MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE: FROM GNOTOBIOTIC EXPERIMENTS TO...

MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE:

FROM GNOTOBIOTIC EXPERIMENTS TO APPLICATIONS

MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE:

FROM GNOTOBIOTIC EXPERIMENTS TO APPLICATIONS

NPC mtg Feb 2012

LARVI09 Keynote on microbial management – Peter BossierGhent University Aquaculture Research Consortium

slide 2 of 39

Conceptual framework

eggs

MCe MCs

juveniles MCe MCs

MCe MCs

larvae

Immuno

stimulant

nutrition

Modulators

composition

Modulators of activity


slide 3 of 39

Experimental approach:

• Gnotobiotic systems– Artemia– Brachionus– Seabass

• Non-gnotobiotic verification


slide 4 of 39

Known micro-

organisms

Host

environment

Hostsimplificati

on

complex gnotobiotic

reality?

MC

How to study host-microbial interactions?


slide 5 of 39

Gnotobiotic Artemia: GARTGnotobiotic Artemia: GART

Instar I

Instar II

Decapsulated cysts

Decapsulation

Axenic conditions

Hydrated cysts

Non-axenic conditions

20-50 nauplii counted for the

Experiments

Gnotobiotic challenge: add VibrioGnotobiotic challenge: add Vibrio


slide 6 of 39

Gnotobiotic Artemia –seabass food chainDAH1

DAH3

DAH14

Artemia

Vibrio anguillarum

DAH7

At 16°C


slide 7 of 39

0102030405060708090

100110120

0 1 2 3 4 5 6 7 8 9 10 11 12

time (day)

surv

ival

(%

)Gnotobiotic Artemia –seabass food

chain

Virulent

Vibrio anguillarum

Strain HI610

serovar O2a

Avirulent

Vibrio anguillarum

Strain 43

serovar 010

20

40

60

80

100

120

0 1 2 3 4 5 6 7 8 9 10 11 12 13

time (day)

surv

ival

(%

)

blank

challenge


slide 8 of 39

Steering host-microbial interactions

• Stimulating the host’s immune response– yeast cell wall-bound glucan– heat shock proteins

• Influencing microbial numbers or activity – polyhydroxybutyric acid– quorum sensing


slide 9 of 39

Yeast cell wall-bound glucanas

immunostimulant?

Yeast cell wall-bound glucanas

immunostimulant?


slide 10 of 39

MannoproteinsMannoproteins

(1,6) glucans(1,6) glucans

(1,3) glucans(1,3) glucans

ChitinChitin

1616%%

7575%%

9%9%

mnn9mnn9 – exp – exp YNBYNB

2%

55%

43%

WTWT- - exp YNBexp YNB

(adapted from EUROCELLWALL project from EU)

Plasma membranePlasma membrane

Since -glucans-glucans are well-known immunostimulants, possibly the mnn9 yeast acts as immunostimulant, allowing Artemia to be protected against pathogens

Yeast mutants with altered cell wall composition


slide 11 of 39

Su

rviv

al (

%)(

da y

6)

Artemia survival in GART

0

10

20

30

40

50

60

70

80

90

WT mnn9 mnn6 fks1 knr4 kre6 gas1 chs3 LVS3

yeast strains

untreated+VC

treated+VC


slide 12 of 39

Non-gnotobiotic: mussel larvae

Survival

14 28 49 39 500

10

20

30

40

50

60

70

80

100% 90/10 80/20 70/30 60/40

%

Survival of 2 week old larvae

Algae100%

60%

Mnn9 yeast0% 40%


slide 13 of 39

Heat shock proteins as

immunostimulants?

Heat shock proteins as

immunostimulants?


slide 14 of 39

DnaK

DnaK (HSP70) overexpression:

E. coli strain YS2

YS1: control strain, no DnaK overproduction

YS2: positive strain, DnaK overproduction by arabinose induction

DnaKWB


slide 15 of 39

Enhanced resistance by DnaK feeding in a Enhanced resistance by DnaK feeding in a Vibrio challengeVibrio challenge

• Survival of Artemia larvae fed either induced or non-induced negative control strain YS1 was low.

• Survival of non-induced YS2 strains as in negative control

• A significant increase in survival in larvae fed with arabinose-induced DnaK overproducing YS2 were exposed to V. campbellii

DnaK

Priming

(g)HOST

HOST + MAMPs

HOST + MAMPs + pathogen

MiC’s, heat shock proteins (DnaK),..

16

Priming the innate immune system:PO expression in Artemia as modulated by DnaK

and Vibrio

YS2(-): E coli not overproducing DnaK (HSP70 homolog)YS2(+): E coli overproducing DnaK (HSP70 homolog)VC: Vibrio campbellii challenge with a delay of 6 h

17


slide 18 of 39

• Exogenous HSPs feeding possibly triggers the Artemia innate immune response, producing anti-inflammatory activity (Phenoloxidase activity) which suppresses infection

• Sofar, no confirmation under non-gnotobiotic conditions

CONCLUSIONS HSPsCONCLUSIONS HSPs


slide 19 of 39

POLY-β-HYDROXYBUTYRATE (PHB) POLY-β-HYDROXYBUTYRATE (PHB)


slide 20 of 39

• Linear polymer of β-hydroxybutyric acid

POLY-β-HYDROXYBUTYRATE (PHB)

OH

O

O

O

O

O

OH

n103-106

Could PHB also be used to protect Artemia from luminescent vibriosis?


slide 21 of 39

H+H+H+

H+

H+

Energy

bacterium

proton pump

fatty acid

• Short-chain fatty acids (SCFA): formic, acetic, propionic, butyric and valeric acid

• Known to inhibit growth of enteric bacteria (Salmonella, Klebsiella, Escherichia coli)

– Acidification of cytoplasm

– Energy needed to keep internal pH optimal

– Effect is pH-dependent (lower pH → higher effect)

SHORT-CHAIN FATTY ACIDS


slide 22 of 39

• Starved nauplii without feed or with PHB particles

No PHB PHB

Light microscopy

Fluorescence microscopy (Nile Blue)

(bar = 250 µm)

The PHB particles are ingested by the nauplii

PHB UPTAKE BY ARTEMIA


slide 23 of 39

0

20

40

60

80

100

120

0 1 2 3

Time (days)

Art

em

ia s

urv

ival (%

) No feedPHB

• Sterile Artemia nauplii: no feed added or only PHB particles (1 g/l) at day 0

Longer survival with PHB particles

The nauplii can obtain

energy from the particles

The particles must be

(partially) degraded in

the gut

EFFECT ON STARVED ARTEMIA


slide 24 of 39

An Artemia – Macrobrachium food chain example

PHB: non-gnotobiotic


slide 25 of 39

241

65A

78a

502

82B

89b

0

10

20

30

40

50

60

70

80

90

100

Day 5 Day 10 Day 15

Rearing time

Su

rviv

al (

%)

Control PHB

Macrobrachium larval survival feeding on PHB enriched Artemia nauplii


slide 26 of 39

a Ax

X

b

B

y

Y

b

B

y

Y

c

C

z

Z

0

10

20

30

40

50

60

70

80

90

Day 10 Day 15 Day 20 Day 28

Rearing time

Su

rviv

al (

%)

PHB combined with a classical HUFA enrichment

• No PHB, no Hufa

• PHB, no Hufa

• No PHB, Hufa

• PHB, Hufa


slide 27 of 39

• PHB particles protect Artemia from luminescent vibriosis

• Positive effects in the aquaculture food chain: but further verification is needed.

CONCLUSIONS PHBCONCLUSIONS PHB


slide 28 of 39

Quorum sensingQuorum sensing


slide 29 of 39

• QS: a mechanism by which bacteria regulate gene

expression in response to their population density by

producing, releasing and detecting small signal molecules

(quorum sensing molecules) (Fuqua et al., 1997).

• QS: process of bacterial cell-to-cell

communication/conversation with signal molecules

What is Quorum Sensing (QS)?What is Quorum Sensing (QS)?


slide 30 of 39

• Many bacterial behaviors are regulated by quorum sensing

– luminescence– Symbiosis– Virulence– Antibiotic production– Biofilm formation

What type of processes are under the control of QS?What type of processes are under the control of QS?


slide 31 of 39

QS molecules: acyl homoserine lactones (AHL)QS molecules: acyl homoserine lactones (AHL)

N-Butyryl-DL-homoserine

lactone (C4-HSL)

N-Hexanol-DL-homoserine

lactone (C6-HSL)

N-Heptanoyl-DL-homoserine

lactone (C7-HSL)

N-Octanoyl-DL-homoserine

lactone (C8-HSL)


slide 32 of 39

Can we demonstrate that quorum

sensing is important in microbial

interference with (larval) stage of

aquatic animals?

What is Quorum sensing (QS)?What is Quorum sensing (QS)?


slide 33 of 39

An Artemia – Macrobrachium food chain example

Quorum sensing: non-gnotobiotic


slide 34 of 39

Effect of AHL mixture in Artemia – Macrobrachium

food chain

Treatments Survival LSI

Control 70.0± 4.2b 5.3 ± 0.4b

AHLmix1 49.2 ± 2.6a 4.8 ± 0.3a

1

• Survival of Macrobrachium larvae on day 7 post-hatch, (mean ± SD, n = 6).

• daily AHL addition of 1 mg/l


slide 35 of 39

Effect of AHL and EC5D on Macrobrachium larviculture:

survivalSurvival rate on day 8

77.7c77.1c77.3c

61.5bc

48.5ab

37.3a

3

13

23

33

43

53

63

73

83

AHL+EC5 EC5 AHL+LVS3 LVS3 AHL Control

Treatment

Su

rviv

al r

ate

(%)

•daily AHL addition of 1 mg/l


slide 36 of 39

Effect of AHL and EC5D on Macrobrachium larviculture: LSI

Larval Stage Index (day 8)

5.0c5.2c

5.1c

4.6b4.6b

4.4a

3.0

3.5

4.0

4.5

5.0

5.5

AHL+EC5 EC5 AHL+LVS3 LVS3 AHL Control

Treatment

LS

I

•daily AHL addition of 1 mg/l

• AHL degradation by pure Bacillus strains isolated from shrimp (LT3, LT12) and sea bass (LCDR16)

ENZYMATIC AHL INACTIVATION

c

b

aa

0

10

20

30

40

50

C– C+ D+ L+

Treatment

Su

rviv

al (

%)

• Use of signal-degrading bacteria as probionts, e.g. in Macrobrachium larvae:

ENZYMATIC AHL INACTIVATION

Untreated

V. harve

yi

AHL degraders

V. harve

yi + A

HL

degraders

Is this effect by AHL degradation?


slide 39 of 39

Quorum sensing: conclusionsQuorum sensing: conclusions

•QS is important in host-microbial interactions in the aquatic environment

•Data on in vivo QS molecule concentration are mostly lacking, necessary to further substantiate QS importance for an aquaculture setting


slide 40 of 39

General conclusionsGeneral conclusions

Quantitative analysis of

the bacterial community

Quantitative analysis of

the bacterial community

Biochemical analysis

e.g.antimicrobial substances

Biochemical analysis

e.g.antimicrobial substances

Host gene expression analysis

Marker genes

Host gene expression analysis

Marker genes

Fish and shellfish Fish and shellfish larvae validationlarvae validationFish and shellfish Fish and shellfish larvae validationlarvae validation

Gnotobiotic Artemiatest

system

Gnotobiotic Artemiatest

system

Quorum sensing analysis Probiotic bacteria

Pathogenic bacteria

Feeds

Antimicrobial PeptidesImmunostimulantsQuorum sensing

analysis Probiotic bacteriaPathogenic

bacteria

Feeds, eg PHB

Antimicrobial PeptidesImmunostimulants

PerformancePerformance

Artemiamodelsystem

GnotobioticArtemiamodel

system

Gnotobiotic

MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE: FROM GNOTOBIOTIC EXPERIMENTS TO...

Documents

Transcript of MICROBIAL MANAGEMENT IN FISH AND SHELLFISH LARVICULTURE: FROM GNOTOBIOTIC EXPERIMENTS TO...