*2.9. Statistics*

Using SPSS 25.0 statistical software, all results were expressed as mean ± standard deviation (*n* = 3). Before analysis, the Kolmogorov - Smirnov and Levene tests were used to test the distribution normality and homogeneity of variance of the original data. One-way ANOVA and Tukey's multiple comparison post hoc test were used to determine whether significant variation existed between the treatments. The *ecr* gene expression and CAT could not achieve normality and homogeneity and were analyzed with the nonparametric Kruskal-Wallis test. A quadratic model was used to fit the SGR to obtain the optimal growth temperature of the juvenile mud crabs. To determine the relationship between temperature, growth, ingestion, OCR, AER, molting, and the expression of *mih* and *ecr*, Pearson correlation analysis and t-test were used. A statistically significant level of *p* < 0.05 was applied in the present study.

#### **3. Results**

#### *3.1. Survival, Growth Performance, Molting, and Feeding*

The survival rate and hepatopancreatic index of the 35 ◦C group were significantly lower than the other groups (*p* < 0.05) (Figure 1A,B). The temperature significantly affected the growth, and SGR showed a parabolic trend (Figures 1C and 2A). Juvenile mud crabs had the best growth performance between 28.5 ◦C and 29.7 ◦C in accordance with the quadratic regression model analysis (Figure 2).

**Figure 1.** Effects of different temperatures (20–35 ◦C) on survival rate (**A**), hepatopancreas index (**B**), and final weight (**C**) of juvenile mud crabs. Values are expressed as mean ± SD (*n* = 3). Different superscripts indicate significant differences between treatments (*p* < 0.05).

**Figure 2.** Relationship between the temperature and the SGR of initial to 1st molt (**A**), 1st to 2nd molt (**B**), 2nd to 3rd molt (**C**), and initial to final (**D**), respectively. Where Xopt means the optimal temperature for the maximum SGR.

The MF of the 30 ◦C (19.80 ± 1.33) group was significantly higher than the 20 ◦C (8.40 ± 1.05), 25 ◦C (13.21 ± 0.51), and 35 ◦C (14.00 ± 1.54) (*p* < 0.05). However, the MI of the 35 ◦C groups (73.58 ± 2.18%) was significantly lower than that of the 20 ◦C (87.13 ± 9.10%), 25 ◦C (93.44 ± 5.16%), and 30 ◦C (86.58 ± 4.36%) groups (*p* < 0.05) (Figure 3).

**Figure 3.** Effects of different temperatures (20–35 ◦C) on the molt frequency and molt increment of juvenile mud crabs. Values are expressed as mean ± SD (*n* = 3). Different superscripts indicate significant differences between treatments (*p* < 0.05).

With the increase in temperature, the average daily food intake showed a trend of first increase and then decrease, and the difference was significant (*p* < 0.05) (Figure 4A). The feed conversion efficiency at 25 ◦C and 30 ◦C was significantly higher than that in the 20 and 35 ◦C groups (*p* < 0.05), the 35 ◦C group was significantly lower than the other three groups (*p* < 0.05), and the FCE in the 30 ◦C group was the highest (45.48 ± 1.21%), the FCE of the 35 ◦C group was the lowest (21.00 ± 3.00%) (Figure 4B).

#### *3.2. Ecdysone Content and Expression of Molting-Related Genes*

The content of ecdysone in crab hemolymph increased significantly with the increasing temperature (*p* < 0.05) (Figure 5A). The expression level of the *ecr* gene in the 35 ◦C group was the highest and was significantly higher than that in the other three groups (*p* < 0.05) (Figure 5B). The expression of the *mih* gene decreased with the increasing temperature (Figure 5C).

#### *3.3. OCR, AER, and O: N*

Between 20–30 ◦C, the OCR increased significantly with the increasing temperature (*p* < 0.05), but no significant difference was found between the 30 ◦C and 35 ◦C groups (*p* > 0.05) (Figure 6A). The AER peaked at 35 ◦C and was significantly higher than the 20–30 ◦C group (*p* < 0.05) (Figure 6B). The O: N showed a parabolic trend and peaked at 30 ◦C (Figure 6C).

**Figure 4.** Effects of different temperatures (20–35 ◦C) on the average daily food intake (**A**) and feed conversion efficiency (**B**) of juvenile mud crabs. Values are expressed as mean ± SD (*n* = 3). Different superscripts indicate significant differences between treatments (*p* < 0.05).

**Figure 5.** The content of ecdysone (**A**) in the hemolymph. Expression of the molting-related gene (*ecr*) (**B**) and molt-inhibiting hormone (*mih*) (**C**) in eyestalks. Values are presented as mean ± SD (*n* = 3). Different superscripts indicate significant differences between treatments (*p* < 0.05).

**Figure 6.** Oxygen consumption rate (OCR) (**A**), ammonia excretion rate (AER) (**B**), and oxygennitrogen ratio (O: N ratio) (**C**) of juvenile mud crabs at different temperatures (20–35 ◦C). Values are expressed as mean ± SD (*n* = 3). Different superscripts indicate significant differences between treatments (*p* < 0.05).

#### *3.4. Antioxidant Capacity*

T-AOC activity was the highest at 25 and 30 ◦C (Figure 7A). The activity of CAT showed a significant upward trend with the increasing temperature (*p* < 0.05) (Figure 7B). The GSH and SOD activities peaked at 30 ◦C (Figure 7C,D). Moreover, the MDA content was significantly lower in the 25 and 30 ◦C groups compared to the 35 ◦C group (Figure 7E).

**Figure 7.** Effects of different temperatures on the activities total of antioxidant capacity (T-AOC) (**A**), catalase (CAT) (**B**), superoxide dismutase (SOD) (**C**), glutathione (GSH) (**D**), and malondialdehyde (MDA) (**E**) content of juvenile mud crabs. Values are expressed as mean ± SD (*n* = 3). Different superscripts indicate significant differences between treatments (*p* < 0.05).

#### *3.5. Hemolymph Index*

In comparison with other groups, the hemolymph of the 35 ◦C group contained significantly more cortisol and LD (*p* < 0.05), and there was no significant difference between the 20–30 ◦C groups (*p* > 0.05) (Figure 8A,B). The uric acid content increased with the increasing temperature (Figure 8F). Glucose and total cholesterol showed a parabolic trend (Figure 8C,E). Significantly higher TG content was found in the 35 ◦C group than in either the 30 ◦C or 20 ◦C groups (*p* < 0.05) (Figure 8D).

#### *3.6. Correlation Analysis*

Correlations between temperature and SGR, MF, MI, OCR, AER, FI, FCE, and relative expression of *mih* and *ecr* parameters were analyzed by Pearson correlation coefficient (Figure 9). Overall, *ecr* gene expression, AER, OCR, and MF were positively correlated with temperature, while *mih* gene expression and MI were negatively correlated. Interestingly, the *mih* gene expression was also negatively correlated with SGR, MF, OCR, AER, and FI.

**Figure 8.** Effects of different temperatures on cortisol (**A**), lactic acid (LD) (**B**), glucose (GLU) (**C**), triglyceride (TG) (**D**), total cholesterol (T-CHO) (**E**), and uric acid (UA) (**F**) content in juvenile mud crabs. Values are presented as mean ± SD (*n* = 3). Different superscripts indicate significant differences between treatments (*p* < 0.05).

**Figure 9.** Correlation analyses among temperature SGR, MF, MI, OCR, AER, FI, FCE, and relative expression of *mih* and *ecr* gene (*n* = 3, 2 individuals per replicate for AER, OCR, *ecr*, and *mih*; *n* = 3 replicate, and 31, 42, 42, and 42 individuals per treatment of SGR, MF, FI, and FCE). "\*" indicates *p* < 0.05.

#### **4. Discussion**

Temperature significantly affected the survival and growth of the mud crab. For crabs reared at 20–30 ◦C, the survival rate was 100%, while high water temperature (35 ◦C) caused mortality. The same trend was also observed in growth performance. According to the quadratic regression analysis, the optimal water temperature for the growth of mud crab was 28.5–29.7 ◦C. A similar study also suggested that the most suitable temperature in Crablet 1 to Crablet 2 phase was 28–32 ◦C [35]. However, another recent research observed that the SGR of the juvenile *S. paramamosain* in the 36–37 ◦C (14.65 ± 0.23% day<sup>−</sup>1) group was significantly higher than the 27–28 ◦C (12.30 ± 0.42% day<sup>−</sup>1), 30–31 ◦C (11.58 ± 0.14% day<sup>−</sup>1), and 33–34 ◦C (10.11 ± 0.06% day−1) groups [35]. The difference could be partly attributed to the fact that the mud crabs were cultured in groups of 30 individuals per tank [36]; thus, temperature-induced cannibalism could potentially contribute to the growth performance. In addition, the speculation was also supported by the markedly lower survival rate (28.9 ± 2.94%) and higher SGR (14.65 ± 0.23% day<sup>−</sup>1) at 36–37 ◦C [35] compared to the present study (80.36 ± 5.92% and 3.00 ± 0.26% day−<sup>1</sup> at 35 ◦C).

The growth of organisms was closely related to feeding [37]. Generally, FI increases with increasing temperature and decreases when the temperature exceeds the optimum temperature range [38]. In this study, both low and high temperatures inhibited FI. Low temperatures inhibit the metabolic capacity of crabs, thereby affecting their appetite and energy balance. On the contrary, long-term high-temperature stress caused heat stress and decreased FI. In the present study, the FCE and FI had a consistent trend, indicating that a suitable temperature not only stimulates FI but also effectively improves the assimilation of food. This result was also observed in turbot (*Scophthalmus maximus*) [39], *Penaeus japonicus* [40], and Atlantic salmon (*Salmo salar*) [41]. Therefore, the decline in FI and FCE could be one of the reasons for the suboptimum growth performance for the 20 and 35 ◦C group.

Molting is essential in crustacean growth [42]. High water temperature inhibited the expression of the *mih* gene but consequently promoted ecdysone synthesis and *ecr* gene expression. These factors could jointly promote the synthesis of the EcR-RXR-ecdysone complex, resulting in a significantly increasing MF. Crustacean growth and molting are highly correlated in most studies [5,43–45]. Interestingly, in this study, both MI and MF dropped simultaneously in the 35 ◦C group compared with the 30 ◦C group. A similar phenomenon was observed in *Penaeus japonicus* [40]. Nonetheless, the underlying physiological process is poorly understood. A possible explanation is crab's metabolic capacity is temperature-dependent.

The metabolic rate of animals, measured by OCR and AER, varies directly with temperature [46,47]. O: N could represent the ratio of protein, fat, and carbohydrate catabolism of aquatic organisms [48]. O: N < 10 suggests the respiratory substrate mainly consisted of protein. On the contrary, a high O: N means the substrate is mainly provided by fats and carbohydrates [49]. The OCR of crabs at 30–35 ◦C was similar, but AER raised dramatically at 35 ◦C, leading to a lower O: N (105.66 ± 16.90) compared with 30 ◦C (143.46 ± 7.97). Though the value is much higher than 10, the results suggest a higher proportion of proteins consumed by respiration than tissue growth at 35 ◦C. The conjecture could further be confirmed by the high hemolymph uric acid level.

The stress response of an organism can be divided into three levels [50]. First-order stress responses include elevated cortisol levels [51]. Secondary stress includes increased energy mobilization, manifested primarily by increases in blood glucose and circulating lipids, which provide the organism with the energy necessary to resist stress [52]. Primary and secondary stress responses trigger tertiary stress responses that ultimately affect animal growth and survival [53]. In this study, a significant increase in cortisol, GLU, TG, and T-CHO in the hemolymph at high temperatures was observed. The results indicate that elevated temperature caused cortisol secretion, increased protein catabolism, and plasma cholesterol, and promoted GLU by inhibiting GLU breakdown. Furthermore, mud crabs raised at 35 ◦C also had higher lactic acid and significantly lower T-AOC, SOD, and GSH, suggesting an inferior thermal resistance [54,55] and antioxidant capacity, leading to downregulated survival rates.

#### **5. Conclusions**

The effects of rearing temperature on mud crab *S. paramamosain* were estimated by growth, molting, feeding intake, energy metabolism, and stress responses. The result showed that the crabs at high temperature (35 ◦C) had a significantly higher ecdysone level and expression of the *ecr* gene but lower MI, MF, and survival rate. The effect could be mediated through respiration, energy metabolism, and antioxidant pathways. It was found that 28.5–29.7 ◦C provided the best conditions for mud crab growth. These findings could help regulate the temperature in mud crab RAS and provide a thread for the thermal adaptation of crustaceans.

**Author Contributions:** Conceptualization, C.S. and J.L.; methodology, J.L.; formal analysis, J.L.; investigation, J.L.; Funding acquisition, C.S.; writing—original draft preparation, J.L.; writing—review and editing, C.S., Y.Y., Z.R., Q.W., Z.M., C.M.; project administration, C.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was sponsored by the National Natural Science Foundation of China (Grant Nos. 32172994 and 31972783), the National Key Research and Development Program of China (Project No. 2019YFD0901000), the Province Key Research and Development Program of Zhejiang (2021C02047), Key Scientific and Technological Grant of Zhejiang for Breeding New Agricultural Varieties (2021C02069-6), 2025 Technological Innovation for Ningbo (2019B10010), China Agriculture Research System of MOF and MARA, SanNongLiuFang Zhejiang Agricultural Science and Technology Cooperation Project (2021SNLF029), K. C. Wong Magna Fund in Ningbo University and the Scientific Research Foundation of Graduate School of Ningbo University (IF2020145).

**Institutional Review Board Statement:** Our study did not involve endangered or protected species. In China, breeding and catching mud crabs, *Scylla paramamosain*, 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 Ningbo University.

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

**Acknowledgments:** Thanks to Qingsong Zhao for providing the site for this experiment.

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

#### **References**

