High immune diversity in farmed Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

As with any other intensively farmed animal species, the Atlantic salmon has been selectively bred and cross-bred to maximize desirable traits. Selection tends to diminish genetic diversity in target populations, which among other negative effects is hypothesized to decrease their capacity to confront a broad variety of pathogens. We have analyzed mitochondrial (mtDNA) as well as major histocompatibility complex (MHC) DNA sequences from individuals collected from a single aquaculture cage in southern Chile. Interestingly, only two mtDNA haplotypes were obtained; however, several different MH alleles were detected, with divergence values that were compared with those of natural populations of salmonids. Thus, it seems evolutionary processes responsible of keeping MH diversity in the wild managed to retain MH variability in farmed Atlantic salmon, maintaining high immune diversity despite the generally lower levels of observed neutral genetic diversity.     

Unmethylated CpG oligodeoxynucleotides activate head kidney leukocytes of Atlantic cod, <Emphasis Type="Italic">Gadus morhua</Emphasis>

Bacterial DNA and synthetic oligodeoxynucleotides (ODNs) that contain unmethylated CpG motifs are strong inducers of immune response in most mammalian organisms. The use of these synthetic CpG motifs in fish, particularly in salmonids and carp, resulted in the modulation of their immune system. However, much less is known in other species of fish such as gadoids including Atlantic cod, Gadus morhua. Using head kidney (HK) leukocytes of cod in an in vitro study, we determined the effects of some established CpG-ODNs on the cellular responses of the fish immunocytes. Incubation of the HK leukocytes with 2 μM concentration of the CpG-ODNs resulted in enhanced respiratory burst. There were differential effects on the activities of acid phosphatase and cellular myeloperoxidase. Only CpG-ODN 1826 triggered a significant increase in the level of both enzymes. On the other hand, the supernatants derived from the HK leukocytes after incubation with different CpG-ODNs did not possess bactericidal activity against Vibrio anguillarum and Aeromonas salmonicida. This study has shown that CpG-ODNs at low concentrations are able to stimulate respiratory burst in cod but have minimal effects on cellular enzymatic activities and antibacterial action.     

The enzyme profiles in the connective tissue attaching pin bones to the surrounding tissue is specific in farmed salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) and cod (<Emphasis Type="Italic">Gadus morhua L</Emphasis>.)

Post mortem storage is a necessary process for removal of pin bones without destruction of fillets, thereby avoiding volume and economic loss. However, the enzymes involved in loosening pin bones during storage have not been studied to a great extent. In this study, the activities and localization of MMPs in the connective tissue (CT) of pin bones dissected from fillet of salmon and cod were investigated. Interestingly, the enzyme activity profile in these two species was different during post mortem storage of fish fillets. Adding MMP inhibitor (GM6001) and serine protease inhibitor (Pefabloc) revealed different effects in the two species, suggesting different regulations in salmon and cod. In situ zymography with the same inhibitors verified MMP and serine protease activity in CT close to pin bone at early post mortem (6 h) in salmon. However, MMP inhibition was not evident in cod in this area at that time point. Immunohistochemistry further revealed MMP9 and MMP13 were located more to the outer rim of CT, facing the pin bone and adipose tissue, while MMP7 was more randomly distributed within CT in salmon. In contrast, all these three MMPs were randomly distributed in CT in cod. In summary, our study reveals different MMP enzyme profiles in salmon and cod in the pin bone area, influenced by serine proteases, and suggests that MMPs and serine proteases must be taken in consideration when studying the conditions for early pin bone removal.     

Responses of Atlantic cod <Emphasis Type="Italic">Gadus morhua</Emphasis> head kidney leukocytes to phytase produced by gastrointestinal-derived bacteria

This study identified phytase-producing bacteria that were previously isolated from the gastrointestinal tract of Atlantic cod, Gadus morhua and determined its effect on head kidney leukocytes. Out of the 216 bacterial strains tested, the two phytase producers were identified as Pseudomonas sp. and Psychrobacter sp. based on their 16S rDNA sequence. Crude phytase from these two bacterial strains was produced employing the shake flask method. Even though the total protein of the crude phytase was not significantly different for the two bacteria, the phytase activity of the crude enzyme produced by Pseudomonas sp. (97.1 ± 16.7 U) was significantly higher than that of the enzyme from Psychrobacter sp. (75.9 ± 2.4 U). When cod head kidney leukocytes were incubated with the crude phytase (50 μg ml⁻¹), it resulted in enhanced cell proliferation, higher myeloperoxidase, and acid phosphatase activities. Extracellular responses—respiratory burst activity and hydrogen peroxide production were not enhanced by the crude enzyme. As a consequence, the growth of two pathogenic bacteria Aeromonas salmonicida and Vibrio anguillarum was not suppressed by the supernatants obtained from head kidney leukocytes incubated with the crude bacterial phytase. Thus, the enzyme from phytase-producing intestinal bacteria of Atlantic cod can stimulate intracellular head kidney leukocyte activities but not the production of extracellular substances that are involved in antibacterial response. These have implications on the potential use of bacterial phytase as feed supplement to boost cellular immune response of the fish and could be employed as a health management strategy in culture systems.     

Growth and blood chemistry of Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis> L.) in relation to salinity and continuous light

In order to study the possible interactive effects of salinity and photoperiod on growth, feed conversion, and blood chemistry in juvenile halibut, 2,604 (initial mean weight 26.8 g ± 0.2 SEM) juvenile halibut were exposed to six different combinations of salinities (13, 21, or 27‰) and photoperiods [continuous light, C and simulated natural photoperiod (65°N), SNP] for 129 days. Improved (10–20%) growth and 10–24% higher feed conversion efficiency were observed at low and intermediate salinities compared to the high salinity groups. Improved feed conversion efficiency (20%) and temporary growth enhancing effects (10%) of continuous light were observed, but effects faded out as day length in the simulated natural photoperiod group increased. No interactive effects of photoperiod and salinity on growth feed conversion or measured blood chemistry variables (blood sodium, pH level, haematocrit, bicarbonate content, and total carbon dioxide). It is suggested that juvenile Atlantic halibut should be reared at low and intermediate salinities and at continuous light, as this will improve growth and increase feed conversion efficiency.     

Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>) larvae can biosynthesis phospholipid de novo from 2-oleoyl-glycerol and glycerol precursors

The dietary requirement of phospholipid (PL) of fish larvae has been suggested to originate in an inefficient ability for de novo biosynthesis of PL based on dietary triacylglycerol (TAG). The main objective of the present study was to investigate whether cod larvae could synthesis PL from sn-2-monoacylglycerol (2-MAG) and glycerol precursors. A tube feeding method was used to deliver equal molar aliquots of 2-oleoyl-[1,2,3-³H]glycerol and [U-¹⁴C] glycerol together with bovine serum albumin (BSA) bound 16:0 (palmitic acid) and 22:6n-3 (docosahexaenoic acid, DHA), with or without choline chloride to the foregut of anesthetized cod larvae and thereafter monitoring the metabolism of these components in the larvae through 4 h following injection. Our results showed that both 2-MAG and glycerol precursors contributed to the de novo synthesis of phosphatidylcholine (PC) and the 2-MAG pathway predominated over the G-3-P (glycerol-3-phosphate) pathway in the synthesis of TAG and PC. The molecular ratio of PC/TAG obtained from the 2-MAG and the G-3-P pathways was 0.44–0.74 and 1.02–2.06 within the first hour of tube feeding, suggesting they might have comparable biosynthesis ability of PC and TAG under the conditions of the present study. Furthermore, supplementation of choline chloride significantly increased PC/TAG ratio (p < 0.05) for both pathways. However, further studies are needed to quantify the enzyme activity involved in the CDP-choline (cytidine diphosphate choline) pathway, and the function of choline either in simulating PC synthesis or TAG catabolism or both needs further investigation.     

Effects of dietary <Emphasis Type="Italic">n</Emphasis>-3 fatty acids on Toll-like receptor activation in primary leucocytes from Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

The shortage of the n-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the international markets has led to increasing substitution of fish oil by plant oils in Atlantic salmon (Salmo salar) feed and thereby reducing the EPA and DHA content in salmon. However, the minimum required levels of these fatty acids in fish diets for securing fish health are unknown. Fish were fed with 0, 1 or 2% EPA or DHA alone or in combination of both over a period, growing from 50 to 400 g. Primary head kidney leucocytes were isolated and stimulated with Toll-like receptor (TLR) ligands to determine if EPA and DHA deficiency can affect expression of important immune genes and eicosanoid production. Several genes related to viral immune response did not vary between groups. However, there was a tendency that the high-level EPA and DHA groups expressed lower levels of IL-1β in non-stimulated leucocytes. These leucocytes were also more responsive to the TLR ligands, inducing higher expression levels of IL-1β and Mx1 after stimulation. The levels of prostaglandin E2 and leukotriene B4 in serum and media from stimulated leucocytes were lower in both low and high EPA and DHA groups. In conclusion, leucocytes from low EPA and DHA groups seemed to be less responsive towards immunostimulants, like TLR ligands, indicating that low levels or absence of dietary EPA and DHA may have immunosuppressive effects.     

Effects of temperature on the final stages of sexual maturation in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Maturing male and female Atlantic salmon (Salmo salar L.) were held under three temperature regimes for 10 weeks between September and December: warm (constant 14–16 °C), ambient (decreasing from 11 to 5 °C), and cold (decreasing from 7 to 3 °C). Blood samples were analyzed for plasma steroid levels, and the fish were inspected for the presence of expressible milt (total volume and spermatocrit) and ovulation weekly. Samples of eggs were dry-fertilized with milt stripped from three males held at the same temperatures and incubated until the eyed stage. In females, levels of plasma testosterone (T) and 17β-oestradiol (E₂) dropped as ovulation approached, concurrent with a rapid increase in levels of plasma 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). In males, levels of T and 11-ketotestosterone (11-KT) peaked 2–3 weeks after the first appearance of expressible milt, while levels of 17,20β-P increased steadily and did not exhibit a definite peak. Exposure of females to cold water amplified and advanced the profiles of all three steroids compared with the ambient group, and increased the survival rates to the eyed egg stage. Cold water had no immediate effect on the male steroid profiles, but later, higher levels of 17,20β-P were evident compared with both the ambient controls and the warm water group, while the effects on 11-KT and T were more variable. Exposure to warm water completely inhibited both milt production and ovulation. Moreover, warm water modulated the steroid profiles of the males with lower 11-KT levels compared with ambient controls and lower 17,20β-P level compared with cold-water-treated males. In females, warm water resulted in total inhibition of the peri-ovulatory peak in 17,20β-P and prevented the normal decline of T and E₂ levels associated with ovulation. The findings of the present study are highly relevant for broodstock management in aquaculture, as well in understanding the impact of climate change/temperature variability on wild salmon spawning.     

Effects of repeated crowding on the stress response and growth performance in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>)

A 64-day growth experiment was conducted in which two groups of Atlantic salmon parr were grown under either control conditions or subjected to a weekly crowding stressor. Subjecting fish to the stressor resulted in a 7.7% reduction in wet weight after 29 days, which was maintained at 7.9% by day 64. This reduction in weight was reflected in a 44% reduction in specific growth rate and 38% increase in feed conversion ratio over the first 29 days of the experiment. Elevation in plasma cortisol was observed in crowded fish on days 1, 29 and 64. Similarly, on days 1 and 29 an increase in both plasma glucose and lactate was detected. On day 64, however, no differences in plasma glucose and lactate were observed, with the magnitude of the cortisol response also significantly reduced. Overall, the relatively moderate impact on growth performance and reduction in magnitude of measured stress parameters at the end of the experiment suggests possible habituation to the applied stressor.     

Cod CGRP and tachykinins in coeliac artery innervation of the Atlantic cod, <Emphasis Type="Italic">Gadus morhua</Emphasis>: presence and vasoactivity

The presence and vasoactive effects of native calcitonin gene-related peptide (CGRP), substance P (SP), and neurokinin A (NKA) were studied on isolated small branches of the coeliac artery from Atlantic cod, Gadus morhua, using immunohistochemistry and myograph recordings, respectively. Immunohistochemistry revealed nerve fibers containing CGRP- and SP/NKA-like material running along the wall of the arteries. CGRP induced vasorelaxation of precontracted arteries with a pD₂ value of 8.54 ± 0.17. Relaxation to CGRP (10⁻⁸ M) was unaffected by l-NAME (3 × 10⁻⁴ M) and indomethacin (10⁻⁶ M) suggesting no involvement of nitric oxide or prostaglandins in the CGRP-induced relaxation. SP and NKA (from 10⁻¹⁰ to 3 × 10⁻⁷ M) contracted the unstimulated arteries at concentrations from 10⁻⁸ M and above in 42% and 33%, respectively, of the vessels. It is concluded that the innervation of the cod celiac artery includes nerves expressing CGRP-like and tachykinin-like material, and that a vasodilatory response to CGRP is highly conserved amongst vertebrates while the response to tachykinins is more variable.     

Stepwise temperature regulation and its effect on growth,feeding and muscle growth patterns of juvenile Atlantic halibut (<Emphasis Type="Italic">Hippoglossus hippoglossus</Emphasis> L.)

To investigate the possible direct effect of a stepwise reduction in temperature with increasing size on growth, feeding parameters and muscle growth patterns of juvenile Atlantic halibut (Hippoglossus hippoglossus L.), 804 juvenile halibut (mean initial weight individuals: 14.2 g ± 0.2 SEM) were reared at constant 9, 12 and 15°C or shifted (T-step, i.e. 15–12°C after 36 days) for 99 days. Despite indications of lower optimal temperature for growth with increasing size, equal end weights were obtained between the constant 12°C, constant 15°C and T-step groups. Best overall growth was observed for the group kept at constant 12°C. The limited effect of the T-step group may relate to the size at movement (too big), the temperatures investigated (close to optimum) and the time and size interval investigated (too narrow). Differences in growth were reflected more by alterations in feed intake (C _T and F%) than by differences in feed conversion efficiencies (FCE). Differences were found with respect to the density of muscle cells, whereas no differences were found between the average muscle cell diameters. The mean diameter of muscle cells tended to increase only slightly with increasing fish weight, while the mean density of muscle cells tended to decrease. Using an optimum temperature of 12°C, an indication of a possible increased rate of hyperplasia in relation to higher growth was seen.     

Recent expansion of Northeast Atlantic and Mediterranean populations of <Emphasis Type="Italic">Melicertus</Emphasis> (<Emphasis Type="Italic">Penaeus</Emphasis>) <Emphasis Type="Italic">kerathurus</Emphasis> (Crustacea: Decapoda)

We analysed the genetic diversity of Melicertus kerathurus (Penaeidae), a commercially valuable penaeid shrimp that is distributed in the Mediterranean Sea and eastern Atlantic Ocean. We examined the polymorphism of a 494 bp DNA segment of the mitochondrial COI region in 173 individuals, sampled in nine Mediterranean and two Atlantic samples, covering the whole range of the species from the tropical waters of the Gulf of Guinea to the eastern part of the Mediterranean Sea. The mean nucleotide and haplotype diversities were π = 0.00275 and h = 0.718, respectively, for the global data set, with the highest values occurring in the African samples and the lowest in the Adriatic Sea. A clear sample differentiation was found (F _st = 0.194), but this did not reflect a geographical pattern and there were only faint traces of an Atlantic–Mediterranean subdivision. Mismatch analysis and a high significant negative value of Tajima’s D suggested that M. kerathurus is not at mutation drift-equilibrium, but underwent a recent expansion after a period of low effective sample size. A postglacial recolonisation of the Mediterranean from an Atlantic refuge could be hypothesised based on these data.     

A moving light stimulus elicits a sustained swimming response in farmed Atlantic salmon, <Emphasis Type="Italic">Salmo salar</Emphasis> L.

The productivity and welfare benefits of sustained swimming in fish are well documented, but are not yet exploited in commercial aquaculture. We report here on a study designed to test the feasibility of inducing sustained exercise in Atlantic salmon (Salmo salar L.) using a novel lighting device that provides an apparently moving light pattern. It was found that such a device could induce sustained swimming in Atlantic salmon held in tanks and that a centrally placed (inner ring) light system was far more effective in this context than one in which lights were placed on the outer wall of tanks. The central configuration was associated with enhanced rates of growth, feed conversion and reduced levels of plasma cortisol. Such developments in fish swimming technologies may assist the sustainability of finfish aquaculture through promotion of sustained exercise leading to improved productivity and welfare.     

Lack of arterial PO<Subscript>2</Subscript> downregulation in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) during long-term normoxia and hyperoxia

Regulation of arterial partial pressure of O₂ (P_aO₂) in Atlantic salmon (Salmo salar) was investigated during resting conditions in normoxic and hyperoxic water. Dorsal aorta cannulated adult Atlantic salmon (1.2–1.6 kg, n = 8) were exposed to 2 week sequential periods of normoxia [16.7 ± 1.1 kPa (mean ± SD)] and hyperoxia (34.1 ± 4.9 kPa) in individual tanks containing seawater (33.7 ± 0.2 ppt) at stable temperature conditions (8.7 ± 0.7°C) and a light regime of L:D = 12:12. Tank design and sampling procedures were optimized to provide suitable shelter and current for the fish, and to allow repeated, undisturbed sampling of blood from free-swimming fish. Fish were sampled regularly through the experimental period. P_wO₂, P_aO₂, blood ion composition (Na⁺, K⁺, Cl⁻), acid–base status (pH, PCO₂, HCO₃ ⁻), haematocrit and glucose were measured. The most frequently observed P_aO₂ values were in the range of 60–80% of P_wO₂, both during normoxia and hyperoxia, and P_aO₂ values were significantly lower during normoxia than during hyperoxia. Blood pH, PCO₂ and HCO₃ ⁻ were significantly elevated during hyperoxia, while, Na⁺, Cl⁻ and Hct were significantly lower. K⁺ and glucose showed no significant differences. This study demonstrates a lack P_aO₂ regulation in Atlantic salmon to low partial pressures, in contrast to previous reports for many aquatic gill breathing animals. Both during normoxia and hyperoxia, P_aO₂ reflects P_wO₂, and alterations in external PO₂ consequently result in proportional arterial PO₂ changes. Physiological adaptation to hyperoxia, as illustrated by changes in several blood parameters, does not include down-regulation of P_aO₂ in Atlantic salmon. The lack of P_aO₂ regulation may make Atlantic salmon vulnerable to the oxidative stress caused by increased free radical formation in hyperoxic conditions.     

Matrilin-1 expression is increased in the vertebral column of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) individuals displaying spinal fusions

We have previously characterized the development of vertebral fusions induced by elevated water temperature in Atlantic salmon. Molecular markers of bone and cartilage development together with histology were used to understand the complex pathology and mechanism in the development of this spinal malformation. In this study, we wanted to use proteomics, a non-hypothetical approach to screen for possible new markers involved in the fusion process. Proteins extracted from non-deformed and fused vertebrae of Atlantic salmon were therefore compared by two-dimensional electrophoresis (2DE) and MALDI-TOF analysis. Data analysis of protein spots in the 2DE gels demonstrated matrilin-1, also named cartilage matrix protein, to be the most highly up-regulated protein in fused compared with non-deformed vertebrae. Furthermore, real-time PCR analysis showed strong up-regulation of matrilin-1 mRNA in fused vertebrae. Immunohistochemistry demonstrated induced matrilin-1 expression in trans-differentiating cells undergoing a metaplastic shift toward chondrocytes in fusing vertebrae, whereas abundant expression was demonstrated in cartilaginous tissue and chordocytes of both non-deformed and fused vertebrae. These results identifies matrilin-1 as a new interesting candidate in the fusion process, and ratify the use of proteomic as a valuable technique to screen for markers involved in vertebral pathogenesis.     

Differences in longitudinal distribution patterns along a Honshu stream of brown trout <Emphasis Type="Italic">Salmo trutta</Emphasis>, white-spotted charr <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis> and masu salmon <Emphasis Type="Italic">Oncorhynchus masou</Emphasis>

Brown trout Salmo trutta were first introduced into Japan in 1892, and they currently naturally reproduce in several rivers in Honshu and Hokkaido, Japan. Although negative impacts of brown trout introductions on native salmonid fishes have been documented in some Hokkaido rivers, studies of ecological interactions between brown trout and native salmonid fishes on Honshu are limited. In this study, we describe the longitudinal distribution patterns of introduced brown trout, white-spotted charr Salvelinus leucomaenis and masu salmon Oncorhynchus masou in a 4 km stretch of a stream in central Honshu. Underwater observations were conducted in all pools within upstream, middle and downstream sections (190–400 m in length) of this stretch in order to estimate the densities of these species. Only white-spotted charr was observed in the upstream section, while brown trout and masu salmon were observed in the middle and downstream sections. Masu salmon densities, however, were much lower than brown trout densities. In the downstream section, white-spotted charr was absent. These results are consistent with results from previous studies of Hokkaido rivers, where it was found that white-spotted charr in low-gradient areas tend to be displaced by brown trout.     

Potential of cinnamon (<Emphasis Type="Italic">Cinnamomum verum</Emphasis>) oil to control <Emphasis Type="Italic">Streptococcus iniae</Emphasis> infection in tilapia (<Emphasis Type="Italic">Oreochromis niloticus</Emphasis>)

In this study, four essential oils—cinnamon oil, leech lime oil, lemongrass oil, and turmeric oil—were examined for their antimicrobial activities against Streptococcus iniae, a bacterium that is pathogenic in fish, in which it causes streptococcosis. Cinnamon oil was the most potent antimicrobial agent among these oils, with a minimal inhibitory concentration (MIC) of 40 μg/ml. By using gas chromatography–mass spectrometry (GC–MS), it was found that the major components of cinnamon oil were cinnamaldehyde (90.24), limonene (2.42%), cinnamyl acetate (2.03%), linalool (1.16%), and α-terpineol (0.87%). Of these compounds, only cinnamaldehyde exhibited antimicrobial activity against S. iniae, with an MIC of 20 μg/ml. In an in vivo trial, no mortality was apparent in fish fed on fish diets supplemented with 0.4% (w/w) of cinnamon oil and with 0.1% (w/w) of oxytetracycline 5 days prior to infection with S. iniae. These results indicate that cinnamon oil had a protective effect on experimental S. iniae infection in tilapia, and thus has the potential to replace the antibiotics used to control this disease.     

Proportions of Atlantic sailfish (<Emphasis Type="Italic">Istiophorus platypterus</Emphasis>) and spearfish (<Emphasis Type="Italic">Tetrapturus pfluegeri</Emphasis>) caught in tuna longline fisheries

The reliability of estimations of catches of pelagic longline fisheries is especially worrying in the case of non-target species such as the sailfish (Istiophorus platypterus) and spearfish (Tetrapturus pfluegeri), because they are often recorded aggregated in onboard maps. For this reason, generalized linear models were used to identify a number of important explanatory variables and their effect on sailfish and spearfish proportions. The response variable (number of sailfish in the aggregate capture) is assumed as binomial using the logit link function. The explanatory variables used were foram year, qrt (quarter), fleet, de (distance from equator) and d (distance from areas above sea level). The selected models showed that the equatorial and coastal regions presented higher densities or catchabilities of sailfish when compared to spearfish, as in the case for fleets of China Taipei and the United States. The lowest mean proportion of sailfish [sailfish/(sailfish + spearfish)] of the third quarter were driven mostly by the low estimations calculated for Japan and Brazil.