Comparative Analysis of Intestinal Characteristics of Largemouth Bass (Micropterus salmoides) and Intestinal Flora with Different Growth Rates

To explore the causes of different growth rates among juvenile populations of largemouth bass, in the present study, a batch of largemouth bass juveniles reared under the same conditions were divided into a fast-growing group and a slow-growing group. We used histological, enzymatic and molecular biology methods to analyze and determine their histomorphological changes, digestive enzyme activity and intestinal floral composition. The fast-growing group had a significantly (p ≤ 0.01) greater intestinal fold height and muscle thickness than the slow-growing group. Lipase activity was significantly (p ≤ 0.01) higher in the fast-growing group than in the slow-growing group. Intestinal microbial analysis showed that the relative abundance of Actinobacteria and Halomonas in the fast-growing group was higher than that in the slow-growing group. This research shows that the differentiation of growth rates in juvenile largemouth bass populations is closely related to intestinal fold status, lipase activity, and intestinal flora.


Introduction
In fishes, the same group of young individuals, exhibits obvious growth rate differentiation after a period of growth, with some individuals outgrowing other individuals one-to two-fold or more. This phenomenon of individual growth rate differentiation in fish has gradually received attention from scholars. Studies by Dobrochna Adamek et al. on Atlantic sturgeon (Acipenser oxyrinchus) showed that individuals belonging to the same population had different growth performances and a large number of differences in body length and weight [1]. Faster-growing individuals are more likely to survive to sexual maturity than slower-growing individuals. Individuals with slower growth usually show lower environmental adaptability and disease resistance [2]. Alm's study on brown trout (Salmo trutta) showed that fast-growing brown trout reached sexual maturity earlier than slow-growing trout [3].
The growth rate of fish of the same species is regulated by exogenous factors such as environmental conditions, diet and culture density [4]. However, under the same culture conditions, the growth rate is mostly regulated by endogenous factors such as the endocrine system and nervous system [5]. Within a given fish species, there are many factors that have an impact on growth, such as feed type, food size and intake and nutrient absorption capacity [6]. Among these factors, nutrient uptake capacity is particularly important for the survival and growth of fish. The intestine plays an important role in vertebrate metabolism, nutrient absorption and immune function [7]. The intestine of fish can generally be divided into three parts: the foregut, midgut and hindgut. The foregut is primarily associated

Materials
Largemouth bass juveniles for the experiment were collected from a farm in Xifeng County (Guizhou, China). A circular pond with a diameter of 14 m was stocked with 20,000 juvenile bass with the following specifications: body length, 3.941 ± 0.236 mm; weight, 0.877 ± 0.052 g. After 92 days of breeding, the juvenile largemouth bass showed appropriate growth differentiation. The same puffed pellet feed was provided to all individuals during the breeding period (Zhuhai Hailong Biotechnology Co., Ltd., Zhuhai, China; crude protein ≥ 49%, crude fat ≥ 6%, crude fiber ≤ 3.5%). Water quality was monitored throughout the culture process and maintained within safe levels for largemouth bass.

Sample Collection
Fish were fasted for 24 h prior to sampling. Nine fish each were randomly selected from the fast-and slow-growing groups (FG and SG groups, respectively) for testing. The specifications of the FG group were as follows: body length, 138.506 ± 5.713 mm; weight, 60.416 ± 4.629 g. The specifications of the SG fish were as follows: body length, 80.950 ± 5.5404 mm; weight, 10.566 ± 1.879 g. Following the sampling under sterile conditions, the intestinal contents of three fish in each group were mixed as a sample in a 2 mL sterile centrifuge tube, immediately frozen with liquid nitrogen and stored at −80 • C for the determination of the intestinal flora. Three replicates were sampled for each group. The intestines of three fish in each group were divided into anterior, middle and posterior segments and fixed with 4% formaldehyde for morphological observation. The remaining intact intestine was placed in a 2 mL sterile centrifuge tube and stored in a freezer at −80 • C for the determination of digestive enzyme activity.

Intestinal Histological Analysis
The foregut, midgut, and hindgut were kept in formaldehyde solution for 24 h, washed, and transferred to 70% ethanol solution. Fixed samples were graded and dehydrated to 100% in various percentages of standard ethanol, cleared in xylene, embedded in paraffin, sectioned at 4 µm intervals, stained with hematoxylin-eosin, dehydrated and cleared and then sealed in neutral gum. The intestinal fold height, fold width, crypt depth and muscular thickness were measured according to the method of Guoxia Wang (2019) for counting goblet cells per fold.

Enzyme Activity Analysis
The intestinal tissues of each group of samples were weighed accurately, mixed proportionally with saline, homogenized mechanically in an ice water bath, and centrifuged (2500 rpm, 10 min). The supernatant was collected and assayed for lipase, amylase and trypsin activities using a commercial kit (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China).
High-throughput sequencing of 16S DNA was performed on the Illumina MiSeq platform (https://docs.qiime2.org/2019.4/tutorials/ accessed on 11 February 2022) by Chengdu Nomi Metabolic Biotechnology Co (Chengdu, China). The data analysis methods were as follows: the primer fragment of the QIIME cutadapt trim-pair excision sequence was used to discard unmatched sequences; DADA2 was then called via QIIME for quality control, denoising, splicing, and chimera removal. The sequences were clustered into operational taxonomic units (amplicon sequence variants, ASVs) with 100% similarity. After completing the denoising of all libraries, the ASV feature sequences were merged, and singleton ASVs were removed. The processed data were used for subsequent diversity analysis. Species annotations were performed on the basis of the Greengenes database using scikit-learn classification.

Data Collection and Statistical Analysis
In the intestinal histological analysis, the images were obtained by biological microscopy (Nexcope), and morphological analysis was performed by ImageView software.
The digestive enzyme activity results were analyzed using SPSS 19.0 software (SPSS USA); p ≤ 0.05 indicated a significant difference, and p ≤ 0.01 indicated an extremely significant difference. Results are expressed as the mean ± standard error (SE). Figures were created by GraphPad Prism 8.0. In the intestinal flora analysis, alpha diversity metrics (Chao1, ACE, Shannon index, Simpson index and coverage) were calculated using QIIME2 software (2019.4). Beta diversity measures were calculated as described above. Other intestinal microbiota analyses were performed using R software (v3.2.0). The gut morphology of the FG and SG groups of juvenile largemouth bass is shown in Figure 1. Overall, the intestinal structure of juvenile largemouth bass in the FG and SG groups was relatively intact, with neat intestinal villi and smooth intestinal mucosa. In the foregut, the FG group had long, dense villi. By contrast, the SG group had sparse intestinal villi with shorter lengths.
In the FG group, the midgut villi were neatly arranged, dense, and intact, whereas in the SG group, the midgut villi were sparse and relatively short, with partial loss. The hindgut villi of the FG group were in good condition, complete, and more numerous and denser than those of the SG group, whereas the hindgut villi of the SG juveniles were sparse and very short.
were created by GraphPad Prism 8.0. In the intestinal flora analysis, alpha diversity metrics (Chao1, ACE, Shannon index, Simpson index and coverage) were calculated using QIIME2 software (2019.4). Beta diversity measures were calculated as described above. Other intestinal microbiota analyses were performed using R software (v3.2.0).

Morphological Characteristics of the Foregut, Midgut and Hindgut
The gut morphology of the FG and SG groups of juvenile largemouth bass is shown in Figure 1. Overall, the intestinal structure of juvenile largemouth bass in the FG and SG groups was relatively intact, with neat intestinal villi and smooth intestinal mucosa. In the foregut, the FG group had long, dense villi. By contrast, the SG group had sparse intestinal villi with shorter lengths.
In the FG group, the midgut villi were neatly arranged, dense, and intact, whereas in the SG group, the midgut villi were sparse and relatively short, with partial loss. The hindgut villi of the FG group were in good condition, complete, and more numerous and denser than those of the SG group, whereas the hindgut villi of the SG juveniles were sparse and very short. (a)

Determination of Morphological Indexes of the Foregut, Midgut and Hindgut
The fold height, fold width, crypt depth, goblet cell number and muscular thickness of each part of the intestine were measured, and the results are shown in Table 1. In the foregut, the fold lengths of the FG group and the SG group were 711.945 ± 143.179 μm and 243.097 ± 32.021 μm, respectively, with an extremely significant difference (p ≤ 0.01). The fold widths of the FG group and SG group were 121.795 ± 30.937 μm and 88.822 ± 16.607 μm, with a significant difference (p ≤ 0.01). The crypt depths of the FG group and SG group were 24.501 ± 4.173 μm and 25.216 ± 1.489 μm, with a significant difference (p ≤ 0.01). The numbers of goblet cells in the FG group and SG group were 10.230 ± 2.888 cells/100 μm and 7.846 ± 1.461 cells/100 μm, respectively, and there was no significant difference between groups (p ≥ 0.05). The muscle thicknesses of the FG group and SG group were 104.589 ± 14.392 μm and 70.897 ± 2.624 μm, respectively, with a significant difference (p ≤ 0.01).

Determination of Morphological Indexes of the Foregut, Midgut and Hindgut
The fold height, fold width, crypt depth, goblet cell number and muscular thickness of each part of the intestine were measured, and the results are shown in Table 1. In the foregut, the fold lengths of the FG group and the SG group were 711.945 ± 143.179 µm and 243.097 ± 32.021 µm, respectively, with an extremely significant difference (p ≤ 0.01). The fold widths of the FG group and SG group were 121.795 ± 30.937 µm and 88.822 ± 16.607 µm, with a significant difference (p ≤ 0.01). The crypt depths of the FG group and SG group were 24.501 ± 4.173 µm and 25.216 ± 1.489 µm, with a significant difference (p ≤ 0.01). The numbers of goblet cells in the FG group and SG group were 10.230 ± 2.888 cells/100 µm and 7.846 ± 1.461 cells/100 µm, respectively, and there was no significant difference between groups (p ≥ 0.05). The muscle thicknesses of the FG group and SG group were 104.589 ± 14.392 µm and 70.897 ± 2.624 µm, respectively, with a significant difference (p ≤ 0.01). In the midgut, the fold lengths of FG group and SG group were 479.917 ± 123.664 µm and 262.311 ± 44.298 µm, respectively, with an extremely significant difference (p ≤ 0.01). The fold widths of the FG group and SG group were 95.035 ± 23.205 µm and 99.601 ± 24.127 µm, respectively, with no significant difference between groups (p ≥ 0.05). The crypt depths of the FG group and SG group were 23.976 ± 4.426 µm and 23.170 ± 4.491 µm, respectively, with no significant difference between groups (p ≥ 0.05). The numbers of goblet cells in the FG group and SG group were 8.481 ± 2.190 cells/100 µm and 7.851 ± 2.582 cells/100 µm, respectively, and there was no significant difference between groups (p ≥ 0.05). The muscle thicknesses of the FG group and SG group were 115.418 ± 42.527 µm and 66.581 ± 17.173 µm, respectively, with an extremely significant difference (p ≤ 0.01).
In the hindgut, the fold lengths of the FG group and SG group were 422.190 ± 87.901 µm and 166.811 ± 36.217 µm, respectively, with an extremely significant difference (p ≤ 0.01). The fold widths of the FG group and SG group were 93.080 ± 20.008 µm and 175.475 ± 16.175 µm, respectively, with an extremely significant difference (p ≤ 0.01). The crypt depths of the FG group and SG group were 25.139 ± 4.500 µm and 40.821 ± 5.996 µm, respectively, with an extremely significant difference (p ≤ 0.01). The numbers of goblet cells in the FG group and SG group were 10.166 ± 3.714 cells/100 µm and 13.222 ± 2.414 cells/100 µm, respectively, with an extremely significant difference (p ≤ 0.01). The muscle thicknesses of the FG group and SG group were 189.381 ± 43.220 µm and 101.991 ± 14.217 µm, respectively, with an extremely significant difference (p ≤ 0.01). Different letters> "A"and "B" indicate significant differences between groups (p ≤ 0.01).

Intestinal Digestive Enzyme Activities of Juvenile Largemouth Bass with Different Growth Rates
The activities of amylase, lipase and trypsin were measured in the intestine of juvenile largemouth bass in the FG and SG groups, and the results are shown in Table 2. There were no significant differences in the activities of amylase and trypsin between the FG and SG groups, whereas the difference in lipase activity was highly significant, with 13.369 ± 0.224 U·g −1 lipase activity in the FG group and 9.756 ± 0.122 U·g −1 lipase activity in the SG group.    Coverage was used to estimate the completeness of sequencing in the alpha diversity analysis. The community coverage for each sample exceeded 99.93%, indicating that the probability of a collection sample not being sequenced was low. The coverage, Shannon index, Simpson index and Chao1 values for each sample from both groups of fish are shown in Figure 3 and were not significantly different between groups (p ≥ 0.05).
Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity was used for beta diversity analysis to detect relationships between microorganisms in different samples. The results showed that the intestinal floras of FG largemouth bass juveniles and SG largemouth bass juveniles were different (Figure 3b). The first principal component (PC1, x axis) and the second principal component (PC2, y axis) explained 43.30% and 24.80% of the variation, respectively, indicating that there were differences between the FG and SG groups, but according to the permutational analysis of variance (PERMANOVA) results, this difference was not significant (p = 0.104). Coverage was used to estimate the completeness of sequencing in the alpha diversity analysis. The community coverage for each sample exceeded 99.93%, indicating that the probability of a collection sample not being sequenced was low. The coverage, Shannon index, Simpson index and Chao1 values for each sample from both groups of fish are shown in Figure 3 and were not significantly different between groups (p ≥ 0.05).
Principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity was used for beta diversity analysis to detect relationships between microorganisms in different samples. The results showed that the intestinal floras of FG largemouth bass juveniles and SG largemouth bass juveniles were different (Figure 3b). The first principal component (PC1, x axis) and the second principal component (PC2, y axis) explained 43.30% and 24.80% of the variation, respectively, indicating that there were differences between the FG and SG groups, but according to the permutational analysis of variance (PERMANOVA) results, this difference was not significant (p = 0.104).

Assessment of Intestinal Floral Differences
The composition of the intestinal flora at the phylum level in the FG and SG groups is shown in Figure 4a. The dominant phyla in the FG and SG groups were Proteobacteria (48.781% vs. 43.929%, respectively) and Firmicutes (19.754% vs. 20.560%, respectively). The abundance of Fusobacteria in the SG group (12.433%) was significantly higher than that in the FG group. The relative abundance of Actinobacteria (9.079%) in the FG group was significantly higher than that in the SG group.
The composition of the intestinal flora in the FG and SG groups at the genus level is shown in Figure 4b. The dominant bacteria of largemouth bass in the FG group were Halomonas (14.182%) and unclassified Enterobacteriaceae (11.405%). The dominant bacteria in the SG group were unclassified Enterobacteriaceae (16.649%) and Clostridium (12.277%).
We used linear discriminant analysis (LDA) to analyze differences in the gut floral taxa of juvenile largemouth bass in the FG and SG groups ( Figure 5). The FG and SG groups were enriched with 77 and 52 bacterial taxa, respectively. Specifically, the FG group included 1 phylum, 5 classes, 9 orders, 23 families and 39 genera, with 10 bacterial taxa showing LDA scores >4. At the phylum and genus levels, the taxa included Actinobacteria, Halomonas and Bifidobacterium. The SG group included 3 phyla, 5 classes, 15 orders, 23 fami-   taxa of juvenile largemouth bass in the FG and SG groups ( Figure 5). The FG and SG groups were enriched with 77 and 52 bacterial taxa, respectively. Specifically, the FG group included 1 phylum, 5 classes, 9 orders, 23 families and 39 genera, with 10 bacterial taxa showing LDA scores >4. At the phylum and genus levels, the taxa included Actinobacteria, Halomonas and Bifidobacterium. The SG group included 3 phyla, 5 classes, 15 orders, 23 families and 15 genera, with 5 bacterial taxa showing LDA scores > 4, including Fusobacteria and Cetobacterium. (a)

Distinct Gut Characteristics of the Fast-Growing Group of Largemouth Bass
In our study, the fold length and muscle thickness of the FG group were significantly greater than those of the SG group in the foregut, midgut and hindgut. Increases in the length and width of the villi expand the absorption area, allowing for better absorption and utilization of nutrients, which in turn promotes fish growth [17]. The thickening of the muscular layer of the intestine accelerates peristalsis and thus enhances intestinal absorption. Li Yingying et al.'s study on the intestine of rhubarb fish with different growth rates showed that the intestinal muscle layer was thicker in the FG group than in the FG group [18]. This result is consistent with the results of our study. In studies of oysters (Ostrea gigas Thunb.), faster-growing oysters had faster feeding and absorption rates than slower-growing oysters [19], and the same results were obtained in a study of mussels (Mytilus edulis) [20]. In studies of juvenile redhead cichlids, those with better growth performance (Vieja melanura) also had greater gut fold heights [21]. Fold height and crypt depth reflect the digestive and absorptive capacities of the small intestine and the maturation rate of epithelial cells, respectively. The fold length/crypt depth ratio reflects the state of digestive and absorptive function of the intestine [22]. The fold length and crypt depth of the FG group were greater than those of the SG group, and the fold length/crypt depth ratio of the FG group was significantly greater than that of the SG group, indicating that the digestive absorption capacity of the FG group was significantly stronger than that of the SG group. The variation in intestinal morphology revealed a difference in intestinal structure between the FG group and FG group, with juvenile fish

Distinct Gut Characteristics of the Fast-Growing Group of Largemouth Bass
In our study, the fold length and muscle thickness of the FG group were significantly greater than those of the SG group in the foregut, midgut and hindgut. Increases in the length and width of the villi expand the absorption area, allowing for better absorption and utilization of nutrients, which in turn promotes fish growth [17]. The thickening of the muscular layer of the intestine accelerates peristalsis and thus enhances intestinal absorption. Li Yingying et al.'s study on the intestine of rhubarb fish with different growth rates showed that the intestinal muscle layer was thicker in the FG group than in the FG group [18]. This result is consistent with the results of our study. In studies of oysters (Ostrea gigas Thunb.), faster-growing oysters had faster feeding and absorption rates than slower-growing oysters [19], and the same results were obtained in a study of mussels (Mytilus edulis) [20]. In studies of juvenile redhead cichlids, those with better growth performance (Vieja melanura) also had greater gut fold heights [21]. Fold height and crypt depth reflect the digestive and absorptive capacities of the small intestine and the maturation rate of epithelial cells, respectively. The fold length/crypt depth ratio reflects the state of digestive and absorptive function of the intestine [22]. The fold length and crypt depth of the FG group were greater than those of the SG group, and the fold length/crypt depth ratio of the FG group was significantly greater than that of the SG group, indicating that the digestive absorption capacity of the FG group was significantly stronger than that of the SG group. The variation in intestinal morphology revealed a difference in intestinal structure between the FG group and FG group, with juvenile fish in the FG group having greater digestive and absorptive capacities. Therefore, the intestinal structural characteristics of largemouth bass juveniles in the FG group reveal that these juveniles have stronger digestion and absorption abilities, which is an important reason for their faster growth.
The digestive capacity of the fish gut can be demonstrated not only by tissue status but also by digestive enzyme activity. Largemouth bass juveniles in the FG group had higher digestive enzyme activities, which improved their ability to digest and absorb lipids and promoted lipid metabolism. This may be another reason for the rapid growth of these largemouth basses.

Differences in Intestinal Floral Levels between the FG Group and SG Group
The fish intestinal flora play an important role in aspects of fish growth, including nutrition, development, immunity and resistance to invasive pathogens [23]. Studies on the intestinal flora of largemouth bass juveniles showed that specific groups can be used to characterize the growth rate. Such groups associated with the FG group appear to be associated with disease-resistance immunity.
At the phylum level, the dominant microbes in the FG and SG groups were Proteobacteria and Firmicutes. In the fish digestive tract, Proteobacteria, Bacteroidetes, Actinobacteria, Fusobacteria and Fusobacterium are generally considered to be the dominant phyla [24]. Proteobacteria are Gram-negative bacteria that help maintain homeostasis in the anaerobic environment of the gut [25,26]. Firmicutes have been shown to participate in fermentation and regulate the absorption of intestinal dietary fats [27].
Notably, the relative abundance of Actinobacteria was significantly higher in the FG group than in the SG group. In general, Actinobacteria, as Gram-positive bacteria, can be used for the production of secondary metabolites, including probiotics and antibiotics, capable of inhibiting pathogenic activity [28,29]. A study of golden pomfret (Trachinotus ovatus) by X Tan et al., showed that the addition of dandelion extract to feed increased the abundance of actinomycetes in the fish intestine, thereby improving the immunity of the fish [30]. In the juvenile largemouth bass studied here, the FG group had more Actinobacteria in their gut than the SG group, indicating that the FG group of juvenile largemouth bass was more resistant to disease than the SG group. In addition, the relative abundance of Fusobacterium was significantly lower in the FG group than in the SG group. The presence of large numbers of Fusobacteria may contribute to colon cancer and is detrimental to the intestinal development of slow-growing individuals [31,32]. The FG group of juvenile largemouth bass had more immune-related flora, which may lead to faster growth.

Differences in Intestinal Flora at the Genus Level between the FG and SG Groups
At the genus level, the dominant flora in the gut of juvenile largemouth bass differed between the FG and SG groups. The relative abundances of Halomonas and Bifidobacterium were significantly higher in the FG group than in the SG group. Halomonas is a common intestinal bacterium that plays an important role in enhancing host immune activity and disease resistance [33]. Bifidobacterium is considered to be beneficialto the host [34]. Itami et al. [35] found that peptidoglycan production by Bifidobacterium thermophilum enhanced disease resistance in Japanese shrimp (kuruma shrimp). We speculated that the growth difference of largemouth bass juveniles may be due to the high abundance of bacteria related to disease resistance in the intestinal flora in the FG group, which confers higher immunity and is more conducive to growth.
Surprisingly, the relative abundance of Cetobacterium was also significantly higher in the gut of the SG group of juvenile fish. Cetobacterium can ferment carbohydrates, producing vitamin B12 for absorption and use by the host [36]. However, in a study of marbled eels (Anguilla marmorata), the opposite result was obtained [37]. The reason for this discrepancy may be that the breeding environment at the sampling site of this study was suitable for the growth of largemouth bass, with an increase in probiotics such as Cetobacterium in the intestinal tract. However, the relatively high abundance of Cetobacterium in the SG group remains to be further explored.

Conclusions
Studies on the morphological structure, digestive enzyme activity and intestinal flora of fast-and slow-growing juvenile largemouth bass have shown that rapid growth is closely related to the following factors. Intestinal fold length and muscle thickness in the fast-growing group were significantly greater than those in the slow-growing group. The lipase activity in juvenile fish of the fast-growing group was higher than that in the slow-growing group. In the fast-growing group, the intestinal tract and disease-resistance immune-related bacteria (Actinobacteria, Halomonas and Bifidobacterium) accounted for a large proportion of the total microbial taxa.  Institutional Review Board Statement: The animal study protocol was approved by the Experimental Animal Ethics Committee of Guizhou University (protocol code EAE-GZU-2021-PO11 and date of approval 10 October 2021).

Data Availability Statement:
The data used to produce the results in this paper can be provided to the corresponding authors if necessary.