Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of resistant and susceptible Atlantic salmon fry challenged with Infectious Pancreatic Necrosis virus

ABSTRACT: Infectious Pancreatic Necrosis (IPN) is a highly contagious birnavirus disease of farmed salmonid fish, which often causes high levels of morbidity and mortality. A large genetic component underlying resistance to this disease has been previously described for Atlantic salmon (Salmo salar L.), which mediates high mortality rates in some families and zero mortality in others. A global comparison of the gene expression profiles of resistant and susceptible Atlantic salmon fry following challenge with the IPN virus was undertaken. Full sibling salmon fry from two IPNV-resistant and two IPNV-susceptible families were challenged with the virus and sampled at 1 day, 7 days and 20 days post-challenge. Significant viral titre was observed in both resistant and susceptible fish at all timepoints, although generally at higher levels in susceptible fish. Microarray interrogations were performed using a custom-designed, oligonucleotide microarray platform (Agilent) with 44 K probes per slide (Salar_2; Agilent Design ID:025520). The design is lodged with ArrayExpress ( under accession number A-MEXP-2065. Dual-label hybridisations were undertaken, with each experimental sample (Cy3 labelled) being competitively hybridised against a pooled reference control (Cy5 labelled) comprising equimolar amounts from each experimental RNA sample. The interrogations comprised 144 separate hybridisations; 2 genotypes (susceptible, resistant) Ã 2 families for each genotype Ã 2 challenge states (control, challenged) Ã 3 timepoints (1, 7, 20 dpi) Ã 4 biological replicates for resistant (2 from each of two tanks) and 8 biological replicates for susceptible (4 from each of two tanks). A preliminary analysis suggested evidence for a segregating QTL in both the susceptible families and therefore twice as many offspring were screened. It was later established that the evidence for QTL segregation in one of the families was inconclusive and therefore comparisons were made at the family level only. The analyses took the unbalanced design into account.

ORGANISM(S): Salmo salar

SUBMITTER: John Taggart

PROVIDER: E-MTAB-4275 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Infectious pancreatic necrosis (IPN) is widespread disease with global distribution and highly contagious, which leads to severe economic problems both freshwater and saltwater, particularly in the Atlantic salmon (Salmo salar). In this work we analyzed and evaluated gene expression profiles in resistant and susceptible families associated to S. salar exposed to IPNV in order to understand the defense mechanisms mounted by resistant fish in combating infection. Experimentally, 29 families with known pedigree and variability were infected by immersion with 1x106 PFU/ml of IPNV, and a gamma frailty model analysis based on Kaplan-Meier mortality curves were obtained to identify and classify families as susceptible or resistant. Samples of head kidney tissue were obtained at day 1 and 5 post-infection from susceptible and resistant families and pooled to analyse their expression pattern with realt-ime PCR and one-color salmon-specific oligonucleotide microarray (21K). The microarray results showed clear differences in both families, where resistant fish presented an over-expression of genes associated with endocrine function, and a down-regulation of genes associated principally with tissue differentiation, metabolism protein degradation, cell signaling and immunological process. Moreover qPCR analysis using as target a number of transcripts associated with immunological processes showed the same trend observing a higher expression in susceptible than resistant families, indicating significant differences for CCL-19, CCRM-NM-3c, IL-12, IFN-M-NM-1, and IFN-M-NM-3. The one-color oligonucleotide microarrays included in this study was performed using pooled samples from each families at two different post-infection time condition. In the case of susceptible families were chosen at 1 day post-infection (dpi) the families 5352 (n = 2) and 5469 (n = 2) and at 5 dpi the families 5629 (n = 2) and 5637 (n = 2), whereas in resistant families were selected at 1 dpi the families 5459 (n =3) and 5761 (n =3), and at 5 dpi the families 5463 (n =3) and 5764 (n =3).

Project description:We evaluated the expression profiles of turbot in spleen, liver and head kidney across five temporal points of the Philasterides dicentrarchi infection process using an 8x15K Agilent oligo-microarray. The microarray included 2,176 different 5-fold replicated gene probes designed from a turbot 3’ sequenced EST database. We were able to identify 221 differentially expressed (DE) genes (8.1% of the whole microarray), 113 in spleen, 83 in liver and 90 in head kidney, in at least one of the 5 temporal points sampled for each organ. Most of these genes could be annotated (83.0%) and functionally categorized using GO terms (69.1%) after the additional sequencing of DE genes from the 5’ end. Many DE genes were related to innate and acquired immune functions. A high proportion of DE genes were organ-specific (70.6%), although their associated GO functions showed notable similarities in the three organs. The most striking difference in functional distribution was observed between the up- and down-regulated gene groups. Up-regulated genes were mostly associated to immune functions, while down-regulated ones mainly involved metabolism-related genes. Genetic response appeared clustered in a few groups of genes with similar expression profiles along the temporal series. The information obtained will aid to understand the turbot immune response and will specifically be valuable to develop strategies of defense to P. dicentrarchi to achieve more resistant broodstocks for turbot industry. We were interested in analyzing the response of turbot as species to P. dicentrarchi in three of the main immune organs (spleen, liver and head kidney) along a temporal series representing the main episodes of infection (1d, 3d, 7d, 15d, 25d). Accordingly, individual samples were pooled at each sampling point to average interindividual variation. A single control point (time 0; non-injected) was used since a very slight effect of injection on gene expression was previously reported in these three organs in turbot. Five fish were sacrificed on day 0 to be used as the unique control in the experiment and one hundred fifty were challenged with a highly virulent strain of P. dicentrarchi as previously described (Paramá et al., 2003). Groups of five fish were sacrificed at 1d, 3d, 7d, 15d and 25d post-challenging. Equal amounts of spleen, liver and head kidney were sampled from each fish, pooled per organ at each sampling point and immediately stored in liquid nitrogen.

Project description:Local adaptation and its underlying molecular basis has long been a key focus in evolutionary biology. There has recently been increased interest in the evolutionary role of plasticity and the molecular mechanisms underlying local adaptation. Using transcriptome analysis, we assessed differences in gene expression profiles for three brown trout (Salmo trutta) populations, one resident and two anadromous, experiencing different temperature regimes in the wild. The study was based on an F2 generation raised in a common garden setting. A previous study of the F1 generation revealed different reaction norms and significantly higher QST than FST among populations for two early life-history traits. In the present study we investigated if similar reaction norm patterns were present at the transcriptome level. Eggs from the three populations were incubated at two temperatures (5 and 8 degrees C) representing conditions encountered in the local environments. Global gene expression for fry at the stage of first feeding was analysed using a 32k cDNA microarray. The results revealed differences in gene expression between populations and temperatures and population M-CM-^W temperature interactions, the latter indicating locally adapted reaction norms. Moreover, the reaction norms paralleled those observed previously at early life-history traits. We were able to identify 90 cDNA clones among the genes with an interaction effect that were differently expressed between the ecologically divergent populations. These included genes involved in immune- and stress response. We observed less plasticity in the resident as compared to the anadromous populations, possibly reflecting that the degree of environmental heterogeneity encountered by individuals throughout their life cycle will select for variable level of phenotypic plasticity at the transcriptome level. Our study demonstrates the usefulness of transcriptome approaches to identify genes with different temperature reaction norms. The responses observed suggest that populations may vary in their susceptibility to climate change. Brown trout populations from three rivers in Denmark were studied: the Karup (KAR), Norring Moellebaek (NOR) and Lilleaa Rivers (LIL). These rivers experience different temperature regimes during the period lasting from egg incubation until fry emergence. During the autumn of 2004 and the winter of 2004/2005, adults from the three populations were collected by electro fishing. The fish were stripped for milt and eggs to establish an F1 generation. In autumn/winter 2008/2009, mature F1s were used to establish F2 offspring for each of the three populations. For KAR and LIL, 20 full-sib families were established, whereas for NOR, 15 full-sib families were established. Eggs from each full sib family were divided into two pools that were incubated at 5 and 8M-BM-!C in separate hatching troughs. At the time of first feeding (ca. 750-780 day-degrees), 10 individuals from each family and temperature were collected and transferred to separate tubes containing RNAlater (QIAGEN, Hilden, Germany). A 32K cDNA microarray developed for salmonids by cGRASP was used for the analyses. Ten families were randomly selected from each population. From each family two individuals were analyzed for each temperature (5 and 8oC, respectively), amounting to a total of 120 individuals. Of the two individuals from each temperature treatment within a family, one was labeled with CY5 and the other with CY3. For each family there were two comparisons of individuals representing different temperatures, but with dyes swapped between comparisons.

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Gene expression profiling of resistant and susceptible Atlantic salmon fry challenged with Infectious Pancreatic Necrosis virus

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