*2.7. Adhesion Behavior*

Movement and ingestion of sea cucumbers greatly relies on the adhesion of mouth tentacles and tube feet. Adhesion behavior was measured according to Tian et al. [11]. Eighteen sea cucumbers from each group were randomly selected and placed in cubic devices (length × width × height: 180 mm × 140 mm × 45 mm; Figure 1E) with eighteen compartments. Each sea cucumber was placed in one compartment. Seawater was added to the devices until the seawater level reached a height of 2 cm. The devices were slowly turned to 180◦ after 10 min. We recorded the time that sea cucumbers fell from the top of the devices, since their adhesion abilities were disabled to support their body weight. The adhesion time was recorded as 600 s if sea cucumbers still adhered to the top of the box after 10 min. Wet body weight (g) of each sea cucumber was measured by using an electronic balance (G & G Co., San Diego, CA, USA) after the recording (*n* = 6). Data are accurate to one decimal place. Adhesion index (*Ai*) was formulated, as described by Tian et al. [11]:

$$A\_i = \frac{T}{W}$$

where *Ai* is the adhesion index, *T* is the adhesion time (s), and *W* is the wet weight of an individual sea cucumber (g).

#### *2.8. Statistical Analysis*

All data were subjected to the analysis of variance distribution and homogeneity of variance using the Kolmogorov–Smirnov test and the Levene test, respectively. The mortality and morbidity of experiments I and II were non-normally distributed and/or heterogeneity of variance. Thus, the data were compared by using the Mann–Whitney *U* test. Crawling frequency, tentacle activity frequency, and the adhesion index were analyzed by using Kruskal–Wallis *H* in both experiments. All statistical analyses were performed using SPSS 22.0 statistical software. The level of significance was considered as *p* < 0.05.

### **3. Results**

#### *3.1. Experiment I*

#### 3.1.1. Mortality and Morbidity

Mortality was significantly higher in group C (43.06 ± 2.56%) than that in group O (5.56 ± 4.12%; Mann–Whitney *U* = 2.961, *p* = 0.003; Figure 2A). Consistently, group C showed significantly higher morbidity (48.61 ± 1.39%) than group O (6.94 ± 3.98%; Mann– Whitney *U* = 3.017, *p* = 0.003; Figure 2B).

**Figure 2.** Mortality (**A**) and morbidity (**B**) of sea cucumbers between groups C and O. The asterisks \*\* mean *p* < 0.01 (mean ± SE, *n* = 6). Group C: sea cucumbers without artificial reefs; Group O: sea cucumbers with artificial reefs.

#### 3.1.2. Crawling Frequency

There was no significant difference in crawling frequency between sea cucumbers of group C (4.44 ± 0.88 times) and individuals outside the artificial reef of group O (3.22 ± 0.50 times; Kruskal–Wallis *H* = 14.332, *p* = 1.000; Figure 3A). Crawling frequency was significantly higher in sea cucumbers inside the artificial reefs of group O (10.83 ± 1.52 times) than that in sea cucumbers outside the artificial reefs of group O (Kruskal–Wallis *H* = 14.332, *p* = 0.001) and group C (Kruskal–Wallis *H* = 14.332, *p* = 0.008; Figure 3A).

**Figure 3.** Crawling frequency (**A**), tentacles activity frequency (**B**) and adhesion behavior (**C**) of sea cucumbers. The asterisks \*, \*\*, \*\*\* mean *p* < 0.05, *p* < 0.01 and *p* < 0.001, respectively (mean ± SE, *n* = 6). Group C: sea cucumbers without artificial reefs in group C; Group O-Out: sea cucumbers outside the artificial reefs in group O; Group O-In: sea cucumbers inside the artificial reefs in group O.

#### 3.1.3. Feeding Behavior

There was no significant difference in tentacle activity frequency between sea cucumbers of group C (10.44 ± 3.51 times) and individuals outside the artificial reefs of group O (8.50 ± 1.87 times; Kruskal–Wallis *H* = 7.705, *p* = 1.000; Figure 3B). Consistently, tentacle activity frequency was not significantly different between sea cucumbers outside (8.50 ± 1.87 times) and inside (18.72 ± 3.26 times) the artificial reefs (Kruskal–Wallis *H* = 7.705, *p* = 0.067; Figure 3B). Tentacle activity frequency was significantly higher in sea cucumbers inside the artificial reefs in group O than that in individuals of group C (Kruskal–Wallis *H* = 7.705, *p* = 0.036; Figure 3B).

#### 3.1.4. Adhesion Behavior

The adhesion index was not significantly different between sea cucumbers outside the artificial reefs of group O (16.66 ± 5.50) and individuals of group C (9.75 ± 6.28; Kruskal– Wallis *H* = 0.561, *p* = 0.065; Figure 3C). Consistently, the adhesion index was not significantly different in sea cucumbers outside and inside the artificial reefs (283.62 ± 61.4) in group O (Kruskal–Wallis *H* = 0.561, *p* = 0.076; Figure 3C). However, sea cucumbers inside the artificial reefs of group O showed significantly higher adhesion index than individuals of group C (Kruskal–Wallis *H* = 0.561, *p* < 0.001; Figure 3C).
