*2.2. Experimental Design*

There were three groups in this study: the control group for *A*. *japonicus* (group C, Figure 1A), the control group for *S*. *intermedius* (group U, Figure 1B) and the integrated multi-trophic aquaculture of *A*. *japonicus* and *S*. *intermedius* (group M, Figure 1C). The stocking density of sea urchins and sea cucumbers in group M was consistent with those in groups U and C, respectively. In group C, three pieces of polyethylene nets (length × width: 100 ×100 mm for each net; 1 mm diameter of mesh size) were tied as substrate under a plastic ball (to float those nets) in a cylindrical plastic bucket (diameter × height: 220 × 280 mm; 37 *A*. *japonicus*/10,638 cm<sup>3</sup> of stocking density). Thirty-seven *A*. *japonicus* were randomly chosen and placed in the plastic bucket according to the culture management commonly used for the seed production of sea cucumbers. In group U, 90 juvenile *S*. *intermedius* (~7 mm of test diameter) were randomly selected and maintained in the plastic cage (length × width × height: 150 × 150 × 130 mm, 3 mm diameter of mesh size) in a cylindrical plastic bucket (diameter × height: 220 × 280 mm; 90 *<sup>S</sup>*. *intermedius*/10,638 cm3 of stocking density) according to the culture practice for the seed production of sea urchins. In group M, culture areas existed for *A*. *japonicus* on top and *S*. *intermedius* at the bottom in the plastic bucket (diameter × height: 220 × 280 mm; 90 *S*. *intermedius* and 37 *A*. *japonicus* /10,638 cm<sup>3</sup> of stocking density). A dismountable plastic box (length × width × height: <sup>150</sup> × <sup>150</sup> × 130 mm, 30 holes of 4 mm diameter/100 cm2), divided into three layers and six compartments in each layer (Figure 1D), was put into the culture areas of *S*. *intermedius*. Five *S*. *intermedius* were randomly selected and put into each compartment of the plastic box (90 *S*. *intermedius* in total for each box).

Each group had 6 replicates under ~30 lx of incandescent light intensity with the photoperiod (12 h light: 12 h dark) in this study from 24 April 2021 to 28 May 2021. The leaf blade of wild fresh kelp *S. japonica* collected from Heishijiao, Dalian (121◦58 E, 38◦87 N) was provided ad libitum to *S*. *intermedius* in group U. Animals in groups C and M were fed a commercial powdered diet (Anyuan Industrial Co., Yantai, China). The feces and dead individuals were removed daily for all the groups. Water temperature was not controlled, ranging from 14.3 to 16.6 ◦C (ambient temperatures). Salinity was 30.7 ± 0.7 ‰, according to the weekly measurement by a portable water quality monitoring meter (Xylem Co., Yellow Springs, OH, USA). All the experimental tanks were kept in still water with aeration, because this does not cause the loss of powdered diet. Two-thirds of the seawater was renewed daily to avoid deterioration of water quality.
