Salmon lice (Lepeophtheirus salmonis) were reared as earlier described [10 (link)]. After development to the adult stage, 15 lice were removed with forceps from their anaesthetised (80 μg/ml benzocaine) salmon hosts (Salmo salar) and 3 lice were stored in RNA later (Ambion). The remaining 12 lice were starved in incubators with flowing seawater for 14 days. After starvation, 3 lice were sampled and stored as described above, and the remaining 9 lice were put in a tank with uninfected salmon where they could settle on their salmon hosts and resume feeding. After 15 days on their new hosts 3 lice were sampled and stored as described above. The experimental procedures were carried out in accordance with national regulations for use of animals in scientific research.
The transcript levels of LsTryp1 [10 (link)] and the reference gene eEF1α [11 (link)] in 1 selected unstarved, starved and refed lice were determined by quantitative real time PCR carried out with 3 parallels at 5 sequential 2-fold dilutions as previously described [10 (link)]. The RNA purification protocol is previously described [10 (link)] and cDNA syntheses were performed using MultiScribe™ according to the manufacturers recommendations (Applied Biosystems). The Q-PCR results were analysed by the 2-ΔΔCT method as earlier described [10 (link)] and a method adjusting for PCR efficiency differences described by Peirson et al. [2 (link)]. The latter analysis was performed partially in the DART-PCR Excel spreadsheet [2 (link)]. When using the 2-ΔΔCT method, at least 2 parallels were required at each dilution. Parallels were removed when the CT value differed more than 0.3 (CT<32) or 0.4 (CT = 32) from the most similar parallel at the same dilution. At least 4 dilutions were required for each stage. The resulting data were calibrated to unstarved lice and analysed as described by Kvamme et al.[10 (link)]. When using the "DART-method", dilutions were removed when PCR efficiency differed significantly (one way ANOVA, α = 0.05) from the other dilutions. The signal corresponding to the initial template concentration (R0) was derived using the average PCR efficiency for LsTryp1 and eEF1α when the PCR efficiencies were not significantly different (one way ANOVA, α = 0.05). When the PCR efficiency differed significantly, R0 was calculated using individual gene specific mean efficiencies. The mean R0 for each dilution of LsTryp1 was normalised to corresponding eEF1α values. The normalised R0 values were calibrated to the values for unstarved lice. 95% confidence intervals (CI) were derived from normalised R0 values.
The transcript levels of LsTryp1 [10 (link)] and the reference gene eEF1α [11 (link)] in 1 selected unstarved, starved and refed lice were determined by quantitative real time PCR carried out with 3 parallels at 5 sequential 2-fold dilutions as previously described [10 (link)]. The RNA purification protocol is previously described [10 (link)] and cDNA syntheses were performed using MultiScribe™ according to the manufacturers recommendations (Applied Biosystems). The Q-PCR results were analysed by the 2-ΔΔCT method as earlier described [10 (link)] and a method adjusting for PCR efficiency differences described by Peirson et al. [2 (link)]. The latter analysis was performed partially in the DART-PCR Excel spreadsheet [2 (link)]. When using the 2-ΔΔCT method, at least 2 parallels were required at each dilution. Parallels were removed when the CT value differed more than 0.3 (CT<32) or 0.4 (CT = 32) from the most similar parallel at the same dilution. At least 4 dilutions were required for each stage. The resulting data were calibrated to unstarved lice and analysed as described by Kvamme et al.[10 (link)]. When using the "DART-method", dilutions were removed when PCR efficiency differed significantly (one way ANOVA, α = 0.05) from the other dilutions. The signal corresponding to the initial template concentration (R0) was derived using the average PCR efficiency for LsTryp1 and eEF1α when the PCR efficiencies were not significantly different (one way ANOVA, α = 0.05). When the PCR efficiency differed significantly, R0 was calculated using individual gene specific mean efficiencies. The mean R0 for each dilution of LsTryp1 was normalised to corresponding eEF1α values. The normalised R0 values were calibrated to the values for unstarved lice. 95% confidence intervals (CI) were derived from normalised R0 values.
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