Aphids are common and serious pests for a wide range of
vegetables and crops. There are many aphid species, the most common of which
are Myzus persicae
(green peach aphid), Lipaphis erysimi
(turnip aphid) and Brevicoryne brassicae
(cabbage aphid). As most
aphids possess distinct morphological features, and the methods of
morphological analysis are simple, intuitive, and easy to use,
morphological classification methods have dominated the
identification of aphid species for a long time (Zhao et al. 2013). However,
aphids are among the
fastest-evolving insects within Hemiptera. Because of their abundant populations, the multidirectionality of evolution, and the directional nature of natural selection,
the morphologies of certain allied aphids can be very similar. Therefore, it can be extremely difficult to
distinguish allied aphid species simply based on external morphology and morphologic degeneration led to
fewer features can be used for taxonomy and phylogenetic analysis (Huang and Qiao 2006). Many researchers have investigated the
classification of insects through mitochondrial
DNA (mtDNA) using
techniques, such as polymerase
chain reaction-restriction fragment length polymorphism (PCR-RFLP), random
amplification of polymorphic DNA (RAPD-PCR) and DNA probes. These studies provided effective means for the
identification of infraspecific taxa as well as the identification and
discrimination of allied species (Lu and Gu 1995; Wei et al. 2010). In particular, RAPD-PCR was widely applied
in studies on the classification of aphid populations (Black et al. 1992; Cenis et
al. 1993) and
the RAPD-PCR marker technique has previously played a major
role in promoting the development of molecular ecology. However, this technique is limited in its ability to
differentiate between heterozygous and homozygous genotypes, and
thus, is ineffective in distinguishing heterozygous genotypes. In addition, RAPD reactions are readily influenced by various
external factors and have poor repeatability, limitations which have hampered the
scope of its applications (Zhang et al. 2000). In
fact, journals such as Molecular Ecology no longer endorse the use of RAPD as a tool for scientific research (Wang et al. 2007a). On the other hand, PCR-RFLP is widely
used in research because of several strengths, including the fact that it does
not require the addition of markers or probes in samples, it is highly
sensitive, low-cost, exhibits high reproducibility, and is very robust.
Furthermore, it is not affected by external
factors, such as age and sex and gene products, and the targets of RFLP are widely distributed
across the genome (Faten et al. 2002; Raboudi et al. 2005; Valenzuela et al. 2007).
Aphids
can damage plants either directly by
feeding on plant sap or indirectly via
the transmission of viruses and diseases (Shew and Lucas 1991). At present, chemical control remains the primary method of
aphid control; however, many aphids have developed differing levels of
resistance to a variety of insecticides in many countries (Harlow and Lampert 1990; Gubran et al. 1992; Hollingsworth et al.
1994; Ahmad et al. 2003; Foster et al. 2007; Wang et al.
2007b),
including neonicotinoid insecticides (Panini et al. 2014). It appeared that overexpression of one or more
P450s was the primary mechanism of neonicotinoid resistance in insect pests (Nauen and Denholm 2005;
Karunker et al. 2008). Moreover, R81T
mutation in the nicotinic acetylcholine receptor (nAChR)
𝛽 subunit
has been proved to be associated with resistance of aphids to neonicotinoid pesticide (Bass
et al. 2011; Puinean et al. 2013). This increased resistance
leads to increased insecticide use, which in turn raises
the cost of pest control and exacerbates environmental pollution. Thus, determination of resistance
development and resistance levels in aphids to each class of insecticide is
pivotal for the formulation of effective aphid control programs. Currently, the most
common resistance detection methods being used are biological and biochemical
methods. However, these approaches have several shortcomings requiring
substantial time and effort to perform as well as needing large numbers of
insect samples. The rapid and effective determination of resistance in pests to
certain classes of insecticides at the molecular level is crucial for insect
resistance management. Previous studies have shown that detection of the R81T mutation in the
acetylcholine receptor gene of the aphid genome is an effective method for
determining the resistance of aphids to neonicotinoids (Panini et al. 2014; Voudouris et al. 2016).
In this study, RFLP and
sequencing analyses were performed
on an aphid mitochondrial COI gene fragment (709 bp) to classify aphid
populations and identify the species found on
eight types of host plants in three regions of Hunan Province. The identified
aphid species subsequently underwent RFLP analysis of a 200 bp acetylcholine
receptor gene fragment containing the R81T
mutation, which confers neonicotinoid resistance. We then determined using RFLP analysis and
toxicity tests whether the aphids living on the eight aforementioned host
plants have neonicotinoid resistance. Our results from this study provide a
theoretical basis for using
RFLP to rapidly
and accurately identify allied aphid species and to
effectively detect neonicotinoid resistance in aphids.
Materials and Methods
Experimental
materials
Aphids
were collected in 2015–2016 from various host plants in the Changsha and Yueyang
regions of Hunan Province (Table 1). The
host plants were tobacco, rapeseed, cabbage, oleander, and radish in Changsha,
cabbage, cowpea, and hyacinth bean in Yueyang. Eight of the collected aphids
were used for DNA extraction, while the remaining aphids were reared on their corresponding
hosts for one generation in our laboratory; the leaf dipping method was then
used to conduct toxicity tests. The susceptible population used as a reference for
the toxicity tests was a population of M. persicae that were reared continuously on tobacco
plants in our laboratory without any insecticides. The rearing conditions were a temperature of 25 ± 1°C, a relative humidity of 70±5% and a light/dark photoperiod of 14 h and 10 h, respectively.
Experimental reagents
DNA extraction was performed using the TIANamp Genomic DNA Kit (Catalog No. DP304,
Tiangen Biotech Co., Ltd., Beijing, China) in
accordance with the manufacturer’s instructions. The Quick Gel
Extraction Kit, DNA markers, ExTaq DNA polymerase, 10×buffer
and dNTPs were all purchased
from Tiangen Biotech (Tiangen
Biotech Co., Ltd., Beijing, China). The
restriction endonucleases, BcoDI and HincII,
and buffers were purchased from New England Biolabs (New Jersey, New York, America) and the primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
Aphid
toxicity tests
Distilled
water containing 0.05% (v/v) Triton X-100 was used to dilute an
acetone-solvated acetamiprid microemulsion (Guizhou Daoyuan
Biotechnology Co., Ltd., Daoyuan, China)
into a graded series of concentrations. Distilled water containing 0.05% (v/v) Triton X-100 was used as a control.
The
leaf-dip method was used to test resistance of wingless aphids
larvae to acetamiprid according to Voudouris et
al. (2016) and revised. A hole punch was used to
punch leaf disks with a diameter of 2 cm from fresh, clean leaves of each host
plant. The disks were soaked in a pre-diluted insecticide solution for 10s,
removed, and dried either in a shaded and well-ventilated area for
approximately 30 min or dried through the removal of excess insecticide
solution using absorbent paper. Next, the disks were placed in a 12-well ELISA
plate pre-coated with 1 mL of 1% agarose gel and then all test disks were placed in
incubator at a temperature of were kept at 18°C and 16:8 h L:D. Apterous adult aphids
(20 per disk) were
carefully placed into the
leaf disks using an ink brush. Five insecticide concentrations were used in
each treatment, with three replicates
for each concentration.
The
ELISA plates were placed in an illuminated incubator at a temperature of 22±1°C and the number of dead insects was counted after 24
h. An aphid was considered dead if no leg movement was observed upon gentle
contact with a brush. A probit analysis program was
used to calculate the LC50 value, toxicity regression line (y= a+bx) and 95% confidence interval of
each tested insecticide.
Genomic DNA extraction from single aphids and amplification of mitochondrial
COI gene segments
A single
aphid was placed
in a 1.5 mL centrifuge tube to which 50 µL of homogenization buffer for DNA extraction was added. A
sterile disposable pestle was used to crush the aphid until the body completely
disintegrated into a cell suspension. Aphid genomic DNA was then extracted and Gel electrophoresis was performed. The gels were
visualized and photographed using 0.05% ethidium
bromide under UV
illumination. Gene Ruler 100 bp DNA Ladder Plus (Tiangen Biotech Co., Ltd., Beijing, China) was used as a molecular weight standard and a UV
spectrophotometer (Thermo, USA) was used
to measure the OD260 and OD260/280 values. Ten single aphid was extracted genomic DNA each
time.
To
amplify the mitochondrial COI gene fragment in aphids, we used the
following primers designed by Valenzuela et al. (2007) in their study on the determination of
allied aphid species: LCO1490(5′-GGTCAACAAATCATAAAGATATTGG-3′) and
HCO2198 (5′-TAAACTTCAGGCTGACCAAAAAATCA-3′). The PCR reaction
mixture for amplification of the 709 bp target sequence consisted
of 1×reaction buffer (3.5 mM MgCl2), 4 mM of dNTPs, 10 μM of both sense and antisense primers, 10ng template DNA, and 0.2 U Taq DNA polymerase (Takara, Japan), with
sterile water to a final volume of 25 μL. The PCR amplification conditions were as follows: 95°C for 6.5 min,
followed by 40 cycles of 94°C for30s, 50°C for 30s and 72°C for 40s, with a final extension at 72°C for 3 min.
Amplification products were separated by electrophoresis using 1.5% agarose
gels, and gels were examined using a gel imaging system.
mtDNA
sequencing and PCR-RFLP
Amplified
COI gene segments were sequenced and MEGA version 7.0
was used for sequence analysis
and construction of the
phylogenetic tree. All sequences were submitted to GenBank, and the New England Biolabs (NEB) cutter tool (NEBcutter, https://www.neb.com/tools-and-resources/interactive-tools) was used to determine the restriction endonuclease
cutting sites of the sequences obtained from each sample (Table 2). For restriction digestions, 20U DraI, 10U HinfI, and 20U SspI,
were added separately to centrifuge tubes containing 4ng of the DNA, which
were incubated overnight at 37°C. Similarly, 20U TaqI was
also added to a centrifuge tube containing 4ng of DNA followed
by overnight digestion at 65°C. Digestion products were separated by
electrophoresis using 2% agarose gels, which were examined using a gel imaging
system.
Detection of the R81T mutation
using PCR-RFLP
The primers designed by Voudouris et al.
(2016) in their study on the molecular detection of neonicotinoid
resistance in aphids (ApF:
5′-TCTAATTATGGGGTTAATTTATAGTCG-3′ and ApR:
5′-ACAGGCGGTCAGGAAGTGTA-3′) were used for PCR amplification of the 280 bp target sequence. The PCR reaction mixture consisted
of 1×reaction buffer (1.8
mM
MgCl2), 0.8
mM of dNTPs, 0.3 μM of the
sense and antisense primer, 10ng template
DNA, and 0.8 U of Taq DNA polymerase. Sterile water
was added to a final volume of 25 μL. PCR thermocycling conditions
were 94°C for 30s, 53°C for
30s and 68°C for 20
s, followed by 29
cycles. Amplification
were separated by electrophoresis using 1.5% agarose
gels, and the gels were evaluated using a gel imaging system. Next, a second
(nested) PCR was performed on the previously amplified 280 bp sequence using the
primers by Voudouris et al. (2016) to generate a 200 bp
target segment containing the R81T
mutation (nAChRF: 5′-CCTGCAGCTATTAAAATATCCA-3′and nAChRR:
5′-ACGTTAGAAAGGAAACTGTTTA-3′).
The PCR reaction mixture consisted of 1×reaction buffer (1.8 mM MgCl2), 0.8 mM of each dNTPs,
0.3 μM of each
of the sense and antisense primers, 10ng
template DNA, and 0.8 U Taq DNA polymerase, with sterile water to a final volume of 25 μL. The PCR amplification conditions were followed
by 29 cycles of 94°C for
30s, 58°C for
15s, and 68°C for 15s. For restriction digestion, 10U BcoDI, and 10U HincII were
separately added to centrifuge tubes containing 8ng of DNA, which
were incubated overnight at 37°C. Digestion
products were separated by electrophoresis using 2.0% agarose gels and the gels
were examined using a gel imaging system.
Results
Identifying
aphid species
PCR
amplification and sequencing of mtCOI in aphids: Using PCR amplification, we obtained approximately 700 bp bands from
genomic DNA extracted from aphids corresponding to the eight types of host plant in
three regions of Hunan Province
(Fig. 1). We found following sequencing and analysis of the PCR fragment that
the nucleotide segment was 709 bp in length, with a shared sequence similarity
of 92.38%
among aphid samples.
RFLP identification of mtCOI in aphids: Restriction digestion of the target COI gene fragments derived from aphid DNA samples was performed using
the four restriction endonucleases, DraI, HinfI, TaqI, and SspI.
Following DraI digestion
and sequencing, we found that the mtCOI PCR products of aphids
from cowpea and hyacinth bean plants in Yueyang,
cabbage, rapeseed, radish, and tobacco plants in Changsha were
cleaved into two Table 1: Sample collection details of the 7 aphid
populations examined in this study
|
Aphid population |
Collection site |
GPS latitude/longitude |
Collection date |
Host plant |
Temperature |
Intensity |
|
1 |
Changsha |
113.08,28.19 |
4th Apr., 2016 |
Tobacco |
14-17°C |
~20 per leave |
|
2 |
Changsha |
113.08,28.19 |
4th Apr., 2016 |
Rapeseed |
14-17°C |
~10 per leave |
|
3 |
Changsha |
113.08,28.12 |
26th Oct., 2016 |
Cabbage |
14-17°C |
~20 per leave |
|
4 |
Changsha |
113.09,28.19 |
18th May, 2016 |
Oleander |
18-24°C |
~10 per leave |
|
5 |
Changsha |
113.08,28.19 |
10th Oct., 2016 |
Radish |
17-24°C |
~15 per leave |
|
6 |
Yueyang |
112.75,28.68 |
15th Nov., 2016 |
Cabbage |
16-18°C |
~20 per leave |
|
7 |
Yueyang |
112.83,28.65 |
15th Nov., 2016 |
Cowpea |
16-18°C |
~15 per leave |
|
8 |
Yueyang |
112.83,28.65 |
15th Nov., 2016 |
Hyacinth
bean |
16-18°C |
~15 per leave |
Table 2: RFLP
fragment patterns (sizes in bp) using four
restriction endonucleases of a 709bp region of the mtCOI gene for identification of
aphid species
|
DraI |
HinfI |
TaqI |
SspI |
Host plant (Region) |
|
87, 622 |
709a |
91, 93, 525 |
108, 275, 326 |
Hyacinth bean and cowpea (Yueyang) |
|
87, 268, 354 |
709 |
91, 93, 525 |
108, 275, 326 |
Cabbage (Yueyang) |
|
87, 622 |
82, 201, 426 |
709 |
15, 108, 203, 383 |
Cabbage, rapeseed, radish, and tobacco (Changsha) |
|
709 |
709 |
91, 93, 525 |
275, 434 |
Oleander (Changsha) |
%20398-404_files/image002.jpg)
Fig. 1: PCR
amplification of a 709bp region of the mtCOI gene in aphids from seven types
of host plants in two regions
of Hunan Province. M: Gene Ruler 100bp DNA Ladder Plus;
Lanes 1-5: aphids
collected from tobacco, rapeseed, cabbage, oleander, and radish plants in
Changsha, respectively; Lanes 6-8: aphids collected from
cabbage, cowpea and hyacinth bean plants in Yueyang,
respectively
a
Undigested PCR product
fragments,
87 and 622 bp in
size. The mtCOI target segment of aphids from cabbage
plants in Yueyang was enzymatically cut into 87, 268
and 354 bp fragments,
while the target segment of aphids from oleander plants show
an undigested 709 bp product,
indicating a lack of DraI restriction sites
(Fig. 2a).
After enzymatic digestion
with HinfI
and sequencing, we found that the mtCOI PCR products
of aphids from cabbage, rapeseed, radish and tobacco
plants in Changsha were
cleaved into three fragments, 82, 201 and 426 bp, while those of aphids from hyacinth bean, cowpea, and cabbage
plants in Yueyang, and oleander plants in Changsha
were not digested (Fig.
2b).
Next, we found following TaqI digestion
and sequencing that the mtCOI target segments of aphids from hyacinth bean, cowpea,
and cabbage plants in Yueyang as well as aphids from
oleander plants in Changsha were digested into 91, 93 and 525 bp fragments. In
contrast, no TaqI digestion sites were found
in the target PCR product of aphids from cabbage, rapeseed, radish, and tobacco
plants in Changsha (Fig. 2c).
After SspI restriction
digestion and sequencing, we found that mtCOI PCR
products of aphids from hyacinth bean, cowpea and cabbage plants in Yueyang generated 108, 275 and 326 bp fragments. Further, the target PCR products of
aphids from cabbage, rapeseed, radish, and tobacco plants in Changsha were enzymatically cut into 15, 108, 203 and 383 bp fragments. We also
found that the mtCOI-derived amplification of aphids
from oleander plants in Changsha contained a single SspI
restriction site resulting in 275 and 434 bp fragments (Fig. 2d).
Nucleotide sequences of the
PCR-amplified 709 bp mtCOI region of aphids collected in this study were compared to
the corresponding 709 bp sequence region of various aphid samples in the NCBI
database using BLAST. A sample was deemed to belong to a corresponding species
in the database when the nucleotide sequence of a sample had 100% similarity
with a sequence of a particular species in the database. In particular, the sequences obtained for aphids
from hyacinth bean, cowpea, and cabbage plantsin Yueyang were identical to the reference NCBI sequence
for Aphis craccivora (KX447141.1), while the sequences obtained for aphids from
cabbage, rapeseed, radish, and tobacco plants in Changsha matched that found
for M. persicae (KU236024.1).
Phylogenetic tree of aphid samples: The
phylogenetic tree of
the aphids examined
in our study was constructed using MEGA version 7.0, based on the topological
structure of the mtCOI gene (Fig. 3). Because aphids from
%20398-404_files/image004.jpg)
Fig. 2: Identification of aphids from seven types of host plants in two regions of Hunan Province by restriction digestion using DraI(a), HinfI(b), TaqI(c), SspI(d). M: D2000 DNA ladder; Lanes 1-8:
target fragment patterns of aphids collected from hyacinth bean, cowpea, and
cabbage plants in Yueyang, cabbage, rapeseed, radish,
tobacco and oleander plants in Changsha, respectively
%20398-404_files/image006.jpg)
Fig. 3: Phylogenetic tree of the aphid
samples characterized in this study. (Maximum Likelihood Tree，2000 replications)
cabbage,
hyacinth bean and cowpea plants in Yueyang and the
known A. craccivora and A. glycines
species clustered in the same group, these species all likely belong to the
same genus. Similarly, aphids from radish, cabbage, tobacco, and rapeseed
plants in Changsha clustered in the same group and thus, may belong to the same genus,
while aphids from oleander plants in Changsha belong to a unique genus.
Molecular detection of insecticide resistance in
aphids
Toxicity tests on aphids: In
this study, we conducted relative acetamiprid
toxicity tests on aphids collected from different regions of Hunan Province
using an insecticide-sensitive strain as a reference, and determined toxicity
regression equations and median lethal concentrations (LC50) for
each aphid sample (Table 3).
Based on the results of the
toxicity tests, we found that the LC50 value of acetamiprid for aphids from all host plants were very low, and thus, indicates that the aphids collected in
this study have minimal
resistance to acetamiprid.
Genotyping
of the R81T mutation in aphid samples: The
280 bp DNA region of Myzus persicae
from Changsha was PCR amplified using the ApF
and ApR primers (Fig. 4).
We then performed nested PCR on the previously amplified 280 bp sequences using the nAChRF and nAChRR primers
obtaining 200 bp target amplification, which contain the R81T mutation (Fig. 5).
Identification of neonicotinoids resistance in aphids: Sequencing
analysis of the 200 bp amplified nested-PCR gene segments, which harbor the
R81T mutation, was performed for all the various aphid samples collected in
this study. Nested PCR amplification was successful from all samples, which
shared 100% sequence similarity.
Restriction
digestion of nested PCR products was performed using BcoDI and HincII, followed
by RFLP analysis
and sequencing. It was found that the R81T-containing PCR amplicon from
all aphid samples contained a BcoDI restriction site
resulting in 93 and 107
bp fragments,
while there was no digestion following HincII incubation because of the absence of restriction
sites in the PCR product (Fig. 6).
Discussion
Currently, morphological analysis is the primary method for
classification of aphids in China. In 1999, Qiao et al. (1999) classified gallnut aphids into 14 species (including
4 subspecies) in 5 genera according to the morphological features of a late
viviparous female aphids, the shape of the gallnuts, and differences in their
summer hosts. However, identification based on morphological analysis is heavily
reliant on the professional knowledge and experience of taxonomists, and
requires specimens with largely intact external morphological features. In
fact, it is often difficult to satisfy
these requirements during actual aphid classification processes. In addition, the morphologies of allied species
and allied genera of aphids are highly similar making
it extremely difficult to distinguish allied species through morphological
identification methods.
Table 3: Acetamiprid
sensitivity of aphid samples from
different regions of Hunan Province
|
Population |
Region |
Toxicity regression equation |
LC50 (mg/L) |
95%
confidence interval (CI (95%)) |
|
Tobacco |
Changsha |
Y=4.89+1.05x |
1.26 |
0.69-2.30 |
|
Rapeseed |
Changsha |
Y=4.70+1.07x |
1.92 |
1.05-3.48 |
|
Oleander |
Changsha |
Y=5.07+0.49x |
0.84 |
0.41-1.70 |
|
Radish |
Changsha |
Y=4.76+0.94x |
1.79 |
0.93-3.45 |
|
Cabbage |
Changsha |
Y=4.70+0.95x |
2.05 |
1.07-3.94 |
|
Hyacinth bean |
Yueyang |
Y=5.17+0.80x |
0.61 |
0.29-1.31 |
|
Cowpea |
Yueyang |
Y=5.19+0.81x |
0.59 |
0.28-1.25 |
|
Cabbage |
Yueyang |
Y=5.26+0.86x |
0.50 |
0.25-1.03 |
The techniques that are currently available for the
molecular classification of aphids include isoenzyme
%20398-404_files/image008.jpg)
Fig. 4: Identification
of a 280bp
gene fragment
containing the R81T mutation in aphids samples. M:
Marker I (DNA ladder); Lanes1-8: target bands of aphids
collected from hyacinth bean and cowpea plants in Yueyang,
cabbage plants in Changsha, cabbage plants in Yueyang,
rapeseed and radish plants in Changsha, and oleander and tobacco plants in
Changsha, respectively
%20398-404_files/image010.jpg)
Fig. 6: RFLP patterns following BcoDI
and HincII digestion of R81T
200 bp target fragments from the aphid samples
collected from host plants
in Yueyang and Changsha. M: DNA ladder; (a) Lanes1-10: cabbage, rapeseed, radish, oleander and tobacco plants in Changsha, respectively. (b) Lanes1-6:
hyacinth bean, cabbage
and cowpea plants in Yueyang
%20398-404_files/image012.jpg)
Fig. 5: Identification
of a 200bp nested PCR product containing the R81T
mutation in aphids collected from seven types of host plants in two regions of Hunan Province. M: Marker I (DNA ladder); Lanes1-8: target bands of aphids
collected from hyacinth bean and cowpea plants in Yueyang,
cabbage plants in Changsha, cabbage plants in Yueyang,
rapeseed and radish plants in Changsha, and oleander and tobacco plants in
Changsha, respectively
analysis,
PCR-RFLP, and RAPD-PCR.
The PCR-RFLP approach
for mitochondrial DNA analysis overcomes limitations
associated with RAPD such as its susceptibility to external factors, and the successful application of PCR-RFLP in the
identification of infraspecific taxa of insects have been reported in the
literature (Bogdanowicz et al. 1993; Cognate et al. 1999; Schroeder et al.
2003; Schroeder and Scholz 2005). Valenzuela et al. (2007) performed PCR-RFLP analysis on mitochondrial COI gene fragments (709
bp) of aphids, in which four
restriction endonucleases (DraI, HinfI,
TaqI and SspI) were used for leading to a single taxon. Based on analyses on the
restriction digestion patterns of the cleaved fragments, they classified 26
aphid haplotypes into 25
species. In
this study, we PCR amplified mitochondrial COI gene fragments (709 bp) of 8 aphid populations collected from seven types of host plants in two regions of Hunan Province. Based on the restriction
digestion patterns using the four restriction enzymes, DraI, HinfI, TaqI, and SspI, we found
that the aphids from oleander plants in Changsha were identical to those in the
study of Valenzuela et al. (2007),
as both aphid populations belong to the same
species, A. nerii. Furthermore,
the nucleotide sequences obtained for these aphids were also identical to the
reference sequence for A. nerii in the NCBI database (KU236024.1). However, aphids from hyacinth bean, cowpea, and
cabbage plants in Yueyang were identified as A. craccivora,
while aphids from cabbage, rapeseed, radish, and tobacco plants in Changsha
were identified as Myzus persicae. The
differences between our study may be attributed to the directional nature of
natural selection during aphid evolution (Feng et al. 2004) as well as the fact that aphid mutations are
induced by different climates, geographical environments, and hosts (Liu et al. 2009).
Biochemical
detection methods are the most prevalent approaches
for resistance detection in aphids, including the residual film method and the
leaf dipping method (Pan et al. 2000; Yu et al. 2016). We used the leaf dipping method to
conduct toxicity tests on the aphids collected in this study, and found that
all tested aphids were sensitive to acetamiprid, with LC50 values
between 0.50–2.05 mg/L (Table
3). However, biochemical detection methods are tedious, require a large number of samples, and
involve a number of uncontrollable factors (Wang and Xia 2004).
Assessment of the knockdown resistance (kdr) and R81T mutation status of individual M. persicae
was performed and described in detail by Panini et al. (2014). Also
based on the
results of the PCR product (200 bp fragment of the acetylcholine receptor gene containing
the R81T mutation) from wild-type genotypes (SS) after digestion by BcoDI, Voudouris et al. (2016) demonstrated that this technique was effective in
determining the presence of strong neonicotinoid resistance in aphids. If an aphid has developed resistance to
neonicotinoids, HincII will be
able to enzymatically cut the 200 bp R81T-containing
gene fragment into 98 and 102 bp fragments, while BcoDI
would not enzymatically
cut the target fragment. In contrast, if an aphid has not developed resistance
to neonicotinoids, BcoDI will be
able to enzymatically cut the 200 bp R81T gene fragment into two fragments, 95
and 105 bp in size, while HincII would not cleave the PCR product because it lacks HincII-sensitive restriction sites. Using PCR-RFLP analysis on the 200 bp R81T gene products, we
found that the products (do not contain a HincII restriction
site) of all aphids were only
digested by BcoDI
into 93 and 107 bp fragments, but not by HincII, which may indicate a lack of neonicotinoid resistance, though the products digested by BcoDI differed from 95 and 105
bp in size.
In addition, the results of toxicity tests indicate that all aphids in this
study have not developed a strong resistance to the neonicotinoid
insecticide acetamiprid. Compared to that found in other
countries, the application of
neonicotinoids in Hunan
Province occurred considerably later.
Conclusion
Based on
these factors as well as our findings in this study, it may be deduced that the
aphids of Hunan Province have yet to develop strong levels of resistance to
acetamiprid.
Acknowledgments
Thanks to all members of the Pest Insecticide and
Control Group for their helpful discussion and assistance in the experiments.
This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.
References
Ahmad M, MI Arif,
I Denholm (2003). High resistance of field populations of the cotton aphid Aphis gossypii Glover (Homoptera: Aphididae) to
pyrethroid insecticides in Pakistan. J Econ Entomol
96:875‒878
Bass C, AM Puinean,
M Andrews, P Cutler, M Daniels, J Elias, VL Paul, AJ Crossthwaite,
I Denholm, LM Field, SP Foster, R Lind, MS
Williamson, R Slater (2011). Mutation of a nicotinic acetylcholine receptor 𝛽 subunit is associated with resistance to neonicotinoid insecticides in
the aphid Myzus persicae.
BMC Neurosci 12; Article 51
Black WC, NM DuTeau, GJ Puterka, JR Nechols, JM Pettorini (1992). Use of the random amplified polymorphic DNA polymerase chain
reaction (RAPD-PCR) to detect DNA polymorphisms in aphids (Homoptera: Aphididae). Bull
Entomol Res 2:151‒159
Bogdanowicz SM, WE Wallner, J Bell, TM
Odell, RG Harrison (1993). Asian gypsy moths (Lepidoptera: Lymantriidae)
in North America: evidence from molecular data. Ann Entomol
Soc Amer 86:710‒715
Cenis JL, P Perez, A Fereres
(1993). Identification of aphid (Homoptera: Aphididae) species and clones by random
amplified polymorphic DNA. Ann Entomol Soc
Amer 5:545‒550
Cognate AI, SJ Seybold, FAH Sperling
(1999). Incomplete barriers to mitochondrial gene flow between pheromone
races of the North American pine engraver, Ipspini
(Say) (Coleoptera, Scolytidae).
Proc Bio Sci 266:1843‒1850
Faten R, M Mohamed, M Mohamed (2002). Polymerase chain
reaction-restriction fragment length polymorphism of ribosomal internal
transcribed spacer region analysis on polyacrylamide gel electrophoresis
reveals two haplotypes coexisting in Myzus persicae. Electrophoresis 2:186‒188
Feng L, XL Huang, GX Qiao (2004). A
study on diversity of aphids in Fujian province, China. Acta Zootaxon Sinica 4:666‒674
Foster SP, G Devine, AL Devonshire (2007). Insecticide
resistance. In: Aphids as Crop Pests, pp: 261‒285. Emden H, R
Harrington (Eds). CAB
International, Wallingford, UK
Gubran EME, R Delorme, D Augé, JP Moreau (1992). Insecticide
resistance in cotton aphid Aphis gossypii
(Glov.) in the Sudan Gezira. Pestic Sci 35:101‒107
Harlow CD, EP Lampert
(1990). Resistance mechanisms in two color
forms of the tobacco aphid (Homoptera: Aphididae). J Econ Entomol
83:2130‒2135
Hollingsworth RG, BE Tabashnik,
DE Ullman, MW Johnson, R Messing (1994). Resistance of Aphis gossypii (Homoptera: Aphididae) to insecticides in Hawaii:
spatial patterns and relation to insecticide use. J Econ Entomol 87:293‒300
Huang XL, GX Qiao (2006). Research status and trends in Aphidology. Acta Entomol Sin 6:1017‒1026
Karunker I, J Benting, B Lueke, T Ponge, R Nauen, E Roditakis, J Vontas, K Gorman, I Denholm, S Morin (2008). Over-expression of cytochrome P450 CYP6CM1 is associated with high
resistance to imidacloprid in the B and Q biotypes of Bemisia
tabaci (Hemiptera: Aleyrodidae). Insect Biochem Mol Biol 38:634‒644
Liu Z, XL Huang, LY Jiang, GX Qiao
(2009). The species diversity and geographical distribution of aphids in
china (Hemiptera, Aphidoidea). Acta Zootaxon Sin 2:277‒291
Lu L, H Gu (1995). The peculiarity of rapdand its
application to insect taxonomy. Acta Entomol
Sin 1:117‒122
Nauen R, I Denholm (2005). Resistance of insect pests to neonicotinoid insecticides: Current status
and future prospects. Arch Insect Biochem Physiol 58:200‒215
Pan WL, ZH Dang, ZL Gao,
HM Jia, KJ Zhang (2000). Studies on resistance of 3 species
of aphids to imidacloprid. Chin J Pestic Sci 4:85‒87
Panini M, D Dradi, G Marani, A Butturini,
E Mazzoni (2014). Detecting the presence of
target-site resistance to neonicotinoids and pyrethroids in Italian populations
of Myzus persicae. Pest Manage Sci 70:931‒938
Puinean AM, J Elias, R
Slater, A Warren, LM Field, MS Williamson, C Bass (2013). Development of a high-throughput real-time PCR assay
for the detection of the R81T mutation in the nicotinic acetylcholine receptor
of neonicotinoid-resistant Myzus persicae. Pest Manag
Sci 69:195‒199
Qiao GX, GX Zhang, F Zhao (1999). Study on
Di Prociphilus gen. nov.
from china, with the description of a new species (HOMOPTERA: PEMPHIGIDAE). Acta Zootaxon Sin 4:393‒396
Raboudi F, M Mezghani, H Makni, M Marrakchi, JD Rouault, M
Makni (2005). Aphid species identification
using cuticular hydrocarbons and cytochrome b gene sequences. J Appl Entomol
2:75‒80
Schroeder
H, H Klotzbach, S Elias, C Augustin,
K Püschel (2003). Use of PCR–RFLP for differentiation of
calliphorid larvae (Diptera, Calliphoridae)
on human corpses. Forensic
Sci Intl 132:76‒81
Schroeder H, F Scholz (2005). Identification of PCR-RFLP haplotypes for
assessing genetic variation in the green oak leaf roller Tortrix viridana L. (Lepidoptera, Tortricidae). Silvae
Genet 54:17‒23
Shew HD, GB Lucas (1991). Compendium of Tobacco
Diseases.
American Phytopathological Society, St. Paul,
Minnesota, USA
Valenzuela I, AA
Hoffmann, MB Malipatil, PM Ridland,
AR Weeks (2007). Identification of aphid species
(Hemiptera: Aphididae: Aphidinae) using a
rapid polymerase chain reaction restriction fragment length polymorphism method
based on the cytochrome oxidase subunit I gene. Aust J Entomol 4:305‒312
Voudouris CC, AN Kati, E Sadikoglou, M
Williamson, PJ Skouras, O Dimotsiou, S Georgiou, B
Fenton, G Skavdis, JT Margaritopoulos
(2016). Insecticide resistance status of Myzus persicae in Greece: long term surveys and new
diagnostics for resistance mechanisms. Pest Manage Sci 72:671‒683
Wang QL, JH Xia
(2004). Advances in insecticide resistance of insects and its management. Plant Prot
6:15‒18
Wang KY, QL Guo, XM
Xia, HY Wang, TX Liu (2007a). Resistance of Aphis gossypii (Homoptera: Aphididae) to selected
insecticides on cotton from five cotton production regions in Shandong, China. J
Pestic Sci 32:372‒378
Wang YM, ZR Shen, LW Gao
(2007b). Microsatellite markers and their application in aphid population
biology. Acta Entomol Sin 6:621‒627
Wei XT, HJ Xiao, H Bai, JX Zhang, Y Li, YC Wang, FS Xue (2010). Identification and genetic diversity of four
geographic populations of Colaphellus bowringi Baly
(Coleoptera: Chrysomelidae) in China by PCR-RFLP. Acta
Entomol Sin 2:209‒215
Yu XQ, S Zhang, SE Song, QR Zhu, CF Xie,
J Ji, XW Gao (2016). Resistance of wheat aphids to six insecticides
and assessment of their field efficacy. Acta Entomol Sin 11:1206‒1212
Zhang SF, XW Yang, JS Ma (2000). A study
on genetic distance among different taxa of aphids (Homoptera: Aphidoidea). Insect Sci
3:235‒242
Zhao C, XF Wang, D Chen, XW Wang, D Xue,
GW Ren (2013). Morphological variations of Myzus persicae
(Hemiptera: Aphididae) from different geographical
populations in China. Acta Entomol Sin
12:1452‒1463