Molecular discrimination between the spider mite Tetranychus evansi Baker & Pritchard, an important pest of tomatoes in southern Africa, and the closely related species T. urticae Koch (Acarina: Tetranychidae)

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Molecular discrimination between the spider mite Tetranychus evansi Baker &
Pritchard, an important pest of tomatoes in southern Africa, and the closely
related species T. urticae Koch (Acarina: Tetranychidae)
M. Knapp1*, B. Wagener1& M. Navajas2
1International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya
2CBGP-INRA, Campus International de Baillarguet, CS 30016, 34988 Montferrier-sur-Lez Cedex, France
During the last two decades, tomato farmers in
southern Africa have been facing increasing prob-
lems with spider mites (Acari: Tetranychidae). In
ZimbabweandZambia,thesemitesarecommonly
referred to as ‘red spider mites’ and identified as
Tetranychus urticae Koch, T. cinnabarinus (Bois-
duval) or Tetranychus sp. (Jensen & Mingochi 1988;
PPRI 1988). Recent work has shown that Tetra-
nychus evansi Baker & Pritchard is responsible
for the spider mite outbreaks on tomato in these
countries (Knapp & Luchen 2000). T. evansi is a pest
of crops of the family Solanaceae (tomatoes, pota-
toes, tobacco and others; Bolland et al. 1998) and
probably originates from South America. It was re-
corded for the first time in Africa from tobacco in
Zimbabwe in 1979 (Blair 1983). T. evansi is now
widespread in southern Africa (Meyer 1996) and
also occurs in Kenya (Knapp 2002), Congo (Bonato
1999), Morocco (El Jaouani 1988) and Tunisia
(Bolland et al. 1998).
The mite is regarded as one of the key arthropod
pests of tomato in Zimbabwe (Sibanda et al. 2000)
causing yield losses of up to 90 % in small-holder
production. In contrast, even though T. urticae is
a common pest of vegetables in southern and
eastern Africa, it is usually not a major problem on
tomato.
Effective control of agricultural pests relies on a
clear understanding of the taxonomic status of the
target species. T. evansi can be distinguished from
T. urticae and other Tetranychus species by different
morphological characters, mainly the shape of the
terminal knob of the aedeagus (a part of the male
genitalia). However, due to the small size of the
Tetranychidae (<0.5 mm), experience is required
to separate the two species using morphological
traits and few specialists in Africa are able to iden-
tify them definitively. This is well reflected in the
frequent misidentification of the species by na-
tional authorities (Jensen & Mingochi 1988; PPRI
1988).
In addition to classical taxonomy, several genetic
markers based on molecular or biochemical tech-
niques have recently been applied to the Tetrany-
chidae to solve taxonomic and systematic ques-
tions, including inter- and intraspecific variation
among populations (see Navajas & Fenton 2000
for a review). Nucleotide variation of mitochon-
drial DNA (mtDNA) and nuclear ribosomal DNA
(rDNA) are the molecular markers most fre-
quently used to investigate differences between
species. Variation between the sequences of the
second internal transcribed spacer (ITS2) of the
ribosomal DNA were used to confirm the species
status of the sibling species Tetranychus pueraricola
Ehara & Gotoh and T. urticae (Gotoh et al. 1998).
Similarly, synonymy between T. kanzawai Kishida
and T. hydrangeae Pritchard & Baker was demon-
strated using the ITS2 sequence together with
cross-breeding experiments (Navajas et al. 2001).
Lee & Lee (1997) and Navajas et al. (1997) analysed
a fragment of the mitochondrial cytochrome
oxidase subunit gene (COI) to distinguish several
species of Tetranychus mites.
In the present study, the ITS2 region of the
nuclear ribosomal DNA was examined by se-
quencing followed by a PCR-restriction-fragment
length-polymorphism (PCR-RFLP) analysis to
discriminate between T. evansi and T. urticae. This
provides an additional tool to properly identify
T. evansi and to differentiate it from the species it is
commonly confused with in southern Africa.
Tetranychus evansi specimens were collected
from cultures kept on tomato (Lycopersicon
esculentum Mill.) plants in green houses at the
Plant Protection Research Institute (PPRI), Harare,
Zimbabwe and at the International Centre of
Insect Physiology and Ecology (ICIPE), Nairobi,
Kenya. T. evansi collected from tomato fields in
Piracicaba, São Paulo State, Brazil, were included
in the study and served as reference. Tetranychus
urticae were collected from beans (Phaseolus
vulgarisL.)atMweaIrrigation Scheme, Kenya,and
*To whom correspondence should be addressed.
E-mail: mknapp@icipe.org
African Entomology
11(2): 300–304 (2003)

froma laboratory culture maintained at the Center
for Biology and Management of Populations
(CBGP), Montpellier, France. All specimens were
preserved in 95 % ethanol.
Five adult females from each location were
examined separately by PCR-RFLP to estimate
intra-strain variation. Total DNA was extracted
from a single mite using the method described by
Collins et al. (1987). The primers 5’-ATA TGC TTA
AAT TCA GCG GG-3’ and 5’-GGG TCG ATG AAG
AAC GCA GC-3’ were used for both DNA amplifi-
cation and direct sequencing of amplicons. An
additional internal primer (5’-GCA TGA GAT TCT
AAG GTT AGT C-3’) was developed for sequenc-
ing. The protocols for the PCR method and the
sequencing are described in detail by Navajas et al.
(1997, 1998). Sequence annotation was carried out
using BioEdit software (Hall 1999) and the align-
ment performed using Clustal W 1.8 (Thompson
et al. 1994). Genetic distance between species was
computed using MEGA 2.0 (Kumar et al. 2001) and
the Kimura 2-parameter model.
For RFLP analyses, amplicons of both species
were digested by the restriction endonucleases
Rsa I, Alu I and Tru1 I. The restriction fragments
were separated by electrophoresis on a 1.5 %
agarose gel or a 3.5 % metaphor gel (7 V/cm, 1–2 h),
stained in ethidium bromide and visualised under
UV light.
The complete ITS2 regions of T. evansi and T. urti-
cae were PCR-amplified with an examined length
of 481 bp for T. urticae and 493–496 bp for T. evansi
(Fig. 1) (EMBL accession numbers: X99881 and
AJ419833).
Between the two species, 30 point mutations (11
transitions and 19 transversions) and 7 insertions/
deletions were observed. The nucleotide diver-
gence between the two species was 7.4 %. Other
comparisons of ITS2 sequences between closely
related tetranychid species displayed lower
nucleotide divergences with 1.2 % between T.
pacificus McGregor and T. mcdanieli McGregor,
1.7 % between T. urticae and T. pueraricola (Gotoh
et al. 1998) and 2.5 % between T. urticae and T.
kanzawai Kishida (Navajas et al. 1997). The higher
nucleotide divergence (7.4 %) between T. evansi
and T. urticae indicates that the two taxa are clearly
distinct.
ITS sequences differ between species but are
generally conserved in length and composition
within species (Hillis & Dixon 1991). However,
intraspecific variation has also been reported in
form of single base point mutations or the inser-
tion of a few base pairs (Navajas et al. 1994, 1999).
In our sequences we found one transversion in the
two sequences of T. urticae and four insertions/
deletions and three point mutations in T. evansi.
In order to define a method to rapidly discrimi-
nate between T. evansi and T. urticae, we developed
a PCR-RFLP assay. This was necessary because dif-
ferences of the sizes of the amplicons of T. urticae
and T. evansi were too small to be detected on
agarose gels. Additionally, sequencing is expen-
sive and labour intensive. Recognition sites of all
three restriction enzymes (Rsa I, Alu I and Tru1I)
are presented in Table 1. Restriction fragments
smaller than 100 bp could not be detected and are
not recorded. The restriction profiles for Rsa Iof
T. urticae (Gotoh et al. 1998) was confirmed in the
present work. In T. evansi only one recognition site
was detected since the second recognition in
T. urticae was disrupted by 2 insertions (Fig. 1).
One unique site at position 363–366 of the ITS2
sequence was detected for Alu IinT. urticae
while in T. evansi two mutations in this position
inactivated the site (Fig. 1). Therefore, two bands
(410 bp and 180 bp) are present in T. urticae
whereas T. evansi was not digested and only one
band appeared (680 bp) (Table 1).
Figure 2 indicates the restriction profile for the
enzyme Tru1IofT. urticae and T. evansi. At position
120 of the ITS2 sequence a point mutation in
T. evansi disrupts the recognition site that is
present in T. urticae. Therefore, only one fragment
Short communications 301
Table 1. Restriction fragment length differences between
Tetranychus urticae
and
T. evansi.
Restriction endonucleases
T. urticae
1
T. evansi
1
Rsa
I 300 bp, 200 bp, 160 bp 310 bp, 350 bp
Alu
I 410 bp, 180 bp 680 bp
Tru
1 I 200 bp, 240 bp 450 bp
1: only fragments >100 bp are indicated.

Short communications 302
5.8S ITS2 70
T. urticae
France CCAACGCACATTGCGGCTTTCGGGTCTTTTCCGAGGTCACATCTGTCTGAGAGTTGAGATGTAAAATAAT
T. urticae
Kenya ----------------------------------------------------------------------
T. evansi
Kenya -T--------------------------------------------------------------------
T. evansi
Zimbabwe -T--------------------------------------------------------------------
T. evansi
Brazil -T--------------------------------------------------------------------
140
T. urticae
France CAACAAAACACTTGCATACTACCATATATGCATTGTTTTTAGAGGATTGT**********ATATTTATAT
T. urticae
Kenya ---------------------------------------G------------------------------
T. evansi
Kenya ---------------------------------------G---T------GCATATATTT----A-G---
T. evansi
Zimbabwe ---------------------------------------G---T------GCATATATTT----A-G---
T. evansi
Brazil ---------------------------------------G---T------GCATATATTT----A-G---
Tru
1 I 210
T. urticae
France GCATGAATCTTGATGTTTTATTCCTTTTCTTAATTGCAATTCGTTGCAATTTAGTAAGGAGAATCTCAAA
T. urticae
Kenya ----------------------------------------------------------------------
T. evansi
Kenya A------------------------------G------TA--T---------------------------
T. evansi
Zimbabwe A------------------------------G------TA--T---------------------------
T. evansi
Brazil A------------------------------G------TA--T---------------------------
Rsa
I280
T. urticae
France TCTACTTGTTTCACATGATAAATTTTGTGTACAATGCATATTTCATCTCTGCAAGCAGTATATATGAATA
T. urticae
Kenya ----------------------------------------------------------------------
T. evansi
Kenya ------------------------------------------------------A--------------T
T. evansi
Zimbabwe ---------------------------*--------------------------A--------------T
T. evansi
Brazil ------------------------------------------------------A--------------T
350
T. urticae
France GATACTAGCATGAGATTCTAAGGTTAGTCGCCTATCTGACGACGCTAAAGTCGTATTGCAGATAACTATG
T. urticae
Kenya ----------------------------------------------------------------------
T. evansi
Kenya -----------------------------------------------------A**-A------CT-**-
T. evansi
Zimbabwe ---------------------------------------------------------A------------
T. evansi
Brazil ---------------------------------------------*-----------A------------
Tru
1 I
Rsa
I
Alu
I420
T. urticae
France GTGATCAACTAACCTGTTAACTGATGAATCTTC*TTGCACTTGT**ATAAATCGTACAAATAGTAGCTAT
T. urticae
Kenya ----------------------------------------------------------------------
T. evansi
Kenya ---------------A----A------------A----------GT-----C-**---------TT----
T. evansi
Zimbabwe ---------------A----A------------A----------GT-----C-**---------TT----
T. evansi
Brazil ---------------A----A------------A----------GT-----C-**---------TT----
Tru
1 I
Tru
1 I 490
T. urticae
France TTCATTCTGTTAAAGCAGACCTAAGAAGTAATGCAAAGGCAAAATTTGTGCAAACATTAAAGTAGATTTA
T. urticae
Kenya ----------------------------------------------------------------------
T. evansi
Kenya ------------------------------T------TA-C---------T-------------------
T. evansi
Zimbabwe ------------------------------T------TA-C---------T-------------------
T. evansi
Brazil ------------------------------T------TA-C---------T-------------------
Tru
1 I
Tru
1 I 28S 560
T. urticae
France CGTTGC*TTGCT*****TGCAAACAACACAAATAAACATTAATCAACTTAATCAATATTTTTGATCTCAG
T. urticae
Kenya ----------------------------------------------------------------------
T. evansi
Kenya -----AAC----ATTAT----TT-----AC----T------------A-*------T-------------
T. evansi
Zimbabwe -----AAC----ATTAT----TT-----AC----T------------A-*------T-------------
T. evansi
Brazil -----AAC----ATTAT----TT-----AC----T------------A-*------T-------------
Fig. 1. Partial 5.8S and complete ITS2 sequence (5’–3’)of
Tetranychus urticae
of two different locations (Montpellier,
France; Mwea, Kenya) and
T. evansi
of three different locations (Nairobi, Kenya; Harare, Zimbabwe; Piracicaba,
Brazil). Identities with
T. urticae
sequence are indicated by dashes, asterisks represent gaps. The recognition sites of
Rsa
I,
Alu
I and
Tru
1 I are depicted in boldface.

Short communications 303
(450 bp) with more than 100 bp is present in T.
evansi, while two fragments (200 bp and 240 bp)
appear in the T. urticae RFLP-pattern.
In conclusion all three restriction endonucleases
(Rsa I, Alu I and Tru1 I) are suitable for distinguish-
ing between T. urticae and T. evansi.
No intraspecific polymorphism such as shown
in the ITS2 sequences of T. evansi could be detected
with PCR-RFLP.
The results presented here clearly show the dif-
ferences between T. urticae and T. evansi obtained
by PCR-RFLP patterns and confirmed by sequence
analysis. For routine analysis, Tetranychus species
are generally identified by morphological charac-
teristics. Molecular techniques provide a reliable
alternative even for non-specialists to distinguish
between species, which are difficult to identify by
classical morphological characters.
ACKNOWLEDGEMENTS
We thank G.J. de Moraes for providing the T.
evansi sample from Brazil, S. Cross-Arteil for
sequencing the samples, and J. Gutierrez for
morphological identification of the reference
specimens. This study was funded by the Bundes-
ministerium für wirtschaftliche Zusammenarbeit
und Entwicklung BMZ (German Federal Ministry
for Economic Cooperation and Development).
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Accepted 10 June 2003
304 African Entomology Vol. 11, No. 2, 2003