The team has found that both fish genes respond actively to pathogenic stress, interact to trigger apoptosis, and can suppress inflammatory signaling. Their findings deepen biological understanding of fish immune mechanisms and provide genetic targets for selective breeding and disease-control strategies in aquaculture.
Black seabream farming has grown rapidly in China, yet Vibrio parahaemolyticus-induced ulcers frequently cause high mortality. Fish defend against pathogens through immune responses that include apoptosis—programmed cell death that removes infected or damaged cells. In mammals, apoptosis is driven by FADD and caspase-8, which form a death-inducing signaling complex (DISC). However, their roles in marine fish remain poorly understood. Inflammatory processes in fish are tightly regulated by the NF-κB pathway, which promotes cytokine release during infection. How apoptotic regulators interact with inflammatory signaling in Acanthopagrus schlegelii has remained unclear, hindering progress in molecular breeding. Addressing these knowledge gaps requires a mechanistic investigation of FADD–caspase-8 function during immune activation.
A study (DOI: 10.48130/animadv-0025-0031) published in Animal Advances on 01 December 2025 by Qingguo Meng & Jiajia Li’s team, Nanjing Normal University, provides new molecular evidence that AsFADD and Ascaspase-8 regulate apoptosis and suppress inflammation in A. schlegelii, offering valuable targets for disease-resistant breeding in aquaculture.
In this study, a combination of molecular cloning, qRT-PCR, overexpression verification, subcellular localization, apoptosis detection, protein interaction assays, and inflammatory pathway analysis was employed to investigate the characteristics and immune functions of AsFADD and Ascaspase-8 in Acanthopagrus schlegelii. First, sequence analysis revealed that the ORF of AsFADD was 576 bp encoding 191 amino acids, while Ascaspase-8 spanned 1,464 bp encoding 487 amino acids. Phylogenetic comparison showed both proteins shared highest homology with Sparus aurata. Next, qRT-PCR was applied to measure tissue transcription patterns, showing AsFADD was mainly expressed in gills and Ascaspase-8 in the brain, and both genes were significantly upregulated in the gills and kidneys after Vibrio parahaemolyticus infection, peaking at 24–48 h for AsFADD and 12–24 h for Ascaspase-8. To assess gene expression in vitro, pEGFP–FADD and pEGFP–caspase-8 plasmids were transfected into RAW264.7 cells, which successfully expressed recombinant proteins verified by fluorescence microscopy and Western blot. Confocal imaging demonstrated AsFADD localized predominantly in the cytoplasm, whereas Ascaspase-8 was mainly nuclear. Apoptosis induction was then evaluated using Western blot, microscopy, and flow cytometry, and overexpression of AsFADD significantly increased cleaved caspase-3/8 and apoptotic cell proportion. Interaction between both proteins was examined using co-immunoprecipitation, confirming intracellular binding of AsFADD and Ascaspase-8. Finally, NF-κB signaling activity and cytokine transcription showed that overexpression of either gene increased IκBα levels and decreased p65 expression, accompanied by reduced transcription of IL-1β, IL-6, IL-10, IL-12, IL-18, IP10, and TNF-α under infection, indicating apoptosis activation suppressed inflammatory factor release.
This work advances the understanding of immune regulation in a key aquaculture species. Identifying AsFADD and Ascaspase-8 as apoptosis-inducing and inflammation-suppressing genes highlights their potential as biomarkers in disease-tolerant breeding programs. By manipulating expression of these genes, selective breeding or gene-editing strategies could enhance survival against Vibrio infection, reducing antibiotic dependency and economic losses. The study also offers molecular clues for designing vaccines and immunostimulants that balance the cell death and inflammation, improving fish resilience under intensive farming conditions.
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References
DOI
10.48130/animadv-0025-0031
Original Source URL
https://doi.org/10.48130/animadv-0025-0031
Funding information
The current work was supported by grants from the Genetic Improvement of Sea Bream and Creation of New Strains (Grant No. PZCZ201744), Jiangsu Provincial Agricultural Project (Grant No. 2023-SJ-030), Major Agricultural Technology Collaborative Promotion Project (Grant No. 2021-ZYXT-09), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (2024).
About Animal Advances
Animal Advances (e-ISSN 3065-7660) is an open-access journal which published by Maximum Academic Press in partnership with Nanjing Agricultural University. The journal is dedicated to delivering cutting-edge discoveries and progress in animal sciences to a diverse audience, encompassing scholars, academicians, and practitioners in the industry.