Study sites

This study was approved by Simon Fraser University, Canada. All procedures performed in accordance with the legal requirements for animal research in Canada, and were conducted under a permit (#1124B-14) issued by Simon Fraser University Animal Care Committee. The study was conducted at nine lagoonal coral reef sites on the north shore of Moorea, French Polynesia. The sites varied in maximum depth (1.5–8 m) and topography, and were chosen because all allowed for safe, repeated passes of a motorboat near the observation sites.

Behavioural observations

Habituation to diver presence and noise

We first conducted a pilot study over 2 days to examine potential habituation by fish engaged in cleaner–client interactions to the presence and noise of a SCUBA diving observer. Four observers watched a total of 12 juvenile cleaner wrasses at one site, which was not part of the main study. We studied juveniles for two reasons: (1) at this location, they are more active cleaners than adults (IMC, SCM, personal observations) and (2) their cleaning behaviour is not yet influenced by sexual motivation, making it more consistent across individuals. Each cleaner was observed, in the absence of any motorboat noise, by a motionless diver hovering or kneeling on the sand 2–3 m (horizontal distance) away for three successive 20-min periods, which mirrors the experimental treatment (see below). During each 20-min period, we recorded the species identity of each visiting client and duration of each inspection (i.e., when the cleaner surveys the body surface, gills or buccal cavity of the fish client).

Effects of motorboat noise on cleaner–client interactions

Following the pilot study, we searched for juvenile cleaner wrasses at eight additional sites, and each diver observed the first cleaner he/she encountered. All observations lasted 60 min, divided into three 20-min periods: (1) a pre-exposure, control period (no motorboat noise); (2) an exposure period (motorboat noise present); and (3) a post-exposure period (no motorboat noise). At the end of the pre-exposure period, all diving observers released a tethered surface marker buoy to signal the start of the exposure period. A research assistant drove (estimated speed: 4–10 knots) one of two small outboard motorboats (boat with 25 hp engine chosen randomly on each day) multiple times past the marker buoys, no closer than 10 m from the buoy (for diver safety) and no further than 100 m. This treatment was expected to generate noise at a broad range of frequencies including 1–3000 kHz, which is audible to most fish species52, at levels of 80–150 dB re 1 μPa (RMS, full spectra), which have been shown to affect behaviour in reef fish (e.g. ref. 6). We recorded the experimental motorboat noise underwater to measure the actual range of frequencies and levels generated around a cleaner (see below).

During each of the three 20-min periods, we recorded the number and species of fish visiting the focal cleaner and the duration of each inspection. We also noted whether the interaction was initiated by the client by adopting a stereotyped incitation posture, which increases the likelihood of being inspected35, whether clients jolted during the interaction, and whether clients chased the cleaner. We moved to a new site after each observation to ensure that cleanerfish were only observed once. Because fish were not marked, a few individual clients might have contributed to the clientele of more than one cleanerfish; however, this is unlikely to have occurred often given the high density of fish at the study sites. Other boats were not observed or heard during observations.

Motorboat noise recording and analysis

Acoustic recordings

To evaluate and compare the range of frequencies and sound levels generated by the diving observer and the passing motorboat during our experiment, we deployed recording equipment ~50 cm from one juvenile cleaner wrasse (which was not observed in the main study) at 4 m depth and a diver with a surface marker, posing as an observer, ~2 m away (horizontal distance). One of two field assistants then drove one of the two motorboats used in our experiment repeatedly near the diver, in an accurate simulation of our experimental treatment. Since some fish can detect pressure and all fish detect particle motion, we recorded both sound pressure and particle acceleration before and during multiple motorboat passes conducted in a 5-min period. Sound pressure was recorded using an omnidirectional hydrophone (HiTech HTI-96-MIN with inbuilt preamplifier; sensitivity -165 dB re 1 V/μPa; frequency range 2 Hz–30 kHz; High Tech Inc., Gulfport MS), and particle acceleration using an accelerometer (M20L, sensitivity 0–3 kHz, manufactured and calibrated by GeoSpectrum Technologies, Dartmouth, Canada). Recordings were made on a laptop via a USB soundcard (MAYA44, ESI Audiotechnik GmbH, Leonberg, Germany). The USB sound card was fully calibrated using pure sine wave signals generated in SAS Lab (Avisoft, Germany), played on a MP3 player, measured in line with an oscilloscope.

Acoustic analysis

Acoustic recordings were analysed in MATLAB v2010a. Fast-Fourier transforms were used to transform time domain recordings into the frequency domain before power spectral density was calculated, to allow comparison of sound levels for each treatment across the frequency range 0–3 kHz. Sound levels are only considered up to 3 kHz due to the upper limit of the sensitivity of the accelerometer. This frequency range is likely to cover the hearing range of most fishes52.

Statistical analysis

For our initial study of habituation to diver presence, we compared one client-focused measure, i.e. the number of client visits, and one cleaner-focused measure, i.e. total inspection duration (obtained by summing the durations of all inspection events for each cleaner), across the three consecutive 20-min observation periods. These measures did not meet the assumptions of parametric testing, even after transformation; therefore, we used non-parametric Wilcoxon’s signed-rank tests with continuity correction for pairwise comparisons of 20-min periods to account for the repeated-measures nature of our observations.

To examine the effect of motorboat noise, we compared seven behavioural metrics of cleaner–client interactions between the 20-min pre-exposure, motorboat-noise exposure and post-exposure periods. The measures were: (1) the number of client visits; (2) total inspection duration (calculated as described above); (3) the proportion of interactions initiated by clients; (4) inspection duration per client; (5) the proportion of jolting clients; (6) the number of jolts per client; and (7) the proportion of jolting clients that chased the cleaner in retaliation. None of these measures met the assumptions of parametric testing, even after transformation, so we used non-parametric Wilcoxon’s signed-rank tests with continuity correction for pairwise comparisons of 20-min periods to account for the repeated-measures nature of our observations. On three occasions where cleaners could not be found by divers after the motorboat exposure, we could only compare the pre- and during exposure periods.

To examine differences in cleaner clientele over time in both the habituation study and the motorboat-noise experiment, we used a permutation-based, non-parametric multivariate analysis of similarity (ANOSIM) using the software PRIMER (Plymouth Routines in Multivariate Ecological Research v. 6.1.13; PRIMER-E Ltd, Plymouth Marine Laboratory, Plymouth, UK)53. We created a visit frequency matrix (client species by cleaner/time period), square-root-transformed the data to reduce the influence of very abundant client species, and computed Bray-Curtis similarity coefficients between pairs of cleaners/time periods54. The ANOSIM procedure was carried out on the similarity matrix. ANOSIM generates an R statistic, which varies between 0 (similarities within and between samples are the same) and 1 (all samples within groups are more similar to each other than to any sample across groups) and is tested for difference from zero with a permutation test (in this study, N = 999 permutations). In the habituation study, we used a one-way ANOSIM to compare clientele among the three consecutive 20-min observation periods; in the motorboat-noise experiment, we used a two-way ANOSIM with motorboat exposure (three groups: before, during and after) and site (eight levels) as main factors. We visualized the differences in client assemblages among time periods with non-metric multidimensional scaling (MDS) plots, in which samples that are more similar in community composition appear closer together than more dissimilar samples. The extent of spatial distortion needed to represent community composition differences in two dimensions is estimated by MDS stress values. Stress values of <0.1 suggest that distances among samples in an MDS plot accurately reflect the extent of community differences54.

Ethics

This study was approved by Simon Fraser University, Canada. All procedures performed in accordance with the legal requirements for animal research in Canada, and were conducted under a permit (#1124B-14) issued by Simon Fraser University Animal Care Committee.

Data accessibility

Data are available as supplementary material.