|
| Status |
Public on Feb 26, 2017 |
| Title |
Control_rep3 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
adult lice (Atlantic Canada)
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
treatment: Control
sampling time (not used): 4
number females in sample pool: 1
number males in sample pool: 1
|
| Treatment protocol |
Fish were infected following standard methods at Atlantic Veterinary College. Following seawater acclimatization, treatment tanks (total = 9 tanks) were randomly assigned to one of three treatment groups and placed on one of the following three diets: control/base feed, low dose (LD), or high dose immunostimulant treated feed (HD). Note that only the control and LD feed groups were used for transcriptome profiling. Fish were maintained on treated feed for 17 days and then sampled (T1). Seven days after T1, fish in all experimental tanks were exposed to L. salmonis copepodids at 10-12 lice/fish (initial exposure). Five days after the initial exposure, fish were sampled (T2). A second and a third lice exposure (also at 10-12 lice/fish) were conducted 14 and 28 days after the first exposure to mimic a more constant low-level exposure as expected in the field. Fish were sampled again at 40 and 47 days post initial infection (T3 and T4, respectively; Figure 1). Samples from T3 and T4, when they had the same number of males and females in the pools of samples, were used for transcriptomics.
|
| Growth protocol |
Egg strings from L. salmonis were harvested from sea cage cultured Atlantic salmon (New Brunswick, Canada) from the BMA2A region (which contains lice with elevated EMB resistance), aerated in 13oC saltwater collected from the sea cage site and maintained at the Atlantic Animal Facility, Atlantic Veterinary College at 33-36 salinity through 10% daily water changes until nauplii hatched and molted to copepodids (~6-8 days).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lice were rapidly enumerated and staged, then flash frozen in dry ice. All tissues were stored at -80oC until required for gene expression analysis. Total RNA was extracted from lice infecting control diet fish and low dose diet fish (LD) from times T3 and T4 (Figure 1) using the TRIzol extraction method including QIAGEN spin column RNA purification and quality evaluation on a BioRad Experion automated electrophoresis machine. These individuals were used to hybridize to an L. salmonis 38K oligonucleotide microarray (Sutherland et al. 2012). Samples were pools of both male and female adult lice collected from the same fish (each sex in equal proportion within each sample; two-four individual per pool). Seven pools were used from the control fish and four pools from the LD.
|
| 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 ensuring each experimental condition was included in the pool.
|
| |
|
| Channel 2 |
| Source name |
adult lice (Atlantic Canada)
|
| Organism |
Lepeophtheirus salmonis |
| Characteristics |
treatment: all condition pool
|
| Treatment protocol |
Fish were infected following standard methods at Atlantic Veterinary College. Following seawater acclimatization, treatment tanks (total = 9 tanks) were randomly assigned to one of three treatment groups and placed on one of the following three diets: control/base feed, low dose (LD), or high dose immunostimulant treated feed (HD). Note that only the control and LD feed groups were used for transcriptome profiling. Fish were maintained on treated feed for 17 days and then sampled (T1). Seven days after T1, fish in all experimental tanks were exposed to L. salmonis copepodids at 10-12 lice/fish (initial exposure). Five days after the initial exposure, fish were sampled (T2). A second and a third lice exposure (also at 10-12 lice/fish) were conducted 14 and 28 days after the first exposure to mimic a more constant low-level exposure as expected in the field. Fish were sampled again at 40 and 47 days post initial infection (T3 and T4, respectively; Figure 1). Samples from T3 and T4, when they had the same number of males and females in the pools of samples, were used for transcriptomics.
|
| Growth protocol |
Egg strings from L. salmonis were harvested from sea cage cultured Atlantic salmon (New Brunswick, Canada) from the BMA2A region (which contains lice with elevated EMB resistance), aerated in 13oC saltwater collected from the sea cage site and maintained at the Atlantic Animal Facility, Atlantic Veterinary College at 33-36 salinity through 10% daily water changes until nauplii hatched and molted to copepodids (~6-8 days).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Lice were rapidly enumerated and staged, then flash frozen in dry ice. All tissues were stored at -80oC until required for gene expression analysis. Total RNA was extracted from lice infecting control diet fish and low dose diet fish (LD) from times T3 and T4 (Figure 1) using the TRIzol extraction method including QIAGEN spin column RNA purification and quality evaluation on a BioRad Experion automated electrophoresis machine. These individuals were used to hybridize to an L. salmonis 38K oligonucleotide microarray (Sutherland et al. 2012). Samples were pools of both male and female adult lice collected from the same fish (each sex in equal proportion within each sample; two-four individual per pool). Seven pools were used from the control fish and four pools from the LD.
|
| 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 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).
|
| Data processing |
For each probe on the microarray, the background median was subtracted from the foreground median. Data analysis was performed using limma in R. The arrays were normalized by within array loess normalization and between array quantile normalization using the Cy3 reference channel. The probes present in the matrix file are those that passed quality and low expression filters: at least three samples in any condition were ≥ 500 for raw fluorescence in both channels, and not saturated in all samples.
|
| |
|
| Submission date |
Jan 24, 2017 |
| Last update date |
Feb 26, 2017 |
| Contact name |
Ben F Koop |
| E-mail(s) |
bkoop@uvic.ca
|
| Phone |
(250) 472-4067
|
| Organization name |
The University of Victoria
|
| Department |
Biology
|
| Lab |
Centre for Biomedical Research
|
| Street address |
PO Box 3020 STN CSC
|
| City |
Victoria |
| State/province |
BC |
| ZIP/Postal code |
V8W 3N5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL15566 |
| Series (1) |
| GSE93998 |
Host-parasite transcriptomics during immunostimulant enhanced rejection of salmon lice Lepeophtheirus salmonis by Atlantic salmon Salmo salar |
|
