Front. Insect Sci. Frontiers in Insect Science Front. Insect Sci. 2673-8600 Frontiers Media S.A. 10.3389/finsc.2025.1536160 Insect Science Original Research Unravelling the complete mitochondrial genomes of Thrips tabaci Lindeman and Thrips parvispinus Karny (Thysanoptera: Thripidae) and their phylogenetic implications Soumia P.S. 1 * Shirsat Dhananjay V. 1 Karuppaiah Vadivelu 1 Divekar Pratap A. 2 Mahajan Vijay 1 1 Crop Protection Section, ICAR-Directorate of Onion and Garlic Research, Pune, Maharashtra, India 2 Division of Crop Protection, ICAR- Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India

Edited by: Tiago Fernandes Carrijo, Universidade Federal do ABC, Brazil

Reviewed by: Menglin Wang, University of California, Berkeley, United States

Alina Mikhailova, University of Münster, Germany

*Correspondence: P.S. Soumia, soumiaps@gmail.com

28 02 2025 2025 5 1536160 28 11 2024 10 02 2025 Copyright © 2025 Soumia, Shirsat, Karuppaiah, Divekar and Mahajan 2025 Soumia, Shirsat, Karuppaiah, Divekar and Mahajan

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Onion (Allium cepa Linnaeus) is an important vegetable crop valued for its nutritional properties and economics worldwide. Onion cultivation faces serious threats from pests and diseases, particularly onion thrips (Thrips tabaci), which cause substantial yield losses. Recently, Black thrips (Thrips parvispinus), an invasive key pest of chili, have been reported to cause severe damage in onion crop and is likely to devastate the onion cultivation in near future. Therefore, this study was conducted to address the knowledge gap concerning the genetic basis and evolutionary history of T. tabaci and T. parvispinus through sequencing of their mitochondrial genomes. T. tabaci and T. parvispinus were collected from different locations in Maharashtra, India, and reared in the laboratory. The mitochondrial genomes of T. tabaci and T. parvispinus were sequenced to a length of 15,277 and 15,285 bp, respectively. Both genomes exhibited similar gene organization with regard to thirteen protein-coding genes and two rRNA genes. T. tabaci contained 19 tRNA genes whereas T. parvispinus contained 18 tRNA genes. The evolutionary positions of T. tabaci and T. parvispinus within the Thysanoptera order were elucidated through phylogenetic analysis of the mitogenomes of 15 thrips species. These findings provide crucial insights into the genetic makeup and evolutionary dynamics of both the thrips species, thereby aiding the development of novel and sustainable pest management strategies to mitigate their impacts on crops in the changing climate scenario.

 invasive pest mitochondrial genome phylogeny Thrips parvispinus Thrips tabaci section-in-acceptance Insect Systematics Introduction

Onion (Allium cepa L.) is widely acknowledged as an important vegetable crop worldwide, extensively grown and consumed for its culinary and nutritional value, and plays a vital role in the economies of many countries (1). Onion cultivation is particularly widespread in India, which is one of the world’s leading producer and exporter (2). However, onion production faces numerous challenges due to the prevalence of various pests and diseases, which lead to substantial yield losses (3). Globally, onion growers are concerned about onion thrips, Thrips tabaci Lindeman (Thysanoptera: Thripidae) (4, 5). In India, this pest is considered of national significance, causing 30% to 40% yield loss in onion crops (6, 7). Thrips infestation not only weakens the plants but also makes them susceptible to secondary infections and diseases, which exacerbates economic impacts on farmers (8, 9). These pests are known vectors for various plant viruses, including the iris yellow spot virus (8, 10), and they also transmit diseases like Stemphylium leaf blight and bacterial leaf blight (3, 11–13). Virus-vector relationship have been well documented in the case of thrips; however, their interaction with fungal pathogens remains largely unexplored (Saini et al., 2024). Moreover, thrips usually exhibit genetic heterogeneity, which might be due to the spatial variation in insecticide efficacy (14). Based on mitochondrial DNA sequences, T. tabaci has been classified into three biotypes: one associated with tobacco and two associated with leek (L1 and L2) (15). Also, due to the anticipated increase in temperature, the lifecycle of T. tabaci is likely to shorten, leading to multiple generations within a single crop season (16). Currently, onion growers rely on chemical pesticides to manage these pests, but it often seems futile due to their overlapping generations, concealed feeding behavior, and growing insecticide resistance (17, 18).

Similarly, Thrips parvispinus (Southeast Asian thrips or black thrips), are known to infest a variety of host plants, including vegetables, ornamentals, and field crops. Recently, T. parvispinus has been found infesting onion crops (19), which could pose a substantial threat in the near future (20). The inclusion of T. parvispinus in the list of onion pests reveals a potential gap in our understanding of thrips species interactions and their impact on onions. A comprehensive understanding of the biology, genetics, and evolutionary relationships of thrips is required to tackle these pests in onion and to devise appropriate management strategies. Recent advancements in molecular biology have enabled researchers to explore the genetic makeup of various organisms, providing insight into their evolutionary histories and ultimately helping in devising novel pest management strategies.

The increasing interest in mitochondrial genomes for phylogenetic studies has led to a surge in published mitogenome sequences, particularly amongst insects. Although the complete mitochondrial genomes of several thrips species, such as Thrips imagines (21), Frankliniella occidentalis (22), Frankliniella intonsa (23), Scirtothrips dorsalis (24), Anaphothrips obscurus (25), Thrips palmi (26), Dendrothrips minowai (27), Thrips hawaiiensis (28), Thrips parvispinus (29) and Aptinothrips stylifer (30), have been sequenced, comprehensive research specifically focusing on onion thrips is unexplored. Insect mitogenomes are small, circular, and consist of 37 genes: 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), and 22 transfer RNA genes (tRNAs), along with a large A+T-rich control region (CR) that regulates transcription and replication (31, 32). Their maternal inheritance, conserved gene content, and rapid evolutionary rate make mitogenomes valuable molecular markers for evolutionary research (33). The significant variation in mitochondrial genome organization observed within the subfamily Thripinae is highly unusual and contrasts with patterns seen in most other animals. The reasons behind the rapid evolution of mitochondrial genomes in Thripinae, as well as the evolutionary dynamics of mitochondrial genomes in other thrips, remain to be explored (25). In this context, the present study on the complete mitogenome of T. tabaci and its phylogenetic implications is of great significance. Furthermore, comparative studies of different thrips mitogenomes will help in elucidating the evolutionary patterns and population dynamics within the Thysanoptera order. Therefore, the study aims to address the knowledge gap by presenting the complete mitochondrial genome sequences of T. tabaci and T. parvispinus. This will offer valuable insights into their evolutionary relationships, population structure, and genetic diversity, ultimately aiding in devising an effective pest management strategy.

 Materials and methods Sample collection

Adults of onion thrips were collected from onion plants and initially reared in the laboratory on French beans at the ICAR-Directorate of Onion and Garlic Research (ICAR-DOGR) in Pune, Maharashtra, India (latitude: 18.84°N, longitude: 73.88°E, 616.29 meters above sea level). After completing their life cycle, adults emerged from individual eggs, were collected and used for further analysis. Meanwhile, Black thrips from chili plants in a farmer’s field (latitude: 18.87°N, longitude: 74.05°E, elevation: 667.84 meters) were collected and preserved in 99% ethanol for further analysis. The species identity of these specimens was confirmed through DNA barcoding of the COX-1 gene, and their sequence information has been submitted in the NCBI GeneBank database with accession numbers PP980527 and PP982736 for T. tabaci and T. parvispinus respectively.

 Sample preparation and DNA isolation

Single adults of T. tabaci and T. parvispinus were macerated in liquid nitrogen, and total genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Germany). The integrity of isolated total DNA was visualized on 1% agarose gel and further quantified using a nanodrop (Bio-Rad, Hercules, California, USA). Mitochondrial DNA was then synthesized from the total DNA using the REPLI-g Mitochondrial DNA Kit (QIAGEN, Germany).

 Sequence assembly, annotation, and analysis

The genome library was constructed with the QIASeq FX DNA kit (QIAGEN, Germany), and sequencing was performed on an Illumina NextSeq 2000 platform using 300-cycle paired-end chemistry, generating primary FASTQ data. These FASTQ files were assessed for total bases, read counts, GC%, Q30, and uncertain base percentages. Reads of high quality were obtained by eliminating adaptor contamination, ambiguous reads, and junk sequences using the fastp tool (v0.12.4) (34). Subsequently, BWA MEM (v0.7.17) was used to align the cleaned reads to the reference sequence (35). Protein-coding and RNA genes were identified from the consensus sequence with SAM tools’ mpileup (36). A de novo assembly using a de Bruijn graph approach was performed to construct longer DNA contigs, and the mitogenome was assembled using the MEGAHIT tool (37) as part of the MitoZ package (38). The quality of assembly was evaluated using the Quast tool (39).

Gene annotation for the mitogenome was conducted with the Prokka annotation tool (40) on the Proksee web server (https://proksee.ca/) (41), producing a circular genome map, GC concentration, and GC skew. For tRNA gene structure prediction and mitogenome assessment, MITOS2 (42) at the Galaxy Europe Web Server (https://usegalaxy.eu/) was used.

The relative synonymous codon usage (RSCU), codon usage, and base composition (A+T contents) of the PCGs were analyzed using MEGA Software (v11.0.13) (43). The GC skewness was computed using the formula GC skew = (G-C)/(G+C); whereas the formula AT skew = (A-T)/(A+T) was used for determining the AT skewness (44). Intergenic spacers and gene overlaps were manually determined. The complete mitochondrial genomes of T. tabaci and T. parvispinus were submitted to the NCBI genome database under accession numbers PQ197393 and PQ197392, respectively.

 Phylogenetic analysis

The evolutionary studies related to T. tabaci and T. parvispinus with the metagenome of 14 other thrips species were analyzed using the NCBI retrieved sequence information. The damsel bug, Alloeorhynchus bakeri, was used as an outgroup species. The nucleotide sequences of 13 protein coding genes of 16 thrips species and one outgroup species were aligned individually using MAFFT 7, the Database of Aligned Structure Homologue (DASH) was utilized to incorporate homologous structures based on amino acid codons (45). Followed by removal of the ambiguously aligned sites, the aligned amino acid sequences were then converted to the nucleic acid sequence. For phylogenetic analysis the sequences of 13 PCGs were concatenated in following order: nad5, nad4, nad4L, nad6, cox1, nad3, cox2, cox3, atp6, atp8, nad1, nad2 and cytB in a single sequence of each species. All the 17 sequences were aligned using CLustalW tool (46). The phylogenetic tree was constructed using the Maximum Likelihood method in MEGA 11 software (43), employing the general time reversible model with gamma distribution (GTR+G) and bootstrap values from 500 iterations.

 Results Structure and composition of mitogenome

The circular genome of T. tabaci is 15,277 bp long ( Figure 1A ), comprising 34 sequence elements (such as 13 PCGs, 19 tRNAs, and 2 rRNA-coding genes). Strand localization analysis revealed 19 genes located on the H-strand (+), whereas 15 genes on the L-strand (–) ( Table 1 ). Gene length in the T. tabaci mitochondrion was 400 bp on average, with minimum and maximum lengths of 57 bp (trnS1) and 1695 bp (nad5), respectively. Base compositions of the complete mitochondrial genome of T. tabaci were 41.3%, 34.8%, 12%, and 11.9% for A, T, G, and C nucleotides. The 13 PCGs and two rRNAs had AT contents ranging from 69.80% to 82.50%, whereas the overall mitogenome had 76.10%. In contrast, the GC content varied from 17.50% to 30.20% for the 13 PCGs and 2 rRNAs, whereas the overall mitogenome had 23.90%.

Circular genome map of (A) T. tabaci and (B) T. parvispinus showing mitogenome sequence features.

Summary of Thrips tabaci mitochondrial genome.

Gene Name Full Name and Function Gene Type Position Length (bp) Strand Intergenic Space Codon Anti-codon
Start End Start Stop
nad5 NADH dehydrogenase subunit 5 PCG 205 1899 1695 – -12 att tag –
trnH Transfer RNA for Histidine tRNA 1888 1948 61 – 10 – – gtg
nad4 NADH dehydrogenase subunit 4 PCG 1959 2966 1008 – 292 att t –
nad4L NADH dehydrogenase subunit 4L PCG 3259 3531 273 – 26 atg taa –
trnC Transfer RNA for Cysteine tRNA 3558 3617 60 + 31 – – gca
nad6 NADH dehydrogenase subunit 6 PCG 3649 4122 474 + 51 att taa –
rrnL 16S ribosomal RNA rRNA 4174 5325 1152 + 22 – – –
COX1 cytochrome c oxidase subunit I PCG 5348 6889 1542 + 65 ata taa –
nad3 NADH dehydrogenase subunit 3 PCG 6955 7305 351 + -1 att taa –
COX2 cytochrome c oxidase subunit II PCG 7305 7964 660 + 11 ata taa –
trnG Transfer RNA for Glycine tRNA 7976 8037 62 + 3 – – tcc
trnK Transfer RNA for Lysine tRNA 8041 8102 62 + 10 – – ttt
COX3 cytochrome c oxidase subunit III PCG 8113 8895 783 + 0 ata taa –
trnN Transfer RNA for Asparagine tRNA 8896 8959 64 + -3 – – gtt
trnT Transfer RNA for Threonine tRNA 8957 9019 63 + -1 – – tgt
trnL1 Transfer RNA for Leucine tRNA 9019 9082 64 + 2 – – tag
trnE Transfer RNA for Glutamic acid tRNA 9085 9147 63 + 8 – – ttc
trnS1 Transfer RNA for Serine tRNA 9156 9212 57 + 6 – – tct
trnQ Transfer RNA for Glutamine tRNA 9219 9286 68 + 37 – – ttg
trnS2 Transfer RNA for Serine tRNA 9324 9387 64 + 0 – – tga
trnD Transfer RNA for Aspertic acid tRNA 9388 9451 64 + 451 – – gtc
trnR Transfer RNA for Arginine tRNA 9903 9968 66 – 18 – – tcg
ATP6 ATP synthase F0 subunit 6 PCG 9987 10607 621 – 50 ata taa –
ATP8 ATP synthase F0 subunit 8 PCG 10658 10860 203 – -54 att t –
rrnS 12S ribosomal RNA rRNA 10807 11413 607 – 116 – – –
trnF Transfer RNA for Phenylalanine tRNA 11530 11594 65 – -1 – – gaa
trnA Transfer RNA for Alanine tRNA 11594 11656 63 – 2 – – tgc
trnM Transfer RNA for Methionine tRNA 11659 11721 63 – -4 – – cat
nad1 NADH dehydrogenase subunit 2 PCG 11718 12641 924 – 6 ata taa –
nad2 NADH dehydrogenase subunit 1 PCG 12648 13628 981 – 73 att taa –
trnY Transfer RNA for Tyrosine tRNA 13702 13765 64 + -3 – – gta
CYTB cytochrome b PCG 13763 14908 1146 – -29 ata tag –
trnI Transfer RNA for Isoleucine tRNA 14880 14943 64 – 2 – – gat
trnP Transfer RNA for Proline tRNA 14946 15008 63 + 268 – – tgg

The AT skewness for the 13 PCGs ranged from 0.426 to 0.033, higher than that of the rRNA genes, rrnS (-0.193) and rrnL (0.180) ( Table 2 ). The 1560 bp of intergenic nucleotides were spread across 23 locations, with individual spacer lengths ranging from 2 to 451 bp. The longest intergenic spacer (451 bp) was found between the trnD and trnR genes. There were overlaps between nine genes, whose lengths varied from 1 to 54 bp. The atp8 and rrnS genes had the lengthiest overlap, 54 bp, as shown in Table 1 and Figure 1A .

Nucleotide composition of Thrips tabaci mitogenome.

Gene T C A G Total GC% AT% GC Skew AT Skew
nad5 53.2 10.9 25.2 10.7 1695 21.60 78.40 -0.009 -0.357
nad4 51.8 10.8 24.4 13 1008 23.80 76.20 0.092 -0.360
nad4l 56.4 8.8 22.7 12.1 273 20.90 79.10 0.158 -0.426
nad6 44.7 9.9 37.8 7.6 474 17.50 82.50 -0.131 -0.084
cox1 37.5 15.9 32.3 14.3 1542 30.20 69.80 -0.053 -0.074
nad3 38.7 12.9 38.7 9.7 351 22.60 77.40 -0.142 0.000
cox2 35.6 16.2 36.2 12 660 28.20 71.80 -0.149 0.008
cox3 39.4 16.4 32.4 11.8 783 28.20 71.80 -0.163 -0.097
atp6 37.7 14.8 36.2 11.3 621 26.10 73.90 -0.134 -0.020
atp8 41.3 14.3 32.5 11.9 126 26.20 73.80 -0.092 -0.119
nad1 37.3 15.4 35.5 11.8 924 27.20 72.80 -0.132 -0.025
nad2 39.1 12 41.8 7.1 981 19.10 80.90 -0.257 0.033
cob 40.6 14.7 32.7 12 1146 26.70 73.30 -0.101 -0.108
rrnS 45.8 12.2 31 11 607 23.20 76.80 -0.052 -0.193
rrnL 32.5 9.8 46.8 10.9 1152 20.70 79.30 0.053 0.180

Likewise, the circular mitogenome of T. parvispinus is 15,285 bp, comprising 13 PCGs, 18 tRNA-coding genes, and 2 rRNA-coding genes, 28 of which are located on the H-strand (+) the remaining 5 on the L-strand (–). Gene length in the T. parvispinus mitochondrion was 428 bp on average, with minimum and maximum lengths of 61 bp (trnS1) and 1695 bp (nad5), respectively ( Figure 1B , Table 3 ). Our result was in accordance with Pakrashi et al. (29), revealing a mitogenome length of 15,067 bp. The mitogenome of T. parvispinus had base compositions of 43.3% A, 30% T, 10% G, and 11.7% C, with an overall AT content of 73.3% and GC content of 21.7%. The AT content across the 13 PCGs and 2 rRNAs ranged from 72.8% to 84%, while the GC content varied from 17% to 27.2%. AT skewness was higher in the PCGs (0.064 to 0.410) compared to the rRNAs (0.161 for rrnS and 0.187 for rrnL) ( Table 4 ). We identified 1,039 bp of intergenic nucleotides across 24 locations, with the longest spacer (347 bp) between the trnE and trnP genes. Seven genes had overlaps, with the largest (35 bp) between the COX2 and ND4 genes ( Table 3 , Figure 1B ).

Summary of Thrips parvispinus mitochondrial genome.

Gene Name Full Name and Function Gene Type Position Length (bp) Strand Intergenic Space Codon Anti-codon
Start End Start Stop
nad5 NADH dehydrogenase subunit 5 PCG 146 1840 1695 – -5 att taa –
nad4 NADH dehydrogenase subunit 4 PCG 1836 3157 1322 – 47 ata t –
nad4L NADH dehydrogenase subunit 4L PCG 3205 3486 282 – 25 atg taa –
trnC Transfer RNA for Cysteine tRNA 3512 3574 63 + 18 – – gca
nad6 NADH dehydrogenase subunit 6 PCG 3593 4090 498 + 100 att taa –
rrnL 16S ribosomal RNA rRNA 4191 5338 1148 + 20 – – –
cox1 cytochrome c oxidase subunit I PCG 5359 6930 1572 + 71 att taa –
nad3 NADH dehydrogenase subunit 3 PCG 7002 7343 342 + -35 att taa –
cox2 cytochrome c oxidase subunit II PCG 7309 8001 693 + 23 ata taa –
trnG Transfer RNA for Glycine tRNA 8025 8087 63 + 3 – – tcc
trnK Transfer RNA for Lysine tRNA 8091 8154 64 + 2 – – ttt
cox3 cytochrome c oxidase subunit III PCG 8157 8945 789 + 4 ttg taa –
trnN Transfer RNA for Asparagine tRNA 8950 9016 67 + -4 – – gtt
trnS1 Transfer RNA for Serine tRNA 9013 9073 61 + 0 – – tct
trnL1 Transfer RNA for Leucine tRNA 9074 9138 65 + 42 – – tag
trnE Transfer RNA for Glutamic acid tRNA 9181 9246 66 + 347 – – ttc
trnP Transfer RNA for Proline tRNA 9594 9659 66 – 34 – – tgg
trnY Transfer RNA for Tyrosine tRNA 9694 9757 64 – 57 – – gta
nad2 NADH dehydrogenase subunit 2 PCG 9815 10825 1011 + 17 att taa –
nad1 NADH dehydrogenase subunit 1 PCG 10843 11772 930 + -4 att taa –
trnM Transfer RNA for Methionine tRNA 11769 11833 65 + 1 – – cat
trnA Transfer RNA for tRNA 11835 11896 62 + -1 – – tgc
trnF Transfer RNA for Phenylalanine tRNA 11896 11960 65 + -2 – – gaa
rrnS 12S ribosomal RNA rRNA 11959 12711 753 + 8 – – –
atp8 ATP synthase F0 subunit 8 PCG 12720 12879 160 + 41 att t –
atp6 ATP synthase F0 subunit 6 PCG 12921 13535 615 + 5 att taa –
trnQ Transfer RNA for Glutamine tRNA 13541 13609 69 + 59 – – ttg
trnS2 Transfer RNA for Serine tRNA 13669 13733 65 + 3 – – tga
trnD Transfer RNA for Aspertic acid tRNA 13737 13801 65 + 1 – – gtc
trnR Transfer RNA for Arginine tRNA 13803 13870 68 + 0 – – tcg
trnT Transfer RNA for Threonine tRNA 13871 13935 65 + 10 – – tgt
trnI Transfer RNA for Isoleucine tRNA 13946 14012 67 + -32 – – gat
cytB cytochrome b PCG 13981 15126 1146 + 101 ata taa –

Nucleotide composition of Thrips parvispinus mitochondrial genome.

Gene Length T C A G GC% AT% GC Skew AT Skew
nad5 1695 54.65 8.40 26.35 10.60 19.00 81.00 0.116 -0.349
nad4 1322 55.45 7.60 25.25 11.70 19.30 80.70 0.212 -0.374
nad4l 282 59.20 6.75 24.80 9.25 16.00 84.00 0.156 -0.410
nad6 498 40.40 9.60 42.00 8.00 17.60 82.40 -0.091 0.019
cox1 1572 36.40 14.20 36.40 13.00 27.20 72.80 -0.044 0.000
nad3 342 38.60 11.70 40.90 8.80 20.50 79.50 -0.141 0.029
cox2 693 34.50 14.30 39.20 12.00 26.30 73.70 -0.087 0.064
cox3 789 36.75 15.50 36.45 11.30 26.80 73.20 -0.157 -0.004
nad2 1011 40.50 11.60 42.50 5.40 17.00 83.00 -0.365 0.024
nad1 930 40.75 12.85 35.40 11.00 23.85 76.15 -0.078 -0.070
atp8 160 38.10 11.30 42.50 8.10 19.40 80.60 -0.165 0.055
atp6 615 39.20 13.30 37.10 10.40 23.70 76.30 -0.122 -0.028
cob 1146 40.00 13.75 35.10 11.15 24.90 75.10 -0.104 -0.065
rrnL 1148 32.35 9.10 47.25 11.30 20.40 79.60 0.108 0.187
rrnS 753 33.30 10.40 46.10 10.20 20.60 79.40 -0.010 0.161
Protein-coding genes and codon usage bias

The mitochondrial genomes of T. tabaci and T. parvispinus exhibit distinct characteristics in their PCGs. The total lengths of the 13 PCGs were 10,584 bp for T. tabaci and 11,055 bp for T. parvispinus. In T. tabaci, the coding sequences ranged from 126 bp (atp8) to 1,542 bp (cox1), with genes such as cox1, cox2, cox3, nad3, and nad6 located on the H-strand, while others like nad1, nad2, nad4, nad4L, nad5, atp6, atp8, and cytB were on the L-strand. Conversely, in T. parvispinus, the coding sequences ranged from 160 bp (atp8) to 1,695 bp (nad5), with most genes situated on the H-strand, except nad4, nad4L, and nad5, which were on the L-strand.

Three start codons (ATT, ATA, and ATG) were identified in the T. tabaci mitogenome. ATT was adopted by genes like nad2, nad3, nad4, nad5, nad6, and atp8, whereas genes like cox1, cox2, cox3, atp6, nad2, and CytB used ATA. However, ATG was used as the start codon by the nad4L gene. In contrast, T. parvispinus utilized four start codons, with ATT being the most prevalent across 8 genes (cox1, atp6, atp8, nad1, nad2, nad3, nad5, and nad6), while 3 genes (nad4, cox2, and cytB) used ATA as their start codon. The ATG and TTG start codons are used by nad4L and cox3, respectively. The ATN start codon is the most common starting codon in most thrips and other insect species (26, 47–49). For stop codons, T. tabaci primarily used TAA, but nad5 and cytB used TAG, and nad4 and atp8 used an incomplete stop codon “T”. Similarly, T. parvispinus mostly used TAA, with nad4 and atp8 also adopting the incomplete “T”. Incomplete termination codons are common in other thrips species (26, 47) and insect mitochondrial genomes and are presumed to be restored through post-transcriptional polyadenylation (48, 49).

The RSCU and amino acid usage in the PCGs of T. tabaci and T. parvispinus are summarized in Figure 2 and Table 5 . In T. tabaci mitogenome, leucine, phenylalanine, isoleucine, serine, lysine, tyrosine, and valine were the most frequent amino acids, whereas tryptophan, methionine, and cyanine were the rarest. In general, UUU, followed by UUA, AUU, AUA, and AAA, was the most frequently used codon. However, in the T. parvispinus mitogenome, isoleucine, phenylalanine, leucine, asparagine, serine, lysine, and tyrosine were the most frequently used amino acids, whereas tryptophan, cyanine, and methionine were the rarest, with UUU, followed by AUU, UUA, AAU, and AUA being the most frequently used codons ( Table 5 ).

Relative synonymous codon use (RSCU) of 13 PCG’s of the T. tabaci and T. parvispinus.

Amino acid usage in the 13 PCG’s of the T. tabaci and T. parvispinus.

Codon T. tabaci T. parvispinus Codon T. tabaci T. parvispinus Codon T. tabaci T. parvispinus Codon T. tabaci T. parvispinus
UUU(F) 30.3 33 UCU(S) 7.5 6.6 UAU(Y) 9 11.8 UGU(C) 3.1 2.6
UUC(F) 6.3 5.2 UCC(S) 2.2 1.8 UAC(Y) 3.5 2.5 UGC(C) 0.6 0.6
UUA(L) 18.5 20.1 UCA(S) 6.2 5.8 UAA(*) 7 5.7 UGA(*) 4.6 4.1
UUG(L) 5.3 4.4 UCG(S) 1.1 1.1 UAG(*) 2.3 1.9 UGG(W) 1.3 1.2
CUU(L) 6.9 5.1 CCU(P) 3.2 2.6 CAU(H) 2.3 3.5 CGU(R) 0.8 1
CUC(L) 1.7 0.5 CCC(P) 0.9 1 CAC(H) 1.7 1.4 CGC(R) 0.3 0.2
CUA(L) 5.7 3.2 CCA(P) 2.8 3.2 CAA(Q) 5.5 3.8 CGA(R) 1.8 2.2
CUG(L) 1.5 0.7 CCG(P) 0.2 0.2 CAG(Q) 2 0.6 CGG(R) 0.5 0.2
AUU(I) 17.8 22.6 ACU(T) 3.5 3.9 AAU(N) 8.5 14.1 AGU(S) 1 2.2
AUC(I) 3.4 4.8 ACC(T) 1.5 1.5 AAC(N) 3.7 3.6 AGC(S) 0.7 1.4
AUA(I) 14.9 13.5 ACA(T) 4.8 6.3 AAA(K) 10.8 13.2 AGA(R) 4.4 5.8
AUG(M) 2.5 2.8 ACG(T) 0.9 0.7 AAG(K) 2.6 2.5 AGG(R) 1.3 1.9
GUU(V) 5.1 5 GCU(A) 2.1 2.2 GAU(D) 2.6 2.8 GGU(G) 2.1 2.5
GUC(V) 1.4 0.8 GCC(A) 0.4 0.3 GAC(D) 1.5 1.1 GGC(G) 0.1 0.2
GUA(V) 2.8 3.9 GCA(A) 1.8 2.6 GAA(E) 6 5.5 GGA(G) 5.9 5.5
GUG(V) 1 0.4 GCG(A) 0.5 0.2 GAG(E) 1 1.2 GGG(G) 1.2 0.5

The symbol * represent the “Stop codons”.

 Transfer RNAs and ribosomal RNAs

The circular mitochondrial genome of T. tabaci contains 19 tRNA genes ranging in size from 57 to 68 bp. In the mitogenome, 13 of the tRNA genes are located on the H-strand (+), while the remaining six are located on the L-strand (–) ( Figure 1A and Table 1 ). The 19 tRNA-coding genes of T. tabaci mitochondrial genome collectively comprised 1,200 bp, representing 7.85% of the entire mitogenome. In T. parvispinus mitogenome, 18 tRNA-coding genes ranging in size from 61 to 69 bp were detected. All of the tRNA-coding genes of T. parvispinus were located on the H-strand (+) with the exception of two genes encoding proline (Pro/P) and tyrosine (Tyr/Y) located on the L-strand (-) of mitogenome ( Figure 1B and Table 3 ). These 18 tRNA-coding genes of T. parvispinus comprise a 1,170-bp region that covers 7.65% of the mitogenome. The characteristic clover leaf secondary structures of tRNA genes were identified in the mitogenomes of both thrips species, with the exception of trnS1, which lacks a dihydrouridine arm ( Figures 3 , 4 ). Three genes encoding tRNAs for leucine (Leu/L2), tryptophan (Trp/W), and valine (Val/V) were lacking in the mitogenome of T. tabaci; however, in addition to these three genes, histidine (His/H) was missing in the T. parvispinus mitogenome.

tRNA structures of the T. tabaci.

tRNA structures of the T. parvispinus.

The rRNA genes rrnL and rrnS collectively constitute 11.51% of the total mitogenome. The rrnS gene (607 bp long with 76.80% A+T content) was situated between the atp8 and tRNA-Phe genes. The rrnL gene (1,152 bp long with 79.30% A+T content) was located between the nad6 and cox1 genes of the T. tabaci mitogenome. However, in the T. parvispinus mitogenome, both the rRNA-coding genes rrnL (1148 bp) and rrnS (753 bp) were found on the H-strand (+) and covered 12.43% (1901 bp) of the whole mitochondrial genome.

 Phylogenetic analysis

Species within the same family, Thripidae, T. palmi, T. imagines, T. hawaiiensis, F. intonsa, F. occidentalis, Dendrothrips minowai, Pseudodendrothrips mori, Neohydatothrips samayunkur, Scirtothrips dorsalis, Anaphothrips obscurus, were grouped together with T. tabaci and T. parvispinus, The species from other thrips family such as Stenurothripidae, Aeolothripidae and Phlaeothripidae were grouped in another cluster. The T. parvispinus was closely related to T. hawaiiensis with 98 bootstrap value. However, T. tabaci was slightly outgrouped from T. hawaiiensis. The F. intonsa, and F. occidentalis were cluster separately from species for genus Thrips ( Figure 5 ). The other cluster included Holarthothrips indicus of the Stenurothripidae family grouped with 51 Bootstrap value with Franklinothrips vespiformis of the Aeolothripidae family, Gynaikothrips uzeli, and Haplothrips aculeatus of the Phlaeothripidae family. The damsel bug, Alloeorhynchus bakeri, was used in the dataset as an outgroup species.

Phylogenetic analysis of 16 Thrips species, with the damsel bug, Alloeorhynchus bakeri as outgroup. (T. tabaci and T. parvispinus marked with red and blue triangles respectively are from current study).

 Control, overlapping, and intergenic spacer regions

The T. tabaci and T. parvispinus mitochondrial genome contains one control in both species, each of which is 451 bp and 347 bp, respectively. In T. tabaci, it is located between the trnD and trnR genes; however, in T. parvispinus, it is situated between the trnE and trnP genes. The percentages of AT in the control T. tabaci and T. parvispinus regions were 62.7% and 76.7%, respectively. T-stretch, GAnT motif, ATnC motif, tandem repeats, and stem loops were found in the control region of the T. tabaci mitogenome. However, the T-stretch followed by the TATA box, GAnT motif, tandem repeats, and stem loop were found in the T. parvispinus mitogenome.

The T. tabaci mitochondrial genomes contain 22 intergenic spacer regions of length 1109 bp that vary in length from 2 to 292 bp. There were 13 major intergenic spacers >10 bp in length observed in the circular genome. The longest intergenic spacer regions (292 bp) were located between nad4 and nad4L genes. However 9 overlapping sequences were found in T. tabaci mitochondrial genome, ranging in size from 1 to 54 bp. Similar to T. tabaci, T. parvispinus mitochondrial genome contained 22 intergenic spacer regions of total 692 bp, varying in length from 1 to 101 bp. There were 14 major intergenic spacers >10 bp in length observed in the T. parvispinus mitogenome. The cytB genes were followed by the longest intergenic spacer region of 101 bp. The 7 overlapping sequences were present in T. tabaci mitochondrial genome, ranging from 1 to 35 bp in size. Amid the nad4 and nad4L genes of the T. tabaci mitogenome, there were 292 bp noncoding nucleotides; however, T. parvispinus revealed 47 bp.

 Discussion

The circular mitogenome of T. tabaci and T. parvispinus from the present study revealed 15,277 bp and 15,285 bp. respectively. A similar trend was observed in most of the thrips species reported earlier. For instance, T. imagines (15,407 bp) (21), T. palmi (15,333 bp) (26), Frankliniella intonsa (15,215 bp) (23), Frankliniella occidentalis (14,889 bp) (22), Scriptothrips dorsalis (15,343 bp) (24), and Anaphothrips obscurus (14,890 bp) (25).

The gene order among these thrips species showed variations, especially within the subfamily Thripinae. In T. tabaci and T. parvispinus the several tRNA genes from both T. tabaci and T. parvispinus have been translocated, showing the variation and arrangement of the gene. Similar variation in the order of the mitogenome genes of the all thrips species was observed. This variation can provide insights into evolutionary processes and mechanisms of genetic diversity. It also highlights the complexity of mitochondrial inheritance and their adaptation to various environments at the genetic level (47). These variations might affect their physiology, behavior, and ecological interactions, making them an interesting subject for evolutionary studies (48). Additionally, understanding these evolutionary patterns can help in pest control strategies, as some thrips species are significant agricultural pests.

The RSCU data analysis revealed that Lysine followed by phenylalanine, leucine, isoleucine, tyrosine, and serine, are the most frequently used amino acids, which is common in most of the thrips species (23, 25, 26). The characteristic clover leaf secondary structures of tRNA genes were identified in the mitogenomes of both thrips species, with the exception of trnS1, which lacks a dihydrouridine (DHU) arm, which is common in most insect species. The DHU arm in the trnS1 secondary structure was missing in marigold thrips (Neohydatothrips samayunkur) (49), green semilooper (Chrysodeixis acutaI) (50), and Indian dammer bee, Tetragonula iridipennis (51).

In both the thrips species, some tRNA-coding genes, namely Leucine (Leu/L2), Tryptophan (Trp/W), and Valine (Val/V) were missing in T. tabaci, and in addition to these three, Histidin (His/H) is missing in T. parvispinus mitogenome. The presence of duplicate copies of tRNA in some thrips species and missing tRNA genes in Gynaikothrips has been reported by Tyagi et al. (52). However in many insect species mitochondrialt tRNA genes reported as lost or missing, on manual annotation found to have unusual secondary structures and contain many nucleotide mismatches (53). In some of the insect species, the truncated tRNA was observed, which formed during their evolution (54). The truncation of tRNA genes poses problems in locating and annotating them due to a high level of nucleotide mismatches (55). In the absence of a well-paired acceptor stem, the 3’ end is not clearly defined. The region downstream from the anticodon stem is extremely variable in sequence and length (54).

The 7 overlapping sequences were present in the T. tabaci mitochondrial genome, ranging from 1 to 35 bp in size. Amid the nad4 and nad4L genes of the T. tabaci mitogenome, there were 292 bp noncoding nucleotides; however, T. parvispinus revealed 47 bp, as in Aeolothrips xinjiangensis (148 bp) (48). Most of the thrips mitogenomes exhibited overlaps of 1-21 bp (mostly around 7 bp) between the nad4 and nad4L. In insect mitogenomes, the nd4 to nd4L regions are transcribed into polycistronic mRNA with either overlaps or no intergenic spacers between them (56–58). However, such a long intergenic spacer might split this polycistronic mRNA into two monocistronic mRNAs (48).

The phylogenetic analysis revealed that the genera Thrips and Frankliniella are closely related, as they cluster together. This close relationship is attributed to their shared homology of paired ctenidia on abdominal segments V-VIII (59). T. tabaci, T. palmi, F. accidentalis, and F. intonsa are known vectors of topoviruses (47). These species cluster together, suggesting they share similar genomic characteristics.

As like the genomic characteristics, the biological similarities are also present in T. tabaci and T. parvispinus. Field emission scanning electron microscopy (FESEM) analysis of both thrips species identified similar types of sensilla, including sensilla basiconica (SBI, SBII, SBIII), sensilla chaetica (SChI, SChII), sensilla trichodea (ST), sensilla campaniformia (SCa), and sensilla cavity (SCav); however, variations in the length of these sensilla were observed between the two species. Also, some morphological characters have the variation in both the thrips species, such as antennae (seven-segmented with forked sensorium on third, and fourth segments), ctenidia (paired ctenidia were present in 5th–8th abdominal segments laterally), and pronotum (two pairs of posteroangular setae) (60).

Mitochondrial genome data have been widely used for phylogenetic, evolutionary studies, and population genetics in insects (61). Among eukaryotes, Thysanoptera, along with other minor paraenopteran insect orders, is regarded as a model for rapid mitochondrial genome evolution (24). In the Thysanoptera order, thrips display exceptional interspecific variation in mitogenomic structure, making them an ideal model for studying mitochondrial evolution (48). In the current study, the several tRNA genes from both T. tabaci and T. parvispinus have been translocated; however, the protein-coding genes and most of the tRNA genes have a similar arrangement, representing that these two species have a low rate of the rearrangement among them, which may be originating from the most recent common ancestors of these thrips species. Similar results were reported by Yan et al. (23) in flower thrips, Frankliniella intonsa, and other thrips species. Mitochondrial DNA plays a crucial regulatory role in insect adaptation to environmental changes, including insecticide resistance (62). Many insecticides target mitochondrial functions, such as oxidative phosphorylation. Mutations in mtDNA can lead to changes in these functions, resulting in resistance to the insecticides (63). Mutations in the mitochondrial-encoded Cytb gene have been implicated in resistance to the novel acaricide Bifenazate in Tetranychus urticae and Panonychus citri (64). Furthermore, a newly identified mutation in the Cytb gene of T. urticae has been associated with resistance to the miticide Acequinocyl (65). However, in the current study, no such mutation was detected in the mitochondrial genes. Also, no report was found in any thrips species to date detecting insecticidal resistance in the thrips species due to mutation in the mitochondrial gene.

The phylogenetic analysis includes species from different thrips families, such as Thripidae, Aeolothripidae, Phlaeothripidae, and Stenurothripidae, showing the phylogenetic diversity among these groups. The diversity within these groups may contribute to the complexity and lower bootstrap values in some parts of the tree. The thrips species clustered together such as T. hawaiiensis and T. parvispinus, with bootstrap values 98, have closely related species T. tabaci with lowest bootstrap value 46, suggesting lower confidence in their relationships. The thrips species in phylogenetic study other than the Thripidae family, Holarthrothrips indicus of Stenurothripidae family, and Franklinothrips vespiformis of Aeolothripidae, Gynaikothrips uzeli and Haplothrips aculeatus of the family Phlaeothripidae shown the lower boot strap values, 51. The lower bootstrap values of these species could be because of the low rate of genetic flow between these species (66).

 Conclusion

Complete mitochondrial genome sequences of T. tabaci and T. parvispinus provide critical insights into their genetic makeup and evolutionary relationships. The mitochondrial genomes of T. tabaci and T. parvispinus were found to be 15,277 bp and 15,285 bp in size, respectively, with similar gene organization, including 13 protein-coding genes, 2 rRNA genes, and 19 tRNA genes in T. tabaci, and 18 tRNA genes in T. parvispinus. The high AT content observed in both species reflects their typical insect mitochondrial genome characteristics, which are essential for understanding their genetic diversity and evolutionary adaptations. Phylogenetic analysis revealed the evolutionary positions of T. tabaci and T. parvispinus within the Thysanoptera order, highlighting the genetic makeup and taxonomy of these species with other thrips species. The findings emphasize the significance of understanding the genetic basis of onion thrips, which can aid in developing targeted pest management strategies to mitigate their impact on onion production. The detailed mitochondrial genome sequences and phylogenetic analyses presented in this study provide a valuable resource for further research on thrips biology, genetics, and pest management.

 Data availability statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.

 Ethics statement

The manuscript presents research on animals that do not require ethical approval for their study.

 Author contributions

PS: Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. DS: Data curation, Formal analysis, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. VK: Data curation, Formal analysis, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing. PD: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. VM: Funding acquisition, Resources, Supervision, Writing – original draft, Writing – review & editing.

 Funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. The author(s) declare that funding for this study, was received from the in-house project of ICAR-Directorate of Onion and Garlic Research, Pune, India with Project code: 052/S/IPP/DOGR/21-26/CPT/003.

 Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Generative AI statement

The author(s) declare that no Generative AI was used in the creation of this manuscript.

 Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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