Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.)

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.     

The production of gynogenetic Atlantic salmon,Salmo salar L.

The production of Atlantic salmon gynogenomes by the combined use of a novel method for sperm irradiation and differently timed high hydrostatic pressure shocks is described. Sperm solutions were exposed to UV irradiation in a temperature-controlled flow-through device. Eggs fertilised with such sperm were exposed to shocks of 9500 psi at 30 min or approximately 7 h after fertilisation in order to produce meiotic and mitotic gynogenomes respectively. Yields of meiotic gynogenomes were generally high (up to 95%); those of mitotic gynogenomes were lower (range 2–20%). Analyses of the offspring by ploidy status and fingerprinting confirmed their gynogenetic origin. Small numbers of mitotic gynogenetic fish were grown on for 2 years in fresh and salt water. S1/S2 ratios were lower in gynogenetic fish and mean age at maturity was greater. Of the presumptive gynogenetic fish subjected to destructive sampling (n = 87) all were female.     

大西洋鲑杀鲑气单胞菌无色亚种的分离鉴定和致病性研究

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Spermatozoa concentration influences cryopreservation success in sea trout (Salmo trutta m. trutta L.)

Variation among individuals is substantial for spermatozoa concentration in fresh milt in sea trout (Salmo trutta m. trutta L.). The objective of the present study was to examine effects of spermatozoa concentration in this species on subsequent cryopreservation success. Milt with high spermatozoa concentration was diluted with seminal plasma to obtain concentrations ranging between 6 and 24 × 10⁹ mL⁻¹ with steps of 2 × 10⁹ mL⁻¹. Diluted milts were cryopreserved in 0.25-mL straws with extender (0.3 M glucose) containing 10% methanol and 10 % (vol/vol) supplement of hen egg yolk. The dilution ratio was 1:3 (milt:cryomedium). Cryopreservation efficacies were assessed according to evaluation of motility of frozen/thawed spermatozoa and quantification of fertilizing ability. Percentage of motility of frozen/thawed spermatozoa was influenced by spermatozoa concentration in the cryomedium (P < 0.05). The highest motility was observed in samples with 3.0 to 4.0 × 10⁹ spermatozoa per mL of cryomedium, which corresponds to 12 to 16 × 10⁹ spermatozoa per mL in fresh milt. Higher sperm concentrations and lower sperm concentrations in cryomedium reduced the effectiveness of cryopreservation when compared with the optimum. Cryopreservation success measured according to fertilization rate was in agreement with results for motility of frozen/thawed spermatozoa, but the optimum could not be determined with statistical precision because of differences in fertilization rate among individual donor males. However, a significant positive correlation was found between postthaw motility and fertilization rate and between cryopreserved spermatozoa velocity and fertilization rate (P < 0.05). In sea trout, cryopreservation efficiency is influenced by spermatozoa concentration in cryomedium. Individual adjustment of the dilution ratio, based on initial spermatozoa density, is recommended in the freezing protocol. Maximum cryoresistance of the cell was obtained when spermatozoa concentration in cryomedium ranged from 3.0 to 4.0 × 10⁹ mL⁻¹.     

Renal,respiratory and ionic regulation in Atlantic salmon (Salmo salar L.) kelts following transfer from fresh water to seawater

Summary Atlantic salmon may return to the sea after spawning in fresh water. These fish, known as kelts, reportedly show a limited ability to hypoosmoregulate. However, this study shows that fresh-water-adapted kelts exposed to seawater demonstrate rapid adaptation (within 48 h) in osmoregulatory parameters to values characteristic of seawater-adapted salmonids. The urine flow rate falls from 1.2 to 0.2 ml·kg^-1·h^-1 within 24 h. Over the same period, urine osmolality increases from 48 mosmol·kg^-1 to become isosmotic with the plasma, and Mg²⁺ secretion by the kidney tubules elevates the urine concentration from 0.5 to 100 mmol·l^-1. As is characteristic for marine teleosts, kelts drink seawater and process the ingested water in the gut to replace body water lost by osmosis to the hyperosmotic medium. Seawater exposure causes a marked hypoxia, arterial oxygen tension falling by 43% within minutes and persisting for at least 4 days at this low level. This is associated with large changes in blood pH and acid-base balance. The physiological mechanisms involved in adaptation to a hyperosmotic external medium are discussed, and the osmoregulatory capacity of kelts is compared with that of salmon at other stages of the life cycle.Abbreviations FW fresh water - GFR glomerular filtration rate - Hb haemoglobin - Hct haematocrit - MCHC mean cell haemoglobin concentration - pH_a pH in arterial blood - P _aO₂ partial pressure of oxygen in arterial blood - SEM standard error of mean - SW seawater - UFR urine flow rate     

Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar)

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.     

Genetic variation in haemolytic activity in Atlantic salmon (Salmo salar L.)

Spontaneous and antibody-dependent haemolytic activity against sheep red blood cells (SRBC) has been studied in serum from Atlantic salmon, Salmo salar L. Considerable increase in haemolytic activity was detected when SRBC were sensitized with haemolysin produced in Atlantic salmon. Haemolytic activity was sensitive both to divalent cations and to heat treatment. Significant reduction in haemolytic activity was detected after absorption with SRBC, indicating the presence of natural antibody against SRBC in Atlantic salmon serum. Persistent haemolytic activity of unsensitized SRBC in serum absorbed to remove natural antibodies was found, which suggests the activation of the alternative complement pathway, while the observed increased haemolytic activity in the presence of specific antibody against SRBC suggests activation of the classical complement pathway. Spontaneous and antibody-dependent haemolytic activity were both analysed in absorbed and non-absorbed sera in a family material of Atlantic salmon. The material consisted of 574 fish belonging to 57 fullsib groups within 20 paternal halfsib groups. Fish with signs of sexual maturity generally showed reduced haemolytic activity. Statistically significant effect of sire on the spontaneous haemolytic activity of both absorbed and nonabsorbed serum, and on antibody-dependent activity, provides evidence of significant additive genetic variation in both the alternative and the classical complement activation in Atlantic salmon. Neither on a phenotypical nor on a family basis were the two traits statistically correlated. The estimated heritabilities were 0.2–0.3 with a standard error of approximately 0.1.     

Mate choice for major histocompatibility complex genetic divergence as a bet-hedging strategy in the Atlantic salmon (Salmo salar)

Major histocompatibility complex (MHC)-dependent mating preferences have been observed across vertebrate taxa and these preferences are expected to promote offspring disease resistance and ultimately, viability. However, little empirical evidence linking MHC-dependent mate choice and fitness is available, particularly in wild populations. Here, we explore the adaptive potential of previously observed patterns of MHC-dependent mate choice in a wild population of Atlantic salmon (Salmo salar) in Québec, Canada, by examining the relationship between MHC genetic variation and adult reproductive success and offspring survival over 3 years of study. While Atlantic salmon choose their mates in order to increase MHC diversity in offspring, adult reproductive success was in fact maximized between pairs exhibiting an intermediate level of MHC dissimilarity. Moreover, patterns of offspring survival between years 0+ and 1+, and 1+ and 2+ and population genetic structure at the MHC locus relative to microsatellite loci indicate that strong temporal variation in selection is likely to be operating on the MHC. We interpret MHC-dependent mate choice for diversity as a likely bet-hedging strategy that maximizes parental fitness in the face of temporally variable and unpredictable natural selection pressures.     

Diel activity pattern of juvenile Atlantic salmon (Salmo salar) in early and late winter

Radiotelemetry was used to investigate the diel activity pattern of juvenile Atlantic salmon (Salmo salar) in early and late winter. Fish were active throughout the diel cycle. However, there was significantly less daytime than nighttime movement and movement declined significantly with increasing fork length. Maximizing winter growth rate, through an overall increase in foraging activity, may reduce the risk of starvation in smaller fish. The results of the present study provide evidence that the activity patterns of juvenile salmonids are quite complex and support the suggestion that individual variation in activity patterns are, at least, partially driven by body size.     

Spatial and temporal variation of genetic diversity and estimation of effective population sizes in Atlantic salmon (Salmo salar, L.) populations from Asturias (Northern Spain) using microsatellites

Rivers in Asturias (northern Spain) constitute the southern limit of the distribution of Atlantic salmon (Salmo salar L.) in Europe, a biological resource facing one of the more serious challenges for conservation today. In this work, eight microsatellite loci have been used to analyse samples collected in 1993 and 1999 from four Asturian rivers (Esva, Narcea, Sella, and Cares), obtaining information about the temporal and the spatial genetic variation in these populations and, in addition, estimations of their effective population sizes. The temporal analysis revealed a general decrease in all the estimated genetic variability parameters when samples from 1993 (mean A ₍₁₉₉₃₎ = 6.47, mean H _O(1993) = 0.472, mean H _E(1993) = 0.530) were compared with those obtained in 1999 (mean A ₍₁₉₉₉₎ = 6.16, mean H _O(1999) = 0.460, mean H _E(1999) = 0.490). This reduction was particularly notable for the case of the Esva river. Our results pointed to a pattern of spatial genetic differentiation inside the Asturian region (F _{ST (1993)} = 0.016 P < 0.01; F _{ST (1999)} = 0.023 P < 0.01). Using the standard Temporal Method we found estimates of N _e^{^} _(Esva) = 75.1 (33.2–267.2); N _e^{^} _(Cares) = 96.6 (40.0–507.5), N _e^{^} _(Sella) = 106.5 (39.1–9396.4) and N _e^{^} _(Narcea) = 113.9 (42.0–3693.3). The use of likelihood-based methods for the N _e^{^} estimations improved the results (smaller CIs) for the Esva and Cares rivers (N _e^{^} _(Esva) = 63.9 (32.3–165.3); N _e^{^} _(Cares) = 76.4 (38.8–202.0) using a Maximum likelihood approach) and suggested the presence of larger populations for the Sella and Narcea rivers (N _e^{^}≈200). These results showed that the Asturian Atlantic salmon populations (in particular Esva and Cares river populations) could be close to the conservation genetic borderline for avoiding inbreeding depression although we discuss some implications of the analysis of temporal genetic change in populations with overlapping generations.     

First access to territorial space and exposure to strong predation pressure: A conflict in early emerging Atlantic salmon (Salmo salar L.) fry

Summary This work aimed to elucidate conflicting factors that may explain the narrow and synchronous emergence in salmonids. Fry are highly vulnerable to predation and stand a better chance of surviving if they emerge synchronously. On the other hand, fry that leave the gravel first should increase their chance of obtaining one of the limited number of feeding territories. The risk involved in early emergence for Atlantic salmon fry was evaluated by exposing them to predatorySalmo trutta. Yolk sac alevins were incubated in an artificial redd in order to catch them by their time of emergence. Early-, peak- and late-emerging fry were then successively marked and transferred to flume tanks in which the predators were either present from the start or not introduced until all fry had been added. When the predators were initially present, the predation pressure differed depending on the time of fry emergence, resulting in survival rates of 11.6, 44.9 and 51% in early-, peak- and late-emerging fry, respectively. By assuming that the predation rate of the three emergence groups was dependent both on time of emergence and fry density the survival rates were calculated to be 7.5, 41.1 and 53.5% in groups I, II and III, respectively. These figures corresponded well to the observed rates. When the predators were added after completed emergence the resulting survival rates were 56.9, 39.7 and 25.2% in early-, peak- and late-emerging fry, respectively. Thus, predation after complete emergence gave a survival probability that varied across the three emergence-date groups, despite being exposed to a predator during the same number of days. The presence of fish predators in combination with a limited territorial space seems to make both early and late emergence hazardous and favours synchronous swimming movements.     

The effects of handling on flounder (Platichthys flesus L.) and Atlantic salmon (Salmo salar L.)

The effect of 9 min of net confinement on two marine teleost species, the flounder and the Atlantic salmon, was investigated in order to gauge how different species respond to the same stress stimulus. Net confinement in both species induced significant elevations in plasma cortisol, glucose, lactate, osmolality, and monovalent ion levels; the responses to net confinement in salmon were generally of a greater magnitude. In both species, handling induced significant alterations in PFFA levels though there were marked species differences in the qualitative and quantitative aspects of this response. Plasma protein levels were significantly elevated only in the flounders. In general, the two species responded similarly to the net confinement with differences only in the absolute levels and durations of the responses.     

IPNV modulation of pro and anti-inflammatory cytokine expression in Atlantic salmon might help the establishment of infection and persistence

IPNV is the agent of a well-characterized acute disease that produces a systemic infection and high mortality in farmed fish species and persistent infection in surviving fish after outbreaks. Because modulation of the host expression of pro and anti-inflammatory cytokines can help establish persistence, in this study, we examined the expression of IL-1β, IL-8, IFNα1 and IL-10 during acute and persistent IPNV infection of Atlantic salmon. Results showed that IPNV infection induces an increase of the IFNα1 and IL-10 mRNA levels in the spleen and head kidney (HK) of fish after acute experimental infection. Levels of the pro-inflammatory cytokines IL-1β and IL-8 did not rise in the spleen although an increase of IL-1β, but not of IL-8, was observed in head kidney. In carrier asymptomatic salmon, cytokine gene expression of IFNα1 in the spleen and IL-10 in head kidney were also significantly higher than expression in non-carrier fish. Interestingly, a decrease of IL-8 expression was also observed. IPNV infection of SHK-1, which is a macrophage-like cell line of salmon, also induced an increase of expression of the anti-inflammatory cytokine IL-10 with no effects on the expression of IL-1β and IL-8. The effects are induced by an unknown mechanism during viral infection because poly I:C and the viral genomic dsRNA showed the opposite effects on cytokine expression in SHK-1 cells. In summary, IPNV always induces up-regulation of the anti-inflammatory cytokine IL-10 in Atlantic salmon. As this is accompanied by a lack of induction of the pro-inflammatory cytokines IL-1β and IL-8, the anti-inflammatory milieu may explain the high frequency, prevalence and persistence of IPNV in salmon. Effects might be part of the viral mechanisms of immune evasion.     

Characterization and PCR multiplexing of novel highly variable tetranucleotide Atlantic salmon (Salmo salar L.) microsatellites

Seven novel and highly variable tetranucleotide microsatellite markers, and conditions for multiplexing and simultaneous genotyping six of these in a single run, are described for Atlantic salmon. These provide a highly informative and cost‐effective set of molecular markers for genetic studies on cultured and wild populations of the species. The primers sets showed cross‐species amplification of appropriately sized amplified products in a number of other salmonid species and suggests the primer sets may have wider application.     

The origins of Atlantic salmon (Salmo salar L.) recolonizing the River Mersey in northwest England

By the 1950s, pollution had extirpated Atlantic salmon in the river Mersey in northwest England. During the 1970s, an extensive restoration program began and in 2001, an adult salmon was caught ascending the river. Subsequently, a fish trap was installed and additional adults are now routinely sampled. In this study, we have genotyped 138 adults and one juvenile salmon at 14 microsatellite loci from across this time period (2001–2011). We have used assignment analysis with a recently compiled pan‐European microsatellite baseline to identify their most probable region of origin. Fish entering the Mersey appear to originate from multiple sources, with the greatest proportion (45–60%, dependent on methodology) assigning to rivers in the geographical region just north of the Mersey, which includes Northwest England and the Solway Firth. Substantial numbers also appear to originate from rivers in western Scotland, and from rivers in Wales and Southwest England; nonetheless, the number of fish originating from proximal rivers to the west of the Mersey was lower than expected. Our results suggest that the majority of salmon sampled in the Mersey are straying in a southerly direction, in accordance with the predominantly clockwise gyre present in the eastern Irish Sea. Our findings highlight the complementary roles of improving water quality and in‐river navigability in restoring salmon to a river and underlines further the potential benefits of restoration over stocking as a long‐term solution to declining fish stocks.     

Ecological determinants and temporal stability of the within-river population structure in Atlantic salmon (Salmo salar L.)

A gene diversity analysis was performed using microsatellite loci in order to (i) describe the extent and pattern of population structure in Atlantic salmon (Salmo salar L.) within a river system; (ii) establish the importance of quantifying the signal:noise ratio in accurately estimating population structure; and (iii) assess the potential usefulness of two evolutionary models in explaining within-river population structure from the ecological and habitat characteristics of Atlantic salmon. We found weak, yet highly significant microscale spatial patterning after accounting for variance among temporal replicates within sites. Lower genetic distances were observed among temporal samples at four sampling sites whereas no evidence for temporal stability was observed at the other three locations. The component of genetic variance attributable to either temporal instability and/or random sampling errors was almost three times more important than the pure spatial component. This indicates that not considering signal:noise ratio may lead to an important overestimation of genetic substructuring in situations of weak genetic differentiation. This study also illustrates the usefulness of the member-vagrant hypothesis to generate a priori predictions regarding the number of subpopulations that should compose a species, given its life-history characteristics and habitat structure. On the other hand, a metapopulation model appears better suited to explain the extent of genetic divergence among subpopulations, as well as its temporal persistence, given the reality of habitat patchiness and environment instability. We thus conclude that the combined use of both models may offer a promising avenue for studies aiming to understand the dynamics of genetic structure of species found in unstable environments.