marine drugs

Article Effects of Chitosan–Gentamicin Conjugate Supplement on Non-Specific Immunity, Aquaculture Water, Intestinal Histology and Microbiota of Pacific White Shrimp (Litopenaeus vannamei)

Fengyan Liang 1,2, Chengpeng Li 1,*, Tingting Hou 1, Chongqing Wen 2, Songzhi Kong 1, Dong Ma 3, Chengbo Sun 2,4,* and Sidong Li 1

1 School of Chemistry and Environmental Science, Guangdong Ocean University, Zhanjiang 524088, China; [email protected] (F.L.); [email protected] (T.H.); [email protected] (S.K.); [email protected] (S.L.) 2 Department of Fisheries, Guangdong Ocean University, Zhanjiang 524088, China; [email protected] 3 Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China; [email protected] 4 Southern Marine Science and Engineering Guangdong Laboratory, Zhanjiang 524025, China * Correspondence: [email protected] (C.L.); [email protected] (C.S.)



Received: 8 July 2020; Accepted: 7 August 2020; Published: 10 August 2020


Abstract: When the aquaculture water environment deteriorates or the temperature rises, shrimp are susceptible to viral or bacterial infections, causing a large number of deaths. This study comprehensively evaluated the effects of the oral administration of a chitosan–gentamicin conjugate (CS-GT) after Litopenaeus vannamei were infected with Vibrio parahaemolyticus, through nonspecific immunity parameter detection, intestinal morphology observation, and the assessment of microbial flora diversification by 16S rRNA gene sequencing. The results showed that the oral administration of CS-GT significantly increased total hemocyte counts and reduced hemocyte apoptosis in shrimp (p < 0.05). The parameters (including superoxide dismutase, glutathione peroxidase, glutathione, lysozyme, acid phosphatase, alkaline phosphatase, and phenoloxidase) were significantly increased (p < 0.05). The integrity of the intestinal epithelial cells and basement membrane were enhanced, which correspondingly alleviated intestinal injury. In terms of the microbiome, the abundances of Vibrio (Gram-negative bacteria and food-borne pathogens) in the water and gut were significantly reduced. The canonical correspondence analysis (CCA) showed that the abundances of Vibrio both in the water and gut were negatively correlated with CS-GT dosage. In conclusion, the oral administration of CS-GT can improve the immunity of shrimp against pathogenic bacteria and significantly reduce the relative abundances of Vibrio in aquaculture water and the gut of Litopenaeus vannamei.

Keywords: Litopenaeus vannamei; chitosan–gentamicin conjugate; nonspecific immunity parameters; intestinal morphology; 16S rRNA gene sequencing; microbiota

1. Introduction Shrimp farming is one of the most important industries in the world [1,2]. Due to high yield, fast growth, and easy adaptability, Pacific white shrimp (Litopenaeus vannamei) have become one of the most profitable species in shrimp farming, with an annual global production of more than 4.1 million tons [3]. This accounts for 73% in China [4]. The Pacific white shrimp belongs to temperate and tropical shrimp. It can survive in the temperature of 10–40 ◦C, and the most suitable temperature range is 25–32 ◦C. It can grow within a salinity range of 28–34%0. Like other shrimps, Pacific white shrimp lack adaptive immune mechanisms and depend on innate immune responses for the detection and elimination of

Mar. Drugs 2020, 18, 419; doi:10.3390/md18080419 www.mdpi.com/journal/marinedrugs Mar. Drugs 2020, 18, 419 2 of 19 pathogens. However, it is estimated that about 1.7 million tonnes of shrimp has been lost in recent years due to the environmental deterioration of aquaculture systems [4]. Among them, Vibrios (including Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus) are responsible for causing diseases in shrimps and human gastroenteritis disease after the consumption of contaminated seafood [5,6]. When the aquaculture water environment deteriorates or the temperature rises (especially in summer), the Vibrio outbreaks result in significant shrimp deaths. To prevent or control Vibrio, a range of strategies are used; for example, antibiotics have been used as the main treatment for Vibrio. However, the excessive or inappropriate use of antibiotics may increase the prevalence of multi-drug-resistant strains, environmental pollution, and residue accumulation. In response to these concerns, alternative strategies have been proposed, including plant extracts [7,8], probiotics [4,9], polysaccharides [10,11], and other feed supplements [12,13]. However, these strategies are ineffective; once the Vibrio break out, the shrimps will still die in large numbers. Chitosan (CS), a cationic polysaccharide, is obtained from the N-deacetylation of chitin [14]. Due to its potential in immune modulation, antioxidant function, biocompatibility, and wide-spectrum antibacterial activity [15], chitosan has been widely applied in aquaculture [16–19]. For example, one study showed that the performance and health of Nile tilapia (Oreochromis niloticus) were significantly improved when chitosan nanoparticles were loaded in basic diets (one gram per 1 kilogram) [20]. Similarly, Khani Oushani et al. found that diets supplemented with 0.05 g kg− of nano-chitosan and clinoptilolite could improve the growth performance and immune parameters of rainbow trout (Oncorhynchus mykiss)[21–24]. However, these studies mentioned above principally aimed to improve the immunity of aquatic animals. However, there are few reports relating to the application of chitosan on aquatic pathogenic bacteria, for the antibacterial activity of chitosan is reported to be poor. Recently, a series of chitosan derivatives with improved antibacterial activity have been reported [25]. Gentamicin (GT) has abundant amine groups and high water solubility, and can penetrate the bacterial cell membrane and inhibit the synthesis of bacterial proteins. In 2019, we successfully synthesized a chitosan–gentamicin conjugate (CS-GT) via oxidation and a Schiff base reaction. Compared to CS, its water-solubility and antimicrobial activity are significantly improved [26]. Our previous research also found that CS-GT-based hydrogels, with antimicrobial activity, cytocompatibility, and hemocompatibility, can accelerate the wound healing of New Zealand rabbits [27]. Particularly, CS-GT can inhibit pathogenic Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus effectively [26]. Therefore, CS-GT may be potentially applied in aquatic farming. The aim of present study was to understand the effects of CS-GT on immunity, intestinal histology, and in vitro and in vivo microbiota, as well as their functions in Litopenaeus vannamei after infection with Vibrio parahaemolyticus. These results are expected to better clarify the mechanisms involved in the oral administration of CS-GT for Litopenaeus vannamei farming and provide useful guidance for chitosan derivative development in aquaculture.

2. Results and Discussion

2.1. Water Physicochemical Quality Water quality monitoring is important in aquaculture. The physical–chemical constituents of the water often directly promote the growth of Litopenaeus vannamei, and ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen are important standards for monitoring water quality. The pH, salinity, total alkalinity, ammonia nitrogen (NH3), nitrate nitrogen (NO3−) and nitrite nitrogen (NO2−) indexes in the CK (infected with Vibrio parahaemolyticus), CS-GT (supplemented with CS-GT after infection with Vibrio parahaemolyticus), GT (supplemented with GT after infection with Vibrio parahaemolyticus), and CS (supplemented with CS after infection with Vibrio parahaemolyticus) groups were measured every day during the experiment. As shown in Figure1, the pH values of all groups were in the range of 7.66 to 8.11. The salinity, total alkalinity, NH3, and NO2− in all the groups were increased, while the NO3− content in all groups was decreased. The overall change trends of NH3 and NO2− were similar to those Mar. Drugs 2020, 18, 419 3 of 19 Mar. Drugs 2020, 18, x 3 of 19 into studiesthose in by studies other expertsby other [28 experts,29]. It [28,29]. is likely It that is likely the excessive that the inputexcessive of feeds input and of massivefeeds and deposition massive ofdeposition fecal matter of fecal during matter the feeding during period—causing the feeding period—causing organic matter organic accumulation, matter decomposition,accumulation, anddecomposition, oxygen consumption, and oxygen resulting consumption, in the resulting bottom of in tank the bottom entering of a tank hypoxic entering state, a hypoxic a large number state, a oflarge anaerobic number bacteria of anaerobic multiplying, bacteria and multiplying, the incomplete and decomposition the incomplete of organic decomposition matter—produced of organic a largematter—produced number of toxic a large and harmfulnumber of physical–chemical toxic and harmful factors physical–chemical such as ammonia factors nitrogen such as and ammonia nitrous nitrogen and nitrous nitrogen [30]. It is worth noting that the concentrations of NH3 and NO2− in the nitrogen [30]. It is worth noting that the concentrations of NH3 and NO2− in the CS-GT group were theCS‐GT lowest group compared were the to lowest other groups.compared to other groups.

Figure 1. Water physical–chemical constituents: (a) pH, (b) salinity, (c) total alkalinity, (d) NH (mg/L), Figure 1. Water physical–chemical constituents: (a) pH, (b) salinity, (c) total alkalinity, (d) NH3 (mg/L), (e) NO2− (mg/L), and (f) NO3− − (mg/L) (mean SD, n = 3). (e) NO2 (mg/L), and (f) NO3 (mg/L) (mean ± ±SD, n = 3). 2.2. Hematological Profile 2.2. Hematological Profile During the experiment, the mortality rates were about 12%, 9%, 7%, and 5% in the CK group, CS group,During CS-GT the experiment, group, and the GT mortality group, respectively. rates were about The hemocytes 12%, 9%, related7%, and to 5% humoral in the CK immunity, group, playCS group, a crucial CS part‐GT group, in immune and defenseGT group, system. respectively. Therefore, The hemocytes hemocytes can related be used to to humoral reflect the immunity, immune responseplay a crucial of the shrimps,part in immune and they defense have been system. reported Therefore, to decrease hemocytes the exposure can ofbe shrimps used to to reflect infectious the pathogensimmune response or environmental of the shrimps, stress, and thereby they have reducing been the reported risk of to secondary decrease infectionsthe exposure [31 ,32of ].shrimps In this study,to infectious the total pathogens hemocyte or countsenvironmental (THCs) ofstress, each thereby group were reducing counted the risk each of day secondary using an infections inverted phase-contrast[31,32]. In this study, microscope. the total As hemocyte shown in counts Figure 2(THCs), the THCs of each of the group supplement were counted groups each significantly day using increasedan inverted compared phase‐contrast to those microscope. in the CK group As shown (p < 0.05). in Figure The THCs 2, the of THCs CS-GT of groupthe supplement were significantly groups significantly increased compared to those in the CK group (p < 0.05). The THCs of CS‐GT group were

Mar. Drugs 2020, 18, 419 4 of 19 Mar. Drugs 2020, 18, x 4 of 19 highersignificantly than those higher of than the GT those and of CS the groups GT and (p CS< 0.05). groups This (p < may 0.05). be This due may to the be supplements, due to the supplements, especially Mar. Drugs 2020, 18, x 4 of 19 CS-GT,especially which CS‐GT, increased which THCsincreased through THCs the through proliferation the proliferation of hemocytes of hemocytes and enhancement and enhancement of their phagocyticof theirsignificantly phagocytic activity. higher activity. The than result those The was ofresult the similar GT was and tosimilar CS that groups of to Zhaithat (p < 0.05).of et Zhai al., This who et may al., found whobe due that found to the enrofloxacin thatsupplements, enrofloxacin (ENR) and(ENR) San-Huang-Sanespecially and San CS‐‐HuangGT, (SHS)which‐San increased promoted (SHS) THCspromoted disease through resistancedisease the proliferation resistance in shrimp of inhemocytes [32 shrimp]. The and hemocyte[32]. enhancement The apoptosishemocyte assaysapoptosisof were their assays phagocytic conducted were activity. with conducted an The Annexin result with was V-FITCan similarAnnexin/PI to Apoptosis that V‐ FITC/PIof Zhai Detection et Apoptosis al., who Kit found at Detection Day that 3 enrofloxacin according Kit at Day to the 3 manufacturer’saccording(ENR) toand the protocol.San manufacturer’s‐Huang As‐San shown (SHS) protocol. in Figurepromoted As3a–d, shown disease the CKin Figureresistance group showed3a–d, in theshrimp the CK most group[32]. hemocyte The showed hemocyte apoptosis, the most whilehemocyteapoptosis the CS-GT apoptosis, assays and werewhile GT groups conducted the CS showed‐GT with and thean GT Annexin least. groups As V showed‐ shownFITC/PI in theApoptosis Figure least.3 e, AsDetection the shown hemocyte Kitin Figureat Day apoptosis 3e,3 the ratehemocyte ofaccording the apoptosis CK to group the manufacturer’s rate was of the the highest, CK protocol. group which wasAs shown was the significantlyhighest, in Figure which 3a–d, higher wasthe CK significantly than group that showed of thehigher the supplement mostthan that hemocyte apoptosis, while the CS‐GT and GT groups showed the least. As shown in Figure 3e, the groupsof the supplement (p < 0.05). There groups was (p no < 0.05). significant There di wasfference no significant in apoptosis difference rate between in apoptosis the supplement rate between groups the hemocyte apoptosis rate of the CK group was the highest, which was significantly higher than that (supplementp > 0.05). The groups results (p indicate > 0.05). that The the results supplementation indicate that can the e supplementationffectively reduce hemocytecan effectively apoptosis reduce in of the supplement groups (p < 0.05). There was no significant difference in apoptosis rate between the Litopenaeushemocytesupplement apoptosis vannamei groups infectedin Litopenaeus(p > 0.05). with The Vibriovannamei results parahaemolyticus indicate infected that with the. Vibrio supplementation parahaemolyticus can effectively. reduce hemocyte apoptosis in Litopenaeus vannamei infected with Vibrio parahaemolyticus.

Figure 2. The total hemocyte counts (THCs) of the shrimp in each group (mean ± SD, n = 6) (different FigureFigure 2.2. TheThe totaltotal hemocytehemocyte countscounts (THCs)(THCs) of of the the shrimp shrimp in in each each group group (mean (mean ± SD,SD, n n= = 6) (di(differentfferent letters indicate significant differences between the different groups at the same time± point (p < 0.05)). lettersletters indicateindicate significantsignificant di differencesfferences between between the the di differentfferent groups groups at at the the same same time time point point ( p(p< <0.05)). 0.05)).

FigureFigure 3. Flow 3. Flow cytometry cytometry detection detection of of hemocyte hemocyte apoptosis in in (a ()a CK,) CK, (b) ( bCS)‐ CS-GT,GT, (c) GT, (c) GT,and and(d) CS (d ) CS groups,groups, and (ande) hemocyte (e) hemocyte apoptosis apoptosis rate rate (mean (mean ± SD,SD, n = 6).6). CK: CK: Infected Infected with with VibrioVibrio parahaemolyticu parahaemolyticuss ± group;group; CS-GT: CS‐GT: Supplemented Supplemented with with CS-GT CS‐GT afterafter infection with with VibrioVibrio parahaemolyticus parahaemolyticus group;group; GT: GT: SupplementedFigureSupplemented 3. Flow withcytometry with GT GT after afterdetection infection infection of hemocytewith VibrioVibrio parahaemolyticusapoptosis parahaemolyticus in (a group;) CK,group; (CS:b) CSSupplemented CS:‐GT, Supplemented (c) GT, with and CS (d with) CS CSgroups, after infectionand (e) hemocyte with Vibrio apoptosis parahaemolyticus rate (meangroup. ± SD, (din =ff 6).erent CK: letters Infected indicate with significantVibrio parahaemolyticu differencess group; CS‐GT: Supplemented with CS‐GT after infection with Vibrio parahaemolyticus group; GT: between the different groups at the same time point (p < 0.05)). Supplemented with GT after infection with Vibrio parahaemolyticus group; CS: Supplemented with CS

Mar. Drugs 2020, 18, x 5 of 19

Mar. Drugsafter 2020infection, 18, 419 with Vibrio parahaemolyticus group. (different letters indicate significant differences5 of 19 between the different groups at the same time point (p < 0.05)).

2.3. Nonspecific Nonspecific Immunity Parameters Antioxidant activities are indicators of the antioxidant status and oxidative stress of aquatic animals. Previous research has showed that, once reactive oxygen species (ROS) production increases, organisms are able to activate a seriesseries ofof antioxidantantioxidant defense systems such as superoxide dismutase (SOD),(SOD), catalase (CAT), andand thethe glutathione triad—glutathionetriad—glutathione (GSH),(GSH), glutathioneglutathione s-transferases‐transferase (GST),(GST), and glutathione peroxidase (GSH-Px)—to(GSH‐Px)—to detoxify the ROS, preventing or repairing oxidativeoxidative damage [[33].33]. SOD SOD is is a a molecular biomarker for for evaluating the oxidative stress status of aquatic organisms, due to it catalyzing the dismutation of superoxide anions to hydrogen peroxide and molecular oxygen,oxygen, forming forming a first-linea first‐line antioxidant antioxidant enzymatic enzymatic defense defense system system [34]. GSH-Px [34]. GSH can‐ removePx can lipidremove peroxides lipid peroxides induced byinduced reactive by oxygen reactive species oxygen and speciesOH, and protecting •OH, protecting the integrity the of integrity cell membrane of cell • structuremembrane and structure function. and GSH function. works with GSH glutathione works with peroxidase glutathione to reduce peroxidase hydrogen to reduce peroxide hydrogen to water, therebyperoxide maintaining to water, thereby the integrity maintaining of red the blood integrity cell membranes of red blood and cell protecting membranes red and blood protecting cells from red damageblood cells by from oxidants damage [35]. by In oxidants this study, [35]. the In this SOD, study, GSH-Px, the SOD, and GSH‐ activitiesPx, and GSH of each activities group of were each measuredgroup were each measured day using each commercial day using commercial detection kits. detection As shown kits. As in Figureshown4 ina–c, Figure the SOD, 4a–c, GSH-PX,the SOD, andGSH GSH‐PX, and activities GSH activities in the CK in group the CK increased group increased after infection after infection from Day from 1 toDay Day 1 to 3; Day the 3; results the results were similarwere similar to those to those of the of previousthe previous study, study, which which showed showed that that the the SOD, SOD, GSH-Px, GSH‐Px, and and GSH GSH activitiesactivities of Litopenaeus vannamei increased after infection compared to those in the control group (healthy(healthy shrimp) [[36].36]. ThisThis maymay be be due due to to the the fact fact that that the the shrimp shrimp can can produce produce a natural a natural immune immune response response and activateand activate the antioxidant the antioxidant system, system, which which play play an important an important role role in clearing in clearing the excessthe excess ROS ROS [36]. [36]. In the In supplementthe supplement groups, groups, the SOD, the GSH-Px,SOD, GSH and‐Px, GSH and activities GSH activities in CS-GT in and CS CS‐GT groups and wereCS groups significantly were highersignificantly than those higher in CKthan and those GT in groups CK and (p < GT0.05). groups The ( antioxidantp < 0.05). The activities antioxidant in the activities CS group in were the theCS highestgroup were compared the highest to those compared in the other to those groups. in the This other may groups. be due This to themay fact be thatdue theto the elevated fact that levels the ofelevated SOD, GSH,levels and of SOD, GSH-Px GSH, activities and GSH upon‐Px activities the oral upon administration the oral administration of CS-GT are of related CS‐GT to are a greaterrelated reductionto a greater in reduction the production in the ofproduction ROS and lipidof ROS peroxidation and lipid peroxidation in Litopenaeus in vannamei Litopenaeus. vannamei.

Figure 4. The antioxidant activities of shrimp in each group (mean SD, n = 6). (a) Superoxide Figure 4. The antioxidant activities of shrimp in each group (mean ±± SD, n = 6). (a) Superoxide dismutase (SOD), (b) glutathione peroxidase (GSH-Px),(GSH‐Px), and (c) glutathione (GSH). (di(differentfferent letters indicate significantsignificant didifferencesfferences betweenbetween thethe didifferentfferent groups groups at at the the same same time time point point ( p(p< < 0.05)).0.05)).

Previous research demonstrated that various immune enzymes including phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (ALP), and lysozyme (LSZ) are generally selected as an indicator to evaluate the immune status and diseasedisease resistanceresistance of shrimpshrimp [[10,11].10,11]. ACP and ALP are composed of many kinds of phosphomonoesterases, which are very important to the crustacean immune system [37]. [37]. Furthermore, Furthermore, ACP ACP is is an an important important component component of of phagocytic phagocytic lysosomes, lysosomes, and and in inthe the phagocytosis phagocytosis and and encapsulation encapsulation of hemocytes, of hemocytes, phagocytic phagocytic lysosomes lysosomes play playa bactericidal a bactericidal role with role withthe release the release of ACP of [32,38]. ACP [32 ALP,38]. is ALP a kind is a of kind phosphomonoesterase of phosphomonoesterase that aids that in aidsdetoxification in detoxification during duringnormal normalliving and living phagolysis and phagolysis and in the and digestion in the digestionand absorption and absorption of many nutrients of many [39]. nutrients Lysozyme [39]. Lysozyme(LSZ) is a component (LSZ) is a component of the innate of immune the innate system immune of invertebrates, system of invertebrates, functioning as functioning an antibacterial as an antibacterialprotein [40]. proteinIt hydrolyzes [40]. Itmucopolysaccharides, hydrolyzes mucopolysaccharides, which are basic which components are basic of components the bacterial of cell the bacterialwall and cellkill wallpathogens and kill [41]. pathogens In many [ 41invertebrates,]. In many invertebrates, the PO cascade the represents PO cascade a critical represents host a defense critical hostresponse. defense PO response. is the terminal PO is enzyme the terminal in the enzyme pro‐PO in activation the pro-PO system activation and involved system and in host involved defense in host defense reactions such as wound healing, cytotoxicity, and phagocytosis [11,42]. In this study, the immune enzyme activities of each group were measured each day using commercial detection kits. Mar. Drugs 2020, 18, x 6 of 19

Mar. Drugs 2020, 18, 419 6 of 19 reactions such as wound healing, cytotoxicity, and phagocytosis [11,42]. In this study, the immune enzyme activities of each group were measured each day using commercial detection kits. As shown Asin shownFigure in5, Figurethe ALP,5, the PO, ALP, and PO, LSZ and activities LSZ activities in the CK in the group CK groupincreased increased from Day from 1 Day to Day 1 to 3, Day while 3, whilethe ACP the ACPactivity activity peaked peaked at Day at 1 Day then 1 decreased then decreased from Day from 2 Day to Day 2 to 3 Day but was 3 but still was higher still higher than at than Day at0. Day The 0.results The resultswere similar were similarto those to in those the previous in the previous study, which study, showed which that showed the ACP, that the ALP, ACP, and ALP, LSM andactivities LSM activities of Litopenaeus of Litopenaeus vannamei vannamei increasedincreased after infection after infection compared compared to those to in those the incontrol the control group group(healthy (healthy shrimp) shrimp) [32]. [32This]. This might might because because of ofVibrioVibrio parahaemolyticus parahaemolyticus infectioninfection leading leading to aa stressstress reactionreaction in in the the shrimp, shrimp, inducing inducing an immunean immune response response [32]. In[32]. the In supplement the supplement groups, groups, the ACP the activity ACP inactivity the CS-GT in the and CS‐ CSGT groups and CS was groups significantly was significantly higher than higher that than in thethat CK in the and CK GT and groups GT groups (p < 0.05) (p < (Figure0.05) (Figure5a); it was 5a); highest it was in highest the CS-GT in the group CS‐GT at Day group 3 ( pat< Day0.05). 3 The(p < ALP 0.05). activity The ALP in the activity supplement in the groupssupplement was significantly groups was higher significantly than that higher in CK than group that (p < in0.05) CK (Figuregroup 5(pb). < The0.05) LSZ (Figure and PO 5b). activities The LSZ inand the PO CS-GT activities and CS in the groups CS‐GT were and significantly CS groups were higher significantly than those inhigher the CK than and those GT groupsin the CK (p

Figure 5. The immune enzyme activities of shrimp in each group (mean SD, n = 6). (a) Acid Figure 5. The immune enzyme activities of shrimp in each group (mean± ± SD, n = 6). (a) Acid phosphatase (ACP), (b) alkaline phosphatase (ALP), (c) lysozyme (LSZ), and (d) phenoloxidase (PO). phosphatase (ACP), (b) alkaline phosphatase (ALP), (c) lysozyme (LSZ), and (d) phenoloxidase (PO). (different letters indicate significant differences between the different groups at the same time point (different letters indicate significant differences between the different groups at the same time point (p < 0.05)). (p < 0.05)). 2.4. Intestinal Histology 2.4. Intestinal Histology The intestine in vertebrates plays a critical role in metabolism, nutrient absorption, and immune functionThe [ 43intestine]. In the in vertebrates present study, plays the a critical intestinal role histology in metabolism, of shrimp nutrient at Day absorption, 3 was observed and immune by stainingfunction with [43]. hematoxylin In the present and eosinstudy, (H&E). the intestinal The microstructure histology of of shrimp the intestine at Day in only3 was infected observed group by wasstaining lesions with (Figure hematoxylin6a); epithelial and cellseosin were (H&E). completely The microstructure detached from of thethe basement intestine membranein only infected and severelygroup was destroyed. lesions Parts (Figure of epithelial 6a); epithelial cells were cells detached were from completely the basement detached membrane from andthe destroyedbasement inmembrane the CS-GT and and GTseverely groups destroyed. (Figure6b,c). Parts In mostof epithelial regions ofcells the intestinewere detached in the CS from group, the epithelial basement cellsmembrane were detached and destroyed from the in the basement CS‐GT membraneand GT groups and severely(Figure 6b,c). destroyed In most (Figure regions6d). of Thethe intestine results indicatedin the CS thatgroup, the epithelial oral administration cells were detached of CS-GT from and GTthe couldbasement enhance membrane the integrity and severely of the intestinaldestroyed epithelial(Figure 6d). cells The and results basement indicated membrane, that the which oral correspondingly administration alleviatedof CS‐GT and intestinal GT could injury. enhance This may the beintegrity due to theof the antibacterial intestinal epithelial effect of CS-GT cells and and basement GT. The membrane, results were which similar correspondingly to those in the previousalleviated

Mar. Drugs 2020, 18, 419x 77 of of 19 intestinal injury. This may be due to the antibacterial effect of CS‐GT and GT. The results were similar studies,to those whichin the previous have shown studies, that dandelionwhich have extract shown can that improve dandelion the extract intestinal can structure improve and the intestinal immunestructure function and intestinal of the immune juvenile goldenfunction pompano of the juvenile (Trachinotus golden ovatus pompano)[44]. (Trachinotus ovatus) [44].

Figure 6. PhotomicrographsPhotomicrographs of of gut gut cross cross-cutting‐cutting in ( ina) CK, (a) CK,(b) CS (b‐)GT, CS-GT, (c) GT, (c )and GT, (d and) CS ( groupsd) CS groups (mean (mean± SD, n = 3).SD, The n magnification= 3). The magnification was 200×, and was the scale 200 bar, and represents the scale 100 bar μm. represents CK: Infected 100 withµm. Vibrio CK: ± × Infectedparahaemolyticu with Vibrios group; parahaemolyticus CS‐GT: Supplementedgroup; CS-GT: with CS Supplemented‐GT after infection with with CS-GT Vibrio after parahaemolyticus infection with Vibriogroup; parahaemolyticus GT: Supplementedgroup; with GT: Supplemented GT after infection with GT with after infectionVibrio parahaemolyticus with Vibrio parahaemolyticus group; CS: group;Supplemented CS: Supplemented with CS after with infection CS after with infection Vibrio with parahaemolyticusVibrio parahaemolyticus group; IEC:group; Intestinal IEC: Intestinalepithelial epithelialcells; BM: cells;Basement BM: Basementmembrane. membrane.

2.5. Microbiota 2.5.1. Alpha Diversity Analysis 2.5.1. Alpha Diversity Analysis A total of 12 water samples and 12 intestinal samples (n = 3) were collected for 16S rRNA gene A total of 12 water samples and 12 intestinal samples (n = 3) were collected for 16S rRNA gene sequencing, respectively. After data quality filtering, an average of 80,150 and 80,104 high-quality sequencing, respectively. After data quality filtering, an average of 80,150 and 80,104 high‐quality reads were obtained from water and gut samples, respectively. For all the water and gut samples, reads were obtained from water and gut samples, respectively. For all the water and gut samples, their rarefaction curves (Figure7a,b) gradually approach the saturation plateau, showing reliable their rarefaction curves (Figure 7a,b) gradually approach the saturation plateau, showing reliable sequencing and sufficient diversity. A Venn diagram of four independent ovals is presented in sequencing and sufficient diversity. A Venn diagram of four independent ovals is presented in Figure Figure7c,d, which are assigned to the four experimental groups, respectively. As shown in Figure7c, 7c,d, which are assigned to the four experimental groups, respectively. As shown in Figure 7c, for the for the water samples, the operational taxonomic units (OTUs) in the CK group (i.e., 619) are higher water samples, the operational taxonomic units (OTUs) in the CK group (i.e., 619) are higher than than those in the supplementation groups. On the contrary, for the gut samples, the OTUs in the CK those in the supplementation groups. On the contrary, for the gut samples, the OTUs in the CK group group (i.e., 536) are lower than those in the CS group (i.e., 591) (Figure7d) but still higher than those in (i.e., 536) are lower than those in the CS group (i.e., 591) (Figure 7d) but still higher than those in the the CS-GT (i.e., 492) and GT (i.e., 445) groups. According to the rank abundance curve (Figure7e,f), CS‐GT (i.e., 492) and GT (i.e., 445) groups. According to the rank abundance curve (Figure 7e,f), the the microbial species richness of all the groups with dietary supplementation was decreased compared microbial species richness of all the groups with dietary supplementation was decreased compared to that in the CK group, except for the gut sample in the CS group. to that in the CK group, except for the gut sample in the CS group. After dietary supplementation, the Chao1 (total species richness), ACE (abundance‐based coverage estimator), and Shannon indices of alpha diversity were all calculated and are shown in Table 1. Conversely, the Simpson indices of alpha diversity were all improved. Generally speaking, there are no significant differences among the four groups for both the water and gut samples, except for the Shannon index of the GT group and Simpson indices of the CS‐GT and GT groups in the water

Mar. Drugs 2020, 18, x 8 of 19

Mar.samples. Drugs 2020 In ,addition,18, 419 all the Good’s coverage indices are approaching 1, indicating that the 8OTUs of 19 obtained from each library can represent the majority of bacteria in the water and gut.

FigureFigure 7. 7.Rarefaction Rarefaction curves curves of of water water ( a()a) and and gut gut ( b(b) samples;) samples; Venn Venn diagram diagram shows shows the the operational operational taxonomictaxonomic unit unit (OTU) (OTU) numbers numbers of waterof water (c) and(c) and gut gut (d) samples;(d) samples; rank rank abundance abundance curve curve of water of water (e) and (e) gutand (f )gut samples (f) samples (mean (meanSD, ± n SD,= 3). n = 3). ±

After dietary supplementation,Table 1. Alpha the diversity Chao1 indices (total species of microbiota richness), in water ACE and (abundance-based gut. coverage estimator), and Shannon indices of alpha diversity were all calculated and are shown in Table1. Richness Estimate Diversity Estimate Conversely,Samples theGroup Simpson indices of alpha diversity were all improved. Generally speaking, there are Chao1 ACE Shannon Simpson Good's Coverage no significant diffCKerences 475.84 among ± 25.31 the a four 478.52 groups ± 45.45 for a both 5.27 the ± 0.27 water a and 0.79 gut± 0.01 samples, a 0.9995 except ± 0.0018 for a the Shannon index ofCS the GT group 461.95 ± and 15.54 Simpson a 432.83 indices ± 44.36 ofa the 5.19 CS-GT + 0.14 anda GT 0.83 groups ± 0.02 a in the 0.9995 water ± 0.0004 samples. a water In addition, all theCS-GT Good’s 435.39 coverage ± 91.77 indices a 411.55 are ± approaching 75.57 a 4.72 1, ± indicating0.57 a 0.88 that ± 0.04 the b OTUs 0.9995 obtained ± 0.0004 froma a a b b a each library can representGT 417.15 the majority ± 5.68 of 404.70 bacteria ± 10.55 in the water 4.17 ± 0.22 and gut. 0.92 ± 0.03 0.9995 ± 0.0002 gut CK 346.18 ± 90.64 a 348.52 ± 88.57 a 4.51 ± 2.57 a 0.78 ± 0.14 a 0.9997 ± 0.0007 a CS 378.42 ± 98.21 a 382.83 ± 96.31 a 4.81 ± 0.32 a 0.72 ± 0.03 a 0.9997 ± 0.0003 a 2.5.2. Beta Diversity Analysis CS-GT 324.53 ± 0.53 a 339.37 ± 13.50 a 4.63 ± 0.56 a 0.81 ± 0.05 a 0.9997 ± 0.0005 a The beta diversityGT was 287.03 used ± 23.05 to analyze a 291.53 the ± 21.06 differences a 4.18 between ± 1.33 a the 0.88 microbial ± 0.17 a communities 0.9997 ± 0.0004 in a the waterValues and gut are samples. expressed Principal as mean coordinates ± SD (n = 3), analysis and values (PCoA) with basedsignificant onthe differences weighted-uniFrac are labeled distancewith for bacterialdifferent profiles letters within was used the same in the row, present a: p > 0.05, study b: p to < 0.05. indicate the community changes in different samples. The PCoA showed that, both in the water and intestinal samples, the CS-GT and GT groups 2.5.2. Beta Diversity Analysis were clustered on the right side of the abscissa, and the distance was closer (Figure8a); the CK and CS groupsThe beta were diversity clustered was on used the left to sideanalyze of thethe abscissa,differences and between the distance the microbial was closer. communities The results in showedthe water that and the gut microbial samples. communities Principal coordinates of the CS-GT analysis and GT (PCoA) groups based were similar,on the weighted-uniFrac as were those of thedistance CK and for CS bacterial groups profiles (Figure8 wasb). It used may in be the related present to the study fact to that indicate both CS-GT the community and GT can changes inhibit in Gram-negative bacteria.

Mar. Drugs 2020, 18, 419 9 of 19

Table 1. Alpha diversity indices of microbiota in water and gut. Mar. Drugs 2020, 18, x 9 of 19 Richness Estimate Diversity Estimate 2.5.2.Samples Beta DiversityGroup Analysis Chao1 ACE Shannon Simpson Good’s Coverage The beta CKdiversity 475.84 was used25.31 toa analyze478.52 the45.45 differencesa 5.27 0.27betweena 0.79 the 0.01microbiala 0.9995 communities0.0018 a in ± ± ± ± ± the water andCS gut samples. 461.95 Principal15.54 a coordinates432.83 44.36 analysisa 5.19 + (PCoA)0.14 a 0.83based0.02 ona the 0.9995weighted0.0004‐uniFraca water ± ± ± ± CS-GT 435.39 91.77 a 411.55 75.57 a 4.72 0.57 a 0.88 0.04 b 0.9995 0.0004 a distance for bacterial profiles± was used in the± present study± to indicate± the community± changes in GT 417.15 5.68 a 404.70 10.55 a 4.17 0.22 b 0.92 0.03 b 0.9995 0.0002 a different samples. The PCoA± showed that, both± in the water± and intestinal± samples, the± CS‐GT and CK 346.18 90.64 a 348.52 88.57 a 4.51 2.57 a 0.78 0.14 a 0.9997 0.0007 a GT groups were clustered on± the right side of± the abscissa,± and the distance± was closer± (Figure 8a); CS 378.42 98.21 a 382.83 96.31 a 4.81 0.32 a 0.72 0.03 a 0.9997 0.0003 a gut ± ± ± ± ± the CK and CSCS-GT groups were 324.53 clustered0.53 a on339.37 the left13.50 sidea of the4.63 abscissa,0.56 a and0.81 the0.05 distancea 0.9997 was 0.0005closer.a The ± ± ± ± ± results showedGT that the 287.03 microbial23.05 communitiesa 291.53 21.06 of thea CS4.18‐GT1.33 anda GT0.88 groups0.17 a were0.9997 similar,0.0004 as awere ± ± ± ± ± thoseValues of the are CK expressed and CS as meangroupsSD (Figure (n = 3), and 8b). values It may with be significant related ditoff erencesthe fact are that labeled both with CS di‐ffGTerent and letters GT can ± inhibitwithin Gram the same‐negative row, a: bacteria.p > 0.05, b: p < 0.05.

FigureFigure 8.8. Principal coordinates coordinates analysis analysis (PCoA) (PCoA) plot plot based based on on weighted weighted-UniFrac‐UniFrac analysis analysis of ofwater water (a) (anda) and guts guts (b) ( bof) of shrimp shrimp (n (n == 3).3). The The abscissa abscissa represents represents one one principal component, thethe ordinateordinate representsrepresents another another principal principal component, component, and and the percentage the percentage represents represents the contribution the contribution of the principal of the componentprincipal component to the sample to the di ffsampleerence. difference. 2.5.3. Microbial Composition and Changes 2.5.3. Microbial Composition and Changes Bacteria present in the aquaculture environment play an important role in the processes of nutrient Bacteria present in the aquaculture environment play an important role in the processes of cycling and mineralization of organic compounds. The microbial composition and changes in the nutrient cycling and mineralization of organic compounds. The microbial composition and changes water in each group were analyzed. At the phylum level (Figure9a), the bacteria were predominantly in the water in each group were analyzed. At the phylum level (Figure 9a), the bacteria were Proteobacteria (62.28–92.22%), Bacteroidetes (11.23–22.56%), Gracilibacteria (5.01–22.35%), Firmicutes predominantly Proteobacteria (62.28–92.22%), Bacteroidetes (11.23–22.56%), Gracilibacteria (5.01– (0.25–11.13%), Verrucomicrobia (0.32–1.13%), and Tenericutes (0.14–1.08%). The results showed that 22.35%), Firmicutes (0.25–11.13%), Verrucomicrobia (0.32–1.13%), and Tenericutes (0.14–1.08%). The Proteobacteria (62.28–92.22%) was the most abundant phylum in all the groups, which was consistent results showed that Proteobacteria (62.28–92.22%) was the most abundant phylum in all the groups, with previous studies [45–47], likely implying that Proteobacteria comprised the largest and most which was consistent with previous studies [45–47], likely implying that Proteobacteria comprised phenotypically diverse division of the prokaryotes [48]. Bacteroidetes was another predominant the largest and most phenotypically diverse division of the prokaryotes [48]. Bacteroidetes was phylum; the higher abundance of Bacteroidetes might be caused by pollutants such as nitrates, another predominant phylum; the higher abundance of Bacteroidetes might be caused by pollutants ammonia, and feces [49]. At the family level (Figure9b), Rhodobacteraceae (53.90–71.49%) was the such as nitrates, ammonia, and feces [49]. At the family level (Figure 9b), Rhodobacteraceae (53.90– most abundant in all the groups, followed by Vibrionaceae (15.38–29.90%), Moraxellaceae (8.76–10.21%), 71.49%) was the most abundant in all the groups, followed by Vibrionaceae (15.38–29.90%), Flavobacteriaceae (5.75–6.86%), and Staphylococcaceae (0.58–1.01%). Rhodobacteraceae showed the Moraxellaceae (8.76–10.21%), Flavobacteriaceae (5.75–6.86%), and Staphylococcaceae (0.58–1.01%). highest abundance in all the groups, especially in the CS-GT group. Rhodobacteraceae, as a specific Rhodobacteraceae showed the highest abundance in all the groups, especially in the CS‐GT group. taxon in aquaculture water, is associated with algae. Some members of Rhodobacteraceae can produce Rhodobacteraceae, as a specific taxon in aquaculture water, is associated with algae. Some members tropodithietic acid (TDA) to inhibit the growth of pathogens [46]. Especially, Vibrio have been regarded of Rhodobacteraceae can produce tropodithietic acid (TDA) to inhibit the growth of pathogens [46]. as opportunistic pathogens causing stress and disease infections [50]. The abundances of Vibrio were Especially, Vibrio have been regarded as opportunistic pathogens causing stress and disease much lower in the GT and CS-GT groups than in the CK group at the genus level (Figure 10). This may infections [50]. The abundances of Vibrio were much lower in the GT and CS‐GT groups than in the be due to the strong bactericidal effect of CS-GT and GT on Gram-negative bacteria. CK group at the genus level (Figure 10). This may be due to the strong bactericidal effect of CS‐GT and GT on Gram‐negative bacteria.

Mar.Mar. Drugs Drugs 20202020, ,1818, ,x 419 1010 of of 19 19 Mar. Drugs 2020, 18, x 10 of 19

Figure 9. Composition and relative abundance of water microbial communities (a) at phylum level FigureFigure 9. 9.Composition Composition and and relative relative abundance abundance of waterof water microbial microbial communities communities (a) at (a phylum) at phylum level andlevel and(b) at(b) family at family level level (mean (meanSD, ± SD, n = n3). = 3). and (b) at family level (mean± ± SD, n = 3).

FigureFigure 10. 10. HeatmapHeatmap analysis of thethe speciesspecies abundance abundance clustering clustering in in the the top top 30 30 at at the the genus genus level level in thein waterFigure (mean 10. HeatmapSD, n = analysis3). of the species abundance clustering in the top 30 at the genus level in the water (mean± ± SD, n = 3). the water (mean ± SD, n = 3). The intestinal microbiota plays an important role in digestion, absorption, metabolism, and defense The intestinal microbiota plays an important role in digestion, absorption, metabolism, and againstThe pathogens intestinal [ 51microbiota]. In this plays study, an the important microbial role compositions in digestion, and absorption, changes in metabolism, the gut in each and defense against pathogens [51]. In this study, the microbial compositions and changes in the gut in groupdefense were against analyzed. pathogens At the [51]. phylum In this levelstudy, (Figure the microbial 11a), the compositions bacteria were and mostly changes Proteobacteria in the gut in each group were analyzed. At the phylum level (Figure 11a), the bacteria were mostly Proteobacteria (37.37–88.87%),each group were Firmicutes analyzed. (4.11–79.88%), At the phylum Tenericutes level (Figure (2.09–16.75%), 11a), the bacteria and Bacteroidetes were mostly (0.42–13.18%).Proteobacteria (37.37–88.87%), Firmicutes (4.11–79.88%), Tenericutes (2.09–16.75%), and Bacteroidetes (0.42–13.18%). Notably,(37.37–88.87%), Proteobacteria Firmicutes (78.34–88.87%) (4.11–79.88%), was Tenericutes most abundant (2.09–16.75%), in the CK and and CS Bacteroidetes groups, while (0.42–13.18%). Firmicutes Notably, Proteobacteria (78.34–88.87%) was most abundant in the CK and CS groups, while (51.82–79.88%)Notably, Proteobacteria was most abundant (78.34–88.87%) in the CS-GT was most and GT abundant groups. Thein the higher CK abundance and CS groups, of Firmicutes while Firmicutes (51.82–79.88%) was most abundant in the CS‐GT and GT groups. The higher abundance inFirmicutes the CS-GT (51.82–79.88%) and GT groups was could most be abundant associated in withthe CS the‐GT presence and GT of groups. untreated The fecalhigher sewage abundance [49]. of Firmicutes in the CS‐GT and GT groups could be associated with the presence of untreated fecal Theof Firmicutes abundance in of the Proteobacteria CS‐GT and GT was groups lower could in the be CS-GT associated and with GT groups, the presence which of may untreated be due fecal to sewage [49]. The abundance of Proteobacteria was lower in the CS‐GT and GT groups, which may be thesewage suppression [49]. The of abundanceVibrio by of GT Proteobacteria and CS-GT. At was the lower family in the level CS‐ (FigureGT and 11GTb), groups, Mycoplasmataceae which may be due to the suppression of Vibrio by GT and CS‐GT. At the family level (Figure 11b), (10.04–81.02%),due to the suppression Rhodobacterales of Vibrio (21.22–41.91%), by GT and Enterococcaceae CS‐GT. At (15.74–22.46%),the family level and Vibrionaceae(Figure 11b), Mycoplasmataceae (10.04–81.02%), Rhodobacterales (21.22–41.91%), Enterococcaceae (15.74–22.46%), (0.7–79.27%)Mycoplasmataceae were the (10.04–81.02%), main families Rhodobacterales in all groups. (21.22–41.91%), Enterococcaceae, Enterococcaceae which have been (15.74–22.46%), used as a and Vibrionaceae (0.7–79.27%) were the main families in all groups. Enterococcaceae, which have probioticand Vibrionaceae for humans (0.7–79.27%) or animals (includingwere the main shrimp) families [52], andin all Rhodobacterales, groups. Enterococcaceae, which are alsowhich known have been used as a probiotic for humans or animals (including shrimp) [52], and Rhodobacterales, which tobeen be potential used as a probiotics probiotic for for humans rainbow or trout animals (Oncorhynchus (including shrimp) mykiss)[ [52],53], wereand Rhodobacterales, more abundant in which the are also known to be potential probiotics for rainbow trout (Oncorhynchus mykiss) [53], were more CS-GTare also and known GT groups. to be potential This study probiotics suggested for that rainbow CS-GT trout and ( GTOncorhynchus may be able mykiss to alter) [53], the were structure more abundant in the CS‐GT and GT groups. This study suggested that CS‐GT and GT may be able to alter ofabundant the intestinal in the microflora CS‐GT and and GT supportgroups. This the colonization study suggested of potential that CS‐ probiotics.GT and GT Moreover, may be ableVibrio to alteris the structure of the intestinal microflora and support the colonization of potential probiotics. the structure of the intestinal microflora and support the colonization of potential probiotics.

Mar. Drugs 2020, 18, 419 11 of 19 Mar.Mar. Drugs Drugs 2020 2020, 18, 18, x, x 1111 of of 19 19 oneMoreover,Moreover, of the mostVibrio Vibrio important is is one one of of diseasesthe the most most inimportant important shrimp aquaculture diseases diseases in in andshrimp shrimp causes aquaculture aquaculture serious mortality and and causes causes in shrimpserious serious worldwidemortalitymortality in [in54 shrimp ].shrimpVibrio worldwide isworldwide widely distributed [54]. [54]. Vibrio Vibrio in is marineis widely widely environments distributed distributed in andin marine marine is typically environments environments among the and and most is is abundanttypicallytypically floraamong among in shrimpthe the most most digestive abundant abundant systems flora flora [55 in ,in56 shrimp ].shrimp Previous digestive digestive studies systems demonstratedsystems [55,56]. [55,56]. that Previous PreviousVibrio is studies highlystudies abundantdemonstrateddemonstrated in the that that guts Vibrio Vibrio of shrimps is is highly highly such abundant abundant as Litopenaeus in in the the vannameiguts guts of of shrimps, shrimpsPeneaus such monodonsuch as as Litopenaeus ,Litopenaeus and Fenneropenaeus vannamei vannamei, , chinensisPeneausPeneaus monodon [monodon46]. As, shownand, and Fenneropenaeus Fenneropenaeus in Figure 12, inchinensis chinensis this study, [46]. [46].Vibrio As As shown shownwas predominant in in Figure Figure 12, 12, in in in the this this CK study, study, and CSVibrio Vibrio groups, was was whilepredominantpredominant being lower in in the the in CK CK the and and GT CS and CS groups, groups, CS-GT while groups;while being being this lower islower because in in the the CS-GTGT GT and and and CS CS‐ GT‐GT cangroups; groups; inhibit this this the is is because growth because ofCSCS Gram-negative‐GT‐GT and and GT GT can can bacteria inhibit inhibit [the26 the]. growth growth of of Gram Gram‐negative‐negative bacteria bacteria [26]. [26].

FigureFigure 11. 11. Composition Composition and and relative relative abundance abundance of of gut gut microbial microbial communities communities (a ())a at) at phylum phylum level level and and ((bb()b) atat) at familyfamily family levellevel level (mean(mean (mean ± ±SD, SD, n n= =3).3). 3). ±

Figure 12. Heatmap analysis of the species abundance clustering in the top 30 at genus level in the gut FigureFigure 12. 12. Heatmap Heatmap analysis analysis of of the the species species abundance abundance clustering clustering in in the the top top 30 30 at at genus genus level level in in the the (mean SD, n = 3). gutgut (mean (mean± ± ±SD, SD, n n= =3). 3). 2.5.4. Microbial Function Prediction 2.5.4.2.5.4. Microbial Microbial Function Function Prediction Prediction The microbial functions of the water and gut were predicted with PICRUSt2 (Phylogenetic InvestigationTheThe microbial microbial of Communities functions functions byof of Reconstructionthe the water water and and of gut gut Unobserved were were predicted predicted States) with and with Tax4Fun2 PICRUSt2 PICRUSt2 (an (Phylogenetic (Phylogenetic open-source RInvestigationInvestigation package that of predictsof Communities Communities the functional by by Reconstruction Reconstruction capabilities of microbialof of Unobserved Unobserved communities States) States) based and and Tax4Fun2 on Tax4Fun2 a 16S dataset) (an (an open open [57],‐ ‐ respectively.sourcesource R R package package The that results that predicts predicts showed the the thatfunctional functional the water capabilities capabilities microbiota of of microbial wasmicrobial enriched communities communities with functions based based on on related a a16S 16S todataset)dataset) glycan [57], [57], biosynthesis respectively. respectively. and The The metabolism, results results showed showed nucleotide that that the the metabolism, water water microbiota microbiota metabolism, was was enriched enriched genetic with with information functions functions relatedrelated toto glycanglycan biosynthesisbiosynthesis andand metabolism,metabolism, nucleotidenucleotide metabolism,metabolism, metabolism,metabolism, geneticgenetic informationinformation processing,processing, replicationreplication andand repair,repair, transcription,transcription, poorlypoorly characterization,characterization, andand thethe

Mar. Drugs 2020, 18, 419 12 of 19

Mar. Drugs 2020, 18, x 12 of 19 processing, replication and repair, transcription, poorly characterization, and the metabolism of cofactors and vitamins (Figure 13a). The intestine microbiota was enriched with functions related to metabolism of cofactors and vitamins (Figure 13a). The intestine microbiota was enriched with the immune system, amino acid metabolism, carbohydrate metabolism, translation, replication and functions related to the immune system, amino acid metabolism, carbohydrate metabolism, repair, energy metabolism, the metabolism of cofactors and vitamins, xenobiotic biodegradation and translation, replication and repair, energy metabolism, the metabolism of cofactors and vitamins, metabolism, and lipid metabolism (Figure 13b). In particular, for the gut, the immune system is more xenobiotic biodegradation and metabolism, and lipid metabolism (Figure 13b). In particular, for the abundant in the CS-GT group than in the CK group, and it is speculated that CS-GT can regulate the gut, the immune system is more abundant in the CS‐GT group than in the CK group, and it is immune-related functions of shrimp. speculated that CS‐GT can regulate the immune‐related functions of shrimp.

FigureFigure 13. 13.Heatmaps Heatmaps of microbialof microbial functions functions in thein the top top 35 of35 level-2 of level for‐2 for water water (a) and(a) and gut gut (b) ( samplesb) samples (mean SD, n = 3). (mean± ± SD, n = 3). 2.5.5. Correlation Analysis between Microbial Communities and Environmental Variables 2.5.5. Correlation Analysis between Microbial Communities and Environmental Variables Canonical correlation analysis (CCA) was performed to determine the correlation between the Canonical correlation analysis (CCA) was performed to determine the correlation between the physical–chemical constituents and microbial communities of the water and guts. For the water physical–chemical constituents and microbial communities of the water and guts. For the water samples, five environmental variables explain 76.50% of the total change in microbial communities samples, five environmental variables explain 76.50% of the total change in microbial communities (Figure 14a). NO3− had the greatest effect on the microbial community, followed by NO2−, pH, (Figure 14a). NO3− had the greatest effect on the microbial community, followed by NO2−, pH, total total alkalinity, and NH3; they were synergistic in their effects on the microbial structure. The five alkalinity, and NH3; they were synergistic in their effects on the microbial structure. The five environmental variables were positively correlated with CK and CS, and negatively with CS-GT and environmental variables were positively correlated with CK and CS, and negatively with CS‐GT and GT; Vibrio was positively correlated with NH3, NO3−, and NO2− concentrations, which were negatively GT; Vibrio was positively correlated with NH3, NO3−, and NO2− concentrations, which were negatively correlated with GT and CS-GT dosage, suggesting that Vibrio was negatively correlated with GT and correlated with GT and CS‐GT dosage, suggesting that Vibrio was negatively correlated with GT and CS-GT dosage. It could be observed from Figure 14b that five environmental variables explained CS‐GT dosage. It could be observed from Figure 14b that five environmental variables explained 75.11% of the total change in intestinal microbial community. NH3, NO3−, and total alkalinity had 75.11% of the total change in intestinal microbial community. NH3, NO3−, and total alkalinity had the the greatest effects on the intestinal microbial community, followed by pH and NO2−. Vibrio was greatest effects on the intestinal microbial community, followed by pH and NO2−. Vibrio was positively correlated with NH3 concentration, which was negatively correlated with GT and CS-GT positively correlated with NH3 concentration, which was negatively correlated with GT and CS‐GT dosage, suggesting that Vibrio was negatively correlated with GT and CS-GT dosage. The results show dosage, suggesting that Vibrio was negatively correlated with GT and CS‐GT dosage. The results that CS-GT can regulate the abundances of Vibrio in water and intestines by adjusting the NO2−, NO3−, show that CS‐GT can regulate the abundances of Vibrio in water and intestines by adjusting the NO2−, and NH3 concentrations. NO3−, and NH3 concentrations.

Mar. Drugs 2020, 18, 419 13 of 19 Mar. Drugs 2020, 18, x 13 of 19

FigureFigure 14. 14.Canonical Canonical correlation correlation analysis analysis (CCA) (CCA) ordination ordination diagrams diagrams of of environmental environmental factors factors among among waterwater ( a(a)) and and gut gut ( b(b)) samples samples (n (n= =3). 3). 3. Materials and Methods 3. Materials and Methods 3.1. CS-GT Synthesis 3.1. CS‐GT Synthesis CS-GT was prepared according to our previous report with modifications [26]. Briefly, 3 g of CS‐GT was prepared according to our previous report with modifications [26]. Briefly, 3 g of CS CS (544 kDa, deacetylation degree: 95%) was firstly dissolved in 200 mL of acetic acid (1 v%) under (544 kDa, deacetylation degree: 95%) was firstly dissolved in 200 mL of acetic acid (1 v%) under magnetic stirring. A volume of 100 mL of sodium periodate solution (0.3 mol/L) was then added into magnetic stirring. A volume of 100 mL of sodium periodate solution (0.3 mol/L) was then added into the CS solution, which was allowed to react in the dark at 30 ◦C for 2 h. A 0.1 mol amount of ethylene the CS solution, which was allowed to react in the dark at 30 °C for 2 h. A 0.1 mol amount of ethylene glycol was then added to terminate the reaction. After dialysis and purification, the as-obtained glycol was then added to terminate the reaction. After dialysis and purification, the as‐obtained oxidized chitosan was dried and collected for further modification. Prior to the graft, 2 g of oxidized oxidized chitosan was dried and collected for further modification. Prior to the graft, 2 g of oxidized chitosan was firstly dissolved in 100 mL of acetic acid solution (1 v%) and then mixed with 2 g of chitosan was firstly dissolved in 100 mL of acetic acid solution (1 v%) and then mixed with 2 g of gentamicin sulfate under stirring for 4 h at 40 ◦C. Subsequently, the mixed solution was cooled to gentamicin sulfate under stirring for 4 h at 40 °C. Subsequently, the mixed solution was cooled to 30 30 ◦C, and its pH value was adjusted to 6.0. Then, 0.4 g of sodium cyanoborohydride (20% of oxidized °C, and its pH value was adjusted to 6.0. Then, 0.4 g of sodium cyanoborohydride (20% of oxidized chitosan) was added, and the reduction reaction was allowed to continue for 2 h at 30 ◦C. After dialysis chitosan) was added, and the reduction reaction was allowed to continue for 2 h at 30 °C. After and lyophilization, the CS-GT was stored in a desiccator for further use. dialysis and lyophilization, the CS‐GT was stored in a desiccator for further use. 3.2. Experimental Design and Diets 3.2. Experimental Design and Diets The Litopenaeus vannamei (body weight: 6.05 0.17 g) used in this study were obtained from East ± IslandThe Marine Litopenaeus Biology vannamei Research (body Base, weight: College 6.05 of Fisheries, ± 0.17 g) used Guangdong in this study Ocean were University obtained (Zhanjiang, from East China).Island Marine The shrimp Biology were Research randomly Base, allotted College to of twelve Fisheries, tanks Guangdong (300 L, polyvinyl Ocean University chloride polymer) (Zhanjiang, at China). The shrimp were 1randomly allotted to twelve tanks (300 L, polyvinyl chloride polymer) at the rate of 30 shrimp tank− . Prior to the experiment, all the shrimp were acclimated to the basal diet −1 (commercialthe rate of 30 feed shrimp pellets tank twice. Prior daily to at the a feedingexperiment, rate ofall 0.5% the shrimp of shrimp were bodies) acclimated and conditions to the basal (30 diet (commercial feed pellets twice daily at a feeding rate of 0.5% of shrimp bodies) and conditions (30± ± 0.5 ◦C, pH 7.8–8.2, and 32%0 salinity) for a week. 0.5 °C,For pH the 7.8–8.2, experimentally and 32%0 salinity) induced for infection a week. implemented according to a previous study [58], For the experimentally induced infection implemented6 according1 to a previous study [58], the the shrimp were exposed to Vibrio parahaemolyticus (10 CFU ml− ) according to pre-experimental 6 −1 results.shrimp were Subsequently, exposed to the Vibrio supplements parahaemolyticus were coated (10 CFU onto ml the) according surface of to commercialpre‐experimental feed pelletsresults. usingSubsequently, peanut oil, the and supplements the final were supplement coated onto content the surface in the experimental of commercial groups feed pellets was designedusing peanut as oil, and the final supplement content in the experimental groups was designed as follows:1 control follows: control check group (i.e., CK group: no supplement), GT group (GT: 10 mg kg− ), CS group check group (i.e.,1 CK group: no supplement), GT group (GT:1 10 mg kg−1), CS group (CS: 2501 mg kg−1), (CS: 250 mg kg− ), and CS-GT group (CS-GT: 50 mg kg− ). There were 40 shrimp tank− in each −1 −1 group.and CS The‐GT residual group (CS food‐GT: and 50 feces mg kg were). removedThere were via 40 a siphon,shrimp whiletank the in each water group. in all theThe tanks residual was food not renewedand feces during were removed the whole via feeding a siphon, trial. while the water in all the tanks was not renewed during the whole feeding trial. 3.3. Water Physicochemical Quality 3.3. Water Physicochemical Quality Water samples were collected daily at 10 a.m. during the whole feeding trial. The pH and salinityWater were samples analyzed were using collected pH and daily salinity at 10 meters, a.m. during respectively. the whole Total feeding alkalinity trial. wasThe pH determined and salinity by were analyzed using pH and salinity meters, respectively. Total alkalinity was determined by analytical titration, while ammonia nitrogen (NH3), nitrite‐nitrogen (NO2−), and nitrate‐nitrogen

Mar. Drugs 2020, 18, 419 14 of 19

analytical titration, while ammonia nitrogen (NH3), nitrite-nitrogen (NO2−), and nitrate-nitrogen (NO3−) concentrations were analyzed using the standard methods reported by Strickland and Parsons [59].

3.4. Hematological Profile

3.4.1. Total Hemocyte Counts (THCs) Initially, anticoagulant solution was prepared by dissolving 0.48 g of citrate, 1.32 g of sodium citrate, and 1.47 g of glucose in 100 mL of double distilled water, and then sterilizing the solution by autoclaving for further use. Six shrimp were randomly selected from each group during the feeding stage. Hemolymph (100 µL) was withdrawn from the ventral sinus of each shrimp using a 1 mL sterile syringe (25 gauge) containing 0.9 mL of anticoagulant solution. A drop of anticoagulant–hemolymph mixture (20 µL) above was transferred onto a hemocytometer for THC measurement using an inverted phase-contrast microscope (Leica DMIL, Munich, Germany). Each sample was repeated in triplicate, and the average was recorded.

3.4.2. Hemocyte Apoptosis Analysis Hemocyte apoptosis assays were conducted with an Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime, Shanghai, China) at Day 3 according to the manufacturer’s protocol. The hemolymph was obtained using similar procedures with the final volume fraction of the anticoagulant solution improved to 50%. The mixture was then centrifuged at 1000 rpm at 4 ◦C for 5 min to obtain the hemocyte microspheres, which were resuspended in PBS (pH 7.4) and adjusted to a cell density of 1 106 cells/mL × for another centrifugation using the same parameters. The final hemocyte microspheres obtained were stained with Annexin V-FITC and PI according to the manufacturer’s instructions. The percentage of apoptotic hemocytes was determined by flow cytometry (BDFACSC anto II, Franklin Lake, NJ, USA) in triplicate.

3.5. Nonspecific Immunity Parameters Six shrimp (body weight: 6.05 0.17 g) were randomly selected from each group at pre-determined ± time intervals (Days 0, 1, 2, and 3) and dissected, with their hepatopancreases immediately stored in liquid nitrogen for antioxidant and immune enzyme activity analysis. Prior to analysis, hepatopancreas samples were homogenized in ice-cold phosphate buffer (1:10 dilution) and then centrifuged for 20 min (4 ◦C, 3000 rpm) to obtain the supernatant for further use. Superoxide dismutase (SOD), glutathione peroxidase (GSH-PX) and glutathione (GSH), lysozyme (LSZ), acid phosphatase (ACP), alkaline phosphatase (ALP), and phenoloxidase (PO) were measured using commercial detection kits (Jianglai Bioengineering Institute, Shanghai, China) according to the manufacturer’s protocols.

3.6. Intestinal Histology Three shrimp were randomly selected from each tank of the four groups at Day 3 and dissected, with the mid-gut immediately stored in Bouin’s solution for later use. After fixation in Bouin’s solution and dehydration in a gradient of ethanol solutions, the mid-gut segments were cleaned in toluene and embedded in paraffin. Cross sections (around 5 µm) of the embedded samples were cut using a rotary microtome and then stained with hematoxylin and eosin (H&E), and imaged with a light microscope (Olympus, Nikon, Tokyo, Japan).

3.7. Microbiota Analysis

3.7.1. DNA Extraction For DNA extraction, approximately 1 L of rearing water (in triplicate) was filtered through a nylon mesh (100 µm pore size) and a polycarbonate membrane (0.22 µm pore size) on Day 3, Mar. Drugs 2020, 18, 419 15 of 19 respectively. The membranes obtained were stored at 80 C for further genomic DNA extraction − ◦ and analysis. Prior to gut sampling, six shrimp (in triplicate) were rinsed twice using 75 v% ethanol, and their gastrointestinal tracts were excised using a sterile toothpick. The gut tissue obtained was then transferred into a sterilized EP tube and immediately stored in liquid nitrogen for genomic DNA extraction and analysis. Total genomic DNA was extracted using the CTAB/SDS method. DNA concentration and purity were assessed on 1% agarose gels. According to the concentration, DNA was diluted to 1 ng/µL using sterile water.

3.7.2. 16S rRNA Gene Sequencing In order to determine the microbial communities of the guts and rearing water, the amplification and sequencing of the V4 region of the bacterial 16S rDNA gene was conducted in triplicate using 515 F and 806 R as primer sets. All the PCR reactions were carried out in 30 µL reactions: 15 µL of Phusion® High-Fidelity PCR Master Mix (New England Biolabs, Massachusetts, MA, USA), 2 µM forward and reverse primers, 10 µL of template DNA (5–10 ng), and 2 µL of H2O. Thermal cycling consisted of an initial denaturation at 98 ◦C for 1 min, followed by 30 cycles of denaturation at 98 ◦C for 10 s, annealing at 50 ◦C for 30 s, elongation at 72 C for 30 s, and, finally, 72 C for 5 min. After amplification, the same volume of 1 ◦ ◦ × TAE loading buffer (contained SYB green) was mixed with PCR products, and electrophoresis was performed on a 2% agarose gel for detection. Samples with a bright main strip between 400 and 450 bp were chosen for further experiments. The PCR products were mixed in equidensitic ratios. Then, the mixture was purified with a Gene JET Gel Extraction Kit (Thermo Scientific, Waltham, MA, USA). Sequencing libraries were generated using an NEB Next® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, Massachusetts, MA, USA) following the manufacturer’s recommendations, and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq platform (Novogene, Beijing, China) and 250 bp paired-end reads were generated.

3.7.3. Sequencing Data Analysis Paired-end reads from the original DNA fragments were merged by using a very fast and accurate analysis tool, Fast Length Adjustment of Short reads (FLASH-1.2.11,McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA), which is designed to merge paired-end reads in the case of overlap between reads1 and reads2. Paired-end reads were assigned to each sample according to the unique barcodes. Sequences were analyzed using the Quantitative Insights Into Microbial Ecology software (QIIME2, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA). Alpha (within samples) and beta (among samples) diversities were analyzed based on in-house Perl scripts in QIIME2. The reads were filtered with QIIME2 and clustered into operational taxonomic units (OTUs) at an identity threshold of 97%. A Venn diagram was generated to identify unique and shared OTUs. Alpha diversity indices including Chao1, Shannon, Simpson, and abundance-based coverage estimator (ACE) were applied to analyze the complexity of species diversity. Chao1 and ACE were used to assess community richness, whereas Shannon and Simpson were used to assess community diversity. The different taxonomic levels were transformed to relative abundance counts to draw bar plots. In order to investigate the similarity of the community structure among different samples, principal coordinates analysis (PCoA) was performed using weighted-uniFrac distances, respectively. PICRUSt2 (Department of Microbiology and Immunology, Dalhousie University, Halifax, NS, Canada) is a biological information software package for predicting metagenome functions based on Marker genes (such as 16S rRNA). Tax4Fun2 (Evolution and Ecology Research Centre, School of Biological, Earth and Environmental Sciences, University of New South Wales, NSW, Australia) is an R Mar. Drugs 2020, 18, 419 16 of 19 program package for function prediction for intestinal, soil, and other environmental samples based on a 16S Silva database [57]. Canonical correspondence analysis (CCA) based on a unimodal model was used to address the relationship between microbial communities and water quality indicators.

3.8. Statistical Analysis All data, presented as mean SD, were subjected to a one-way analysis of variance (ANOVA) ± using Statistical Package for the Social Sciences (IBM SPSS v20.0, Inc., 2010, Chicago, IL, USA), followed by the testing of mean differences using Tukey’s multiple comparison tests. The value of p < 0.05 was set for statistical significance.

4. Conclusions Our results show that the total hemocyte counts, antioxidant activities, immune enzyme activities, and intestinal morphology of Litopenaeus vannamei were improved after dietary supplementation with a chitosan–gentamicin conjugate. Meanwhile, the abundances of Vibrio in the water and guts of Litopenaeus vannamei were significantly reduced after dietary supplementation with the chitosan–gentamicin conjugate. This study provides a new research foundation and ideas for the future development of chitosan conjugates in aquaculture.

Author Contributions: Data curation, D.M.; Funding acquisition, C.S.; Investigation, T.H.; Methodology, S.L.; Supervision, C.W.; Validation, S.K.; Writing—original draft, F.L.; Writing—review & editing, C.L. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Enhancing School with Innovation of Guangdong Ocean University (230419108), the Guangdong Provincial Natural Science Foundation of China (2020A151501003), the Project of Zhanjiang Science & Technology Plan (2019A01014), The Fund of Southern Marine Science and Engineering Guangdong Laboratory (Zhanjiang) (013S19006-007), The Project of 2019 Annual Guangdong Provincial Special Financial Fund (Grant No.2319412525), and the Fangchenggang Science and Technology Plan Project (Grant No. AD19008017). Conflicts of Interest: The authors declare no conflicts of interest.

References

1. Xu, W.; Xu, Y.; Huang, X.; Hu, X.; Xu, Y.; Su, H.; Li, Z.; Yang, K.; Wen, G.; Cao, Y. Addition of algicidal bacterium CZBC1 and molasses to inhibit cyanobacteria and improve microbial communities, water quality and shrimp performance in culture systems. Aquaculture 2019, 502, 303–311. [CrossRef] 2. Alfiansah, Y.R.; Hassenruck, C.; Kunzmann, A.; Taslihan, A.; Harder, J.; Gardes, A. Bacterial abundance and community composition in pond water from shrimp aquaculture systems with different stocking densities. Front. Microbiol. 2018, 9, 2457. [CrossRef][PubMed] 3. Zeng, S.; Hou, D.; Liu, J.; Ji, P.; Weng, S.; He, J.; Huang, Z. Antibiotic supplement in feed can perturb the intestinal microbial composition and function in Pacific white shrimp. Appl. Microbiol. Biotechnol. 2019, 103, 3111–3122. [CrossRef][PubMed] 4. Amoah, K.; Huang, Q.C.; Tan, B.P.; Zhang, S.; Chi, S.Y.; Yang, Q.H.; Liu, H.Y.; Dong, X.H. Dietary supplementation of probiotic Bacillus coagulans ATCC 7050, improves the growth performance, intestinal morphology, microflora, immune response, and disease confrontation of Pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2019, 87, 796–808. [CrossRef][PubMed] 5. Jiao, L.; Dai, T.; Zhong, S.; Jin, M.; Sun, P.; Zhou, Q. Vibrio parahaemolyticus infection influenced trace element homeostasis, impaired antioxidant function, and induced inflammation response in Litopenaeus vannamei. Biol. Trace Elem. Res. 2020, 1–9. [CrossRef] 6. Thadtapong, N.; Salinas, M.B.S.; Charoensawan, V.; Saksmerprome, V.; Chaturongakul, S. Genome Characterization and Comparison of Early Mortality Syndrome Causing Vibrio parahaemolyticus pirABvp Mutant from Thailand With V. parahaemolyticus pirABvp+ AHPND Isolates. Front. Mar. Sci. 2020, − 7, 290. [CrossRef] Mar. Drugs 2020, 18, 419 17 of 19

7. Rattanavichai, W.; Cheng, W. Effects of hot-water extract of banana (Musa acuminata) fruit’s peel on the antibacterial activity, and anti-hypothermal stress, immune responses and disease resistance of the giant freshwater prawn, Macrobrachium rosenbegii. Fish Shellfish Immunol. 2014, 39, 326–335. [CrossRef] 8. Karnjana, K.; Soowannayan, C.; Wongprasert, K. Ethanolic extract of red seaweed Gracilaria fisheri and furanone eradicate Vibrio harveyi and Vibrio parahaemolyticus biofilms and ameliorate the bacterial infection in shrimp. Fish Shellfish Immunol. 2019, 88, 91–101. [CrossRef] 9. Interaminense, J.A.; Vogeley, J.L.; Gouveia, C.K.; Portela, R.S.; Oliveira, J.P.; Silva, S.; Coimbra, M.R.M.; Peixoto, S.M.; Soares, R.B.; Buarque, D.S.; et al. Effects of dietary Bacillus subtilis and Shewanella algae in expression profile of immune-related genes from hemolymph of Litopenaeus vannamei challenged with Vibrio parahaemolyticus. Fish Shellfish Immunol. 2019, 86, 253–259. [CrossRef] 10. Liu, X.L.; Xi, Q.Y.; Yang, L.; Li, H.Y.; Jiang, Q.Y.; Shu, G.; Wang, S.B.; Gao, P.; Zhu, X.T.; Zhang, Y.L. The effect of dietary Panax ginseng polysaccharide extract on the immune responses in white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2011, 30, 495–500. [CrossRef] 11. Deng, B.; Wang, Z.P.; Tao, W.J.; Li, W.F.; Wang, C.; Wang, M.Q.; Ye, S.S.; Du, Y.J.; Wu, X.X.; Wu, D. Effects of polysaccharides from mycelia of Cordyceps sinensison growth performance, immunity and antioxidant indicators of the white shrimp, Litopenaeus vannamei. Aquac. Nutr. 2015, 21, 173–179. [CrossRef] 12. Tello-Olea, M.; Rosales-Mendoza, S.; Campa-Cordova, A.I.; Palestino, G.; Luna-Gonzalez, A.; Reyes-Becerril, M.; Velazquez, E.; Hernandez-Adame, L.; Angulo, C. Gold nanoparticles (AuNP) exert immunostimulatory and protective effects in shrimp (Litopenaeus vannamei) against Vibrio parahaemolyticus. Fish Shellfish Immunol. 2019, 84, 756–767. [CrossRef][PubMed] 13. Fierro-Coronado, J.A.; Angulo, C.; Rubio-Castro, A.; Luna-González, A.; Cáceres-Martínez, C.J.; Ruiz-Verdugo, C.A.; Álvarez-Ruíz, P.; Escamilla-Montes, R.; González-Ocampo, H.A.; Diarte-Plata, G. Dietary fulvic acid effects on survival and expression of immune-related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus. Aquac. Res. 2018, 49, 3218–3227. [CrossRef] 14. Pereira, L.A.; da Silva Reis, L.; Batista, F.A.; Mendes, A.N.; Osajima, J.A.; Silva-Filho, E.C. Biological properties of chitosan derivatives associated with the ceftazidime drug. Carbohydr. Polym. 2019, 222, 115002. [CrossRef] 15. Xing, K.; Xing, Y.; Liu, Y.; Zhang, Y.; Shen, X.; Li, X.; Miao, X.; Feng, Z.; Peng, X.; Qin, S. Fungicidal effect of chitosan via inducing membrane disturbance against Ceratocystis fimbriata. Carbohydr. Polym. 2018, 192, 95–103. [CrossRef][PubMed] 16. Wang, S.H.; Chen, J.C. The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. Fish Shellfish Immunol. 2005, 19, 191–204. [CrossRef] 17. Kamali Najafabad, M.; Imanpoor, M.R.; Taghizadeh, V.; Alishahi, A. Effect of dietary chitosan on growth performance, hematological parameters, intestinal histology and stress resistance of Caspian kutum (Rutilus frisii kutum Kamenskii, 1901) fingerlings. Fish Physiol. Biochem. 2016, 42, 1063–1071. [CrossRef] 18. Ahmed, F.; Soliman, F.M.; Adly, M.A.; Soliman, H.A.M.; El-Matbouli, M.; Saleh, M. Recent progress in biomedical applications of chitosan and its nanocomposites in aquaculture: A review. Res. Vet. Sci. 2019, 126, 68–82. [CrossRef] 19. Chen, J.; Chen, L. Effects of chitosan-supplemented diets on the growth performance, nonspecific immunity and health of loach fish (Misgurnus anguillicadatus). Carbohydr. Polym. 2019, 225, 115227. [CrossRef] 20. Abdel-Tawwab, M.; Razek, N.A.; Abdel-Rahman, A.M. Immunostimulatory effect of dietary chitosan nanoparticles on the performance of Nile tilapia, Oreochromis niloticus (L.). Fish Shellfish Immunol. 2019, 88, 254–258. [CrossRef] 21. Khani Oushani, A.; Soltani, M.; Sheikhzadeh, N.; Shamsaie Mehrgan, M.; Rajabi Islami, H. Effects of dietary chitosan and nano-chitosan loaded clinoptilolite on growth and immune responses of rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2020, 98, 210–217. [CrossRef] 22. Beyazit, N.; Cakran, H.S.; Cabir, A.; Akiscan, Y.; Demetgul, C. Synthesis, characterization and antioxidant activity of chitosan Schiff base derivatives bearing (-)-gossypol. Carbohydr. Polym. 2020, 240, 116333. [CrossRef][PubMed] 23. Ghazaie, M.; Ghiaci, M.; Soleimanian-Zad, S.; Behzadi-Teshnizi, S. Preparing natural biocomposites of N-quaternary chitosan with antibacterial activity to reduce consumption of antibacterial drugs. J. Hazard. Mater. 2019, 371, 224–232. [CrossRef][PubMed] 24. Salama, A.; Hasanin, M.; Hesemann, P. Synthesis and antimicrobial properties of new chitosan derivatives containing guanidinium groups. Carbohydr. Polym. 2020, 241, 116363. [CrossRef][PubMed] Mar. Drugs 2020, 18, 419 18 of 19

25. Barbosa, H.F.G.; Attjioui, M.; Leitao, A.; Moerschbacher, B.M.; Cavalheiro, E.T.G. Characterization, solubility and biological activity of amphihilic biopolymeric Schiff bases synthesized using chitosans. Carbohydr. Polym. 2019, 220, 1–11. [CrossRef] 26. Yan, T.; Li, C.; Ouyang, Q.; Zhang, D.; Zhong, Q.; Li, P.; Li, S.; Yang, Z.; Wang, T.; Zhao, Q. Synthesis of gentamicin-grafted-chitosan with improved solubility and antibacterial activity. React. Funct. Polym. 2019, 137, 38–45. [CrossRef] 27. Yan, T.; Kong, S.; Ouyang, Q.; Li, C.; Hou, T.; Chen, Y.; Li, S. Chitosan-Gentamicin Conjugate Hydrogel Promoting Skin Scald Repair. Mar. Drugs 2020, 18, 233. [CrossRef] 28. Zheng, X.; Wang, Y.; Zhang, D. Effects of microbial products and extra carbon on water quality and bacterial community in a fish polyculture system. Aquac. Res. 2019, 50, 1220–1229. [CrossRef] 29. Cole, A.J.; Tulsankar, S.S.; Saunders, B.J.; Fotedar, R. Effects of pond age and a commercial substrate (the water cleanser™) on natural productivity, bacterial abundance, nutrient concentrations, and growth and survival of MARRON (CHERAX CAINII Austin, 2002) in semi-intensive pond culture. Aquaculture 2019, 502, 242–249. [CrossRef] 30. Ray, A.J.; Seaborn, G.; Leffler, J.W.; Wilde, S.B.; Lawson, A.; Browdy, C.L. Characterization of microbial communities in minimal-exchange, intensive aquaculture systems and the effects of suspended solids management. Aquaculture 2010, 310, 130–138. [CrossRef] 31. Chen, G.F.; Huang, J.; Song, X.L. General situation of the immunological capability of shrimp. J. Fish. China 2004, 28, 209–215. 32. Zhai, Q.; Li, J. Effectiveness of traditional Chinese herbal medicine, San-Huang-San, in combination with enrofloxacin to treat AHPND-causing strain of Vibrio parahaemolyticus infection in Litopenaeus vannamei. Fish Shellfish Immunol. 2019, 87, 360–370. [CrossRef][PubMed] 33. Arojojoye, O.A.; Oyagbemi, A.A.; Afolabi, J.M. Toxicological assessment of heavy metal bioaccumulation and oxidative stress biomarkers in clarias gariepinus from Igbokoda River of South Western Nigeria. Bull. Environ. Contam. Toxicol. 2018, 100, 765–771. [CrossRef][PubMed] 34. Xie, J.J.; Liu, Q.Q.; Liao, S.; Fang, H.H.; Yin, P.; Xie, S.W.; Tian, L.X.; Liu, Y.J.; Niu, J. Effects of dietary mixed probiotics on growth, non-specific immunity, intestinal morphology and microbiota of juvenile pacific white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2019, 90, 456–465. [CrossRef] 35. Couto, N.; Malys, N.; Gaskell, S.J.; Barber, J. Partition and turnover of glutathione reductase from saccharomyces cerevisiae: A proteomic approach. J. Proteome Res. 2013, 12, 2885–2894. [CrossRef] 36. Duan, Y.; Zhang, J.; Dong, H.; Wang, Y.; Liu, Q.; Li, H. Oxidative stress response of the black tiger shrimp Penaeus monodon to Vibrio parahaemolyticus challenge. Fish Shellfish Immunol. 2015, 46, 354–365. [CrossRef] 37. Yang, C.; Kong, J.; Wang, Q.; Liu, Q.; Tian, Y.; Luo, K. Heterosis of haemolymph analytes of two geographic populations in Chinese shrimp Fenneropenaeus chinensis. Fish Shellfish Immunol. 2007, 23, 62–70. [CrossRef] 38. Feng, N.N.; Sun, Y.M.; Wen, R.; Zhang, C.S.; Fu-Hua, L.I. Analysis on the activity of immune related enzymes in survived Exopalaemon carinicauda from WSSV infection. Mar. Sci. 2014, 38, 75–79. 39. Zhang, M.; Wang, L.; Guo, Z.Y.; Wang, B.J. Effect of lipopolysaccharide and Vibrio anguillarum on the activities of phosphatase, superoxide dismutase and the content of hemocyanin in the serum of Fenneropenaeus chinesis. Mar. Sci. 2004, 28, 25–30. 40. Sotelo-Mundo, R.R.; Islas-Osuna, M.A.; De-La-Re-Vega, E.; Hernández-López, J.; Yepiz-Plascencia, G. cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei. Fish Shellfish Immunol. 2003, 15, 325–331. [CrossRef] 41. Li, J.; Xu, Y.; Jin, L.; Li, X. Effects of a probiotic mixture (Bacillus subtilis YBand Bacillus cereus YB on disease resistance and non-specific immunity of sea cucumber, Apostichopus japonicus (Selenka). Aquac. Res. 2015, 46, 3008–3019. [CrossRef] 42. Newton, K.; Peters, R.; Raftos, D. Phenoloxidase and QX disease resistance in Sydney rock oysters (Saccostrea glomerata). Dev. Comp. Immunol. 2004, 28, 565–569. [CrossRef] 43. Wang, C.; Wang, X.; Xiao, S.; Bu, X.; Lin, Z.; Qi, C.; Qin, J.G.; Chen, L. T-2 toxin in the diet suppresses growth and induces immunotoxicity in juvenile Chinese mitten crab (Eriocheir sinensis). Fish Shellfish Immunol. 2020, 97, 593–601. [CrossRef][PubMed] Mar. Drugs 2020, 18, 419 19 of 19

44. Tan, X.; Sun, Z.; Zhou, C.; Huang, Z.; Tan, L.; Xun, P.; Huang, Q.; Lin, H.; Ye, C.; Wang, A. Effects of dietary dandelion extract on intestinal morphology, antioxidant status, immune function and physical barrier function of juvenile golden pompano Trachinotus ovatus. Fish Shellfish Immunol. 2018, 73, 197–206. [CrossRef] [PubMed] 45. Wang, L.; Zhang, J.; Li, H.; Yang, H.; Peng, C.; Peng, Z.; Lu, L. Shift in the microbial community composition of surface water and sediment along an urban river. Sci. Total Environ. 2018, 627, 600–612. [CrossRef] 46. Cardona, E.; Gueguen, Y.; Magre, K.; Lorgeoux, B.; Piquemal, D.; Pierrat, F.; Noguier, F.; Saulnier, D. Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system. BMC Microbiol. 2016, 16, 157. [CrossRef] 47. Fan, L.; Wang, Z.; Chen, M.; Qu, Y.; Li, J.; Zhou, A.; Xie, S.; Zeng, F.; Zou, J. Microbiota comparison of Pacific white shrimp intestine and sediment at freshwater and marine cultured environment. Sci. Total Environ. 2019, 657, 1194–1204. [CrossRef] 48. Zhang, M.; Pan, L.; Huang, F.; Gao, S.; Su, C.; Zhang, M.; He, Z. Metagenomic analysis of composition, function and cycling processes of microbial community in water, sediment and effluent of Litopenaeus vannamei farming environments under different culture modes. Aquaculture 2019, 506, 280–293. [CrossRef] 49. Beyersmann, P.G.; Tomasch, J.; Son, K.; Stocker, R.; Goker, M.; Wagner-Dobler, I.; Simon, M.; Brinkhoff, T. Dual function of tropodithietic acid as antibiotic and signaling molecule in global gene regulation of the probiotic bacterium Phaeobacter inhibens. Sci. Rep. 2017, 7, 730. [CrossRef] 50. Amoah, K.; Huang, Q.C.; Dong, X.H.; Tan, B.P.; Zhang, S.; Chi, S.Y.; Yang, Q.H.; Liu, H.Y.; Yang, Y.Z. Paenibacillus polymyxa improves the growth, immune and antioxidant activity, intestinal health, and disease resistance in Litopenaeus vannamei challenged with Vibrio parahaemolyticus. Aquaculture 2019, 734563. [CrossRef] 51. Jang, W.J.; Lee, J.M.; Hasan, M.T.; Lee, B.J.; Lim, S.G.; Kong, I.S. Effects of probiotic supplementation of a plant-based protein diet on intestinal microbial diversity, digestive enzyme activity, intestinal structure, and immunity in olive flounder (Paralichthys olivaceus). Fish Shellfish Immunol. 2019, 92, 719–727. [CrossRef] [PubMed] 52. Swain, S.M.; Singh, C.; Arul, V. Inhibitory activity of probiotics Streptococcus phocae PI80 and Enterococcus faecium MC13 against Vibriosis in shrimp Penaeus monodon. World J. Microbiol. Biotechnol. 2009, 25, 697–703. [CrossRef] 53. Sharifuzzaman, S.M.; Rahman, H.; Austin, D.A.; Austin, B. Properties of Probiotics Kocuria SM1 and Rhodococcus SM2 Isolated from Fish Guts. Probiotics Antimicrob. Proteins 2018, 10, 534–542. [CrossRef] [PubMed] 54. Tsai, C.-Y.; Santos, H.M.; Hu, S.-Y.; Sang, C.Y.; Yanuaria, C.A.S.; Lola, E.N.G.; Tayo, L.L.; Nitura, K.M.A.; Liu, C.H.; Chuang, K.P. LpxD gene knockout elicits protection to Litopenaeus vannamei, white shrimp, against Vibrio parahaemolyticus infection. Aquac. Int. 2019, 27, 1383–1393. [CrossRef] 55. Huynh, T.G.; Hu, S.Y.; Chiu, C.S.; Truong, Q.P.; Liu, C.H. Bacterial population in intestines of white shrimp, Litopenaeus vannamei fed a synbiotic containing Lactobacillus plantarum and galactooligosaccharide. Aquac. Res. 2019, 50, 807–817. [CrossRef] 56. Liu, H.; Wang, L.; Liu, M.; Wang, B.; Jiang, K.; Ma, S.; Li, Q. The intestinal microbial diversity in Chinese shrimp (Fenneropenaeus chinensis) as determined by PCR–DGGE and clone library analyses. Aquaculture 2011, 317, 32–36. [CrossRef] 57. Koo, H.; Hakim, J.A.; Morrow, C.D.; Eipers, P.G.; Davila, A.; Andersen, D.T.; Bej, A.K. Comparison of two bioinformatics tools used to characterize the microbial diversity and predictive functional attributes of microbial mats from Lake Obersee, Antarctica. J. Microbiol. Methods 2017, 140, 15–22. [CrossRef] 58. Thanigaivel, S.; Vijayakumar, S.; Mukherjee, A.; Chandrasekaran, N.; Thomas, J. Antioxidant and antibacterial activity of Chaetomorpha antennina against shrimp pathogen Vibrio parahaemolyticus. Aquaculture 2014, 433, 467–475. [CrossRef] 59. Strickland, J.D.H.; Parsons, T.R. A Practical Handbook of Seawater Analysis, 2nd ed.; Fisheries Research Board of Canada, Bulletin: Ottawa, ON, USA, 1972; p. 167.

© 2020 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 (http://creativecommons.org/licenses/by/4.0/).