Journal Pre-proof

Metagenomics in bioflocs and their effects on gut microbiome and immune responses
in Pacific white shrimp

Surapun Tepaamorndech, Intawat Nookaew, Shawn M. Higdon, Pannita Santiyanont,

Metavee Phromson, Kanittha Chantarasakha, Wuttichai Mhuantong, Vetthachai
Plengvidhya, Wonnop Visessanguan
PII: S1050-4648(20)30606-9
DOI: https://doi.org/10.1016/j.fsi.2020.08.042
Reference: YFSIM 7264

To appear in: Fish and Shellfish Immunology

Received Date: 24 March 2020

Revised Date: 19 August 2020
Accepted Date: 24 August 2020

Please cite this article as: Tepaamorndech S, Nookaew I, Higdon SM, Santiyanont P, Phromson M,
Chantarasakha K, Mhuantong W, Plengvidhya V, Visessanguan W, Metagenomics in bioflocs and
their effects on gut microbiome and immune responses in Pacific white shrimp, Fish and Shellfish
Immunology (2020), doi: https://doi.org/10.1016/j.fsi.2020.08.042.

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Credit author statement
ST: Conceptualization, Methodology, Software, Formal analysis, Investigation, Resources, Data curation,

Writing-Original Draft, Writing-Review &Editing, Visualization, Supervision, Funding acquisition. IN:

Methodology, Software, Formal Analysis, Investigation, Resources. SH: Writing-Original Draft, Writing-

Review &Editing. PS: Software, Formal Analysis, Writing-Review &Editing, Visualization. MP:

Investigation, Resources. KC: Investigation, Project administration. WM: Software, Formal analysis.

VP: Conceptualization, Resources. WV: Conceptualization, Resources.

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1 Metagenomics in bioflocs and their effects on gut microbiome and immune responses in Pacific

2 white shrimp

4 Surapun Tepaamorndecha*, Intawat Nookaewb, Shawn M. Higdonc, Pannita Santiyanonta, Metavee

5 Phromsona, Kanittha Chantarasakhaa, Wuttichai Mhuantonga, Vetthachai Plengvidhyaa, Wonnop

6 Visessanguana

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a
8 National Center for Genetic Engineering and Biotechnology (BIOTEC), Phahonyothin Rd., Pathumthani

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9 12120, Thailand
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10 Department of Biomedical Informatics, University of Arkansas for Medical Sciences, Little Rock, AR

11 72205, USA
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12 Department of Plant Sciences, University of California, Davis, CA, 95616, USA
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*
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Corresponding author:

15 Surapun Tepaamorndech, Ph.D.

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16 National Center for Genetic Engineering and Biotechnology (BIOTEC)

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17 113 Phahonyothin Rd., Pathumthani 12120, Thailand

18 Email: surapun.tep@biotec.or.th

19

20 Conflict of interest: No conflict of interest is claimed

21

22

23

24

25

26
27 Abstract

28 Biofloc systems generate and accumulate microbial aggregates known as bioflocs. The presence

29 of bioflocs has been shown to change gut bacterial diversity and stimulate innate immunity in shrimp. The

30 microbial niche of bioflocs may therefore have the potential to drive shifts in the shrimp gut microbiota

31 associated with stimulation of innate immunity. We performed shotgun metagenomic analysis and 16S

32 rRNA-based amplicon sequencing to characterize complex bacterial members in bioflocs and the shrimp

33 digestive tract, respectively. Moreover, we determined whether biofloc-grown shrimp with discrete gut

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34 microbiomes had an elevation in local immune-related gene expression and systemic immune activities.

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35 Our findings demonstrated that the bacterial community in bioflocs changed dynamically during Pacific

36 white shrimp cultivation. Metagenomic analysis revealed that Vibrio comprised 90% of the biofloc

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population, while Pseualteromonas, Photobacterium, Shewanella, Alteromonas, Bacillus, Lactobacillus,
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38 Acinetobacter, Clostridium, Marinifilum, and Pseudomonas were also detected. In the digestive tract,
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39 biofloc-grown shrimp maintained the presence of commensal bacteria including Vibrio, Photobacterium,

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Shewanella, Granulosicoccus, and Ruegeria similar to control shrimp. However, Vibrio and

41 Photobacterium were significantly enriched and declined, respectively, in biofloc-grown shrimp. The
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42 presence of bioflocs upregulated immune-related genes encoding serine proteinase and prophenoloxidase
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43 in digestive organs which are routinely exposed to gut microbiota. Biofloc-grown shrimp also

44 demonstrated a significant increase in systemic immune status. As a result, the survival rate of biofloc-

45 grown shrimp was substantially higher than that of the control shrimp. Our findings suggested that the

46 high relative abundance of vibrios in bioflocs enriched the number of vibrios in the digestive tract of

47 biofloc-grown shrimp. This shift in gut microbiota composition may be partially responsible for local

48 upregulation of immune-related gene expression in digestive organs and systemic promotion of immune

49 status in circulating hemolymph.

50 1. Introduction

51 Biofloc technology has been used in aquaculture because of various benefits to marine animals.

52 The biofloc system is operated with no water exchange leading to accumulation of nitrogen wastes from

53 animal excretion [1]. Nitrogen containing wastes cooperating with an exogenous carbon source accelerate

54 microbial growth and deposit substantial levels of the microbial aggregates known as bioflocs [2, 3]. In

55 shrimp cultivation, the presence of bioflocs was shown to significantly stimulate the innate immunity of

56 shrimp [4-7]. As a result, biofloc-grown shrimp become resistant to pathogen infection and mortality.

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57 Additional experiments demonstrated that bioflocs triggered innate immune mechanisms against shrimp

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58 infection. The effect of bioflocs on shrimp innate immunity has been proposed to act through ingestion of

59 biofloc microflora [8, 9]. Therefore, microbes residing in bioflocs may play important roles in stimulating

60 the immune system of shrimp.

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61 Increased efforts to investigate the biofloc microbiome stem from their ability to confer
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62 immunomodulatory effects on marine animals. Previous attempts to characterize biofloc microbiota

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identified complex bacterial populations as the predominating microbial components [3, 6, 10]. First,

64 enrichment cultures were performed to characterize the composition of biofloc microbial communities.
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65 Vibrio, Lactobacillus, and Bacillus were isolated and applied as probiotics in aquacultures[11]. Other
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66 genera including Halomonas, Providencia, Nitratireductor, and Pseudoalteromonas were also detected

67 [12]. Nevertheless, culture dependent approaches present a limitation to characterizing the complete

68 microbial population present in bioflocs, where the cultivation approach commonly reveals only a small

69 bacterial portion of which only 0.1% are estimated to have been characterized [13]. To address this,

70 microbial community analysis based on DNA sequencing was subsequently applied to elucidate the

71 complexity of bacterial niches in bioflocs. For example, analysis of water collected from the biofloc

72 system with Litopenaeus stylirostris cultivation using 16S rRNA amplicon sequencing revealed that

73 Proteobacteria, Bacteroidetes, and Cyanobacteria were found to be the bacterial taxa of the highest

74 relative abundance in the biofloc system [14].

 75 The gut microbiota is known to be associated with human and animal health [15, 16]. In

76 mammals, alterations of gut bacterial communities are considered to be an early response prior to

77 physiological changes in the host [17, 18]. Similar to mammals, vibrio infected shrimp showed gut

78 microbiome shifts before disease symptoms were observed [19]. Other factors including growth, diet and

79 habitat have been reported to alter bacterial diversity in the shrimp digestive tract [20-23]. A recent study

80 demonstrated that gut bacterial communities of biofloc-grown shrimp were not identical to those of

81 shrimp cultivated in clear seawater [14]. While previous microbiome studies have primarily focused on

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82 the key factors shifting bacterial community in the shrimp gastrointestinal (GI) tract [19-27], functional

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83 aspects of the gut microbiome on shrimp health remain largely uncharacterized.

84 Although bioflocs have been shown to alter the shrimp gut microbiota and promote innate

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immunity in the host, these findings were accumulated from different investigations that varied in shrimp
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86 breed, growth stage, cultivation time, and experimental factors [4-7, 14]. This study aimed to decipher the
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87 bacterial complexity in bioflocs, investigate their potential to modulate the gut microbiome of Pacific

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white shrimp, and identify causal links between microbiome shifts and host innate immunity. We

89 evaluated bacterial similarity using community fingerprints followed by shotgun metagenomic analysis to
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90 acquire in-depth characterization of bacterial members present in bioflocs. Subsequently, analyzing

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91 amplicon sequencing libraries targeting the 16S rRNA gene enabled community profiling for bacteria

92 present in the shrimp GI tract. Performing gene expression analysis and biochemical evaluations also

93 determined local and systemic innate immunity, respectively. Our findings not only improve the current

94 understating of biofloc effects on the shrimp gut microbiome, but also exemplify the potential application

95 of gut microbiome alteration to promote shrimp health.

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97 2. Materials and methods

98 2.1 Shrimp cultivation and tissue collection

99 Pacific white shrimp at the postlarva (PL) stage 8 to 12 were purchased from a hatchery farm in

100 the coastal region of Thailand. During acclimation in a 2,000 L tank, the postlarvae were screened for
101 pathogen contamination as described previously [28]. The study design was illustrated in Fig. 1A. Shrimp

102 at an average body weight of 0.9 ± 0.1 g were transferred to 220 L tanks in an indoor facility (100

103 shrimp/tank). Three tanks were randomly assigned to operate with biofloc systems as described

104 previously [29]. Briefly, molasses was supplemented as a carbon source to stimulate biofloc formation.

105 The carbon to nitrogen ratio was maintained at 15. The levels of bioflocs were measured using an Imhoff

106 cone every week. Cultivation water was collected and centrifuged at 10,000×g, 4ºC for 5 min, and stored

107 as biofloc samples at -80ºC until use. Bioflocs collected from each of the three cultivation tanks at 6-week

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108 cultivation were designated as BF-1, -2, and -3, respectively. The biofloc samples were collected at three,

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109 five, and six weeks during shrimp cultivation. Three additional tanks were operated as controls using the

110 clear water system. No carbon source was supplemented. Suspensions were continuously removed using a
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111 filtration unit. Water temperature, pH, dissolved oxygen (DO), and salinity were monitored every two
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112 days. Alkalinity, ammonia, ammonium, and nitrite were measured weekly. Both systems were similarly
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113 controlled as follows. Aeration was generated using air blowers with DO >5 mg/L. Water temperature
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114 and pH were maintained at 29 ± 1ºC and 8 ± 0.5, respectively. Alkalinity was maintained from 130 to 150

115 mg/L. Ammonia, ammonium, and nitrite were controlled < 0.1, 1.0, and 1.0 mg/L, respectively. Salinity
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116 was adjusted to 15 g/L. Water change was prohibited during shrimp cultivation. Water was only filled to
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117 replenish evaporation loss. Shrimp were fed a diet consisting of 38% crude protein (Wave, Inteqc) four

118 times a day for 6 weeks. The survival rates were recorded at the end of the study. Shrimp were

119 anesthetized on ice before hemolymph and tissue collection. Shrimp GI tract and hepatopancreas were

120 harvested, snap-frozen in liquid nitrogen, and stored at -80ºC until use. All animal experiments were

121 conducted in accordance with National Institutes of Health guidelines for the Care and Use of

122 Experimental Animals and were approved by Institutional Animal Care and Use Committee of BIOTEC.

123

124 2.2 DNA isolation and automated ribosomal intergenic spacer analysis (ARISA)

125 Shrimp GI tract containing feces was homogenized using a Dounce tissue grinder (Sigma-

126 Aldrich). DNA was isolated from bioflocs and fecal lysates using a Quick-DNA Fecal/Soil Microbe kit
127 (Zymo Research). DNA extraction was processed according to the manufacturer’s instruction. DNA

128 integrity and concentration were determined using gel electrophoresis, and a NanodropTM 2000

129 spectrophotometer (ThermoFisher Scientific), respectively. The DNA samples were stored at -20ºC until

130 use. Bacterial communities were examined using ARISA as described previously [30]. TSF primer (5′-

131 GTCGTAACAAGGTAGCCGTA-3′) tagged with 5′-FAM, and ITSeub primer (5′-

132 GCCAAGGCATCCACC-3′) were used to amplify ITS fragments. The PCR condition included 94°C for

133 3 min, 30 cycles of 94°C for 45 s, 55°C for 45 s, 72°C for 90 s and a final extension at 72°C for 5 min.

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134 The amplicons were subjected to fragment analysis using a 3730xl DNA analyzer (ThermoFisher

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135 Scientific) with a Genescan™ 1200 LIZ™ dye size standard (ThermoFisher Scientific). Visual alignment

136 with the standard was performed using Peakstudio, version 2.2 [31]. Size compensation was used as

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previously described [32]. DNA fragments sized from 300 to 1250 bp with fluorescence intensities higher
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138 than 100 fluorescence units were used in downstream analysis [33].
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2.3 Library preparation and metagenomic analysis

141 DNA isolated from bioflocs was subjected to shotgun sequencing analysis. DNA concentration
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142 was measured using a Qubit Fluorometer (ThermoFisher Scientific). DNA libraries were constructed
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143 using the Nextera XT library preparation kit (Illumina) according to the manufacturer’s protocol. DNA

144 inputs were fragmented using transposome and tagged with adaptor sequences. Fragmented DNA with

145 adaptors were PCR-amplified and cleaned up using Agencourt AMPure XP beads (Beckman). Library

146 quantity was determined using the 2100 Bioanalyzer system (Agilent Technologies). Paired-end libraries

147 with an insert size of 150 bp were normalized, diluted, and sequenced using a short-read NextSeq 500

148 (Illumina). The taxonomic composition was analyzed using the MEDUSA pipeline as described

149 previously [34].

150

151 2.4 16S rRNA gene amplification, Nanopore sequencing, and data analysis
152 DNA samples were obtained from the ZymoBIOMICS microbial community standard (Zymo

153 Research) or shrimp feces. DNA concentration was analyzed using a Qubit Fluorometer (ThermoFisher

154 Scientific). 16S barcoded primers (Oxford Nanopore Technologies) were used with ExTaq DNA

155 polymerase (Takara) in PCR amplification. The PCR condition was performed according to the

156 manufacturer’s instruction. 16S rRNA amplicons were purified using Agencourt AMPure XP beads

157 (Beckman). DNA libraries were constructed and sequenced using a 16S Barcoding Kit and FLO-MIN106

158 R9 Version (Oxford Nanopore Technologies), respectively. Base calling was performed using Albacore.

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159 High quality reads with Q score ≥ 8, and sequence length ≥ 1000 bp were blasted to Ezbiocloud database

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160 and individually summed into each bacterial taxon [35]. Due to limitations in species identification of

161 long-read Nanopore sequencing of the 16S rRNA gene, bacterial taxonomic classifications were only

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assigned to the genus rank in this report [36]. Heatmaps were generated based on the relative abundance
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163 levels within the bacterial populations using Heatmapper [37].
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2.5 Gene expression analysis

166 Shrimp GI tract and hepatopancreas samples were homogenized in TRIzol (Thermo Fisher
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167 Scientific) using a Dounce tissue grinder (Sigma-Aldrich). RNA was purified according to the
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168 manufacturer’s instructions. cDNA was synthesized from total RNA samples using an iScript cDNA

169 synthesis kit (BioRad) and diluted with distilled water before use. Quantitative PCR was performed using

170 a SsoAdvancedTM SYBR® Green supermix (BioRad) on a CFX384 Real-time System (BioRad). Specific

171 primers were adopted from previous reports [38-41]. The reactions were performed in duplicate with

172 melting temperature analysis. The amount of specific target amplicons was normalized to that of the

173 housekeeping Actb amplicons. The relative expression of the target genes was calculated using a 2-∆∆CT

174 method.

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176 2.6 Analysis of systemic innate immunity

177 Hemolymph was collected from the ventral sinus using a syringe coated with the anticoagulant,

178 10% (w/v) formalin in 0.45 M NaCl. Total hemocyte count and the phenoloxidase activity in hemocytes

179 were performed as described previously [30]. Plasma superoxide dismutase and total antioxidant activity

180 were examined using commercial assay kits (Cayman Chemical). Plasma protein was measured using the

181 BCA assay (Millipore). The protein concentration was used to normalize the phenoloxidase, superoxide

182 dismutase and total antioxidant activity. A microplate reader (SparkTM 10M, TECAN) was used to

183 measure optical density in all colorimetric assays.

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185 2.7 Statistical analysis

186 Biological replicates of bioflocs were obtained from three cultivation tanks at each sampling time

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and used in bacterial community fingerprint and metagenomics analysis (Fig. 1A). Physicochemical
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188 parameters were also measured from all the cultivation tanks and computed from the means of each
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189 measurement. Shrimp were randomly sampled from each cultivation tanks and used in metagenomics or

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biochemical analysis. Three to nine shrimp were used in each experiment as indicated in Fig. 1A. The

191 results were shown as the mean ± S.D. The student’s t test was used to compare physicochemical data, the
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192 levels of gut bacterial taxa, gene expression, immune-related activity, and survival of shrimp cultivated in
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193 the biofloc and control groups. Cluster analysis of ARISA was computed based on Bray-Curtis similarity

194 matrix and plotted using non-metric multidimensional scaling (NMDS). All analyses showed stress values

195 below 0.1 indicating adequate ordination of the data [42]. Statistical significance was tested using analysis

196 of similarities (ANOSIM). ANOSIM-R is the degree of group separation with a score of 0 to 1 indicating

197 no to complete separation, respectively [43]. SIMPER was also used to estimate % dissimilarity

198 between/among groups. NMDS, ANOSIM, and SIMPER were performed using PAST3 software [44].

199 Differences were considered statistically significant at p < 0.05.

200

201 3. Results

202 3.1 Biofloc formation and alterations in biofloc bacterial community

203 The presence of bioflocs was observed one week after the cultivation system was established. The

204 biofloc levels were constantly elevated and accumulated in the cultivation system until shrimp were

205 harvested at 6 weeks (Fig. 1B). Physicochemical data collected during shrimp cultivation demonstrated

206 that pH and water temperature in the biofloc system were slightly, but significantly lower than those in

207 the control system (Table 1). We also detected a significant decrease in the ammonia (NH3)

208 concentrations of the biofloc system when compared to the controls. A downward trend in the levels of

209 alkalinity and nitrite (NO2-) was also observed. The degree of bacterial community similarity in three

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210 biofloc tanks was analyzed at 3, 5, and 6 weeks. As shown in Fig. 1C, a NMDS plot illustrated a close

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211 similarity in bacterial diversity among the tanks at the same week. In contrast, bacterial communities were

212 distinctly separated over cultivation time. ANOSIM indicated that bacterial communities in bioflocs

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collected at different weeks were remarkably dissimilar (R = 0.942, p = 0.004). The mean rank within and
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214 between groups was 5.778 and 22.740, respectively. SIMPER reported 65.64% overall average
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215 dissimilarity among groups. These results indicated that bacterial communities in bioflocs were

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significantly shifted during shrimp cultivation.

217
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218 3.2 Metagenomic analysis of bioflocs during shrimp cultivation

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219 Bacterial diversity in bioflocs was characterized after 6 weeks of cultivation when shrimp were

220 simultaneously harvested for analyzing innate immune responses. Sequencing of biofloc DNA samples

221 including BF-1, -2, and -3 generated 93,691,977 total sequencing reads across all three libraries. Raw

222 reads were analyzed using the MEDUSA pipeline, which identified 1,139 bacterial genera. As shown in

223 Fig 2A, bacterial taxa were categorically similar in each sample. Vibrio was the most abundant genus

224 found in all the tanks. The average percent richness of Vibrio was 90.22±10.89% of the total bacterial

225 population. These results indicated that other genera were considerably less abundant than Vibrio. We

226 observed deviations of some minor genera in BF-1 compared to that of BF-2 and BF-3.

227 Pseudoalteromonas had the most discrete detection levels at 6.47%, 0.353%, and 0.317% in BF-1, BF-2

228 and BF-3, respectively. The average number of Photobacterium, Shewanella, Alteromonas, Bacillus,
229 Lactobacillus, Acinetobacter, Clostridium, Pseudomonas and Paraglaciecola was 0.32±0.17%,

230 0.24±0.33%, 0.19±0.29%, 0.18±0.15%, 0.16±0.14%, 0.14±0.14%, 0.14±0.14%, 0.11±0.11%, and

231 0.08±0.11%, respectively. We also characterized vibrios at the species level. V. parahaemolyticus was the

232 most prominent taxon in BF-1 with a relative abundance level of 26.19% (Fig. 2B), whereas V. campbellii

233 was the most enriched species detected in BF-2 and BF-3 at 71.66% and 71.96%, respectively. Other

234 species detected in all three biofloc tanks included V. vulnifucus, V. alginolyticus, V. harveyi, and V.

235 jasicida. These results revealed the diversity of bacterial populations in bioflocs, where Vibrio spp.

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236 including V. parahaemolyticus, V. campbellii, V. vulnifucus, V. alginolyticus, V. harveyi, and V. jasicida

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237 were the most dominant taxa during shrimp cultivation.

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239 3.3 Shrimp gut microbiome

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240 Community fingerprinting analysis illustrated that the gut bacterial populations of shrimp
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241 cultivated in the biofloc and control systems were positioned closely (Fig. 3A). However, ANOSIM

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showed the significant mean rank within and between groups at 36.240 and 54.270, respectively (R =

243 0.389, p = 0.009). Additionally, the calculated ANOSIM-R values between 0.5 and 0.75 indicated that
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244 bioflocs had a moderate effect on shrimp gut microbiome composition. The overall mean dissimilarity of
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245 gut bacterial diversity in both systems was 63.61%. These results pointed out a significant shift in gut

246 bacterial community composition for shrimp cultivated in the presence of bioflocs. We characterized the

247 bacterial community in shrimp GI tracts using long-read Nanopore technology. To validate Nanopore

248 sequencing and taxonomic analysis of the 16S rRNA amplicons, we analyzed a mock community

249 containing eight bacterial taxa and found each bacterium to be classified correctly at the genus level (data

250 not shown). In the shrimp microbiome study, nanopore sequencing of the 16S rRNA amplicons generated

251 46,032 reads with an average read length of 1,411 bp. Taxonomic analysis of the amplicons revealed five

252 dominant taxa that included Vibrio, Photobacterium, Shewanella, Granulosicoccus, and Ruegeria (Fig.

253 3B). All of those bacteria showed ≥ 1% of the total bacterial population. Regardless of the cultivation

254 systems, Vibrio was the most dominant member in shrimp GI tract. The proportion of Photobacterium
255 and Shewanella was significantly larger than those of Granulosicoccus, and Ruegeria. The average

256 proportions of 16S rRNA reads classified as Vibrio, Photobacterium, Shewanella, Granulosicoccus, and

257 Ruegeria were 80.14±10.49%, 4.77±4.05%, 10.35±6.59%, 0.10±0.17%, and 0.77±1.13%, respectively, in

258 biofloc-cultivated shrimp, while those of the same taxa accounted for 54.53±0.93%, 31.23±15.21%,

259 10.96±12.72%, 1.26±0.79%, and 0.08± 0.09%, respectively, in the control shrimp. Comparative heatmap

260 analysis of the relative abundances for each identified bacterial member in both cultivation systems

261 revealed that the levels of Vibrio in the GI tract of shrimp samples from the biofloc system were

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262 remarkably higher than those of the controls (Fig. 3C). In contrast, the relative abundance of

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263 Photobacterium found in shrimp GI tracts collected from the biofloc system was significantly lower than

264 that of those from the control system. Granulosicoccus exhibited the trend of decreased relative

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abundance in the presence of bioflocs but did not reach the significance threshold (p = 0.067). These
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266 findings showed that while major bacterial types in shrimp GI tracts were similar across treatment groups,
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267 the relative abundances of Vibrio and Photobacterium shifted significantly between the cultivation

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systems.

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270 3.4 Immune-related gene expression analysis

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271 We further determined immune related gene expression in shrimp GI tracts which have direct

272 contact with shrimp gut microbiota. As shown in Fig 4A, the mRNA level of the gene encoding lectin in

273 biofloc-grown shrimp was 1.80-fold higher than that of the control. Moreover, compared to the control,

274 the presence of bioflocs showed 3.13- and 2.60-fold upregulation of the prophenoloxidase and serine

275 proteinase genes, respectively. We also analyzed gene expression in shrimp hepatopancreas, a food

276 digestive and absorptive organ connecting to GI tract. Our results demonstrated that the mRNA level of

277 prophenoloxidase, and serine proteinase in the biofloc system were 3.78-, and 4.17-fold greater than that

278 of the control, respectively (Fig 4B). No significant differences in the expression of other genes were

279 observed between the two groups in both GI tract and hepatopancreas samples. These results showed

280 consistent upregulation of prophenoloxidase and serine proteinase in digestive organs, which suggested a
281 functional link between the shifted gut microbiome and the expression of genes related to the shrimp

282 immune system in digestive organs.

283

284 3.5 Shrimp innate immunity and survival

285 In addition to gene expression analysis, we investigated whether the levels of systemic immune

286 status differed between shrimp that were cultivated in the presence and absence of bioflocs. As shown in

287 Fig. 5A, shrimp cultivated in the biofloc system displayed a significant increase in total hemocyte counts

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288 compared to the controls. Biofloc grown shrimp with high loads of commensal vibrios in the GI tract

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289 showed 1.5-fold elevation in total hemocyte counts. No difference in the phenoloxidase activity was

290 detected between two groups (Fig. 5B). We also quantified their respective levels of antioxidant activities.

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Compared to the control shrimp, biofloc-grown shrimp showcased 1.80- and 2.05-fold increases in
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292 plasma superoxide dismutase (SOD) and total antioxidant activity, respectively (Fig. 5C and 5D).
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293 Recording the number of shrimp present at the end of the study indicated that shrimp cultivated in the

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biofloc system displayed a significantly higher survival rate than that of those grown under the control

295 condition (Fig. 5E). Collectively, these results showed that the presence of bioflocs promoted the
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296 hemocyte number, increased antioxidant activity, and led to an increase in shrimp survival.
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298 4. Discussion

299 Bioflocs ingested by shrimp contain a niche of microbes [2, 3, 45]. Shrimp cultivated in the

300 biofloc system showcase a number of health benefits, most notably of which include an increase in innate

301 immunity [4-7]. While the presence of bioflocs was shown to affect the composition of bacterial

302 populations in the shrimp GI tract [14], the degrees to which bioflocs shift the gut microbiome and

303 enhance innate immunity in shrimp have never been investigated concurrently. Our findings showed that

304 bioflocs contained Vibrio spp. as the predominant taxon. In addition, the relative abundance of vibrios

305 was also significantly enriched in shrimp GI tracts and found to be coincident with an increase in local

306 immune related gene expression, and systemic immune activities. These results suggested that bioflocs
307 significantly modified the shrimp gut microbiota and, potentially, had priming effects on shrimp innate

308 immunity.

309 Cooperation and competition among bacteria could be expected in bioflocs due to fluctuations in

310 nutrient availability and physicochemical conditions, as well as differences in the capability of microbial

311 proliferation, growth, and colonization. Our community fingerprints of bioflocs demonstrated constant

312 alterations in bacterial community composition over time during shrimp cultivation. The dynamic

313 changes in each microbial population were previously reported in both biofloc aggregates and water [10,

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314 46]. To successfully establish in bioflocs, bacteria were likely to be found as microflora in water, shrimp,

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315 or feed, and later introduced into the cultivation system. In order for bacterial populations to expand and

316 establish dominance within the community, bacterial flora must compete for utilization of carbon and

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nitrogen sources present in the system. Our metagenomic analysis found that Vibrio spp. were the most
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318 dominant taxon in bioflocs. These findings were expected as vibrios are known to be the prevalent
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319 microflora in shrimp, and these marine bacteria are likely to thrive under the conditions in which shrimp

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were cultivated [47]. Additionally, vibrios were previously shown to be capable of assimilating ammonia

321 and nitrate accumulated in the biofloc system [48, 49]. The toxicity of these nitrogenous compounds to
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322 shrimp and their ability to suppress shrimp immunity is well-established. In our study, the abundance of
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323 vibrios in bioflocs may have been responsible for a decrease in ammonia and nitrate concentrations that

324 partially promoted shrimp immune activities and survival. The major vibrio species in bioflocs were

325 characterized as V. parahaemolyticus and V. campbellii, both of which were previously shown to cause

326 acute hepatopancreatic necrosis disease (AHPND) [50]. A plasmid containing the pirABvp toxin gene is

327 essential for an AHPND outbreak. However, absence of plasmids encoding the gene among eighty

328 percent of known V. parahaemolyticus strains indicated that the majority of these bacteria are non-

329 virulent [51]. Other bacterial taxa identified in our study included Pseualteromonas, Photobacterium,

330 Shewanella, and Alteromonas. These bacteria were consistently found to comprise ≥0.05% of total

331 bacteria classified within biofloc populations. These results collaborated similar findings among a number

332 of other culture dependent studies [11, 46, 52].

333 Environmental factors significantly impact the composition of the microbial communities in

334 bioflocs. A previous metagenomic study showed that Leucotrix spp. was identified as the predominant

335 taxon rather than a Vibrio spp. in the water of the biofloc system [14]. We hypothesized that dissimilarity

336 in the dominant taxon of the community may result from stark differences across cultivation conditions.

337 Cardona et al. constructed an outdoor biofloc system using seawater from a lagoon. As a result, both algae

338 and the algae dependent bacterium, Leucotrix, were detected at high levels of relative abundance.

339 Contrariwise, our study established the biofloc system using artificial seawater in an indoor facility. These

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340 differences highlight the need for consistency in procedural aspects of biofloc utilization for shrimp

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341 cultivation. In this study, Vibrio, Pseudoalteromonas, Photobacterium, Shewanella, and Alteromonas

342 accounted for 93.35% of total bacterial abundance in bioflocs, which are taxa commonly found in

343
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seawater or in association with marine animals [53]. This bacterial group is likely to be associated with
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344 shrimp and simultaneously transferred into the cultivation system. In addition, these marine bacteria have
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345 the potential to flourish in artificial marine habitats [54, 55]. Moreover, this bacterial group may be

346
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considered as a bacterial inoculum that plays a crucial role in microbial community-forming bioflocs [56].

347 The addition of molasses also promoted vibrio proliferation in the biofloc system during shrimp
ur

348 cultivation [57]. However, dominance of Vibrio, Pseudoalteromonas, Photobacterium, Shewanella, and
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349 Alteromonas in bioflocs may not be profoundly abundant during the early periods of biofloc formation. In

350 fact, ARISA indicated dissimilarity of bacterial community across samples from different cultivation

351 times. Therefore, these abundant bacterial taxa were likely introduced as low-level shrimp flora that rose

352 in population size due to favorable environmental factors, especially salinity, of the artificial marine

353 habitat. Despite these findings, further investigations are needed to monitor species changes over time

354 during biofloc formation.

355 Another focus of this study was the effect of bioflocs on the shrimp GI tract microbiota. The

356 observed dissimilarity of gut bacterial fingerprints between the biofloc and clear water systems over time

357 supported previous investigations, which suggested that the presence of selective pressures leads to the

358 recruitment of specific microbial inhabitants within the shrimp GI tract [14, 53]. Although five major taxa
359 including Vibrio, Photobacterium, Shewanella, Granulosicoccus and Ruegeria were detected in both our

360 biofloc and control systems, proportions of some bacterial taxa remained significantly dissimilar between

361 groups. Interestingly, while Vibrio and Photobacterium have been characterized as the dominant members

362 of the shrimp GI tract [14, 23, 53, 58, 59], results from our study suggested that shrimp cultivation in

363 bioflocs contributed significantly to both Vibrio enrichment and a lowering of Photobacterium density in

364 the shrimp digestive tract. Despite the presence of their pathogenic strains, most of shrimp inhabitant

365 Vibrio spp. were determined to be non-pathogenic [26]. Moreover, the use of bioflocs as a dietary

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366 supplement directly ingested by shrimp has been proposed repeatedly [29, 56, 60]. To this point, our

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367 results also suggest that vibrios may be directly delivered as a dietary component into the shrimp GI tract

368

369
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as a means to outcompete Photobacterium in biofloc-grown shrimp. This is further supported by previous

studies that demonstrated shrimp ingestion of bacterial species with high environmental abundance
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370 resulted in alterations to their gut microbiome, and that some vibrios had an in vitro inhibitory effect on
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371 the growth of photobacteria [61, 62].

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372 The beneficial effects of Vibrio as commensal bacteria in marine animals are underrepresented

373 and require additional investigation, as this marine bacterium has been suggested to be a source of various
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374 antimicrobial compounds [63]. For example, several non-pathogenic strains of Vibrio were shown to
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375 potentially inhibit pathogen growth [64-66]. This provides support for the concept that deposition of

376 commensal vibrios may mitigate the potential risk of infection by lowering or inhibiting colonization of

377 virulent strains. On the other hand, biofloc-modified shrimp gut microbiota, which exhibited an increase

378 in Vibrio population, may be associated with shrimp immune stimulation. The local effects of microbiota

379 on gut immune development have been established in mammals [67, 68]. We performed gene expression

380 analysis in the shrimp GI tract where its microbiota was altered. To our knowledge, these results indicated

381 the first reported upregulation of immune related genes in the shrimp GI tract in concordance with

382 heightened commensal vibrios density. The genes encoding lectin, serine proteinase, and

383 prophenoloxidase all function in the phenoloxidase cascade [69, 70]. Upregulation of the serine
384 proteinase and prophenoloxidase encoding genes was also detected in shrimp hepatopancreas at similar

385 levels. Interestingly, bioflocs are comprised by many viable microbes, but also contain dead bacterial

386 cells and their respective compartments. Routine exposures to membrane components isolated from gram

387 negative bacteria may prime the immune responses of an invertebrate animal [71]. Hence, the abundance

388 of vibrio membrane components in shrimp GI tracts may be a key factor to stimulating immune-related

389 gene expression.

390 In mammals, intestinal bacteria play important roles in hematopoiesis and innate immune cell

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391 development [72-74]. The number and activity of the innate immune cells are influenced by the type and

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392 content of gut microbiota in the digestive tract. For example, previous investigations in mice found that

393 reduction of the host intestinal microbiota leading to the reduced production of stimulating molecules

394
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associated with neutrophil formation [73]. As a result, the number of neutrophils and innate immune
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395 progenitor cells declined. Moreover, innate immune cell activities could be depleted in mice with poor
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396 colonization of intestinal bacteria. In germ-free mice, a signaling cascade triggering inflammatory

397
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responses and interferon gene expression was impaired in non-mucosal lymphoid NK cells [74]. A lack of

398 bacterial peptidoglycan recognition also suppressed the ability of neutrophils to eliminate pathogens [72].
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399 These aspects of impaired immunity increase host susceptibility to pathogen infection.
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400 We sought to determine whether effects similar to those observed in mice also occurred in

401 shrimp. Considering that hemocytes are considered to be innate immune cells in shrimp [75], our results

402 agree with those of previous studies, where a high number of circulating hemocytes was observed in

403 biofloc-grown shrimp[4, 5]. Indeed, bioflocs not only raised relative vibrio accumulation, but also

404 promoted total bacterial population density in shrimp GI tracts [76]. Increased number of commensal

405 bacteria plays an important role in modulating the systemic innate immunity of shrimp in a similar

406 fashion to the trend observed in mice. Intestinal microbiota richness in biofloc grown shrimp may

407 stimulate production of hematopoietic growth factors. Previously, shrimp intestines were shown to

408 express genes involved with the synthesis of astakine, which is an ancient cytokine that may induce the

409 proliferation and differentiation of hemocytes [77]. In contrast, the phenoloxidase activity was not
410 different in shrimp hemocytes. This difference may be due to the fact that shrimp haemolymph contain a

411 very low number of bacteria [78]. The availability of microbes or their components in hemolymph may be

412 insufficient to promote hemocyte phenoloxidase. We also observed an increase in the antioxidant systems

413 including the superoxide dismutase and total antioxidant activity. This was likely due to an elevation in

414 both the hemocyte number, and antioxidant activities, which conferred health benefits and supported the

415 high survival of biofloc grown shrimp. However, further investigation is required to elucidate whether the

416 shrimp gut microbiome systemically regulates the production of hemocyte, influences antioxidant

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417 activities, and, potentially, determine the associated mechanisms.

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418 In summary, bioflocs demonstrated dynamic changes in bacterial population and contained

419 vibrios as the dominant member during Pacific white shrimp cultivation. The presence of bioflocs

420
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preserved bacterial categories, but significantly promoted an abundance of Vibrio, and suppressed
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421 Photobacterium density in the shrimp GI tract. We observed upregulation of genes involved in the
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422 phenoloxidase cascade in digestive organs of biofloc-grown shrimp containing shifted gut microbiome. In

423
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addition, biofloc-grown shrimp showed an elevation in hemocyte number and antioxidant activity, which

424 possibly contributed to high survival rate.

ur

425
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426 Acknowledgements

427 This study was supported by BIOTEC (P-16-50847).

428

429 References

430 [1] M. Emerenciano, G. Gaxiola, G. Cuzon, Biofloc Technology (BFT): A Review for Aquaculture

431 Application and Animal Food Industry, in: M.D. Matovic (Ed.), Biomass Now - Cultivation and

432 Utilization, IntechOpen2013.

433 [2] M. de Jesús Becerra-Dorame, L.R. Martinez-Cordova, M. Martínez-Porchas, J. Hernández-López,

434 J.A. López-Elías, F. Mendoza-Cano, Effect of using autotrophic and heterotrophic microbial-based-
435 systems for the pre-grown of Litopenaeus vannamei, on the production performance and selected

436 haemolymph parameters, Aquac Res 45(5) (2014) 944-948. https://doi.org/10.1111/are.12033.

437 [3] Z.Y. Ju, I. Forster, L. Conquest, W. Dominy, W.C. Kuo, F. David Horgen, Determination of

438 microbial community structures of shrimp floc cultures by biomarkers and analysis of floc amino

439 acid profiles, Aquac Res 39(2) (2008) 118-133. https://doi.org/10.1111/j.1365-2109.2007.01856.x.

440 [4] W.J. Xu, L.Q. Pan, Enhancement of immune response and antioxidant status of Litopenaeus

441 vannamei juvenile in biofloc-based culture tanks manipulating high C/N ratio of feed input,

of
442 Aquaculture 412-413 (2013) 117-124. https://doi.org/10.1016/j.aquaculture.2013.07.017.

ro
443 [5] J. Ekasari, M. Hanif Azhar, E.H. Surawidjaja, S. Nuryati, P. De Schryver, P. Bossier, Immune

444 response and disease resistance of shrimp fed biofloc grown on different carbon sources, Fish

445
-p
Shellfish Immunol 41(2) (2014) 332-339. https://doi.org/10.1016/j.fsi.2014.09.004.
re
446 [6] S.K. Kim, Z. Pang, H.C. Seo, Y.R. Cho, T. Samocha, I.K. Jang, Effect of bioflocs on growth and
lP

447 immune activity of Pacific white shrimp, Litopenaeus vannamei postlarvae, Aquac Res 45(2)

448
na

(2014) 362-371. https://doi.org/10.1111/are.12319.

449 [7] E. Cardona, D. Saulnier, B. Lorgeoux, L. Chim, Y. Gueguen, Rearing effect of biofloc on
ur

450 antioxidant and antimicrobial transcriptional response in Litopenaeus stylirostris shrimp facing an
Jo

451 experimental sub-lethal hydrogen peroxide stress, Fish Shellfish Immunol 45(2) (2015) 933-939.

452 https://doi.org/10.1016/j.fsi.2015.05.041.

453 [8] A.S. Ninawe, J. Selvin, Probiotics in shrimp aquaculture: Avenues and challenges probiotics in

454 shrimp aquaculture: Avenues and challenges A.S. Ninawe et al, Crit Rev Microbiol 35(1) (2009)

455 43-66. https://doi.org/10.1080/10408410802667202.

456 [9] V.J. Smith, J.H. Brown, C. Hauton, Immunostimulation in crustaceans: Does it really protect

457 against infection?, Fish Shellfish Immunol 15(1) (2003) 71-90. https://doi.org/10.1016/S1050-

458 4648(02)00140-7.

459 [10] A.J. Ray, G. Seaborn, J.W. Leffler, S.B. Wilde, A. Lawson, C.L. Browdy, Characterization of

460 microbial communities in minimal-exchange, intensive aquaculture systems and the effects of
461 suspended solids management, Aquaculture 310(1-2) (2010) 130-138.

462 https://doi.org/10.1016/j.aquaculture.2010.10.019.

463 [11] G.S. Ferreira, N.C. Bolívar, S.A. Pereira, C. Guertler, F.N. Vieira, J.L.P. Mouriño, W.Q. Seiffert,

464 Microbial biofloc as source of probiotic bacteria for the culture of Litopenaeus vannamei,

465 Aquaculture 448 (2015) 273-279. https://doi.org/10.1016/j.aquaculture.2015.06.006.

466 [12] N.A. Kasan, N.A. Ghazali, M. Ikhwanuddin, Z. Ibrahim, Isolation of potential bacteria as inoculum

467 for biofloc formation in pacific whiteleg shrimp, Litopenaeus vannamei culture ponds, Pakistan J

of
468 Biol Sci 20(6) (2017) 306-313. https://doi.org/10.3923/pjbs.2017.306.313.

ro
469 [13] G.T. Hill, N.A. Mitkowski, L. Aldrich-Wolfe, L.R. Emele, D.D. Jurkonie, A. Ficke, S. Maldonado-

470 Ramirez, S.T. Lynch, E.B. Nelson, Methods for assessing the composition and diversity of soil

471
-p
microbial communities, Appl Soil Ecol 15(1) (2000) 25-36. https://doi.org/10.1016/S0929-
re
472 1393(00)00069-X.
lP

473 [14] E. Cardona, Y. Gueguen, K. Magré, B. Lorgeoux, D. Piquemal, F. Pierrat, F. Noguier, D. Saulnier,

474
na

Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris

475 in a biofloc system, BMC Microbiol 16(1) (2016) 1-9. https://doi.org/10.1186/s12866-016-0770-z.

ur

476 [15] I. Sekirov, S.L. Russell, L.C.M. Antunes, B.B. Finlay, Gut Microbiota in Health and Disease,
Jo

477 Physiol rev 90 (2010) 859–904. https://doi.org/10.1152/physrev.00045.2009.

478 [16] C.M. Guinane, P.D. Cotter, Role of the gut microbiota in health and chronic gastrointestinal

479 disease: Understanding a hidden metabolic organ, Ther Adv Gastroenter 6(4) (2013) 295-308.

480 https://doi.org/10.1177/1756283X13482996.

481 [17] R.A. Koeth, Z. Wang, B.S. Levison, J.A. Buffa, E. Org, B.T. Sheehy, E.B. Britt, X. Fu, Y. Wu, L.

482 Li, J.D. Smith, J.A. Didonato, J. Chen, H. Li, G.D. Wu, J.D. Lewis, M. Warrier, J.M. Brown, R.M.

483 Krauss, W.H.W. Tang, F.D. Bushman, A.J. Lusis, S.L. Hazen, Intestinal microbiota metabolism of

484 L-carnitine, a nutrient in red meat, promotes atherosclerosis, Nat Med 19(5) (2013) 576-585.

485 https://doi.org/10.1038/nm.3145.
486 [18] H. Wu, E. Esteve, V. Tremaroli, M.T. Khan, R. Caesar, L. Mannerås-Holm, M. Ståhlman, L.M.

487 Olsson, M. Serino, M. Planas-Fèlix, G. Xifra, J.M. Mercader, D. Torrents, R. Burcelin, W. Ricart,

488 R. Perkins, J.M. Fernàndez-Real, F. Bäckhed, Metformin alters the gut microbiome of individuals

489 with treatment-naive type 2 diabetes, contributing to the therapeutic effects of the drug, Nat Med

490 23(7) (2017) 850-858. https://doi.org/10.1038/nm.4345.

491 [19] W.Y. Chen, T.H. Ng, J.H. Wu, J.W. Chen, H.C. Wang, Microbiome Dynamics in a Shrimp Grow-

492 out Pond with Possible Outbreak of Acute Hepatopancreatic Necrosis Disease, Sci Rep 7(1) (2017).

of
493 https://doi.org/10.1038/s41598-017-09923-6.

ro
494 [20] S. Chaiyapechara, W. Rungrassamee, I. Suriyachay, Y. Kuncharin, A. Klanchui, N.

495 Karoonuthaisiri, P. Jiravanichpaisal, Bacterial Community Associated with the Intestinal Tract of

496 P. monodon in Commercial Farms,

-p Microb Ecol 63(4) (2012) 938-953.
re
497 https://doi.org/10.1007/s00248-011-9936-2.
lP

498 [21] J.M. Harris, The presence, nature, and role of gut microflora in aquatic invertebrates: A synthesis,

499
na

Microb Ecol 25(3) (1993) 195-231. https://doi.org/10.1007/BF00171889.

500 [22] C.Z. Wang, G.R. Lin, T. Yan, Z.P. Zheng, B. Chen, F.L. Sun, The cellular community in the
ur

501 intestine of the shrimp Penaeus penicillatus and its culture environments, Fisheries Sci 80(5)
Jo

502 (2014) 1001-1007. https://doi.org/10.1007/s12562-014-0765-3.

503 [23] Z. Huang, X. Li, L. Wang, Z. Shao, Changes in the intestinal bacterial community during the

504 growth of white shrimp, Litopenaeus vannamei, Aquac Res 47(6) (2016) 1737-1746.

505 https://doi.org/10.1111/are.12628.

506 [24] W. Rungrassamee, A. Klanchui, S. Chaiyapechara, S. Maibunkaew, S. Tangphatsornruang, P.

507 Jiravanichpaisal, N. Karoonuthaisiri, Bacterial Population in Intestines of the Black Tiger Shrimp

508 (Penaeus monodon) under Different Growth Stages, PLOS ONE 8(4) (2013).

509 https://doi.org/10.1371/journal.pone.0060802.

510 [25] F. Cornejo-Granados, A.A. Lopez-Zavala, L. Gallardo-Becerra, A. Mendoza-Vargas, F. Sánchez,

511 R. Vichido, L.G. Brieba, M.T. Viana, R.R. Sotelo-Mundo, A. Ochoa-Leyva, Microbiome of Pacific
512 Whiteleg shrimp reveals differential bacterial community composition between Wild, Aquacultured

513 and AHPND/EMS outbreak conditions, Sci Rep 7(1) (2017). https://doi.org/10.1038/s41598-017-

514 11805-w.

515 [26] W. Yu, J.H. Wu, J. Zhang, W. Yang, J. Chen, J. Xiong, A meta-analysis reveals universal gut

516 bacterial signatures for diagnosing the incidence of shrimp disease, FEMS Microbiol Ecol 94(10)

517 (2018). https://doi.org/10.1093/femsec/fiy147.

518 [27] J. Xiong, Progress in the gut microbiota in exploring shrimp disease pathogenesis and incidence,

of
519 Appl Microbiol Biot 102(17) (2018) 7343-7350. https://doi.org/10.1007/s00253-018-9199-7.

ro
520 [28] W. Rungrassamee, A. Klanchui, S. Maibunkaew, N. Karoonuthaisiri, Bacterial dynamics in

521 intestines of the black tiger shrimp and the Pacific white shrimp during Vibrio harveyi exposure, J

522
-p
Invertebr Pathol 133 (2016) 12-19. https://doi.org/10.1016/j.jip.2015.11.004.
re
523 [29] P. De Schryver, R. Crab, T. Defoirdt, N. Boon, W. Verstraete, The basics of bio-flocs technology:
lP

524 The added value for aquaculture, Aquaculture 277(3-4) (2008) 125-137.

525
na

https://doi.org/10.1016/j.aquaculture.2008.02.019.

526 [30] S. Tepaamorndech, K. Chantarasakha, Y. Kingcha, S. Chaiyapechara, M. Phromson, M.

ur

527 Sriariyanun, C.P. Kirschke, L. Huang, W. Visessanguan, Effects of Bacillus aryabhattai

Jo

528 TBRC8450 on vibriosis resistance and immune enhancement in Pacific white shrimp, Litopenaeus

529 vannamei, Fish Shellfish Immunol 86 (2019) 4-13. https://doi.org/10.1016/j.fsi.2018.11.010.

530 [31] J. McCafferty, R. Reid, M. Spencer, T. Hamp, A. Fodor, Peak Studio: A tool for the visualization

531 and analysis of fragment analysis files, Env Microbiol Rep 4(5) (2012) 556-561.

532 https://doi.org/10.1111/j.1758-2229.2012.00368.x.

533 [32] M.V. Brown, M.S. Schwalbach, I. Hewson, J.A. Fuhrman, Coupling 16S-ITS rDNA clone libraries

534 and automated ribosomal intergenic spacer analysis to show marine microbial diversity:

535 Development and application to a time series, Environ Microbiol 7(9) (2005) 1466-1479.

536 https://doi.org/10.1111/j.1462-2920.2005.00835.x.
537 [33] A.C. Yannarell, E.W. Triplett, Geographic and environmental sources of variation in lake bacterial

538 community composition, Appl Environ Microb 71(1) (2005) 227-239.

539 https://doi.org/10.1128/AEM.71.1.227-239.2005.

540 [34] F.H. Karlsson, I. Nookaew, J. Nielsen, Metagenomic Data Utilization and Analysis (MEDUSA)

541 and Construction of a Global Gut Microbial Gene Catalogue, PLOS Comput Biol 10(7) (2014)

542 e1003706. https://doi.org/10.1371/journal.pcbi.1003706.

543 [35] S.H. Yoon, S.M. Ha, S. Kwon, J. Lim, Y. Kim, H. Seo, J. Chun, Introducing EzBioCloud: A

of
544 taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies, Int J

ro
545 Syst Evol Microbiol 67(5) (2017) 1613-1617. https://doi.org/10.1099/ijsem.0.001755.

546 [36] R. Winand, B. Bogaerts, S. Hoffman, L. Lefevre, M. Delvoye, J. Van Braekel, Q. Fu, N.H.C.

547
-p
Roosens, S.C.J. De Keersmaecker, K. Vanneste, Targeting the 16s rRNA gene for bacterial
re
548 identification in complex mixed samples: Comparative evaluation of second (illumina) and third
lP

549 (oxford nanopore technologies) generation sequencing technologies, Int J Mol Sci 21(1) (2020).

550
na

https://doi.org/10.3390/ijms21010298.

551 [37] S. Babicki, D. Arndt, A. Marcu, Y. Liang, J.R. Grant, A. Maciejewski, D.S. Wishart, Heatmapper:
ur

552 web-enabled heat mapping for all, Nucleic acids res 44(W1) (2016) W147-W153.
Jo

553 https://doi.org/10.1093/nar/gkw419.

554 [38] P.F. Ji, C.L. Yao, Z.Y. Wang, Immune response and gene expression in shrimp (Litopenaeus

555 vannamei) hemocytes and hepatopancreas against some pathogen-associated molecular patterns,

556 Fish Shellfish Immunol 27(4) (2009) 563-70. https://doi.org/10.1016/j.fsi.2009.08.001.

557 [39] C.C. Yang, C.L. Lu, S. Chen, W.L. Liao, S.N. Chen, Immune gene expression for diverse

558 haemocytes derived from pacific white shrimp, Litopenaeus vannamei, Fish Shellfish Immunol

559 44(1) (2015) 265-271. https://doi.org/10.1016/j.fsi.2015.02.001.

560 [40] W.N. Wang, J. Zhou, P. Wang, T.T. Tian, Y. Zheng, Y. Liu, W.j. Mai, A.L. Wang, Oxidative

561 stress, DNA damage and antioxidant enzyme gene expression in the Pacific white shrimp,
562 Litopenaeus vannamei when exposed to acute pH stress, Comp Biochem Phys C 150(4) (2009)

563 428-435. https://doi.org/10.1016/j.cbpc.2009.06.010.

564 [41] J. Zhou, L. Wang, Y. Xin, W.N. Wang, W.Y. He, A.L. Wang, Y. Liu, Effect of temperature on

565 antioxidant enzyme gene expression and stress protein response in white shrimp, Litopenaeus

566 vannamei, J Therm Biol 35(6) (2010) 284-289. https://doi.org/10.1016/j.jtherbio.2010.06.004.

567 [42] K.R. Clarke, Non‐parametric multivariate analyses of changes in community structure, Aust J Ecol

568 18(1) (1993) 117-143. https://doi.org/10.1111/j.1442-9993.1993.tb00438.x.

of
569 [43] A. Ramette, Multivariate analyses in microbial ecology, FEMS Microbiol Ecol 62(2) (2007) 142-

ro
570 160. https://doi.org/10.1111/j.1574-6941.2007.00375.x.

571 [44] Ø. Hammer, D.A.T. Harper, P.D. Ryan, Past: Paleontological statistics software package for

572
-p
education and data analysis, Palaeontol Electron 4(1) (2001) XIX-XX
re
573 [45] D.D. Kuhn, G.D. Boardman, A.L. Lawrence, L. Marsh, G.J. Flick Jr, Microbial floc meal as a
lP

574 replacement ingredient for fish meal and soybean protein in shrimp feed, Aquaculture 296(1-2)

575
na

(2009) 51-57. https://doi.org/10.1016/j.aquaculture.2009.07.025.

576 [46] H. Manan, J.H.Z. Moh, N.A. Kasan, S. Suratman, M. Ikhwanuddin, Identification of biofloc
ur

577 microscopic composition as the natural bioremediation in zero water exchange of Pacific white
Jo

578 shrimp, Penaeus vannamei, culture in closed hatchery system, Appl Water Sci 7(5) (2017) 2437-

579 2446. https://doi.org/10.1007/s13201-016-0421-4.

580 [47] J. Vandenberghe, L. Verdonck, R. Robles-Arozarena, G. Rivera, A. Bolland, M. Balladares, B.

581 Gomez-Gil, J. Calderon, P. Sorgeloos, J. Swings, Vibrios associated with Litopenaeus vannamei

582 larvae, postlarvae, broodstock, and hatchery probionts, Appl Environ Microb 65(6) (1999) 2592-

583 2597

584 [48] E. Bueno, B. Sit, M.K. Waldor, F. Cava, Anaerobic nitrate reduction divergently governs

585 population expansion of the enteropathogen Vibrio cholerae, Nat Microbiol 3(12) (2018) 1346-

586 1353. https://doi.org/10.1038/s41564-018-0253-0.

587 [49] G.M. Dunn, J.N. Wardell, R.A. Herbert, C.M. Brown, Enrichment, enumeration and

588 characterisation of nitrate-reducing bacteria present in sediments of the River Tay Estuary,

589 Proceedings of the Royal Society of Edinburgh. Section B. Biological Sciences 78(3-4) (2011) s47-

590 s56. https://doi.org/10.1017/S026972700000302X.

591 [50] X. Dong, D. Bi, H. Wang, P. Zou, G. Xie, X. Wan, Q. Yang, Y. Zhu, M. Chen, C. Guo, Z. Liu, W.

592 Wang, J. Huang, pirAB(vp) -Bearing Vibrio parahaemolyticus and Vibrio campbellii Pathogens

593 Isolated from the Same AHPND-Affected Pond Possess Highly Similar Pathogenic Plasmids, Front

of
594 Microbiol 8 (2017) 1859-1859. https://doi.org/10.3389/fmicb.2017.01859.

ro
595 [51] J. Vadivelu, S.D. Puthucheary, A. Mitin, C.Y. Wan, B. Van Melle, J.A. Puthucheary, Hemolysins

596 and plasmid profiles of Vibrio parahaemolyticus, SE Asian J Trop Med 27(1) (1996) 126-131

597
-p
[52] P.S.S. Anand, M.P.S. Kohli, S. Kumar, J.K. Sundaray, S.D. Roy, G. Venkateshwarlu, A. Sinha,
re
598 G.H. Pailan, Effect of dietary supplementation of biofloc on growth performance and digestive
lP

599 enzyme activities in Penaeus monodon, Aquaculture 418-419 (2014) 108-115.

600
na

https://doi.org/10.1016/j.aquaculture.2013.09.051.

601 [53] M.Z.H. Md Zoqratt, W.W.H. Eng, B.T. Thai, C.M. Austin, H.M. Gan, Microbiome analysis of
ur

602 Pacific white shrimp gut and rearing water from Malaysia and Vietnam: implications for
Jo

603 aquaculture research and management, PeerJ 6 (2018) e5826. https://doi.org/10.7717/peerj.5826.

604 [54] N.V. Patin, Z.A. Pratte, M. Regensburger, E. Hall, K. Gilde, A.D.M. Dove, F.J. Stewart,

605 Microbiome dynamics in a large artificial seawater aquarium, Appl Environ Microb 84(10) (2018).

606 https://doi.org/10.1128/AEM.00179-18.

607 [55] A. LaPointe, S.M. Gibbons, A. Frazier, J. Hampton-Marcell, J. Gilbert, Aquarium microbiome

608 response to ninety-percent system water change: Clues to microbiome management, Zoo Biol 34(4)

609 (2015) 360-367. https://doi.org/10.1002/zoo.21220.

610 [56] M. Martínez-Porchas, F. Vargas-Albores, Microbial metagenomics in aquaculture: a potential tool

611 for a deeper insight into the activity, Rev Aquacult 9(1) (2017) 42-56.

612 https://doi.org/10.1111/raq.12102.
613 [57] C.M. do Espírito Santo, I.C. Pinheiro, G.F.A. de Jesus, J.L.P. Mouriño, F.D.N. Vieira, W.Q.

614 Seiffert, Soybean molasses as an organic carbon source in the farming of Litopenaeus vannamei

615 (Boone, 1931) in a biofloc system, Aquac Res 48(4) (2017) 1827-1835.

616 https://doi.org/10.1111/are.13020.

617 [58] J.T. Tzuc, D.R. Escalante, R. Rojas Herrera, G. Gaxiola Cortés, M.L.A. Ortiz, Microbiota from

618 Litopenaeus vannamei: Digestive tract microbial community of Pacific white shrimp (Litopenaeus

619 vannamei), SpringerPlus 3(1) (2014) 1-10. https://doi.org/10.1186/2193-1801-3-280.

of
620 [59] W. Rungrassamee, A. Klanchui, S. Maibunkaew, S. Chaiyapechara, P. Jiravanichpaisal, N.

ro
621 Karoonuthaisiri, Characterization of intestinal bacteria in wild and domesticated adult black tiger

622 shrimp (Penaeus monodon), PLOS ONE 9(3) (2014).

623 https://doi.org/10.1371/journal.pone.0091853.
-p
re
624 [60] R. Crab, T. Defoirdt, P. Bossier, W. Verstraete, Biofloc technology in aquaculture: Beneficial
lP

625 effects and future challenges, Aquaculture 356-357 (2012) 351-356.

626
na

https://doi.org/10.1016/j.aquaculture.2012.04.046.

627 [61] Y. Zhao, C. Duan, X.X. Zhang, H. Chen, H. Ren, Y. Yin, L. Ye, Insights into the gut microbiota of
ur

628 freshwater shrimp and its associations with the surrounding microbiota and environmental factors, J
Jo

629 Microbiol Biotechn 28(6) (2018) 946-956. https://doi.org/10.4014/jmb.1710.09070.

630 [62] M.J. Eickhoff, B.L. Bassler, Vibrio fischeri siderophore production drives competitive exclusion

631 during dual-species growth, Mol Microbiol (2020). https://doi.org/10.1111/mmi.14509.

632 [63] M. Mansson, L. Gram, T.O. Larsen, Production of bioactive secondary metabolites by marine

633 Vibrionaceae, Mar Drugs 9(9) (2011) 1440-1468. https://doi.org/10.3390/md9091440.

634 [64] D.J. Burks, S. Norris, K.M. Kauffman, A. Joy, P. Arevalo, R.K. Azad, H. Wildschutte,

635 Environmental vibrios represent a source of antagonistic compounds that inhibit pathogenic Vibrio

636 cholerae and Vibrio parahaemolyticus strains, MicrobiologyOpen 6(5) (2017).

637 https://doi.org/10.1002/mbo3.504.
638 [65] J. Thompson, S. Gregory, S. Plummer, R.J. Shields, A.F. Rowley, An in vitro and in vivo

639 assessment of the potential of Vibrio spp. as probiotics for the Pacific White shrimp, Litopenaeus

640 vannamei, J Appl Microbiol 109(4) (2010) 1177-1187. https://doi.org/10.1111/j.1365-

641 2672.2010.04743.x.

642 [66] S. Direkbusarakom, M. Yoshimizu, Y. Ezura, L. Ruangpan, Y. Danayadol, Vibrio spp., the

643 dominant flora in shrimp hatchery against some fish pathogenic viruses, J Mar Biotechnol 6(4)

644 (1998) 266-267

of
645 [67] J.A. Hall, N. Bouladoux, C.M. Sun, E.A. Wohlfert, R.B. Blank, Q. Zhu, M.E. Grigg, J.A.

ro
646 Berzofsky, Y. Belkaid, Commensal DNA Limits Regulatory T Cell Conversion and Is a Natural

647 Adjuvant of Intestinal Immune Responses, Immunity 29(4) (2008) 637-649.

648 https://doi.org/10.1016/j.immuni.2008.08.009.
-p
re
649 [68] S. Uematsu, K. Fujimoto, M.H. Jang, B.G. Yang, Y.J. Jung, M. Nishiyama, S. Sato, T. Tsujimura,
lP

650 M. Yamamoto, Y. Yokota, H. Kiyono, M. Miyasaka, K.J. Ishii, S. Akira, Regulation of humoral

651
na

and cellular gut immunity by lamina propria dendritic cells expressing Toll-like receptor 5, Nat

652 Immunol 9(7) (2008) 769-776. https://doi.org/10.1038/ni.1622.

ur

653 [69] X.W. Wang, J.X. Wang, Diversity and multiple functions of lectins in shrimp immunity, Dev comp
Jo

654 immunol 39(1-2) (2013) 27-38

655 [70] H. Liu, P. Jiravanichpaisal, L. Cerenius, L.L. Bok, I. Söderhäll, K. Söderhäll, Phenoloxidase is an

656 important component of the defense against Aeromonas hydrophila infection in a crustacean,

657 Pacifastacus leniusculus, J Biol Chem 282(46) (2007) 33593-33598.

658 https://doi.org/10.1074/jbc.M706113200.

659 [71] A. Lu, Q. Zhang, J. Zhang, B. Yang, K. Wu, W. Xie, Y.X. Luan, E. Ling, Insect prophenoloxidase:

660 The view beyond immunity, Front Physiol 5 JUL (2014).

661 https://doi.org/10.3389/fphys.2014.00252.
662 [72] T.B. Clarke, K.M. Davis, E.S. Lysenko, A.Y. Zhou, Y. Yu, J.N. Weiser, Recognition of

663 peptidoglycan from the microbiota by Nod1 enhances systemic innate immunity, Nat Med 16(2)

664 (2010) 228-231. https://doi.org/10.1038/nm.2087.

665 [73] H.S. Deshmukh, Y. Liu, O.R. Menkiti, J. Mei, N. Dai, C.E. O'Leary, P.M. Oliver, J.K. Kolls, J.N.

666 Weiser, G.S. Worthen, The microbiota regulates neutrophil homeostasis and host resistance to

667 Escherichia coli K1 sepsis in neonatal mice, Nat Med 20(5) (2014) 524-530.

668 https://doi.org/10.1038/nm.3542.

of
669 [74] S.C. Ganal, S.L. Sanos, C. Kallfass, K. Oberle, C. Johner, C. Kirschning, S. Lienenklaus, S. Weiss,

ro
670 P. Staeheli, P. Aichele, A. Diefenbach, Priming of Natural Killer Cells by Nonmucosal

671 Mononuclear Phagocytes Requires Instructive Signals from Commensal Microbiota, Immunity

672
-p
37(1) (2012) 171-186. https://doi.org/10.1016/j.immuni.2012.05.020.
re
673 [75] Y.L. Song, C.Y. Li, Shrimp immune system - Special focus on penaeidin, J Mar Sci Tech-Taiw
lP

674 22(1) (2014) 1-8. https://doi.org/10.6119/JMST-013-0813-1.

675
na

[76] M.R. Pilotto, A.N.A. Goncalves, F.N. Vieira, W.Q. Seifert, E. Bachère, R.D. Rosa, L.M. Perazzolo,

676 Exploring the Impact of the Biofloc Rearing System and an Oral WSSV Challenge on the Intestinal
ur

677 Bacteriome of Litopenaeus vannamei, Microorganisms 6(3) (2018) 83.

Jo

678 https://doi.org/10.3390/microorganisms6030083.

679 [77] X. Lin, M. Novotny, K. Söderhäll, I. Söderhäll, Ancient cytokines, the role of astakines as

680 hematopoietic growth factors, J Biol Chem 285(37) (2010) 28577-28586.

681 https://doi.org/10.1074/jbc.M110.138560.

682 [78] X.W. Wang, J.D. Xu, X.F. Zhao, G.R. Vasta, J.X. Wang, A shrimp C-type lectin inhibits

683 proliferation of the hemolymph microbiota by maintaining the expression of antimicrobial peptides,

684 J Biol Chem 289(17) (2014) 11779-11790. https://doi.org/10.1074/jbc.M114.552307.

685
686 Figure legends
687
688 Fig. 1. Biofloc formation and bacterial community fingerprints during shrimp cultivation. A. A schematic
689 diagram of the study. Shrimp postlarvae (PL) 8-12 were acclimatized and transferred to the biofloc and
690 control system. Bioflocs were collected to compare similarity of bacterial community at 3, 5, and 6 weeks
691 of shrimp cultivation. Shrimp were cultivated for 6 weeks and harvested to examine gut microbiome,
692 immune-related gene expression, and innate immune activities. The survival rate was also recorded. B.
693 The levels of bioflocs measured using an Imhoff cone. Values are the mean ± S.D., n = 3 per group. *, p <
694 0.05, ** p < 0.01. C. Cluster analysis of Bray-Curtis dissimilarity using ASIRA. The yellow dots, pink
695 triangles, and red squares represented bacterial population isolated from bioflocs collected at 3, 5, and 6
696 weeks, respectively. n = 3 per group.
697
698 Fig. 2. Bacterial characterization in bioflocs. The biofloc and control system were used to cultivate
699 shrimp for 6 weeks in which bioflocs were harvested for DNA purification, and metagenomic analysis. A.

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700 Proportions of bacterial taxa at the genus level found in each biofloc samples: BF-1, BF-2, and BF-3, n =
701 3. Pie charts and bar inserts illustrate proportions of bacterial genus present in at least one biofloc sample

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702 at > 0.2% relative abundance. B. The relative levels of Vibrio spp. present in each biofloc, n = 3. Bar plots
703 show proportions of vibrio species present in at least one biofloc sample at > 1% relative abundance.
704 Bacterial taxa and vibrio species present less than 0.2% and 1.0% were grouped into Other in A. and B.,
705
706
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707 Fig. 3. Analysis of shrimp gut microbiome. Shrimp were cultivated in the biofloc and control system for 6
708 weeks and their GI tract with fecal contents were collected for DNA purification. A. NMDS plot of Bray-
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709 Curtis dissimilarity using ASIRA. The blue dots, and green triangles represented gut bacterial community
710 isolated from shrimp in both the systems, respectively. n = 7 per group. B. Proportions of gut bacterial
711 genera present in shrimp cultivated in the biofloc and control system. Bar plots show proportions of
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bacterial species present in at least one biofloc sample at > 1% relative abundance. Taxa present at less
713 than 1.0% were grouped into the “Other” category. n = 3 per group. C. Cluster analysis and heatmap of
714 bacteria genus found in shrimp intestine. The relative levels of gut bacteria in shrimp were compared
715
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between two groups. n = 3 per group. *, p < 0.05, ** p < 0.01.

716
717 Fig. 4. Gene expression analysis in shrimp tissue. GI tract and hepatopancreas were collected from shrimp
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718 cultivated in the biofloc and clear water system for 6 weeks. A. Expression of immune related genes in
719 the shrimp GI tract. B. Expression of immune related genes in the shrimp hepatopancreas. The levels of
720 the targeted genes including lectin, prophenoloxidase, lipopolysaccharide (LPS)- and β-1,3-glucan-
721 binding protein, serine proteinase, penaeidin 3, and heat shock protein 70 were measured using a SYBR-
722 based qPCR. Actb was used as an internal control. The PCR reaction was performed duplicated. Values
723 are the mean ± S.D., n ≥ 4 per group. *, p < 0.05, ** p < 0.01.
724
725
726 Fig. 5. Immune-related activities in shrimp hemolymph. Hemolymph was collected from shrimp
727 cultivated in the biofloc and clear water system for 6 weeks. A. Total hemocyte count (THC). B.
728 Hemocyte phenoloxidase (PO) activity. C. Serum superoxide dismutase (SOD) activity. D. The serum
729 total antioxidant activity. E. The survival rate of shrimp. Values are the mean ± S.D., n ≥ 3 per group. *, p
730 < 0.05, ** p < 0.01.
731
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749 Table 1. Water quality parameters of Pacific white shrimp cultivation water in the control and biofloc
750 system for 6 weeks. pH, and dissolved oxygen (DO) were measured every two days. Alkalinity, ammonia
751 (NH3), ammonium (NH4+), and nitrite (NO2) were measured weekly. Values are the mean ± S.D., n = 3
752 tanks per group. Student’s t test was used for calculation of P values *, p < 0.05, ** p < 0.01.
753
Parameters Control system Biofloc system P-value

pH 8.16 ± 0.09 7.44 ± 0.77 0.001**

DO (mg/L) 5.88 ± 0.24 5.75 ± 0.33 0.171

Alkalinity (mg/L) 116 ± 14.88 107 ± 26.00 0.404

NH3 (mg/L) 0.02 ± 0.02 0.01 ± 0.00 0.015*

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NH4+ (mg/L) 0.23 ± 0.19 0.32 ± 0.26 0.449

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NO2 (mg/L) 0.25 ± 0.22 0.20 ± 0.23 0.686

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Highlights
• Bacterial community in bioflocs dynamically changed during Pacific white shrimp cultivation.

• Metagenomic analysis revealed that Vibrio was the major bacterial population in bioflocs.

• Bioflocs maintained the presence of shrimp gut bacteria, but shifted the bacterial proportions.

• Alteration of gut microbiome was associated with promotion of both local and systemic
immunity.

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