DOI: 10.5061/dryad.xsj3tx9f3
title: Differential learning by native versus invasive predators to avoid distasteful cleaning mutualists

description: 1. Cleaning symbioses on coral reefs are mutually beneficial interactions
 between two individuals, in which a ‘cleaner’ removes and eats parasites
 from the surface of a ‘client’ fish. A suite of behavioural and
 morphological traits of cleaners signal cooperation with co-evolved
 species, thus protecting the cleaner from being eaten by otherwise
 predatory clients. However, it is unclear whether cooperation between
 cleaners and predatory clients is innate or learned, and therefore whether
 an introduced predator might consume, cooperate with, or alter the
 behaviour of cleaners. 2. We explored the role of learning in cleaning
 symbioses by comparing the interactions of native cleaner fishes with both
 naïve and experienced, non-native and native fish predators. In so doing,
 we tested the vulnerability of the predominant cleaners on Atlantic coral
 reefs, cleaning gobies (Elacatinus spp.), to the recent introduction of a
 generalist predator, the Indo-Pacific red lionfish (Pterois volitans). 3.
 Naïve juveniles of both invasive (P. volitans) and native predators
 (Cephalopholis spp. groupers) initially attacked cleaning gobies and
 hyperventilated from a putative toxin on the gobies’ skin during
 laboratory experiments. After 1 to 5 such encounters, invasive lionfish
 often approached the cleaner closely, then turned away without striking.
 Consistent with learned avoidance, invasive lionfish rarely interacted
 with cleaning gobies in the wild, either antagonistically or
 cooperatively, and did not affect gobies’ abundance. Native predators
 showed little evidence of learning during early encounters; they
 repeatedly attacked the cleaner during laboratory experiments and
 hyperventilated less violently than did lionfish. However, consistent with
 learned cooperation, native predators rarely antagonised and were
 frequently cleaned by gobies in the wild. 4. We demonstrate that rapid,
 learned avoidance protects a distasteful cleaning mutualist from an
 invasive predator. The behavioural plasticity of this invader likely
 contributes to its success across its invaded range. Additionally, our
 results suggest that the cleaner’s chemical defence most likely evolved as
 a way to deter predation and reinforce cooperation with naïve individuals
 of native species.
description: Research Question 1: <i>How do invasive and native
 predators interact with cleaners?</i> 
 Field We explored how predators interact
 with two common cleaners on Atlantic coral reefs, the cleaner goby
 (<i>Elacatinus genie</i>, dominant cleaner on shallower reefs
 &lt;12 m depth, but exists 0-30 m: Colin 1975) and the sharknose goby
 (<i>E. evelynae</i>, dominant cleaner on deeper reefs
 &gt;12 m depth, but exists 0-50 m: Colin 1975).  In the Bahamas, we
 observed cleaning stations at natural patch reefs where both invasive
 lionfish and native predators were present.  A species was considered a
 potential predator of cleaning gobies if most of their diet is fish
 (Randall 1967).  From a distance of at least 3 m, in 10-minute intervals,
 and during daylight hours (07h00 to 20h00), SCUBA divers documented the
 nature (cleaning, predation, etc.) and duration of all interactions
 between cleaning gobies and other fishes that occurred at a focal cleaning
 station.  Cleaning behaviour was presumed when a cleaning goby made
 contact with another fish’s body for at least one second.  We conducted
 our observations on 4 coral patch reefs (depth: 6-20 m; surface area:
 200-1300 m<sup>2</sup>) in the northern Exuma Sound in the
 Bahamas (24°47'10"N, 76°19'33"W) during the summer of
 2013.  Observation time totaled 24 hr, 10 min, 13 sec, with
 observation-effort spread both across reefs (no less than 4.5 hr at a
 reef, with an average of 6.0 hr per reef) and within reefs (no fewer than
 10 cleaning stations per reef).  This resulted in no fewer than 37
 observations of cleaning at a reef (76.0±17.4, mean±SEM) at an average
 frequency of one cleaning interaction every 4.75 min. 
 Laboratory             We conducted a
 laboratory experiment to determine how invasive lionfish and two native
 groupers – graysby (<i>Cephalopholis cruentata</i>) and coney
 (<i>C. fulva</i>) – interact with the cleaner goby
 (<i>E. genie</i>), during the summer of 2011 at Lee Stocking
 Island, the Bahamas (23°46'00"N, 76°06'00"W) and
 Little Cayman, Cayman Islands (19°41'56"N, 80°3'38"W).
  Graysby and coney are common native mesopredators, and are similar to
 lionfish in size and diet (Morris & Akins, 2009; Randall, 1967).
  Divers used SCUBA and hand nets to capture juvenile (&lt;15 cm total
 length TL) lionfish, graysby, and coney (hereafter “predators”) on nearby
 reefs at 3-15 m depth.  Predators were held in indoor aquaria at least 72
 hours prior to their experimental trial and fed live mosquitofish
 (<i>Gambusia </i>sp.) once daily except for the 24 hours
 preceding each trial.             Divers used SCUBA and
 handnets to capture two goby species: the cleaner goby and the bridled
 goby (<i>Coryphopterus</i> <i>glaucofraenum</i>). 
 The bridled goby is a known common prey of both lionfish (Albins &
 Hixon, 2008) and native groupers (Randall, 1967) and was therefore an
 indicator of predator hunger during lab trials. 
             To account for order of exposure to prey, we randomly
 assigned predators to one of two groups – (1) bridled goby then cleaner
 goby, or (2) cleaner goby then bridled goby – such that each predator was
 offered exactly two gobies less than one-third their total length
 (lionfish consume prey up to one-half their own length in the wild: Morris
 & Akins 2009).  On the day of the trial, individual predators were
 placed in a transparent, 208-L indoor aquarium (122 x 33 x 51 cm) and
 allowed to acclimate for at least 10 minutes.  A trial began when we
 released the first goby into the aquarium with the predator.  We observed
 all subsequent behaviour of the predator for 20 minutes, deemed during
 preliminary trials as sufficient time for a predator to detect a goby of
 either species.  We made observations from a distance of 2 m for lionfish,
 and from behind a viewing blind for grouper (observer could see the
 grouper, but the grouper could not see the observer) because grouper
 seldom hunted when people were visible.  If the first goby was eaten, then
 we waited 10 minutes for digestion before placing the second goby into the
 aquarium with the same predator.  If the first goby was uneaten at the end
 of 20 minutes, then we removed it from the aquarium, and replaced it with
 the second goby.  The trial then followed the same protocol as described
 above.              There were no instances of
 cleaning, so all analyses focused on predation behaviour, for which we
 excluded all trials in which the predator did not strike at either goby
 (n=11 of 42 lionfish, 9 of 32 graysby, and 18 of 30 coney).  We then
 calculated the proportion of trials in which a predator ate a cleaner goby
 and compared this proportion among predator species using Fisher’s exact
 tests.  We also used binary logistic regression to determine whether
 predation on the cleaner goby was affected by eating the bridled goby, the
 order of exposure to the prey species, the total lengths of the predator
 and the cleaner goby, and the region (Bahamas or Cayman Islands) where the
 trial was conducted.  A unique regression model was created for each
 predator species in <i>R</i> v3.2.1 (R Core Team,
 2020).   Research Question
 2: <i>Does the interaction of invasive and native predators with
 cleaners change over time with repeated exposures?</i>
             To determine whether novel and native interactions
 change over short time scales, we repeatedly exposed individual lionfish
 and graysby to a cleaner goby (a different goby each trial) over a
 two-week period and monitored the predators’ behaviours for any changes in
 response to cleaner versus bridled gobies.  This work was done at
 laboratory facilities in southern Eleuthera in the Bahamas
 (24°49'53"N, 76°19'43"W) during the summers of 2013,
 2014, and 2015, and followed approximately the same protocol as described
 above (differences described below).  To minimise the likelihood of
 individual predators having prior experience with the cleaner goby and
 therefore maximise our ability to detect changes in the predators’
 behaviours, we captured predators from small, isolated patch reefs (³100 m
 from nearest reef) without cleaner gobies.            
 To determine if the cleaner goby is distasteful to predators, as has been
 previously suggested (Colin, 1975; Lettieri & Streelman, 2010), we
 quantified predators’ gill ventilation rates before and after goby
 consumption.  On the day of each trial, we allowed each predator to
 acclimate to the observation aquarium for at least 10 minutes, then
 determined their baseline gill ventilation rate by counting the number of
 times their gill opercula beat during a 10-second interval.  We then
 introduced the first goby to the tank and proceeded with the trial as
 described for the previous lab experiment.  Immediately after a predator
 ate a goby, we again determined the predator’s gill ventilation rate, and
 continued doing so every minute thereafter for no less than 3 minutes, and
 until the predator’s ventilation rate returned to its baseline level. 
 After completing a trial, we returned each predator to its holding
 aquarium where it was fasted until its next trial, approximately 48 hours
 later.  To allow for learning, we exposed individual predators to a
 cleaner goby no fewer than four times, collecting predators’ gill
 ventilation rates before and after goby consumption for each
 trial. We conducted
 a separate experiment at Lee Stocking Island in 2007, during which we
 repeatedly exposed juvenile lionfish (n=9) to a cleaner.  We used a
 similar method as previously described except that lionfish were offered a
 cleaner goby a total of 8 times at an interval of once every 2, 4, or 6
 days, with trials lasting 1 minute.             To
 assess predator learning, we first excluded from analyses all trials in
 which the predator did not strike at either goby species, and all
 individuals that ate during no more than one trial (n=0 of 29 lionfish and
 3 of 15 graysby).  We then divided the predators into two mutually
 exclusive groups: those that struck at a cleaner goby at least once, and
 those that did not (i.e., the predator struck at the bridled goby but
 never at a cleaner goby).  Of those predators that struck at a cleaner at
 least once, we calculated the proportion of individuals that developed an
 aversion to the cleaner (i.e., learned not to eat the goby).  A predator
 was considered to have developed an aversion if after striking at or
 eating a cleaner goby in an initial trial (1) it did not strike at a
 cleaner for 3 trials in a row, or (2) it approached a cleaner in hunting
 posture then turned away without striking, even if the lionfish was hungry
 (as demonstrated by eating a bridled goby during the same trial).  To test
 for learning, we used Cochran’s Q test (and when significant,
 <i>p</i> ≤ 0.05, pairwise McNemar’s tests with Bonferroni
 adjustments for multiple comparisons) to compare the proportions of each
 predator species that struck at a cleaner over time, beginning with the
 first trial in which a predator struck at a cleaner.
             Gill ventilation rates were quantified and compared
 in two ways: the number of gill opercular beats per minute upon consuming
 a goby, and the number of minutes after consuming a goby that it took for
 the predator’s gill ventilation rate to return to “normal,” defined as
 within 6 gill opercular beats/minute of the baseline level.  We used
 rank-sum tests to compare gill ventilation rates among predator
 species.   Research Question
 3: <i>Does the invasive predator affect wild populations of
 cleaners?</i>             To determine whether
 invasive lionfish affect densities of <i>Elacatinus </i>spp.
 cleaning gobies in the wild, we conducted a manipulative experiment on 8
 coral patch reefs in the northern Exuma Sound in the Bahamas (24°47’10”N,
 76°19’33”W) during the summer of 2013.  Reefs were surrounded by sand and
 seagrass and the nearest hard substrate was at least 80 m away.  We paired
 reefs by similarity in size (surface areas 200-1300
 m<sup>2</sup>), depth (6-20 m), vertical relief, and benthic
 community (coral percent cover) to create 4 experimental reef pairs.  We
 randomly assigned one reef in each pair to have periodic lionfish removals
 (“low-lionfish”; approaching 0 lionfish/m<sup>2</sup>), and
 the other reef to have periodic lionfish additions (“high-lionfish”; about
 0.04 lionfish/m<sup>2</sup>, similar to unmanipulated
 densities in the Bahamas, mean±SD: 0.039±0.014
 lionfish/m<sup>2</sup>: Green & Côté 2009).
             Before manipulating lionfish densities, we conducted
 full reef censuses of the two cleaning gobies present at these reefs: the
 cleaner goby (<i>E. genie</i>) and the sharknose goby
 (<i>E. evelynae</i>).  After baseline goby censuses were
 complete, we manipulated lionfish densities (low vs. high) for the next 10
 weeks.  We conducted full-reef censuses of cleaning gobies, lionfish,
 graysby, and coney 5-6 times over 10 weeks, at approximately 2-week
 intervals. At the end of the experiment, lionfish were removed from all
 experimental reefs.             We used a linear mixed
 effects model (LME) to assess the effect of lionfish on changes in
 cleaning goby density, with <i>lionfish treatment</i> (low-
 vs. high-lionfish densities) as a categorical fixed effect,
 <i>time</i> in days (day 0 was the time of baseline census) as
 a continuous fixed effect, the interaction between treatment and time as a
 fixed effect (<i>treatment*time</i>), and
 <i>reef</i> (8 reefs) as a random effect (with weighted terms
 to allow for variance among reefs) (Bolker et al., 2009).  We first fitted
 models with and without (1) random effects (<i>reef </i>and
 <i>reef pair</i>, 4 reef pairs), (2) weighted terms to allow
 variance to differ among reefs, and (3) AR1 structures to allow for
 temporal autocorrelation within reefs, using restricted maximum likelihood
 estimation (REML).  We chose the best-performing model per Akaike’s
 Information Criterion (AIC) and p-values from likelihood ratio tests
 (LRTs) (see Table S1 in Supporting Information).  Residuals from the final
 model (full fixed effects + random effect of <i>reef +
 </i>weighted variance among reefs) indicated that all assumptions
 were met.  We conducted our analyses using the statistical software
 <i>R</i> v3.2.1 (R Core Team, 2020) with the associated
 packages <i>nlme</i> v3.1-118 (Pinheiro et al., 2016) and
 <i>MASS</i> v7.3-35 (Venables & Ripley,
 2002).   <i>Ethics and
 Permits</i> Oregon State University's
 Institutional Animal Care and Use Committee (IACUC; ACUP 3886), the
 Department of Marine Resources of the Bahamas, and the Marine Conservation
 Board of the Cayman Islands Department of the Environment all approved our
 work.  
 <i>Methods References</i>
 Albins, M. A., & Hixon, M. A. (2008).
 Invasive Indo-Pacific lionfish <i>Pterois volitans</i> reduce
 recruitment of Atlantic coral-reef fishes. <i>Marine Ecology
 Progress Series</i>, <i>367</i>, 233–238.
 https://doi.org/10.3354/meps07620 Bolker,
 B. M., Brooks, M. E., Clark, C. J., Geange, S. W., Poulsen, J. R.,
 Stevens, M. H. H., & White, J.-S. S. (2009). Generalized linear
 mixed models: a practical guide for ecology and evolution. <i>Trends
 in Ecology & Evolution</i>, <i>24</i>(3),
 127–135. Colin, P. (1975). <i>The
 Neon Gobies</i>. Neptune City, NJ: T.F.H. Publications,
 Inc. Green, S. J., &
 Côté, I. M. (2009). Record densities of Indo-Pacific lionfish on Bahamian
 coral reefs. <i>Coral Reefs</i>, <i>28</i>(1),
 107–107. https://doi.org/10.1007/s00338-008-0446-8
 Lettieri, L. B., & Streelman, J. T. (2010).
 Colourful stripes send mixed messages to safe and risky partners in a
 diffuse cleaning mutualism. <i>Journal of Evolutionary
 Biology</i>, <i>23</i>(11), 2289–2299.
 https://doi.org/10.1111/j.1420-9101.2010.02098.x
 Morris, J. A., & Akins, J. L. (2009). Feeding
 ecology of invasive lionfish (<i>Pterois volitans</i>) in the
 Bahamian archipelago. <i>Environmental Biology of Fishes</i>,
 <i>86</i>(3), 389–398.
 https://doi.org/10.1007/s10641-009-9538-8
 Pinheiro, J., Bates, D., DebRoy, S., &
 Sarkar, D. (2016). <i>nlme: Linear and Nonlinear Mixed Effects
 Models. R package version 3.1-128</i>. Retrieved from
 http://cran.r-project.org/package=nlme
 R Core Team. (2020). <i>R: A language and
 environment for statistical computing</i>. Retrieved from
 http://www.r-project.org
 Randall, J. E. (1967). Food Habits of Reef Fishes of
 the West Indies. <i>Studies in Tropical Oceanography</i>,
 <i>5</i>, 665–847. 
 Venables, W. N., & Ripley, B. D. (2002).
 <i>Modern Applied Statistics with S.</i> (4th ed.). New York,
 New York: Springer.
description: Please see README.md for a description of the files associated
 with these data and analyses.