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The endangered and range-restricted Maugean skate ( Zearaja maugeana) is subjected to large environmental variability coupled with anthropogenic stressors in its endemic habitat, Macquarie Harbour, Tasmania. However, little is known about the basic biology/physiology of this skate, or how it may respond to future environmental challenges predicted from climate change and/or increases in human activities such as aquaculture. These skate live at a preferred depth of 5–15 m where the dissolved oxygen (DO) levels are moderate (55% air saturation), but can be found in areas of the Harbour where DO can range from 100% saturation to anoxia. Given that the water at their preferred depth is already hypoxic, we sought to investigate their response to further decreases in DO that may arise from potential increases in anthropogenic stress. We measured oxygen consumption, haematological parameters, tissue–enzyme capacity and heat shock protein (HSP) levels in skate exposed to 55% dissolved O 2 saturation (control) and 20% dissolved O 2 saturation (hypoxic) for 48 h. We conclude that the Maugean skate appears to be an oxyconformer, with a decrease in the rate of O 2 consumption with increasing hypoxia. Increases in blood glucose and lactate at 20% O 2 suggest that skate are relying more on anaerobic metabolism to tolerate periods of very low oxygen.
Despite these metabolic shifts, there was no difference in HSP70 levels between groups, suggesting this short-term exposure did not elicit a cellular stress response. The metabolic state of the skate suggests that low oxygen stress for longer periods of time (i.e. 48 h) may not be tolerable and could potentially result in loss of habitat or shifts in their preferred habitat.
Given its endemic distribution and limited life-history information, it will be critical to understand its tolerance to environmental challenges to create robust conservation strategies. IntroductionHypoxia occurs naturally in many aquatic ecosystems including coastal waters with high nutrient run-off, or systems characterized by strong density-driven stratification and limited mixing (; ). Both climate change and anthropogenic influences can further impact the severity or duration of hypoxia through increasing eutrophication of coastal waters and decreases in the solubility of oxygen due to increasing water temperature. Indeed, the IPCC predicts that the frequency, severity and duration of hypoxic events will increase in the future.
Hypoxia is a serious threat to aquatic fauna and can limit viable habitat to hypoxia sensitive species. In fact, hypoxia can result in mass mortality or loss of biodiversity. For range-restricted species, hypoxic events pose a severe threat as avoidance of hypoxic water in a restricted geographical space may be limited. Understanding how individual aquatic animals cope with hypoxic stress can help with conservation efforts and predicting future ecological impacts (; ).A prime example of the impacts of anthropogenic induced hypoxia can be found in Macquarie Harbour, Tasmania; home to the endemic Maugean skate ( Zearaja maugeana).
Macquarie Harbour is 280 km 2 and is highly stratified; the surface layer is heavily tannined preventing light penetration much below the surface, is predominantly freshwater and exhibits seasonal thermal fluctuations. The middle layer is brackish with little thermal variation and reduced dissolved oxygen (DO), and the bottom layer is nearly marine with a salinity of 31 ppt, with little to no thermal variation, and in some areas, has limited oceanic input and minimal DO (0–20%). The saline stratification of Macquarie Harbour greatly reduces operating costs of salmonid aquaculture as the pathogen responsible for amoebic gill disease cannot survive in freshwater.
The Tasmanian salmonid aquaculture industry has been growing steadily with increasing interest in farming within Macquarie Harbour due to the aforementioned benefits. However, salmonid farming is well known to have impacts on surrounding water quality and can decrease DO in the water surrounding the sites due to increased nutrient loading (;; ).Little is known about the biology of the Maugean skate ( Zearaja maugeana), which is listed as endangered under the Threatened Species Protection Act (Tasmania), the Environmental Protection and Biodiversity Conservation Act (Commonwealth) and is listed on the International Union for the Conservation of Nature Red List of Threatened Species. It is endemic to only two remote estuaries in western Tasmania, Australia; Macquarie Harbour and Bathurst Harbour.
However, the status of the Bathurst Harbour population is currently uncertain, with no individuals recorded from the area for over 20 years , whilst the population in Macquarie Harbour is believed to be 3200 individuals. As a member of the Rajiformes order, the Maugean skate is expected to have a conservative life history, reaching sexual maturity at a late age (;;; ).
On the other hand, recent preliminary ageing data suggests that the Maugean skate may be short-lived for a rajiform and mature at a relatively young age. It has been shown to have an asynchronous, discontinuous reproductive cycle , however, fecundity has not yet been established. The relative lack of life-history data combined with its endemic distribution, low genetic diversity and limited diet provide challenging conditions for conservation of this species. In addition, throughout the last century Macquarie Harbour has seen increasing anthropogenic stress such as the damming of major tributaries for hydroelectricity, mining effluent, commercial/recreational fishing and salmonid aquaculture, which can potentially introduce several environmental stressors for this species. Based on acoustic telemetry, the preferred depth for the Maugean skate is between 5 and 15 m where ranges of DO are generally 30–80%, temperature from 12 to 15°C and salinity from 18 to 24 ppt.
Some individuals have, however, been detected to depths of at least 55 m suggesting some level of tolerance to the wide array of environmental conditions here, including severe hypoxia (. Animal collection and careTwelve Maugean Skate (average mass = 1.72 ± 0.27 kg; 8 female, 4 male) were captured at 10 m depth by gillnet in the Table Head and Swan Basin regions of Macquarie Harbour, Tasmania during November of 2014. Gillnets were standard monofilament “graballs” (50 m long by 33 mesh drop, 114 mm stretched mesh), the type commonly used by recreational fishers. Gillnets were set during the morning hours and retrieved within 2–3 h to ensure that the skate were in good condition. Upon capture, skate were measured (total length), sexed and tagged with PIT tags and then placed in a 250 L tank with water that had been pumped from 10 m depth to ensure water chemistry was consistent with the depth of capture for the majority of individuals. Within 2 h of capture the 12 skate were transported to an onshore laboratory and transferred to a 1200 L tank containing water pumped from 10 m depth from the area of capture. Oxygen saturation at this depth of the harbour is 55% O 2 and this was maintained in the holding tank and used as our control.
The temperature of the holding tank was not regulated, and fluctuated with the daily atmospheric temperature between 15 and 18°C for the duration of the experiment. Water circulation within the tank was maintained by a large pump on the bottom and oxygen levels were monitored using a HQ40d DO metre and LDO probe (HACH) and maintained at 55% by regulated injection of either nitrogen or oxygen. Skate were acclimated to these conditions for 24 h prior to experimentation. Skate were not fed for the duration of the experiment to remove the effect of the energetic cost associated with digestion (i.e. Specific dynamic action) during respirometry (see review by ).All procedures were undertaken with the approval of the University of Tasmania Animal Ethics Committee (permit A13468), scientific permits 13 125 and 14 139 issued under Section 14 of the Living Marine Resource Management Act 1995 and permits TFA 13 982, 14 019 and 14 253 issued under Regulation 4 of the Threatened Species Protection Regulations 2006 and Section 29 of the Nature Conservation Act 2002.
These permits covered the capture, possession and biological sampling of an endangered species, the deployment of research fishing gear and the deployment of moorings within the Macquarie Harbour, including the World Heritage Area. Experimental protocolAfter the 24-h acclimation period, half of the skate were transferred to a separate 1200 L experimental tank containing the same water as the control tank but maintained at an oxygen saturation of 20% O 2, representative of areas of low oxygen saturation in the harbour.
DO was maintained at 20% by monitoring O 2 content and bubbling in N 2 as required. Both control (55% O 2; three male, three female; average mass 1.63 ± 0.4 kg) and treatment (20% O 2; one male, five female; average mass 1.82 ± 0.4 kg) groups were held at their respective DO levels for a total of 48 hours. After the first 24 h, a 0.5–1 mL blood sample was taken from the caudal vasculature immediately posterior to the cloaca with a 22-gauge heparinized syringe prior to the start of metabolic measurements. This process took 1 min per animal. Each skate (fasted for 48 h) was then transferred to an individual respirometer to determine their metabolic rate across the full range of oxygen saturation (75–20%; see below). A second blood sample was taken at the completion of the respirometry tests and the skate were returned to their respective tanks and allowed to recover for 24 h. A third caudal blood sample was taken prior to skate being euthanized via cranial puncture.
Heart, liver, rectal gland, gill and white muscle (WM) tissue were dissected, weighed and frozen immediately in liquid nitrogen for later metabolite and enzymatic analysis. Haematological analysisHaematocrit, haemoglobin, glucose and lactate were measured in whole blood immediately after each blood sample was taken. Haematocrit was measured in duplicate in a SpinCrit Microhaematocrit centrifuge (SpinCrit). Haemoglobin was measured using HemoCue Hb210+ system in accordance with the manufacturer’s instructions and values were corrected according to for fish.
The mean cell haemoglobin concentration (MCHC), a measure of the mean haemoglobin within each red blood cell, was calculated by dividing the corrected haemoglobin values by the haematocrit values. Whole blood glucose and lactate were measured using hand held OneTouch Ultra2 glucometer (LifeScan, Milpitas, California) and a Lactate Pro™ (Arkray Global Business, Inc.), respectively. The remaining blood samples were spun at 13 000 rpm for 4 min to separate red blood cells and plasma. The plasma was separated into a clean microcentrifuge tube, the buffy coat removed from the remaining red blood cells and both fractions were stored at −80°C until further analysis. RespirometryRespirometry measurements were conducted in large flat plastic trays appropriate for the size of the skate to ensure they remained still. Each skate was transferred to an individual tray containing water from their respective tanks and a small pump to maintain water circulation, and allowed to rest for 1 h prior to the start of the measurements.
At the start of the trial, O 2 saturation was raised to 75% and the trays were then sealed with gas impermeable, translucent plastic sheets (previously tested for 24 h over a 0% O 2 tank with no oxygen transfer). Oxygen consumption was recorded using a Fibox O 2 probe (PreSens Fibox) throughout the duration of the trial.
The O 2 level was allowed to drop as the skate consumed the oxygen from 75 to 20% (1–2 h), which represents the typical range of oxygen saturation that the skate are found in. Below 20% DO, O 2 consumption was negligible, and the trials were concluded. The average slope of O 2 consumption from each 5% increment was used to calculate routine aerobic metabolic rate (ṀO 2; mgO 2kg −1h −1), considering background O 2 consumption rate (measured as oxygen depletion in the empty respirometer), the volume of the tray, skate mass, temperature and barometric pressure. The values for each skate at each DO increment were then averaged. Tissue assaysWe used heart, liver and WM tissue to assess the metabolic state of the skate.
We used tissue glycogen as an indicator of energy storage that could be used for anaerobic energy production, tissue lactate as an indicator of anaerobic metabolism , citrate synthase and lactate dehydrogenase as indicators of aerobic and anaerobic metabolism, respectively. We also measured heat shock protein HSP70 in all five tissues collected as a measure of hypoxia-induced cellular stress.Frozen tissues were powdered under liquid N 2 using a mortar and pestle and kept at −80°C until further analysis. For citrate synthase and lactate dehydrogenase, powdered tissue was homogenized in 20x w:v of ice cold homogenisation buffer (100 mM potassium phosphate, 5 mM EDTA and 0.1% Triton x-100 at pH 7.2). Tissue lactate and glycogen were isolated as described below. All assays were performed in triplicate at room temperature in 96 well clear bottom plates using a SpectraMax Plus 384 spectrophotometer (Molecular Devices, Sunnyvale, CA), and we collected data using Softmax Pro 4.7.1 software (Molecular Devices, Sunnyvale, CA).Citrate Synthase (CS).
CS was measured according to. The CS assay buffer contained 20 mM Tris (pH 8.0), 0.1 mM 5,5-dithiobis (2-nitrobenzoic acid) and 0.3 mM acetyl-CoA. The reaction was initiated by the addition of 0.5 mm oxaloacetate, and absorbance was measured for 5 min at 412 nm. Control samples were assayed without oxaloacetate to control for background hydrolase activity.Lactate Dehydrogenase (LDH). LDH was measured using a modified assay from. The assay buffer consisted of 50 mM TRIS-HCl, 2 mM sodium pyruvate and 0.15 mM NADH.
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Absorbance was measured at 340 nm for 3 min.Tissue Lactate. Tissue lactate was extracted and measured as in ). Powdered tissues were homogenized in 1:4 w:v 8% PCA containing 1 mM EDTA using a PowerGen 125 homogenizer (Fisher Scientific, Ottawa, Canada). Samples were centrifuged at 16 438 x g for 4 min at 4°C. The supernatant was neutralized with 2 mM KOH containing 0.4 mM imidazole. Samples were centrifuged again at 16 438 x g for 1 min at 4°C.
The final supernatant was used for lactate quantification relative to an L-lactic acid standard curve. The assay buffer contained 0.16 M glycine, 0.13 M hydrazine, 1.9 M NAD + and 10 U lactate dehydrogenase. The reaction was run to completion at room temperature (35 min) and absorbance was measured at 340 nm.Tissue Glycogen. Tissue glycogen was extracted and fully hydrolysed to glucose according to and the hydrolysates were kept at −80°C until further analysis.
Glucose was measured according to using a glucose standard curve. The assay media contained 250 mM imidazole, 5 mM MgSO 4, 10 mM ATP, 0.8 mM NADP +. G6PDH (25 μL of 10 U/mL) was added to the wells and incubated at room temperature for 10 min to eliminate any endogenous glucose-6-phosphate. The plate was read at 340 nm to establish background absorbance before hexokinase (25 μL of 10 U/mL) was added to each well and incubated for 25 min.
Absorbance was read again at 340 nm and glucose was calculated according to the standard curve.HSP70. All five tissues collected were ground in liquid nitrogen to a fine powder, and a 15x weight-to-volume ratio was used to dilute the samples in 1x homogenisation buffer (50 mM Tris Base, 70 mM SDS) with the addition of 1X Protease Inhibitor Cocktail (PIC004.1, Bioshop Canada). We used a PowerGen125 homogenizer at 50% power to subject the samples to 20 s bursts, and then spun solutions at 14 800 x g for 10 min. We assayed the supernatants for soluble protein concentration using the detergent-compatible DC assay against a BSA standard (Bio-Rad).
Protein extracts were prepared for electrophoresis based on equivalent total protein content (15 μg/well) using 1x sample buffer (Life Technologies) and 50 mM DTT, then heated 5 min at 70 °C. We separated proteins in a Bolt 4–12% Bis Tris SDS-PAGE gel (Life Technologies) with 200 V power for 34 min, then transferred to polyvinylidene difluoride (PVDF, Bio-Rad) membrane for 60 min at 30 V. Each gel had a three-point serial dilution of a reference fish to give a standard curve for comparison. We blocked membranes overnight at 4 °C in 5% w/v fat-free milk powder dissolved in TBS-T (Tris, 20 mM; NaCl, 137 mM; Tween-20, 0.1% v/v), then incubated in 1:10 000 rabbit anti-HSP70 antibody for 1 h (Agrisera AS05083A; recognizes both constitutive and inducible isoforms) and finally in 1:20 000 goat anti-rabbit IgG HRP conjugated antibody (Abcam ab6721) for 1 h.
Membranes were rinsed with TBS-T solution five times after each antibody incubation. Chemi-luminescent images were obtained using ECL Select reagent (GE Healthcare) and a VersaDoc™MP 400 System (Bio-Rad). Band densities for samples were determined against the standard curve using the ImageLab software (v 4.0, Bio-Rad).Protein. We measured tissue soluble protein using a DC Protein Assay kit (Bio-Rad) using bovine serum albumin as a standard. Statistical analysisWe used R (version 3.6.0, R Core Team 2019) statistical software for our analysis.
To determine whether acclimation to low DO had an impact on metabolic response to acute hypoxia, we compared two linear mixed models (R package lme4) of the relationship between metabolic rate and ambient DO, one with both ambient DO and acclimation treatment (hypoxic or normoxic; Model 1) and one with only ambient DO included as the predictor variable (Model 2). Individual ID was included as a random effect in both models to account for repeated metabolic rate measurements for each individual. Models were compared using the Akaike Information Criterion (AIC), the significance of parameters ( α = 0.05) of each of the models was estimated using the Wald Chi-squared test. Visual inspection of model residuals was used to ensure assumptions of homoscedasticity and normality of residuals were not violated.
Models were fitted in the R software environment for statistical computing (R Core Team, 2014). A repeated-measures two-way ANOVA and Holm-Sidak post-hoc tests were used to test for differences in haematological factors between treatments and over time. T-tests were used to test for differences in tissue enzymes, metabolites and HSP70 expression between the control and hypoxic groups. P ≤ 0.05 was considered to be statistically significant. HaematologyDespite differences in oxygen consumption, there was no significant difference in haemoglobin or haematocrit between groups or after 48 h in their respective treatment groups.
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However, the MCHC was significantly lower in the hypoxic group (; P = 0.005) at 24 and 48 h. Whole blood lactate, a measure of anaerobic activity, was not detectable in the control group at either 24 or 48 h and was significantly higher in the hypoxic group at both time points (; P = 0.003). There was a significant effect of time ( P = 0.0068), but not oxygen, on the whole blood glucose concentration.
Whole blood glucose declined over time in both control and hypoxic groups. MetabolitesWe measured tissue lactate and glycogen (as glucose) in the heart, WM and liver to further understand the metabolic effects of hypoxia. We found that the concentration of glycogen did not differ between treatments in the heart or liver, but was significantly lower in the WM of the hypoxic group (; P = 0.0232). In contrast, the concentration of lactate did not differ between treatments in the WM but was significantly higher in the heart ( P = 0.0026) and liver ( P = 0.0019) of the hypoxic group ( and F, respectively). Despite changes in blood and tissue lactate, tissue LDH did not change in any of the tested tissues between control and hypoxic skate.
We measured CS as a proxy for aerobic capacity and found that there was no change in the heart or muscle, but a significant increase in CS activity in the liver of the hypoxic group (; P = 0.0091). DiscussionConservation efforts for endangered range-restricted species require comprehensive knowledge of the biology of the animals and the environmental conditions in which they are found, particularly when their environment is subject to anthropogenic impact. For the Maugean skate, there has been a strong focus on describing the environmental conditions of Macquarie Harbour, which are characterized by low DO bottom waters , and how these conditions are degrading under anthropogenic pressures (;, ). Until relatively recently, there had been limited investigation into their biology.
However, the recent decline in DO in Macquarie harbour (; ) has prompted numerous research endeavours to understand the biology of the endangered Maugean skate, and the effects of environmental disturbances on the population (;;;, ). This study is the first to describe the metabolic characteristics of the Maugean skate and its physiological response to environmentally relevant hypoxia.The Maugean skate appears to be an oxyconformer ( sensu) as indicated by the linear decline in the rate of oxygen uptake in both control and hypoxic groups. Hypoxia tolerance in elasmobranchs ranges from species capable of surviving extremely low levels of DO (. We thank the Tasmanian Salmonid Growers Association and the three individual salmon aquaculture companies Tassal, Petuna and Huon Aquaculture for providing the Macquarie Harbour DO data. Institute for Marine and Antarctic Studies (IMAS), University of Tasmania staff and students Edward Forbes, David Moreno and Kay Weltz assisted with fieldwork.
David Moreno also produced. We thank Sustainable Timber Tasmania employees Theresa Weller, for approving our use of the Strahan facility as our laboratory, and Leigh Clark, for assisting us whilst on site. Funding to conduct the experiments was provided by the Winifred Violet Scott Charitable Trust, with laboratory analysis funded by the Natural Sciences and Engineering Research Council Discovery Program (SC; #06177).
Funding for the field component was provided by the Australian Government through the Fisheries Research and Development Corporation (Project 2013/008) and IMAS.
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