There are many proteins involved in the synthesis and export of lipids that are folded or/and bounded at the ER. Hence, the ER also plays a key role in the homeostasis of lipid metabolism [43]. The overload of lipid metabolism leads to continuous ERs, which leads to lipid synthesis as a result of the protein metabolism in the ER [43,44]. In the present study, 4-PBA exhibited fat lowering effects that are dependent on ER metabolism remolding. The fat-lowering effect of 4-PBA also contributed to the alleviation of oxidative stress [45].

It is reported that there is an interplay between ERs and oxidative stress [23]. The overexpression of UPR components, such as CHOP and ATF4, directly contribute to ROS synthesis in the ER [33]. Moreover, the impaired ER can translate Ca2+ to mitochondria through the calcium release channel and mitochondrial-associated membranes, which exaggerates the production of ROS via the electron transport chain (ETC) [46]. In the present study, the expression of *CHOP* and *ATF4* were downregulated by dietary 4-PBA. Moreover, the analysis of TEM showed that HFD-induced mitochondrial damage was also alleviated. Based on these phenomena, we postulate that 4-PBA also inhibits ROS formation in both the ER and mitochondria.

There are many factors in the aquaculture environment that can be triggers of oxidative stress [6,47]. Notably, the highly intensive aquaculture industry has boomed in recent decades, as there has been a high demand for fish products [48]. Thus, fish often suffer ammonia nitrogen stress in the highly intensive culture [12]. The high ammonia nitrogen concentration can cause serious tissue damage and high mortality, which is a high-risk factor during fish farming [49–51]. Ammonia nitrogen stress can rapidly gather in the blood and tissue of fish and act as a potent cause of oxidative stress [52]. Fish that were fed the 4-PBA supplementation diet had a lower mortality rate and serum transaminases activities after the acute ammonia nitrogen challenge. This indicates that dietary 4-PBA can improve the tolerance of fish to ammonia stress. Further analysis showed that 4-PBA can enhance antioxidative abilities and reduce the peroxidation of proteins and lipids. Moreover, 4-PBA downregulated the gene expression of UPR factors. This indicates that 4-PBA supplementation exhibits the protective effect of ER homeostasis in fish that are exposed to high ammonia nitrogen concentrations.

#### **5. Conclusions**

In summary, the present study showed that ERs played an important role in the oxidative stress induced by a HFD or stress caused by high ammonia nitrogen concentrations. Furthermore, 4-PBA can attenuate the stress of the ER and excess fat deposition caused by the HFD feeding. Moreover, the supplementation of 4-PBA can increase the tolerance of fish that have suffered ammonia stress.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/antiox11071276/s1, Table S1: Formulation and Proximate Composition of Experimental Diets; Table S2: Sequences of Primers Used for RT-qPCR.

**Author Contributions:** Conceptualization, K.L., L.L. and Y.D.; methodology, L.W., L.X., N.D. and Y.W.; formal analysis, Y.D., L.L. and T.X.; investigation, Y.D. and L.L.; writing—original draft preparation, Y.D.; writing—review and editing, Y.W. and K.L.; visualization, Y.D.; supervision, K.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Natural Science Foundation of China (32072984), the Natural Science Foundation of Fujian Province (2020J01664), and China Agriculture Research System (CARS-47).

**Institutional Review Board Statement:** The animal study protocol was approved by the Ethics Committee of Animal Research Institute Committee guidelines, Jimei University, China (No. 2020-29, Approval date: 15 June 2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Conflicts of Interest:** K.L. has received research grants from the Fujian Aonong Biological Science and Technology Group Co., Ltd. Moreover, L.W., L.X., N.S. and Y.W. are employees of the Fujian Aonong Biological Science and Technology Group Co., Ltd.

#### **References**

### *Article* **Taurine Alleviates Cadmium-Induced Toxicity via Genetically Specific Strategies in Two Strains of Gibel Carp (***Carassius gibelio***)**

**Wenjie Xu 1,†, Hongyan Li 1,†, Liyun Wu 1,2, Junyan Jin 1,\*, Dong Han 1, Xiaoming Zhu 1, Yunxia Yang 1, Haokun Liu 1 and Shouqi Xie 1,2,3**

**Abstract:** Our previous studies in gibel carp (*Carassius gibelio*) have shown that cadmium (Cd) exposure elicits deleterious effects depending on the genetic background, and thus we hypothesized that mitigation via nutritional intervention may vary between strains. Therefore, two gibel carp strains (the A and F strains) were fed diets supplemented with 0% or 1% taurine for 8 weeks prior to 96 h Cd exposure, and the responses of antioxidant pathways, endoplasmic reticulum (ER) stress, autophagy, and apoptosis were investigated. The results showed that taurine supplementation had no effect on the growth performance of gibel carp. After Cd exposure, histological damage to mitochondria and ER, induction of oxidative stress and antioxidant responses, occurrence of ER stress, and apoptotic signals were observed in the livers. Upon the diet effects, taurine supplementation alleviated the ER-stress-induced autophagy and apoptosis after Cd exposure and stimulated antioxidant pathways. Regarding the difference between strains, taurine played a protective role in alleviating Cd toxicity through the antioxidant response, ER stress, and autophagy in the F strain, whereas such effects were achieved by the attenuation of apoptosis in the A strain. Taken together, our results demonstrate the potential use of taurine in the mitigation of heavy metal toxicity in aquatic organisms.

**Keywords:** taurine; Cd exposure; strain; autophagy; apoptosis

#### **1. Introduction**

Owing to widespread environmental pollution, the diverse hazardous impacts of exposure to toxic heavy metals on living organisms are becoming a global issue of great concern [1]. Cadmium (Cd) is one of the most abundant environmental pollutants in the biosphere, and it can be both toxic and carcinogenic [2,3]. Compared to other animals, aquatic species are vulnerable to Cd toxicity via the dietborne as well as the waterborne routes [4,5]. Therefore, aquatic toxicological evaluation of the effects of Cd has been widely investigated in teleosts under chronic or acute exposure in species such as gilthead sea bream, tilapia, yellow perch, and gibel carp (*Carassius gibelio*) [6–9].

Cadmium is reported to elicit deleterious effects via neurotoxicity, immunotoxicity, induction of oxidative stress, damage to organ structure, and cellular dysfunction [2,10]. Much effort has been made to investigate the mechanism of Cd toxicity and to develop a safe therapeutic approach to mitigating the toxic effects [1]. Some chemopreventive agents such as garlic extract containing specific organosulfur compounds have been used to protect against the toxic effects of Cd in both animal models and cell lines [11,12]. Cd

**Citation:** Xu, W.; Li, H.; Wu, L.; Jin, J.; Han, D.; Zhu, X.; Yang, Y.; Liu, H.; Xie, S. Taurine Alleviates Cadmium-Induced Toxicity via Genetically Specific Strategies in Two Strains of Gibel Carp (*Carassius gibelio*). *Antioxidants* **2022**, *11*, 1381. https://doi.org/10.3390/ antiox11071381

Academic Editors: Bo Liu, Changyou Song and Cunxin Sun

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

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

exposure disrupts the cellular oxidative homeostasis [13] that is regulated by various enzymatic or non-enzymatic antioxidants. Importantly, oxidative stress and glutathione (GSH) depletion are crucial components of Cd toxicity in aquatic organisms [14]. Therefore, nutrients with antioxidant properties have been applied to ameliorate the hepatotoxicity by modulating antioxidant pathways, such nutrients include vitamin C, vitamin E, carotenoids, and selenium [1,15].

Taurine (TAU, 2-amino ethanesulfonic acid), as a semi-essential amino acid, is a derivative of a sulfur-containing amino acid that has multiple functions in fish physiology [16]. Taurine is usually supplemented as an additive in the diet of aquatic animals for the promotion of growth as well as boosting the reproduction system, immune functions, and antioxidant effects [17]. The mechanism of the antioxidant activity of taurine was reported to be associated with enhanced mitochondrial function that protects the mitochondria from excessive superoxide [18]. In addition, taurine has been considered as a promising candidate for the improvement of liver function, and it has been reported as possessing tissue protective effects in treating oxidant-induced injury [19,20]. In mammalian models, taurine has been reported to alleviate the toxic effects of copper, lead, aluminum, and cadmium [21–23]. Similarly, administration of taurine affected hepatic metabolism and reduced Cd contamination in red sea bream and catfish [16,24]. Nevertheless, the mechanisms underlying the ameliorative effects of taurine against Cd poisoning in teleost fish are still not fully elaborated.

Previous studies have shown that exposure to Cd caused different toxic effects in gibel carp (*Carassius gibelio*) A strain (CAS III) and F strain (CAS V), regardless of whether via the dietborne or waterborne routes [9,25]. Specifically, these two strains of gibel carp showed genetically based metabolic strategies in response to Cd toxicity, verifying the fact that differences in genetic background may be an important cause of metabolic differences between fish strains. To ascertain the potential of taurine in the prevention of Cd poisoning and to explore whether these effects would vary between the two strains, experiments were performed with taurine supplementation via the diet route in the present study. We assessed the liver functions of the two strains, because the liver is the center of intermediary metabolism and plays vital roles in detoxifying processes [26,27].

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

#### *2.1. Experimental Procedures*

The experimental scheme is illustrated in Figure 1. Gibel carp used in this trial were obtained from the hatchery of the Institute of Hydrobiology, the Chinese Academy of Sciences, Wuhan, Hubei, China. The healthy and uniformly sized gibel carp A (4.61 ± 0.03 g) and F (4.58 ± 0.04 g) strains were fed diets supplemented with 0% (Control) or 1% TAU for 8 weeks (Figure 1, Phase 1). Diets were formulated in the laboratory according to the procedures described by Li et al. [28]. The diet formulation and approximate composition are shown in Table 1. Therefore, four groups of fish were obtained: the A strain fed the control diet (A0), the A strain fed a 1% TAU supplemented diet (A1), the F strain fed the control diet (F0), and the F strain fed a 1% TAU supplemented diet (F1).

After the 8-week feeding trial, a challenge test was conducted with fish from each of the four groups by exposing the fish to acute waterborne Cd (11.9 mg/L) (Figure 1, Phase 2). Cd exposure was performed in a static aquarium system with continuous aeration for 96 h, with 10 fish per tank and triplicate replicates for each tank. The concentration of Cd was set based on the value shown by a preliminary experiment that identified the 96 h median lethal concentration (LC50) [25]. CdCl2·2.5 H2O was added to the water by diluting a stock solution according to methods described by Li et al. [25]. During the acute exposure experiment, water in the system was refreshed daily. This experiment was implemented following the guiding principles for the care and use of laboratory animals and was approved by the Institute of Hydrobiology, Chinese Academy of Sciences.

**Figure 1.** Experimental design. Control: control diet; TAU: diet supplemented with taurine.

**Table 1.** Ingredients and proximate composition of the experimental diets (g/kg).

1 Fish meal was purchased from American Seafood Company, Seattle, Washington, USA. 2 Soybean and rapeseed meal were purchased from Coland Feed Co. Ltd., Wuhan, Hubei, China. 3 Vitamin premix (mg/kg diet): vitamin A, 1.65; vitamin D, 0.025; vitamin E, 50; vitamin K, 10; ascorbic acid, 100; thiamin, 20; riboflavin, 20; pyridoxine, 20; cyanocobalamine, 0.02; folic acid, 5; calcium pantothenate, 50; inositol, 100; niacin, 100; biotin, 0.1; cellulose, 645.2. 4 Mineral premix (mg/kg diet): NaCl, 500; MgSO4·7H2O, 8155.6; NaH2PO4·2H2O, 12,500.0; KH2PO4, 16,000.0; CaHPO4·H2O, 7650.6; FeSO4·7H2O, 2286.2; C6H10CaO6·5H2O, 1750.0; ZnSO4·7H2O, 178.0; MnSO4·H2O, 61.4; CuSO4·5H2O, 15.5; CoSO4·7H2O, 0.5; KI, 1.5; corn starch, 753.7.

#### *2.2. Sample Collection*

At the end of the 96 h Cd waterborne experiment, fish were anesthetized with MS-222 solution (Aminobenzoate methanesulfonate, 0.06 g/L, Sigma, St. Louis, MO, USA). The livers of two fish from each tank were dissected immediately on ice, with one part frozen in liquid nitrogen and then stored at −80 ◦C, and one part fixed in 2.5% glutaraldehyde solution and 4% paraformaldehyde.

#### *2.3. Transmission Electron Microscopy (TEM) Observation*

The liver samples of the two strains were dissected into 1 mm3 cubes and then fixed immediately in 2.5% glutaraldehyde solution. The samples were then rinsed with 0.1 M phosphate buffer solution (pH = 7.4) three times (15 min each time). Postfixation was conducted with 1% osmium tetroxide for 2 h, and then the fixed samples were washed three times with 0.1 M phosphate buffer solution (pH = 7.4). The dehydration was performed in a graded ethanol series followed by acetone. After that, samples were infiltrated with acetone:SPI-Pon 812 resin (1:1) followed by acetone:SPI-Pon 812 resin (1:2) and SPI-Pon 812 resin. Subsequently, the samples were embedded in SPI-Pon 812 resin for 48 h at 60 ◦C. Ultra-thin sections (80–100 nm) were stained with uranyl acetate and lead citrate. Finally, observations were conducted using a transmission electron microscope (Tecnai G2 20 TWIN, FEI, Hillsboro, OR, USA).

#### *2.4. TUNEL Analysis*

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analyses were performed according to the procedure described by Li et al. [25]. The liver samples were fixed in 4% paraformaldehyde and then embedded in paraffin. After that, the samples were cut into 5 μm sections and deparaffinized in dimethylbenzene. The samples were dehydrated in a graded ethanol series, repaired with proteinase K, and permeabilized with Triton X-100/PBS solutions. DNA fragmentation was determined using TdT and dUTP reagents (1:9) for 2 h incubation followed by staining with 4 ,6-diamidino-2-phenylindole (DAPI, 0.3 mmol/L) for 10 min. The samples were examined under a Nikon Eclipse Ti-SR inverted microscope.

#### *2.5. Chemical and Biochemical Analyses*

Cd concentrations in water samples were measured in accordance with the National Standards of the Republic of China (GB/T 7475-1987, Water quality determination of copper, zinc, lead and cadmium—atomic absorption spectrometry). In summary, sample digestion was conducted by adding hydrogen peroxide and concentrated nitric acid to the samples. After adding palladium nitrate, the samples were tested via inductively coupled plasma optical emission spectroscopy (ICP-OES, PerkinElmer Optima 8000, Waltham, MA, USA).

The activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), and the contents of malondialdehyde (MDA) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The activity of caspase 3 (Casp3) in livers was tested using a commercial kit (Caspase 3 Activity Assay Kit, Be-yotime Biotechnology, Shanghai, China).

#### *2.6. qRT-PCR Analysis*

Total RNA from liver samples was extracted using TRIzol reagent (Ambion, Life Technologies, Austin, TX, USA) according to the manufacturer's instructions. The RNA integrity and purity were assessed by agarose gel electrophoresis and NanoDrop spectrophotometer determination, respectively. The cDNA was synthesized by reverse transcription using an M-MLV First-Strand Transcriptase kit (Invitrogen, Carlsbad, CA, USA). All quantitative real-time PCR (qRT-PCR) assays were performed on a LightCycler 480 System (Roche, Jena, Thüringen, Germany). The primers used for quantitative RT-PCR are shown in Table 2. The housekeeping gene *tubulin* was chosen to normalize the relative quantification of target genes according to the methods described by Pfaffl [29].

**Table 2.** Sequences of the primers used for qRT-PCR analysis in gibel

#### *2.7. Statistical Analysis*

Results are presented as means ± standard errors. Normality and homoscedasticity of the data were assessed by Shapiro–Wilk and Levene tests. Two-way analysis of variance (ANOVA) was conducted with SPSS 26.0 (Chicago, IL, USA), and *p* < 0.05 was considered as a significant difference. Independent t-tests were conducted to examine the differences between pre- and post-challenge test groups. Gene expression heatmap of genes related to antioxidation, ER stress, autophagy, and apoptosis were created using heatmapper (http://www.heatmapper.ca/ accessed on 4 July 2022).

#### **3. Results**

#### *3.1. Growth Performance*

No significant differences in final body weight (FBW) or specific growth rate (SGR) were observed in the two strains of gibel carp fed diets with taurine supplementation (Figure 2). However, dietary taurine supplementation significantly decreased the feed efficiency (FE) and increased the feed conversion ratio (FCR) in both strains. The F strain presented significantly higher FBW, SGR, and FE and lower FCR than the A strain (*p* < 0.05). During the experiment, the survival rate of fish was 100%.

**Figure 2.** Specific growth rate and feed efficiency of two gibel carp strains fed the control diet and diet supplemented with taurine. Control: control diet, white bars; TAU: diet supplemented with taurine, black bars. FBW: final body weight; SGR: specific growth rate (%/d) = 100 × [ln (final weight) − ln (initial weight)]/day; FE: feeding efficiency (%) = (100 × body weight gain)/dry feed intake; FCR: feed conversion ratio = (100 × dry feed intake)/body weight gain. Bars with different uppercase letters (A, B) represent significant differences between the A and F strains (*p* < 0.05). Bars with different upper-case letters (X, Y) represent significant differences between the control diet group and the taurine diet group (*p* < 0.05).

#### *3.2. Histological Observation*

Ultrastructural images of the liver in the two gibel carp strains exposed to Cd are shown in Figure 3. Cd exposure induced ultrastructural alterations in the two gibel carp strains, as shown by the degenerated cristae and swelling of mitochondria. Meanwhile, irregular parallel stacked endoplasmic reticulum and plaque accumulation within hepatocytes were detected by transmission electron microscopy.

**Figure 3.** Representative histological transmission electron microscopy (TEM) images of gibel carp (A and F strains) after 96 h cadmium exposure. Control: control diet; TAU: diet supplemented with taurine.

A terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) was conducted to assess the apoptosis index in the livers of gibel carp (Figure 4). The results showed that apoptosis signals increased significantly after Cd exposure (*p* < 0.05), while dietary taurine supplementation decreased the apoptosis index compared with the control group. Moreover, the apoptosis index in the A strain fed the control diet was the highest (*p* < 0.05).

**Figure 4.** Representative DAPI and TUNEL double staining and image quantification results from the livers of gibel carp (A and F strains) after 96 h cadmium exposure. Positive apoptotic cells appear in green, and normal nuclei appear in blue. Control: control diet, white bars; TAU: diet supplemented with taurine, black bars. Bars with different lowercase letters (a, b) indicate the interaction effect and represent significant differences among groups (*p* < 0.05). Bars with \* indicate significant changes between diets in the same strain (*p* < 0.05). The magnification factor is 200×, and the scale bar is 100 μm.

#### 176

#### *3.3. Activities of the Antioxidant and Caspase Enzymes*

Before Cd exposure, the enzyme activities of T-AOC, SOD, GSH-Px, Casp3, and contents of GSH showed no significant variation among treatments (Figure 5). Dietary taurine supplementation significantly enhanced the activities of CAT in the A strain compared to other groups (*p* < 0.05), while MDA contents were significantly lower in the F strain than in the A strain (*p* < 0.05). After Cd exposure, no significant differences were found among groups in the activities of T-AOC, SOD, or Casp3 or in GSH content. The A strain had significantly higher GSH-Px activities and MDA contents than the F strain (*p* < 0.05). For the diet effects, dietary taurine supplementation elevated the CAT activities, whereas taurine decreased the MDA content after Cd exposure in both strains.

**Figure 5.** Antioxidant indices and Casp3 activity in the livers of gibel carp (A and F strains) before cadmium exposure (white bars) and after cadmium exposure (black bars).  Bars with different uppercase letters (A, B) represent significant differences between the A and F strains (*p* < 0.05). Bars with different uppercase letters (X, Y) represent significant differences between the control diet group and the taurine diet group (*p* < 0.05). Bars with different lowercase letters (a, b) indicate the interaction effect and represent significant differences among all groups (*p* < 0.05). Bars with \* indicate significant changes between before and after cadmium exposure (*p* < 0.05).

Cd exposure significantly inhibited the activities of CAT in both strains of gibel carp and reduced the content of MDA in the F strain. For both gibel carp strains, Cd exposure suppressed the activity of T-AOC, whereas the activity of Casp3 and the contents of GSH were elevated. However, the F strain fed the diet with taurine supplementation did not show significant differences in the activities of T-AOC or Casp3 or in GSH content. Among all groups, only the F strain given dietary taurine supplementation showed significant reduction in the activity of GSH-Px (*p* < 0.05).

#### *3.4. Antioxidant Pathways and Metallothionein Levels*

The expression levels of antioxidant genes and metallothionein are shown in Figure 6. Prior to Cd exposure, the gene expression levels of *prx2*, *hsp70*, and *mt* showed no significant differences among groups (*p* > 0.05). Dietary taurine supplementation significantly upregulated the expression of *bach1* and *nrf2* in both strains compared to the control group (*p* < 0.05). The F strain showed significantly higher mRNA levels of *keap1* than the A strain (*p* < 0.05).

**Figure 6.** Expression levels of genes related to antioxidation and metallothionein (*mt*) in the livers of gibel carp (A and F strains) before cadmium exposure (white bars) and after cadmium exposure (black bars).  Bars with different uppercase letters (A, B) represent significant differences between the A and F strains (*p* < 0.05). Bars with different uppercase letters (X, Y) represent significant differences between the control diet group and the taurine diet groups (*p* < 0.05). Bars with different lowercase letters (a, b) indicate a significant interaction effect and represent the differences among all the groups (*p* < 0.05). Bars with \* indicate significant changes between before and after cadmium exposure (*p* < 0.05).

After Cd exposure, no significant variation was observed in the mRNA levels of *bach1*, *keap1*, *nrf2*, or *hsp70* among all groups (*p* > 0.05). The A strain showed significantly higher mt mRNA levels than the F strain (*p* < 0.05). Interactions were identified in the expression of *prx2*, with the highest levels found in the F strain given dietary taurine supplementation (*p* < 0.05). Cd exposure enhanced the expression levels of *nrf2* and *mt* in both strains, while the A strain showed significant elevation of the mRNA level of *bach1* (*p* < 0.05). The expression of *prx2* was significantly upregulated after Cd exposure (*p* < 0.05), while no significant differences were observed in the F strains (*p* > 0.05). The expression of *keap1* was significantly higher after Cd exposure (*p* < 0.05), except for the F strain fed with the control diet.

#### *3.5. ER Stress*

As shown in Figure 7, no significant differences were observed in the expression of *xbp1* or *eif2a* among all groups before Cd exposure (*p* > 0.05). Dietary taurine supplementation significantly upregulated the mRNA levels of *ire1*, *perk*, and *chop* in the livers of both strains compared to the control group. The F strain had markedly higher levels of *atf6* than the A

strain (*p* < 0.05). Diets interacted with strains to affect the expression of *atf4* in the livers of gibel carp, with the F strain showed the highest levels among all groups (*p* < 0.05).

**Figure 7.** Expression levels of genes related to ER stress in the livers of gibel carp (A and F strains) before cadmium exposure (white bars) and after cadmium exposure (black bars).  Bars with different uppercase letters (A, B) represent significant differences between the A and F strains (*p* < 0.05). Bars with different uppercase letters (X, Y) represent significant differences between the control diet and taurine diet groups (*p* < 0.05). Bars with different lowercase letters (a, b) indicate the interaction effect and represent the differences among all groups (*p* < 0.05). Bars with \* indicate significant changes between before and after cadmium exposure (*p* < 0.05).

After Cd exposure, no significant variation was observed in the expression of *ire1*, *perk*, or *chop* among all groups (*p* > 0.05). The F strain showed significantly higher expression levels of *atf6*, *eif2a*, and *atf4* than the A strain (*p* < 0.05). Interactions were observed in the mRNA level of *xbp1*, with the highest level found in the A strain fed the control diet (*p* < 0.05). Cd exposure significantly induced higher mRNA levels of *ire1*, *perk*, *atf6*, *xbp1*, *eif2a*, and *atf4* in the livers of both strains, whereas the A strain fed the control diet had the higher *chop* mRNA levels (*p* < 0.05).

#### *3.6. Autophagy and Apoptosis*

Hepatic mRNA levels related to autophagy and apoptosis were investigated in both strains (Figure 8). Prior to Cd exposure, no significant differences were found in the expression of *atg12*, *atg5*, *beclin1*, or *bcl2* (*p* > 0.05). The mRNA levels of *lc3b* in the A strain were significantly lower than in the F strain among all groups (*p* < 0.05). Dietary taurine supplementation elevated the expression of *ero1α* and *bax* (*p* < 0.05). The diets and strains interacted to affect the expression of *casp9* and *casp3*, with the highest levels found in the A strain fed the taurine diet (*p* < 0.05).

**Figure 8.** Expression levels of genes related to autophagy and apoptosis in the livers of gibel carp (A and F strains) before cadmium exposure (white bars) and after cadmium exposure (black bars).  Bars with different uppercase letters (A, B) represent significant differences between A and F strains (*p* < 0.05). Bars with different upper-case letters (X, Y) represent significant differences between the control diet group and the taurine diet group (*p* < 0.05). Bars with different lowercase letters (a, b) indicate the interaction effect and represent the differences among all groups (*p* < 0.05). Bars with \* mean significant changes between before and after cadmium exposure (*p* < 0.05).

After Cd exposure, the expression of *atg12*, *atg5*, *beclin1*, *bcl2*, and *casp9* showed no significant differences among all groups (*p* > 0.05). The A strain showed significantly higher levels of *lc3b*, *ero1α*, and *bax* and significantly lower levels of *casp9* than the F strain (*p* < 0.05). Cd exposure significantly upregulated the mRNA levels of *atg12*, *atg5*, and *ero1α* in the livers of both strains. However, the upregulated levels of *lc3b* were only found in the F strain fed the taurine diet. The increased expression of *beclin1* was found in the A strain fed the control diet and the F strain fed the taurine diet (*p* < 0.05). The A strain subjected to the taurine diet had significantly higher hepatic mRNA levels of *casp3* and *casp9*. Cd exposure induced significant upregulation of the expression of *bax* among all groups (*p* < 0.05) except for the A strain fed the diet with taurine supplementation (*p* > 0.05).

#### *3.7. Heatmap Cluster Analysis*

The mean values of molecular (gene expression) signatures of all treatments are presented in the clustering heatmap (Figure 9). Obvious differences were observed in the two strains before or after Cd exposure. Molecular expression of genes involved in antioxidant response, ER stress, and autophagy in the F strain after Cd exposure was not in a cluster with other treatments, especially in the F strain fed the taurine diet.

**Figure 9.** Gene expression heatmap of genes related to antioxidation, ER stress, autophagy, and apoptosis in the livers of gibel carp.

#### **4. Discussion**

Taurine has been reported to have beneficial effects on the growth performance of aquatic animals fed low fish meal diets in species such as black carp and white shrimp [30,31]. In the Phase 1 period of the present study, no significant effects on FBW or SGR were observed in either strain of gibel carp subjected to 8 weeks of taurine supplementation. Consistent with our results, the positive effects of dietary taurine supplementation (0, 0.5, 1.0, 1.5, and 2.0%) on growth improvement in yellowtail disappeared after six weeks of feeding, although higher final body weight was observed in fish fed a taurine diet for three weeks [32]. Dietary taurine supplementation significantly decreased FE and increased FCR in both strains. When the dietary taurine level exceeds the basic nutritional requirement, this can lead to feed intake reduction, as has been reported in Nile tilapia [33]. Therefore, the effects of dietary taurine supplement on growth of aquatic animals may be dose or time dependent.

As a semi-essential amino acid, taurine has physiological functions in the antioxidant and anti-apoptosis responses. Being a non-essential heavy metal, Cd exerts its effects and causes damage to tissues primarily through peroxidation and apoptosis [34]. Reports have shown that dietary taurine supplementation could mitigate Cd toxicity in catfish and red sea bream [16,24]. In the present study, histological observations showed that 96 h Cd exposure caused degenerated cristae, swelling of mitochondria, and irregular parallel stacked endoplasmic reticulum, and plaques in the cytoplasm of hepatocytes of both strains, suggesting that Cd damaged the mitochondria and endoplasmic reticulum in the liver cells of gibel carp. Meanwhile, Cd triggered apoptosis signals, as shown by the TUNEL results. Nevertheless, the apoptosis index was significantly lower in both strains fed diets with taurine supplementation compared to the control groups. Therefore, dietary taurine supplementation could apparently mitigate Cd-induced hepatic damage in gibel carp as in catfish and red sea bream [16,24]. Metallothionein (mt) is considered as a biomarker in the Cd detoxification process, as it can combine with Cd to form a Cd-MT complex [35]. In the present study, hepatic mRNA levels of *mt* increased significantly after Cd exposure,

indicating that Cd triggered the protective proteins to counteract the damage to the liver. To further elucidate the potential protective effects of taurine in gibel carp against Cd toxicity, we investigated the antioxidant response, ER stress, autophagy, and apoptosis.

Induction of oxidative stress is one of the toxicological mechanisms involved in heavy metal stress in fish, where the production of ROS (reactive oxygen species) causes oxidative damage to cells. Previous studies have shown that the hepatic enzyme activity of SOD increased significantly in rainbow trout after 7 days of waterborne Cd exposure [36]. Meanwhile, 21 days of waterborne Cd exposure enhanced the hepatic enzyme activity of SOD in catfish [37]. In the present study, Cd exposure elevated the SOD activity in gibel carp fed the control diet. However, no significant differences were found in hepatic SOD activities in fish fed diets with taurine supplementation, implying the protective role of taurine against Cd exposure in gibel carp. MDA is considered as a biomarker of the lipid peroxidation level under oxidative stress [38]. Dietary taurine supplementation significantly decreased MDA levels in the liver, which is consistent with the results for hepatic SOD activity. Cd exposure suppressed the enzyme activity of CAT in the livers of both strains, but the F strain showed higher levels than the A strain. Moreover, Cd exposure inhibited the activities of T-AOC while elevating the activity of Casp3 and the contents of GSH. However, no significant differences were observed in the activities of T-AOC or Casp3 or in the content of GSH in the F strain fed the taurine diet. Additionally, even though the activity of GSH-Px showed no variation among groups, the lowest level was found in the F strain fed the diet with taurine supplementation. Overall, taurine may exert its protective function against Cd poisoning more efficiently in the F strain than in the A strain.

Nuclear factor erythroid 2-related factor 2 (nrf2) is a key transcriptional factor involved in the regulation of the cellular antioxidant response [39]. Nrf2 regulates downstream antioxidant-related genes such as *keap1*, *prx2*, *bach1*, and *hsp70* to alleviate oxidative stress in organisms [40]. In the present study, the nrf2 signaling pathway was activated, as indicated by upregulation of *nrf2* mRNA levels in both strains fed the taurine supplemented diet. In zebrafish, the nrf2 pathway demonstrated protective effects by mitigating Cd-induced cellular oxidative damage [41]. Before Cd exposure, the expression levels of *nrf2* and *bach1* were significantly higher in both strains fed diets with taurine supplementation than in the control groups, indicating that dietary taurine could enhance the antioxidant potential of gibel carp, while such beneficial effects were not observed after the Cd exposure. Moreover, Cd exposure downregulated the expression levels of *prx2*, except in the F strain fed the diet with taurine supplementation. Taken together, the results suggest that taurine had a protective role against Cd-induced damage in both strains, especially in the F strain.

The endoplasmic reticulum is a dynamic organelle that is responsible for folding and assembly of proteins [42]. ER stress and its downstream signaling pathways play a crucial regulatory role in response to heavy-metal-induced toxic effects [43]. Previous studies had indicated that Cd waterborne could induce ER stress in both strains of gibel carp [25]. In the present study, the expression levels of the ER-stress-related genes *ire1*, *perk*, *atf6*, *xbp1*, *eif2α*, and *atf4* were increased after Cd exposure. In other words, all branches of the regulatory pathways of PERK-eIF2a-ATF4, IRE1-XBP1, and ER stress transducers ATF6 were induced after Cd exposure, suggesting the occurrence of ER stress in gibel carp exposed to Cd. The phosphorylation dependence of PERK induces dissociation of Nrf2/Keap1 complexes, thereby triggering the transcription of downstream genes involved in antioxidant pathways [44]. The expression level of *perk* had a variation trend similar to that of *nrf2*. Meanwhile, hepatic histological alterations such as swelling of mitochondria and irregular parallel stacks of ER were triggered by Cd exposure, observations that confirmed the ER stress in gibel carp.

Autophagy refers to a catabolic process in which cytoplasmic constituents and organelles in the lysosome are degraded to maintain homeostasis as an adaptative response to stressful conditions [45]. Autophagic pathways can be triggered through induction of ER sensors under long-lasting ER stress [46]. It has been reported that Cd exposure could cause

such a stress response, eliciting ER-stress-mediated autophagic and apoptosis processes in both strains of gibel carp [9,25]. Moreover, the formation of autophagosomes requires two ubiquitin-like conjugation pathways: one involves the formation of the multimeric complex of ATG5-ATG12-ATG16 conjugation; the other results in the conjugation of phosphatidylethanolamine (PE) to LC3b for the expansion of autophagic membranes [45,47]. In the present study, Cd exposure upregulated the hepatic mRNA levels of *atg5* and *atg12* in both strains regardless of the diet effect, suggesting that autophagic processes may be triggered by the increasing level of the ATG5-ATG12 complex. The mRNA levels of *lc3b* were only elevated in the F strain fed with the taurine diet, implying that more conjugation pathways were stimulated in the F strain. Thus, stronger autophagy may have been triggered in the F strain fed the taurine diet. Furthermore, Beclin-1 is a critical regulator of autophagy, because it participates in the formation of autophagosomes [48]. The transcriptional levels of *beclin1* were increased in the A strain fed the control diet and the F strain fed the taurine diet. Taken together, the results suggest that Cd exposure induced the autophagic process, and stronger autophagy responses were observed in the F strain fed the taurine diet.

Autophagy may play a protective role in cell survival, and extensive autophagy may trigger apoptosis as an independent pathway of cell death [49]. Apoptosis is also known as a cellular biomarker of metal-induced physiological alterations in aquatic animals [50]. Cd exposure was reported to induce apoptosis in topsmelt, purse red common carp, and gibel carp [25,51,52]. Apoptosis can be triggered by three main pathways, one of which is upstream caspase activation and includes the enzymes Caspase 9 and Caspase 3 [53]. In the present study, the transcriptional levels of *casp9* and *casp3* were inhibited in the A strain fed the diet supplemented with taurine. The mRNA levels of *casp3* were not consistent with the Casp3 activities, possibly due to a feedback response; a similar result has been reported in *Litopenaeus vannamei* [54]. Meanwhile, the apoptotic index in the A strain after Cd exposure was higher than in other groups as shown by the TUNEL results, suggesting that Cd exposure caused higher levels of apoptosis, and dietary taurine supplementation manifested its antioxidant effects through the regulation of the caspase gene in the A strain. In the apoptotic process, bcl2 is a member of the anti-apoptosis protein family, while bax and ero1α have opposite functions [47,55]. In the present study, no significant variation in *bcl2* expression was found after exposure to Cd, while the expression of *ero1α* was significantly elevated, indicating that apoptosis was induced by Cd exposure. The mRNA levels of *bax* increased after Cd exposure in all groups, but unaltered mRNA levels of *bax* were found in the A strain fed taurine, implying that dietary taurine supplementation alleviated the Cd toxicity by attenuating apoptosis in the A strain compared to the F strain.

Hierarchy cluster heatmap analysis showed that significant differences were observed in the two strains before or after Cd exposure, which verified the effects induced by Cd exposure as mentioned above. Expression levels of genes involved in antioxidant response, ER stress, and autophagy in the F strain post Cd exposure was not in cluster with other treatments, especially in the F strain fed the taurine diet, which was in line with previous results. Differential responses between the A and F strains of gibel carp were investigated in our previous studies owing to their genetic differences produced by selection [9,25]. The A strain was produced from eggs of gibel carp D strain and the sperm of gibel carp A strain, while the F strain was produced by the eggs of gibel carp E strain via stimulation with blunt snout bream sperm [56,57]. Therefore, a partial genome from the blunt snout bream may have been introduced into the genome of the F strain; this may have caused genetic differences between the A and F strains that led to differential genomic expression between the two strains upon Cd exposure. In the present study, even the growth performance was not significantly improved by dietary taurine supplement, but the detoxication of taurine might help to increase the survival rate of fish and raise fish quality, thereby improving the economic benefits.

#### **5. Conclusions**

Our study found that Cd exposure induced damage and oxidative stress in the livers of both strains of gibel carp, thereby triggering the occurrence of ER stress and the downstream responses of autophagy and apoptosis. Dietary taurine supplementation had no significant effect on the growth performance of gibel carp but did alleviate the Cd toxicity in both strains via specific genetic pathways. Dietary taurine played a protective role in mitigating Cd toxicity in the F strain through the antioxidant response, ER stress response, and autophagy, while in the A strain taurine alleviated cadmium toxicity by attenuation of apoptosis. In conclusion, the present study has provided evidence for the use of taurine in intervention or therapy for Cd poisoning in fish; thus, providing useful information for selective breeding in aquaculture.

**Author Contributions:** Conceptualization, investigation, methodology, validation, visualization, writing—review and editing, funding acquisition, W.X.; Conceptualization, data curation, methodology, formal analysis, writing—original draft, H.L. (Hongyan Li); Data curation, L.W.; Conceptualization, funding acquisition, project administration, supervision, writing—review and editing, J.J.; Supervision, D.H., X.Z., Y.Y. and H.L. (Haokun Liu); Funding acquisition, investigation, supervision, S.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was financially supported by the China Agriculture Research System of MOF and MARA (CARS-45-09), the National Natural Science Foundation of China (32122089; U19A2041; 32102811; 31972805), the National Key R&D Program of China (2018YFD0900605; 2019YFD0900200) and the Strategic Priority Research Program of Chinese Academy of Sciences (XDA24010206).

**Institutional Review Board Statement:** All animal care and experimental procedures were approved by the Experimental Animal Ethics Committee of Institute of Hydrobiology, Chinese Academy of Sciences (approval ID: IHB20140724).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** The authors thank Guanghan Nie for his technical support with the research system.

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

#### **References**

### *Article* **A Novel Disease (Water Bubble Disease) of the Giant Freshwater Prawn** *Macrobrachium rosenbergii* **Caused by** *Citrobacter freundii***: Antibiotic Treatment and Effects on the Antioxidant Enzyme Activity and Immune Responses**

**Caiyuan Zhao 1, Huagen Wen 2, Shengsheng Huang 2, Shaoping Weng 2 and Jianguo He 1,2,\***

**Abstract:** The giant freshwater prawn, *Macrobrachium rosenbergii*, is an important and economical aquaculture species widely farmed in tropical and subtropical areas of the world. A new disease, "water bubble disease (WBD)", has emerged and resulted in a large loss of *M. rosenbergii* cultured in China. A water bubble with a diameter of about 7 mm under the carapace represents the main clinical sign of diseased prawns. In the present study, *Citrobacter freundii* was isolated and identified from the water bubble. The optimum temperature, pH, and salinity of the *C. freundii* were 32 ◦C, 6, and 1%, respectively. A challenging experiment showed that *C. freundii* caused the same typical signs of WBD in prawns. Median lethal dose of the *C. freundii* to prawn was 104.94 CFU/g. According to the antibiogram tests of *C. freundii*, florfenicol and ofloxacin were selected to evaluate their therapeutic effects against *C. freundii* in prawn. After the challenge with *C. freundii*, 86.67% and 72.22% survival of protective effects against *C. freundii* were evaluated in the oral florfenicol pellets and oral ofloxacin pellets feding prawns, respectively, whereas the mortality of prawns without fed antibiotics was 93%. After antibiotic treatment and *C. freundii* infection, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), malondialdehyde (MDA), acid phosphatase (ACP), alkaline phosphatase (ALP), and lysozyme (LZM) in the hemolymph and hepatopancreas of the prawns and the immune-related gene expression levels of Cu/Zn-SOD, CAT, GPx, GST, LZM, ACP, anti-lipopolysaccharide factor, crustin, cyclophilin A, and C-type lectin in hepatopancreas were all significantly changed, indicating that innate immune responses were induced by *C. freundii*. These results can be beneficial for the prevention and control of *C. freundii* in prawns.

**Keywords:** *Macrobrachium rosenbergii*; water bubble disease; *Citrobacter freundii*; antibiotic treatment; antioxidant enzyme activity

#### **1. Introduction**

The giant freshwater prawn, *Macrobrachium rosenbergii*, is a member of arthropods, belonging to genus *Macrobrachium*, which is the largest freshwater and the most favored prawn for aquaculture in tropical and subtropical areas of the world [1–3], with annual yields of 290,708 metric tons [4]. *M. rosenbergii* has already been developed into an important economically aquaculture species in China [5–8]. The outbreaks of bacterial diseases in *M. rosenbergii* aquaculture have been reported, such as muscle necrosis disease caused by *Enterococcus* or *Pseudomonas aeruginosa* [9–11], *Aeromonas* species (*A. veronii* and *A. caviae*) and *Vibrio* species (*V. alginolyticus* and *V. parahaemolyticus*) [12,13] caused high-mortality

**Citation:** Zhao, C.; Wen, H.; Huang, S.; Weng, S.; He, J. A Novel Disease (Water Bubble Disease) of the Giant Freshwater Prawn *Macrobrachium rosenbergii* Caused by *Citrobacter freundii*: Antibiotic Treatment and Effects on the Antioxidant Enzyme Activity and Immune Responses. *Antioxidants* **2022**, *11*, 1491. https:// doi.org/10.3390/antiox11081491

Academic Editors: Bo Liu, Changyou Song and Cunxin Sun

Received: 14 June 2022 Accepted: 26 July 2022 Published: 29 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

diseases. A new epidemic disease, referred to as "water bubble disease" (WBD), recently occurred in cultured *M. rosenbergii* in China, and it caused a mortality of over 30% in diseased prawns.

As *M. rosenbergii* is an invertebrate, it lacks the typical acquired immunity of vertebrates and entirely relies on its innate immunity system to resist infection by various pathogens [14]. Enhanced knowledge about the immune system of *M. rosenbergii* is crucial for disease management [15]. The innate immune system provides defense against invading bacteria through AMPs, enzymes, and cellular components [16]. Antiproteases, myeloperoxidase, and lysozyme (LZM) have been used to learn about and determine the innate immune status of an organism [17]. During pathogen invasion, host cells produce abundant reactive oxygen species (ROS) to kill the invading pathogens. However, a considerably high level of ROS in the body will cause the destruction and damage of DNA and other biological macromolecules [18]. Several major antioxidant enzymes include glutathione peroxidase (GPx), glutathione S-transferase (GST), catalase (CAT), malondialdehyde (MDA), and superoxide dismutase (SOD), which play important roles in anti-oxidization damage and related coping mechanisms [19,20]. Acid phosphatase (ACP), and alkaline phosphatase (ALP) are symbols of macrophage activation, important components of the lysosome system, and play important roles in the innate immune system by engulfing antigens and LZM intracellularly [21]. C-Type lectin (CTL), cyclophilin A (CypA), and AMPs, such as anti-lipopolysaccharide factor (ALF) and crustins, play important roles in the innate immunity for the recognition of effector molecules and clearance of bacteria, fungi, and viruses [22–29]. Antibiotics are commonly used in aquaculture to control the diseases caused by bacteria [30]. Antibiotics at therapeutic levels are mainly administered for short periods of time via the oral route (the most common route for the delivery occurs by mixing the antibiotic with commercial feed) to groups of prawn. Antibiotics legally used in aquaculture must be authorized by Food and Drug Administration (FDA), and abide by the rules for antibiotic use, comprising permissible routes of delivery, dose forms, withdrawal times, tolerances, and dose rates and limitations of specific species. In Norway, antibiotics were sold in pharmacies or in feed plants approved by the Norwegian Medicines Agency, and the amount of antibiotics used and retain records in prescriptions of veterinarian were mandatory to report [31].

In the present study, we aimed to isolate and identify the pathogen associated with WBD in *M. rosenbergii* using challenge experiments, molecular methods and antibiotic treatment. The activities of antioxidant enzymes (i.e., SOD, CAT, GPx, GST, and MDA), ACP, ALP, and LZM and the immune-related gene expression levels of Cu/Zn-SOD, CAT, GPx, GST, LZM, ACP, ALF, crustin, CypA, and CTL in *M. rosenbergii* after *C. freundii* challenge were investigated. The results of the present study will expound the pathogenesis of bacteria in *M. rosenbergii* and facilitate the further prevention and control of *C. freundii* in *M. rosenbergii*.

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

#### *2.1. Strain Isolation and Identification*

In November 2019, 30 *M. rosenbergii* (mean body weight 13.5 ± 2.3 g, body length 13.1 ± 1.2 cm) with WBD were collected in Zhaoqing, Guangdong, China. The water bubble diseased *M. rosenbergii* were dissected aseptically, and liquid sample of the water bubble was streaked onto a Luria-Bertani (LB) agarose plate (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agarose powder 15 g/L, pH 7.4) (Sangon Biotech, Shanghai, China). The inoculated plates were incubated for 48 h at 28 ◦C, and single colonies were obtained and labeled GDZQ201912. After incubation in liquid LB culture (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7.4) for 24 h, the bacteria were collected and stored at −80 ◦C in 50% glycerol (*v*/*v*).

Biochemical characterization of the isolated strain was identified by Gram staining, shape, and motility, followed by biochemical tests and carbohydrate utilization test. The biochemical tests were performed with GEN III MicroPlate on BIOLOGY (Biology, Vacaville, CA, USA). Transmission electron microscopy (TEM) (JEOL JEM-1400, JEOL, Tokyo, Japan) was used to observe the isolated strain at 120 kV.

#### *2.2. Gene Sequencing and Phylogenetic Analysis*

Genomic DNA of the bacterial isolate was extracted by using FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, Nanjing, China). The 16S rRNA gene of the bacterium was amplified for polymerase chain reaction (PCR) analysis in accordance with the method of Lee et al. [32]. The following amplification primers were used: F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and R (5 -CGGTTACCTTGTTACGACTT-3 ) (NR\_176804.1). The PCR program was carried out as follows: denaturation at 95 ◦C for 10 min, 30 cycles of denaturation at 95 ◦C for 30 s, annealing at 53 ◦C for 30 s, extension at 72 ◦C for 80 s, and a final extension at 72 ◦C for 10 min. PCR products were run on a TBE-agarose gel, and the DNA was purified using a TaKaRa MiniBEST Universal Genomic DNA Extraction Kit (Takara, Kusatsu, Japan) and sub-cloned into the pMD19-T Easy Vector (Takara, Japan) for sequencing.

The 1416 bp PCR product was sequenced and blast analyzed. The 16S rRNA gene sequence of GDZQ201912 was aligned with the 16S rRNA gene sequences of other *Citrobacter* (KC210829.1, FN997639.1, MG011554.1, AP022399.1, AP022486.1, and AP022513.1), *Edwardsiella* (FJ405305.1, EF467289.1, GQ180182.1, FJ405309.1, GQ180181.1, and KC309472.1), *Vibrio* (MT071600.1, MT269596.1, MT307282.1, and MT505697.1), and *Aeromonas* species (EU770272.1, KF358430.1, MK182872.1, and MK182893.1) retrieved from the National Center for Biotechnology Information database. The phylogenetic tree based on 16S rRNA gene sequences of the bacteria cells was constructed via the neighbor-joining (NJ) method with the MEGA program (version 7) using a maximum composite likelihood model. The robustness of the NJ tree was assessed using the approximate likelihood-ratio test and bootstrapping with 1000 replicates. Bootstrap values were shown at each node.

#### *2.3. Effects of Temperature, pH, and Salinity on the Growth of Citrobacter freundii*

The isolated strain was inoculated into the LB liquid medium and homogenized. Different temperatures (24 ◦C, 28 ◦C, 32 ◦C, and 42 ◦C) were used to test their effects on the growth of *C. freundii* at pH 7.0 and salinity 1%. Different pH (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0) were used to test their effects on the growth of *C. freundii* at 28 ◦C temperature and 1% salinity. Different salinities (0%, 1%, 2.0%, 3%, 4%, 5%, 6%, and 7%) were used to test their effects on the growth of *C. freundii* at 28 ◦C temperature and pH 7.0. A 210 μL LB liquid medium containing the isolated strain (102 CFU/mL) of different temperatures, pH, and salinities was added to each well of 96-well plates. Then, the bacteria were sampled regularly in triplicate to determine the optical density at a wavelength of 600 nm (OD600) per 30 min and cultured for 24 h.

#### *2.4. Antibiotic Susceptibility Test*

The antibiotic susceptibility test was performed using 106 CFU/mL isolated strain in the disk diffusion method as described by Lalitha [33]. The isolated strain was tested following the antimicrobial agents (disk content indicated in parentheses): penicillin (10 μg), doxycycline (30 μg), florfenicol (30 μg), norfloxacin (10 μg), ofloxacin (5 μg), amoxicillin (20 μg), ampicillin (10 μg), cefepime (30 μg), ceftriaxone (30 μg), neomycin (30 μg), gentamicin (10 μg), streptomycin (10 μg), clindamycin (2 μg), lincomycin (2 μg), tetracycline (30 μg), novobiocin (30 μg), polymyxin B (300 μg), and rifampin (5 μg) (HANGWEI, Beijing, China). The sizes of the inhibition zones were measured and recorded triplicately. The experimental results were judged in accordance with the clinical and laboratory standards institute standards [34].

#### *2.5. Experimental Challenge*

Prawns were obtained from an aquaculture farm in Guandong and acclimated in a recirculating-water aquarium system filled with aerated freshwater for a week. The prawns were fed twice per day with commercial pellet feed (Table 1). Water was maintained at 28 ± 1 ◦C using a water heater. Healthy prawns (11.00 ± 1.38 g) were used in the following challenge tests. The challenge tests included two groups: a control group, in which 30 prawns were injected intramuscularly with 100 μL phosphate-buffered saline (PBS) at the junction between the 3rd and 4th abdominal segments, and an infected group, in which 30 prawns were injected with 100 μL bacteria inocula (106.76 CFU/g) at the same junction. The 30 prawns of the normal or infected group were stocked in three 36 L aquaria (10 animals per aquarium). The survival rates and clinical signs of the infected and control groups were recorded daily.

**Table 1.** Ingredient composition and proximate analysis of commercial feed used for *M. rosenbergii*.

a Mineral premix contains: Mn, 11.0%; Zn, 10.0%; Fe, 8.0%; I, 0.36%; Co, 0.10%; Ca (carrier). b Vitamin premix contains: vitamin B1, 1.63%; riboflavin, 1.72%; pyridoxine, 1.2%; nicotinic acid, 9.97%; folie acid, 0.34%; vitamin B12, 0.003%; pantotbenic acid, 3.89%; ascorbic acid, 43.69%; vitamin A, 1700 IU/kg; vitamin D3 1200 IU/kg; vitamin E, 66.138 IU/kg; etboxyquin, 0.66%. c Acid hydrolysis.

#### *2.6. Median Lethal Dose Determination*

In total, 240 healthy prawns (8.00 ± 0.79 g) were used and stocked in 36 L aquaria (10 animals per aquarium). The *C. freundii* (108.01 cells/g) were used for the challenge experiments. For each trial, 10 animals were injected intramuscularly with the *C. freundii* (100 μL) at 10-fold serial dilutions (100–10−6). Animals serving as negative controls were injected with the same volume of PBS. Each treatment was carried out in triplicate. The inoculum and PBS were injected at the junction between the third and fourth abdominal segments. Dead, moribund animals, and clinical signs in each treatment group were recorded and observed at 24 h intervals and examined by the method described in Section 2.1 to ensure the accuracy of the median lethal dose (LD50). The LD50 was calculated using the Behrens-Kärber method [35].

#### *2.7. Antibiotic Treatment*

Based on the results of drug sensitivity tests on GDZQ201912, florfenicol and ofloxacin were selected for the evaluation of their therapeutic effects against *C. freundii* in *M. rosenbergii* by common oral pellets containing antibiotics. Florfenicol and ofloxacin were mixed in the pellets based on the 20.0 mg/kg prawn body weight dosage. All of the pellets were coated on the surface feed using edible oil. In total, 360 healthy prawns (7.00 ± 1.03 g) were used and stocked in 36 L aquaria (10 animals per aquarium). The experiment was divided into two groups depending on the different oral antibiotic mixed pellets and time. Group I prawns was treated with antibiotic pellets at the same time with *C. freundii* injection to evaluate their therapeutic effects against *C. freundii*, and group II was treated with antibiotic pellets at the time of typical signs appear after the challenge with *C. freundii* to evaluate the therapeutic effects of the pellets against *C. freundii*. Each group underwent six trials: feeding with common pellets after *C. freundii* injection, feeding with florfenicol mixed pellets after

*C. freundii* injection, feeding with ofloxacin mixed pellets after *C. freundii* injection, feeding with common pellets after PBS injection, feeding with florfenicol mixed pellets after PBS injection, and feeding with ofloxacin mixed pellets after PBS injection. Each treatment included 10 prawns and had 3 replicates, and the experimental observation period was 14 days. The concentration of *C. freundii* used to injected prawns was 100-fold LD50 of *C. freundii* in *M. rosenbergii* and the same volume described in Section 2.6. The prawns of PBS injection trials were injected with the same volume of PBS. The relative percent survival (RPS) of each trial was counted by the formula: (1% immunized mortality/% control mortality) × 100% [36].

#### *2.8. Immune and Anti-Oxidization Parameter Changes in Hemolymph and Hepatopancreas*

The hemolymph and hepatopancreas were also collected from group I in the treatment with antibiotics at 1-, 3-, 5-, 7-, 9-, 11-, and 14-days for the following immune enzyme activity assay. The determination of ACP, ALP, CAT, GPx, GST, LZM, MDA, and SOD activities in the hemolymph and hepatopancreas was performed following the protocols of the commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

#### *2.9. Expressions of Immune-Related Genes in Hepatopancreas*

The total RNA of hepatopancreas was extracted using a high-purity total RNA Rapid Extraction Kit (Promega, Madison, WI, USA), in accordance with the manufacturer's protocol. The final RNA was resuspended in 50 mL diethyl pyrocarbonate water and stored at −80 ◦C. Easy Script One-Step gDNA Removal and cDNA Synthesis SuperMix (AccurateBiology, Changsha, China) were used to synthesize first-strand cDNA following the manufacturer's instructions. Quantitative PCR (qPCR) was operated on a LightCycler 480 (Roche Applied Science, Basel, Switzerland) with a final 10 μL reaction volume including 0.5 μL of each primer (10 mM), 5 μL SYBR® Green Realtime PCR Master Mix (AccurateBiology, China), 1 μL cDNA, and 3 μL DNase/RNase-free water. The immune-related gene expression levels of ACP, ALF, CAT, crustin, CTL, Cu/Zn-SOD, CypA, GPx, GST, and LZM were evaluated using qPCR to determine the changes after *C. freundii* infection in prawns, with β-actin acting as the house-keeping gene. The PCR cycling conditions and calculation of the relative expression of each gene were performed as described elsewhere [37]. Table 2 shows the primers used to amplify the above genes.

**Table 2.** Primers used for quantitative real-time PCR (qRT-PCR).

**Table 2.** *Cont.*

#### *2.10. Statistical Analysis*

All data were statistically analyzed using SPSS 21.0 software (IBM, New York, NY, USA), and the mean ± standard deviation was determined. *p*-value < 0.05 and <0.01 was considered significant after each measured datum was subjected to Least-significant Difference test. Different graphemes meant significant differences, and the same graphemes meant no significant difference. All columns in this study were drawn with GraphPad Prism 6.0 (San Diego, CA, USA).

#### **3. Results**

#### *3.1. Clinical Symptoms of WBD in M. rosenbergii*

A total of 30 infected *M. rosenbergii* and 30 healthy *M. rosenbergii* were used to examine the clinical signs of *C. freundii* infection. Compared with normal prawns, *C. freundii*infected prawns showed a 7 mm-diameter bubble under the carapace of the diseased prawn (Figure 1), loss of appetite, inactivity, and weight loss. The 30 infected prawns all had a water bubble under the carapace. *C. freundii* infection caused mass mortality of the infected prawns after the onset of clinical symptoms and the rapid spread of the disease between prawns.

**Figure 1.** *Macrobrachium rosenbergii* infected with *Citrobacter freundii* showing typical signs of WBD under the carapace (red arrow).

#### *3.2. Molecular Identification and Biochemical Characterization*

The *C. freundii* strain GDZQ201912 was a motile, rod shaped, Gram-negative bacterium (Figure 2A). *C. freundii* was rod-shaped with blunt edges at both ends by TEM (Figure 2B). Ten individuals were randomly measured. The width and length of the bacteria were 0.67–0.76 and 1.88–2.07 μm, respectively.

**Figure 2.** Morphological characteristics and TEM observation of *C. freundii* strain GDZQ201912. (**A**) Gram-stained strain GDZQ201912 observed by light micrograph (1000×). Scale bar 50 μm. (**B**) TEM observation of strain GDZQ201912. Scale bar 1 μm.

The 16S rRNA gene amplified from strain GDZQ201912 was sequenced and BLAST analyzed against the non-redundant database, which showed 99.66% identity with *C. freundii* (GenBank Accession Number: CP060662.1). Tables 3 and 4 present the physiological and biochemical characteristics of strain GDZQ201912, respectively. The molecular sequence and biochemical analyses revealed that the isolated bacterium was *C. freundii*.

**Table 3.** Morphological properties and conventional tests of strain GDZQ201912 isolated from *Macrobrachium rosenbergii*.

"+" positive reaction, "−" negative reaction; "V" variable (positive or negative reaction). \* Reference strain data compiled from Bergey's manual [38].

**Table 4.** Carbon source utilization reactions of strain GDZQ201912.

(+) positive and (−) negative. \* Reference strain data compiled from Bergey's manual [38].

#### *3.3. Phylogenetic Analysis*

A phylogenetic tree was constructed using the NJ method by the MEGA 7 software based on the 16S rRNA gene sequences of strain GDZQ201912, and the phylogenetic tree revealed the relationship of strain GDZQ201912 with other *Citrobacter* and pathogenic bacterium species. Bootstrap values above 50% are shown at the nodes. The 16S rRNA gene sequence of strain GDZQ201912 was found to be evolutionarily close to *C. freundii* with the highest bootstrap value (Figure 3).

**Figure 3.** Phylogenetic tree analysis of *Citrobacter* sp. and other pathogenic bacterium species based on 16S rRNA nucleotide sequences. The tree was generated using NJ method by the MEGA 7 software. Bootstrap values above 50% are shown at the nodes. The isolate strain GDZQ201912 identified in this study is indicated by the red shaded diamond.

#### *3.4. Effects of Temperature, pH, and Salinity on the Growth of C. freundii*

Figure 4 shows the effects of temperature, pH, and salinity on the growth of *C. freundii*. *C. freundii* exhibits a wide adaptability to temperature. It can grow well in a range from 24 ◦C to 42 ◦C (Figure 4A). The optimum temperature was 32 ◦C. *C. freundii* has a wide pH range and can grow normally at pH 4–9, whose optimal value was around pH 6 (Figure 4B). The maximum salt tolerance of *C. freundii* was 6%, whose optimum value was 1% (Figure 4C). Our results indicated that *C. freundii* has a wide range of temperature, pH, and salinity tolerance.

**Figure 4.** Growth (OD600) of strain GDZQ201912 at different temperatures (**A**), pH (**B**), and salinities (**C**).

#### *3.5. Experimental Challenge*

Prawns challenged with strain GDZQ201912 died from the third day post-injection (dpi), and no death occurred in the control groups (Figure 5). The challenged prawns exhibited the typical signs of WBD as described previously. The diseased prawns were similar to those observed in naturally infected prawns in 2019. The bacteria re-isolated from the water bubble of challenged prawns and were reconfirmed as *C. freundii*.

**Figure 5.** Cumulative mortality curves of the experimentally infected *M. rosenbergii* with strain GDZQ201912. Controls 1, 2, 3, and three replicates of healthy prawns injected with PBS; Groups 1, 2, 3, and three replicates of healthy prawns injected with bacterial inoculum. Each trial contained 10 animals.

#### *3.6. LD50 of Strain GDZQ201912 in M. rosenbergii*

Figure 6 shows the cumulative mortality rate of *M. rosenbergii* after post-infection with *C. freundii*. For each trial, 10 prawns were injected with bacteria inoculum at 100–10−6 dilutions, and the control group was injected with same volume of PBS. The control prawns showed no mortality during the experimental challenge. Among *M. rosenbergii* injected with serial dilutions of *C. freundii*, prawns injected with 100, 10−1, and 10−2 dilutions showed disease signs from 2, 3, and 4 dpi, respectively. Prawns injected with 10−3 and 10−4 dilutions exhibited disease symptoms from 5 dpi, whereas no death occurred among those injected with 10−5 and 10−6 dilution during the experimental challenge. The LD50 of *C. freundii* was 104.94 CFU/g (Figure 6).

**Figure 6.** Cumulative mortality curves for the determination of LD50 in *M. rosenbergii* challenged with *C. freundii* at different concentrations.

#### *3.7. Antibiogram Tests of Strain GDZQ201912*

Antibiogram tests of strain GDZQ201912 revealed its resistance to different antibiotics, such as penicillin, ampicillin, clindamycin, lincomycin, and novobiocin, whereas it showed sensitivity to doxycycline, florfenicol, norfloxacin, ofloxacin, amoxicillin, cefepime, ceftriaxone, neomycin, gentamicin, streptomycin, tetracycline, polymyxin B, and rifampin (Table 5). Among the 13 sensitive antibiotics, strain GDZQ201912 was the most sensitive to cefepime, followed ceftriaxone, ofloxacin, and florfenicol in fourth.

**Table 5.** Antimicrobial susceptibility analysis of *C. freundii* isolates from *M. rosenbergii* (strain GDZQ201912).

"+ + +": Highly sensitive; "+ +": Moderately sensitive; "±": Drug resistance.

#### *3.8. Antibiotic Treatment*

The *M. rosenbergii* mortalities caused by florfenicol and ofloxacin treatment at different time points after the intramuscular challenge with *C. freundii* or PBS were monitored for 14 days (Figure 7). Prawns fed with common pellets after *C. freundii* infection died from 5 dpi, and the death reached the maximum on 8 dpi with mortalities of 93.0%. In Group I, prawns fed with florfenicol mixed pellets after *C. freundii* infection died from 8 dpi, and the death reached the maximum on 8 dpi with mortalities of 19.40%; prawns fed with ofloxacin mixed pellets after *C. freundii* infection died from 7 dpi, and death reached the maximum on 8 dpi with mortalities of 32.83%. In Group II, prawns fed with florfenicol and ofloxacin mixed pellets after *C. freundii* infection all died from 5 dpi, and the death reached the maximum on 7 dpi with mortalities of 35.67% and 57.47%, respectively. No death was observed among prawns fed with common pellets and mixed pellets of florfenicol and ofloxacin after PBS injection. The evaluation of the therapeutic effects of antibiotic pellets revealed that the RPS of florfenicol and ofloxacin in group I against *C. freundii* in prawns were 86.67% and 72.22%, respectively (Figure 7A). The RPS of florfenicol and ofloxacin in Group II against *C. freundii* in prawns were 69.17% and 47.53%, respectively (Figure 7B). Both florfenicol and ofloxacin initially showed effective protection for *M. rosenbergii* against *C. freundii* (and especially florfenicol).

**Figure 7.** RPS of florfenicol and ofloxacin treatment at different time points after the challenge intramuscular with 100-fold LD50 *C. freundii* strain GDZQ201912 for 14 days. (**A**) RPS of prawns treated with antibiotic pellets at the same time with *C. freundii* challenge. (**B**) RPS of prawns treated with antibiotic pellets after death occurred post *C. freundii* challenge.

#### *3.9. Immune and Antioxidant Enzyme Activity Changes in Hemolymph*

The enzymatic activities of SOD, CAT, GPx, GST, MDA, ACP, ALP, and LZM of *M. rosenbergii* were measured at 1-, 3-, 5-, 7-, 9-, 11-, and 14-days after the *C. freundii* strain GDZQ201912 and florfenicol challenge. In hemolymph, the results revealed that the SOD activity in the groups fed with common and florfenicol mixed pellets significantly increased from three days post *C. freundii* infection, rapidly declined at five and seven days (*p* < 0.05, Figure 8A), and increased again to normal levels at 9-, 11-, and 14- days compared with the PBS control (fed with common pellets) group (*p* > 0.05, Figure 8A). No significant differences were observed between the SOD activities in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 8A).

The CAT activity in *M. rosenbergii* fed with common pellets significantly increased from three-, five-, and nine-days post *C. freundii* infection and reached the peak value at 5 dpi compared with the PBS control (fed with common pellets) group (*p* < 0.05, Figure 8B). This value significantly reduced to the lowest at 7-dpi. During this period, the CAT activity of prawns fed with florfenicol mixed pellets significantly increased from three and five days post *C. freundii* infection and reached the peak value at 5-dpi compared with the PBS control (fed with common pellets) group (*p* < 0.05, Figure 8B). No significant differences were observed between the CAT activities in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 8B).

The GPx activity in *M. rosenbergii* fed with common pellets significantly increased at 3-, 5-, 7-, 9-, 11-, and 14-days post *C. freundii* infection and reached the peak value at 5-dpi compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8C). During this period, the groups fed with florfenicol mixed pellets (*C. freundii* infection) presented similar trend levels of enzymatic activity, which were lower than that observed for the group fed with common pellets (*C. freundii* infection) and reached the peak value at 5 dpi compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8C). No significant differences were observed between the GPx activities in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 8C).

**Figure 8.** Changes in non-specific immune parameters of *M. rosenbergii* hemolymph at 1-, 3-, 5-, 7-, 9-, 11-, and 14-days after *C. freundii* infection. (**A**) SOD, (**B**) CAT, (**C**) GPx, (**D**) GST, (**E**) MDA, (**F**) ACP, (**G**) ALP, and (**H**) LZM activities. Data are presented as mean ± standard error of the mean (SEM) (*n* = 3); different graphemes (a, b, c, d, e, f, g, h and i) indicate significant difference values of non-specific immune parameters at *p* < 0.05.

The GST activity of prawns fed with common mixed pellets significantly increased at 3-, 5-, 7-, 9-, 11-, and 14-days post *C. freundii* infection and reached the peak value at 3 dpi compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8D). During this period, the groups fed with florfenicol mixed pellets (*C. freundii* infection) presented similar trend levels of enzymatic activity, which were lower than that observed for the group fed with common pellets (*C. freundii* infection) and reached the peak value at 3-dpi compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8D). No significant differences were observed between the GST activities in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 8D).

The MDA activity of prawns fed with common mixed pellets significantly increased at three, five, seven, and nine days post *C. freundii* infection and reached the peak value at 3-dpi compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8E). During this period, the groups fed with florfenicol mixed pellets (*C. freundii* infection) presented similar trend levels of enzymatic activity, which were lower than that observed for the group fed with common pellets (*C. freundii* infection) and reached the peak value at 3-dpi, compared with the PBS control (fed with common pellets) group (*p* < 0.05, Figure 8E). No significant differences were observed between the MDA activity in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 8E).

The ACP activity in *M. rosenbergii* fed with common pellets significantly increased from 3-days post *C. freundii* infection and peaked at 7-dpi (556.34 U/L) compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8F). Subsequently, this value declined at 9-, 11-, and 14-days. During this period, the two groups fed with florfenicol pellets (PBS and *C. freundii* infected) presented similar trend levels of enzymatic activity, which were lower than that observed for the group fed with common pellets and reached the peak values of 177.67 and 376.34 U/L on 5- and 7- dpi, respectively (*p* < 0.01). No significant differences were observed in this parameter in the PBS control (fed with common pellets) group (*p* > 0.05, Figure 8F).

The ALP activity of prawns fed with common and florfenicol mixed pellets significantly increased at three-, five-, seven-, and nine-days after *C. freundii* infection, with peak values of 98.67 and 64.67 U/L, respectively, at three and seven days compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8G). No significant differences were observed between ALP activities in the PBS control (fed with common pellets) group (*p* > 0.05, Figure 8G).

The LZM activity in *M. rosenbergii* fed with common pellets significantly increased from 3-, 5-, 7-, 11-, and 14-days post *C. freundii* infection and peaked 450.00 U/mL at 7 days compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 8H). This value declined to normal level at 9-dpi (*p* > 0.05, Figure 8H). During this period, the groups fed with florfenicol mixed pellets (*C. freundii* infection) presented similar trend levels of enzymatic activity, which were lower than that observed for the group fed with common pellets and reached the peak value at seven days (*p* < 0.05, Figure 8H). No significant differences were observed between the LZM activities in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 8H).

#### *3.10. Immune and Antioxidant Enzyme Activity Changes in Hepatopancreas*

The results revealed that the SOD activity in *M. rosenbergii* (fed with common and florfenicol mixed pellets) significantly increased at 3-days, decreased at 5- and 7-days post *C. freundii* infection (*p* < 0.05, Figure 9A), and increased again to normal levels at 11 and 14 days compared with the PBS control (fed with common pellets) group (*p* > 0.05, Figure 9A). No significant differences were observed between the SOD activities in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 9A).

The CAT activity in *M. rosenbergii* (fed with common and florfenicol mixed pellets) significantly increased at three and five days and reached the peak value at 3- and 5 dpi, respectively, while prawns fed with common pellets decreased at seven days post *C. freundii* injection compared with the PBS control (fed with common pellets) group

(*p* < 0.05, Figure 9B). No significant differences were observed between the CAT activities in the PBS control (fed with common pellets) groups (*p* > 0.05, Figure 9B).

The GPx activity in *M. rosenbergii* (fed with common and florfenicol mixed pellets) significantly increased at 3-, 5-, 7-, 9-, and 11-days post *C. freundii* injection and both reached the peak value at 5-dpi compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 9C). No significant differences were observed between the GPx activities in the PBS control (fed with common pellets) group (*p* > 0.05, Figure 9C).

The GST activity of prawns (fed with common and florfenicol mixed pellets) significantly decreased at 3-, 5-, 7-, 9-, and 11-days post *C. freundii* injection, compared with the PBS control (fed with common pellets) group (*p* < 0.05, Figure 9D). No significant differences were observed between the GST activities in the PBS control (fed with common pellets) and *C. freundii* infection groups (fed with florfenicol mixed pellets) (*p* > 0.05, Figure 9D).

The MDA activity of prawns (fed with common and florfenicol mixed pellets) significantly increased at 3-, 5-, and 7-days post *C. freundii* injection, compared with the PBS control (fed with common pellets) group (*p* < 0.05, Figure 9E). No significant differences were observed between the MDA activities in the PBS control (fed with common and florfenicol mixed pellets) groups (*p* > 0.05, Figure 9E).

The ACP activity in the hepatopancreas of *M. rosenbergii* fed with common and florfenicol mixed pellets significantly increased at 3-, 5-, 7-, 9-, 11-, and 14-days post *C. freundii* infection and reached the peak value at 5- and 7-dpi, respectively, compared with the PBS control (fed with common pellets) group (*p* < 0.05, Figure 9F). No significant differences were observed between the ACP activities in the PBS control (fed with common pellets and florfenicol mixed pellets) groups (*p* > 0.05, Figure 9F).

The ALP activity of prawns (fed with common and florfenicol mixed pellets) significantly increased at three- and five-days post *C. freundii* injection, compared with the PBS control (fed with common pellets) group (*p* < 0.05, Figure 9G). No significant differences were observed between the ALP activities in the PBS control (fed with common and florfenicol mixed pellets) groups (*p* > 0.05, Figure 9G).

The LZM activity in *M. rosenbergii* fed with common pellets significantly increased at three-, five-, and seven-days post *C. freundii* infection and reached the peak value at 5-dpi compared with the PBS control (fed with common pellets) group (*p* < 0.01, Figure 9H). This value declined to normal levels at 9-, 11-, and 14-dpi. During this period, the groups fed with florfenicol mixed pellets (*C. freundii* infection) presented similar trend levels of enzymatic activity, which were lower than that observed for the prawns fed with common pellets and reached the peak value at 7-days (*p* < 0.05, Figure 9H). No significant differences were observed between the LZM activities in the PBS control (fed with common and florfenicol mixed pellets) groups (*p* > 0.05, Figure 9H).

#### *3.11. Expression of Immune-Related Genes in Hepatopancreas after the Challenge with C. freundii*

Immunity-related genes (Cu/Zn-SOD, CAT, GPx, GST, LZM, ACP, ALF, crustin, CypA and CTL) mRNA expressions were determined in the hepatopancreas of *M. rosenbergii* at 1-, 3-, 5-, 7-, 9-, 11-, and 14-days after the challenge with *C. freundii* (Figure 10). These genes were up-regulated at different levels following the challenge with *C. freundii* in the groups fed with common and florfenicol mixed pellets compared with the PBS control groups (fed with common and florfenicol mixed pellets). The mRNA expressions of several genes (CAT, GPx, GST, LZM, ACP, ALF, and CypA) in *M. rosenbergii* fed with common pellets reached their peak at five days after the challenged with *C. freundii* (Figure 10B–G,I), whereas the mRNA expressions of crustin (Figure 10H) and CTL (Figure 10J) peaked at 3 and 7-dpi, respectively. The mRNA expressions of several genes (CAT, GPx, GST, crustin and CTL) in *M. rosenbergii* fed with florfenicol mixed pellets reached their peak at 5-dpi (Figure 10B–D,H,J, respectively). Meanwhile, the mRNA expressions of LZM, ACP, ALF and CypA genes peaked at 7-dpi (Figure 10E–G,I, respectively). In addition, the mRNA expression of Cu/ZnSOD in prawns (fed with common and florfenicol mixed pellets) were

up-regulated at 3-dpi and down-regulated at 5- and 7-dpi, with the lowest peak value observed at 7-dpi (Figure 10A). No evident difference was reported between the PBS control groups (fed with common pellets).

**Figure 9.** Changes in non-specific immune parameters of *M. rosenbergii* hepatopancreas at 1-, 3-, 5-, 7-, 9-, 11-, and 14-days after *C. freundii* infection. (**A**) SOD, (**B**) CAT, (**C**) GPx, (**D**) GST, (**E**) MDA, (**F**) ACP, (**G**) ALP, and (**H**) LZM activities. Data are presented as mean ± SEM (*n* = 3); different graphemes (a–g) indicate significant difference values of non-specific immune parameters at *p* < 0.05.

**Figure 10.** Analysis of immune-related gene expressions in hepatopancreas of *M. rosenbergii* in response to *C. freundii* challenge by RT-PCR at 1-, 3-, 5-, 7-, 9-, 11-, and 14-dpi. (**A**) Cu/ZnSOD, (**B**) CAT, (**C**) GPx, (**D**) GST, (**E**) LZM, (**F**) ACP, (**G**) ALF, (**H**) Crustin, (I) Cyp A, and (J) CTL gene expression levels of immune-related genes were measured by qRT-PCR, and measurement was performed in triplicate for each sample. Expression values were normalized to those of β-actin using the Livak (2−ΔΔCT) method, and the expression level detected at 1 day was set as 1.0. Data are presented as mean ± SEM (*n* = 3); \* *p* < 0.05, \*\* *p* < 0.01.

#### **4. Discussion**

Diseases are a major constraint in the sustainable development of aquaculture all over the world. The outbreak and spread of bacterial infections in aquaculture farms pose a major concern. *C. freundii* is an opportunistic pathogen that is commonly found in water, soil, animals and has been reported to cause numerous diseases in aquaculture [39–41]. In recent years, an increasing number of studies reported *C. freundii* causing diseases and serious mass deaths in various aquatic products, such as Chinese mitten-handed crab [42], red swamp crayfish [14], grass carp [24], and crucian carp [43]. In addition, *C. freundii* can cause infections in amphibians, reptiles, birds, and mammals [36,44–48]. In the present study, *C. freundii* isolated from diseased *M. rosenbergii* was confirmed to be the cause contributing to prawn mortality, with the typical signs of water bubble under the carapace.

Phylogenetic analysis revealed that our isolated strain GDZQ201912 showed a very close association with other strains of *C. freundii* submitted from China and UK and formed separate clades with other *Citrobacter* sp., such as *C. portucalensis* (AP022399.1, AP022486.1, and AP022513.1). The physiological and biochemical profiles of strain GDZQ201912 were similar to those of other isolates reported by earlier works [49,50]. Variations in the biochemical test, such as those of tyrosine, putrescine [38], indole, citrate, H2S, malonate, raffinose, and dulcitol, were also described by earlier studies [17,49].

In the case of pathogenic bacteria, such as *C. freundii*, multidrug resistance is one of the most prominent problems [50]. In this study, the isolated strain was resistant to four different β-lactam antibiotics, including ampicillin, clindamycin, lincomycin, and penicillin. Numerous studies demonstrated the existence of β-lactamase in *C. freundii* [51–54]. The resistant feature against β-lactam antibiotics of our strain indicated the existence of β-lactamase gene, which benefits the strain. The *C. freundii* resistant to tetracycline and oxytetracycline has been isolated from mammals, poikilothermic hosts, poultry, catfish, and rainbow trout [55–60].

The innate immune system defense against invading pathogens relies on AMPs, enzymes, and cellular components [16]. During this process, host cells will produce abundant ROS, such as superoxide anion (O2 •−), hydrogen peroxide (H2O2), hydroxyl radicals (•OH), and singlet oxygen (1O2), and reactive oxygen intermediates to kill the invading pathogens; however, excessive levels of ROS in the body will also cause the destruction and damage of DNA and other biological macromolecules [18]. Crustaceans can rely on complex enzymatic antioxidant systems to alleviate and restore the oxidative damage, SOD, GPx, GST, CAT, MDA, and so on [61]. SODs conduct the first step in eliminating the oxidative damage caused by ROS from cells. The SOD activity first increased at 1.5 h but decreased significantly at 12–48 h in the hepatopancreas of *P. monodon* after *V. parahaemolyticus* infection [62]. In *L. vannamei*, the SOD activity showed remarkable decreases in the hemolymph after 6–96 h and recovery after 96 h post-*V. alginolyticus* injection [63]. The SOD activity was also significantly reduced in *Probopyrus ringueleti*infected *Palaemontes argentinus* [64]. In addition, the Cu/ZnSOD showed an up-regulated expression in the hemocytes of *M. rosenbergii* at 3 and 6 h after *Lactococcus garvieae* infection, whereas down-regulation was observed in the hepatopancreas at 3 h after *L. garvieae* infection [65]. In *M. nipponense*, the ecCuZnSOD mRNA expression level showed a drastic decrease at 12 h after *A. hydrophila* injection [66]. In *M. rosenbergii*, the cytMn-SOD transcript also decreased in the hepatopancreas at 3 h after *L. garvieae* injection [67]. The expression of icCu/Zn-SOD decreased in the first 8–16 h and then recovered after the challenge with *Listonella anguillarum* and *Micrococcus luteus* in *Chlamys farreri* [68]. The relative expression level of icCu/Zn-SOD mRNA increased rapidly at 6 h post infection with *Vibrio anguillarum* in *Venerupis philippinarum*, followed by a remarkable decrease, and then increased again [69]. In the present study, a similar expression profile was found in the hepatopancreas of *M. rosenbergii* after *C. freundii* infection, in which the relative expression level of Cu/ZnSOD mRNA was up-regulated first and then decrease extremely. Then, the expression level recovered, and SOD activities in hepatopancreas and hemolymph were restored. CAT plays an important role in the antioxidant enzyme defense system to alleviate ROS. The CAT activity in the hepatopancreas of *M. rosenbergii* and CAT mRNA expression increased

markedly at 6 and 24 h post injection with *V. parahaemolyticus* [70]. The CAT activity exhibited a significant rise at the early stage of white spot syndrome virus infection in *P. monodon* [71]. The CAT activity in the hepatopancreas of *M. rosenbergii* significantly decreased at 5 days after spiroplasma MR-1008 infection and showed a recovery at 15 days; however, the mRNA expression level was up-regulated and peaked at 12 days [72]. Similar trends were observed in the CAT activity and mRNA expression level in the present study. GST and GPx are involved in different mechanisms for the host defense against oxidative damage [73]. In *Fenneropenaeus chinensis*, the mRNA expression levels of *FcTheta*GST, *FcMu*GST, and *Fc*GPx in the hepatopancreas were up-regulated after the challenge with *V. anguillarum* for 24 h, whereas the GST activity decreased [74]. In *L. vannamei*, the GPx activity and mRNA transcription increased significantly at 12 h after the challenge with *V. anguillarum* [75]. In *P. monodon*, the GPx activity of hepatopancreas increased significantly at 6 h and reached the peak level at 12 h after the *V. parahaemolyticus* challenge; the MDA activity significantly increased from 6 h to 24 h with a peak at 6 h, whereas the GST activity decreased significantly at 3 h [62]. The GPx activity in the gill of *F. chinensis* increased at 6 h and remained at high levels up to 24 h [74]. In addition, the results of increased mRNA expression levels and activities (GPx, GST, CAT, and MDA) during *C. freundii* infection in *M. rosenbergii* were in agreement with the histopathological damage in hepatopancreas.

LZM is an important enzyme of the innate immune defense system against bacterial infection, and it can cause bacterial cell disintegration by hydrolyzing the bacterial cell wall [76,77]. Lysosomal enzymes, which are produced by phagocytes from crayfish *Procambarus clarkii*, can efficiently degrade and eliminate foreign materials, as had been demonstrated by Franchini and Ottaviani [78]. In *P. monodon* and *M. rosenbergii*, the lysosomal activity showed a significant increase after *V. vulnificus* infection and increased in *L. vannamei* infected with *Micrococcus lysodeikticus* [79,80]. ACP and ALP are symbols of macrophage activation, important components of the lysosome system, and play important roles in the innate immune system to engulf antigens intracellularly [21]. The ACP activity increased substantially within 48 h in the hemolymph of *C. farreri* after the treatment with *V. anguillarum* [81]. In *L. vannamei*, the ACP and ALP activities significantly increased with *V. parahaemolyticus* and *M. lysodeikticus* infections, respectively [82]. In addition, the ACP and ALP activities in *M. rosenbergii* were significantly enhanced after the challenge with a novel pathogen spiroplasma MR-1008 [72]. In the present study, the enzyme activities of ACP, ALP, and LZM in the hepatopancreas and hemolymph were all enhanced after *M. rosenbergii* infection with *C. freundii*; the enzyme activities all increased significantly in the hepatopancreas after *T. chinensis* infection in *M. nipponense* [83].

AMPs are the main effector molecules in the innate immunity of crustaceans, and they act as frontline effectors to defend against invading bacteria, fungi, and viruses [26,27,84]. The major AMPs, such as penaeidins, crustins, and ALF, have been identified in shrimp [29]. In *P. monodon*, the mRNA expression level of crustin-like peptide significantly increased at 24 h post-infection with *V. harveyi* and recovered at 72 h [85]. The mortality of crustindepleted *L. vannamei* by RNA interference (RNAi) had a remarkable increase within 48 h post-infection with *V. penaeicida* [86]. In *Marsupenaeus japonicus*, *Mj*Cru I-1 can enhance the hemocyte phagocytosis, increase shrimp survival rate with *V. anguillarum* and *Staphylococcus aureus* infection, and weaken the bacterial clearance by knockdown of *Mj*Cru I-1 [84]. In *L. vannamei*, *Lv*CrustinB was up-regulated with *V. parahaemolyticus* infection, and the knockdown of *Lv*CrustinB can increase the shrimp mortality rate [87]. The expression level of *Fc*ALF2 in *Fenneropenaeus chinensis* significantly increased after the injection with *M. lysodeikticus* and *V. anguillarum* [88]. The in vivo function of *Lv*ALF1 in response to *V. penaeicida* and *Fusarium oxysporum* infections was measured by RNAi; the *Lv*ALF1-depleted *L. vannamei* caused a significant increase in the mortality of infected shrimps [89]. With *C. freundii* infection, the ALF mRNA expression significantly increased in crayfish *P. clarkii* [14]. The expressions of ALF and crustin increased in the hepatopancreas of *M. rosenbergii* after *C. freundii* infection in the present study; in addition, the ALF expression significantly increased in *L. vannamei* after the *V. anguillarum* and *M. lysodeikticus* challenge [90].

CypA acts as the receptor for the immunosuppressive agent cyclosporin A and belongs to the superfamily of peptidyl-prolyl cis-trans isomerases [91]. The mRNA expression of *bts*CypA was up-regulated in the hepatopancreas of *P. monodon* after stimulation by lipopolysaccharide [92]. After *V. anguillarum* challenge, the expression level of *Cf*CypA was up-regulated in the gonads of *C. farreri* and reached the peak at 4 h post-injection [93]. The mRNA expression level of *Es*CypA increased in the fungal *Pichia pastoris*-infected *E. sinensis* [24]. The significantly up-regulated *Vp*CypA2 mRNA expression level was observed in the hemocytes of *V. philippinarum* after the challenge with *L. anguillarum* [94]. The expression of CypA increased in the hepatopancreas of *M. rosenbergii* after *C. freundii* infection in the present study; the CypA mRNA expression level was significantly upregulated in the hepatopancreas of *P. clarkii* after the challenge with *C. freundii* [14].

CTLs are key recognition proteins and play important roles in shrimp innate immunity for the recognition and clearance of pathogens [22–25]. *Pm*CL1 expression was significantly upregulated in the hepatopancreas and gill of *P. monodon* after the infection with *V. harveyi* and *V. anguillarum*, respectively [95]. The expressions of hepatopancreas-specific CTL (*Fchs*L) and CTLs (*Fc*lectin, *Fc*Lec3 and *Fc*Lec4) from *F. chinensis* were up-regulated following the challenge of shrimp with *V. anguillarum* or *S. aureus* [96–99]. In *L. vannamei*, the expressions of *Lv*CTL3 and *Lv*PLP (the perlucin-like protein, a typical CTL) were up-regulated after *V. parahaemolyticus* infection; two CTLs (*Lv*Lectin-1 and *Lv*Lectin-2) showed significantly increased expressions after the *L. anguillarum* challenge [100–102]. Two novel lectins (*Mn*CTLDcp2 and *Mn*CTLDcp3) from *M. nipponense* were up-regulated in the heart after the challenge with *A. hydrophila* [103]. The expression of CTL increased in the hepatopancreas of *M. rosenbergii* after *C. freundii* infection in the present study, and a significantly up-regulated CTL mRNA expression level was observed in the hepatopancreas of *P. clarkii* after the challenge with *C. freundii* [14].

In general, when *M. rosenbergii* was infected with *C. freundii*, CTL, as one of patternrecognition receptor, immediately recognized the non-self material that had entered the prawn body. With *C. freundii* infection, enzymes involved in the antioxidant antibacterial system (SOD, CAT, GPx, GST, and MDA) were synthesized rapidly in the hepatopancreas and hemolymph, and the phosphatase enzymes (ACP and ALP) and LZM also actively participated in this immune response. In addition, the expressions of immune-related genes (Cu/Zn-SOD, CAT, GPx, GST, LZM, ACP, ALF, crustin, CypA, and CTL) in the hepatopancreas were significantly up- or down- regulated, which suggested that the immediate immune response system in *M. rosenbergii* offers defense against *C. freundii* infection. The results of this study indicated that all of these antioxidant enzymes and genes play important roles in the *M. rosenbergii* immune responses. Furthermore, the hepatopancreas are important immune tissues in the defense against *C. freundii*.

Antibiotics are natural or synthetic compounds and have become the main means for the prevention and treatment of infectious diseases due to their low cost, good curative effect, and simple operation [104,105]. Florfenicol, oxytetracycline, oxytetracycline hydrochloride, ormetoprim, and sulfamethazine were the five antibiotics approved by the FDA [106]. In the present study, florfenicol and ofloxacin were selected to evaluate their therapeutic effects against *C. freundii* in *M. rosenbergii*. Ofloxacin was forbidden in aquaculture and its use in the present study was just for scientific research. Florfenicol is a broad-spectrum and effective antibacterial, belonging to a part of the chloramphenicol family of drugs, that is widely used to control susceptible bacterial diseases in fish and shrimp farming [107–111]. Florfenicol and ofloxacin can stop bacteria from multiplying by interfering with bacterial protein production and DNA replication, respectively [31]. The increasd immune and antioxidant responses in *M. rosenbergii* after *C. freundii* challenge were results of *C. freundii* activated the host's innate immune system. With antibiotics feeding, *C. freundii* in *M. rosenbergii* cannot multiplying well owing to the protein production or DNA replication interfering. Then, the innate immune system of *C. freundii* infected *M. rosenbergii* was more efficient against bacteria with the higher survival rate, immune and antioxidant responses. *C. freundii* can break the innate immune system of *M. rosenbergii* treated without antibiotics, damage the capital functional organs and cause death. Florfenicol was effective in the treatment of pseudotuberculosis in Yellowtail *Seriola quinqueradiata* [112], vibriosis in Goldfish *Carassius auratus* [113], and furunculosis in Atlantic Salmon *Salmo salar* [114], *V. anguillarum* infection in Atlantic cod *Gadus morhua* [106], vibriosis in Black Tiger Shrimps *P. monodon* [111], *V. harveyi* infection in *L. vannamei* [110]. Due to the adverse environmental consequences, alternatives to antibiotics have been developed, including probiotics, phage therapy, essential oils (EOs), and Chinese herbal medicine [31]. *Pseudomonas synxantha* and *Pseudomonas aeruginosa*-fed *Penaeus latisulcatus* showed a higher tolerance to the *V. harveyi* challenge [115]. Four phages that had lytic activity against *V. harveyi* were isolated, and were demonstrated to be effective in controlling the population of *V. harveyi* in hatchery systems and improved the survival of *P. monodon* [116]. The usage of EOs from *Cinnamosma fragrans* enhanced the survival of *P. monodon* larvae with *Vibrio penaeicidae* and *Vibrio splendidus* challenge and decreased bacterial concentration [117]. For the WBD in *M. rosenbergii*, we also conducted alternatives therapies, such as Chinese herbal medicine. We found strain GDZQ201912 was sensitive to *Terminalia chebula Retz*, *Scutellaria baicalensis Georgi*, *Caesalpinia sappan Linn*, *Rhus chinensis Mill*, and *Schisandra chinensis* as well as the evaluations of their therapies of WBD, survival rate, immune and antioxidant responses of *M. rosenbergii* (Unpublished data).

#### **5. Conclusions**

Overall, in the present study, the pathogenic bacterium *C. freundii* was isolated from WBD *M. rosenbergii* with a mass mortality, and the cause of WBD in *M. rosenbergii* was identified with the typical sign under the carapace. The TEM observation, physiological and biochemical characteristics, LD50, and antimicrobial susceptibility of the *C. freundii* isolate were studied. The therapeutic effects of antibiotics against WBD were also evaluated, and florfenicol was the most efficient antibiotic to defend against *C. freundii*. In addition, the results of the present study provide insights into the histopathological changes, antioxidant enzymatic activity, and changes in the expressions of immune-related genes of *M. rosenbergii* challenged with *C. freundii*, revealing the antibacterial immune responses in giant river prawn. These results will facilitate the development of therapies and management of the disease caused by this pathogen in aquaculture systems.

**Author Contributions:** Conceptualization, J.H.; methodology, C.Z.; software, H.W.; validation, C.Z., H.W. and S.H.; formal analysis, C.Z.; investigation, S.W.; resources, J.H.; data curation, C.Z.; writing—original draft preparation, C.Z.; writing—review and editing, J.H.; visualization, C.Z.; supervision, J.H. and C.Z.; project administration, S.W.; funding acquisition, J.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Key Research and Development Program of China (2018YFD0900501), Key-Area Research and Development Program of Guangdong Province (2019B020217001) and Supported by the earmarked fund for CARS-48.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in the article.

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

#### **References**

### *Article* **The Assessment of Dietary Organic Zinc on Zinc Homeostasis, Antioxidant Capacity, Immune Response, Glycolysis and Intestinal Microbiota in White Shrimp (***Litopenaeus vannamei* **Boone, 1931)**

**Jinzhu Yang 1, Tiantian Wang 1, Gang Lin 2, Mingzhu Li 3, Yanjiao Zhang 1,\* and Kangsen Mai 1**

**Abstract:** This study aimed to assess dietary organic zinc on zinc homeostasis, antioxidant capacity, immune response, glycolysis and intestinal microbiota in white shrimp (*Litopenaeus vannamei* Boone, 1931). Six experimental diets were formulated: Control, zinc free; S120, 120 mg·kg−1 zinc from ZnSO4·7H2O added into control diet; O30, O60, O90 and O120, 30, 60, 90 and 120 mg·kg−1 zinc from Zn-proteinate added into control diet, respectively. The results showed that organic zinc significantly promoted zinc content and gene expression of ZnT1, ZIP11 and MT in the hepatopancreas and enhanced antioxidant capacity and immunity (in terms of increased activities of T-SOD, Cu/Zn SOD, PO, LZM, decreased content of MDA, upregulated expressions of GST, G6PDH, ProPO, LZM and Hemo, and increased resistance to *Vibrio parahaemolyticus*). Organic zinc significantly upregulated GluT1 expression in the intestine, increased glucose content of plasma and GCK, PFK and PDH activities of hepatopancreas, and decreased pyruvate content of hepatopancreas. Organic zinc improved intestinal microbiota communities, increased the abundance of potentially beneficial bacteria and decreased the abundance of potential pathogens. Inorganic zinc (S120) also had positive effects, but organic zinc (as low as O60) could achieve better effects. Overall, organic zinc had a higher bioavailability and was a more beneficial zinc resource than inorganic zinc in shrimp feeds.

**Keywords:** organic zinc; zinc homeostasis; antioxidants; immunity; glycolysis; intestinal microbiota; *Litopenaeus vannamei* Boone, 1931

#### **1. Introduction**

As an essential microelement for animals, zinc is well known for its key role in various physiological processes such as organism growth and metabolism of proteins, carbohydrates and lipids [1,2]. Zinc is also a cofactor or component of various enzymes related to antioxidants, immune response and regulation, such as alkaline phosphatase (AKP), metallothionein (MT), superoxide dismutase (SOD) and copper/zinc superoxide dismutase (Cu/Zn SOD) [3]. In aquafeeds, zinc sulfate is traditionally used to meet the demand of aquatic animals due to its lower price and easy accessibility [4,5]. However, the disadvantages of zinc sulfate have been noticed with its low bioavailability and potential environmental problems. On the one hand, plant-based ingredients have been widely used in aquafeeds [6–8]. It is known that plant-based ingredients contain antinutritional factors that reduce the bioavailability of minerals [9,10]; for example, phytate is easily chelated to Zn2+, forming stable chelates that are not absorbed intestinally [3,9]. Thus, overdosage of

**Citation:** Yang, J.; Wang, T.; Lin, G.; Li, M.; Zhang, Y.; Mai, K. The Assessment of Dietary Organic Zinc on Zinc Homeostasis, Antioxidant Capacity, Immune Response, Glycolysis and Intestinal Microbiota in White Shrimp (*Litopenaeus vannamei* Boone, 1931). *Antioxidants* **2022**, *11*, 1492. https://doi.org/ 10.3390/antiox11081492

Academic Editors: Bo Liu, Changyou Song and Cunxin Sun

Received: 13 June 2022 Accepted: 28 July 2022 Published: 29 July 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**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/).

zinc addition is common in aquafeeds [11,12], which increases the risk of water pollution in intensive aquaculture zones and their surroundings. On the other hand, sulfate salts are a common type of inorganic mineral (e.g., ZnSO4, CuSO4, MnSO4) in feed addition [13]. The high concentration of SO4 2− is toxic to aquatic organisms. SO4 2− can be reduced into sulfide under anaerobic conditions, which is toxic to a number of aquatic organisms as well [14,15].

Organic zinc, formed by the chelation of Zn2+ with amino acids (e.g., glycine, methionine) and/or partially hydrolyzed proteins, provides a new option for zinc supplementation in aquafeeds [16]. It has been reported that organic zinc is more easily absorbed by the intestinal epithelium than inorganic zinc [17,18], and it can prevent the chelation of Zn2+ with phytate and protect the micronutrients (e.g., vitamins, fatty acids) from oxidation by Zn2+ [19]. In channel catfish (*Ictalurus punctatus*), organic zinc had over 3 times the potency of inorganic zinc in improving growth and increased resistance to *Edwardsiella ictaluri* [20,21]. Similarly, there was also a higher bioavailability of organic zinc than inorganic zinc in beluga sturgeon (*Huso huso*) [22], pangasius catfish (*Pangasianodon hypophthalmus*) [23] and juvenile abalone (*Haliotis discus hannai* Ino.) [24]. In triploid rainbow trout (*Oncorhynchus mykiss*), organic zinc reduced the zinc requirement and enhanced antioxidant capacity compared to inorganic zinc [25,26]. In white shrimp (*Litopenaeus vannamei* Boone, 1931), compared to inorganic zinc, organic zinc promoted growth performance and enhanced immunity and resistance to *Vibrio harveyi* better [27–29]. Glycolysis is a cellular process that breaks down glucose into pyruvate. Pyruvate can enter the tricarboxylic acid cycle (TAC) for further ATP production. Glycolysis is also an important method to metabolize glucose in most organisms. Previous studies have described that zinc plays a role in stimulating glycolysis in mice [30] and rats [31,32]; however, in aquaculture, relevant information is limited. The intestinal microbiota plays key roles in host health, nutrition metabolism and immune response. Studies have found zinc could increase the abundance of potential probiotics in weanling pigs [33] and improve the microbial population of broiler chickens [34]. However, information on the effect of zinc on the intestinal microbiota of aquatic animals is also limited.

White shrimp is a major species in aquaculture around the world and has great economic value due to its great texture, rapid growth rate and good adaptability to the environment [35]. At present, the increasing farming density and scale pose a challenge to the health and disease resistance of shrimp [36]. Organic zinc is considered to have immuneenhancing effects, which is beneficial for shrimp health. Most previous studies of organic zinc have focused on growth performance and biochemical parameters. This study aimed to assess dietary organic zinc (Zn-proteinate) on zinc homeostasis, antioxidant capacity, immune response, glycolysis, intestinal microbiota and resistance to *V. parahaemolyticus* in white shrimp, which will provide a comprehensive assessment of organic zinc application in shrimp culture.

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

#### *2.1. Experimental Diets*

Six isonitrogenous and isolipidic experimental diets were formulated to contain different dosage forms of zinc, resulting in the following six dietary treatments: Control, dietary Zn free; S120, dietary 120 mg·kg−1 zinc from ZnSO4·7H2O added into control diet; O30, O60, O90, O120, dietary 30, 60, 90, 120 mg·kg−1 zinc from Zn-proteinate added into control diet, respectively. All the ingredients were thoroughly mixed and pelleted with an approximate diameter of 2 mm. After that, feeds were dried until constant weight at 55 ◦C in a ventilated oven. Feeds were then stored at −20 ◦C until use. The formulation, chemical composition and zinc content in feeds are shown in Table 1.

**Table 1.** Formulation and proximate compositions of experimental diets.

1 Fish meal, shrimp shell meal, brewer yeast, etc., were purchased from Qingdao Great-seven Nutr-tech Co., Ltd., Qingdao, China. 2 Choline chloride, amino acid, ZnSO4·7H2O and Y2O3 were purchased from Shanghai Macklin Biochemical Co., Ltd., Shanghai, China. 3 Vitamin premix and mineral premix were purchased from Qingdao Master Biotech Co., Ltd., Qingdao, China. Vitamin premix contains (kg−1): vitamin A acetate, 714,000 IU; vitamin D3, 266,000 IU; DL-α-tocopherol acetate, 8.6 g; menadione, 1.0 g; thiamine mononitrate, 1.0 g; riboflavin, 1.4 g; pyridoxine hydrochloride, 1.2 g; cyanocobalamin, 0.004 g; D-calcium pantothenate, 4.0 g; nicotinamide, 6.8 g; folic acid, 0.28 g; D-biotin, 0.012 g; inositol, 7.6 g; L-ascorbic acid-2-phosphate, 16.6 g; mineral premix contains (kg−1): Mg, 12.5 g; Fe, 4.0 g; Mn, 2.0 g; Cu, 1.25 g; Co, 0.05 g; Se, 0.015 g; I, 0.05 g. 4 Bioplex Zn® was provided by Beijing Alltech Biological Products (China) Co., Ltd., Beijing, China.

Analysis of the chemical compositions of feeds was performed following standard protocols [37]. Dry matter was measured by drying samples to a constant weight at 105 ◦C, crude protein was determined by measuring nitrogen (N × 6.25) using the Kjeldahl method (FOSS 8400, Denmark, Sweden), crude lipid was determined by mineral ether extraction using the Soxhlet method (BUCHI 36880, Flawil, Switzerland) and ash content was determined by incineration of samples at 550 ◦C in a muffle furnace.

The moisture was calculated with the following equation:

$$\text{Moisture} \left(\% \right) = 100 \times \left(\text{W}\_1 - \text{W}\_2 \right) / \text{W}\_{1\prime} \tag{1}$$

W1: Wet weight of matter; W2: Dry weight of matter. The ash content was calculated with the following equation:

$$\text{Ash (\%)}=100 \times \text{W}\_{\text{3}}/\text{W}\_{4\prime} \tag{2}$$

W3: Ash weight after incineration; W4: Dry weight before incineration.

#### *2.2. Feeding Trial and Sample Collection*

The feeding trial was carried out at Huanghai Aquaculture Co., Ltd., Shandong, China. White shrimp were purchased from a local farm. Before the feeding trial, shrimp were acclimated to a commercial diet for 2 weeks with flowing water. Shrimp were then fasted for 24 h and weighted. A total of 960 shrimp (initial body weight of 2.37 ± 0.01 g) were randomly distributed to 24 cylindrical fiberglass tanks (with 200 L of seawater) in an indoor rearing system with flow-through seawater. A total of 40 shrimp were cultured in each tank. The six diets were randomly assigned to tanks (4 replications each group). During the feeding trial, shrimp were fed 4 times daily (05:30, 11:00, 16:30 and 21:30). The daily feeding quantity was at 4–6% of body weight and adjusted according to previous feeding responses. Two-thirds of seawater was exchanged twice daily. The water condition was detected once a week, and the results were as follows: temperature was 26.4–28.0 ◦C; salinity was 31–33‰; pH was 8.2–8.4; dissolved oxygen was higher than 7 mg·L−1; nitrite was lower than 0.005 mg·L−1; nitrate was lower than 15 mg·L−1; ammonia was lower than 0.02 mg·L−1.

After 8 weeks of feeding, shrimp were fasted for 24 h; all surviving shrimp were collected, and then the body length and weight of each shrimp were measured. After that, 12 shrimp of similar size from each tank were randomly selected. The hemolymph was collected from the caudal vein using 1 mL syringes and diluted immediately at a hemolymph-to-anticoagulant ratio of 1:1.5 (10 mmol·L−1 EDTA-Na2, 450 mmol·L−1 NaCl, 10 mmol·L−1 KCl, 10 mmol·L−1 HEPES, pH 7.3) [38]. Approximately 50 μL of hemolymph was used for the hemocyte count using a hemocytometer (XB-K-25, Yuhuan County Qiujing Medical Instrument Factory, Zhejiang, China). The rest was centrifuged at 4 ◦C, 500 g·min−1 for 10 min. The supernatant was then collected into 0.2 mL PCR tubes (Cat. No.: PCR02C, Axygen™, Union City, CA, USA) and stored at −80 ◦C for biochemical analysis. After that, the hepatopancreas was quickly removed and then transferred to 1.8 mL sterile RNase-free cryogenic tubes (Cat. No.: 377267, Nunc™, Rochester, NY, USA), frozen in liquid nitrogen and stored at −80 ◦C for analysis of enzyme activity and gene expression. Intestines were then removed and transferred to 1.8 mL sterile RNase-free cryogenic tubes (Cat. No.: 377267, Nunc™, Rochester, NY, USA), frozen in liquid nitrogen and stored at −80 ◦C, of which 6 were used for gene expression analysis and the other 6 for microbiota analysis. The muscle and carapace of 2 shrimp were randomly selected and stored at −20 ◦C for zinc concentration analysis.

#### *2.3. Vibrio Parahaemolyticus Challenge*

After the feeding trial, 30 shrimp of each group were randomly selected and separated into 3 tanks (with 50 L of seawater). Each shrimp was injected intramuscularly with 0.1 mL of *Vibrio parahaemolyticus* (5 × 106 CFU/mL). Based on pre-experiments, mortality was recorded for 7 days. During the challenging trial, temperature was 26.4–28.0 ◦C, salinity was 31–33‰, pH was 8.2–8.4, dissolved oxygen was higher than 7 mg·L−1, nitrite was lower than 0.005 mg·L−1, nitrate was lower than 15 mg·L−1 and ammonia was lower than 0.02 mg·L−1.

#### *2.4. Growth Performance and Hemocyte Count*

Growth performance was calculated by using the following equation:

Weight gain rate (WGR,%) = 100 × (final body weight − initial body weight)/initial body weight (3)

Specific growth rate (SGR,%·day−1) = 100 × (Ln final body weight − Ln initial body weight)/days (4)

Feed intake (FI,%·day−1) = 100 × feed intake/[(initial body weight + final body weight)/2]/days (5)

Feed efficiency (FE) = (final body weight − initial body weight)/feeds consumed (6)

Condition factor (CF, 100 g·cm−3) = 100 × final body weight/finial body length3 (7)

Hemocyte count was measured with a hemocytometer under a light microscope (Nikon, E 600, Tokyo, Japan). The number of blood cells per mL of hemolymph was calculated according to the formula below:

Hemocyte count in 1 mL hemolymph = *A*/5 × 25 × 10000 × *B* (8)

*A*: the total hemocyte count in 5 medium squares; *B*: dilution ratio of the sample.

#### *2.5. Zinc Accumulation Analysis*

Zinc concentrations in the diets, muscle and carapace were analyzed by an inductively coupled plasma optical emission spectrometer (ICP-OES, PE 2100DV, Perkin Elmer, Boston, MA, USA). This analysis was completed by the Beijing ZKGX Research Institute of Chemical Technology (Material Lab, Beijing, China). Zinc concentrations in the plasma and hepatopancreas were determined with a commercial assay kit (E011-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

#### *2.6. Biochemical Analysis*

Plasma of shrimp was used for biochemical analysis directly. The hepatopancreas was weighted, thawed and homogenized (1:9) in ice-cold 0.9% NaCl solution. After centrifugation (2500 rpm, 15 min, 4 ◦C), the supernatant was collected for biochemical analysis. The activities of acid phosphatase (ACP, A060-2-1), AKP (A059-2-2), catalase (CAT, A007-1-1), phenoloxidase (PO, H247), total antioxidant capacity (T-AOC, A015-2-1) and the contents of glucose (Glu, A154-1-1), malondialdehyde (MDA, A003-1-2) of plasma were determined with commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The content of pyruvate (A081-1-1) and the activities of glucokinase (GCK, H439-1), phosphofructokinase (PFK, H244), pyruvate dehydrogenase (PDH, H262- 1-2) of hepatopancreas were determined with commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The activities of total superoxide dismutase (T-SOD, S0101M) and Cu/Zn SOD (S0103) of plasma and hepatopancreas and the content of total protein (P0012) of hepatopancreas were determined with commercial assay kits (Beyotime Biotechnology, Shanghai, China). The activity of lysozyme (LZM) of plasma was determined by using commercial Shrimp ELISA kits and following the manufacturer's instructions (CK-E94755, Shanghai Elisa Biotech Co., Ltd., Shanghai, China).

#### *2.7. RNA Extraction and qPCR*

The total RNA of hepatopancreas and intestine were extracted using the MolPure® Cell/Tissue Total RNA Kit (19221ES50; Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China). The concentration and quality of RNA were assessed with NanoDrop™ 2000 spectrophotometers (Thermo Scientific™, Waltham, MA, USA). The integrity of extracted RNA was determined by electrophoresis on a 1.2% (*w*/*v*) agarose gel. Reverse transcription of 1000 ng of RNA was conducted using Hifair® III 1st Strand cDNA Synthesis SuperMix for qPCR (11141ES60; Yeasen, Shanghai, China).

The qPCR was performed in a 20 μL volume: 1 μL of cDNA template (≤50 ng); 0.4 μL of forward primer (10 μM); 0.4 μL of reverse primer (10 μM); 8.2 μL of RNase-free ddH2O (P071-01, Vazyme Biotech Co., Ltd., Nanjing, China); 10 μL of SYBR® Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biotechnology (Hunan) Co., Ltd., Hunan, China). A two-step qPCR program was used: 95 ◦C for 30 s, followed by 40 cycles of 95 ◦C for 5 s and 60 ◦C for 30 s. Finally, melting curve analysis was used to ensure the specification of the PCR product. Specific gene primers were designed in NCBI, synthesized by Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China. The specificity and amplification efficiency of primers were assessed (Table S1). β-Actin, 18S ribosomal RNA and elongation factor 1-alpha were selected as candidate housekeeping genes. BestKeeper [39] and

NormFinder [40] tools were used to assess the most stable expression gene. Finally, β-actin was assessed as the best housekeeping gene in the present experiment (Tables S2 and S3).

All qPCR analyses were performed in CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Moreover, a no-template control (NTC) (0 μL of cDNA template, 0.4 μL of forward primer (10 μM); 0.4 μL of reverse primer (10 μM); 9.2 μL of RNase-free ddH2O; 10 μL of SYBR® Green Premix Pro Taq HS qPCR Kit) was set in each qPCR plate reaction to ensure no extraneous nucleic acid contamination. The gene expression levels were normalized using the relative quantitative method (2−ΔΔCq) referencing β-actin of shrimp [41].

#### *2.8. Intestinal Microbiota DNA Extraction and Sequencing*

Genomic DNA of intestinal microbiota was extracted using the QIAamp PowerFecal® Pro DNA Kit (51804, Qiagen, Hilden, Germany) on a super-clean bench following the manual. Primer 515F/806R was used to amplify the V4 region of the 16S rRNA gene. PCR reaction and quality control were performed by Novogene Genomics Technology Co., Ltd., Beijing, China. Sequencing was conducted on an Illumina NovaSeq platform provided by Novogene Genomics Technology Co., Ltd., Beijing, China. After sequencing, raw data were merged with Fast Length Adjustment of SHort reads (FLASH) [42] and then assigned to each sample with unique barcodes. To obtain effective reads for further analysis, Cutadapter was used to cut adapter, barcode and primer sequences and filter low-quality reads [43], and the UCHIME algorithm was used to detect and remove chimeric sequences [44]. After dereplication, abundance sort and discarding singleton reads, sequences were clustered to operational taxonomic units (OTUs) with ≥97% similarity using UPARSE [45]. The representative sequence for each OUT was screened for further annotation using the Silva Database (version 138) based on the Ribosomal Database Project (RDP) classifier [46]. Alpha diversity (OTUs, Chao1, ACE, Shannon, Simpson and PD whole tree) and beta diversity (principal coordinates analysis (PCoA) and unweighted pair group method with arithmetic mean (UPGMA) clustering) were calculated with Quantitative Insights Into Microbial Ecology (QIIME) and displayed with R software (version 4.1.0) [47].

#### *2.9. Statistical Analysis*

Statistical software SPSS 22.0 for Windows (IBM SPSS Corporation, Chicago, IL, USA) was used for the data analysis. Results were analyzed by one-way analysis of variance (ANOVA). Tukey's multiple-range test was used for the multiple comparisons of group means. Differences were regarded as significant when *p <* 0.05.

Microbiota sequence data analysis proceeded on the NovoMagic cloud platform provided by Novogene Genomics Technology Co., Ltd., Beijing, China. An analysis of molecular variance (AMOVA) test was employed to assess the difference of microbiota composition within or between groups using the adegenet package in R software (version 4.1.0). MetaStat analysis was used to identify the differential abundant taxa between groups. The *p*-value was adjusted by the Benjamini-Hochberg false discovery rate (FDR), and differences were regarded as significant when *Q* < 0.05 (*Q*, adjusted *p*-value) [48].

#### **3. Results**

#### *3.1. Growth Performance*

As shown in Table 2, the FBW, WGR and SGR of shrimp fed the O60 diet were significantly higher than those of shrimp fed the control diet (*p <* 0.05). S120, O30, O90 and O120 were intermediate in the FBW, WGR and SGR, with no significant differences observed (*p >* 0.05). No significant differences were observed in the FI, FE, CF and total hemocyte number of shrimp among all groups (*p >* 0.05).

**Table 2.** Effects of organic and inorganic zinc on growth performance of *Litopenaeus vannamei* Boone, 1931 \*.

\* Values represent are means ± S.E. of 4 replicate tanks. IBW, initial body weight, FBW, final body weight, WGR, weight gain rate, SGR, specific growth rate, FI, feed intake, FE, feed efficiency, CF, condition factor. a,b Different superscript letters within a row denote significant differences as evaluated by Tukey's test (*p <* 0.05).

#### *3.2. Zinc Accumulation and Zinc Transport*

As shown in Figure 1A, no significant differences were observed in the zinc concentrations of shrimp muscle and carapace among all groups (*p >* 0.05). The zinc concentrations in the hepatopancreas and plasma of the control group were lower than in all other diets (*p <* 0.05). The zinc concentrations in the hepatopancreas and plasma of shrimp fed the S120 diet were significantly lower than those of shrimp fed the O60, O90 and O120 diets (*p <* 0.05). The zinc concentration in plasma was the highest in the O120 group (*p <* 0.05).

**Figure 1.** Effects of organic and inorganic zinc on zinc accumulation of *Litopenaeus vannamei* Boone, 1931 tissues (**A**) and gene expressions of zinc transport in hepatopancreas of *L. vannamei* (**B**). ZnT1, zinc transporter 1; ZIP11, zinc transporter ZIP11, MT, metallothionein. Values represented are means ± S.E. of 4 replicate tanks. a,b,c,d Value bars not sharing the same superscript letter are significantly different (*p <* 0.05).

The zinc-transport-related gene expressions in the hepatopancreas (Figure 1B) showed that, compared with the control diet, the expression of ZnT1, ZIP11 and MT was significantly upregulated by the S120, O60, O90 and O120 diets, the O60 and O120 diets, and the O60, O90 and O120 diets, respectively (*p <* 0.05). Compared with the S120 diet, the expression of ZnT1 and MT was significantly upregulated by the O60, O90 and O120 diets and the O60 diet, respectively (*p <* 0.05).

#### *3.3. Antioxidant Capacity*

In plasma, as shown in Figure 2A, the activities of T-SOD, Cu/Zn SOD, CAT and T-AOC of shrimp fed the O60, O90 and O120 diets were significantly higher than those of shrimp fed the control diet (*p <* 0.05). The activities of T-SOD, Cu/Zn SOD and CAT of shrimp fed the O90 and O120 diets were significantly higher than those of shrimp fed the S120 diet (*p <* 0.05). The activities of T-SOD, Cu/Zn SOD and T-AOC of shrimp fed the control diet were significantly lower than those of shrimp fed the S120 diet (*p <* 0.05). The MDA content of the control group was the highest among all groups (*p <* 0.05). The MDA contents of the O90 and O120 groups were significantly lower than those of the S120 group (*p <* 0.05). In the hepatopancreas, as shown in Figure 2B, the activities of T-SOD and Cu/Zn SOD of shrimp fed the S120, O60, O90 and O120 diets were significantly higher than those of shrimp fed the control diet (*p <* 0.05). The activity of T-SOD of the O120 group was the highest among all groups (*p <* 0.05).

**Figure 2.** Effects of organic and inorganic zinc on antioxidant capacity of *L. vannamei*. (**A**) enzyme activities of plasma; (**B**) enzyme activities of hepatopancreas; (**C**) gene expressions of hepatopancreas. SOD, super dismutase; CAT, catalase; T-AOC, total antioxidant capacity; MDA, malondialdehyde; Gpx, glutathione peroxidase; GST, glutathione S-transferase; G6PDH, glucose-6-phosphate dehydrogenase. Values represented are means ± S.E. of 4 replicate tanks. a,b,c,d,e Value bars not sharing the same superscript letter are significantly different (*p <* 0.05).