Bacterial-mediated RNAi and functional analysis of Natalisin in a moth

Abstract

The neuropeptide natalisin (NTL) has been determined to play essential roles in reproduction in two Diptera and one Coleoptera species. Whether NTL has similar or even different functions in Lepidoptera remains to be determined. Here, we cloned the NTL transcript in the common cutworm moth Spodoptera litura. This transcript encodes a 438-amino acid protein. Twelve putative Sl-NTL neuropeptides were defined by cleavage sites. These NTL peptides share a DDPFWxxRamide C-terminal motif. The expressions of Sl-NTL is low during the egg and larval stages, which increased to a higher level during the pupal stage, and then reached the maximum during the adult stage. Moreover, the expression pattern during the pupal stage is similar between sexes while during the adult stage, it is dimorphic. To explore the function of Sl-NTL and assess its potential as a target for pest control, we knocked down the expression of Sl-NTL in both sexes by using bacteria-mediated RNAi. This technique significantly down regulated (reduced up to 83%) the expression of Sl-NTL in both sexes. Knocking down Sl-NTL expression did not significantly affect its development, survival and morphology but significantly reduced adults’ reproductive behavior (including female calling, male courtship, mating and remating patterns and rates) and reproductive output (offspring gain reduced more than 70%).

Interference Efficiency and Effects of Bacterium-mediated RNAi in the Fall Armyworm (Lepidoptera: Noctuidae)

“…Moreover, bacterium-mediated RNAi also has been suggested as a cost-effective method for gene function analysis ( Xiang et al 2009 , Zhang et al 2012 , Xu et al 2016 , Wang et al 2021 ). Therefore, we further injected pupae and adults with bacterially expressed dsRNAs of the four target genes, which induced faster (tested 24 h after injection) and greater reduction (up to 76.36%) in gene expression ( Table 2 ).…”

“…Expressed dsRNAs were extracted from bacteria using RNAiso Plus (TaKaRa, China) and the lengths of dsRNA confirmed via 1.5% agarose gel electrophoresis. The production level achieved using this bacterial incubation system was ~7–9 μg dsRNA/mL culture ( Wang et al 2021 ), as estimated by comparing the brightness of the dsRNA band and quantified RNA marker.…”

“…Bacterial cultures of the four target genes and control EGFP were used for preparation of cells for feeding. In brief, 30 ml IPTG-induced bacterial culture was centrifuged for 3 min at 10,000 g and resuspended in 1 ml DEPC-water ( Wang et al 2021 ). Second-instar larvae (3-d-old) individually caged in plastic cells (4 × 4 × 4 cm) were fed with 20 µl of the prepared bacterial cell solution (dropped on food) once a day and kept for 8 d ( Wang et al 2021 ).…”

“…The use of bacterially expressed dsRNA is significantly more cost-effective than producing dsRNA in vitro with a kit, particularly for large-scale gene function analysis and pest control applications ( Fraser et al 2000 , Solis et al 2009 , Xu et al 2016 , Zhang et al 2019b ). Previous studies on bacterium-mediated RNAi have validated its cost-effectiveness in gene function exploration and significant potential for agricultural applications ( Solis et al 2009 , Xu et al 2016 , Goodfellow et al 2019 , Zhang et al 2019b , Wang et al 2021 ). To our knowledge, however, there is no report on the use of this technique in S. frugiperda so far.…”

Methods for the Cost-Effective Production of Bacteria-Derived Double-Stranded RNA for in vitro Knockdown Studies

“…The system is mainly used for direct feeding of induced bacteria to research animals in a process termed bacterium-mediated RNA interference (bmRNAi) ( Timmons and Fire, 1998 ; Goodfellow et al, 2019 ; Kunte et al, 2020 ). Recent publications reaffirm its useability in the important lepidopteran pest genera Spodoptera and Helicoverpa ( Vatanparast et al, 2021 ; Wan et al, 2021 ; Wang et al, 2021 ).…”