Discovery and Characterization of the ddx41 Gene in Atlantic Salmon: Evolutionary Implications, Structural Functions, and Innate Immune Responses to Piscirickettsia salmonis and Renibacterium salmoninarum Infections.

The innate immune response in Salmo salar, mediated by pattern recognition receptors (PRRs), is crucial for defending against pathogens. This study examined DDX41 protein functions as a cytosolic/nuclear sensor for cyclic dinucleotides, RNA, and DNA from invasive intracellular bacteria. The investigation determined the existence, conservation, and functional expression of the ddx41 gene in S. salar. In silico predictions and experimental validations identified a single ddx41 gene on chromosome 5 in S. salar, showing 83.92% homology with its human counterpart. Transcriptomic analysis in salmon head kidney confirmed gene transcriptional integrity. Proteomic identification through mass spectrometry characterized three unique peptides with 99.99% statistical confidence. Phylogenetic analysis demonstrated significant evolutionary conservation across species. Functional gene expression analysis in SHK-1 cells infected by Piscirickettsia salmonis and Renibacterium salmoninarum indicated significant upregulation of DDX41, correlated with increased proinflammatory cytokine levels and activation of irf3 and interferon signaling pathways. In vivo studies corroborated DDX41 activation in immune responses, particularly when S. salar was challenged with P. salmonis, underscoring its potential in enhancing disease resistance. This is the first study to identify the DDX41 pathway as a key component in S. salar innate immune response to invading pathogens, establishing a basis for future research in salmonid disease resistance.

Alternative splicing in Atlantic salmon head kidney and SHK-1 cell line during the Piscirickettsia salmonis infection: A comparative transcriptome survey.

Piscirickettsia salmonis, an intracellular bacterium in salmon aquaculture, is a big challenge because it is responsible for 54.2% of Atlantic salmon mortalities. In recent years, the high relevance of Alternative Splicing (AS) as a molecular mechanism associated with infectious conditions and host-pathogen interaction processes, especially in host immune activation, has been observed. Several studies have highlighted the role of AS in the host's immune response during viral, bacterial, and endoparasite infection. In the present study, we evaluated AS transcriptome profiles during P. salmonis infection in the two most used study models, SHK-1 cell line and salmon head kidney tissue. First, the SHK-1 cell line was exposed to P. salmonis infection at 0-, 7-, and 14-days post-infection (dpi). Following, total RNA was extracted for Illumina sequencing. On the other hand, RNA-Seq datasets of Atlantic salmon head kidney infected with the same P. salmonis strayingwase used. For both study models, the highest number of differentially alternative splicing (DAS) events was observed at 7 dpi, 16,830 DAS events derived from 9213 DAS genes in SHK-1 cells, and 13,820 DAS events from 7684 DAS genes in salmon HK. Alternative first exon (AF) was the most abundant AS type in the three infection times analyzed, representing 31% in SHK-1 cells and 228.6 in salmon HK; meanwhile, mutually exclusive exon (MX) was the least abundant. Notably, functional annotation of DAS genes in SHK-1 cells infected with P. salmonis showed a high presence of genes related to nucleotide metabolism. In contrast, the salmon head kidney exhibited many GO terms associated with immune response. Our findings reported the role of AS during P. salmonis infection in Atlantic salmon. These studies would contribute to a better understanding of the molecular bases that support the pathogen-host interaction, evidencing the contribution of AS regulating the transcriptional host response.

Effect of cortisol on the immune-like response of rainbow trout (Oncorhynchus mykiss) myotubes challenged with Piscirickettsia salmonis.

Salmonids are a species of high commercial value in Chilean aquaculture, where muscle is the final product of the industry. Fish can be affected by stress during intensive cultures, increasing susceptibility to infections. Recently, we reported that muscle is an important focus of immune reactions. However, studies have shown the immunosuppressive effect of stress only in lymphoid organs, and few studies have been conducted on muscle and immunity. Hence, we determine the effects of cortisol on the immune-like response of fish myotubes challenged with Piscirickettsia salmonis by three trials. First, rainbow trout primary culture of muscle was cultured and treated with cortisol (100 ng/mL) for 3 and 4 h. Second, myotubes were challenged with P. salmonis (MOI 50) for 4, 6 and 8 h. And third, muscle cell cultures were pretreated with cortisol and then challenged with P. salmonis. The mRNA levels of glucocorticoid pathway and innate immunity were evaluated by qPCR. Cortisol increased the klf15 levels and downregulated the innate immune-related tlr5m gene and antimicrobial peptides. P. salmonis challenge upregulated several immune-related genes. Finally, cortisol pretreatment followed by P. salmonis challenge differentially modulated stress- and immune-related genes. These data suggest that fish muscle cells possess an intrinsic immune response and are differentially regulated by cortisol, which could lead to bacterial outbreaks in muscle under stress conditions.

Transcriptome Profiling of Atlantic Salmon (Salmo salar) Parr With Higher and Lower Pathogen Loads Following Piscirickettsia salmonis Infection.

Salmonid rickettsial septicemia (SRS), caused by Piscirickettsia salmonis, is one of the most devastating diseases of salmonids. However, the transcriptomic responses of Atlantic salmon (Salmon salar) in freshwater to an EM-90-like isolate have not been explored. Here, we infected Atlantic salmon parr with an EM-90-like isolate and conducted time-course qPCR analyses of pathogen load and four biomarkers (campb, hampa, il8a, tlr5a) of innate immunity on the head kidney samples. Transcript expression of three of these genes (except hampa), as well as pathogen level, peaked at 21 days post-injection (DPI). Multivariate analyses of infected individuals at 21 DPI revealed two infection phenotypes [lower (L-SRS) and higher (H-SRS) infection level]. Five fish from each group (Control, L-SRS, and H-SRS) were selected for transcriptome profiling using a 44K salmonid microarray platform. We identified 1,636 and 3,076 differentially expressed probes (DEPs) in the L-SRS and H-SRS groups compared with the control group, respectively (FDR = 1%). Gene ontology term enrichment analyses of SRS-responsive genes revealed the activation of a large number of innate (e.g. "phagocytosis", "defense response to bacterium", "inflammatory response") and adaptive (e.g. "regulation of T cell activation", "antigen processing and presentation of exogenous antigen") immune processes, while a small number of general physiological processes (e.g. "apoptotic process", development and metabolism relevant) was enriched. Transcriptome results were confirmed by qPCR analyses of 42 microarray-identified transcripts. Furthermore, the comparison of individuals with differing levels of infection (H-SRS vs. L-SRS) generated insights into the biological processes possibly involved in disease resistance or susceptibility. This study demonstrated a low mortality (~30%) EM-90-like infection model and broadened the current understanding of molecular pathways underlying P. salmonis-triggered responses of Atlantic salmon, identifying biomarkers that may assist to diagnose and combat this pathogen.

Host genetic variation explains reduced protection of commercial vaccines against Piscirickettsia salmonis in Atlantic salmon.

Vaccination is a widely used control strategy to prevent Piscirickettsia salmonis causing disease in salmon farming. However, it is not known why all the currently available commercial vaccines generally fail to protect against this pathogenic bacteria. Here, we report, from two different populations, that between-family variation is a strong intrinsic factor that determines vaccine protection for this disease. While in some full-sib families, the protection added by vaccination increased the survival time in 13 days in comparison with their unvaccinated siblings; in other families, there was no added protection by vaccination or even it was slightly negative. Resistance to P. salmonis, measured as days to death, was higher in vaccinated than unvaccinated fish, but only a moderate positive genetic correlation was obtained between these traits. This disputes a previous hypothesis, that stated that both traits were fully controlled by the same genes, and challenges the use of unvaccinated fish as gold standard for evaluating and selecting fish resistant to P. salmonis, particularly if the offspring will be vaccinated. More studies are necessary to evaluate if variation in the host immune response to vaccination could explain the between-family differences in resistance observed in vaccinated fish.

Single-Step Genome-Wide Association Study for Resistance to Piscirickettsia salmonis in Rainbow Trout (Oncorhynchus mykiss).

One of the main pathogens affecting rainbow trout (Oncorhynchus mykiss) farming is the facultative intracellular bacteria Piscirickettsia salmonis Current treatments, such as antibiotics and vaccines, have not had the expected effectiveness in field conditions. Genetic improvement by means of selection for resistance is proposed as a viable alternative for control. Genomic information can be used to identify the genomic regions associated with resistance and enhance the genetic evaluation methods to speed up the genetic improvement for the trait. The objectives of this study were to i) identify the genomic regions associated with resistance to P. salmonis; and ii) identify candidate genes associated with the trait in rainbow trout. We experimentally challenged 2,130 rainbow trout with P. salmonis and genotyped them with a 57 K single nucleotide polymorphism (SNP) array. Resistance to P. salmonis was defined as time to death (TD) and as binary survival (BS). Significant heritabilities were estimated for TD and BS (0.48 ± 0.04 and 0.34 ± 0.04, respectively). A total of 2,047 fish and 26,068 SNPs passed quality control for samples and genotypes. Using a single-step genome wide association analysis (ssGWAS) we identified four genomic regions explaining over 1% of the genetic variance for TD and three for BS. Interestingly, the same genomic region located on Omy27 was found to explain the highest proportion of genetic variance for both traits (2.4 and 1.5% for TD and BS, respectively). The identified SNP in this region is located within an exon of a gene related with actin cytoskeletal organization, a protein exploited by P. salmonis during infection. Other important candidate genes identified are related with innate immune response and oxidative stress. The moderate heritability values estimated in the present study show it is possible to improve resistance to P. salmonis through artificial selection in the rainbow trout population studied here. Furthermore, our results suggest a polygenic genetic architecture for the trait and provide novel insights into the candidate genes underpinning resistance to P. salmonis in O. mykiss.

Genomic Prediction Accuracy for Resistance Against Piscirickettsia salmonis in Farmed Rainbow Trout.

Salmonid rickettsial syndrome (SRS), caused by the intracellular bacterium Piscirickettsia salmonis, is one of the main diseases affecting rainbow trout (Oncorhynchus mykiss) farming. To accelerate genetic progress, genomic selection methods can be used as an effective approach to control the disease. The aims of this study were: (i) to compare the accuracy of estimated breeding values using pedigree-based best linear unbiased prediction (PBLUP) with genomic BLUP (GBLUP), single-step GBLUP (ssGBLUP), Bayes C, and Bayesian Lasso (LASSO); and (ii) to test the accuracy of genomic prediction and PBLUP using different marker densities (0.5, 3, 10, 20, and 27 K) for resistance against P. salmonis in rainbow trout. Phenotypes were recorded as number of days to death (DD) and binary survival (BS) from 2416 fish challenged with P. salmonis A total of 1934 fish were genotyped using a 57 K single-nucleotide polymorphism (SNP) array. All genomic prediction methods achieved higher accuracies than PBLUP. The relative increase in accuracy for different genomic models ranged from 28 to 41% for both DD and BS at 27 K SNP. Between different genomic models, the highest relative increase in accuracy was obtained with Bayes C (∼40%), where 3 K SNP was enough to achieve a similar accuracy to that of the 27 K SNP for both traits. For resistance against P. salmonis in rainbow trout, we showed that genomic predictions using GBLUP, ssGBLUP, Bayes C, and LASSO can increase accuracy compared with PBLUP. Moreover, it is possible to use relatively low-density SNP panels for genomic prediction without compromising accuracy predictions for resistance against P. salmonis in rainbow trout.

MicroRNA-based transcriptomic responses of Atlantic salmon during infection by the intracellular bacterium Piscirickettsia salmonis.

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as key regulators in diverse biological processes across taxa. However, despite the importance of these transcripts, little is known about their role during the immune response in salmonids. Because of this, we use deep sequencing technologies to explore the microRNA-based transcriptomic response of the Atlantic salmon (Salmo salar) to the intracellular bacteria Piscirickettsia salmonis, one of the main threats to salmon aquaculture in Chile. Hence, 594 different miRNAs were identified from head kidney and spleen transcriptomic data. Among them, miRNA families mir-181, mir-143 and mir-21 were the most abundant in control groups, while after infection with P. salmonis, mir-21, mir-181 and mir-30 were the most predominant families. Furthermore, transcriptional analysis revealed 84 and 25 differentially expressed miRNAs in head kidney and spleen respectively, with an overlapping response of 10 miRNAs between the analyzed tissues. Target prediction, coupled with GO enrichment analysis, revealed that the possible targets of the most regulated miRNAs were genes involved in the immune response, such as cortisol metabolism, chemokine-mediated signaling pathway and neutrophil chemotaxis genes. Among these, predicted putative target genes such as C-C motif chemokine 19-like, stromal cell-derived factor 1-like, myxovirus resistance protein 2 and hepcidin-1 were identified. Overall, our results suggest that miRNA expression in co-modulation with transcription activity of target genes is related to putative roles of non-coding RNAs in the immune response of Atlantic salmon against intracellular bacterial pathogens.

Novel insights into the response of Atlantic salmon (Salmo salar) to Piscirickettsia salmonis: Interplay of coding genes and lncRNAs during bacterial infection.

Despite the high prevalence and impact to Chilean salmon aquaculture of the intracellular bacterium Piscirickettsia salmonis, the molecular underpinnings of host-pathogen interactions remain unclear. Herein, the interplay of coding and non-coding transcripts has been proposed as a key mechanism involved in immune response. Therefore, the aim of this study was to evidence how coding and non-coding transcripts are modulated during the infection process of Atlantic salmon with P. salmonis. For this, RNA-seq was conducted in brain, spleen, and head kidney samples, revealing different transcriptional profiles according to bacterial load. Additionally, while most of the regulated genes annotated for diverse biological processes during infection, a common response associated with clathrin-mediated endocytosis and iron homeostasis was present in all tissues. Interestingly, while endocytosis-promoting factors and clathrin inductions were upregulated, endocytic receptors were mainly downregulated. Furthermore, the regulation of genes related to iron homeostasis suggested an intracellular accumulation of iron, a process in which heme biosynthesis/degradation pathways might play an important role. Regarding the non-coding response, 918 putative long non-coding RNAs were identified, where 425 were newly characterized for S. salar. Finally, co-localization and co-expression analyses revealed a strong correlation between the modulations of long non-coding RNAs and genes associated with endocytosis and iron homeostasis. These results represent the first comprehensive study of putative interplaying mechanisms of coding and non-coding RNAs during bacterial infection in salmonids.

Transcriptional response of Atlantic salmon families to Piscirickettsia salmonis infection highlights the relevance of the iron-deprivation defence system.

BACKGROUND: Piscirickettsiosis or Salmonid Rickettsial Septicaemia (SRS) is a bacterial disease that has a major economic impact on the Chilean salmon farming industry. Despite the fact that Piscirickettsia salmonis has been recognized as a major fish pathogen for over 20 years, the molecular strategies underlying the fish response to infection and the bacterial mechanisms of pathogenesis are poorly understood. We analysed and compared the head kidney transcriptional response of Atlantic salmon (Salmo salar) families with different levels of susceptibility to P. salmonis infection in order to reveal mechanisms that might confer infection resistance. RESULTS: We ranked forty full-sibling Atlantic salmon families according to accumulated mortality after a challenge with P. salmonis and selected the families with the lowest and highest cumulative mortalities for microarray gene expression analysis. A comparison of the response to P. salmonis infection between low and high susceptibility groups identified biological processes presumably involved in natural resistance to the pathogen. In particular, expression changes of genes linked to cellular iron depletion, as well as low iron content and bacterial load in the head kidney of fish from low susceptibility families, suggest that iron-deprivation is an innate immunity defence mechanism against P. salmonis. To complement these results, we predicted a set of iron acquisition genes from the P. salmonis genome. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed that most of these genes form part of the Fur regulon of P. salmonis. CONCLUSIONS: This study revealed, for the first time, differences in the transcriptional response to P. salmonis infection among Atlantic salmon families with varied levels of susceptibility to the infection. These differences correlated with changes in the abundance of transcripts encoding proteins directly and indirectly involved in the immune response; changes that highlighted the role of nutritional immunity through iron deprivation in host defence mechanisms against P. salmonis. Additionally, we found that P. salmonis has several mechanisms for iron acquisition, suggesting that this bacterium can obtain iron from different sources, including ferric iron through capturing endogenous and exogenous siderophores and ferrous iron. Our results contribute to determining the underlying resistance mechanisms of Atlantic salmon to P. salmonis infection and to identifying future treatment strategies.

Patterns of Piscirickettsia salmonis load in susceptible and resistant families of Salmo salar.

The pathogen Piscirickettsia salmonis produces a systemic aggressive infection that involves several organs and tissues in salmonids. In spite of the great economic losses caused by this pathogen in the Atlantic salmon (Salmo salar) industry, very little is known about the resistance mechanisms of the host to this pathogen. In this paper, for the first time, we aimed to identify the bacterial load in head kidney and muscle of Atlantic salmon exhibiting differential familiar mortality. Furthermore, in order to assess the patterns of gene expression of immune related genes in susceptible and resistant families, a set of candidate genes was evaluated using deep sequencing of the transcriptome. The results showed that the bacterial load was significantly lower in resistant fish, when compared with the susceptible individuals. Based on the candidate genes analysis, we infer that the resistant hosts triggered up-regulation of specific genes (such as for example the LysC), which may explain a decrease in the bacterial load in head kidney, while the susceptible fish presented an exacerbated innate response, which is unable to exert an effective response against the bacteria. Interestingly, we found a higher bacterial load in muscle when compared with head kidney. We argue that this is possible due to the availability of an additional source of iron in muscle. Besides, the results show that the resistant fish could not be a likely reservoir of the bacteria.

Multiple tissue transcriptomic responses to Piscirickettsia salmonis in Atlantic salmon (Salmo salar).

The bacterium Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia (SRS), a severe disease that causes major economic losses to the Atlantic salmon aquaculture industry every year. Little is known about the infective strategy of P. salmonis, which is able to infect, survive within, and replicate inside salmonid macrophages as an intracellular parasite. Similarly there is little knowledge concerning the fish host's response to invasion by this pathogen. We have examined the transcriptional response of postsmolt Atlantic salmon (Salmo salar) to P. salmonis at 48 h following infection in three tissues, liver, head kidney, and muscle, using an Atlantic salmon oligonucleotide microarray (Salar_2, Agilent 4x44K). The infection led to a large alteration of transcriptional activity in all the tissues studied. In infected salmon 886, 207, and 153 transcripts were differentially expressed in liver, head kidney, and muscle, respectively. Assessment of enrichment for particular biological pathways by gene ontology analysis showed an upregulation of genes involved in oxidative and inflammatory responses in infected fish, indicative of the activation of the innate immune response. The downregulation of genes involved in the adaptive immune response, G protein signaling pathway, and apoptotic process in infected fish may be reflective of mechanisms used by P. salmonis to survive, replicate, and escape host defenses. There was also evidence of differential responses between studied tissues, with protein metabolism being decreased in muscle of infected fish and with a concomitant increase being shown in liver.