Stefano Peruzzi

Background : Pancreas disease, caused by salmonid alphavirus subtype 3 (SAV3), remains a significant threat to Atlantic salmon (Salmo salar L.) aquaculture in Norway despite the widespread use of inactivated vaccines. Molecular adjuvants represent a promising strategy to improve vaccine efficacy by enhancing immune responses. Methods: In this study, we evaluated the immunological and protective effects of two transcription factors, gata3 and eomes encoded by plasmid DNA adjuvants in combination with a low-dose inactivated SAV3 vaccine. Atlantic salmon were immunized with inactivated virus alone or in combination with either gata3- or eomes-encoding plasmids, followed by SAV3 challenge. Vaccine performance was assessed by growth, antibody response (ELISA and virus neutralization assay), and histopathological scoring of heart and pancreas. Results: The effects of gata3 and eomes were context-dependent: eomes reduced pancreas lesions at 4 weeks post-challenge, while both plasmid-adjuvant groups showed improved growth at 10 weeks post-challenge compared to the plasmid control. Although antibody responses were increased in all vaccinated groups at 6 weeks post-immunization, the plasmid control performed comparably to the GATA3 or EOMES groups, and no significant differences were detected in the virus neutralization test. Interestingly, the empty plasmids given to control fish exhibited immunostimulatory activity, warranting further investigation. In vitro and in vivo fluorescence microscopy confirmed expression of the transcription factors in transfected cells and at the injection site. Conclusions: Our results suggest that transcription factor-encoding plasmids can provide selective adjuvant effects, while also highlighting the potential that plasmid DNA can have as adjuvants in fish vaccine development.

In this study, we report the genome size and karyotype of the spotted wolffish (Anarhichas minor), a species classified at risk in Canada and not commonly the target of directed commercial fisheries elsewhere but increasingly significant for aquaculture. Despite the scientific and commercial interest, there is a lack of cytogenetic and genomic information about this species. Flow cytometric analysis determined an average haploid nuclear DNA content of 0.68 pg (665 Mb), with no significant differences observed between male and female fish. Karyotyping revealed a diploid number (2n) of 48 chromosomes, predominantly consisting of two-armed chromosomes, with no evidence of sex-linked chromosomal heteromorphism. These findings provide critical baseline data for interpreting and validating genome sequencing efforts and lay the groundwork for further cytogenetic studies of A. minor.

The induction of triploidy, a strategy to mitigate unwanted pre-harvest sexual maturation and a genetic containment measure for escaped farmed Atlantic salmon (Salmo salar), may give rise to challenges because of the distinct environmental and dietary requirements of sterile triploid fish. Smoltification is a critical phase in the life cycle of Atlantic salmon, so knowledge about parr–smolt transformation in triploids is important for the salmon farming industry. This study covered an investigation of hepatic expression of growth hormone receptor (GHrec) and insulin-like growth factor-I (IGF-I) genes, both of which are intimately involved in the regulation of osmoregulation and growth. Additionally, hepatic presence and location of IGF-I mRNA were examined using RNAscope^®, an advanced in situ hybridization technique. Triplicate groups of juvenile diploid and triploid salmon were reared at low temperature (10 °C) and fed either a standard diet or one enriched with hydrolyzed fish proteins from the start of feeding onwards. Liver samples were collected from three fish per tank each month from October to December (2454–3044 degree-days post-start feeding), the period encompassing smoltification, and hepatic expression of IGF-I and GHrec genes was quantified by real-time PCR. The results indicated that neither ploidy nor diet significantly influenced IGF-I or GHrec gene expression, suggesting that, under our conditions, triploidy and diet did not adversely affect this molecular pathway linked to growth and osmoregulation. IGF-I gene expression exhibited significant temporal variation, correlating with the progression of smoltification, while GHrec gene expression showed a similar, albeit non-significant, trend. Triploids exhibited IGF-I and GHrec gene expression patterns comparable to diploids, and both the temporal changes and lack of difference between triploids and diploids were mirrored in the quantification of IGF-I mRNA within the liver cells. The potential applicability to a commercial aquaculture setting requires further investigation.

The genome size of organisms impacts their evolution and biology and is often assumed to be characteristic of a species. Here we present the first published estimates of genome size of the ecologically and economically important ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae). Four independent L. salmonis genome assemblies of the North Atlantic subspecies Lepeophtheirus salmonis salmonis, including two chromosome level assemblies, yield assemblies ranging from 665 to 790 Mbps. These genome assemblies are congruent in their findings, and appear very complete with Benchmarking Universal Single-Copy Orthologs analyses finding > 92% of expected genes and transcriptome datasets routinely mapping > 90% of reads. However, two cytometric techniques, flow cytometry and Feulgen image analysis densitometry, yield measurements of 1.3–1.6 Gb in the haploid genome. Interestingly, earlier cytometric measurements reported genome sizes of 939 and 567 Mbps in L. salmonis salmonis samples from Bay of Fundy and Norway, respectively. Available data thus suggest that the genome sizes of salmon lice are variable. Current understanding of eukaryotic genome dynamics suggests that the most likely explanation for such variability involves repetitive DNA, which for L. salmonis makes up ≈ 60% of the genome assemblies.

The genome size of organisms impacts their evolution and biology and is often assumed to be characteristic of a species. Here we present the first published estimates of genome size of the ecologically and economically important ectoparasite, Lepeophtheirus salmonis (Copepoda, Caligidae). Four independent L. salmonis genome assemblies of the North Atlantic subspecies Lepeophtheirus salmonis salmonis , including two chromosome level assemblies, yield assemblies ranging from 665 – 790 Mbps. These genome assemblies are congruent in their findings, and appear very complete with Benchmarking Universal Single-Copy Orthologs analyses finding >92% of expected genes and transcriptome datasets routinely mapping >90% of reads. However, two cytometric techniques, flow cytometry and Feulgen image analysis densitometry, yield measurements of 1.3-1.6 Gb in the haploid genome. Interestingly, earlier cytometric measurements reported genome sizes of 939 and 567 Mbps in L. salmonis salmonis samples from Bay of Fundy and Norway, respectively. Available data thus suggest that the genome sizes of salmon lice are variable. Current understanding of eukaryotic genome dynamics suggests that the most likely explanation for such variability involves repetitive DNA, which for L. salmonis makes up ≈60% of the genome assemblies.

Disease resistance of fish larvae may be improved by bath treatment in water containing immunostimulants. Pattern recognition receptors, such as TLR3, TLR7, and MDA5, work as an “early warning” to induce intracellular signaling and facilitate an antiviral response. A single bath of newly hatched larvae, with Astragalus, upregulated the expression of IFNα, IFNc, ISG15, MDA5, PKR, STAT1, TLR3, and TLR7 immune genes, on day 4 post treatment. Similar patterns were observed for Hyaluronic acid and Poly I:C. Increased expression was observed for ISG15, MDA5, MX, STAT1, TLR3, TLR7, and RSAD2, on day 9 for Imiquimod. Metabolic gene expression was stimulated on day 1 after immunostimulant bath in ULK1, MYC, SLC2A1, HIF1A, MTOR, and SIX1, in Astragalus, Hyaluronic acid, and Imiquimod. Expression of NOS2 in Poly I:C was an average fourfold above that of control at the same timepoint. Throughout the remaining sampling days (2, 4, 9, 16, 32, and 45 days post immunostimulant bath), NOS2 and IL1B were consistently overexpressed. In conclusion, the immunostimulants induced antiviral gene responses, indicating that a single bath at an early life stage could enable a more robust antiviral defense in fish. Additionally, it was demonstrated, based on gene expression data, that cell metabolism was perturbed, where several metabolic genes were co-regulated with innate antiviral genes.

Triploid, sterile Atlantic salmon (Salmo salar) could make a contribution to the development of the farming industry, but uncertainties about the performance and welfare of triploids have limited their adoption by farmers. In this study, we compared the ontogeny of digestive tract morphology and enzyme activities (pepsin, trypsin, chymotrypsin, alkaline phosphatase and aminopeptidase) of diploid and triploid Atlantic salmon. Fish were fed diets based on fishmeal (STD) or a mix of fishmeal and hydrolysed fish proteins (HFM) whilst being reared at low temperature from start-feeding to completion of the parr-smolt transformation. Fish weights for each ploidy and feed combination were used to calculate thermal growth coefficients (TGCs) that spanned this developmental period, and the data were used to examine possible relationships between enzyme activities and growth. At the end of the experiment, faeces were collected and analyzed to determine the apparent digestibility coefficients (ADCs) of the dietary amino acids (AAs). Digestive tract histo-morphology did not differ substantially between ploidies and generally reflected organ maturation and functionality. There were no consistent differences in proteolytic enzyme activities resulting from the inclusion of HFM in the diet, nor was there improved digestibility and AA bioavailability of the HFM feed in either diploid or triploid fish. The triploid salmon had lower ADCs than diploids for most essential and non-essential AAs in both diets (STD and HFM), but without there being any indication of lower intestinal protease activity in triploid fish. When trypsin-to-chymotrypsin activity and trypsin and alkaline phosphatase (ALP) ratios (T:C and T:ALP, respectively) were considered in combination with growth data (TGC) low T:C and T:ALP values coincided with times of reduced fish growth, and vice versa, suggesting that T:C and T:ALP may be used to predict recent growth history and possible growth potential.

Triploid Atlantic salmon (Salmo salar L.) is seen as one of the best solutions to solve key issues in the salmon farming industry, such as the impact of escapees on wild stocks and pre-harvest sexual maturation. However, the effects of triploidy on salmon smoltification are poorly understood at the molecular level, even though smoltification is a very sensitive period that has a major influence on survival rate and performance of farmed salmon. In this study, we have compared the liver transcriptomes of diploid and triploid Atlantic salmon at three ontogeny stages: fry, parr and smolt. In diploid fish, a total of 2,655 genes were differentially expressed between fry and parr, whereas 506 genes had significantly different transcript levels between parr and smolts. In triploids, 1,507 and 974 genes were differentially expressed between fry and parr, and between parr and smolts, respectively. Most of these genes were down-regulated and 34 genes were differentially expressed between ploidies at the same stage. In both ploidy groups, the top differentially expressed genes with ontogeny stage belonged to common functional categories that can be related to smoltification. Nucleotide and energy metabolism were significantly down-regulated in fry when compared to parr, while immune system processes were significantly down-regulated in parr when compared to smolts. The close resemblance of enriched biological processes and pathways between ploidy groups suggests that triploidy is regulated by genome dosage compensation in Atlantic salmon. Histological analysis revealed that areas of vacuolization (steatosis) were present only in fry and parr stages, in contrast to a compact cellular histology with glycogen granules after smoltification. There was no significant difference in vacuolization between ploidy groups at the fry stage but the liver of diploid parr had a 33.5% higher vacuolization area compared to their triploid counterparts. Taken together, our data provide novel insights into the changes that occur at the molecular and histological level in the liver of both diploid and triploid Atlantic salmon prior to and during smoltification.