India is the largest producer and also consumer of blackgram. It is referred as the “king of the pulses” due to its delicious taste and numerous other nutritional qualities (Vadivel et al., 2023). It is rich in nutritional quality with 24-27% protein, 1% fat, 57% carbohydrate, 3.8% fibre and 4.8 % ash. It is grown in both summer and winter seasons (Mohanlal et al., 2023). Furthermore, it is fed to milch cows in particular as nutrient-rich fodder. Globally India is the largest producer of black gram, accounting for more than 70% of production followed by Myanmar and Pakistan (Bharathi et al., 2025). Thrips, major sucking insect pests in blackgram causing considerable damage by sucking cell sap and also as vectors of GBNV which causes bud necrosis. A management technique requires accurate pest identification as a basic initial step. A technical person’s diagnosis depends on their ability to quickly and accurately identify taxa. Without the presence of adults, it is typically impossible to identify larval Thysanoptera to species level. A constraint in thrips morphological identification is that larval stages cannot be identified with most available keys and exhibit fewer characters of diagnostic value than the adults (Glover et al., 2010). Species identification using morphological features has some significant limitations as the species might have minimal or no phenotypic changes but have high genetic variability. Morphological identification overlooks cryptic features, which are common in many groups and the use of keys requires a high level of expertise as misdiagnosis is common (Hebert et al. 2003; Armstrong and Ball, 2005). The most reliable identification is obtained by the combination of various techniques. The time-consuming nature of the classical morphological procedures does not make them unappreciated; particularly as appropriate identification using morphological keys is typically a necessary initial step in the validation of the more recent methods. Morphological identification is much cheaper economically than molecular identification as the materials and equipment used in morphological identification require less expenditure (Hillis and Davis, 1987; Wiens, 2004). Morphological keys may still be essential for specimen identification down to the genus level. Alternatively, these keys may be optional. Majority of thrips are host-plant specific, hence molecular and morphological identification techniques need to be used in a complementary manner to clearly identify the species of the specimens. Therefore, a more general, simple, accurate and large-scale identification method would be helpful to facilitate identification of thrips species occurring in a cropping system where multiple species co-exist, and the population dynamics are influenced by numerous factors (Kadirvel et al., 2013). Molecular techniques provide powerful tools for the study of insect population ecology and insect systematics. In addition, analysis of molecular markers that can discriminate closely related species and monitor specific populations in the field. The outward traits of a species can vary within the species or overlap with those of other species, making the morphological examination method of adult identification challenging. The ‘‘DNA barcoding’’ is a method based on DNA sequencing of a standard gene region (Hebert et al., 2003). It can be helpful in species diagnosis because sequence divergences are usually much lower among individuals of a species than between closely related species (Hebert et al., 2003). However, studies on thrips infesting blackgram are scanty especially on  their identification using morphology and molecular strategies together are very few. Keeping this in view current study was designed.

The present investigation entitled “Identification and molecular characterization of T. palmi (Karny, 1925) a potential GBNV vector in blackgram using ITS2 marker” has been conducted in the laboratory of the Department of Entomology, Agricultural College, Bapatla, Guntur district, A.P., India during 2019-2022. Thrips were randomly collected from predominant blackgram cultivating areas duly covering all climatic zones of A.P., India. Thrips were collected by simply beating the plants on black tray and carefully transferred to vials containing alcohol, glycerin acetic acid (AGA) mixture having 10 parts of 60% ethyl alcohol with one part of glycerin and one part of acetic acid. Additional specimens collected were immediately transferred to -20°C to carryout molecular studies. Permanent mounts in natural Canada balsam were prepared for microscopic examination using Maceration and dehydration protocol (Mound and Kibby, 1998). Specimens were identified by following the taxonomic keys given by Cluever and Smith (2017), labeled neatly and then percentage of species composition was worked out. Further species confirmation was done through molecular characterization using stored buffer samples. From all the 35 locations, two samples each for T. palmi were subjected to molecular characterization through PCR. Further a representative sample from each district was selected and utilized for characterization studies. Single thrips specimens collected from each location was morphologically identified based on the taxonomic keys and quickly transferred to 1.5 mL centrifuge tubes with proper labeling. The DNA was extracted from a single thrips specimen using the salting out protocol given by Sunnucks and Hales (1996) with slight modifications (Supplementary material). Concentration of the DNA was examined, diluted and stored at -20°C for further PCR analysis. Thrips palmi specific marker ITS2 (Internal transcribed spacer 2-located in the 5.8S region flanking the ITS2 region of ribosomal DNA) FP:GTGAACTG CAGGACACAT RP:CACC TGAA CAGAGGTCGG was employed. The PCR was carried out Initial denaturation 94°C~10 minutes; Denaturation 94°C~60 seconds; Annealing 55°C~60 seconds; Extension 72°C~60 seconds (total 35 cycles), Final extension 72°C~15 minutes; Hold ~4°C. The migration pattern of the DNA fragments in the agarose gel was visualized in a UV light transmitted gel documentation system (SYNGENE Gene flash, U.K.). After this, PCR products were processed for purification using QIAGEN QIAquick PCR Purification Kit (cat. No. 28104). A total of 07 (seven) samples were sequenced bidirectionally using Sanger di-deoxy chain termination method at Barcode Biosciences Pvt. Limited, Bengaluru. To investigate the genetic relationship across the collected samples a phylogenetic tree was constructed using all identified haplotypes. The homologous sequences were selected based on the similarity percentage between present study and sequences available at NCBI. Complete DNA alignment was done using MEGA software (version 11.0). Neighbor Joining tree (NJ) method with 1000 bootstrap was employed. Twenty-three Gene sequences (ITS2) aligned against the current study sequences. The final dataset of thirty (30) sequences was aligned, edited using Bioeditv 7.0 software. To test the reciprocal monophyletic criteria for species identification, the generated sequence data set was further studied for genetic divergence through haplotype analysis. Diversity was estimated in terms of segregating sites, nucleotide and haplotype diversity along with Tajima’s D statistic, which tests for neutrality and recent population expansion or contraction, using DNASP6.

Specimens were identified based on the photo based key given by Cluever and Smith (2017) as follows.
1 Wings (brachypterous or macropterous) or wing buds present ……………………..2
2′ Wings fully formed, with setae present ............… ………………………... Adult, 4
4′ Abdominal segment X conical; female with saw-like ovipositor ……..Terebrantia, 6
6′ If ctenidia are present on abdominal tergites V–VII, ctenidium on tergite VIII  posterior to spiracle ; anterior  margin of prothorax lacking major setae  antennae 7-, 8-, or 9-segmented . ..15
15′ Lateral margins of abdominal tergites VI–VI lacking closely spaced rows of microtrichia; cilia of forewing fringe wavy; ocellar III setae not arising within the ocellar triangle………...16
16′ Abdominal tergites V–VIII with paired ctenidia laterally; antennae 7-or 8 segmented………………………… ……………………………………………………19
19′ Tergites III-VIII lacking craspedum; pronotum transverse; antennae 7-or 8-segmented………………… ……………………………………………………………………….20
20′ Metanotum with median pair of setae arising posterior to anterior margin; antennae 7 segmented……… ………………………………………………………………………22
22′ Abdominal sternites lacking discal setae, setae present only at posterior margin; row of setae on 1^st vein with spaces between setal bases much greater than length of each seta……………………………… ………………………… …………………...23
23′ Metanotal campaniform sensilla present; microtrichia lacking on lateral thirds of tergites IV–VI ……………………………………………………………..Thrips palmi (Karny)
 
Thrips palmi (Karny)
 
A clear yellow body with no dark areas on the head, thorax or abdomen (slightly thickened-blackish body setae); antennal segments I and II pale, III yellow with apex shaded and sensorium forked (Plate 1), IV-VII brown but usually with base of IV-V yellow; forewings uniformly slightly shaded, prominent setae dark.


       
Antennae always seven-segmented (Plate 2).     

 

Post ocular setae II and IV much smaller than remaining setae.
Ocellar setae III standing either just outside the ocellar triangle or touching the tangent lines connecting the anterior ocellus and each of the posterior ocelli (Plate 3).



Pronotum with transverse carina parallel to posterior margin, median area weakly transversely reticulate; 2 pairs of long posteroangular setae, outer longer than inner, one pair of anteroangular setae moderately prominent (Plate 4).



Metascutum with sculpture converging posteriorly; median pair of setae behind anterior margin; paired campaniform sensilla present.
Forewing first vein with three (occasionally two) distal setae (Plate 5).



Abdominal tergite II with four lateral marginal setae (Plate 6).



Abdominal tergites III to IV with setae S2 dark and subequal to S3.
Abdominal tergite VIII with posteromarginal comb in female complete, in male broadly developed posteriorly.
Abdominal tergite IX usually with two pairs of campaniform sensilla (pores).
Abdominal sternites without discal setae or ciliate microtrichia.
Abdominal pleurotergites without discal setae.
Male: Sternites III-VII each with a narrow transverse glandular area.

From Table 1, it was evident that T. palmi was found in all locations with 65.01, 61.39, 49.25, 44.00, 76.54, 76.13 and 67.13 per cent in Srikakulam, Vizianagaram, Krishna, Guntur, Prakasam, Kurnool and Chittoor districts of A.P., respectively. In Chittoor district Megalurothrips typicus (Bagnall), Ayyaria chaetophora (Karny), Phibalothrips peringueyi (Faure) and some Tubulifera thrips were also observed in meager numbers.


 
Confirmation of Thrips palmi using ITS2 marker
 
Internal Transcribed Spacer (ITS) is a challenging marker, technically it is present in multiple distinct copies and has likelihood of containing high intra and inter-genomic variation. This marker is useful for species identification in taxon specific studies as it produces alignment overlaps in the genus-specific range (Dentinger et al. 2011; Stern et al., 2012). However, the fact that ITS2 sequences are potential markers for general phylogenetic studies and have been widely used for phylogenetic tree reconstructions both at genus and species levels which makes them ideal for species differentiation (Miao et al., 2008; Schultz and Wolf, 2009). The ITS-based markers have been used by various researchers for species-level identification of thrips (Farris et al., 2010; Grazia et al., 2016; Toda and Komazaki, 2002; Kumar et al., 2017). All the 70 thrips samples collected from 35 geographic locations produced an amplicon size of ~570bp with ITS2 marker. The results are in agreement with Sumit et al., (2020) who have reported that T. palmi was identified from the samples collected from brinjal, lettuce and tomato using multiplex PCR assay with designed ITS2 primer without any cross reactivity. Other workers Nakahara and Minoura (2015) amplified the internal transcribed spacer 2 region (ITS2) of nuclear ribosomal DNA using five specific primers for 71 individuals of the four thrips species viz., T. palmi, T. tabaci, F. intonsa and F. occidentalis that were frequently found in Japanese quarantine inspection sites based on species-specific single bands (470 bp, 410 bp, 370 bp, 280 bp). Yeh et al., (2014) obtained 43 ITS1 sequences ranging from 800-1200 bp for 15 thrips species and deposited in NCBI (AB904169-AB904212) and also reported that multiplex PCR using specific primers based on ITS1 sequences is a simple, reliable and cost-effective diagnostic tool in the identification of thrips (T. tabaci, F. intonsa and S. dorsalis). Farris et al., (2010) studied 432 S. dorsalis specimens representing 15 geographic populations and reported that 12 populations displayed 100% amplification of ITS2 fragment ranging from 131 to 135 bp and these included the populations from India, Japan, U.S.A, Barbados, Israel and Venezuela.
 
Sequencing and homology studies:
 
Seven representative T. palmi samples (Table 1) were sequenced bidirectionally using sanger sequencing method.  Homologous sequences across the globe were retrieved from NCBI database using BLASTN tool and subjected to homology studies. Our sequences had shown similarity of 97 to 100 per cent with data sets throughout the world. The NCBI data base sequences MN889880, KU884558, FM956428, FM956427, FM956422 from India, AB775442 from China showed 100% similarity with present T. palmi isolates. KF680274, KU884557, KU884556 sequences have shown 99 per cent similarity where as KF680275 (India), KT885219 and KT885218 (U.S.A), AB775439 and AB775435 (China) have shown 98 per cent similarity. MN1942020 (India), KT885216 (U.S.A), AM932178 and AM932146, AM932140, AM932157 (U.K), AB063341 (Japan), AB775437, AB775436 (China), KM877305 and LC416224 (Taiwan) have shown 97 per cent similarity. The sequences of the present study were aligned using MEGA 11.0 (Molecular Evolutionary Genetic Analysis) with known reference sequences in NCBI website. Sequences generated in this study were annotated and submitted to the global database (GenBank) to acquire the unique accession numbers Table 1. Phylogeny tree was constructed using neighbor joining (NJ, ML) method depicted cohesive clustering of the identified seven sequences of T. palmi along with the database sequences (23 homologous sequences with similarity of 97 to 99%) with 1000 bootstrap replicates. The phylogenetic tree represented four distinct clades of the present dataset Clade-1, Clade II, Clade-III and Clade IV (Fig 1) with a bootstrap value >80% highlighting a significant rate of phylogenetic relationships among the species studied. Clade I showed cohesive clustering of present study specimens (accession number MZ427914 to MZ42720) with other reported isolates from Indian sub-continent. Further, T. palmi isolates from foreign countries were also clustered in clade I on different node. Isolates of T. palmi from China, U.K, U.S.A were very closely clustered in another node under clade I (T. palmi group), whereas the clade II comprised of T. nigropilus species of U.K, clade III comprised of T. tabaci species of U.K and clade IV comprised of T. flavus species of U.K. The out-group sequences were procured from NCBI website (https://www.ncbi.nlm.nih.gov). Out group is more distantly related group of organisms that serves as a reference group when determining the evolutionary relationships of the ingroup. Out-group serves as a point of comparison for the in-group and specifically allows for the phylogeny to be rooted.  The phylogeny revealed the close relationship among India, china, U.K and U.S.A populations of T. palmi. Further, T. palmi species has close association with T. nigropilosus rather than with T. tabaci and T. flavus populations. Both the species were present in different clades under same cluster. Similar findings of distinct species-wise groups of T. palmi, T. tabaci, F. occidentalis, S. dorsalis and an unclassified group were also reported by (Kadirvel et al., 2013). Higher intra specific genetic variation was observed in case of   S. dorsalis and   T. palmi followed by T. tabaci   and F. occidentalis. Genetic divergence and haplotype analysis study revealed that the seven ITS2 sequences of T. palmi of present study combined with 17 sequences from GenBank i.e., four geographic regions (India, U.K, U.S.A and China) revealed 22 haplotypes which were clustered in a network according to genetic diversity existed among them. The ITS2 sequence with 552 nucleotide region was selected for the present haplotype analysis and finally 533 nucleotides were used excluding sites with gaps or missing data. Data pertaining to haplotype and genetic diversity is presented in Table 3. Data presented in the Table 2 and 3, (Fig 2) reveals that the present study sequences were formed into seven haplotypes namely Hap_1 to Hap_7 where as other ITS2 sequences from India were formed into seven haplotypes i.e. Hap_8 to Hap_14. Sequences from other countries viz. U.S.A and U.K were formed into Hap_15. The sequences from country U.K were formed into three different haplotypes i.e., Hap_16, Hap_17, Hap_18 and Hap_22. Sequences from China were formed into three haplotypes i.e. Hap_19, Hap_20, Hap_21. The haplotype network generated in the present study is in accordance with the neighbor joining and maximum likelihood tree obtained earlier in this study. A total of 48 sequences were used for construction of haplotype network and these sequences were grouped into 22 haplotypes. A maximum of 88 segregating sites were observed with Nucleotide diversity (p) 0.02836 and standard deviation of nucleotide diversity (p) 0.00302. Haplotype diversity was recorded as 0.968 with a standard deviation of 0.009. Estimated mutations among the sequences were 96. Nucleotide diversity (pi) values range between 1 (very diverse) and 0 (not diverse) and hence present study showed pie value 0.00302 which revealed the existence of low genetic polymorphism among the ITS2 sequences of T. palmi. Tajimas D statistic was also estimated i.e., -1.07506 (Not significant, P>0.10; values greater than +2 or less than -2 are likely to be significant). A negative Tajima’s D signifies an excess of low frequency polymorphisms relative to expectation, indicating population size expansion where as a positive Tajima’s D signifies low levels of low and high frequency polymorphisms, indicating a decrease in population size and/or balancing selection.  However, such type of interpretation could not be made as D-value is statistically not significant.

The precise identification of species is a first step in development of management strategies for any pest. Species identification and subsequent understanding of vector specificity play a major role in management of vector transmitted diseases. Since thrips are very tiny insects, species identification by morphological observation is very difficult task and it may lead to confusion about the vector status. Hence, morphological identification coupled with molecular characterization gives accurate identification. As thrips are very minute, their total genomic DNA yield will be generally low. To overcome these possible problems of insufficient template, specific primers were used to amplify a smaller fragment of the targeted gene. The specific primer ITS2 and gene fragment-based DNA barcoding in the current study enabled proper identification of species T. palmi. The presence of remaining species were also in considerable range among the surveyed locations but not consistent.

The present study was conducted by the corresponding author during doctoral degree programme.
 
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The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
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The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript. Informed consent. All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.