Fig 2: 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.

Fig 3: 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].