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Abstract:Intestinal flora plays an important role in maintaining intestinal health, promoting intestinal development, resisting pathogen invasion, regulating the body's energy absorption and lipid metabolism. In ichthyological research, due to the wide variety, large differences in diet, and complex and changeable living environment, the research on the intestinal flora of fish is facing great challenges. On the basis of summarizing the composition of common fish gut microorganisms, this paper focuses on the research progress of fish bait composition, aquatic environmental factors, species, genotype, development stage, breeding mode and feeding strategy on the impact of fish intestinal flora, and analyzes the existing problems in the current study, in order to provide a certain reference for the study of fish intestinal flora.
The gut microbiome is regarded as a large and complex ecosystem in vivo, which together with the host constitutes a "superaorganism", whose composition changes are closely related to environmental factors and host individuals, and play an important role in maintaining intestinal health, promoting intestinal development, resisting pathogen invasion, regulating the body's energy absorption and lipid metabolism [1-2]. Studies of homo sapiens and mice (Mus musculus) have shown that food composition[3], environmental factors [4], physiological status[5] and genetic factors [6] can affect the structure and quantity of intestinal flora, which in turn affects the body's nutritional metabolism, immune regulation, neuroendocrine and other functions. Compared with terrestrial mammals, in ichthyological research, fish species, large differences in diet, and complex and changeable living environments have brought great challenges to the study of fish intestinal flora. However, with the continuous development of cultureable technology and microbiome sequencing technology, the study of fish intestinal flora has gradually become a hot research in the field of aquatic zoology. Therefore, based on the main factors affecting the intestinal microecological environment of major farmed fish, it is of great significance to carry out research on the influence of different acting factors on the intestinal flora of fish, and then to further elucidate the interaction mechanism between intestinal flora and host, which is of great significance for understanding the function of fish intestinal flora and promoting the healthy development of aquaculture industry.
1 Composition of the fish intestinal flora
In the classification of vertebrates, fish occupy the most important taxonomic position and are rich in ecological diversity. There are significant differences in the microbial composition of different tissues and organs (skin, gills, intestines) and their excreta (feces) in fish [7] (Figure 1). Previously, the understanding of the gut microbiome relied heavily on culturable techniques, and the resulting pure culture flora was relatively homogeneous [8], leading to the widespread belief in the academic community that the composition of the gut microbiome in fish was simpler than that of mammals. With the continuous improvement and improvement of molecular sequencing technology, it was found that the composition of fish intestinal flora is equally complex. Compared with terrestrial animals, aerobic bacteria, facultative anaerobic bacteria, and obligate anaerobic bacteria are the main microbial groups in the fish gut, containing about 107 to 1011 bacteria per gram of intestinal contents,[9] including Proteobacteria, Fusobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Wartymic microbacteriology (Verrucomicrobia) [10-11] etc. Among them, the proteobacter phylum, the pachylobacter phylum and the actinomycete phylum are the main microbial taxa. Due to the diversity of their feeding habits, living environments and different stages of development, fish have spatio-temporal differences in the composition of intestinal flora. Currently, here
Figure 1 Major microbial taxa of different tissues of fish (Llewellyn et al.[7])
Fig. 1 The major microbiota in different tissues of fish(Llewellyn et al.[7])
Marine fish with more research include the Ontario salmon (Salmo
salar), Atlantic bass morhua, Salvelinus alpinus and Scophthalmus maximus, freshwater fish including Danio rerio, rainbow trout (Oncorhynchus mykiss), Ictalurus punctatus, and grass carp (Ctenopharyngodon idella) and cyprinus carpio, etc. (Table 1).
Studies of freshwater carnivorous fish Siniperca chuatsi showed an absolute dominance of the Phylum Proteus, with the Enterobacteriaceae, Aeromonadaceae and Pseudomonadaceae
It is a dominant flora [12-14]. Among them, protease-producing and lipase-producing microorganisms, such as Halomonas and Cetobacterium, which are closely related to the digestion of carnivorous fish, are more abundant. The research results on freshwater herbivorous fish such as grass carp are not consistent, and some scholars have found that the phylum Proteus, pachylossus and actinomycetes are the main dominant taxa, the bacteroides phylum
Abundance was low [15-16], and it has been found that Phylum Pachylobacteria (27.1%), Clostridium phylum (26.5%), and Phylum Proteus (13.9%) are the dominant taxa [17], which may be related to differences in feed composition and sequencing methods of study subjects [16]. Among them, cellulose-degrading bacteria include Anoxybacillus, Enterobacter, and Enterococcus
Genus (Enterococcus), Klebsiella spp., Bacillus brevibacillus, Leuconobacterium spp., Leuconobacterium spp
(Leuconostoc), Clostridium, Actinomyces, Citrobacter, and Aeromonas [11,14,18], the abundance of these microbial taxa increases significantly after grass carp ingest plant fibers. Studies of freshwater omnivorous fish carp have shown that Clostridium, Clostridium, Planctomcetes, and γ Proteobacteria are the dominant microbial groups, while Verrucomicrobiae, Clostridia, and Bacilli are smaller [19–20].
In the study of the intestinal flora of marine fish, because its living environment is completely different from that of freshwater fish, there are also obvious differences in the composition of the intestinal flora. The results of a study of the intestinal flora of Ontario salmon using high-throughput sequencing technology showed that the proteobacter phylum, pachylosbacter, Clostridium, and Actinomycetes phylum were the main taxa in the intestinal digestient, and the microbial taxa abundance of the digestion was significantly higher than that of the intestinal mucosa [21]. Ringø et al. [22] studies of intestinal cultureable microorganisms of Atlantic cod show that Cold Bacillus, Cyclotropia spp., and Carnivorous Bacillus are the main taxa. Xing Mengxin et al. [23] against Da Ling
Turbot gut microbial studies have shown that the Phylum Proteus is the dominant taxon, of which Vibrio spp. has the highest proportion. Compared with freshwater fish, it was found that some microbial groups such as Vibrio, Carnivorous, Alteromonas, Flaviflorum and Pallidus accounted for a relatively high proportion of the intestinal microorganisms of marine fish, and some taxa may be unique to marine fish.
2 Effect of bait factors on fish intestinal flora
Studies have shown that different food components can significantly change the composition of intestinal microorganisms in humans, mice and livestock and poultry animals, which in turn has an impact on the body's nutritional metabolism, immune function, growth and development, and even nerve conduction. At present, there are relatively few research reports on the effects of different bait components on the composition of fish intestinal flora. With the continuous development of high-throughput sequencing technology, the influence of feed components on the composition of the intestinal flora of farmed fish will be clearer, which is essential for an in-depth understanding of the interaction of intestinal flora and fish physiological functions.
2.1 Effects of fats, proteins and carbohydrates on fish intestinal flora
Due to the shortage of animal protein sources, especially fishmeal resources, the industry generally reduces the cost by increasing the fat content in feed to exert a "protein saving effect" and replacing fish oil with vegetable oil, thereby achieving the purpose of promoting the rapid growth of fish. At the same time, however, it has brought problems such as excessive fat accumulation and immune damage to fish[37], and has also had an impact on the composition of the intestinal flora. It has been found that feeding zebrafish on a high-fat diet leads to a significant decrease in the diversity of their intestinal flora, with significant enrichments of the genus Purple, Pseudomonas, and Cetaceans, while the α Proteus phylum, especially Rhizobiales, decreased significantly, and the body length of zebrafish was significantly reduced compared with the control group [38-39]. Lésel et al. [40] Fed rainbow trout on feeds with different fat content, it was found that the intestinal flora of rainbow trout fed at high lipid levels was more structured and more abundant than those of the low-level fat group, Aeromonas, Xanthobacterium, Pseudomonas and Coryneforms [40]. Semova et al. [41] modeled on sterile zebrafish to confirm that intestinal flora can promote the absorption of fat in the intestinal epithelium and liver tissue and promote the formation of lipid droplets, of which the pachychia phylum plays a key role, while the bacteria of the phylum Bacteroides and phylum Proteus do not increase the number of lipid droplets. In addition, different types of fat sources can also cause changes in the composition and abundance of fish intestinal flora. The addition of polyunsaturated fatty acids (linoleic acid, linolenic acid) or high unsaturated fatty acids (HUFA) to feed to onshore salmon found a significant increase in the number of lactobacillus bacteria in the intestinal contents and feces of the linolenic acid and HUFA groups [35,42]; The Northern Red Spot Salmon, which was fed soybean oil as a fat source, isolated from Acinetobacter, Carnagee, Corynebacter, and Cooterella microbiota in all replicates in the experimental group, but were not found in the control group [43]. After feeding rainbow trout for 60 days with sunflower oil, flaxseed oil, rapeseed oil, marine animal oil and commercial feed, it was found that there was no Enterococcus species in the rapeseed oil group, but Marinilactibacillus psychrotolerans-like, which were unique to the group, and Shewanella putrefaciens and Sarelians ( Serratia)[42]。 The above studies show that different types of intestinal flora in the fish intestine play different physiological roles in the process of lipid metabolism, and the fat content and type of the diet can change the composition, function and fluidity of the intestinal mucosa, change the binding site of intestinal myxoflora, and then affect the body's lipid metabolism. The impact of high-fat diet on human intestinal flora has been very in-depth research, but for aquaculture fish, due to the limited popularity of sterile model animals, it is unclear which types of microorganisms play a key role in the process of fat metabolism, and the specific metabolic pathways through which they affect the body's lipid metabolism process also need to be further studied.
The replacement of aquatic feed protein sources, especially the replacement of fishmeal with plants, animal by-products or new animal proteins as protein sources, has long become a hot topic for scientists at home and abroad, and fishmeal has been replaced
The question of whether generations affect the gut health of fish, especially the balance of gut microbes, is worth paying attention to. In terms of plant protein source substitution, the substitution of 30% soy protein for fishmeal, with the help of pure culture methods, found that there was no significant effect on the composition of the intestinal flora of Carassius auratus gibelio, Ontario salmon and rainbow trout, but compared with the fishmeal group, the overall number of flora increased, the abundance of Proteus phylum decreased significantly, and the number of phylum Pachycetes increased significantly, and the abundance of Corynebacteriaceae, Lactobacillus, Vibrio, and Streptococcus had different degrees of changes [44-46]. Among them, the abundances of Sphingosine monocytogenes, Kluyvera and Peptostreptoccus were significantly changed, and the abundance of Sphingosine monocytogenes was significantly negatively correlated with the total number of intestinal flora, while the opposite of Digestive Streptococcus was the opposite. The reasons for the analysis may be closely related to the oxygen content and redox potential in the intestine, in the fishmeal group, the total number of bacteria in the intestine is small, the oxygen content is more sufficient, the aerobic bacteria of the genus Sphingosineomum are the dominant flora, and when soy protein replaces fishmeal, the plant protein is fermented in the intestine and consumes oxygen, the intestine is in an anaerobic state, and the anaerobic bacteria are the dominant flora at this time. In summary, the current research results based on pure culture technology show that soybean protein has not significantly changed the microecological balance of the fish intestine after replacing fishmeal, and to a certain extent, it has led to the formation of anaerobic states in the intestine, which can resist the invasion of pathogenic bacteria, so it can be used as a good alternative protein source for fishmeal. In addition, the feeding of grass carp with ryegrass as a protein source showed that the composition of the intestinal flora was significantly different from that of the commercial feed group, and the carbohydrate, amino acid, and lipid synthesis and metabolic pathways were significantly altered, indicating that the intestinal flora played an important role in the utilization of non-digestible polysaccharides and the maintenance of nutritional and physiological homeostasis in grass carp [17]. In terms of replacing fishmeal with new animal protein sources, recently, domestic and foreign scholars have carried out a large number of studies on the digestion, absorption, growth, meat quality and immunity of farmed fish nutrients, such as silkworm pupae [47] and hermetia illucens[48], but the impact on the intestinal flora of fish has not been reported. In addition, it should also be noted whether the addition of traditional protein ingredients such as Antarctic krill (containing chitin), lees protein feed (DDGS), concentrated peas, feather powder, chicken powder and other traditional protein ingredients to feed will also have an impact on the balance of fish intestinal microecology.
In terms of glucose metabolism, fish are seen as inherently "diabetic" sufferers with an intolerance to high sugar, which scientists have tried to explain from the perspective of evolution [49] and molecular biology [50]. Industrial applications are also trying to reduce feed costs by increasing the sugar content or adding different sugar sources in fish feed. However, it is unclear how different sugar sources interact with the intestinal flora of fish and how they affect the sugar metabolism process of fish. The only references show that The Norwegian tongue-toothed perch (Dicentrarchus labrax) was fed with lupine flour (non-starch polysaccharide) and corn starch (amylose polysaccharide) as sugar sources, and compared with the control and corn starch groups, the intestinal conditional pathogenic bacteria in the sea bass in the lupine powder group were significantly suppressed, and the abundance of Vibrio spp. was significantly reduced, while the abundance of Clostridium spp. was significantly improved [51]. Geurden et al. [52] fed rainbow trout on feeds with different glucose content, and although the diversity of the gut flora changed, there were no significant differences. At present, other common sugar sources in feed also include tapioca flour, potato, dextrin, wheat bran, etc., but whether the fish intestinal flora can use different sugar sources for energy metabolism, which key taxa play a role, or due to the lack of certain taxa, fish have low ability to use sugar, these problems are not clear, and related research needs to be carried out in depth.
2.2 Effects of additives on fish intestinal flora and host nutrient metabolism
Amino acids, vitamins, minerals on the impact of fish intestinal flora Amino acids, vitamins and minerals as important nutrients in the body's metabolism, most of them can not be synthesized by themselves in teleost fish, need to rely on exogenous additions to meet the body's growth, development, metabolism needs. To C. carpio var. Jian's findings showed that the addition of appropriate amounts of methionine (Met), isoleucine (Ile), pantothenic acid (VB5), biotin (VB7), ascorbic acid (VC) and phosphorus to the feed can effectively inhibit the growth of E. coli (E. coli), aeromonas, promote the proliferation of lactic acid bacteria and Bacillus bacillus, and improve the activity of intestinal digestive enzymes [53-58]. The addition of copper (copper-carrying montmorillonite) ions in moderation reduces the number of intestinal aerobic bacteria in nile-hatched non-crucian carp (Oreochromis niloticus niloticus) and inhibits the growth of microorganisms of Aeromonas, Vibrio, Pseudomonas, Xanthobacter, Acinetobacter, and Enterobacteridae [59]. Studies have also shown that the addition of nano-copper to zebrafish feed significantly inhibits the growth of beneficial flora in the intestine, but does not cause intestinal epithelial damage and inflammation [60]. Therefore, the type and dosage of mineral elements in the feed may be crucial for the composition of the fish intestinal flora and the homeostasis of the intestine.
The impact of probiotics and prebiotics on the intestinal flora of fish In recent years, the development direction of aquaculture has changed from quantitative increase to qualitative improvement. This has been followed by the rapid development of related probiotics, prebiotic scientific research and industry. At present, the main probiotics used in aquatic feed include lactic acid bacteria, bacillus, C. bifermentans and yeasts, common prebiotics include fructooligosaccharides, galacto-oligosaccharides, dextrans, mannans, xylose oligosaccharides and the like. Ringø et al. [42] and Merrifield et al. [61] reviewed the studies in 2014 and 2016, respectively, and this section only analyzes and elaborates on the studies conducted since 2016. Lactic acid bacteria and yeast are currently the most widely used probiotics in aquaculture[62-66], Giorgia et al. [67] with a new micro-ecological preparation L. rhamnosus rhamnosus fed Nile-hatched non-crucian larvae, the abundance of intestinal lactic acid bacteria significantly increased, gene expression related to muscle growth and appetite significantly upregulated, the endocrine system was effectively activated, and potential pathogens such as Microbacteriaceae ( Microbacteriaceae ) Legionellaceae and Weeksellaceae have significantly reduced abundance. In zebrafish studies, the addition of Lactobacillus rhamnosus inhibits food-promoting genes, upregulating the expression of anorexia genes, while inhibiting gene expression associated with cholesterol and triglyceride anabolism [38]. Similarly, after yeast feeding, the abundance of lactic acid bacteria in the intestines of Arctic red-spotted salmon and rainbow trout increased, and resistance to pathogens was significantly improved [66,68]. Liu et al. [69] further demonstrated that when the hybridization mouth incubation of non-crucian carp stopped eating Lactobacillus plantarum JCM1149, the diversity of the intestinal flora α decreased significantly, the abundance of the pachylobacteria phylum decreased significantly, and the actinomycetes phylum increased significantly, while the hybridization hatching of non-crucian susceptible aeromonas, intestinal bile acids, amino acids and glucose metabolism was disordered. The above research suggests that the use of probiotics in the aquaculture process has a significant effect on the body's nutritional metabolism and immune regulation, but it cannot be used as a drug, but should be based on the long-term stabilization of the animal's intestinal micro-ecological balance and the improvement of the animal's body health. In addition, prebiotics as a carbon and nitrogen source for the effective proliferation of intestinal probiotics, and their use alone or in combination with probiotics is also the current mainstream research. The results showed that the addition of short-chain fructooligosaccharides and xylose oligosaccharides to the feed did not alter the intestinal microbial diversity of Norwegian tongue-toothed perch, but the abundance of lactic acid bacteria was significantly increased[70], and the combination of galactose oligosaccharides and Pediococcas acidilactici (Pediococcas acidilactici) significantly increased the intestinal lactic acid bacteria abundance and short-chain fatty acids, especially butyric acid content of rainbow trout juveniles [71]. Taken together, most of the probiotics used in the above studies were fish heterologous strains, while there were almost no indigenous probiotic strains isolated from the fish themselves. Alonso et al. [72] Recently isolated nine strains of lactic acid bacteria with broad-spectrum antimicrobial activity from the guts of 13 marine fish species and demonstrated that they could be used as potential aquatic probiotics. Therefore, it is of great significance for the aquaculture industry to isolate fish-derived probiotics from the intestines of fish with different diets and environments and carry out related taxonomic and basic biological research.
Effects of Chinese herbal medicine or plant extract additives on the intestinal flora of fish Studies in humans and mice have shown that Chinese herbal medicine can play an important role in alleviating obesity and type 2 diabetes by influencing the intestinal flora and then the body's nutrient metabolism and immunity through its metabolites [73-74]. The basic mechanisms of action include the biological activity of metabolites produced by the intestinal flora during the conversion of phytochemicals; the alteration of the composition of the intestinal flora, alleviating the biological disorders of the body in the pathological state; and the regulation of the interaction (synergy or antagonism) of complex phytochemicals by the intestinal flora [73]. However, at present, in the field of aquatic animal research, there are still very few relevant studies at home and abroad. The effects of five different Chinese herbal medicines on the intestinal flora of Carp were measured by cultureable methods, and the results showed that the number of bacteria in the intestine increased significantly, the bacillus spp. and Acinetobacter spp. increased significantly, while the aeromonas spp., o-omosa, Pseudomonas spp., and Vibrio spp. decreased significantly [75]. The results of the study of heterogeneous silver crucian carp also showed that after the feeding of Chinese herbal medicines, the richness and diversity of the intestinal flora increased significantly, and at the genera level, the aeromonas and Shivas related to pathogenic bacteria decreased significantly, while the Lactobacillus, Lactococcus and Bacillus spp. increased significantly [76]. For plant extracts, the active ingredients are mostly glycosides, acids, polyphenols, polysaccharides, terpenes, flavonoids, alkaloids, etc. Studies of rainbow trout have shown that the addition of carvacrol or thymol to the feed reduces the number of anaerobic bacteria in the intestine and the number of lactic acid bacteria in the thymol group decreases [77]. However, most of the above studies are based on cultureable methods, with the development of high-throughput sequencing technology, especially three-generation sequencing technology, combined with culture-free means to carry out metagenomic, macrotradome and macro metabolome research will help us to understand the complex pathways of Chinese herbal medicine and plant extracts in depth.
3 Effects of aquatic environmental factors on fish intestinal flora and host metabolism
In addition to food components, aquatic environmental factors are also important influencing factors, such as temperature, salinity, pH, ammonia nitrogen, nitrite, water weight metal ions, nanoparticles and organic pollutants. When the water environment factors change, the intestinal microecological environment of fish will also fluctuate, which will affect the body's immunity and basic metabolic capacity. At the same time, certain significant changes in the gut microbiome due to fluctuations in aquatic environmental factors can also be used as biomarkers to detect changes in the aquatic environment. A study of the Colossoma macropomum found that low pH (pH4.0) cultured water bodies significantly reduced the abundance of Xanthobacterium in the intestine, and the ratio of pachychophytes to Phytophthora in feces was significantly increased, which showed a beneficial effect overall, but the intestinal flora tended to control levels after two weeks of culture [78]. The exposure of different concentrations of copper ions to carp showed that the diversity of α and β of the intestinal flora changed significantly, and the abundance of some short-chain fatty acid-producing bacteria such as Allobaculum and Blautia decreased significantly, while the expression of intestinal epithelial tight junction proteins ZO-1 and Occludin decreased, the expression of genes related to lipid metabolism and synthesis in the body decreased, and the expression of decomposition-related genes increased [20]. Similarly, when zebrafish are fed nano-copper, the beneficial bacteria in their intestines are significantly suppressed and the digestive system is damaged [60]. When Atlantic cod was exposed to marine crude oil pollutants, the diversity of its intestinal flora was also significantly reduced, and the abundance of Deferribacterales was significantly increased in the high concentration exposure group, while the abundance of Fusobacteriales and Alteromonadales decreased, and it is speculated that Ferritobacterium can be used as an iconic organism for marine pollution [34]. The adaptability and tolerance of different fish to the water environment are different, and the impact of changing water environment factors on the niche of fish gut microorganisms is not consistent. All in all, water pollutants can destroy the fish intestinal micro-ecological environment, reduce immunity, interfere with the normal metabolism of the body, and moderate pH, salinity and other physical and chemical factors will be beneficial to the growth of intestinal probiotics, bringing positive effects to fish. However, the current research in this field is still relatively weak, and further in-depth analysis is needed.
4 Effects of other factors on the intestinal flora of fish
4.1 Effects of species, genotype and developmental stage on fish intestinal flora
In addition to exogenous factors such as feed and water environment that can affect the intestinal flora of fish, endogenous factors such as different fish species, different genotypes of the same species, and different stages of development of the same individual can also have an impact on the composition of the fish intestinal flora. The analysis of the intestinal flora of silver crucian carp, grass carp, silver carp and turbot cultured in the same water body found that there were significant differences in the intestinal prokaryote microbiota between different species, while the eukaryotic taxa was basically the same [79]. The main intestinal microbial groups of transgenic carp with rapid growth characteristics were Proteus phylum, Fusobacterium phylum, Bacteroides phylum, and Phylum Pachyderma, which were significantly different from wild-type carp, and the ratio of Bacteroides to Pachyderma phylum decreased significantly, energy acquisition increased, and growth rate accelerated [28]. The results of studies of the intestinal flora at different stages of development of the spotted forktail catfish[26] and zebrafish[80] confirmed that the intestinal microbial taxa at each stage of physiological development after the hatching of fish embryos is not the same, and this change does not depend on the breeding environment and changes in the bait. At the beginning of the development stage, the composition of the intestinal flora is more consistent with the breeding environment, and with the increase of age, the structure of the intestinal flora is more complex, and the differences between individuals are gradually increasing. At present, in the process of researching the intestinal flora of fish at home and abroad, there is less consideration for endogenous influencing factors, such as whether the genetic background is consistent, whether the time stage of sampling is representative, whether the different sex ratios of different fish are considered, and these factors may interfere with the final experimental conclusions, so it needs to be paid great attention to.
4.2 Effects of culture patterns and feeding strategies on fish intestinal flora
With the continuous development of the aquaculture industry, new farming models and farming technologies are constantly being innovated, and it is worth paying attention to whether changes in these models and farming technologies will affect the intestinal microecological balance of farmed fish. Factory high-density aquaculture is the direction of future aquaculture development, Wong et al. [81] analyzed the intestinal flora of rainbow trout under high-density culture conditions, and the results showed that the intestinal microbial taxa did not change significantly, and had no effect on feed conversion rate, weight gain rate and meat quality. Ingerslev et al. [82] fed rainbow trout with different open baits and found that the open bait of farmed aquatic animals is very critical to the colonization of their intestinal microorganisms, which will affect the intestinal microecological balance of fish throughout the subsequent developmental stage. However, in the culture stage, if the Asian sea bass is treated with starvation, the intestinal bacteroides phylum is significantly enriched, while the abundance of the β-Proteus phylum is significantly reduced, the functional genes associated with antibacterial activity in the microbiome are significantly enriched, and the expression of host immune-related functional genes is significantly upregulated [83]. The above research results suggest that in the artificial breeding process of fish, the use of appropriate breeding modes and feeding strategies can effectively improve the intestinal microecological environment of fish and improve the health level of fish.
5 Research methods and strategies of fish intestinal flora
The study of the composition of the fish intestinal flora can be easily achieved with the help of next-generation sequencing technology (NGS), but deeper research on the molecular mechanisms of the fish intestinal flora and host in terms of nutrient metabolism, immune stimulation, endocrine regulation and other aspects needs to be carried out in depth. Scholars at home and abroad have carried out relevant work in combination with NGS technology (metagenomic, macro transcriptome, macro metabolome), microbial pure culture technology [84-85], sterile zebrafish model [41, 86-87], fecal bacteria transplantation [88] technology and electron microscopy technology [87], and the main research methods and strategies are shown in Figure 2. The selection of microbial samples, the use of DNA extraction methods, and sequencing platform analysis need to be determined according to different experimental designs and purposes. 6 Future research direction of fish intestinal flora
With the continuous deepening of the research level of the gut microbiome, scientists have realized the importance of the gut microbiome for the health of the body. In the future research process of fish intestinal flora, there are several points worth paying attention to: (1) with the rapid development of three generations of sequencing technology, the research of fish intestinal flora should gradually expand from the current qualitative research at the genus level to the qualitative, quantitative and functional research at the species level; (2) cultureable technology is the cornerstone of microbial research, and the research on culturable technology should be increased to obtain more fish intestinal indigenous pure cultures, especially probiotics; (3) in terms of research methods and strategies, attention should be paid to distinguishing between different fish sexes and genetic backgrounds (4) The impact of intestinal flora on the health of the body In addition to basal metabolism, research on fish behavior and neuroendocrine needs to be strengthened.