*4.5. Effects of Crabs on Aquatic Vascular Plants in Paddy Fields*

Large plants control zooplankton, provide habitats for fish that feed on zooplankton, and provide shelter for phytoplankton [39]. In the early stages of this experiment, the main submerged plant in the paddy field environment was *S. polyrhiza*. The growth of the submerged plants, including *V. spiralis* and *Potamogeton* sp., increased, and the biomass of submerged plants also increased. Shading also increases with the rapid growth of emergent plants [40]. Crabs began feeding on the submerged plants, which reduced their growth until no submerged plants were detected from 15 September. On 7 October, the emergent plants decayed and died, decreasing the biomass.

Farmers have traditionally used chemical weeding machines to remove large weeds from rice fields. Over time, weed resistance and herbicide damage have become increasingly serious problems [41], since crabs are omnivorous and feed on large plants, such as aquatic vascular plants [42]. Even if there is an excess of animal food in the environment, crabs will still consume aquatic plants, especially submerged plants [43]. However, crabs rarely feed on emergent plants, which allows the emergent plants to grow and absorb fertilizer from the crab pond sediment [44]. Lv et al. [45] found that the fresh and dry weights of weeds in the experimental group without crabs being provided artificial feed were significantly lower than those in the crab feeding group. Other studies have also shown that weed control by rice crabs is more effective than traditional weed control methods used in rice production [19,46].

#### *4.6. Changes in Benthic Animals in Rice–Crab Co-Culture*

Benthic animals are the main food source for crabs [47]. Xu et al. [48] found that crabs affect habitat structure in two ways: feeding and reducing competition with aquatic plants by preying on attached organisms, thereby promoting the growth of aquatic plants. At the beginning of the experiment, the local benthic animals were in the culture period, and the biomass showed an upward trend, which is consistent with Li et al. [46]. As crabs grew, the predation of benthic animals by crabs increased, and the biomass of the benthic animals decreased. The stress of predation by crabs caused *Branchiura* sp., *Limnodrilus* sp., and other benthic animals that reproduce via burrowing, to increase in numbers, causing an overall increase in the benthic animal biomass.

In this experiment, the crabs fed with high-protein compound feed were larger and had a stronger predation ability on benthic animals than the representative crabs. On 2 September, the biomass of benthic animals increased with the level of dietary protein. The biomass of benthic animals in the control group was significantly higher than that of the three experimental groups. Xu et al. [48] found that the stocking of crabs reduced the benthic animal diversity in the environment based on the lakes where crabs were stocked, outside the lake enclosure, in natural lake waters, and in lakes where fish were stocked. Benthic species diversity decreased significantly, and the production volume and density decreased by more than 60% compared with those of the control water body. The

results vary from the results in our experiment, which may be caused by the different environments, sampling times, and stocking densities of the crabs. First, lake and paddy field environments are quite different. Second, unlike Xu et al.'s [48] experiment, which took four samples twice a year for two years, we monitored the dynamics of zoobenthos nine times over a period of nearly five months (from May to October). By comparing the culture densities, Xu et al. [48] showed that the over farming of crabs causes the high variations. Conversely, our experiment used normal crab culture density.

#### **5. Conclusions**

In this study, we evaluated how different levels of protein in crab feed could affect the performance of crabs and the environment in rice–crab co-culture in paddy fields. The results showed that feed with 15% protein level compound diet can not only meet the nutritional requirements of crabs but also reduce the cost of cultivation and improve the water quality of the paddy field. The discharged water had low ammonia nitrogen and nitrite content, and no eutrophication was observed. Consequently, the water could be recycled. These findings provide a scientific basis for feed formulation for juvenile crabs in rice–crab co-culture.

**Author Contributions:** Conceptualization, X.L. (Xiaodong Li), J.W. and Y.Y.; data curation, Y.Y.; formal analysis, Y.Y. and X.L. (Xiaochen Liang); funding acquisition, X.L. (Xiaodong Li); investigation, X.L. (Xiaochen Liang), Y.W., X.L. (Xueshen Liu), J.M. and Y.Y.; methodology, N.S. and Y.Y.; project administration, Y.Y. and N.S.; resources, X.L. (Xiaodong Li) and N.S.; supervision, X.L. (Xiaodong Li) and N.S.; validation, X.L. (Xiaodong Li); writing—original draft preparation, Y.Y.; writing—review and editing, X.L. (Xiaodong Li). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Liaoning Province Key R&D Planning Project (2019JH2/10200006), National Key R&D Program of China (2018YFD0901702), Liaoning Province Key R&D Guidance Program (201802120), Shenyang Agricultural University High Level Introduction of talent Program (880416005), and Liaoning Province "The Open Competition Mechanism to Select the Best Candidates" Project, Grant (2021JH1/10400040). The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Institutional Review Board Statement:** Our study did not involve endangered or protected species. In China, breeding and catching Chinese mitten crabs, *Eriocheir sinensis*, in rice fields does not require specific permits. All efforts were made to minimize animal suffering and discomfort. The animal study protocol was approved by the Animal Ethics Committee of Shenyang Agriculture University.

**Data Availability Statement:** The data presented in this study are not publicly available but are available upon request from the corresponding author.

**Acknowledgments:** The author would like to acknowledge my mentor Li for imparting knowledge to me and helping me revise the article carefully. We would also like to thank all employees of the Panjin Guanghe Crab Industry Co., Ltd. for patiently helping us and providing us with an excellent test environment. We thank teacher Hu, who is a teacher and a friend, for helping me to revise the article and overcome many difficulties. We are very grateful to Tian for his suggestions on the revision of the article and teaching us a lot of knowledge. Thanks to Jiang for helping me contact the company to polish the article. Thanks to my employees at the company's R and D center for their experimental help and guidance. The author would also like to acknowledge Zuo and Liu from Dalian Ocean University for their great help with the formula and production of the feed required for the experiment. Thanks also to Liu and Zheng who gave me practical guidance and help with the culture. I would also like to thank MSA Bi who provided guidance on testing techniques. Thanks to my junior Liang for assisting me in data processing and analysis.

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

#### **Appendix A**


**Table A1.** The cost of each experimental feed.

**Table A2.** Phytoplankton in each enclosure with the average wet weight.




**Table A3.** Phytoplankton in each enclosure with the average wet weight.




**Table A4.** Species and biomass of the phytoplankton in each enclosure and grouped by crab diet (*n* = 3).



**Table A4.** *Cont.*

Note: 0.00 mg·L−<sup>1</sup> is provided when the average biomass is less than 0.005 mg·L−<sup>1</sup> or not detected.

**Table A5.** Composition of the predominant species of phytoplankton in the enclosures of each treatment group (*n* = 3).



#### **Table A5.** *Cont.*

**Table A6.** Biomass and species of zooplankton in the enclosures of each treatment group (*n* = 3).




Note: 0.00 mg·L−<sup>1</sup> is the term used when the average biomass is less than 0.005 mg·L−<sup>1</sup> or is not detected.

**Table A7.** Composition of the predominant zooplankton species in the enclosures of each treatment group (*n* = 3).



**Table A7.** *Cont.*

**Table A8.** Species and biomass of benthos in the enclosures of each treatment group (*n* = 3; *x* ± SD).



**Table A8.** *Cont.*

#### **References**

