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Muhammedali V. C.
Muhammedali V. C.
Muhammedali V. C.
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International Research
Journal of
Vol. 6(10), 8-10, October (201
7
International Science Community
Association
Short Communication
DNA barcoding for identification of
Tettigoniidae) from Northern Kerala using Cytochrome Oxidase Subunit I
V.C. Muhammedali
1
Department of Biotechnology, SAS SNDP Yogam College Konni, Pathanamthitta, Kerala 689 691, India
2
Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala 673 635, India
Received 8
th
August
Abstract
Grasshoppers are widely distributed in all ecosystems. Tettigoniidae
crickets in the order Orthoptera. Approximately 6,400 species of Tettigoniids are found around the world. Conocephalus
dorsalis, Tettigoniidae family, was collected from rice fields of Northern Kerala
grasses, seeds and small insects including pests. They are predators on rice field, controlling pest populations. The present
study deals with species identification and revealed phylogenetic history of C. dorsalis u
(COI) gene encoded as mitochondria. The COI gene of C. dorsalis, 589bp are sequenced and obtained was deposited in the
NCBI GenBank.
Keywords: Conocephalus dorsalis
, mitochondrial COI gene, DNA barcoding.
Introduction
Orthoptera is one of the largest order having over 20,000
species worldwide. Tettigoniidae is a major family of long
horned grasshoppers, placed in the suborder Ensifera
have long and thread like antenna which may exceed their own
body length. Tettigoniids play an important role in the
ecosystem. Many species are exclusively predatory which make
the balance between pests and their natural enemies. Some
Tettigoniids
are considered as insect pests by commercial crop
growers and are applying pesticide to limit the population
Conocephalus dorsalis
is widely distributed over central Europe
to Western Siberia. They are small green bush cricket, size from
11-18 mm in len
gths. They are characterized by strong hind
limbs for leaping, long and thread like antenna, the wings are
short, abdomen entirely blackish and sometimes dark brown
colour. It is an omnivorous insect but feeds mainly on seed
heads, grasses and also capture
s small insects like pests
are reports from Indonesia have reported that
predators on rice fields
4
. Therefore, they will benefit in reducing
pest population and are naturally quite important. They colonize
wetlands, reed edges, fen
meadows and ditches. The nymphs are
arises from May to June and the adults from July to mid
October.
DNA barcoding is one of the most important taxonomic method
which helps in the identification of organisms using short
genetic markers.
The barcode region, cytochrome oxidase
Journal of
Biological Sciences _______________________
____
7
)
Association
DNA barcoding for identification of
Conocephalus dorsalis
Tettigoniidae) from Northern Kerala using Cytochrome Oxidase Subunit I
Gene
V.C. Muhammedali
1
, V.P. Akhilesh
2
and C.D. Sebastian
2*
Department of Biotechnology, SAS SNDP Yogam College Konni, Pathanamthitta, Kerala 689 691, India
Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala 673 635, India
drcdsebastian@gmail.com
Available online at:
www.isca.in, www.isca.me
August
2017, revised 5
th
October 2017, accepted 9
th
October 2017
Grasshoppers are widely distributed in all ecosystems. Tettigoniidae
is a family of grasshoppers including katydids or bush
crickets in the order Orthoptera. Approximately 6,400 species of Tettigoniids are found around the world. Conocephalus
dorsalis, Tettigoniidae family, was collected from rice fields of Northern Kerala
. It is an omnivorous insect that feeds
grasses, seeds and small insects including pests. They are predators on rice field, controlling pest populations. The present
study deals with species identification and revealed phylogenetic history of C. dorsalis u
sing cytochrome oxidase subunit I
(COI) gene encoded as mitochondria. The COI gene of C. dorsalis, 589bp are sequenced and obtained was deposited in the
, mitochondrial COI gene, DNA barcoding.
Orthoptera is one of the largest order having over 20,000
species worldwide. Tettigoniidae is a major family of long
horned grasshoppers, placed in the suborder Ensifera
1
. They
have long and thread like antenna which may exceed their own
body length. Tettigoniids play an important role in the
ecosystem. Many species are exclusively predatory which make
the balance between pests and their natural enemies. Some
are considered as insect pests by commercial crop
growers and are applying pesticide to limit the population
2
.
is widely distributed over central Europe
to Western Siberia. They are small green bush cricket, size from
gths. They are characterized by strong hind
limbs for leaping, long and thread like antenna, the wings are
short, abdomen entirely blackish and sometimes dark brown
colour. It is an omnivorous insect but feeds mainly on seed
s small insects like pests
3
. There
are reports from Indonesia have reported that
C. dorsalis are
. Therefore, they will benefit in reducing
pest population and are naturally quite important. They colonize
meadows and ditches. The nymphs are
arises from May to June and the adults from July to mid
DNA barcoding is one of the most important taxonomic method
which helps in the identification of organisms using short
The barcode region, cytochrome oxidase
subunit I gene (COI) was proposed by Paul Hebert
based on intra specific and inter specific variation. The sequence
data is also used to develop barcode libraries for identification
of unknown species by mat
ching sequences with the known
species. Phylogenetic tree of related individuals are clustered
together and established species relationships
is proposed as a powerful tool for taxonomic studies and the
bioidentification of organisms.
Phyl
ogenetic analysis using mitochondrial COI gene sequence
were extensively carried out in various insect groups like
grasshopper,
Microcentrum rhombifolium
Ceriagrion coromandelianum
8
, cigarette beetle
serricorne
9
, leaf hopper
Thaia
Herpetogramma stulasis
11
.
Materials and methods
Sample collection and preservation
present study was collected from rice fields of Parappanangadi,
Northern Kerala. Sampling was done manually, using sweep net
method
and the sample was transferred to
contained tube.
The collected sample was identified
morphologically and which preserved as cooling storage at
C.
DNA extraction, a
mplification,
Mitochondrial genomic DNA of cytochrome
gene (COI) was extracted from one of the thoracic leg. The
____
_________ ISSN 2278-3202
Int. Res. J. Biological Sci.
8
Conocephalus dorsalis
(Orthoptera:
Tettigoniidae) from Northern Kerala using Cytochrome Oxidase Subunit I
Department of Biotechnology, SAS SNDP Yogam College Konni, Pathanamthitta, Kerala 689 691, India
Molecular Biology Laboratory, Department of Zoology, University of Calicut, Kerala 673 635, India
is a family of grasshoppers including katydids or bush
crickets in the order Orthoptera. Approximately 6,400 species of Tettigoniids are found around the world. Conocephalus
. It is an omnivorous insect that feeds
grasses, seeds and small insects including pests. They are predators on rice field, controlling pest populations. The present
sing cytochrome oxidase subunit I
(COI) gene encoded as mitochondria. The COI gene of C. dorsalis, 589bp are sequenced and obtained was deposited in the
subunit I gene (COI) was proposed by Paul Hebert
5
in 2003,
based on intra specific and inter specific variation. The sequence
data is also used to develop barcode libraries for identification
ching sequences with the known
species. Phylogenetic tree of related individuals are clustered
together and established species relationships
6
. DNA barcoding
is proposed as a powerful tool for taxonomic studies and the
ogenetic analysis using mitochondrial COI gene sequence
were extensively carried out in various insect groups like
Microcentrum rhombifolium
7
, damsel flies
, cigarette beetle
Lasioderma
Thaia
subrufa
10
and moth
Sample collection and preservation
: C. dorsalis used in
present study was collected from rice fields of Parappanangadi,
Northern Kerala. Sampling was done manually, using sweep net
and the sample was transferred to
70% of alcohol
The collected sample was identified
morphologically and which preserved as cooling storage at
-20
mplification,
sequencing: The
Mitochondrial genomic DNA of cytochrome
oxidase subunit I
gene (COI) was extracted from one of the thoracic leg. The
International Research Journal of Biological Sciences ________________________________________________ISSN 2278-3202
Vol. 6(10), 8-10, October (2017) Int. Res. J. Biological Sci.
International Science Community Association 9
tissue was homogenized and the genomic DNA was extracted to
using genomic DNA kit (Origin genomic DNA kit).
Approximately 2 ng of genomic DNA was amplified for COI
gene using forward primer, 5'- GGT CAA CAA ATC ATA
AAG ATA TTG G-3' and the reverse primer 5'- TAA ACT
TCA GGG TGA CCA AAA AAT CA- 3'. The PCR reaction
mixture contained 2 ng of 1µ l genomic DNA, 1µ l each forward
and reverse primer at a concentration of 10µM, 2µl dNTPs
(2Mm), 10µl of 10X reaction buffer, 1µ l Taq polymerase
(5U/µl) and 84 µl water. The PCR profile involved initial
denaturation step of 5 minutes at 95ºC, followed by 30 cycles of
10 second at 95ºC, 1 minute 50ºC and 1 minute at 72ºC and
ending with final phase of 72ºC for 3 minutes. The amplified
product of COI gene was analyzed on 2% TAE - agarose gel
electrophoresis for the confirmation. The remaining portion of
amplified product was column purified using GeneJET PCR
purification kit (Fermentas Life Science). The purified PCR
product of COI was sequenced from both ends using forward
and reverse primers by Sanger’s dideoxy chain termination
sequencing method, with ABI 3730XL Automated Sequencer
12
.
Sequences were aligned to using the MEGA6 software package
and the estimation of residue and pair wise distances were using
the Clustal W tool of MEGA6 software. The final sequences
were used for its similarity using BLAST programme of NCBI
GenkBank. The Sequence divergences were estimated and
Neighbour Joining tree was developed to exhibit evolutionary
history of species.
Results and discussion
In the present study, COI gene of Conocephalus dorsalis
yielded as 589bp size of fragment. The sequence obtained was
deposited in the NCBI GenBank (GenBank Accession: KX
503055). BLAST analysis revealed sequence similarity between
the species and C. dorsalis has 97% similarity with Acrida
exaltata (GenBank Accession: GU226877) from Tamilnadu,
India, which doesn’t belongs to same family. The two species of
Atractomorpha lata (GenBank Accession: KF966602) and
Atractomorpha sinensis (GenBank Accession: KJ889692) from
USA showed 91% similarity with the present result. The
percentage of COI evolutionary divergence of C. dorsalis with
other related species is presented in Table-1. Phylogenetic
analysis also depicted that C. dorsalis is closer to Othopteran
grasshopper species and a phylogenetic tree (Figure-1)
constructed using Neighbour Joining method revealed
monophyletic lineage. The majority of insects under order
Orthoptera showed 85 to 97% of sequence similarity to that C.
dorsalis COI gene sequences.
The percentage of COI evolutionary divergence of C. dorsalis
showed 9% evolutionary divergence between Atractomorpha
sinensis (KJ889692.1) and Atractomorpha lata (KF 966602.1).
C. dorsalis shows 15% evolutionary divergence between
Cibolacris parvileps (JQ 513033.1), Melanoplus sanguinipes
(KR 148046.1), Sinopodisma housanda (KC 139912.1) and
Acrida wellemsei (KJ 8889501.1).
The study established that DNA barcoding can provide the
complement taxonomical studies of organisms. The
combination of DNA sequenced data with the classical
taxonomy will serve as a model which can revealed on many
disciplines. It would enhance the rate of species identification,
which in turn helps in conservation of insect biodiversity. The
results reveal that mitochondrial COI gene permits the
unambiguous identification of grasshopper species. The partial
sequenced gene of C. dorsalis was more diverge from the sister
taxon viz. Acrida exaltata. This is also supported by the
geographical aspects. The barcode generated for C. dorsalis in
the present study can be used for accurate identification of the
organism.
Table-1: Evolutionary divergence between Conocephalus
dorsalis and other related species.
Species % of divergence
GU226877.1 Acrida exaltata 3%
KJ889692.1 Atractomorpha sinensis 9%
KF 966602.1 Atractomorpha lata 9%
KP 641752.1 Ichtyotettix mexicanus 15%
JQ 513033.1 Cibolacris parvileps 15%
KR 148046.1 Melanoplus sanguinipes 15%
KR 145454.1 Melanoplus femurrubrum 15%
KC 139912.1 Sinopodisma housanda 15%
KC 139912.1 Sinopodisma iushiensis 15%
KC 139921.1 Sinopodisma tsinlingensis 15%
KJ 8889501.1 Acrida wellemsei 15%
KM 532301.1 Opeia obscura 15%
KM 816659.1 Notostaurus albicornis 15%
KR 005939.1 Dociostaurus kraussi 15%
Conclusion
The present study indicates that the COI sequence of C. dorsalis
can be used as taxonomical studies and bioidentification system
of the species, which is one of the dominating species in rice
fields due to its high predation on pest species. The COI
sequence of the C. dorsalis revealed that 3% to 15% of
sequence divergence with many other grasshopper species.
Phylogenetically C. dorsalis is closer to Acridida exaltata
showing 97% of similarity.
International Research Journal of Biological Sciences ________________________________________________ISSN 2278-3202
Vol. 6(10), 8-10, October (2017) Int. Res. J. Biological Sci.
International Science Community Association 10
Figure-1: Phylogenetic status of Conocephalus dorsalis using Neighbor joining method.
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Dociostaurus tartarus (KM816676.1)
Dociostaurus kraussi(KR005939.1)
Notostaurus albicornis (KM816659.1)
Opeia obscura(KM532301.1)
Acrida wil lemsei (KJ889697.1)
Acrida wil lemsei (KJ889504.1)
Acrida wil lemsei (KJ889501.1)
Sinopodi sma tsinlingensis (KC139931.1)
Sinopodi sma lushiensis (KC139921.1)
Sinopodi sma houshana (KC139912.1)
Mel anoplus femurrubrum (KR145454.1)
Mel anoplus sp. (KT708372.1)
Mel anoplus sanguinipes (KR148046.1)
Cibolacris parviceps (JQ513033.1)
Ichthyotettix mexicanus (KP641752.1)
Atractomorpha lata (KF966602.1)
Atractomorpha sinensis (KJ889692.1)
Conocephalus dorsalis Kerala (KX 503055)
Acrida exaltata (GU226877.1)
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