Fig. 1: The exposure to fish water induces anxiety-like behaviors, persisting for up to 18 h and in a different context.

A timeline of the experiment is presented above the data. A-B Aerial Respiration. A Naïve snails (N = 20) were placed in hypoxic artificial pond water (PW) for 30 min (beige bars), and the total breathing time was recorded. Three hours later, the total breathing time was recorded in hypoxic fish water (FW) (green bar). A significant increase in aerial respiration was observed in FW compared to artificial PW. This anti-predatory behavior persisted 18 h later when aerial respiration was recorded again in artificial PW (beige bars). B Control snails (N = 11) exposed only to artificial PW (beige bars) showed no significant differences in total breathing time. C-D Righting Time. C Naïve snails (N = 20) were placed in a vulnerable position with their ventral part exposed, and the righting time was recorded after exposure to PW (beige bar) and 3 h later to FW (green bar). Snails significantly decreased their righting time following FW exposure. This reduced righting time persisted when tested 18 h later in artificial PW. D Control snails (N = 10) exposed only to artificial PW (beige bars) showed no significant differences in righting time. E-F Escape Behavior. E Naïve snails (N = 20) were placed in a lid containing artificial PW. The lids were placed on filter paper soaked with artificial PW, and the number of escapes was recorded for 30 min (beige bar). Three hours later, the lids were placed on filter paper soaked with FW, and the number of escapes was recorded again (green bar). A significant reduction in the number of escapes was found following FW exposure. This reduced escape behavior persisted when tested 18 h later in artificial PW. F Control snails (N = 10) exposed only to artificial PW (beige bars) showed no significant differences in escape behavior. The data demonstrate that exposure to FW induces significant anti-predatory behaviors in snails, evidenced by increased aerial respiration, decreased righting time, and reduced escape behavior. These responses persisted even after the initial FW exposure, indicating a lasting behavioral change. Control snails that were not exposed to FW did not show significant changes in any of the measured behaviors, confirming that the observed effects were specific to the FW exposure. Data shown in Fig. 1A were analyzed using the Friedman test followed by Dunn’s post-hoc test, whereas data shown in Fig. 1B-F were analyzed using repeated measures ANOVA (RM ANOVA) followed by Tukey post hoc tests B-F. **** p < 0.0001, *** p < 0.001, * p < 0.05, ns = not significant (p > 0.05). The solid line represents the mean, and the error bars represent the standard error of the mean (SEM.).