Molecular and Functional Characterization of Neuropeptide F Receptor in Pomacea canaliculata: Roles in Feeding and Digestion and Communication with the Insulin Pathway

Abstract

The invertebrate neuropeptide F (NPF) signaling plays versatile roles in diverse biological activities and processes. Still, whether and how it mediates feeding and digestion in Pomacea canaliculate remain gaps in our knowledge. Herein, we first identified and characterized PcNPFR via bioinformatics analysis in P. canaliculate, which is a polyphagous herbivore with a voracious appetite that causes devastating damages to ecosystem functioning and services in colonized ranges. Double stranded RNA (dsRNA)-based RNA interference (RNAi) and exogenous rescue were utilized to decipher and substantiate underlying mechanisms whereby NPFR executed its modulatory functions. Multiple sequence alignment and phylogeny indicated that PcNPFR harbored typical seven transmembrane domains (7 TMD) and belonged to rhodopsin-like GPCRs, with amino acid sequence sharing 27.61-63.75% homology to orthologues. Spatio-temporal expression profiles revealed the lowest abundance of PcNPFR occurred in pleopod tissues and the egg stage, while it peaked in male snails and testes. Quantitative real-time PCR (qRT-PCR) analysis showed that 4 µg dsNPFR and 10^-6 M trNPF (NPFR agonist) were optimal doses to exert silencing and rescue effects, accordingly with sampling time at 3 days post treatments. Moreover, the dsNPFR injection (4 µg) at 1/3/5/7 day/s delivered silencing efficiency of 32.20-74.01%. After 3 days upon dsNPFR knockdown (4 µg), mRNA levels of ILP7/InR/Akt/PI3Kc/PI3K_R were significantly downregulated compared to dsGFP controls, except FOXO substantially upregulated at both transcript and translation levels. In addition, the activities of alpha-amylase, protease and lipase were significantly suppressed, accompanied by decreased leaf area consumption, attenuated feeding behavior and diminished feeding rate. Moreover, expression trends were opposite and proxies were partially or fully restored to baseline levels post exogenous compensation of trNPF, suggesting phenotypes specifically attributable to PcNPFR RNAi but not off-target effects. PcNPFR is implicated in both feeding and digestion by modulating the ISP pathway and digestive enzyme activities. It may serve as a promising molecular target for RNAi-based antifeedants to manage P. canaliculate invasion.

Keywords: ISP pathway; NPF signaling; Pomacea canaliculate; RNAi; feeding and digestion.

Figure 1

Amino acid (aa) sequence alignments (A) and neighbor-joining phylogeny of PcNPFR (B) against orthologs from other species. Identical residues among receptors were shaded in black, while 80% and 60% conserved substitutions in pink and cyan, respectively. Horizontal bars denoted TM helix 1–7. Typical rhodopsin-like GPCR motifs were marked by red boxes. In the phylogenetic tree, dots at branch nodes represented bootstrap values (50%). A scale bar indicated that the average number of aa substitutions per site was 0.2. The D. melanogaster neuropeptide F receptor was chosen as the outgroup. The aligned NPFR/NPYR homologs were retrieved from the NCBI database under the following accession numbers: XP_025096430.1 [Pomacea canaliculata], XP_005089880.1 [Aplysia californica], XP_055874125.1 [Biomphalaria glabrata], XP_046372073.2 [Haliotis rufescens], XP_041369772.1 [Gigantopelta aegis], XP_050395368.1 [Patella vulgata], GFO06936.1 [Plakobranchus ocellatus], GFR99678.1 [Elysia marginata], XP_022289835.1 [Crassostrea virginica], XP_048746931.2 [Ostrea edulis], XP_052224883.1 [Dreissena polymorpha], XP_006509657.1 [Mus musculus], NP_524245.3 [Drosophila melanogaster], XP_054206094.1 [Homo sapiens].

Figure 1

Amino acid (aa) sequence alignments (A) and neighbor-joining phylogeny of PcNPFR (B) against orthologs from other species. Identical residues among receptors were shaded in black, while 80% and 60% conserved substitutions in pink and cyan, respectively. Horizontal bars denoted TM helix 1–7. Typical rhodopsin-like GPCR motifs were marked by red boxes. In the phylogenetic tree, dots at branch nodes represented bootstrap values (50%). A scale bar indicated that the average number of aa substitutions per site was 0.2. The D. melanogaster neuropeptide F receptor was chosen as the outgroup. The aligned NPFR/NPYR homologs were retrieved from the NCBI database under the following accession numbers: XP_025096430.1 [Pomacea canaliculata], XP_005089880.1 [Aplysia californica], XP_055874125.1 [Biomphalaria glabrata], XP_046372073.2 [Haliotis rufescens], XP_041369772.1 [Gigantopelta aegis], XP_050395368.1 [Patella vulgata], GFO06936.1 [Plakobranchus ocellatus], GFR99678.1 [Elysia marginata], XP_022289835.1 [Crassostrea virginica], XP_048746931.2 [Ostrea edulis], XP_052224883.1 [Dreissena polymorpha], XP_006509657.1 [Mus musculus], NP_524245.3 [Drosophila melanogaster], XP_054206094.1 [Homo sapiens].

Figure 2

Stage- (A,B) and tissue-specific (C,D) expression patterns of PcNPFR. Protein and mRNA levels of embryonic stage and pleopod tissues were set as calibrators and relative expressions of PcNPFR were normalized to GAPDH. Different lowercase letters denoted significant differences—p < 0.05.

Figure 3

Screening optimal doses and sampling time for RNAi and restoration treatments. (A) Survival curve of juvenile snails and (B) mRNA levels of PcNPFR post knockdown. (C) Survival curve of juvenile snails and (D) mRNA levels of PcNPFR upon trNPF rescue. Asterisk * indicated significant differences at the same time point as compared to dsGFP group (equal amount of dsRNA or trNPF administered); p < 0.05.

Figure 4

Effects of dsNPFR treatment and trNPF injection on feeding activity. (A) Representative picture depicting spatial distribution of snails, with individuals that ceased ingestion after 2 h outlined by red rectangle. (B) Leaf morphology and area of L. sativa devoured by snails after 8 h. (C) The time taken by snails to utterly consume four leaves. Each group comprised three replicates, with 10 juvenile snails and 4 leaves of equal area per replicate. Different lowercase letters denoted significant differences—p < 0.05.

Figure 5

Effects of silencing and compensatory treatments on activities of α-amylase (A), cellulase (B), protease (C), and lipase (D) in digestive glands. Different lowercase letters represented significant differences between treatments and control—p < 0.05.

Figure 6

Effects of NPFR silencing and trNPF rescue on expression profiling of the ISP pathway. (A) qRT-PCR quantification of ISP-related genes. Transcript levels of dsGFP group were set to 1 as calibrator and fold changes in target genes were subjected to normalization using GAPDH as internal reference. (B) Representative blotting images of NPFR, t-FOXO (total FOXO) and p-FOXO (phosphorylated FOXO). The relative protein level was calculated as ratio of band intensity, viz., target protein divided by GAPDH. Protein levels of dsGFP group were set to 1 as calibrator.

Figure 7

A schematic diagram of inter-organ modulation orchestrated by NPF–NPFR signaling and the ISP pathway in feeding and digestion. The insulin receptor (InR) binds with insulin-like peptides (ILP7) and initiates PI3K/Akt cascade to phosphorylate and represses the transcription factor FOXO and its downstream targets. Upon PcNPFR silencing (left panel), PcNPFR expressions were depleted (Figure 3B and Figure 6B) with ILP7/InR/PI3K/Akt transcript levels (Figure 6A), digestive enzymes (Figure 5) and feeding activity (Figure 4) all suppressed, except for the upregulation of p-FOXO and t-FOXO at protein and mRNA level (Figure 6). These were partially or fully reversed post trNPFR rescue (right panel), with opposite molecular, biochemical alterations and behavioral phenotypes.