Early Life Intervention Using Probiotic Clostridium butyricum Improves Intestinal Development, Immune Response, and Gut Microbiota in Large Yellow Croaker (Larimichthys crocea) Larvae

Marine fish larvae are vulnerable during the early life period. The early intervention using probiotics may be a promising method to improve growth of fish larvae. In this study, a 30-day feeding trial was conducted to evaluate the effects of early life intervention using probiotic Clostridium butyricum (CB) on growth performance, intestinal development, immune response and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. Four isonitrogenous and isolipidic diets were formulated with the supplementation of four different levels of CB (5 × 109 CFU g−1), 0.00% (Control), 0.10% (CB1), 0.20% (CB2), and 0.40% (CB3). Results showed that larvae fed diets with CB had significant higher final length than the control group. Meanwhile, larvae fed the diet with 0.10% CB had significant higher final weight and specific growth rate (SGR) than the control group. However, no significant difference in survival rate was observed among dietary treatments. CB supplementation significantly increased the height of intestinal villus and the length of intestinal enterocyte. Similarly, CB supplementation significantly increased the expression of tight zonula occludens-2 (zo-2) and ornithine decarboxylase (odc) than the control group. Larvae fed the diet with 0.20% CB had significant higher lipase and leucine-aminopeptidase (LAP) activity than the control group. Moreover, CB supplementation significantly improved immune enzyme activities than the control group. Sequencing of bacterial 16S rRNA V4-5 region indicated that dietary CB altered intestinal microbiota profile and decreased intestinal microbial diversities of larvae. CB supplementation could effectively increase the abundance of CB, and decrease the abundance of some potential pathogenic bacteria in larval gut. These results revealed that early life intervention using 0.10–0.20% CB could promote growth of large yellow croaker larvae probably through promoting intestinal development, improving immune enzyme activities and modulating gut microbiota.


INTRODUCTION
Marine fish gut tract is not fully developed during the early stage of life (1). Fish larvae undergo major morphological, cellular and functional changes of gut tract during the first stage of life (2). The successful development of gut tract is important for fish larvae to digest and absorb food (3). Furthermore, gut microbiota is not fully assembled in marine fish digestive tract during the early stage of life (4). The microbes that reside in the gut tract have profound influence on host immune system priming, protection and development, as well as nutrient supplementation to the host (5,6). The rate and trajectory of acquisition of gut microbiota have a considerable impact on later health outcomes (7).
In recent years, many studies have indicated that probiotic treatment in the early life is necessary for digestive physiology functions, gastrointestinal tract immune and microbiota development (8,9). Clostridium butyricum (CB) is a grampositive, obligate anaerobic and endospore-forming probiotic, which is part of the normal gut bacteria for both mammals and aquatic animals (10,11). CB could improve gastrointestinal function, which has been used for the clinical treatment of newborn animals (12)(13)(14). In recent years, research studies in regard to CB were carried out in aquatic species, including tilapia (15), shrimp (16), and prawn (17). However, to our best knowledge, the role of dietary CB in marine fish larvae is still uncertain.
Large yellow croaker (Larimichthys crocea) is an economic important marine fish species in south China (18). Similarly to most marine fish species, large yellow croaker larvae had an undifferentiated and undeveloped gut and accessory organs (19). The production of large yellow croaker is often hampered by high mortality in larval stage (20). Therefore, the present study was conducted to investigate the effects of early life intervention using CB on the growth performance, immune response, intestinal development and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. By using a marine fish larvae model, the present study might provide novel insights into probiotic treatment in early life intervention.

Animal Ethics
All procedures of this study were conducted on fish, and animal care was performed strictly followed the Management Rule of Laboratory Animals (Chinese Order No. 676 of the State Council, revised 1 March 2017).

Diet Formulation
Large yellow croaker larvae were fed with four isonitrogenous (52% crude protein) and isolipidic (19% crude lipid) diets, which were supplemented with different levels of CB (0.00% as the control group, 0.10% as CB1 group, 0.20% as CB2 group, and 0.40% as CB3 group) (Supplementary Table 1). The additive CB was purchased from Vland Biotech Co., Ltd., China, containing cells at 5 × 10 9 colony forming units (CFU) g −1 . Micro-diet (MD) was manufactured by micro-bonding technology (20). The particle size of the formulated diets ranged from 250 to 380 mm for fish larvae between 14 and 24 days after hatch (DAH) and 380-500 mm for fish larvae between 25 and 47 DAH.

Experimental Procedure
The objective animal of this study were purchased from the Ningde Fufa Fishery Company Limited, Ningde, China, and were reared at the State Key Laboratory of Large Yellow Croaker, Ningde, China. All fish larvae in the hatchery were fed with rotifers, Brachionus plicatilis (0.5-2.0 × 10 4 individual L −1 ) from 3 to 7 DAH, Artemia nauplii (1.0-1.5 × 10 3 individual L −1 ) from 6 to 11 DAH, and Calanus sinicus from 10 to 14 DAH, and then the larvae were weaned onto experimental diets. Ten thousand larvae (17 DAH) were randomly allocated in 12 blue plastic tanks (water volume 1,000 L) to conduct the experiment. Different experimental diets were randomly allocated to triplicate groups of larvae. During the rearing period, water temperature, pH, and salinity were strictly controlled during the rearing period, ranging from 22 to 24°C, 7.9 to 8.3, 21 to 24‰, respectively. The water volume was renewed about 60-150% daily. Larvae were reared under 16h light:8h dark dial cycle photoperiod. All the larvae were manually fed to satiation with the experimental diets seven times daily (6:00, 8:00, 11:00, 14:00, 17:00, 20:00, and 22:00) during the whole experiment period (from 17 DAH to 47 DAH).

Sample and Dissection
Before the experiment was conducted, initial body weight and body length of 150 randomly collected larvae (17 DAH) were measured. At the end of the experiment, larvae were fasted for 24 h before sampling. Survival rate was calculated by comparing the remaining larvae in each tank to the initial number. Final body weight and final body length of 1,000 larvae randomly collected from each tank were measured. Thirty larvae were collected from each tank and preserved in 4% paraformaldehyde for 24 h and transferred to 75% alcohol for hematoxylin and eosin (H&E) staining. Ninety larvae from each tank were dissected on ice to obtain visceral mass, which contains a crude mixture of pancreas, liver, heart, spleen and intestine, for immune enzyme activity assay. Pancreatic and intestinal segments (PS and IS) of sixty larvae were separated on a glass plate maintained at 0°C under a dissecting microscope for digestive enzyme activity assay (20). The whole intestine of sixty larvae from each tank was separated aseptically under a dissecting microscope for the analysis of intestinal microflora and gene expression assays.

Digestive Enzyme Activities Assay
Four stages (22,27,37, and 47 DAH) of large yellow croaker larvae were chosen to reflect digestive enzyme activities during different growth stages (1). PS and IS (0.1-0.2 g) of 22, 27, 37, and 47 DAH larvae were weighed and homogenized in 2 ml 0°C normal saline, followed by centrifuging (2,500g, 10 min), then the supernatant was collected for further assay. Purified brush border membranes (BBMs) from homogenate of the intestinal segment were obtained according to a method described by published paper (21). Activities of leucine-aminopeptidase (LAP) were assayed in BBM according to published paper (22). Trypsin activity was assayed following published paper (23). Amylase activity was assayed with a-Amylase Assay Kit (Nanjing Jiancheng Bio-Engineering Institute, China). Lipase activity was assayed with Lipase Assay Kit (Nanjing Jiancheng Bio-Engineering Institute, China). Protein was determined by the Total protein quantitative assay kit (Nanjing Jiancheng Bio-Engineering Institute, China).

Immune Enzyme Activities Assay
The visceral mass was weighed and homogenized in phosphatebuffered saline. The proportion of tissue (g) and saline (ml) was 1:9. The homogenate of larvae was then centrifuged (4,000g, 15 min), and the supernatant was collected for the following assays. Immune enzymes kits (Nanjing Jiancheng Bio-Engineering Institute, China) were used to evaluate the activity of acid phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT), lysozyme (LZM), and superoxide dismutase (SOD) in the collected visceral mass.

Intestinal Histology Analysis
The intestinal micromorphology was determined based on the method described by published paper (19). Briefly, the whole larvae were washed and dehydrated with gradient alcohol, and then paraffin-embedded, sectioned, and stained with hematoxylin and eosin.

Calculations and Statistical Analysis
The growth parameters were calculated as follows: Where N t is final number of larvae in each tank and N i is initial number of larvae in each tank at the beginning of the experiment; W t and W i are the final and initial body weights, respectively; d is the experimental period in days.
Statistical analysis was performed in SPSS 16.0 (SPSS Inc., USA). Data from each treatment were subjected to one-way analysis of variance (ANOVA) followed by Tukey's multiplerange test. For statistically significant differences, P <0.05 was applied. Results were presented as mean ± S.E. (Standard error of means).

Early Life Intervention With CB Improved Growth of Fish Larvae
No significant difference in survival rate was observed among dietary treatments (P > 0.05) ( Table 1), whereas the highest value was recorded in larvae fed diets with 0.10% CB, followed by 0.20% CB, 0.40% CB and 0.00% CB, respectively. With the increase of CB level in diets, the growth performance firstly increased and then decreased. CB supplementation significantly improved final body length than the control group (P < 0.05) ( Table 1). Meanwhile, larvae fed the diet with 0.10% CB showed significant higher final body weight and SGR than the control group (P < 0.05) ( Table 1).

Early Life Intervention With CB Improved Gut Tract Development
Early Life Intervention With CB Improved the Intestinal Morphology CB supplementation effectively improved the intestinal morphology of large yellow croaker larvae (Supplementary Figure S1). Larvae fed diets with CB had significant higher villus height and enterocyte height than the control group (P < 0.05) ( Table 2). Similarly, significant higher muscular thickness was observed in fish fed the diet with 0.10% CB than the control group (P < 0.05) ( Table 2).

Early Life Intervention With CB Increased Gene Expression of zo-2 and odc
The markers for epithelial proliferation and differentiation were selected (odc, pcna, zo-1, zo-2 and occludin) to compare intestinal development among larvae fed diets with different levels of CB. CB supplementation significantly increased mRNA expression of zo-2 and odc than the control group (P < 0.05) ( Figure 1). However, no significant difference in mRNA expression of zo-1, occludin, and pcna was observed among dietary treatments (P > 0.05) (Figure 1).

Early Life Intervention With CB Improved Lipase and LAP Activities
No significant difference in the activity of trypsin and amylase in IS and PS was observed among dietary treatments (P > 0.05) ( Table 3). Compared with the control group, 0.20% CB supplementation not only increased lipase activity of 37 DAH larvae in PS and IS, but also increased lipase activity of 47 DAH larvae in IS (P < 0.05) ( Table 3). Similarly, 0.20% CB supplementation significantly increased LAP activity of 47 DAH larvae in BBM than the control group (P < 0.05) ( Table 3).   With the increase of CB level in diets, immune enzyme activities firstly increased and then decreased. CB supplementation significantly improved LZM activity than the control group (P < 0.05) ( Table 4). Furthermore, 0.10 and 0.20% CB supplementation significantly increased AKP and CAT activities than the control group (P < 0.05) ( Table 4). 0.10% CB supplementation significantly increased ACP activity than the control group (P < 0.05) ( Table 4). However, no significant difference in the activity of SOD was observed among dietary treatments (P > 0.05) ( Table 4). Similarly, no significant difference in mRNA expression of inflammatory factors (il-1b, il-6, il-8, ifng and cox-2) was observed among dietary treatments (P > 0.05) (Supplementary Figure 2). The units of enzyme activity is U/g protein. 3 The unit of enzyme activity is U/mg protein. 4 PS, pancreatic segments; IS, intestinal segments; BBM, brush border membrane. 5 LAP, leucine-aminopeptidase. The unit of enzyme activity is U/g protein. 3 The unit of enzyme activity is U/mg protein. 4 ACP, acid phosphatase; AKP, alkaline phosphatase; CAT, catalase; LZM, lysozyme; SOD, superoxide dismutase.  Figure 3).
A Venn diagram showed that 106 OTUs were shared by group Control, CB1, CB2 and CB3, and the number of unique OTUs in group Control, CB1, CB2 and CB3 was 312, 104, 80 and 121, respectively (Figure 2A). The alpha diversity index results indicated that the inclusion of dietary CB led to lower richness of observed species number in large yellow croaker larvae gut microbiota (P < 0.05) ( Table 5). Alpha diversity including phylogenetic diversity whole tree (PD whole tree) index and abundance-based coverage estimator (ACE) index were markedly decreased in the gut microbiota of larvae in CB groups (P < 0.05) ( Table 5). Meanwhile, larvae fed the diet with 0.20% CB showed significant lower Shannon index in the gut microbiota (P < 0.05) ( Table 5). To analyze the extent of similarities in microbial communities, PCA was conducted to determine the beta diversity ( Figure 2B). The PCA results showed that the intestine bacterial in the control group and CB groups were separated ( Figure 2B). Similarly, the gut microbiota structure from groups CB1, CB2, and CB3 were similar and clustered within one higher branch, whereas the control group was distinct from the CB groups and formed another branch ( Figure 2C).

Component Changes of Gut Microbiota in Response to CB Supplementation
At the phylum level, Proteobacteria, Firmicutes and Actinobacteria were detected as predominant bacterial phyla in the intestine of large yellow croaker from all groups ( Figure 3A). At the genus level, Leisingera, Pseudomonas and unidentified Clostridiales were detected as predominant bacterial genera in the intestine of large yellow croaker larvae from all groups ( Figure 3B). At the species level, Pseudomonas psychrotolerans, Clostridium butyricum, Alcaligenes faecalis composed dominant species of larval gut microbiota communities ( Figure 3C).
To compare the differences in gut microbial community composition between the control group and CB groups, LEfSe  analysis was performed (Figure 4). The results revealed that there were significant differences in taxonomic distribution of intestinal microbiota communities between the control group and CB groups. CB supplementation significantly increased the relative abundance of class Clostridia, order Closrtidiales, family unidentified Clostridiales, genus unidentified Clostridiales and specie Clostridium butyricum, while decreased the relative abundance of class Bacilli, family Moraxellaceae and genus Acinetobacter (P < 0.05) (Figures 4A-F). Furthermore, dietary CB at 0.10% level significantly increased the relative abundance of phylum Firmicutes, order Vibrionales, family Vibrionaceae, genus Aliivibrio and specie Aliivibrio fischeri, but significantly decreased the relative abundance of phylum Actinobacteria, class unidentified Actinobacteria, orders Bacillales and Pseudomonadales (P < 0.05) ( Figures 4A, D). Dietary CB at 0.20% level significantly decreased the relative abundance of phylum Actinobacteria, class unidentified Actinobacteria, orders Bacillales, Lactobacillales and unidentified Gammaproteobacteria, family Burkholderiaceae, genus Alcaligenes and specie Alcaligenes faecalis (P < 0.05) (Figures 4B, E). Dietary CB at 0.40% level significantly increased the relative abundance of genera Ralstonia and Nautella and specie Nautella italica (P < 0.05) ( Figures 4C, F).

DISCUSSION
There has been increasing evidence suggesting the maturation of intestinal tract, immune system and gut microbiota in the early life is significant for growth and development of marine fish larvae (34,35). Early infancy is considered as an ideal window for gut microbial colonization (36,37). Therefore, early intervention using probiotics during the critical period may be a promising method to enhance growth of fish larvae. In the present study, appropriate CB supplementation could enhance growth performance of large yellow croaker larvae, which were consistent with some previous findings in Miichthys miiuy (38), Penaeus monodon (39) and Litopenaeus vannamei (40). The normal development of intestinal tract during the early life has a considerable impact on later health outcomes (1). The maturation process of gut tract can be enhanced, stopped or delayed depending on diet composition (19). In this study, CB supplementation could enlarge the villus height, enterocyte height, and muscular thickness of larval gut tract, which was consisted with some previous studies on tilapia (15), pacific white shrimp (40) and weaned piglets (41). Furthermore, the expression of zo-2 and odc was higher in larvae fed diets with CB than the control group. Odc plays important roles in the proliferation, migration, and differentiation of cells (42); zo-2 participates in tight junction structural integrity by binding to the actin cytoskeleton (43). These results suggested that CB could promote intestinal development of larvae by accelerating the proliferation of intestinal epithelial and maintaining the integrity of the intestinal barrier.
Moreover, digestive enzyme activities could be used as an indicator of development rate, food acceptance and digestive capacity (1). Previous research has indicated that CB could improve digestive function through producing a range of supplemental digestive enzymes (10,39). In this study, 0.20% CB supplementation not only increased lipase in PS and IS of 37 DAH larvae, but also increased lipase in IS of 47 DAH larvae, which were consistent with the previous studies in broiler chickens (44), piglets (41), Macrobrachium rosenbergii (17) and Litopenaeus vannamei (40). Furthermore, the onset of BBM enzymes in intestine was considered as crucial in the maturation process of the digestive function (45). The present study found that 0.20% CB supplementation could improve activities of LAP in BBM, which demonstrated that appropriate dietary CB could promote the transition of larval digestive functions to adult mode (1).
The immune system during the early life is immature, which has been considered as a major restriction to healthy growth of fish larvae (46,47). Probiotics can act as non-specific immune factors and have a positive effect on host immune system (35,48) CB supplementation significantly affected nearly all immune enzyme activities (ACP, AKP, CAT and LZM), indicating CB supplementation could improve immune status of larvae. Intriguingly, no significant differences in the survival rate and mRNA expression of inflammatory factors were observed among dietary treatments. Zhao et al. (49) found CB could decrease cytokine levels (ifn-g, il-1b, il-8, and tnf-a) in intestinal tissues of chickens with Salmonella infection, but no significant differences in cytokine levels were observed between CB treatment group and normal diet group (49). Above all, combining the results of the present study and current research, we speculated dietary CB could effectively decrease inflammatory factor level to protect host from pathogens.
Gut microbiota plays a crucial role in health and well-being of marine fish starting from hatch (4,8). The current study indicated that modulation of intestine microbiota in large yellow croaker towards potentially more beneficial microbial community could be achieved through CB supplementation. The present study showed CB had a strong effect on the overall structure of intestinal microbiota in larvae. Similarly, compared with the control group, observed species number, PD whole tree index and ACE index were lower in the gut of larvae fed diets with CB supplementation. These results indicated that CB supplementation could regulate the structure of gut microbiota and decrease intestinal microbial diversity and richness, representing the positive effect of CB on gut microbiota development during the early life of larvae (35,50).
Moreover, early life intervention using CB supplementation improved the composition of gut microbiota. CB supplementation at 0.10 and 0.20% levels significantly decreased the relative abundance of phylum Actinobacteria, indicating 0.10 and 0.20% CB may have a more profound effect on the intestinal community of larvae than 0.40% CB. Intriguingly, the present study found CB supplementation could increase the relative abundance of CB, which indicated larval intestine could be colonized by CB. Previous research has indicated that CB decreased the number of pathogens in the host gut tract by creating an oxygen-free environment, producing prebiotics and generating butyric acid (10). The present study found that CB significantly downregulated the relative abundance of genus Acinetobacter, and CB at 0.20% significantly decreased the relative abundance of genus Alcaligenes and specie Alcaligenes faecalis. Acinetobacter was associated with bacteremia, pulmonary infections, meningitis, diarrhea, and nosocomial infections (51). And Alcaligenes faecalis was an opportunistic pathogen, which could be resistant to common antibiotics and closely related with nosocomial infections (52). These results revealed that CB could colonize in the fish larval gut tract, and play an inhibitory effect on opportunistic pathogens.

CONCLUSION
This study demonstrated that early life intervention using dietary CB could improve growth performance of large yellow croaker larvae probably through promoting intestinal development, improving immune enzyme activities, and modulating gut microbiota. The optimal CB (containing cells at 5 × 10 9 CFU g −1 ) supplementation dosage is 0.10-0.20%. Probiotic treatment in early life intervention may be a promising method to promote the maturation of the intestinal tract, immune system, and gut microbiota.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI BioProject, accession no: PRJNA686247.

ETHICS STATEMENT
The animal study was reviewed and approved by the Committee on the Ethics of Animal Experiments of Ocean University of China.

AUTHOR CONTRIBUTIONS
KM, QA, and ZY designed the research. ZY, QL, and YH conducted the research. SG, WH, CY, and ZY analyzed the data. ZY wrote the manuscript. QA and YG provided language help. All authors contributed to the article and approved the submitted version.