Gene Annealing Temperature (°C) Standard Curve Efficiencies Protocol CYP18 A1-like 60 1.02 3 Step Trypsin-1 65 0.99 3 Step Tissue plasminogen activator precursor-like 60 1.07 3 Step Cytochrome p450 isoform-1 like Protein 65 1.07 3 Step Peroxinectin-like 60 0.99 3 Step Leukocyte receptor cluster member 9-like 65 1 3 Step Glycene receptor α-2 like 65 0.99 3 Step Nicotinic acetylcholine receptor subunit-like 65 1.06 3 Step Ribosomal Protein S20 61 0.92 2 Step Glyceraldehyde 3-phosphate dehydrogenase 65.1 0.97 2 Step Elongation Initiation Factor 1 65.1 0.91 2 Step

Table 2: Quantitative PCR protocols used to amplify reference genes and genes of interest in Lepeophtheirus salmonis. Three step protocols consist of: One cycle of 95°C for 10 minutes, forty cycles of 95°C for 15 seconds, TAnnealing°C for 15 seconds and 72°C for 15 seconds, and one melt curve. Two step protocol consisted of: One cycle of 95°C for 10 minutes, forty cycles of 95°C for 15 seconds, TAnnealing°C for 30 seconds and one melt curve.

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