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Spider mites belonging to the family Tetranychidae are economically important
pests of various agricultural and horticultural crops. Recently, spider mites emerged as
serious pests of vegetables, banana and rice in Kerala. Though identification of spider
mites is primarily based on external morphology, the existence of cryptic species
makes precise identification difficult. In this context, the present study entitled “DNA
barcoding of spider mite (Prostigmata: Tetranychidae) was conducted in Centre for
Plant Biotechnology and Molecular Biology (CPBMB) and Department of
Agricultural Entomology, College of Horticulture, Vellanikkara with an objective to
generate DNA barcodes for different species of spider mites on major crops of Kerala
and to find out genetic variability among them.
A purposive survey was conducted in four districts of Kerala viz., Thrissur,
Palakkad, Ernakulam and Malappuram during the period of February-May and
November- December 2016 and February-May 2017. Spider mites associated with ten
different crops viz., cowpea, brinjal, okra, ashgourd, pumpkin, banana, papaya,
Adenium, Dahlia and tapioca from 11 different localities were collected. The
specimens were maintained in acarology laboratory as sixteen separate isolines
assigning unique accession number. Permanent slides were prepared separately for
adult female and male mites from each isoline for morphological characterization. The
chaetotaxy of hysterosoma and legs as well as empodial characters of female were
used for generic level identification. The shape of aedeagal knob in male was the
characteristic feature utilised for species level identification.
The study revealed that the spider mites collected from different crops
belonged to three species of a single genus Tetranychus namely, T. truncatus, T.
okinawanus and T. udaipurensis. The host range of T. truncatus includes tapioca,
banana, pumpkin, Dahlia and cowpea T. okinawanus was recorded on papaya,
ashgourd, Adenium, cowpea and brinjal, while T. udaipurensis was found only on
okra, banana and tapioca.
For molecular characterisation and identification, DNA was isolated from all
the sixteen accessions using the modified CTAB method. The DNA was assessed for
quality and quantity with NanoDrop spectrophotometer (ND-1000). The absorbance
ratio A260/A280 for all accessions was in the range of 1.8-2.0 and concentration was
obtained in the range 106-359 ng/μL indicating good quality. The COI loci was
amplified with UBC6 (forward) and RCOI (reverse) primers while ITS2 loci was
amplified with the primer ITS2 KAU (forward) and R ITS2 KAU (reverse primer).
The reaction mixture proposed by Li et al. (2010) was used in study and for ITS2 loci
the annealing temperature was standardised by gradient PCR (Polymerase Chain
Reaction). The amplification was confirmed by two per cent Agarose Gel
Electrophoresis (AGE) and the amplicon size obtained for ITS2 and COI were in the
range of 600-700 bp and 800-900 bp, respectively. The PCR products were sequenced
by outsourcing at AgriGenome Lab. Pvt. Ltd. Kochi.
The forward and reverse sequences obtained after sequencing were merged
with the CAP3 sequence assembler to obtain contigs. Fourteen out of sixteen
sequences formed contigs. Using MEGA 7 software the sequences were analysed for
the presence of stop codons. Stop codons were removed with BioEdit software.
Nucleotide BLAST (BLASTn) was done for all the accessions and query coverage (%)
and identity (%) were obtained in the range of 97-99 for most accessions. The
BLASTn result was in consensus with the morphology based species determination.
The nine COI sequences and five ITS2 sequences were aligned with Clustal W of
MEGA 7 and barcode gaps were identified for both the loci. The sequences were
aligned in MEGA 7 using Kimura 2 parameter (K2P) and pairwise distances between
the sequences were analysed which showed that intraspecific divergences were always
less than one, however accessions PmMk0927 showed maximum divergence within T.
truncatus (0.812- 0.849) and accession BrTv0356 showed maximum divergence
within T. okinawanus (0.835). Divergence of T. truncatus sequences from T.
okinawanus (ITS2) and T. udaipurensis (COI) were found to be 0.113 and 0.140-
0.142 respectively. The distance summary of sequences when computed with the
sequence analysis tool provided by BOLD (Barcode of Life Data systems) showed a
sequence divergence within species in the range of 0.00 – 0.31 per cent for ITS2
sequences representing T. truncatus and T. okinawanus and 0.96 – 2.88 per cent for
COI sequences representing T. truncatus and T. udaipurensis.
A phylogenetic tree was constructed with the fourteen sequences in MEGA 7
using the neighbour joining method and it was seen that the COI and ITS2 sequences
branched out into two monophyletic groups. The accessions PmMK0927 (Pumpkin,
Manjakunnu) and BrTv0356 (Brinjal, Tavannur) formed out group for COI and ITS2
clusters respectively, as these sequences formed shorter contigs. The sequences were
submitted to BOLD (Barcode of Life Data Systems) for generation of barcodes and
also to GenBank (NCBI - National centre for Biotechnology Information).
The study brings to light the occurrence of T. truncatus and T. okinawanus as
the predominant species of spider mites on crops of Kerala. The study recorded four
new hosts for T. okinawanus, a species reported only recently from India, indicating its
potential to widen the host range. This is suggestive that this alien species has potential
to turn invasive. Three new hosts were also recorded from Kerala for T. truncatus and
T. udaipurensis. The pairwise distance analysis, distance summary analysis and
phylogenetic tree are indicative that T. truncatus is closely related to T. okinawanus at
ITS2 loci and to T. udaipurensis at COI loci. Present study confirms that ITS2 and
COI loci of spider mite DNA provides species level resolution and thus can be used as
reliable tool to differentiate closely related species of spider mites.