Fig. 3: EVs production is regulated by biotic and abiotic factors.

a Map of the sampling sites in HRW (n = 13). b The box plots showed the particle number (up) and protein content (down) of EVs in HRW, in which Wilcoxon tests were performed. US: upstream; MS: midstream; DS: downstream. c Heatmaps showed pairwise correlations between the characteristics of EVs and biotic as well as abiotic factors in HRW. The color gradient indicated Spearman’s correlation coefficients with an asterisk (see notes below). d Structural equation modeling analysis showed the influence of biotic and abiotic factors on EV particle number. e The EVs production rates during the logarithmic growth phase of XL92 (Flavobacterium sp.) strain (three repetitions) at different pH, salinity, and temperature when a single variable was controlled. Log 1, Log 2, and Log 3 represent the first, second, and third stages of logarithmic growth, respectively. The significant asterisk markers were represented as *P < 0.05, **P < 0.01, ***P < 0.001. f The proportions of the EVs taxonomic sources in HRW using FEAST (fast expectation-maximization microbial source tracking) algorithm. g Scanning electron microscopy (SEM) images (scale bar, 100 nm) of the XL92 (Flavobacterium sp.) strain isolated from sampling sites. The red arrows pointed to the EVs which were bubbling. Notes: APS average particle size, HRW Houxi River watershed, Tem water temperature, Tur turbidity, chl-a chlorophyl a, DO dissolved oxygen, ORP oxidation reduction potential, Con conductivity, Sal salinity, Trans transparency, TN total nitrogen, NH4_N ammonium nitrogen, NO3_N nitrate nitrogen, NO2_N nitrite nitrogen, TP total phosphorus, PO4_P phosphate phosphorus, TDS total dissolved solids, ACD algae cell density.