Abstract
RNA interference (RNAi) technology has been used in the development of approaches for pest control. The presence of some essential genes, the so-called “core genes,” in the RNAi machinery is crucial for its efficiency and robust response in gene silencing. Thus, our study was designed to examine whether the RNAi machinery is functional in the South American (SA) fruit fly Anastrepha fraterculus (Diptera: Tephritidae) and whether the sensitivity to the uptake of double-stranded RNA (dsRNA) could generate an RNAi response in this fruit fly species. To prepare a transcriptome database of the SA fruit fly, total RNA was extracted from all the life stages for later cDNA synthesis and Illumina sequencing. After the de novo transcriptome assembly and gene annotation, the transcriptome was screened for RNAi pathway genes, as well as the duplication or loss of genes and novel target genes to dsRNA delivery bioassays. The dsRNA delivery assay by soaking was performed in larvae to evaluate the gene-silencing of V-ATPase, and the upregulation of Dicer-2 and Argonaute-2 after dsRNA delivery was analyzed to verify the activation of siRNAi machinery. We tested the stability of dsRNA using dsGFP with an in vitro incubation of larvae body fluid (hemolymph). We identified 55 genes related to the RNAi machinery with duplication and loss for some genes and selected 143 different target genes related to biological processes involved in post-embryonic growth/development and reproduction of A. fraterculus. Larvae soaked in dsRNA (dsV-ATPase) solution showed a strong knockdown of V-ATPase after 48 h, and the expression of Dicer-2 and Argonaute-2 responded with an increase upon the exposure to dsRNA. Our data demonstrated the existence of a functional RNAi machinery in the SA fruit fly, and we present an easy and robust physiological bioassay with the larval stages that can further be used for screening of target genes at in vivo organisms’ level for RNAi-based control of fruit fly pests. This is the first study that provides evidence of a functional siRNA machinery in the SA fruit fly.
Highlights
The South American fruit fly (SA fruit fly), Anastrepha fraterculus, is a major polyphagous pest of fruit crops
Our project made available more than 84,000 new entries related to the developmental of A. fraterculus and generated a database of 143 novel and different target genes to doublestranded RNA (dsRNA) bioassays
This transcriptome database is a handy tool for research on the SA fruit fly, especially in studies with a focus on RNA interference (RNAi)
Summary
The South American fruit fly (SA fruit fly), Anastrepha fraterculus, is a major polyphagous pest of fruit crops This fruit fly species occurs from the Southern United States (Texas) and Mexico to Argentina and is associated with 116 plant species in Brazil alone (Zucchi, 2008). Molecules of doublestranded RNA (dsRNA) are RNAi triggers that promote the post-transcriptional down-regulation of a target-gene (Elbashir et al, 2001). Some features, such as high target gene specificity and lack of environmental persistence, make RNAi technique desirable for crop protection (Huvenne and Smagghe, 2010; Zotti et al, 2018)
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