Environmental DNA Monitoring for Detection and Spread of Prawn ( Macrobrachium rosenbergii ) Disease Organisms in Hatcheries and Natural Conditions

ABSTRACT Pathogen outbreaks by Macrobrachium rosenbergii nodavirus (MrNV), extra small virus (XSV), M. rosenbergii golda virus (MrGV), and pathogenic Vibrio spp. ( V. parahaemolyticus , vulnificus , harveyi ) have devastated Bangladesh's freshwater prawn aquaculture, causing mass mortalities and multimillion‐dollar losses due to delayed conventional tissue diagnostics. We collected 1–5 L water samples biweekly or quarterly (July 2023–April 2025) from 10 hatcheries and 10 linked natural sites, filtered (0.45 μm), preserved in ethanol at 4°C, and extracted eDNA using Invitrogen PureLink kits (A260/280 1.7–1.9). Custom TaqMan qPCR assays targeted MrNV RdRp (112 bp), XSV capsid‐UTR (145 bp), MrGV ORF1a‐ORF3 (189 bp), and Vibrio toxR (98 bp), with plasmid standards (91%–97% efficiency, R 2  ≥ 0.997), LOD 95 6–14 copies/reaction (CLSI EP17‐A2), and internal controls. Positives (Ct ≤ 37) were validated in triplicate, Sanger‐sequenced ( n  = 152, ABI 3730xl, BigDye v3.1), BLASTn‐analyzed (85%–100% identity, e  ≤ 10 −60 ), and phylogenetically assessed (IQ‐TREE, 5000 bootstraps). Hatcheries showed 4.7‐fold higher median loads (log 10 copies/L, Wilcoxon p  < 10 −10 ) than natural sites: MrNV 1300 copies/L (94% prevalence, 82%–99% CI) versus 450 (41%, 29%–54% CI); Vibrio 1600 (88%) versus 1400 (76%); XSV/MrGV 850/450 versus 320/280—with frequent co‐infections (SparCC ρ  = 0.72, e.g., MrNV‐XSV in 84% hatcheries). Negative binomial GLMMs ( R 2 marg  = 0.65, R 2 cond  = 0.77) confirmed hatchery effluent spillover (coef. = 2.03, p  < 10 −11 ), modulated by distance/salinity ( n  = 10 pairs), with biosecurity correlations r  = −0.62 ( p  = 0.01); eDNA performed optimally at pH 7–8.5, salinity 0.01–36 ppt, 26.5°C–31°C. Variants included MrNV SNPs (6.4 ± 2.1, F ST  = 0.018 hatchery vs. 0.037 natural), novel XSV 6‐nt deletion (23% natural), MrGV substitutions (V134I, N212D, G345S), and Vibrio clades (F ST  = 0.29–0.48). These results validate eDNA as a non‐invasive early‐warning tool, recommending routine hatchery screening, UV/chlorine treatment, and natural monitoring to curb reservoirs and sustain production.