The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages

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.

Keywords: Anastrepha fraterculus; Diptera; RNA interference; gene silencing; transcriptome.

FIGURE 1

Percentage of Anastrepha fraterculus contigs assigned to a certain gene ontology term as predicted by QuickGO from EBI. Top 10 terms are shown.

FIGURE 2

Copy number of the ten RNAi-related genes and SID-1 found in Anastrepha fraterculus transcriptome by Trinity and in other insect species (showed by Dowling et al., 2016). The number of copies showed in A. fraterculus is compared to Drosophila. (=) Same and (+) duplication.

FIGURE 3

Phylogenetic trees of siRNA pathway genes, Dicer 2 (Dcr-2) and Argonaute 2 (Ago-2). MEGA X was used to construct the phylogenetic trees with Neighbor-Joining method. Anastrepha fraterculus sequence from transcriptome was marked with a red triangle. All accession numbers are shown in Supplementary Table S4.

FIGURE 4

Phylogenetic tree of target gene of silencing, V-ATPase. MEGA X was used to construct the phylogenetic tree with Neighbor-Joining method. Anastrepha fraterculus sequence from transcriptome was marked with a red triangle. All accession numbers are shown in Supplementary Table S4.

FIGURE 5

Relative mRNA expression of V-ATPase in Anastrepha fraterculus larvae after 24, 48, and 72 h of soaking in dsRNA (500 ng/μl). The mRNA levels were normalized using α-tubulin and actin as reference genes. The columns represent the mean ± SE (n = 3). ^*p ≤ 0.05 (t-test).

FIGURE 6

Mortality cumulative of Anastrepha fraterculus larvae (n = 57) after soaking in dsRNA solution (500 ng/μl) from V-ATPase (dsVTP) and GFP control (dsGFP) at 2, 4, and 7 days.

FIGURE 7

Relative mRNA expression of Dicer-2 (A) and Argonaute-2 (B) in Anastrepha fraterculus larvae in response to dsGFP soaking after 24, 48, and 72 h (500 ng/μl). Nuclease-free water was used as control. The mRNA levels were normalized using α-tubulin and actin as reference genes. The columns represent the mean ± SE (n = 3). ^*p ≤ 0.05 (t-test).