Development of a plausible ‘pan-specific primer’ targeting fish CYP1A gene for a rapid streamlined assessment of aquatic ecosystem integrity

Aquatic ecosystems, one of the vital resources of earth’s biosphere, are deviously threatened by anthropogenic pollution from compounds like polycyclic aromatic hydrocarbons (PAHs), heavy metals, and persistent organic pollutants. Though their early detection at trace levels in the aquatic environment is critical, fish CYP1A gene expression, induced via the AhR pathway, stands as a dominant and reliable sensitive biomarker. However, lack of a broad-range primer has limited large-scale ecological monitoring across diverse fish taxa. Addressing this gap for maintaining the healthy status of aquatic ecosystems, the present study aims to develop and validate a pan-specific primer targeting the fish CYP1A gene for multi-species biomonitoring in Northeast India, especially Assam. Seasonal samples of five freshwater fish species that includes Indian major carps, exotic carp, and indigenous fishes, were collected from an ecologically important site, the Deepor Beel, the only Ramsar site in Assam. Fish specimens were collected in two batches for each season: one batch was immediately dissected on-site for liver tissue isolation to extract RNA, while the other batch was acclimatized in the laboratory for 15 days to serve as the control group for further analyses. Following RNA isolation and cDNA synthesis, primers were designed and optimized via gradient PCR and qRT-PCR. The designed primer efficiently amplified CYP1A across species with 295 bp product size and sensitivity up to 0.1 ng/µL cDNA (LOD = 10⁻⁴). Significant upregulation of CYP1A expression was observed in the wild species compared to the control, indicating pollution-induced stress. This optimized primer enables time and cost-effective, species-independent assessment of aquatic pollution and can streamline conservation strategies in biodiverse regions. Future applications may expand its use to establish it as a universal tool for ecological risk assessment.