Fish gut microbiota: Factors affecting fish gut

microbiome and their physiological role in fish

G. Sathishkumar
U-18-TN-03-007-M-F-010
Fish Nutrition and Feed Technology
 Introduction Microbiome
Microbiota

 Collection of microorganisms (bacteria,

fungi and virus) that are present in
particular environment

Metagenomics

 A technique for investigating genetic

 Collection of genomes of the
content from microbes
microorganisms within in the microbiota
 Intestinal microbiota
Gut microbiota of freshwater
107 to 1011 bacteria/gram of Gut microbiota of marine fishes
fish
Acinetobacter, Aeromonas, intestinal content
Alcaligenes, Alteromonas,
Flavobacterium, Lactococcus,
Aeromonas, Flavobacterium,
Pseudomonas, obligate anaerobes
Carnobacterium, Moraxella,
(Bacteroides, Clostridium and
Micrococcus, Pseudomonas and
Fusobacterium) and members of family
Vibrio
Enterobacteriaceae
Gómez and Balcazar, 2008
Depending on the duration
Transient microbiota Persistent microbiota

Nutritional ingestion mechanism Lives in the host intestinal membrane

Do not last long time within in the environment Symbiotic relation to the host
 Microbial composition of gut microbiota in herbivore, carnivore and omnivore fish
Bacterial species Fish species References
Herbivores
Clostridium sp., Eubacterium Butterfish Odax pullus Clements et al. (2007)
Vibrio sp., Photobacterium, Bacteroidetes Parrotfish Chlorurus sordidus Smriga et al. (2010)
Non-vibrio Proteobacteria, Firmicutes Surgeonfish Acanthurus nigricans Moran et al. (2005)
Zooplanktivores
Pseudomonas, Alteromonas, Psychrobacter Herring Clupea harengis Hansen et al. (1992)
Achromobacter, Vibrio sp., Pseudomonas Sardines Sardinella longiceps Karthiayani and Mahadeva Iyer (1967)
Psychrobacter, Vibrio sp., Shewanella Atlantic mackerel Scomber scombrus Svanevik & Lunestad (2011)
Carnivores
Acinetobacter junii, Mycoplasma, Atlantic salmon Salmo salar Holben et al. (2002)
Lactobacillus sp., P. phosphoreum,
Lactococcus sp., Bacillus sp.
Pseudomonas sp. Gilthead seabream Sparus aurata Floris et al. (2013)
Bacillus, Vibrio, Delftia, Psychroacter, Grouper Epinephelus coioides Sun et al. (2009)
Acinetobacter, Pseudomona
Omnivores
Clostridium, Mycoplasma, Photobacterium, Pinfish Lagodon rhomboides Ransom (2008); Givens et al. (2015)
Propionibacterium, Staphylococcus,
Pseudomonas, Corynebacterium
 Importance of fish gut microbiome in aquaculture

Aquaculture
• As fish increasingly
demanded as food supply
chain
• Satisfy the consumer
• Increase the growth of
requirement
fish production
• Protection against
pathogen

Antimicrobial resistant

Diseases
• Gut microbiome can
Antibiotic dependence resolve this problems
Aquaculture
Bacterial infection: issues in aquaculture
Vibriosis
industry
Symptoms:
Aeromonas 1. Dark discolouration
salmonicida of skin and fins
1. Furnuclosis 2. Serious haemorrhage
2. Furnuclosis is in spleen, body cavity
externally indicated by and in the intestine – V.
haemorrhages that anguillarum
occur in the base of the 3.Other species – V.
Photobacteria
fish fin due to the lack
Flavobacterium Vulnificus,V.
Photobacterium
of hunger,
columnarae lethargic parahemolyticus, V.
damselae disease is a
movement
1. Complete necrosis and salmonicida, V.
most serious
melanosis. Photobacteria and carchariae, V. ordalii,
and gill lesions as are Vibriosis – caused cataclysmic disease in
consequence of this V. damsela aquaculture
disease
almost all infectiousand V. alginolitycus
Symptoms:
2. Dark discoloration in diseases in aqua Edwardsiella
1. White tubercles and
the centre of the fin and industry 1. Haemorrhagic
lesions onandthe internal
is boundLactococcosis
by reddish and Enteric redmouth enteric septicaemia organ.
Streptococcosis
tinge disease 2 Edwardsiella
2. Necrosis of spleen,
Symptoms: 1. Rainbow trout ictaluris and and liver.
kidney
1. Dark pigmentation,2. Yersinia ruckeriEdwardsiella tarda –
exophthalmos, 3. At early phase ofisolated from catfish
haemorrhage, infection – death rate is3. Symptoms -
congestion and erratic slower Complete necrosis and
swimming lethargy 4. Death rate is skin lesions of the
major internal organs
increased – stress due
to high stocking and muscles
 Antimicrobial
Effects of antibiotic usage in aquaculture industry resistant
microbes
• Increased the usage of antibiotics leads to random
Erythromycin
chromosomal alteration of microbes (Spontaneous
Sulfamethoxazole
mutation)
Norfloxacin and • Increased exposure to antibiotics continuously minimize
Trimethoprim the native microbes further increase the no.of resistant
microbes

Antibiotics can reduce the bacterial infections 407 bacterial strains including Gram-positive
cocci (GPC), enterobacteria (ENT) and
non-fermenting Gram-negative rods (NFR)
Results
• GPC are unaffected due to
Tested for antibiotic resistance over minimal
penicillin and azithromycin
inhibitory concentration (MIC) analysis
• NFR and ENT - gentamicin,
ampicillin and sulbactam
Meek et al., 2015
 • Increased the epithelial proliferation,
• Promote the nutrient metabolism and
• Digestion
• Direct interactions b/w
commensals and pathogen
• Promote the functions of
• Compete with nutrients and niche Gut mucosal barrier and
• Commensals – Bacteriocin and microbes enhance the innate immune
toxins
responses
• Modify the host environment e.g.
pH

Defense mechanisms
 SCFA, indoles, Physiological roles of gut microbiota Metabolites
propionate,
butyrate and
acetate 1. Enterocytes and
regulate their
intestinal barrier
digestion and metabolism functions
2. Absorptive capacity
Gut microbiota (e.g.,
monosaccharide
absorption)
3. Nutrient absorption
GIT neurotransmitters and storage (altered
(Serotonin, catecholamine, the enzymatic activity
Dopamine and and fat storage)
norepinephrine) 4. Influencing the
metabolism (e.g.,
cholesterol
Gut motility function and
metabolism and
hormone
adipogenesis)
release as well as feeding
behavior
Gut microbiota on feed intake Butt and Volkoff, 2019
 Gut microbiome on metabolism
Fish species Effects
Grass carp • Many biosynthesis and metabolism pathways of CHO,
amino acids and lipids change as the composition of
microbiota changes

Zebra fish • Promote the epithelial absorption of fatty acids

• Increased the lipid accumulation in the intestinal
epithelium
• Increased the expression of gene related to lipid
metabolism compared to germ free fish

Japaneous flounder fed

• Higher weight gain and feed efficiency
Bacillus clausii
• This could be attributed to increased the food intake and
improved the nutrient digestibility
 Stress in fish can be caused by
Gut microbiome on stress response  Poor water quality,
 High levels of
Stress particulates,
• Mediated by several hormones  Suboptimal photoperiod,
• Bi-directional communication between the brain  Oxygen levels,
and peripheral organs  Temperature,
Hypothalamic-pituitary-adrenal (HPA) axis  High population density,
 Poor diet/ malnutrition,
 Transportation and
Corticotrophin-releasing hormone (CRH)
Zebrafish handling

Enhancing the microbiota reduces Secretion of adrenocorticotropic hormone Induces an increased

anxiety-like behavior and decreases (ACTH) from the anterior pituitary sloughing off of
the stress response mucus
Secretion of adrenal glucocorticoids
Lowering CRH expression and Removal of
cortisol levels autochthonous bacteria
Prepare the body to cope with stress (protective role
against pathogen
Change the structure of intestinal Absorption of nutrients, Infections by opportunistic
Stress mucosa and induce alterations in the gut immunity pathogens
intestinal mucus
 Zebra fish Gut microbiome on reproduction
Birth to sexual maturation Ornamental live bearer and Gold fish

Lactobacillus rhamnosus
Supplemented with probiotics
Alters the gut microbiota
+ GSI
+ Fecundity and fry production
Accelerate the larval development
+ Length and weight of fry
by growth and sex differentiation

Adult female fed L. rhamnosus

• + no. of vitellogenic follicle
• + GSI
• + no. of ovulated eggs
• + expression levels of reproductive hormones (Kisspeptinss,
GnRH3, Leptin) compared to control fish
The mechanism mediating the actions of
gut microbiota in fish reproduction is
still under investigation
 Gut microbiome on immune responses
• Microbiota protects the host from colonization and proliferation of environmental pathogen,
process known as “colonization resistance”
• Although mechanism behind this resistance are not clear, it has been suggested that commensal
bacteria are compete with pathogen for niche space and produce and secrete antimicrobial
peptides.
 Water environment

Rearing conditions
Environmental
Seasonal changes factors
Pollutants & toxins

Factors affecting fish gut microbiota

Genetics

Sex
Host – specific
Age/life stages intrinsic factors
Diet/feeding
habits
Butt and Volkoff, 2019
 Environmental factors
Rearing conditions
Microbes free Water environment

Rainbow trout
• FW fish – Aeromonas sp and Pseudomonas sp • Indoor RAS
• MW fish – Vibrio sp • Cage

Black molly – Salinity – Changes the bacterial flora

Different microbial
Rainbow trout – Temperature – Microbial composition composition
Microbes -environment

Seasonal changes Pollutants & toxins

Water composition and Temperature
quality • Tilapia - gut bacterial load in winter
• Hybrid tilapia – Pseudomonas, Pesticides,
micrococcus and Flavobacterium - heavy metals
Winter and antibiotics
 Host – specific intrinsic factors
Genetics
• Rainbow trout - some bacterial groups associated with specific families,
perhaps due to different habits or different diets
• Channel catfish & blue fish – constant environmental conditions – similar gut
microbiota – environmental conditions can overcome difference in host
genetics
Age/life stages
Sex
• Atlantic salmon – gut microbiota composition
• Three spine stickleback vary b/w embryonic stages and hatchlings
• Eurasian perch • Malaysian mahseer – larval, juvenile and adult
stage have higher microbiota than fingerlings
• Fish of different life stages and ages:
• Different nutrient requirements
• Different diets and feeding habitats
 Feeding habitats
• Herbivores – Clostridium, Citrobacter Starvation/Feeding
• Omnivores – Leptotrichia, Cetabacterium and • Fasting – alter the shape of fish
Halomonas gut microbiota
• Carnivores – Clostridium, Cetabacterium and • Zebra fish & Leopard coral
Halomonas grouper – gut microbiota
• Gut microbiota vary within species of the same diversity and richness are higher
trophic levels under feeding conditions than
fasting conditions
Diets • Grouper
Mullet (Mugil Guar gum (non-starch Induce alteration in • Fasting – Proteobacteria
liza) polysaccharides) the bacterial
quantity and • Feed condition – Firmicutes
Rainbow trout Soy proteins • Asian seabass – Fasting –
composition
Enrichment of Bacteroidetes
and depletion of
Zebra fish Dietary nucleotides Alter the microbial Betaproteobacteria
composition
Channel catfish Different diets with Minimal effects on
 Gut microbiota manipulation and applications
Gnotobiotic fish
Gnotobiotic fish
• Animals with a known microbiota composition
Prebiotics
• Germ free animals and animals that have been inoculated
Probiotics
with known microorganisms
Symbiotic
Proteins
Proteins
• Source, quantity and chemical structure of proteins can
influence the gut microbiota
• Short peptides – can directly manipulate gut microbiota by
providing suitable substrates
• Peptides – AMP’s
 Effects of prebiotics in aquaculture • These bacteria are considered
beneficial to the health and
• Non-digestible compounds
development of the host by
• Metabolized by common bacteria such as
reducing the existence of intestinal
Lactobacillus and Bifidobacterium that
pathogens
promote health
Environment friendly feed additive
Prebiotics
Carbohydrates
• Inulin
• Fructooligosaccharides (FOS)
• Galactooligosaccharides (GOS)
• Arabinoxylo-oligosaccharides
• Monosaccharides,
• oligosaccharides and (AXOS)
• Chitooligosaccharide (COS)
• polysaccharides based on molecular
• Mannanoligosaccharides (MOS)
size and polymeration
• Levan
• Beta-glucan
Inulin
• Fructose polymer composed of β -D-
fructofuranoses attached by β -2-1-linkage bond
• Sugar-based oligosaccharides
• Stimulates normal bacterial flora in the gut
• Defeat the pathogens and enhances the immune
system
Fructooligosaccharides (FOS)
• Short and medium β -D- fructans chains in which
fructosyl units are bound to a terminal glucose unit
by β-(2–1) glycosidic linkage
Galactooligosaccharides (GOS)
• Composed of 2–20 galactose and glucose
molecules, which can be produced by enzyme
lactose treatments
Arabinoxylan-oligosaccharides (AXOS)
• Beta-glucan @ 0.4% - Nile tilapia- increased
• Hydrolysis product of
arabinoxylans
Chitooligosaccharide (COS)
• Chitooligosaccharide is a type of
oligosaccharides obtained through chemical and
enzymatic chitosan hydrolysis
Mannanoligosaccharides (MOS)
• Mannanoligosaccharides derived from the yeast
cell wall (Saccharomyces cerevisiae)
Levan
• Levan is another fructose polymer
Beta glucan
• Group of β-D-glucose polysaccharides naturally
occurring in the cell walls of cereals, bacteria,
and fungi
the Lactobacillus, Phycicoccus and
Rikenellaceae (Xu et al., 2020)
 Isolation heterotrophic
bacteria

Physiological
Biosafety to host
characterization
Non-pathogenic live
microorganism
Stability in GI tract
Probiotics
Lactobacillus, Lactococcus, Leuconostoc,
Beneficial impact on the host Enterococcus, Carnobacterium,
intestinal health Shewanella, Bacillus, Aeromonas, Vibrio,
Enterobacter, Pseudomonas, Clostridium,
and Saccharomyces genera
Commercialized
Prebiotics and probiotics may also be
administered in combinations, known as
Effects of probiotics in aquaculture symbiotics
 Probiotics used in aquaculture
Probiotic sp and aquatic specises
Bacillus subtilis – Nile tilapia
Saccharomyces cerevisiae and Bacillus
subtilis – Nile tilapia
Pediococcus acidilactici – Rainbow trout
Saccharomyces cerevisiae and
Lactobacillus casei – Juvenile barramundi
Rummeliibacillus stabekisii – Nile tilapia
Bacillus licheniformis and Bacillus subtilis
– Nile tilapia
Results References
• Modulates the gut bacterial composition and Deng et al.,2021
increase the beneficial bacteria's
• Retained the probiotics in their guts and lower Opiyo et al., 2019
microbial load was realized in their muscle
• Modulated the intestinal microbiota, Al-Hisnawi et al., 2019
• Upregulated the pro-inflammatory cytokines
(il1 and il8) and igt gene expression and
• Increased the serum lysozyme activity
• Positive influence on microbiota diversity Siddik et al., 2021
• Decreased abundance of pathogenic
Staphylococcus and Corynebacterium
• Increased the probiotic bacterias (Bacillus and Tan et al., 2019
Lactobacillus spp.)
• Reduced the pathogens (Streptococcus and
Staphylococcus spp.)
• Increased probiotic phyla Tachibana et al., 2020
(Firmicutes, Fusobacteria and Bacteroidetes)
• Decreased the Proteobacteria
Microbiota Objective Advantages Disadvantages Examples of host
manipulation species examined
Gnotobiotic Establish a germfree Examine the effects of Complex procedures Zebrafish
host or a known specific gut required to produce and
microbiota microorganisms on host maintain gnotobiotes
composition
Antibiotic Inhibit or eliminate Bacterial disease Potential to disrupt Zebrafish, Mosquito
targeted gut prevention/treatment microbial communities Fish, Black molly
microbiota and increase disease
bacteria or bacterial susceptibility, and
pathogens bioaccumulation
Probiotic Establish beneficial Use in aquaculture, Zebrafish, Rainbow
gut microbiota improve fish health trout, Malaysia
bacteria (digestion/growth), masheer
Prebiotic Stimulate growth of health Rainbow trout, Nile
probiotic bacteria management, disease No known disadvantages tilapia, Common
prevention & carp fry
Overall safe
Symbiotic Establish prebiotic Improve fish health Nile tilapia
and (better than the
probiotic bacteria application of probiotics
and prebiotics)
 Conclusion

 The fish gut microbiota influence the overall health of the host fish with regards to
overall physiology, digestion, stress responses, reproduction and immune system.
 Manipulating the gut microbiota of fish is great for aquaculture to improve growth.
 However, the future of probiotics and prebiotics faces several challenges, including the
appropriate mode of treatment (oral or in water) and dose, the characterization of the
mechanism of action of individual probiotic organisms and quality control and
regulation.
 Future scope

• Study the microbiome interaction for developing probiotics

• Study and isolate of stress resistant microbes for expansion of aquaculture

• Studies at species and subspecies level for the development of probiotics

• Characterizing the commensal bacterial community of cultured animals and its genetic
potential is important as a changing environment can modify the bacterial community,
structure, and function
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