Download expression of key genes related to unspecific

Cod Farming in the Nordic Countries
Grand Hotel, Reykjavík
21 September 2011
EXPRESSION OF KEY GENES
RELATED TO UNSPECIFIC IMMUNE
RESPONSES IN COD LARVAE
FOLLOWING PROBIOTIC
TREATMENT
EE Thorarinsdottir, J Johannsdottir,
Cod conference 21. september 2011
Aims of the study
• Adapt the RT-qPCR method for quantitative analysis of the
expression of selected unspecific immune related genes in cod
larvae
•
•
membrane bound IgM
g-type lysozyme
• Evaluate the effects of probiotic treatment using a mixture of
two bacterial isolates
•
•
Arthrobacter sp.
Enterococcus sp.
The research was a part of the MSc. studies of Eydis Elva
Thorarinsdottir at the University of Akureyri (MSc. in May 2010).
Jónína Þ. Jóhannsdóttir
21.09.2011
2
Introduction – RT-qPCR
• reliable, sensitive and accurate
• the method of choice for the quantitative detection of mRNA gene
expression levels
• frequently applied to measure how various compounds or
experimental conditions affect organisms at the molecular level
• Requires:
•
understanding of the PCR theory
•
different detection chemistries
•
different quantification methods and normalisation strategies
Jónína Þ. Jóhannsdóttir
21.09.2011
3
Introduction
• The production of cod larvae is restricted
•
•
•
high mortalities
high bacterial numbers
poor larval quality (deformities)
• The innate immune system represents the
first line of defence
•
•
natural IgM
Lysozyme
• Probiotics
•
•
•
•
immunomodulation
normalised intestinal microbiota composition
enhancing the nutritional value of feed
inhibit growth of pathogenes
Jónína Þ. Jóhannsdóttir
21.09.2011
4
Materials and methods
Probiotic treatment
• Bathing of larvae
• Enrichment of the live prey
•Real time PCR
•geNorm was used to evaluate the
expression
stability
of
three
housekeeping genes (RPL4, EF1-α
and ARP3) across the samples
tested.
• Normalized expression levels for
the target genes (mIgM and g-type
lysozyme) were calculated according
to Vandesompele et al. (2002).
Jónína Þ. Jóhannsdóttir
21.09.2011
5
Results
IgM
• RPL4 and EF1-α used for
normalization of GOI
• Poor larval survival (6%)
• Increased expression for
GOI prior to and at the
onset of metamorphosis
Lysozyme
• Probiotic treatment
resulted in stimulation of
mIgM expression in larvae
at 26 and 36 dph.
Jónína Þ. Jóhannsdóttir
21.09.2011
6
The experiments were carried out at the Icelandic Marine
Research Institute as a part of the Topcod and Optilar projects,
financed by The Technology Development Fund and AVS R&D
Fund of Ministry of Fisheries in Iceland.
Jónína Þ. Jóhannsdóttir
21.09.2011
7