Impact of ozone treatment on dissolved organic matter in land-based recirculating aquaculture systems studied by Fourier transform ion cyclotron resonance mass spectrometry.

In land-based recirculating aquaculture systems (RAS), the accumulation of dissolved organic matter (DOM) can have detrimental effects on water quality impacting the system performance, microbial community, and consequently fish health and welfare. Ozone is used in the RAS water treatment process to improve water quality and remove DOM. However, little is known about the molecular composition of DOM in RAS and its transformation when exposed to ozone. In this study, we performed a detailed molecular characterization of DOM in RAS and explored its transformation induced by ozonation of RAS waters. Ultra-high resolution (UHR) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) was used to characterize the DOM matrix of RAS waters (pump-sump and tanks) and to evaluate its transformation by ozonation. The analysis of DOM extracted from makeup water and feed samples allowed for the determination of DOM sources in RAS prior to ozonation. The CHO and unsaturated group of compounds were the most abundant class found in water samples. On the contrary, the DOM from feed samples was unique and consisted mainly of CHO, CHON and unsaturated group of compounds. After the ozonation of RAS waters, humic-like and unsaturated compounds [positive oxygen subtracted double bond equivalent per carbon (DBE-O)/C)] were decomposed, particularly the CHO-DOM that contained fewer -CH2- features. Fulvic-like compounds and several hundred saturated compounds [negative (DBE-O)/C)] were formed post ozonation, particularly the CHON and CHONS group of compounds that were associated with fish diets, makeup waters and transformation products from the ozonation of the RAS waters. This study showed that the high accuracy of the ultra-high resolution FTICR MS can be applied to characterize and monitor the changes of DOM at a molecular level in RAS waters. To our knowledge, this is the first study where FTICR MS was incorporated for the characterization of DOM and its sources in RAS.

Intermittent administration of peracetic acid is a mild environmental stressor that elicits mucosal and systemic adaptive responses from Atlantic salmon post-smolts.

BACKGROUND: Fish encounter oxidative stress several times during their lifetime, and it has a pervasive influence on their health and welfare. One of the triggers of oxidative stress in fish farming is the use of oxidative disinfectants to improve rearing conditions, especially in production systems employing recirculation technology. Here we report the physiological and morphological adaptive responses of Atlantic salmon (Salmo salar L.) post-smolts to intermittent exposure to a potent oxidative agent peracetic acid (PAA). Fish reared in semi-commercial scale brackish water recirculating aquaculture system (RAS) were exposed to 1 ppm PAA every 3 days over 6 weeks. Mucosal and systemic responses were profiled before exposure, 22 and 45 days during the intermittent PAA administration. RESULTS: Oxidative stress was likely triggered as plasma antioxidant capacity increased significantly during the exposure period. Adaptive stress response to the periodic oxidant challenge was likewise demonstrated in the changes in plasma glucose and lactate levels. PAA-induced alterations in the transcription of antioxidants, cytokines, heat shock proteins and mucin genes showed a tissue-specific pattern: downregulation was observed in the gills and olfactory rosette, upregulation occurred in the skin, and no substantial changes in the liver. Further, PAA exposure resulted in histological changes in key mucosal organs (i.e. olfactory rosette, skin and gills); pathological alterations were predominant in the gills where cases of epithelial lifting, hypertrophy and clubbing were prevalent. In addition, intermittent PAA administration resulted in an apparent overproduction of mucus in the nasal mucosa. Lastly, PAA did not dramatically alter the ability of salmon to mount a physiological stress response in the presence of a secondary stressor, though some subtle interference was documented in the kinetics and magnitude of plasma cortisol and glucose response post-stress. CONCLUSIONS: The present study collectively demonstrated that intermittent oxidant exposure was a mild environmental stressor that salmon could mount strong adaptive responses at systemic and mucosal levels. The results will be valuable in optimising the rearing conditions of post-smolts in RAS, especially in adopting water treatment strategies that do not considerably interfere with fish health and welfare.

Consequences of continuous ozonation on the health and welfare of Atlantic salmon post-smolts in a brackish water recirculating aquaculture system.

This study investigated the biological consequences of 45-day continuous ozonation on Atlantic salmon (Salmo salar) post-smolts in a brackish water recirculating aquaculture system (RAS). There was no significant difference in survival, operational welfare indicators, and average weight at termination between the ozone-treated and control groups. Plasma biochemical analyses revealed that the creatinine level was significantly higher in the ozone-treated group than in the control at termination. Histological evaluation of skin health showed no significant difference between the two groups. On the other hand, quantitative histopathology disclosed that the ozone group exhibited a better gill health status than did the control group, particularly at the end of the trial. Mucosal transcriptomics revealed a distinct response profile between the gills and skin. At day 45, there were no differentially expressed genes (DEG) identified in the skin, in contrast to 242 ozone-induced DEGs in the gills. Assessment of the transcriptomic profiles over time revealed that temporal effects were of greater impact in skin compared to gills, regardless of the treatment. The treatment did not result in metabolomic dysregulation and the overall profile lent support to the transcriptomics data that temporal effects had a greater influence on the changes observed. Exposure to handling-confinement stress revealed that ozone treatment did not alter the ability of post-smolts to respond to a secondary stressor. In summary, the suite of health and welfare indicators collectively indicated that continuous ozonation resulted in minimal physiological perturbations in salmon post-smolts. The results are expected to contribute to optimising the rearing conditions for post-smolts in RAS.

A Novel Miniaturized Biosensor for Monitoring Atlantic Salmon Swimming Activity and Respiratory Frequency.

The advanced development of sensor technologies has led to the emergence of fish biosensors that are currently used for research and commercial purposes. AEFishBIT is a miniaturized biosensor attached to fish operculum that measures physical activity and respiration frequencies. In this study, we determined the effect of the tagging method and evaluated the use of this biosensor to monitor post-smolt Atlantic salmon in a tank-based system. The use of piercing fish tag had a negative impact on the gills and operculum, unlike the identical protocols used in gilthead sea bream and European sea bass. In contrast, a surgical thread did not show any apparent tissue damage. Two data recording schedules were considered to monitor immediate early reaction to fish handling and light regime changes (records every 15 min over 2 days) or adaptation to new light conditions (records every 30 min over 4 days). Data showed stabilization of physical activity 8 h post-tagging, with different steady states for the activity/respiratory ratio after changes in light intensity that reflected a different time course adaptation to new light conditions. High correlations were observed between AEFishBIT and video recording data. These findings supported the use of AEFishBIT as a promising tool for smart sensing of Atlantic salmon.

Stress Impairs Skin Barrier Function and Induces α2-3 Linked N-Acetylneuraminic Acid and Core 1 O-Glycans on Skin Mucins in Atlantic Salmon, Salmo salar.

The skin barrier consists of mucus, primarily comprising highly glycosylated mucins, and the epithelium. Host mucin glycosylation governs interactions with pathogens and stress is associated with impaired epithelial barrier function. We characterized Atlantic salmon skin barrier function during chronic stress (high density) and mucin O-glycosylation changes in response to acute and chronic stress. Fish held at low (LD: 14-30 kg/m3) and high densities (HD: 50-80 kg/m3) were subjected to acute stress 24 h before sampling at 17 and 21 weeks after start of the experiment. Blood parameters indicated primary and secondary stress responses at both sampling points. At the second sampling, skin barrier function towards molecules was reduced in the HD compared to the LD group (Papp mannitol; p < 0.01). Liquid chromatography-mass spectrometry revealed 81 O-glycan structures from the skin. Fish subjected to both chronic and acute stress had an increased proportion of large O-glycan structures. Overall, four of the O-glycan changes have potential as indicators of stress, especially for the combined chronic and acute stress. Stress thus impairs skin barrier function and induces glycosylation changes, which have potential to both affect interactions with pathogens and serve as stress indicators.

Characterizing changes of dissolved organic matter composition with the use of distinct feeds in recirculating aquaculture systems via high-resolution mass spectrometry.

Recirculating aquaculture systems (RAS) are a new alternative to traditional aquaculture approaches, allowing full control over the fish production conditions, while reducing the water demand. The reduction of water exchange leads to an accumulation of dissolved organic matter (DOM) that can have potential effects on water quality, fish welfare and system performance. Despite the growing awareness of DOM in aquaculture, scarce scientific information exists for understanding the composition and transformation of DOM in RAS. In this study, a non-targeted approach using ultra-performance liquid chromatography coupled to a hybrid quadrupole-time of flight mass spectrometer (UPLC-QTOF-MS) was used to characterize compositional changes of low molecular weight (LMW) DOM in RAS, when operated under two different feed types. A total of 1823 chemicals were identified and the majority of those contained a CHON chemical group in their structure. Changes in the composition of LMW-DOM in RAS waters were observed when the standard feed was switched to RAS feed. The DOM with the use of standard feed, consisted mainly of lignin/CRAM-like, CHO and CHOS chemical groups, while the DOM that used RAS feed, was mainly composed by unsaturated hydrocarbon, CHNO and CHNOS chemical groups. The Bray-Curtis dissimilarity cluster demonstrated differences in the composition of DOM from RAS and was associated to the type of feed used. When the RAS feed was used, the Kendrick mass defect plots of -CH2- homologous units in the pump-sump (after the water treatment) showed a high removal capacity for CHNO, CHNOS and halogenated chemicals with high Kendrick mass defect, KMD > 0.7. To our knowledge, this is the first report of LMW-DOM characterization of RAS by high-resolution mass spectrometry (HRMS).

The Effects of Ozone on Atlantic Salmon Post-Smolt in Brackish Water-Establishing Welfare Indicators and Thresholds.

Ozone is a strong oxidant, and its use in aquaculture has been shown to improve water quality and fish health. At present, it is predominantly used in freshwater systems due to the high risk of toxic residual oxidant exposure in brackish water and seawater. Here, we report the effects of ozone on Atlantic salmon (Salmo salar) post-smolts (~100 g), in a brackish water (12 ppt) flow-through system. Salmon were exposed to oxidation reduction potential concentrations of 250 mV (control), 280 mV (low), 350 mV (medium), 425 mV (high) and 500 mV (very high). The physiological impacts of ozone were characterized by blood biochemical profiling, histopathologic examination and gene expression analysis in skin and gills. Fish exposed to 425 mV and higher showed ≥33% cumulative mortality in less than 10 days. No significant mortalities were recorded in the remaining groups. The skin surface quality and the thickness of the dermal and epidermal layers were not significantly affected by the treatments. On the other hand, gill histopathology showed the adverse effects of increasing ozone doses and the changes were more pronounced in the group exposed to 350 mV and higher. Cases of gill damages such as necrosis, lamellar fusion and hypertrophy were prevalent in the high and very high groups. Expression profiling of key biomarkers for mucosal health supported the histology results, showing that gills were significantly more affected by higher ozone doses compared to the skin. Increasing ozone doses triggered anti-oxidative stress and inflammatory responses in the gills, where transcript levels of glutathione reductase, copper/zinc superoxide dismutase, interleukin 1ß and interleukin were significantly elevated. Heat shock protein 70 was significantly upregulated in the skin of fish exposed to 350 mV and higher. Bcl-2 associated x protein was the only gene marker that was significantly upregulated by increasing ozone doses in both mucosal tissues. In conclusion, the study revealed that short-term exposure to ozone at concentrations higher than 350 mV in salmon in brackish water resulted in significant health and welfare consequences, including mortality and gill damages. The results of the study will be valuable in developing water treatment protocols for salmon farming.

A salty start: Brackish water start-up as a microbial management strategy for nitrifying bioreactors with variable salinity.

Nitrifying biofilms developed in brackish water are reported to be more robust to salinity changes than freshwater biofilms. This makes them a promising strategy for water treatment systems with variable salinity, such as recirculating aquaculture systems for Atlantic salmon. However, little is known about the time required for nitrification start-up in brackish water or the microbial community dynamics. To investigate the development of nitrifying biofilms at intermediate salinity, we compared the startup of moving bed biofilm reactors with virgin carriers in brackish- (12‰ salinity) and freshwater. After 60 days, the brackish water biofilm had half the nitrification capacity of the freshwater biofilm, with a less diverse microbial community, lower proportion of nitrifiers, and a significantly different nitrifying community composition. Nitrosomonas and Nitrosospira-like bacteria were the main ammonia oxidizers in the brackish water biofilms, whereas Nitrosomonas was dominant in freshwater biofilms. Nitrotoga was the dominant nitrite oxidizer in both treatments. Despite the lower nitrification capacity in the brackish water treatment, the low ammonia and nitrite concentration with rapidly increasing nitrate concentration indicated that complete nitrification was established in both reactors within 60 days. The results suggest that biofilms develop nitrification in brackish water in comparable time as in freshwater, and brackish start-up can be a strategy for bioreactors with varying salinity.

Biofilms remember: Osmotic stress priming as a microbial management strategy for improving salinity acclimation in nitrifying biofilms.

With increasing freshwater scarcity and greater use of seawater, fluctuating salinities are becoming common in water treatment systems. This can be challenging for salinity-sensitive processes like nitrification, especially in recirculating aquaculture systems (RAS), where maintaining nitrification efficiency is crucial for fish health. This study was undertaken to determine if prior exposure to seawater (priming) could improve nitrification in moving bed biofilm reactors (MBBR) under salinity increase from freshwater to seawater. The results showed that seawater-primed freshwater MBBRs had less than 10% reduction in nitrification activity and twice the ammonia oxidation capacity of the unprimed bioreactors after seawater transfer. The primed biofilms had different microbial community composition but the same nitrifying taxa, suggesting that priming promoted physiological adaptation of the nitrifiers. Priming may also have strengthened the extrapolymeric matrix protecting the nitrifiers. In MBBRs started up in brackish water (12‰ salinity), seawater priming had no significant impact on the nitrification activity and the microbial community composition. These bioreactors were inherently robust to salinity increase, likely because they were already primed to osmotic stress by virtue of their native salinity of 12‰. The results show that osmotic stress priming is an effective strategy for improving salinity acclimation in nitrifying biofilms and can be applied to water treatment systems where salinity variations are expected.

Molecular and physiological responses to long-term sublethal ammonia exposure in Atlantic salmon (Salmo salar).

The objective of this study was to determine the underlying physiological and molecular responses to long-term sublethal ammonia exposure in Atlantic salmon (Salmo salar) parr. Previous studies have predominately focused on mechanisms during acute, short-term exposure. For that purpose Atlantic salmon parr were exposed to four ammonia concentrations between 4 and 1800 µmol l(-1) total ammonia nitrogen (TAN), and subjected to two feeding regimes for 15 weeks. Elevated environmental ammonia and full feeding strength caused an initial increase in plasma ammonia levels ([T(amm)]) after 22 days of exposure, which thereafter declined and remained similar to the control animals towards the end of the study. On the other hand, a progressive decrease in plasma urea levels was evident throughout the entire exposure period and depended on the concentration of environmental ammonia, with the largest decrease in urea levels observed at the highest ammonia concentrations (1700 and 1800 µmol l(-1) TAN). We hypothesized that the successful adaptation to long-term elevated ammonia levels would involve an increased capacity for carrier-facilitated branchial excretion. This hypothesis was strengthened by the first evidence of an up-regulation of branchial transcription of the genes encoding the Rhesus (Rh) glycoproteins, Rhcg1 and Rhcg2, urea transporter (UT) and aquaporin 3a (Aqp3a), during long-term exposure. Of the Rhesus glycoprotein (Rh) mRNAs, Rhcg1 was up-regulated at all tested ammonia levels, while Rhcg2 showed a concentration-sensitive increase. Increased transcription levels of V-type H(+)-ATPase (H(+)-ATPase) were observed at the highest ammonia concentrations (1700 and 1800 µmol l(-1) TAN) and coincided with an up-regulation of Rhcg2 at these concentrations. Transcription of UT and Aqp3a was increased after 15 weeks of exposure to low ammonia levels (470 and 480 µmol l(-1) TAN). A significant increase in brain glutamine (Gln) concentration was observed for full fed Atlantic salmon after 22 days and in fish with restricted feeding after 105 days of exposure to 1800 and 1700 µmol l(-1) TAN, respectively, without any concomitant decrease in brain glutamate (Glu) concentrations. These results suggest that Gln synthesis is an ammonia detoxifying strategy employed in the brain of Atlantic salmon parr during long-term sublethal ammonia exposure. Full feed strength had an additive effect on plasma [T(amm)], while the restricted feeding regime postponed the majority of the observed physiological and molecular responses. In conclusion, Atlantic salmon parr adapts to the long-term sublethal ammonia concentrations with increased branchial transcription levels of ammonia and urea transporting proteins and ammonia detoxification in the brain.

Evolution and differential expression of a vertebrate vitellogenin gene cluster.

BACKGROUND: The multiplicity or loss of the vitellogenin (vtg) gene family in vertebrates has been argued to have broad implications for the mode of reproduction (placental or non-placental), cleavage pattern (meroblastic or holoblastic) and character of the egg (pelagic or benthic). Earlier proposals for the existence of three forms of vertebrate vtgs present conflicting models for their origin and subsequent duplication. RESULTS: By integrating phylogenetics of novel vtg transcripts from old and modern teleosts with syntenic analyses of all available genomic variants of non-metatherian vertebrates we identify the gene orthologies between the Sarcopterygii (tetrapod branch) and Actinopterygii (fish branch). We argue that the vertebrate vtg gene cluster originated in proto-chromosome m, but that vtg genes have subsequently duplicated and rearranged following whole genome duplications. Sequencing of a novel fourth vtg transcript in labrid species, and the presence of duplicated paralogs in certain model organisms supports the notion that lineage-specific gene duplications frequently occur in teleosts. The data show that the vtg gene cluster is more conserved between acanthomorph teleosts and tetrapods, than in ostariophysan teleosts such as the zebrafish. The differential expression of the labrid vtg genes are further consistent with the notion that neofunctionalized Aa-type vtgs are important determinants of the pelagic or benthic character of the eggs in acanthomorph teleosts. CONCLUSION: The vertebrate vtg gene cluster existed prior to the separation of Sarcopterygii from Actinopterygii >450 million years ago, a period associated with the second round of whole genome duplication. The presence of higher copy numbers in a more highly expressed subcluster is particularly prevalent in teleosts. The differential expression and latent neofunctionalization of vtg genes in acanthomorph teleosts is an adaptive feature associated with oocyte hydration and spawning in the marine environment.

Genomic and proteomic analyses reveal non-neofunctionalized vitellogenins in a basal clupeocephalan, the Atlantic herring, and point to the origin of maturational yolk proteolysis in marine teleosts.

Oocyte hydration is a unique event in oviparous marine teleosts that provides the single-celled egg with an essential pool of water for survival during early development in the saline oceanic environment. A conserved mechanism of maturational yolk proteolysis of a neofunctionalized vitellogenin (VtgAa) has been shown to underlie the hydration event in all teleosts that spawn pelagic eggs (pelagophils), and is argued to be a key adaptation for teleost radiation in the oceanic environment 55 Ma. We have recently shown that a small pool of free amino acids (FAAs) significantly contributes to the osmolarity of the ovulated egg in an ancestral marine teleost, the Atlantic herring that spawns benthic eggs (benthophil). To determine whether multiple forms of vtg exist and whether neofunctionalization of the gene products are related to the egg FAA pool in this species, genomic sequences conserved between the exons of Atlantic herring and zebrafish were amplified. This approach identified a small polymorphic intron between exons 9 and 10 in Atlantic herring and demonstrated that two closely related major vtg transcripts (chvtgAc1 and chvtgAc2) are expressed during oogenesis. A separate polymerase chain reaction-based approach identified a more ancestral phosvitinless transcript (chvtgC). Proteomic analyses of the translated products of the major vtg forms demonstrated that the yolk proteins are similarly processed during deposition, and oocyte maturation and reveal that vtgs have duplicated but not neofunctionalized in this species. Phylogenetic analyses consistently clustered the transcripts and proteins as the basal sister group to the Ostariophysi in full congruence with the Clupeocephalan rank, and suggest that expansion of ostariophysan vtgAo1 and vtgAo2 genes occurred in a lineage-specific manner after separation from the Clupeiformes. Three-dimensional modeling of the ChvtgAc1 sequence against the resolved lamprey lipovitellin module revealed that the tertiary structure is highly conserved, with most substitutions occurring on the outside of the molecule. The data indicate that the phosvitin domain, the smallest yet reported for teleosts, and an N-terminal fragment of the lipovitellin light chain contribute to the FAA pool. The present findings thus show that yolk proteolysis and the generation of an organic osmolyte pool of FAAs was an adaptive response to spawning in seawater also for the Clupeiformes, but that this process was not evolutionarily successful in terms of biodiversity until vtg gene neofunctionalization occurred in the Acanthomorpha.

Goldsinny wrasse (Ctenolabrus rupestris) is an extreme vtgAa-type pelagophil teleost.

During oocyte maturation in the goldsinny wrasse (Ctenolabrus rupestris) extensive proteolysis of yolk proteins generates a large pool of free amino acids that drive hydration of the pelagic egg. By cloning hepatic vitellogenins (vtg) and using mass spectrometry, N-terminal microsequencing, and Western-immunoblotting to identify the yolk proteins (Yp), we show that multiple forms of vitellogenin mRNAs (vtgAa, vtgAb, and vtgC) are expressed in the liver, but only a single major class of the Yps derived from vtgAa predominates in the oocytes. Some Yps derived from vtgAb and vtgC appear also to be incorporated in the oocytes and eggs, but only at background levels. During oocyte hydration the vtgAa-derived lipovitellin heavy chain (LvH-Aa) and its cleavage variants are completely degraded leaving only a processed lipovitellin light chain (LvL-Aa) fragment as the major yolk protein for embryonic development. The maturational cleavage site of the LvL-Aa is identified as two amino acids downstream from the conserved Tyr(1168) of VtgAa in Atlantic halibut. In addition, although a beta'-component (approximately 18 kDa) is present in the oocytes, it is not fully degraded during the hydration process.