Hydrogen peroxide treatment in Atlantic salmon induces stress and detoxification response in a daily manner

Daily variation in the absorption, metabolism and excretion of toxic substances will ultimately determine the actual concentration to which the cells and tissues are exposed. In aquaculture, Atlantic salmon (Salmo salar) can be frequently exposed to hydrogen peroxide (H2O2) to treat topical skin and gill infections, particularly in relation to parasitic infections (e.g. sea liceLepeophtheirus salmonisand amoebic gill disease caused byNeoparamoeba perurans). It is well accepted that the time of administration influences pharmacodynamics and pharmacokinetics of drugs which in turn affects their efficacy and toxicity. Consequently, a better understanding of drug side effects as a function of time of day exposure would help to improve treatment efficacy and fish welfare. To this end, salmon were exposed to H2O2(1500 mg/L) for 20 min at six different times of the day during a 24-h cycle and we investigated the time-dependent effects of exposure on physiological stress (glucose, lactate and cortisol) and antioxidant enzyme expression (gpx1, cat, Mn-sodandhsp70) in liver and gills. In addition, at each sampling point, 8 control fish were also sampled. Our results revealed that the time of administration of H2O2caused significant differences in the induction of both physiological and oxidative stress responses. Glucose and lactate were higher in the treated fish during daytime whereas cortisol levels appeared to be systematically increased (>1000 ng/mL) after H2O2treatment irrespective of exposure time, although differences with control levels were higher during the day. In liver, gene expression of antioxidant enzymes displayed daily rhythmicity in both treated and control groups and showed higher mRNA expression levels in salmon treated with H2O2at ZT6 (6 h after lights onset). In gills, rhythmic expression was only found forgpx1in the control fish and forhsp70andMn-sodin the treated groups. However, in the treated salmon, higher gene expression levels of all the investigated enzymes were also observed at ZT6-10. Clock gene expression showed rhythmicity only in the liver in accordance with the daily rhythm of enzyme expression observed in this tissue. Altogether, this study provides first evidence of chronotoxicity in Atlantic salmon treated with H2O2and suggests increased sublethal toxic effect during the first half of the day. These results have direct relevance to the salmon and broader aquaculture industry by optimising the timing of treatment administration, opening the door to chronotherapy to treat fish diseases

Development of a water-stable agar-based diet for the supplementary feeding of cleaner fish ballan wrasse (Labrus bergylta) deployed within commercial Atlantic salmon (Salmon salar) net-pens

The aim of this project was to develop a water-stable and palatable diet for the supplementary feeding of wrasse deployed in salmon sea-pens using a gelling agent mixed with a manufactured dry-feed component. Three binders (gelatine from cold water fish skin, beef gelatin and agar-agar) were compared for water-gel strength over a range of concentrations. Gel formed using agar was found to be significantly stronger than the other binders tested. An experimental aqua-feed made using a grinded, dry ingredient mix binded with 20g/L agar solution at 1/1.6 (w/v) ratio and offered as blocks within individual feeders was water-stable for 7 days when deployed fresh or following a week of preservation at -20°C. Farmed ballan wrasse in tanks fed on the agar-based diet within 2 days of deployment. Wild wrasse stocked in salmon sea-pens at low density (1.2-2.1%), up to 4 weeks prior the start of the trial and not previously fed a manufactured diet first ingested the agar feed within 2 weeks and total feed intake significantly increased afterwards. Feed intake was significantly higher from feeders placed within a small feeding shelter made of artificial kelp than within the large wrasse shelter. No nutrient leaching after water immersion and no alterations in the fatty acid profile after preparation of the experimental feed was found. A manufactured grinded ingredient mix binded with 20g/L agar solution at a 1/1.6 (w/v) ratio and offered within static feeders is proposed as the basis of a novel supplementary feeding methodology for cleaner fish wrasse deployed in salmon sea-pens. This methodology has the potential to facilitate wrasse feeding and to allow the monitoring of feed intake to safeguard the health, welfare and delousing activity of the biological stock over the salmon rearing cycle

Temperature-induced testicular germ cell loss and recovery in Nile tilapia Oreochromis niloticus

Water temperature is a critical external factor influencing gonadal development in fish. This research aimed to study the impact of elevated temperature on testicular germ cell survival and reproductive capacity of Nile tilapia. Male Nile tilapia were exposed to high temperatures of either 36 (HT1) or 37°C (HT2) for 3000 degree-days (DD) and thereafter returned to the control temperature of 27°C (CT) for 2200 DD. The deleterious effects on testicular germ and somatic cells were observed histologically, characterised by vacuolisation, atrophy and the loss of spermatogenic cells in testes with a more severe impact of HT2 compared to HT1. Interestingly, serum 11-ketotestosterone (11-KT) and testosterone (T) levels tended to be higher during the heat treatments than CT. Expression levels of germline-specific genes piwil1, piwil2 and nanos2 and Bcl-2 family genes, bcl-xLb and baxa were significantly reduced during the heat treatment compared to CT, more so in the HT2, while the levels of nanos3 and gfra1 transcripts were only significantly reduced in HT2, implying a significant loss of spermatogonial stem cell (SSC) and spermatogonia in HT2. The effect of HT2 is further evidenced by the significantly reduced sperm density and fertilisation rate compared to CT and HT1 at the end of the recovery period but complete sterility was not induced by HT2. Overall, the present study showed significant effects of HT2 on germ cell survival with histological changes in testes, reduced milt quality, increased 11-KT, and decreased expression of germline-specific genes, SSC marker genes and Bcl-2 family genes in testes which could therefore be potential target genes for sterilisation by genome editing

Expression pattern of nanos, piwil, dnd, vasa and pum genes during ontogenic development in Nile tilapia Oreochromis niloticus

Primordial germ cells (PGCs) are specified by maternally provided determinants in fish. PGCs migrate then into prospective gonadal sites during early development and give rise to germ cell lineage. PGC disrupted animals do not sexually mature which has a range of commercial as well as environmental benefits. To find potential target genes for sterilisation of Nile tilapia, relative mRNA abundance patterns and tissue distribution of four nanos, two piwil, dnd1, vasa and three pum genes were investigated during ontogenic development from unfertilised eggs to newly hatched larvae and in adult tissues, respectively. The ontogenic pattern of RNA abundance revealed that all the investigated gene transcripts are maternally deposited to varying degrees, except for nanos2 which is not expressed in eggs. The ontogenic patterns of relative RNA abundance could be grouped into three categories. The first one, including nanos3, piwil1, piwil2, dnd1 and vasa, showed abundant transcript levels during early developmental stages which are then degraded during the period of maternal to zygotic transition between blastula and gastrula stages with a reduction in expression of four to five orders of magnitude by hatching stage. Another, including pum2 and pum3, showed similar patterns to the first group, but the transcript levels are reduced by only two orders of magnitude. The third group, including nanos1a, nanos1b and pum1, was characterised by a zygotic increase. nanos2 had no detectable transcripts until hatching stage. The tissue screening of nanos1a, nanos1b, pum1, pum2 and pum3 showed that they are expressed in various tissues, implying their potential pleiotropic effects in these tissues apart from gonads. In contrast, nanos3, piwil1, piwil2, dnd1 and vasa appeared to be exclusively expressed in gonads (both ovary and testis), and nanos2 showed testis-specific expression. Based on these results nanos3, piwil1, piwil2, dnd1 and vasa were prioritised among the 11 selected genes as potential target genes for sterilisation in Nile tilapia as they have no significant zygotic expression during embryogenesis, they are expressed exclusively in gonads and maternally deposited. These features suggest a potential role of these genes in the specification and maintenance of PGCs during the ontogenic development of Nile tilapia

The potential of alternative lighting-systems to suppress pre-harvest sexual maturation of 1+ Atlantic salmon (Salmo salar) post-smolts reared in commercial sea-cages

The aim of this study was to compare the efficiency of new candidate lighting-technologies (50W ‘blue’ light-emitting-diode (B, λmax = 465 nm); 232 W ‘green’ hot cathode, (G, λmax = 546 nm); 400 W ‘red’ tungsten-halogen, (R, λmax = 667 to 740 nm)) against a standard 400 W ‘white’ metal-halide used as control technology (C, broad spectrum) at suppressing sexual maturation of 1+ Atlantic salmon (Salmo salar) in sea-cages. A total of seven experimental set-ups were tested on a commercial-scale in three trials using a standardised photoperiod regime in the form of continuous artificial-light (LL) applied from winter to summer solstice during the second year at sea. The experimental stocks were raised under an ambient thermal regime that was similar across all trials. Technical performances (spectral output, light-attenuation and irradiance distance) of the individual light-units were measured and light-perception was assessed by quantifying plasma melatonin levels. Body-size parameters (BW, FL, K) were measured at the switch-on and turn-off of the photoperiod regimes. Maturation rates were estimated at the end of the light-treatments and at harvest. The B-unit provided the shortest effective irradiance distance (distance from the light-bulb to the minimum irradiance suppressing plasma melatonin to basal day-time level = 0.016 W m-2) but the longest relative to its energy consumption; while the G- and R-units did not offer a comparative advantage over the C-unit in that regard (B>C>G>R). Nocturnal plasma-melatonin and maturation rate decreased proportionally to the light-intensity provided using a range of technologies emitting distinct spectral profiles. Light-intensity rather than light-spectral composition appeared to be the prime parameter negatively affecting sexual maturation. Maximal suppression of maturation was observed in treatments depressing nocturnal plasma melatonin to a 1.2-fold but not to a 1.7-fold increase compared to daytime levels, confirming that a threshold level of light-irradiance is necessary to obtain the desired effect. Results suggest that this can be achieved under standard commercial practices by applying, over the photoperiod regime presently used, continuous artificial-illumination with an (electrical) energy consumption of 0.28 Wh m-3 generating a mean-irradiance of 0.012 W m-2 and providing a minimum volume of effective irradiance equivalent to 12% of the rearing-environment. Such a low volume of biologically effective irradiance was likely sufficient due to the strong photic attraction already reported in Atlantic salmon. Maximal suppression of pre-harvest sexual maturation can be achieved in the Atlantic salmon on-growing industry using alternative light-technologies. Present data provides methods and threshold values favouring the implementation of photoperiod-manipulation to suppress pre-harvest maturation at the most advantageous scale and cost

Body size dimorphism of sea-reared Atlantic salmon (Salmo salar L.): implications for the management of sexual maturation and harvest quality

Body size dimorphism between immature and early sexually recruited cohorts of farmed Scottish Atlantic salmon were investigated with the view to optimize the practical management of early maturation over the second-year at sea. Mixed-sex smolts from a single strain and freshwater source were stocked into four discrete commercial sites and sampled at harvest from June to December 2007, 15 to 22 months post-sea transfer. Individuals were sexed and their maturity status determined based on gonado-somatic-index (GSI) and oocyte leading stage. Whole body weight (BW), fork length (FL) and Fulton condition factor (K) were measured and flesh quality analyzed. The immature mixed-sex population and each gender analyzed separately had an isometric weight-length relationship (WLR) but exhibited seasonal variations in K. Body size of immature Atlantic salmon were consistently sexually dimorphic with males exhibiting a higher BW (+13.4%) and FL (+5.9%) but a lower K (-5.0%) than females. Individuals at an early stage of sexual maturation had a significantly higher BW (+35.2%) and K (+20.6%) than the immature cohort in June and July. During this period BW, FL and K together or BW alone were strong and standard indicators of early maturity in our discrete sites. Body size dimorphism described in this study show that sex-ratio is an important parameter of farmed Atlantic salmon populations which is likely to vary following weight-grading and that population composition (sex-ratio and maturation rate) affects the seasonality in K typically observed at harvest. Importantly, the commitment of Atlantic salmon into maturation in spring can be rapidly and accurately estimated in a number of discrete populations by using simple weight-length morphological indicators characterized in a single rearing unit. Following maturation rate estimation, weight-grading implemented according to the predicted stock morphological structure could be used to selectively harvest a high proportion of maturing individuals at a stage where their flesh quality remains optimal. This could be applied as a powerful and practical on-site maturation management tool in the salmon industry as well as in other commercially important fish species

Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system

The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0

Light- and clock-control of genes involved in detoxification

Circadian regulation of hepatic detoxification seems to be amongst the key roles of the biological clock. The liver is the major site for biotransformation, and in mammals, it contains several clock-controlled transcription factors such as PAR basic leucine zipper proteins (bZIP) and basic-helix-loop-helix (bHLH)-PAS family that act as circadian regulators of detoxification genes. This investigation explored the existence of daily and circadian expression of transcription factors involved in detoxification, as well as the temporal profile of a set of their target genes in zebrafish liver. In our study, zebrafish were able to synchronize to a light-dark (LD) cycle and displayed a diurnal pattern of activity. In addition, the expression of clock genes presented daily and circadian rhythmicity in liver. Apart from hlfa, the expression of PAR bZIP transcription factors also displayed daily rhythms, which appeared to be both light-dependent and clock-controlled, as circadian rhythms free-ran under constant conditions (continuous darkness, DD). Under LD, tefb, dbpa and dbpb expression peaked at the end of the darkness period whereas tefa showed peak levels of expression at the onset of the photophase. In addition, these four genes exhibited circadian expression under DD, with higher expression levels at the end of the subjective night. The expression of the bHLH-PAS transcription factor arh2 also showed circadian rhythmicity in zebrafish liver, peaking in the middle of the subjective night and approximately 3-4 hours before peak expression of the PAR bZIP genes. Regarding the detoxification genes, the major target gene of AhR, cyp1a, showed daily and circadian expression with an acrophase 2 hours after ahr2. Under LD, abcb4 also showed daily rhythmicity, with an acrophase 1-2 h after that of PAR bZIP factors during the transition between darkness and light phases, when zebrafish become active. However, the expression of six detoxification genes showed circadian rhythmicity under DD, including cyp1a and abcb4 as well as gstr1, mgst3a, abcg2 and sult2_st2. In all cases, the acrophases of these 3 genes were found during the second half of the subjective night, in phase with the PAR bZIP transcription factors. This suggested that their expression is clock-controlled, either directly by core clock genes or through transcription factors. This study presents new data demonstrating that the process of detoxification is under circadian control in fish. Results showed that time of day should be considered when designing toxicological studies or administering drugs to fish

Research and development of stock management strategies to optimise growth potential in on-growing of Atlantic cod, Gadus morhua, and Atlantic halibut, Hippoglossus hippoglossus

Aquaculture is an essential developing sector for world food production, however the attainment of sexual maturity during commercial on-growing is a major bottleneck to industry expansion. Sexual maturation brings a commercial loss due to reduced growth performance as well as reduced immune function. Furthermore, serious concerns exist over potential genetic interaction with native stocks through broadcast spawning or spawning interaction by escapees. In the north Atlantic region, the Atlantic cod (Gadus morhua) and Atlantic halibut (Hippoglossus hippoglossus) are key aquaculture species in which industry expansion is limited by pre-harvest sexual maturation. However, through a species specific combination of modern technologies and refinement in management practices it is possible that this sexual maturation can be controlled and on-growing potential enhanced. Thus the overall aim of this thesis was to conduct novel research that will improve our understanding of the underlying mechanisms that regulate sexual maturation, whilst also advancing the optimisation of technologies for the management of maturation in cod and halibut. In Atlantic cod, owing to the inconsistent inhibition of maturation in commercial conditions, ever increasing intensities of light and in some cases narrow spectrum technologies are being used to try to combat this problem. Firstly, this PhD project investigated the potential welfare impacts of high intensity artificial lighting which have not been studied to date (Chapter 2). The work specifically investigated the effect of traditional metal halide and novel green cathode lighting on the stress response, innate immunity, retina structure, feeding activity and light perception of Atlantic cod. Results indicated that although acute responses to light were observed, there were no clear significant long term effects of any of the lighting treatments on these parameters. Regarding light perception, interestingly even when subjected to high intensity constant lighting (metal halide mean tank intensity: 16.6 watts m-2), cod still demonstrated a day/night rhythm in melatonin release which suggests perception of the overlying ambient photoperiod. The second trial of this PhD project investigated the efficacy of shading of ambient photoperiod in addition to constant lighting to inhibit maturation of cod outdoors (Chapter 3). This aimed at improving the performance of artificial lighting regimes in the open cage system during commercial on-growing by reducing the relative difference between day/night light intensities. The trial was conducted over a one year period where a low and high shade treatment were tested in outdoor tanks. Shading increased the relative night time illumination to 6.6% and 31.3% of daytime levels respectively, compared to <2% in an unshaded set-up. Both shading treatments were effective at suppressing sexual development in cod as confirmed through measurements of gonadosomatic index, histological analysis of gonadal development, oocyte diameter measurements and sex steroid profiles as well as measurements of growth. In addition to research at the applied level in Atlantic cod, this thesis has also extended to the fundamental level and explored one of the potential mechanisms relaying photoperiod signal to the endogenous regulation of sexual maturation in cod, namely the kisspeptin system (Chapter 4). Partial sequences for the signal peptide Kiss2 and its receptor Kissr4 were isolated and described showing similarity to other teleost species such as the medaka, Oryzias latipes and stickleback, Danio rerio. Novel molecular qPCR assays were designed and developed to measure the expression of both genes in male and female cod over a maturation cycle and compared to cod under constant lighting which remained immature. Interestingly, expression patterns of kiss2 and kissr4 did not reveal any clear association with season or photoperiod treatment. However, pituitary expression of gonadotropins (FSH, follicle stimulating hormone; LH, luteinising hormone) did show a differential expression in relation to treatment from early winter approximately 4-6 months after the photoperiod change. These new results are in contradiction with the hypothesis that the kisspeptin system would be involved in the initiation of gametogenesis, as shown in mammals. However, the FSH/LH data defines a window during which time kisspeptin or another GnRH stimulating mechanism must be active, this compels the need further investigation. In Atlantic halibut farming, all-female production removes the concerns of production losses through sexual maturation. Accordingly, this thesis investigated the potential/feasibility of generating monosex populations by FACS (fluorescence activated cell sorting) semen sexing based on cellular DNA content, as proven in terrestrial agriculture. Results however did not show any clear differences between the DNA of sperm in a range of species tested (Atlantic halibut, cod, sea bass, perch) suggesting that this technique may not be applicable in such species. The project also focussed on the production of a population of sex reversed halibut broodstock (neomales) that will generate, in the long term, a basis for traditional monosex population generation in the UK. Two in feed MDHT (17α-methyldihydrotestosterone) treatments were tested with the aim to reduce the use of hormone. Results were very successful with a hormone treatment of 5ppm MDHT generating a 97% phenotypic male population thus suggesting the presence of sex-reversed halibut which can be used for future monosex production. Overall, this work aimed to develop and/or refine potential remediation techniques for sexual maturation in two key commercially important farmed marine fish species, cod and halibut, as well as further our understanding on the regulation of puberty. The knowledge gained from this work provides a means to optimise the techniques employed in the industry and has the potential to increase production and profitability without compromising farmed animal welfare, thus ultimately promoting the sustainable expansion of the Atlantic cod and halibut aquaculture.EThOS - Electronic Theses Online ServiceScottish Aquaculture Research Forum (027)GBUnited Kingdo

Removal of the adhesive gum layer surrounding naturally fertilised ballan wrasse (Labrus bergylta) eggs

Commercial production of ballan wrasse (Labrus bergylta) as a cleaner fish for the removal of sea lice (Lepeophtheirus salmonis) from farmed salmonids (Salmo salar) has increased due to its proven efficiency. One bottleneck in commercial hatchery production is working with the benthic adhesive eggs, which makes disinfection and incubation of eggs challenging; therefore, this study aimed to find a chemical or enzymatic treatment and process to remove the adhesive gum layer. Naturally spawned eggs were collected from artificial spawning substrates up to 24h post spawning from wild caught broodstock kept in captivity at the Marine Harvest, Machrihanish facility. Four treatments were tested: tannic acid (0.2, 0.1, and 0.05%), sodium sulfite (2, 1, and 0.5%),l-cysteine (2, and 1%), and enzyme alcalase&reg; (4.0, 3.0, 2.0, 1.0, and 0.5%)in vitro. Eggs were exposed for 25min while being continually agitated, and the proportion of &ldquo;degummed&rdquo; eggs was counted at the end of each time period. Enzyme alcalase&reg; was the only treatment that proved successful in degumming eggs, with the time to complete degumming (&ge;96%) inversely related to enzyme concentration. Complete degumming occurred between 15 and 30min for all enzyme alcalase&reg; dose rates. Mean hatch rates for eggs treated with enzyme alcalase &reg; were not compromised by the treatment and in the highest dose tested were actually found to be higher in treated eggs (78.9&plusmn;2.4%) than controls (71.3&plusmn;3.3%). The use of enzyme alcalase&reg; has proven effective in degumming ballan wrasse eggs without affecting hatch rates. However, translation of this method toin situdegumming and thus removal of eggs from spawning substrate on farm remains to be standardised