Meher Un Nissa

Ammonia accumulation is a major constraint in intensive Macrobrachium rosenbergii aquaculture, yet the mechanisms linking multi-organ injury to tissue deterioration remain unclear. Juvenile prawns were exposed for 7 days to 0, 2, and 50 mg L⁻¹ total ammonia nitrogen (TAN) to evaluate integrated physiological, histological, transcriptomic, and metabolomic responses in gill, hepatopancreas, and muscle. Survival decreased from 100% in controls to 89% and 75% at 2 and 50 mg L⁻¹, respectively, accompanied by gill lamellar disruption, hepatopancreatic tubule degeneration, and muscle fiber separation. RNA-seq identified 4,944 differentially expressed genes with strong organ-specific patterns. The hepatopancreas showed enhanced lysosomal activity and redox defenses, the gills exhibited disrupted nitrogen and lipid metabolism, and the muscle displayed impaired glycolysis and altered structural protein signaling. Changes in CD63, CssYP2J, GLUL, and AIFM1 expression, together with altered CAT, GS, and GDH activities, indicated adaptive detoxification at moderate TAN. However, high TAN overwhelmed these responses, leading to glutamine detoxification failure, lipid peroxidation, and malondialdehyde accumulation. Gill metabolomics revealed TAN-dependent alterations in carbon, amino acid, and phospholipid pathways, particularly glycerol-phosphocholine, citric acid, and citrulline metabolism, highlighting metabolite–gene interactions within nitrogen and membrane lipid networks. Overall, even sublethal TAN acted as a systemic stressor, reprogramming metabolic and antioxidant pathways across organs, with muscle emerging as the most vulnerable tissue. These findings refine ammonia toxicity thresholds and support maintaining TAN below 2 mg L⁻¹ to protect prawn health and aquaculture sustainability.

The rapid evolution of CRISPR/Cas technology has transformed genome editing across biological systems in which zebrafish have emerged as a powerful vertebrate model for functional genomics and disease research. Due to its transparency, genetic similarity to humans, and suitability for large-scale screening, zebrafish is an appropriate system for translating molecular discoveries into biomedical and environmental applications. Thereby, this review highlights the recent progress in zebrafish gene editing, targeting innovations in ribonucleoprotein delivery, PAM-flexible Cas variants, and precision editors. These approaches have greatly improved editing accuracy, reduced mosaicism, and enabled efficient F0 phenotyping. In the near future, automated microinjections, optimized guide RNA design, and multi-omics validation pipelines are expected to enhance reproducibility and scalability. Although recent innovations such as ribonucleoprotein delivery, PAM-flexible Cas variants, and precision editors have expanded the zebrafish genome-editing toolkit, their benefits are often incremental and context-dependent. Mosaicism, allele complexity, and variable germline transmission remain common, particularly in F0 embryos. Precision editors enable defined nucleotide changes but typically exhibit modest efficiencies and locus-specific constraints in zebrafish. Consequently, rigorous validation, standardized workflows, and careful interpretation of F0 phenotypes remain essential. This review critically examines both the capabilities and limitations of current zebrafish gene-editing technologies, emphasizing experimental trade-offs, reproducibility challenges, and realistic use cases.

Aeromonas hydrophila causes motile Aeromonas septicemia in fish, severely impacting common carp (Cyprinus carpio) and leading to significant losses in aquaculture. Live attenuated vaccines are considered effective for preventing bacterial infections in aquaculture, but specific vaccines for common carp are relatively few. Hemolysin is a major virulence factor of A. hydrophila, causing the lysis of red blood cells and other eukaryotic cells by disrupting their membranes. In this study, we deleted two hemolysin-related genes, hlyIII and ahh1, in A. hydrophila and investigated the phenotype, virulence, immunogenicity, and ability to confer immune protection in common carp. The ∆hlyIII, ∆ahh1, and ∆hlyIII∆ahh1 mutant strains exhibited significantly diminished hemolytic activity, attenuated virulence, and increased antibiotic susceptibility compared to the wild-type (WT) strain. RNAseq analysis revealed 490 differentially expressed genes in the ∆hlyIII∆ahh1 mutant, including 347 upregulated and 143 downregulated genes. KEGG enrichment analysis and qRT-PCR validation indicated downregulation of genes associated with the two-component system, quorum sensing, bacterial chemotaxis, and catabolic antimicrobial peptide (CAMP) resistance pathways. The LD50 value of ∆hlyIII∆ahh1 (8.13 × 10^8 CFU/mL) was 25.7 times higher than that of the WT (3.16 × 10^7 CFU/mL). Fish immunized by immersion with ∆hlyIII∆ahh1 showed enhanced IgM responses and upregulated cytokine gene expression levels (TNF-α, IL-1β, IL-16). Additionally, ∆hlyIII∆ahh1 conferred the highest relative percent survival (RPS) of 81.1% in common carp following two immersion immunizations. These findings suggest that ∆hlyIII∆ahh1 could serve as a potential vaccine candidate for A. hydrophila infections in common carp, benefiting the aquaculture industry by providing a means for effective immersion vaccination.

N-ethyl-N-nitrosourea (ENU) mutagenesis is a useful method for the genetic engineering of plants, and the production of functional mutants in animal models including mice and zebrafish. Grass carp reovirus (GCRV) is a haemorrhagic disease of grass carp which has caused noteworthy losses in fingerlings over the last few years. To overcome this problem, we used ENU mutant grass carp in an attempt to identify functional resistance genes for future hereditary rearing projects in grass carp. This study used ENU-mutated grass carp to identify genetic markers associated with resistance to the haemorrhagic disease caused by GCRV. Bulked segregant analysis (BSA) was performed on two homozygous gynogenetic ENU grass carp groups who were susceptible or resistant to GCRV. This analysis identified 466,162 SNPs and 197,644 InDels within the genomes of these mixed pools with a total of 170 genes annotated in the associated region, including 49 genes with non-synonymous mutations at SNP sites and 25 genes with frame shift mutations at InDel sites. Of these 170 mutated genes, 5 randomly selected immune-related genes were shown to be more strongly expressed in the resistant group as compared to the susceptible animals. In addition, we found that one immune-related gene, EPHB2, presented with two heterozygous SNP mutations which altered the animal’s responded to GCRV disease. These SNPs were found in the intron region of EPHB2 at positions 5859 (5859^G > A) and 5968 (5968^G > A) and were significantly (p = 0.002, 0.003) associated with resistance to GCRV. These SNP sites were also shown to correlate with the GCRV-resistant phenotype in these ENU grass carp. We also evaluated the mortality of the different ENU fish genotypes in response to GCRV and the SNPs in EPHB2. The outcomes of these evaluations will be useful in future selections of GCRV-resistant genes for genetic breeding in grass carp. Our results provide a proof of concept for the application of BSA-sequence analysis in detecting genes responsible for specific functional genotypes and may help to develop better methods for marker-assisted selection, especially for disease resistance in response to GCRV.