3.2.1. Mortality and Morbidity

Mortality was significantly higher in group C (30.56 ± 3.51%) than that in group O (4.17 ± 2.84%; Mann–Whitney *U* =2.879 *p* = 0.004; Figure 4A). Morbidity was significantly higher in group C (43.06 ± 2.56%) than that in group O (6.94 ± 4.52%; Mann–Whitney *U* =2.961, *p* = 0.003; Figure 4B).

**Figure 4.** 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.

#### 3.2.2. Crawling Frequency

There was no significant difference in crawling frequency between sea cucumbers of group C (1.67 ± 0.35 times) and individuals outside the artificial reefs in group O (2.56 ± 0.56 times; Kruskal–Wallis *H* = 6.908, *p* = 0.762; Figure 5A). Consistently, crawling frequencies of sea cucumbers outside (2.56 ± 0.56 times) and inside (3.67 ± 0.60 times) the artificial reefs in group O (Kruskal–Wallis *H* = 6.908, *p* = 0.417; Figure 5A) were not significantly different in the experiments. Significantly higher crawling frequency occurred in sea cucumbers inside the artificial reefs in group O than individuals in group C (Kruskal– Wallis *H* = 6.908, *p* = 0.026; Figure 5A).

**Figure 5.** Crawling frequency (**A**), tentacle activity frequency (**B**), and adhesion behavior (**C**) of sea cucumbers. The asterisks \*, \*\* mean *p* < 0.05 and *p* < 0.01, respectively (mean ± SE, *n* = 6). Group C: sea cucumbers without the 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.

#### 3.2.3. Feeding Behavior

There was no significant difference in feeding behavior of sea cucumbers cultured inside artificial reefs of group O, outside artificial reefs of group O, and in group C (*p* > 0.05; Figure 5B).

#### 3.2.4. Adhesion Behavior

The adhesion index was significantly higher in sea cucumbers inside the artificial reefs of group O (206.44 ± 47.12) than that in the individuals of group C (11.41 ± 5.37; Kruskal–Wallis *H* = 13.007, *p* = 0.001; Figure 5C). The adhesion index of sea cucumbers of group C (11.41 ± 5.37) was not significantly different from that of those outside the artificial reefs in group O (105.28 ± 28.26) (Kruskal–Wallis *H* = 13.007, *p* = 0.053; Figure 5C). Consistently, no significant difference was found in the adhesion indexes between sea cucumbers outside and inside the artificial reefs in group O (Kruskal–Wallis *H* = 13.007, *p* = 0.732; Figure 5C).

#### **4. Discussion**

The survival of sea cucumbers is constrained by high temperatures. A temperature of 25 ◦C is the threshold for severe heat stress in sea cucumbers, leading to a significantly increased risk of death and disease in sea cucumbers [6]. Air exposure is unavoidable in seed production, which not only causes mechanical damage [27] but also disrupts the immune system of sea cucumbers [9]. Furthermore, our previous study found that the negative effects of air exposure and high temperature on sea cucumbers were synergistic [11]. This present study found that artificial reefs significantly reduced mortality and morbidity of sea cucumbers exposed to the air at 25 ◦C. Specifically, in such conditions, the morbidity and mortality of sea cucumbers not cultured in artificial reefs were 7-fold and 8-fold the morbidity and mortality of those cultured in artificial reefs, respectively. This suggests that artificial reefs are an effective approach to improving the survival of sea cucumbers under the combined stress of high temperature and air exposure. Fitness-related behavior is a common indicator for evaluating the condition of sea cucumbers. The typical characteristics of sea cucumbers in poor conditions are anorexia, decreased adhesion capacity, and poor movements ability [17]. Tian et al. [11] found that artificial reefs improved fitness-related behaviors of sea cucumbers after they were exposed to high temperatures and the air. However, it is unknown how artificial reefs improve fitness-related behaviors. This present study found that sea cucumbers cultured inside the artificial reefs had significantly better feeding, crawling, and adhesion behaviors than those cultured without artificial reefs. Sea cucumbers exposed to the air and/or high temperatures reduce feeding or even stop feeding behavior [11,27,28]. Sea cucumbers cultured inside artificial reefs may acquire more energy by frequent feeding [28] and, consequently, may better cope with combined stressors. The adhesion of the tube feet of sea cucumbers affects the subsequent movement such as crawling behavior [29]. It is important to note that the fitness-related behaviors (feeding, crawling, and adhesion behaviors) of sea cucumbers cultured outside artificial reefs are not significantly different from those of sea cucumbers without artificial reefs. This suggests that the condition of the sea cucumbers inside the artificial reefs is better under the combined stressors. Therefore, artificial reef improves the survival and fitness-related behaviors of sea cucumbers after they are exposed to high temperatures and the air, thereby increasing the survival rate.

Skin ulcer syndrome is one of the common diseases with high infectivity and high mortality in seed production of sea cucumbers [17]. High temperatures reduce the immunity and disease resistance of sea cucumbers [30,31] and, thus, greatly increases the possibility of infection. The present study found that artificial reefs significantly reduced the mortality and morbidity of sea cucumbers during disease outbreaks at high temperatures. Specifically, during disease outbreaks at high temperatures, the morbidity and mortality of sea cucumbers not cultured in artificial reefs were 6-fold and 7-fold the morbidity and mortality of those cultured in artificial reefs. This suggests that artificial reefs improve the survival in disease environments and probably affect the spread of skin ulcer syndrome. In disease challenge assays, sea cucumbers cultured inside artificial reefs had significantly better adhesion and crawling behaviors, while there was no significant difference in the fitness-related behaviors between the sea cucumbers cultured outside and without artificial reefs. Decreased adhesion and movement ability are the symptoms of skin ulcer

syndrome [17]. Hence, we speculated that sea cucumbers cultured outside and without artificial reefs were more affected by diseased sea cucumbers, although their skins were not ulcerated. Chemosensory cues are important for a variety of fundamental behavioral processes [32,33]. Lobsters use chemical cues from diseased individuals to determine whether the shelter is safe, and healthy lobsters rarely share the shelters with diseased lobsters [34]. Diseased sea cucumbers emit certain chemical cues for warning [33,35]. Sea cucumbers show escaping behavior after receiving alarm cues from diseased individuals [35,36]. Artificial reefs are possible places for escape due to their attraction to sea cucumbers [20,37]. Frequent physical contact with diseased individuals increases the probability of disease transmission [19]. Diseased sea cucumbers probably release chemical cues that promote the escape of individuals with strong movement to the crevices of artificial reefs. In the limited space, sea cucumbers that did not enter the reef inevitably contacted with diseased individuals, which increased the extent to which they were affected. In the present study, each crevice is accessible to only one sea cucumber. Sea cucumbers cultured with artificial reefs probably showed less contact with diseased and affected individuals after staying in porous artificial reefs, which reduced the extent of the affection. Seed production is an intensive aquaculture mode, and isolation of diseased sea cucumbers is expensive. Therefore, the artificial reef is a cost-effective approach to increasing the possibility of the survival of sea cucumbers by reducing the frequency of contact with diseased and affected individuals in seed production. Notably, our experiment is based on a laboratory experiment. Therefore, conducting a further field experiment is necessary.

In conclusion, we encourage aqua-farmers to use artificial reefs to decrease disease transmission and, thus, reduce the mortality and morbidity of small sea cucumbers in seed production at high temperatures. The present novel finding provides valuable insights into the improved management for the seed production of sea cucumbers in summer.

**Author Contributions:** Conceptualization, H.W., C.Z. and Y.S.; methodology, H.W., G.W., J.D. and C.Z.; software, H.W. and G.W.; validation, H.W., G.W., F.H. and R.T.; formal analysis, H.W., G.W. and F.H.; investigation, H.W., G.W. and R.T.; data curation, H.W. and G.W.; writing—original draft preparation, H.W. and G.W.; writing—review and editing, C.Z. and Y.S.; visualization, H.W.; supervision, C.Z., Y.S., J.D. and Y.C.; project administration, C.Z. and Y.S.; funding acquisition, C.Z., J.D., Y.S. and Y.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of Liaoning Province (2022-MS-352), High-level talent support grant for innovation in Dalian (2020RD03), Modern Fisheries Technology Mission in Changhai, Liaoning (2022JH5/10400015) and Liaoning Province "Xingliao Talents Plan" project (XLYC2002107).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study is available on request from the corresponding authors.

**Acknowledgments:** We appreciate Wei Tang for her editorial suggestions. We thank Mingfang Yang, Xiaomei Chi, Yushi Yu, and Xiang Li for their assistance in this experiment.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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