Cortisol is a key stress biomarker in humans and animals, including fishes. In aquafarming, stress monitoring using cortisol quantification can help to optimize aquaculture practices for welfare and productivity enhancement. However, most current methods for cortisol detection rely on invasive tissue sampling. In this work, we developed a gold nanoparticle (AuNP)-based cortisol sensor to address the demand of detecting picomolar ranges of cortisol from complex fish tank water matrices as a non-invasive alternative for more effective stress monitoring. We first identified a DNA aptamer with effective binding to cortisol and then conjugated the thiol-labelled aptamer to AuNPs together with a blocker molecule (CALNN) to form an Au-Apt-CALNN conjugate that is stable in fish tank water. The cortisol detection principle is based on magnesium chloride (MgCl₂)-induced particle aggregation, where the cortisol-bound aptamer on the AuNPs folds into a tertiary structure and provides greater protection for Au-Apt-CALNN against MgCl₂-induced aggregation due to steric stabilization. At an optimum MgCl₂ concentration, the differential stability of particles with and without cortisol binding offers a limit of detection (LOD) of 100 pM for cortisol within a 35 min reaction. The aptasensor has been validated on recirculating aquaculture system (RAS) fish tank water samples by the HPLC method and was able to detect changes in water cortisol induced by two different stress paradigms. This on-site deployable and non-invasive sensor offers opportunities for more efficient and real-time fish stress monitoring for the optimization of aquaculture practices.

Scale drop disease virus (SDDV) is a major viral pathogen causing high mortality in farmed Asian seabass (Lates calcarifer), yet its impacts on host-associated microbiota remain poorly understood. In this study, juvenile L. calcarifer were intraperitoneally challenged with 10⁷ viral copies (0.1 ml SDDV), resulting in 94.2% cumulative mortality over a 24-day experimental period. A total of 36 intestinal content samples were collected at defined time points relative to challenge (dpc) from clinically healthy (H) and sick (S) fish, representing five stages: pre-infection baseline (H0 dpc), early infection (H6 dpc), peak mortality (H10 dpc and S10 dpc), declining mortality (S15 dpc) and recovery (H24 dpc). Histopathological examination revealed progressive intestinal pathology, characterised by multifocal vesicle formation and epithelial necrosis, consistent with SDDV-associated enteropathy. 16S rRNA gene sequencing demonstrated pronounced intestinal dysbiosis during infection, with a significant collapse of alpha diversity at peak mortality (10 dpc; Kruskal-Wallis test with Dunn's post-hoc comparisons, p < 0.05) where Chao1 decreased from 942 at baseline to 416 and Shannon from 4.3 to 1.9. At peak disease, the gut microbiome was overwhelmingly dominated by Vibrio (>80% relative abundance), suggesting secondary opportunistic expansion associated with virus-induced intestinal damage. Co-occurrence network analysis further revealed fragmentation of microbial interactions during peak infection, followed by partial restoration of network connectivity and re-emergence of taxa such as Photobacterium and Rhodobacteraceae during recovery from infection. Collectively, these findings demonstrated that SDDV infection induced severe intestinal pathology accompanied by dynamic restructuring of the gut microbiome in L. calcarifer, linking viral epithelial damage to destabilization of the intestinal microbial homeostasis and secondary Vibrio overgrowth. Distinct microbiome features, including reduced community diversity and Vibrio dominance at peak disease, as well as recovery-associated taxa in surviving fish, may represent potential microbial indicators of disease progression and intestinal recovery, providing a reference framework for future microbiome-informed health management strategies aimed at mitigating SDDV impacts in L. calcarifer aquaculture.