Temporal Variability of Bioindicators and Microbial Source-Tracking Markers over 24 Hours in River Water

With increasing contamination in aquatic ecosystems, effective monitoring is crucial to preserve biodiversity and protect public health. This study quantified bioindicators (red swamp crayfish (Pcla), Genji-firefly (Lcr2), Ayu fish (Paa), and caddisfly (Sma)), microbial source tracking markers (ruminants (BacR), pigs (Pig2Bac), and humans (gyrB)), and a fecal indicator bacterium (Escherichia coli (sfmD)) using quantitative PCR on river water samples collected every 2 h between 21 and 22 July 2023 (from the Omo and Bingushi Rivers in Yamanashi Prefecture, Japan). Initially, the optimal filter sizes of 1.0, 0.65, and 0.22 µm were evaluated, where the 0.65 µm filter yielded higher Paa concentrations (Kruskal–Wallis test, p < 0.05) and was used subsequently. BacR and Paa exhibited 100% detection in the Omo (13/13) and Bingushi (13/13) Rivers with concentrations of 5.0 log10 and 5.5 log10 copies/L, respectively. These concentrations were used to assess 24 h temporal variability, but no significant fluctuations or cyclical trends between morning, afternoon, evening, and night were observed in either river. The BacR–Paa pair exhibited perfect positive detection correlation (Φ = 1.0) and complete similarity (Jaccard Index = 1.0), but a moderate negative correlation of mean concentrations highlights the importance of considering habitat overlaps and behavioral synchronicity.