Vol.4, No.9, 483-490 (2013) Agricultural Sciences
RNAi of MiASB caused high mortality of
Meloidogyne incognita juveniles and
inhibited the nematode disease
Yonghong Huang1,2*, Mei Mei1,3*, Baoming Shen1,3, Zhenchuan Mao1, Bingyan Xie1#
1Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China;
#Corresponding Author: xiebingyan@caas.cn, albertluke@126.com
2Key Laboratory of Biology and Genetic Resource Utilization of Fruit Tree in South Subtropics, Ministry of Agriculture/Fruit
Research Institute, Guangdong Academy of Agricultural Science, Guangzhou, China
3College of Horticulture and Landscape, Hunan Agriculture University, Changsha, China
Received 27 April 2013; revised 28 May 2013; accepted 10 June 2013
Copyright © 2013 Yonghong Huang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The southern root-knot nematode, Meloidogyne
incognita, is one of the most prevalent and
damaging plant-parasitic nematodes in the
world and causes serious damages to agricul-
tural production. W e cloned a mitoc hondrial ATP
synthase b subunit gene fragment of M. incog-
nita (MiASB) based on the nematode genomics
prediction. By soaking in the MiASB dsRNA so-
lution, the hatching of RNAi treated eggs was
reduced by 60% compared to negative control
and by 64% compared to untreated control.
Mortality of RNAi treated second stage juvenile
(J2) was 8.6 times higher than that of negative
control and 26 times higher than the untreated
control. Inoculating the RNAi treated egg mass-
es and J2 to tomato seedlings showed the pa-
thogencity was significantly reduced. For the
RNAi treated egg masses, the amount of root
galls on silence treated seedlings was reduced
by 92% compared to that on the negative control
seedlings, and reduced by 93% compared to that
on untreated control seedlings. For the treated
J2, the amount of root galls on silence treated
seedlings was reduced by 83% and 86% com-
pared to negative and untreated control seed-
lings, respectively. The study revealed the Mi-
ASB silence had a positive effect on prevention
and control of root-knot nematode disease, and
also showed that the MiASB may be involved in
the pathogenesis of nematode, which provided
new ideas and ways to the researc h of nematode
pathology and nematode disease control.
Keywords: Root-knot Nematode; Meloidogyne
Spp.; Mitochondrial ATP Synthase; RNA
Interference; dsRNA
Root-knot nematodes (RKN), genus Meloidogyne, are
root endoparasites that can develop on a wide range of
plant species. The infective second stage juvenile (J2)
penetrates the root tip and migrates intercellularly until it
reaches the differentiating vascular cylinder [1]. It in-
duces root-knot, stunts nutrient deficiency and disrupts
the physiology of the host plants through their reproduc-
tion and feeding within plant roots, leading to a signify-
cant reduction in crop yield and deterioration of product
quality [2,3]. Crop infestation by RKN causes 70 billion
U.S. dollars of crop losses annually in fruit and vegetable
production [4]. The RKN is being controlled in the fields
mainly based on cultural practices, host-plant resistance
and the application of synthetic pesticides. Nematode
control has become difficult in recent years owning to
the withdrawal or restricted use of effective synthetic
pesticides for environmental problems and human and
animal health concerns [3,5-7], which urges the need for
alternative control methods. Strategies based on the spe-
cific blocking of parasitism gene products involving in
the success of infection would offer attractive alterna-
tives to reduce nematode populations in the field [8].
RNA interference (RNAi) is a mechanism for post-
transcriptional gene silencing. This technique uses the
fact that exposure of an organism to double-stranded
RNA (dsRNA) from a gene of interest causes posttrans-
criptional silencing of the endogenous gene and allows a
null phenotype to be mimicked [9]. Since it was firstly
*These authors contribute equally to the article.
Copyright © 2013 SciRes. OPEN ACCES S
Y. H. Huang et al. / Agricultural Sciences 4 (2013) 4 83-490
discovered over two decade ago in Caenorhabditis ele-
gans [10], RNAi have been proved to be a widespread
phenomenon shared by many phyla [11]. In C. elegans,
targeting the dsRNA into the nematode intestine by
microinjection or ingestion allows the silencing of genes
expressed in distal tissues. In Nippostrongylus brasilien-
sis, dsRNA uptake into the nematode body was induced
by the cationic lipid polymer lipofectin. In Brugia malayi,
a low-volume culture system was developed to expose
females to dsRNA [1]. Nowadays, RNAi has been devel-
oped as a powerful tool for studying gene function in a
variety of organisms [12].
Mitochondria and chloroplasts serve as power stations
of living cells. By generating the biological energy cur-
rency ATP, ATP synthases play a decisive role in this
process [13]. Mitochondrial ATP synthase (also named
F1F0-ATP synthase or complex V) is located in the inner
mitochondrial membrane together with respiratory chain
complexes I-IV [14] and is a rotating nanomotor in pro-
karyotic and eukaryotic cells that uses a transmembrane
electrochemical ion gradient as an energy source to con-
vert ADP and Pi to ATP [15]. F1F0-ATP synthase con-
sists of up to 18 different subunits [16], each of which is
the essential components for ATP synthase to generate
In preliminary studies, based on the genomic data of C.
elegans, M. hapla, M. incognita and RNAi data in
Wormbase, we predicted some functional genes in Meloi-
dogyne nematode, one of which was ASB encoding the b
subunit of the F0 proton channel portion of F1F0-ATP
synthase. In this study, we cloned a 650 bp ASB fragment
from M. incognita (MiASB). Using RNAi by soaking it in
the dsRNA solution of MiASB, we investigated the ef-
fects of MiASB on the hatching of the egg masses and the
mortality of Meloidogyne nematode juveniles, and also
studied its inhibitory effect on nematode disease caused
by M. incognita.
2.1. Nematode Collection
Nematode M. incognita was extracted from the pure
culture that was previously initiated by egg masses and
propagated on tomato (Solanum lycopersicum) in the
glasshouse. Egg masses were handpicked using sterilized
forceps from heavily infected roots 45 days after incuba-
tion. The egg masses were surface-sterilized (1% NaOCl)
for 1 min followed by washing with sterile distilled water.
Some of the egg masses was placed on 15 mesh sieves (8
cm in diameter) containing crossed layers of tissue paper
in Petri-dishes with sterile distilled water just deep
enough to contact the egg masses and incubated 25˚C to
26˚C to obtain freshly hatched J2. Emerged J2 were col-
lected daily and stored at 4˚C. J2 up to 4 days old were
used in experiments [17].
2.2. Plant Material
Tomato (Solanum lycopersicum cv. Lichun) was used
in the experiment. Seeds of tomato were soaked in warm
water for 6 h, then were placed on the sterile filter paper
in a Petri dish for germinating at 25˚C in dark. The ger-
minated seeds were sown in pots containing culture sub-
strate composed of peat and vermiculite at 2 to 1 ratio in
greenhouse. Seedlings with 3 - 4 true leaves were used
for further experiments.
2.3. Molecular Cloning of MiASB Fragment
Total RNA was isolated from M. incognita J2 using
Trizol (Invitrogen), following the manufacturer’s proto-
col. The first strand was synthesized using SuperScript
TM III First-Strand Synthesis System for RT-PCR (In-
vitrogen) according to manufacturer’s instructions. A 650
bp fragment of MiASB was amplified by PCR using Mi-
ASB-specific primers including MiASB-F (5’-TGT TGG
TTT TCT TGG GTT GCT TTG-3’) designed according
to the MiASB (GeneBank Accession: JQ247982), a gene
we cloned in the preliminary studies based on the pre-
dicted MiASB according to the nematode genomics using
bioinformatics methods.
Polymerase chain reaction (PCR) was performed using
2 μl of the first-strand reaction as a template. Amplifica-
tions were performed with Taq DNA polymerase (2.5 U,
Promega) using 1 mM of each of the primers and 200
mM of each of the deoxy nucleotide triphosphates
(dNTP). The conditions were as follows: 94˚C, 3 min;
followed by 35 cycles of 94˚C, 30 s; 53˚C, 30 s; 72˚C, 60
sand a final cycle of 72˚C, 10 min. The resulting PCR
products were analyzed by agarose gel electrophoresis.
Bands with the correct size were excised from the gel,
purified using TaKaRa MiniBEST Agarose Gel DNA
Extraction Kit Ver.3.0 and cloned into pGM-T Vector
(TA Cloning Kit, Tiangen) according to the manufac-
turer’s instructions, forming the vector pGM-MiASB. The
pGM-MiASB was transformed into the competent cells of
E. coli DH5α and incubated at 37˚C overnight. The white
clones grown on the LB medium containing Ampicil-
lin/X-gal/IPTG were picked and the plasmids were ex-
tracted and sent to Invitrogen (Beijing) Co., Ltd., Beijing,
China for sequencing.
2.4. Plasmid Recombinant and in Vitro
Transcription to Produce dsRNA
The pGM-MiASB was digested with restriction en-
donuclease Eco RI (TaKaRa), the resulting products of
which were analyzed by agarose gel electrophoresis,
bands with the small size were excised from the gel, pu-
Copyright © 2013 SciRes. OPEN ACCES S
Y. H. Huang et al. / Agricultural Sciences 4 (2013) 4 83-490 485
rified using TaKaRa MiniBEST Agarose Gel DNA Ex-
traction Kit Ver.3.0, ligated to the LITMUS 28 i plasmid
(NEB, USA)digested with the exactly same restriction
endonuclease using T4 DNA ligase (Invitrogen), result-
ing in the plasmid LITMUS-MiASB. and then trans-
formed into competent cells of E. Coli DH5α and incu-
bated at 37˚C overnight. LITMUS-MiASB plasmid from
a single positive colony was confirmed by sequencing.
To insert T7 RNA polymerase site into the two ends of
the MiASB, 1 μl of the identified LITMUS-MiASB plas-
mid were used as a template to perform another PCR.
Amplifications were performed with Taq DNA poly-
merase (2.5 U, Promega) using 2 mM of T7 primer
(5’-TAA TAC GAC TCA CTA TAG G-3’) and 10 mM of
each of the deoxy nucleotide triphosphates (dNTP), with
an initial denaturation step of 94˚C for 5 min, followed
by 35 cycles of 94˚C for 30 s, 55˚C for 30 s, 72˚C for 50
s, and a final elongation step of 72˚C for 10 min. After
amplification, the PCR products (T7-MiASB-T7) were
separated by electrophoresis on agarose gel and purified
using TaKaRa MiniBEST Agarose Gel DNA Extraction
Kit Ver.3.0. Then the purified T7-MiASB-T7 fragment
was used to generatedds RNAs using in vitro Transcrip-
tion T7 Kit (TaKaRa) following the manufacturer’s in-
structions. The synthesized dsRNAs was stored at 70˚C
for further experiments.
2.5. RNAi of MiASB by Soaking
RNAi experiment was performed in a 24-microwell
plate according to the previous studies [1,9,18] with
slight modification. Uniform single egg mass or ap-
proximately 2000 freshly J2 were deposited in each well
and socked in M9 buffer solution (43.6 mM Na2HPO4,
22 mM KH2PO4, 18.7 mM NH4Cl, and 8.6 mM NaCl)
containing 1% resorcinol, 50 mM octopamine, 3 mM
spermidine, 0.05% gelatin, and dsRNA at 2 mg/ml in the
dark at room temperature on a rotator (RNAi treatment).
One control samples were incubated in the same solution
but without dsRNA (Negative control). And the other
control samples were incubated in the sterile water and
also no dsRNA was added (Untreated control). For the
egg RNAi treatment, half of the egg masses was treated
just for 1 day and the total cumulative number of juve-
nile hatched from the egg masses were counted one days
later (1-day-treatment), and the other half of the egg
masses were treated for 3 days and total cumulative
number of juvenile from these egg masses were counted
3 days later (3-day-treatment) to estimate the RNAi ef-
fect on the hatching of the egg masses. The cumulative
numbers of dead juvenile in 1-day- and 3-day-treatments
were also recorded at the corresponding time point, re-
spectively, to essay the RNAi effect on the mortality of
For the J2 RNAi treatment, Total J2 and the dead J2
was counted 6 h after the treatmentto determine RNAi
effect on the mortality of J2. The juvenile that was
straight and did not move even after mechanical prod-
ding were defined as dead.
2.6. Infection of Plants
The RNAi treated egg masses (1-day-treatment and
3-day-treatment) and RNAi treated J2 were applied close
to the roots of seedlings using a sterilized micropiette. 3
RNAi treated egg masses or approximately 6000 RNAi
treated J2 were inoculated to each seedling. The two
corresponding controls (negative control and untreated
control) were also inoculated according to the RNAi
treatment. These treated seedlings were cultured in the
greenhouse. The seedlings were uprooted 45 days later,
and root galls were recorded to evaluate the effects of the
MiASB RNAi on the disease caused by M. incognita.
3.1. Generation of Mi A SB dsRNA
A 650 bp fragment of MiASB was obtained by PCR
using total RNA of M. incognita J2 as template. The
identity of the cloned MiASB fragment with the corre-
sponding parts of the MiASB (GeneBank) accession:
JQ247982) was as high as 100%.To generate the dsRNA
of MiASB (Figure 1), the recombinant pLITMUS-Mi-
ASB was firstly constructed by linearizing pGM-MiASB
and pLITMUS-28i with Eco RI, respectively, and ligated
with T4 DNA ligase. Then pLITMUS-MiASB were used
to performed PCR as a template with T7 Primer and the
T7 polymerase site was inserted into two ends of the
MiASB and formed the fragment T7-MiASB-T7. After
that, in vitro transcription was catalyzed by T7 poly-
merase with fragment T7-MiASB-T7 as template andds
RNA of MiASB was finally generated.
3.2. MiASB RNAi Inhibited Hatching of M.
incognita Egg Masses
On the first day, juveniles were hatched from the egg
masses, the amounts of which were slightly but not sig-
nificantly different (F = 0.98, P = 0.3975). On the third-
days, the cumulative juvenilea mounts in the RNAi
treatment was significantly lower than those in negative
and untreated controls (F = 14.9, P = 0.003). The average
amountin MiASB treatment was 63, which was reduced
by 60% and 64% compared to negative and untreated
controls, respectively (Figure 2). The juvenile amount
innegative control was slightly, but not significantly,
higher than untreated control treatment, which showed
the reagents supplemented in the RNAi solution hardly
influenced the hatching of the egg masses, but RNAi of
MiASB significantly inhibited the hatching of egg masses
Copyright © 2013 SciRes. OPEN ACCES S
Y. H. Huang et al. / Agricultural Sciences 4 (2013) 4 83-490
Copyright © 2013 SciRes.
La c
Mi Asb
La c
f1 ori
MiAsb T7
Figure 1. Generation of the dsRNA: A: pGM-MiASB and pLITMUS-28i were lin-
earized with Eco RI and the respective resulting production were ligated with T4 DNA
ligase to construct pLITMUS-MiASB; B: The pLITMUS-MiASB were used to perform
PCR as a template with T7 primer, the result of which was T7 polymerase site was in-
serted into two ends of the MiASB and forming the fragment T7-MiASB-T7. C: In vitro
transcription was catalyzed by T7 polymerase with fragment T7-MiASB-T7 as tem-
plate and the dsRNA of MiASB was generated.
of hatched juveniles in the RNAi treatment was 5.9 and
25.5 times higher than the negative and untreated con-
trols (F = 15.01, P = 0.0003). On the third day, 62.8 of
the 63.3, 6.8 of the 158.4 and 5.3 of the 176.7 hatched
juvenilesdied in the RNAi treatment, negative and untreat-
ed controls, respectively. The cumulative percentage of
juvenile mortality in the MiASB RNAi treatment was 23
times higher than negative control and 33 times higher
than the untreated control, which were significantly dif-
of M. incognita.
3.3. MiASB RNAi Caused High Mortality of M.
incognita Juveniles Hatched from the
Egg Masses
RNAi of MiASB significantly inhibited the hatching of
M. incognita egg masses, and also caused high mortality
of the juveniles (Figure 3). On the first day, the mortality
Y. H. Huang et al. / Agricultural Sciences 4 (2013) 4 83-490 487
ferent (F = 6417.04, P < 0.0001).
3.4. MiASB RNAi Caused High Mortality of M.
incognita J2
M. incognita J2 was soaked in the dsRNA solution di-
rectly and were died. 6 h after treatment, about 434 of the
2000 J2 was found dead in the RNAi treatment, but 50 of
the 2000 J2 in the negative control and 17 of 2000 J2 in
untreated control were dead, respectively. The J2 mortal-
ity of RNAi treatment was 8.6 and 26 times higher than
the negative and untreated controls, respectively (Figure
4), and were significantly different (F = 157.34, P <
3.5. MiASB RNAi Treated Egg Masses
Reduced Root Galls on Tomato
RNAi treated egg masses were inoculated to the to-
Figure 2. The number of juveniles hatched from MiASB RNAi
treated egg masses of M. incognita was significantly lower than
those hatched from the negative and untreated controls 3 days
Figure 3. The mortality of juveniles hatched from MiASB
RNAi treated egg masses was significantly higher than those
hatched from the negative and untreated controls.
mato seedlings. 45 days later, the root galls were induced
on all the tomato seedlings. For the 1-day-treatment, the
number of root galls were between 18 and 30 in the
RNAi treated seedlings, and those ranged from 48 to 112
for the two controls. The average number of root galls on
RNAi treated seedling sreduced by 70.6% and 74.7%
compared to the negative and untreated control seedlings,
respectively (Figure 5) and were significantly different
(F = 37.1, P < 0.0001). For the 3-day-treatment, the root
gall amounts on the RNAi treated seedling were between
0 and 10, but those on the two controls ranged from 30 to
79. The average number of root galls on negative control
and untreated control seedlings were 51.7 and 59, re-
spectively, and that on the silenced seedlings was only
3.8 (Figure 5). Statistical analysis showed a significant
difference existed among the number of root galls on the
three differently treated seedlings (F = 38.1, P < 0.0001).
The amount of root galls on the RNAi treated seedlings
reduced by 92% and 93% compared to the negative and
untreated control seedlings, respectively.
Figure 4. The mortality of J2 in the MiASB RNAi treatment
was significantly higher than those of the negative and un-
treated controls 6 h later.
Figure 5. The root galls induced on the tomato seedlings in-
oculated with MiASB RNAi treated egg masses were signifi-
cantly reduced compared to those inoculated with the negative
and untreated control egg masses 45 days later.
Copyright © 2013 SciRes. OPEN ACCES S
Y. H. Huang et al. / Agricultural Sciences 4 (2013) 4 83-490
3.6. MiASB RNAi Treated M. incognita J2
Reduced Root Galls on Tomato
MiASB RNAi treated J2 were inoculated to the tomato
seedlings. 45 days later, the root galls were induced on
all the seedlings. But the number of root galls grown on
negative control and untreated control seedlings was lar-
ger and the sizes were bigger compared to those on the
MiASB RNAi treated seedlings (Figure 6). The root gall
amounts on RNAi treated seedlings were between 38 and
57, but those on the two controls ranged from 240 to 368.
The average number of root galls on negative control and
untreated control seedlings was 273 and 321, respectively,
and that on the RNAi treated seedlings was only 46. Sta-
tistical analysis showed a significant difference existed
among the number of root galls on the three differently
treated seedlings (F = 30.64, P = 0.0007) (Figure 7). The
amount of root galls on RNAi treated seedlings reduced
by 83% and 86% compared to that on the negative and
untreated control seedling, respectively.
Plant parasitic nematodes have been so far refractory
to transformation or mutagenesis, and no reverse genetic
tool is available to analyze the roles of genes in parasit-
ism [1]. With the discovery of gene expression control
via small interfering RNA (siRNA) and micro RNA
(miRNA) molecules, biologists are exploring genes and
developmentfrom a new perspective [19]. Understanding
of this ubiquitous phenomenon has revealed RNAi as a
powerful tool to manipulate gene expression and analyze
gene function [10]. Induction of RNAi by delivering
double-stranded RNA (dsRNA) has been very successful
in the model nonparasitic nematode C. elegans, and
RNAi has been used to systematically analyze 90% of
the predicted genes on chromosome I [20], 96% of the
predicted genes on chromosome III [21], approximately
2500 nonredundant cDNAs [22], and the function of 350
oocyte cDNAs [23], 86% of the 19,427 predicted genes
Figure 6. Phenotype of underground parts of the differently
treated tomato seedlings. The number of root galls grown on
the negative control seedlings (B) and untreated seedlings (C)
were larger and the sizes were bigger compared to those on
MiASB RNAi treated seedlings (A).
Figure 7. The root galls induced on the tomato seedlings in-
oculated with MiASB RNAi treated J2 were significantly re-
duced compared to those inoculated with the negative and un-
treated control J2 45 days later.
of C. elegans and identified mutant phenotypes for 1722
genes [24], 98% of all genes predicted in the C. elegans
genome and developed a phenotypic profiling system
[25]. These projects have rapidly advanced the under-
standing of gene function in C. elegans. Subsequently,
RNAi has been applied successfully to animal parasitic
nematodessuch as root-knot nematode [1,26,27], cyst
nematodes [9,18,19,28] and Haemonchus contortus [29].
In the present research, based on the previously re-
searches, by soaking the egg masses and J2 of M. incog-
nita in dsRNA of MiASB, we found RNAi treatment sig-
nificantly inhibited the hatching of the eggs and caused
high morality of the juveniles, which showed we suc-
cessfully triggered RNAi in parasitic nematode M. in-
The silencing of root-knot nematode genes by soaking
in dsRNA highly depends on dsRNA uptake by nonfeed-
ing J2 [1]. No in vitro culture system is yet available for
plant parasitic nematodes, and the only free-living stage
is the infective J2 [9]. The invasive J2 did not take up
dsRNA from solution, so a major challenge in applying
RNAi to plant parasitic nematodes is getting the dsRNA
ingested by the nonfeeding J2. Urwin et al. [18] (2002)
useda neurotransmitter octopamine to induce feeding in
invasive parasitic cyst nematode J2, allowing uptake of
dsRNA from solution, and used this method to knock out
several genes. Rosso et al. [1] successfully induced up-
take by adding resorcinol. In our study, based on the pre-
vious studies, we supplemented 1% resorcinol and 50
mM octopamine to solve the problem. Incubating J2 in
1% resorcinol for 4 h resulted in active pharyngeal up-
take and did not show detrimental effects on nematode
infectivity and development, but long incubation times
showed a deleterious effect on the nematodes [1]. In the
present research, we incubated the egg masses and juve-
nilesin 1% resorcinol for 1 day, even for 3 days which
Copyright © 2013 SciRes. OPEN ACCES S
Y. H. Huang et al. / Agricultural Sciences 4 (2013) 4 83-490 489
were far longer than 4 h. To avoid the experiment devia-
tion and improve experiment accuracy, we designed two
controls, one was negative control to counteract the ef-
fect of the reagent including resorcinol and other ele-
ments supplemented in the solution. The other was sterile
water to replace M9 buffer solution and the supplements.
But the results showed no significant difference in the
hatching inhibition and juveniles mortality between the
two controls.
In F1F0-ATP synthase, the static peripheral stalk (δb2)
do not appear to be a part of the main stalk, but it has
been suggested that they form a second connection, a
stator that fixes the catalytic α3β3 subcomplex to the
subunit to allow rotation of central stalk and c-ring (cn)
[30]. And synthesis of ATP depends upon rotation of the
central stalk (γε) and c-ring (cn) [15]. In the present re-
search, the nematode egg masses and juveniles were
soaked in the dsRNA solution of MiASB, which triggered
RNAi in the eggs and the juvenile bodies, resulting in the
inhibition of egg hatching and high morality of juveniles,
and further reduced the pathogencity. In the process of
growth and development, nematodes need ATP as energy
to perform various activities including infection. Once
the RNAi of MiASB was triggered in the nematode, it
caused the reduction of b subunit in ATP synthases, fur-
ther impaired the formation of proton channel portion of
ATP synthase, finally impacted the production of ATP. In
return, the reduced ATP production slowed down the
metabolism of the nematode, leading to the low hatching
ability, high mortality and reduced infection, which fi-
nally resulted in the low nematode disease.
In conclusion, we successfully triggered RNAi in the
M. incognita, and RNAi of MiASB significantly reduced
the nematode disease. The finding of this research sug-
gested MiASB may be associated with the pathogencity
of the nematode, and it provided new ideas and ways to
further study on nematodes pathogenic mechanisms and
prevention and control of nematodes.
The research was supported by the Major State Basic Research De-
velopment Program of China (Grant No.2009CB119000), Special Fund
for Agro-scientific Research in the Public Interest (Grant No.
201103018) and National Natural Science Foundation of China (Grant
No. 31030057). We sincerely thank anonymous peer reviewers for the
coming suggestions.
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