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Transcriptome Profiling and In Silico Analysis of the Antimicrobial Peptides of the Grasshopper Oxya chinensis sinuosa


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Antimicrobial peptides/proteins (AMPs) are present in all types of organisms, from microbes and plants to vertebrates and invertebrates such as insects. The grasshopper Oxya chinensis sinuosa is an insect species that is widely consumed around the world for its broad medicinal value. However, the lack of available genetic information for this species is an obstacle to understanding the full potential of its AMPs. Analysis of the O. chinensis sinuosa transcriptome and expression profile is essential for extending the available genetic information resources. In this study, we determined the whole body transcriptome of O. chinensis sinuosa and analyzed the potential AMPs induced by bacterial immunization. A high-throughput RNA-seq approach generated 94,348 contigs and 66,555 unigenes. Of these unigenes, 36,032 (54.14%) matched known proteins in the NCBI database in a BLAST search. Functional analysis demonstrated that 38,219 unigenes were clustered into 5,499 Gene Ontology (GO) terms. In addition, 26 cDNAs encoding novel AMPs were identified by an in silico approach using public databases. Our transcriptome data set and AMP profile greatly improves our understanding of O. chinensis sinuosa genetics and provides a huge number of gene sequences for further study, including genes of known importance and genes of unknown function.
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November 2016
Vol. 26
No. 11
J. Microbiol. Biotechnol. (2016), 26(11), 1863–1870
Research Article
Transcriptome Profiling and In Silico Analysis of the Antimicrobial
Peptides of the Grasshopper Oxya chinensis sinuosa
In-Woo Kim
, Kesavan Markkandan
, Joon Ha Lee
, Sathiyamoorthy Subramaniyam
, Seungil Yoo
Junhyung Park
*, and Jae Sam Hwang
Department of Agricultural Biology, National Institute of Agricultural Sciences, Rural Development Administration, Wanju 55365,
Republic of Korea
Theragen ETEX Bio Institute, Theragen Etex Inc., Suwon 16229, Republic of Korea
Oxya chinensis sinuosa
is a grasshopper species belonging
to the phylum Arthropoda (Order: Orthoptera; Family:
Acrididae; subfamily: Oxyinae) that is edible and widely
consumed. There are approximately 1,900 edible insects,
and among these, insects belonging to the Orthoptera are
the fourth most commonly consumed at its mature stage [3,
19]. Orthopteran insects are also commonly used for
entomotherapy [5], and are used to treat various human
ailments, including enuresis in women, scorpion stings,
anemia, violent headaches, foot inflammation, fertility,
hypertension, asthma, stroke, and skin diseases [7]. These
conditions are ameliorated by the combination of
biochemicals, including proteins, minerals, and fatty acids,
that are present in insect hemocytes and fat body mass.
Antimicrobial peptides (AMPs) are small peptides/
proteins (~100 aa) that are secreted/triggered by the host
innate immune system in response to external microbial
infection. AMPs are important components of the host
defense system in all invertebrates. In addition, AMPs are
considered as an alternative to conventional antibiotics
[18]. Recently, AMPs have been predicted in insect
transcriptomes, and these transcriptome profiles revealed
that defensins, cecropins, and attacins are widely distributed
in insects [15]. In the genomic era, most of the bases present
Received: August 11, 2016
Revised: August 19, 2016
Accepted: August 30, 2016
First published online
September 2, 2016
Corresponding authors
Phone: +82-63-238-2974;
Fax: +82-63-238-3833;
Phone: +82-31-888-9318;
Fax: +82-31-888-9335;
E-mail: junhyung.park@
These authors contributed
equally to this work.
upplementary data for this
paper are available on-line only at
pISSN 1017-7825, eISSN 1738-8872
2016 by
The Korean Society for Microbiology
and Biotechnology
Antimicrobial peptides/proteins (AMPs) are present in all types of organisms, from microbes
and plants to vertebrates and invertebrates such as insects. The grasshopper
Oxya chinensis
is an insect species that is widely consumed around the world for its broad medicinal
value. However, the lack of available genetic information for this species is an obstacle to
understanding the full potential of its AMPs. Analysis of the
O. chinensis sinuosa
and expression profile is essential for extending the available genetic information resources. In
this study, we determined the whole-body transcriptome of
O. chinensis sinuosa
and analyzed
the potential AMPs induced by bacterial immunization. A high-throughput RNA-Seq
approach generated 94,348 contigs and 66,555 unigenes. Of these unigenes, 36,032 (54.14%)
matched known proteins in the NCBI database in a BLAST search. Functional analysis
demonstrated that 38,219 unigenes were clustered into 5,499 gene ontology terms. In addition,
26 cDNAs encoding novel AMPs were identified by an in silico approach using public
databases. Our transcriptome dataset and AMP profile greatly improve our understanding of
O. chinensis sinuosa
genetics and provide a huge number of gene sequences for further study,
including genes of known importance and genes of unknown function.
Oxya chinensis
, transcriptome, antimicrobial peptides, insect-derived
1864 Kim et al.
J. Microbiol. Biotechnol.
in cells are sequenced by next-generation sequencing (NGS)
technologies and annotated using bioinformatics methods
[10]. To uncover the hidden benefits of insects in ecology
and human health, the insect research community has
collectively undertaken two molecular meta-data projects,
the 1K Insect Transcriptome Evolution (1KITE) and 5,000
Insect Genome Project (i5k) [17]. The largest sequenced
insect genome (~6 Gb) belongs to an Orthopteran (
) [23]. Currently, only a few
studies have been published, on heavy metal stresses and
nutrient supplements. Specifically,
Oxya hyla hyla
Oxya chinensis
have been studied as potential heavy metal
bioindicators in industrial agricultural fields [1, 3, 27], and
O. fuscovittata
O. hyla hyla
have been
studied as
nutrient supplements for fish [9] and poultry birds [19].
O. chinensis sinuosa
has long been used as a food
source for the South Korean population and it possesses
antioxidant and antimicrobial properties [9], we sought to
examine its transcriptome.
Therefore, in the present study,
we set out to obtain high-throughput data of the
O. chinensis
transcriptome using Illumina-based NGS. In addition,
we obtained the first transcriptome data for a member of
the Oxyinae subfamily, and predicted the AMPs as
preliminary data to support future molecular studies.
Materials and Methods
Insects and Treatment
O. chinensis sinuosa
were obtained from Jeonnam
Agricultural Research & Extension Services, South Korea. For
immunization, each grasshopper was injected with log phase
Escherichia coli
(4 × 10 colony forming units) suspended in 10
l of
autoclaved 10 mM sodium phosphate buffer (pH 7.4). Non-
immunized and immunized grasshoppers were reared at 25°C ±
1°C for 18 h before total RNA isolation.
E. coli
Strain and Growth Conditions
E. coli
KACC 13821 (ATCC 11775) was purchased from the
Korean Agricultural Culture Collection (KACC). Bacteria were
cultivated overnight in tryptic soy broth (TSB; Difco, USA) in a
shaking incubator (200 rpm, 37°C) until the stationary phase.
Then, the bacteria were cultivated in fresh TSB medium under the
same conditions until log phase (for 3 h). Bacteria were stored in
15% glycerol at −70°C until use.
Library Preparation and Sequencing
Total RNA was extracted from each sample with the RNeasy
Lipid Tissue Kit (Qiagen, Germany) according to the manufacturer’s
instructions after treatment with RNase-free DNase I (Qiagen) to
eliminate genomic DNA. The concentration and integrity of the
RNA were assessed with a Thermo Scientific NanoDrop 8000
Spectrophotometer and Agilent 2100 Bioanalyzer, respectively
(Agilent Technologies, USA). RNA with an OD
1.8 and an
RNA integrity number
7.0 was used in subsequent experiments.
Equal amounts of high-quality RNA from tissues were then
pooled for cDNA synthesis and sequencing. The cDNA library
was prepared with ~2.5
g of total RNA according to the Illumina
TrueSeq RNA Sample Preparation Kit (Illumina) protocol. The
library was then amplified, and the final library yielded ~400 ng
of cDNA, with an average fragment size of ~300 bp. The resulting
cDNA libraries (for all four samples) were then paired-end
sequenced (2 × 150 bp) with NextSeq (Illumina).
Preprocessing, De Novo Assembly, and Annotations
Paired-end sequence files from four samples (Fastq: R1, R2)
were obtained and subjected to processing using Trimmomatic-
0.32, with the following parameter settings: leading, 5; trailing, 5;
sliding window, 4:15; and minlen, 30. Processed sequences were
checked for bacterial contamination using an in-house bacterial
database that was constructed from NCBI GenBank. Preprocessed
clean reads were mapped to the bacterial database using Bowtie2
with default parameters, and the mapped reads and their
respective pairs were removed. From this point on, these
sequences are called preprocessed. Total preprocessed sequences
from NextSeq were pooled and assembled with Trinity assembler
ver. 2.0.6 [11] using default values. To remove redundant
sequences, CD-HIT-EST [13] was used, with a 95% sequence
similarity cutoff. Finally, transcripts greater than 500 bp were
selected for inclusion in the reference transcriptome. The
reference transcriptomes were subjected to functional annotation
using BLASTX mapping (e-value cutoff 1e ) against the UniProt
KB (Metazoa) database, and the Gene Ontology (GO) terms and
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway
maps were determined using Blast2GO [6]. GO annotations were
classified using the WEGO Web server [25].
Identification of Differentially Expressed genes
Differentially expressed genes (DEGs) were measured by
counting tags in immunized samples and comparing the number
with that in non-immunized
O. chinensis sinuosa
samples and were
normalized using the RNA Sequence Expected Maximization
method [16]. Reads from all samples were mapped to the
reference transcriptome, and differential expression was assessed
using Trinity utility scripts (
and as instructed (http:
// From the edgeR statistics files,
regulated transcripts across libraries were filtered with default
parameters (
., 1
log (FC), FDR < 0.01) using Python scripts. To
determine the differential expression pattern from the GenBank
datasets, the same procedures were followed.
Antimicrobial Peptide Prediction and Classification
The deduced amino acid sequences were subjected to AMP
prediction using a modified bioinformatics strategy. Peptide
Antimicrobial Peptides in the Grasshopper Oxya chinensis sinuosa 1865
November 2016
Vol. 26
No. 11
characteristics such as (i) molecular propensity (physicochemical
properties) (ii) aggregation propensity (in vitro and in vivo), and
(iii) AMP prediction were assessed by using a predefined
bioinformatics strategy with the given parameters [24]. In
addition, the allergenic propensity of the peptides was predicted
using Allerdictor [8, 22]. Finally, the AMPs were mapped with the
CAMP database [22] and classified as novel or known. To classify
the AMPs as novel, sequences were matched to the CAMP
database with two programs, PatMatch (no mismatch) for
20 amino acids [19] and BLASTP (1E-05) for sequences
20 amino acids. The BLAST results were filtered using a
similarity score
90. Similar sequences, using the given cutoff,
were considered as known AMPs, and the others were considered
novel AMPs.
Data Deposition
The raw reads from
O. chinensis sinuosa
were submitted to the
NCBI Sequence Read Archive under Accession No. SRP080832.
Results and Discussion
Transcriptome Sequencing and Assembly
The de novo transcriptome of
O. chinensis sinuosa
processed as depicted in Fig. 1. To obtain a more
comprehensive view of the transcriptome, pooled cDNA
samples from the non-immunized control and
E. coli
O. chinensis sinuosa
were isolated. Illumina
sequencing produced an average of 40,809,315 and
40,232,081 clean reads, representing 5.52 Gb and 5.41 Gb of
the non-immunized control and immunized samples,
respectively (Table 1). The quality of the transcriptome
sequence was high, with a Q percentage (percentage of
sequences with a sequencing error rate lower than 1%) of
85.74% and 45.16% G+C. These short reads were assembled
into 94,348 contigs with a mean length of 1,439 bp. We
obtained 66,555 unigenes, with a mean size of 1,286 bp
(range, 500–26,614 bp), implying that the pipeline used to
assemble the
O. chinensis sinuosa
transcriptome libraries
Fig. 1.
Workflow of the transcriptome assembly and analysis
Oxya chinensis sinuosa
high-throughput sequencing data.
Tab le 1 .
Sequencing, assembly, and annotation statistics.
A. Sequencing
Control (non-immunized) Immunized
Total sequenced bases 13,720,373,521 100% 13,550,796,905 100%
Total preprocessed bases 11,053,358,720 81% 10,834,378,027 80%
B. Assembly
Contigs Bases
Trinity 94,383 100% 135,864,252 100%
CD-HIT-EST 64,047 67.8% 82,380,545 60.6%
C. Annotation
No. of Contigs %
No. of hits 30,523 45.6%
BLAST 36,032 54.1%
Gene Ontology 18,991 29.6%
KEGG 3,115 4.8%
1866 Kim et al.
J. Microbiol. Biotechnol.
was satisfactory (Fig. 1). Of these unigenes, 28,049 (42.14%)
were longer than 1,000 bp. BLAST searching matched
36,032 unigenes (54.14%) to known genes, suggesting that
the assembly quality was high (Table 1). The remaining
unigenes could not be matched to any known gene, as
there is currently no genome information available for
O. chinensis sinuosa
. Recently, the transcriptome profiles of
some insect species in the Acrididae family, including
Schistocerca gregaria
Locusta migratoria
, were investigated using cDNA libraries [2, 4, 14].
Fig. 2.
Annotation of the
Oxya chinensis sinuosa
) Histogram of the GO classifications. The
O. chinensis sinuosa
transcriptome was annotated in three ontology categories: “Biological Processes,”
“Cellular Component,” and “Molecular Function.” (
) Species distribution of the BLASTX matches to transcriptome unigenes against the nr
protein database (cutoff value E < 10 ) and the proportion of matches in each species.
Antimicrobial Peptides in the Grasshopper Oxya chinensis sinuosa 1867
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In addition, the antennal transcriptome of the odorant-
binding proteins in the grasshopper
Oedaleus asiaticus
also been studied [26]. However, there has been no
published research on bacteria-immunized grasshoppers.
The current transcriptome sequencing of
O. chinensis sinuosa
provided significant amounts of information (66,555 unigenes)
compared with that available for other grasshoppers, revealing
more detailed genetic and gene expression data that were
produced using whole-body transcriptome sequencing.
Annotation of Predicted Proteins
To determine the putative annotations of the reference
transcripts, distinct sequences were first searched by
BLASTX against the GenBank non-redundant protein
database, with an E-value cutoff of 10 using Blast2GO, as
described in the Materials and Methods section. A total of
36,032 distinct sequences (54.14% of the unigenes) were
matched to known genes that encode functional proteins.
The percentage of unigenes matching known proteins was
much higher than those reported in the whole-body
transcriptomes of
S. gregaria
L. migratoria
[2, 4]. Of the
total unigenes, almost half (30,523) shared no significant
similarity to known genes; therefore, these may be novel or
fast-evolving sequences. Studying these transcripts will
provide information about insects in the family Acrididae.
In addition, our results will also be useful for future
studies, such as the cloning and characterization of
O. chinensis sinuosa
genes. In the functional annotation,
5,499 GO terms were assigned, which were subsequently
categorized into three level 2 category observations;
biological processes (13,736), cellular component (8,834),
and molecular function (15,649) (Fig. 2A). Within the
biological processes category, genes encoding metabolic
processes (16.8%) and cellular processes (16.7%) were the
most enriched. Proteins related to cells (14.4%) and cell
part (14.3%) were enriched in the cellular component
category. With regard to the category of molecular
function, catalytic (27.0%) and binding (26.6%) were the
most highly represented categories. It is not very surprising
that numerous sequences were classified into every GO
category. These are more general GO terms that comprise
the basic processes required to maintain a living organism.
The top BLASTX hit showed that
O. chinensis sinuosa
highly homologous to
Zootermopsis nevadensis
followed by
Acyrthosiphon pisum
O. chinensis sinuosa
shared only 2% homology with
Locusta migratoria
(Fig. 2B).
Our data are the first ever transcriptome profile of an
organism belonging to the genus
and the above
results suggest more representative collections of
O. chinensis
genes in this study.
Analysis of Differentially Expressed Genes
Pairwise comparisons of the non-immunized control and
O. chinensis sinuosa
for differential expression
analysis revealed a total of 2,887 DEGs (Table S1). The MA
plot showed significant DEGs (blue) against all nonsignificant
DEGs (red) (Fig. 3). Among the identified DEGs, 1,800 were
expressed at significantly higher levels in the control,
whereas 1,087 genes were expressed at significantly higher
levels in the immunized sample. In addition, 2,117 genes
showed more than a 10-fold difference between the non-
immunized control and immunized samples (Fig. 3). Of the
DEGs, 1,053 could not be annotated using any database,
and 419 of these were more highly expressed in immunized
Fig. 3.
MA plot of differentially expressed genes in the transcriptome of non-immunized control and
E. coli
O. chinensis sinuosa
Data are the individual gene responses plotted as log fold-change versus base mean fold-change >2 (
< 0.05), with negative changes representing
downregulated genes and positive changes representing upregulated genes.
1868 Kim et al.
J. Microbiol. Biotechnol.
samples. Of the 2,887 DEGs, 512 were annotated as
homologous genes in
Zootermopsis nevadensis
, a dampwood
termite that is closely related to
O. chinensis sinuosa
. The
number of genes with higher expression levels in immunized
O. chinensis sinuosa
was lower than that in the control, which
clearly showed that the bacterial infection was significant.
In Silico Identification of AMPs
Insects produce a greater number of AMPs than any
other taxonomic group, and the number of individual
AMPs produced by each insect species varies substantially
[18, 21]. A vast number of studies have been performed
that have identified more than 100 insect-specific AMPs by
high-throughput, forward genetics approaches and various
screening procedures [12]. Our comprehensive transcriptome
dataset from non-immunized control and bacteria-immunized
O. chinensis sinuosa
was screened extensively for cDNAs
encoding AMPs using public databases (Table 2). We
identified 26 novel AMPs (non-allergen) belonging to
diverse families and functional classes (Table S2). Among
these, nine are involved in various biological functions,
whereas the remaining 17 have unknown/uncharacterized
functions. Furthermore, our DEG results revealed that
most of the AMPs were differentially expressed, and this
was visualized by preparing a heat map to compare the
normalized mapped read (RPKM) values of each AMP
between the non-immunized control and immunized
samples (Fig. 4).
High-throughput analyses, such as RNA-Seq, have led to
the discovery of not only many immune-related genes but
also the corresponding networks and pathways involved in
pathogen recognition, signal transduction, and effector
functions, including AMPs [20]. Previous reports have
identified several potential AMPs in insects of various
orders [18]. Recently, the transcriptome of the ladybird
Harmonia axyridis
, was analyzed for AMPs induced
E. coli
Micrococcus luteus
, and
Saccharomyces cerevisiae
[21]. Several novel AMPs in
Periplaneta americana
identified in an
E. coli
-immunized de novo
and were validated in vitro. Indeed, most of these AMPs
are involved in defense and protein binding [15].
In conclusion, transcriptome sequencing and annotation
can be used to provide remarkable information for analyzing
the molecular basis of the economically important traits of
an organism. Here, we described the first comprehensive
investigation of the
O. chinensis sinuosa
transcriptome. In
this study, we characterized the transcriptome of
E. coli
O. chinensis sinuosa
and identified a significant
number of AMPs through an in silico approach. The
transcriptome data assembled in this study will be a valuable
resource for future studies, including gene expression and
annotation studies of the
O. chinensis sinuosa
Tab le 2 .
In silico functional characterization of identified AMPs.
Filter Propensity Method Cutoff No. of total peptide Seq
Step 1 Molecular Pepstats Total peptides (
100) 58,696
Charge >0(+) 55,073
12 41,207
Aggregation TANGO
(in vivo)
500 42,866
25 41,612
100 18,134
Homologs BLAST Known (similarity >90%) 1,627
Novel Novel (no BLAST score) 57,069
Allergen Allerdictor Non-allergen 58,261
Filter 1 total (common) 9,015
Step 2 Aggregation Aggrescan (in vitro) -40
60 8,065
RF AMP 1,908
ANN AMP 2,934
DA AMP 2,024
(3 out of 4)
3 1,496
Final peptides (Filter 2 common) 1,346
Antimicrobial Peptides in the Grasshopper Oxya chinensis sinuosa 1869
November 2016
Vol. 26
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Promising insect-derived AMPs are currently being
employed in various medicinal applications. Therefore, our
future work will focus on producing
-derived AMPs
in the quantities required for topical therapeutic applications.
This research was supported by a grant (PJ01099301) from
the Agenda Program, Rural Development Administration.
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... The promising pharmaceutical effect of psacotheasin accelerated our interest to further identify putative AMPs from P. hilaris. We utilized next generation sequencing technology, a modern age tool, to divulge putative AMPs [14][15][16]. In silico screening for AMPs was done on transcriptome of the yellow spotted long horned beetle immunized with bacterial and yeast strains. ...
... We aimed to identify putative AMPs from the transcriptome of immunized as well as non-immunized P. hilaris utilizing in silico strategies. Previous analysis of AMPs through computation methods has an added scientific insight in understanding AMPs [14][15][16]. Insects are diversified organisms with strong environmental adaptation and a well-built defense system against invading harmful bacteria, fungus, parasites, and viruses [7]. AMPs are multi-targeted which can be competently used to combat the increasing global burden of multidrug resistant bacteria. ...
... After screening the synthesized peptides, we finally selected 13 AMPs (Table 3) and tested their antimicrobial activities against E. coli, S. aureus and C. albicans using a radial diffusion assay ( Figure 3). We found antimicrobial activity in 13 synthetic peptides (Ph 1, 2, 3,4,8,10,12,14,16,20,22,24,26), which increased in a dose-dependent manner. Remarkably, the peptides Ph 1, 2, 22, 26 showed stronger antimicrobial activity than melittin and Ph 3, 4 showed similar antimicrobial activity with melittin in E. coli. ...
Full-text available
Antimicrobial peptides (AMPs) are the frontline innate defense system evolutionarily preserved in insects to combat invading pathogens. These AMPs could serve as an alternative to classical antibiotics to overcome the burden of treating multidrug resistant bacteria. Psacotheasin, a knottin type AMP was isolated from Psacothea hilaris and shown to exhibit antimicrobial activity, especially against fungi through apoptosis mediated cell death. In this study, we aimed to identify novel probable AMPs from Psacothea hilaris, the yellow spotted longicorn beetle. The beetle was immunized with the two bacterial strains (E. coli and S. aureus), and the yeast strain C. albicans. After immunization, total RNA was isolated and sequenced in Illumina platform. Then, beetle transcriptome was de novo assembled and searched for putative AMPs with the known physiochemical features of the AMPs. A selection of AMP candidates were synthesized and tested for antimicrobial activity. Four peptides showed stronger activity against E. coli than the control AMP, melittin while one peptide showed similar activity against S. aureus. Moreover, four peptides and two peptides showed antifungal activity stronger than and similar to melittin, respectively. Collectively one peptide showed both antibacterial and antifungal activity superior to melittin; thus, it provides a potent antimicrobial peptide. All the peptides showed no hemolysis in all the tested concentrations. These results suggest that in silico mining of insects' transcriptome could be a promising tool to obtain and optimize novel AMPs for human needs.
... Moreover, they have also been used to explore the molecular mechanisms of fungal drug-resistance or fungi-host interactions Barad et al., 2016;Wang et al., 2016). The fungal response mechanisms to antimicrobial chemicals, such as iturins, C 12 -OOWW-NH 2 , and other antimicrobial substances have been successfully investigated (Muthaiyan et al., 2008;Pietiäinen et al., 2009;Kim et al., 2016;Le et al., 2016;Sun et al., 2017;Wang et al., 2017;Zhao et al., 2017;Jiang et al., 2020;Li et al., 2020). ...
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Alternaria solani is an airborne fungus and the primary causal agent of potato early blight worldwide. No available fungicides that are both effective and environmentally friendly are usable to control this fungus. Therefore, biological control is a potential approach for its suppression. In this study, Bacillus subtilis strain ZD01’s fermentation broth strongly reduced A. solani pathogenicity under greenhouse conditions. The effects of strain ZD01’s secondary metabolites on A. solani were investigated. The exposure of A. solani hyphae to the supernatant resulted in swelling and swollen sacs, and the ZD01 supernatant reduced A. solani conidial germination significantly. Matrix-assisted laser desorption/ionization time of flight mass spectrometry and pure product tests revealed that fengycins were the main antifungal lipopeptide substances. To elucidate the molecular mechanism of the fengycins’ biological control, RNA sequencing analyses were performed. A transcriptome analysis revealed that 304 and 522 genes in A. solani were differentially expressed after 2-h and 6-h fengycin treatments, respectively. These genes were respectively mapped to 53 and 57 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, the most enriched KEGG pathway analysis indicated that the inhibitory mechanisms of fengycins against A. solani regulated the expression of genes related to cell wall, cell membrane, transport, energy process, protein synthesis and genetic information. In particular, cell wall and cell membrane metabolism were the main processes affected by fengycin stress. Scanning and transmission electron microscope results revealed hyphal enlargement and a wide range of abnormalities in A. solani cells after exposure to fengycins. Furthermore, fengycins induced chitin synthesis in treated cells, and also caused the capture of cellular fluorescent green labeling and the release of adenosine triphosphate (ATP) from outer membranes of A. solani cells, which may enhance the fengycins ability to alter cell membrane permeability. Thus, this study increases the transcriptome data resources available and supplies a molecular framework for B. subtilis ZD01 inhibition of A. solani HWC-168 through various mechanisms, especially damaging A. solani cell walls and membranes. The transcriptomic insights may lead to an effective control strategy for potato early blight.
... The most common insect species utilized as food and medicine belong to orders Coleoptera, Lepidoptera, Hymenoptera, Orthoptera, and Hemiptera [13]. Among them, Oxya chinensis sinuosa (rice field grasshopper) which belongs to order Orthoptera of Acrididae family [14] is widely utilized in Korea and other Asian countries since millennia because of its high nutritional and medicinal value [15]. Traditionally, O. chinensis sinuosa is reported to cure several ailments including asthma, bronchitis, cough, paralysis, and seizures [16]. ...
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Oxya chinensis sinuosa (rice field grasshopper) is an edible insect with numerous health beneficial properties, traditionally being used to treat many ailments in Korea and other countries. O. chinensis sinuosa has been used from centuries, however, a little is known about the chemical functionality of its bioactive compounds. Therefore, this study examined the anti-inflammatory and cathepsin C inhibitory activities of N-acetyldopamine dimer (2R, 3S)-2-(3′,4′-dihydroxyphenyl)-3-acetylamino-7-(N-acetyl-2″-aminoethyl)-1,4-benzodioxane (DAB1) isolated from O. chinensis sinuosa. Results showed that DAB1 reduced the expression of pro-inflammatory mediator (iNOS, COX-2) and cytokines (TNF-α, IL-1β, and IL-6), and curtailed the nuclear translocation of NF-κB by inhibiting the phosphorylation of IκBα in lipopolysaccharide stimulated macrophages. Additionally, DAB1 inhibited cathepsin C activity at the cellular level, supported by in vitro assay (Ki, 71.56 ± 10.21 µM and Kis, 133.55 ± 18.2 µM). Moreover, combinatorial molecular simulation and binding free energy analysis suggested a significant stability and binding affinity of cathepsin C-DAB1 complex via formation of hydrogen bond and hydrophobic interactions with the catalytic residues (Gln228, Thr379, Asn380, and Hie381). Also, essential dynamics analysis showed DAB1 induced non-functional motions in cathepsin C structure. Collectively, DAB1 was concluded as anti-inflammatory and cathepsin C inhibiting agent and could be used in the drug development against respective diseases.
... We have aimed to identify putative AMPs from the transcriptome of immunized as well as non-immunized L. migratoria utilizing in silico strategies. Previous analysis of AMPs through computation methods has an added scientific insight in understanding AMPs (Kim et al., 2016a(Kim et al., , 2016bYoo et al., 2014). Insects are diversified organism with strong environmental adaptation and well-built defense system against invading harmful bacteria, fungus, parasites, and viruses (Wu et al., 2018). ...
Evolutionary immune system upgradation is done by all living organism to survive from the invading pathogens. Insects has strong defense system comprising anatomical barrier, humoral antimicrobial peptide (AMP) production and cellular immune components. Locusta migratoria (L. migratoria) is an agricultural pest insect migrating long distance might encounter distinct pathogens. Owing to this, we aimed in identifying AMPs present in the L. migratoria and elucidated the antimicrobial activity. The migratory locust was immunized with Escherichia coli, Staphylococcus aureus, Candida albicans mixture, then RNA was isolated and sequenced. The L. migratoria transcriptome was de novo assembled using trinity and screened for AMPs propensity specifically molecular and aggregation properties. The novelty is tested by blasting in known AMP databases. Finally, obtained novel putative AMPs were then tested for antimicrobial and hemolytic activity. The prediction resulted in 3,524 putative AMPs which was further screened to ten AMPs for final testing. Two peptides showing promising antimicrobial effects were obtained. All the analyzed peptides showed no hemolysis confirms its non-toxicity. Thus, our peptides could be promising drug candidate and can be used as an alternative to antibacterial or antifungal therapy with further validations.
... AMP prediction from the insect transcriptome using the computational approach is our long-standing idea from which we have continuously thrived in testing [20,25,26]. The natural environment of insect livelihood is rich in harmful bacteria, fungus, parasites, and viruses, thus there is a need for an efficient defense system [3]. ...
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An insect’s innate immune system is the front line of defense against many invading microorganisms. One of the important components of this defense system is antimicrobial peptides (AMPs). Papiliocin is a well-studied antimicrobial peptide (AMP) isolated from the swallowtail butterfly, Papilio xuthus, and it was previously reported to be effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, particularly in drug resistant Gram-negative bacteria. Hence, we aimed to identify novel AMPs from Papilio xuthus using its transcriptome. We immunized the swallowtail butterfly with Escherichia coli, Staphylococcus aureus, Candida albicans, and the total RNA was isolated. De novo transcriptome assembly and functional annotations were conducted, and AMPs were predicted using an in-silico pipeline. The obtained 344,804,442 raw reads were then pre-processed to retrieve 312,509,806 (90.6%) total clean reads. A total of 38,272 unigenes were assembled with the average length of 1010 bp. Differential gene expression analysis identified 584 and 1409 upregulated and downregulated genes, respectively. The physicochemical, aggregation, and allergen propensity were used as filtration criteria. A total of 248 peptides were predicted using our in-house pipeline and the known AMPs were removed, resulting in 193 novel peptides. Finally, seven peptides were tested in vitro and three peptides (Px 5, 6, and 7) showed stronger antimicrobial activity against Gram-negative bacteria and yeast. All the tested peptides were non-allergens. The identified novel AMPs may serve as potential candidates for future antimicrobial studies.
The farming of insects for food has many advantages, since insects are readily available, can be grown in small areas, are efficient at the conversion of food to biomass, and have a minimal contribution to environmental degradation. We review the prospects for farming insects using data obtained from next-generation genome-wide DNA sequencing to guide the selection of desirable traits. This approach is more efficient than traditional breeding processes and promises to facilitate the development of insect strains with phenotypes which make them desirable as food sources. We also review some of the potential problems associated with the mass production of insects and suggest some factors which must be taken into consideration as inbreeding depression, adaptability, and allergens. Insects are a cheap, easily available source of nutrients such as proteins and fats, and the efficient mass production of insect-based foods may help to ameliorate problems of the food supply.
Heavy metals are major environmental pollutants that affect organisms across different trophic levels. Herbivorous insects play an important role in the bioaccumulation, and eventually, biomagnification of these metals. Although effects of heavy metal stress on insects have been well-studied, the molecular mechanisms underlying their effects remain poorly understood. Here, we used the RNA-Seq profiling and isobaric tags for relative and absolute quantitation (iTRAQ) approaches to unravel these mechanisms in the polyphagous pest Spodoptera litura exposed to lead (Pb) at two different concentrations (12.5 and 100 mg Pb/kg; PbL and PbH, respectively). Altogether, 1392 and 1630 differentially expressed genes (DEGs) and 58, 114 differentially expressed proteins (DEPs) were identified in larvae exposed to PbL and PbH, respectively. After exposed to PbL, the main up-regulated genes clusters and proteins in S. litura larvae were associated with their metabolic processes, including carbohydrate, protein, and lipid metabolism, but the levels of cytochrome P450 associated with the pathway of xenobiotic biodegradation and metabolism were found to be decreased. In contrast, the main up-regulated genes clusters and proteins in larvae exposed to PbH were enriched in the metabolism of xenobiotic by cytochrome P450, drug metabolism-cytochrome P450, and other drug metabolism enzymes, while the down-regulated genes and proteins were found to be closely related to the lipid (lipase) and protein (serine protease, trypsin) metabolism and growth processes (cuticular protein). These findings indicate that S. litura larvae exposed to PbL could enhance food digestion and absorption to prioritize for growth rather than detoxification, whereas S. litura larvae exposed to PbH reduced food digestion and absorption and channelized the limited energy for detoxification rather than growth. These contrasting results explain the dose-dependent effects of heavy metal stress on insect life-history traits, wherein low levels of heavy metal stress induce stimulation, while high levels of heavy metal stress cause inhibition at the transcriptome and proteome levels.
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Zophobas atratus which is also referred as giant meal worm, belongs to the largest darkling beetle family. The rich protein and fat content in the giant meal worm made it as excellent protein source in pet feed and thus instigated the commercial rearing. Insect antimicrobial peptides (AMPs) have therapeutic potential with wide range of activity against bacteria, fungus, parasites, and viruses. However, there is a lack of study of AMPs from Zophobas atratus. We have immunized the giant meal worm with Escherichia coli, Staphylococcus aureus, Candida albicans and total RNA was isolated and sequenced. De novo transcriptome assembly and functional annotations was done. The AMPs were predicted using in silico pipeline from transcriptome data. A total of 355,771,565 raw reads with 26.6 gigabytes was obtained. After trimming and low-quality sequences removal, 328,196,384 (92.2%) total clean reads was obtained. A total of 47,635 unigenes were assembled with the average length of 1109 bp. AMP pipeline predicted a total of 752 peptides of which 177 were novel AMPs all of which are non-allergens. Based on the α-helix secondary structure requirement for higher efficiency 14 peptides were synthesized and tested in vitro to determine the minimum inhibitory concentrations. Eventually, the peptides Za 7, Za 15, Za 22 were selected as the prime candidates for the development of antimicrobial agents and none of them showed hemolytic activity up to 100 μg/mL. Our data provides an insight in Zophobas atratus transcriptome derived AMPs and understanding its antimicrobial activity.
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Oxya chinensis sinuosa (Ocs) is consumed as representative edible insects in Asia, but its function in various immune systems remains unclear. This study aimed to demonstrate the immunomodulatory effect, particularly on the innate and adaptive immune response, of Ocs protein (Ocs-P) and to investigate its function as a potent anticancer immunostimulant when administered during the progression stage of colon carcinoma in tumor-bearing mice. Our in vitro results demonstrated that Ocs-P treatment induces phenotypic alteration (increased expression of surface molecules and production of T Helper type I-polarizing (Th1-polarizing) cytokines and decreased antigen uptake ability) of dendritic cells (DCs) through the activation of Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B-dependent (NF-κB-dependent) signaling pathways. Additionally, Ocs-P-stimulated DCs initiated differentiation of naive T cells into IFN-γ-producing Th1-type T cells effectively and activated cytotoxic CD8+ T cell response. In colon carcinoma-bearing mouse models, oral administration of Ocs-P inhibited tumor growth and restored the expression of decreased surface molecules in lineage-CD11c+MHC-II+ splenic DCs. Furthermore, Ocs-P administration enhanced the generation of multifunctional CD4+ and CD8+ T cells expressing Th1-type cytokines (TNF-α, IFN-γ, and IL-2) and the degranulation marker (CD107a). Collectively, these results suggest that Ocs-P demonstrates an immunostimulatory effect and may induce powerful anticancer immunity.
The contamination of Aspergillus carbonarius causes decreases and great decay of agricultural products, and threatens the human and animal health by producing mycotoxins, especially ochratoxin A. Bacillus subtilis has been proved to efficiently inhibit the growth of A. carbonarius. Revealing the major active compound and the mechanisms for the antifungal of B. subtilis are essential to enhance its antifungal activity and control the quality of antifungal products made of it. In this study, we determined that iturin A is the major compound that inhibits Aspergillus carbonarius, a widespread fungal pathogen of grape and other fruits. Iturin A significantly inhibited growth and ochratoxin A production of A. carbonarius with minimal inhibitory concentrations (MICs) of 10 μg/mL and 0.312 μg/mL, respectively. Morphological observations revealed that iturin A caused swelling of the fungal cells and thinning of the cell wall and membrane at 1/2 MIC, whereas it inhibited fungal spore germination and caused mitochondrial swelling at higher concentrations. A differential transcriptomic analysis indicated that the mechanisms used by iturin A to inhibit A. carbonarius were to downregulate the expression of genes related to cell membrane, transport, osmotic pressure, oxidation-reduction processes, and energy metabolism. Among the down-regulated genes, those related to the transport capacity were most significantly influenced, including the increase of energy-related transport pathways and decrease of other pathways. Notably, the genes related to taurine and hypotaurine metabolism were also decreased, indicating iturin A potentially cause the occurrence of osmotic imbalance in A. carbonarius, which may be the intrinsic cause for the swelling of fungal cells and mitochondria. Overall, iturin A produced by B. subtilis played important roles to inhibit A. carbonarius via changing the fungal cell structure and causing perturbations to energy, transport and osmotic pressure metabolisms in fungi. The results indicated a new direction for researches on the mechanisms for lipopeptides and provided useful information to develop more efficient antifungal agents, which are important to agriculture and biomedicine.
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Epacromius coerulipes (Ivanov) is one of the most widely distributed locusts. To date, the main methods to kill locusts still rely on chemical controls, which can result in the selection of locusts with resistance to chemical pesticides. Butene-fipronil is a new pesticide that was discovered by the structural modification of fipronil. This pesticide has been used to control various agricultural pests and has become an important pesticide product to control pests that exhibit resistance to other pesticides, including locusts. To extend its useful half-life, studies of the initiation and progression of resistance to this pesticide are needed. Herein, two E. coerulipes strains, a pesticide-sensitive (PS) and a pesticide-resistant (PR) strain, were chosen to undergo de novo assembly by paired-end transcriptome Illumina sequencing. Overall, 63,033 unigenes were detected; the average gene length was 772 bp and the N50 was 1,589 bp. Among these unigenes, similar to 25,132 (39.87% of the total) could be identified as known proteins in bioinformatic databases from national centers. A comparison of the PR and PS strains revealed that 2,568 genes were differentially expressed, including 1,646 and 922 genes that were upand down-regulated, respectively. According to the Gene Ontology (GO) database, among biological processes the metabolic process group was the largest group (6,900 genes, 22.47%) and contained a high frequency of differentially expressed genes (544 genes, 27.54%). According to the Clusters of Orthologous Groups (COG) categories, 28 genes, representing 2.98% of all genes, belonged to the group of genes involved in the biosynthesis, transportation, and catabolism of secondary metabolites. The differentially expressed genes that we identified are involved in 50 metabolic pathways. Among these pathways, the metabolism pathway was the most represented. After enrichment analysis of differential gene expression pathways, six pathways-ribosome; starch, and sucrose metabolism; ascorbate and aldarate metabolism; drug metabolism-cytochrome P450; metabolism of xenobiotics by cytochrome P450; and glutathione metabolism-showed a high degree of enrichment. Among these pathways, drug metabolism-cytochrome P450, metabolism of xenobiotics by cytochrome P450, and glutathione metabolism have been associated with pesticide metabolism. Furthermore, 316 unigenes in the E. coerulipes transcriptome encode detoxifying enzymes and 76 unigenes encode target proteins of pesticides. Among these genes, 23 genes that encode detoxifying enzymes in the resistance group were found to be up-regulated. The transcriptome sequencing results of E. coerulipes established a genomics database of E. coerulipes for the first time. This study also establishes a molecular basis for gene function analysis of E. coerulipes. Moreover, it provides a theoretical resource for mechanistic studies on pesticide resistance through the screening and investigation of resistance genes.
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Cockroaches are surrogate hosts for microbes that cause many human diseases. In spite of their generally destructive nature, cockroaches have recently been found to harbor potentially beneficial and medically useful substances such as drugs and allergens. However, genomic information for the American cockroach (Periplaneta americana) is currently unavailable; therefore, transcriptome and gene expression profiling is needed as an important resource to better understand the fundamental biological mechanisms of this species, which would be particularly useful for the selection of novel antimicrobial peptides. Thus, we performed de novo transcriptome analysis of P. americana that were or were not immunized with Escherichia coli. Using an Illumina HiSeq sequencer, we generated a total of 9.5 Gb of sequences, which were assembled into 85,984 contigs and functionally annotated using Basic Local Alignment Search Tool (BLAST), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) database terms. Finally, using an in silico antimicrobial peptide prediction method, 86 antimicrobial peptide candidates were predicted from the transcriptome, and 21 of these peptides were experimentally validated for their antimicrobial activity against yeast and gram positive and -negative bacteria by a radial diffusion assay. Notably, 11 peptides showed strong antimicrobial activities against these organisms and displayed little or no cytotoxic effects in the hemolysis and cell viability assay. This work provides prerequisite baseline data for the identification and development of novel antimicrobial peptides, which is expected to provide a better understanding of the phenomenon of innate immunity in similar species.
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Antimicrobial peptides (AMPs) are short proteins with antimicrobial activity. A large portion of known AMPs originate from insects, and the number and diversity of these molecules in different species varies considerably. Insect AMPs represent a potential source of alternative antibiotics to address the limitation of current antibiotics, which has been caused by the emergence and spread of multidrug-resistant pathogens. To get more insight into AMPs, we investigated the diversity and evolution of insect AMPs by mapping their phylogenetic distribution, allowing us to predict the evolutionary origins of selected AMP families and to identify evolutionarily conserved and taxon-specific families. Furthermore, we highlight the use of the nematode Caenorhabditis elegans as a whole-animal model in high-throughput screening methods to identify AMPs with efficacy against human pathogens, including Acinetobacter baumanii and methicillin-resistant Staphylococcus aureus. We also discuss the potential medical applications of AMPs, including their use as alternatives for conventional antibiotics in ectopic therapies, their combined use with antibiotics to restore the susceptibility of multidrug-resistant pathogens, and their use as templates for the rational design of peptidomimetic drugs that overcome the disadvantages of therapeutic peptides. The article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.
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Locusts are one of the world's most destructive agricultural pests and represent a useful model system in entomology. Here we present a draft 6.5 Gb genome sequence of Locusta migratoria, which is the largest animal genome sequenced so far. Our findings indicate that the large genome size of L. migratoria is likely to be because of transposable element proliferation combined with slow rates of loss for these elements. Methylome and transcriptome analyses reveal complex regulatory mechanisms involved in microtubule dynamic-mediated synapse plasticity during phase change. We find significant expansion of gene families associated with energy consumption and detoxification, consistent with long-distance flight capacity and phytophagy. We report hundreds of potential insecticide target genes, including cys-loop ligand-gated ion channels, G-protein-coupled receptors and lethal genes. The L. migratoria genome sequence offers new insights into the biology and sustainable management of this pest species, and will promote its wide use as a model system.
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Antimicrobial peptides (AMPs) are known to have family-specific sequence composition, which can be mined for discovery and design of AMPs. Here, we present CAMPR3; an update to the existing CAMP database available online at It is a database of sequences, structures and family-specific signatures of prokaryotic and eukaryotic AMPs. Family-specific sequence signatures comprising of patterns and Hidden Markov Models were generated for 45 AMP families by analysing 1386 experimentally studied AMPs. These were further used to retrieve AMPs from online sequence databases. More than 4000 AMPs could be identified using these signatures. AMP family signatures provided in CAMPR3 can thus be used to accelerate and expand the discovery of AMPs. CAMPR3 presently holds 10247 sequences, 757 structures and 114 family-specific signatures of AMPs. Users can avail the sequence optimization algorithm for rational design of AMPs. The database integrated with tools for AMP sequence and structure analysis will be a valuable resource for family-based studies on AMPs.
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To study the accumulation and contamination of heavy metals (i.e., Cd, Cr, Cu, Ni, and Zn) in soil, air, and water, few insect species were assayed as ecological indicators. Study area comes under industrial zone of district Gujrat of Punjab, Pakistan. Insects used as bioindicators included a libellulid dragonfly (Crocothemis servilia), an acridid grasshopper (Oxya hyla hyla), and a nymphalid butterfly (Danaus chrysippus) near industrial zone of Gujrat. Accumulation of Cd was highest in insect species followed by Cu, Cr, Zn, and Ni at í µí± < 0.05. Hierarchical cluster analysis (HACA) was carried out to study metal accumulation level in all insects. Correlation and regression analysis confirmed HACA observations and declared concentration of heavy metals above permissible limits. Metal concentrations in insects were significantly higher near industries and nallahs in Gujrat and relatively higher concentrations of metals were found in Orthoptera than Odonata and Lepidoptera. The total metal concentrations in insects were pointed significantly higher at sites S3 (Mid of HalsiNala), S9 (End of HalsiNala), and S1 (Start of HalsiNala), whereas lowest value was detected at site S6 (Kalra Khasa) located far from industrial area. HACA indicates that these insect groups are potential indicators of metal contamination and can be used in biomonitoring.
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Growth of the ornamental fish industry is being hindered by the scarcity of low cost feed; hence alternative protein supplements should be explored. In this context the present study aims to evaluate whether the grasshopper Oxya fuscovittata could be used as a supplement for fish meal in the diets of Poecillia sphenops, which is one of the most common ornamental fishes worldwide. The present work is divided into three phases: In the first phase proximate composition of the grasshopper is obtained and five diets are prepared where fish meal is gradually replaced by Oxya meal and named as control, D1, D2, D3 and D4. All the diets are formulated on iso-nitrogenous basis where the protein percentage is fixed at 400 g/kg. The second phase deals with feeding trial and in the third phase all the data of the feeding trial are subjected to a linear model. The feeding trial shows that the control, D1 and D2 fed fishes have almost similar results. The linear model proves that the variation in the indices are mainly due to replacement of fish meal by Oxya meal, not due to the variations of rice husk and mustard oil cake that are also used to formulate the diets of the present study. From the results two Oxya supplemented diets, i.e. D1 and D2 are proved to be almost equivalent to the control diet. Hence it is concluded that Oxya meal is able to replace 25% to 50% of fish meal from the diets of P. sphenops.
Since the completion of the human genome project in 2003, extraordinary progress has been made in genome sequencing technologies, which has led to a decreased cost per megabase and an increase in the number and diversity of sequenced genomes. An astonishing complexity of genome architecture has been revealed, bringing these sequencing technologies to even greater advancements. Some approaches maximize the number of bases sequenced in the least amount of time, generating a wealth of data that can be used to understand increasingly complex phenotypes. Alternatively, other approaches now aim to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions. These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.