Biomarker Platform for Commercial Aquaculture Feed Development project: - PowerPoint Presentation

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Biomarker Platform for Commercial Aquaculture Feed Development project: - PPT Presentation

New findings and methods for feed engineering to face present and future challenges in salmon aquaculture Presenter Albert Caballero Solares PhD Collaborators X Xue 1 JR Hall 2 ID: 749319

abp diet biomarker veg diet abp veg biomarker project gapp levels microarray liver dha epa transcriptome 3lc1 med immune

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Biomarker Platform for Commercial Aquaculture Feed Development project: New findings and methods for feed engineering to face present and future challenges in salmon aquaculture

Presenter:Albert Caballero Solares, Ph.DCollaborators:X. Xue1, J.R. Hall2, K. Eslamloo1, N.C. Smith1, S.M. Inkpen1, T. Katan1, J. Santander1, C.C. Parrish1, R.G. Taylor2 and M.L. Rise11Department of Ocean Sciences, Memorial University of Newfoundland, 1 Marine Lab Road, St. John's, NL A1C 5S7, Canada. 2Cargill Innovation, 4335 Dirdal, NorwaySlide2

Biomarker Platform for Commercial Aquaculture Feed Development projecta Genomic Applications Partnership Program (GAPP)

Funded by the Government of Canada through Genome Canada and Genome AtlanticTeam:Slide3

GAPP Biomarker Project: Experimental WorkflowSlide4

GAPP Biomarker Project: Experiments conducted

Feeding trial I(Raw materials)Feeding trial II(ω3/ω6 ratios)Nutrition

Immunity

HK RNA

Fish injected with PBS, poly(I:C)

qPCR

Macrophage RNA

Treated with PBS, poly(I:C)

Microarray/qPCR/Lipid composition

Hepatocytes RNA

Treated

tBHP

Microarray

qPCR

Liver RNA

(FM/FO

)

Microarray

qPCR

Lipid composition

Liver RNA

(EPA+DHA)

Microarray

qPCR

Lipid composition

Liver RNA

(

ω3/ω6

ratios

)

Microarray

qPCR

Lipid composition

HK RNA

Fish injected with PBS, poly(I:C), ASAL antigen

qPCR

Macrophage RNA

Treated with PBS, poly(I:C), LPS

Microarray/qPCR/Lipid composition

Feeding trial III

(

ISAv

challenge)Raw materialsFFI supplement

HK RNAISAv-exposed salmon (cohabitation)MicroarrayqPCR

Feeding trial IV

(SRS challenge)

No dietary component

HK RNASRS-exposed salmon (IP-injection)MicroarrayqPCR

In vitro FFI testing

Antioxidants

Primary HK macrophage cultures

Primary hepatocyte cultures

Immunostimulants

Primary HK macrophage culturesSlide5

GAPP Biomarker Project: Feeding trial ISlide6

GAPP Biomarker Project: Feeding trial I (2015)

Diet 1MARFO VOFM ABP VEG

Diet 2

Med-MAR

ABP

VEG

Diet 3

ABP

VEG

Diet 4

VEG

ABP

VEG

Diet 5

3LC0

ABP

VEG

Diet 6

3LC1

FM ABP VEG

Diet 7

ω3LC1.4FM ABP VEG

+ Lipid and fatty acid composition

iver transcriptome

Agilent 44K salmonid oligonucleotide microarray

epatocyte response to oxidative stressAgilent 44K salmonid oligonucleotide microarray

Antiviral immune response

(head kidney)

eal-time

quantitative polymerase chain reaction

Effects of FM/FO replacement on Slide7

GAPP Biomarker Project: FM/FO replacement on liver transcriptome

Diet 1MAR

FO VO

ABP VEG

Diet 2

Med-MAR

ABP

VEG

Diet 3

ABP

VEG

Diet 4

VEG

ABP

VEG

Diet 5

ω3LC0FM ABP

VEGVODiet 6ω3LC1FM ABP

VEG

FO VO

Diet 7ω3LC1.4FM ABP VEG

VOABP: down-regulated glycolysis correlated with feed intake (p<0.05)  and

growth (p<0.05)VO-diets (ABP, VEG): up-regulated cholesterol synthesisVO-diets (ABP, VEG): up-regulated FA mobilization for β-oxidation

cholesterol synthesis found

inversely correlated to saturated FA in liver

(Manuscript in prep.)Slide8

GAPP Biomarker Project: FM/FO replacement on liver transcriptome

Diet-responsive antiviral transcriptsHigher constitutive levels in the liver of salmon fed VEG diet

Diet 1

MAR

ABP

VEG

Diet 2

Med-MAR

ABP

VEG

Diet 3

ABP

VEG

Diet 4

VEG

ABP VEGFO

VODiet 5ω3LC0FM ABP VEG

Diet 6ω3LC1FM

ABP VEGFO VO

Diet 7

3LC1.4FM ABP VEGFO

VO(Manuscript in prep.)Slide9

GAPP Biomarker Project: FM/FO replacement on HK transcriptome

Head kidney: diet-responsive antiviral transcriptsStronger poly(I:C) induction in the head kidney of salmon fed VEG dietSlide10

GAPP Biomarker Project: Feeding trial I (2015)

Diet 1MARFO VOFM ABP VEG

Diet 2

Med-MAR

ABP

VEG

Diet 3

ABP

VEG

Diet 4

VEG

ABP

VEG

Diet 5

3LC0

ABP

VEG

Diet 6

3LC1

FM ABP VEG

Diet 7

ω3LC1.4FM ABP VEG

Antiviral immune response (macrophages)

Agilent 44K salmonid oligonucleotide microarray

iver transcriptome

Agilent 44K salmonid oligonucleotide microarray

Effects of EPA+DHA levels on+ Lipid and fatty acid composition Slide11

GAPP Biomarker Project: EPA+DHA levels on liver transcriptome



dietary EPA and DHA



desaturases

Diet 1

Marine

ABP

VEG

Diet 2

Med-MAR

ABP

VEG

Diet 3

ABP

VEG

Diet 4

VEG

FM ABP VEGFO VO

Diet 5

ω3LC0FM ABP VEG

VODiet 6ω3LC1

ABP VEGFO

VODiet 7ω3LC1.4FM ABP VEG

FO VO

Figures from Xi Xue (in prep.)Slide12

GAPP Biomarker Project: EPA+DHA levels on liver transcriptome



dietary EPA and DHA



interferon pathway.

Diet 1

Marine

ABP

VEG

Diet 2

Med-MAR

ABP

VEG

Diet 3

ABP

VEG

Diet 4

VEG

FM ABP VEGFO VO

Diet 5

ω3LC0FM ABP

VEGVODiet 6ω3LC1

ABP VEG

FO VODiet 7ω3LC1.4FM ABP VEG

FO VO

Figures from Xi

Xue

(in prep.)Slide13

GAPP Biomarker Project: Feeding trial II (2016)

Diet 1High ω3ω3:ω63:1

Diet

2Med ω

Diet 3

Balanced

Diet 4

Med

1:2

Diet 4

High

1:3

iver transcriptome

Agilent 44K salmonid oligonucleotide microarray

Antiviral immune response (head kidney)

eal-time quantitative polymerase chain reactionEffects of ω3/ω6 levels on Antiviral immune response (macrophages)Agilent 44K salmonid oligonucleotide microarraySlide14

GAPP Biomarker Project: effects of ω3/ω6 levels on

Figures from Tomer Katan (in prep.)Muscle FA compositionSlide15

GAPP Biomarker Project: effects of ω3/ω6 levels on

Figures from Tomer Katan (in prep.)Liver transcriptome: 44K microarray experimentCommon Reference(equal contribution of RNA from each individual)

High

3 diet

High

6 diet

Serine protease HTRA1 precursor

cell proliferation and apoptosis.

Integrin beta-5 precursor

immune function.

Peroxisomal proliferator-activated receptor A-interacting complex 285

kDa

protein

responsive to virus and

pIC

Cytochrome c oxidase subunit 1 and 2

mitochondrial respiratory chain complex.

Preliminary data:

Diet 1

High

3:1

Diet 2Med ω3

3:ω

62:1

Diet 3Balanced

ω3:ω

61:1

Diet 4

Med ω6

ω6

1:2

Diet 4

High

1:3Slide16

GAPP Biomarker Project: effects of ω3/ω6 levels on

HK immune response to viral and bacterial PAMPsDiet 1High ω3ω3:ω6

3:1

Diet

Med

Diet 3

Balanced

Diet 4

Med

1:2

Diet 4

High

1:3Slide17

GAPP Biomarker Project: effects of ω3/ω6 levels on

HK immune response



6 FAs in diet



immune response

= ARA

levels across diets

but

(Manuscript in prep.)Slide18

GAPP Biomarker Project: effects of ω3/ω6 levels on

HK immune response



6 FAs in diet



cathepsin

and



Lysozyme C-2

(Manuscript in prep.)Slide19

GAPP Biomarker Project: effects of ω3/ω6 levels on

HK inflammatory response



6 FAs in diet:

pro-inflammatory

However, evidence of

fast recovery

(Manuscript in prep.)Slide20

GAPP Biomarker Project: Transcriptome & desired phenotypes

Diet-responsive genes related tonutritionDiet-responsive genes related toimmunityGrowth

Composition

(e.g. EPA &DHA)

Resistance to pathogensSlide21

GAPP Biomarker Project: Transcriptomic data obtainedSlide22

GAPP Biomarker Project: Selecting predictive gene biomarkers

gck, pfkfb5,6pgd,adssl1c,acac, idi1,

sqs,

dgat2a,

dgat2b,

fabp3a

fabp3b,

sgk2a

sgk2b

htra1a

htra1b

mxb

mxa

ifit5

mhcI

, lect2a, lect2b

igd

igma

igmb

, mtco1

Stepwise discriminant

analysis

(qPCR data of

25 transcripts)

Changes in relevant metabolic pathways

Changes in immune transcriptome

Included

DiscardedSlide23

Grouping variable

Biomarkers includedFunction% of varianceWilks' LambdaAccuracy (%)λ

χ2

p-value

Diet

sqs

gck

, sgk2b,

igd

79.5

0.116

57.1

8.0

<0.001

20.5

0.524

17.1

3.0

0.001

Growth (High, Low)

sgk2b, ifit5, mxb, htra1a

100

0.40224.6

4<0.00184

EPA+DHA (High, Low)

fabp3a, igd1

100

0.717

7.98

0.019

Results from discriminant analyses after categorizing individuals by

diet

growth

, and

muscle EPA+DHA levels

GAPP Biomarker Project: Predict desirable phenotypesSlide24

GAPP Biomarker Project: Multiplex platformsSlide25

GAPP Biomarker Project: Liver multiplexes

Liver multiplex I and II: primers for 48 gene biomarkersGenomeLab GeXPTM Analysis systemGood correlation of single and multiplex dataSlide26

GAPP Biomarker Project: Liver multiplexes

Validate previous findingsConfirm hypothesisExplore other pathwaysAnd,4. Generate predictive modelsSlide27

Results from discriminant analyses after categorizing individuals by diet, growth, and muscle EPA+DHA levels

GAPP Biomarker Project: Predict desirable phenotypesGrouping variableBiomarkers includedFunction

% of varianceWilks' Lambda

Accuracy (%)

p-value

Diet

pparb1,

apoaI

76.7

0.318

23.5

4.0

<0.001

23.3

0.722

6.7

1.0

0.010

Growth (High, Low)

pparb1,

igmb

, igfb5b1, pfkfb1, srebp2

100

0.866

27.1

<0.001

EPA+DHA (High, Low)

srebp1,

igmb

100

0.705

11.70

0.003

Slide28

GAPP Biomarker Project: ConclusionsSlide29

Thank you!

Biomarker Platform for Commercial Aquaculture Feed Development project: - PowerPoint Presentation

Biomarker Platform for Commercial Aquaculture Feed Development project: - PPT Presentation

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