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Background <p>Environmental salinity is a major ecological stressor influencing immune regulation in tilapia, yet the molecular mechanisms underlying salinity-associated immune variation among species remain poorly understood. Tumor necrosis factor-beta (<i>TNF-β</i>) is a central cytokine in immune signaling and stress responses in teleost fish, making it a strong candidate for examining molecular mechanism underlying adaptation across habitats with contrasting salinity conditions.</p> Methods <p><i>TNF-β</i> was amplified from cDNA of wild tilapia species collected from brackish and freshwater environments using PCR (expected amplicon ~ 875&#xa0;bp), followed by Sanger sequencing and nucleotide sequences trimmed to ~ 565&#xa0;bp coding region. cDNA quality was verified using GAPDH as a reference gene. <i>TNF-β</i> expression was quantified by quantitative real-time PCR and analysed using the comparative ΔΔCt method. Sequence variation was assessed through multiple sequence alignment, SNP detection, and amino acid translation. Phylogenetic relationships were inferred using the Maximum Likelihood approach with 1,000 bootstrap replicates. SNP nucleotide base composition was statistically evaluated using Chi-square tests.</p> Results <p>Multiple sequence alignment identified 37 coding single-nucleotide polymorphisms (SNPs), including both synonymous and non-synonymous substitutions. While most species showed a complete open reading frame of 179 amino acids, the freshwater <i>Coptodon zillii</i> sequence had a premature stop codon, leading to a truncated protein. Although <i>TNF-β</i> nucleotide composition varied descriptively among habitats, Chi-square analysis found no significant association with habitat type, supporting overall sequence conservation. <i>TNF-β</i> transcript levels were lower in brackish-water species and higher in freshwater <i>C. zillii</i>, indicating relative differences in gene expression among species. Phylogenetic analysis revealed a relative similarity to other cichlid species <i>TNF-β</i> sequences, with clustering consistent with species relationships rather than habitat preferences.</p> Conclusion <p>This study reveals a conserved <i>TNF-β</i> coding structure across tilapia species, with limited sequence and expression variation that may reflect ecological and species-specific influences. While the functional significance of these polymorphisms remains unclear, <i>TNF-β</i> represents a promising candidate gene for future research on immune responses and environmental stress adaptation in cichlid fishes.</p>

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Molecular characterization and comparative analysis of the TNF-β gene in tilapia species from contrasting salinity environments

  • 期刊论文

  • 发表日期：

    2026年2月24日

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  • J. B. Joseph,
  • O. O. Ogunruku,
  • A. S. Adesina,
  • Y. F. Taiwo,
  • O. R. Oguntade,
  • O. R. Osoniyi

摘要

Background

Environmental salinity is a major ecological stressor influencing immune regulation in tilapia, yet the molecular mechanisms underlying salinity-associated immune variation among species remain poorly understood. Tumor necrosis factor-beta (TNF-β) is a central cytokine in immune signaling and stress responses in teleost fish, making it a strong candidate for examining molecular mechanism underlying adaptation across habitats with contrasting salinity conditions.

Methods

TNF-β was amplified from cDNA of wild tilapia species collected from brackish and freshwater environments using PCR (expected amplicon ~ 875 bp), followed by Sanger sequencing and nucleotide sequences trimmed to ~ 565 bp coding region. cDNA quality was verified using GAPDH as a reference gene. TNF-β expression was quantified by quantitative real-time PCR and analysed using the comparative ΔΔCt method. Sequence variation was assessed through multiple sequence alignment, SNP detection, and amino acid translation. Phylogenetic relationships were inferred using the Maximum Likelihood approach with 1,000 bootstrap replicates. SNP nucleotide base composition was statistically evaluated using Chi-square tests.

Results

Multiple sequence alignment identified 37 coding single-nucleotide polymorphisms (SNPs), including both synonymous and non-synonymous substitutions. While most species showed a complete open reading frame of 179 amino acids, the freshwater Coptodon zillii sequence had a premature stop codon, leading to a truncated protein. Although TNF-β nucleotide composition varied descriptively among habitats, Chi-square analysis found no significant association with habitat type, supporting overall sequence conservation. TNF-β transcript levels were lower in brackish-water species and higher in freshwater C. zillii, indicating relative differences in gene expression among species. Phylogenetic analysis revealed a relative similarity to other cichlid species TNF-β sequences, with clustering consistent with species relationships rather than habitat preferences.

Conclusion

This study reveals a conserved TNF-β coding structure across tilapia species, with limited sequence and expression variation that may reflect ecological and species-specific influences. While the functional significance of these polymorphisms remains unclear, TNF-β represents a promising candidate gene for future research on immune responses and environmental stress adaptation in cichlid fishes.