The base ration for all trials was an Atlantic salmon extruded diet (Corey Feed Mills Ltd, 136 Hodgson Road, Fredericton, NB, Canada E3C 2G4). Emamectin benzoate (SLICE TM) premix and its quality control were provided by the manufacturer (Intervet Schering-Plough Animal Health Corporation, Summit, New Jersey, USA) to Northeast Nutrition Inc. (Truro, Nova Scotia) and Corey Feed Mills Ltd. Ivermectin (Ivomec 0.6% w/w/ DIN 01913085) premix and its quality control were also provided by the manufacturer (Merial Canada Inc., Baie-D'Urfe, Quebec, Canada) to Northeast Nutrition Inc. Medicated diet was prepared according to industry standards by the source feed mill. Prior to the initiation of the trial, the Toxicology and Analytical Services Laboratory (TASL), University of Prince Edward Island, and Maxxam Analytics, Ontario, Canada tested nominal concentrations of EMB and IVM, respectively. The nominal concentration was used to calculate the daily ration required to attain a dose of 50 µg kg-1 (1x), 100 µg kg-1 (2x), 150 µg kg-1 (3x) EMB or 50 µg kg-1 (1x IVM) body weight. Emamectin benzoate was fed for 7 consecutive days whereas ivermectin was offered twice over a seven day period for four consecutive weeks. Fish in the treatment groups were fed the medicated diet at a rate of 1.1% kg-1 body weight for the 1x EMB, 1.2% kg-1 body weight for the 3x EMB (per day for seven consecutive days) and 1.0% body weight for IVM at all applicable feeding days.
Growth protocol
Atlantic L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm located in BMA-2b in the Bay of Fundy, New Brunswick. Eggs were hatched in a static sea water hatch system and grown to copepodids at Huntsman Marine Science Centre, St Andrew's, NB, Canada.
Extracted molecule
total RNA
Extraction protocol
Lice were flash frozen individually and and kept at -80 degrees Celcius until RNA extraction. Pools of 6 preadult I lice (n = 48 pools) were included in the study (n = 6 for each treatment and sex combination, except n = 5 for control males and n = 7 for male 3xEMB). Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), on-column DNase digestion (QIAGEN) followed by RNEasy column purification as per manufacturers’ instructions (QIAGEN). Purified total RNA was tested by agarose gel and automated electrophoresis (Experion; Bio-Rad) and spec.
Label
Cy5-CTP
Label protocol
Labeled cRNA was generated from 825ng total RNA using Low-Input Quick Amp kits (Agilent; v6.5). A Cy3-cRNA reference pool to hybridize alongside samples was created for each of the above experiments, ensuring each experimental condition was included in the pool.
The base ration for all trials was an Atlantic salmon extruded diet (Corey Feed Mills Ltd, 136 Hodgson Road, Fredericton, NB, Canada E3C 2G4). Emamectin benzoate (SLICE TM) premix and its quality control were provided by the manufacturer (Intervet Schering-Plough Animal Health Corporation, Summit, New Jersey, USA) to Northeast Nutrition Inc. (Truro, Nova Scotia) and Corey Feed Mills Ltd. Ivermectin (Ivomec 0.6% w/w/ DIN 01913085) premix and its quality control were also provided by the manufacturer (Merial Canada Inc., Baie-D'Urfe, Quebec, Canada) to Northeast Nutrition Inc. Medicated diet was prepared according to industry standards by the source feed mill. Prior to the initiation of the trial, the Toxicology and Analytical Services Laboratory (TASL), University of Prince Edward Island, and Maxxam Analytics, Ontario, Canada tested nominal concentrations of EMB and IVM, respectively. The nominal concentration was used to calculate the daily ration required to attain a dose of 50 µg kg-1 (1x), 100 µg kg-1 (2x), 150 µg kg-1 (3x) EMB or 50 µg kg-1 (1x IVM) body weight. Emamectin benzoate was fed for 7 consecutive days whereas ivermectin was offered twice over a seven day period for four consecutive weeks. Fish in the treatment groups were fed the medicated diet at a rate of 1.1% kg-1 body weight for the 1x EMB, 1.2% kg-1 body weight for the 3x EMB (per day for seven consecutive days) and 1.0% body weight for IVM at all applicable feeding days.
Growth protocol
Atlantic L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm located in BMA-2b in the Bay of Fundy, New Brunswick. Eggs were hatched in a static sea water hatch system and grown to copepodids at Huntsman Marine Science Centre, St Andrew's, NB, Canada.
Extracted molecule
total RNA
Extraction protocol
Lice were flash frozen individually and and kept at -80 degrees Celcius until RNA extraction. Pools of 6 preadult I lice (n = 48 pools) were included in the study (n = 6 for each treatment and sex combination, except n = 5 for control males and n = 7 for male 3xEMB). Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), on-column DNase digestion (QIAGEN) followed by RNEasy column purification as per manufacturers’ instructions (QIAGEN). Purified total RNA was tested by agarose gel and automated electrophoresis (Experion; Bio-Rad) and spec.
Label
Cy3-CTP
Label protocol
Labeled cRNA was generated from 825ng total RNA using Low-Input Quick Amp kits (Agilent; v6.5). A Cy3-cRNA reference pool to hybridize alongside samples was created for each of the above experiments, ensuring each experimental condition was included in the pool.
Hybridization protocol
Samples were hybridized to oligonucleotide microarrays as per manufacturers' instructions for Low-Input Quick Amp kits (Agilent; v6.5). Arrays were designed using previously annotated ESTs from both Pacific and Atlantic L. salmonis (Yasuike et al. 2012; eArray design ID 024389; Agilent).
Scan protocol
Scanned at 5 micrometer resolution on a ScanArray Express (Perkin Elmer) and quantified on Imagene (v8.1; BioDiscovery).
Description
Ivermectin
Data processing
For each probe on the microarray, the background median was subtracted from the foreground median. Data analysis was performed in GeneSpring GX13 (Agilent). Each array was normalized using an intensity-dependent Lowess normalization. Only entities on the array passing quality control filter criteria for each sex and treatmet combination were retained: at least 66% of the samples in any one condition had raw signal ≥ 500 in both channels and no poor quality flags.
