Accepted Manuscript Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2 on immunology and growth performance of Hybrid sturgeon Hui Fei, Guo-dong Lin, Cheng-cai Zheng, Meng-meng Huang, Shi-Chao Qian, Zhenju Wu, Cong Sun, Zhen-guang Shi, Jian-you Li, Bing-nan Han PII: S1050-4648(18)30506-0 DOI: 10.1016/j.fsi.2018.08.031 Reference: YFSIM 5489 To appear in: Fish and Shellfish Immunology Received Date: 13 July 2018 Revised Date: 14 August 2018 Accepted Date: 15 August 2018 Please cite this article as: Fei H, Lin G-d, Zheng C-c, Huang M-m, Qian S-C, Wu Z-j, Sun C, Shi Z-g, Li J-y, Han B-n, Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2 on immunology and growth performance of Hybrid sturgeon, Fish and Shellfish Immunology (2018), doi: 10.1016/ j.fsi.2018.08.031. 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. ACCEPTED MANUSCRIPT Effects of Bacillus amyloliquefaciens and Yarrowia lipolytica lipase 2 2 on immunology and growth performance of Hybrid sturgeon 3 Hui Fei a,e*,Guo-dong Lin a,e, Cheng-cai Zheng a,e, Meng-meng Huang a,e , Shi-Chao 4 Qian b, Zhen-ju Wu b, Cong Sun a,e, Zhen-guang Shi c, Jian-you Li d, Bing-nan Han a,f RI PT 1 5 a. College of Life Sciences, Zhejiang Sci-Tech University, 310018, Hangzhou, China 7 b. Hangzhou Biopeptide Biotech Co., Ltd. 310012, Hangzhou, China 8 c. Yunnan Amur sturgeon group Co., Ltd. 654200, Huize, China 9 d. Huize Dian Ze aquaculture Co., Ltd. 654200, Huize, China 10 e. Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, 11 Zhejiang Sci-Tech University, Hangzhou 310018, China. 12 f. Qingdao Master Biotechnology Co., Ltd. 266000, China. TE D M AN U SC 6 13 14 Key words ： Bacillus amyloliquefaciens; Yarrowia lipolytica lipase 2; Hybrid 16 sturgeons; Growth performance; Innate immunity 18 19 AC C 17 EP 15 20 *Correspondence to: 21 Dr. Hui Fei 22 Tel./fax: +86 571 868 48367. 23 E-mail: firstname.lastname@example.org (H, Fei) ACCEPTED MANUSCRIPT 24 Abstract: A 12-weeks feeding trial was performed to investigate the possible effects of 26 supplementation of Hybrid sturgeon diet with Bacillus amyloliquefaciens (GB-9) and 27 Yarrowia lipolytica lipase2 (YLL2) single or combined on immune response and 28 growth performance of Hybrid sturgeon (Acipenser schrenkii ♂and Acipenser baeri 29 ♀). For this aim, Hybrid sturgeons were fed with four experimental diets namely: Diet 30 1 (0-control), Diet 2 (5.0g/kg GB-9), Diet 3 (4.0g/kg YLL2), and Diet 4 (5.0g/kg 31 GB-9 + 4.0g/kg YLL2), respectively. After fed with varied diets, growth performance, 32 mucosal immune response, leukocytes immune response and serum immunological 33 response were measured. The results indicated that supplementations of GB-9 + 34 YLL2 resulted in a significant increase in final weight, Docosahexaenoic acid (DHA) 35 and Eicosapentenoic acid (EPA) concentration, compared with that of control (p < 36 0.05). 37 leukocytes phagocytosis activity and reactive oxygen species level，and serum 38 alternative complement pathway activity, peroxidase and lysozyme activity were 39 significantly higher in supplemented groups compared to the control (p < 0.05). The 40 highest values were recorded in fish fed both YLL2 and GB-9 with respect to the 41 individual application. The present results suggested that the combination of these 42 supplementation could be considered as potential feed-additives for aquaculture 43 farmed fish. TE D M AN U SC RI PT 25 AC C EP For innate immunity, the results showed that skin mucus lysozyme activity, 44 45 1. Introduction ACCEPTED MANUSCRIPT Sturgeons, especially Hybrid sturgeons (Acipenser schrenkii ♂ and Acipenser baeri 47 ♀), as a high-quality protein and unsaturated fatty acid resource [1, 2]，have been 48 large-scale cultured and shown a high market value. Sturgeon aquaculture 49 is developing rapidly, China has become the largest sturgeon culture region since 50 2000 . However, the available information on sturgeon about dietary lipid is limited, 51 which is an important factor in intensive fish farming and successful mass production 52 . Hybrid sturgeons are subjected to stress condition that damage fish immune 53 systems, leading to increased infection of pathogens and the emergence of diseases 54 . M AN U SC RI PT 46 In recent years, dietary administration of functional (biological activity) feed 56 additives has been suggested as an environmental friendly alternative approach to 57 enhance immune response and increase the growth performance of fish [4-5]. Bacillus 58 subtilis, one of the most studied probiotics in fish and shrimp, has been reported to 59 have various beneficial properties, including immunostimulant and disease resistance 60 substances [6-9]. While most research evaluate the effect of administering probiotics 61 individually [7-9], modulation of immune responses using a combination of lipase and 62 probiotics has rarely been investigated in fish. Therefore, the present investigation 63 was carried out to study the effect of dietary supplementation with the probiotic 64 bacterium, Bacillus amyloliquefaciens (GB-9), Yarrowia lipolytica lipase 2 (YLL2) 65 and their combination on the immunology and growth performance of hybrid 66 sturgeon. 67 AC C EP TE D 55 YLL2 is a typically lipase which has the characteristics of tolerance to the ACCEPTED MANUSCRIPT gastrointestinal environment, good biological safety, stable at low pH and superior 69 temperature performance. YLL2 has been reported to have various beneficial 70 properties when supplementing in juvenile fish diets, including reducing the amount 71 of fish oil adding in diet, promoting the secretion of digestive enzymes [10-12]. More 72 interestingly, YLL2 could efficiently hydrolyze the crude fish oil to produce 73 polyunsaturated fatty acids, especially DHA and EPA, which would improve 74 fecundity, egg hatchability, cell viability , immune functions and the overall quality of 75 the fish [4, 13, 14]. M AN U SC RI PT 68 Probiotics GB-9 are active microorganisms that are beneficial to the host and are 77 commonly used as immunomodulators . Several reports suggest that 78 supplemented probiotics can improve growth, immune responses and alleviate fish 79 resistance [16-19]. The most commonly used probiotics in aquaculture are Bacillus, 80 Lactobacillus and Saccharomyces [20, 21]. Bacillus sp. is a non-pathogenic aerobic 81 gram-positive bacterium that enhances physical condition and GI microbial 82 populations either by oral administration or by oral administration to bacterial water 83 of the fish [15, 22]. GB-9 is a non-pathogenic aerobic Gram-positive bacterium 84 belonging to the genus B. subtilis, which produces various bacteriostatic substances as 85 probiotics including antimicrobial proteins, lipopeptides, antibiotics, macrolides, 86 oligopeptidases, polyketides, peptides and polyketides [23-27]. While GB-9 is added 87 to fish diet, it plays a beneficial role in the intestine by inhibiting the growth of 88 harmful intestine bacteria through producing antimicrobial substances, inhibiting the 89 growth of competing bacteria. AC C EP TE D 76 ACCEPTED MANUSCRIPT 90 2. Materials and methods 91 2.1 Juvenile hybrid sturgeons 2-month juvenile hybrid sturgeons (5.0 g mean body weight) were reared in in-door 93 plastic pool at aquatic farm of Yunnan Amur Sturgeon Group Co., Ltd., China. 400 94 juvenile fish were randomly divided into four groups of 100 fish per plastic pool. 95 Average stocking density was 0.5 kg/m3. The juvenile fish were fed every 3 h to the 96 apparent satiation observed from July 2017 to October 2017 under natural 97 photoperiod. Water temperature ranged between 10 and 12 ℃ during the experimental 98 period. 99 100 2.2 Microorganisms and Lipase M AN U SC RI PT 92 GB-9 strain (GB-9) was provided by Zhejiang Science and Technology University, 102 China, and stored in 20% glycerol. The mixture bacterial liquid was inoculated into 103 5.0 mL LB medium (5 g L-1 peptone，10 g L-1 yeast extract，10 g L-1 NaCl in 1.0 L 104 sterile water ), inoculation amount of 1%, 37 ℃, 220 rpm cultured for 12 h, then 105 transferred to 1.0 L LB medium, inoculated for 12h under the same condition. After 106 fermentation, the products was sent to spray drying (input temperature of 170 ℃, 107 output temperature of 85 ℃, evaporation capacity 2 L/h) with corn starch as an 108 auxiliary material (Wproducts：Wcorninlet =1:10), to harvest GB-9 preparation (2×109 109 CFU/g). AC C EP TE D 101 110 Yarrowia lipolytica lipase 2 (YLL2) was kindly provided by Dr. Jinyong Yan 111 (College of Life Science & Technology, Huazhong University of Science and ACCEPTED MANUSCRIPT Technology, China) [28, 29]. Yarrowia lipolytica was inoculated into 50 mL medium 113 (10 g L-1 Yeast Extract , 20 g L-1 Peptone, 50 g L-1 Sucrose) in 500 mL conical flask, 114 inoculation amount of 2%, 28 ℃, 250 rpm cultured for 84 h, after centrifugation for 30 115 min (5000r/min), the supernatant was sent to spray drying (input temperature of 116 150 ℃, output temperature of 80 ℃, evaporation capacity 2 L/h) with corn starch as 117 an auxiliary material (Wproducts：Wstarch =1:5), The enzyme activity of YYL2 mixture 118 powder was 4125 U/mg, assayed according to published method . SC RI PT 112 120 M AN U 119 2.3 Diet For preparation of the experimental diets, the basal diet was supplemented with 122 different levels of GB-9 and YLL2 as follows: Control group, (Diet 1-Control); 123 Group1: 5.0 g/kg of GB-9 (Diet 2); Group2: 4.0g /kg YLL2 (Diet 3) and Group3: 5.0 124 g/kg of GB-9 + 4.0 g/kg YLL2 for Diet 4 (Table 1).The GB-9 concentration (5.0 g/kg, 125 107 CFU/g fodder ) employed was based in previous studies carried . The YLL2 126 concentration (4.0 g/kg, 16.5 U/g fodder) determined by basal diet composition (13.5g 127 crude lipid/100g fodder). At this concentration, the YLL2 could hydrolyze the crude 128 lipid with relatively high efficiency and maintain relatively low cost of use. EP AC C 129 TE D 121 Briefly, normal pellet diet was crushed, mixed with tap water (where GB-9 was 130 added at the desired concentration) and made again into pellets, while YLL2 was 131 mixed with tap water and then added at the desired concentration and incubated for 1 132 h in order to help hydrolyze the crude lipid. 133 ACCEPTED MANUSCRIPT 134 135 2.4 Growth performance The growth performance of hybrid sturgeons fed different experimental diets was calculated at the end of the feeding trial, based on the following formulae: 137 2.4.1 Determination of SR, WG, SGR, FCR RI PT 136 Survival rate (SR, %) = 100 × number of final fish / number of initial fish; 139 Weight gain (WG) = final weight (g) - initial weight (g); 140 Specific growth rate (SGR, % day−1) = 100 × [ln (final body weight) − ln (initial 142 143 144 body weight)] / days; M AN U 141 SC 138 Total amount of the feed consumed (g) = total amount of the fed food − collected uneaten feed/leaching loss rate; Feed conversion ratio (FCR) = feed given (dried weight) / weight gain (wet weight). 146 2.4.2 Concentration of DHA and EPA TE D 145 In the total lipid extract, concentration of DHA and EPA of hybrid sturgeon was 148 determined. Total lipids were extracted and measured through dichloromethane 149 method [31, 32]. Fatty acid methyl esters were prepared by acid-catalyzed 150 transmethylation of total lipids using boron trifluoride-methanol , and were 151 analyzed in a Thermo Scientific Trace GC Ultra gas chromatograph. The 152 chromatograph was equipped with a Restek-Rtx-225, 60.0 m × 250 µm capillary 153 column. Nitrogen was used as carrier gas (1.4 mL min-1) and the thermal gradient was 154 a constant temperature of 100℃ for 13 min, 100-180 ℃ at 10 ℃ min-1, 180-200 ℃ at 155 1℃ min-1 and 200-230 ℃ at 4 ℃ min-1. Injector and flame ionization detector AC C EP 147 ACCEPTED MANUSCRIPT 156 temperatures were 270 ℃ and 280 ℃, respectively. The fatty acid concentration of 157 hybrid sturgeon was determined with artificial fatty acid as a standard. 158 2.5 Measurement of innate immune response RI PT 159 At the end of the feeding trial (12 weeks), 12 randomly selected fish from each 161 group were selected for the immunological assays, and skin mucus was collected by 162 scrapping the mucus off the fish's skin by clean slides. After processing with PBS 163 (0.01 mM, pH 7.4 ) with EDTA and PFSM, the mucus samples were centrifuged to 164 get supernatant and stored at -80 ℃ for use [34, 35]. Briefly, whole blood samples 165 were obtained from the caudal vein of each specimen with a 23-gauge needle and 10.0 166 mL syringe. After clotting at 4 ℃, each sample was centrifuged (5000 g, 25min, 4 ℃) 167 and the serum was collected and frozen at -80 ℃ until further analysis. Leukocytes 168 were isolated from peripheral blood with 10.0 mL syringe (containing anticoagulant)， 169 each sample was centrifuged (100 g, 10 min, 4 ℃) to remove erythrocytes. Leukocytes 170 were extracted following the method by Van et. al., [34, 35]. The protein 171 concentration of skin mucus and serum was assayed with BCA method. 172 2.5.1 Fish 50% complement hemolysis (CH50) level M AN U TE D EP AC C 173 SC 160 The CH50 was assayed by using Fish 50% complement hemolysis (CH50) ELISA 174 Kit (KeShun. Bio, Shanghai, China). Briefly, 50 µL standard liquid was added to 175 standard well, then10 µL testing sample (skin mucus and serum), 40 µL diluent was 176 added to the testing sample well, while blank well add nothing. After that, 100 µL 177 HRP-conjugate reagent was added to each well, subsequently incubated for 60 ACCEPTED MANUSCRIPT minutes at 37 ℃. After incubation, each well was washed for five times, 50 µL 179 chromogen solution A and 50 µL chromogen solution B was added to each well. After 180 incubated for 15 minutes at 37 °C, 50 µL terminal solution was added to each well, 181 consequently determined at OD450. All above assays were repeated on three occasions. 182 2.5.2 Alternative complement pathway activity (ACH50) RI PT 178 The activity of the alternative complement pathway was assayed using sheep red 184 blood cells (300 µL SRBC, Biomedics) as targets [36, 37]. The degree of haemolysis 185 and the lysis curve for each specimen were obtained. The volume of serum producing 186 50% haemolysis (ACH50) was determined and the number of ACH50 units/mL 187 obtained for each experimental group. 188 2.5.3 Reactive oxygen species level (ROS level) M AN U SC 183 Leukocyte were adjusted to the concentration of 3×106 cells/mL (V=1.0 mL) with 190 PBS buffer solution, the activity oxygen fluorescent probe was added until the final 191 concentration reached 2.0 µM, incubated on rotary equipment for 50 min at room 192 temperature without light [38-40]. After cells were washed three times with PBS, the 193 fluorescence intensity was detected on the flow cytometer (BD-FACSAria II). Setting 194 the assay with the parameters of PE-Texas Red-A, the average fluorescence intensity 195 (MFI) of P1 PE-Texas Red-A was analyzed as the result. 196 2.5.4 Lysozyme activity (LZM) AC C EP TE D 189 197 The method described by Parry et al. was used for determination of lysozyme 198 activity . The lysozyme activity of mucus and serum was measured and expressed 199 as mg/mL. ACCEPTED MANUSCRIPT 200 2.5.5 Peroxidase activity (POD) Peroxidase activity was measured with peroxidase assay kit . The optical 202 density was read at 420 nm in a cuvette (optical path=1.0cm). Standard samples 203 without serum or skin mucus were used as blanks. Peroxidase activity was defined as 204 follows: 205 Peroxidase-activity (U/mL) 206 = 207 OD − OD blank T otal reaction v olume （ mL） × ÷ Reaction ti me × Diluting factor × 1000 12 × Optical path （1.0cm ） Sample （ mL） SC 2.5.6 Phagocytosis activity M AN U 208 RI PT 201 Phagocytic ability was defined as the percentage of cells with one or more ingested 210 fluorescent beads within the phagocytic cell population [43, 44]. All samples 211 (leukocytes) were analyzed in a flow cytometer (BD-FACSAria II) with an argonion 212 laser adjusted to 488 nm. The quantitative study of the flow cytometric results was 213 made using the statistical option of the Lysis Software Package (BD-FACSAria II). 215 216 EP AC C 214 TE D 209 2.6 Statistical analysis All data were subjected to one-way analysis of variance (ANOVA) to test the 217 difference between diets. Multiple comparisons among means were conducted using 218 Duncan's procedure when significant difference was identified. Difference was 219 regarded as significant when p < 0.05. All statistical analyses were performed by 220 SPSS 18.0 software package. ACCEPTED MANUSCRIPT 221 222 3.Result 223 3.1 Growth performance The growth performances of juvenile hybrid sturgeons fed the diets containing 225 alternative supplementation over a period of 12 weeks are showed in Table 3. There 226 was a statistically significant increase of Group 3 and Group 2 in the specific growth 227 rate (SGR), weight gain (WG), final weight (FW) compared with that of control (p < 228 0.05; Table 3). The highest SGR and WG values were observed in fish fed both 229 dietary GB-9 and YLL2 (Table 3). However, no significant differences in these 230 parameters were observed in fish fed GB-9 alone (p > 0.05; Table 3). The FCR was 231 significantly lower in fish fed 5.0 g/kg GB-9 + 1.0 g/kg YLL2 than in other groups, 232 while control group presented the highest values (p < 0.05). Survival rate of the fish 233 showed significant differences between the Group3 and control group (Table 3). 235 SC M AN U TE D 3.2 Protein concentration of skin mucus and serum EP 234 RI PT 224 In order to investigate the influence of supplemented with different levels of GB-9 237 and YLL2, protein concentration was evaluated. The result showed that there were no 238 significant differences in skin mucus among the groups, while protein concentration 239 of serum was increased compared with that of control (Table. 4). AC C 236 240 241 242 3.3 Mucosal immune response The mucosal immunity of hybrid sturgeon fed on different supplemented diets was ACCEPTED MANUSCRIPT evaluated after 12 weeks of the treatment. Among the mucosal immune response, 244 lysozyme was the most affected by the assayed diets. The result showed that 245 lysozyme activity significantly increased following 12 weeks of the feeding trial with 246 the GB-9 + YLL2 diets compared to the control diet (Fig. 1). The highest values 10.57 247 ± 0.53 (µg/mL) was recorded for fish fed both GB-9 and YLL2 supplementations. 248 However, no significant difference of peroxidase activity and CH50 was recorded 249 between experimental groups (Fig. 2 and Fig. 3). No alternative complement pathway 250 activity (ACH50) was observed after assayed for 2.5 h in all groups. M AN U SC RI PT 243 251 252 3.4 Leukocytes immunological response To elucidate the cellular immune response, phagocytosis rates and ROS levels were 254 determined between control group and supplemented groups after 12-weeks feed trail. 255 It has been reported that phagocytosis plays an important role in the host-defense 256 mechanisms and contributes to inflammation and the immune response. 257 Phagocytosis activity was analyzed by flow cytometry, which showed that the 258 phagocytic percentage of hybrid sturgeon leukocytes fed on different supplemented 259 diets was promoted with varying degrees compared with that of the control (Fig. 4), 260 indicating that YL2 and GB-9 enhanced the phagocytosis activity of leukocytes. 261 Reactive oxygen species (ROS) is of fundamental importance for host defense and 262 cellular signaling . As shown in (Fig. 5), ROS levels showed that hybrid sturgeon 263 treated with supplement in diets increased as compared to control. 264 AC C EP TE D 253 ACCEPTED MANUSCRIPT 265 3.5 Serum immunological response To elucidate the serum immunological response, peroxidase activity, 50% 267 complement hemolysis (CH50) level, lysozyme activity, and alternative complement 268 pathway activity (ACH50) were observed between control group and supplemented 269 groups after 12-weeks feed trail. RI PT 266 Peroxidase may play an important role in increasing host defenses against 271 pathogens . lysozyme is likely the most powerful bacteriolytic protein because 272 of 273 defence against bacterial infections . The alternative complement pathway 274 comprises plasma proteins, which represent the major humoral defense mechanism 275 against infection. To determine the effect of supplement diet in immunity, peroxidase 276 activity, lysozyme activity and alternative complement pathway were performed in 277 serum of hybrid sturgeon. Results (Fig. 6, Fig. 7 and Fig. 8) showed that YYL2 and 278 GB-9 induced the upregulation of peroxidase, lysozyme and alternative complement 279 pathway activity, compared with that of control. SC 270 EP TE D M AN U the ability to cleave the bacterial peptidoglycan and contributes to host The complement system plays a critical role in the identification and removal of 281 foreign substances and immune complexes and in the stimulation of inflammatory 282 response. The 50% hemolytic complement (CH50) assay is the most conventional 283 method for functional activity of the classical complement pathway. In our data, 284 YYL2 and GB-9 as immunostimulant agent, downregulated CH50 level of hybrid 285 sturgeon serum compared with that of control. 286 AC C 280 ACCEPTED MANUSCRIPT 287 4. Discussion According to the latest statistics, the global aquaculture production reached 73.8 289 million metric tons in 2014 . Taking into account this production level and the 290 main cost intensive aquaculture (even as high as 84% of the total production cost), 291 improving feed efficiency and additives are most beneficial to ensure the profitability 292 of the aquaculture industry . Hence, it has become a new trend to add feed 293 additives to increase the utilization rate of aquaculture feeds . Probiotics are 294 considered as potential feed additives in aquaculture. It is demonstrated that GB-9 can 295 improve the immune status and disease resistance of Nile tilapia . Sen Liu et al. 296 showed that the supplementation of exogenous lipase can improve the growth, gut 297 function, and health status of juvenile grass carp . Aly et al. reported that Bacillus 298 B. subtilis increased WG and SGR of Oreochromis O. niloticus . However, the 299 study of Bacillus and exogenous lipase supplement in hybrid sturgeon is limited. TE D M AN U SC RI PT 288 It has been shown that Bacillus strains in the diet can increase disease resistance 301 by stimulating cellular and humoral immune functions, including phagocytic activity, 302 lysozyme activity and peroxidase activity [53-55]. Peroxidase may play critical role in 303 increasing host defenses against pathogens . Lysozyme is an indispensable 304 bactericidal cationic enzyme that hydrolyzes the peptidoglycan layers of bacterial cell 305 walls 306 N-acetylglucosamine and is increased in the sera of fish during infection with various 307 variable microorganisms [56, 57]. It activates phagocytosis and the complement 308 system and is an important factor in the natural immune response in fish against AC C EP 300 by splitting glycosidic bonds between N-acetylmuramic acid and ACCEPTED MANUSCRIPT pathogenic infections . In our data, lysozyme activity of skin mucus and serum 310 and peroxidase activity of serum are greatly enhanced, when GB-9 is added at 4×107 311 CFU/g. Phagocytosis plays an important role in the host-defense mechanisms and 312 contributes to innate immunity [44, 59, 60]. During phagocytosis, phagocytic cells 313 produce a high degree microbiocidal reactive oxygen specificity (ROS) and attack 314 invading pathogens . This ROS-producing activity is known as respiratory burst 315 activity and is commonly used as an index to assess the defensive ability of defense 316 pathways-causes . Our data showed respiratory burst activity and phagocytosis 317 activity of hybrid sturgeon leukocytes, which fed with GB-9 increased significantly 318 compared with that of control. M AN U SC RI PT 309 Al Marzooqi and Leeson reported that supplementing lipase can improve the fat 320 digestion of broilers . Fat digestion can produce fatty acids in the intestines of 321 mammals and fish . It has been reported that dietary medium-chain fatty acids 322 may affect bacterial metabolites and thus affect the intestinal health of weaned piglets 323 .Therefore, exogenous lipase supplementation may affect the fish's gut by raising 324 fat digestion to improve health levels, which requires further investigation [66, 67]. In 325 this study, the addition of exogenous lipase YLL2 to diets can significantly improve 326 the growth performance of fish, increase the content of DHA and EPA, help maintain 327 the physical barrier of the intestine and enhance the immune ability, including 328 lysozyme activity of skin and serum, the peroxidase and alternative complement 329 pathway activity of serum, the ROS-producing activity and phagocytosis activity of 330 leukocytes. AC C EP TE D 319 ACCEPTED MANUSCRIPT Taken together, it has been proved that supplementation of probiotics and natural 332 substances combined may exhibit higher up-regulating effects on Nile tilapia immune 333 parameters and growth performance than that of separately supplementation . In 334 our study, supplementation of GB-9 and YLL2 together improved the growth 335 performance and promoted the immune stimulation of hybrid sturgeon significantly, 336 especially enhanced the lysozyme activity of skin and serum, phagocytosis activity of 337 leukocytes and alternative complement pathway activity. This might be due to the 338 DHA and EPA hydrolyzed by YYL2 improved the poor establishment of the GB-9 in 339 the gastrointestinal tract of hybrid sturgeon and might have promoted the growth of 340 GB-9 . M AN U SC RI PT 331 Regarding to the survival rate, group 4 showed the maximum survival rate 342 compared with other experimental groups. The survival rate exhibited some relevance 343 with the ROS level, phagocytosis activity and skin mucus lysozyme activity. It could 344 be inferred that the higher level of skin mucus lysozyme activity could help hybrid 345 sturgeon eliminate the pathogenic bacterium from external environment, the higher 346 level of phagocytosis activity and ROS could promote the leukocyte migration, help 347 eliminate local invading bacteria in hybrid sturgeon . EP AC C 348 TE D 341 In addition, the alternative complement pathway comprises plasma proteins, 349 which represent the major humoral defense mechanism against infection. In teleost 350 fish, the complement system plays a key role in the adaptive immune response . 351 Panigrahi et al. showed that serum complement C3 increased significantly with the 352 increase of B. licheniformis . In our study, the fact the ACH50 increased after fed ACCEPTED MANUSCRIPT with lipase, supported previous reports. Sun et al. proved B. pumilusor B. Diets 354 containing C. clausii can activate the complement system during the initial feeding 355 . However, after 12-weeks days of feeding, the levels of C3 in the probiotic group 356 did not differ from those in the control group. In our study, CH50 level showed no 357 significant difference compared with that of control in skin mucus, while 358 downregulated in serum compared with that of control. Our data supported previous 359 reports that persistent activation of complement may lead to host adverse reactions 360 and immunosuppression [55, 72]. However, the impact of probiotics on the 361 complement system and its related immune functions remains to be further 362 investigated. M AN U SC RI PT 353 In short, current research showed that dietary consumption of YLL2 and GB-9 can 364 significantly increase the growth performance, protein concentration and serum 365 immune response of hybrid sturgeon. This study may constitute a new strategy for 366 fish feeding supplementation of exogenous lipase and Bacillus. However, the precise 367 mechanism of how YLL2 and GB-9 stimulate growth and immune system of Hybrid 368 sturgeon is not clarified as yet and further research on this aspect is needed. 370 371 EP AC C 369 TE D 363 Acknowledgements The authors wish to thank Zhejiang Provincial Natural Science Foundation of 372 China (No. LQ18B060005); Zhejiang Sci-Tech University Foundation 373 (No.11612932611702); Special Fund for Agro-scientific Research in the Public 374 Interest of Zhejiang province (LGN18C190011); Project for Jiaozhou Excellent ACCEPTED MANUSCRIPT Innovation Team (18-CX-1) 376 Reference 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 1. Rajeev R P, Zhu X, Lei W, et al. 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The complement system. Curr Biol. 1998 8:R259-61. AC C 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 ACCEPTED MANUSCRIPT 593 Table 1 594 The formulation of experimental groups (g/kg diet). mealc Wheat flourd Soy lecithin Mineral c Premixe GB-9 YLL2 460 460 460 460 200 200 200 200 260 260 260 50 50 50 30 30 30 / 5.0 / / extruded pellets. b 598 c 599 d 600 e 30 / 5.0 4.0 4.0 YiHai Kerry Investment Company Limited, Shandong, China; Guchan Group, Beijing, China; Beijing Enhalor Biotech Ltd. Co. Beijing, China Table 2 603 Proximate compositions of experimental groups (g/kg diet). EP 602 Proximate composition (% dry matter) Group 1 Group 2 Group 3 Group 4 Dry matter (% diet) Crude protein 92.7 42.0 93.6 42.4 95.1 42.5 97.8 42.7 Crude lipid Ash Lys 13.5 63.5 1.60 13.6 64.6 1.60 13.5 63.7 1.60 13.6 64.8 1.60 AC C 606 50 Fishmeal were supplied by FuShen Fish meal Co., Qingdao, China 601 605 260 All diets were produced at Yunnan Amur Sturgeon Group Co., Ltd. China, as 597 604 Group 4 M AN U 596 a Group 3 TE D 595 Group 2 RI PT Fish meal Soybean Group 1 b SC Ingredientsa ACCEPTED MANUSCRIPT Table 3 608 Effect of dietary supplementation on growth performance and feed utilization of 609 juvenile hybrid sturgeon, A. schrenkii ♂ and A. baeri ♀, fed experimental diets for 12 610 weeks. RI PT 607 Diet 1 Diet 2 Diet 3 Diet 4 IW (g) 5.0 ± 0.05 5.0 ± 0.07 5.0 ± 0.08 5.0 ± 0.03 FW (g) 57.5 ± 1.5c 61.3 ± 1.7b 75.2 ± 1.6b 80.9 ± 1.8a WG (g) 52.5 ± 1.4c 56.3 ± 1.8b 70.2 ± 2.1b 75.9 ± 1.5a SR (%) 90 96 SGR (%) 2.91 ± 0.02c 2.98 ± 0.03b FCR 1.41 ± 0.02a 1.35 ± 0.00b 1.99 β EPA(%) 1.11 93 97 3.22 ± 0.03b 3.31± 0.02a 1.26 ± 0.00b 1.23 ± 0.02b 2.35 5.95 6.22 1.51 3.01 3.37 M AN U DHA(%)α SC Index IW=initial weigh fish-1; FW=Final weight fish-1; SGR =Specific growth rate fish-1; 612 FCR = Feed conversion ratio; SR = Survival rate; 613 Data are mean ± SEM. Means not bearing the same superscript letters in the same row 614 are significantly different (p < 0.05). 615 DHA (%)α means the concentration of DHA in hybrid sturgeon muscle; EPA(%)β 616 means the concentration of EPA in hybrid sturgeon muscle. 617 EP TE D 611 Table.4 619 The protein concentration of skin mucus and serum 620 AC C 618 Groups Protein concentration of skin mucus (mg/mL) Protein concentration of serum (mg/mL) Control group 4.91 15.90 Group 1 Group 2 Group 3 4.65 4.77 4.51 17.12 21.25 23.66 ACCEPTED MANUSCRIPT Figure legends 622 Fig. 1. Skin mucus lysozyme activity of hybrid sturgeon after 12 weeks of feeding 623 trial fed on different diets (mean ±S.E., n=6): Control Group (0-control), Group 1 (5.0 624 g/kg of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). 625 The bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 626 one-way ANOVA). RI PT 621 SC 627 Fig. 2. Skin mucus peroxidase activity of hybrid sturgeon after 12 weeks of feeding 629 trial fed different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 630 g/kg of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). 631 The bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 632 one-way ANOVA). TE D M AN U 628 633 Fig. 3. Skin mucus CH50 level of hybrid sturgeon after 12 weeks of feeding trial fed 635 different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of 636 GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The 637 bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 638 one-way ANOVA). AC C 639 EP 634 640 Fig. 4. Phagocytosis rate of hybrid sturgeon leukocyte after 12 weeks of feeding trial 641 fed different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg 642 of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The ACCEPTED MANUSCRIPT 643 bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 644 one-way ANOVA). 645 Fig. 5. ROS levels of hybrid sturgeon leukocyte after 12 weeks of feeding trial fed 647 different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of 648 GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The 649 bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 650 one-way ANOVA). M AN U SC RI PT 646 651 Fig. 6. Peroxidase activity of hybrid sturgeon serum after 12 weeks of feeding trial 653 fed different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg 654 of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The 655 bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 656 one-way ANOVA). EP 657 TE D 652 Fig. 7. Lysozyme activity of hybrid sturgeon serum after 12 weeks of feeding trial fed 659 different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of 660 GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The 661 bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 662 one-way ANOVA). AC C 658 663 664 Fig. 8. Alternative complement pathway activity of hybrid sturgeon serum after 12 ACCEPTED MANUSCRIPT weeks of feeding trial fed different diets (mean ± S.E., n=6): Control Group 666 (0-control), Group 1 (5.0 g/kg of GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg 667 of GB-9+4.0 g/kg YLL2). The bars with different superscripts (a, b, and c) are 668 significantly different (p < 0.05, one-way ANOVA). RI PT 665 669 Fig. 9. CH50 level of hybrid sturgeon serum after 12 weeks of feeding trial fed 671 different diets (mean ± S.E., n=6): Control Group (0-control), Group 1 (5.0 g/kg of 672 GB-9), Group 2 (4.0 g/kg YLL2), Group 3 (5.0 g/kg of GB-9+4.0 g/kg YLL2). The 673 bars with different superscripts (a, b, and c) are significantly different (p < 0.05, 674 one-way ANOVA). M AN U SC 670 AC C EP TE D 675 676 677 AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT 1. The supplementations of GB-9 and YLL2 resulted in a significant increase in final weight, DHA and EPA concentration of Hybrid sturgeon. 2. The skin mucus lysozyme activity, leukocytes phagocytosis activity and reactive RI PT oxygen species level were significantly higher in supplemented group. 3. The serum alternative complement pathway activity, peroxidase and lysozyme activity were significantly higher in supplemented group. SC 4. The combination of exogenous lipase and Bacillus could be considered as AC C EP TE D M AN U potential feed-additives for aquaculture farmed fish.