Agilent quality metrics were used to remove low quality features such as saturated or non-uniform features and population outliers. The expression data was then normalised using Lowess normalisation of the log2-expression ratios without baseline transformation.
Raw microarray images were captured using the Axon Genepix 4200A scanner with Genepix Pro 6.1 image acquisition software, using 40% laser power, 5µm pixel size resolution and auto photo-multiplier tube (auto-PMT) function with 0.05 saturation tolerances. The images were then processed using the Agilent Feature Extraction (FE) software version 18.104.22.168.
A total of 300 ng test aRNA, labelled with Cy 3, was competitively hybridised to a 15K custom L. salmonis microarray with 300 ng Cy5 labelled reference pool aRNA, according to the Agilent technologies Two-colour Microarray-based gene expression analysis protocol version 6.5
500ng total RNA was used for amplification of synthetic RNA with incorporation of the modified nucleotide 5-(3-aminoallyl)-UTP (aaUTP)using the AminoAllyl MessageAmp II kit (Ambion, UK), the synthetic RNA was then labelled with cyanine 3 or 5 mono-reactive dye (GE Healthcare, UK) using a dye coupling reaction. Unincorporated dye was removed by column purification (Illustra Autoseq™ G-50 spin columns; GE healthcare, UK), and then dye incorporation was assessed by spectrophotometry (NanoDrop ND-1000, Thermo Scientific, USA) and fluorescent gel electrophoresis.
A common reference pool was created through pooling equal amounts of all aRNA test samples to be used in the experiment, prior to labelling this pool with cyanine 5 mono-reactive dye (GE Healthcare, UK) using a dye coupling reaction.
Four whole adult male salmon lice, for each treatment group, were ground under liquid nitrogen and total RNA was purified using the modified TRI-reagent method as recommended for samples with high polysaccharide content (Chomczynski and Mackey, 1995). The total RNA was resuspended in nuclease-free water and further purified using RNeasy columns (Qiagen, UK).
Whole salmon lice were preserved in RNA stabilisation solution (4.54M ammonium sulphate, 25mM trisodium citrate, 20mM EDTA, pH 5.4) prior to storage at -70°C for total RNA extraction.
Atlantic salmon were anaesthetised with 2-phenoxyethanol (100 mg/L ), adult male salmon lice were then removed from host fish and allowed to recover in aerated seawater for ~2 hours prior to the exposure experiment. Salmon lice from both strain were subjected to short term (1 and 3 hours) exposures to 200 µg/L EMB, a concentration that would result in >95% immotility in S lice after 24 hours but have no effects in PT lice. In addition, the experiment comprised seawater and solvent (0.01% (v/v) PEG300) controls. For each combination of strain, exposure period and treatment, three pooled samples consisting of four salmon lice each were collected for later RNA extraction
Total RNA was sourced from salmon lice that were culture on the host at the University of Stirling's Marine Environmental Reasearch Laboratory (MERL). Salmon lice were maintained at MERL on Atlantic salmon (S. salar) with an initial weight of 500–1000 g in circular tanks supplied with fresh seawater at ambient temperature, using a photoperiod corresponding to natural day length. To propagate salmon louse cultures, egg strings were allowed to hatch and develop to copepodids, which were used to inoculate a tank containing fresh host fish. An emamectin benzoate (EMB) susceptible strain S was established in 2003 using salmon lice from a Scottish farm site where no chemical control agents other than hydrogen peroxide had been used. A moderately EMB-resistant salmon louse strain PT was established in December 2008 using salmon lice from another Scottish production site with reports of variable EMB treatment efficacies. These two strains have since been cultured under identical laboratory conditions.