Appl Entomol Zool (2017) 52:661–665

DOI 10.1007/s13355-017-0512-1

TECHNICAL NOTE

Non‑destructive direct polymerase chain reaction (direct PCR)

Received: 5 June 2017 / Accepted: 25 July 2017 / Published online: 18 August 2017
© The Japanese Society of Applied Entomology and Zoology 2017

Abstract Spider mites (Acari: Tetranychidae) include seri- Keywords Non-destructive DNA extraction · Invasive
ous agricultural pests and some species have spread globally species · DNA barcoding · C1-J-1718 · COI REVA
as invasive species.. For this reason, rapid and simple iden-
tification of spider mite species is necessary for agricultural
field and plant quarantine inspection. DNA sequence-based Introduction
molecular techniques can rapidly identify spider mites. How-
ever, extracting DNA from minute invertebrates is difficult, Spider mites (Acari: Tetranychidae) are one of the most
expensive and time consuming. Here, we describe a poly- important agricultural pest groups throughout the world
merase chain reaction (PCR) technique in which the whole (Migeon and Dorkeld 2006–2017; Navajas et al. 2010;
body of a spider mite adult is non-destructively soaked in Zhang 2003). This group comprises more than 1300 species,
PCR solution. The mite is then removed intact and can be of which more than 100 species can be considered as agri-
used as a voucher specimen, leaving the PCR solution as the cultural pests. In particular, Tetranychus urticae Koch, Olig-
template and avoiding the need for a DNA extraction kit. For onychus coffeae (Nietner) and Panonychus citri (McGregor)
this study, we used six common spider mite species from cause serious economic losses worldwide (Migeon and Dor-
four genera [Tetranychus urticae Koch (red form), Tetra- keld 2006–2017). Rapid identification of spider mite species
nychus kanzawai Kishida, Tetranychus parakanzawai Ehara, is essential for the management of these pests. However,
Oligonychus gotohi Ehara, Eotetranychus smithi Pritchard because of their tiny size (<0.5 mm) and limited number of
& Baker and Panonychus citri (McGregor)]. A portion of distinctive morphological characters, morphological iden-
the mitochondrial cytochrome c oxidase subunit I (COI) tification of spider mites requires expertise and experience
gene was amplified with a universal primer pair from all (Gotoh et al. 2009; Wauthy et al. 1998; Zhang and Jacobson
individuals examined and sequenced. This method short- 2000). Furthermore, species-level identification of spider
ened the time for molecular identification from 9 to 5 h and mites needs microscopic examination to determine the shape
eliminated the cost of commercial kits for DNA extraction of the male genitalia (aedeagus) (Ehara 1999). Therefore, a
[ca. 600 yen (~5.3 USD) per sample]. non-morphological method for the identification of spider
mites would be best suited to agricultural field and plant
quarantine inspections.
Molecular-based techniques have been used to assist
* Tetsuo Gotoh morphological identifications (Ben-David et al. 2007;
tetsuo.gotoh.acari@vc.ibaraki.ac.jp Darling and Blum 2007; Navajas et al. 1992; Navajas and
1
Fenton 2000; Ros and Breeuwer 2007). Navajas and Bour-
Laboratory of Applied Entomology and Zoology, Faculty
sot (2003) successfully separated two very closely related
of Agriculture, Ibaraki University, Ami, Ibaraki 300‑0393,
Japan Tetranychus species, Tetranychus urticae and Tetranychus
2 turkestani (Ugarov and Nikolski), by using the sequences
Department of Life Science and Medical Bioscience, School
of Advanced Science and Engineering, Waseda University, of the internal transcribed spacer 2 (ITS2) region of nuclear
Shinjuku‑Ku, Tokyo 162‑8480, Japan ribosomal RNA genes. Matsuda et al. (2012, 2013) showed

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662 Appl Entomol Zool (2017) 52:661–665

that almost all Japanese Oligonychus (17 of 18 species) and Another study shortened the time for identification of mos-
all Japanese Tetranychus (13 species) spider mites could be quitoes from 13 to 5 h (Werblow et al. 2016). Furthermore,
identified by mitochondrial cytochrome c oxidase subunit I in both studies, specimens were recovered with intact mor-
(COI) gene sequences amplified by a universal primer pair, phological features. However, direct PCR did not work well
C1-J-1718 (Simon et al. 1994) and COI REVA (Gotoh et al. with some taxonomic groups, such as the Formicidae (ants),
2009). Subsequently, a portion of the COI gene sequences Dytiscidae (diving beetles) and Odonata (dragonflies), which
was found to be useful as a molecular barcode for the identi- has slowed its acceptance as an identification method (Wong
fication of 38 species belonging to 12 genera of spider mites et al. 2014).
(Matsuda et al. 2014). Furthermore, for the identification In the present study, we examined the applicability of
of Tetranychus mites, polymerase chain reaction-restriction non-destructive direct PCR for six spider mite species
fragment length polymorphism (PCR-RFLP) targeting ITS belonging to four genera. We amplified the COI gene
region sequences was designed (Arimoto et al. 2013; Osak- sequences with individual whole spider mites as the PCR
abe et al. 2008). Most recently, a real-time PCR assay with template and observed the morphological features of spider
specific probes targeting the ITS1 region sequences of T. mites after PCR. Our study demonstrates that direct PCR is
urticae was developed for the identification of this serious an effective way of identifying spider mites.
pest species (Li et al. 2015).
In order to use DNA sequence-based identification meth-
ods in the field and for plant quarantine inspections, the Materials and methods
methods must be rapid and inexpensive. Presently, at least
4 h are needed for DNA extraction from insects (Wong et al. Mites
2014) and the cost of using a spin column-based extrac-
tion kit [e.g., QIAamp DNA Micro Kit (Qiagen, Valencia, Six species of common red spider mites [T. urticae (red
CA)] is about 600 yen (~5.3 USD) per sample. Reducing the form), T. kanzawai Kishida, T. parakanzawai Ehara, Oli-
cost of DNA extraction is essential for the spread of DNA gonychus gotohi Ehara, Eotetranychus smithi Pritchard &
sequence-based identification. Baker and P. citri] were selected for this study (Table 1).
Direct PCR is an alternative rapid and low-cost PCR The three Tetranychus species were reared on common bean
strategy that uses individual samples directly without DNA (Phaseolus vulgaris L.) leaves, O. gotohi was reared on Jap-
extraction and purification, i.e., it can eliminate the cost of anese stone oak [Lithocarpus edulis (Makino) Nakai] leaves,
DNA extraction (Werblow et al. 2016; Wong et al. 2014). E. smithi was reared on mulberry (Morus bombycis Koidz.)
Wong et al. (2014) used this method to identify midges, and leaves and P. citri was reared on satsuma mandarin (Citrus
shortened the time required for identification from 10 to 6 h. unshiu Marc.). The leaves were placed on a water-saturated

Table 1  Collection records of spider mites used in this study

Tetranychus Tetranychus urti- 27 August 2011 Matsukawa, Dianthus sp. 0219 AB736079 Matsuda et al.
cae Koch (red Nagano, Japan (2013)
form)
Tetranychus kan- 19 May 1993 Kanaya, Shizuoka, Camellia sinensis 0158 AB736043 Matsuda et al.
zawai Kishida Japan (2013)
Tetranychus para- 5 June 1993 Ami, Ibaraki, Pueraria montana 0155 AB736060 Matsuda et al.
kanzawai Ehara Japan (2013)
Oligonychus Oligonychus 1 July 2007 Tsuchiura, Ibaraki, Lithocarpus edulis 0116 AB683668 Matsuda et al.
gotohi Ehara Japan (2012)
Eotetranychus Eotetranychus 14 August 2007 Minami-Shima- Rosa multiflora 0545 AB981235 Matsuda et al.
smithi Pritchard bara, Nagasaki, (2014)
& Baker Japan
Panonychus Panonychus citri 6 May 1993 Ishioka, Ibaraki, Ilex crenata 0226 AB981208 Matsuda et al.
(McGregor) Japan (2014)
a
Scientific names of host plants follow the Plant List version 1.1 (2013)
b
Voucher specimens are preserved at the Laboratory of Applied Entomology and Zoology (Faculty of Agriculture, Ibaraki University) under the
serial voucher specimen numbers
c
Cytochrome c oxidase subunit I gene

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Appl Entomol Zool (2017) 52:661–665 663

polyurethane mat in a plastic dish (90-mm diameter, 20-mm was allowed for last strand elongation. PCR products were
depth). The mites were reared at 25 °C and under a 16:8-h run on 1% agarose gels to confirm the presence of DNA
light:dark photoperiod until PCR analysis. bands with the expected size [approximately 900 base pairs
(bp)]. The PCR product was cleaned up with a MinElute
Direct PCR and sequencing PCR Purification Kit (Qiagen) and directly sequenced in
both directions using a BigDye Terminator Cycle Sequenc-
The PCR mixture was composed of 25 μl of Premix Ex Taq ing Kit version 3.1 (Applied Biosystems, Foster City, CA)
Hot Start Version (Takara, Shiga, Japan), 1 μl of both 10 pM and an ABI 3130xl automated sequencer.
primer of C1-J-1718 [5′-GGA GGA TTT GGA ATT GAT
TAG TTC C-3′ (Simon et al. 1994)] and COI-REVA [5′- Morphological observation of exoskeletons
GAT AAA AAC GTA ATG AAA ATG AGC TAC-3′ (Gotoh
et al. 2009)] and 23 μl of sterile distilled water for the total To check for any morphological damage of the spider mite
volume of 50 μl. The PCR mixture was pipetted into a PCR body after direct PCR, we selected males of T. urticae, as
tube and the PCR tube was placed on ice. To obtain a spider it is such a well-known species. After direct PCR, six ran-
mite, the tip of a stainless needle was placed near a live domly selected T. urticae males were mounted in Hoyer’s
adult on a rearing leaf disk, allowing the mite to cling to the medium and their morphology was observed under phase-
needle. The mite was then submerged in the PCR mixture contrast and differential interference-contrast microscopes
directly, i.e., without any pre-treatment cleaning procedure. to check for damage.
The mites were serially placed in the PCR solutions. About
1 min per mite was required, thus it took about n minutes to
transfer n mites. Then aliquots of the solution were taken for Results and discussion
the reaction in the same order. Thus, for an assay of n mites,
the mites were in the PCR solution for about n minutes (in Direct PCR using the whole body of a single spider mite
our experiments, 6–36 individuals were used for one PCR with a universal COI primer pair produced a single and clear
assay). The low temperature of the PCR mixture slowed the band of the expected size (approximately 900 bp) for each of
movement of spider mites so they could not escape from the 30 individuals of both sexes of each species. Representative
tube. Thirty replicates of direct PCR were examined for each PCRs are shown in Fig. 1. In both females and males, the
sex of each species. DNA bands amplified by direct PCR were generally equal
As a positive control, DNA was extracted from one adult to or stronger than those of the positive controls (Fig. 1).
female of each species with QIAamp DNA Micro Kit (Qia- Male spider mites, because of their smaller size, produce
gen) according to the recommended protocol. Briefly, a only about half as much DNA as females (Shim et al. 2016).
female was placed in a 1.5-ml microcentrifuge tube, and pro- However, the success rate of direct PCR was 100% for both
tein lysis buffer with proteinase K was added. The tube was sexes. The success rate of direct PCR was not affected by
incubated at 56 °C overnight. The DNA was then cleaned the sample preparation time prior to PCR. All amplicons
up with the kit, yielding 20 μl of DNA extract. An aliquot obtained by direct PCR were sequenced successfully. All
of 1 μl of DNA extract was used as the PCR template. PCR sequences were identical to the COI sequences that we previ-
amplification was performed with the following profile: ously obtained with commercial kits (Matsuda et al. 2012,
3 min at 94 °C, followed by 35 cycles of 10 s at 98 °C, 30 s 2013, 2014). This indicates that organic substances released
at 52 °C and 1 min at 72 °C. An additional 10 min at 72 °C into the PCR reactions by direct PCR did not interfere with

Fig. 1  Typical band patterns obtained with direct polymerase chain (All-Purpose Hi-Lo™ DNA Marker; Bionexus, Raleigh, NC). Lanes
reaction (direct PCR) using the whole body of an individual spider 1 single female; lanes 2 single male; lanes 3 distilled water (negative
mite with a universal mitochondrial cytochrome c oxidase subunit I control); lanes 4 DNA extraction from single female using QIAamp
(COI) primer pair (C1-J-1718 and COI-REVA). M Molecular marker DNA Micro Kit (Qiagen, Valencia, CA) (positive control)

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664 Appl Entomol Zool (2017) 52:661–665

Fig. 2  Morphological features

the downstream direct-sequencing reaction. Using direct Acknowledgments We are grateful to Dr. Y. Kitashima for kindly
PCR shortened the time required for molecular identification helping us with this study. This work was supported in part by Japan
Society for the Promotion of Science KAKENHI grants (Grant-in-Aid
from 9 h (4 h for DNA extraction, 1.5 h for PCR amplifica- for Scientific Research B, JP25292033 and JP17H03775).
tion, 0.5 h for electrophoresis, 0.5 h for PCR clean-up and
2.5 h for direct-sequencing) to only 5 h for identification
of a spider mite sample. Likewise, it eliminated the cost
of commercial kits for DNA extraction [ca. 600 yen (~5.3 References
USD) per sample].
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