salmon type: Pacific Canada age: pre-adult lice Sex: mixed pool emb sensitivity: sensitive exposed to emb (ppb): 50
Treatment protocol
Atlantic: Stock emamectin benzoate (EMB; PESTANAL®, Sigma-Aldrich) was prepared to 100 mg/L in methanol, then diluted to working concentrations in seawater. EMB dilutions were prepared to final concentrations of 0, 0.1, 25, 300 or 1000 parts per billion (n = 4 individuals for each population/sex/dose combination). Lice were randomly distributed to beakers containing the above concentrations, and exposed for 24 hours. Pacific: As with Atlantic, but to final concentrations of 0, 25, or 50 ppb EMB (n = 6 pools per condition). Pacific: As described for Atlantic, except that lice were flash frozen together and treated with on-column DNase (QIAGEN) instead of TURBO™ DNase, and quality tested with agarose gel electrophoresis and by spectrophotometry (NanoDrop-1000) for purity and quantity.
Growth protocol
Atlantic: L. salmonis individuals were obtained from Atlantic salmon farms in Back Bay, New Brunswick, Canada (high EMB resistance) or near Grand Manan, New Brunswick, Canada (low EMB resistance). Lice collected from salmon farms were grown on fish in house at Atlantic Veterinary College (University of Prince Edward Island) for approximately 80-90 days, after which the third set of egg strings were extruded. Lice hatched from these adults (F1 generation) were grown on Atlantic salmon in house until molt to pre-adult stage. At this point, Atlantic salmon were sedated with Tricaine Methylsulfonate (MS-222) to prevent damage to lice during removal, and lice were collected and placed in Petri dishes with seawater (10 oC, 33 parts per thousand salinity). Petri dishes were swirled and any lice not adhering to the side were not used. Pacific: L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm near Campbell River, British Columbia (BC) in March 2009. Eggs were hatched, grown to copepodids, and then grown on Atlantic salmon in house at Pacific Biological Station, Nanaimo, BC, Canada. Pre-adult lice were removed from Atlantic salmon sedated with Metomidate hydrochloride (Aquacalm, Syndel Laboratories Ltd.) and held briefly in cold seawater for less than 1 hour. Approximately 25 pre-adult I and II stage male and female individuals were randomly distributed into each of 24 beakers containing 500 ml filtered seawater (10 oC; 30 parts per thousand salinity; one air stone per beaker).
Extracted molecule
total RNA
Extraction protocol
Atlantic: Lice were flash frozen individually and kept at -80 degrees Celcius until RNA extraction (n = 77 individuals). Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), treated with TURBO™ DNase treatment (Life Technologies) 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.
Atlantic: Stock emamectin benzoate (EMB; PESTANAL®, Sigma-Aldrich) was prepared to 100 mg/L in methanol, then diluted to working concentrations in seawater. EMB dilutions were prepared to final concentrations of 0, 0.1, 25, 300 or 1000 parts per billion (n = 4 individuals for each population/sex/dose combination). Lice were randomly distributed to beakers containing the above concentrations, and exposed for 24 hours. Pacific: As with Atlantic, but to final concentrations of 0, 25, or 50 ppb EMB (n = 6 pools per condition). Pacific: As described for Atlantic, except that lice were flash frozen together and treated with on-column DNase (QIAGEN) instead of TURBO™ DNase, and quality tested with agarose gel electrophoresis and by spectrophotometry (NanoDrop-1000) for purity and quantity.
Growth protocol
Atlantic: L. salmonis individuals were obtained from Atlantic salmon farms in Back Bay, New Brunswick, Canada (high EMB resistance) or near Grand Manan, New Brunswick, Canada (low EMB resistance). Lice collected from salmon farms were grown on fish in house at Atlantic Veterinary College (University of Prince Edward Island) for approximately 80-90 days, after which the third set of egg strings were extruded. Lice hatched from these adults (F1 generation) were grown on Atlantic salmon in house until molt to pre-adult stage. At this point, Atlantic salmon were sedated with Tricaine Methylsulfonate (MS-222) to prevent damage to lice during removal, and lice were collected and placed in Petri dishes with seawater (10 oC, 33 parts per thousand salinity). Petri dishes were swirled and any lice not adhering to the side were not used. Pacific: L. salmonis egg strings were obtained from adult female lice infecting Atlantic salmon in a salmon farm near Campbell River, British Columbia (BC) in March 2009. Eggs were hatched, grown to copepodids, and then grown on Atlantic salmon in house at Pacific Biological Station, Nanaimo, BC, Canada. Pre-adult lice were removed from Atlantic salmon sedated with Metomidate hydrochloride (Aquacalm, Syndel Laboratories Ltd.) and held briefly in cold seawater for less than 1 hour. Approximately 25 pre-adult I and II stage male and female individuals were randomly distributed into each of 24 beakers containing 500 ml filtered seawater (10 oC; 30 parts per thousand salinity; one air stone per beaker).
Extracted molecule
total RNA
Extraction protocol
Atlantic: Lice were flash frozen individually and kept at -80 degrees Celcius until RNA extraction (n = 77 individuals). Total RNA was extracted from frozen tissue using TRIzol reagent® (Life Technologies), treated with TURBO™ DNase treatment (Life Technologies) 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
pacific_EB50 Slide_5_SLICE_Block4.txt
Data processing
For each probe on the microarray, the background median was subtracted from the foreground median. Data analysis was performed in GeneSpring GX11 (Agilent). Each array was normalized using an intensity-dependent Lowess normalization. All entities on the array are presented in the attached matrix file here, however, for the analysis, quality control filters for each of the three experiments retained probes that pass the following criteria in at least 65% of the samples in any one condition: raw signal ≥ 500 in both channels and no poor quality flags.
Transcriptomic responses to emamectin benzoate in Pacific and Atlantic Canada salmon lice Lepeophtheirus salmonis with differing levels of drug resistance
Data table header descriptions
ID_REF
VALUE
lowess normalized log2 ratio (sample/reference pool)
