Vibrio anguillarum is a Gram-negative marine pathogen causative agent of vibriosis in a wide range of hosts, including invertebrates and teleosts. Lumpfish (Cyclopterus lumpus), a native fish of the North Atlantic Ocean, is utilized as cleaner fish to control sea lice (Lepeophtheirus salmonis) infestations in the Atlantic salmon (Salmo salar) aquaculture industry. V. anguillarum is one of the most frequent bacterial pathogens affecting lumpfish. Here, we described the phenotype and genomic characteristics of V. anguillarum strain J360 isolated from infected cultured lumpfish in Newfoundland, Canada. Koch’s postulates determined in naïve lumpfish showed lethal acute vibriosis in lumpfish. The V. anguillarum J360 genome was shown to be composed of two chromosomes and two plasmids with a total genome size of 4.56 Mb with 44.85% G + C content. Phylogenetic and comparative analyses showed that V. anguillarum J360 is closely related to V. anguillarum strain VIB43, isolated in Scotland, with a 99.8% genome identity. Differences in the genomic organization were identified and associated with insertion sequence elements (ISs). Additionally, V. anguillarum J360 does not possess a pJM1-like plasmid, typically present in virulent isolates from the Pacific Ocean, suggesting that acquisition of this extrachromosomal element and the virulence of V. anguillarum J360 or other Atlantic isolates could increase.

Lumpfish is utilized as a cleaner fish to biocontrol sealice infestations in Atlantic salmon farms. Aeromonas salmonicida, a Gram-negative facultative intracellular pathogen, is the causative agent of furunculosis in several fish species, including lumpfish. In this study, lumpfish were intraperitoneally injected with different doses of A. salmonicida to calculate the LD₅₀. Samples of blood, head-kidney, spleen, and liver were collected at different time points to determine the infection kinetics. We determined that A. salmonicida LD₅₀ is 10² CFU per dose. We found that the lumpfish head-kidney is the primary target organ of A. salmonicida. Triplicate biological samples were collected from head-kidney, spleen, and liver pre-infection and at 3- and 10-days post-infection for RNA-sequencing. The reference genome-guided transcriptome assembly resulted in 6246 differentially expressed genes. The de novo assembly resulted in 403,204 transcripts, which added 1307 novel genes not identified by the reference genome-guided transcriptome. Differential gene expression and gene ontology enrichment analyses suggested that A. salmonicida induces lethal infection in lumpfish by uncontrolled and detrimental blood coagulation, complement activation, inflammation, DNA damage, suppression of the adaptive immune system, and prevention of cytoskeleton formation.

Aeromonas salmonicida, a widely distributed aquatic pathogen causing furunculosis in fish, exhibits varied virulence, posing challenges in infectious disease and immunity studies, notably in vaccine efficacy assessment. Lumpfish (Cyclopterus lumpus) has become a valuable model for marine pathogenesis studies. This study evaluated several antigen preparations against A. salmonicida J223, a hypervirulent strain of teleost fish, including lumpfish. The potential immune protective effect of A. salmonicida bacterins in the presence and absence of the A‐layer and extracellular products was tested in lumpfish. Also, we evaluated the impact of A. salmonicida outer membrane proteins (OMPs) and iron‐regulated outer membrane proteins (IROMPs) on lumpfish immunity. The immunized lumpfish were intraperitoneally (i.p.) challenged with 10⁴ A. salmonicida cells/dose at 8 weeks‐post immunization (wpi). Immunized and non‐immunized fish died within 2 weeks post‐challenge. Our analyses showed that immunization with A. salmonicida J223 bacterins and antigen preparations did not increase IgM titres. In addition, adaptive immunity biomarker genes (e.g., igm, mhc‐ii and cd4) were down‐regulated. These findings suggest that A. salmonicida J223 antigen preparations hinder lumpfish immunity. Notably, many fish vaccines are bacterin‐based, often lacking efficacy evaluation. This study offers crucial insights for finfish vaccine approval and regulations.

MicroRNAs (miRNAs) are endogenous small RNA molecules involved in the post-transcriptional regulation of protein expression by binding to the mRNA of target genes. They are key regulators in teleost development, maintenance of tissue-specific functions, and immune responses. Lumpfish (Cyclopterus lumpus) is becoming an emergent aquaculture species as it has been utilized as a cleaner fish to biocontrol sea lice (e.g., Lepeophtheirus salmonis) infestation in the Atlantic Salmon (Salmo salar) aquaculture. The lumpfish miRNAs repertoire is unknown. This study identified and characterized miRNA encoding genes in lumpfish from three developmental stages (adult, embryos, and larvae). A total of 16 samples from six different adult lumpfish organs (spleen, liver, head kidney, brain, muscle, and gill), embryos, and larvae were individually small RNA sequenced. Altogether, 391 conserved miRNA precursor sequences (discovered in the majority of teleost fish species reported in miRbase), eight novel miRNA precursor sequences (so far only discovered in lumpfish), and 443 unique mature miRNAs were identified. Transcriptomics analysis suggested organ-specific and age-specific expression of miRNAs (e.g., miR-122-1-5p specific of the liver). Most of the miRNAs found in lumpfish are conserved in teleost and higher vertebrates, suggesting an essential and common role across teleost and higher vertebrates. This study is the first miRNA characterization of lumpfish that provides the reference miRNAome for future functional studies.

The development of effective vaccines is a critical step in effective disease management in aquaculture. This study introduces a novel Multiepitope Chimeric Vaccine (MCV) designed to enhance immunity in lumpfish against Vibrio anguillarum, Aeromonas salmonicida, Yersinia ruckeri, Moritella viscosa and Piscirickettsia salmonis. Epitopes from major toxins and virulence factors were selected to construct the MCV in silico. Structural validation showed 96.7% of residues in favored regions, confirming stability. Codon optimization yielded a G+C content of 54.61% and a Codon Adaptation Index (CAI) of 1, indicating strong expression potential in Escherichia coli. Immune simulations predicted robust B- and T-cell responses, suggesting induction of both humoral and cell-mediated immunity. Experimental vaccination of lumpfish (n = 35/group) with E. coli-expressed MCV led to significantly elevated IgM levels at four- and six-weeks post-vaccination (p ≤ 0.05, p ≤ 0.01, respectively). Upon pathogen challenge, vaccinated groups showed delayed mortality against V. anguillarum, A. salmonicida, and P. salmonis, though survival differences were not statistically significant across treatments. These results highlight the immunogenicity potential of the MCV and its capacity to elicit targeted immune responses. However, further optimization is necessary to improve protective efficacy and survival outcomes. This study lays a foundation for the application of multiepitope vaccines in lumpfish aquaculture and supports ongoing efforts toward sustainable disease control strategies.