Functional genomics and stress responses in aquacultured fish

Functional genomics and stress responses in aquacultured fish

Lluís Tort

Research Group in Stress and Immunophysiology in AquacultureUniversitat Autonoma de Barcelona

Dpt. Cell Biology, Physiology and ImmunologyUnitat Fisiologia AnimalBergen, Sept 2007

AQUAGENOME MEETING. Bergen, 20-21 september 2007

Research in stress responses using genomic technology

through 2 approaches:


The wholistic approach using microarrays to search for molecular signatures and gene patterns following stress treatments.

The gene descovery/gene indication approach to identify specific genes as indicators of a stressed status.

Available genomic platforms related to stress and fish A number of initiatives, under public EU funds, have been undertaken to build genomic know-how and platforms, most of them including somehow stress assessment in fish.

BassmapBridgemapMarine genomicsStressgenesAquagenomeAquafuncAquafirstEurocarpWealthFastfishReprofishAquabreedingEadgeneWellfish

There is a real need to share the information obtained and perhaps to agree in building technological genomic platforms to reach efficiency in EU genomic research and development


-Is it suitable to determine the stress response in fish through genomics? -Are we taking advantage of this approach?-What has been achieved so far?


Gracey, Somero et al., 2001 hypoxiaKawano et al., 2003 cortisolTon et al., 2003 hypoxiaGracey, Cossins et al., 2004 coldSarropoulou et al., 2005 cortisolKrasnov et al., 2005 handlingMackenzie et al., 2006 cortisol-LPSKassahn et al., 2007 heatCairns et al., 2007 handlingMomoda et al., 2007 hypoxia/handlingWiseman et al., 2007 handling

Selection of published papers on genomic responses after environmental and husbandry stressors in fish.


HYPOXIAGracey, Somero et al., 2001

Ton et al., 2003

TEMPERATUREGracey, Cossins et al.,2004 (cold)

Kassahn et al., 2007 (heat)

HANDLINGKrasnov et al., 2005Cairns et al., 2007

Momoda et al., 2007Wiseman et al., 2007

CORTISOL TREATMENTKawano et al., 2003

Sarropoulou et al., 2005Mackenzie et al., 2006

STRESSORS

Gracey, Troll and Somero, 2001Hypoxia-induced gene expression in the euryoxic fish Gillicthys mirabilisPNAS, 98(4).

- Stressor: Hypoxia, 90 minutes hypoxia 8 to 0,8 mg/L oxygen- Species: Gillicthys mirabilis- Tissue: liver, muscle, brain- Timing: 90 minutes hypoxia 8 to 0,8 mg/L oxygen


- Stressor: Hypoxia (5% oxygen)- Species: zebrafish- Tissue: embryos- Timing: 24h hypoxia

Ton et al., 2003Gene expression profile in zebrafish exposed to hypoxia during developmentPhysiol. Genomics, 14.

RESULTS ON HYPOXIA

-Reduction in protein synthesis and locomotion involved genes in muscle-Increase in anaerobic processes and gluconeogenesis in liver-Reduction in cell growth, cell proliferation and protein synthesis-Increase in defense and chaperone molecules

- Stressor: Cold (30ºC to 23ºC or 10ºC at 7ºC/day)- Species: carp- Tissue: gill, kidney, brain, heart, muscle, liver, intestine- Timing: 21 days

Gracey, Cossins et al., 2004Coping with cold. Transcriptomic analysis in carpsPNAS, 101.


Kassahn et al., 2007Identification of early gene response to heat stress.Molecular Ecology, 16

- Stressor: Heat treatment- Species: coral reef fish (Pomacentrus moluccensis)- Tissue: kidney- Timing: 3 hours

TEMPERATURE

-There is a common gene response after temperature changes but also specific changes depending on the tissues-Decrease of energy consumption processes after heat and increase of stress related responses (HsP, chaperones).-Modulation of energetic resource driving in the brain and resource processing in the liver

- Stressor: acute handling- Species: trout- Tissue: brain and kidney- Timing: 1-24h


Gene expression in trout after handlingstressKrasnov et al., 2005. BMC Genomics, 6

Cairns, Pottinger et al., 2007Assessment of gene expression in trout in response to handling and confinementComp. Biochem. Physiol. D

- Stressor: Handling and confinement- Species: trout- Tissue: liver- Timing: early and late: 2h-21 days(Stressgenes)

- Stressor: hypoxia and handling- Species: trout- Tissue: liver- Timing: 0,5-3-24h post stress

Momoda, Schreck et al., 2007Liver gene expression in trout during the stress response to hypoxia and handlingComp. Biochem. Physiol, D

- Stressor: acute, 3 min handling.- Species: trout- Tissue: liver- Timing: 1-24h

Wiseman, Vijayan et al., 2007.Gene expression during recovery from an acute stressor (handling)Comp. Biochem. Physiol. D


HANDLING

BRAIN: Increase in metalloproteins and energy driving processes (catabolism)Decrease of immune defense genes

LIVER: Decrease of HsPUpregulation of immune related genesGenes involved in energetics and reprogramming liver machineryResponsive genes to corticosteroids

- Stressor: Cortisol (1micromolar)- Species: carp- Tissue: head kidney macrophages- Timing: 8h


Kawano et al., 2003Analysis of gene expression in carp treated with cortisolComp. Biochem. Physiol. B. 136

- Stressor: cortisol (implant 10mg/kg)- Species: seabream- Tissue: embryos, and kidney (juveniles)- Timing: 72h

Sarropoulou et al., 2005Gene expression profile in seabream during development and detection of stress (cortisol) related genes.Physiol. Genomics, 23.

-Stressor: LPS (10ug/mL)cortisol (600ng/mL)

- Species: trout- Cells: head kidney macrophages- Timing: 6-24 h

Transcriptional analysis in response to LPS treatment and cortisol in troutMackenzie et al., 2006Molecular Immunology, 43


CORTISOL

Upregulation of energetic metabolism (enolase, fructose phosphatases)Upregulation of protein biosynthesisUpregulation of ion transport (Na-K-ATPases)Down regulation of cell reorganization and growth (Iron metabolism)

Opposite action on genes regulated by pathogens-Inhibition of 53% of the genes activated by LPS-Activation of 78% of the genes suppressed by LPS

Searching for the stress response in fish through genomics- Overall rediscovery of the importance of the metabolic and energetic rearrangements after stress.- Immune-regulated genes- Cell growth/cell proliferation related genes- Genes discovered or rediscovered

But still relevant challenges to take into acount:-Adequate biological model:

fish (scope of the responses)environmental/stress tolerance capacities, behaviourbackground

tissues/ cells (active vs. reactive) regulatory organs responding organs

-Treatment -Timingintensity time of responsesstressor nature time of sampling


time

magnitude

seconds minutes hours days monthsAdrenalineNervousGene expression

CortisolImmuneOsmotic

MetabolicImmune

Performance: disease resistancegrowthreproduction

4) time course of the physiologic stress response

Different cells and tissuesinvolved.Adequate timingshould be chosen.


brain muscle


Regulatory organsPortals of entry/environment contactImmune responding organsReaction/recovery organs

Environmental conditions

Time-course of changes in plasma cortisol level during chronicconfinement in LR and HR trout (Pottinger et al., 2002)

0 2 3 45 7 24 48 168 336

Plas

ma c

ortis

ol (n

gm

l)

0

50

100

150

200

250

300

350

400

HR

LR

hours

Variability of parameters depending on the individual / group differences


Individual variation in basal and post-stress levels(Martins et al., 2006)

78% Homology human enolase

New stress indicators: Gene discovery. An example: Enolase

Phylogeneticanalysis

ENOLASEENOLASENOLASEE

Heat shock protein, Iida H. et al., 1985 (Nature)Lens t Protein,Wistow, G.J. et al., 1988 (J. Cell. Biol.)Cancer, Autoimmune disorders, Bacterial diseases

Surface receptor /plasminogenMiles et al., 1991 (Biochem.J)

Transcriptional RegulatorHomology with DNA union protein Ray etal.1995 (Cancer Res.)

Glycolisis

2-fosfoglicerate

Fosfoenolpiruvate+H2O

2-fosfo-D gliceratehidrolase/ENOLASE


Enolase expressed differentially after chronic stress or LPS injection (Ribas et al. 2004. Aquaculture)Enolase has been shown to be regulated (up regulated in 84% of experiments) after metaanalysis of 34 experiments with microarrays

Enolase

S18

C24 24 C72 72

Red muscle Brain Gut LiverHead Kidney HeartWhite muscle

C24 24 C72 72 C24 24 C72 72 C24 24 C72 72 C24 24 C72 72 C24 24 C72 72 C24 24 C72 72


Genomics-Stress-Physiology-Aquaculture

-The stress response is an evolutive phenomenon that involves allphysiological compartments and all regulatory processes.-Severe/acute stressors may induce molecular signatures that may be identified, but chronic subacute (aquaculture-related) stressors may just modulate processes (more difficult to identify).-We probably need to better distinguish between regulatory processes and reactive processes, when identifying new stress indicators.

-In aquaculture it will be necessary to address the precise specific models to avoid a bulk of non-relevant genomic information.

-There is a need for a stronger link between genetics and genomics in aquaculture industry to look for specific stress-susceptible / stress-resistant / stress responsive strains.


Documents

Functional genomics and stress responses in aquacultured fish