sufficient data from a wide variety of genetic The PCR profile consisted of an initial denaturation step of 2 minutes at 95°C, followed by 30 cycles of 5s at 95°C, 45s at 50°C and 45s at 72° C and ending with a final phase *Corresponding author. ° Email address: [email protected] of 72 C for 3 minutes. The PCR products were resolved Mobile: 91-9447648961 Volume 3; Issue 5; May, 2015 Int.J.Curr.Biotechnol. 12 Table – 1: Composition of nucleotides in each position of codon of the COI sequence of the Trichogrammatidae species

Nucleotide Frequencies in percentage Species Name T/U C A G T1 C-1 1-1 G-1 T-2 C-2 A-2 G-2 T3 C-3 A-3 G-3 T. brasiliensis (DQ177919) 42.1 12.1 32.2 13.6 38 9.3 33.9 18.6 43 26.3 12.7 17.8 45 0.8 50.0 4.2 T. platneri (KJ084938) 42.9 12.4 30.8 13.8 38 9.3 34.7 17.8 43 26.3 13.6 16.9 47 1.7 44.1 6.8 T. mwanzai (KP142716) 43.2 11.9 31.9 13.0 38 9.3 34.7 17.8 43 26.3 16.9 13.6 48 0 44.1 7.6 T. evanescens (KP127627) 43.2 12.1 31.6 13.0 37 9.3 34.7 18.6 42 26.3 16.9 14.4 50 0.8 43.2 5.9 T. hebbalensis (KP090265) 44.4 12.1 31.1 12.4 38 9.3 34.7 17.8 43 26.3 15.3 15.3 52 0.8 43.2 4.2 T. chilotraeae (KP090266) 43.2 12.1 32.2 12.4 38 9.3 34.7 17.8 43 26.3 14.4 16.1 48 0.8 47.5 3.4 T. brassicae (KM998974) 42.9 12.7 31.9 12.4 38 9.3 34.7 17.8 43 26.3 13.6 16.9 47 2.5 47.5 2.5 T. pretiosum (KM998973) 43.5 11.9 31.6 13.0 38 9.3 34.7 17.8 43 26.3 13.6 16.9 49 0 46.6 4.2 T. cordubensis (KM232610) 43.5 12.4 30.5 13.6 38 9.3 34.7 17.8 43 26.3 13.6 16.9 49 1.7 43.2 5.9 T.embryophagum (KM105169) 42.1 12.1 33.3 12.4 38 9.3 35.6 16.9 43 26.3 16.1 14.4 45 0.8 48.3 5.9 T. cacoeciae (KM242285) 42.1 12.7 33.3 11.9 38 9.3 35.6 16.9 43 26.3 16.1 14.4 45 2.5 48.3 4.2 T. dendrolimi (KC411497) 44.1 12.1 31.1 12.7 39 9.3 33.9 17.8 43 26.3 13.6 16.9 50 0.8 45.8 3.4 T. ostriniae (DQ177914) 42.9 12.1 31.1 13.8 38 9.3 33.9 18.6 43 26.3 12.7 17.8 47 0.8 46.6 5.1 T. armigera 02(KP994541) 44.9 11.9 29.4 13.8 41 9.3 31.4 18.6 43 25.4 13.6 17.8 51 0.8 43.2 5.1 T. armigera (KP994542) 44.9 11.9 29.4 13.8 41 9.3 31.4 18.6 43 25.4 13.6 17.8 51 0.8 43.2 5.1 T. achaeae (KP994548) 44.6 11.9 32.5 11.0 38 9.3 34.7 17.8 43 26.3 16.1 14.4 53 0.0 46.6 .8 T. bactrae (KP994547) 45.5 11.6 29.7 13.3 40 9.3 32.2 18.6 43 25.4 13.6 17.8 53 0.0 43.2 3.4 T. chilonis (KP994546) 42.4 12.1 32.8 12.7 37 10.2 34.7 17.8 43 26.3 13.6 16.9 47 0 50.0 3.4 T. danaidiphaga (KP994545) 44.6 11.9 30.8 12.7 38 9.3 34.7 17.8 43 26.3 13.6 16.9 53 0 44.1 3.4 T. japonicum (KP994544) 43.8 11.9 32.2 12.1 38 9.3 37.3 15.3 43 26.3 16.1 14.4 50 0 43.2 6.8

13 Int.J.Curr.Biotechnol. Volume 3; Issue 5; May, 2015 Table – 2: Pattern of Nucleotide Substitution by Maximum Composite Likelihood

A T C G A - 11.53 3.2 11.77 T 8.33 - 1.42 3.41 C 8.33 5.12 - 3.41 G 28.75 11.53 3.2 -

Figure - 1: Phylogenic relationship of Trichogrammatidae family members isolated from Kerala by Neighbor joining and Maximum parsimony methods

(a) Neighbor joining method

88 Trichogramma embryophagum(KM105169) 43 Trichogramma cacoeciae(KM242285) 19 Trichogramma japonicum(KP994544) 10 Trichogramma mwanzai (KP142716) 13 Trichogramma hebbalensis(KP090265) 6 Trichogramma chilotraeae(KP090266) Trichogramma platneri (KJ084938) 2 Trichogramma achaeae (KP994548)

99 Trichogramma evanescens (KP127627) 4 Trichogramma cordubensis (KM232610) 25 Trichogramma dendrolimi (KC411497) 29 Trichogramma chilonis(KP994546) Trichogramma brassicae(KM998974) Trichogramma ostriniae (DQ177914) 19 36 Trichogramma brasiliensis(DQ177919) 60 Trichogramma pretiosum (KM998973) Trichogramma danaidiphaga(KP994545) Trichogrammatoidea bactrae (KP994547) 41 99 Trichgrammatoidea armigera 02(KP994541) 100 Trichgrammatoidea armigera 01 (KP994542)

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Volume 3; Issue 5; May, 2015 Int.J.Curr.Biotechnol. 14 (b) Maximum Parsimony method

Trichogramma embryophagum(KM105169) Trichogramma cacoeciae(KM242285) Trichogramma japonicum(KP994544) Trichogramma hebbalensis(KP090265) Trichogramma chilotraeae(KP090266) Trichogramma chilonis(KP994546) Trichogramma achaeae (KP994548) Trichogramma evanescens (KP127627) Trichogramma cordubensis (KM232610) Trichogramma dendrolimi (KC411497) Trichogramma ostriniae (DQ177914) Trichogramma brasiliensis(DQ177919) Trichogramma pretiosum (KM998973) Trichogramma danaidiphaga(KP994545) Trichogramma platneri (KJ084938) Trichogramma mwanzai (KP142716) Trichogrammatoidea bactrae (KP994547) Trichgrammatoidea armigera 02(KP994541) Trichgrammatoidea armigera 01 (KP994542) Trichogramma brassicae(KM998974) on a 1% TAE-agarose gel, stained with Ethidium bromide were plotted for each codon position, respectively and photographed using a gel documentation system. amongst all taxa to examine COI sequences for saturation After ascertaining the PCR amplification of the (Tamura et al., 2013). Phylogenetic analyses were corresponding COI fragment, the remaining portion of performed using neighbor-joining (NJ) and maximum the PCR products were column purified using Ultraclean parsimony (MP) methods implemented in MEGA6 PCR Clean-up Kit (Mo Bio Laboratories, Inc. California) software (Tamura and Kumar, 2004). as per the manufacturer’s instructions. The purified PCR products were sequenced from both ends using the Results and Discussion forward and reverse primers used for the PCR using The PCR amplification of partial COI gene sequences is Sanger’s sequencing method (Sanger, 1975). found to be novel and have been deposited in the NCBI GenBank (Accession Nos. KP 994548, KP 994547, KP Phylogeny analysis 994546, KP 994545, KP 994544, KP 994542 and KP 994541). The mitochondrial DNA COI genes in seven members The PCR amplification of partial COI gene sequences of belong to six species from Family Trichogrammatidae Trichogrammatidae family shows little variation in their were sequenced and those of 13 species in the Family nucleotides and A+T % is very high compared to the Trichogrammatidae were downloaded from NCBI G+C %. The composition of nucleotides of the family GenBank database for comparison. The nucleotide Trichogrammatidae showed clear bias to nucleotide ‘AT’. sequences of the COI gene were used for species The nucleotide composition analysis revealed the high determination and calculating nucleotide composition AT content in the COI gene of Trichogrammatidae and phylogenetic analysis. The forward and reverse species. The average percentage of nucleotides A, T, G, sequences obtained were trimmed for the primer C present in the COI sequence are in the concentrations sequences, assembled by using ClustalW and the A - 31.5, T - 43.5, G - 12.9, C - 12.1 respectively (Table 1). consensus was taken for the analysis. The nucleotide sequence and peptide sequence were searched for its High concentration of nucleotide ‘T’ in the third position similarity using BLAST programme of NCBI of codon results in a preference of polar and hydrophobic (www.ncbi.nlm.nih.gov/) and Inter and intra specific amino acids in the membrane associated proteins. There genetic diversity were calculated using Kimura 2- is 13.2 % increase in average concentration of nucleotide parameter model with the pair wise deletion option and ‘G’ in first position of codon compared to third position. the difference in the nucleotide in codon usage partial In Table 2, each entry shows the probability of COI sequence of Trichogrammatids was analyzed using substitution (R) from one base (row) to another base MEGA6 software. In this study, the p-distance versus (column). For simplicity, the sum of ‘R’ values is made the absolute number of transitions and transversions equal to 100. Rates of different transitional substitutions

15 Int.J.Curr.Biotechnol. Volume 3; Issue 5; May, 2015 are shown in bold and those of transversionsal Yousuf, M. and Shafee, S.A. 1988. Taxonomy of Indian substitutions are shown in italics. The nucleotide Trichogrammatidae (Hymenoptera: Chalcidoidea). frequencies are 31.47% (A), 43.55% (T/U), 12.10% (C), International Journal of Systematic Entomology, 4: 55- and 12.88% (G). The analysis involved 20 nucleotide 200. sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. The NJ tree and the MP tree is some difference in their clades (Figure1). Conclusion The COI developed in this study can be used for the taxonomy and phylogeny analysis of the Family Trichogrammatidae. Variation in the nucleotide sequence is a fundamental property of all organisms which used for its identification. This study reveals that the former region is capable of differentiating the variation of Trichogrammatidae species found in world. Acknowledgements The financial assistance from University Grants Commission, New Delhi and Kerala State Council for Science Technology and Environment under Research Projects and Ministry of Minority Affairs, Government of India in the form of MANF are gratefully acknowledged. References Bakkendorf, O. 1934. Biological investigations on some Danish hymenopterans egg-parasites, especially in homopterous and heteropterous eggs, with taxonomic remarks and descriptions of new species. Ent. Meddel, 19: 1-96. Chandrasekhar, N., Neetha, N.V., Linda Koshy Vaidyan and Moinak Banerjee. 2008. Deciphering the molecular phylogenetics of Family Hyblaeidae and inferring the phylogeographical relationships using DNA barcoding. Journal of Genetics and Molecular Biology, 19: 158-167. Flanders, S.E. 1937. Starvation of developing parasites as an evolution of immunity. Journal of Economic Entomology, 30: 970-971. Noyes, J.S. 2012. Universal chalcidoidea database. Worldwide Web electronic publication. www.nhm.ac.uk/ entomology/index.hmt.

Rukhsana, K. and Sebastian, C.D., 2014. Genetic structure and phylogeny analysis of coconut black headed caterpillar, (: Oecophoridae). Asian Journal of Biological and Life Sciences, 3:163-166.

Salma, Begum and Shoeba, B. Anis. 2014. Checkist of Indian Trichogrammatidae (Hymenoptera: Chalcidoidea). International Journal of Entomological Research.2: 7-14 Tamura, K. and Nei, M. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution 10:512- 526.

Tamura, K., Nei, M., and Kumar, S. 2004. Prospects for inferring very large phylogenies by using the neighbor- joining method. Proceedings of the National Academy of Sciences (USA) 101:11030-11035. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution30: 2725-2729.

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