**1. Introduction**

Throughout tropical and temperate waters, the mud crab (*Scylla paramamosain*) is a common and economically important marine crab [1,2]. There is a long history of mud crab farming in China, Japan, and the Philippines [3]. China's 2020 mud crab production is 159,433 tonnes [4]. Mud crab is mainly cultured in ponds, but the unit output is low due to serious cannibalism. In recent years, aquaculturists have tried to culture the mud crab in recirculating aquaculture system (RAS). Unlike traditional pond culture, RAS can prevent cannibalism from the early developing stages and effectively improve the survival rate of mud crabs, even in the nursery. Although researchers have studied the factors such as tank bottom area [5] and tank color [6], they are still insufficient compared to the rapid development of the industry.

**Citation:** Liu, J.; Shi, C.; Ye, Y.; Ma, Z.; Mu, C.; Ren, Z.; Wu, Q.; Wang, C. Effects of Temperature on Growth, Molting, Feed Intake, and Energy Metabolism of Individually Cultured Juvenile Mud Crab *Scylla paramamosain* in the Recirculating Aquaculture System. *Water* **2022**, *14*, 2988. https://doi.org/10.3390/ w14192988

Academic Editors: Xiangli Tian and Li Li

Received: 20 August 2022 Accepted: 12 September 2022 Published: 23 September 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/).

Temperature is a ubiquitous factor in the life history of aquatic animals, and the high specific heat and heat conduction of water create a challenging thermal environment for aquatic animals [7]. The temperature of the water plays a major part in the survival and growth of crustaceans, according to a number of studies. For example, the mortality rate of ornamental red cherry shrimp (*Neocaridina heteropoda heteropoda*) at 32 ◦C was significantly higher than at 24 and 28 ◦C [8]. The survival rate of *Macrobrachium amazonicum* was also affected by temperature, and the survival rate at 28 ◦C was higher than that at 30 and 32 ◦C [9]. On growth, the red king crab (*Paralithodes camtschaticus*) grows exponentially with temperature [10]. Crustaceans achieve faster growth by increasing the molt increment (MI) or molt frequency (MF). Synthesized in the Y organ, ecdysone diffuses across the cell membrane and releases into the hemolymph, where it is converted to 20-HE and binds to the EcR-RXR-ecdysone complex to regulate molting [11]. However, the *mih* gene inhibits molting by inhibiting ecdysone in the hemolymph [12].

According to the principle of thermodynamics, the increase in temperature stimulates the metabolic process of the organism [13]. Ambient temperature can significantly affect aquatic animals' metabolic levels and physiological regulation mechanisms. The optimal temperature strongly supports the physiological and biochemical processes of the organisms. At the same time, it can provide maximum energy efficiency [14,15]. The Oxygen consumption rate (OCR) of most crustaceans in the thermophilic range increases gradually with increasing temperature [16]. When the temperature is too high, the metabolic level of crustaceans decreases [17]. For example, in the southern rock lobster (*Jasus edwardsii*), the OCR positively correlates to the temperature at 18–22 ◦C. However, when the temperature increased further, the metabolic level decreased instead [17]. Furthermore, since ammonia production is the result of amino acid deamination, ammonia excretion rate (AER) can be used to estimate protein utilization by aquatic organisms [18]. Compared with fishes, crustaceans had an open-vessel circulatory system and transported nutrients through the hemolymph [19]. Hemolymph metabolites could reflect the morphological and physiological adaptation of crustaceans to the environment [20]. Total cholesterol (T-CHO), triglyceride (TG), and glucose (GLU) in hemolymph can evaluate the energy metabolism of crustaceans [21].

An increase in temperature is associated with a higher metabolic rate (Q10 effect), resulting in increased oxygen consumption, increased flux at the electron transport chain level, and more reactive oxygen species (ROS) [22]. ROS can oxidize surrounding molecules, impair cellular functions, and lead to oxidative stress [23,24]. Oxidative enzymes and non-enzymatic antioxidants are used by aquatic organisms to remove excess ROS. The antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) are among them. Non-enzymatic antioxidants include fat-soluble vitamins (e.g., alpha-tocopherol) and small water-soluble molecules such as glutathione (GSH) [25]. Total antioxidant capacity (T-AOC) reflects the metabolic ability of antioxidant enzymatic and non-enzymatic systems under external stress [26]. Malondialdehyde (MDA), as the end product of lipid peroxidation, reflects the degree of cellular oxidative damage [27]. Studies have shown that long-term stress in crustaceans inhibits antioxidant enzyme activity and produces more MDA [5,28,29], accompanied by reduced food intake (FI) [30].

Therefore, this study aimed to evaluate the optimal temperature range for juvenile mud crabs in RAS in terms of growth, molting, energy metabolism, antioxidant capacity, and stress response. This study could help define the management protocol of mud crab and support the design of crab RAS.

#### **2. Material and Methods**

#### *2.1. Experimental Design*

Four temperatures, i.e., 20, 25, 30, and 35 ◦C, were set with four independent RAS. Each RAS consisted of 7 square tanks (0.5 × 0.3 × 0.2 m3) with six compartments (0.1 × 0.1 × 0.13 m3) in each tank. Thus, each RAS had 42 compartments with one crab per compartment. The

crabs were distributed to three replicates for each treatment, with 14 crabs per replicate. The experiment lasted eight weeks at Ningbo University.

#### *2.2. Experimental Animal and Rearing Conditions*

The experiment was conducted in July–August 2021 in the Intelligent Aquaculture Laboratory (Ningbo City, Zhejiang province, China). Selection of healthy and uniformly sized juvenile mud crabs bought from mud crab nursery farm (Ningbo City, Zhejiang province, China). Before the experiment, the crabs were domesticated in the laboratory for one week. During this period, the commercial feed was overfed daily at 20:00 (Table 1). The excessive commercial feed was siphoned, and 1/3 of isothermal seawater was exchanged at 18:00 every day.


**Table 1.** Specific primers were used for real-time PCR in this study.

*ecr*: Ecdysone receptor, *mih*: Molt-inhibiting hormone.

When the experiment started, the 168 juvenile *S. paramamosain* with complete appendages and good vitality were weighed and then distributed to 4 RAS (weight: 0.36 ± 0.09 g). Starting from the room temperature of 28 ◦C, the RAS was adjusted to 20 ◦C (20.11 ± 0.43 ◦C), 25 ◦C (24.98 ± 0.23 ◦C), 30 ◦C (30.08 ± 0.11 ◦C), and 35 ◦C (34.88 ± 0.39 ◦C) at a rate of 1 ◦C d<sup>−</sup>1.

During the experiment, 20 and 25 ◦C were achieved with a refrigerating machine (AO LING HENG YE, LA-160, China). 30 and 35 ◦C groups were achieved by the heater (SUN SUN, AR-450, China). The same feeding protocol was used as described above, and the residual feed was counted daily. The seawater salinity was 25 ppt, and the photoperiod was 14L:10D.

#### *2.3. Sampling*

At the end of the 8-week breeding experiment, the crabs were starved for 24 h and then sampled. The crabs were anesthetized on ice, weighed, and then dissected. The eyestalk, hepatopancreas, and muscle were isolated with tweezers quickly. The hemolymph was collected with a disposable syringe from the pericardial sinus of the crab and kept at 4 ◦C overnight, then centrifuged at 3500 RCF for 15 min. The supernatant and all the other tissues were stored at −80 ◦C.

#### *2.4. Ecdysone Content and Molting-Related Genes*

Six crabs in each group were randomly selected to extract hemolymph to determine ecdysone content using a crab ecdysone-specific enzyme-linked immunosorbent assay kit (Enzyme Link Biotechnology, Shanghai, China). Every two crabs as a repeat. Most of the mud crab's hormones were secreted and synthesized by the eyestalk. Therefore, the eyestalks of 2 crabs were selected in each replicate of each treatment to examine the relative expression of molting-related genes (total of 6/treatment). After adding liquid nitrogen to the mortar, the eyestalk was ground into powder, and the powder was added to 1 ml of Trizol reagent (Invitrogen, Waltham, MA, USA). After rapid shaking and mixing, put into liquid nitrogen flash freezing, and the total RNA was extracted after. The total RNA product was aspirated and subjected to 1% agarose denaturing gel electrophoresis to detect RNA integrity. Synthesis of cDNA using HiFiScript cDNA synthesis kit (CW Biotech. Co. Lid., Shanghai, China) with total RNA as a template by reverse transcription. The expression of *mih* and *ecr* were detected by real-time PCR (LightCycler480 II, ROCHE, Basel, Switzerland) with *β-actin* as an internal reference gene. The relative expression levels of *ecr* and *mih* gene were calculated by 2−ΔΔCT method [31].

#### *2.5. OCR and AER*

The OCR and AER were measured on the sampling day. The water used in the experiments was fully aerated to saturation and was recorded as initial dissolved oxygen by YSI Pross handheld multiparameter water quality analyzer (USA). Six juvenile crabs of similar body weight and intact appendages from each treatment were carefully transferred to conical flasks with 0.1 L of aerated water. To prevent gas exchange, the mouth of the conical flasks was utterly sealed with plastic film. The conical flasks were sufficiently immersed in each RAS to keep a constant temperature. To exclude the interference of water respiration, a control group without crab was set for each treatment group. The experiment lasted for 60 min, and the final dissolved oxygen was measured [32]. The experimental method of AER is the same as OCR. The experiment lasted for 6 h. The AER was calculated according to the ammonia nitrogen concentration change before and after the experiment (HACH, 2604545) [33].

#### *2.6. Antioxidant Capacity*

Six crabs were randomly selected from each group, and every two crabs were used as one replicate for hepatopancreas antioxidant capacity measurement. The samples were centrifuged at 3500 rcf at 4 ◦C for 15 min after averaging in ice-cold physiological saline. T-AOC (A015-2-1), MDA (A003-1-2), SOD (A007-1-1), CAT (007-1-1), and GSH (A006-2-1) were analyzed using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions [28].

#### *2.7. Hemolymph Indexes*

Six crabs were randomly selected from each group, the hemolymph samples were determined, and every two crabs were used as one replicate. The determination of cortisol in crab hemolymph with a crab's specific cortisol ELISA kit (Enzyme-linked Biotechnology, Shanghai, China). T-CHO (A111-1-1), UA (C012-2-1), GLU (A154-1-1), TG (A110-1-1), and LD (A109-2-1) content were determined by commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) [34].

#### *2.8. Data Collection and Calculation*

Survival rate (%) = 100 × (final number of crabs)/(initial number of crabs) Specific growth rate (SGR, % day<sup>−</sup>1) = 100 × (Ln Wf − Ln Wi)/t Feed conversion efficiency (FCE, %) = (Wf − Wi) × 100/FC Molt frequency (MF) = Σ((Cn − 1) × Nn )/Nt OCR = [(O1 − O2) × V]/(Wf × T) AER = [(N1 − N2) × V]/(Wf × T) O: N ratio = (OCR/16)/(AER/14) FI (% body weight d<sup>−</sup>1) = FC/[T × (Wf + Wi)/2] × <sup>100</sup>

Wf, final weight (g); Wi, initial weight (g); t, duration of the experiment (d); Cn, the developmental stage of crab; Nn, the number of molting stages; Nt, total number of survival crabs; O1, dissolved oxygen in the blank group (mg L<sup>−</sup>1); O2, test group dissolved oxygen (mg L−1); V, the volume of water in a beaker (L); T, metabolism time (h); N1, ammonia nitrogen in the control group (mg L−1); N2, ammonia nitrogen in the experimental group (mg L<sup>−</sup>1); FC, the weight of food ingested during the experiment (dry weight, g).
