Species selectivity of clients by the neon goby cleaner fish, Gobiosoma oceanops

Candidate: Mico Tatalovic Project Supervisor: Dr. Theresa Burt de Perera

Species selectivity of clients by the neon goby cleaner fish, Gobiosoma oceanops

Submitted in partial fulfilment of the requirements for a BA in Biological Science at the University of Oxford, 2006

ABSTRACT The neon goby (Gobiosoma oceanops J.) is an obligate marine cleaner fish that lives in the Caribbean. The gobies clean a number of client fish of various species that have striking colour patterns. In the first part of the study I investigated whether these patterns influenced the neon gobies’ decision to inspect, by presenting them with a choice of two wooden model fish painted with different colour patterns. These were: lateral stripe, no stripe; yellow blotch, no yellow blotch; black outlines, no outlines, enlarged black outlines; natural sized eye, enlarged eye. I then recorded the amount of time that a cleaner fish spent with each wooden model. The results indicated that neon gobies had no preference for any of the colour patterns tested.

This could be due to the gobies having more complex perception mechanisms than simple recognition of colour patterns visible to humans, or due to the stress related to unnatural conditions the gobies encountered in the aquaria.

In the second part of the study, I observed neon gobies in s itu on the coral reef patch in Honduras by snorkelling, at two different times of day: in the morning and the afternoon to examine daily variation in the composition of the neon gobies’ clientele. These observations suggested that fewer species posed and got inspected in the morning than the afternoon. Since parasites are most abundant on clients in the morning it is possible that proximate and ultimate causes of clients’ visits to cleaning station may differ, affecting cleaners’ choice.

Word count: 6850

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CONTENTS

1. INTRODUCTION………………………………………………………………......1 -1.1 Cleaning symbioses...…………………………………………...………..1 -1.2 Aims and hypotheses……………………………………………...….......4 2. MATERIALS............................................................................................................6 -2.1 Wooden models………………………..………….............……………..6 -2.2 Neon gobies……………………..…………………………………….....6 -2.3 Aquaria and other laboratory equipment…………..………………….....7 -2.4 Snorkelling equipment……..………………………………………….....8 3. METHODS................................................................................................................9 -3.1 Field site…………...…………………………………………………......9 -3.2 Aquaria experiments…………………………………………………......9 -3.3 Snorkelling observations…………………….………………………….10 -3.4 Analysis of data…………………………………………………………11 4. RESULTS -4.1 Results from the aquaria experiments………………………..………....13 -4.2 Results from the in situ observations……………..………….………....22 5. DISSCUSSION........................................................................................................26 -5.1 Experiments..............................................................................................26 -5.2 Observations.............................................................................................30 6. SUMMARY.............................................................................................................36 7. ACKNOWLEDGEMENTS……………………………………………………….37 8. REFERENCES.........................................................................................................38 9. APPENDICES -Gantt chart 1 -Gantt chart 2 -Project management -Safety form, parts 1-3 -Certificate for passing the Coral Reef Ecology course -Minitab output

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1. INTRODUCTION 1.1 Cleaning symbioses Cleaning symbioses occur in a wide range of animal taxa. Examples include birds cleaning various reptiles (e.g. Geospiza finch species), deer (Aphelocoma jay species) or ungulates (Buphagus oxpecker species); crabs cleaning turtles (Planes crab species); shrimps cleaning fish (Periclimenes species); and fish cleaning fish and turtles (e.g. Thalassoma species) (Poulin and Grutter, 1996). However, most cleaning symbioses described so far occur in aquatic environments, both freshwater and marine. In marine environments, cleaning symbioses are found both in temperate and tropical seas, but research so far has mainly focused on the coral reef species.

On coral reefs, cleaning stations are usually positioned in prominent places (e.g. coral heads), occupied by one or more cleaners of one or more species, and are relatively permanent features: up to several generations of cleaners may choose the same cleaning station as their habitat. Cleaners may be obligate or facultative, depending on species. So for example Gobiosoma oceanops J. is an obligate cleaner throughout its life, feeding mainly on clients’ ectoparasites and mucus whereas facultative cleaners tend to be juveniles of species such as french angelfish (Pomacanthus paru B.) or spanish hogfish (Bodianus rufus L.) that feed on other items as well and cease to be cleaners as adults (Deloach & Humann 2003). Cleaners advertise their availability to clients by positioning themselves in a visible place at the station. Clients visit the stations and often pose to initiate the inspection by the cleaner. These inspection bouts are usually several seconds long and the cleaner may remove ectoparasites (mainly gnathiid isopods (Darcy et al. 1974, Deloach & Humann 2003)) from the client’s body surface, whilst the client remains calm during the interaction although it may occasionally jolt (Bshary, 2001). Jolts may indicate 1

instances of the cleaner biting the client’s tissues rather than removing the ectoparasites (Bshary, 2001) or may be the client’s signal that it is about to leave the cleaning station (Trivers, 1971). The clients’ poses are species specific but often involve the fish hovering vertically with its head pointing up or down (Losey, 1971).

Cleaners exhibit dancing movements prior to and during the inspection and these also may involve tactile stimulation of the client; the tactile stimulation may act as a risk management strategy towards piscivorous clients by the cleaners (Grutter, 2004).

However, there are some fish that do not pose, yet the cleaners attempt to swim towards them and inspect them (personal observation) and also those that do pose but cleaners do not clean them (Arnal, 2000). The reasons for these behaviours are still unknown but it seems that cleaners are using signals other than posing to choose some clients and that posing frequency may depend on how attractive a client is to a cleaner based on these other signals (Sheridan, 2002).

Cleaning symbioses have been viewed as mutualistic selfless cooperations (Poulin, R. & Grutter, 1996), as an example of reciprocal altruism (Trivers, 1971) and more recently as behaviour parasitism (Poulin, R. & Grutter, 1996). Removal of the ectoparasites is central to the first two views whereas the third view concentrates on client mucus ingestion by the cleaners. The most recent theoretical background to understanding cleaning symbioses has been the biological market theory where goods are seen as being exchanged between the cleaner and the client (Bshary 2001; Bshary & Schäffer, 2002; Grutter 2004). In the light of this theory cleaning interactions provide opportunity for both cleaners and clients to cheat. Cleaners can cheat by ingesting mucus and taking bites out of clients whereas clients can cheat by eating the cleaner if the client is a piscivore. Clients can also punish cleaners for not cooperating by chasing them (Bshary & Grutter 2002a), which could be energetically costly for

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both parties. Cleaners, on the other hand, may apply pre-conflict management strategies to avoid being eaten (Grutter, 2004). Although the piscivorous clients may eat the cleaners they generally do not do so. This fact has been exploited by some other fish species that are mimics of cleaner fish (e.g. wrasse blenny (Hemiemblemaria simulus L. & H.) mimics the juvenile blue head wrasse (Thalassoma bifasciatum B.) species to avoid being eaten by piscivores and exploit them as a food source (mucus) pretending to be cleaners (Deloach & Humann, 2003).

An interesting question that is still unanswered is what determines how attractive the client is to the cleaner. One possibility is that cleaners recognize clients’ parasite load and choose clients that are most infected (Gorlick, 1984), although some results have questioned the role of parasite loads as the only proximate cause of cleaning behaviour (Côté and Molloy, 2003). Another possibility is that the clients are chosen on the basis of the nutritional quality of their mucus (Gorlick, 1980; Bshary & Grutter, 2002a, 2003) but presumably mucus quality would have to be related to some signal that allows cleaners to recognize clients with high-quality mucus. However, there is no information on potential colour cues that cleaners may use in recognition and choice between clients. This is important for several reasons. Firstly, cleaners have been shown to be able to recognize familiar clients (Tebbich, Bshary & Grutter, 2002); more specifically, they can distinguish clients that have access to only one cleaning station from those that have access to more than one (Bshary & Grutter, 2002b; Bshary & Schäffer, 2002). Secondly, cleaners seem to be able to recognize piscivorous clients as well as their satiation level (Bshary & Würth 2001; Grutter, 2004). The ability to accurately recognize these clients may be visually based and colour may play a large role in this recognition process. This is supported by studies that show that client fish use colour cues to identify cleaners. Clients pose to cleaner

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models that possess basic coloration pattern of obligate cleaners—most often a lateral black stripe (Stummer et al, 2004; Arnal, Verneau & Desdevises, 2005), but use of ultraviolet colour cues may also be important (Losey, 2003). Experiments where naïve clients, the clients that have never encountered a cleaner before, are exposed to cleaners have shown that some clients have an innate posing response to cleaners whereas others need to learn what the cleaners look like before they pose to them (Losey, 1995). Furthermore, Deloach and Humann (2003) report that client bluestriped grunt (Haemulon sciurus S.) have an innate response (opening mouth to be cleaned) to the bright colours (orange and white horizontal stripes and black vertical bars) of an adult porkfish (Anisotremus virginicus L.) that only cleans as a juvenile. This response indicates that the colour pattern of cleaners may be recognized by clients and in this case may even represent a response to supernormal stimulus— an adult fish has larger colour surfaces than the juvenile cleaner. So if client fish use colours to recognise and respond to cleaners and if cleaners are able to recognize and respond to individual clients then perhaps they also use colour cues to do so. I tested this possibility by investigating client selectivity of cleaner fish Gobiosoma oceanops (neon goby) in Honduras, Central America.

1.2 Aims and hypotheses Aims To investigate how colour patterns influence the cleaner’s (Gobiosoma oceanops) choice of clients. To investigate the cleaners’ behaviour in situ and assess the extent of choice the cleaner has at the cleaning stations in the morning and afternoon.

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Hypotheses 1) The cleaner may prefer clients with contrasting colour features such as the black lateral stripe or yellow round blotch.

2) The cleaner may prefer clients with enlarged eyes or enlarged colour contrasts (supernormal stimuli).

3) The visiting and posing frequencies of different client species may differ in the morning and the afternoon hence limiting the cleaners’ choice.

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2. MATERIALS 2.1 Wooden models Wooden models of two reported client species for the neon goby (Colin, 1975) and one Indo-Pacific client species, were made in the Zoology department in Oxford.

These models were painted, using a non-toxic water-resistant paint.

To test hypothesis 1, three species of clients were used as models: clown wrasse (Halichoeres maculipinna M. & T.), blue chromis (Chromis cyanea P.) and achilles tang (Acanthurus achilles S.). The clown wrasse has a black lateral stripe running from its nose to its tail. Two models were made for this species: one containing the lateral black stripe and one without the stripe. Both models were 15 cm long. The blue chromis is a blue fish with contrasting black outlines along its back, tail and rear underside. To test the importance of the contrasting outlines two models were made: one with the normal outlines and one lacking the black outlines. Both models were 15 cm long. The achilles tang has a large yellow blotch at the base of its tail that contrasts with the deep blue colour of the rest of its body. Two models were made: one with the yellow blotch and one without the blotch. Both models were 20 cm long.

To test hypothesis 2, three blue chromis models were used: a model with the normal colour pattern, a model with an enlarged eye surface (2x original radius) and a model with enlarged black outlines. All three models were 15 cm long.

2.2 Neon gobies The gobies were caught from the coral reefs within the Cayos Cochinos Marine Reserve Area using a plastic net bought from a local supplier. Fourteen fish were caught in total. They varied in size from 1 to 4.5 cm. Neon gobies are obligate

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cleaners throughout their life so the size differences were not considered to be important for this study (Patrick Denning, field supervisor, personal communication).

The sex of the fish was not assessed as both sexes act as obligate cleaners and should show preferences for the same clients (Patrick Denning, field supervisor, personal communication). The fish were held in separate holding tanks between experiments.

They were left for 24 h to acclimatise to the new environment before the experiments started. The fish were not fed during the experiments (6 days in total) in order not to interfere with their motivation for cleaning (Lenke (1982) found no effect of cleaner (Labroides dimidiatus) satiation on propensity to clean dummy clients, however, the neon goby is a different species). Four fish died during the experiments. All the surviving fish were returned to their natural habitat.

2.3 Aquaria and other laboratory equipment Six aquaria were used: five of them as holding tanks and one as an experimental tank, and all of them were the same size. The tanks were 44.4 cm long and 30 cm deep. They were divided into three sections each of 14.8 cm in length using a black marker pen across the outside of all three tanks. The same markings were drawn on both holding and experimental tanks. The water was at ambient temperature, between 27-29 째C. Water in the experimental tank was changed every morning before the experiments. Water in the holding tank was refreshed during the night with an automatised inflow of sea water and an outflow of excess water.

Two stopwatches were used to measure time. Plastic nets were used for handling the fish. A transparent plastic tube was used to confine the fish to the middle of the tank. Transparent fishing line and transparent tape were used to attach the

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models to the sides of the tank. Dead coral found on the beach was put in the holding tanks when the experiments were not taking place to enrich the fishes’ environment.

2.4 Snorkelling equipment A short wetsuit, fins, mask and snorkel were used for snorkelling observations. The times were taken using a wrist stopwatch and recorded on a white board with a pencil.

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3. METHODS 3.1 Field site The research was carried out in Cayos Cochinos Marine Reserve Area in Honduras between 14th of July and 24th of August 2005. Research was divided into two phases: 1) Experiments in the aquaria using wooden models of the clients.

2) Snorkelling observations on the coral reef.

The aquaria experiments were based in the ‘wet lab’ at the Operation Wallacea field station on the Cayo Menor island. The snorkelling observations were made on the small coral reef just off the south coast of Cayo Menor island around the corner from the field station. The reef was 40 m away from the shore and was pristine except for some mechanical damage caused by boats on the edges of the reef and bleaching in the middle of the reef due to its shallow position. Twenty-eight cleaning stations were observed once, and they were situated within a depth range of 1.35-3.90 m.

3.2 Aquaria experiments The models were introduced into opposite sides of the tank after which a single neon goby was transferred from a storage tank into the experimental tank. Its movement was restricted with a transparent plastic tube placed in the middle of the tank. After two minutes the neon goby would resume a calm swimming or resting position. The tube was then removed and the amount of time spent in the three different parts of the tank was recorded during a five-minute interval. Each fish was tested twice for the same set of models, and the models were placed in the opposite ends of the tank on the second trial. This controlled for any biases a fish

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might have had for the sides of the tank and avoided the problem of pseudoreplication (Grafen & Hails, 2002). Each model set was tested on all the available cleaner fish.

The models were suspended from the top of the tank using a transparent nylon line, which was taped to the back side of the wooden models. Both sides of the models were painted the same colour.

Diagram 1. The experimental set up (side view). neon goby Model 1

Model 2

3.3 Snorkelling observations Twenty-eight cleaning stations were chosen randomly across the coral reef site. Water depth, visibility, coral species, and number of neon gobies were recorded.

When two gobies were present on the station the behaviour of both was noted.

Observations were carried out during two different time periods: morning (7.2010.15) and afternoon (15.00-17.15). Each station was observed for one 35-minute session. The total observation time was 14 hours: 7 hours in the morning and 7 hours in the afternoon. Observations were made following the method of C么t茅 and Molloy (2003): I floated 2 -2.5 m from the station, facing the incoming current to minimize net movement and started observations after a 5-minute delay to allow the fish to become accustomed to my presence. All fish swimming within 1m of the cleaning

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station were recorded as well as whether the client species posed or not, duration of pose, number and duration of inspections by the cleaner, and unusual behaviours by any of the fish present. One 35-minute observation of a juvenile French angelfish (Pomacanthus paru) was also carried out following the same method.

3.4 Analysis of data There was an error in performing the tank experiments: all the fish were presented with the models in the same order (i.e. model A left in trial 1 and right in trial 2) in trials 1 and 2, rather than the order being randomised among fish. This would be problematic if the fish was less willing to examine a model in the second trial (for example if the interest of the fish was reduced): the model and side would be confounded. Because of this, paired t-tests were used to test whether the fish were equally likely to examine the models in both trials. If there were significant differences for both time spent in a side and with a model in the two trials I could not average the results from the two trials due to the confounding effect of the side and the model. However, if either one of the t-tests for time spent with a model and in a side in the two trials gave a non-significant result I carried on with the analyses as if the experiment was a priori randomised. The time spent with one model of the pair was used as a measure of preference. The time spent with one model of the pair was averaged across the two trials and compared to the expected time using paired t-tests.

The expected time spent with a model A was calculated by adding the average time spent with the model A in the two trials ((time with A in trial 1+time with A in trial 2)/2) to the average time spent with a model B in the two trials ((time with B in trial 1+time with B in trial 2)/2) and dividing that number by 2. This was the expected average time spent with model A that was compared to the observed average time

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spent with the model A ((time with A in trial 1+time with A in trial 2)/2) by paired ttests or its non-parametric equivalent Wilcoxon Signed Rank test (when the assumptions of the parametric test did not hold) (Dytham, 2003). The same procedure was done to test preference for side of the tank.

The observational results were tabulated and presented in a graphical form.

Statistical analyses were not used for this part of the results because it was not possible to cohere to the assumptions of statistical tests such as the independence of data points. For example, I was not able to track individual fish throughout the observations so a single fish may have contributed to several data points.

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4. RESULTS 4.1 Results from the aquaria experiments Preliminary tests indicated that the side and model were not confounded due to experimental design error and the average values from the two trials were used to test for model and side preference. The results of the preliminary tests on time in the left side and with a model in the two trials are presented for each pair of the models tested as well as the main results of average time spent with a model compared to expected time spent with a model if there was no preference for either of the two models. The results for average time spent in the left side of the tank were given when there was a significant preference for a side.

Testing hypothesis 1: Models tested: lateral stripe versus no lateral stripe Halichoeres maculipinna

SQRT(time (s))

Figure 4.1: Square root transformed mean time the gobies spent in the left compartment in trial 1 and trial 2. Paired t-test indicates that the difference was not significant (t=-0.14, N=14, P=0.893). Bars are one standard error from the mean.

10 9 8 7 6 5 4 3 2 1 0 trial 1

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trial 2

Figure 4.2: Mean time the gobies spent with the model with no horizontal stripe in trial 1 and trial 2. The paired t-test indicates that the times were not significantly different (t=-0.16 N=14, P=0.879). Bars are one standard error from the mean.

140 120

Time (s)

100 80 60 40 20 0 trial 1

trial 2

The time neon gobies spent with the lateral stripe (normal looking) model was not significantly different from the time expected under the assumption of no preference between models (Paired t-test, t=0.33, N=14, P=0.746). This indicates that there was no preference for the model with the stripe.

Figure 4.3: Mean time the gobies spent with the model with no hor izontal stripe and expected mean time with the same model if there was no preference (Paired t-test, t=0.33, N=14, P=0.746). Bars are one standard error from the mean.

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Models tested: yellow blotch versus no yellow blotch Acanthurus achilles Figure 4.4: Mean time the gobies spent in the left compartment in the trial 1 and trial 2. Paired t-test indicates that the times were not significantly different (t=0.74, N=10, P=0.477). Bars are one standard error from the mean.

160 140

Time (s)

120 100 80 60 40 20 0 trial 1

trial 2

Figure 4.5: Median time the gobies spent with the yellow blotch model in trial 1 and trial 2. Wilcoxon Signed Rank test indicates that the times spent with the yellow blotch model by the fish was not significantly different in the trial 1 and trial 2 (T=20.0, N=6, P=0.059). Bars are indicating interquartile range.

250

Time (s)

200 150 100 50 0 trial 1

15

trial 2

The time neon gobies spent with the yellow blotch (normal looking) model was not significantly different from the time expected under the assumption of no preference between models (Paired t-test, t=0.32, N=10, P=0.753). However, the time spent in the left part of the tank was significantly longer than the time expected under the assumption of no preference for side (Paired t-test, t=2.42, N=10, P=0.039; data square root transformed). This indicates that there was no preference for the model with the yellow blotch but that there was preference for the left side.

Figure 4.6: Mean time the gobies spent with the yellow blotch model and expected mean time spent with the same model if there was no preference (Paired t-test, t=0.32, N=10, P=0.753). Bars are one standard error from the mean.

Figure 4.7: Mean time the gobies spent in the left compartment of the tank and expected mean time spent in the left compartment if there was no preference (Paired t-test, t=2.42, N=10, P=0.039. Bars are one standard error from the mean.

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Models tested: normal black markings versus no black markings Chromis cyanea Figure 4.8: Median time the gobies spent in the left compartment in the trial 1 and trial 2. Wilcoxon Signed Rank test indicates that the times were not significantly different (T=24.0, N=10, P=0.76). Bars are indicating interquartile range.

300

Time (s)

250 200 150 100 50 0 trial 1

trial 2

Time (s)

Figure 4.9: Mean time the gobies spent with normal black markings model in trial 1 and trial 2. Paired t-test indicates that the times were not significantly different (t=2.01, N=13, P=0.067). Bars are one standard error from the mean.

200 180 160 140 120 100 80 60 40 20 0 trial 1

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trial 2

The time neon gobies spent with the normal black markings model was not significantly different from the time expected under the assumption of no preference between models (Paired t-test, t=-0.32, N=13, P=0.757; data log transformed).

However, the time spent in the left part of the tank was significantly longer than under the assumption of no preference for side (Wilcoxon signed-ranks test, T=75.0, N=13, P=0.043). This indicates that there was no preference for the normal black markings model but that there was preference for the left side.

Figure 4.10: Mean time the gobies spent with the normal black markings model and expected mean time with the same model if there was no preference (Paired t-test, t=-0.32, N=13, P=0.757). Bars are one standard error from the mean.

150

Time (s)

125 100 75 50 25 0 Normal black markings model

Expected

Figure 4.11: Mean time the gobies spent in the left compartment and the expected mean time in the left compartment (Wilcoxon signed-ranks test, T=75.0, N=13, P=0.043). Bars are one standard error from the mean.

200

Time (s)

150 100 50 0 Left

Expected

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Testing hypothesis 2: Models tested: wide (enhanced) black edges versus normal black edges Chromis cyanea Figure 4.12: Median time the gobies spent in the left compartment in trial 1 and trial 2. Wilcoxon Signed Rank test indicates that the times did not significantly differ (T=19.0, N=7, P=0.447). Bars are indicating interquartile range.

350 300

Time (s)

250 200 150 100 50 0 trial 1

trial 2

Figure 4.13: Median time the gobies spent with the enhanced edges model in trial 1 and trial 2. Wilcoxon Signed rank test indicates that the times differed significantly (T=56.0, N=11, P=0.045). Bars are indicating interquartile range.

350 300

Time (s)

250 200 150 100 50 0 trial 1

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trial 2

The time neon gobies spent with the enhanced black edges model was not significantly different from the time expected under the assumption of no preference between models (Paired t-test, t= 0.16, N=12, P=0.876).

Figure 4.14: Mean time the gobies spent with the wide (enhanced) edges model and the expected mean time with the same model if there was no preference (Paired ttest, t= 0.16, N=12, P=0.876. Bars are one standard error from the mean.

150

Time (s)

125 100 75 50 25 0 Enhanced black edges model

Expected

Models tested: enlarged black eye (2xr) versus normal sized black eye Chromis cyanea Figure 4.15: Mean time the gobies spent in the left compartment in the trial 1 and trial 2. Paired t-test indicates that the times were not significantly different (t=0.92, N=10, P=0.380). Bars are one standard error from the mean.

140 120

Time (s)

100 80 60 40 20 0 trial 1

20

trial 2

Figure 4.16: Square root transformed mean time the gobies spent with the enlarged eye model in the trial 1 and trial 2. Paired t-test indicates that the times were significantly different (t=2.98, N=10, P=0.015). Bars are one standard error from the mean.

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SQRT(time (s))

10 8 6 4 2 0 trial 1

trial 2

The time neon gobies spent with the normal looking model was not significantly different from the time expected under the assumption of no preference between models (Paired t-test: t=-0.76, N=10, P=0.467).

Figure 4.17: Mean time the gobies spent with the enlarged eye model and the expected mean time with the same model if there was no preference (Paired t-test: t=-0.76, N=10, P=0.467). Bars are one standard error from the mean.

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4.2 Results from the in situ observations Testing hypothesis 3: All of the fish species that posed in the afternoon (18 species) were inspected, whereas 2 of the 14 species that posed in the morning were not inspected. Although a similar number of species passed by the cleaning station in both time periods (26 in the a.m. and 27 in the p.m.), more species posed in the afternoon. As far as the individual fish are concerned, 78% of those that posed in the a.m. got inspected, whilst 77% of all fish that posed in the p.m. got inspected. So almost the same percentage of individual fish that posed were inspected at the two time periods.

Figure 4.18: Number of fish species that visited, posed and got inspected at the neon goby cleaning stations in the morning and the afternoon.

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Table 4.1: Identity of fish species that visited cleaning stations in the two time periods.

Fish family Pomacentridae Pomacentridae

Fish species, a.m.

Dusky damselfish Yellowtail damselfish Pomacentridae Threespot damselfish Pomacentridae Sergeant major Pomacentridae Queen angelfish Pomacentridae Acantharidae Ocean surgeonfish Acantharidae Blue tang Chaetodontidae Chaetodontidae Foureye butterflyfish Chaetodontidae Scaridae Scaridae

Fish species, p.m.

Dusky damselfish Yellowtail damselfish Threespot damselfish Sergeant major Queen angelfish French angelfish Ocean surgeonfish Blue tang Banded butterflyfish Foureye butterflyfish Spotfin butterflyfish Striped parrotfish Stoplight parrotfish

Latin species name Chaetodon capistratus Microspathodon chrysurus Stegastes planifrons

Bluelip parrotfish Caesar grunt French grunt

Cryptotomus roseus Haemulon carbonarium Haemulon flavolineatum Haemulon chrysargyreum Haemulon album Haemulon aurolineatum Gramma loreto Hypoplectrus chlorurus Cephalopholis fulva Lutjanus apodus Ocyurus chrysurus Thalassoma bifasciatum Halichoeres radiatus Holocentrus species Carangoides ruber Sphyraena barracuda Aetobatus narinari Aulostomus maculatus Acanthostracion quadricornis

Scaridae Haemulidae Haemulidae

Striped parrotfish Stoplight parrotfish Caesar grunt French grunt

Haemulidae

Smallmouth grunt

Smallmouth grunt

Haemulidae Haemulidae Grammatidae Serranidae Serranidae Lutjanidae Lutjanidae Labridae Labridae Holocentridae Carangidae Sphyraenidae Myliobatidae Aulostonidae Ostraciidae

White grunt Tomtate Fairy basslet Yellowtail hamlet Schoolmaster Yellowtail snapper Bluehead wrasse Pudding wife Bar jack Great barracuda Spotted eagle ray Trumpetfish Scrawled cowfish

White grunt Fairy basslet Yellowtail hamlet Coney Schoolmaster Yellowtail snapper Bluehead wrasse Squirrelfish Bar jack Trumpetfish -

Total

26

27 23

Abudefduf saxatilis Holacanthus ciliaris Pomacanthus paru Acanthurus bahianus Acanthurus coeruleus Chaetodon striatus Chaetodon capistratus Chaetodon ocellatus Scarus iseri Sparisoma viride

Figure 4.19: Number of individual fish that visited (defined as swimming within 1m of the cleaning station), posed and were inspected at the neon goby cleaning station in the morning and the afternoon.

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Figure 4.20: Time spent posing and being inspected in the morning and the afternoon for species observed excluding french grunt, caesar grunt and schoolmaster. These three species spent much longer posing and being inspected than the others.

Table 4.2: Instances of neon gobies switching one client in favour of another one.

Switch from Smallmouth grunt Ocean surgeonfish (x2) French grunt Banded butterflyfish

Switch to Caesar grunt Caesar grunt (x2) Ocean surgeonfish Ocean surgeonfish

Table 4.3: Identity of fish species that were chased away by territorial dusky damselfish while approaching the station, posing or being inspected at the station.

Fish Number of times chased Activity preceding away the chase Foureye butterflyfish 3 Swimming by Spotfin butterflyfish 2 Swimming by Stoplight butterflyfish 1 Swimming by Yellowtail snapper 2 Swimming by Ocean surgeonfish 1 Being inspected Striped parrotfish 1 Swimming by Sergeant major 1 Posing Yellowtail damselfish 2 Being inspected or swimming by 25

5. DISCUSSION 5.1 Experiments The results from the experiments in aquaria show that the neon gobies had no significant preference for any of the models. These results indicate that hypotheses 1 and 2 cannot be accepted, at least not for the set of colour cues tested by this research.

There are several reasons why this might be so: 1) Neon gobies do not use colour cues at all when choosing clients.

2) Neon gobies use other cues (e.g. chemical, odour) in addition to visual cues when choosing clients.

3) Neon gobies do use visual cues when choosing clients but not the exact cues tested in these experiments.

4) Neon gobies use visual cues but they did not recognize the models as real fish so did not see them as potential clients.

5) Neon gobies were put in an unnatural situation and were too stressed to react in a meaningful way.

On the other hand, time spent in the left compartment was significantly higher than the expected time spent in the left compartment for three out of five analysed experiments. It is unclear why this should be so as the experimental design aimed at minimising external influences and differences within the experimental tank, however two possible explanations are as follows: 1) The light intensity was higher on the right side of the tank due to a window that was situated 1.5 m away from the experimental tank.

2) The inner surface of the tank’s far left corner might have been lined with a thicker layer of transparent glue that was keeping the tank sides together and neon gobies used this as a preferred substrate.

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The nature of neon goby’s vision is still unknown; there are no studies that present any evidence of colour vision in neon gobies. However, given the variety of colours that reef fish exhibit it seems probable that neon gobies have colour vision.

Poulin and Grutter (1996) quote good vision as one of the necessary characteristics fish need to have to evolve into obligate cleaners. The eyes of the neon goby appear qualitatively to be large with respect to rest of its body and this may be another indication of good vision in neon goby. Given that colour diversity plays a role in various fish species interactions and could have evolved in response to more than one selective pressure (Marshall, 2000) and also considering the large number of interactions a cleaner fish goes through every day with different species that all have distinct colour patterns it seems probable that neon gobies have evolved colour vision to recognize clients. Nevertheless, though plausible, this might not be the case. The results of this study suggest neon gobies do not use colour cues to select clients and therefore may be indicative of the absence of colour vision in neon gobies, yet other factors may account for the same results and the ability of neon gobies to perceive colours will have to be left for future studies.

If other cues are used by neon gobies to recognize fish as clients, cues such as body movement or odour, then my experiments may not be the best way of examining the cleaner’s species selectivity. The stiff wooden models did not move in the same way a real fish would move next to a cleaner and also all of the models were presented to the cleaners in a horizontal posture; both slight movements by the client and its bodily orientation during the pose may play large role in both initial recognition of a fish as a potential client and subsequent choice to inspect that fish. It has been shown that some behaviour in some fish species is governed primarily by olfactory cues, for example female association preferences in wild guppies (Shohet &

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Watt, 2004). It may be the case that neon gobies choose their clients on the basis of olfaction alone or in combination with other factors. A further study with live clients presented in the same (olfaction allowed) or adjacent (olfaction prevented) tanks might be useful for elucidating this issue.

Neon gobies might be using visual cues in their choice of clients but they might not be using contrasting outlines of the fish that were the main area of interest in the present study. Furthermore, they may be using colour cues in combination with UV colour cues; painting the models so the colours are visible to humans and seem equivalent to the colours of the wild client fish might not have the same effect on neon gobies and they may perceive the model colours differently due to a different vision spectrum. UV colour vision may be present in many reef fish and may play an important role in communication (Losey, 2003); unfortunately, UV vision was beyond the scope of my research. Furthermore, neon gobies could be using colour to distinguish ectoparasites from the background of the client body colour, again something that further research should look into.

The wooden models may not have been realistic enough for the neon gobies to recognize them as clients; this may have been due to the lack of other cues normally associated with a client fish and may further indicate the absence of innate response to colour stimuli and point towards more complex perception of the client fish by the cleaner. However, other studies with models presented to fish have yielded results (e.g. Grutter, Glover & Bshary, 2005). Maybe then, the fish were too stressed to act naturally in the experimental environment. In the wild, neon gobies have established cleaning stations where they advertise on a certain substratum such as brain corals but in the experimental tanks the only substratum present was smooth glass and neon gobies were introduced to a new environment shortly before their behaviour was

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tested; perhaps this did not give them enough time to establish the behaviour they would normally show at a cleaning station. Future research with these and similar fish study subjects may be improved if it allows cleaners to establish their cleaning stations within the tank before models are introduced into the cleaning stations.

The preference for the left side is curious since the experimental tank had symmetric left and right sides except for a small 1cm high circular (radius 0.5cm) protrusion on the left side that was inherent in the tank design and could not be removed. However, fish did not spend much time at this protrusion and it is unlikely to be the cause of preference for the left side. The window to the left side of the tank was further away from the tank than the window on the right side but shutters were closed on both windows and the main light source was a semi-transparent roof cover which let in a lot of light. Nevertheless, the light that did still reach the tank from the window through the shutters may have played a role in deterring gobies from the right side of the tank.

When fish spent a lot of time in the left compartment they seemed to mainly occupy the far bottom edge or the far corner of the tank and on subsequent examination of these places a layer of glue was found that was holding the tank together. Although transparent and small, this layer may have been a preferable substrate to the smooth glass for neon gobies and it may have caused significant preference for the left side. However, it is unclear why this was not the case in all of the experiments since the same fish were tested in all experiments.

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5.2 Observations Côté and Molloy (2003) found that clients were most often observed at the station in the afternoon when they had fewer ectoparasites, however my data show that number of fish species visiting stations were similar (26 and 27 in the a.m. and p.m. respectively) and that the number of fish visiting the stations was higher in the morning (230 and 191 in the a.m. and p.m. respectively). However, the proportion of fish species and individuals that posed in the afternoon was higher than in the morning (54% of species in the morning and 66% in the afternoon; 28% of fish individuals in the morning (78% of which got inspected) and 52% in the afternoon (77% of which got inspected)) which agrees with Côté and Molloy’s (2003) conclusion that the proximate cause of visiting cleaners might not be the number of parasites a client carries.

My observational data only notes 12 instances of clients queuing and a further 5 cases of cleaners switching mid-cleaning to a client of a different species. This may be an indication of the rarity with which neon gobies are offered choice of clients on the coral reefs around Cayo Menor. An alternative explanation may be that the fish were afraid of me; some species may be underrepresented and others may be absent from my results if they would normally visit cleaning stations in the absence of human observers. This may be due to similarity of a human snorkeller to predators or because of general cautious behaviour by some client fish when encountering a new situation. However, the study reef is often visited by student snorkellers working on various projects so it is not clear how afraid the fish were, although the possibility exists that they were.

Perhaps different species of cleaner fish have been exposed to different degrees of selection pressure to deal with choices between clients. In the 14 hours of

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observations neon gobies only had to choose between clients of different species 17 times. Given that 164 fish posed at the station in the same period of time, this suggests that only in around 10% of all potential inspection instances do neon gobies have a choice of clients. It is difficult to see how much a neon goby can benefit from making a choice or how much it has to lose if it makes a wrong choice in this 10% of its interactions.

Nevertheless, the fact that out of 164 instances of client posing, interaction followed in only 127 instances, or in 77.4% of the possible inspections, might suggest that a single interaction might not be of great importance to the cleaner. On the other hand, perhaps inspecting a client that does not carry enough ectoparasites or whose mucus is of low nutritional quality involves higher costs than benefits to the neon goby and this is the reason why they miss out on such a large percentage of posing clients. Following the same line of argument, one may see that a client carrying a large

number

of

ectoparasites

and/or

high

quality

mucus

might

offer

disproportionately greater benefits than costs to the cleaner. This would mean that neon gobies might be selected for making an appropriate choice in those 10% of cases when they have a choice. However, to make any substantial conclusions on costs and benefits associated with neon gobies’ choice, further studies quantifying energetic and other costs of inspection and benefits associated with inspecting different species of clients would have to be completed. If choice of client was one of major selective pressures operating on neon gobies, then one would expect to see effective fish species recognition abilities in the neon gobies.

Grutter (2001) found that experimentally manipulated clients carrying more parasites spent more time next to a cleaner than control clients; she presented this as evidence for parasitic load being a proximate cause of clients seeking cleaners. My

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results seem to contradict this conclusion since neon gobies ignore such a large proportion of clients that pose. If the clients were indeed posing because they had high levels of parasite infestation, then neon gobies would be expected to inspect them, which they did not do in 22.6% of all observed cases. Higher frequencies of posing than being inspected for most client species might be due to their seeking tactile stimulation rather than removal of parasites by the cleaners. This is also supported by an observation of terminal phase striped parrotfish posing inside sea plumes for 6s (pseudoposing (Deloach and Humann, 2003)) in the absence of any cleaners.

Bshary (2001) found that cleaners switched to larger clients significantly more often than to smaller clients, however, when he examined the data within each category of clients, size ceased to influence the cleaners’ choice. Furthermore, Bshary (2001) demonstrated that the overall significance in clients’ size was due to cleaners’ preference for harmless floaters (fish that have access to more than one cleaning station) over harmless residents (fish that only have access to one cleaning station), since floaters tend to be longer than residents (40 out of 51). Interestingly, cleaners also switched more often from a larger resident to a smaller floater than the other way around. These observations were also corroborated by the finding that resident clients had to queue significantly more often than floaters when both were present at the station and also that even a larger resident has to queue significantly more often while a smaller floater is being inspected rather than the other way around (Bshary, 2001).

Bshary’s (2001) data support the idea that the cleaners’ selectivity of clients is based on their recognition of the clients as being either floaters or residents, rather than simply choosing the bigger client. Although size has been found to be correlated to parasite levels (Sikkel et al., 2000) it may not be as important in client species

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selectivity as other factors (Bshary, 2001) although some evidence suggests that species with higher parasite levels do indeed visit cleaning stations more often than those with lower levels (Côté & Morand, 2001). The latter research was done on cleaning stations with gobies from the genus Evelynae in Barbados so it may be more indicative of what is happening with neon gobies in Honduras than the research with Labroides wrasses in the Indo-Pacific. The five instances of client switching that I observed included gobies switching from smallmouth grunt and ocean surgeonfish to caesar grunt and from french grunt and banded butterflyfish to ocean surgeonfish. In all of these instances the preferred client was larger, however other factors may play a more important role; for example, in all cases the cleaner had already associated with the client for 15-39s so it may have been close to ending the inspection anyway, and indeed in the case of the french grunt being switched for an ocean surgeonfish, the latter had posed for 5s previous to the switch. So, the small number of actual switching between clients and also the influence of other factors that have not been controlled (e.g. whether the fish is a floater or resident, the fish’s parasite load, etc.) prevents me from making any strong conclusions about which client species are preferred by neon gobies in the wild.

An interesting observation I made was that most neon goby stations are shared with resident territorial dusky damselfish. These fish acted violently towards visiting fish when they were present. They chased away 12 different fish from 8 different species that were either present at the s tation (e.g. an ocean surgeonfish and a yellowtail damselfish being inspected by a goby and a sergeant major posing) or arriving at the station. This may have a negative influence on neon gobies’ feeding opportunities and the extent to which it does so may be a topic of a future research.

Arnal and Côté (1997) found that significantly fewer client species and individuals

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visited gobies’ (Elacatinus spp.) cleaning stations that were within dusky damselfish territories than those that were not. On the other hand, they also found that dusky damselfish encountered increased costs due to lower foraging rate, more time spent chasing intruders and increased risk of egg predation (Arnal & CôtÊ, 1998). This poses questions of the potential benefit to these two fish species of sharing a territory; further research may elucidate whether gobies and dusky damselfish share territories because of necessity, in spite of the costs, or whether there are actual benefits that compensate the costs.

Two out of the eight species observed as being chased away by the dusky damselfish were never observed being inspected by the neon gobies: banded butterflyfish and foureye butterflyfish. An interesting topic for future research might include examining the parasite levels and parasite species composition of these clients to try and establish whether it is absence of gnathiid parasites on their bodies or the aggression of the dusky damselfish that discourages them from visiting neon goby stations. It may be possible for clients to visit different cleaner species to have different types of parasites removed. I have observed french grunt clients on several occasions at the neon goby stations posing by hovering horizontally in front of the goby; however I have also observed a french grunt posing vertically in a head-down position to a juvenile french angelfish cleaner. Perhaps visits to different cleaners include different posing behaviour by the clients; this might indicate that the speciesspecific cleaning interactions have been evolving separately due to similar selection pressures: different types of parasites. Parasites may play an important role in structuring interactions involved in the cleaning symbioses since they are a living and therefore evolving third party in these interactions (Grutter, 2002). This is exemplified in the research done by Jones, Grutter and Cribb (2004) that suggests a

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novel way of transmission of parasites (Digenea) through cleaning behaviour: a labrid cleaner may become infected by the parasite having ingested it off the client. My research ignored the effect that parasite presence might have on cleaner choice.

Nevertheless, I have not come across research describing colour changes that may result from high levels of parasites on the client fish but it seems reasonable to assume that high parasite levels may indeed have an effect on colour. Colour changes have been reported for some clients when visiting cleaning stations but have never been decisively researched (Trivers, 1971; Deloach & Humann, 2003).

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6. SUMMARY This project looked at the species selectivity of clients by cleaner fish neon goby, Gobiosoma oceanops, in the Cayos Cohinos Marine Reserve Area off the north coast of Honduras. The study incorporated two components: a laboratory based set of choice experiments and an in situ set of observation. The experiments presented the cleaners with a choice of two wooden models painted differently in an attempt to elucidate potential color cues that the cleaners use when choosing a client. The snorkeling observations included 28 different cleaning stations at two time periods (morning and afternoon) and recorded number of clients, number of client species, time that each client spent posing and/or being inspected and unusual behaviors in an attempt to look into the pattern of client availability to cleaners at different times of day and the extent of choice the cleaners have. The choice experiments yielded no positive preferences for any of the models which may be due to more complex perception mechanisms of neon gobies than simple recognition of color patterns visible to humans or due to the stress related to unnatural conditions the gobies encountered in the aquaria. The snorkeling observations found similar numbers of fish species visiting stations in both time periods as well as more individual fish visiting in the morning. The posing frequency was higher in the afternoon for both number of species and number of individuals that posed and also more fish got inspected in the afternoon. This finding seems to contradict the idea that fish visit stations when they have highest level of parasites, which occurs in the early morning due to gnathiid parasites activity patterns. The percentage of times neon gobies were presented with a choice of clients in the wild was 10% and the extent to which this has an influence on their fitness is unclear from the present study.

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7. ACKNOWLEDGEMENTS I would like to thank all the foundations that helped me fund the expedition: Keble Association, Barcapel Foundation, Alan Palgrave Brown Foundation and City of Rijeka; Operation Wallacea staff for making this and similar research projects possible, and Patrick Denning who supervised my project in the field and caught all the fish. I would especially like to thank Dr. Theresa Burt de Perera for patiently supervising my project at the University. I am grateful to Christopher Taylor and James Fox for snorkelling with me.

Picture 1: Two neon gobies, Gobiosoma oceanops, advertising on a brain coral (photo: James Fox)

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