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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