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The impact of a moderate chronic temperature increase on spleen immune-relevant gene transcription depends on whether Atlantic cod (Gadus morhua) are stimulated with bacterial versus viral antigens

Publication: Genome
31 August 2013

Abstract

Exposure to elevated temperature is an inherent feature of Atlantic cod (Gadus morhua) sea-cage culture in some regions (e.g., Newfoundland) and may also become an increasingly prevalent challenge for wild fish populations because of accelerated climate change. Therefore, understanding how elevated temperatures impacts the immune response of this commercially important species may help to reduce the potential negative impacts of such challenges. Previously, we investigated the impacts of moderately elevated temperature on the antiviral responses of Atlantic cod (Hori et al. 2012) and reported that elevated temperature modulated the spleen transcriptome response to polyriboinosinic polyribocytidylic acid (pIC, a viral mimic). Herein, we report a complementary microarray study that investigated the impact of the same elevated temperature regime on the Atlantic cod spleen transcriptome response to intraperitoneal (IP) injection of formalin-killed Aeromonas salmonicida (ASAL). Fish were held at two different temperatures (10 °C and 16 °C) prior to immune stimulation and sampled 6 and 24 h post-injection (HPI). In this experiment, we identified 711 and 666 nonredundant ASAL-responsive genes at 6HPI and 24HPI, respectively. These included several known antibacterial genes, including hepcidin, cathelicidin, ferritin heavy subunit, and interleukin 8. However, we only identified 15 differentially expressed genes at 6HPI and 2 at 24HPI (FDR 1%) when comparing ASAL-injected fish held at 10 °C versus 16 °C. In contrast, the same comparisons with pIC-injected fish yielded 290 and 339 differentially expressed genes (FDR 1%) at 6HPI and 24HPI, respectively. These results suggest that moderately elevated temperature has a lesser effect on the Atlantic cod spleen transcriptome response to ASAL (i.e., the antibacterial response) than to pIC (i.e., antiviral response). Thus, the impacts of high temperatures on the cod’s immune response may be pathogen dependent.

Résumé

L’exposition à des températures élevées est une caractéristique inhérente à l’élevage en mer de la morue de l’Atlantique dans certaines régions (e.g. Terre-Neuve) et pourrait présenter des défis croissants pour les populations sauvages de poissons en raison des changements climatiques. Ainsi, comprendre comment les températures plus élevées affectent les réponses immunitaires chez cette importante espèce commerciale pourrait aider à réduire les possibles impacts négatifs de ces changements. Précédemment, les auteurs avaient étudié les impacts d’un accroissement modéré de la température sur les réponses antivirales de la more de l’Atlantique (Hori et al. 2012) et avaient rapporté qu’une température élevée modifiait la réponse transcriptomique de la rate à l’acide polyriboinosinique polyribocytidylique (pIC, un composé mimétique des virus). Les auteurs rapportent ici une analyse microarray complémentaire documentant l’impact des mêmes températures élevées sur la réponse transcriptomique de la rate de la morue de l’Atlantique à l’injection intrapéritonéale (IP) de l’Aeromonas salmonicida (ASAL) tué à la formaline. Les poissons ont été maintenus à deux températures (10 °C ou 16 °C) avant la stimulation immunitaire et ont été échantillonnés 6 et 24 heures après injection (HPI). Dans cette expérience, les auteurs ont identifié 711 et 666 gènes non-redondants affectés par l’ASAL à 6HPI et 24HPI respectivement. Ceux-ci incluaient plusieurs gènes antibactériens connus, dont l’hepcidine, la cathelcidine, la ferritine et la chaîne lourde de l’interleukine 8. Cependant, seuls 15 gènes exprimés différemment à 6HPI et 2 gènes à 24HPI (à un FDR de 1 %) ont été identifiés en comparant les poissons injectés à l’ASAL et maintenus à 10 °C versus 16 °C. Par contre, les mêmes comparaisons entre poissons injectés au pIC avaient produit 290 et 339 gènes exprimés différemment (à un FDR de 1 %) à 6HPI et à 24HPI respectivement. Ces résultats suggèrent qu’une élévation de température modérée a un impact moindre sur la réponse transcriptomique de la rate chez la morue de l’Atlantique à l’injection d’ASAL (réponse antibactérienne) qu’à l’injection de pIC (réponse antivirale). Ainsi, les impacts de températures élevées sur le système immunitaire de la morue pourraient dépendre de l’agent pathogène. [Traduit par la Rédaction]

Introduction

A sharp decline in wild populations of Atlantic cod (Gadus morhua) (Myers et al. 1997; Rose 2007) has greatly reduced the productivity of this species’ capture fishery in recent years (i.e., from ∼1.5 million tonnes in the 1960s to ∼60 000 tonnes in 2008 in the Northwest Atlantic) (FAO 2011). The Atlantic cod has historically been very important to the economy of several countries and regions, including Atlantic Canada, Norway, and Iceland, and this drastic decline in landings led to the development of Atlantic cod aquaculture as an alternative commercial source of this species for seafood markets (Brown et al. 2003; Rosenlund and Skretting 2006). However, the Atlantic cod aquaculture industry remains in its infancy, partially because of our incomplete understanding of this species’ biology (Kjesbu et al. 2006; Rosenlund and Halldórsson 2007), including but not limited to the lack of a comprehensive knowledge of how the Atlantic cod is affected by changes in its environment.
Fluctuations in ocean temperature occur both daily and seasonally, especially during the spring and summer months in Newfoundland, Canada (Gollock et al. 2006; Pérez-Casanova et al. 2008). Fish held in net-pens in this region may not be able to completely avoid nonoptimal temperatures during these periods, and thus, they may be chronically exposed to elevated temperature and suffer high rates of mortality (Gollock et al. 2006; Pérez-Casanova et al. 2008). However, average maximum summer water temperatures often observed at the cage-sites (∼16–18 °C) were not found to be lethal for Atlantic cod in laboratory experiments (A.K. Gamperl, unpublished), and this suggests that the observed cage-site mortalities were not directly related to temperature.
Recently, our research group demonstrated that moderately elevated temperature (16 °C vs. 10 °C) modulates the Atlantic cod antiviral response to polyriboinosinic polyribocytidylic acid (pIC, a viral mimic) by shifting the timing of the mRNA expression of many transcripts, including some encoding key antiviral proteins (e.g., IRF1, STAT1, SCYA123, IL-8, Viperin) (Hori et al. 2012). In that study, we identified 290 genes at 6 h post-injection (HPI) and 339 genes at 24HPI that were differentially expressed between fish stimulated with pIC at 10 °C versus 16 °C. Furthermore, we found that only 41 genes responded significantly to pIC-injection at 10 °C (as compared with saline-injected controls) at the 6HPI sampling point, whereas 656 genes responded significantly at 16 °C at this time point. These results suggest that increased temperature hastens the cod’s antiviral immune response, and thus, it may have a major impact on this species’ disease susceptibility. These findings are consistent with Raida and Buchmann (2007) who showed a faster and more intense response of key immune genes in the spleen of rainbow trout (Oncorhynchus mykiss) following stimulation with a Yersinia ruckeri bacterin at elevated temperatures (15 °C and 25 °C vs. 5 °C), and they are in general agreement with numerous studies that have shown that elevated water temperature modulates the immunity and (or) immune-related gene expression of fish (e.g., Magnadóttir et al. 1999; Alishahi and Buchmann 2006; Varsamos et al. 2006; Raida and Buchmann 2007; Pérez-Casanova et al. 2008; Jeffries et al. 2012; Avunje et al. 2013; Polinski et al. 2013). However, these studies do not present a consistent picture of how temperature affects fish immunology and disease resistance. For example, Varsamos et al. (2006) showed that variations in water temperature increased the susceptibility of sea bass (Dicentrachus labrax) to nodavirus. Whereas, Jeffries et al. (2012) hypothesized that the enhanced survival of wild Pacific sockeye salmon (Oncorhynchus nerka) when challenged with temperature elevations could be, in part, related to increased immune-related transcript expression, and it has been demonstrated that exposure to constant elevated temperatures can increase the efficacy of vaccination in Atlantic salmon (Salmo salar) (Raida and Buchmann 2007) and channel catfish (Ictalurus punctatus) (Martins et al. 2011). Collectively, these data suggest that the effect of elevated temperature on the fish’s immune system and disease resistance may be pathogen and (or) species dependent.
Given this background, the objective of this work was to investigate the impacts of an elevated temperature regimen on the Atlantic cod response to intraperitoneal (IP) injection of formalin-killed typical Aeromonas salmonicida (ASAL) and to compare this data set to the antiviral response data published in Hori et al. (2012). We chose the Gram-negative bacterium A. salmonicida for this study because it is the aetiological agent of typical furunculosis in Atlantic cod (Samuelsen et al. 2006); this disease causes great losses in the aquaculture of other fish species, especially Atlantic salmon (Reith et al. 2008), and atypical strains of A. salmonicida are an emerging issue for Atlantic cod aquaculture (Lund et al. 2008). Further, understanding the impacts of elevated temperature on the immune response of Atlantic cod to bacterial antigens may be relevant to this species’ conservation and management as ocean water temperatures are predicted to rise because of accelerated climate change (Hansen et al. 2006; Roemmich et al. 2012).
In both studies, we used the Atlantic Cod Genomics and Broodstock Development Project (CGP) 20 000 (20K) 50-mer oligonucleotide microarray (Booman et al. 2011; Bowman et al. 2011), which was previously shown to be a valuable tool for investigating the impacts of immune challenges on Atlantic cod (Booman et al. 2011; Hori et al. 2012).

Materials and methods

All experiments reported herein were conducted in accordance with the Canadian Council for Animal Care guidelines and were approved by the Memorial University of Newfoundland’s Institutional Animal Care Committee.

Experimental design

The IP injection of fish with ASAL was performed in parallel with the IP injection of fish with pIC, which were used in our previous publication describing the impacts of elevated temperature on the pIC response of juvenile Atlantic cod (∼60 g) (Hori et al. 2012). Detailed information on experimental procedures (e.g., passive integrated transponder (PIT)-tagging) and design can be found in Hori et al. (2012, see Fig. 1 and methods section therein). Briefly, fish in the elevated temperature groups (denoted by @16 °C) were exposed to a chronic elevation in temperature (from 10 °C to 16 °C at ∼1 °C every 5 days) and were held at 16 °C for ∼1 week, whereas fish in the temperature control groups (denoted by @10 °C) remained at 10 °C for this entire period. Thereafter, fish in all groups were stimulated with formalin-killed typical ASAL or phosphate-buffered saline (PBS) as described below. In the present manuscript, we do not include data on the groups prior to immune stimulation (i.e., constitutive gene expression), as those data were previously published in Hori et al. (2012). Lastly, we would like to point out that the PBS-injected groups in the present experiment were the same control groups used in our previous pIC work. Therefore, we will not present any comparisons between the PBS-injected groups, as they were previously addressed in Hori et al. (2012).

Immune stimulation

Formalin-killed typical ASAL was obtained in the form of a vaccine (Furogen dip, Novartis, PE). The vaccine was pelleted by centrifugation (2000g for 10 min at 4 °C) and washed with ice-cold 0.2 μm filtered PBS three times. Following the third wash, the pelleted cell debris was resuspended in ice-cold PBS to an optical density of 1.0 at 600 nm wavelength (OD600). Fish in the elevated temperature group and fish in the temperature control group were injected with 4 μL of ASAL solution per gram of wet mass solution following the injection procedures described in Hori et al. (2012). Fish were then sampled 6HPI and 24HPI.

Tissue sampling and RNA extractions

Tissue sampling, RNA extraction, DNase-I treatment, and column purification of RNA samples were performed as previously described in Hori et al. (2012). Six individuals from each group at each temperature and time point were used for microarray analysis.

Microarray hybridizations

The platform used in this experiment was the CGP 20K Atlantic cod oligonucleotide microarray (GEO accession No. GPL10532), which was designed based on a collection of over 150 000 ESTs, and all targets hybridized to the microarrays were labeled with either ALEXA 647 (experimental individuals) or ALEXA 555 (common reference) using the SuperScript Direct Labeling Kit (Life Technologies, Carlsbad, Calif.) (see Booman et al. (2011) for specific details).
Figure 1 depicts the overall microarray experimental design and summarizes the microarray results. For each of the four treatment–temperature combinations at each time point, six microarrays were used to compare each individual to the common reference (composed of an equal contribution of RNA from every individual involved in the overall meta-experiment—identical to the common reference used in Hori et al. (2012)). However, one array from the ASAL 24HPI@10 °C group had to be excluded from the analysis because it was missing an entire subgrid. Sample group designations follow the same conventions as in Hori et al. (2012) and indicate treatment (PBS or ASAL), sampling time point (6HPI or 24HPI), and holding temperature (@10 °C or @16 °C). For example, ASAL 24HPI@10 °C indicates a fish held at 10 °C that was sampled 24 h after it was injected with ASAL.
Fig. 1.
Fig. 1. Microarray experimental design and summary of findings. For complete gene lists, including fold-changes, d values, raw p values, and standard deviations, refer to Supplemental Tables S1–S3.2
2
Supplementary data are available with the article through the journal Web site at Supplementary Material.

Microarray data acquisition and analysis

All data acquisition, preprocessing, and analysis were performed as in Booman et al. (2011) and Hori et al. (2012). Briefly, hybridized microarrays were scanned using a ScanArray Gx Plus (Perkin Elmer, Woodbridge, Ont.) (5 μm resolution and 90% laser power), and fluorescence data were extracted from TIFF images using Imagene v7.5 (BioDiscovery, El Segundo, Calif.). Data normalization, threshold setting, and quality filtering were performed in R, using the scripts previously published in Booman et al. (2011). The significance analysis of microarrays (SAM) algorithm (Tusher et al. 2001) was used to identify genes that were differentially expressed between sample groups in pairwise comparisons (e.g., PBS 6HPI@10 °C vs. ASAL 6HPI@10 °C), using a false discovery rate (FDR) cutoff of 0.01 (i.e., 1%). Prior to SAM analysis, probes absent in more than 25% of the arrays in this study (i.e., missing from more than 11 arrays out of the total of 47) were removed from the final data set. This resulted in a final set of 11 063 probes used for analysis. Imputation was performed using LSImpute (Bø et al. 2004; Celton et al. 2010) as described previously (Hori et al. 2012).
The resulting gene lists were re-annotated using Blast2GO v2.6.4 (Conesa et al. 2005) based on the ESTs or contigs from which the informative probes were designed—detailed information on probe design can be found in Booman et al. (2011). For this, Blastx was used within Blast2GO with an E-value threshold of 10−5 and high scoring pairs (HSP) >33. Then, gene ontology (GO) terms were mapped to the Blastx results using Blast2GO, and mapping was used for GO term enrichment analysis (Fisher’s exact test, FDR < 5% or p < 0.05).
Hierarchical clustering and heatmaps were constructed in R using the mosaic function of the ClassDiscovery v2.9.0 package (distributed with OOMPA v2.15.1; http://bioinformatics.mdanderson.org/main/OOMPA:Overview). We used “uncentered Pearson correlation” as the distance measure for the hcclust function. To facilitate the comparison between the current data set and our previous work, we used the expression data from the pIC-responsive genes (i.e., genes that were differentially expressed between PBS- and pIC-injected fish), which were previously identified in Hori et al. (2012), and re-analyzed it using the current clustering methods. For each time point, ASAL-responsive probes (i.e., differentially expressed between PBS- and ASAL-injected fish) at 10 °C and at 16 °C were combined into a nonredundant probe list for overall clustering (Figs. 2A and 3A). The same was done for the pIC-responsive genes (Figs. 2B and 3B). These nonredundant lists were used to avoid bias towards genes that were only present in one list (i.e., only differentially expressed at one temperature). This approach allowed us to compare the same types of gene lists (i.e., nonredundant pIC- or ASAL-responsive genes) between the two experiments, which would not have been possible with the clusterings published in Hori et al. (2012). The resulting hierarchical clusterings for the pIC data differ from those previously published in Hori et al. (2012), because the current clustering is based on pIC-responsive genes and the previous clustering was based on genes that were differentially expressed between pIC-injected fish held at 10 °C and those that were held at 16 °C. For genes overlapping between the present study and Booman et al. (2011) and Feng et al. (2009) (Fig. 4), we used the 45 features that were identified as ASAL-responsive in at least two of the studies by matching probe identifiers (IDs) or gene name.
Fig. 2.
Fig. 2. Hierarchical clustering of all samples based on (A) a set of 711 nonredundant ASAL-responsive genes (significantly dysregulated by ASAL stimulation at least at one temperature at 6HPI, FDR 1%) or (B) a set of 668 nonredundant pIC-responsive genes (significantly dysregulated by pIC stimulation at least at one temperature at 6HPI, FDR 1%). Colored bars underneath the heat maps represent clusters of individuals that were injected with an immune-stimulant (ASAL or pIC, yellow) or PBS (light green).
Fig. 3.
Fig. 3. Hierarchical clustering of all samples based on (A) a set of 666 nonredundant ASAL-responsive genes (significantly dysregulated by ASAL stimulation at least at one temperature at 24HPI, FDR 1%) or (B) a set of 1075 nonredundant pIC-responsive genes (significantly dysregulated by pIC stimulation at least at one temperature at 24HPI, FDR 1%). Colored bars underneath the heat maps represent clusters of individuals that were injected with an immune-stimulant (ASAL or pIC, yellow) or PBS (light green).
Fig. 4.
Fig. 4. Hierarchical clustering of all samples based on the 45 probes (representing 33 unique genes) identified as ASAL-responsive in the current investigation and in the spleen of Atlantic cod stimulated with an IP-injection of formalin-killed atypical ASAL (Booman et al. 2011 and (or) Feng et al. 2009). Different clusters of genes are marked in green, red, and blue on the left side of the heat map. One outlier sample from the PBS 24HPI@16 °C is marked with an asterisk (*).

Results

Spleen transcriptome response to ASAL stimulation

To identify ASAL-responsive genes at 10 °C and 16 °C, we compared each ASAL group to its time- and temperature-matched PBS group using SAM. This resulted in the following four gene lists (Fig. 1; Supplemental Tables S1A–S1D): 259 up-regulated and 37 down-regulated (296 total) in ASAL 6HPI@10 °C compared with PBS 6HPI@10 °C; 485 up-regulated and 21 down-regulated (506 total) in ASAL 6HPI@16 °C compared with PBS 6HPI@16 °C; 397 up-regulated and 111 down-regulated (508 total) in ASAL 24HPI@10 °C compared with PBS 24HPI@10 °C; and 156 up-regulated and 41 down-regulated (197 total) in ASAL 24HPI@16 °C compared with PBS 24HPI@16 °C. Among these genes were several well-known antibacterial genes, such as hepcidin, cathelicidin, interleukin 8, interleukin 11a, ferritin heavy subunit, goose-type lysozyme, interleukin 12 receptor, and small inducible cytokine (SCYA123) (Table 1; Supplemental Table S1). When the lists of ASAL-responsive genes at both temperatures for each time point were combined, we identified 711 nonredundant ASAL-responsive probes at 6HPI (Fig. 2A) and 666 nonredundant ASAL-responsive probes at 24HPI (Fig. 3A). At 6HPI, 91 out of the 711 nonredundant genes were significantly responsive to ASAL at both temperatures, while at 24HPI, 39 out of 666 nonredundant genes were significantly responsive to ASAL at both temperatures (Supplemental Tables S2A–S2B). Only four probes (20K probe identifiers 37609, 42243, 44096, and 53585 representing hepcidin, an unknown transcript, aquaporin 12, and another unknown transcript, respectively) were significantly responsive to ASAL at all time points and temperatures. For detailed information on these informative probes, including raw p values, d values, and standard deviations, refer to Supplemental Tables S1A–S1D.
Table 1.
Table 1. Selected antibacterial genesa identified in the spleen of Atlantic cod that showed a significant response to ASAL-stimulation.
a
Genes were selected based on our previous work on the Atlantic cod spleen transcriptome response to stimulation with atypical ASAL by matching either their probe ID or gene name. With exception of probes 37206 and 38598, all other genes were identified as differentially expressed in response to ASAL in the microarray experiment reported in Booman et al. (2011).
b
Fold-changes are only presented for genes whose expression was significantly different in at least one comparison (e.g. PBS 6HPI@10 °C vs. ASAL 6HPI@10 °C), otherwise a dash (—) is used. Values are given as generated by siggenes. Detailed information on these probes can be found in Supplemental Table S1.
c
QPCR validation for these probes and (or) same-named genes was previously published in the referred papers for Atlantic cod stimulated with formalin-killed atypical ASAL at 10 °C.
d
These genes were represented by more than one same-named gene in the overlap between the present microarray experiment and Booman et al. (2011). Only one representative probe was included in this table.
e
This gene name follows the nomenclature of Borza et al. (2010) for Atlantic cod small inducible cytokines.
f
This probe was annotated as “Bactericidal permeability increasing protein/lipopolysaccharide binding protein variant b (Gadus morhua)” in the array, but it had no significant (i.e., E value < 10−5) Blastx results.
g
This probe was annotated as “Syndecan (Tetraodon nigroviridis)” in the array, but it had no significant (i.e., E value < 10−5) Blastx results.
To confirm the current results, we compared our gene lists to previous results from our group in which Atlantic cod were IP-injected with formalin-killed atypical ASAL at 10 °C (Feng et al. 2009; Booman et al. 2011). Of the 82 probes reported in Booman et al. (2011) as responding significantly to IP injection with ASAL (FDR 1%), 31 were also identified in the present work; of those, eight probes were validated as ASAL-responsive by QPCR in Booman et al. (2011) (Table 1). We also identified 14 additional probes in the present work that had same-named genes that were identified in the microarray results of Booman et al. (2011) and (or) that were SSH-identified and validated by QPCR as ASAL-responsive in Feng et al. (2009). Among these 14 were interferon regulatory factor 1 (IRF1), cathelicidin, hepcidin, small inducible cytokine (SCYA123), interleukin 8 (IL8), and ferritin heavy subunit. These 45 probes (31 identified by 20K probe identifier, and 14 identified by associated gene name) that were identified previously as being ASAL-responsive represent 33 unique genes (Table 1). A list of these genes, their current Blastx annotations, and fold-change values for the comparisons where these probes were found to significantly respond to ASAL can be found in Table 1. The transcript expression levels of these 45 probes for all samples can be found in Fig. 4.

Impacts of exposure to elevated temperature on the spleen transcriptome response to ASAL

Even though the lists of genes differentially expressed in response to ASAL at the two studied holding temperatures and time points, as compared with their respective temperature- and time-matched PBS controls, had relatively different numbers of informative probes (see above, e.g., 506 genes significantly responding to ASAL at 16 °C vs. 296 genes significantly responding to ASAL at 10 °C for the 6HPI sampling time point), the direct comparisons between the ASAL-injected groups at 10 °C and 16 °C revealed very few genes that were differentially expressed between these groups at an FDR of 1%. We only identified 15 genes whose expression was significantly different when comparing the ASAL 6HPI@10 °C versus ASAL 6HPI@16 °C groups and only two genes when comparing the ASAL 24HPI@10 °C versus ASAL 24HPI@16 °C groups (Fig. 1; Table 2). These genes are presented in Table 2, and detailed information on these probes can be found in Supplemental Tables S3A–S3B.
Table 2.
Table 2. Probes that showed significantly different (FDR 1%) levels of expression in fish stimulated with ASAL at 10 °C or 16 °C.
a
Fold-changes are reported as generated by the SAM algorithm.
b
This gene name follows the nomenclature of Borza et al. (2010) for Atlantic cod small inducible cytokines.
When clustering individuals based on expression of the nonredundant ASAL-responsive genes (711 at 6HPI and 666 at 24HPI, Figs. 2A and 3A), the ASAL-injected fish showed a distinct bacteria-responsive transcript expression signature and clustered in a separate branch from the PBS-injected fish. The same clustering pattern was found when clustering was performed based on the 45 probes (representing 33 unique genes) that were overlapping between the current work and our previous studies using atypical ASAL stimulation (with the exception of one PBS-injected fish that clustered with the ASAL-injected fish, marked with an asterisk (*) on Fig. 4) (Feng et al. 2009; Booman et al. 2011). In contrast, clustering of individuals based on the expression of the nonredundant pIC-responsive genes (668 at 6HPI and 1075 at 24HPI, Figs. 2B and 3B) revealed that all pIC 6HPI@10 °C fish showed a nonstimulated transcript expression signature and clustered together with PBS-injected fish. All other pIC-injected fish (i.e., pIC 6HPI@16 °C, pIC 24HPI@10 °C, and pIC 24HPI@16 °C) showed a distinct virus-responsive transcript expression signature and clustered in a separate branch from the PBS-injected fish and pIC 6HPI@10 °C fish (Figs. 2 and 3). Finally, we used Blast2GO to compare the GO term distribution in the lists of genes that were ASAL-responsive only at 10 °C to those responsive only at 16 °C for each time point (205 genes at 10 °C and 415 genes at 16 °C for the 6HPI sampling time point; 469 genes at 10 °C and 158 genes at 16 °C for the 24HPI sampling time point). However, this analysis did not identify any significantly enriched GO terms in any comparison using either an FDR of 5% or a p value of 0.05 as cutoffs.

Discussion

Genes responsive to ASAL

We identified a set of 1239 nonredundant informative probes that responded significantly (FDR 1%) to ASAL at least at one time point and one temperature (i.e., the union of the 711 and 666 ASAL-responsive probes at 6HPI or 24HPI, respectively) (see Supplemental Tables S1A–S1D). To further confirm our data set, we compared the probe identifiers and (or) gene names of 20K microarray probes identified as ASAL-responsive to those reported in Booman et al. (2011) and Feng et al. (2009) for Atlantic cod stimulated with formalin-killed atypical ASAL at 10 °C. Of the 12 QPCR-validated ASAL-responsive genes presented in Booman et al. (2011), we identified eight in the present data set as the same 20K microarray probes used for QPCR primer design in the previous study (fold-changes for the comparisons in which these genes were identified can be found in Table 1, and their expression levels in all samples can be found in Fig. 4). In addition to these genes, ferritin heavy subunit and IRF1, which were SSH-identified and QPCR-validated as ASAL-responsive in Feng et al. (2009), were among the ASAL-responsive probes in the present study (fold-changes for the comparisons in which these genes were identified can be found in Table 1 and their expression levels in all samples can be found in Fig. 4). Based on these results, we are confident that our data set is accurate and that the genes reported in this manuscript represent a trustworthy catalog of ASAL-responsive (i.e., antibacterial) genes in Atlantic cod. Ferritin and hepcidin are genes known to be involved in iron homeostasis (Solstad et al. 2008; Feng et al. 2009) and represent strong candidates for future marker development of robust immune response against ASAL. They were both significantly induced by IP injection with ASAL in the current study, and their response to bacterial antigens has been validated by QPCR in two previous studies (Feng et al. 2009; Booman et al. 2011). Furthermore, studies have shown that iron homeostasis plays an important role in ASAL pathogenesis in Atlantic cod and Atlantic salmon (Reith et al. 2008; Feng et al. 2009; Beaz-Hidalgo and Figueras 2013). Many studies have also shown that the genes SCYA123 and IL8 can be important in the response of Atlantic cod to both bacteria and viruses (Seppola et al. 2008; Borza et al. 2010; Feng et al. 2009; Rise et al. 2010; Booman et al. 2011; Holen et al. 2012; Hori et al. 2012). However, to our knowledge, this is the first time that several other known Atlantic cod antiviral genes, including DHX58, sacsin, and IRF10 (Rise et al. 2008; Hori et al. 2012), have been shown to also be responsive to stimulation with bacterial antigens.

Temperature effects on the cod’s immune response

In the current study, we show that elevated temperature only had a minimal effect on the Atlantic cod spleen transcriptome response to ASAL at 6HPI and 24HPI. Only 15 genes were differentially expressed between fish held at 10 °C versus 16 °C at 6HPI, and only two genes were identified when this comparison was made at 24HPI (Fig. 1; Table 2). This result is in distinct contrast to what our research group previously reported for pIC stimulation. In Hori et al. (2012), 290 and 339 genes were differentially expressed between pIC-stimulated fish held at 10 °C versus 16 °C at 6HPI and 24HPI, respectively. Further, we found that there were 296 significant ASAL-responsive probes when comparing ASAL 6HPI@10 °C versus PBS 6HPI@10 °C, and 506 probes when comparing ASAL 6HPI@16 °C versus PBS 6HPI@16 °C. This is in contrast to the pIC data published in Hori et al. (2012) where there were only 41 significant pIC-responsive probes when comparing pIC 6HPI@10 °C versus PBS 6HPI@10 °C, and 656 probes when comparing pIC 6HPI@16 °C versus PBS 6HPI@16 °C.
This difference between the two studies was also observed in the results from the clustering and GO term analyses. Fish injected with pIC at 10 °C and sampled at 6HPI had spleen transcript expression profiles that clustered with those of fish that were not stimulated with pIC (i.e., showed a nonstimulated transcript expression signature) (Hori et al. 2012), a result that was further confirmed in the current study by clustering these samples based on the nonredundant lists of pIC-responsive probes (Figs. 2B and 3B). This transcript expression clustering result was not seen in ASAL-stimulated fish held at 10 °C and sampled at 6HPI. Rather, all ASAL-stimulated fish grouped together when clustered based on a nonredundant set of ASAL-responsive genes (i.e., showed a distinct antibacterial transcript expression signature), and clustered separately from nonstimulated fish at both time points (Figs. 2A and 3A). In Hori et al. (2012), several GO terms were enriched in the list of pIC-responsive genes at 16 °C compared with those at 10 °C for the 24HPI sampling point and these included unfolded protein binding, regulation of interferon-gamma-mediated signaling, and regulation of cytokine-mediated signaling pathway. In the current study, a comparable analysis showed that there were no GO terms enriched in the list of ASAL-responsive genes at 16 °C compared with those at 10 °C. Overall, these findings clearly demonstrate that, in terms of regulation of gene expression, the Atlantic cod immune response following pIC injection is much more temperature-dependent than is this species’ immune response to ASAL.
Some genes known to be important in the fish innate immune response were responsive to both ASAL (present study) and pIC (Hori et al. 2012). Of these, interferon regulatory factors (IRFs) 1 and 10, interleukin-8 variant 5 (IL8), SCYA123, and DHX58 (alias LGP2)(Rise et al. 2008; Feng et al. 2009; Rise et al. 2010; Polinski et al. 2013) all showed a clear shift in the timing of up-regulation when pIC-injected fish were held at 16 °C versus at 10 °C, with the fish held at 16 °C showing a significantly higher response than fish held at 10 °C at 6HPI and the opposite being true at 24HPI (Hori et al. 2012). In contrast, only two of these genes, SCYA123 and IL8, showed a difference in the timing of up-regulation at the different temperatures following ASAL injection (Table 2). Microarray probe 44453 representing SCYA123 was ∼6-fold more highly expressed in ASAL 6HPI@16 °C as compared with ASAL 6HPI@10 °C (Table 2). Microarray probe 38638 representing IL8 was ∼5-fold more highly expressed in ASAL 24HPI@10 °C as compared with ASAL 24HPI@16 °C (Table 2). Although the overall impact of elevated temperature on the spleen transcriptome response to ASAL was less than the impact of elevated temperature on the pIC response, the temperature-dependence of SCYA123 and IL8 transcript expression responses to bacterial antigens (i.e., ASAL) could still impact the ability of the fish to respond to bacterial pathogens.
Interestingly, our finding that the cod’s splenic immune gene expression response to pathogenic bacterial antigens (i.e., ASAL) was only minimally affected by increased temperature is not consistent with much of the literature in this research area. For example, while differences in the up-regulation of known inflammatory genes (e.g., TNFα and IL8) in bluefin tuna (Thunnus maccoyii) peripheral blood leukocytes (PBLs) stimulated with LPS were not found at 6HPI, 12HPI, or 24HPI when the fish were held at elevated versus optimal temperatures, differences were apparent at 3HPI (Polinski et al. 2013). IP injection of Yersinia ruckeri bacterin into rainbow trout (Oncorhynchus mykiss) at elevated temperature (15 °C and 25 °C vs. 5 °C) resulted in significantly increased and earlier expression of several key immune related genes (e.g., IL1β, IL10, IFNγ) in the spleen (Raida and Buchmann 2007). In addition, Lund et al. (2008) reported an increase in mortality of Atlantic cod challenged with four strains of atypical A. salmonicida when inoculated at 14 °C versus 10 °C. Furthermore, vaccination of Atlantic salmon against ASAL leads to a greater antibody production at 10 °C compared with 2 °C, but with similar levels of pathogen protection (Eggset et al. 1997). Finally, rainbow trout challenged with the myxozoan Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease (PKD), experienced greater mortality at 19 °C compared with either 16 °C or 14 °C (Bettge et al. 2009). Collectively, these results suggest that the impact(s) of elevated temperature on fish immunity and immune-related gene expression may be related to the fish species, the tissue type, the antigen or pathogen, and (or) the particular temperature range over which responses are compared.
In conclusion, we have shown that the overall impact of moderately elevated temperature on the Atlantic cod immune-relevant gene expression response to ASAL is less than the impact of elevated temperature on this species’ response to pIC. This is a novel finding and could affect the Atlantic cod’s ability to fight bacterial infections when exposed to elevated temperatures. In keeping with the hypothesis that modulation of immune-related gene transcription can affect immune robustness of fish, Jeffries et al. (2012) suggested that the differential expression of key immune genes may underlie the survival of wild sockeye salmon exposed to elevated temperatures. However, the literature is equivocal with regard to the effects of temperature on fish responses to bacterial pathogens (including A. salmonicida), and thus, experiments must be performed to determine if differences in the expression profiles observed in Hori et al. (2012) and the present manuscript correlate significantly with mortality during live pathogen challenges at elevated temperatures.

Acknowledgements

This research was funded through the Genome Atlantic, Genome Canada, and the Atlantic Canada Opportunities Agency (ACOA) supported Atlantic Cod Genomics and Broodstock Development Project; an NSERC Discovery Grant to M.L.R.; Canada Foundation for Innovation (CFI) funding in support of M.L.R.’s Canada Research Chair (tier 2 in Marine Biotechnology); and Memorial University-based fellowships and scholarships to T.S.H. We would also like to acknowledge the assistance provided by Luis O.B. Afonso and Stewart C. Johnson with injections and sampling. Lastly, we would also like to acknowledge the staff of the Dr. Joe Brown Aquatic Research Building for their help with fish husbandry.

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Supplementary Material

Supplementary data (gen-2013-0090suppla.pdf)
Supplementary data (gen-2013-0090supplb.pdf)
Supplementary data (gen-2013-0090supplc.pdf)

Information & Authors

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Published In

cover image Genome
Genome
Volume 56Number 10October 2013
Pages: 567 - 576
Editor: R. Danzmann

History

Received: 15 May 2013
Accepted: 30 August 2013
Accepted manuscript online: 31 August 2013
Version of record online: 31 August 2013

Notes

This article is part of a Special Commemorative Issue marking the one-year anniversary of “Genomics: The Power and the Promise”.

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Key Words

  1. temperature
  2. Aeromonas salmonicida
  3. aquaculture
  4. Atlantic cod (Gadus morhua)
  5. microarray
  6. poly IC
  7. viral

Mots-clés

  1. température
  2. Aeromonas salmonicida
  3. aquaculture
  4. morue de l’Atlantique (Gadus morhua)
  5. microarray
  6. polyIC
  7. viral

Authors

Affiliations

Tiago S. Hori*
Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada.
A. Kurt Gamperl
Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada.
Gord Nash
Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada.
Marije Booman
Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada.
Ashoktaru Barat
Directorate of Coldwater Fisheries Research, Anusandhan Bhawan, Industrial Area, Bhimtal, 263136, India.
Matthew L. Rise
Department of Ocean Sciences, Memorial University of Newfoundland, St. John’s, NL A1C 5S7, Canada.

Notes

*
Present address: Ocean Sciences Centre, Memorial University of Newfoundland, 1 Marine Lab Road, St. John’s, NL A1C 5S7, Canada.

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