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Neuropeptides play crucial roles in regulating various physiological processes in vertebrates. In this study, we identified a novel neuropeptide-encoding gene, nwk, in the genomes of some vertebrate species. The nwk cDNA was subsequently cloned from the brain of zebrafish. The Nwk precursor comprises 88 amino acids, with a putative mature peptide (Nwk-22) of 22 amino acids. Sequence analysis revealed that Nwk-22 is relatively conserved across vertebrate species. Nwk is predominantly expressed in the brain, with positive mRNA cells identified in the TPp and preoptic area. Intraperitoneal injection of Nwk-22 suppressed food intake and downregulated the mRNA expression of the orexigenic factor agouti-related peptide (agrp) in zebrafish. Additionally, a CRISPR/Cas9 approach was used to generate nwk mutant zebrafish. The nwk -/- zebrafish exhibited increased food consumption compared to wild-type controls. Furthermore, Nwk-22a injection in nwk -/- fish also suppressed agrp expression while stimulating the expression of the anorexigenic gene pomca, further supporting the anorexigenic role of Nwk. Taken together, these findings suggest that Nwk functions as an anorexigenic factor, reducing food intake by downregulating orexigenic genes like agrp and upregulating anorexigenic genes like pomc in zebrafish., Competing Interests: Declaration of competing interest The authors declare neither competing interests nor conflict of financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Toll-like receptor 5 (TLR5), serving as a sensor of bacterial flagellin, mediates the innate immune response to actively engage in the host's immune processes against pathogen invasion. However, the mechanism underlying TLR5-mediated immune response in fish remains unclear. Despite the presumed cell surface expression of TLR5 member form (TLR5M), its trafficking dynamics remain elusive. Here, we have identified Epinephelus coioides TLR5M as a crucial mediator of Vibrio flagellin-induced cytokine expression in grouper cells. EcTLR5M facilitated the activation of NF-κB signaling pathway in response to flagellin stimulation and exerted a modest influence on the mitogen-activated protein kinase (MAPK)-extracellular regulated kinase (ERK) signaling. The trafficking chaperone Unc-93 homolog B1 (EcUNC93B1) participated in EcTLR5M-mediated NF-κB signaling activation and downstream cytokine expression. In addition, EcUNC93B1 combined with EcTLR5M to mediate its exit from the endoplasmic reticulum, and also affected its post-translational maturation. Collectively, these findings first discovered that EcTLR5M mediated the flagellin-induced cytokine expression primarily by regulating the NF-κB signaling pathway, and EcUNC93B1 mediated EcTLR5M function through regulating its trafficking and post-translational maturation. This research expanded the understanding of fish innate immunity and provided a novel concept for the advancement of anti-vibrio immunity technology., Competing Interests: Declaration of competing interest All authors declare no conflict of interest that could have appeared to influence the work reported in this article. This article does not contain any studies with human participants or animals performed by any of the authors., (Copyright © 2023 Elsevier B.V. All rights reserved.)