*3.2. Water Quality*

The physical and chemical parameters of water quality in each enclosure during the experimental period was assessed (Figure 3). Generally, the parameters of water quality in each enclosure were consistent with the fishery water quality standard of the People's Republic of China (GB 11607-89). During the experiment, the water temperature ranged from 24.5 ◦C to 28.2 ◦C, and the salinity from 0.493‰ to 1.743‰. No significant difference was found in dissolved oxygen, ammonia, and nitrate levels in all enclosures (Figure 3).

#### *3.3. Phytoplankton in the Paddy Fields and Variations with Time*

#### 3.3.1. Phytoplankton Biodiversity

A total of 54 species of phytoplankton from seven phyla were detected in four treatments during the experiment (Table A4). Seven phyla were present in all treatments (Figure 4). Bacillariophyta was the dominant group, with 19 species present, and accounted for 35.19% of the phytoplankton species observed. Chlorophyta was the phylum with the second highest number of species (18 species) and accounted for 33.33% of the total number of species. Other groups included Cyanophyta (9 species; 16.67%) and Euglenophyta (5 species; 9.26%). The phyla Cryptophyta, Chrysophyta, and Pyrrophyta were represented by one species each and accounted for 1.85% of the total species.

The rank of phytoplankton biomass of each group, from nine sampling events, was: Chlorophyta (32.48 mg/L, accounting for 26.96% of the total biomass), Eugelenophyta (29.19 mg/L; 24.23%), Chrysophyta (26.85 mg/L; 22.29%), Cyanobacteria (0.26 mg/L; 15.15%), Bacillariophyta (13.68 mg/L; 11.36%), and Cryptophyta (3.06 mg/L; 2.54%). Pyrrophyta was detected only once at a very low proportion.

**Figure 3.** Water quality parameters of each enclosure during the experiment. The *y*-axis of NO2 −-N is the secondary coordinate axis (right), and others correspond to the left primary coordinate axis.

**Figure 4.** Species of phytoplankton observed in different treatment groups during the experiment.

The phytoplankton diversity in each group was analyzed using the Shannon–Wiener diversity index (Figure 5), with an overall average of 1.07. The diversity index of the Co group was 0.59 to 1.43, with an average of 1.03. The diversity index of the T15 group was 0.46 to 1.62, with an average of 0.95. The diversity index of the T30 group was 0.54 to 1.65, with an average of 1.02. The diversity index of the T45 group was 0.93 to 1.62, with an average of 1.26.

**Figure 5.** Shannon–Wiener diversity distribution indexes in different treatment groups during the experiment.

3.3.2. Variation Trends in Phytoplankton Biomass over Time

The phytoplankton biomass of different treatment groups over times was analyzed statistically. The overall average biomass of each experimental group showed a downward trend with time. However, an abnormal increase occurred for those in Co group on 15 August (Figure 6). On 25 May, the biomass in T45 was significantly higher than that in Co (*p* < 0.05). On 30 June, the biomass in T15 was significantly lower than that in Co and T45, while those in Co and T15 was significantly higher than that in T45 on 15 August (*p* < 0.05). The phytoplankton biomass in Co group was the lowest among all groups on 15 October (*p* < 0.05).

**Figure 6.** Phytoplankton biomass in different treatment groups during the experiment. Different lowercase letters in each group represent significant differences (*p* < 0.05).

3.3.3. Succession and Population Changes in the Dominant Phytoplankton Species

The dominant phytoplankton species were calculated, and the results are presented in Figure 7. If Y > 0.02, then the species was considered dominant. After the quantification and calculation analysis, there were five phyla of dominant zooplankton. The species composition and dominance of each phytoplankton species varied within different sampling time intervals in each treatment group (Table A5).

**Figure 7.** Dominance phytoplankton at phyla level in different treatment groups during the experiment.

*Chromulina pygmaea* and *Chlorella pyrenoidosa* were highly dominant species in different treatment groups during the experiment. The diversity of dominant phytoplankton species in the Co group increased over time. Comparatively, the dominant species of each feeding group were relatively simple. In the later stage of the experiment, the degree of dominance of *Chroococcus* in the T30 and T45 groups was higher than that in the Co and T15 groups (Figure 8). On 2 September, the degree of dominance of *Chroococcus* in T45 was significantly higher than in the other groups (*p* < 0.05).

**Figure 8.** Dominance of *Chroococcus* sp. in different treatment groups during the experiment.

*3.4. Species, Quantities, and Changes in Zooplankton*

3.4.1. Zooplankton Species Diversity

A total of 50 species of zooplankton were detected during the experiment (Figure 9). Protozoa had the highest number of species, with 23 species detected, and accounted for

46% of the total number of species. Rotifera had 15 species detected, accounting for 30% of the total species. Cladocera had eight species, accounting for 16% of the total species. Copepoda had four species, accounting for 8% of the total species.

**Figure 9.** Species of zooplankton in different treatment groups during the experiment.

The zooplankton species in different treatment groups during the experiment are listed in Table A6. The average biomass of Copepoda was the largest at 31.61 mg/L and accounted for 50.00% of the overall zooplankton biomass. That of Cladocera was 29.29 mg/L and accounted for 46.33% of the total biomass. These two groups accounted for 96.32% of the total average biomass. The average biomass of Protozoa was 1.76 mg/L, accounting for 2.79% of the total. The zooplankton group with the lowest average biomass was the rotifers, with only 0.56 mg/L, accounting for 0.89% of the total average biomass.

The zooplankton diversity in each group was analyzed using the Shannon–Wiener diversity index (Figure 10). The overall average value was 1.69. The diversity index was from 1.19 to 2.16 for the Co group (1.74 average), 1.15 to 2.31 for the T15 group (1.67 average), 1.00 to 2.18 for the T30 group (1.61 average), and 1.27 to 2.08 for the T45 group (1.73 average), respectively.

**Figure 10.** Shannon–Wiener diversity indexes of the zooplankton in different treatment groups during the experiment.

3.4.2. Variation Trends of the Zooplankton Biomass over Time

The total average zooplankton biomass in the paddy field fluctuated over time. There was an upward trend from the beginning of the experiment to 15 July, which then decreased to 15 August and increased to September 15 before decreasing again (Figure 11). The biomass of zooplankton in T45 was significantly lower than those in T15 and T30 on 30 June (*p* < 0.05), while the biomass in T45 was significantly higher than those in other groups on 29 July (*p* < 0.05).

**Figure 11.** Zooplankton biomass in different treatment groups during the experiment.

3.4.3. Succession of Dominant Zooplankton Species and Community Changes

The dominant zooplankton species in the paddy fields during the experiment are shown in Table A7 and Figure 12. When dominance value (Y) > 0.02, the species was considered dominant. The dominant species in different treatment groups consisted of 32 species belonging to 4 zooplankton groups (i.e., Rotifera, Copepoda, Cladocera and Protozoa), respectively. The biomasses of the dominant zooplankton species were relatively low in the four treatment groups on 25 May and 7 October. The number of dominant species in T15 was the lowest on 15 August. The numbers of dominant species were 6 to 10 in Co group, 4 to 11 in T15 group, 4 to 9 in T30 group, and 6 to 9 in T45 group, respectively, from 30 June to 26 September.

#### *3.5. Species, Quantities, and Changes in Aquatic Vascular Plants*

The species diversity and biomass of the aquatic vascular plants in the enclosures of each treatment group are shown in Figure 13. Seven aquatic vascular plants were detected in the four treatment groups, i.e., *Vallisneria spiralis, Monochoria vaginalis, Sparganium stenophyllum, Spirodela polyrhiza*, *Potamogeton* sp., *Elodea nuttallii*, and *Scirpus validus*.

*Sparganium stenophyllum* was the most dominant species, with an average biomass of 535.71 g/m2, accounting for 52.02% of the total aquatic vascular plant biomass. The following dominant species was *S. validus* with 287.12 g/m2, accounting for 27.88% of the total biomass. Meanwhile, the biomasses for *M. vaginalis*, *V. spiralis*, *E. nuttallii*, *S. polyrhiza* and *Potamogeton* sp. were 82.79, 79.55, 24.09, 15.41 and 5.12 g/m2, and accounted for 8.04%, 7.72%, 2.34%, 1.50% and 0.50% of the total biomass, respectively.

The changes of the submerged-plant biomass in different treatment groups over time are shown in Figure 14. The statistical analysis revealed significant variations in the biomass of submerged plants in each treatment group with different times. The overall submerged-plant biomass rapidly decreased to 30 June before rapidly increasing to 25 July and then increased gradually to 29 July and 15 August. The biomass in the T45 group was significantly higher than the other groups (*p* < 0.05). Afterwards, submerged-plant biomass rapidly decreased again. No submerged plant was found in all treatment groups from 15 September to 7 October.

The biomass of the emergent plants in different treatment groups over time are shown in Figure 15. The statistical analysis revealed significant variations in the biomass of emerged plants at different times. Generally, the biomass increased throughout the experiment and only decreased in the last sample collection. The emergent plants biomass in the Co group was significantly higher than that in the T45 group on July 15 and July 29, while the biomass in the T15 and T30 groups was significantly higher than that in the T45 group on July 29 (*p* < 0.05).

 **Figure 12.** Composition of the predominant zooplankton species in different treatment groups during the experiment.

**Figure 13.** Species and biomass of aquatic vascular plants in different treatment groups during the experiment.

**Figure 14.** Biomass of the submerged plants in different treatment groups during the experiment.

**Figure 15.** Biomass of the emergent aquatic plants in different treatment groups during the experiment.

*3.6. Variations in the Benthic Animal Species and Quantities with Time*

The diversity and biomass of benthic animals in different treatment groups are shown in Table A8 and Figure 16. Five taxa were found in the nine samples from the four treatment groups, i.e., *Gyraulus* sp., *Euconulus* sp., *Limnodrilus* sp., *Branchiura* sp., and Insecta.

The biomass of benthic animals in different treatment groups changed over time. Generally, they initially increased from 23 May to 29 July, then decreased rapidly to 15 August, followed by a gradual decline through September until the end of the experiment (Figure 17). On 2 September, the biomass of the benthic animals in the Co group was significantly higher than that in the T45 and T30 groups (*p* < 0.05). No significant difference was found among various treatment groups at the same sampling time.

**Figure 16.** Species and biomass of benthos in different treatment groups during the experiment.

**Figure 17.** Biomass of the zoobenthos in different treatment groups during the experiment.

#### **4. Discussion**

*4.1. Effects of Diets with Different Protein Levels on the Growth Performance and Yield of Juvenile Crabs*

Protein is one of the most important nutritional components in the diet of crabs, and the level required varies depending on growth stages [16]. The five separate measurements of morphological parameters of the crabs revealed that the high-protein compound feed resulted in significantly higher crab carapace length, width, and height and body weight compared with those in the low-protein group. Feeding with a high-protein compound feed had a significantly positive effect on the weight gain rate, final body weight, and specific growth rate of juvenile crabs. These results support the findings of Zhang [6]. However, the T30 group in this experiment may have escaped and/or had "milky disease" [17]. The rate of disease varied according to the original health status of the crabs and may have resulted in the observed differences in survival rates; however, there was no significant difference in the crab yield between the different diet treatments. These results do not correspond with the growth rate results. Therefore, the contribution of natural food to crab growth may be underestimated in the rice–crab co-culture mode.

Integrated agricultural and aquaculture systems can effectively contribute to green and sustainable agricultural development and ensure food security [18]. In 2017, the "General Principles of Technical Specifications for Rice and Fishing Integrated Planting and Culture," issued by the Chinese Agriculture and Rural Affairs Bureau, highlighted the fact that animals raised in aquaculture should make full use of natural bait present in the environment (in this case the rice paddies), reducing the use of fish feed. The results of this experiment strongly support this statement. The lower temperature in the paddies is more favorable to the growth of crabs compared with the temperature in monoculture crab systems, which can reduce crab sexual precocity [19].

In this experiment, the sexual precocity rate in the T45 group was approximately 10%, which may be due to the high protein content. Chen et al. [20] demonstrated that when the protein content in the feed is too high, excess protein is converted into fat and stored in the hepatopancreas, resulting in sexual precocity in crabs. Sexual precocity during the breeding process reduces culture efficiency. Studies have shown that the survival rate of adult crabs cultured with precocious crab species in the second year is already extremely low [21]. Therefore, it is important to prevent the sexual precocity of crabs in production.

#### *4.2. Changes in Physical and Chemical Properties of Paddy Water Environment*

There were no obvious changes in water temperature, pH, salinity, ammonia nitrogen, or nitrite nitrogen over the course of this experiment. There were also no significant differences between the experimental groups. The ammonia nitrogen and nitrite content of the water was low. However, there was a downward trend in dissolved oxygen levels in the water, which may have been caused by a variety of factors. The daily photosynthesis of plants is the main source of dissolved oxygen in water [22]. Animal respiration in the water releases large amounts of organic matter. Additional organic matter is produced during decay (after death) and after feeding, which leads to an increase in the respiration in water and sediments, and is also the main destination of dissolved oxygen [23]. The crabs were placed in the experimental enclosures on 29 May. On the same day, the dissolved oxygen in the water began to decrease, indicating that crab respiration was the main factor causing the low dissolved oxygen in the rice–crab co-culture. As the experiment progressed, the shading effect of large vascular plants (including the rice) led to a decrease in phytoplankton photosynthesis. This is also one of the reasons for the decrease in dissolved oxygen levels. In the later stages of the experiment, the plankton species and biomass and the biomass of zooplankton increased, while the biomass of the phytoplankton decreased. Consequently, there were more aerobic biological factors and less oxygen-producing organisms in the environment, resulting in a decrease in the dissolved oxygen levels in the water. The levels of dissolved oxygen ranged from 3.04 to 8.75 mg/L, which is lower than the normal dissolved oxygen requirements of crabs (5 mg/L). In low dissolved oxygen conditions, crabs tend to escape. Crabs also crawl to the shore in the later culture stage. The dissolved oxygen content of the water in rice–crab co-culture is lower than that in conventional rice fields [24,25]. This may cause a stress response in the crabs and is, therefore, one of the shortcomings of breeding crabs in rice paddies.

#### *4.3. Effects of Diets with Different Protein Levels on the Phytoplankton in Paddy Fields*

As primary producers, phytoplankton also act as a natural food source for crabs in rice– crab co-culture systems. Through the experiment, the aquatic organisms and water quality factors affected and correlated with each other. Species diversity is a basic characteristic of biological communities and is an important indicator of a healthy system [26]. In this experiment, the overall average Shannon–Wiener diversity index of the phytoplankton in the paddy field was 1.07, indicating that the phytoplankton diversity in the paddy field environment was not extremely diverse but superior to that in polluted waters. Studies have shown the Shannon–Wiener diversity index for phytoplankton in the rice–crab coculture mode is higher than that in conventional rice fields [5,27]. This is due to the reduction in the use of chemical fertilizers and pesticides in rice–fish symbiosis [27,28]. Thus, biodiversity in the paddy fields has been well protected [29,30].

The rice growth and the subsequent shading effect of the rice reduced the lightreceiving area of the water in the paddy field. This weakened the photosynthesis by phytoplankton. Furthermore, the physical and chemical factors involved in water quality and organisms in the water environment interact with each other [30,31]. Individual phytoplankton are small and varied, and different species can affect the environment in diverse ways. Adaptability of the species differs, and most can intuitively reflect the changes in water physicochemical factors after environmental changes. Rice will absorb nutrients and ions in the paddy field, effectively regulating the physical properties and chemicals in the environment, and can inhibit the absorption of nutrients by phytoplankton. Therefore, it was expected that the total average biomass of phytoplankton would show a decreasing trend with time. When the phytoplankton productivity is low, the consumption of phytoplankton by zooplankton is an important factor affecting phytoplankton growth [32]. For example, the sample collected on 15 August revealed that the phytoplankton biomass had abnormally increased. When the data were combined with the analysis of changes in zooplankton biomass, the zooplankton biomass had decreased significantly at that time. Reduced grazing pressure on phytoplankton results in abnormally elevated phytoplankton biomass. In the natural environment, there are many reasons for an increase in phytoplankton biomass. For example, an increase in nutrient concentration can lead to similar results. The water quality monitoring results showed that the nutrient index in the water was not significantly different from that in other periods; therefore, there was no increased nutrient concentration.

The results of this experiment revealed that the diversity of the dominant phytoplankton species in the control group presented an increasing trend. However, it did not vary among the feeding groups. This may be because the addition of exogenous nutrients in the feed led to the eutrophication of the water body, resulting in a decrease in biodiversity. Water eutrophication destroys the ecosystem balance and can even lead to the collapse of the entire aquatic system [33]. When the nutrients in the water increased, Cyanophyta phytoplankton gradually became the dominant species at the expense of other species, indicating that Cyanophyta are indicator organisms for water eutrophication [34]. In this experiment, the dominance of *Chroococcus* in the T45 group was significantly higher than that of the other three groups on 2 September, after which it decreased with no significant difference, indicating that high protein levels could cause water eutrophication. However, in a paddy field environment, water eutrophication is not a concern owing to the self-purification function of rice.

#### *4.4. Effects of Different Protein Levels in the Crab Diet on Zooplankton in Paddy Fields*

In the rice–crab co-culture environment, zooplankton can feed on phytoplankton, which is also the main food of crabs. Zooplankton is an important link between energy flow and material cycling in the ecosystem [35]. Zooplankton species and community structure are affected by environmental factors. A total of 51 species of zooplankton were identified in this experiment, and the Shannon–Wiener diversity index was 1.73. Previous research has shown that the Shannon–Wiener diversity index of Cladocera and Rotifers in a water environment under rice–crab co-culture is higher than that of conventional rice fields [36]. In addition, owing to the purification effect of rice, zooplankton in crab paddy fields is highly diverse.

The results of this experiment showed that the zooplankton biomass initially increased and then decreased before increasing again. Combined with the analysis of the changes in phytoplankton in the paddy field, the biomass of the phytoplankton was relatively high in the early stage of the experiment. Then, the zooplankton fed on the phytoplankton and grew rapidly; its biomass also increased. As crabs grew, they preyed on the zooplankton, and the zooplankton biomass decreased. Horn et al. [37] tracked zooplankton body length and found that not only the maximum body length of zooplankton decreases, but the average body length and length frequency distribution of zooplankton also shifted to that of smaller individuals under predation pressure. The results showed that predation

pressure on zooplankton by crabs led to the miniaturization of zooplankton. In the later stage of this experiment, the miniaturization of zooplankton, combined with the larger size and mouthparts of crabs, reduced the crab predation on zooplankton, so the biomass of zooplankton increased.

The dominant zooplankton species in the early stages of the experiment, on 25 May and 30 June, were rotifers, especially *Polyarthra trigla*, which is consistent with Zhang's [6] results. Rotifers, Cladocera, and copepods all competitively feed on phytoplankton in paddy fields. According to Gilbert [38], when there is competition between Cladocera and rotifers, Cladocera has an advantage. Therefore, the existence of Cladocera affects the diversity and quantity of rotifers. As the experiment progressed, some Cladocera species gradually became dominant. In the later stage of this experiment, the dominant species of zooplankton in the crab paddy field were small and mainly existed in the state of copepod nauplii, with the dominant species being mainly protozoa.
