Sea urchins
Sea urchins (~ 2 cm of test diameter) were transported from Dalian Haibao Fishery Company (121° 22′ E, 38° 77′ N) to the Key Laboratory of Mariculture and Stock Enhancement in North China’s Sea, Ministry of Agriculture and Rural Affairs (121° 56′ E, 38° 87′ N) in November 2020 and maintained in the fiberglass tank of 139 L (750 × 430 × 430 mm) of the recirculating system (Huixin Co., China) at ~ 14 °C. During the experiment period, the salinity of the recirculated seawater was 30.07–30.25‰. Sea urchins were fed the kelp Saccharina japonica ad libitum with aeration before the experiments. The seawater was changed every three days to remove feces and algal debris.
Conspecific alarm cues
Conspecific alarm cues were made from crushing one M. nudus in 50 mL of fresh seawater and then filtered using a fine silk net (260 μm of mesh size)6. Fresh conspecific alarm cues were prepared before each behavioral experiment. Five mL of conspecific alarm cues was used to simulate the natural conditions, in which a fraction of body fluids from a sea urchin instantly released into the surrounding water6.
Experiment 1: whether M. nudus are attracted by the kelp exposed to conspecific alarm cues
A piece of wild fresh S. japonica (~ 5 g) was placed on the side of the device with the raceways (70 × 6 × 5 cm) and 5 mL of conspecific alarm cues were subsequently added above it in the experimental group (Fig. 4A). Five mL of conspecific alarm cues were added in the control group, while the kelp was not involved (Fig. 4B). The sea urchin was individually placed 15 cm away from the kelp for each group (Fig. 4A,B). Foraging behavior of sea urchins was recorded for 20 min using a digital video recorder (FDR-AXP55, SONY, Japan). The danger area (b area) refers to the position 15 cm away from the kelp and the escape area (a area) refers to the position 15 cm away from the sea urchin (Fig. 4A,B). The time of sea urchins spent in the danger (a area) and escape (b area) areas was calculated individually for each group. The experiment was repeated 20 times using different sea urchins for each group (N = 20). The seawater was changed and the experimental device was washed for each trial to avoid potential non-experimental effects.
Foraging devices for the experiments with Mesocentrotus nudus. Experiment 1: the kelp was attracted by sea urchins (A,B); the positions of a, c and f refer to the escape areas, while positions of b, d and e refer to the danger areas. Experiment 2: five mL of conspecific alarm cues were put in the middle of the device (C). Experiment 3 (D–F): foraging behavior of M. nudus at 70 cm in the first trial (D); five mL of conspecific alarm cues were put in the middle of the device in the second trial (E); the position of g refers to the danger area; different amounts of conspecific alarm cues (5 mL and 0.5 mL) were put in the left and right of the devices in the third trial (F); the positions of h and i refer to the safety areas close to the areas with more and less conspecific alarm cues, respectively. This figure was created using the Adobe Photoshop (version CS5) software.
Experiment 2: whether foraging behavior of fasted M. nudus is affected when they encountered conspecific alarm cues
This experiment investigated the effects of conspecific alarm cues on the foraging behavior of fasted sea urchins. The experiment group was set to simulate that conspecific alarm cues appear on the way to kelp beds (Fig. 4C). Sea urchins were fasted for 7, 14 and 21 days in the experiment groups.
An acrylic device with the raceways (70 × 6 × 5 cm) was designed according to the method of our previous study17 with some revisions. Five mL of conspecific alarm cues were added in the middle of the device, the wild fresh S. japonica (~ 10 g) was placed 15 cm away from the left side of the device, while the sea urchin was placed on 15 cm away from the right of the device (Fig. 4C). Foraging behavior of sea urchins was recorded for 20 min using a digital video recorder (FDR-AXP55, SONY, Japan). Arriving at the kelp within 20 min was defined as successfully foraging18. The danger area (e area) refers to the position between conspecific alarm cues and the sea urchin, while the escape area (f area) refers to the 15 cm position away from the right side of the sea urchin (Fig. 4C). The time of sea urchins spent in the e and f areas was calculated individually for all the groups. The individual experiment was repeated 20 times with different sea urchins for each group (N = 20).
Experiment 3: whether foraging ability of M. nudus links to their responses to conspecific alarm cues
An acrylic device with the raceways (70 × 6 × 5 cm) was designed for the measurement of foraging behavior of sea urchins. Experiment 3 included three trials.
In the first trial, we measured the foraging ability of sea urchins. Ten grams of the kelp was placed on one side of the tank and one sea urchin was placed on the other side of the tank (Fig. 4D). Foraging behavior was recorded for 20 min using a digital video recorder (FDR-AXP55, SONY, Japan). Movement and velocity of sea urchins were calculated using the software ImageJ (version 1.51 n). The experiment was individually repeated 31 times using different sea urchins (N = 31). At the end of this foraging experiment, 31 sea urchins were recorded in individual cylindrical plastic cages in the tank. All the sea urchins were subsequently measured for the second and third trials (N = 31). To avoid potential fatigue influences, each behavioral experiment was carried out after another 24 h.
The second trial was carried out to test the hypothesis that sea urchins with strong foraging ability are affected when conspecific alarm cues appear on the way to the kelp. A piece of wild fresh kelp (~ 10 g) was placed on the left side of the device (70 × 6 × 5 cm), while 5 mL of conspecific alarm cues were put in the middle of the device. The individual was placed on the right of the device (Fig. 4E). Foraging behavior of sea urchins was recorded for 20 min using a digital video recorder (FDR-AXP55, SONY, Japan). The danger area (g area) refers to the 15 cm position away from conspecific alarm cues. The time of the 31 sea urchins spent in the g area was individually calculated.
We investigated whether different amounts of conspecific alarm cues around the kelp affected the sea urchins with strong foraging ability in the third trial. More conspecific alarm cues (5 mL) and a piece of wild fresh kelp (~ 10 g) were placed on the left side of the device (70 × 6 × 5 cm), while less conspecific alarm cues (0.5 mL) and wild fresh kelp (~ 10 g) were placed on the right side of the device (Fig. 4F). Sea urchins were individually placed in the middle of the device. Foraging behavior of sea urchins was recorded for 20 min using a digital video recorder (FDR-AXP55, SONY, Japan). The number of sea urchins that successfully foraged to areas with more and less conspecific alarm cues was recorded. The area h refers to the safety area that was 15 cm position away from the sea urchin, which was close to the side with more conspecific alarm cues. The area of i refers to the safety area that was 15 cm position away from the sea urchin, which was close to the side with less conspecifics alarm cues. The time spent in the h and i areas was individually calculated in 31 sea urchins.
Statistical analysis
Normal distribution and homogeneity of variance of the data were analyzed using the Kolmogorov–Smirnov test and Levene test, respectively. Independent-sample t-test was carried out to compare the differences of duration in danger (b and d) and escape (a and c) areas in experiment 1. One-way ANOVA was used to analyze the duration in danger (e) and escape (f) areas of the sea urchins fasted for 7, 14 and 21 days. Pairwise multiple comparisons were carried out using LSD test when significant differences were found in the ANOVAs. Correlations between foraging velocity and duration in the g, h and i areas were analyzed using Pearson correlation analysis. A probability level of P < 0.05 was considered significant. All data analyses were performed using SPSS 21.0 statistical software. Graphs 2−4 were performed using Origin 9.0 software (OriginLab, USA).
Source: Ecology - nature.com
