Study organisms and collections

The study was conducted from October through to December 2010 In the laboratory facilities and reefs around Lizard Island Research Station (14°38′S, 145°28′E) on the northern Great Barrier Reef, Australia. Settlement stage damselfish (family Pomacentridae) were collected from light traps that had been deployed overnight about 50 m from the reef edge. The study species, Pomacentrus amboinensis, is an abundant and very common damselfish species that settles on the reefs during the summer months after a pelagic larval phase of 15–23 days28. Light traps catch the fish at the end of their larval phase, as they are entering the reefs at night to settle, therefore ensuring fish are naïve to reef-based, bottom-dwelling predators. Within 6 hours of settlement P. amboinensis will metamorphose and lose the transparent colour typical of the pelagic larval stage and gain the bright yellow body coloration and conspicuous black dorsal eye spot representing the juvenile stage of this species29. The predator used as the stimulus was the dusky dottyback, Pseudochromis fuscus, which is one of the most abundant meso-predators on the shallow reefs throughout the Indo-Pacific30. This particular species is responsible for consuming a large amount of the newly settled and juvenile damselfish during the summer recruitment season31 and is found in areas where P. amboinensis settle. A herbivorous goby, Amblygobius phalanea, was used as an experimental control to test for the effect of exposing P. amboinensis to visual and chemical cues of any heterospecific fish32. This fish has a similar body shape and size to the predatory dottyback and is often found in areas of the reef were recruits settle. Both species were caught on the reefs surrounding Lizard using a dilute clove oil anaesthetic and a handnet. Research was conducted under James Cook University ethics approval A1593 and A1720.

Laboratory study and experimental design

Individual P. amboinensis were exposed to a combination of olfactory and visual cues of a predator (P. fuscus), a non-predator (A. phalanea) or a blank control (receiving no cue sources). The growth, development and behaviour of P. amboinensis were assessed over a 6 week period. Naïve prey fish that had been collected with light traps (were brought back to the laboratory and placed in 60 L flow-through tanks (density: 50fish/tank) over a period of 10 days and fed Artemia nauplii ad libitum 3 times per day (ensuring all fish used in the experiment had an analogous baseline body condition at the start of the experiment). All P. amboinensis individuals were then conditioned to recognize the sight and olfactory cues of P. fuscus by placing the predator inside a transparent plastic bag in their tank for 30 minutes, while simultaneously injecting previously collected odour cues of the predator and skin extract cues of P. amboinensis. This is a training procedure found to increase the probability of survival in the ambon damselfish33 and is necessary to make sure that prey can recognise the cues of the predator species. It also ensured that all fish had the same baseline predator experience before the commencement of the study. Individual prey then had their morphology and shape photographically recorded against a scale before being transferred into a series of specially-designed 18 L PVC predator–prey tanks (64.2 × 11.5 × 18 cm). The tanks had a 7.5 L main section (containing either a predator or a herbivore) and 6 individually isolated prey compartments (1.5 L: 10.7×13×18 cm). The main compartment was separated from each of the 6 prey compartments by transparent Perspex that contained a series of small holes. The fish in the six prey compartments were visually isolated from each other using grey PVC partitions. Water flowed from the main predator/herbivore compartment to each of the prey compartments and then out the side of each of the prey compartments. This arrangement ensured that the prey fish in each of the six compartments were also chemically isolated from one another (see supplementary material, Fig. S1). The bottom of both the predator/herbivore compartment and the prey compartment was covered by a 1.5 cm layer of sand and the predator/ herbivore section had one plastic tube (12×5 cm) placed in the centre to provide shelter. A small coral skeleton (Pocillopora sp. ~4×5×5 cm) was placed at the back of each prey compartment to provide a refuge. The tanks were situated outside to ensure that animals received all natural temporal cues and the water was supplied by a flow through system from the ocean so organisms were given all the same environmental cues as that of fish residing in the wild. This design ensured that the individual prey in each compartment received all the olfactory diet cues as well as visual cues from the main section, ensuring all prey could both smell and see either the predator or herbivore (n = 36 fish/treatment), but that the prey could not see or smell each other. The chemical and visual isolation allowed us to consider the fish in each compartment as independent samples. Prey were fed twice daily with a standardized amount of boosted (DHA Selco) Artemia sp. nauplii (5 ml with ~550 Artemia/ ml) while predators were fed two damselfish individuals morning and night, which is an accurate representations of what P. fuscus consume in their natural environment31 ensuring that the cue stimulus provided to P. amboinensis was realistic. Gobies were given a combination of dry fish food pellets (INVE Aquaculture Nutrition NRD pellets; containing no fish products) and small crustaceans. Predators and herbivores were replaced every weeks, ensuring that significant effects could not be attributed to individual predators/herbivores. In addition to this there was an experimental control were individual prey were placed in separate 1.5 L compartments (10.7×13×18 cm) that received no cue sources (n = 21). After 6 weeks individual P. amboinensis were removed from their compartments and photographed against a scale (10×10 mm) for morphological measurements. Shape and size of fish were analysed from digital photographs using the software Optimas 6.5. Five variables were measured: standard length, body depth, total area of ocellus, diameter of ocellus (black and white) and entire diameter of the visible eye.

Monitoring prey behaviour

One week after the commencement of the experiment, a mirror (80 × 40 cm) was suspended over each tank at 45° so that focal fish could be observed undisturbed from above. A wire grid (2×2 cm) was also placed on the top of each chamber so that movement and location of individuals could be accurately quantified as the number of times fish crossed a line on the grid. Water flow was stopped and individual P. amboinensis were fed Artemia sp. nauplii. One minute later the fish had their behaviour assessed for a 2 min period. The mirror and grid were then removed. This procedure was repeated after 5 weeks for all treatments. The behaviour of individual fish in each of the 7 experimental treatments was quantified by recording: total number of feeding strikes (successful or otherwise), activity (quantified as the number of times a fish crossed a line on the grid that had been suspended over the tank) and % time spent within shelter (defined as being inside the branches of the coral shelter).

Field survival

After being photographed prey fish from each treatment were transferred onto individual patch reefs in the field. Patch reefs (25×15×20 cm) were placed 2 meters away from the main reef and 3 metres apart and were made up of healthy Pocillopora damicornis colonies (a hard bushy coral), which is the preferred settlement site for P. amboinensis. Individual fish were transferred onto separate patch reefs and left to acclimate with a cage on top for 1 h, before having the cage removed (sample size ranged from 14–27 per treatment). Following the acclimation time, individual fish had their survival monitored twice a day (morning and afternoon) for 4 days after release by SCUBA divers12. Fish were assumed to be caught by a predator when missing from the patch reef. Cage controls that allowed fish to swim away found that there was no movement from patches, suggesting that when a fish was missing it was due to predation rather than migration.