The specific host plant DNA detection suggests a potential migration of Apolygus lucorum from cotton to mungbean fields
The specific host plant DNA detection suggests a potential migration of Apolygus lucorum from cotton to mungbean fields
Qian Wang 0 1
Wei-Fang Bao 1
Fan Yang 1
Bin Xu 0 1
Yi-Zhong Yang 0 1
0 College of Horticulture and Plant Protection, Yangzhou University , Yangzhou , China , 2 State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences , Beijing , China
1 Editor: J Joe Hull, USDA Agricultural Research Service , UNITED STATES
The polyphagous mirid bug Apolygus lucorum (Heteroptera: Miridae) has more than 200 species of host plants and is an insect pest of important agricultural crops, including cotton (Gossypium hirsutum) and mungbean (Vigna radiata). Previous field trials have shown that A. lucorum adults prefer mungbean to cotton plants, indicating the considerable potential of mungbean as a trap crop in cotton fields. However, direct evidence supporting the migration of A. lucorum adults from cotton to mungbean is lacking. We developed a DNA-based polymerase chain reaction (PCR) approach to reveal the movement of A. lucorum between neighboring mungbean and cotton fields. Two pairs of PCR primers specific to cotton or mungbean were designed to target the trnL-trnF region of chloroplast DNA. Significant differences in the detectability half-life (DS50) were observed between these two host plants, and the mean for cotton (8.26 h) was approximately two times longer than that of mungbean (4.38 h), requiring weighted mean calculations to compare the detectability of plant DNA in the guts of field-collected bugs. In field trials, cotton DNA was detected in the guts of the adult A. lucorum individuals collected in mungbean plots, and the cotton DNA detection rate decreased successively from 5 to 15 m away from the mungbean-cotton midline. In addition to the specific detection of cotton- and mungbean-fed bugs, both cotton and mungbean DNA were simultaneously detected within the guts of single individuals caught from mungbean fields. This study successfully established a tool for molecular gut-content analyses and clearly demonstrated the movement of A. lucorum adults from cotton to neighboring mungbean fields, providing new insights into understanding the feeding characteristics and landscape-level ecology of A. lucorum under natural conditions.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This research was supported a grant
from the Key Project for Breeding Genetically
Modified Organisms (2016ZX08012-004). The
funder had no role in study design, data collection
and analysis, decision to publish, or preparation of
Competing interests: The authors have declared
that no competing interests exist.
Transgenic Bacillus thuringiensis (Bt) cotton was widely adopted for management of
Helicoverpa species infestation in northern China during the late 1990s. As a result of reduced
insecticide applications, the mirid bug Apolygus lucorum (Hemiptera: Miridae) has become the most
dominant insect pest in the Yangtze River and Yellow River cotton-growing regions of China
]. As a polyphagous herbivorous pest, A. lucorum has more than 200 recorded host plant
species and causes economic injury to cotton and other crops, including fruit trees and tea
plants [3±5]. Such a broad host range together with its remarkable reproduction ability,
longdistance dispersal capacity  and relatively inefficient control by predators [
] increases the
destruction by these pests. To date, the suppression of mirid bug populations in the field still
relies heavily on chemical pesticide applications, leading to problems associated with resistance
development, pest resurgence and environmental pollution; thus, an environmentally friendly
pest management strategy is required, such as ªpush-pullº habitat management that utilizes
repellent plants and attractant trapping crops to manipulate the population density of pests on
the target crop [
Large-scale field trials have shown that A. lucorum population abundance significantly
differs among distinct habitats and host plants [
]. Apolygus lucorum adults typically exhibit clear
preferences for particular plant species or plant growth stages [
]. From April to mid-June, A.
lucorum reportedly prefers to settle and oviposit on Medicago sativa L and Vicia faba L. [
During the summer, the population density of A. lucorum on mungbean (Vigna radiata L.)
appears to be significantly higher than that on cotton and other crops [
]. Combining the
results from both field cages and open-field plots, Geng et al. (2012) reported that adult
preference and nymph development are greater on mungbean than on cotton [
]. Additionally, the
densities of these insects are significantly lower on cotton plots with mungbean strips than on
cotton plots without mungbean [
]. These results suggest that mungbean cultivated near
cotton fields might attract A. lucorum adults. Two-choice bioassay studies using a Y-shaped
olfactometer demonstrate that A. lucorum adults prefer mungbean to cotton [
]. However, this
conclusion is based on laboratory observation and might not necessarily reflect reality under
natural conditions. Direct evidence supporting the migration of A. lucorum from cotton to
mungbean in open fields is lacking. In addition, studies identifying the host plants and
determining dietary choices of this mirid bug rely heavily on conventional methods such as visual
inspection. Because of the inconspicuous nature of symptoms after A. lucorum feeding, visual
inspection for maceration symptoms to estimate feeding status might lead to false positive
results in field surveys. Hence, a more accurate approach to assessing diet and to obtain direct
information on the landscape-level movement of A. lucorum should be developed.
Diverse approaches have been developed to assess movement or identify arthropod host
species [13±16]. Nevertheless, all of these useful methods appear to have inherent limitations.
Molecular gut content analysis can overcome conventional methodological hurdles and
provides an appealing alternative to address these issues [
]. DNA-based gut content analyses
are commonly used to track the trophic interactions between predators and prey [19±22].
Because multiple candidate DNA barcodes, including the internal transcribed spacer regions
(ITS) of nuclear ribosomal DNA [23±25], the ribulose bisphosphate carboxylase gene large
subunit (rbcL) [26±29], and the regions between tRNA for leucine (trnL) and tRNA for
phenylalanine (trnF) (trnL-F regions) [30±35] on chloroplast DNA, have been successfully used to
differentiate plant taxa, different DNA-based approaches have been developed to analyze the
gut contents of herbivorous insects . Diagnostic PCR employing general and specific
primers [24, 25, 36±38] and the continual improvement of next generation sequencing (NGS) [
allows the identity of phytophagous insect host plants to be recognized at the family [
] and even species levels [
23, 24, 42,43
In this study, we designed specific primers targeting the trnL-trnF region and established
two PCR assays for detecting the DNA of the two host plant species G. hirsutum and V.
radiata. The specificity and sensitivity of both PCR assays were assessed. Subsequently, this newly
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developed plant DNA detection approach was employed to assess potential A. lucorum adult
movement from cotton to mungbean fields under natural conditions.
Assessment of the specificity of the designed plant primers
Plant-specific primers for G. hirsutum and V. radiata (Table 1) were designed based on an
alignment of the trnL-trnF region among different host plant species of A. lucorum. To
confirm their species specificity, the primers were tested against 31 non-target plants (Table 2).
The results showed that all extracted plant DNA samples, except for the negative controls,
exhibited a similar 120 bp band when general plant primers targeting the trnL region were
used (S1 Fig), indicating the successful plant DNA extraction. The results of a cross-reactivity
test revealed that the primers designed for cotton and mungbean amplified bands of 236 bp
and 199 bp, respectively, and sequencing verification confirmed that these products were
indeed cotton and mungbean. In addition, fragments at the same position were not observed
when other plant DNA extracts were used (S2 Fig). These combined results indicate that the
designed primers are species specific.
Analysis of the plant DNA detectability half-life
The PCR assay did not show clear amplification using general plant primers targeting trnL,
suggesting that no host plant DNA remained in the gut and that adult A. lucorum were hungry
enough to be readily fed. As shown in Fig 1, PCR amplicons were most consistently observed
from adult A. lucorum immediately removed from plants, and declined with increasing
digestion time. The maximum-life for plant detection did not reach 100% at time = 0 h and the
value was significantly longer for cotton DNA than that for mungbean DNA. A negative
exponential equation was constructed based on the detection curve, from which the detectability
half-life (DS50) was calculated as 8.26 h for cotton, which was approximately two times longer
than that of mungbean (4.38 h) (t = 4.53, df = 4, P = 0.01) (Fig 1A and 1B).
Plant DNA detection rates in field-collected bugs and distance effects
To assess whether adult A. lucorum feed on mungbean and/or cotton in open fields and
whether adult bugs in cotton fields migrate to mungbean plots, we collected adult A. lucorum
from mungbean fields and performed molecular gut-content analyses using our newly
developed species-specific DNA detection system. The average number of adult bugs specifically fed
mungbean was estimated at 17.75, while the average number was estimated at 8.75 for those
specifically fed cotton and 9.0 for those simultaneously feeding on both plants (n = 60 for each
repetition). In total, the weighted detection rate was 44.64% for mungbean and 15.65% for
cotton (Fig 2A). The simultaneous detection rate for both of these plants shown in the black part
of each column in Fig 2A accounted for 50.70 percent of all identified cotton, which was much
higher than the proportion (33.64%) of that in all the detection rates of mungbean (Fig 2A).
To evaluate the effect of distance on plant DNA detection rates, adult A. lucorum in
mungbean plots 5, 10 and 15 m away from the mungbean-cotton midline were sampled and
assessed. The results of this analysis revealed that the weighted detection rate of mungbean
DNA in adult A. lucorum guts collected in mungbean plots was not significantly different for
the three distances (F2, 9 = 1.24, P = 0.3332). However, the weighted detection rate of cotton
DNA in adult A. lucorum guts varied for the three tested distances (F2, 9 = 26.45, P = 0.0002),
with the highest detection rates observed at 5 m, followed by 10 m and 15 m (Fig 2B).
In this study, we successfully developed a PCR-based method to detect plant DNA ingested by
A. lucorum, and the high specificity and sensitivity to distinguish between cotton and
mungbean indicates that this is a feasible approach for assessing the movement of mirid bugs at the
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Fig 1. Detection of cotton (A) and mungbean (B) DNA in the guts of A. lucorum adults at different times after ingestion. Error bars
at each point on the curves represent the standard error of three replicates. The model for the relationship between ingestion time (x) and %
positive (y) was y = 100%×e(2.27±0.26x)/(1+e(2.27±0.26x)), F = 251.18, df = 2,5, P<0.0001, (A) and y = 100%×e(1.53±0.37x)/(1+e(1.53±0.37x)),
F = 139.22, df = 2,5, P<0.0001 (B).
Fast-evolving sequences of the chloroplast genome region make it a suitable DNA barcode
to identify plants [
]. PCR assays using specific/general primers targeting chloroplast DNA
have been directly applied to examine the food choices of herbivorous insects with chewing
feeders [34±37] and sucking feeders on both plant cells [
] and on phloem sap [
when using chloroplast DNA for insect diet assessment, a trade-off occurs between
amplification success and host plant resolution. Hereward and Walter suggested that the chloroplast
trnL intron was not successfully amplified from target plant DNA in the green mirid bug
Creontiades dilutus because of degradation by extra-oral digestion [
]. The region of P6 loop
of the trnL intron is reportedly more robust for amplification from degraded DNA but exhibits
lower host species resolution [
]. The mirid bug A. lucorum in this study resembles C. dilutus
and Lygus spp. in feeding behavior; all of them perform extra-oral digestion and lacerate and
macerate plant cells using stylet-probing movement and watery salivary discharge [
addition, an objective of our study was to discriminate cotton from mungbean, the two defined
host plants of A. lucorum [3±6], thereby both the amplification success and the resolution
require fully consideration. We used two pairs of specific primers targeting the trnL-trnF
region of chloroplast DNA to develop a DNA-based molecular diagnostic system. The
sequence of the trnL-trnF region has been previously reported to show a high degree of
variation across families and thus provides robust resolution when applied to systematic studies
below the family level [
]. As expected, positive amplicons were observed, and the designed
primers were confirmed to be specific for cotton and mungbean (S1 and S2 Figs).
Our feeding experiment revealed that the maximum-life for plant detection did not reach
100% at time = 0 h, which resembled previous reports [
] and may be attributed to either
the effect of extra-oral digestion in A. lucorum or the pretended ingestion of plant tissues. The
detectability of cotton and mungbean DNA was significantly different, with cotton exhibiting
a longer detectable timeÐits DS50 value was approximately two times higher than that of
mungbean (Fig 1A and 1B). This result is consistent with findings for the Agriotes click beetle
larvae , which suggest that plant identity can affect post-feeding DNA detection. Here, our
results indicate that the mungbean digestion rate is higher than that of cotton. When feeding
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Fig 2. Detection rates of host plant DNA in mungbean field-collected bugs and the effect of distance.
(A) The weighted DNA detection rates for mungbean and cotton in A. lucorum adult guts collected from
mungbean fields were 44.64% and 15.65%, respectively. The black part of each column represents the
percentage of simultaneously detected cotton and mungbean DNA. (B) Effect of different distances on the
weighted DNA detection rates of mungbean and cotton in adult A. lucorum collected from mungbean plots.
Error bars represent the standard error, and different letters (a) and (α, û and χ) indicate no difference or a
significant difference (p<0.05), respectively, among the tested samples.
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on the plants, mirid bugs typically discharge lytic salivary enzymes such as salivary
polygalacturonases, proteases, amylases and glucose dehydrogenase that facilitate extra-oral digestion of
host tissues [
]. Moreover, persuasive evidence indicates that mirid bugs like Lygus hesperus
Knight prefer to feed on squares that have been preconditioned by salivary enzymes released
during early stylet-probing activities [
]. In A. lucorum, previous experiments have shown
that protease and amylase activities are significantly higher in mungbean-fed adults than in
cotton-fed adults [
]. Additionally, the difference in digestion rates correlates to A. lucorum
preference and performance for the two host plants, as both longevity and fecundity were
improved in mirid bugs that fed on mungbean compared to those that fed on cotton [
Therefore, the different digestion rates for cotton and mungbean by A. lucorum adults might
be partly ascribed to either distinct effects on catalytic digestive enzyme activity or a different
relative fitness between these two host plants. However, these inferences require further
Molecular gut content analyses of ingested plant DNA can provide new insight into the
landscape-level ecology of polyphagous insects. Recently, this technique has been
demonstrated to be suitable for assessment on which host plants a polyphagous potato psyllid
Bactericera cockerelli fed before the emergence of target host crops [
]. Our study revealed that the
detectability of plant DNA decreased gradually with digestion time (Fig 1A and 1B), suggesting
that the detection of both cotton and mungbean DNA was negatively correlated with the
duration of digestion. Nevertheless, a weighted detection rate was used, which can correct for field
data and compensate for different digestion times [
]. According to this method, the
weighted detection rates of field-collected bugs were comparable, and this finding could be
important for confirming the presence of A. lucorum host plant species in open fields as well as
for assessing their potential movement at the landscape level.
The mirid bug, A. lucorum resembled other mirid species like C. dilutus and Lygus spp. by
exhibiting high mobility [
], and their observed occurrence on plants did not necessarily
suggest regular feeding. The positive detection of ingested cotton and mungbean DNA in the guts
of field-collected bugs indicated that A. lucorum adults in the field can feed on both cotton and
mungbean, which was consistent with previous conclusions that both of them are A. lucorum
host plants [3±6, 9]. In addition, the positive detection of cotton DNA in the guts of mirid
bugs caught on mungbean plots suggested movement from cotton to the adjacent mungbean.
This inference is more persuasive when the successively decreasing DNA detection rates for
cotton from 5 to 15 m is compared with similar detection rates over distances for mungbean
(Fig 2B). Likewise, based on molecular gut content analyses, Hereward and Walter proposed
that the mirid bug C. dilutus often fed on host plant species other than the one from which it
had been collected, indicating potential movements and multiple host usage of a mirid bug
]. Our newly developed specific molecular diagnostic approach supported this inference; in
addition to the specific detection of mungbean or cotton DNA, both mungbean and cotton
DNA was detected in the gut of a single individual bug (Fig 2A). Interestingly, the ratio of bugs
showing mixed feeding on both cotton and mungbean to bugs that were specifically cotton-fed
was approximately 1:1, suggesting that half of the A. lucorum adults moved from cotton to the
mungbean field fed on mungbean. This feeding habit was similar to another Hemipteran,
Nezara viridula L, which moved from one plant species to another during feeding [
Velascol and Walter proposed that host-switching enhances survival and reproduction of N. viridula
]. However, whether the ecological significance of A. lucorum movement resembles that in
N. viridula or whether A. lucorum adult benefits from a combination diet under natural
conditions still requires further investigation.
In conclusion, this study developed a molecular approach to identify remaining host plant
DNA from the gut of a polyphagous mirid bug, A. lucorum. Our findings herein provide direct
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evidence supporting the movement of A. lucorum adults from cotton to mungbean fields and
are a significant step toward exploiting the full potential of DNA-based molecular detection
techniques to study the landscape-level ecology of A. lucorum under natural conditions.
Materials and methods
Insect rearing and plant collection
To perform the feeding experiment, a colony of A. lucorum has been established. Briefly, A.
lucorum mirid bugs were obtained from a colony and reared on fresh green bean (Phaseolus
vulgaris) pods in the laboratory at the Langfang Experimental Station at the Institute of Plant
Protection of the Chinese Academy of Agricultural Sciences (IPP-CAAS). The colony was
maintained at 25±1ÊC with 60±10% RH and a 16:8 h L:D photoperiod. To design specific
primers and develop the molecular diagnostic system, the following host plants were selected:
cotton, mungbean and 31 non-target plant species across 16 families (Table 2). These
non-target plant species were all A. lucorum host plants and widely distributed across northern China.
All plant specimens were collected at Langfang Experimental Station in the summer of 2014
and stored at -80ÊC until their use.
Plant DNA extraction
Plant DNA was extracted from 1 cm diameter leaf discs using the DNeasy Plant Mini Kit
(QIAGEN, Hilden, Germany) following the manufacturer's protocol, and all the extracted
DNA samples were quantified using a spectrophotometer (NanoDrop™ 1000, Thermo Fisher
Scientific, USA) and stored at -20ÊC until use. To check for cross-sample contamination
among extractions, two negative controls were included. General primers located in the trnL
chloroplast DNA regionÐc B49317 primer (5’-CGAAATCGGTAGACGCTACG-3’, for the
trnL (UAA) 5' exon [
]) and trnL 110R primer (5’-GATTTGGCTCAGGATTGCCC-3’, for
the trnL (UAA) intron [
]) function as a positive control to verify whether the plant DNA
was well extracted, because they result in amplicons with similar sizes (approximate 120 bp) in
different plant species.
Primer design and specific assessment
The trnL-trnF region of the host cotton, mungbean and other non-target plants was aligned
using the BioEdit sequence alignment editor 184.108.40.206 [
], and specific primers for cotton and
mungbean were designed using Primer Premier 5 version 5.00 [
]. Plant sequences were
obtained from GenBank, including sequences for G. hirsutum (HQ696725), V. radiate
(JX233513) and the non-target plants. The specificity of the primers was tested by amplifying
DNA from the leaves of those 31 non-target plant species by PCR (Table 2). The PCR products
were analyzed on 2.0% agarose gels, purified using the Axygen Gel Extraction kit (Axygen)
and cloned into the pGEM-T easy vector (Promega, Madison, WI, USA). Positive clones were
selected by PCR and sequenced using an ABI3730XL automated sequencer (Applied
Biosystems) with standard M13 primers.
To test the sensitivity of the newly established molecular detection system, a feeding
experiment was performed. Before the feeding trial, 3-day-old adult A. lucorum specimens were
starved for 48 h to confirm no plant tissues remains within their guts (at 25±1ÊC). Tender
cotton and mungbean leaves were separately placed in a Petri dish (10 cm diameter, 2.6 cm
height). Thereafter, each adult bug was separately introduced into each Petri dish for 3 h at
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25ÊC and then observed every 10 min (a mirid bug seen with its stylet inserted into the leaf at
least three times was considered to have fed) [
]. After feeding, individuals were maintained
at 25ÊC for 0, 2, 4, 8, 12, 16, 20 and 24 h (10 individuals per time point as a repetition, a total of
three repetitions), and they were then frozen at -80ÊC until evaluation via PCR.
According to Wallinger et al. (2013) , the sensitivity of the PCR assays was assessed via
serial dilution of template DNA (either the plant DNA template or the mirid bug DNA
template extracted from samples in the feeding experiment). The results showed that no significant
difference in primer efficiency within the same dilution ratio. No positive amplicons were
observed at the lowest concentration tested by either the cotton or mungbean primers. A 4 μL
extracted bug DNA solution (10 ng/μL) is able to provide positive amplicons for both cotton
and mugbean primers.
Insect DNA extraction
DNA from adult A. lucorum was extracted followed a CTAB-based protocol described by
Wallinger et al. . To avoid DNA amplification from plant material that may have adhered
to the body surface of the mirid bugs, each adult mirid bug was cleaned following a modified
method described in previous studies [
35, 56, 57
]. Briefly, each individual insect was placed in
1 mL of 1±1.5% sodium hypochlorite (Beijing Chemical Works, Beijing, China) for 5 s. Each
individual was then rinsed twice with molecular-grade water. Our preliminary trial showed
that this method successfully removed external plant DNA contamination and did not destroy
the ingested plant DNA in the gut of A. lucorum. Two extraction-negative controls were
included in each batch of 24 samples to check for cross-sample contamination.
PCR amplifications were performed in 20 μL reaction mixtures containing 4 μL DNA solution
(10 ng/μL), 2 μL 10× Taq buffer (TransGen Biotech, Beijing, China), 0.4 μL dNTP (2.5 mM),
0.2 μL Easy Taq (5 units/μL) (TransGen Biotech), 0.75 μL each primer (10 μM), and 11.9 μL
autoclaved distilled water. The PCR reactions were performed in Veriti 96-Well Thermal
Cyclers (Applied Biosystems, USA). The thermo cycling protocol began with an initial
denaturing step of 95ÊC for 10 min, followed by 35 cycles of 95ÊC for 30 s, 56ÊC for 30 s and 72ÊC
for 1 min, and a final extension of 72ÊC for 10 min. The PCR products (6 μL) were then
separated using a 2% agarose gel in TAE buffer (40 mmol/L Tris-acetate, 2 mmol/L Na2EDTA
H2O) and visualized with a UV trans-illuminator. Two positive (cotton or mungbean plant
DNA) and two negative controls (PCR-grade water instead of extracted insect DNA) were
included in each PCR assay to determine amplification success and DNA carry-over
To assess the movement of A. lucorum adults from cotton to mungbean fields, field plots of
cotton and mungbean were established at the Langfang Experimental Station of IPP-CAAS
(39.53ÊN, 116.70ÊE) in Hebei Province, China in 2014. Cotton and mungbean plots pairs (20
m x 30 m, each) comprised a 20 m adjoining midline edge area (S3 Fig). Four replicates of
these two host plant block (20 m x 60 m) were considered. The four blocks were arranged in a
straight line from south to north, with 10 m intervals between adjacent blocks. A road and a
wall were situated along the western side of the field blocks, a road was situated along the
eastern side of the blocks, and roads and walls were situated at the northern and southern ends of
the group of blocks. All the blocks received the same fertilizer and irrigation, and insecticide
was not applied during the experimental period.
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Molecular detection of field-collected samples and distance effects
To confirm whether adult A. lucorum in mungbean plots migrated from the adjacent cotton
plots and to evaluate the detection rate, we collected the adult A. lucorum in late July with
sweep netting (38 cm diameter) in mungbean fields. The sampling range of each plot was 15
m away from the cotton-mungbean midline. Approximate sixty lively and undamaged adult
bugs caught in each mungbean plot were subjected to molecular analysis using both
mungbean- and cotton-specific primers. To assess the effect of distance, adult A. lucorum within
each plot of mungbean were collected by sweep netting (38 cm diameter) along parallel lines
5, 10, and 15 m away from the cotton-mungbean midline. Each line was sampled by one
hundred sweep nets (38 cm diameter), and the sampling depth was 20 m, i.e., the length of
each designed plot. To avoid adult dispersal, sampling was performed simultaneously at
each distance. Subsequently, approximately thirty lively and undamaged A. lucorum adults
from each line were randomly selected for analysis. For molecular detection, each individual
was transferred to a 1.5 mL microcentrifuge tube, transported back to the laboratory (within
1 h) on ice, and immediately frozen in liquid nitrogen. All the selected mirid bugs were
stored at -80ÊC until their DNA was extracted. PCR assays were performed as previously
The effect of digestion time (i.e., time post-feeding) on plant DNA detection success was tested
for the species-specific plant primers using a logistic regression with a binomial distribution
(PROC GENMOD). SAS 9.30 software was used to estimate the DS50 values [
] for the two
plant species, which were compared using t-tests.
We used a previously described method [
] to weight the plant detection rates and
better interpret the extent of plant DNA in the gut contents under natural conditions. Briefly, the
weighted detection rates were corrected based on the detection rates, the shorter DS50 of plant
DNA was assigned an importance weighting value of 1.0, and the importance weighting value
of the DNA of the other plant was obtained using this benchmark DS50 as the numerator and
the DS50 of the other plant as the denominator. The corrected detection rate was calculated by
multiplying the proportion of plant DNA in the field-collected A. lucorum adults by the
importance weighting value. The weighted detection rates of the samples collected at different
distances from the mungbean-cotton midline were compared via a one-way analysis of variance
S1 Fig. PCR amplification from all of the extracted plant DNA samples using general
primers located in the trnL chloroplast DNA region. Lines 1±4, negative controls for DNA
extraction; lines 5±6, negative controls for PCR amplification; lines 7±72, all of the plant
species samples with two biological replicates. M is a 50 bp DNA ladder.
S2 Fig. PCR amplifications from all of the extracted plant DNA samples using mungbean
V. radiata (A) and cotton G. hirsutum (B) specific primers targeting the trnL-trnF region.
Each sample includes two biological replicates. Lines 1 and 2, negative controls; lines 3 and 4,
the targeted host plant species, mungbean or cotton; lines 5±66, 31 non-targeted host plants
similar to the list in Table 2. M is a 50 bp DNA ladder.
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S3 Fig. Schematic diagram of the designed cotton and mungbean field plots.
This research was supported a grant from the Key Project for Breeding Genetically Modified
Organisms (2016ZX08012-004). We greatly thank the anonymous reviewers for their valuable
comments. This manuscript has been edited by the native English-speaking experts of
American Journal Experts.
Conceptualization: QW YZY.
Data curation: QW WFB YZY.
Formal analysis: QW FY YZY.
Funding acquisition: YZY.
Investigation: QW WFB.
Methodology: QW BX.
Project administration: YZY.
Resources: QW YZY.
Software: QW YZY.
Validation: QW YZY.
Visualization: QW YZY.
Writing ± original draft: QW.
Writing ± review & editing: QW YZY.
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