ISSN: 0976-1675

Res. Jr. of Agril. Sci. 11(5): 1115-1120,

Sept-Oct 2020
www.rjas.org Research
Paper
Research Journal of Agricultural Sciences

© Centre for Advanced Research in Agricultural Sciences DI: 6173-0606-215

DNA Barcoding of Lepidopteron Moths from Marathawada

Region of Maharashtra
Ravindra Fakirrao Pathre*, Digamber D Bhutekar and Sharad Devidasrao Jadhav
Department of Zoology,

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 Arts, Science and Commerce College, Ambad, District Jalna - 431 213, Maharashtra, India

Received: 06 June 2020; Revised accepted: 14 July 2020

ABSTRACT
The present study is the first effort to describe diversity and molecular phylogeny of moths from
Marathawada region of Maharashtra. A total 55 moth specimens were collected and sequenced from different
sampling stations across the region. Our 47 sequences matched with COI sequences already deposited with
BOLD. But, 4 sequences did not match with any species but correctly matched with deposited sequences of
genus. Our 4 sequences are new record to BOLD but correctly matched with NCBI database. Nearest neighbor
distances were greater than 3% for all the species but for two pair of specimens: 1) Agrotis ipsilon
(EDBLM004/EDBLM005) vs Agrotis munda (EDBLM034) it was 2.8, Agrotis munda (EDBLM035) vs Agrotis
ipsilon (EDBLM005) it was 2.8. The largest nearest neighboring distance of 14.47% was observed in Hydrilodes
metisalis vs Agrotis munda. In present study, we have produced DNA sequences for 21 moth species from
Marathwada region of Maharashtra, which may help future conservation efforts and in construction of DNA
library of moth species from here.

Key words: DNA barcoding, Marathwada, 55 specimens of moths, Agrotis munda

L
epidoptera except Antarctica found in all terrestrial 2011, Hausmann et al. 2011, 2013, Huemer et al. 2014, Liu
habitats (Gullan and Cranston 2005). Among them, et al. 2014, Zahiri et al. 2014). Many studies have shown
butterflies have been discussed more due to colorful wings that DNA barcoding could resolve the taxonomic problems
(Wang and Fang 2007). Earlier in Maharashtra, there have in lepidopteran systematic (Hajibabaei et al. 2006, Burns et
been very few moth surveys carried out. Hampson et al. al. 2008, Mutanen et al. 2012, Jiang et al. 2017).
(1891) recorded about 611 species of moths from Nilgiris, Marathawada region is well known to world by its
Maharashtra. The moths from Sanjay Gandhi National park, Ajanta and Ellora caves. It is most diverse region of
Boriwali, Mumbai were studied by Vaylure et al. (2012). Maharashtra connected with Sahyadri mountain ranges.
Gurule et al. (2013) studied 728 species of moths from Moreover, majority of maize, pulses and cotton production
North Maharashtra. Nimbalkar et al. (2015) studied 49 in county is accounted by this region and moth species are
species of moths from Marathwada region of Maharashtra. important pest here. However so far, this diversity of insects
Kalawate et al. (2018) collected 99 species of moths from is largely noted by morphological analysis only. Till now,
northern Western Ghats of Maharashtra. In moths, due to the nobody has done DNA barcoding of moths in the region. So,
complex morphological characters, identification is difficult this study aims to produce DNA sequences of moth species.
(Janzen et al. 2005, Hausmann et al. 2009, Huemer and Also, this information on moths would be helpful in quick
Mutanen 2012). Now in species identification, DNA identification and effective management of some pest
barcoding has been proved to be useful (Hebert et al. 2003), species.
Moreover, many workers are studying region-specific
lepidopteron diversity (Dinca et al. 2011, deWaard et al.
MATERIALS AND METHODS
*Corresponding author: Ravindra Fakirrao Pathre, Assistant Professor and Head, Department of Zoology, Arts, Science
and Commerce College, Ambad, District Jalna - 431 213, Maharashtra
e-mail: [email protected]  Contact: +91- 7588794162

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 DNA Barcoding of Lepidopteron Moths from Marathawada Region of Maharashtra
Marathawada is more diverse region with eight (8) 2µl DNA template, 10 pmol of each primer and 200 µM of
districts. The temperature ranges between 7.8°C (winter) to dNTP and 0.2 µl of Taq polymerase (Banglore, Genei).
42.8°C (summer) as per seasons (Indian meteorological Thermo-cycling was as follows: First cycle of 1 min at 94°C
department, regional office Pune, India). The area receives followed by five cycles of 94°C for 1 min, 45°C for 1 min
rain from Southeast monsoons. This area has a tropical 30 s, 72°C for 1 min 15 s, then 30 cycles of 94°C for 1 min,
climate, specially a tropical wet and dry climate with 51°C for 1 min 30 s, 72°C for 1 min 15s, with last step of
dryness of seven months and rainfall from June to 72°C for 5 min. The products were checked by 1% agarose
September. gel and then purified using PEG-NaCl method. Finally,
All moth samples were collected from various sampling using an automated sequencer (3730 DNA analyzer, ABI,
stations during the May 2016 to May 2017. Moths were Hitachi), products was sequenced by both, the forward and
collected through light traps, using 85-Watt CFL bulb, reverse primers.
which is convenient method. White cloth sheet hanging
between two vertical poles. All specimens collected in jars Data analysis
and killed using ethyl acetate. Before pinning and spreading ClustalW nucleotide sequence alignment and assembly
on the board a leg clip or a tip of abdomen from specimen was carried out using MEGA7. The nucleotide sequences
was cut and stored in Ethanol (90% alcohol). All ethanol were searched for its similarity using BOLD
preserved samples were stored in refrigerator for molecular (www.boldsystems.org) and NCBI blast. Sequence
analysis. Subsequent to isolation of DNA, moths were divergence values within and among species, were
spread and preserved according to standard entomological employed using the Kimura two parameter (K2P) model and
methods at department of Zoology Arts, Science and using analytical functions on BOLD V3. A phylogeny was
Commerce College Ambad. Identification was based on inferred using maximum likelihood tree based on K2P
wing shape and color pattern described in available model (MEGA7) in which insect Flatidae sequence was
keys/identification guides. used as outgroup and nucleotide composition values also
obtained. Earlier studies have revealed that the most
DNA extraction, PCR and sequencing different species of Lepidoptera show >3% sequence
DNA extraction, polymerase chain reaction (PCR) divergence at COI (Hebert et al. 2003) and researchers have
amplifications and sequencing were performed at the Paul used a 3% pairwise threshold for species delimitation
Hebert Centre for DNA Barcoding and Biodiversity, Dr. (Strutzenberger 2011). For the barcode-based identity
Babasaheb Ambedkar Marathawada University, analysis, we also used a threshold of 3% divergence. A
Aurangabad, Maharashtra, India. Specimen DNA from leg barcode gap analysis was performed using BOLD.
or tip of abdomen was isolated using automated DNA Sequences were submitted to BOLD project code
isolation machine. Then, using polymerase chain reaction [EDBLM]. Sequences from moth species from this region
(PCR) (ABI thermocycler) obtained DNA was amplified. were compared with the sequence of the conspecifics from
These primers were used for amplification of COI gene: other geographical areas. It was to check intra and
LEP F1 50 ATT CAACCAATCATAAAGATAT 30 and interspecific divergences in such widely distributed area and
LEP R1 50 TAAA CTTCTGGATGTCCAAAAA 30. For check if any cryptic or overlooked species showing deep
PCR reaction, total volume of 25µl was taken which contain divergence.

Table 1 Showing details of 55 moth specimens studied and species identification by BOLD system
Sample Id Family Subfamily Genus Species
ACCAB2 Noctuidae Noctuinae Agrotis Agrotis ipsilon
ACCAB-B2 Agrotis Agrotis ipsilon
GTS6 Agrotis Agrotis segetum
MRBK5 Agrotis Agrotis segetum
MCA02 Agrotis Agrotis munda
MCA2-2 Agrotis Agrotis munda
MCA2-1 Agrotis Agrotis munda
CCA11 Amphipyrinae Sesamia Sesamia inferens
KTGH2 Sesamia Sesamia inferens
MRBK3 Sesamia Sesamia inferens
ACCAB1 Noctuinae Spodoptera Spodoptera litura
TWR2 Spodoptera Spodoptera exigua
TWR2-1 Spodoptera Spodoptera exigua
ACC3 Athetis Athetis recluse
CCA10 Athetis Athetis recluse
CCA6 Athetis Athetis recluse
ACCB1 Heliothinae Helicoverpa Helicoverpa armigera
ACCB2 Helicoverpa Helicoverpa armigera
ACCB3 Helicoverpa Helicoverpa armigera

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 DNA Barcoding of Lepidopteron Moths from Marathawada Region of Maharashtra
TWR3 Ogdoconta (NCBI Blast)
CCA02 Geometridae Ennominae Chiasmia -
GRB2 Sterrhinae Scopula species -
CCA13 - Chiasmia species -
MRBK2 Ennominae Cleora Cleora tenebrata
PRA8 Cleora Cleora tenebrata
TWR1 Chaismia Chaismia multistrigata
GRB10 Sterrhinae Traminda Traminda mundissima
CCA12 Traminda Traminda mundissima
GRB1 Ennominae Isturgia Isturgia disputaria
HBA1 Isturgia Isturgia disputaria
PRAB2 Erebeidae Arctiinae Creatonotos Creatonotos gangis
CCA04 - Acantholipes -
ACC6 Arctiinae Amata Amata cysseus
ACCBC1 Amata Amata passalis
GBS4 Herminiinae Hydrillodes Hydrillodes metisalis
CCA01 Arctiinae Utetheisa pulchella (NCBI Blast)
MRBK8 Utetheisa Utetheisa pulchelloides
MRBKB5 Utetheisa Utetheisa pulchella
JRA2 Utetheisa Utetheisa pulchella
MRBKB1 Utetheisa Utetheisa pulchella
MRBKB2 Utetheisa Utetheisa pulchella
MRBKB3 Utetheisa Utetheisa pulchella
MRBKB4 Utetheisa Utetheisa pulchella
PRA2 Calpinae Culasta Culasta indecisa
JRA1 Erebinae Pandesma Pandesma guenauadi
MRBK4 Erebinae Mocis Mocis trifasciata
HBA3 - Melipotis jucunda (NCBI Blast)
PRA6 Erebinae Spirama Spirama retorta
GTSB3 Lymantriinae Euproctis Euproctis lunata
GTSB2 Euproctis Euproctis lunata
GTS3 Euproctis Euproctis lunata
JRAB2 Lymantriinae Lymantria Lymantria incerta
PRA4 Nolidae - Selepa species (NCBI Blast)
GBS5 Crambidae Glaphyriinae Noorda Noorda blitealis
TWR2-2 Pyrilidae Galleriinae Lamoria Lamoria anella
CCA15 Insect Flatidae as outgroup

Table 2 Barcode gap analysis for moth species

Name of species Mean Intra-Sp Max Intra-Sp to distance with nearest neighbor
Noorda blitealis N/A N/A Agrotis munda 11.02
Amata cysseus N/A N/A Mocis trifasciata 10.49
Creatonotos gangis N/A N/A Athetis reclusa 10.14
Hydrillodes metisalis N/A N/A Agrotis munda 9.09
Mocis trifasciata N/A N/A Agrotis munda 8.92
Pandesma guenauadi N/A N/A Mocis trifasciata 9.09
Spirama retorta N/A N/A Pandesma guenauadi 10.59
Utetheisa pulchella 0.32 0.92 Utetheisa pulchelloides 3.94
Utetheisa pulchelloides N/A N/A Utetheisa pulchella 3.94
Cleora tenebrata 0.32 0.32 Mocis trifasciata 12.09
Isturgia disputaria 1.25 1.25 Utetheisa pulchelloides 11.9
Traminda mundissima 0.5 0.5 Spodoptera exigua 13.4
Agrotis ipsilon 1.12 1.12 Agrotis munda 2.8
Agrotis munda 0.43 0.49 Agrotis ipsilon 2.8
Agrotis segetum 0 0 Agrotis ipsilon 4.32
Athetis reclusa 0.57 0.71 Agrotis munda 7.84
Helicoverpa armigera 0.21 0.32 Athetis reclusa 7.89
Sesamia inferens 1.15 1.23 Agrotis munda 6.91
Spodoptera exigua 1.14 1.14 Spodoptera litura 7.31

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 Pathre et al. 2020 Res. Jr. of Agril. Sci.
11(5)
Spodoptera litura N/A N/A Spodoptera exigua 7.31
Lamoria anella N/A N/A Hydrillodes metisalis 14.47

RESULTS AND DISCUSSION

We collected 456 moth specimens belongs to 112
morphologically identifiable species, 88 genera, 9 super
families and 15 families. A total of 55 COI sequences (Table
1) for 21 species were generated. Our 47 sequences matched
with COI sequences already deposited with BOLD. But, 4
sequences not matched with any species but correctly
matched with deposited sequences of genus. A Further, our
4 sequences are new record to BOLD but matched with
NCBI database. The ClustalW alignment showed 331
conserved sites, 327 variable sites, 230 parsimony
informative sites, and 97 singleton sites. All the amplified
sequences were 632 bp (mean) with no insertions, deletions,
and stop codons. The overall GC content was 29.78
(SE=0.14). GC content at codon positions 1 was 40.02
(SE=0.19), at 2 was 41.92 (SE=0.07), at 3 was 7.48
(SE=0.38) (Table 3). All the sequences were submitted to
the BOLD with the project name EDBLM. From the total, 4
sequences represent new record and did not match to the
BOLD sequences but correctly matched with NCBI
sequences. Genetic divergence increased with taxonomic
rank. Intraspecific divergence ranged from 0.0 to 1.25 with a
mean of 0.49% (SE=0.01%) (Table 2), while for intragenic
distance ranged from 2.8% to 7.67 with a mean of 4.53%
(SE=0.05). The distance within families ranged from 6.91%
to 17.13 with mean of 10.35% (SE=0.01).
Barcode gap analysis revealed intra and interspecific
sequences distance in species (Table 2-3) (21, species, 40
sequences analyzed). Here, low intraspecific distance
(<3%) suggest low species resolution, thus leading to
species overlap. Intraspecific distances could not be
determined for the 10 species with just a single
representative. Gaikwad et al. (2011) studied butterflies
from Western Ghats of Maharashtra. Vikas Kumar (2019)
Fig 1 Showing maximum likelihood tree based on Kimura also studied Geometridae moths from Namdapha National
two parameter (K2P) model using MEGA7 Park, Eastern Himalaya.

Table 3 Showing distance summary of 55 sequences

n Taxa Comparisons Min Dist (%) Mean Dist (%) Max Dist (%) SE Dist (%)
Within species 30 11 33 0 0.49 1.25 0.01
Within genus 17 3 24 2.8 4.53 7.67 0.05
Within family 38 3 207 6.91 10.35 17.13 0.01

Table 4 Showing nucleotide frequency distribution

Min Mean Max SE
G% 13.83 14.45 15.2 0.04
C% 13.7 15.34 19.76 0.15
A% 28.57 30.85 35.56 0.19
T% 30.7 39.36 41.95 0.23
GC % 28.38 29.78 33.74 0.14
GC % Codon Pos 1 37.13 40.02 43.38 0.19
GC % Codon Pos 2 40.61 41.92 43.35 0.07
GC % Codon Pos 3 3.77 7.48 18.18 0.38

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 DNA Barcoding of Lepidopteron Moths from Marathawada Region of Maharashtra
Nearest neighbor distances were greater than 3% for all species (Nolidae) formed monophyletic clade with Culasta
the species but for two species pairs: 1) Agrotis ipsilon indecisa (Erebediae). Noordae blitealis (Crambidae) found
(EDBLM004/EDBLM005) vs Agrotis munda (EDBLM034) grouped with Chiasmia species (Geometridae). The present
it was 2.8, Agrotis munda (EDBLM035) vs Agrotis ipsilon tree shows that Lamoria anella (Pyralidae) is ancestor of all
(EDBLM005) it was 2.8. The max-intraspecific distance other families.
was observed with four species, Agrotis ipsilon (1.12), In present work, genus Agrotis formed closed clustering
Spodoptera exigua (1.14), Isturgia disputaria (1.25), with each other and with species Sesamia inferens. Athetis
Sesamia inferens (1.23) respectively. The specimens of reclusa clustered closely with Helicoverpa armigera. All
Isturgia disputaria showed maximum intra-species these species are morphological different each other but
divergence 1.25% collected from different regions while in formed clade with district families show presence of cryptic
Agrotis segetum it was zero (0%) even when they were species in the region. In present study, we have produced
collected from distinct geographical area. The largest nearest first DNA based identification of moths from Marathwada
neighboring distance of 14.47% was observed in Hydrilodes region of Maharashtra, which may help effective
metisalis vs Agrotis munda. The average nearest neighbor management of some pest species of moth from here.
distance was 8.39% (SE=0.16). The maximum likelihood
tree with the highest log likelihood (-6050.81) was obtained Acknowledgement
(Fig 1). Family Noctuidae formed monophyletic clade with First author is thankful to all those who helped in the
four species, Chaismia and Scopula (Geometridae) and collection of moths. We would also like to thank the
Creatonotos gangis and Acantholipes (Erebediae). Selepa unknown referee for their valuable suggestions.

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