Shrimp Gut Microbial Ecology,

Immunity and Functional Feed

IngredientsJohn A. Hargreaves, Ph.D.

Aquaculture Consultant, USSEC

Presentation outline

• Overview of shrimp digestion system

• Bacterial community composition of shrimp midgut

• The shrimp gut and the immune system

• Options for regulating shrimp gut health

Soonthornchai et al. 2009

Digestive system summary:foregut

• Foregut: mouth, esophagus, cardiac and pyloric stomach chambers, all covered with a cuticle layer.

• food ingested via the mouth moves through the esophagus into the cardiac stomach chamber

• after cutting, crushing, mixing by the lateral teeth systems and filtering by a cardiac setal screen in the cardiac chamber, processed food is drained into the pyloric chamber.

• In the pyloric region, processed material is further sorted by the ampullary setal screen into a liquid form for further digestion in the hepatopancreas and particles for subsequent transport into the midgut region.

Digestive system summary: midgut

• Nutrient absorption occurs through the midgut

• Epithelial midgut cells not covered by a cuticle, unlike foregut and hindgut

– Vulnerable to attack by bacteria

• Epithelial midgut cells arrange in microvilli (folds to increase surface area for nutrient absorption)

• Midgut is location of shrimp intestinal bacterial community

• Additonally, a large number of bacteria are attached to the peritrophic membrane and food particles in the midgut.

Soonthornchai et al. 2015

P. Jiravanichpaisal

P. Jiravanichpaisal

Bacterial community in digestive tract of shrimp

Durand et al. 2009

Gut epibionts (b) between microvilli (mv) of shrimp gut epithelium.

P. Jiravanichpaisal

Control mid-gut AHPND (EMS) infected mid-gut

With infection:Colonizing bacteria cover the epithelium of the anterior midgut.

Heavy destruction of the epithelial layers by bacteria exposed of the basement membrane underneath.

Densely packed-bacteria cover the epithelium of the anterior midgut.

Digestive system summary: peritrophic membrane

• The midgut region secretes the peritrophic membrane that wraps material coming from the pyloric stomach chamber

• The peritrophic membrane is continuously formed on the surface of epithelial midgut cells

– stages 1 and 2, PM is closely attached to the microvilli of epithelial midgut cells

– PM in stage 3 is detached

Digestive system summary: peritrophic membrane

• It is composed of acellular material produced by the midgut epithelium and consists of proteins and glycosaminoglycans embedded in a chitinous matrix

• Peritrophic matrix : lattice of chitin fibrils; glycans fill interstitial spaces, creating a molecular sieve

• The pore size of midgut peritrophic membrane can be as small as 20 nm

Chandran et al. 2015

Digestive system summary: peritrophic membrane

• The PM separates ingested food from the gut epithelium and seems to have an important role in the protection against microbial invasion

• physical barrier: protects midgut epithelium from– abrasive food particles

– digestive enzymes

– infectious pathogens

• the PM may play a defensive role by preventing contact between bacteria and the gut epithelium

• biochemical barrier: sequestering or inactivating toxins

• compartmentalizes digestive processes: efficient nutrient acquisition and reuse of hydrolytic enzymes

Thuong et al. 2016

Digestive system summary: hindgut

Hindgut is a simple cuticle-lined tube that functions in expelling the peritrophic membrane, containing the feces.

Presentation outline

• Overview of shrimp digestion system

• Bacterial community composition of shrimp midgut

• The shrimp gut and the immune system

• Options for regulating shrimp gut health

Powerful new analytical tools

• PCR-DGGE (PCR-denaturing gradient gel electrophoresis) analysis

• 16S rDNA pyrosequencing analysis (“barcode”)

• In situ hybridization (FISH)

• Bioinformatics / gene libraries

• Methods based on traditional culture of bacteria are limited in their usefulness

Bacterial concentration averaged 0.5 to 32 million CFU/g

Chaiyapechara et al. 2011

Wang et al. 2014

Multi-dimensional scale plot to compare bacterial communities from different sources

Bacterial community in intestines of healthy shrimp most closely resemble that of sediment

Chaiyapechara et al. 2011

The composition of bacteria in shrimp intestine is distinct from pond water

DGGE profiles of shrimp intestine and water from different farms

Cardona et al. 2016

Bacteria in water more diverse than fish intestinesIntestinal microbial community between CW and BFT only 27% similar

Cardona et al. 2016

Bacterial communities of shrimp intestine in CW was similar to waterBacterial communities of shrimp intestine in BFT was very different to community in water

Multidimensional scaling plot

Chaiyapechara et al. 2011

The bacterial community composition of individual shrimp from different farms generally clustered together

Multidimensional scaling plot comparing intestinal bacterial profiles of shrimp from different farms

Chaiyapechara et al. 2011

Suo et al. 2017

Cladogram of the intestinal microbiota of white shrimp that are the top 20 most abundant at the genus level. Node size is proportional to average abundance; color intensity indicates relative concentration of the cluster.

Shrimp intestinal bacterial communities are dominated by phylum Proteobacteria

Chaiyapechara et al. 2011

Proteobacteria in shrimp intestinal bacterial community are dominated by Vibrio, Photobacterium or Aeromonas

Chaiyapechara et al. 2011

Gut bacteria of black tiger shrimp from a farm in Bali were dominated by Proteobacteria

Oetama et al. 2016

Frequency distribution of bacterial phyla in black tiger shrimp intestines from wild-caught (WC) and domesticated (DB) shrimp

Rungrassamee et al. 2014

Shrimp intestinal conditions exert stronger selective pressure for bacterial community establishment than rearing environment

Rungrassamee et al. 2014

Unique and shared OTUs in intestines of black tiger shrimp from pyrosequencing: WC = wild-caught and DB = domesticated broodstock.

Frequency distribution of different groups of bacteria in intestines of black tiger shrimp of different ages

Rungrassamee et al. 2013

Frequency distribution of different groups of Gammaproteobacteria in intestines of black tiger shrimp of different ages

Rungrassamee et al. 2013

Rungrassamee et al. 2013

Top five most abundant bacterial genera in shrimp intestines of four growth stages.

Dendrogram analysis of DGGE profile of predominant bacteria in black tiger shrimp of different ages

Rungrassamee et al. 2013

Bacterial community composition of Pacific white shrimp gut at different ages

Huang et al. 2014

Intenstinal microbial community dynamics in L. vannamei

• The community was dominated by Flavobacteriaceae during middle growth stages and Vibrionaceae during the late growth stages.

• Rhodobacteraceae and Flavobacteriaceaewere present in all growth stages and likely form the intestinal core microbiome.

• The intestinal bacterial community of white shrimp undergoes dynamic changes at the OTU (species) level during growth. Huang et al. 2014

Enzyme producingcapabilities of shrimp gut bacteria

• Vibrio and Pseudoalteromonas genera are normal flora in the shrimp digestive system

• Pseudoalteromonas is a common genus in the marine environment and produces several chemical compounds, including amylases, β-galactosidases, phospholipases, antimicrobial compounds and proteases

• Vibrio species are broadly distributed in marine environments and are associated with a wide variety of organisms.

• Many are known to be pathogens, some in shrimp, while others have been tested as probiotics (e.g. V. alginolyticus, V. fluvialis and V. campbellii)

• Most of the identified strains produce proteases, amylases, lipases/esterases and chitinases, indicating that L. vannamei bacterial flora includes some groups exhibiting multienzymatic activity.

Tzuc et al. 2014

Presentation outline

• Overview of shrimp digestion system

• Bacterial community composition of shrimp midgut

• The shrimp gut and the immune system

• Options for regulating shrimp gut health

P. Jiravanichpaisal

Aspects of gut immunity in non-vertebrates

• pathogen recognition

• activation of immune cells

• effector mechanisms of host defense

• the gut microbiota plays a role in regulation of the immune system

Interactions between the immune system and the gut microbiota

• Normal bacterial flora provide protection against competing microbes, but also regulate shrimp innate and adaptive immunity

• the interplay between shrimp and the gut microbiota is important for the maintenance of homeostasis and for protecting shrimp against pathogens

• the composition of the gut microbiota can be modulated according to shrimp developmental stage

• the presence of a normal gut microbiota can also influence shrimp development

Mechanisms of mucosal immunity in the gut

• Gut possesses physical and physiological processes to prevent colonization (low pH, peristalsis, antimicrobial molecules)

• Receptors in gut recognize pathogens (pepditoglycan recognition proteins)

• Activation of immune cells in the gut

– hemocyte recruitment to gut epithelium after infection with Vibrio

– multilayered capsule of hemocytes to confine infection

P. Jiravanichpaisal 2012

Shrimp Intestinal Immune System

Mechanisms of immunity in the gut

• Lysozymes– hydrolytic enzymes

• Reactive oxygen species (ROS)– microbicidal role and modulate signal transduction

pathways; regulation of growth of gut microbiota

• Antimicrobial peptides (AMPs)– V. harveyi infection induces AMP gene expression

Vibrio harveyi has the capacity to induce expression of some immune-related genes in shrimp after bacterial immersion.

In the juvenile gut, small changes in expression of the antimicrobial peptide (AMP) genes were observed.

The expression of C-type lectin was affected most.

Soonthornchai et al. 2010

Relative expression of six genes in the midgut (AM) and hindgut (PM) of shrimp during V. harveyi challenge by immersion

Mechanisms of immunity in the gut

• Lysozymes– hydrolytic enzymes

• Reactive oxygen species (ROS)– microbicidal role and modulate signal transduction

pathways; regulation of growth of gut microbiota

• Antimicrobial peptides (AMPs)– V. harveyi infection induces AMP gene expression

• Melanization and the proPO system– Leads to melanization of pathogens; gut defense

mechanism

– Beta glucan recognition protein

Amparyup et al. 2012

Presentation outline

• Overview of shrimp digestion system

• Bacterial community composition of shrimp midgut

• The shrimp gut and the immune system

• Options for regulating shrimp gut health

Options to regulate shrimp gut health

• Probiotics – gut inoculation

• Prebiotics – nutrients to promote development of selected bacteria

• Immunostimulants

• Botanical (phytogenic) substances –modulation of microflora

• Gut acidifiers (organic acids)

Probiotics

• Improve “balance” of bacteria in gut

• Compete with pathogenic bacteria for “space” (adhesion sites)

• Produce antimicrobial substances in gut

• Lower pH through acid production

• Reduce load of Vibrio in gut

Ninawe and Selvin 2009

Probiotics – evidence of beneficial effects

• Adding strains of Vibrio and Bacillus to water promotes shrimp growth

• A mixture of strains of Vibrio alginolyticus, B. subtilis, Roseobacter gallaeciensis, and Pseudomonas aestumarina to the feed of white shrimp for 28 days significantly reduces FCR and promotes growth

• A mixture of photosynthetic bacteria and B. subtilis added to the feed of white shrimp for 28 days promotes shrimp growth and improves protease, amylase, cellulase and lipase activities

• Feeding a Bacillus strain to shrimp can increase the number of hemocytes and the antibacterial and phenoloxidase activity, and can significantly improve survival rates after a challenge with Vibrio harveyi.

• Feeding a Bacillus strain increases the phenoloxidase and phagocytic activity and the bacterial clearance rate and improves the survival rate of shrimp challenged with V. alginolyticus

Encarnacao 2010

Vargas-Albores et al. 2017

Classification of bacteria in a probiotic used for shrimp production

Vargas-Albores et al. 2017

Intestinal microbiome of shrimp fed a probiotic

Vargas-Albores et al. 2017

Taxanomic profile of intestinal bacterial diversity of shrimp grown with or without a probiotic

Luis-Villasenor et al. 2013

Composition of intestinal bacterial community of individual white shrimp inoculated with probiotics: Bacillus mix (M5-M20), Alibio (A5-A20) and Control (C5-C20).

Luis-Villasenor et al. 2012

5 d

10 d

15 d

20 d

M = Bacillus mixA = Commercial probiotic (Alibio)C = No probiotics

Used 8 g white shrimp

Potential functional components of probiotics for shrimpPhotosynthetic bacteria

purple non-sulfur bacteria

Antagonistic bacteria

e.g. Vibrio-antagonistic strains

Microorganisms for nutritional and enzymatic contribution to digestion

lactic acid bacteria and yeasts

Bacteria for improving water quality

nitrifiers, aerobic denitrifiers

Bdellovibrio

lyses Gram-negative bacterial cells

Components of commercial probioticsLactic acid producing bacteria (Lactobacillus)

Non lactic acid producing bacteriaBacillus subtilis, Bacillus licheniformis, Bacillus sulfidophilus, Bacillus amyloliquefaciens

Non-pathogenic yeasts (Saccharomyces)

Photosynthetic bacteria

Nitrifying bacteria

Aerobic denitrifying bacteria

Phytogenics

• Plant-derived, natural compounds incorporated into diets that improve animal productivity

• Classification: herbs, botanicals, essential oils, oleoresins

• Wide variety of mode of action:– anti-oxidant, anti-microbial, anticarcinogenic, analgesic, insecticidal,

antiparasitic, anticoccidial, growth promoters, appetite enhancement, stimulant of secretion of bile and digestive enzyme activity, laxative and anti-diarrheal, hepatoprotection

• anise, basil, bay laurel, clove, coriander, cinnamon, dill, fennel, garlic, ginger, mustard, oregano, parsley, pepper peppermint, rosemary, tarragon, thyme

• thymol, eugenol, cucurmin, piperin, capsicum, cinnamaldehyde, carvacrol

Immunostimulants in crustaceans

• cell wall fragments, beta-glucans, peptidoglycans, lipopolysaccharides and nucleotides

• enhances non-specific immune response

• increases phagocytosis of pathogens by activating phagocytic cells in the hemolymph

• increases antibacterial and antiseptic properties of hemolymph

• activates the prophenoloxidase system

• mediates signal recognition and phagocytosis

Main immunostimulants in aquaculture

• polysaccharides (e.g. ß-glucans, pepditoglycans, chitosan)

• nutrients

• oligosaccharides

• herbs

• antibacterial peptides

• microorganisms

ß-glucans

• mainly exist in cell walls of bacteria and yeast

• recognized by the immune system of aquatic animals as a foreign molecular pattern

• Increases shrimp growth when added to feed

• Improves intestinal immune response

• Improves hemocyte phagocytosis, phenoloxidase activity and respiratory burst activity

• Enhances the resistance of shrimp to WSSV

Organic acids

• Improved activity of digestive enzymes and creates an impaired environment for pathogens

• shift in the dominant hierarchies of bacteria through the lysing of Gram-negative bacteria.

Functional feed ingredients• digestibility enhancers

• gut health promoters

• antimicrobial compounds

• quorum sensing inhibitors (quorum quenchers)

• botanical extracts and phytobiotic compounds

• short and medium chain fatty acids

• organic acids

• enzymes

• natural emulsifiers

Inhibition of Vibrio activity – quorum sensing• Cell-to-cell communication• Bacteria sense and respond to environmental changes and

to each other through extracellular signal molecules• Signal molecules regulate gene expression for

virulence/pathogenicity, biofilm formation, toxin secretion, bioluminescence, etc.

Gonzalez and Keshavan 2006

LuxSLuxQ

LuxP

LuxM

LuxN CqsS

CqsA

LuxU

LuxO

LuxR

sRNA’s + Hfq

σ54

HAI-1AI-2

OH

O

CAI-1

Promoter of

target genes

OOB

-

O

OH

OH

OH OH

NO

OH

H OH O

Quorum sensing signaling systems in Vibrio

Quorum quenching• disruption of quorum sensing

• targets: signal production, signal molecules, signal detection

• signal-degrading bacteria (Bacillus strain) used as probionts

• plant extract (cinnamaldehyde) – quorum sensing disrupter

Bossier et al.

0

10

20

30

40

50

60

Surv

ival

(%

)

Inhibition of Vibrio growthPoly-β-hydroxybutyrate (PHB)

bacterial growth inhibitor

PHB particles in water protects Artemia from vibriosis

Bossier et al.

Some practical aspects

• In vitro versus in vivo effects

• Isolation of locally adapted strains of probiotics

• Proper activation of probiotics

• Adding sufficient doses

• Adding to feed without significant change to feed properties/quality

• Developing capacity on farm to culture bacteria (agar plates/slants)

In Vitro Antagonism Test

Luis-Villasenor et al. 2012Zone of inhibition

Take aways• Gut bacterial communities tend to be dominated by

Vibrios

– Difference between “good” and “bad” Vibrios often a matter of concentration (QS)

• Gut communities are dynamic but ability to manipulate is limited and short term

– Bacillus colonization is minor

– Greater benefit likely associated with enzyme production

• Gut plays an important role in immune function

• Economic evaluation of using functional feed ingredients needed

• Value of probiotics increases with production intensity