RNA sequencing, selection of reference genes and demonstration of feeding RNAi in Thrips tabaci (Lind.) (Thysanoptera: Thripidae)

Abstract

Background Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time. Results From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of CDS identified showed positive Blast hits. The homologs of most of the core RNAi machinery genes were identified in this transcriptome. To select reference genes for reverse-transcriptase real-time quantitative PCR (RT-qPCR) experiments, 14 housekeeping genes were identified in the transcriptome and their expression was analyzed by (RT-qPCR). UbiCE in adult, 28s in nymphs and SOD under starvation stress were identified as the most stable reference genes for RT-qPCR. Feeding dsSNF7 and dsAQP caused 16.4- and 14.47-fold reduction in SNF7 and AQP mRNA levels respectively, when compared to their levels in dsGFP fed control insects. Feeding dsSNF7 or dsAQP also caused 62 and 72% mortality in T. tabaci . Interestingly, simultaneous feeding of dsRNAs targeting SNF7 or AQP and one of the RNAi pathway genes ( Dicer - 2/Aubergine/Staufen ) resulted in a significant reduction in RNAi of target genes. These data suggest the existence of robust RNAi machinery in T. tabaci . Conclusion The current research is the first report of the assembled, analyzed and annotated RNAseq resource for T. tabaci , which may be used for future molecular studies in this insect. Reference genes validated across stages and starvation stress provides first-hand information on stable genes in T. tabaci. The information on RNAi machinery genes and significant knockdown of the target gene through dsRNA feeding in synthetic diet confirms the presence of efficient RNAi in this insect. These data provide a solid foundation for further research on developing RNAi as a method to manage this pest. Electronic supplementary material The online version of this article (10.1186/s12867-019-0123-1) contains supplementary material, which is available to authorized users.

Using de novo transcriptome assembly and analysis to study RNAi in Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae)

“…In C. elegans , Staufen has been known to play a role in RNAi and recently, robust RNAi efficiency in beetles has been attributed to coleopteran-specific Staufen ( StauC ), which is absent in other insect orders 28,72 . Recent study with Thrips tabaci also suggest the role of Staufen in RNAi 73 . However, Staufen , which is found in other insect species has no role in this process 28 .…”

Transcriptome analysis unravels RNAi pathways genes and putative expansion of CYP450 gene family in cotton leafhopper Amrasca biguttula biguttula(Ishida)

“…Staufen may be contributing to RNAi in insect's species other than coleopterans, which lack specific StauC or its homologue as its knockdown has hampered the RNAi mechanism in our studies. Earlier studies with Thrips tabaci (Singh et al , 2019a) and P. solenopsis (Singh et al, 2019b) also reveal the role of Staufen protein in RNAi mechanism.…”

Frontiers Approaches to the Diagnosis of Thrips (Thysanoptera): How Effective Are the Molecular and Electronic Detection Platforms?

“…In thrips, HTS has unveiled the mitochondrial genomes (mitogenomes) of T. imaginis Bagnall, F. occidentalis , F. intonsa , S. dorsalis , A. obscurus , T. palmi , O. loti (Haliday), and N. samayunkur (Kudo) [ 226 , 227 , 228 , 229 , 230 , 231 , 232 , 233 ]. The transcriptomes of F. occidentalis [ 234 , 235 , 236 , 237 ], F. fusca [ 238 , 239 ], T. palmi [ 240 ], T. tabaci [ 241 ], and F. tritici [ 239 ] have been revealed through HTS. Furthermore, regulatory microRNAs have been identified for T. palmi and T. tabaci [ 242 , 243 ].…”