Experimental animals

All research presented here was conducted under permission of the Research Ethics Committee: Animal Care and Use of Stellenbosch University (SU-ACUM14-00006) and is in accordance with the relevant guidelines and regulations. Specimens (80) of the Puffadder shyshark H. edwardsii were caught in the harbour basin of the False Bay Yacht Club in Simons Town, South Africa (34.07°S, 18.33°E) in austral spring. Sharks were caught by SCUBA divers by hand after setting out bait (sardines) in perforated 5 l plastic bottles. Caught sharks were collected in nets until they were transferred into an 800 l tank on a car trailer. Water in the tank was continuously provided with oxygen (technical) from a cylinder. After capture, sharks were transported within an hour to holding tanks at the Research Aquarium of the Department of Agriculture, Forestry and Fisheries (DAFF) in Cape Town.

In Cape Town, they were weighed and maintained in round flow-through holding tanks (4500 l) for four months prior to experimentation (pH ranged from 7.9 to 8.1; T A from 8.4 to 16.8 °C). They were fed rations of 5% of average body mass with pieces of squid once a week. Sharks were not fed in the week of experimentation.

Experimental procedures

For acute exposure, 66 larger sharks (179 ± 52 g, 67% male) were acclimatized for 48 h prior to experimentation in smaller round tanks (∅ = 1.2 m, h = 1 m, 1130 l). Tanks were well mixed by propellers and aerated by compressed air. At the start of the trial, individuals were distributed between two replicate control- (normocapnic) or hypercapnic tanks (141 l rectangular glass tanks) with corresponding pH levels of ~7.3 and ~8.0, respectively. Weight of animals did not differ significantly between replicate groups (ANOVA, p = 0.993). For blood sampling, individuals were removed from tanks at the respective time points of incubation, i.e. after 1.5, 3, 6 and 24 hours, alternating between replicate tanks. Each time point represents samples from both replicates of the same treatment (3 per replicate for hypercapnic treatment, 2 or 3 for normocapnic treatment. For the starting value (time 0), blood was collected from sharks from the same batch. These sharks were not incubated thereafter. To test recovery, some sharks were transferred after 24 h of exposure into tanks with normocapnic conditions and sampled after a further 8 h (i.e. a total of 32 h from the start of the trial). After sampling, sharks were transferred back into the acclimation tank and not used further. Blood was sampled (see below) only once from each shark. Analyses were carried out in the statistical software environment R (Ver. 3.0.1.), including the nlme package (Ver. 3.0.1.)33. Differences between parameters were tested for each interval against the base value (0 h) for the normocapnic (control) group and the hypercapnic (treatment) by means of ANOVA. Response variables were modelled as a function of the interaction between sampling time and treatment with linear-mixed effects models. Tank was initially included as a random effect, but found to increase the AIC value when tested against a fixed-effects model and consequently dropped. Filtered seawater for the system was provided by the main water storage tank of the research aquarium. It supplied each replicate via a header (mixing) tank. In two of the header tanks, pH was adjusted by its own CO 2 supply for hypercapnic treatment. This was accomplished by using a pH controller (7074/2, TUNZE, Germany) containing a solenoid valve (7074.111) and a pH electrode (7070.110) attached to a 9 kg CO 2 bottle (technical). A level of pH of 7.3 was selected as this level is predicted by the year 23007. The experiment was carried out in a room with stable ambient air temperature (ranging from 16–18 °C) so that additional control of seawater temperature was unnecessary. During the acute experiment, seawater conditions were tested five times in each replicate tank (summarised in Table 1) and did not differ significantly between replicates of each treatment (pH, T; ANOVA).

Table 1 Physicochemical seawater parameters recorded during acclimation, normocapnia, hypercapnia and recovery in acute* and chronic* experimental treatments of adult H. edwardsii. Full size table

Subsequently, the remaining 13 smaller sharks were taken from the holding tank (see above) for chronic exposure. They were weighed (w) total length (L) measured and tagged (barbed dart tags (D-tag; 89 mm, Ø 1.4 mm; Hallprint Pty Ltd, South Australia) left of the first dorsal fin (121.4 ± 34.2 g, 85% male, see Results for more details). Thereafter they were transferred into normocapnic- or hypercapnic replicate tanks as described above, except that rectangular 1000 l plastic tanks were used here. All tanks were well mixed and aerated. ANOVA revealed no difference in w and L between the four replicate groups (p = 0.993; 0.914). In the two weeks prior to chronic exposure, seawater temperature in the holding tanks was in the range of 15.7–16.3 °C. Sharks of both treatments were acclimatized for a week to around 18 °C after which the pH for hypercapnic treatment was lowered in two steps in five days from approximately 8.1 to 7.3, using a pH control system as described above. The experimental pH of 7.3 was selected as this level is predicted to be reached by the year 23007. It will possibly be reached earlier in the BCLME as it is close to values already attained over short periods during severe upwelling periods and after decay of algal blooms10. Seawater temperature was allowed to fluctuate with the incoming seawater. Similar seawater temperatures were recorded in all replicate tanks throughout experimentation. Seawater parameters were measured daily with the exception of A T which was measured thrice a week (summarised in Table 1) and did not differ significantly between replicates of the same treatment (pH, T; ANOVA). Sharks remained under experimental conditions for 63 days (~9 weeks). To record growth and to adjust the food rations, sharks were re-weighed and measured after 4, 6 and 9 weeks. Paired t-tests were carried out to test for significant changes in length and mass within treatments. Differences at the end of incubation (blood parameters, elemental composition and physical damage of denticles) were modelled with linear-mixed effects models that included treatment as fixed and Tank as random effect. Equally to the acute experiment, Tank was found to increase the AIC value when tested against a fixed-effects model and consequently dropped (R Ver. 3.0.1.)33.

Seawater pCO 2 , [CO 3 2−] and [HCO 3 −] were calculated using measured pH, salinity, ambient temperature (T A ) and total alkalinity (A T )34 as constants in CO2SYS software35. Oxygen concentration was determined using a Multi 350i meter set (WTW, Germany). Water quality was monitored by measuring NH 3 concentration (Ammonia test kit, Sera, Germany) and never exceeded 0.09 mg l−1.

Sampling

At the given intervals during the acute experiment and at termination of the chronic experiment, sharks were removed from their tanks, heads (eyes) were covered by a seawater-soaked cloth to reduce stress and prevent curling of the tail and animals placed upside down on a seawater-soaked cloth. In addition, head and tail were held tight by hand to avoid movement. Approximately 1 ml blood was immediately withdrawn from the caudal vein by syringe with a hypodermic needle (Neomedic 1 ml, 26 G) into a 2 ml syringe treated with heparin before the animals were carefully returned to the tank. All animals from the chronic experiment were subsequently sacrificed using ethylene glycol monophenyl ether (C 8 H 10 C 2 , 0.8 ml l−1). Skin samples were then taken dorso-laterally next to the first dorsal fin and frozen at −20 °C for electron-microscopic- and elemental analysis.

Analysis of denticles

Micrographs of shark skin areas and denticles were obtained by scanning electron microscopy (SEM) using a Leo 1430VP (Zeiss, Germany) of gold-platinum-sputtered samples whereas elemental composition of the outer denticle surface was analysed using energy-dispersive X-ray spectroscopy (EDX) with an ESEM Quanta 400 FEG instrument (Thermo Scientific, USA) after sputtering with gold and palladium (80:20)36,37. On the resulting SEM micrographs, ratios of damaged and intact denticles were quantified by counting.

Blood acid-base balance

The blood pH was measured within 20 s after sampling using an Orion 3 star pH meter equipped with an Orion 8220 BNWP micro pH electrode (Thermo Scientific, USA). Calibration was performed with NBS precision buffers (Applichem, Germany) at the same temperature as that of ambient seawater of the experimental tanks. A blood subsample (50 µl) was immediately injected into a de-gassing (magnetic stirrer) chamber containing 200 µl of 100 mM H 2 SO 4 and liberated total CO 2 (cCO 2 ) determined as described previously38. From measured pH and cCO 2 values, pCO 2 , and [HCO 3 −] were calculated using derivatives of the Henderson Hasselbalch equation (I and II). The required solubility coefficient αCO 2 and dissociation constant pK’ 1 of carbonic acid were obtained from Boutilier et al.39 for catsharks (Scyliorhinus canicula and S. stellaris).

$$pC{O}_{2}=\frac{cC{O}_{2}}{{10}^{pH-p{K^{\prime} }^{1}}\times \alpha C{O}_{2}+\alpha C{O}_{2}}$$ (1)

$$HC{O}_{3}^{-}=cC{O}_{2}-\alpha C{O}_{2}\times pC{O}_{2}$$ (2)

Ca2+ and Mg2+ concentrations were determined spectrophotometrically by commercial kits (Diaglobal, Germany) in undiluted small subsamples.

Haematocrit

Subsamples of 500 μl blood were immediately transferred into an EDTA pre-treated K2E reaction vessel (BD Microtainer, USA) for measurement of haematocrit. The vessels were closed and the samples shaken to ensure mixing of EDTA. Thereafter, 80 iu/ml sodium-heparinised micro haematocrit capillaries (Marienfeld, Germany) were completely filled, sealed with plasticine and subsequently spun at room temperature for 5 min in a Haematospin 1300 centrifuge (Lasec, SA). Haematocrit was subsequently quantified using a Micro Haematocrit reader (Hawksley, UK).