2.3.2. Growth Performance and Yield of Crabs

The megalopae of crabs were collected and weighed to ensure all enclosures contained the same number of crabs (160/g) on 25 May. The megalopae were cultured in the enclosures for 46 days until they reached the feeding phase. The experiment was initiated after the crabs were measured. The average body length, width and height was 1.08 ± 0.08, 1.16 ± 0.09 and 0.53 ± 0.05 cm, respectively, and average body weight was 0.63 ± 0.13 g. During the experiment, the body length, width, height, and weight of the crabs were measured on 10 July, 28 July, 17 August, 8 September, and 8 October. All crabs were humanely harvested at the end of the experiment. Crabs were caught using a plastic bucket inserted into a hole dug in the bottom of the enclosure. The frequency of collection depended on the number of crabs. All specimens were counted, measured, and weighed. Precocious puberty was assessed by comparing the abdomen, junction, villi, gonad, color, and crab patterns with those of the representative crab specimens [12]. Growth performance indicators were calculated using the following formulae:

Survival rate/% = nt/n0 × 100%,

Weight gain rate/% = (mt − m0)/m0 × 100%,

Specific growth rate/%/d = (ln mt − ln m0)/t × 100%,

Total output/g·m−<sup>2</sup> = W/S,

Net output/g·m−<sup>2</sup> = (W <sup>−</sup> W0)/S,

where n0 represents the initial number of crabs, nt represents the final number of crabs, mt represents the final average body weight, m0 represents the initial average body weight, t represents the total number of days of the experiment, W represents the final total weight of crabs in an enclosure, W0 represents the initial total weight of crabs in an enclosure, and S represents the area of the enclosure (6 m × 6.7 m = 40.2 m2).

#### 2.3.3. Qualitative and Quantitative Analysis of Phytoplankton

The sampling and measurement methods used to assess the phytoplankton were based on those of Zhang [6]. Briefly, 1 L of water was collected by five-point sampling at each point and mixed in a bucket. A lugol solution (10–15 mL) was then evenly mixed into the water. After 48 h, the sample was concentrated by siphonage, fixed at 100 mL volume, and then put into an iodometric bottle for qualitative analysis. The qualitative and quantitative analyses followed Li et al. [13], and Zhao [14], respectively. The specific gravity of phytoplankton is approximately 1. Therefore, the volume was directly converted into wet weight, and the phytoplankton biomass was calculated (Table A2).

## 2.3.4. Qualitative and Quantitative Analyses of Zooplankton

The sampling and measurement of the zooplankton were based on methods of Zhang [6]. The qualitative and quantitative methods followed those described in Section 2.3.3, and the zooplankton biomass was calculated (Table A3).

#### 2.3.5. Qualitative and Quantitative Analyses of Aquatic Vascular Plants

The aquatic vascular plants were sampled by selecting two points that were consistent for each enclosure. A 30 cm × 30 cm iron frame was used to divide the sampling area. The plants (except rice) were uprooted, species were identified, and plant wet weight was determined.

#### 2.3.6. Qualitative and Quantitative Analysis of Benthic Animals

The quantification of the benthic animals was conducted at the same sampling points as those mentioned in Section 2.3.5 at a depth of approximately 10 cm using a self-made barrel dredger [15]. The benthic animals were screened using a sieve with an aperture of 0.2–2 mm and then wet-weighed, identified, and counted with precision.

### *2.4. Statistical Analysis*

The experimental data were collated using Excel. The homogeneity of variance test and one-way ANOVA were performed using SPSS 24.0. Any significant differences between groups were further analyzed using Duncan's multiple comparison tests. The results were expressed as the mean ± standard deviation. In all analyses, a probability value less than 0.05 was considered significant (*p* < 0.05).

Dominance (Y) was calculated according to the formula:

$$\mathbf{Y} = m\mathbf{i}/\mathbf{N} \times f\mathbf{i}$$

where Y is the degree of dominance, *ni* is the number of individuals of species *i*, N is the total number of individuals, and *fi* is the frequency of occurrence of species *i* at five sampling points within an enclosure.

The Shannon–Wiener diversity index (H ) was calculated using the formula:

$$\mathbf{H}' = -\sum [(\mathbf{n}i/\mathbf{N}) \times \ln(\mathbf{n}i/\mathbf{N})]\_{\prime\prime}$$

where *ni* is the number of individuals of species *i*, and N is the total number of individuals of the species.

#### **3. Results**

#### *3.1. Growth Performance and Yield of Crabs*

The morphological parameters of the crabs in the high- and low-protein diet groups varied significantly at each measurement (Figure 2). At the end of the experiment, the carapace length, width, and height of both T30 and T45 groups were significantly higher than those of the Co group (*p* < 0.05), and the carapace length and width were significantly higher than those in the T15 group (*p* < 0.05). The final body weight and weight gain rate of the crabs in the T45 group were significantly higher than those of the Co group (*p* < 0.05). The growth rate of the crabs in the Co group was significantly lower than that of the T30 and T45 groups (*p* < 0.05).

**Figure 2.** Comparison of morphological parameters of crabs among different protein-content groups. I, the initial size in the beginning of experiment; F, the final size at end of experiment; CL, carapace

length; CW, carapace width; CH, carapace height. Different letters indicate the significant difference among treatment groups (*p* < 0.05).

The final body weight of the crabs ranged from 8.30 g to 17.28 g (Table 2). The final body weight of the crabs that were fed diets increased significantly with the increase of protein content (*p* < 0.05). The body weight increase rate varied from 9641.86% to 20,181.73% and significantly increased with the increase in dietary protein content (*p* < 0.05). The specific growth rate of the crabs varied from 3.30%/d to 3.85%/d and significantly increased as the dietary protein content increased (*p* < 0.05).

**Table 2.** Effects of different dietary protein content levels on the growth performance and yield (*n* = 3; *x* ± SD) of juvenile Chinese mitten crabs.


Note: Values in each row with different superscripts are significantly different (*p* < 0.05).

The total and net yields of crabs varied from 43.86 g/m<sup>2</sup> to 60.41 g/m2 and 38.10 g/m<sup>2</sup> to 54.64 g/m2, respectively. The highest total and net yields were for crabs in the T45 group, followed by those of the T15 and Co groups; the lowest was for those of the T30 group. There was no significant variation in the total and net yield of crabs in the different experimental groups (*p* > 0.05). Towards the end of the experiment, approximately 10% of crabs in the T45 group experienced precocious puberty.
