*2.3. Mortality and Morbidity*

Black-mouth disease, which is characterized by the blackened peristomial membrane (Figure 1E), is one of the most serious diseases in *S*. *intermedius* aquaculture [24]. The performance of sea urchins without disease is shown in Figure 1E. Sea cucumbers with skin ulceration syndrome are indicated by the white spots on the integument, and these spots quickly fill the whole integument and consequently leads to death [25] (Figure 1F). The performance of sea cucumbers without disease is shown in Figure 1F. Mortality and morbidity were evaluated at the end of the experiment.

**Figure 1.** Experimental facilities used for group C (**A**), group U (**B**), and group M (**C**). Group C: the control group for sea cucumbers. Group U: the control group for sea urchins. Group M: the integrated multi-trophic aquaculture of sea cucumbers and sea urchins. A dismountable plastic box divided into three layers and six compartments in each layer was used to culture sea urchins in group M (**D**). Sea urchins with the performance of black-mouth disease and without the disease performance (**E**). Sea cucumbers with the performance of skin ulceration syndrome and without the disease performance (**F**). Red circles indicate the ulcerative skin in sea cucumbers (**F**). Aristotle's lantern reflex of sea urchin (**G**). Feeding behavior of sea cucumbers refers to the process, by which the tentacles reach the food sediments and deliver them to the mouth (**H**). Crawling behavior indicates the movement ability of sea cucumbers, and it was divided into five stages (**I**).

#### *2.4. Growth*

Final growth traits were assessed at the end of the experiment. Sea cucumbers (both inside and outside nets) from groups C and M were randomly selected for the subsequent measurement. Sea urchins of group M were randomly selected from ten haphazardly chosen compartments for the following measurement [22]. Test diameter and lantern length of *S*. *intermedius* were measured using a digital vernier caliper (Mahr Co., Ruhr, Germany). Body and lantern were weighted wet by an electric balance (G & G Co., San Diego, CA, USA). To evaluate the growth traits of *A*. *japonicus*, they were randomly selected and placed in a small tank (length × width × height: 227 × 157 × 61 mm) filled with fresh seawater according to Broeke et al. [26]. The software ImageJ 1.51 n was used to measure their naturally relaxed body length as a polygonal line after being photographed

by a digital video (Canon Co., Shenzhen, China). Wet body weight was assessed using an electric balance (G & G Co., San Diego, CA, USA). The average of all the ten animals was considered as one value for each of the six replicates (*n* = 6).

### *2.5. Aristotle's Lantern Reflex of S. intermedius*

Aristotle's lantern reflex is defined as one cycle of the teeth from opening to closing, indicating the ability to obtain food in *S*. *intermedius* [24]. A simple device with three compartments (length × width × height: 30 × 20 × 18 mm for each compartment) with agar film (2 g kelp powder with 3 g agar powder for feeding of sea urchins) at the bottom was used to assess Aristotle's lantern reflex according to Ding et al. [27] (Figure 1G). Five *S*. *intermedius* were randomly selected from each group and put into the experimental device at the end of the rearing experiment. The number of Aristotle's lantern reflex within 10 min were counted using a digital camera (Canon Co., Shenzhen, China). The average of all the five *S*. *intermedius* was considered as one value for each of the six replicates (*n* = 6).

#### *2.6. Feeding and Crawling Behaviors of A. japonicus*

Feeding behavior, which is closely related to the food intake of *A*. *japonicus* [28], was evaluated according to Sun et al. [29]. It refers to the process by which the tentacles reach the food sediments and deliver them to the mouth in *A*. *japonicus* [29] (Figure 1H). Completing one cycle was recorded as one tentacle activity frequency in this study.

Crawling behavior indicates the movement ability of *A*. *japonicus* [30,31]. Crawling behavior includes the processes below according to Lin [32]: (1) sea cucumbers remain at the quiescent condition; (2) they contract from the anus to the back; (3) the contraction gradually transits to the ostium; (4) the contraction subsides, and sea cucumbers remain at the quiescent condition (Figure 1I). Completing one cycle was recorded as one crawling frequency in this study.

Four *A*. *japonicus* of each replicate were randomly selected and individually put into experimental tanks (length × width × height: 70 × 60 × 60 mm) filled with a commercial powdered diet (Anyuan Industrial Co., Yantai, China) at the end of the experiment. Tentacle activity frequency and crawling frequency were recorded within one hour using a digital camera (Canon Co., Shenzhen, China). The average of all the four *A*. *japonicus* was considered as one value for each of the six replicates (*n* = 6)

#### *2.7. Statistical Analysis*

The normal distribution and homogeneity of variance were analyzed using the Kolmogorov–Smirnov test and Levene test, respectively. Body weight and Aristotle's lantern reflex of *S*. *intermedius*, body weight and tentacles activity frequency of *A*. *japonicus* were analyzed using the Mann–Whitney *U* test, because of the non-normal distribution and/or heterogeneity of variance. Differences in the rest traits between groups were compared using the independent sample *t*-test. The replicate means were calculated for all variables. All statistical analyses were performed using SPSS 19.0 statistical software. *p* < 0.05 was considered statistically significant.
