*4.3. Feeding Behavior and Body Size of S. intermedius*

Sea urchin seeds (>10 mm of test diameter) are further used for longline culture and stock enhancement [12]. It takes about three months for juvenile *S*. *intermedius* (3–4 mm of test diameter) to become qualified seeds (10–20 mm of test diameter) by feeding fresh kelp in land-based nursery tanks [10]. The slow growth of *S*. *intermedius* along with the high cost of diets greatly hinders the development of sea urchin aquaculture. The present study found that group M showed a significantly larger body size compared with those in group U after 34 days, suggesting that the IMTA system displays a great potential for seed production (>10 mm of test diameter) in large quantities. A significantly higher rate of Aristotle's lantern reflex was consistently found in group M than that in group U in this study. Aristotle's lantern reflex, which refers to the process of grasping and jawing food using the teeth [44], is commonly used to represent the ability of food consumption in sea urchins [27]. For example, Hu et al. [24] found that superior Aristotle's lantern reflex led to significantly higher food consumption in sea urchins. Thus, it is probable that the improved feeding behavior of *S*. *intermedius* contributed to the better utilization of the powdered diets in this new IMTA system.

Furthermore, although fresh kelp can be used for *S*. *intermedius* culture, this approach is inefficient and not environmentally sustainable [45], because it displays a low level of energy protein and varies seasonally in nutrients [46,47]. Formulated feed (e.g., sea cucumber diet), which can be supplied stably with high nutrients [48,49], greatly improved somatic growth during the juvenile stages of *S*. *intermedius* in this study. Most importantly, *S*. *intermedius* can utilize wasted sea cucumber diets deposited in the bottom of tanks. Thus, there is no need for additional supplementation of fresh kelp in the culture period. This greatly decreases the cost in *S*. *intermedius* aquaculture.

#### *4.4. Survival of S. intermedius*

The present study found that mass mortality and morbidity of *S*. *intermedius* occurred in both groups M and U. However, group M showed significantly lower mortality and morbidity compared to those in group U. Mass mortality and morbidity of sea urchins are consistent with Lawrence et al. [10], in which 80–90% of *S*. *intermedius* died due to the increasing water temperature. Black-mouth disease, which is caused by opportunistic bacteria, such as the genus *Bacillus firmus* [50], is one of the most serious diseases threatening the survival of *S*. *intermedius* in aquaculture [24]. The optimum temperature for the growth of both the pathogenic bacteria *B*. *firmus* and sea urchins *S*. *intermedius* is ~15 ◦C [12,50], which consequently results in the disease outbreak during the production season of sea urchins. Our previous study documented that eliminating interactions in

multi-layer cultures greatly improved the survival after disease challenge assays in cultured *S*. *intermedius* [22]. However, it remains unclear whether this approach is applicable in the IMTA system. Here, the present results indicate that segregation in multi-layer culture is essential for *S*. *intermedius* culture in the IMTA system. It greatly contributes to improving the survival of *S*. *intermedius* when disease outbreaks, although the potential differences were not evaluated in pathogenic bacteria between groups in the present study.

In addition, there were various seaweeds in the powdered diets of sea cucumbers [17]. Dietary supplementation with seaweed promotes the activities of immunocytes and immunologic factors [51], because seaweeds have antiviral and antimicrobial activities [52,53]. For example, dietary supplementation with seaweeds *Sargassum whitti* and *Ulva prolifera* facilitates the levels of lysozyme in fishes *Mugil cephalus* and *Scophthalmus maximus*, respectively [54,55]. Therefore, another explanation is that sea urchins being fed a sea cucumber diet is probably beneficial to their survival in the new IMTA, which further highlights the superiority of this new system.

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

**Funding:** This research was funded by the National Key Research and Development Program of China (2018YFD0901604), a research project for marine economy development in Liaoning province (for Jun Ding), the National Natural Science Foundation of China (41506177), a grant Chinese Outstanding Talents in Agricultural Sciences (for Yaqing Chang) and a grant for innovative talents in universities in Liaoning Province (for Chong Zhao).

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

**Informed Consent Statement:** Not applicable.

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

**Acknowledgments:** We thank John Lawrence, Yaqing Chang and Konrad Wojnarowski for academic and editorial suggestions, and thank Mingfang Yang, Xiaomei Chi, Yongchao Li, Peng Ding, Yihai Qiao and Xiang Li for their assistance.

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