Bigeyes were collected by SCUBA divers using soft-meshed nets from a cave on the Leigh coastline (36°17′23.70“S, 174°49′11.97”E), north-east New Zealand, where a large number of bigeyes are known to take refuge during the day. These fish were transported to the nearby Leigh Marine Laboratory where they were held in a large, black polyethylene holding tank (circular 2800 L, 1.5 m in diameter) with flow-through ambient seawater. They were allowed 3 weeks to acclimate to their artificial “cave”, where they were monitored daily and fed 3 times a week. All experiments were carried out in accordance with the University of Auckland’s ethics committee approval number 001150.

Experimental setup

Individual experiments took place by transferring selected fish into a white polyethylene tank (circular 1500 L, 1.84 m in diameter) supplied with flow-through ambient seawater. A GoPro Hero3 (GoPro Inc.) camera was mounted 1 m above the centre of the tank, below which a Soundtrap 202 hydrophone (frequency response 20 Hz–60 kHz, Oceaninstruments Ltd) was submerged midway in the water column. Digitally recorded sounds were played from an MP3 player connected to a 222 W amplifier and delivered into the tank via a J9 speaker (Naval Undersea Warfare Center, Underwater Sound Reference Division, Newport, US) (projector 40 Hz to 20 kHz) suspended midway in the water column at the margin of the tank and projecting into the tank. All external light was eliminated from the experimental tank with illumination provided by a small number of dim red LED lights.

A recording was taken in the experimental tank while fish were absent and water flow was off and the minimum power level of ambient noise was measured at an average received level of 100 dB dB re 1 μPa, due to transferred noise from the nearby coast, water pumps and electrical equipment.

Sound files

Two pre-recorded sound files were used in this experiment: ambient reef sound and bigeye vocalisations. The ambient reef sound file consisted of a 10 minute sound clip taken from a recording of North Reef in the Cape Rodney to Okakari Point Marine Reserve nearby to where the fish for the experiment were collected. This recording did not contain any bigeye vocalisations and is representative of ambient sound experienced by these fish in their natural habitat. The 10 minute bigeye vocalisation file was composed of repeated 20 second segments (Fig. 6) of bigeye calls recorded during experiments on captive fish reported in18.

Figure 6 Waveform (a) and spectrogram (b) of 20 second sample from recording of bigeye vocalisations from captive fish and used in the vocalisation playback experiment. Full size image

Sound files were played at averaged received levels well above the tank’s ambient sound level. Average received levels were calculated from recordings taken at 12 points over a grid laid out within the tank, with the sound level adjusted via the output control on the amplifier. Ambient reef sound was played at average received levels in the tank of 125 dB, 130 dB and 135 dB re 1 μPa, while the bigeye vocalisation track was played at an average received level of 135 dB re 1 uPa.

Playback experiments

Four groups of 24 randomly selected fish were carefully transferred from the holding tank to the experimental tank and left for at least 24 hours. Experiments began at dusk the following day, at which time the water flow to the tank was turned off to reduce background noise. Each group of fish were exposed to only one received sound level per night and were only exposed to the three received ambient levels and vocalisation playback once, with at least 24 hours separating the start of each experiment to eliminate the effects of previous sound exposure.

Ambient Sound playback

Ambient reef sound was played at one of the three received levels for 10 minutes followed by 10 minutes of silence (sound on and sound off), which repeated seven times. The hydrophone constantly recorded all ambient, playback and biological sound while the GoPro took an overhead digital image of the tank every 60 seconds enabling the position of all individual fish to be identified.

Vocalisation playback

Following the same methods as the ambient sound playback, the four groups of fish were exposed to vocalisation playback at least 24 hours after the final ambient sound experiment.

Data analyses

Shoal area was determined as the percentage of the total two-dimensional area of the tank occupied by the shoal of fish and was taken as a measure of group cohesion. Digital images were analysed using ImageJ (http://imagej.nih.gov/ij/) in which the outer edges of the outer most members of the shoal were connected and the area enclosed within this margin was calculated as a percentage of total available tank area. This method was used by Domenici, et al.42 who found it to be as effective a measure of group cohesion as the total three-dimensional area, or volume, of the shoal. Average shoal area was calculated for 70 images taken during silent control periods and while playback sound was on.

Sound recordings from the tank during the experimental period were edited in Audacity using a low pass filter and then run through PAMGuard (www.pamguard.org), which was configured to count the number of bigeye calls. The number of calls was determined for each of the seven sound on and sound off periods and then a mean calculated for both the sound on and sound off periods. Bigeye calls recorded included a range of fused and unfused pop sounds. Radford, et al.18 defined unfused pops as those separated by at least 100 ms, but for maximum accuracy in PAMGuard this was set to 150 samples with a maximum click length of 1500 samples. For this reason, some fused calls were counted as single pops, but this approach was consistent across all sound recordings that were analysed.

For vocalisation playback experiments, the number of calls emitted from the speaker in each 10 minute segment (1717) was subtracted from the number of calls detected in the experimental recordings to determine the number of calls produced by the fish in the tank.

Statistical analysis

Paired t-tests were used to determine any differences between sound on and sound off periods for the shoal area and for the number of bigeye calls during both ambient sound and bigeye vocalisation playback experiments. Where non-normality of the data was detected a priori Wilcoxon signed rank test was performed. Data are presented as means ± S.E.