Ethics statement

Research was carried out under approval of the University of Adelaide animal ethics committee (permit: S-2013-095) and according to the University’s animal ethics guidelines. Egg collections around the Gulf St. Vincent were carried out with permission of the South Australian Government Department of Primary Industry and Regions SA (permit: 9902595).

Study species and sample collection

The study species Heterodontus portusjacksoni (Meyer, 1793) is an ideal model species because it is robust to handling stress that could affect their physiology55. It is a medium-sized benthic oviparous shark endemic throughout the southern half of Australia56. It is known to aggregate in groups as juveniles, however, this is also influenced by habitat57. It breeds annually, between the months of September and November, laying a pair of eggs every 10–12 days over 2–3 month period27 and the incubation can last up to a year. H. portusjacksoni lays large eggs containing a single embryo with an average weight of 155.5 g56. A total of 98 eggs were collected from Gulf St. Vincent, South Australia, over two collection dates (7th and 28th June 2013) via snorkelling.

Egg and shark rearing

The collected eggs were held in a temperature-controlled laboratory until hatching. Developmental stages56 were determined for all collected eggs and showed that they were of similar stage (stage 14—at least 7.5 months). The eggs were placed in 40 L tanks containing natural filtered seawater which was partially exchanged every 2–3 days. The tanks were placed in water baths with temperatures maintained using heater chiller units (TR15 Aquarium chillers, TECO refrigeration technologies, Ravenna, Italy) and 300 W glass heaters. Pumps were connected to the chiller units which ensured an even temperature distribution throughout the water baths. The eggs were left to acclimatize over a period of seven days where temperature was steadily increased by 1 °C to the elevated temperature treatment. The eggs were kept in either control (~400 μatm) or elevated CO 2 (~1000 μatm)16,58 crossed with control (~16 °C) or elevated temperature (~19 °C) (Table S4). Eggs were evenly distributed over 4 tanks per treatment with a max density of 9 eggs per tank. Exposure time of the embryos varied from an average of 108 days for the elevated temperature treatment to 143 days for the lower temperature treatments (hatching rate was affected by temperature which affected embryonic exposure time).

Upon hatching the juvenile sharks were relocated to new tanks with the exact same treatment set-up as described above, again with 4 tanks per treatment. The sharks were placed into large tubs of 100 L or 150 L in volume. The number of sharks in each of the tanks ranged from 1–8 for the 150 L tanks and 1–4 for the 100 L tanks (differing numbers due to differences in hatching time and because at some point 33 sharks were removed for the subsequent mesocosm experiment). Sharks were kept in their respective treatments for at least 2 months. Water parameters (Table S4) were measured daily. Tanks received water changes every other day (minimum 40% of total volume). Sharks were fed ad libidum with thawed frozen prawns daily.

A PEGAS 4000 MF Gas Mixer (Columbus Instruments, Columbus, Ohio) was used to achieve different CO 2 concentrations in the seawater by bubbling the CO 2 enriched air directly into the tanks. The gas mixer was connected to a CO 2 tank and an air compressor. Temperature and pH NBS of each tank was measured daily using a pH and temperature meter (Mettler Toledo SevenGo™ SG2) calibrated with fresh buffers each day. Additionally, oxygen and salinity were also measured daily within the tanks. Total alkalinity of seawater was estimated by Gran titration (888 Titrando, Metrohm, Switzerland) from water samples taken weekly from each of the treatment tanks. Alkalinity standards were accurate within 1% of certified reference material from Dr A. Dickson (Scripps Institution of Oceanography; Langdon et al. 2000). Average seawater pCO 2 (Table S4) was calculated using CO2SYS with the constants of Mehbrach et al.59 refit by Dickson and Milero60. The variability in pCO 2 is higher than for pH because it was calculated using weekly measurements of total alkalinity, whereas pH was measured on a daily basis.

Hatching rate, feeding and growth measurements in the laboratory

The tanks holding the eggs were checked daily for new hatchlings. As soon as new hatchlings were observed, their weight and sex was recorded as well as a photo taken of each individual for future identification. The newly hatched shark was then placed into a new tank with the same CO 2 and temperature treatment as it experienced while still in the egg. The sharks were measured each week for changes in weight (±1 g) and also photographed to aid identification of individuals to track their growth for the duration of the experiment. Sharks were fed ad libidum with mussels and prawn meat during the first month after hatching and afterwards with prawn meat alone. Food consumption was recorded daily by comparing the difference in weight of food offered and food remaining after 30 minutes of feeding. Thirty minutes was selected as the end period because this was well beyond the time it took sharks to feed to satiation (usually ~10 min). Because multiple sharks were kept in a tank, food consumption was calculated at the level of tanks and divided by the number of sharks in the respective tank. This was deemed as a fair representation of individual shark consumption rates because leftover food in the tanks indicated they were all fully fed and competition for food resources was unlikely to take place because food was not limiting. Although Port Jackson sharks usually feed at night, our sharks were conditioned to feed during the day directly upon hatching and therefore we expect this to represent true demand of food intake.

Growth in mesocosm experiments

After the laboratory experiment, a subset of the sharks was relocated to a mesocosm setup in South Australia. Three sharks were placed in each of the 12 mesocosm tanks (2,000 L volume each) which were manipulated to mimic a shallow temperate reef habitat (n = 3 sharks for each control and treatment mesocosm, see Table S4). The mesocosms had the same crossed design of elevated CO 2 and temperature as the laboratory experiments with 3 replicate mesocosm per treatment (Table S4). Each mesocosm had the same biological set up which included 5 kelp plants (Ecklonia radiata) with an average weight of 250 g per plant, a single spiny rock lobster (Jasus edwardsii) of ~2 kg in weight, 1 crab (Ozius truncatus), 15 snails (Turbo undulatus), 6 urchins (Heliocidarcis erythrogramma) and amphipods (>1,000). The kelp, snails and urchins were replenished 3 times over the duration of the experiment (68 days) as needed. The snails, crab, lobster and urchins were too large for the sharks to consume and their primary food source was the amphipods that successfully populated and reproduced within the tanks. Turf algae started growing naturally and covered the major part of the substratum in the mesocosms. The mesocosms had a flow-through system using natural seawater filtered through a sand filter. Temperatures were manipulated using external heater/chiller units (TC60 Aquarium chillers, TECO refrigeration technologies, Ravenna, Italy). The same thermal mass flow meter/controller as in the laboratory experiments was used to achieve an elevated CO 2 concentration in the seawater of the mesocosm via bubbling of enriched air directly into the tanks and both temperature and pH were measured daily.

The sharks were measured individually for total weight and photographed (to aid with the identification and tracking of individual growth for the duration of the experiment) prior to placement in the mesocosms. Sharks were re-measured after 61 days and after 68 days at the end of the experiment. During the first two weeks of the experiment, the sharks in both high temperature treatments were fed 2 g of fresh prawn meat, whereas sharks in both ambient temperature treatments were fed 1 g of meat each. These were similar to the food intake quantities as measured in the laboratory prior to placement into the mesocosms. This served as an acclimation period during which the sharks could familiarize themselves with the natural prey items in the mesocosms. After 2 weeks the feeding was standardized to 1 g per shark for all treatments. Due to the lowered food provisioning and due to their continuing increase in growth, the sharks increased their reliance on foraging on natural prey in the mesocosms such as amphipods. Observations showed shark foraging in-between the turf algae (which occupied most of the substratum and vertical tank walls of the mesocosms). Biomass of amphipods was not enhanced in the control treatments compared to the elevated CO 2 /temperature treatments (single sampling event of total weight and numbers of all amphipods found on the kelp: Control = 0.06 g, n = 124, Temperature = 0.06 g, n = 56, CO 2 = 0.09 g, n = 86 and T × CO 2 = 0.05 g, n =114) and could therefore not have been responsible for the observed reductions in growth rates in the latter treatments. There were no large differences in growth between the three sharks in one tank (this was the same for all tanks and no shark was observed to compete while feeding individually).

Hunting behaviour in mesocosm experiments

After an average 36-day exposure (range: 35–38 days because sharks were introduced into the mesocosms over a 4 day interval) to the experimental treatments in the mesocosms, the effect of elevated CO 2 on shark prey hunting behaviour through olfaction was tested. Prior to the day of testing the sharks were not fed, although they were still able to obtain prey (amphipods) from the tanks. Nevertheless, the sharks showed high degree of motivation towards the food offered in the olfactory trial the next day. The olfaction tests consisted of placing two equally sized (33 × 23 × 5 cm) sand-filled trays within each of the mesocosms. One tray (i.e. the food tray) had a combination of prawn meat (4 equally sized pieces of approx. 1 g each) and 5 fresh cockles of equal size (still in their shell but opened), buried into the sand. The control tray contained no food but had 5 empty and cleaned out cockle shells buried in the sand to reduce any visual bias of the slightly exposed top ends of the shell in the food tray. Both trays were placed near each other (average distance of 5 cm between the trays) and the shark responses were recorded using a GoPro HD HERO3 video camera (white edition) for a period of 40 min. The recordings were then analysed to determine the length of time it took for each shark to locate the hidden food and to determine the number of sharks that responded to the introduction of the prey. The timer started counting from the moment the trays were lowered onto the bottom of the mesocosm until the time each shark found the hidden prey items in the tray and started retrieving them from the sand or until the end of the experiment (after 40 minutes). Although Port Jackson sharks are nocturnal feeders these sharks were accustomed since birth to being fed during the day and responded actively when food was offered. It was possible to distinguish individual sharks within each mesocosm due to the markings on their upper bodies between the eyes, first dorsal and pectoral fins. These areas showed the most variation in patterning between sharks and remained consistent from hatching (photos were taken weekly after hatching).

Statistical analysis