First Report of Tetranychus truncatus Ehara (Acari: Tetranychidae) Infesting Groundnut (Arachis hypogaea L.) in India through Morphological and Molecular Insight

Groundnut (Arachis hypogaea L.) belongs to the family Fabaceae is a leguminous plant that is widely cultivated and valued for its high-oil content and edible seeds. Groundnut is the fourth most important source of edible oil and a third most important source of vegetable protein in the world. Groundnut is not only an important oilseed crop of India, but also an important agricultural export commodity. Globally, Groundnut covers 315 lakh hectares with the production of 536 lakh tonnes with the productivity of 1701 kg per hectare (FAOSTAT, 2021), with annual all-season coverage of 55.71 lakh hectares, globally. India ranks first in Groundnut area under cultivation and is the second largest producer in the world with 102 lakh tonnes with productivity of 1831 kg per hectare during 2020-21. 

Several arthropod pests may cause severe loss in the agro-ecosystem reducing the productivity of groundnut (Lourencao et al., 2001; Mound, 2005; Mound and Ng, 2009; Riley et al., 2011; Seeman and Beard, 2011; Biswas and Das, 2011; Biswas, 2014). High infestations of mites, may impair the development and reduce yield of the crop, increase the production costs (Flechtmann and Knihinicki, 2002; Mendonca et al., 2011), contribute to the contamination of the environment and affect humans, due to the intensive use of pesticides to control these organisms (Chhillar et al., 2007; Roy et al., 2011; Silva and Gondim Junior, 2016). Among the mites, 31 species of Tetranychidae family are associated with groundnut crop, of which 22 belong to the genus Tetranychus (Migeon and Dorkeld 2023). The red spider mite, Tetranychus (cinnabarinus) urticae was reported as a minor pest in groundnut by Gupta and Sandhu (1969). The two-spotted spider mite, T. urticae is a destructive pest of groundnut and considered as the most harmful species in this crop in several countries (Campbell et al., 1974; Johnson et al., 1982; Heagle et al., 1994; Nandagopal and Gedia, 1995; Ashley et al., 2006). Initially, T. truncatus was reported on groundnut by Ho et al., (1997). In India it has been reported on Morus in Karnataka (Srinivasa et al., 2012) and on ornamental plants in Kerala (Prakash et al., 2022) but to date as far as reported it has not been found damaging cultivated groundnut. The groundnut plants, on infestation by T. truncatus initially exhibit white spots on upper surface of leaves due to feeding by the mites on the lower surface, which turn into chlorotic spots followed by drying of leaves. On severe infestation profuse webbing is formed over the foliage, giving it a carpet like appearance (Plate 1).

Infested plant parts were collected directly from the groundnut crop based on the symptoms and were examined under LEICA Stereo zoom microscope. Some mites were maintained separately as isoline cultures for the purpose of DNA analysis. Live mite specimens, both adult males and females, were slide mounted separately in Hoyer’s medium for microscopic study. The mites were mounted dorsally, ventrally and laterally for clear examination of taxonomic characters. Females (dorsal and ventral) and males (lateral) were also mounted to identify the species. After mounting, the slides were processed by gently warming the slides at 45-50°C for 2-3 days in a hot air oven and then sealed all around and preserved. Measurements and photographs were taken with a Leica DM750 phase contrast microscope equipped with a drawing tube, at 400x or 1000x under differential interference contrast optics (DIC). Morphological terminologies were followed as per Lindquist (1985). The material used for the description is from Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu. Measurements are given in micrometers. Leg setal numbers are written as the total number of setae followed by the number of sensory setae (solenidia + eupathidia) in parentheses. Abbreviations: sp.- species; cx- coxa; tr- trochanter; fe- femur; ge- genu; ti- tibia.


 
Molecular characterization of mites
 
DNA extraction and PCR analysis
 
The DNA isolation of T. truncatus isolines was obtained by using the HotShot method (Montero Pau et al., 2008). Five fresh adult females from the same colony were chosen and placed in a 1.5 ml Eppendorf tube with 20 microliters of alkaline lysis buffer (To prepare 50 ml of alkaline lysis buffer (pH 12), 125 μl of 10N NaOH and 20 μl of 0.5 M Na2 EDTA (pH 8) were added in 50 ml of ddH₂O). The samples were then crushed for two minutes with a toothpick or micro pestle and kept in hot water at 90°C for 30 minutes. After cooling for 10 minutes at 4°C, an equal volume of neutralizing buffer (To prepare 100 ml of neutralizing lysis buffer (pH 5), 630 mg of Tris- HCL was added in 100 ml of ddH₂O) was added and vortexed. Before being used, these samples were kept in a deep freezer (-80°C).
 
The PCR amplification
 
Amplification of ITS2 region was carried out using specific primers. Primer set for the ITS2 region is as follows: forward (5´- ATATGCTTAAATTCAGCGGG-3´) and reverse (5´- GGGTCGATGAAGAACGCAGC-3´) primers which amplified the complete ITS2 region. The PCR was performed in a volume of 50 µl (Eppendorf) in the Mastercycler ® containing 27 µl 2X Master mix (Takara), 10 µl of Nuclease free water, 7 µl of template DNA and 3 µl of each primer. The mixtures were amplified by the following conditions: an initial denaturation at 94°C for 4 min followed by 35 cycles at 94°C for 1 min and 72°C for 1 min and a final elongation step at 72°C for 1.30 min.
 
Analysis of DNA samples
 
The quality of DNA sample was evaluated using a 1 per cent agarose gel. In a microwave, agarose and 1X TAE buffer were combined and melted. Ethidium bromide (5 µl) was added after the liquid had been cooled to 60°C and was gently stirred to prevent the formation of bubbles. The fluid was then poured into the gel mould that contained the comb and left until it set. The gel buffer reservoir in the electrophoresis apparatus was filled with 1X TAE buffer and emptied into the gel electrophoresis tank about 2 mm above the gel surface. The appropriate gel slots were loaded with the contents and electrophoresis was performed at 70 V. The gel was examined and documented using a gel documentation unit after the run (GELSTAN 4X Advanced). PCR products were analyzed by 1% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution in “Gelstan 4X Advanced” system and software gel i.
 
Sequencing and data analysis
 
The purified PCR product of the amplified fragment of T. truncatus was sequenced at Syngenome Private Limited, Tamil Nadu and sequenced through double pass sequencing in both forward and reverse directions. The nucleotide sequence was compared to identify the similarity between them by the Basic Local Alignment Search Tool (BLAST). The MUSCLE algorithm was used to multiple align the ITS2 sequences of tetranychid mites (Edgar, 2004). The multiple alignments were further trimmed to derive a uniform length of alignment at both ends for further phylogenetic tree construction. Pairwise distance among the trimmed ITS2 sequences of tetranychid mites was computed using MEGAX (Tamura et al., 2007). The sequenced ITS2 was confirmed by a sequence similarity search against the nucleotide database (nt) using the BLAST program and it was further submitted into the NCBI GenBank database.

Description
 
Tetranychus truncatus (Ehara, 1956). J. Faculty Sci. Hokkaido Univ. Ser. VI, Zool., 12: 507.
 
Materials examined (not types) India
 
5 ♂♂, TNAU-Coimbatore, Tn-GN-007-011, 09.ix.2023 and 6♂♀♀, TNAU- Coimbatore, Tn-GN-001-006, 09.ix.2023 on Arachis hypogaea. The specimens are retained in the Acarology laboratory, Department of Agricultural Entomology, Tamil Nadu Agricultural University Coimbatore, TN, India.
 
Diagnosis
 
Female
 
Body, idiosoma length excluding gnathosoma (v2-f2) 332-370 µm, width (sc2-sc2) 204-289 µm (Plate 2a and 2b). The mites were red in colour (Plate 1).
 
Dorsum
 
Idiosomal setae longer than the interval between their longitudinal bases and gradually tapers distally (Plate 3a). Dorsal striae between setae e1-e1 mixed longitudinal and oblique; dorsal striae between setae f1-f1 longitudinal; dorsal striae between setae e1 and f1 transverse, forming a diamond-shape medially (Plate 3b); ventral striae without lobes; pregenital striae entire, unbroken (Plate 3c). Peritreme hook 27-28 μm long with slightly expanded bulbous tip forming a distinct ‘U’ shaped (Plate 2e). Dorsal setae of prodorsum barbed. Opisthosomal setae long, pubescent (except setae h2, h3 thin, smooth. Empodia with 6 proximoventral hairs, empodia I-IV each with a spur (>2 μm long) (Plate 6a); tarsus I with sockets of 4 tactile setae proximal to proximal pair of duplex setae; Dduplex 24-26 (Plate 2d); tarsus III with 1 proximal tactile seta.




 
Gnathosoma (Palp)
 
Terminal sensillum or spinneret (eupathidion su ζ), equal in length and breadth with a pit at tip (Plate 4b) and slightly shorter (5 μm) than both eupathidia ul'ζ and ul"ζ (6 to 9 μm) (Plate 4c); dorsal sensillum (solenidion w), slightly taller (6.1 μm) than, terminal sensillum and tapering distally (Plate 4a). Trochanter is glabrous, palpal chaetotaxy is from trochanter to tibia, 0-1 (dPFe)-1 (l"PGe)-3 (dPTi, l'PTi, l"PTi). The dorsal seta (dPFe), on the femur is long and thin.


 
Legs
 
Setal counts: cx 2,2,1,1; tr 1,1,1,1; fe 10,6,4,4; ge 5,5,4,4; ti 10,7,6,7; ta 18,16,10,10. Tarsus I with two sets of duplex setae (solenidion on proximal 55-63, solenidion on distal 85-93), 1 solenidion, 3 eupathidia and 10 tactile setae; with 4 tactile setae proximal and 1 solenidion adjacent, to proximal duplex setae (Plate 2d).
 
Male (n=5)
 
Body, idiosoma length excluding gnathosoma (v2-f2) 197-210 µm, width (sc2-sc2) 145-156 µm (Plate 2c). Colour in life red.
 
Dorsum
 
Peritreme with hook (length of hook 26-28) and slightly enlarged bulbous tip. Striae on prodorsum finely spaced. Dorsal setae of prodorsum barbed and slender. Opisthosomal setae long and pubescent (except setae h2, h3 thin, smooth).
 
Venter
 
Setae thin, mostly smooth (generally anterior setae smooth, posterior setae barbed).
 
Gnathosoma (Palp)
 
Eupathidia (ul'ζ, ul"ζ) approximately equal in size (Plate 5c and 5d), solenidion ù slightly shorter (Plate 5b). Terminal sensillum or spinneret (eupathidion su ζ), twice as long as wide (4.1 in length and 2.7 in width) (Plate 5a). The trochanter is glabrous, palpal chaetotaxy is from trochanter to tibia, 0-1 (dPFe)-1 (l"PGe)- 3 (dPTi, l'PTi, l"PTi). The femoral seta d is formed as a short, stout, partly sunken spine-like process exhibiting sexual dimorphism.


 
Legs
 
Setal counts: cx 2,2,1,1; tr 1,1,1,1; fe 10,6,4,4; ge 5,5,4,4; ti 13,7,6,7; ta 20,16,11,11. Tarsus I with two sets of duplex setae (proximal solenidion 47-51, distal solenidion 59-61), 3 solenidia, 3 eupathidia and 10 tactile setae; with 4 tactile setae and 3 solenidia proximal to proximal set of duplex setae; Empodia I-IV each with obvious dorsal spur 4 μm long; empodia I and II claw-like (uncinate) (Plate 6c and 6d), empodia III and IV each with proximoventral hairs long and free (Plate 6b).


 
Aedeagus
 
Aedeagus with terminal knob, anterior and posterior projection short and pointed; flat to slightly convex dorsal surface and a medial indentation (Plate 6e).
 
Remarks
 
The infestation was high during the summer months. Symptom of damage caused by T. truncatus, is more similar to that of other Tetranychus spp. and is hard to differentiate unless proper mounting of the specimens and careful examination of the morphological characters done particularly that of male aedeagus. Reports of T. truncatus from North-western Himalayan regions of Jammu and Kashmir and Himachal Pradesh by Rather (1983) claims to be the first report of this species in India. Despite Gupta and Gupta (1994) stating it could be T. urticae, which was known to infest Dahlia. Nearly two decades later, Srinivasa et al., (2012) reported T. truncatus to infest Morus sp. from Karnataka. In Kerala, T. truncatus was reported by Bennur et al. (2015) on vegetable crops; on cucumber and amaranthus grown in polyhouses by Lenin and Bhaskar, 2016; on cowpea, pumpkin, tapioca, banana and Dahlia by Arunima (2017); on banana (Nendran) by (Bhaskar and Lenin, 2018). Recently T. truncatus was recorded on marigold, cock’s comb and rose from different localities of Thrissur district, Kerala by Prakash et al. (2022).
 
Hosts
 
Occurrence of T. truncatus has been recorded from 104 host plant species, of which major reports have been on Morus sp (Migeon and Dorkeld, 2023). In India, reports of incidence and damage has been given by Srinivasa et al., (2012) and Prakash et al., (2022).
 
Distribution
 
Tetranychus truncatus has so far been reported from 14 countries including India viz., Japan, China, Korea, Bangladesh, Indonesia, Phillipines, Taiwan, Thailand, Vietnam, Iran, Guam,  Burkina Faso and Northern Mariana Is. In India, reports of its occurrence have been claimed from Karnataka and Kerala (Migeon and Dorkeld, 2023).
 
Molecular characterization of Tetranychid mites
 
Molecular sequence analysis was performed to supplement the morphological description and taxonomic identity of tetranychid mites based on the sequence information obtained for the Internal Transcribed Spacer region 2 (ITS2). ITS2 gene, the most commonly used species-specific primer, was amplified, yielding an amplicon size of ~620 bp (Fig 1). The PCR products were purified and sequenced. The resulting sequences were searched against GenBank nucleotide database for deriving their annotation using nBLAST program. The results of BLAST analysis showed that Tetranychus truncatus, had 99 per cent similarity with GENBANK sequences (Fig 2).

Red spider mite Tetranychus truncatus has been reported on 14 host plants from 104 countries worldwide. In India, reports have been claimed from Karnataka on Morus sp. and Kerala on few vegetables and ornamental plants. At present, the incidence of red spider mites on groundnut was found to be severe and identified the species, T. truncatus based on morphological characters and molecular tools. The severity of the damage caused and the adaptability of the mite to warm and dry conditions indicate that this species as a potential pest in Tamil Nadu.

The authors are grateful to the Network Coordinator, AINP on Agricultural Acarology, UAS, Bengaluru and Department of Agricultural Entomology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University for providing all facilities to carry out the work.

There is no Conflict of Interest pertaining to this study.