Vasco Felipe Cardoso Neves Mota

Dulce (Palmaria palmata) is a high-value macroalga that is increasingly being cultivated, with strong potential for waste valorisation in nutrient-rich aquaculture systems (RASs). This study evaluated P. palmata growth in, and nutrient uptake from, commercial Atlantic salmon RAS effluent. A 12-week bench-scale experiment cultivated wild-collected P. palmata (average 10 g fresh weight, FW). These were grown in 1 L glass beakers at three effluent dilutions (25%, RAS25; 50%, RAS50; 100%, RAS100) and in seawater (SW) using 10 replicates. The water samples were analysed for ammonium nitrogen (NH₄-N), nitrate nitrogen (NO₃-N), and orthophosphate (PO₄-P) using a spectrophotometer. RAS25 and RAS50 exhibited 100% survival and maintained a dark red colour, with RAS50 achieving the highest specific growth rate (0.49 ± 0.13% day⁻¹), significantly higher than that of SW and RAS25. In contrast, RAS100 led to complete disintegration by 4–8 weeks with significant colour degradation. SW also exhibited reduced colour and 50% mortality by week 12. Sori’s presence was highest in RAS25/RAS50 (up to 80% at week 8 in RAS50), low in SW (10%), and absent in RAS100. The NH₄-N uptake was notably 3× higher than that of NO₃-N (0.16 vs. 0.05 mg g FW⁻¹ day⁻¹), without differences among the groups. The PO₄-P uptake was significantly higher for RAS50 (0.07 mg g FW⁻¹ day⁻¹) than for RAS100. P. palmata performed best in the diluted RAS effluents, as undiluted conditions led to acute tissue disintegration; the nitrogen and phosphorus uptake from the RAS effluents demonstrates significant potential for nutrient valorisation.

Sexual maturation of Atlantic salmon males is marked by dramatic endocrine changes and rapid growth of the testes, resulting in an increase in the gonad somatic index (GSI). We examined the association of gonadal growth with serum sex steroids, as well as pituitary and testicular gene expression levels, which were assessed with a DNA oligonucleotide microarray. The testes transcriptome was stable in males with a GSI < 0.08% despite the large difference between the smallest and the largest gonads. Fish with a GSI ≥ 0.23% had 7–17 times higher serum levels of five male steroids and a 2-fold increase in progesterone, without a change in cortisol and related steroids. The pituitary transcriptome showed an upregulation of the hormone-coding genes that control reproduction and behavior, and structural rearrangement was indicated by the genes involved in synaptic transmission and the differentiation of neurons. The observed changes in the abundance of testicular transcripts were caused by the regulation of transcription and/or disproportional growth, with a greater increase in the germinative compartment. As these factors could not be separated, the transcriptome results are presented as higher or lower specific activities (HSA and LSA). LSA was observed in 4268 genes, including many genes involved in various immune responses and developmental processes. LSA also included genes with roles in female reproduction, germinal cell maintenance and gonad development, responses to endocrine and neural regulation, and the biosynthesis of sex steroids. Two functional groups prevailed among HSA: structure and activity of the cilia (95 genes) and meiosis (34 genes). The puberty of A. salmon testis is marked by the predominance of spermatogenesis, which displaces other processes; masculinization; and the weakening of external regulation. Results confirmed the known roles of many genes involved in reproduction and pointed to uncharacterized genes that deserve attention as possible regulators of sexual maturation.

Introduction: The use of recirculating aquaculture systems (RAS) to produce Atlantic salmon smolts has resulted in exceptionally high fish growth rates. However, there are potential negative trade-offs between fast growth and key physiological processes, such as inadequate smoltification and early sexual maturation, which can both be linked to body energy reserves.Methods: This study determined the effect of i) dietary fat levels on Atlantic salmon whole-body fat and fatty acids composition, growth performance and smoltification and ii) a previous dietary regime on seawater growth performance and male early sexual maturation. In freshwater RAS, salmon parr (~19g) were fed 3 fat levels (20, 24, and 28%) over a 14-week period at 12°C. Subsequently, in seawater flow-through systems, smolt (~96g) were fed a control diet (26%) for 12 weeks at 2 temperatures (12 and 16°C).Results: Dietary fat levels resulted in differences in k-factor, fish whole-body fat and fatty acids composition; the low fat diet resulted in fish with a 23% lower whole-body fat when compared to the other two dietary groups at the end of the freshwater phase. These differences in whole-body fat faded at the end of the seawater phase. During the freshwater phase, all three feeds resulted in a comparable growth performance and smoltification indicator values, including k-factor, gill Na⁺, K⁺-ATPase, blood serum chloride and smolt index score. In contrast, water temperature resulted in fish body weight differences, where groups reared at 16°C were larger than those reared at 12°C. Nevertheless, both temperature regimes supported a similar thermal growth coefficient. Smolts grown at 16°C showed a higher level of the sex steroids androstenedione compared to those at 12°C. However, 11-ketotestosterone and testosterone levels did not differ despite a trend for higher levels at 16°C. Furthermore, testis histology at the final sampling indicated that some individuals showed initial signs of maturation (stage 3).Discussion: In conclusion, varying dietary fat levels (20 - 28%) during the freshwater phase did not influence smoltification or male early sexual maturation during the subsequent grow-out phase. However, a temperature increase from 12 to 16°C resulted in larger fish and appeared to stimulate early male maturation in some fish individuals.

Introduction: Maturation in seawater-adapted (smoltified) male Atlantic salmon is undesirable yet frequently reported in fish weighing up to 1 kg reared under intensive conditions in land-based facilities. Although the photoperiod regulates both smoltification and maturation, the optimal post-smoltification regimen for the prevention of maturation is unclear.Methods: In this study, mixed-sex Atlantic salmon weighing 95 g were exposed to three different photoperiod regimes: continuous daylength, gradually decreasing daylength, and short-to-long daylength after the induction of smoltification by the “winter signal” (12 h:12 h light:dark). Fish were maintained under these conditions in triplicate for 18 weeks in a brackish water flow-through system at 12°C and with a salinity of 20 ppt. This initial phase was followed by a grow-out period (1 kg–2.5 kg) in seawater (32-ppt salinity) at 13°C for 11 weeks, with exposure to decreasing daylength. Throughout the 29-week study, body condition, gonad development, and morphological traits were assessed. Additionally, during exposure to brackish water, serum 11-ketotestosterone (11-KT) levels were quantified, and the testes were examined histologically. Upon termination of the study, the maturation rate was determined.Results: This study exclusively detected maturation among male salmon. Under continuous light exposure, the number of fish initiating maturity steadily increased from week 8 and continued to increase in the subsequent samplings. In the short-to-long daylength group, a subset of male fish showed elevated 11-KT levels at week 14, although their gonadal development and morphological changes were not evident until week 18. Male fish exposed to decreasing daylength showed no substantial increase in 11-KT levels, although a few fish exhibited advanced gonad development by week 18. At the end of the trial, markedly lower percentages of maturing and fully mature male fish were observed among those fish previously exposed to decreasing daylength. The maturation rates at study termination were 44.4% for the short-to-long daylength group, 29.4% for the continuous daylength group, and 4.3% for the decreasing daylength group.Discussion: These results show that photoperiod can be utilized to regulate maturation in male Atlantic salmon. In addition, decreasing the photoperiod after smoltification seems to limit early sexual maturation without compromising body size development. Thus, decreasing the photoperiod could be utilized to limit maturation in the production of post-smolt salmon or salmon in land-based facilities.

Ultrafiltration (UF) membranes are used to successfully remove waterborne virus and bacteria from wastewater and drinking water. However, UF membrane application in aquaculture water treatment is limited. In this study we evaluate the performance of a capillary polyethersulfone UF membrane to remove two benchmark waterborne fish pathogens: i) the infectious pancreatic necrosis virus - IPNV, which is an unenveloped icosahedral virus, and ii) the bacterium Aeromonas salmonicida, which is a Gram-negative, facultative anaerobic bacilli. Moreover, the UF membrane bench-scale unit was tested at two temperatures according to salmonid aquaculture: low (4 - 7°C) and high (16 - 19°C). Sterilised natural seawater was spiked with laboratory cultured pathogens, the water was filtrated, and the membrane permeate collected. Both pathogen solution and permeate were evaluated using a cell culture method to estimate the colony-forming units (CFU/ml) for bacteria presence, a median tissue culture infectious dose (TCID₅₀/ml) assay for virus presence, and real-time quantitative polymerase chain reaction (RT-qPCR) for both bacteria and virus presence. The membrane permeate was negative for both virus and bacteria for all analysis and for both low and high temperatures. The results from this bench-scale study are encouraging for the application of UF membrane technology in aquaculture water treatment to prevent virus and bacteria outbreaks. Further studies should validate this UF membrane technology results in commercial aquaculture conditions.

Oxidative stress is a condition caused by an imbalance in the occurrence of reactive oxygen species in the cells and tissues of organisms. An ultra-performance liquid chromatography–electrospray ionization tandem mass spectrometry (UPLC–ESI–MS/MS) method was developed for the simultaneous determination of two oxidative stress biomarkers, 8-hydroxydeoxyguanosine (8OHDG) and dityrosine (DIY), in the gills, skin, dorsal fin, and liver tissue of Atlantic salmon (Salmo salar) parr. The use of target analyte-specific ¹³C and ¹⁵N internal standards allowed quantification of each target analyte to be performed through the standard solvent calibration curve. The relative recoveries [mean ± (relative standard deviation%)] of 8OHDG and DIY were 101 ± 11 and 104 ± 13% at a fortified concentration of 10 ng/mL (8OHDG) and 500 ng/mL (DIY), respectively, ensuring the accuracy of the extraction and quantification. The chromatographic separation was carried out using a gradient elution program with a total run time of 5 min. The limits of detection (LODs) were 0.11 and 1.37 ng/g wet weight (w.w.) for 8OHDG and DIY, respectively. To demonstrate the applicability of the developed method, it was applied in 907 tissue samples that were collected from Atlantic salmon parr individuals reared in an experimental land-based recirculating aquaculture system (RAS) treated with peracetic acid. Moreover, the possibility of using the dorsal fin as an alternative matrix for the minimally invasive assessment of oxidative stress in Atlantic salmon parr was introduced. To our knowledge, 8OHDG and DIY were used for the first time as biomarkers for biomonitoring the fish health (oxidative stress) of Atlantic salmon parr in RAS.

Peracetic acid (PAA) is an oxidative disinfectant with a broad spectrum of antimicrobial activity and low environmental impact. In this study, we investigated the physiological impacts of PAA application in Atlantic salmon (Salmo salar) parr reared in freshwater recirculating aquaculture systems over a 4-week period. PAA at a target concentration of 1 mg/L was administered either in pulse (every 3 days) or continuous. The group that did not receive PAA served as a control. Fish tissue samples were collected for histology, gene expression, and biochemical analyses at day 0 and after 2 and 4 weeks of exposure. The expression of genes encoding for antioxidant defence in the olfactory organs, skin, and gills changed during the trial, but the temporal effects were more pronounced than inter-treatment impacts. The glutathione group of antioxidant genes was more responsive to PAA. In most cases, an upregulation was observed. Significantly lower levels of reactive oxygen species were identified in the plasma and skin mucus of the two PAA-exposed groups at week 4; nonetheless, significantly increased levels of total antioxidant capacity were only observed in the skin mucus of fish from the continuous treatment group. Additional markers of oxidative stress (i.e., 8-oxo-2′-deoxyguanosine and o,o'-dityrosine) were analysed in the skin, gills, liver, and dorsal fins. These markers were unaffected by the two PAA treatments. Sporadic reversible structural alterations were observed in the three mucosal organs; the changes were time-dependent, and the effects of PAA treatment were minimal. The number of mucous cells varied over time but not within treatments except in the skin of the pulse group at week 4 where a reduction was observed. The ratio of acidic and neutral mucous cells in the skin and gills were affected by PAA treatments especially in the pulse group. Overall, this study revealed that Atlantic salmon parr mobilised mucosal and systemic antioxidant defences against the oxidative disinfectant PAA, but it was evident that the mode of application did not impose a strong influence. The minimal effects of PAA application on the indicators of health and welfare underscore the potential use of PAA as a routine disinfectant in recirculating aquaculture systems.