**1. Introduction**

The Chinese mitten crab (*Eriocheir sinensis*) is the most commonly farmed crab species in China. In 2020, the General Office of the Ministry of Agriculture and Rural Affairs proposed the implementation of "five major actions," including the promotion of ecological and healthy farming modes. The new rice–crab co-culture mode integrates the culturing of rice and crabs with ecological, economical, and social benefits [1]. The food chain in the ecosystem of this mode is quite complex, creating a more stable ecosystem than that in single-species aquaculture. Crabs are at the top of this food chain and feed on plankton, weeds, and benthic animals in rice fields, ensuring an efficient matter circulation and a smooth energy flow through the whole system [2]. Furthermore, this mode produces a double harvest of rice and crabs [3].

The ecological environment in rice–crab co-culture may be affected by several factors. For instance, high-density culture adversely affects phytoplankton and benthic animals [4].

**Citation:** Yu, Y.; Wan, J.; Liang, X.; Wang, Y.; Liu, X.; Mei, J.; Sun, N.; Li, X. Effects of Protein Level on the Production and Growth Performance of Juvenile Chinese Mitten Crab (*Eriocheir sinensis*) and Environmental Parameters in Paddy Fields. *Water* **2022**, *14*, 1941. https://doi.org/ 10.3390/w14121941

Academic Editors: Abasiofiok Mark Ibekwe and Thomas Hein

Received: 2 April 2022 Accepted: 14 June 2022 Published: 16 June 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Zhang et al. [5] demonstrated that the phytoplankton biomass in crab culture was significantly higher than that in rice culture or rice co-culture. The daily activities of crabs change the physical and chemical environment of water bodies, which indirectly affects the plankton community structure [6]. These studies showed that the feeding behavior of crabs on plankton and benthic animals influenced the rice–crab co-culture ecosystem.

The quality and output of the mode are not the only important factors; it is also necessary to minimize expenses. Feed costs are the main expense in aquaculture. Almost all crab artificial feeds use fish meal (FM) as the main protein source [2]. However, there is a limited supply of FM, and it is costly [7], necessitating the use of expensive compound feeds. The optimum crude protein for the growth of juvenile crabs is 347.8 g/kg under the indoor individual Chinese mitten crab system [8]. Xu et al. found that a certain amount of fish meal replaced by soybean meal effectively reduced the cost of feed and had no effect on the growth performance, related enzyme activities and genes expression of Chinese mitten crabs [9]. These experiments showed that the protein in the feed played a key role in the growth performance of crabs and cost of feed.

After more than 30 years of development, China's rice–crab co-culture area comprised approximately 1.386 × 105 hm2 in 2019, which accounted for 5.94% of the national rice and fishery planting area and produced a yield of 6.18 × 104 tons [10]. However, although paddy fields are rich in natural nutrients in rice–crab co-culture systems, there is a lack of details on the nutritional requirements, feed costs, and environmental response mechanisms of crabs, especially regarding the interaction between crabs and the ecosystem in paddy fields. In this study, we investigated the effect of three different protein levels in compound feeds on crabs, plankton, aquatic vascular plants, and benthic animals in a rice–crab co-culture system. We aimed to comprehensively analyze the protein requirements of juvenile Chinese mitten crabs under a rice–crab co-culture system. The findings from this research will provide a reference for the optimization of the feeding strategy in the rice–crab co-culture system.

#### **2. Materials and Methods**

#### *2.1. Experimental Animals and Experimental Paddy Field Management*

The experimental animals were obtained from Panjin Photosynthetic Crab Co., Ltd. (Panjin, China) and raised in the nursery pond at their facility. Crabs with complete appendages and of uniform size were randomly selected for our experiment. The experimental paddy field was routinely managed, and a base fertilizer was applied once, prior to rice planting.

#### *2.2. Experimental Design*

The paddy-field crab–culture experiment was conducted in the paddy field at Panjin Photosynthetic Crab Industry Co., Ltd. from 25 May to 8 October 2020 (Figure 1; E 121◦50 38.73–121◦50 41.85, N 40◦54 1.07). Water samples were collected nine times, including a sample in May prior to the initiation of the experiment and to any rice field being stocked with crabs. Subsequently, samples were collected twice a month throughout the experiment. The experimental design involved a total of 12 enclosures (6 m × 6.7 m). The enclosures were divided into three experimental groups and a control group, with three repetitions for each group. Crabs were stocked in each enclosure (the macrophthalmia size was 160/g at 30,000/hm2), including the control group, which was not supplied with any artificial food.

The feed used in the experimental group was designed by the research group with FM and soybean meal as the main protein sources, and fish oil was used as the main fat source (Table 1). Three types of isolipid feeds with different protein contents were formulated by simultaneously increasing the FM and soybean meal content (FM: soybean meal = 2). The feed protein levels were 15%, 30%, and 45%. The various solid raw materials were accurately weighed according to the required formula ratio and, then, were fully mixed according to the principle of step-by-step enlargement. Subsequently, the artificial feed

was pulverized through a 100-mesh sieve, the oil was added, and all ingredients were stirred to an even consistency. Finally, water was added (30%), and the feed was mixed again. A double helix A pellet mill (DES-TS1280, Jinan Dingrun Machinery Equipment Co., Ltd., Jinan, China) was used to press the feed into 3 mm diameter pellets. The pellets were naturally air-dried and packaged and sealed in plastic bags. The bags were stored in a refrigerator at −20 ◦C. Crabs were fed at a rate of 10% of their body weight. A detailed list of contents of each experimental feed is shown in Table 1, and the feed costs are presented in Table A1.

**Figure 1.** Experimental design field of the crab–rice field. Inlet is where the enclosure received water; the outlet is where the enclosure drained water. Co is the control group with no artificial feed supplied, and T15, T30, and T45 represent the treatment groups fed with experimental feeds of 15%, 30%, and 45% protein content, respectively.


**Table 1.** Detailed list of contents of each experimental feed.

#### *2.3. Test Methods*

2.3.1. Monitoring Water Quality in the Paddy Fields

During the experiment, physical and chemical water quality indicators were recorded and monitored in each enclosure. The indicators included temperature (oxygen dissolving instrument, YSI550-A, Vasey Instrument Company, Exton, PA, USA), pH (pH meter, PHB-1, Shanghai Thunder Magnetic, Shanghai, China), salinity (Pen salinity meter, AR-8012, Xima Instrument Co., Ltd., Dongguan, China), dissolved oxygen (oxygen dissolving instrument, YSI550-A, Vasey Instrument Company), ammonium nitrogen (visible spectrophotometer, V-1100, Shanghai Meitong Instrument Co., Ltd., Shanghai, China), and nitrite nitrogen [11].
