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. 2016 Sep 29;11(9):e0163635.
doi: 10.1371/journal.pone.0163635. eCollection 2016.

The First Report of miRNAs from a Thysanopteran Insect, Thrips palmi Karny Using High-Throughput Sequencing

K B Rebijith  1 , R Asokan  1 , H Ranjitha Hande  1 , N K Krishna Kumar  2
Affiliations

The First Report of miRNAs from a Thysanopteran Insect, Thrips palmi Karny Using High-Throughput Sequencing

K B Rebijith et al. PLoS One. .

Abstract

Thrips palmi Karny (Thysanoptera: Thripidae) is the sole vector of Watermelon bud necrosis tospovirus, where the crop loss has been estimated to be around USD 50 million annually. Chemical insecticides are of limited use in the management of T. palmi due to the thigmokinetic behaviour and development of high levels of resistance to insecticides. There is an urgent need to find out an effective futuristic management strategy, where the small RNAs especially microRNAs hold great promise as a key player in the growth and development. miRNAs are a class of short non-coding RNAs involved in regulation of gene expression either by mRNA cleavage or by translational repression. We identified and characterized a total of 77 miRNAs from T. palmi using high-throughput deep sequencing. Functional classifications of the targets for these miRNAs revealed that majority of them are involved in the regulation of transcription and translation, nucleotide binding and signal transduction. We have also validated few of these miRNAs employing stem-loop RT-PCR, qRT-PCR and Northern blot. The present study not only provides an in-depth understanding of the biological and physiological roles of miRNAs in governing gene expression but may also lead as an invaluable tool for the management of thysanopteran insects in the future.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Length distribution of mappable reads obtained from T. palmi deep-sequencing.
Reads with ≥ 18 nt to ≤ 26 nt were considered for miRNA mapping.
Fig 2
Fig 2. Stem loop structures of two miRNAs and their star strands.
(A) The secondary structure of tpa-miR-6489 and tpa-miR-6489*. (B) The secondary structure of tpa-miR-6493 and tpa-miR-6493*. Both miRNAs and its star reads were marked by black bars. The secondary structure was predicted by employing RNA fold WebServer (www.rna.tbi.univie.ac.at/lgi-bin/RNAfold.cgi).
Fig 3
Fig 3. Various hairpin secondary structures of the ten novel pre-miRNAs of T. palmi.
The mature miRNAs are indicated by yellow shades. The secondary structure was predicted using RNA fold WebServer.
Fig 4
Fig 4
(A) to (D). Homology, phylogeny and weblogo analysis of T. palmi miRNAs. 4(A) to (D) 1. Homology in the seed region of the T. palmi miRNA with respect to its counterpart from other insect species. Sequence conservation of the T. palmi mature miRNAs including the seed region over a wide range of insects. The first three letters of each miRNAs indicating the name of the species. 4(A) to (D) 2. Phylogenetic trees (ML tree, RaxML.v.7.0.4) of four families of precursor miRNA sequences from various members of the animal kingdom. 4(A) to (D) 3. T. palmi pre-miRNAs weblogo. The pre-miRNA sequence logos for the T. palmi, in which mature miRNA is indicated by blue bars. Each logo consists of stacks of symbols, one for each nucleotide position in the sequence. The height indicates the sequence conservation at that nucleotide position and the height of symbols within the stack indicates the relative frequency of each nucleotide at that position. Fig 4(B) 3 indicated the possible presence of miR-281* which was not evident from the NGS raw reads. However, the presence of miR-281* has been validated by stem-loop RT-PCR.
Fig 5
Fig 5
Gene Ontology (GO) classification of the putative target genes for the T. palmi miRNAs against ESTs (A) and transcriptome (B) sequences of F. occidentalis. GO terms were assigned to each target gene based on the annotation and were summarized into three main GO categories viz. (i) biological process (BP) (ii) molecular function (MF) and (iii) cellular component (CC). Only top ten subcategories are presented in the case of GO for transcriptome sequences.
Fig 6
Fig 6. The synteny analyses using Circos (Krzywinski et al. 2009).
Map of the Western Flower Thrips, F. occidentalis scaffolds linking T. palmi miRNAs and their targets prepared using Circos. The outer circle represents the highlights of 10 novel miRNA in blue and 40 known miRNA represented in red colour. The inner lines in red colour represent known miRNAs and their targets (839 targets) and blue lines represent 11 novel miRNAs and their targets (33 targets) across 300 scaffolds of F. occidentalis genome.
Fig 7
Fig 7. Validation of selected conserved and novel miRNAs from T. palmi.
(A) Stem-loop RT-PCR analyses of nine conserved and four novel miRNAs from T. palmi. The products were resolved on 3% agarose gel in 1X TBE stained with ethidium bromide. HyperLadder™ 25bp (Bioline, USA) employed as a marker. (B) Stem-loop RT-qPCR analysis of spatiotemporally expressed T. palmi miRNAs in larva and adults. ‘*’ and ‘**’ means a statistically significant difference at level p < 0.05 and p < 0.001 respectively for these miRNAs in the larva and adult T. palmi. The error bars indicate standard deviation for three biological replications. (C) Small RNA Northern blot validation. Both conserved (tpa-miR-750, tpa-miR-92b, tpa-miR-2796) and novel (tpa-miR-N3, tpa-miR-N4 and tpa-miR-N10) miRNAs were validated by small RNA northern analysis employing small RNA isolated from pooled T. palmi.
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Grants and funding

This work was supported by Out Reach Program on Management of Sucking Pests (ORP-SP) of Horticultural Crops’ funded by Indian Council for Agricultural Research (ICAR), New Delhi (ICAR-ORP-SP-2012-2017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

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