Effects of dietary administration of Lactococcus lactis HNL12 on growth, innate immune response, and disease resistance of humpback grouper (Cromileptes altivelis)

Accepted Manuscript Effects of dietary administration of Lactococcus lactis HNL12 on growth, innate immune response, and disease resistance of humpback grouper (Cromileptes altivelis) Yun Sun, Mingwang He, Zhenjie Cao, Zhenyu Xie, Chunsheng Liu, Shifeng Wang, Weiliang Guo, Xiang Zhang, Yongcan Zhou PII:

S1050-4648(18)30512-6

DOI:

10.1016/j.fsi.2018.08.039

Reference:

YFSIM 5497

To appear in:

Fish and Shellfish Immunology

Received Date: 16 June 2018 Revised Date:

10 August 2018

Accepted Date: 17 August 2018

Please cite this article as: Sun Y, He M, Cao Z, Xie Z, Liu C, Wang S, Guo W, Zhang X, Zhou Y, Effects of dietary administration of Lactococcus lactis HNL12 on growth, innate immune response, and disease resistance of humpback grouper (Cromileptes altivelis), Fish and Shellfish Immunology (2018), doi: 10.1016/j.fsi.2018.08.039. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Effects of dietary administration of Lactococcus lactis

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HNL12 on growth, innate immune response, and disease

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resistance of humpback grouper (Cromileptes altivelis) Yun Suna,b, Mingwang Hea,c, Zhenjie Caoa,c, Zhenyu Xiea,b, Chunsheng Liuc,

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Shifeng Wanga,b, Weiliang Guoa, Xiang Zhang a,c,*, Yongcan Zhoua,c,*

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a

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P.R. China

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b

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PR China

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State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University,

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Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan University,

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Marine Science, Hainan University, PR China

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Hainan Provincial Key Laboratory for Tropical Hydrobiology and Biotechnology, College of

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* To whom correspondence should be addressed

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College of Marine Sciences

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Hainan University

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58 Renmin Avenue

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Haikou 570228

PR ChinaPhone and Fax: 86-898-66256125

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Email: [email protected] (Y.C. Zhou) [email protected] (X. Zhang)

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Abstract

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Lactic acid bacteria are a common group of probiotics that have been widely studied and used in

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aquaculture. In the present study, we isolated Lactococcus lactis HNL12 from the gut of wild

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humpback grouper (Cromileptes altivelis) and explored its probiotic properties. For this purpose, L.

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lactis HNL12 was added to the commercial fish feed. The results showed that HNL12 had high

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auto-aggregation ability and strong tolerance to simulated gastrointestinal stress. When C. altivelis

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consumed a diet containing 0 (control), 106, 108, or 1010 CFU/g HNL12 for four weeks, all of the

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groupers fed a diet with HNL12 had significantly increased percent weight gain (PWG), especially

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those fed with 108 CFU/g, which had a PWG of 231.45%. Compared to the control, fish fed with L.

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lactis HNL12 exhibited significantly increased survival rates following injection with Vibrio

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harveyi after one month. Immunological analysis showed that C. altivelis fed with HNL12 had (i)

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enhanced respiratory burst activity of head kidney macrophages, superoxide dismutase, acid

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phosphatase, and lysozyme activities of serum; (ii) an improved survival rate from 36% to 70%;

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and (iii) upregulated expression of a broad spectrum of immunity. Meanwhile, de novo

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transcriptome assembly yielded 89,314 unigenes, which were annotated by at least one of the

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reference databases (Nr, Swiss-Prot, GO, COG and KEGG). A total of 307 genes showed

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significantly different expression between the groups fed with or without added HNL12. GO and

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KEGG enrichment analyses of the significantly different expression gene categories and pathways

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were related to infectious diseases, antigen processing and presentation, and other immune system

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responses. These results indicate that L. lactis HNL12 is effective for enhancing the growth,

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immunity, and disease resistance of C. altivelis; this study also provides insight into the use of

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probiotics for commercial applications.

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Key words: Lactococcus lactis, Cromileptes altivelis, Growth, Innate immunity, Protection, Transcriptomes

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1. Introduction

The humpback grouper (Cromileptes altivelis) is a commercially important marine finfish in

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China with a strong demand on the global market [1]. C. altivelis, in the order Perciformes and

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family Epinephelidae, is mainly distributed in the tropical Indian Ocean and the Pacific [2,3]. The

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fish are consumed for their delicious flavor and high nutritional value, thus they have a high

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market price reported to reach US $50/kg [4]. However, the expanding market demand has led to

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overexploitation of the humpback grouper [5]. The low survival rate, slow growth rate, and

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increased prevalence of diseases have limited the development of artificial cultures of humpback

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grouper [6,7]. Therefore, it is essential to develop strategies that are beneficial for improving the

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immune response and growth performance of C. altivelis while maintaining stringent food safety

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standards.

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Recently, an increasing number of studies have focused on dietary supplements [8,9].

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Probiotics, living organisms which when administered in adequate amounts confer a health benefit

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on the host, are regarded as potential feed additives in the aquaculture industry [10-12]. Dietary

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probiotics can produce inhibitory compounds, improve the microbial balance, and enhance the

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metabolism, digestion, and absorptive abilities of host [13,14]. As a result, dietary probiotics have

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been widely used, replacing antibiotics and chemical disinfectants in aquaculture to provide

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nutrients and enzymatic contribution to digestion, inhibit pathogenic microorganisms, and

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promote the growth and innate immune response of a host [15,16]. Lactic acid bacteria, such as

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Lactobacillus spp., and Lactococcus spp., are a common group of probiotics that have been widely

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studied for their probiotic properties in aquaculture [17-19]. Lactococcus lactis is a spherical-shaped, Gram positive, catalase negative lactic acid

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bacterium, which is widely used as a probiotic microorganism [20]. It is well known for reduction

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of antibiotic-associated diarrhea [21], improvement of digestion [22], and immune modulatory

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effects [23] in animals. Kim et al. demonstrated that L. lactis BFE920 showed strong antibacterial

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activity against Streptococcus iniae, and enhanced feed efficiency and weight gain in olive

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flounder farming [18]. L. lactis WFLU12 has been shown to confer protection against

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Streptococcus parauberis in olive flounder through competitive exclusion and increased innate

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immune responses [19].

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Although many studies have used probiotics in animals grown in aquaculture, most have not

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yet been studied in depth. To our knowledge, there is no report about the use of probiotics in C.

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altivelis. In this study, we first isolated an L. lactis strain (named HNL12) from the gut of wild

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humpback grouper and explored its probiotic properties. We conducted a comparative

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transcriptome profiling analysis among C. altivelis fed diets with or without added L. lactis. The

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aim of the current study was to examine the effects of L. lactis HNL12 as a feed additive on the

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growth performance, innate immune parameters, and protection against Vibrio harveyi in

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humpback grouper.

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2. Materials and Methods

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2.1.1 Probiotic bacteria

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L. lactis HNL12 was isolated from the gut of wild humpback grouper. The bacterium was

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identified by partial sequencing of the 16S rDNA gene as previously described [24]. For routine

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use, the strain was cultured at 30 °C with shaking at 180 rpm for 24 h in de Man, Rogosa and

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Sharpe (MRS) broth.

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2.1.2. Auto-aggregation

The extent of auto-aggregation ability was assayed according to the previously described

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technique with some modifications [25]. Briefly, HNL12 was cultured overnight and centrifuged

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at 4,000 rpm for 10 min. The pellet was washed with PBS buffer (pH 7.4) and resuspended in the

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same buffer twice to a final optical density (OD) of approximately 1.00 at 600 nm. The 4 mL

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resuspensions were vortexed for 10 s and left to stand at room temperature. Variation of

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absorbance at 600 nm of cellular suspensions was measured at 1, 2, 3, 4, 5, and 10 h. The

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percentage of auto-aggregation was calculated by the following expression: auto-aggregation (%)

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= [1-(At/A0)] × 100, where At means absorbance at different times (1, 2, 3, 4, 5, or 10 h) and A0

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indicates absorbance at 0 h.

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2.1.3. Tolerance to simulated gastrointestinal stress

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a previous study [26]. Briefly, simulated gastric fluid (SGF) was prepared in PBS by

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supplementing pepsin (0.3 mg/mL) and NaCl (0.5%, w/v) adjusted to pH 2, 3, or 4, and then

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filtered using a 0.22 µm filter membrane. Similarly, simulated intestinal fluid (SIF) was prepared

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by adding pancreatin (0.1 mg/mL, Sigma) and 0.3% (w/v) Oxgall (Solarbio, Beijing, China) in

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PBS adjusted to pH 6.8 or 8 and filtered using a 0.22 µm filter membrane. The bacteria HNL12

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was harvested overnight and centrifuged at 4,000 rpm for 10 min for cell collection. The cells then

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were washed twice with PBS and adjusted to an approximately concentration of 108 CFU/mL.

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Bacteria cells were inoculated (5%, v/v) into SGF or SIF and incubated together at 30 °C for 0, 2,

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and 4 h. Then at the different time intervals, 100 µL of bacterial cell suspension was coated onto

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MRS agar medium to count the number of surviving cells. The survival rate was calculated as

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follows: survival rate (%) = (Nt/N0) × 100. Where Nt is the number of surviving cells at 2 or 4 h,

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and N0 is the number at 0 h.

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2.2. Preparation of the feed with L. lactis HNL12

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Commercial feed (51.7% crude protein, 8.4% crude lipid, 12.9% ash, and 9.0% moisture;

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Hayashikane Sangyo Co., Ltd., Feed Business Division, Chofu Plant, Japan) was purchased as the

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basal component of the diet. Three experimental feeds containing different doses of L. lactis

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HNL12 (106, 108, and 1010 CFU/g diet) were produced. The final dose of 106, 108, and 1010 CFU/g

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of L. lactis HNL12 was diluted with phosphate-buffered saline solution (PBS; pH 7.2), thoroughly

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homogenized, and then sprayed uniformly onto the basal diet. The feed supplemented with the

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same volume of PBS was used as the control. The test diets were dried at 25 °C with the aid of an

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air conditioner and stored at 4 °C until use. The experimental diets were prepared fresh each week

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to ensure the quality of the experimental diets.

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2.3. Fish management and experimental design

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Healthy juvenile C. altivelis (average weight: 3.97 ± 0.54 g), which were used for the growth

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measurements and mortality, were purchased from the Hong Yuan fishery company of Sanya

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(Hainan Province, China) and were maintained in the company. Prior to the start of the feeding

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trial, the fish were fed with commercial feed for 2 weeks. After acclimation, a total of 120 fish

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were randomly distributed into four groups (30 fish per group) with recirculating aerated seawater.

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The four groups were fed with 106, 108, and 1010 CFU/g of L. lactis HNL12, and PBS as the

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control. Fish were fed their respective diets twice a day (7:00 am and 18:00 pm) at a 3% feed rate

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of body weight for 4 weeks. The experiments were repeated once. Meanwhile, healthy young C.

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altivelis (average weight: 23.98 ± 0.71 g) were used for the analysis of non-specific immune

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responses. Fish were grouped and fed as described above (25 fish per group).

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2.4. Growth performance

Ten randomly selected fish from each group were measured and weighed at the beginning of

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the test period (initial weight). At the end of the 4-week feeding trial, ten randomly selected fish

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from each treatment were measured and weighed (final weight). Percent weight gain (PWG, %)

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was calculated as PWG (%) = 100 × [(Final weight - Initial weight) / Initial weight]. Specific

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growth rate (SGR, %) was determined according to the following formula: SGR (%) = 100 × [ln

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(Final weight) - ln (Initial weight)] / Experimental days.

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2.5. Non-specific immune responses

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2.5.1. Sampling

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During each week of the feeding trail, five young C. altivelis were randomly selected for

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analysis of non-specific immune parameters from each group described as above. Before sampling,

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fish were anesthetized using tricaine methanesulfonate (Sigma-Aldrich, St. Louis, MO, USA).

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Blood samples were drawn from each fish using a 1 mL syringe and transferred into the Eppendorf

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tubes. All tubes were stored at 4 °C overnight and then centrifuged at 800 × g for 15 min at 4 °C.

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Then the serum was removed and put into the new tubes to analyze the immune parameters.

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Macrophages from the head kidney were collected following previously described methods [27].

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Cell viability was calculated using the trypan blue exclusion test. The concentration of harvested

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cells was adjusted to 2 × 106 cells/mL and was used for the analysis of respiratory burst activity.

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2.5.2. Respiratory burst (RB) activity

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Respiratory burst activity of head kidney macrophages (HKMs) was determined with the

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nitroblue tetrazolium (NBT, Sigma Aldrich) assay as described in previously studies [28].

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Spectrophotometric results were measured at 630 nm and KOH/DMSO was used as a blank.

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2.5.3. Serum immune activities

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Superoxide dismutase (SOD), acid phosphatase (ACP), and serum lysozyme (LZM) activities

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in the serum were measured with commercial kits (Nanjing Jiancheng Bioengineering Institute,

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Nanjing, Jiangsu, China) [29] according to the manufacturer's instructions.

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V. harveyi strain QT520, was obtained from our laboratory, which had been reported that displayed strong pathogenicity to T. ovatus [30,31].

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2.6. Challenging with V. harveyi

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One month after the feeding trial of a diet with or without L. lactis HNL12 began, 25 fish

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were removed from each group, and infected via intraperitoneal with 100 µL of V. harveyi that had

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been cultured in LB medium to midlogarithmic phase and resuspended in PBS to 1 × 106 CFU/mL.

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Mortality was monitored after the injection for at least 15 days. Relative percent of survival (RPS)

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was calculated according to the following formula: RPS = [1 - (% mortality in treatment fish / %

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mortality in control fish)] ×100 [28]. The experiments were repeated once.

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2.7 Sampling, mRNA isolation, cDNA library preparation, and sequencing

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To investigate the immune response of fish fed a diet with or without added L. lactis,

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sequenced and assembled transcriptomes were performed based on our obtained results. Briefly,

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fish (average weight: 20.18 ± 0.65 g) were fed with or without 108 CFU/g of L. lactis HNL12.

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After 14 days of feeding, head kidneys were randomly collected from six fish. After fish were

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anaesthetized, head kidneys from each fish were removed, immediately frozen, and kept in liquid

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(Invitrogen, USA) following the manufacturer’s protocols, and then the extracted RNA was

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treated with RNase-free DNase I (Omega, USA) to remove genomic DNA contamination. Gel

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electrophoresis (1.5% agarose gel) and absorption spectroscopy were used to verify the quality

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and integrity of the mRNA fractions. After measuring the RNA concentration of each sample with

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an Agilent 2100 Bioanalyzer (Agilent, USA) and Qubit (Life, USA), equal amounts of RNA from

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the six fish per group were pooled for the next gene expression analysis.

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After the RNA was tested, a cDNA library was constructed. Briefly, mRNA was enriched

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using Oligo (dT) magnetic beads and then cut into short fragments. The fragmented mRNA was

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used as a template with six random hexamers to synthesize first strand cDNA. The complementary

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strand was synthesized with buffer, dNTPs, DNA polymerase I, and RNase H. After several

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purification steps, adaptor addition, and cDNA length selection, the suitable template was

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amplified by PCR. The resulting paired-end cDNA library was sequenced on an Illumina HiSeq

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2000 platform by Beijing Novogene Bioinformatics.

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2.8 Data filtering, de novo assembly, and gene annotation

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Raw reads were trimmed by removing adapter sequences and ambiguous nucleotides to obtain

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high-quality clean reads. After that, clean reads from the two groups were assembled into contigs

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using Trinity software following the manufacturer’s recommended guidelines. These contigs were

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handled by Corset software to obtain unigenes and further annotated against the non-redundant

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(NR) protein database, UniProtKB/SwissProt, and Gene Ontology (GO), Kyoto Encyclopedia of

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Genes and Genomes (KEGG), and EuKaryotic Orthologous Groups (KOG) databases. GO

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annotation was performed using Blast2GO version 2.5. WEGO software was used to classify GO

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functions of all unigenes. KEGG pathways were analyzed using the KEGG Automatic Annotation

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Server (KASS).

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2.9. Identification of differentially expressed contigs

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High quality clean reads from each sample were mapped to the unigenes using RSEM

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software. The number of aligned clean reads in different unigenes/treatment conditions was

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normalized by the RPKM (Reads per kilobase of exon model per million mapped reads) method.

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Differentially expressed genes were analyzed using the DEGseq package in R. Stringent criteria

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with a q value ≤ 0.005 and absolute fold changes ≥ 1 were used to define differentially expressed

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genes. In order to analyze over-represented functional genes and pathways, GO and KEGG

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enrichment analyses were performed using hypergeometric tests; the corrected p-value < 0.05 was

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taken as a threshold.

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2.10. Quantitative RT-PCR (qPCR)

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To examine the expression of immunerelated genes induced by L. lactis HNL12 and validate

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the transcriptome results, qRT-PCR was carried out. RNA extraction and cDNA synthesis were

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conducted as described above. qRT-PCR was conducted using the SYBR ExScript qRT-PCR Kit

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(Takara, Dalian, China) in an QuantStudioTM 6 FLEX (ABI, USA) as previously described [32].

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The primers used for qRT-PCR were designed based on the assembled gene sequences using

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Premier 6 software (Premier Biosoft International, CA, USA) (Table S1). The assay was analyzed

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in three replicates.

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2.11. Statistical analyses

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All Statistical analyses were conducted using SPSS 17.0 software (SPSS. Inc., Chicago, IL, USA). The results were analyzed with ANOVA or long-rank test. Results were considered

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statistically significant when probability (p) values were less than 0.05.

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3. Results

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3.1. HNL12 auto-aggregation ability and tolerance to simulated gastrointestinal stress

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Auto-aggregation ability is related to the adhesion ability of bacteria to epithelial cells.

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According to the auto-aggregation results obtained at different incubation times, L. lactis HNL12

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showed a high auto-aggregation percentage of 35.57%–79.2% (Fig. 1). Table 1 shows the ability

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of L. lactis HNL12 to tolerate simulated gastrointestinal stress. At pH = 2, the cell numbers of L.

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lactis HNL12 were (1.053±0.017)×108, (5.693±0.082)×107, and (3.850±0.081)×107 after 0, 2, and

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4 h in SGF, respectively. At pH = 3, the cell numbers were (1.117±0.015)×108, (7.168±0.041)×107,

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and (5.340±0.133)×107 after 0, 2, and 4 h in SGF, respectively. Moreover, the cell numbers were

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(1.085±0.062)×108, (1.054±0.023)×108, and (1.034±0.009)×108 at pH = 4 after 0, 2, and 4 h in

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SGF, respectively. In addition, after 2 h of incubation in SIF at pH = 6.8 and 8.0, the cell numbers

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of L. lactis HNL12 gently decreased. However, after 4 h in SIF at pH = 6.8, the number of cells

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increased to 144.29%. The results of SGF and SIF suggest that L. lactis HNL12 has a strong

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survivability capacity under simulated gastrointestinal stress.

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3.2. Growth performance

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After four weeks of feeding, fish consuming a diet of 108 CFU/g L. lactis HNL12 had the

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highest final length and final weight (Table 2) compared with the other three groups. Percent

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weight gains (PWG) of fish fed with 108 and 1010 CFU/g L. lactis HNL12 were 231.45±38.54 and

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208.35±21.23, respectively, which were significantly higher (p < 0.05) than the control and 106

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groups, especially the control group (PWG = 160.61±23.91). In accordance with the PWG results,

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the specific growth rate (%) (SGR) of fish fed with the diet containing 108 and 1010 CFU/g L.

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lactis HNL12 showed significantly higher (p < 0.05) results, compared to the control group and

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fish fed with 106 CFU/g L. lactis HNL12-containing diet (Table 2).

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[Table 2]

3.3. Immune system parameters

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Immune response analysis showed that macrophages and serum from all fish fed with HNL12

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(106,108, and 1010 CFU/g ) exhibited significantly enhanced respiratory burst activity, serum ACP

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activity, and serum LZM activity compared to the fish fed the control diet for two weeks (Fig. 2).

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Serum from the groups fed with 108 and 1010 CFU/g HNL12 exhibited significantly higher SOD

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activity at one week compared to that of the fish fed the control diet (Fig. 2).

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[Fig. 2]

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3.4 Protective effect of HNL12 After one month of consuming the diet with or without L. lactis, the fish were injected with V. harveyi and monitored for mortality. The mean survival rates of fish fed with added 106, 108, 1010

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CFU/g L. lactis diets, were 56%, 70%, and 68%, respectively, while the mean survival rate of fish

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fed the control diet was 36% (Fig. 3); hence, the RPS rates of fish fed with added 106, 108, 1010

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CFU/g L. lactis diets were 30.98%, 53.14%, and 50.20% in the control feeding treatment,

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respectively. [Fig. 3]

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3.5. Sequencing and de novo transcriptome assembly

To obtain the transcriptome expression profile, head kidneys were taken from the two groups

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of C. altivelis fed with commercial feed with or without added L. lactis 14 days after the start of

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the trial. The extracted RNA was high quality and had high integrity. cDNA libraries were

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constructed from pooled head kidney RNA and sequenced on the Illumina HiSeq 2000 platform.

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Using Trinity, a total of 283,721 transcripts (min length: 201 bp, max length: 17,631 bp, N50:1389)

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were assembled. After clustering, 167,834 sequences (min length: 201 bp, max length: 17,631 bp,

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N50:1754) were defined as unigenes and used for downstream analyses.

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3.6. Gene identification and annotation

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Overall, a total of 89,314 unigenes were annotated by at least one of the reference databases

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(Nr, Swiss-Prot, GO, COG, and KEGG). Significant BLASTx hits were found for 77,892

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sequences (87.2%) in the Nr database, 64,493 sequences (72.2%) were annotated in Swiss-Prot,

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60,405 sequences (67.6%) were annotated in the Go database, and 41,956 sequences (47.0%) were

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annotated in the KEGG database. Significant BLASTx hits were found for 29,398 sequences

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(32.9%) in the KOG database (Table 3).

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3.7. Identification and analysis of differentially expressed genes

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The clean reads in different libraries were mapped using RSEM software to the corresponding

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assembled consensus sequences. The total reads of the fish fed with L. lactis were 58,163,162

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(42,636,591 mapped). In the group fed the control diet, the total number of reads was 58,783,614

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(43,182,656 mapped). Differential expression analyses were carried out among the two groups fed

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with or without added L. lactis. A total of 307 genes showed significantly different expression

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between the two groups, including 169 up-regulated genes and 138 downregulated genes in the

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group fed with L. lactis compared to the control group. GO and KEGG enrichment analyses were

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conducted to evaluate significantly differently expressed gene categories and pathways, which

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resulted in a total of 9 GO terms and 8 KEGG pathways with a p-value <0.05 and FDR <0.05.

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These GO terms included immune system process, oxaloacetate decarboxylase activity, sodium

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ion transmembrane transport, sodium ion export, immune response, MHC protein complex, MHC

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class II protein complex, antigen processing and presentation, regulation of G-protein coupled

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receptor protein signaling pathway, and response to external stimulus immune system process (Fig.

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4). The dominant KEGG pathway subcategories were Staphylococcus aureus infection, systemic

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lupus

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graft-versus-host disease, hematopoietic cell lineage4, and allograft rejection (Table 4).

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erythematosus,

pertussis,

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presentation,

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3.8. Expression of immune-related genes by qRT-PCR and RNA-seq To examine the expression of immune related genes induced by L. lactis HNL12 and validate

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the gene expression profile, seven immune genes, including major histocompatibility complex

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(MHC) Iα, MHC IIα, MHC IIβ, CC chemokine (CC), CXC chemokine (CXC),

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complement component 1 subcomponent q (C1q), and complement receptor 1 (CR1), were

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selected for qRT-PCR analysis. Primers were designed based on the assembled unigenes and

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yielded single production after amplification (Table S1). The expression of all the examined genes

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from the fish fed with L. lactis HNL12 were significantly upregulated than the control fish (Figure

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5). Meanwhile, the results showed a similar trend as the expression pattern to RNA-Seq for all

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seven genes. The correlation between qRT-PCR and RNA-Seq results was 0.84, providing

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evidence that the de novo transcriptome assembly was accurate (Figure 6).

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[Fig. 5] [Fig. 6]

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4. Discussion

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The use of probiotics in human and animal nutrition is well documented [12-16]. Recently,

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the use of probiotics in aquatic animal feed has received increasing attention due to the demand of

346

environmentally friendly health management in aquaculture [17-19]. L. lactis is well known for its

347

diverse properties beneficial to hosts [20-23]. In this study, we isolated the L. lactis strain HNL12,

348

which we found to be beneficial to the growth, innate immunity, and protection of humpback

349

grouper from V. harveyi infection.

ACCEPTED MANUSCRIPT Lactic acid bacteria are very efficient at metabolizing a large variety of small fractions of

351

lactic acid, various enzymes, lipids, and proteins [33,34]. These metabolic products can stimulate

352

immune responses in humans and animals, and improve the immune system [23,35]. Lactic acid

353

bacteria use carbohydrates in the host intestine to ferment various compounds, producing lactic

354

acid, which inhibits the survival of pathogenic bacteria in the intestine [36,37]. Meanwhile, higher

355

enzyme activities and some metabolic products in the digestive tract enhance digestive capabilities

356

and growth performance of the host [38,39].

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Studies on L. lactis have reported on its capacity to improve the growth and health of aquatic

358

animals [40,41]. In Paralichthys olivaceus, for example, many studies have demonstrated that

359

feeding L. lactis to P. olivaceaus improved the fish’s growth rate, innate immunity, and survival

360

rate of P. olivaceaus infected with Streptococcus iniae [18,42,43]. In Pagrus major, Dawood et al.

361

found that consumption of feed supplemented with L. plantarum and L. lactis could affect the

362

growth performance and immune response of the fish [44]. Zhou et al. had reported that

363

Oreochromis niloticus fed a diet containing L. lactis RQ516 significantly improved growth and

364

survival rate than the control fish [45]. In addition, L. lactis has been shown to have beneficial

365

effects in Gadus morhua, Salmo salar, Salmo trutta, and other teleosts [47-50]. The application of

366

L. lactis in fish has achieved relatively positive results. In our study, PWG, SGR, and survival rate

367

after injected with V. harveyi of fish fed with 108 and 1010 CFU/g L. lactis HNL12 were

368

significantly higher than the control group, but no significant difference was found between 108

369

and 1010 groups. Similarly, Kim et al. had reported 1.25×108 CFU/g L. lactis BFE920 were

370

effective for olive flounder [18]. While Nguyen et al. had reported that the growth of P. olivaceus

371

fed with 109 CFU/g L. lactis WFLU12 was significantly improved after 4 weeks of feeding [19]. It

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ACCEPTED MANUSCRIPT 372

is likely that the optimal supplementary dose of different L. lactis stains using in teleosts is a little

373

different. It is well known that non-specific immunity first plays a defensive role when the pathogen

375

invades the body [51]. Previous studies have shown that Lactobacillus spp. play a role in

376

activating macrophages, lymphocytes, and NK cells to stimulate production of cytokines [52,53].

377

Additionally, phagocytes are more easily activated in a cytokine-rich environment [54]. In the

378

present study, the C. altivelis fed a diet with added L. lactis showed significant inductions of RB,

379

SOD, ACP, and serum LZM activities in HKMs or serum, suggesting that a diet supplemented

380

with HNL12 could induce non-specific immunity in the humpback grouper. In line with these

381

results, qRT-PCR showed that after consuming diets with added L. lactis, the immune genes MHC

382

Iα, MHC IIα, MHC IIβ, CC, CXC, C1q, and CR1, in C. altivelis were significantly higher than

383

those in the control fish. This is most likely due to the enhanced activation of non-specific

384

immunity, which promoted the fish’s immune system against invading pathogens. Similarly, in P.

385

olivaceaus, Heo et al. showed that the lysozyme activity, serum peroxidase activity, and blood

386

respiratory activity were significantly increased by feeding with a mixture of lactic acid bacteria

387

[18]. In red sea bream fed with lactic acid bacteria, the complement pathway, peroxidase, and

388

mucus secretion were significantly increased compared with those of the control group [44]. In

389

Epinephelus coioide, the lysozyme activity, respiratory bursts of fish fed L. plantarum were

390

significantly higher than those of fish fed the control diet [46]. Balcázar et al. found significantly

391

higher phagocytic activity and respiratory bursts of head kidney leucocytes in Oncorhynchus

392

mykiss fed L. lactis- and L. mesenteroides-containing diets [50]. In addition, many studies have

393

shown that L. lactis could activate phagocytosis and energy metabolism of macrophages and could

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ACCEPTED MANUSCRIPT 394

also promote the expression of some immune genes [55]. All of the abovementioned studies

395

indicate that Lactobacillus can activate the innate immune system. Once the innate immune system is activated, the status of activation is controlled and

397

maintained at a constant level (homeostasis) in healthy conditions [18,56]. Bacterial infection

398

challenges the health status of the fish [52,57,58]. Nguyen et al. found that feeding with L. lactis

399

could significantly enhance the survival rate of olive flounder against Streptococcus [43]. Aly et al.

400

demonstrated that the lactic acid bacteria treated group improved the resistance ability against P.

401

fluorescens in Tilapia nilotica [59]. Harikrishnan et al. reported that diet mixed with Lactobacillus

402

sakei could increase the protection against S. iniae and Streptococcus parauberis in kelp grouper

403

[60]. Similar results were observed in Gadus morhua, Salmo salar, Litopenaeus vannamei,

404

Fenneropenaeus chinensis, and other aquaculture species [47,48,52,57,58]. In the present study,

405

dietary administration of 108 CFU/g L. lactis HNL12 for a four-week period significantly

406

increased the survival rate of humpback grouper from 36% (control group) to 70% (L. lactis-fed

407

group) against V. harveyi, thus the RPS is 53.14%.

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In addition, we sequenced and assembled transcriptomes to further investigate the immune

409

response of fish fed a diet with or without added L. lactis. We obtained 307 genes with

410

significantly different expression between the two groups. GO enrichment analysis indicated that

411

some immune pathways had advantages in the MHC, MHC class II protein complex, and antigen

412

processing and presentation. The dominant KEGG pathways included S. aureus infection,

413

systemic lupus erythematosus, antigen processing and presentation, graft-versus-host disease, and

414

allograft rejection, and the MHC was up regulated in these pathways. Similarly, previous studies

415

have shown that L. lactis could up regulate the expression of MHC, CD40, or CD80 [58,61].

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ACCEPTED MANUSCRIPT These results suggest that L. lactis may promote the presentation of antigens and activate MHC I

417

and II pathway. In line with the transcription group analysis, the non-specific immune responses

418

and the expression of immune genes are most likely due to the enhanced activation of the immune

419

system, which promoted a strong immune response to the invading pathogens, ultimately

420

preventing illness.

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In conclusion, L. lactis HNL12 improves the growth rate, stimulates macrophage activation,

422

secretes extracellular enzymes, and improves immunity and disease resistance against V. harveyi in

423

C. altivelis. These results indicate that administering 108 CFU/g L. lactis HNL12 is an ideal

424

method to increase the growth and immune system response of humpback grouper.

425 426

Acknowledgments

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This research was supported financially by the National Natural Science Foundation of China

428

(No. 31560725, No. 41666006, No. 31660744), Key Research Project of Hainan Province

429

(ZDKJ2016011), National Marine Public Welfare Research Project of China (No. 201405020-4),

430

Postdoctoral Science Foundation of Hainan Province (BSH-RST-2018001).

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References:

433 434 435 436 437 438 439 440 441 442 443 444 445

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tract of wild shrimp on growth and survival of white shrimp (Litopenaeus vannamei) challenged with Vibrio harveyi, Fish Shellfish Immunol. 32 (2012) 170–177. [53] H.Z. Far, C. Saad, H.M. Daud, S.A. Harmin, S. Shakibazadeh, Effect of Bacillus subtilis on the growth and survival rate of shrimp (Litopenaeus vannamei), Afr. J. Biotechnol. 8 (2009) 3369–3376. [54] M.W. Boule, C. Broughton, F. Mackay, S. Akira, A. Marshak-Rothstein, I.R. Rifkin, Toll-like receptor 9 dependent and independent dendritic cell activation by chromatin-immunoglobulin G complexes, J. Exp. Med. 199 (2004) 1631–1640. [55] K. Sekine, J. Ohta, M. Onishi, T. Tatsuki, Y. Shimokawa, T. Toida, et al., Analysis of antitumor properties of effector cells stimulated with a cell wall preparation (WPG) of Bifidobacterium infantis, Biol. Pharm. Bull. 18 (1995) 148–153. [56] S. Gordon, F.O. Martinez, Alternative activation of macrophages: mechanisms and functions, Immunity 32 (2010) 593–604. [57] G. Dash, R.P. Raman, K.P. Prasad, M. Makesh, M.A. Pradeep, S. Sen, Evaluation of paraprobiotic applicability of Lactobacillus plantarum in improving the immune response and disease protection in giant freshwater prawn, Macrobrachium rosenbergii (de Man, 1879), Fish Shellfish Immunol. 43 (2015) 167–174. [58] W. Wang, M. Li, W. Fang, A.K. Pradhan, Y. Li, A predictive model for assessment of decontamination effects of lactic acid and chitosan used in combination on Vibrio parahaemolyticus in shrimps, Int. J. Food Microbial. 167 (2013) 124–130. [59] S.M. Aly, Y.A.G. Ahmed, A.A.A. Ghareeb, M.F. Mohamed, Studies on Bacillus subtilis and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections, Fish Shellfish Immunol. 25 (2008) 128–136. [60] R. Harikrishnan, C. Balasundaram, M.S. Heo, Lactobacillus sakei BK19 enriched diet enhances the immunity status and disease resistance to streptococcosis infection in kelp grouper, Epinephelus bruneus, Fish Shellfish Immunol. 29 (2010) 1037–1043. [61] M. Bahey-El-Din, P.G. Casey, B.T. Griffin, C.G. Gahan, Lactococcus lactis-expressing listeriolysin O (LLO) provides protection and specific CD8(+) T cells against Listeria monocytogenes in the murine infection model, Vaccine 26 (2008) 5304–5314.

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Tables

617 618 619 620 621 622

Table 1 Survival bacterial number of Lactococcus lactis HNL12 under simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Results are expressed as mean ± SD (n=3). Gastrointestinal stress

pH value

Treatment time 0h

2h

2.0 3.0 4.0 6.8 8.0

SIF

EP

Control

(3.850±0.081)×107 (5.340±0.133)×107 (1.034±0.009)×108 (1.541±0.018)×108 (8.018±0.013)×107

106

108

1010

Initial length (cm)

8.43±0.49a

8.34±0.61a

8.40±0.45a

8.45±0.57a

Final length (cm)

9.23±0.39a

9.87±0.38b

10.37±0.61c

9.93±0.51b

Initial weight (g)

3.96±0.30a

4.01±0.58a

3.98±0.36a

3.94±0.35a

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632 633 634 635 636 637 638 639 640

(5.693±0.082)×107 (7.168±0.041)×107 (1.054±0.023)×108 (9.070±0.036)×107 (8.567±0.188)×107

Table 2 Weight gain, percent weight gain (%) (PWG), specific growth rate (%) (SGR) of Chromileptes altivelis fed with diets containing different experimental doses of Lactococcus lactis HNL12 (0, 106, 108, and 1010 CFU/g diet) for 4 weeks.

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623 624 625 626 627 628 629 630 631

(1.053±0.017)×108 (1.117±0.015)×108 (1.085±0.062)×108 (1.068±0.009)×108 (1.142±0.103)×108

M AN U

SGF

4h

SC

Bacterial number (CFU/ml)

RI PT

616

Final weight (g)

10.29±0.70a

11.08±0.77a

13.18±1.14b

12.16±0.84b

PWG (%)

160.18±17.80a

176.18±19.25a

231.45±28.73b

208.35±21.23b

SGR (%)

3.41±0.24a

3.62±0.24a

4.27±0.32b

4.01±0.24b

Note: Data (mean ± SE) at the same sampling time with different letters significantly differ (p < 0.05) among treatments.

ACCEPTED MANUSCRIPT 641 642 643 644 645 646 647

Table 3 Statistics of annotation results for Cromileptes altivelis head kidney transcriptome unigenes.

SC DEG number

Corrected P-value

Staphylococcus aureus infection

8

8.4*10-6

Systemic lupus erythematosus

7

3.3*10-4

Pertussis

6

8.6*10-3

4

0.019

Antigen processing and presentation

6

0.028

Graft-versus-host disease

3

0.041

Hematopoietic cell lineage

4

0.041

Allograft rejection

3

0.048

AC C

Prion diseases

EP

Pathway name

658 659 660 661 662 663 664 665 666 667

87.2 72.2 67.6 47.0 32.9 100

Table 4 KEGG classification of significant pathways of Cromileptes altivelis fed with Lactococcus lactis HNL12.

TE D

648 649 650 651 652 653 654 655 656 657

77892 64493 60405 41956 29398 89314

M AN U

Annotated in NR Annotated in swiss-prot Annotated in GO Annotated in KEGG Annotated in KOG Total Unigenes

Percentage (100%)

RI PT

Number of Unigenes

ACCEPTED MANUSCRIPT 668

Fig. 1. Auto-aggregation percentages of Lactococcus lactis HNL12 under different incubation times.

SC

RI PT

Fig. 2. The effect of different experimental doses of Lactococcus lactis HNL12 (0, 106, 108, and 1010 CFU/g diet) on innate immune parameters in Chromileptes altivelis. Macrophages respiratory burst (RB) activity (A), serum superoxide dismutase (SOD) activity (B), serum acid phosphatase (ACP) activity (C), serum lysozyme (LZM) activity (D). Each bar represents the mean value from five determinations with the standard deviation (SD). Different letters denoted the data at the same sampling time with significantly differ (p < 0.05) among treatments.

M AN U

Fig. 3. Survival percentages of different administrating fish. Chromileptes altivelis fed with 106, 108, 1010 CFU/g L. lactis HNL12, or control (PBS) for 4 weeks and then were challenged with Vibrio harveyi. Survival percentages were monitored daily. "*" represents significance between the survivals of the different administrating fish and control fish, which was determined with Chi-sqare test. *p < 0.05.

TE D

Fig. 4. Enriched GO terms between Chromileptes altivelis fed with or without Lactococcus lactis HNL12.

Fig. 5. qRT-PCR analysis of the expression of immune-related genes in fish fed with 108 CFU/g L. lactis HNL12. Cromileptes altivelis were fed with 108 CFU/g L. lactis HNL12, and the expression of major histocompatibility complex (MHC) Iα, MHC IIα, MHC IIβ, CC chemokine (CC), CXC chemokine (CXC), complement component 1 subcomponent q (C1q), and complement receptor 1 (CR1) in head kidney were determined by quantitative real-time RT-PCR. The fold change of gene expression was normalized to β-actin gene and relative to the control group samples. qRT-PCR data were reported as means ± SD (N = 3), and the statistical significance is analyzed. **p < 0.01, *p < 0.05.

EP

670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703 704 705 706 707 708 709 710

Figure Legends

AC C

669

Fig. 6. Effect of Lactococcus lactis on the expression of immune genes and qPCR validation of putative DEGs. Fold changes of selected genes, major histocompatibility complex (MHC) Iα, MHC IIα, MHC IIβ, CC chemokine (CC), CXC chemokine (CXC), complement component 1 subcomponent q (C1q), and complement receptor 1 (CR1), were given either according to RNA-seq or qPCR results. The fold change of gene expression was normalized to β-actin gene and relative to the control group samples. qRT-PCR data were reported as mean ± SD.

ACCEPTED MANUSCRIPT

Fig.1

TE D EP AC C

717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741

M AN U

SC

RI PT

711 712 713 714 715 716

ACCEPTED MANUSCRIPT

Fig.2

749 750 751 752 753 754 755 756 757 758 759 760 761 762

AC C

748

EP

TE D

M AN U

SC

RI PT

742 743 744 745 746 747

ACCEPTED MANUSCRIPT

Fig. 3

EP AC C

768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787

TE D

M AN U

SC

RI PT

763 764 765 766 767

ACCEPTED MANUSCRIPT

Fig. 4

AC C

794 795 796 797 798 799 800 801 802 803 804 805 806 807 808

EP

TE D

M AN U

SC

RI PT

788 789 790 791 792 793

ACCEPTED MANUSCRIPT

Fig. 5

M AN U

SC

RI PT

809 810 811 812 813 814

EP AC C

816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838

TE D

815

ACCEPTED MANUSCRIPT

Fig. 6

M AN U

SC

RI PT

839 840 841 842 843 844 845

EP AC C

847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868

TE D

846

ACCEPTED MANUSCRIPT 869 870 871 872 873

Supplement data

875 Table S1 Primers used in this study. Primer sequences (5'-3')

MHCIIα-F

GGCCAGCCTGTCTTTCAGTCCA

MHCIIα-R

CAGCAGTCTGCCAGCCGATGTT

MHCIIβ-F

TCCCTCCTCTTCATCAGCCTCT

MHCIIβ-R

GCGTTCTTCACACCGTACTCAGT

MHCIα-F

TCGACCAAATGAAGACGACACC

MHCIα-R

ACAAGACAACCAGCACCACACC

CC-F

GCTCTTTTCTTCCTTCTGCTCC

CC-R

GTTTTTTGTGTGACGGCTTCTT

CXC-F

GCAGTCTACCCAAAGAGCCCCA

CXC-R

ACAGCCTCACATCACGACCCAA

C1q-F

CTCCAACCAGCAGAGATCCTTC

CR1-F CR1-R β-actin-F

878

M AN U

CCCTTCACCAGCTTCAGACACA

TGACCTTCACCTGCAGTCAGG TTGAACGTATCCGACGTCACA CGCTGACAGGATGCAGAAGG

AC C

β-actin-R

TE D

C1q-R

SC

Primer name

EP

876 877

RI PT

874

TGAAGTTGTTGGGCGTTTGG

ACCEPTED MANUSCRIPT

Highlights: 1. Lactococcus lactis HNL12 which was isolated from the gut of wild humpback grouper, has a strong survivability capacity under simulated

RI PT

gastrointestinal stress. 2. Cromileptes altivelis fed with Lactococcus lactis HNL12 had better growth and enhanced non-specific immunity of Cromileptes altivelis than

SC

control group.

M AN U

3. Lactococcus lactis HNL12 as feeding could improve the disease resistance against Vibrio harveyi for Cromileptes altivelis. 4. Transcriptome analysis provides insights into the immune responsive pathways and genes in the head kidney of Cromileptes altivelis fed with

AC C

EP

TE D

Lactococcus lactis HNL12.