85202026

International Journal of Research in Advent Technology, Vol.8, No.6, June 2020
E-ISSN: 2321-9637
Available online at www.ijrat.org
1
doi: 10.32622/ijrat.85202026
Abstract: Dengue disease is a mosquito vector borne
viral disease which is transmitted mostly by the Aedes
aegypti mosquito vector species. Other viral diseases
such as chikungunya, yellow fever and zika are also
caused by this vector. After feeding on a DENV-infected
person, the virus replicates in the mosquito midgut then it
distributes to other tissues. Medicinal plant species
contains wide series of phytochemicals such as primary
and secondary metabolites that produce biological
activities and defenses against mosquitoes. In the current
study, a methanol leaf extract of Aegle marmelos was
assessed for larvicidal efficacy and midgut alteration of
third instar Ae. aegypti larvae. After the larvicidal
bioassay, the probit analysis identified Lc50 in this
extract that killed the larvae at the concentration of 49
ppm. Proteomic analysis and in-silico studies revealed
that the predicted protein could disrupt the larvae midgut
because of the Aegle marmelos natural bioactive
compounds and secondary metabolites. Our approach is
to identify the inhibitor proteins that bound to midgut of
Ae. aegypti larvae after treatment with the Aegle
marmelos bioactive compounds by using computational
proteomic analysis.
Index Terms - Dengue, Aedes mosquito, MALDI,
midgut & proteome.
I. INTRODUCTION
Dengue, Dengue Shock Syndrome and Dengue
Hemorrhagic Fever (DHF) are an important vector borne
viral diseases. WHO said the prevalence of dengue has
grown-up dramatically worldwide in these decades and
the largest number of dengue cases ever reported was in
2019.
Manuscript received April, 2020; revised May 2020, and published
on June, 2020
Sugapriya Menaga Paulraj & Sakkanan Ilango, 1
Post Graduate and
Research Department of Zoology,
Maheswaran Baskaran & Geetha Paramasivam, 2
Department of
Biotechnology, Ayya Nadar Janaki Ammal College (Autonomous),
Sivakasi, India.
Joseph Sebastinraj, 3
Jamal Mohamed College (Autonomous),
Tiruchirappalli, Tamil Nadu, India.
Email: menagadurai@gmail.com
There are 2.5 billion people Worldwide are at
risk of dengue fever (Guzman et al., 2010). In India, a
sum of 136,422 cases and 132 deaths were recorded in
2019 which is the maximum number of cases when
compared to previous year. Dengue virus contains, four
serotypes are DENV1, DENV2, DENV3 and DENV4
and also several subtypes. One serotype can produce
lifelong immunity but it is merely a partial immunity
besides the other serotypes of reinfection. All these
serotypes have interaction with the host and displayed
unique features based upon its response (Ekta and Neha,
2014). The structural features of these virus comprises of
three structural proteins such as capsid, membrane and
envelope and seven (NS1, NS2A, NS2B, NS3, NS4A,
NS4B and NS5) non-structural proteins.
World's insecticide market of phytochemicals
was reported as one per cent (Ghosh et al., 2012).
Phytochemicals may help as these are relatively safe,
cheap, environment friendly, biodegradable and readily
available in all over the world. Controlling this medically
important vector is really a challenge of emerging
resistance to chemical pesticides. Medicinal plants
contain a wide variety of phytochemicals such as
alkaloids, polyines, coumarins, peptides, flavonoids,
terpenoids, polyphenolics and saponins that have
demonstrated therapeutic effect against a wide range of
viruses, their entry and replication (Idrees and Ashfaq,
2013).
The plant Aegle marmelos belongs to Rutaceae
family, also known as bael, a spiny tree. Native to India,
it is an important medicinal herb and widely used in
medicinal systems (Atul, 2012). These plants consists
various phytochemicals and are responsible for its
medicinal value. Hence this study, aims at investigating
the larvicidal efficacy of the leaves methanol extract of
Aegle marmelos on the III instar Ae. aegypti larvae.
Bradford method was used to estimate the infected gut
protein concentrations (Stephenson, 2010). Further the
plant extract that exhibits larvicidal activity was used to
study proteomic analysis of the larvae midgut and its
related proteins were predicted by computational
analysis. We have isolated the protein present in
Insilico and Proteomic Analysis of Dengue Vector
Midgut Proteins Treated by Aegle marmelos
Bioactive Compounds
Sugapriya Menaga Paulraj, Maheswaran Baskaran, Sakkanan Ilango,
Geetha Paramasivam and Joseph Sebastinraj

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doi: 10.32622/ijrat.85202026
mosquito midgut using SDS-PAGE after intoxication of
Ae. aegypti larvae with plant extract Aegle marmelos to
discover novel inhibitor proteins. In this contest,
proteomics analysis might be very useful for the
identification and development of new drug targets to
advance the insecticide resistance on midgut larvae.
The proteomic analysis was conducted to find
Ae. aegypti midgut proteins after intoxicated with the
plant extract with the help of Mass spectrometry (Whiten
et al., 2018). Our approach combined with SDS PAGE
and MALDI to identify the DENV inhibitor proteins that
interacted with the Ae. aegypti midgut.
II. MATERIAL AND METHODS
1) Plant Material Collection and Extraction
The studied plant leaves of Aegle marmelos was
freshly collected from Sivakasi, Viudhunagar District in
the state of Tamil Nadu, India. The studied plant was
identified with voucher specimen. The leaf samples were
washed and rinsed thrice with sterile water to remove the
contaminants and then shade dried at room temperature
to remove the moisture. Then, the dried samples were
made into fine powder. The fine powders were extracted
twice with 95 % methanol solvent in Soxhlet apparatus,
to obtain the phytochemicals. The extract was then
concentrated under reduced pressure in the rotary
vacuum evaporator until the solvents evaporated
completely at 450
C to get semisolid mass of crude
extracts and then freeze dried at -800
C to obtain solid
residue (George, 2008).
2) Larvae Collection & Larvicidal bioassay
The mosquito larvae (Aedes aegypti) were
collected from the Indian Council of Medical Research
(ICMR), Madurai, Tamil Nadu and India. Mosquito
culture was maintained at the temperature of 28 to 29°C,
80 to 85% relative humidity under the light: dark
photoperiod cycle of 14:10 h. The larvae were reared in
plastic tray containing tap water and fed brewer’s yeast
and dog biscuits powder in the ratio of 1:2. The water has
been changed on each alternate day.
According to the guidelines of World Health
Organizationm, the larvicidal bioassay was performed on
third instar larvae (WHO, 2005). To 150ml of
de-chlorinated tap water taken in a beaker appropriate
volume of 1% stock solution of Aegle marmelos
methanol extract fractions were added and mixed to
obtain different concentrations. Third instar larvae of
Ae.aegypti in 25 numbers were released to each
concentration and provided with larval feed and test was
conducted in five replicates. There are two controls were
maintained (one with 150ml water alone and the other
with 150ml of water containing maximum volume of
acetone in the test sample). Primary larvicidal screening
was carried out with 100, 500 & 1000 ppm
concentrations to identify the active range for the further
bioassay with the extract. Afterwards of the preliminary
analysis, a test range of 50,100, 150, 200 and 250 ppm
were fixed to identify the Lc50 and Lc90 values and
outright of per cent mortality recorded after 24 and 48hrs
of exposure.
3) Statistical Data analysis
Considering the percentage mortality of the
larvae after 24 and 48hrs in different concentrations,
Lc50 of the test fractions, we calculated using probit
analysis and IBM SPSS Statistics 23 software. Per cent
mortality was calculated based on the Abbott’s formula
(Abbott, 2010) and the statistical analysis was carried out
based on the log-dose response (Finney, 1971). The
significant difference in Lc50, Lc90, and 95% Fiducial
limits and also the slope values are calculated.
4) Preparation of midgut protein extracts
In this study, the proteomic study on the third
instar larvae of Aedes aegypti mosquito vector was
carried out after intoxication with various concentration
of Aegle marmelos methanol extract for 24 and 48hr
period. After 48 hr the dead larvae from the treatment
were collected. For the midgut sample preparation of
SDS, larvae midguts were collected under a microscope
by dissection using ER Buffer consist of protease
inhibitor in the conc. of 1 μL/mL (Sigma P9599) (English
and Reddy, 1989). The midgut was completely separated
from other mosquito parts by standard procedure (Butler
and Deana, 2014). Centrifugation of midgut sample was
done at 12,000 rpm for 15 mins at ice cold condition. In
to new eppendorf tubes the supernatants were collected
and stored in -80°C till further electrophoretic analysis.
The photographs of both control and tested larvae midgut
was captured in the light microscope attached with a
digital camera. Protein concentration in the midgut was
estimated by Bradford assay which was compared with
standard BSA protein (Bradford, 1976).
5) Protein profiling
SDS-PAGE analysis of both the control and
treated Aedes midgut larvae extract was performed by
using the standard protein isolation (Laemmli, 1970). For
sample preparation, 50 mg of protein extract was mixed
with sample loading buffer, kept in water bath at 60 to
65°C for 2min and electrophoresed on 15% of separating
gel and 4% stacking gel mix (Pandiarajan, et al., 2011).
The SDS – PAGE gel was stained with Coomassie
Brilliant Blue R-250 for six hour. Washed the gel twice

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with the double distilled water and kept in a destaining
solution for the appearance of bands in the gel.
6) In-gel protein digestion
The identified band in SDS-PAGE gel was
excised; destaining was carried out with methanol and
incubated with 200mM ammonium bicarbonate.
Dehydration of the gel was done with acetonitrile
solution, dried under vacuum followed by rehydration
and tryptic digestion.
7) Protein Identification by (MALDI-TOF) &
Bioinformatics analyses
In-gel Trypsin digestion and Mass spectrometry
analysis was performed in the Molecular Biophysics
Unit, IISC, Bangalore, India. The m/z ratio peaks
obtained from the MALDI MS analysis was subjected to
online MASCOT search software tool (Matrix Science
Inc., Boston, U.S.A.) to obtain the peptide sequences.
The search parameters were set as following, the fixed
modification is set to carbamidomethyl C (Cysteine) and
variable modification is set to oxidized M (Methionine),
missed trypsin cleavage site is set to 1.
In the mascot search engine, predicted protein should
significant with a p-value less than 0.05. Identified
sequences were searched against with Drosophila
organisms in Swissprot database sequence for the
functions detection. Vectorbase database and BLAST
server was used to validate the protein and the protein
interactions were predicted by using STRING database.
III. RESULTS AND DISCUSSION
The primary phytochemical screening of the
methanolic extract of the Aegle marmelos leaves showed
the presence of various primary and secondary
metabolites in the extract such as protein, phenols,
tannins, steroids and titerpene compounds. The current
study indicates that the most active ingredient isolated
can be act as a best larvicidal agent against the third instar
larvae of Aedes aegypti and also showed development
distortion. This insect feed on these secondary
metabolites encountering toxic substances with
non-specific effects on molecular targets and in turn
affects physiology in many different ways at different
receptor sites (Ghosh et al., 2012).. High level mortality
was noticed in the Aegle marmelos and which may be due
to the phytochemicals in the extracts which arrests the
metabolic activities of larvae. The larvicidal efficacy of
A. marmelos against the Aedes aegypti third instar larvae
was fixed at 50, 100, 150, 200 and 250 ppm. The highest
toxicity in the bioactive compounds observed at 24 hours
Lc50 values are 59 and 49 and at 48 hours Lc90 values
are 114ppm and 108 ppm respectively (Table 1).
Table 1: Larvicidal Toxicity of Aegle marmelos methanol leaf Extract The Dengue Vector, Aedes aegypti
Per cent Mortality (ppm) Lc50
(ppm)
95%
(LCL-UCL)
Lc90
(ppm)
95%
(LCL-UCL)
Slope ±SE χ2
(df=3) Reg. equation
50 100 150 200 250
24hr 08 32 60 88 96 59 50.56 - 68.11 114 95.95-150.97 4.504±.682 2.23* y = 0.928x - 12.8
48hr 20 44 68 88 100 49 39.68 - 57.39 108 88.11-150.59 3.711±.592 3.82* y = 0.816x + 2.8
Control-Nil mortality, UCL & LCL - Upper & Lower confidence Limit, X2
- Chi-square value, df - degrees of freedom, *Significant at P < 0.05 level.
No significant mortality for control assays. The
methanol leaf extract of Aegle marmelos treated larvae
revealed the damage disruption around the midgut The
midgut region was completely disrupted with shrunken
bodies. Thus this extract obviously led to disruptions in
growth of Ae. aegypti third instar larvae. A. pinnata
might have contributed in body effects of larvae and their
study indicated that the potential application of this plant
phytochemicals as mosquito larvicidal agent (Zulkrnin et
al., 2018). The S. terebinthifolius plant derived bioactive
compounds have been reported for larvicidal effects in
the mosquito midgut (Procópio et al., 2015), similar to
the Aegle marmelos leaf extract. The damage to midgut
cells of Ae. aegypti larvae caused by the Aegle marmelos
methanol leaf extract may have digestive dysfunction in
the larval midgut, and distortion in the growth of larval
development.
Primarily, the A. marmelos treated larvae
midgut protein sample was quantified using Bradford
method BSA as standard (Table 2) and the quantification
of protein was plotted in the Fig.1. Ae. aegypti midgut
protein of larvae was analyzed quantitatively by using
Colorimeter and the OD value was measured at 595nm.
The present investigation showed that the protein
concentration was found to vary in the treated and
untreated Ae. aegypti midgut proteins. The mean protein
concentration was effectively recorded for both untreated
larvae (1237.2μg/mL) and Aegle marmelos leaf extract
treated larvae (1078.6μg/mL). In the present study, total
protein content was significantly higher in untreated
larvae than the Aegle marmelos leaf extract treated. The
results showed that concentration of protein seen higher
in untreated larvae reflects a high rate of protein synthesis
in the control. Thus, the present findings suggest that
there is a disruption taking within the midgut protein

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synthesis. Ratten (2010) reviewed the mode of action of
secondary metabolites on insect body and documented
several physiological disruptions.
Table 2 Quantification of Aegle marmelos treated
Ae. aegypti midgut protein by Bradford’s method
Test
tube
Conc.
µg/ml
BSA
(µl)
Distilled
H20 (µl)
OD
(595 nm)
S1 10 100 1900 Bradford
Reagent3ml
0.035
S2 20 200 1800 0.662
S3 30 300 1700 0.695
S4 40 400 1600 1.466
S5 50 500 1500 1.864
T1 1237.2 100 1900 1.116
T2 1078.6 100 1900 0.978
S1 to, S5 – Standard protein BSA; T1– Untreated; T2– Treated
with Aegle marmelos; Control: 5ml Bradford’s reagent;
Fig. 1 Aegle marmelos treated Ae. aegypti third instar
larvae midgut protein quantified by Bradford’s
method
In this study, the proteomic analysis of Aedes
aegypti third instar larvae midgut was evaluated after
treated with various concentrations of Aegle marmelos
leaf extract that kills the larvae at 48hrs of incubation. To
know the peptide responsible for the larvae structure
modification, we decided to separate the midgut protein
extract in 15% SDS-PAGE. The size of the band obtained
in the gel was between 20 to 66 kDa in range. This result
is corroborated with (Abbas et al., 2013), they reported
that six protein bands were identified ranged from 16.6 to
75 kDa in molecular weight.
Previous studies of midgut epithelial cell
structure and function have revealed some information
on the cell types defined in D. melanogaster are also
established in Ae. aegypti (Fernandes et al., 2014). The
pathway induced in midgut of DENV2-primered
mosquitoes were identified as Notch transcription
(Serrato et al., 2018) and it is an important defensive
mechanism against dengue virus infection.The 57 kDa
protein possesses dengue viral binding protein property
and it has proven by previous studies in the Aedes
midgut. The DS3 strains of Ae. aegypti total purified
protein resolved in SDS showed that they were in the
range of 57 and 67 kDa (Muñoz et al., 2013). Based on
this, we also focused on the ~57 kDa fragment was eluted
from the SDS-PAGE gel (Fig. 2) exposed to MALDI-MS
analysis followed by tryptic digestion and the
chromatogram results displayed in the Fig. 3.
Fig. 2: Protein profile of Aegle marmelos methanol
leaf extracts treated and untreated Aedes aegypti
Midgut.
Mosquito midgut proteins were separated by SDS-PAGE
with CBB stained gel. On the right side, shows molecular
weights of the proteins and the left shows marker protein.
Fig. 3 MALDI MS tryptic digested peptide of Aedes
aeygpti midgut protein treated by Aegle marmelos
plant extract and the peptide masses were used for
protein database searches
The peptide masses list were searched against
the protein sequence database, and predicted protein as
the product of gene Cyp313a1 of cytochrome P450
sequence of Drosophila melanogaster which is a 56 kDa
protein (Fig.4). The experimentally determined
seventeen masses cover only 17 per cent of the protein
sequence. Table 3 shows the observed and calculated
masses of peptide and their sequence assignments.
Among the 17 peptide masses identified, 8 peptides
covered in the predicted protein Drosophila sequence.
662.354
832.374
1567.842
1479.894
582.386
634.321
752.406
1724.957
2045.171
550.663
684.340
1439.913
522.612
1881.049
854.358
927.564
804.341
678.353
1589.821
1501.859
1163.696
2512.347
1537.889
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Intens.[a.u.]
600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600
m/z

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The obtained sequence was compared with the
non-redundant protein sequence database specified
with the Aedes aegypti organism using BLASTP
software.
The results confirmed that the predicted
protein belongs to Aedes aegypti cytochrome P450
sequence with 42 per cent identity Fig. 5.1 to 5.3. Further,
the longest matched peptide as query sequence in fasta
format (NCIGSKYAMMSSKFALCR) was subjected to
a Vectorbase BLASTP tool for similar sequence
identification from Aedes species sequences stored. The
analysis was based on Vectorbase database Ae. aegypti
sequences and related similarity. Interestingly the query
is also perfectly aligned with the eight cytochrome P450
peptides of Aedes aegypti sequences with more than 55
per cent identity and score. Among these, four peptide
sequences AAEL012772-PA, AAEL017136-PB,
AAEL012766-PA and AAEL003748-PA showed
biologically significant result once viewed their E-values
(Table 4). By mascot searches, Muñoz et al. (2013)
identified the 57 – 67 kDa proteins were enolase,
beta-ARK, translation elongation factor EF-1 alpha/ Tu
and cadherin. Peptide Mass Fingerprint data analysis
couldn’t show the same mass of different peptides (Sechi
and Chait, 1998; He et al., 2008).
But the protein identified by this current study
does not match with any other Aedes aegypti midgut
proteins studied so far this means that the Aegle
marmelos plant leaf extract might altered or influence the
protein present in the midgut membrane. The total mass
chromatogram of Aedes aegypti third instar larvae treated
with Aegle marmelos plant leaf extract midgut proteins
obtained, after that MALDI/MS spectrum of mascot
search identified four upregulated proteins with
Drosophila melanogaster are AT-rich binding protein,
Bomanin, Accessory gland-specific peptide and
Eukaryotic translation initiation factor at peaks 662.354,
832.374, 1479.894 & 1567.842 m/z respectively (Table
5). Structural and functional networks of protein-protein
interactions of identified proteins were analyzed and
predicted using STRING 11 analysis software. STRING
analysis can be used to understand the cellular machinery
at the system level and this information can be implicated
in modeling, annotation and pathway studies (Fig. 6).
Fig. 4 MALDI peptide masses analyzed by Mascot server against Swissprot database shows that the predicted
protein has 17% sequence coverage with Drosophila Cytochrome P450; Matched peptide sequences shown in bold red
Input: BLAST Query Sequence in Fasta format retrieved from Swissprot Database
>sp|Q9VFJ0|CA131_DROME Probable cytochrome P450 OS=Drosophila melanogaster GN=Cyp313a1
MLTINLLLAVGALFWIYFLWSRRRLYFLMLKIPGPIGLPILGSSLENIITYKRKLSFRTKYLNKYGSTILTWMGPVPFIVTRDPKVVEDIFSSPD
CHNKSQHIVNAITSCMGNGLLGKQDPHWLDRRKHFNPSFKQDLLLSFFHIFDAETKVLMNLLDTYVDKGEIDVVPEMLRWSFKIAAQTT
MGSEVKHDEHFKNGSLVESFESLISHSTLNILMPLVQNRMISKICGYDKLRADNFSRIQKMLDNVVNKKVNPLPKTDSDPESNIVINRAM
ELYRKGDITYMDVKSECCIMIAAGYDTSALTVYHALFLLANHPEHQEAVFEELNGVFPDAGHFGITYPDMQKLDYLERVIKETLRLIPAIPIT
ARETKNDVRLSNGVLIPKGVVIGIDMFHTHRNPEVWGPDADNFNPDNFLAENMEQKHPYAYIPFARGKRNCIGSKYAMMSSKFALCRI
LRNYKISTSTLYKDLVYVDNMTMKLAEYPRLKLQRRG

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Table 3: Molecular Masses for Tryptic Peptides Identified from MALDI Search against Mascot server for the Aedes
aegypti Midgut protein Treated with Aegle marmelos plant extract
Mass (Dab
)
Seq.
position
Observed Expected Calculated Start End M Peptide Sequencea
522.612 521.6047 521.2962 55 58 0 K.LSFR.T
568.172 567.1647 566.2853 175 178 0 R.WSFK.I;K.IAAQTTMc
GSEVKHDEHFK.N
650.079 649.0717 649.3911 54 58 1 R.KLSFR.T
651.298 650.2907 649.3911 54 58 1 R.KLSFR.T
666.075 665.0677 665.3319 450 454 0 K.FALCR.I
678.353 677.3457 677.3166 437 442 0 R.NCIGSK.Y
752.406 751.3987 750.4388 55 60 1 K.LSFRTK.Y
754.396 753.3887 754.332 228 233 0 K.ICGYDK.L
832.374 831.3667 832.3459 443 449 0 K.YAMMc
SSK.F
861.133 860.1257 860.4352 370 376 1 R.ETKNDVR.L
876.344 875.3367 875.429 129 135 0 K.HFNPSFK.Q
927.564 926.5567 926.5299 25 31 0 R.LYFLMc
LK.I
1439.913 1438.9057 1438.7378 152 163 0 K.VLMc
NLLDTYVDK.G
1479.894 1478.8867 1479.6672 443 454 1 K.YAMMc
SSKFALCR.I
2045.171 2044.1637 2043.9684 179 196 1 K.IAAQTTMc
GSEVKHDEHFK.N
a The peptides cover 17% sequence coverage and 17 peptides were predicted. The peptide mass accuracy (better than ±1.2 Da) and identify the protein in the
Swissprot protein sequence database; b Monoisotopic, neutral masses; c Met is oxidized.; M - Missed cleavage
Table 4 : Similarity Search of Cyt P450 mascot predicted protein against the Aedes aegypti
Sequence database at Vectorbase (Aegle marmelos Treated)
Blast Query for Vectorbase :
> Cyt P450 peptide(fasta)
NCIGSKYAMMSSKFALCR
Similar Peptide Gene E-value Score Identity
AAEL012772-PA CYP325G3 0.023 68 70.60%
AAEL000320-PA CYP325T1 0.36 59 68.80%
AAEL017136-PB CYP325V1 0.053 65 66.70%
AAEL012766-PA CYP325G2 0.096 63 64.70%
AAEL006044-PA CYP325Q1 0.28 60 62.50%
AAEL003748-PA CYP9AE1 0.045 66 61.10%
AAEL005775-PA CYP325R1 0.53 57 56.30%
AAEL007812-PA CYP4H32 0.46 58 55.60%
Fig. 5.1 Setting up of BLASTP search of the protein sequence predicted by MALDI Mascot search against the NCBI
Aedes Aegypti (taxid:7159) organism Non-redundant Protein sequence database

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Fig. 5.2 BLASTP Output: a) Query details; b) A Graphical overview of all the similar sequences;c) List of BLASTP
hits that produce significant alignments with our query sequence and contains links to the NCBI databases
Fig. 5.3 a) BLASTP alignment between the peptide sequence predicted (Query) with the Aedes aegypti Cytochrome
P450 sequence (Sbjct); b) Aedes aegypti Cytochrome P450 retrieved from NCBI Sequence database
Table 5: Peptides identified as up-regulated in midgut of Aegle marmelos treated Ae. aegypti larvae
Peptide mass
(Da)
Peptide sequence
Similar Sequence
Drosophila melanogaster
Score
Sequence
coverage
pI
662.354 FKYKSRMELHRVVHSKER AT-rich binding protein 57 5% 6.75
832.374 K.VCNIRGD
Bomanin-068 - immune-induced
peptide toll signalling
19 17% 7.82
1479.894 R.KPTKFPIPSPNPR.D Accessory gland-specific peptide 19 23% 10.1
1567.842 K.GNDDDIQDGLVHIR.I EK Translation initiation factor 17 12% 6.82

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Fig. 6 STRING PPI network of Cytochrome P450 protein
identified in the of Aegle marmelos Treated Ae. Aegypti
larvae midgut. ID numbers denote sequence accession
numbers in the Swissprot. Interaction networks are
shown in the confidence view with color lines.
Green colour depicts neighbourhood; Red colour: Gene fusion; Pink
colour:Experiments; Light green colour: Text mining; Blue
colour:Cooccurrence; Dark blue colour: Coexpression; Purple
colour:Homology; and circle nodes indicated different proteins. Interaction
networks are shown in evidence view
The available association network was studied for
the protein, i.e. AAEL012772-PA (Cytochrome P450)
matched with monooxygenase and Heme peroxidase has
homologs with Aedes, Culex and Anopheles species with the
p-value of 0.566. Detailed functional analysis of these
proteins was carried out using GO and other bioinformatics
algorithms. The biological process of the identified peptide
involved in oxidation-reduction process and the molecular
function depicts the monooxygenase activity.
The xenobiotics reduction or oxidation process of
cytochrome P450 monooxygenase (CYP) gene was studied
(Saavedra et al., 2014) and it’s represented in Aedes aegypti
detoxification genes. P450s are involved in drug metabolism
(Werck and Feyereisen, 2000) and insecticide resistance. The
pyrethroid influence in CYP4 expression may cause adverse
effect in A. albopictus (Avicor et al., 2014). Ilango et al.
(2007) reported that C-methylated flavone from C.
lanceolatus is responsible for the larvicidal activity. The
active compounds identified will play a role in
developmental duration of the mosqutoes such as C.
quinquefasciatus and A. stephensi (Ilango et al., 2016).
IV. CONCLUSION
At present, there is no potent medicine for dengue
making the laboratory oriented investigation system is an
important need and an essential tool to control the Aedes
aegypti mosquito vector control and also environmental
friendly. Dengue virus replication mainly occurs in mosquito
midgut and plays a mail role in the transmission of this
disease to humans. Ecofriendly, cost effective control of this
mosquito by natural bioactive compound is necessary.
In-silico proteomic analysis is essential for developing novel
vector control strategies, and to identify dengue viral protein
receptors on the midgut which will help in future research. In
conclusion, our study revealed that Aegle marmelos leaf
extract had potencial application as larvicide.
The computational proteomic analysis highlights
the potential effect of Aegle marmelos leaf extract against the
Ae. aegypti third instar larvae and exhibited an induction of
structural disorganization in the midgut epithelial cells. The
identified phytochemicals and their ability to control this
vector species is either by their insecticidal property or by
growth disruption. These phytoextracts could be purified
further and used as biological insecticides instead of
synthetic chemicals, which currently is the major means of
mosquito vector control and also environmental friendly.
Further studies will be indispensable to validate the
pharmacological properties and make potent drug at cheap
cost from of natural phytochemicals.
CONFLICT OF INTEREST
The authors declare that they have no conflict of
interests.
ACKNOWLEDGMENT
The authors magnanimously thank TNSCST, Tamil
nadu for providing financial support to carry out this work
under SPS Scheme. We also thank the Management and
Principal, Ayya Nadar Janaki Ammal College, Sivakasi for
provided great support by making the availability of various
institutional facilities.
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AUTHORS PROFILE
Sugapriya Menaga Paulraj, M.Sc. M.Phil., PGDCA,
ADBI is having fourteen years of teaching experience
and currently working as Assistant Professor in the
Department of Biotechnology and Bioinformatics in
Ayya Nadar Janaki Ammal College [Autonomous,
CPE, Affiliated to MKU, Re-accredited (3rd
Cycle) with A grade with CGPA
3.67 out of 4.0 by NAAC, DBT STAR College] in Sivakasi, Virudhuunagar
district. The author received four TNSCST Projects from the Government of
Tamil Nadu and organized Workshops and Seminars on Bioinformatics and
Proteomics.

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