Figures and data in Salmonids elicit an acute behavioral response to heterothermal environments

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34 figures, 5 videos, 20 tables and 1 additional file

Figures

Figure 1 with 4 supplements

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Lock-exchange tank used to expose fish to an acute heterothermal environment.

(A) Side-view of the experimental tank during the acclimation phase (15 min), with fish in the left compartment exposed to water at their acclimation temperature (12 °C) separated by a closed gate from the dyed water of a different temperature in the right compartment. Red ellipses indicate the locations of four trout parr tested simultaneously. Fish were tracked continuously based on video recordings of the lateral view of the entire tank (Figure 1—figure supplements 1–3). (B) The treatment phase was initiated by rapidly removing the gate, allowing the cold water to slowly intrude below the warmer water (1–2 min), thereby exposing the fish to a heterothermal environment. A sharp thermal interface (dashed line) separated upper, warmer water from lower, colder water. Within the first two minutes, the thermal interface moved laterally and afterwards spanned the entire lateral extent of the tank. Over time, the interface increasingly stabilized around the central depth of the tank (Figure 1—figure supplement 4). The movement of the interface was much slower than fish swimming speeds (Figure 1—figure supplement 4 and Appendix 4—figure 2). The frame used here for illustration was captured in a cold-water treatment, 11 min after gate removal. (C) Trajectory of a single fish over 18 min (treatment TR3, $T_{bottom} = 6^{\circ} C$ , $T_{top} = 12^{\circ} C$ ). The trajectory is color-coded according to the temporal phase of the experiment (see D). (D) The different phases of the experiments. After 15 min acclimation, the gate was removed and it took approximately 2 min to establish a horizontal thermal interface spanning the entire tank length. To allow the analysis of temporal trends in fish behavior, the dataset was divided into four periods of equal duration: acclimation, and experimental phases p1–p3. For a list of treatments, see Appendix 1—table 1.

Figure 1—figure supplement 1

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Experimental setup and animals.

(A) Photograph showing a side-view of the experimental tank in phase 2 (approximately 10 min after removal of the central gate) with four individual fish. The camera used for imaging is visible in the foreground. The dark casing obstructs the entry of natural light. Experimental animals (B); central gate to partition the tank; (C) right side of the tank with three temperature probes (white arrows) and dissolved oxygen probe (red arrow) (D).

Figure 1—figure supplement 2

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Side view of experimental setup for cold (A) and warm (B) treatment (TR2 and TR7).

The thermal interface line (black) was derived using a custom-made image analysis pipeline.

Figure 1—figure supplement 3

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Image processing steps to derive fish position and thermal interface.

(A) Rotated and cropped RGB image of the side camera (GoPro hero7). (B) Subsection of RGB image. (C) Red channel. The turquoise dot indicates the fish’s center of gravity for this time instance, derived from tracking in TRex. (D) Green channel. Red line indicates the thermal interface, derived by custom-made Python scripts.

Figure 1—figure supplement 4

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Movement of thermal interface.

Transient movement of the thermal interface/line during experimental phases (p1, p2, and p3) in cold water Treatments (TR1-TR4, **A-D**). Colored lines depict time steps of 10 s. The water surface is located at Y = 4 cm. The instantaneous position of the thermal interface is shown for each experimental phase (acclim., p1, p2, and p3) at a reduced temporal resolution of 0.2 Hz (the temporal resolution in the experiments was 24 Hz). The interface was approximated as a line ( $X, Y$ ) for each frame. During the acclimation period (acclim: grey dots) a sealed gate was located at X = 100 cm. The interface is colored by the respective temporal phase of the experiment (see A).

Figure 2 with 3 supplements

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Fish avoid the cold, but not the warm (see also Figure 1—figure supplements 1–3).

(A) Side-view of the (two-dimensional) swimming trajectory of a fish (colored line) in relation to the thermal interface (dashed black line). Black dots along the trajectory indicate the center of gravity of the fish during an example segment of 12 s. Colors indicate elapsed time, from blue to yellow. (B) Definition of warm- and cold-water excursions within a trajectory based on the fish’s instantaneous vertical distance to the thermal interface, $D (t)$ . An excursion starts and ends when the fish crosses the thermal interface. (**C, F**) Probability density functions of normalized fish depth $Y_{norm}$ during the final 6 min (p3) of the experiments for all cold-water (TR1–TR4, C) and all warm-water (TR6–TR9, F) treatments. Data were aggregated at the treatment level, hence each line represents data from 20 fish, tested in groups of four individuals in five separate experiments. $T_{bottom}$ and $T_{top}$ indicate the water temperature below and above the thermal interface. The depth was normalized so that $Y_{norm} = 0$ is the water surface and $Y_{norm} = 1$ indicates the bottom of the tank. (**D, G**) Probability density functions of fish depth for TR2 (D) and TR7 (G), separated according to the four phases of analysis (acclimation phase and experimental phases p1–p3; see Figure 1D). Data were aggregated at the treatment level as in C and F. Data for other treatments are shown in Figure 2—figure supplement 2. (**E, H**) Vertical distance to the interface, (D) as a function of time for each cold-water (light blue shaded region) and warm-water excursion (light pink shaded region) for TR2 ( $N = 980$ ) (E) and TR7 ( $N = 430$ ) (H) aggregated for all three experimental phases (p1–p3). $N$ is the total number of cold-water excursions performed by the 20 fish. The vertical extent of the thermal gradient thickness is indicated as blue and red (see Methods). Data for other treatments are shown in Appendix 8—figures 2 and 3. For a list of treatments, see Appendix 1—table 1.

Figure 2—figure supplement 1

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Cumulative time spent in incoming cold water (A) and warm water (B).

Thick lines represent the averaged cumulative time spent in cold water on the replicate level. Thin lines depict the 84th and 16th percentiles across experimental replicates. Two cold shocked individuals (in TR2) were excluded from the average as outliers. Fish displayed opposing responses to incoming warm and cold water over time. Namely, fish decreased the occupancy of cold water, while in warm treatments fish enhanced occupancy of warm water (see also Figure 2—figure supplement 3).

Figure 2—figure supplement 2

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Vertical occupation.

Probability density functions of fish depth for all cold-water (TR1–TR4, **A–D**) and all warm-water (TR6–TR9, **E–H**) treatments ( $N = 20$ fish in each case), separated according to the four phases of analysis (acclimation, and the three experimental phases p1–p3). The data were aggregated at the treatment level; hence each line represents data from 20 fish, tested in groups of four individuals in five separate experiments. $T_{b o t t o m}$ and $T_{t o p}$ indicate the water temperature below and above the thermal interface. The depth was normalized so that $Y_{norm} = 0$ is the water surface and $Y_{norm} = 1$ indicates the bottom of the tank.

Figure 2—figure supplement 3

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Time spent in acclimation temperature.

The average time fraction spent in 12 °C was calculated for each fish and phase (p1, p2, and p3) as $T_{cold} / 6$ min for incoming cold water (TR1–TR4, A) and incoming warm water (TR6–TR9, B). Each dot represents the average of this quantity over four individuals that were tested simultaneously within one experimental replicate. The Friedman-test suggests that there are significant temporal differences within TR1 (p = 0.022; Appendix 8—table 1). Significance levels are indicated above the plot (Dunn-test: *: $0.01 < p \leq 0.05$ ).

Figure 3

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Fish avoid cold water by directional upward turning.

(A) Example trajectory (12 s) with identified vertical turning points (pink triangles, downward; green triangles, upward). (B) Turning points were identified as sign changes of the time series of the vertical velocity component, $v_{y} (t)$ . The same trajectory as in A is shown. (C) Boxplots showing the number of cold-water excursions for each cold temperature treatment. Each dot represents the median number of cold-water excursions $\tilde{N}$ of four individuals tested simultaneously within one experiment. Red lines indicate the median of five experiments. Whiskers extend to the full range of observations. The temperature treatments TR2 and TR4 differ significantly (ANOVA, p < 0.001), with pairwise t-test results as indicated (* $0.01 < p \leq 0.05$ ; ** $0.001 < p \leq 0.01$ , see also Appendix 8—tables 4 and 5). (**D, E**) Swimming trajectories (gray lines) and vertical turning points (color-coded as in panel A) plotted with respect to the vertical distance $\hat{D}$ to the thermal interface (black dashed line) for cold-water treatment TR2 (D) and warm-water treatment TR7 (E). The trajectories shown are from the central region ( $80 < X < 120 cm$ ) of the tank, for 20 fish (5x4 fish per experiment). Turning points were filtered based on a vertical displacement threshold of dz >3 cm to detect turning activity that resulted in significant vertical displacement (Appendix 9—figure 2). Data for other treatments are shown in Appendix 9—figures 3 and 4. (**F, G**) Difference of probability density distributions of upward turns ( $ρ_{up}$ ) and downward turns ( $ρ_{down}$ ) for the entire width of the tank ( $0 < X < 200 cm$ ) as a function of the distance from the thermal interface (located at $\hat{D} = 0$ ), for all cold (F) and warm treatments (G). The same y-axes as in panels D and E apply. The tank bottom is located at around $- 10 cm > \hat{D} > - 20 cm$ , while the water surface is located at around $10 cm < \hat{D} < 20 cm$ (the variability being related to the initial period of time in which the thermal interface oscillates). The underlying normalized histograms are in Appendix 9—figure 2C and E. The vertical extent of the thermal gradient thickness is indicated as blue and red (see Methods).

Figure 4 with 2 supplements

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Fish reduce the duration and depth of cold-water excursions in larger measure the colder the water temperature below the thermal interface.

(A) Fish perform cold-water excursions in which they cross the thermal interface and swim into colder water, then turn upward to return to the warmer water. Individual cold excursions were characterized by their duration, $Ω$ , and the maximum vertical distance to the thermal interface, $D_{max}$ . (B) Fish exhibit shorter durations of cold-water excursions when encountering lower water temperatures below the interface ( $T_{bottom}$ ). The decreasing trend was statistically significant (Mann–Kendall test: for $\tilde{\tilde{Ω}}$ , $p < 0.001$ ; for ${\tilde{\tilde{D}}}_{max}$ , $p < 0.001$ ; Appendix 9—table 1). Boxplots show the median duration of cold-water excursions for fish groups across all phases (p1, p2, and p3) within each treatment. Points represent the median of a single experiment. TR2, one outlier not shown (out of range). (C) For colder water temperatures, fish penetrate less deeply into the cold lower water. The decreasing trend was statistically significant (Mann–Kendall test: ${\tilde{\tilde{D}}}_{max}$ , $p < 0.001$ ; Appendix 9—table 2). Underlying distributions and histograms are shown in Appendix 8—figure 5. Equivalent plots for warm treatments are shown in Appendix 8—figure 6A and B. (**D, E**) Probability density distributions of the durations $Ω$ of warm- and cold-water excursions for TR1 (D) and TR3 (E), respectively. The distributions of the warm- and cold-water excursion durations become increasingly distinct over exposure time for TR3 but not TR1. Excursions at 12 °C (above the interface) are shown in black, while excursions in the lower, colder water are depicted in the respective color. Treatment phases are indicated by line type (p1–p3). Values of $n$ indicate the total number of excursions of each type within that phase for 20 fish (as the excursions alternate, $n$ is equal for cold and warm excursions). Underlying histograms of D and E and other treatments are shown in Figure 4—figure supplements 1 and 2.

Figure 4—figure supplement 1

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Probability density function of the duration of warm- and cold-water excursions for cold (**A–D**) and warm treatments (**E–H**) and across treatment phases (p1, p2, and p3).

As a reference for comparison, black lines depict trajectory excursions in 12 °C. Colored lines show respective distributions in treatment water temperatures below (**A–D**) or above the thermal interface (**E–H**).

Figure 4—figure supplement 2

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Underlying probability density distributions of the durations $Ω$ of warm- and cold-water excursions for TR1 (A) and TR3 (B), respectively.

The distributions of the warm- and cold-water excursion durations become increasingly distinct over exposure time for TR3 but not TR1. Excursions at 12 °C (above the interface) are shown in black, whereas excursions in the lower, colder water are depicted in the respective color (green: $T_{b o t t o m} = 10^{\circ} C$ ; blue: $T_{b o t t o m} = 6^{\circ} C$ ). Treatment phases are indicated by line type (p1–p3). Values of $N$ indicate the total number of excursions of each type within that phase for 20 fish (as the excursions alternate, $N$ is equal for cold and warm excursions). Underlying distributions are shown as histograms.

Figure 5 with 2 supplements

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Fish swim more rapidly in the cold.

(A) Mean swimming speed when above (in warmer water) and below (in colder water) the thermal interface. Speed was standardized by the average swimming speed of each individual during the treatment phases (p1–p3). Data were aggregated at the replicate level; hence $\bar{\bar{v}}_{n, t}$ represents the average response of the fish group (four fish) within one replicate. Symbols represent the results of pairwise t-tests (ns, $p > 0.05$ ; *, $0.01 < p \leq 0.05$ ; ***, $0.0001 < p \leq 0.001$ , see also Appendix 4—table 3). Whiskers extend to the full range of observations and red lines indicate the median. The equivalent data for warm treatments are shown in Figure 5—figure supplement 1 and absolute swimming speeds are shown in Appendix 4—figure 2. (**B, C**) Probability density functions of averaged swimming speed during cold-water excursions (B) and warm-water excursions (C) in cold treatments (TR1–TR4). Speed was standardized by the average swimming speed of each individual within each phase and ${\bar{v}}_{n, p}$ represents the average of this quantity over the duration of each individual excursion. The total numbers of cold-water excursions are TR1, $n = 889$ ; TR2, $n = 389$ ; TR3, $n = 611$ ; and TR4, $n = 452$ ; with identical numbers of warm-water excursions for each treatment. A kernel density estimator was fitted to the normalized histogram after ${log}_{10}$ -transforming the data. Equivalent Figures for warm treatments are shown in Figure 5—figure supplement 2. Excursion durations were scattered against average normed swimming speed (see Appendix 11—figure 1).

Figure 5—figure supplement 1

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Mean swimming speed when above (in warmer water) and below (in colder water) the thermal interface.

Speed was standardized by the average swimming speed of each individual during the treatment phases (p1–p3). Data were aggregated at the replicate level; hence ${\bar{\bar{v}}}_{n, t}$ represents the average response of the fish group (four fish) within one replicate. Symbols represent the results of t-tests (ns, $p > 0.05$ ; *, $0.01 < p \leq 0.05$ ; ***, $0.0001 < p \leq 0.001$ , see also Appendix 4—table 4). Whiskers extend to the full range of observations and blue lines indicate the median.

Figure 5—figure supplement 2

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Averaged phase normalized swimming speed during warm water excursions (A) and cold-water excursions (B) in warm treatments (TR6, TR7, TR8, and TR9).

Data has been transformed to $l o g_{10}$ and a kernel density estimator was fitted to the normalized histogram.

Appendix 1—figure 1

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Side view of temporal progression of gravity current for cold treatment 4 (TR4).

A stationary vertical stratification (i.e. the water is practically still) is reached only after about t = 10 min.

Appendix 1—figure 2

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Cold treatments.

Time traces of temperature [°C] and dissolved oxygen concentration [mg/L] in experiments during acclimation (left) and treatment phase (right). Temperature (Probe 1-6) was measured at three different depths (red, orange, blue) at both side walls of the experimental tank for all experiments (upper plots). Dissolved oxygen was measured on both sides of the tank. All measurements were continuously obtained at a frequency of 5 Hz.

Appendix 1—figure 3

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Warm treatments.

Time traces of temperature [°C] and dissolved oxygen concentration [mg/L] in experiments during acclimation (left) and treatment phase (right). Temperature (Probe 1-6) was measured at three different depths (red, orange, blue) at both side walls of the experimental tank for all experiments (upper plots). Dissolved oxygen was measured on both sides of the tank. All measurements were continuously obtained at a frequency of 5 Hz.

Appendix 2—figure 1

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Tracking success rate for each individual fish during experimental phases (p1, p2, and p3).

Low values are explained by fish occluding over longer periods of time (without moving) or being located behind the dissolved oxygen probes. For these cases, a second camera perspective was used to resolve the occlusions, and data gaps were closed using linear interpolation. The open-source tracking software ‘TRex’ (67) was used to track fish at a temporal resolution of 24 frames/s.

Appendix 3—figure 1

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Fish tracking for cold treatments.

Trajectories for all fish during acclimation phase (**A–D**) and treatment phases (**E–F**).

Appendix 3—figure 2

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Fish tracking for warm treatments.

Trajectories for all fish during acclimation phase (**A–D**) and treatment phases (**E–F**).

Appendix 4—figure 1

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Example time traces illustrating the derivation of fish swimming speed (V) and normed swimming speed V_norm from the raw tracking output.

(A) Raw trajectory output from tracking software (TRex) for a single fish during 1 min of treatment 3 (T_bottom = 6 °C). (**B, C**) Horizontal and vertical velocity components (v_x, v_y) derived from raw output (blue line) and after applying the spline smoothing (Lüthi et al., 2005). (D) Absolute 2D-swimming speed V based on smoothed velocity components. (E) Normalized swimming speed V_norm speed derived as V_norm = V(t) / V_mean. The dashed line indicates the average swimming speed during the entire treatment duration (p1, p2, and p3 combined).

Appendix 4—figure 2

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Probability density of absolute swimming speeds per experimental phase (ac: acclimation; p1, p2, and p3) and treatment.

Cold treatments (**A–D**) and warm treatments (**E–H**). Each line indicates the normalized distributions of N = 6 min x 60 s x 24 fps x 20 fish = 172’800 observations.

Appendix 5—figure 1

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Cold treatments.

Boxplots of time fraction spent in upper half (Y_norm = 0–0.5) and lower half (Y_norm = 0.5–1) of the tank across experimental phases (acclim.: acclimation; p1, p2, and p3) and treatments (TR1, TR2, TR3, and TR4).

Appendix 5—figure 2

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Warm treatments.

Boxplots of time fraction spent in upper half (Y_norm = 0–0.5) and lower half (Y_norm = 0.5–1) of the tank across experimental phases (acclim.: acclimation; p1, p2, and p3) and treatments (TR6, TR7, TR8, and TR9).

Appendix 6—figure 1

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2D-swimming trajectories for TR3 (**A, C**) and its control without blue dye (TR5, **B, D**) during 6 min acclimation (**A, B**) and later experimental phases (p1, p2, and p3, **C, D**).

Both treatments had identical temperature conditions (T_left = 12 °C; T_right = 6 °C).

Appendix 6—figure 2

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Vertical relocation in control experiment.

(**A, B**) Probability density functions of fish depth during *experimental phases* (*acclim*.: acclimation; p1, p2, and p3) of the experiments for cold-water treatment 3 (TR3, A, N = 16 fish) and its control without the addition of blue dye (TR5, B, N = 16 fish). Both cases had identical thermal conditions (T_bottom = 6 °C, T_top = 12 °C) above and below the thermal interface. The depth was normalized so that Y_norm = 0 is the water surface and Y_norm = 1 indicates the bottom of the tank. (C) Boxplots aggregated on the fish group level did not show significant differences with regards to the vertical relocation across experimental phases.

Appendix 7—figure 1

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Image operations in Python (OpenCV) to derive the instantaneous position of the thermal interface.

As the lock exchange flow is primarily a 2D phenomenon and the width of the tank is narrow (20 cm) as compared to its length (200 cm), the temperature interface was approximated as a 2D line (X(t),Y(t)). The interface coordinates were derived for each time frame using a custom-made image analysis pipeline (Python, OpenCV).

Appendix 7—figure 2

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The theoretical large-scale gravitational velocity u₀ plotted against the cold current propagation speed *u_p* obtained from experimental videos of cold-water treatments (TR1–TR4).

(A) A linear regression fit (red dashed line) is shown. (B) The Kolmogorov length η as a function of the temperature contrast ΔT. The black dots represent experimental data, while the red dashed line indicates a power-law fit. (C) Temporal evolution of the thermal interface thickness, influenced by turbulence and diffusion. A 2D numerical simulation was conducted over a 20 minute period, using an initial interface thickness estimated as $δ ⋍ 3.7 η$ (see Methods). The spatial domain was discretized into 1 mm² grid cells (dx = dy = 0.001 m) and a constant thermal diffusivity of $D = 1.43 e - 07 m^{2} / s$ was assumed. Results indicate that, over the course of the experiment, the gradient thickness $δ_{k}$ increased over time, rising to values larger than the length of the individual fish.

Appendix 8—figure 1

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The temporal progression of the vertical distance to the thermal interface line, D(t), is shown for four individuals in an experiment of cold treatment 3 (TR3) (A) and warm treatment 8 (TR8) (B).

The vertical gate was removed at time *t = 0*. Negative values of D(t) depict times when the center of gravity of the tracked fish was below the thermal interface; hence, in the colder water and vice versa. Each sign change of D(t) depicts an interface crossing. Data gaps within the first two minutes arise from the fact that the gravity current did not yet fully span the entire horizontal dimension of the tank (i.e. in this period was excluded from our analysis, fish may neither be located below nor above but laterally displaced from the thermal interface). Shortly after the current reaches the wall, maximum distances $| D (t) |$ of up to 20 cm are possible (see also Appendix 13—Video 1). After the vertical stratification becomes stationary ( $t \approx 10$ min), the interface is approximately located at half the tank depth (h = 14 cm), and values for $| D (t) |$ are below 14 cm.

Appendix 8—figure 2

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Cold treatments, TR1–TR4 (**A–D**).

Temporal evolution of the fish’s instantaneous vertical distance to the thermal interface D(t) within the first 25 s for each cold- and warm-water interval for all cold treatments. At t = 0 fish crosses the thermal interface line upwards (warm segment; upper half) or downwards (cold segment; lower half). The end of an interval occurs when the interface line was crossed again in the opposite direction.

Appendix 8—figure 3

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Warm treatments, TR6–TR9 (**A–D**).

Temporal evolution of the fish’s instantaneous vertical distance to the thermal interface D(t) within the first 25 ss for each cold- and warm-water interval for all warm treatments. At t = 0, fish crosses the thermal interface line upwards (upper half) or downwards (lower half). The end of an interval occurs when the interface line was crossed again in the opposite direction.

Appendix 8—figure 4

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Number of performed cold-water excursions (A) and warm-water excursions (B) over time (p1–p3).

Boxplots show the median; whiskers extend to the full data range, and the boxes are limited by the 25th and 75th percentiles. Each dot represents the median across four individuals tested simultaneously.

Appendix 8—figure 5

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Probability density function of maximum distance to interface line D_max for cold (A) and warm (B) excursions for all treatments.

Underlying normalized histograms are shown in thin colored lines.

Appendix 8—figure 6

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Warm-water excursions during warm treatments (TR6-TR9).

For each quantity, the median of four individuals (tested simultaneously) was considered; hence, each dot represents a single experiment. (A) Boxplots of the maximum (upwards) distance to the interface of warm-water excursions. The median of $D_{max}$ was applied to all excursions performed by an individual fish and for all phases (p1, p2, and p3). (B) Boxplots of the median duration of cold-water excursions for fish. The median of $D_{max}$ was applied to all excursions performed by an individual fish and for all phases (p1, p2, and p3). Each dot represents a single experiment. (C) Boxplot of the median number of warm-water excursions (upwards) for fish groups across warm treatments and experimental phases (p1, p2, and p3). Each dot represents the median response of four fish in a single experiment. Blue lines indicate the median; boxplots are limited by the 25th and 75th percentiles. Whiskers extend to the full range of observations.

Appendix 9—figure 1

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Derivation of vertical turning points.

(A) To smooth the fish’s vertical position signal (red line), a fifth-order polynomial was fitted to a moving window of 50 consecutive time points (black line). This is a standard practice in particle tracking to filter out noise (e.g. Lüthi et al., 2005). Therefore, the SAVGOL function (python: scipy.signal.savgol_filter) was applied on the raw output (black line) from the tracking software. Upward turning (green triangles) was detected as a sign change (from negative to positive) of the derivative of the smoothed signal. Downward turning (red triangles) was detected via sign change (from positive to negative). (B) Occurrence of turning points after gate removal (at t = 0) for an individual fish in cold treatment (TR3).

Appendix 9—figure 2

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Occurence frequency of vertical turning points.

(A) Normalized histogram of the vertical distance dz between all consecutive turning points for all treatments (depicted by colors). A threshold of dz = 3 cm was used to differentiate turning events that result in small, cruising-related vertical displacements (**B, D**) from vertical turns that resulted in large vertical displacement (dz >3 cm) (**C, E**).

Appendix 9—figure 3

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Cold treatments.

Swimming trajectories (black lines) relative to the thermal interface (red line) for all fish across cold treatments (TR1 – TR4, **A–D**). The horizontal axis depicts the horizontal position in centimeters; the vertical axis depicts the instantaneous distance from the thermal interface. For D > 0, fish is located at a distance D [cm] above the interface and for D < 0, the fish is located below the temperature interface. Red and green triangles depict large downward and upward turns, respectively (see Appendix 9—figure 2 for derivation).

Appendix 9—figure 4

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Warm treatments.

Swimming trajectories (black lines) relative to the thermal interface (red line) for all fish across cold treatments (TR6–TR9, **A–D**). The horizontal axis depicts the horizontal position in centimeters; the vertical axis depicts the instantaneous distance from the thermal interface. For D > 0, fish is located at a distance D [cm] above the interface and for D < 0, the fish is located below the temperature interface. Red and green triangles depict large downward and upward turns, respectively (see Appendix 9—figure 2 for derivation).

Appendix 9—figure 5

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Violin plots showing the vertical distance from the thermal interface at which up-turning events occurred during cold-water treatments (TR1–TR4, **A–D**).

Each black dot represents a single up-turning event, and the red line indicates the median distance. Data are grouped by experimental phase (p1–p3). The dashed line denotes the position of the thermal interface (0 cm), and the red shaded area indicates the estimated thickness of the vertical thermal gradient (see Methods).

Appendix 10—figure 1

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Extreme swimming speeds above V = 20 cm/s for TR1–TR4 (**A-D**).

Black histograms show all observations above the thermal interface (T_top = 12 °C). Colored histograms display all observations below the thermal interface (in colder water). Dashed vertical lines depict literature thresholds for burst swimming speeds of rheophilic fish based on temperature (orange line: T = 12°; red line: T = 4 °C), body length (3.6 cm), and swimming durations (5 s) as proposed by Ebel, 2014 (see above). High swimming speeds were most frequent during cold-water occupation in colder treatments (TR2–TR4).

Appendix 11—figure 1

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Normalized swimming speed ${\bar{v}}_{n,p}$ averaged over excursions plotted as a function of the duration Ω of each cold (blue points) and warm (red points) water excursion for all cold (**A–D**) and all warm treatments (**E–H**).

To control for temporal trends, speed was standardized by the average swimming speed of each individual within each phase. Fitting a 2D Kernel density estimator to the point clouds (red and blue lines) reveals for cold treatments with $T_{Bottom} \leq 8^{\circ} C$ (TR2–TR4, **B–D**), clusters separate with respect to swimming speed (y-axis) and duration (x-axis).

Appendix 12—figure 1

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Simulated body temperature after cold-water excursions.

(A) Violin plots of simulated body temperature T_body after each performed cold-water excursion for cold-water treatments (TR1-TR4). Each dot is one excursion, the red line indicates the median, and n is the number of performed excursions by 5x4 = 20 fish within each treatment. The model (see *Methods*) assumed fish body mass of m = 4 gramm, initial body temperature of T₀ = 12 °C and a cooling rate coefficient of k = 1.373 min-1. (B) Box plot of the median body temperature ( ${\tilde{\tilde{T}}}_{body}$ ) after cold-water excursions for each experimental replicate. The red line indicates the median across experimental replicates.

Author response image 1

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The colored area indicates the temporal growth of thermal interface thickness.

Videos

Appendix 13—video 1

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posterframe for video — Tracking of 1 fish in cold treatment (TR2).

Side-view of the tank with individual fish swimming-trajectory and gravity current for single experimental replicate (TR2, cold-water in light blue). Individual fish movement trajectory is shown in red and its instantaneous vertical distance to the thermal interface $D (t)$ in the lower plot for the progression of the entire treatment (p1, p2, and p3) immediately after gate-removal. Playback is 20 x real speed.

Appendix 13—video 2

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Appendix 13—video 3

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Appendix 13—video 4

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Appendix 13—video 5

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Tables

Appendix 1—table 1

Definition of treatments.

We contrasted water at the acclimation temperature (12 °C) with four cold treatments (TR1–TR4) and four warm treatments (TR6–TR9); hence, we varied the incoming water temperature in both directions. Initial water temperature ( $T$ ) and density ( $ρ$ ) in the left (subscript: _left) and right (subscript: _right) compartments of the tank for each treatment. Treatment 5 (TR5) was the control treatment, conducted without the addition of dye (see Appendix 6).

Treatment	T_left [°C]	T_right [°C]	ρ_left [kg/m³]	ρ_right [kg/m³]	Density ratio: γ [-]
TR1	12	10	999.5	999.7026	0.99979734
TR2	12	8	999.5	999.8512	0.99964875
TR3	12	6	999.5	999.9432	0.99955677
TR4	12	4	999.5	999.9749	0.99952509
TR5 (control)	12	6	999.5	999.9432	0.99955677
TR6	12	14	999.5	999.2474	1.00025279
TR7	12	16	999.5	998.946	1.00055458
TR8	12	18	999.5	998.5986	1.00090266
TR9	12	20	999.5	998.2071	1.00129522

Appendix 2—table 1

Tracking parameters used for fish tracking in TRex software.

TGrabs parameters	TRex parameters
enable_difference = true reset_average = true use_adaptive_threshold = true adaptive_threshold_scale = 2 dilation_size = –3 track_max_individuals = 4 average_samples = 3,000 averaging_method = mean threshhold = 150 meta_real_width = 202 blob_size_ranges = [[0.1,6]] approximate_length_minutes = 15 cam_framerate = 24	blob_size_ranges = [[0.1,6]] track_max_reassign_time = 5 speed_extrapolation = 3 frame_rate = 24 gui_focus_group = [0,1,2,3] heatmap_ids = [0,1,2,3] track_max_reassign_time = 0.5 heatmap_resolution = 9 huge_timestamp_ends_segment = false midline_start_with_head = true midline_stiff_percentage = 0.4 outline_approximate = 15 output_csv_decimals = 2 output_posture_data = true output_heatmaps = true posture_closing_steps = 1 track_end_segment_for_speed = false track_max_speed = 40 track_max_individuals = 4 track_max_reassign_time = 5 blob_split_max_shrink = 0.2 output_invalid_value = nan track_ignore = [[[0,0],[0,16],[1840,0],[1840,16]]] auto_quit = true gui_auto_scale_focus_one = true

TGrabs parameters

TRex parameters

enable_difference = true
reset_average = true
use_adaptive_threshold = true adaptive_threshold_scale = 2
dilation_size = –3
track_max_individuals = 4
average_samples = 3,000
averaging_method = mean
threshhold = 150
meta_real_width = 202
blob_size_ranges = [[0.1,6]] approximate_length_minutes = 15
cam_framerate = 24

blob_size_ranges = [[0.1,6]] track_max_reassign_time = 5
speed_extrapolation = 3
frame_rate = 24
gui_focus_group = [0,1,2,3] heatmap_ids = [0,1,2,3]
track_max_reassign_time = 0.5
heatmap_resolution = 9
huge_timestamp_ends_segment = false midline_start_with_head = true midline_stiff_percentage = 0.4
outline_approximate = 15
output_csv_decimals = 2
output_posture_data = true
output_heatmaps = true
posture_closing_steps = 1
track_end_segment_for_speed = false track_max_speed = 40
track_max_individuals = 4
track_max_reassign_time = 5
blob_split_max_shrink = 0.2
output_invalid_value = nan
track_ignore = [[[0,0],[0,16],[1840,0],[1840,16]]]
auto_quit = true
gui_auto_scale_focus_one = true

Appendix 4—table 1

Shapiro-Wilk test for cold treatments.

Group averaged and normed swimming speeds ${\bar{\bar{v}}}_{n, t}$ for different temperatures (above and below interface).

	TR1		TR2		TR3		TR4
Temperature [°C]	12	10	12	8	12	6	12	4
Replicate
1	1.0726	0.9593	1.4859	1.1885	0.8875	1.4206	0.9857	1.0257
2	1.0231	0.9907	0.9691	1.1946	0.9896	1.2103	0.928	1.489
3	0.9545	1.1524	0.9309	1.4012	0.9202	1.1159	1.0319	1.0756
4	1.0015	0.9802	0.981	1.2883	0.9433	1.3381	0.9652	1.279
5	0.9436	1.0808	0.9747	1.1839	0.9841	1.3986	0.917	1.3978
p-value	0.714	0.297	0.002	0.079	0.612	0.465	0.743	0.576
test_stat	0.947	0.877	0.63	0.799	0.932	0.909	0.951	0.927

Appendix 4—table 2

Shapiro-Wilk test for warm treatments.

Group averaged and normed swimming speeds for different temperatures (above and below interface).

	TR6		TR7		TR8		TR9
Temperature [°C]	12	14	12	16	12	18	12	20
Replicate
1	1.0107	1.0181	0.9482	1.0876	0.9654	1.0185	0.9535	1.1097
2	0.9723	1.027	0.8855	1.1742	1.1352	0.9216	0.959	1.0755
3	0.985	1.0255	0.9288	1.1218	0.8776	1.0737	0.9735	1.3444
4	0.9409	1.0696	0.9546	1.0416	0.8878	1.0693	0.9914	1.0356
5	0.9811	0.9994	0.8443	1.1643	0.9375	1.0068	1.0333	0.9775
p-value	0.839	0.396	0.409	0.678	0.15	0.361	0.387	0.247
test_stat	0.965	0.897	0.9	0.942	0.834	0.891	0.896	0.865

Appendix 4—table 3

t-test results for cold treatments.

ns: not significant; *: $0.01 < p \leq 0.05$ ; ***: $0.0001 < p \leq 0.001$ :

Treatment	T_top [°C]	T_bottom [°C]	p-Value	Significance
TR1	12	10	0.4603	ns
TR2	12	8	0.1438	ns
TR3	12	6	0.0004	***
TR4	12	4	0.014	*

Appendix 4—table 4

t-test results for warm treatments.

Treatment	Ttop [°C]	Tbottom [°C]	p-Value	Significance
TR6	14	12	0.0147	*
TR7	16	12	0.0002	***
TR8	18	12	0.3199	ns
TR9	20	12	0.0859	ns

Appendix 5—table 1

Friedman test within each treatment,<time-fraction spent in 12 °C >, Tr depicts the treatment temperature initially in the right compartment of the tank.

Treatment	T_r [°C]	Source	W	ddof1	Q	p-unc
TR1	10	Phase	0.76	2	7.6	0.022*
TR2	8	Phase	0.04	2	0.4	0.819
TR3	6	Phase	0.76	2	7.6	0.022*
TR4	4	Phase	0.48	2	4.8	0.091
TR6	14	Phase	0.76	2	7.6	0.094
TR7	16	Phase	0.84	2	8.4	0.015*
TR8	18	Phase	0.52	2	5.2	0.074
TR9	20	Phase	0.28	2	2.8	0.247

Appendix 5—table 2

TR1, Dunn-test.

Phase	1	2	3
1	1.0	0.04*	0.049*
2		1.0	1.0
3			1.0

Appendix 5—table 3

TR3, Dunn-test.

Phase	1	2	3
1	1.0	0.774	0.688
2		1.0	1.0
3			1.0

Appendix 5—table 4

TR7, Dunn-test.

Phase	1	2	3
1	1.0	0.688	0.537
2		1.0	1.0
3			1.0

Appendix 8—table 1

Friedman test, result, and median number of excursions over time for all treatments.

Treatment	T_r [°C]	Source	W	ddof1	Q	p-unc
TR1	10	Phase	0.760	2	7.600	0.022
TR2	8	Phase	0.443	2	4.429	0.109
TR3	6	Phase	0.200	2	2.000	0.368
TR4	4	Phase	0.178	2	1.778	0.411
TR6	14	Phase	0.074	2	0.737	0.692
TR7	16	Phase	0.040	2	0.400	0.819
TR8	18	Phase	0.520	2	5.200	0.074
TR9	20	Phase	0.640	2	6.400	0.041

Appendix 8—table 2

TR1, Dunn-test (post-hoc).

Phase	1	2	3
1	1.0	0.537	0.312
2		1.0	1.0
3			1.0

Appendix 8—table 3

TR9, Dunn-test (post-hoc).

Phase	1	2	3
1	1.0	1.0	0.308
2		1.0	1.0
3			1.0

Appendix 8—table 4

Median number of cold-water excursions $\tilde{N}$ of four fish tested simultaneously within each experimental replicate.

Shapiro-Wilk-test results.

Replicate	TR1	TR2	TR3	TR4	TR6	TR7	TR8	TR9
1	40.5	2.5	18.0	21.5	56.5	48.5	61.0	70.5
2	61.0	20.5	24.5	24.5	61.5	46.5	44.5	66.5
3	50.0	31.0	34.5	28.0	65.5	51.5	60.0	68.5
4	69.0	11.5	43.0	24.0	62.5	51.0	75.0	97.0
5	34.5	32.5	27.5	16.0	70.0	43.5	67.5	87.0
test_stat	0.96	0.93	0.98	0.95	0.99	0.93	0.96	0.85
p-value	0.82	0.58	0.95	0.77	0.98	0.64	0.78	0.19

Appendix 8—table 5

One-way ANOVA test result.

f-Statistic	p-Value
21.5	2.02e-10

Appendix 8—table 6

Pairwise t-test result.

Pairs	p-Value
TR1, TR2	0.0063
TR1, TR3	0.0230
TR1, TR4	0.0029

Appendix 8—table 7

Percentage of all captured cold and warm excursions shorter than 10 s for all phases (p1, p2, and p3) of each treatment.

Treatment	Short cold excursions (Ω < 10 s) [%]	Short warm excursions (Ω < 10 s) [%]
TR1	75.4	69
TR2	91.2	38.1
TR3	94	49.1
TR4	92.2	42.7
TR6	81	76.3
TR7	73.1	71.4
TR8	72.5	82.7
TR9	88.2	81.6

Appendix 9—table 1

Mann–Kendall test.

Input data: median distance ${\tilde{\tilde{D}}}_{max}$ (in centimeters) and median durations $\tilde{\tilde{Ω}}$ (in seconds) across all treatments and experimental replicates.

Treatment	T_bottom [°C]	${\tilde{\tilde{D}}}_{max}$ [cm]	Med( ${\tilde{\tilde{D}}}_{max}$ )	$\tilde{\tilde{Ω}}$ [s]	Med( $\tilde{\tilde{Ω}}$ )
TR1	10	10.87		7.02
	10	9.83		5.45
	10	9.50		6.04
	10	10.37		4.10
	10	9.91	9.91	6.94	6.04
TR2	8	14.52		276.22
	8	5.79		4.04
	8	7.38		3.94
	8	6.31		4.30
	8	4.97	6.31	2.88	4.04
TR3	6	4.86		3.25
	6	4.97		3.23
	6	5.98		3.48
	6	3.90		2.56
	6	5.00	4.97	2.79	3.23
TR4	4	4.14		2.84
	4	2.94		2.19
	4	5.27		2.63
	4	5.24		2.59
	4	4.28	4.28	1.95	2.59

Appendix 9—table 2

Mann–Kendall test result.

A p-value lower than some significance level (common choices are 0.10, 0.05, and 0.01) indicates statistically significant evidence that a trend is present in the data. We found in both cases p < 0.001.

Test results	(T_bottom, $\tilde{\tilde{Ω}}$ )	(T_bottom, ${\tilde{\tilde{D}}}_{m a x}$ )
Trend	Decreasing	Decreasing
p-value	5.71E-07	4.27E-06
Test statistic	–292	–263
z-value	-5	–4.60
tau-value	–0.77	–0.69
s-value	–292	–263.00
Slope	–0.30	–0.36
Intercept	7.85	9.55

Appendix 12—table 1

Input to body-cooling model.

Summary of fish body temperature at the beginning of cold-water excursion (T_b) and ambient water temperature (T_a) during cold-water excursions.

Treatment	T_b [°C]	T_a [°C]
1	12	10
2	12	8
3	12	6
4	12	4

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Robert Naudascher
Stefano Brizzolara
Jonasz Slomka
Robert M Boes
Markus Holzner
Luiz GM Silva
Roman Stocker

(2026)

Salmonids elicit an acute behavioral response to heterothermal environments

eLife 13:RP99126.

https://doi.org/10.7554/eLife.99126.3

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Lock-exchange tank used to expose fish to an acute heterothermal environment.

Experimental setup and animals.

Side view of experimental setup for cold (A) and warm (B) treatment (TR2 and TR7).

Image processing steps to derive fish position and thermal interface.

Movement of thermal interface.

Fish avoid the cold, but not the warm (see also Figure 1—figure supplements 1–3).

Cumulative time spent in incoming cold water (A) and warm water (B).

Vertical occupation.

Time spent in acclimation temperature.

Fish avoid cold water by directional upward turning.

Fish reduce the duration and depth of cold-water excursions in larger measure the colder the water temperature below the thermal interface.

Probability density function of the duration of warm- and cold-water excursions for cold (A–D) and warm treatments (E–H) and across treatment phases (p1, p2, and p3).

Underlying probability density distributions of the durations Ω of warm- and cold-water excursions for TR1 (A) and TR3 (B), respectively.

Fish swim more rapidly in the cold.

Mean swimming speed when above (in warmer water) and below (in colder water) the thermal interface.

Averaged phase normalized swimming speed during warm water excursions (A) and cold-water excursions (B) in warm treatments (TR6, TR7, TR8, and TR9).

Side view of temporal progression of gravity current for cold treatment 4 (TR4).

Cold treatments.

Warm treatments.

Tracking success rate for each individual fish during experimental phases (p1, p2, and p3).

Fish tracking for cold treatments.

Fish tracking for warm treatments.

Example time traces illustrating the derivation of fish swimming speed (V) and normed swimming speed Vnorm from the raw tracking output.

Probability density of absolute swimming speeds per experimental phase (ac: acclimation; p1, p2, and p3) and treatment.

Cold treatments.

Warm treatments.

2D-swimming trajectories for TR3 (A, C) and its control without blue dye (TR5, B, D) during 6 min acclimation (A, B) and later experimental phases (p1, p2, and p3, C, D).

Vertical relocation in control experiment.

Image operations in Python (OpenCV) to derive the instantaneous position of the thermal interface.

The theoretical large-scale gravitational velocity u0 plotted against the cold current propagation speed up obtained from experimental videos of cold-water treatments (TR1–TR4).

The temporal progression of the vertical distance to the thermal interface line, D(t), is shown for four individuals in an experiment of cold treatment 3 (TR3) (A) and warm treatment 8 (TR8) (B).

Cold treatments, TR1–TR4 (A–D).

Warm treatments, TR6–TR9 (A–D).

Number of performed cold-water excursions (A) and warm-water excursions (B) over time (p1–p3).

Probability density function of maximum distance to interface line Dmax for cold (A) and warm (B) excursions for all treatments.

Warm-water excursions during warm treatments (TR6-TR9).

Derivation of vertical turning points.

Occurence frequency of vertical turning points.

Cold treatments.

Warm treatments.

Violin plots showing the vertical distance from the thermal interface at which up-turning events occurred during cold-water treatments (TR1–TR4, A–D).

Extreme swimming speeds above V = 20 cm/s for TR1–TR4 (A-D).

Normalized swimming speed v¯n,p averaged over excursions plotted as a function of the duration Ω of each cold (blue points) and warm (red points) water excursion for all cold (A–D) and all warm treatments (E–H).

Simulated body temperature after cold-water excursions.

The colored area indicates the temporal growth of thermal interface thickness.

Tracking of 1 fish in cold treatment (TR2).

Tracking of four fish in cold treatment (TR2).

Tracking of one fish in warm treatment (TR7).

Tracking of four fish in warm treatment (TR7).

Derivation of normed swimming speed.

Definition of treatments.

Tracking parameters used for fish tracking in TRex software.

Shapiro-Wilk test for cold treatments.

Shapiro-Wilk test for warm treatments.

t-test results for cold treatments.

t-test results for warm treatments.

Friedman test within each treatment,<time-fraction spent in 12 °C >, Tr depicts the treatment temperature initially in the right compartment of the tank.

TR1, Dunn-test.

TR3, Dunn-test.

TR7, Dunn-test.

Friedman test, result, and median number of excursions over time for all treatments.

TR1, Dunn-test (post-hoc).

TR9, Dunn-test (post-hoc).

Median number of cold-water excursions N~ of four fish tested simultaneously within each experimental replicate.

One-way ANOVA test result.

Pairwise t-test result.

Percentage of all captured cold and warm excursions shorter than 10 s for all phases (p1, p2, and p3) of each treatment.

Mann–Kendall test.

Mann–Kendall test result.

Input to body-cooling model.

MDAR checklist

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Underlying probability density distributions of the durations $Ω$ of warm- and cold-water excursions for TR1 (A) and TR3 (B), respectively.

Example time traces illustrating the derivation of fish swimming speed (V) and normed swimming speed V_norm from the raw tracking output.

The theoretical large-scale gravitational velocity u₀ plotted against the cold current propagation speed u_p obtained from experimental videos of cold-water treatments (TR1–TR4).

Probability density function of maximum distance to interface line D_max for cold (A) and warm (B) excursions for all treatments.

Normalized swimming speed ${\bar{v}}_{n,p}$ averaged over excursions plotted as a function of the duration Ω of each cold (blue points) and warm (red points) water excursion for all cold (A–D) and all warm treatments (E–H).

Median number of cold-water excursions $\tilde{N}$ of four fish tested simultaneously within each experimental replicate.