Key to Megalurothrips usitatus (Bagnall) given by (Mound and Yongfoo 2009)
Never dark brown and reticulate; major setae not long and capitate. Pronotum never with more than five pairs of major setae. Abdominal tergites without numerous microtrichia occupying lateral thirds, with a few microtrichia near lateral margins. Major setae on head, pronotum and forewings setaceous Antennal segment II external margin not prolonged, segment I not swollen. Pronotum with at least one pair of prominent posteroangular setae Pronotal anterior margin with 1 or 2 pairs of setae that are much longer than discal setae. Forewing first vein with setal row widely interrupted; tergites without ctenidia forewing second vein with many equally spaced setae; tergite VIII either with no comb or with comb interrupted medially; tergites and sternites without prominent reticulation. Ocellar setae pair III arising close to anterolateral margins of ocellar triangle; tergite VIII posterior margin on lateral thirds with well-developed comb
Megalurothrips usitatus.
Megalurothrips usitatus (Bagnall)
Both sexes fully winged. Body dark brown, tarsi, apices of mid and hind tibiae, also most of fore tibiae yellow; hind tibiae with 2 stout dark apical setae (Plate 1). Fore wings brown with basal quarter pale and an extensive pale area sub-apically (Plate 2). Antennal segments I-II brownish yellow, III yellow, IV and sometimes V yellow at base; fore wing light brown, pale sub-basally and with sub-apical pale band (Plate 3). Antennae 8-segmented, I with pair of dorso-apical setae; III-IV with constricted apical neck, sensorium forked, VIII almost twice as long as VII (Plate 4). Head conspicuously transversely striate/reticulate at posterior (Plate 5), ocellar setae III long, arising just inside triangle; postocular setae not long (Plate 6). Pronotum sometimes 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 7). Mesonotum with transverse reticulation, lateral setae not long (Plate 8). Metanotum reticulate medially, median setae long, at anterior margin, campaniform sensilla present. Mesosternal furca with spinula, metafurca without spinula.
Tarsi all 2-segmented. Fore wing first vein with long row of setae before distinct sub-apical gap followed by 2 setae; second vein with complete row of setae; postero-marginal cilia wavy. Abdominal tergites II-VIII with no sculpture medially but lateral thirds with sub-parallel lines (Plate 9), median setae small (Plate 10); VIII with postero-marginal comb of small microtrichia laterally (Plate 11), discal area antero-mesead of spiracle with 2 or more rows of strong microtrichia; tergite X with incomplete longitudinal split. Sternites without discal setae, three pairs of long marginal setae, setal pair S1 on VII arise in front of margin (Plate 12). Male similar to female but smaller and paler, pronotum usually yellow; legs sometimes almost yellow; tergite IX with pair of short stout setae posterolaterally; sternites with no pore plates (Plate 13).
The species identification of thrips was confirmed by sending specimens to ICAR-NBAIR (Germplasm Collection and Characterization). Dr. Laurence Mound, an honorary research fellow at CSIRO in Australia, provided further confirmation. Precise identification, vector specificity plays major role in management of vector transmitted diseases. Thrips species level identification is mostly based on morphological characters, for instance color; antennae segment arrangement, body structural design, chaetotaxy, ocelli, thoracic features
etc. On the other hand, the morphology-based detection from time to time could be difficult due to their minute size, sexual dimorphism, high degree of similarity in various developmental stages and polymorphism (in color, wing development, body size
etc.), co-existence of multiple species on the same plant. The problem of identification can be aggravated when their appearances are varied in many ways within the species (
Mound and Kibby, 1998,
Natasa and Tradan, 2012).
Megalurothrips sp. is an economic importance pest and generally they attack various Fabaceae crop such as
Glycine,
Arachis and
Vigna (
Palmer, 1987;
Sani and Umar, 2017;
Tang et al., 2015; Zafirah and Azidah, 2018). Pest status of
M.
usitatus on blackgram in India is not yet reported and available literature in this aspect is also scanty. Hence, the present study concentrated on identification and molecular characterization of
M.
usitatus to know the species status. Following the identification of every specimen, the percentage of species composition was calculated and is shown in (Table 1). Districts Srikakulam, Vizianagaram, Krishna, Guntur, Prakasam, Kurnool and Chittoor districts showed 31.43, 28.14, 35.58, 47.31, 20.30, 21.14, 28.81 mean per cent of
M.
usitatus, respectively. Pittalavanipalem mandal of Guntur district and R. Amudalavalasa mandal of Srikakulam district have recorded with highest mean per cent of
M.
usitatus among the all other mandals
i.
e. 66.67 and 56.3, respectively.
Identification and confirmation of M. usitatus using DNA barcodes
Molecular markers that are developed with mitochondrial DNA (mtDNA) are useful to discriminate closely related species. The mitochondrial COI sequence was validated to identify and classify thrips species and to understand the phylogenetic relationship. In the present study, amplification was observed at 655 bp for 70 individual specimens using mtCOI marker (Plate 14-15 of supplementary information). Present results are in accordance with
Chakraborty et al., (2019) who studied 43
Scirtothrips spp specimens across India and identified using SEM utilizing the morphometric key and were further confirmed using mtCOI markers based on species specific amplification (648bp) and contributed six novel barcode sequences of three
Scirtothrips species from India. Another finding by
Rabeena et al., (2020) on the utilization of mtCOI based universal primer in identification of
F.
schultzei collected from tomato growing hot spot regions of Tamil Nadu and Karnataka. Specific amplicon size of ~638 bp was detected and the samples were sequenced and deposited in NCBI. Further
Singha et al., (2019) identified 11 specimens of
Frankliniella occidentalis (Pergande) collected from Karnataka, India and studied using
mtCOI universal primer and generated four sequences specific to the collected specimens and submitted in NCBI.
Studies on sequencing and homology
The sequences of
Megalurothrips usitatus found in this study exhibited 99-100% similarity with the NCBI sequences. The accessions from Bangladesh, Pakistan and India exhibited 100% similarity with the sequences of the current study, where as 99 per cent similarity was observed with accessions from India, Bangladesh, Pakistan, China, Indonesia. The generated DNA sequences of the species were annotated and submitted to the global database (GenBank) to acquire the unique accession numbers. List of samples, geographic location along with accession numbers presented in the (Table 1). With sufficient bootstrap support and posterior probability, the calculated Neighbor Joining phylogeny (30 sequences) has showed cohesive grouping of the generated
M.
usitatus sequences. All of the
M.
usitatus sequences of this investigation (MZ392030, MZ436473 to MZ436477, MZ478649) were clearly closely clustered with other
M.
usitatus derived from Bangladesh under Clade I (Fig 1). The
M.
usitatus group was generated by the cohesive clustering of the remaining sequences of
M.
usitatus from several countries, including China, Indonesia, Bangladesh and one sequence from IIHR in Bangalore, India, under clade I. Furthermore, this phylogenic tree showed that
Megalurothrips distalis (Karny) and
Megalurothrips pecularis (Bagnall) split off as distinct branches in clade II and separated as a separate cluster from clade I. Phylogenic tree analysis made it abundantly evident that
M.
usitatus is distantly connected to
Megalurothrips typicus (Bagnall) and closely related to
M.
pecularis and
M.
distalis. Using phylogenetic analysis, the NCBI sequences of
M.
typicus and the mtCOI sequences of
T.
tabaci and
T.
palmi, were also used to examine species-level diversity. Clade III emerged as the
T.
tabaci group, containing sequences from China, India and Australia, three distinct nations. Clade IV of the
T.
palmi group originated in southern India and Pakistan. Similar findings of distinct species-wise groups of
T.
palmi,
T.
tabaci,
F.
occidentalis,
S.
dorsalis and an unclassified group were also reported by
Kadirivel 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. Findings of the present study were in line with
Zafiraha et al., (2020) developed a phylogenic tree using the sequences of
M.
uistatus and
M.
typicus,
M.
distalis based on COI gene marker and reported that
M.
usitatus group formed as clade I where as
M.
usitatus Lineage II,
M.
distalis,
M.
typicus were formed as clade II. Typically
M.
usitatus Linaeage II was formed under subclade II and also reported that the inter specific distances between both
M.
usitatus Lineage I and Lineage II ranged from 8.78 to 9.63 per cent, suggesting the presence of cryptic and non-monophyly lineages between two morphoforms of
M.
usitatus in Peninsular Malaysia.
Genetic divergence and haplotype analysis
mtCOI sequences of this study (07 sequences of
M.
usitatus from Andhra Pradesh, Annexure II), nine GenBank-sourced sequences of three different geographic regions (India, Indonesia, Bangladesh and China) combined has revealed 06 haplotypes which were clustered in a network according to genetic diversity. For the current haplotype analysis,
mtCOI sequence containing 657 nucleotides was chosen and 628 nucleotides total-excluding gaps or missing data sites-were employed. Data revealed that Srikakulam, Vizianagaram, Guntur, Chittoor, of present study and sequences from Bangladesh were formed into Hap_1 where as other mtCOI sequences from Krishna, Prakasam and Kurnool of present study were formed into Hap_2. Six sequences from Bangladesh were formed into Hap_3. Other sequences from Indonesia was formed into Hap_4. Sequences from India and China were formed into Hap_5 and Hap_6, respectively (Fig 2). This haplotype network was in support with our previously constructed neighbor joining and maximum likelihood trees of present study. These results were supported by
Tyagi et al., (2017) who analysed 85
T.
palmi mtCOI sequences from India, resulted in eight haplotypes (H9-H10, H126-H131) forming three distinct clades in both the NJ and BA tree. These three clades were also represented by three MOTUs (
T.
palmi Ia1,
T.
plami IIa1 and
T.
palmi Ib2) in ABGD, GMYC and bPTP analysis. Further analysis stated that a total of 32 sequences were used for haplotype network and these sequences were grouped into six haplotypes. A maximum of nine segregating sites were observed with nucleotide diversity (p) 0.00509 and standard de
viation of nucleotide diversity (p)
i.
e., 0.00055. Haplotype diversity was recorded as 0.750 with a standard deviation of 0.056. Estimated mutations among the sequences were nine. Tajimas D statistic was 1.31414 (Not significant, P>0.10) (Table 2) which revealed the existence of low genetic polymorphism among the COI sequences of
M.
usitatus. Virus transmission ability of
M.
usitatus on blackgram is yet to be discovered in the further research programmes especially in blackgram crop where bud necrosis is often causing huge losses.
