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

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Volume 3; Issue 5; May, 2015 Int.J.Curr.Biotechnol. 12
Rukhsana Kokkadan and Sebastian Chempakassery Devasia, Phylogeny and Molecular Taxonomy of Family Trichogrammatidae
(Insecta: Hymenoptera) Derived from Mitochond rial DNA Sequence Analysis, Int.J.Curr.Biotechnol., 2015, 3(5):12-16.
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.
A R T I C L E I N F O A B S T R A C T
Article History:
Received 5 May 2015
Received in revised form 13 May 2015
Accepted 25 May 2015
Available online 30 May 2015
Key words:
Trichogram mati dae , DNA barcodi ng,
molecular phylogeny, COI gene.
Family Trichogrammatidae include egg parasitoid of different insect pest and the
present study reveals molecular barcoding and phylogeny analysis of seven species
in Trichogrammatidae family. The adult Trichogrammatids gave excellent control of
egg of many insect pests. We have PCR amplified and sequenced the partial fragment
of mitochondrial cytochrome oxidase subunit I (COI) gene of these seven species
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.
The results obtained can be used for the accurate taxonomic identification and phy-
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
The family Trichogrammatidae is one of the most widely
di s t r i but ed and bi ologica l l y div er s e grou p of
Hymenoptera. The members of this group are minute and
are easily recognized by their three segmented tarsi (Salma
and Sh oeba, 2014). The trichogrammatids fauna is
represented by 151 species in 31 genera (Noyes, 2012).
Trichogrammatids are economically important group
because of these groups are egg parasitoid of different
insect pest (Yousuf and Shafee, 1987). The taxonomic
study of Trichogrammatids is using morphology such as
modifications in antennal, wing, male genital characters
are laborious, time consuming and very difficult.
The eggs of these species have a little elongated and
sometimes slightly expanded central with both ends
smoothly rounded (Flanders, 1937) or there may be a
peduncle at one end (Silvestri, 1916; Bakkendorf, 1934).
The first instar larva is usually one of two types, either
sacciform or mymariform. The mature larva is robust,
distinctly segmented. Pupation takes place within the
remains of the host egg, the adult parasitoid emerging
by biting a hole in the chorion of the egg.
Using mitochondrial cytochrome oxidase subunit I (COI)
gene sequences depicted the phylogeny of many insects.
The information about phylogenetic relationships of the
Family Trichogrammatidae is not readily available for the
lack of sufficient data from a wide variety of genetic
markers. The genetic diversity and phylogeny of Opisina
arenos ell a (Rukh sa na and Sebasti an, 2014) and
Hyblaeidae family (Chandrasekhar et al., 2008) are also
studied. The focus of the current study was to decipher
th e systemati c positi on and ph ylogeny of family
Trichogrammatidae using mitochondrial COI gene
sequences.
Materials and Methods
Sample collection and DNA extraction
The seven specimen vouchers of six Trichogramma
species were collected from the different areas of Kerala,
India. The insects were stored at -20°C until the DNA was
extracted. The genomic DNA from the adults was isolated
using GeNei Ultrapure Mammalian Genomic DNA Prep
Kit (GeNei, Bangalor e) as per the manufacturer ’s
instruction.
PCR amplification and DNA sequencing
The mitochondrial cytochrome oxidase subunit I (COI)
genes of these species were amplified using the forward
primer with DNA sequence 5'- GGTCAACAAATCATA
AAGATATTGG-3' and reverse primer with DNA sequence
5'- TAAACTTCAGGGTGACCAAAAAATCA-3'. The
PCR reaction mixture consisted of 2 ng of genomic DNA,
1µl each forward and reverse primers at a concentration
of 2.5 µM, 2.5 µl of dNTPs (2mM), 2.5 µl of 10X reaction
buffer 2µl of Taq polymerase (1U/µl) and 11.8 µl H2O.
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
of 72°C for 3 minutes. The PCR products were resolved
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*Corr esp onding aut ho r.
Em ail addr es s: drc dseb asti an@ gm a il.c om
Mobile: 91-9447648961
IS SN : 232 1 - 83 71

13
Int.J.Curr.Biotechnol. Volume 3; Issue 5; May, 2015
Species Name
Nucleotide Frequencies in percentage
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
Table – 1: Composition of nucleotides in each position of codon of the COI sequence of the Trichogrammatidae
species

Volume 3; Issue 5; May, 2015 Int.J.Curr.Biotechnol. 14
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 -
Table – 2: Pattern of Nucleotide Substitution by Maximum Composite Likelihood
Figure - 1: Phylogenic relationship of Trichogrammatidae family members isolated from Kerala by Neighbor joining
and Maximum parsimony methods
Trichogramma embryophagum(KM105169)
Trichogramma cacoeciae(KM242285)
Trichogramma japonicum(KP994544)
Trichogramma mwanzai (KP142716)
Trichogramma hebbalensis(KP090265)
Trichogramma chilotraeae(KP090266)
Trichogramma platneri (KJ084938)
Trichogramma achaeae (KP994548)
Trichogramma evanescens (KP127627)
Trichogramma cordubensis (KM232610)
Trichogramma dendrolimi (KC411497)
Trichogramma chilonis(KP994546)
Trichogramma brassicae(KM998974)
Trichogramma ostriniae (DQ177914)
Trichogramma brasiliensis(DQ177919)
Trichogramma pretiosum (KM998973)
Trichogramma danaidiphaga(KP994545)
Trichogrammatoidea bactrae (KP994547)
Trichgrammatoidea armigera 02(KP994541)
Trichgrammatoidea armigera 01 (KP994542)
100
99
88
99
60
43
19
41
10
13
36
29
25
19
4
2
6
0.01
(a) Neighbor joining method

15 Int.J.Curr.Biotechnol. Volume 3; Issue 5; May, 2015
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)
(b) Maximum Parsimony method
on a 1% TAE-agarose gel, stained with Ethidium bromide
and photographed using a gel documentation system.
Aft er ascer tain ing th e PCR ampl ificati on of th e
corresponding COI fragment, the remaining portion of
the PCR products were column purified using Ultraclean
PCR Clean-up Kit (Mo Bio Laboratories, Inc. California)
as per the manufacturer’s instructions. The purified PCR
products were sequenced from both ends using the
forward and reverse primers used for the PCR using
Sanger’s sequencing method (Sanger, 1975).
Phylogeny analysis
The mitochondrial DNA COI genes in seven members
belong to six species from Family Trichogrammatidae
were sequenced and those of 13 species in the Family
Trichogrammatidae were downloaded from NCBI
GenBank database for comparison. The nucleotide
sequences of the COI gene were used for species
determination and calculating nucleotide composition
and phylogenetic analysis. The forward and reverse
sequences obta ined were trimmed for the pr imer
sequen ces, a ssembled by u sing ClustalW and the
consensus was taken for the analysis. The nucleotide
sequence and peptide sequence were searched for its
si m i l ar ity us i n g BLAS T prog r a mme of NCB I
(www.ncbi.nlm.nih.gov/) and Inter and intra specific
genetic diversity were calculated using Kimura 2-
parameter model with the pair wise deletion option and
the difference in the nucleotide in codon usage partial
COI sequence of Trichogrammatids was analyzed using
MEGA6 software. In this study, the p-distance versus
the absolute number of transitions and transversions
were plotted for each codon position, respect ively
amongst all taxa to examine COI sequences for saturation
(Tamur a et al., 2013). Phylogen etic analyses were
performed using neighbor-joining (NJ) and maximum
parsimony (MP) methods implemented in MEGA6
software (Tamura and Kumar, 2004).
Results and Discussion
The PCR amplification of partial COI gene sequences is
found to be novel and have been deposited in the NCBI
GenBank (Accession Nos. KP 994548, KP 994547, KP
994546, KP 994545, KP 994544, KP 994542 and KP 994541).
The PCR amplification of partial COI gene sequences of
Trichogrammatidae family shows little variation in their
nucleotides and A+T % is very high compared to the
G+C %. The composition of nucleotides of the family
Trichogrammatidae showed clear bias to nucleotide ‘AT’.
The nucleotide composition analysis revealed the high
AT content in the COI gene of Trichogrammatidae
species. The average percentage of nucleotides A, T, G,
C present in the COI sequence are in the concentrations
A - 31.5, T - 43.5, G - 12.9, C - 12.1 respectively (Table 1).
High concentration of nucleotide ‘T’ in the third position
of codon results in a preference of polar and hydrophobic
amino acids in the membrane associated proteins. There
is 13.2 % increase in average concentration of nucleotide
‘G’ in first position of codon compared to third position.
In Table 2, each entry sh ows th e proba bi lity of
substitution (R) from one base (row) to another base
(column). For simplicity, the sum of ‘R’ values is made
equal to 100. Rates of different transitional substitutions

Volume 3; Issue 5; May, 2015 Int.J.Curr.Biotechnol. 16
are sh own in bo ld a n d th ose of trans ver sion sa l
substitutions are shown in italic s. The nucleotide
frequencies are 31.47% (A), 43.55% (T/U), 12.10% (C),
and 12.88% (G). The analysis involved 20 nucleotide
se quen c es. Co don pos i tion s i n cl u ded we r e
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 phylogen y an alysis of the Fami ly
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
Th e finan ci a l assistan ce from Univer si ty Gra nts
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 for m of MAN F a r e gratefully
acknowledged.
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