Molecular identification of edible insects of Nagaland

Abstract

Entomophagy practices should rely on proper identification of insects, which are usually classified relying on morphological keys and traditional knowledge practices. This may lead to misidentification of edible insects. Hence, the present study, DNA barcoding was used to identify and documentation of edible insects. In the primary study 7 edible insects were collected and identify by morphological and confirmed by DNA barcoding method and the species level confirm through phylogenic analysis using BLAST. COI gene sequence was submitted to Gene Bank under the accession Number Choroedocus illustris (MN82982), Chondracris rosea (MN829822), Samia ricini (MN829823), Tagasta indica (MN829824): Tessaratoma papillosa (MT009020), Batocera rubus larva (MT089705) and Odontotermes longignathus (MT009019).

Full text

203
International Journal of Entomology Research
www.entomologyjournals.com
ISSN: 2455-4758
Received: 30-01-2021, Accepted: 03-03-2021, Published: 11-04-2021
Volume 6, Issue 2, 2021, Page No. 203-208
Molecular identification of edible insects of Nagaland
Lipoktola Tzudir1, Murali Markandan2*
1 Department of Zoology, Immanuel College, Lengrijan, Dimapur, Nagaland, India
2 PG and Research Department of Zoology, St Joseph University, Dimapur, Nagaland, India
Abstract
Entomophagy practices should rely on proper identification of insects, which are usually classified relying on morphological
keys and traditional knowledge practices. This may lead to misidentification of edible insects. Hence, the present study, DNA
barcoding was used to identify and documentation of edible insects. In the primary study 7 edible insects were collected and
identify by morphological and confirmed by DNA barcoding method and the species level confirm through phylogenic
analysis using BLAST. COI gene sequence was submitted to Gene Bank under the accession Number Choroedocus illustris
(MN82982), Chondracris rosea (MN829822), Samia ricini (MN829823), Tagasta indica (MN829824): Tessaratoma
papillosa (MT009020), Batocera rubus larva (MT089705) and Odontotermes longignathus (MT009019).
Keywords: COI gene sequences, DNA barcode, edible insects
Introduction
Insect as food is common among the ethnic people of
Northeast India mainly among tribes of Arunachal Pradesh,
Assam, Manipur and Nagaland. Studies have revealed that
almost 255 insect species are used as food by different tribes
of India. Documentation of edible insect in various state of
India have shown that Arunachal Pradesh (158 Species of
edible insect), Manipur (41 species of edible insects),
Assam (38 Species of edible insects) and Nagaland (42
Species of edible insects) Meghalay (16 species) Kerala 5
species [1, 2]. The native people inhabiting in the north
eastern state of India consume edible insect species at their
different development stages (Adults, larva stages, pupa
Eggs).These people use their traditional knowledge to
determine which species to be eaten and at what stage. The
People of different tribes select the edible insects on the
basis of their traditional idea, taste and also regional and
seasonal availability [3]. They likewise have a tremendous
customary information on the viable use of consumable
insects which are obtained through experience and normally
passed on by oral conventions as a monitored mystery of
specific families [4]. In the studies, Molecular tools used for
identification of edible insects. Over the recent three
decades, Mitochondrial DNA has been broadly analyzed [5]
and demonstrated to be a significant tool in species
delimitation as it has biological properties making it
appropriate as a marker for molecular biodiversity. Limited
DNA sequencing of the mitochondrial gene, for example,
Cytochrome C oxidase I (COI) and other molecular makers
have been utilized to recognize and find new species. A few
investigations have indicated that a 648bp fragment of COI
can be utilized as a DNA barcode to identification and
recognize individual species [6]. Fragments of COI has been
appeared to give high goals to recognize obscure species,
along these lines expanding scientific classification based on
biodiversity gauges [7]. Nonetheless, DNA barcoding proved
to be a versatile tool with a variety of application, for
example, by facilitating the association between different
developmental stages in insects [8]. The methods of
identification of edible insects and practices of
entomophagy may not be known to other people and may
disappear with that group of tribal. In the present study, the
aim was to collect information about edible insect in certain
Naga tribes and then the identified edible insect was
confirmed by Molecular methods of DNA barcoding
techniques.
Material and Methods
Insects were collected from local market in Dimapur,
Nagaland and preserved in 100 % ethanol. DNA was
extracted from body tissues using CTAB or Kit based
methods. Genes were amplified using PCR. Each PCR
reaction for testing the amplification efficiency and
development of multiplex PCR assays for DNA barcode
primers contained 1μl DNA template (25 ng), 2μl 10X
reaction buffer, 0.5μl MgCl2 (50pM), 1μl dNTPs mix
(10mM), 1μl forward primer (10pM), 1μl reverse primer
(10pM), 0.5μl Taq polymerase (5 U/pi) and the final volume
25μl will be adjusted with molecular grade water. Primers
are standard primers available for COI gene amplification.
COIF- GGTCAACAAATCATAAAGATATTGG- Tm
510C
COIR- TAAACTTCAGGGTGACCAAAAAATCA- Tm
530C
Phylogenetic Tree Analysis
A Phylogenetic tree was drawn based on distance neighbor
tree-joining method and Maximum Likelihood method in
Geneious v.9.0.2 [9] and also used BLAST analysis tool from
NCBI. FAST format of the COI gene sequence were
submitted in Gene Bank to get accession number from
NCBI (https://www.ncbi.nlm.nih.gov/). The analysis of
Sequence composition was performed with help of available
tools in BOLD. Barcode sequences of the sampled specimen
are available online in the dataset of MMLT20 present in

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the Database of BOLD. (http://www.boldsystems.org).
Result
Entomophagy is an age old practice that continues to this
day in many tribe’s people in Nagaland, A total seven edible
insects belonging to 7 families of 4 orders (Orthoptera,
Lepidoptera, Coleoptera and Hemiptera) were collected
from Dimapur, Nagaland. DNA extracted from tissue
sample of 7 edible species. Most of amplified sequences
were up to 450 to 680 bp length. BLAST was used to check
homology of between the retrieved sequences in Gene Bank
library. This help to identify sequence similarity across
genomes. Analysis of homology using BLAST revealed that
Sample-2 were 98.45% similar with voucher insects of
Choroedocus illustris (KY83924.1) (Table-1, Fig-1) and
Sample-3 have 98.67% match with the voucher specimens
of Chondracris rosea (MK007252.1) (Table-2, Fig-2).
Sample 4 COI sequences were analyzed in BLAST to
revealed 100% pair wise matched with voucher samples of
Samia ricini (MN120775.1) in NCBI (Table-3, Fig-3).
Another BLAST analysis disclosed that the observed COI
sequence of sample -5 showed 95.98% homology with
voucher specimen of Tagasta indica (NC045930.1) (Table -
4, Fig-4). It indicates that observed sample 5 is Tagasta
indica. In the table,-6, BLAST analysis showed that the
observed sequence of sample 6 has 92.54 % homology with
voucher specimen sequences in Gene bank from India. It
reveals that the observed sample is Odontotermes
longignathus (Table-5, Fig-5). BLAST result disclosed that
the observed sample COI sequence of Tessaratoma sp
showed 80.58 homology with voucher specimen of
Tessaratoma papillosa (NC037742.1). It indicates that the
observed samples are Tessaratoma papillosa (Table-6, Fig-
6). In table- 7, BLAST analysis showed that the observed
sequence of samples 8 has 92.47 % similar with voucher
specimen sequence of Batocera rubus (KT314213.1) in the
gene bank. The nucleotide sequences of the COI for the 7
species of edible insects were submitted to Gen Bank and
subjected to a homology search using BLAST. A
Phylogenetic tree based on COI sequence of the edible
insects was constructed using neighbor tree joining and
maximum likelihood methods. All edible insects COI
sequence was submitted in NCBI under the accession
number provided in table-8. Phylogenic of NJ analysis also
depicted three species closely related Choroedocus illustris,
Chondracris rosea and Tagasta indica. In the clad A,
Chondracris rosea is resoled as a sister group to
Choroedocus illustris and Tagasta indica as well as forms a
monophyletic group (Fig-7). The pair wise distance was
calculated by the MEGA X software. The inter specific
nucleotide divergence between the seven edible species
range from 6.76% to 17.72 (Table-9). The highest distance
of 17.72 was obtained between Tagasta indica and
Tessaratoma papillosa the shortest distance of 7.47 was
obtained between Choroedocus illustris and Chondracris
rosea. Sequences composition analysis by using BOLD
system the highest mean value has registered in “T” bas pair
33.27 and followed by 26.16 (Adenine), 22.92
(Guanine),17.63 (Cytosine) (Tabl-10).DNA barcode images
depicted in table-11.
Discussion
In Nagaland 42 to 60 insect species have been documented
as edible insects by morphological identification methods,
belonging to different family and orders. In the present
study, molecular identification of selected edible insects
(Grasshoppers: Choroedocus illustris, Chondracris rosea,
Tagasta indica, Samia ricini (silk worm), Tessaratoma
papillosa (Sting Bugs), Batocera rubus larva (wood borers)
and Odontotermes longignathus (wings termites) was done
by DNA barcoding method. These results could be
explained by the findings of Sanket Tembe et al., (2014) [10]
who also done selected Sting Bugs species identified by
DNA barcoding methods and states that DNA barcode
method is very useful and quick method for the insects to be
identified at the species level [10]. Similarly, Shama Paraveen
et al., (2015) have also done molecular identification of
different life stage (Egg, different instar stages and adults)
of Tessaratoma javanica (Thun bug) by DNA barcoding
method. The first report revealed 670 bp COI gene
sequences from tissues samples of Tagasta indica in India.
Three grasshopper have been done COI gene sequences
screen from tissues samples [11]. The present findings
supported that of Muhammedali et al. (2017) [12] who have
done for 589 bp of COI gene sequence screened from
Grasshopper Conocephlus dorsalis and received accession
number KX503055 from Genebank [12]. Additional, Sanket
tembe et al., (2014) [10] who have added databases of
mitochondrial cytochrome C oxidase I (mtCOI) sequences
from the forty three species of indigenous true bugs. COI
gene sequence screen from larva stages of Samia ricini (Silk
worm) [10] and Batocera rubus larva stages parallel result
support from the observations of Ekrem et al., 2010, who
have reported that DNA barcoding allows the inclusion of
all life stages in biodiversity assessments (Ekrem etal. 2010)
[13].
Table 1: BLAST analysis of Choroedocus illustris with voucher specimens in Gene bank
S. No
Specimen Name
Total Score
Query Cover
E value
Per identification
Accession
1
Choroedocus illustris
1067
87
0.0
98.45
KY83924.1
2
Choroedocus illustris
1056
86
0.0
98.44
KY837806

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205
Fig 1: Molecular phylogentic analysis by NJ methods of edible insects of Choroedocus illustris
Table 2: BLAST analysis of Chondracris rosea with voucher specimens in Gene bank
S. No
Specimen Name
Total Score
Query Cover
E value
Per identification
Accession
1
Chondracris rosea
1067
93 %
0.0
98.62%
MK007252.1
2
Chondracris rosea
1067
99%
0.0
96.90%
GU249619.1
Fig 2: Molecular phylogentic analysis by NN methods of edible insects of Chondracris rosea
Table 3: BLAST analysis of Samia ricini with voucher specimens in Gene bank
S. No
Specimen Name
Total Score
Query Cover
E value
Per identification
Accession
1
Samia ricini
1210
99%
0.0
100%
MN120775.1
2
Samia ricini
1213
100
0.0
99.84%
AH015037
3
Samia ricini
1213
100
0.0
99.84%
AB015866.1
Fig 3: Molecular phylogentic analysis by NN methods of edible insects of Samia ricini

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Table 4: BLAST analysis of Tagasta indica with voucher specimens in Gene bank
S. No
Specimen Name
Total Score
Query Cover
E value
Per identification
Accession
1
Tagasta indica
1092
96%
0.0
95.98%
NC045930.1
Fig 4: Molecular phylogentic analysis by NJ methods of edible insects of Tagasta indica
Table 5: BLAST analysis of Odontotermes longignathus with voucher specimens in Genebank
S. No
Specimen Name
Total Score
Query Cover
E value
Per identification
Accession
1
Odontotermes longignathus
500
77%
3e-137
92.50%
MN205551.1
2
Odontotermes longignathus
500
77%
3e-137
92.50
KY224665.1
3
Odontotermes longignathus
496
72%
4e-136
92.78
KJ934560.1
Fig 5: Phylogentic analysis by NJ methods of edible insects of Odontotermes longignathus
Table 6: BLAST analysis of Tessaratoma papillosa with voucher specimens in Genebank
S. No
Specimen Name
Total Score
Query Cover
E value
Per identification
Accession
1
Tessaratoma papillosa
193
40%
1e-30
80.58
NC037742.1
2
Tessaratoma papillosa
193
40%
1e-30
80.58
AY252948.1
Fig-6: Phylogenic analysis by NJ methods of edible insects of Batocera rubus

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Table 7: BLAST analysis of Batocera rubus with voucher specimens in Gene bank
S. No
Specimen Name
Total Score
Query Cover
E value
Per identification
Accession
1
Batocera rubus
265
59
1e-66
92.47
KT314213.1
2
Batocera rubus
265
59
1e-66
92.47
KX09090.1
3
Batocera rubus
211
58
1e-50
87.57
MK689189.1
Fig 7: Neighbor joining (NJ) tree of the seven edible insects based on COI gene sequences.
Table 8: List of species name and Gene Bank accession numbers
S.No
Order / Family
Species Name
Gene Bank Accession number
1
Orthropoda/
Choroedocus illustris (Grasshopper)
MN829821
2
Acrididae
Chondracris rosea (Grasshopper)
MN829822
3
Orthoptera/ pyrgomorphidae
Tagasta indica (Short leg Grasshopper)
MN829824
4
Lepidoptera/ Saturniidae
Samia ricini (Silk worm)
MN829823
5
Isoptera/ Termitidae
Odontotermes longignathus (Wings Termite)
MT009019
6
Hemiptera/ Tessaratomidae
Tessaratoma papillosa (Sting bugs)
MT009020
7
Coleoptera/ Cerambycidae
Batocera rubus (wood borer)
MT089705
Table 9: Percentage Pair wise distances between edible insects based on COI gene sequences.
1
2
3
4
5
6
1.MT089707 Odontotermes longignathus
2.MT089705 Batocera rubus
6.76
3.MT009020 Tessaratoma papillosa
13.74
9.69
4.MN829824 Tagasta indica
14.46
17.72
9.91
5.MN829823 Samia ricini
14.06
12.22
9.60
12.41
6.MN829822 Chondracris rosea
8.55
12.47
12.08
10.15
10.43
7.MN829821 Choroedocus illustris
10.34
9.37
13.67
7.65
10.53
7.47
Table 10: Sequence composition, Summary statistics for nucleotide frequency distribution are provided in the table below.
Nucleotides
Min
Mean
Max
SE
G %
16.57
22.92
29.27
4.49
C %
16.16
17.63
19.10
1.04
A %
21.31
26.16
31.04
3.44
T %
33.26
33.27
33.28
0.01
GC %
35.67
40.46
45.43
3.45
GC % Codon pos1
45.29
46.42
47.55
0.799
GC % Codon Pos 2
40.62
41.44
424.25
.057
GC % Codon Pos 3
21.08
33.78
46.48
8.98
Table 11: List of DNA barcode of edible species
Choroedocus illustris

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Chondracris rosea
Samia ricini
Tagasta indica
Tessaratoma papillosa
Batocera rubus
Odontotermes longignathus
Conclusion
In this study, we have analyzed and documented only 7
edible insects that were collected from Nagaland, but there
about 2000 species that have been documented in India. The
comprehensive data generated from present study would be
useful in further understanding of the biodiversity of edible
insects associated with other regions of country and this
study would certainly have implication for edible insects for
development of diagnostic guide at molecular level.
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