Development of a modified protocol for extraction of environmental DNA from water samples to assess the presence of fish species in wetland ecosystems.

Accurate fisheries assessment is challenged by limitations in traditional catch estimation methods, suggesting the need for advanced molecular tools such as environmental DNA (eDNA) analysis. This approach has transformed non-invasive biodiversity monitoring worldwide, yet standardized protocols remain underdeveloped in India, limiting practical application. The absence of validated, field-applicable eDNA extraction protocols optimized for complex aquatic environment like wetlands hinders reliable fish diversity assessment. This study validates a modified eDNA extraction protocol using mesocosm and field experiments. Water samples (300 mL) were filtered through glass fiber filters (1.5 μm), preserved in Longmire's and phosphate-buffered saline (PBS) lysis buffers, and processed using a modified phenol-chloroform isoamyl alcohol (PCI) extraction method. eDNA yield was quantified over 0, 10, and 20-day storage intervals and log transformed for analyses, eDNA degradation, and inhibitor modeling was performed. Amplification efficiency was compared for Ward barcode (COI) and Ac12S (12S rRNA) primers. The modified protocol outperformed the conventional protocol. PBS-preserved samples showed initial high yields (log10 mean ± SD, 3.06 ± 0.02 at Day 0) but degraded rapidly (λ = 0.102 day⁻1, half‑life = 6.8 days), falling to 2.18 ± 0.42 by Day 20. Longmire's buffer maintained statistically stable yields across storage (p > 0.05 for all pairwise comparisons), with no significant degradation. Ward barcode consistently amplified Longmire's preserved samples at 50 to 400 ng/μL; PBS samples showed no amplification. Ac12S showed limited amplification in both buffers. Application of the modified protocol in Harike wetland field samples confirmed its sensitivity and practical utility, detecting fish eDNA of Cyprinus carpio, Pethia conchonius, and Lepidocephalichthys thermalis. The findings suggest that buffer selection and standardized extraction are critical for reliable eDNA-based diversity surveys. The optimized workflow enhances flexibility for sampling in resource-limited areas, facilitating non-invasive surveys in protected wetlands.