Hypoxic acclimation leads to metabolic compensation after reoxygenation in Atlantic salmon yolk-sac alevins.

Hypoxia is common in aquatic environments and has substantial effects on development, metabolism and survival of aquatic organisms. To understand the physiological effects of hypoxia and its dependence on temperature, metabolic rate ( [Formula: see text] ) and cardiorespiratory function were studied in response to acute hypoxia (21→5kPa) at different measurement temperatures (Ta; 4, 8 and 12°C) in Salmo salar alevins that were incubated under normoxic conditions (PO2=21kPa) or following hypoxic acclimation (PO2=10kPa) as well as two different temperatures (4°C or 8°C). Hypoxic acclimation lead to a developmental delay manifested through slower yolk absorption. The general response to acute hypoxia was metabolic depression (~60%). Hypoxia acclimated alevins had higher [Formula: see text] s when measured in normoxia than alevins acclimated to normoxia. [Formula: see text] s were elevated to the same degree (~30% per 4°C change) irrespective of Ta. Under severe, acute hypoxia (~5kPa) and irrespective of Ta or acclimation, [Formula: see text] s were similar between most groups. This suggests that despite different acclimation regimes, O2 transport was limited to the same degree. While cardiorespiratory function (heart-, ventilation rate) was unchanged in response to acute hypoxia after normoxic acclimation, hypoxic acclimation led to cardiorespiratory changes predominantly in severe hypoxia, indicating earlier onset and plasticity of cardiorespiratory control mechanisms. Although [Formula: see text] in normoxia was higher after hypoxic acclimation, at the respective acclimation PO2, [Formula: see text] was similar in normoxia and hypoxia acclimated alevins. This is indicative of metabolic compensation to an intrinsic [Formula: see text] at the acclimation condition in hypoxia-acclimated alevins after re-exposure to normoxia.

Hypoxia during incubation does not affect aerobic performance or haematology of Atlantic salmon (Salmo salar) when re-exposed in later life.

Hypoxia in aquatic ecosystems is becoming increasingly prevalent, potentially reducing fish performance and survival by limiting the oxygen available for aerobic activities. Hypoxia is a challenge for conserving and managing fish populations and demands a better understanding of the short- and long-term impacts of hypoxic environments on fish performance. Fish acclimate to hypoxia via a variety of short- and long-term physiological modifications in an attempt to maintain aerobic performance. In particular, hypoxia exposure during early development may result in enduring cardio-respiratory modifications that affect future hypoxia acclimation capacity, yet this possibility remains poorly investigated. We incubated Atlantic salmon (Salmo salar) in normoxia (~100% dissolved oxygen [DO, as percent air saturation]), moderate hypoxia (~63% DO) or cyclical hypoxia (100-25% DO daily) from fertilization until 113 days post-fertilization prior to rearing all groups in normoxia for a further 8 months. At ~11 months of age, subsets of each group were acclimated to hypoxia (50% DO) for up to 44 days prior to haematology, aerobic metabolic rate and hypoxia tolerance measurements. Hypoxia exposure during incubation (fertilization to 113 days post-fertilization) did not affect the haematology, aerobic performance or hypoxia tolerance of juvenile salmon in later life. Juveniles acclimated to hypoxia increased maximum aerobic metabolic rate and aerobic scope by ~23 and ~52%, respectively, when measured at 50% DO but not at 100% DO. Hypoxia-incubated juveniles also increased haematocrit and haemoglobin concentration but did not affect acute hypoxia tolerance (critical oxygen level and DO at LOE). Thus, while Atlantic salmon possess a considerable capacity to physiologically acclimate to hypoxia by improving aerobic performance in low oxygen conditions, we found no evidence that this capacity is influenced by early-life hypoxia exposure.

Physiological effects of dissolved oxygen are stage-specific in incubating Atlantic salmon (Salmo salar).

Oxygen availability is highly variable during salmonid incubation in natural redds and also in aquaculture incubation systems. Hypoxia generally decreases growth and aerobic metabolism prior to hatching, in parallel with eliciting physiological modifications that enhance oxygen delivery. However, it is less-well known whether developmental hyperoxia can drive the opposite effect. Moreover, there is insufficient understanding of stage-specific developmental windows during which ambient oxygen availability may be of greater or lesser impact to incubating embryos. Here, we tested the effects of hypoxia (50% dissolved oxygen: DO, % air saturation) and hyperoxia (150% DO) on the growth, routine aerobic metabolism ([Formula: see text]) and hypoxia tolerance (O2crit) of Atlantic salmon (Salmo salar) during seven developmental windows throughout incubation. Embryos exposed to hyperoxia (150% DO) did not differ from the normoxic group in growth, [Formula: see text] or O2crit at any developmental window. In contrast, embryos exposed to hypoxia grew slower and had a lower [Formula: see text], but had higher hypoxia tolerance (lower O2crit) than normoxic and hyperoxic counterparts. Interestingly, these differences were only apparent when the embryos were measured prior to hatching. Larvae (alevins) incubated in hypoxia following hatching grew similarly to normoxia-incubated alevins. Our results provide evidence that Atlantic salmon embryos are most sensitive to hypoxia prior to hatching, probably due to increasing (absolute) oxygen requirements concurrent with restricted oxygen diffusion through the egg. Moreover, the similarities between normoxia- and hyperoxia-incubated salmon demonstrate that embryos are not oxygen-limited under normoxic conditions.

Greenlip Abalone (Haliotis laevigata) Genome and Protein Analysis Provides Insights into Maturation and Spawning.

Wild abalone (Family Haliotidae) populations have been severely affected by commercial fishing, poaching, anthropogenic pollution, environment and climate changes. These issues have stimulated an increase in aquaculture production; however production growth has been slow due to a lack of genetic knowledge and resources. We have sequenced a draft genome for the commercially important temperate Australian 'greenlip' abalone (Haliotis laevigata, Donovan 1808) and generated 11 tissue transcriptomes from a female adult abalone. Phylogenetic analysis of the greenlip abalone with reference to the Pacific abalone (Haliotis discus hannai) indicates that these abalone species diverged approximately 71 million years ago. This study presents an in-depth analysis into the features of reproductive dysfunction, where we provide the putative biochemical messenger components (neuropeptides) that may regulate reproduction including gonad maturation and spawning. Indeed, we isolate the egg-laying hormone neuropeptide and under trial conditions induce spawning at 80% efficiency. Altogether, we provide a solid platform for further studies aimed at stimulating advances in abalone aquaculture production. The H. laevigata genome and resources are made available to the public on the abalone 'omics website, http://abalonedb.org.

Resistance to amoebic gill disease (AGD) is characterised by the transcriptional dysregulation of immune and cell cycle pathways.

Amoebic gill disease (AGD) is a parasite-mediated proliferative gill disease capable of affecting a range of teleost hosts. While a moderate heritability for AGD resistance in Atlantic salmon has been reported previously, the mechanisms by which individuals resist the proliferative effects remain poorly understood. To gain more knowledge of this commercially important trait, we compared gill transcriptomes of two groups of Atlantic salmon, one designated putatively resistant, and one designated putatively susceptible to AGD. Utilising a 17k Atlantic salmon cDNA microarray we identified 196 transcripts that were differentially expressed between the two groups. Expression of 11 transcripts were further examined with real-time quantitative RT-PCR (qPCR) in the AGD-resistant and AGD-susceptible animals, as well as non-infected naïve fish. Gene expression determined by qPCR was in strong agreement with the microarray analysis. A large number of differentially expressed genes were involved in immune and cell cycle responses. Resistant individuals displayed significantly higher expression of genes involved in adaptive immunity and negative regulation of the cell cycle. In contrast, AGD-susceptible individuals showed higher expression of acute phase proteins and positive regulators of the cell cycle. Combined with the gill histopathology, our results suggest AGD resistance is acquired rather than innately present, and that this resistance is for the most part associated with the dysregulation of immune and cell cycle pathways.

Transcriptome analyses of amoebic gill disease-affected Atlantic salmon (Salmo salar) tissues reveal localized host gene suppression.

The transcriptome response of Atlantic salmon (Salmo salar) displaying advanced stages of amoebic gill disease (AGD) was investigated. Naïve smolt were challenged with AGD for 19 days, at which time all fish were euthanized and their severity of infection quantified through histopathological scoring. Gene expression profiles were compared between heavily infected and naïve individuals using a 17 K Atlantic salmon cDNA microarray with real-time quantitative RT-PCR (qPCR) verification. Expression profiles were examined in the gill, anterior kidney, and liver. Twenty-seven transcripts were significantly differentially expressed within the gill; 20 of these transcripts were down-regulated in the AGD-affected individuals compared with naïve individuals. In contrast, only nine transcripts were significantly differentially expressed within the anterior kidney and five within the liver. Again the majority of these transcripts were down-regulated within the diseased individuals. A down-regulation of transcripts involved in apoptosis (procathepsin L, cathepsin H precursor, and cystatin B) was observed in AGD-affected Atlantic salmon. Four transcripts encoding genes with antioxidant properties also were down-regulated in AGD-affected gill tissue according to qPCR analysis. The most up-regulated transcript within the gill was an unknown expressed sequence tag (EST) whose expression was 218-fold (+/- SE 66) higher within the AGD affected gill tissue. Our results suggest that Atlantic salmon experiencing advanced stages of AGD demonstrate general down-regulation of gene expression, which is most pronounced within the gill. We propose that this general gene suppression is parasite-mediated, thus allowing the parasite to withstand or ameliorate the host response.

Developmental Hypoxia Has Negligible Effects on Long-Term Hypoxia Tolerance and Aerobic Metabolism of Atlantic Salmon (Salmo salar).

Exposure to developmental hypoxia can have long-term impacts on the physiological performance of fish because of irreversible plasticity. Wild and captive-reared Atlantic salmon (Salmo salar) can be exposed to hypoxic conditions during development and continue to experience fluctuating oxygen levels as juveniles and adults. Here, we examine whether developmental hypoxia impacts subsequent hypoxia tolerance and aerobic performance of Atlantic salmon. Individuals at 8°C were exposed to 50% (hypoxia) or 100% (normoxia) dissolved oxygen (DO) saturation (as percent of air saturation) from fertilization for ∼100 d (800 degree days) and then raised in normoxic conditions for a further 15 mo. At 18 mo after fertilization, aerobic scope was calculated in normoxia (100% DO) and acute (18 h) hypoxia (50% DO) from the difference between the minimum and maximum oxygen consumption rates ([Formula: see text] and [Formula: see text], respectively) at 10°C. Hypoxia tolerance was determined as the DO at which loss of equilibrium (LOE) occurred in a constantly decreasing DO environment. There was no difference in [Formula: see text], [Formula: see text], or aerobic scope between fish raised in hypoxia or normoxia. There was some evidence that hypoxia tolerance was lower (higher DO at LOE) in hypoxia-raised fish compared with those raised in normoxia, but the magnitude of the effect was small (12.52% DO vs. 11.73% DO at LOE). Acute hypoxia significantly reduced aerobic scope by reducing [Formula: see text], while [Formula: see text] remained unchanged. Interestingly, acute hypoxia uncovered individual-level relationships between DO at LOE and [Formula: see text], [Formula: see text], and aerobic scope. We discuss our findings in the context of developmental trajectories and the role of aerobic performance in hypoxia tolerance.

Automated Non-invasive Video-Microscopy of Oyster Spat Heart Rate during Acute Temperature Change: Impact of Acclimation Temperature.

We developed an automated, non-invasive method to detect real-time cardiac contraction in post-larval (1.1-1.7 mm length), juvenile oysters (i.e., oyster spat) via a fiber-optic trans-illumination system. The system is housed within a temperature-controlled chamber and video microscopy imaging of the heart was coupled with video edge-detection to measure cardiac contraction, inter-beat interval, and heart rate (HR). We used the method to address the hypothesis that cool acclimation (10°C vs. 22°C-Ta10 or Ta22, respectively; each n = 8) would preserve cardiac phenotype (assessed via HR variability, HRV analysis and maintained cardiac activity) during acute temperature changes. The temperature ramp (TR) protocol comprised 2°C steps (10 min/experimental temperature, Texp) from 22°C to 10°C to 22°C. HR was related to Texp in both acclimation groups. Spat became asystolic at low temperatures, particularly Ta22 spat (Ta22: 8/8 vs. Ta10: 3/8 asystolic at Texp = 10°C). The rate of HR decrease during cooling was less in Ta10 vs. Ta22 spat when asystole was included in analysis (P = 0.026). Time-domain HRV was inversely related to temperature and elevated in Ta10 vs. Ta22 spat (P < 0.001), whereas a lack of defined peaks in spectral density precluded frequency-domain analysis. Application of the method during an acute cooling challenge revealed that cool temperature acclimation preserved active cardiac contraction in oyster spat and increased time-domain HRV responses, whereas warm acclimation enhanced asystole. These physiologic changes highlight the need for studies of mechanisms, and have translational potential for oyster aquaculture practices.

Oligonucleotide primers for PCR amplification of coelomate introns.

Abstract Seven novel oligonucleotide primer pairs for polymerase chain reaction amplification of introns from nuclear genes in coelomates were designed and tested. Each pair bound to adjacent exons that are separated by a single intron in most coelomate species. The primer sets amplified introns in species as widely separated by the course of evolution as oysters (Mollusca: Protostoma) and salmon (Chordata: Deuterostoma). Each primer set was tested on a further 6 coelomate species and found to amplify introns in most cases. These primer sets may therefore be useful tools for developing nuclear DNA markers in diverse coelomate species for studies of population genetics, phylogenetics, or genome mapping.

18S ribosomal DNA-based PCR identification of Neoparamoeba pemaquidensis, the agent of amoebic gill disease in sea-farmed salmonids.

Neoparamoeba pemaquidensis is a parasomal amoeboid protozoan identified as the agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar reared in sea-pens in Tasmania, Australia, and coho salmon Oncorhynchus kisutch farmed on the west coast of the USA. Outbreaks of AGD caused by immunologically cross-reactive paramoebae have also been reported in sea-farmed salmonids in several other countries. Complete 18S rDNA sequences were determined for respective paramoebae isolated from infected gills of salmon from Tasmania and Ireland, and N. pemaquidensis isolates from the USA and UK, including representative free-living isolates. Alignments over 2110 bp revealed 98.1 to 99.0% sequence similarities among isolates, confirming that paramoebae implicated in AGD in geographically distant countries were homologous and belonged to the same species, N. pemaquidensis. The results supported previous findings that N. pemaquidensis exists as a widely distributed, amphizoic marine protozoan. Partial 18S rDNA sequences were obtained for the ultrastructurally similar species, N. aestuarina, and for the morphologically similar but non-parasomal amoeba Pseudoparamoeba pagei. N. aestuarina had 95.3 to 95.7% sequence similarities with N. pemaquidensis strains, which distinguished 2 closely related but separate species. Neoparamoeba spp. were not analogous to P. pagei or to other marine Gymnamoebia. We designed 4 oligonucleotide primers based on elucidated 18S rDNA sequences and applied them to single-step and nested 2-step PCR protocols developed to identify N. pemaquidensis to the exclusion of apparently closely related and non-related protistan taxa. Nested PCR was able to detect the AGD parasite from non-purified, culture-enriched net microfouling samples from Atlantic salmon sea-pens in Tasmania, and confirmed that N. pemaquidensis was also responsible for AGD in chinook salmon O. tshawytscha in New Zealand. Our sequence and PCR analyses have now shown that AGD affecting 3 different salmonid species farmed in 4 countries are associated with N. pemaquidensis. A species-specific diagnostic PCR provides for the first time, a highly specific detection and identification assay for N. pemaquidensis that will facilitate future ecological and epidemiological studies of AGD.

Respirometry: correcting for diffusion and validating the use of plastic multiwell plates with integrated optodes.

Polystyrene multiwell plates with integrated optodes act as multiple closed-system respirometers that enable the simultaneous measurement of oxygen consumption in small animals. However, the diffusion of oxygen through polystyrene needs to be taken into consideration. Here we provide an equation that accounts for the empirically determined rate of oxygen through a polystyrene well when calculating the instantaneous rate of oxygen consumption. Furthermore, we describe a novel method of calibrating a small respirometer for accuracy using micro-osmotic pumps containing an oxygen scavenger, which is delivered at a constant rate and therefore yields a constant rate of oxygen consumption in an airtight system.

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.