Marine Biology (2001) 138: 777±784
Ó Springer-Verlag 2001
C. Arnal á S. Morand
Importance of ectoparasites and mucus in cleaning interactions in the Mediterranean cleaner wrasse Symphodus melanocercus
Received: 28 July 2000 / Accepted: 8 November 2000
Abstract In ®sh cleaning associations, the net bene®ts gained by clients and cleaners from cleaning have still not been clearly evaluated. In particular, the role of ectoparasitism and the importance of client mucus characteristics remain unclear for most cleaner ®sh species. This paper investigates the cleaning behaviour of the Mediterranean cleaner wrasse Symphodus melanocercus, based on observations, cleaner gut contents, client ectoparasites and mucus characteristics. We showed that this ®sh is a specialised cleaner ®sh, similar to some other tropical cleaner species. Gnathiid isopod larvae and caligid copepods represented a large proportion of the items preyed on by S. melanocercus. Although their feeding activity was related to their client ectoparasite load, it was also signi®cantly linked to client mucus load, which would indicate that the cleaning behaviour of S. melanocercus is not purely altruistic. Finally, as client visit to cleaning stations is related to their ectoparasitism, we propose that ectoparasite removal is likely to be a bene®t for the client ®shes of S. melanocercus.
Introduction The behaviour of cleaner ®shes, which remove ectoparasites, mucus and scales from the body of client ®shes, has been widely investigated (Gorlick et al. 1978; Losey 1987; Poulin and Grutter 1996; Sayer et al. 1996; CoÃte in press). Many species have been described as
Communicated by S. A. Poulet, Rosco C. Arnal (&) á S. Morand Centre de Biologie et d'Ecologie Tropicale et MeÂditerraneÂenne, Laboratoire de Biologie Animale (UMR 5555 CNRS), Universite de Perpignan, 66860 Perpignan Cedex, France e-mail:
[email protected] Fax: +33-468-662281
cleaner ®shes (van Tassel et al. 1994). They may be obligate cleaner ®shes, cleaning throughout their life-span, or facultative cleaner ®shes, cleaning only as juveniles or as females. Among obligate cleaner ®shes, variations in ectoparasite removal and feeding on benthic animals have been observed (Grutter 1997a). Within a species, cleaners may be very specialised, relying almost exclusively on ectoparasites, or nonspecialised, feeding on benthic animals and ectoparasites and mucus from their client's body surface (Grutter 1997a, 1999a). The net bene®ts gained by clients and cleaners from cleaning are yet to be evaluated properly. Although most of the cleaner ®sh removal experiments failed to ®nd any change in client health or behaviour (Youngbluth 1968; Gorlick et al. 1987; Grutter 1997b; but see Losey 1972 and Limbaugh 1961), two studies revealed that cleaners have an impact on their client's ectoparasite size and abundance (Gorlick et al. 1987; Grutter 1999b). Reliable data on ®sh ectoparasite load (in particular for Gnathidae and Caligidae) are, however, scarcely available, and the role of ectoparasitism has still not been clearly demonstrated from ®eld observations. Cleaner ®shes ingest mucus from the body surface of their client ®shes (Gorlick 1980). However, mucus is an amorphous material which cannot be clearly recognised in stomach content analysis. Thus, the importance of mucus in cleaning interactions should be assessed using another methodology. Gorlick (1980) found a qualitative agreement between the preference of the cleaning wrasse Labroides phthirophagus for speci®c clients according to their mucus load and energetic value. The importance of client mucus characteristics in cleaning remains unclear. This is regrettable considering that mucus may be a more reliable food source for cleaner ®shes than ectoparasites (Gorlick 1980), and may present an important glycoprotein source (Nakagawa 1988; Shephard 1994). The majority of past studies have concentrated on tropical cleaner ®shes. To our knowledge, from 1968 only 13% of the publications on cleaning have involved
778
temperate cleaner ®shes (Arnal, personal observation). This can be explained by the fact that temperate cleaner ®shes are generally considered to be less specialised than tropical ones (Limbaugh 1961; van Tassel et al. 1994; Zander and Nieder 1997; Galeote and Otero 1998), and that temperate regions oer less favourable conditions for ®eld observations (Potts 1968; Breen 1996). Furthermore, the costs and bene®ts of cleaning interactions between temperate cleaner and their client ®shes remain to be evaluated (Losey 1987; Poulin and Grutter 1996). Among cleaner ®sh species, the Labridae family is well represented, with 61 and 10 species in tropical and temperate waters, respectively (CoÃte in press). In the Mediterranean Sea, the most conspicuous cleaner wrasse is Symphodus (Crenilabrus) melanocercus (Risso, 1810). Few studies have described the cleaning behaviour of this cleaner wrasse, and many of them are mostly descriptive (Wahlert and Wahlert 1961; Potts 1968; Lejeune and Voss 1979, 1980; Zander and Nieder 1997). Symphodus melanocercus and its client ®shes show behaviour during cleaning interactions similar to that of tropical cleaner wrasses (Potts 1968). They live in ®xed territories, or ``cleaning stations'', which they defend aggressively against conspeci®cs (Potts 1968; Lejeune and Voss 1980; Zander and Nieder 1997). The relationship begins with a client's visit to the cleaning station. This may be initiated either by the cleaner ®sh, soliciting ®shes by swimming towards them, or by client ®shes which come to the cleaning station without being solicited (Wahlert and Wahlert 1961; Potts 1968; Lejeune and Voss 1979). The frequency of client visits is generally related to their density (Grutter and Poulin 1998; Arnal et al. 2000). Visiting clients may adopt a stereotyped horizontal, head-stand or tail-stand posture, considered ``pose behaviour'' (Potts 1968; Lejeune and Voss 1979). Then, S. melanocercus may inspect its client ®shes, picking at their body surface (Potts 1968). At any moment, the interaction may be terminated by the client ¯eeing away or, less frequently, by the client becoming aggressive toward the cleaner, probably when cleaner bites are aversive (Potts 1968; Lejeune and Voss 1979; Zander and Nieder 1997). Stomach content analyses of S. melanocercus have rarely been performed (Potts 1968; Lejeune and Voss 1979) and exploitable numerical data are not available. It appears, therefore, dicult to assess, for S. melanocercus, its level of specialisation in cleaning and to compare its feeding behaviour to that of tropical cleaner ®sh species. In this paper we investigate the cleaning behaviour of S. melanocercus, based on behavioural observations and on direct measurement of cleaner gut contents, client ectoparasites and mucus characteristics. We assessed three questions: (1) What is the cleaning activity of S. melanocercus? (2) What is the composition of the diet of S. melanocercus? (3) Are the behavioural patterns of cleaner ®sh and their client species, in the Mediterranean Sea, related to the client's mucus characteristics and/or ectoparasite load?
Materials and methods Data collection Study site and behavioural observations The study was carried out at Banyuls-sur-Mer (Mediterranean Coast, France), in July 1999. All observations were made at Ile Grosse, near the Laboratoire Arago. The study site was located at a depth ranging from 10.4 to 12.4 m, over a bottom covered by large boulders and pebbles. We identi®ed six cleaning stations at the beginning of the study (i.e. cleaner wrasse territories) and mapped them. We carried out observations by SCUBA-diving, between 0830 and 1800 hours. A single diver recorded all data directly on underwater paper from a distance of 3±5 m from the cleaner ®sh (according to the visibility). A period of 3±5 min prior to data recording allowed the ®sh to become used to the presence of the diver. We made a total of 1380 min of observations of cleaning stations. Each cleaning station was observed 8±11 times for a total of 200±265 min. We recorded, for each cleaning station, the number and species of clients solicited or visiting without being solicited by the cleaner. We considered that cleaners solicited their clients, when they left their cleaning station, swimming toward them. For each cleaning event, we noted whether the client posed, whether the interaction resulted in an inspection, the inspection duration, and the number of bites delivered to the client. Finally, we noted whether or not the interaction was terminated by the client ¯eeing away (Table 1). Assessment of ®sh density Fish density was obtained from visual counts. This was made by a single diver, swimming slowly along a 50 m transect line, and recording on a plastic slate all the client ®sh species present within 2 m on either side of the line (see Leum and Choat 1980; Galzin 1987, for similar methods). The sampling time was separated into three periods, four replicate counts were performed at 0930, 1400 and 1700 hours; we observed a minimum delay of 15 min between each count. Transect lines were positioned in the area where behavioural observations occurred, to assess more precisely the densities of client ®sh. We thus obtained 12 transects of 100 m2 (50 ´ 2 m) each (Table 1). Gut content analysis Symphodus melanocercus (n 20) were collected haphazardly by divers, following Grutter (1994). We used a 8 ´ 1.5 m barrier net with a 8 mm mesh; ®sh where captured with a hand net and placed in small plastic bags with an overdose of anaesthetic. Fish were preserved whole immediately after the dive (a maximum of 30 min after capture) in 10% formalin. Five ®shes were collected in each of the four collection periods: 0800±1000, 1000±1200, 1400±1600 and 1600±1800 hours, to control for daily variations in cleaner food consumption (Grutter 1996, 1997a; Arnal and CoÃte 2000). Each ®sh was weighed (mg) and measured (SL). Gut contents were categorised following Grutter (1997a) into: gnathiid isopod larvae, caligid copepod adults and pre-adults, other parasitic copepods (Peniculus ®stula and copepod larvae), ®sh scales, and benthic organisms (molluscs, cnidarian polyps), and free-living copepods. Gnathiid heads, with or without an attached body, were used to estimate the number of gnathiids, following Grutter (1996). Although mucus is sometimes considered in gut content analysis of cleaner ®shes (e.g. Grutter 1997a, 1999a), we could not readily distinguish it from other digested material in this study. We used two methods to assess cleaner wrasse gut contents (Hyslop 1980): the percentage cover of each food category was estimated visually in each of the 28 squares (1.5 ´ 1.5 cm) of a marked 9 cm diameter dish, then, discrete items were counted and removed from the dish to prevent double-counting.
779 Assessment of client ectoparasitism Client ®sh species (n 10) were chosen from behavioural observations but also according to their abundance on the reef, in order to collect the most abundant species. Fish collection took place at the same location as behavioural observations, at the end of July 1999. Fish were collected (n 5 per species) as described in the previous section. Fish were captured with a hand net and placed as quickly as possible in a sealed plastic bag. In the laboratory, ®sh were then removed from the bag, and the contents of the plastic bag rinsed and kept for latter ®ltration. Fish were soaked in 0.4% anaesthetic chlorobutanol (SIGMA, T-5138) for 90 min, and then the ®sh body surface and gills were rinsed with seawater (Grutter 1995). All liquids were ®ltered through ®lter paper (60 lm mesh). Filter and ®ltered materials were preserved in 10% formaldehyde in seawater. These were later examined under a binocular microscope (250 ´ )500 ´ magni®cation) and ectoparasites were isolated for identi®cation. According to gut content analysis, this method allowed us to record the major ectoparasite species eaten by S. melanocercus: Gnathia spp. and Caligus spp. Free-living copepods were identi®ed by Prof. A. Raibault (Station MeÂditerraneÂenne de l'Environnement Littoral, SeÁte, France). However, these copepods may live as epibionts on ®sh bodies (A. Raibault, personal communication), and to our knowledge, no studies have investigated this phenomenon. Note that the presence of epibionts may be attributable to the seawater taken during ®sh collection, as the bag contents were not ®ltered separately. We only estimated, for each ®sh species, the number of Gnathia spp., of Caligus spp., the
Table 1 Behavioural patterns and density (mean number per 100 m2) of 18 client species [Visit number of client visits; Solic. number of client solicitations by cleaner; Pose number of client inspection events; Inspect. number of inspections; Durat. inspection
number of copepod larvae and the number of non-parasitic copepods (Table 2); other ectoparasites (e.g. monogeneans) were not taken into account. Assessment of client mucus load and quality Client ®sh species (n 15) were collected (n 3 per species), as described in previous sections; ®sh where captured with a hand net and transported alive to the laboratory where they were maintained in aerated tanks with running seawater for a maximum of 6 h. As they were used for mucus removal, ®shes were killed, one by one, with a blow to the head. To assess mucus load and quality of each ®sh, we followed the method of Gorlick (1980). Each ®sh was rinsed in fresh water for 5 s to remove excess salt water from its body surface, and was then submerged for 60 s in a beaker containing tap water heated to 50 °C. This temperature was sucient to cause denaturation and coagulation of the surface mucus which turned a milky white colour. Fish were then suspended by the jaws over a beaker, their body surface was gently scraped using a scalpel to remove surface mucus (caution was taken not to remove ®sh epidermis). Surface mucus was collected in 5 ml tubes. The water/ mucus mixture was then dried in a 70 °C incubator chamber. Dried mucus was weighted (DW). The body surface of each ®sh (cm2) was estimated by ®tting aluminium foil to half of the client's body and ®ns; then we used a weight/area relationship with pieces of foil of known area. Mucus load (mg DW) was thus expressed per square centimetre of ®sh area (Table 3). Finally, the gross chemical com-
duration (s); Bite number of bites taken by cleaner wrasse on client's body; Flee number of cleaning events stopped by client's ¯eeing, all values on behaviour per 15 min observation]
Species
Density
Visit
Solic.
Pose
Inspect.
Durat.
Bite
Flee
Chelon labrosus Chromis chromis Coris julis Crenilabrus rupestris Diplodus annularis Diplodus puntazzo Diplodus sargus Diplodus vulgaris Labrus merula Mullus surmuletus Oblada melanura Sarpa salpa Serranus cabrilla Spicara maena Symphodus mediterraneus Symphodus melanocercus Symphodus ocellatus Symphodus tinca
0.41 29.75 19.58 1.66 1.91 1.33 5.16 5.41 0.66 11.16 1.41 4.91 5.33 3.41 5.41 3.25 3.75 14.41
0.04 16.24 3.01 0.30 0.32 0.23 8.97 0.57 0.14 0.41 0.13 0.34 1.51 0.86 1.49 0.08 0.76 7.98
0.04 0.33 0.38 0.07 0.02 0.03 0.83 0.43 0.07 0.39 0.10 0.15 0.58 0.16 0.04 0.01 0.02 1.25
0.00 15.76 2.62 0.23 0.30 0.19 8.97 0.13 0.06 0.02 0.02 0.19 0.92 0.47 1.44 0.05 0.74 6.64
0.00 14.91 2.74 0.28 0.17 0.19 7.89 0.29 0.09 0.15 0.05 0.34 1.33 0.75 0.98 0.08 0.44 7.27
0.00 32.44 6.34 0.80 0.57 1.51 27.01 1.07 0.91 0.78 0.28 1.60 7.19 1.28 2.09 0.25 0.76 33.70
0.00 10.24 1.68 0.25 0.19 0.56 8.76 0.35 0.40 0.25 0.08 0.61 1.95 0.33 0.55 0.10 0.19 11.76
0.04 0.59 0.14 0.02 0.03 0.02 1.96 0.42 0.04 0.32 0.13 1.13 0.34 0.43 0.02 0.00 0.11 0.41
Table 2 Mean number (SE) of Gnathia spp., Caligus spp., other parasitic copepods (Peniculus ®stula and copepod larvae) and non-parasitic copepods per ®sh collected from ten ®sh species (n = 5 ®sh per species), at Banyuls-surMer, Mediterranean Sea
Fish species
Gnathia spp.
Caligus spp.
Other copepods
Non-parasitic copepods
Chromis chromis Coris julis Diplodus sargus Diplodus vulgaris Mullus surmuletus Oblada melanura Sarpa salpa Serranus cabrilla Symphodus mediterraneus Symphodus tinca
0.4 1.8 0.4 0 1.2 0 0 1.2 2.2 5.6
2.6 0.2 3.2 0.4 0.4 0 0 1.2 0.6 0.2
9.6 0.2 1.0 0 0.4 0.4 0 43.8 1.6 1.8
0.4 0.2 0 2.6 1.6 2.6 1.1 0 3.4 1.9 0.4 0.2 1 0.6 4.4 2.3 0.6 0.4
0.2 0.7 0.2 0.6 0.8 0.7 2.8
1.1 0.2 1.0 0.4 0.2
0.5 0.6 0.2
4.2 0.2 0.8 0.2 0.2 22.6 0.5 0.6
780 Table 3 Mean (SE) client mucus load, mucus protein richness and calori®c value of 15 client ®sh species of Symphodus melanocercus, at Banyuls-sur-Mer, Mediterranean Sea. The index
of mucus quality was derived by averaging the ranks of mucus protein richness and mucus calori®c values, see ``Materials and methods ± Data analysis'' (n number of ®sh sampled)
Species
n
Protein (% DW)
Calories (mg)1 DW)
Index of mucus quality
Mucus load (mg cm)2)
Chromis chromis Coris julis Diplodus annulatis Diplodus puntazzo Diplodus sargus Diplodus vulgaris Labrus merula Mullus surmuletus Oblada melanura Sarpa salpa Serranus cabrilla Spicara maena Symphodus mediterraneus Symphodus ocellatus Symphodus tinca
3 3 3 3 3 3 1 3 3 3 3 3 3 3 3
40.83 67.91 43.75 52.70 57.75 59.21 38.05 41.54 41.25 49.37 51.16 38.98 65.42 63.75 64.00
3.14 5.42 2.47 3.08 3.81 4.18 5.02 6.98 2.34 3.55 3.51 2.37 5.19 4.64 4.97
4 14.5 4.5 6.5 9 10 6.5 10 2.5 7 7 2 13.5 11 12
1.94 0.47 1.40 2.51 1.05 1.42 2.08 1.55 0.21 2.42 1.30 0.59 2.33 0.43 1.55
3.70 9.96 1.65 2.20 9.79 0.58
1.43 1.30 1.65 5.34 1.27 2.32 5.12 2.17
0.10 0.35 0.17 0.17 0.75 0.29
0.11 0.05 0.66 0.42 0.05 0.22 0.43 0.55
position of dried mucus was estimated by carbon±nitrogen (CN) analysis. The samples obtained above were analysed in a CHN analyser (Service Central d'Analyse CNRS) for percentage of carbon (C%), nitrogen (N%) and ash (ash%). Protein content of client mucus was estimated according to Holland et al. (1991): protein (% DW) 6.25 ´ N%. Direct calorimetric measurement was not feasible so we used a substitute following Gorlick (1980). We thus approximated caloric content of client mucus as: calories/g DW 1351 + 106(C%) ) 21.1(ash%) (Table 3). Data analysis Phylogenetic relatedness among client species To take into consideration the phylogenetic relatedness among client species, we used a principal coordinate analysis method (PCoA) (Diniz-Filho et al. 1998; Legendre and Legendre 1998). This method allowed us to obtain a Euclidean representation (the principal coordinates in a Cartesian coordinate system) of a phylogeny presented as a distance matrix. The phylogeny of the 15 client ®shes was inferred from the taxonomy of Sasal et al. (1997). We obtained a (15 ´ 15) distance matrix (Euclidean distances) using the software PAUP 4.0 (Swoord 1999). From this distance matrix, we computed a PCoA (Legendre and Anderson 1999) and obtained 15 eigenvalues and the principal coordinates (eigenvectors) corresponding to the positive eigenvalues. To obtain the best possible Euclidean approximation of the original distances, we selected only the signi®cant eigenvalues using the broken-stick model (Diniz-Filho et al. 1998; Legendre and Legendre 1998). In our case, we selected only the ®rst of the 15 eigenvalues, which represented 64.3% of the variance. In¯uence of mucus characteristics and ectoparasitism In this study, for each client species, we considered seven dependent variables: the visit tendency (the residuals of the regression of total visit number, with and without being solicited, to cleaning stations versus client density; n 18, r2 0.657, b 1.162, P < 0.0001), the number of solicitations by cleaners (the residuals of the regression of solicitation number versus client density; n 18, r2 0.394, b 0.687, P 0.005), the pose tendency (the residuals of the regression of pose number versus visit number; n 18, r2 0.903, b 1.203, P < 0.0001), the inspection tendency (the residuals of the relationship of inspection number versus visit number; n 18, r2 0.961, b 1.129, P < 0.0001), the cleaner inspection duration (the residuals of the relationship of inspection duration versus inspection number; n 18, r2 0.918, b 1.003,
0.34 0.10 0.22 0.39 0.45 0.79
0.97 0.14 0.43 0.33 0.24 0.35 0.21 0.45
P
