Search Results (19)

Chile is the second-largest producer of Atlantic salmon (Salmo salar), a key industry facing challenges such as infestations by Caligus rogercresseyi, which cause significant economic losses. However, there are no studies exploring how environmental variables in marine culture centers affect salmon’s immune response, considering their poikilothermic characteristics. This study analyzed the effect of the seasonal temperature on the skin transcriptome of Atlantic salmon infested with C. rogercresseyi in fjord-based farms in the Aysén Region during autumn and spring. Two different centers were analyzed (autumn: Farm-A; spring: Farm-S) to ensure the same age of the fish sampled in each season. RNA sequencing (RNA-seq) and functional network analysis revealed notable seasonal transcriptomic differences. In autumn, 253 differentially expressed genes (DEGs) were found, primarily associated with stress response and negative regulation of T-cell proliferation, suggesting an impaired response against the ectoparasite. In spring, 103 DEGs were detected, associated with protein synthesis and the activation of immune mechanisms, including complement activation, granulocyte chemotaxis, and antigen presentation. This is the first study to evaluate the transcriptomic response of healthy skin in C. rogercresseyi-infested salmon under farming conditions, highlighting the importance of considering seasonal variations for the development of more effective management strategies in the aquaculture industry. Full article

Background/Objetives: Sea lice (Caligus rogercresseyi) pose a major threat to Atlantic salmon (Salmo salar) aquaculture by compromising fish health and reducing production efficiency. While genetic variation in parasite load has been reported, the molecular mechanisms underlying this variation remain unclear. Methods: two sea lice challenge trials were conducted, achieving high infestation rates (47.5% and 43.5%). A total of 85 fish, selected based on extreme phenotypes for lice burden (42 low, 43 high), were subjected to transcriptomic analysis. Differential gene expression was integrated with allele-specific expression (ASE) analysis to uncover cis-regulatory variation influencing host response. Results: Sixty genes showed significant ASE (p < 0.05), including 33 overexpressed and 27 underexpressed. Overexpressed ASE genes included Keratin 15, Collagen IV/V, TRIM16, and Angiopoietin-1-like, which are associated with epithelial integrity, immune response, and tissue remodeling. Underexpressed ASE genes such as SOCS3, CSF3R, and Neutrophil cytosolic factor suggest individual variation in cytokine signaling and oxidative stress pathways. Conclusions: several ASE genes co-localized with previously identified QTLs for sea lice resistance, indicating that cis-regulatory variants contribute to phenotypic differences in parasite susceptibility. These results highlight ASE analysis as a powerful tool to identify functional regulatory elements and provide valuable candidates for selective breeding and genomic improvement strategies in aquaculture. Full article

Aquaculture is currently the fastest-growing sector in animal production, with an average annual growth rate of 7.5% since 1970. In Chile, the industry is largely driven by salmonid farming, with Salmo salar (Atlantic salmon) accounting for over 65% of national production. This species has shown the most significant growth within the sector. This growth is achieved by having high-density farming, which results in high levels of stress due to overcrowding and the appearance of pathogens such as the Infectious Salmon Anemia (ISA) virus, Bacterial Kidney Disease (BKD), Caligus sea lice (Caligus rogercresseyi), and Piscirickettsiosis (SRS) caused by Piscirickettsia salmonis, among others. This study evaluated the toxicity of the two most commonly used antibiotics in the salmon industry—oxytetracycline and florfenicol—at four concentrations each, using primary liver cell cultures of Atlantic salmon (Salmo salar). Oxidative stress was assessed through enzymatic activity and gene expression of oxidative markers, including cytochrome P450, catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx). Samples were analyzed at 1, 3, 6, 12, and 48 h post-exposure. These findings reveal time- and dose-dependent oxidative responses in salmon liver cells to OTC and FLO, providing critical insights into the sublethal cellular effects of antibiotics commonly used in aquaculture, which indicates the presence of a high amount of free radicals in the liver cells, indicating toxicity of both antibiotics. Full article

Proteins present in blood samples from Atlantic salmon (Salmo salar) infected with salmon lice (Lepeophtheirus salmonis) were analyzed using liquid chromatography–high-resolution mass spectrometry. Bioinformatic analyses revealed 1820 proteins, of which 58 were assigned to lice. Among these, peroxiredoxin-2, an antioxidant protein, was found relevant with respect to blood feeding of the parasite. The three-dimensional structure analysis of the protein revealed a surface amino acid sequence of interest. A 13-amino-acid peptide was selected as a potential antigen due to its predicted solubility, antigenicity, probable non-allergenic, and non-toxic nature. This peroxiredoxin-2-derived peptide was synthesized, combined with a commercially available adjuvant, and used for vaccination. The test vaccine demonstrated a 60–70% protection rate against early-stage Lepeophtheirus salmonis infection in a challenge trial in Norway. Additionally, the vaccine was tested against salmon lice (Caligus rogercresseyi) in Chile, where a remarkable 92% reduction in the number of adult lice was observed. Thus, in combination with the selected adjuvant, the peptide showed antigenic potential, making it a suitable candidate for future vaccine development. The approach described holds promise for the development of peptide vaccines against various ectoparasites feeding on blood or skin secretions of their hosts. Full article

Currently, one of the primary challenges in salmon farming is caligidosis, caused by the copepod ectoparasites Caligus spp. The infection process is determined by the copepod’s ability to adhere to the fish skin through the insertion of its chitin-composed filament. In this study, we examined several antimicrobial peptides previously identified in salmonid mucosal secretions, with a primary focus on their potential to bind to chitin as an initial step. The binding capacity to chitin was tested, with hepcidin and piscidin showing positive results. Further assessments involving cytotoxicity in salmonid cells RTgill-W1, SHK-1, RTS-11, and RT-gut indicated that the peptides did not adversely affect cell viability. However, hemolysis assays unveiled the hemolytic capacity of piscidin at lower concentrations, leading to the selection of hepcidin for antiparasitic assays. The results demonstrated that the nauplius II stage of C. rogercresseyi exhibited higher susceptibility to hepcidin treatments, achieving a 50% reduction in parasitic involvement at 50 µM. Utilizing fluorescence and scanning electron microscopy, we observed the localization of hepcidin on the surface of the parasite, inducing significant spherical protuberances along the exoskeleton of C. rogercresseyi. These findings suggest that cysteine-rich AMPs derived from fish mucosa possess the capability to alter the development of the chitin exoskeleton in copepod ectoparasites, making them therapeutic targets to combat recurrent parasitic diseases in salmon farming. Full article

The immune response of Atlantic salmon to sea lice has been extensively studied, but we still do not know the mechanisms by which some fish become resistant and others do not. In this study, we estimated the heritabilities of three key proteins associated with the innate immunity and resistance of Salmo salar against the sea louse Caligus rogercresseyi. In particular, we quantified the abundance of 2 pro-inflammatory cytokines, Tnfα and Il-8, and an antioxidant enzyme, Nkef, in Atlantic salmon skin and gill tissue from 21 families and 268 individuals by indirect ELISA. This covers a wide parasite load range from low or resistant (mean sea lice ± SE = 8.7 ± 0.9) to high or susceptible (mean sea lice ± SE = 43.3 ± 2.0). Our results showed that susceptible fish had higher levels of Nkef and Tnfα than resistant fish in their gills and skin, although gill Il-8 was higher in resistant fish, while no significant differences were found in the skin. Furthermore, moderate to very high heritable genetic variation was estimated for Nkef (h² skin: 0.96 ± 0.14 and gills: 0.97 ± 0.11) and Tnfα (h² skin: 0.53 ± 0.17 and gills: 0.32 ± 0.14), but not for Il-8 (h² skin: 0.22 ± 0.12 ns and gills: 0.09 ± 0.08 ns). This work provides evidence that Nkef and Tnfα protein expressions are highly heritable and related to resistance against sea lice in Atlantic salmon. Full article

Salmon aquaculture is constantly threatened by pathogens that impact fish health, welfare, and productivity, including the sea louse Caligus rogercresseyi. This marine ectoparasite is mainly controlled through delousing drug treatments that have lost efficacy. Therein, strategies such as salmon breeding selection represent a sustainable alternative to produce fish with resistance to sea lice. This study explored the whole-transcriptome changes in Atlantic salmon families with contrasting resistance phenotypes against lice infestation. In total, 121 Atlantic salmon families were challenged with 35 copepodites per fish and ranked after 14 infestation days. Skin and head kidney tissue from the top two lowest (R) and highest (S) infested families were sequenced by the Illumina platform. Genome-scale transcriptome analysis showed different expression profiles between the phenotypes. Significant differences in chromosome modulation between the R and S families were observed in skin tissue. Notably, the upregulation of genes associated with tissue repairs, such as collagen and myosin, was found in R families. Furthermore, skin tissue of resistant families showed the highest number of genes associated with molecular functions such as ion binding, transferase, and cytokine activity, compared with the susceptible. Interestingly, lncRNAs differentially modulated in the R/S families are located near genes associated with immune response, which are upregulated in the R family. Finally, SNPs variations were identified in both salmon families, where the resistant ones showed the highest number of SNPs variations. Remarkably, among the genes with SPNs, genes associated with the tissue repair process were identified. This study reported Atlantic salmon chromosome regions exclusively expressed in R or S Atlantic salmon families’ phenotypes. Furthermore, due to the presence of SNPs and high expression of tissue repair genes in the resistant families, it is possible to suggest mucosal immune activation associated with the Atlantic salmon resistance to sea louse infestation. Full article

The development of vaccines against sea lice in salmon farming is complex, expensive, and takes several years for commercial availability. Recently, transcriptome studies in sea louse have provided valuable information for identifying relevant molecules with potential use for fish vaccines. However, the bottleneck is the in vivo testing of recombinant protein candidates, the dosage, and the polyvalent formulation strategies. This study explored a cell-based approach to prospect antigens as candidate vaccines against sea lice by comparison with immunized fish. Herein, SHK-1 cells and Atlantic salmon head kidney tissue were exposed to the antigen cathepsin identified from the sea louse Caligus rogercresseyi. The cathepsin protein was cloned and recombinantly expressed in Escherichia coli, and then SHK-1 cell lines were stimulated with 100 ng/mL cathepsin recombinant for 24 h. In addition, Atlantic salmons were vaccinated with 30 ug/mL recombinant protein, and head kidney samples were then collected 30 days post-immunization. SHK-1 cells and salmon head kidney exposed to cathepsin were analyzed by Illumina RNA sequencing. The statistical comparisons showed differences in the transcriptomic profiles between SHK-1 cells and the salmon head kidney. However, 24.15% of the differentially expressed genes were shared. Moreover, putative gene regulation through lncRNAs revealed tissue-specific transcription patterns. The top 50 up and downregulated lncRNAs were highly correlated with genes involved in immune response, iron homeostasis, pro-inflammatory cytokines, and apoptosis. Also, highly enriched pathways related to the immune system and signal transduction were shared between both tissues. These findings highlight a novel approach to evaluating candidate antigens for sea lice vaccine development, improving the antigens screening in the SHK-1 cell line model. Full article

Due to the reduced efficacy of delousing drugs used for sea lice control in salmon aquaculture, fish vaccines have emerged as one of the most sustainable strategies in animal health. Herein, the availability of C. rogercresseyi and Salmo salar genomes increases the capability of identifying new candidate antigens for lice vaccines using RNA sequencing and computational tools. This study aimed to evaluate the effects of two recombinant antigens characterized as peritrophin and cathepsin proteins on the transcriptome profiling of Atlantic salmon during a sea lice infestation. Four experimental groups were used: Peritrophin, cathepsin, and peritrophin/cathepsin (P/C), and PBS as the control. C. rogercresseyi female, S. salar head kidney, and skin tissue samples were sampled at 25 days post-infestation (dpi) for Illumina sequencing and RNA-seq analysis. Differential gene expression, gene ontology, and chromosomal expression analyses were performed. Furthermore, the dual RNA-seq analysis approach was performed to simultaneously explore host and pathogen transcriptomes, identifying functional associations for vaccine design. The morphometry of female sea lice exposed to immunized fish was also evaluated. The RNA-Seq analysis exhibited prototype-dependent transcriptome modulation, showing a conspicuous competition for metal ions during the infestation. Moreover, Dual RNA-seq analysis revealed vaccine-dependent gene patterns in both the host and the pathogen. Notably, significant morphometric differences between lice collected from immunized and control fish were observed, where cathepsin and P/C showed 57% efficacy. This study showed the potential of two proteins as lice vaccines for the salmon industry, suggesting novel molecular mechanisms between host–parasite interactions. Full article

Caligus rogercresseyi is the main ectoparasite that affects the salmon industry in Chile. The mechanisms used by the parasite to support its life strategy are of great interest for developing control strategies. Due to the critical role of insect peritrophins in host–parasite interactions and response to pest control drugs, this study aimed to identify and characterize the peritrophin-like genes present in C. rogercresseyi. Moreover, the expression of peritrophin-like genes was evaluated on parasites exposed to delousing drugs such as pyrethroids and azamethiphos. Peritrophin genes were identified by homology analysis among the sea louse transcriptome database and arthropods peritrophin-protein database obtained from GenBank and UniProt. Moreover, the gene loci in the parasite genome were located. Furthermore, peritrophin gene expression levels were evaluated by RNA-Seq analysis in sea louse developmental stages and sea lice exposed to delousing drugs deltamethrin, cypermethrin, and azamethiphos. Seven putative peritrophin-like genes were identified in C. rogercresseyi with high homology with other crustacean peritrophins. Differences in the presence of signal peptides, the number of chitin-binding domains, and the position of conserved cysteines were found. In addition, seven peritrophin-like gene sequences were identified in the C. rogercresseyi genome. Gene expression analysis revealed a stage-dependent expression profile. Notably, differential regulation of peritrophin genes in resistant and susceptible populations to delousing drugs was found. These data are the first report and characterization of peritrophin genes in the sea louse C. rogercresseyi, representing valuable knowledge to understand sea louse biology. Moreover, this study provides evidence for a deeper understanding of the molecular basis of C. rogercresseyi response to delousing drugs. Full article

The sea louse Caligus rogercresseyi genome has opened the opportunity to apply the reverse vaccinology strategy for identifying antigens with potential effects on lice development and its application in sea lice control. This study aimed to explore the efficacy of three sea lice vaccines against the early stage of infestation, assessing the transcriptome modulation of immunized Atlantic salmon. Therein, three experimental groups of Salmo salar (Atlantic salmon) were vaccinated with the recombinant proteins: Peritrophin (prototype A), Cathepsin (prototype B), and the mix of them (prototype C), respectively. Sea lice infestation was evaluated during chalimus I-II, the early-infective stages attached at 7-days post infestation. In parallel, head kidney and skin tissue samples were taken for mRNA Illumina sequencing. Relative expression analyses of genes were conducted to identify immune responses, iron transport, and stress responses associated with the tested vaccines during the early stages of sea lice infection. The vaccine prototypes A, B, and C reduced the parasite burden by 24, 44, and 52% compared with the control group. In addition, the RNA-Seq analysis exhibited a prototype-dependent transcriptome modulation. The high expression differences were observed in genes associated with metal ion binding, molecular processes, and energy production. The findings suggest a balance between the host’s inflammatory response and metabolic process in vaccinated fish, increasing their transcriptional activity, which can alter the early host–parasite interactions. This study uncovers molecular responses produced by three vaccine prototypes at the early stages of infestation, providing new knowledge for sea lice control in the salmon aquaculture. Full article

In this work, two microencapsulation techniques were used to protect and improve the absorption of emamectin benzoate (EB), which is an antiparasitic drug used to control Caligus rogercresseyi. EB has a low aqueous solubility, which affects its absorption in the intestine of Salmo salar. Microparticles were produced by spray drying and ionic gelation, using Soluplus^® (EB–SOL) and sodium alginate (EB–ALG) as polymers, respectively. Studies were conducted on dissolution/permeation, apparent permeability (Papp), apparent solubility (Sapp), and absorption using synthetic and biological membranes. Based on these results, the amount of EB in the microparticles needed to achieve a therapeutic dose was estimated. The EB–ALG microparticles outperformed both EB–SOL and free EB, for all parameters analyzed. The results show values of 0.45 mg/mL (80.2%) for dissolution/permeation, a Papp of 6.2 mg/mL in RS–L, an absorption of 7.3% in RS, and a Sapp of 53.1% in EM medium. The EB–ALG microparticles decrease the therapeutic dose necessary to control the parasite, with values of 3.0⁻² mg/mL and 1.1⁻² mg/mL for EB in EM and RS, respectively. The Korsmeyer–Peppas kinetic model was the best model to fit the EB–ALG and EB–SOL dissolution/permeation experiments. In addition, some of our experimental results using synthetic membranes are similar to those obtained with biological membranes, which suggests that, for some parameters, it is possible to replace biological membranes with synthetic membranes. The encapsulation of EB by ionic gelation shows it is a promising formulation to increase the absorption of the poorly soluble drug. In contrast, the spray-dried microparticles produced using Soluplus^® result in even less dissolution/permeation than free EB, so the technique cannot be used to improve the solubility of EB. Full article

The role of trypsin genes in pharmacological sensitivity has been described in numerous arthropod species, including the sea louse Caligus rogercresseyi. This ectoparasite species is mainly controlled by xenobiotic drugs in Atlantic salmon farming. However, the post-transcriptional regulation of trypsin genes and the molecular components involved in drug response remain unclear. In particular, the miRNA bantam family has previously been associated with drug response in arthropods and is also found in C. rogercresseyi, showing a high diversity of isomiRs. This study aimed to uncover molecular interactions among trypsin genes and bantam miRNAs in the sea louse C. rogercresseyi in response to delousing drugs. Herein, putative mRNA/miRNA sequences were identified and localized in the C. rogercresseyi genome through genome mapping and blast analyses. Expression analyses were obtained from the mRNA transcriptome and small-RNA libraries from groups with differential sensitivity to three drugs used as anti-sea lice agents: azamethiphos, deltamethrin, and cypermethrin. The validation was conducted by qPCR analyses and luciferase assay of selected bantam and trypsin genes identified from in silico transcript prediction. A total of 60 trypsin genes were identified in the C. rogercresseyi genome, and 39 bantam miRNAs were differentially expressed in response to drug exposure. Notably, expression analyses and correlation among values obtained from trypsin and bantam revealed an opposite trend and potential binding sites with significant ΔG values. The luciferase assay showed a reduction of around 50% in the expression levels of the trypsin 2-like gene, which could imply that this gene is a potential target for bantam. The role of trypsin genes and bantam miRNAs in the pharmacological sensitivity of sea lice and the use of miRNAs as potential markers in these parasites are discussed in this study. Full article

Infection processes displayed by pathogens require the acquisition of essential inorganic nutrients and trace elements from the host to survive and proliferate. Without a doubt, iron is a crucial trace metal for all living organisms and also a pivotal component in the host–parasite interactions. In particular, the host reduces the iron available to face the infectious disease, increasing iron transport proteins’ expression and activating the heme synthesis and degradation pathways. Moreover, recent findings have suggested that iron metabolism modulation in fish promotes the immune response by reducing cellular iron toxicity. We hypothesized that recombinant proteins related to iron metabolism could modulate the fish’s immune system through iron metabolism and iron-responsive genes. Here a chimeric iron transport protein (IPath^®) was bioinformatically designed and then expressed in a recombinant bacterial system. The IPath^® protein showed a significant chelating activity under in vitro conditions and biological activity. Taking this evidence, a vaccine candidate based on IPath^® was evaluated in Atlantic salmon challenged with three different fish pathogens. Experimental trials were conducted using two fish groups: one immunized with IPath^® and another injected with adjutant as the control group. After 400 accumulated thermal units (ATUs), two different infection trials were performed. In the first one, fish were infected with the bacterium Aeromonas salmonicida, and in a second trial, fish were exposed to the ectoparasite Caligus rogercresseyi and subsequently infected with the intracellular bacterium Piscirickettsia salmonis. Fish immunized with IPath^® showed a significant delay in the mortality curve in response to A. salmonicida and P. salmonis infections. However, no significant differences between infected and control fish groups were observed at the end of the experiment. Notably, sea lice burden reduction was observed in vaccinated Atlantic salmon. Transcriptional analysis evidenced a high modulation of iron-homeostasis-related genes in fish vaccinated with IPath^® compared to the control group during the infection. Moreover, increasing expression of Atlantic salmon IgT was associated with IPath^® immunization. This study provides evidence that the IPath^® protein could be used as an antigen or booster in commercial fish vaccines, improving the immune response against relevant pathogens for salmon aquaculture. Full article

Caligus rogercresseyi, commonly known as sea louse, is an ectoparasite copepod that impacts the salmon aquaculture in Chile, causing losses of hundreds of million dollars per year. This pathogen is mainly controlled by immersion baths with delousing drugs, which can lead to resistant traits selection in lice populations. Bioassays are commonly used to assess louse drug sensitivity, but the current procedures may mask relevant molecular responses. This study aimed to discover novel coding genes and non-coding RNAs that could evidence drug sensitivity at the genomic level. Sea lice samples from populations with contrasting sensitivity to delousing drugs were collected. Bioassays using azamethiphos, cypermethrin, and deltamethrin drugs were conducted to evaluate the sensitivity and to collect samples for RNA-sequencing. Transcriptome sequencing was conducted on samples exposed to each drug to evaluate the presence of coding and non-coding RNAs associated with the response of these compounds. The results revealed specific transcriptome patterns in lice exposed to azamethiphos, deltamethrin, and cypermethrin drugs. Enrichment analyses of Gene Ontology terms showed specific biological processes and molecular functions associated with each delousing drug analyzed. Furthermore, novel long non-coding RNAs (lncRNAs) were identified in C. rogercresseyi and tightly linked to differentially expressed coding genes. A significant correlation between gene transcription patterns and phenotypic effects was found in lice collected from different salmon farms with contrasting drug treatment efficacies. The significant correlation among gene transcription patterns with the historical background of drug sensitivity suggests novel molecular mechanisms of pharmacological resistance in lice populations. Full article